Polímeros: Ciência e Tecnologia 1st. issue, vol. 32, 2022

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Políímeros meros Pol

Prof. Ailton de Souza Gomez GomeS Emeritus Emeritus Professor, Professor, IMA/UFRJ IMA/UFRJ

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VOLUME XXXII XXXII -- Issue Issue II -- Jan./Mar., Jan./Mar., 2022 2022 VOLUME

São São Paulo Paulo 994 994 St. St. São São Carlos, Carlos, SP, SP, Brazil, Brazil, 13560-340 13560-340 Phone: Phone: +55 +55 16 16 3374-3949 3374-3949 Email: Email: abpol@abpol.org.br abpol@abpol.org.br 2021 2021

DESCUBRA o conjunto de instrumentos que conduzem a percepções mais profundas sobre as PROPRIEDADES e ESTRUTURA DO POLÍMERO em cada etapa






ISSN 1678-5169 (Online)

Emeritus Professor Ailton de Souza Gomes We are all in grief because our dear Professor Ailton de Souza Gomes has passed away last December (12/24/2021). He was a reference in Polymer teaching and research in Brazil and renowned internationally. In 1968, he obtained his PhD in Chemistry from the University of Pennsylvania. Soon afterwards, he was invited to participate in a bilateral program between the National Academy of Science (NAS) and CNPq, called at that time National Research Council, for developing postgraduate teaching in chemistry in Brazil. Thus, he carried out a post-doctorate in the research group of Professor Charles G. Overberger, whose focus was the field of Polymer. Back in Brazil, Professor Ailton undoubtedly participated in the creation of the Institute of Macromolecules. By bringing his experience in this area to the recently created “Grupo de Polímeros” and having worked intensively at the Chemistry Institute of Federal University of Rio de Janeiro UFRJ since 1970 as a professor, he contributed to the consolidation and development of the Group which, in 1972, became the Macromolecular Nucleus and later, in 1976, “Instituto de Macromoléculas”. In the following year, he completed his Post-Doctorate at the Imperial College of Science and Technology as a fellow of the British Council. In 1978, he took up the position of coordinator of the Post-Graduate Course in Science and Technology of Polymers which was created in 1977. During his management, he organized the syllabus of the Postgraduate Course disciplines, which characterizes its interdisciplinarity and justifies its name which intended to bridge Polymer Science and Technology.


Throughout his professional career, he trained more than a generation of professors and researchers who are scattered all over the country, in federal and state universities, in research centers (CENPES), research institutes (INPI, INT) and industries. The 27 doctoral students he supervised are examples of leadership and professionalism in their respective academic fields. Many have reached outstanding positions as Professors, namely José Augusto Marcondes Agnelli (DEMA/UFSCAR), Juan Carlos Rueda Sanchez (PUC/Peru), Leni Akcelrud (UFPR), Raquel Santos Mauler (IQ/UFRGS), Suzana Lieberman (UFRGS), and the late Professor Fernanda Margarida Barbosa Coutinho (IMA/ UFRJ and UERJ). In addition to his interest in the field of polymers, he had always been a visionary and an innovative researcher, working on current and cutting-edge research fields. Being a tireless researcher he carried out investigation on nanostructured polymeric materials, nanocomposites for prolonged drug release and membranes for use in fuel cells and nanostructured polymeric materials. In addition to working in the academic field, he also had professional experience in the industry. Thus, between 1972 and 1976, as a Petroleum Chemist at PETROBRAS, he participated in the installation of the Polymer Laboratories at the company’s Research Center (CENPES).It involved everything from the organization of the laboratory and research line guidelines up to the specification and purchase of equipment. In 1977, he created the Postgraduate Program in Science and Technology of Polymers, which awarded the highest rating at CAPES since



2013. Additionally, during this period, he was also involved in strategic research projects for PETROQUISA. In one of them, he was the direct supervisor of the development of Ziegler-Natta catalysts for isoprene polymerization. Even on unpaid leave, he continued to assist his students in carrying out the research they had been developing until the completion of their graduate works. In 1987, he returned from unpaid leave and was transferred to the Instituto de Química at UFRJ. On this occasion, in order to increase the partnership between the private sector and the public university, he held, on a part-time basis, the position of Petroleum manager, of the recently opened Ultra Group Research Center (Mauá/SP). He acted as a researcher and consultant in other projects, always aiming to contribute to the scientific and technological development of the country. In 1992 he returned to IMA and in 1994 he became a Full Professor therein. He was elected Director of this Institute in 1995 and served at the institution for twelve years. He created the Lato Sensu Post-Graduate Course in Plastics and Rubbers in 2000. His well-known competence was demonstrated by the activities he had carried out and recognized through honors, titles and awards, such as: - Teaching Fellowship – University of Pennsylvania, Philadelphia/PA (USA), 1966; -

Associate Researcher – The University of Michigan, Ann Arbor/MI (USA), 1969;

- Member of CNPq/NAS – Program of Postgraduate Research and Teaching in Chemistry in Brazil, from 1969 to 1976; -

Visiting Professor, Case Western Reserve University, Cleveland/OH (USA), 1989;


Queen’s Fellowship – Scholarship of Queen Elizabeth II, of England, for post-doctoral studies, 1977;

- Eloisa Mano Award from the Brazilian Polymer Association, ABPol, 2007.

In addition to his academic and technical activities, he also acted as advisor and consultant to government agencies CNPq, CAPES, FAPESP, FAPERJ, among others; as a member of the Editorial Board of the Polímeros – Ciência


e Tecnologia journal (since 1991); as an editorial consultant for the international journals such as: Journal of Applied Polymer Science, Polymer International, Journal of Membrane Science, Electrochimica Acta; as editor of Macromolecular Symposia (n. 245-246), in 2006 and representative of Brazil in the Polymers Division of IUPAC (2006-2008). Considering his participation in administrative positions, since 1993 he was Director of the largest polymer association in Brazil - Associação Brasileira de Polímeros, ABPol, and later his President (1995-1997), participating in the activities of the Association either in the organization of the III Brazilian Congress of Polymers, in Rio de Janeiro (1995) or in the creation of the Southeast Regional and later the South Regional. Professor Ailton also participated in the organizing committee of the I, II, III and IV Polymer Seminars (SEMPOL), held in 1978, 80, 82 and 84, respectively. He was responsible for coordinating the V Brazilian Polymer Congress, in Águas de Lindóia / SP (1999), and coordinator of the organizing committees of the IV Latin American Polymer Symposium, in Gramado / RS (1994); World Polymer Congress – MACRO 2006/41st International Symposium on Macromolecules, in Rio de Janeiro/RJ (2006). He was the representative of Brazil in the coordination of SLAP 2008, held in Lima, Peru and Macro 2012 at Virginia Tech, in the United States. He was part of the organizing committee of the European Polymer Federation EPF 2013, held in Pisa, Italy in June 2013. His entrepreneurial character and professional experience in the industry brought innovations to the University, generating new horizons for research, contributing significantly to the scientific community in Brazil and abroad. To conclude may I emphasize how important and participative his family was, in the person of his wife Teresa and his four children, who stood by his side, supporting him through his long and brilliant academic career until the moment he left us.

Maria Inês Bruno Tavares Director, Instituto de Macromoléculas Professora Eloisa Mano, Universidade Federal do Rio de Janeiro – UFRJ, Rio de Janeiro, RJ, Brasil


ISSN 0104-1428 (printed) ISSN 1678-5169 (online)

P o l í m e r o s - I ss u e I - V o l u m e X X X I I - 2 0 2 2 Indexed


“ C h e m ic a l A b s t r a c t s ” — “ RA P RA A b s t r a c t s ” — “A l l - R u s s i a n I n s t i t u t e ­T e c h n ic a l I n f o r m a t i o n ” — “ L a t i n d e x ” — “ W e b o f S ci e n c e ”


S ci e n c e


Polímeros E d i t o r i a l C o u nci l

Editorial Committee

Antonio Aprigio S. Curvelo (USP/IQSC) - President

Sebastião V. Canevarolo Jr. – Editor-in-Chief

Members Ailton S. Gomes (UFRJ/IMA), Rio de Janeiro, RJ (in memoriam) Alain Dufresne (Grenoble INP/Pagora) Bluma G. Soares (UFRJ/IMA) César Liberato Petzhold (UFRGS/IQ) Cristina T. Andrade (UFRJ/IQ) Edson R. Simielli (Simielli - Soluções em Polímeros) Edvani Curti Muniz (UEM/DQI) Elias Hage Jr. (UFSCar/DEMa) José Alexandrino de Sousa (UFSCar/DEMa) José António C. Gomes Covas (UMinho/IPC) José Carlos C. S. Pinto (UFRJ/COPPE) Júlio Harada (Harada Hajime Machado Consutoria Ltda) Luiz Antonio Pessan (UFSCar/DEMa) Luiz Henrique C. Mattoso (EMBRAPA) Marcelo Silveira Rabello (UFCG/UAEMa) Marco Aurelio De Paoli (UNICAMP/IQ) Osvaldo N. Oliveira Jr. (USP/IFSC) Paula Moldenaers (KU Leuven/CIT) Raquel S. Mauler (UFRGS/IQ) Regina Célia R. Nunes (UFRJ/IMA) Richard G. Weiss (GU/DeptChemistry) Rodrigo Lambert Oréfice (UFMG/DEMET) Sebastião V. Canevarolo Jr. (UFSCar/DEMa) Silvio Manrich (UFSCar/DEMa)

A ss o ci at e E d i t o r s Alain Dufresne Bluma G. Soares César Liberato Petzhold José António C. Gomes Covas José Carlos C. S. Pinto Paula Moldenaers Richard G. Weiss Rodrigo Lambert Oréfice

D e s k t o p P u b l is h in g


“Polímeros” is a publication of the Associação Brasileira de Polímeros São Paulo 994 St. São Carlos, SP, Brazil, 13560-340 Phone: +55 16 3374-3949 emails: abpol@abpol.org.br / revista@abpol.org.br http://www.abpol.org.br Date of publication: September 2021

Financial support:

Available online at: www.scielo.br

Polímeros / Associação Brasileira de Polímeros. vol. 1, nº 1 (1991) -.- São Carlos: ABPol, 1991Quarterly v. 32, nº 1 (Jan./Mar 2022) ISSN 0104-1428 ISSN 1678-5169 (electronic version)

Website of the “Polímeros”: www.revistapolimeros.org.br

1. Polímeros. l. Associação Brasileira de Polímeros. Polímeros, 32(1), 2022




Editorial Section Editorial................................................................................................................................................................................................E1 News....................................................................................................................................................................................................E5 Agenda.................................................................................................................................................................................................E6 16th Brazilian Polymer Conference - 16th CBPol Ouro Preto, MG, October 24 to 28, 2021 Kátia Monteiro Novackand Rodrigo Lambert Oréfice ..................................................................................................................................... 1-5

O r i g in a l A r t ic l e A review on research, application, processing, and recycling of PPS based materials Larissa Stieven Montagna, Marcel Yuzo Kondo, Emanuele Schneider Callisaya, Celson Mello, Bárbara Righetti de Souza, Ana Paula Lemes, Edson Cocchieri Botelho, Michelle Leali Costa, Manoel Cléber de Sampaio Alves, Marcos Valério Ribeiro and Mirabel Cerqueira Rezende............................................................................................................................................................................ 1-12

Effect of hollow glass microspheres addition on density reduction and mechanical properties of PA6/glass fibers composites Thaysa Rodrigues Mendes Ferreira, Matheus de Alencar Lechtman, Filipe Lauro Dias and Aline Bruna da Silva....................................... 1-9

Incorporation of Aloe vera extract in bacterial nanocellulose membranes Lya Piaia, Camila Quinetti Paes Pittella, Samara Silva de Souza, Fernanda Vieira Berti and Luismar Marques Porto................................ 1-8

Scaffold based on castor oil as an osteoconductive matrix in bone repair: biocompatibility analysis Fabianne Soares Lima, Luis Felipe Matos, Isnayra Kerolaynne Pacheco, Fernando Reis, João Victor Frazão Câmara, Josué Junior Araujo Pierote, José Milton Matos, Alessandra Ribeiro, Walter Moura and Ana Cristina Fialho............................................. 1-5

CO2 adsorption by cryogels produced from poultry litter wastes

Lídia Kunz Lazzari, Daniele Perondi, Ademir José Zattera and Ruth Marlene Campomanes Santana........................................................... 1-7

Cellulose nanocrystals into Poly(ethyl methacrylate) used for dental application Andressa Leite, Hamille Viotto, Thais Nunes, Daniel Pasquini and Ana Pero................................................................................................. 1-7

Ecological structure: production of organic impregnation material from mussel shell and combustion

Hüseyin Tan, Murat Şirin and Hasan Baltaş.................................................................................................................................................... 1-8

Fatigue damage propagation and creep behavior on sisal/epoxy composites Mateus da Silva Batista, Linconl Araujo Teixeira, Alisson de Souza Louly, Sayra Oliveira Silva and Sandra Maria da Luz......................... 1-9 13

C ss-NMR Singular value decomposition and fitting for sorghum proteins conformation elucidation

Tatiana Santana Ribeiro, Juliana Aparecida Scramin, José Avelino Santos Rodrigues, Rubens Bernardes Filho, Luiz Alberto Colnago and Lucimara Aparecida Forato.............................................................................................................................................................................. 1-6

Determination leaching of boron from Oriental beech wood coated with polyurethane/polyurea (PUU) hybrid and epoxy (EPR) resins

Çaglar Altay, Hilmi Toker, Mustafa Kucuktuvek, Mehmet Yeniocak, İlknur Babahan Bircan and Ergun Baysal............................................. 1-9

Cross-link density measurement of nitrile rubber vulcanizates using dynamic shear test Gustavo Ninho Campos, Ana Carolina Ribeiro Coimbra, Arianne Aparecida da Silva, Elisson Brum Dutra da Rocha, Felipe Nunes Linhares, Cristina Russi Guimarães Furtado and Ana Maria Furtado de Sousa...................................................................... 1-6

Effect of non-thermal argon plasma on the shear strength of adhesive systems Isabella de Almeida Guimarães Passos, Juliana das Neves Marques, João Victor Frazão Câmara, Renata Antoun Simão, Maíra do Prado and Gisele Damiana da Silveira Pereira................................................................................................................................................................. 1-7 Cover: Prof. Ailton de Souza Gomez Emeritus Professor, IMA/UFRJ Arts by Editora Cubo.


Polímeros, 32(1), 2022

NEW LIGHTWEIGHT MATERIAL IS STRONGER THAN STEEL The new substance is the result of a feat thought to be impossible: polymerizing a material in two dimensions. Using a novel polymerization process, MIT chemical engineers have created a new material that is stronger than steel and as light as plastic, and can be easily manufactured in large quantities. The new material is a two-dimensional polymer that self-assembles into sheets, unlike all other polymers, which form one-dimensional, spaghetti-like chains. Until now, scientists had believed it was impossible to induce polymers to form 2D sheets. Such a material could be used as a lightweight, durable coating for car parts or cell phones, or as a building material for bridges or other structures, says Michael Strano, the Carbon P. Dubbs Professor of Chemical Engineering at MIT and the senior author of the new study. “We don’t usually think of plastics as being something that you could use to support a building, but with this material, you can enable new things,” he says. “It has very unusual properties and we are very excited about that.” The researchers have filed for two patents on the process they used to generate the material, which they describe in a paper appearing in Nature. MIT postdoc Yuwen Zeng is the lead author of the study. Two dimensions Polymers, which include all plastics, consist of chains of building blocks called mers. These chains grow by adding new molecules onto their ends. Once formed, polymers can be shaped into three-dimensional objects, such as water bottles, using injection moulding. Polymer scientists have long hypothesized that if polymers could be induced to grow into a two-dimensional sheet, they should form extremely strong, lightweight materials. However, many decades of work in this field led to the conclusion that it was impossible to create such sheets. One reason for this was that if just one mer rotates up or down, out of the plane of the growing sheet, the material will begin expanding in three dimensions and the sheet-like structure will be lost. However, in the new study, Strano and his colleagues came up with a new polymerization process that allows them to generate a two-dimensional sheet of polyaramide. For the monomer building blocks, they use a compound called melamine, which contains a ring of carbon and nitrogen atoms.

Polímeros, 32(1), 2022

Under the right conditions, these monomers can grow in two dimensions, forming disks. These disks stack on top of each other, held together by hydrogen bonds between the layers, which make the structure very stable and strong. “Instead of making a spaghetti-like molecule, we can make a sheet-like molecular plane, where we get molecules to hook themselves together in two dimensions,” Strano says. “This mechanism happens spontaneously in solution, and after we synthesize the material, we can easily spin-coat thin films that are extraordinarily strong.” Because the material self-assembles in solution, it can be made in large quantities by simply increasing the quantity of the starting materials. The researchers showed that they could coat surfaces with films of the material, which they call 2DPA-1. “With this advance, we have planar molecules that are going to be much easier to fashion into a very strong, but extremely thin material,” Strano says. Light but strong The researchers found that the new material’s elastic modulus is between four and six times greater than that of bulletproof glass. They also found that its yield strength, or how much force it takes to break the material, is twice that of steel, even though the material has only about one-sixth the density of steel. Matthew Tirrell, dean of the Pritzker School of Molecular Engineering at the University of Chicago, who was not involved in the study, says that the new technique “embodies some very creative chemistry to make these bonded 2D polymers. An important aspect of these new polymers is that they are readily processable in solution, which will facilitate numerous new applications where high strength to weight ratio is important, such as new composite or diffusion barrier materials.” Another key feature of 2DPA-1 is that it is impermeable to gases. While other polymers are made from coiled chains with gaps that allow gases to seep through, the new material is made from mers that lock together like LEGOs, and molecules cannot get between them. “This could allow us to create ultrathin coatings that can completely prevent water or gases from getting through,” Strano says. “This kind of barrier coating could be used to protect metal in cars and other vehicles, or steel structures.” The research was funded by the Center for Enhanced Nanofluidic Transport (CENT) an Energy Frontier Research Center sponsored by the U.S. Department of Energy Office of Science, and the Army Research Laboratory. Source: Massachusetts Institute of Technology – news.mit.edu




June Polymer Sourcing & Distribution – 2022 Date: June 28-30, 2022 Location: Hamburg, Germany Website: www.ami.international/events/event?Code=C1186 2nd Global Conference on Advances in Polymer Science and Nanotechnology Date: June 27-28, 2022 Location: Berlin, Germany Website: polymerscience.peersalleyconferences.com European Advanced Materials Congress Date: June 25 - July 2, 2022 Location: Genoa, Italy Website: www.advancedmaterialscongress.org/europe/ EPF – European Polymer Congress Date: June 26 - July 1, 2022 Location: Prague, Czech Republic Website: www.epf2022.org

July 49th World Polymer Congress – MACRO2022 Date: July 17-21, 2022 Location: Winnipeg, Canada Website: www.macro2022.org PVC Formulation Asia - 2022 Date: July 19-20, 2022 Location: Bangkok, Thailand Website: www.ami.international/events/event?Code=C1178 84th Prague Meeting on Macromolecules – Frontiers of Polymer Colloids Date: July 24-28, 2022 Location: Prague, Czech Republic Website: www.imc.cas.cz/sympo/84pmm/ 8th International Conference on Chemical and Polymer Engineering (ICCPE’22) Date: July 31 - August 2, 2022, (hybrid) Location: Prague, Czech Republic Website: cpeconference.com

August Baltic Conference Series Date: August 15-17, 2022 Location: Stockholm, Sweden Website: www.advancedmaterialscongress.org/baltic-spring/ The Global Meet on Bio-Polymers and Polymer Science (GMBPPS2022) Date: August 25-27, 2022 Location: Paris, France Website: primemeetings.org/2022/polymer-science Advanced Energy Materials & Technology Congress Date: August 28-31, 2022 Location: Stockholm, Sweden Website: www.advancedmaterialscongress.org/energy/ International Conference on Nanomaterials & Nanotechnology Date: August 28-31, 2022 Location: Stockholm, Sweden Website: www.advancedmaterialscongress.org/icnano/ 7th International Conference and Exhibition on Polymer Chemistry Date: August 30-31, 2022 Location: London , United Kingdom Website: polymerchemistry.euroscicon.com/

September Polymer Physics Meeting — Retirement Conference for Dame Athene Donald Date: September 12-13, 2022 Location: Cambridge - United Kingdom


Website: events.iop.org/polymer-physics-meeting-retirementconference-dame-athene-donald Advances in Polyolefins (APO-22) Date: September 18-21, 2022 Location: Rohnert Park (Northern California), United States Website: www.polyacs.net/22apo 1st Baltic Symposium on Polymer and (Bio)Materials Science - “From Materials Design to Advanced Structures” Date: September 22-23, 2022 Location: Szczecin, Poland Website: balticbiomat.zut.edu.pl/

October Advanced Materials World Congress Date: October 1-8, 2022 Location: Venice, Italy Website: www.advancedmaterialscongress.org/world/ POLYMER CONNECT - Polymer Science and Composite Materials Conference Date: October 3-5, 2022 Location: Rome, Italy Website: polymersconference.yuktan.com/ Polymers and Nanotechnology Date: October 16 - 19, 2022 Location: Napa, United States Website: www.polyacs.net/21polynano POLYMAT — International Conference on Polymers and Advanced Materials Date: October 16 - 21, 2022 Location: Huatulco, Oaxaca, Mexico Website: polymat.iim.unam.mx/ 7th International Conference on Multifunctional, Hybrid and Nanomaterials Date: October 19 - 22, 2022 Location: Genoa, Italy Website: www.elsevier.com/events/conferences/internationalconference-on-multifunctional-hybrid-and-nanomaterials Polymers in Medicine and Biology Date: October 23 - 26, 2022 Location: Napa, United States Website: www.polyacs.net/21polynano PRE 2022 — 11th International Workshop on Polymer Reaction Engineering Date: October 23 - 27, 2022 Location: Arizona, United States Website: engconf.us/conferences/chemical-engineering/polymerreaction-engineering-xi/ NuMat2022: The Nuclear Materials Conference Date: October 24 - 28, 2022 Location: Ghent, Belgium Website: www.elsevier.com/events/conferences/the-nuclearmaterials-conference American Advanced Materials Congress Date: October 29 – November 5, 2022 Location: Miami, United States Website: www.advancedmaterialscongress.org/america/

2023 January POLY-CHAR 2023 Date: January 22-27, 2023 Location: Auckland, New Zealand Website: www.poly-char2023.org/

March 18th International Plastics and Petrochemicals Trade Exhibitions Date: March 21-24, 2023 Location: Riyadh, Saudi Arabia Website: saudi-pppp.com/saudi-plastics-petrochem

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38th International Conference of the Polymer Processing Society Date: March 22-23, 2023 Location: St.Gallen, Switzerland Website: www.pps-38.org/

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April 24th International Conference on Wear of Materials Date: April 16-20, 2023 Location: Alberta, Canada Website: www.wearofmaterialsconference.com/


ABPol Associates Sponsoring Partners

Polímeros, 32(1), 2022


ISSN 1678-5169 (Online)

16th Brazilian Polymer Conference - 16th CBPol Ouro Preto, MG, October 24 to 28, 2021

The Brazilian Polymer Conference (CBPol), organized biannually since 1991 by the Brazilian Polymer Association, is the most important scientific event in the field of polymers in the country, bringing together professionals from universities, research institutes, government agencies and companies interested in the development of technologies for the production, characterization and application of polymers. The 16th edition of CBPol (16th CBPol) intended to transport the event to a more extensive international scope. For this, the submitted papers were all mandatorily written in English, as well as the oral and poster presentations had to give priority to the use of the English language. The 16th CBPol was initially planned to be held in the Minas Gerais city of Ouro Preto. However, uncertainties and concerns about the COVID19 pandemic forced its establishment in virtual form. Thus, the 16th Brazilian Congress of Polymers was held virtually (remotely) from October 24 to 28, 2021. Despite being held remotely, the event kept the theme associated with its initial headquarters

(Ouro Preto/MG). In addition, the opening ceremony was broadcast live from the historic center of Ouro Preto with the presence of members of the organizing committee, such as Profa. Kátia Novack (Chair), Prof. Marco Aurelio De Paoli (ABPol´s president) and Prof. Cláudio Gouvêa dos Santos, among others (Figure 1). Following the speeches, awards were given to remarkable Brazilian researchers: Prof. José Carlos Costa da Silva Pinto (UFRJ) received the ABPol “Profa Eloisa Mano” Polymer Science prize, Prof. Juliano Elvis de Oliveira (UFLA) received the “ABPol Young Researcher” prize and the ABPol “Polymer Technology” prize was awarded to Eng. José Ricardo Roriz Coelho (ABIPLAST) (Figure 2). The ceremony was closed with a concert performed by the Trio Amaranto, a vocal group from Belo Horizonte (Figure 3). Another non-technical highlight of the congress was short videos that were broadcast in between sessions of the congress with subjects such as: how to make “pão-de-queijo”, how to speak “mineirês” and how to become a real “mineiro”, regionalism of the people from Minas Gerais State.

1. The Opening Cerimony broadcast online. From left to right, Prof. Cláudio Gouvêa dos Santos (Member of organizing committee), Prof. Kátia Novack (Member of organizing committee), Prof. Marco Aurelio De Paoli (ABPol’s president) and Prof. Máximo Eleotério Martins (Adjunct Pro-Dean of Administration at the Federal University of Ouro Preto)

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Novack, K. M., & Oréfice, R. L.

2. The ABPol 2021 awardees. (a) Prof. José Carlos Costa da Silva Pinto (UFRJ), ABPol “Profa Eloisa Mano” Award. (b) Eng. José Ricardo Roriz Coelho (ABIPLAST), ABPol Technology Award. (c) Prof. Juliano Elvis de Oliveira (UFLA) ABPol Young Researcher Award. 2/5

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3. The concert was performed by Trio Amaranto.

The congress had 15 plenary presentations lasting 50 minutes, which featured highly prestigious speakers in the field of polymers from Brazil and abroad. In addition to the plenary sessions, 5 keynote lectures of 30 minutes and 129 oral presentations of 20 minutes each were held. The oral presentations were shown simultaneously in 5 rooms throughout the days of the event. Three (3) poster sessions of 100 posters each were held with so-called e-posters, in which the posters were viewed online together with a short presentation recorded by the authors, which remained available for conversation via the platform chat during the presentation. Seven (7) technical lectures were held during the event, covering subjects such as polymer analysis and processing techniques. During the event, 456 scientific and technical papers associated with polymers were presented. Around 198 teaching and research institutions and companies in the polymer area were represented at the event. In addition, papers from 23 different countries were presented, including countries in America (Central, South and North), Europe, Asia and Africa. In Brazil, papers from 20 different states in the 5 regions of the country were presented. These numbers demonstrate the impact of the event and its importance in national and international contexts. Of the 456 papers presented at the 16ºCBPol, 289 (63%) were presented by students, and of these papers, 62 were presented by undergraduate or technical education students and 227 by graduate students (master and PhD). These numbers clearly show a high level of participation of students in the event. The presentation of papers at the event allowed the students to be exposed to a select and extensive audience that evaluates, comments, criticizes and congratulates the works done, which are considerably important for their future carriers. In addition, the event expanded and amplified knowledge in areas of great importance to the country (polymer materials) for these Polímeros, 32(1), 2  022

students and other participants in subjects not normally addressed in technical and higher education courses. Over the years, CBPol has always played a leading role in informing and discussing issues related to polymer science and technology. The following renowned researchers participated in the 16th CBPol as invited and keynote speakers: 1. Alejandro J. Müller - University of the Basque Country UPV/EHU (Spain) – “Application of the SSA thermal fractionation technique to thermoplastic polyurethanes and recycled polyolefin blends” 2. Amin Shavandi - Université Libre Bruxelles (Belgium) – “Restore and Regenerate Our Natural Resources: High-value Biobased Products for Biomaterials Applications” 3. André Studart - ETH Zürich (Switzerland) – “3D Printing of Polymers into Biologically-Inspired Structures” 4. Anthony Brennan - University of Florida (USA) – “Recent Advances In Antifouling Technologies” 5. Denise Petri – USP (Brazil) – “Polysaccharides as platforms for new functional materials” 6. Filomena Barreiro – Instituto Politécnico de Bragança (Portugal) – “Microencapsulation: a world of possibilities” 7. Jaime C. Grunlan - Texas A&M University (USA) – “Water-Based Multifunctional Nanocoatings from Polyelectrolyte Complexation: Opportunities & Challenges” 8. Jose Covas – Universidade do Minho (Portugal) – “Inprocess characterization of polymer nanocomposites during extrusion “ 3/5

Novack, K. M., & Oréfice, R. L. 9. Mohammad Arjmand – The University of British Columbia (Canada) – “Advanced Polymer-based Electromagnetic Shields” 10. Patrícia Patrício – CEFET-MG (Brazil) - “Integrated technologies using polymers that contribute to the reduction of waste from different industrial sectors” 11. Richard Weiss – Georgetown University (USA) – “Complex Polymeric Materials Through Self-Assembly Initiated by Small Molecules: Reversible Gels, Ionomers, and Rubbery Polymers” 12. Rosario Bretas – UFSCar (Brazil) – “Electrospinning: An Old Technique For New Applications”

13. Sabu Thomas – Mahatma Gandhi University (India) 14. Sadhan C. Jana – The University of Akron (USA) – “Design of Polymer-Derived Nanoparticles, Nanorods And Porous Microparticles” 15. Silvia Bettini – UFSCar (Brazil) – “Industrial citrus waste: an opportunity to obtain biodegradable and flexible compounds” 16. Ulrich Wiesner – Cornell University (USA) – “Polymer Self-Assembly based Functional Materials”

Figure 4 shows images captured during the online conference as an example on how the virtual presentations and other activities occurred.

4. Two moments taken during the online event. 4/5

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It is worth mentioning that a commission established by the scientific committee of the 16th CBPol evaluated all posters presented by students and awarded the three best posters in the undergraduate/technical education, master’s and PhD categories. To this end, at least two different evaluators had contact with the paper, presentation and student of each poster. After this evaluation, the evaluators filled out an online questionnaire that was used to define the best posters. Winners received books and coupons from scientific publishers.

Organically Modified With Oregano Essential Oil For Active Packaging Applications, Superviser: Larissa Nardini Carli.

Best Poster Awards - PhD Students 1° - BISMARK NOGUEIRA DA SILVA, Universidade Federal de Juiz de Fora (UFJF), Title: Preparation Of Silver Nanoparticles Using Polyaniline/Cellulose Acetate Blend Films For Sers Application, Superviser: Celly M. S. Izumi.

Best Poster Awards - Undergraduate Students

2° - LUCIANA MENTASTI, Centro de Investigaciones en Física e Ingeniería del Centro de la Provincia de Buenos Aires (CIFICEN) - INMAT, Argentina, Title: Luminescent YVO4:Eu3+: from nanoparticles to nanocomposites.

1° - HEMILLY CIRQUEIRA MARTINS, Fundação Universidade Federal do Tocantins (UFT), Title: Myrcene Release By Chitosan Loaders Crosslinked With Sodium Tripolyphosphate Or Glutaraldehyde, Adviser: Ana Maria da Silva Maia. 2° - VANESSA ZIMMER KIEFFER, Universidade Federal do Rio Grande do Sul (UFRGS), Title: Influence Of Physical Treatment In Curaua Fiber On Post-Consumption Hdpe Composites, Superviser: Ruth Marlene Campomanes Santana. 3° - YAN FARIA GUIMARÃES SILVA, Univerdade Federal de São Carlos (UFSCar), Title: PLA/SEP nanocomposites: Effect of nanoparticle content on the short-term mechanical properties, Superviser: Juliano Marini.

Best Poster Awards - Master Students 1° - MATHEUS FERNANDES FLORES, Universidade de São Paulo (USP) - Instituto de Química de São Carlos, Title: Solubilization of cellulose by supercritic CO2 in the presence of superbase DBU, Superviser: Antonio Aprigio da Silva Curvelo. 2° - CHAIANE NEUMANN, Instituto Federal de Educação, Ciência e Tecnologia do Rio Grande do Sul, Title: Interaction Between Type I And Ii Coagents And Polybutadiene Isomers On Crosslinking Systems Using Organic Peroxide, Superviser: Edson Francisquetti. 3° - PÂMELA ROSA OLIVEIRA, Universidade Federal de Santa Catarina (UFSC), Title: Kaolin Nanoclays

Polímeros, 32(1), 2  022

3° - VANESSA OLIVEIRA CASTRO, Universidade Federal de Santa Catarina (UFSC), Title: Electrical And Morphological Properties Of Aligned Plga/ Ntcs Mats For Tissue Engineering, Superviser: Claudia Merlini.

We would like to thank the organizing and scientific committee as well as the symposia coordinators and the ad hoc referees, who played a key role in achieving the excellent scientific quality of the conference. We also thank the funding agencies that supported the event, CNPq and FAPEMIG, as well as the sponsors and exhibitors. We also gratefully acknowledge ABPol, its board and employee (Mr. Marcelo P. Gomes) and the APTOR team, for all of their support in the organization of the event. Finally, we thank all of the participants of the 16th CBPol who contributed significantly to the success of the conference. We look forward to see you all at the 17th CBPol!

Kátia Monteiro Novack1 and Rodrigo Lambert Oréfice2 Chair, Departamento de Química – DEQUI, Universidade Federal de Ouro Preto – UFOP, Ouro Preto, MG, Brasil 2 Scientific Coordinator, Departamento de Engenharia de Metalúrgica e de Materiais – DEMET, Universidade Federal de Minas Gerais – UFMG, Belo Horizonte, MG, Brasil



ISSN 1678-5169 (Online)


A review on research, application, processing, and recycling of PPS based materials Larissa Stieven Montagna1* , Marcel Yuzo Kondo2 , Emanuele Schneider Callisaya2 , Celson Mello2 , Bárbara Righetti de Souza1 , Ana Paula Lemes1 , Edson Cocchieri Botelho2 , Michelle Leali Costa2,3 , Manoel Cléber de Sampaio Alves2 , Marcos Valério Ribeiro2  and Mirabel Cerqueira Rezende1  Laboratório de Tecnologia de Polímeros e Biopolímeros – TecPBio, Universidade Federal de São Paulo – UNIFESP, São José dos Campos, SP, Brasil 2 Departamento de Materiais e Tecnologia – DMT, Universidade de São Paulo – UNESP, Guaratinguetá, SP, Brasil 3 Laboratório de Estruturas Leves – LEL, Instituto de Pesquisas Tecnológicas – IPT, São José dos Campos, SP, Brasil 1


Abstract Among the engineering thermoplastics, poly(phenylene sulfide) (PPS) stands out for its excellent properties and mainly for processing at lower temperatures. The requirements requested by industries can be made by improving mechanical strength, weight reduction, and durable components by reinforcing the PPS matrix with fiberglass (FG) and carbon fiber (CF). This review intends to present the most current research related to the physical, mechanical, and thermal properties of PPS and the PPS/FG and PPS/CF composites most currently used by the aerospace, automotive, and energy industries. In addition to presenting the feasibility of mechanical and thermal recycling processes for PPS-based waste to reinsert a high market value thermoplastic into the industrial production cycle, thus contributing to the minimization of waste destined for landfills and incinerated or even improperly disposed of in the environment. Keywords: applications, carbon fiber, composites, fiberglass, poly(phenylene sulfide). How to cite: Montagna, L. S., Kondo, M. Y., Callisaya, E. S., Mello, C., Souza, B. R., Lemes, A. P., Botelho, E. C., Costa, M. L., Alves, M. C. S., Ribeiro, M. V., & Rezende, M. C. (2022). A review on research, application, processing, and recycling of PPS based materials. Polímeros: Ciência e Tecnologia, 32(1), e2022005.

1. Introduction The diversified aerospace, automotive, and energy industries have similar requirements and interest in highperformance materials that are lightweight but strong enough to take high loads. Thus, the use of engineering thermoplastics, such as poly(phenylene sulfide) (PPS), poly(ether-ether-ketone) (PEEK), poly(aryl-ether-ketone) (PAEK), and poly(ether-imide) (PEI), has been arousing great interest in these industrial sectors[1]. PPS has stood out among these engineering thermoplastics due to its low processing temperatures (~280 – 320 ºC)[2]. Furthermore, PPS is a semi-crystalline polymer formed by alternating sulfur atoms and aromatic rings. Due to this configuration and the stability of the molecular structure, unique characteristics are granted, such as good thermal stability, low thermal degradation[3], high flame resistance, superior mechanical properties (high modulus, tensile strength, good dimensional stability, good fatigue resistance), good chemical stability (resistant to solvents), and low moisture absorption. These properties make PPS an exciting and essential engineering polymer[2,4]. In 1888, Friedel and Crafts discovered the PPS. In 1967, the production methodology was developed, and only

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in 1972 was PPS commercialized by Phillips Petroleum Company[3,5]. Since then, it has been produced and used widely and commonly in several areas, such as in aerospace[6], automotive[7], and wind energy industries[8]. PPS-based materials, such as composites and blends, have been widely used to meet high-performance requirements, for example, in aerospace structures such as wings, tails, fuselages[9]; in the automotive industry, in motor vehicle engine compartments, pump housings, lamp and headlight bases, and in the renewable energy sector[1], in wind turbine blades[10]. To meet the requirements of industries, thermoplastics can be reinforced with fiberglass (FG) and carbon fiber (CF). So, it is possible to obtain the structure lightness, excellent mechanical resistance, and, consequently, more durable components due to its high strength-to-weight ratio[11,12]. PPS can also be used in blends when a physical or chemical mixture of different polymers is sought to obtain a material with similar or superior properties, thus complementing each component or even achieving a specific set of functional properties. Therefore, through the appropriate choice of matrices, it is possible to obtain a material with greater ease



Montagna, L. S., Kondo, M. Y., Callisaya, E. S., Mello, C., Souza, B. R., Lemes, A. P., Botelho, E. C., Costa, M. L., Alves, M. C. S., Ribeiro, M. V., & Rezende, M. C. of processing, more tenacity, greater strength, lightness, and low cost, among other qualities[13]. The thermal properties of PPS-based materials must be understood because its great importance in the conformation and processing steps of components. Proper processing parameters are essentials in obtaining good mechanical and physical properties of the produced components. Machining of PPS-based matrix composites with fiber-reinforcement, mainly FG and CF, has been studied in recent years[14-16]. Lack of experience in machining fiber-reinforced PPS is a factor that affects the quality of the machined pieces, which can harm finished parts in terms of mechanical strength, fatigue resistance, and dimensional accuracy. The anisotropic property of reinforced PPS changes the material removal mechanisms during the machining, compared to conventional metal machining. The main problem is the delamination of the layers that form the composite. Searching optimal cutting parameters for minimal delamination occurrence with minimal tool wear and higher production are the biggest challenges in composite material machining research. The excellent characteristics of engineering thermoplastics, especially PPS, have increased their demand and, consequently, the generation of waste from the production process to the end of the product’s useful life. Composites and blends based on PPS have high technology in the manufacturing process combined with processing complexity. Recycling often becomes unfeasible, resulting in the destination of sanitary landfills and incineration of these residues[17]. However, due to environmental pressures, industries that consume these materials have been showing interest in recycling, aiming not only at waste and environmental concerns but also at the possible recovery of valuable capital from these materials. It is currently possible to recycle PPS-based materials through mechanical and thermal recycling, which may be

a viable, lucrative, and ecologically correct solution for the disposal of waste based on PPS, in addition to minimizing the destination of these wastes in landfills and incineration[18,19]. These solutions can an economic return from these residues, which may be reinserted in the production process of the same sectors generated or even used as raw material for other industries, as indicated in Figure 1. Thus, this review article focused on the feasibility and possible application of the PPS in components in the aeronautics, automobile, and renewable energy industries. Furthermore, the present review article reports the latest studies (2018–2022) related to the application, processing, thermal properties, welding, and feasibility of recycling PPS, aiming to show viable and profitable recycling processes for PPS waste with a high market value.

2. Applications PPS is a high-performance thermoplastic and semicrystalline polymer with excellent mechanical and thermal properties. Alternating para-substituted rings of phenylene and sulfur atoms form its linear and rigid chemical structure (Figure 2), which confers its particular characteristics, as per example, its high melting temperatures, being between 270-290 °C; glass transition, approximately 90 °C; of thermal decomposition around 508 °C, and coefficient of linear thermal expansion of 49 µm/m°C[3,20]. The exceptional properties of engineering thermoplastics combine high performance, relatively easy processing for structural parts with complex geometry, lightness, and consequently reduced consumption of fossil fuels, which leads to a reduction in the release of greenhouse gases into the environment, in addition, to being corrosion-resistant. Thus, due to the numerous qualities of thermoplastics, the demand and replacement of many metals and metallic

Figure 1. Feasibility of the economic and environmental cycle of PPS-based components. 2/12

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A review on research, application, processing, and recycling of pps based materials alloy components have gradually increased day by day[21]. Figure 3 shows the possible applications of PPS-based materials in the aerospace, automotive, and energy industries.

2.1 Aerospace Currently, the aerospace sector leads the consumption and application of thermoplastic and thermoset composites. Approximately 50% of polymer composites are applied in aircraft structural parts, and 40% are manufactured carbon composites[6,9]. Boeing 787 was the first aircraft that used composites as the primary material in constructing its structure, mainly in the main wing and fuselage. The payoff of included materials composites, about 50% composites by weight (80% by volume), is a 20% reduction in fuel consumption compared to similar-sized conventional aircraft[22,23]. A few years after the presentation of the Boeing 787, Airbus launched the A350, made up of precisely 53% of composite materials, distributed among fuselage, wings, landing gear, windows, doors, keel beam, and the empennage, consisting mainly of reinforced polymers with CF, and with

Figure 2. Chemical structure of PPS.

savings of up to 25% in fuel consumption[24,25]. Other Airbus aircraft, such as the A320 and A340, feature 15%, and the A380 features 25% composite materials, mainly PPS/CF. Polymeric composites are applied in ailerons, rudder, flaps, spoilers, elevator, vertical, and horizontal stabilizers, wing panels (leading and trailing edges), landing gear doors, nacelles, flap rail fairing, and wing box, and on the A380, it is the first aircraft ever equipped with a central wing box made of composite material. In both aircraft mentioned, PPS is used in the composition, since currently the PPS is widely applied in aircraft components, such as interior parts, passenger seats, overhead cabinets, aerodynamic stabilizers, and wing trailing edge panels[9]. Other aircraft that have PPS in the components materials is the Fokker 50 and Gulfstream G650 use PPS/CF composites on critical control surfaces (rear rudder and elevator)[6,26]. These are some of the examples of the growing use of these composite materials in aircraft structures.

2.2 Automotive Polymers are widely used in the automotive industry due to their numerous qualities highlighting lightweight, good dimensional stability, excellent fracture and fatigue resistance, ease of processing, and corrosion resistance. Approximately 24% of thermosets and 50% of thermoplastics are used in this sector; polypropylene (PP)[27] and polyurethane (PU)[28,29] are the most used thermoplastics in the automotive industry[7,30,31]. GF and CF reinforced thermoplastics are always widely used in some external components of automobiles, such as crash boxes, leaf springs, bumper beams, fenders, spoilers, and spare wheel wells and are intended to reduce the weight of automotive components further, in addition to having the advantages of reduced manufacturing cost, due to shorter processing time[32]. The reinforcement of thermoplastics with CF is intended to facilitate the integration of parts, low cost,

Figure 3. Possible applications of PPS-based materials in the aerospace, automotive, and energy industries. Polímeros, 32(1), e2022005, 2022


Montagna, L. S., Kondo, M. Y., Callisaya, E. S., Mello, C., Souza, B. R., Lemes, A. P., Botelho, E. C., Costa, M. L., Alves, M. C. S., Ribeiro, M. V., & Rezende, M. C. and weight reduction. Regarding the application of CF in thermoplastics, advances in manufacturing technology are required to obtain an excellent cost-benefit ratio for large quantity production, in addition to the high cost[33]. PPS is relatively still little used; however, it is possible to observe an increase in automakers based on scientific research[34]. As one of the results of these studies, Bosch verified the feasibility of applying PPS (Ryton® PPS, Solvay) in an active vacuum brake booster connecting the piston for use in sports vehicle braking systems. With this replacement, Bosch verified a cost reduction of 84%, in addition to a weight reduction of 78%[21,35]. Moran, Lake, and Dole[34] used PPS (Fortron®, Ticona) to manufacture pumps used with harsh fluids at elevated temperatures. The authors chose to use PPS due to its excellent mechanical properties, resistance to cracking and deformation, in addition to its resistance to high temperatures (200 ºC), and because of its chemical stability, since the pump was in contact with corrosive substances. PPS is already being used in structural components, such as motor vehicle engine compartment, fuel rail, pump impellers, thermostat housing, throttle body, ignition coil bobbin, micro-precision injection molded parts, encapsulation of computer chips, and other sensitive electronic components, lamp and headlight bases, pump housings[1]. Furthermore, in the automotive sector, blends are widely applied. According to the study carried out by Begum, Rane, and Kanny[36], the automotive industry is responsible for the greater use of polymer blends being applied to the exterior, interior, and underhood components of automobiles.

2.3 Energy In the renewable energy industry, wind turbine blades have always been made of polymeric composites, approximately 80% of which are thermosets, due to the high strength, stiffness, and ease of processing processes[37]. The wind turbine blades require that the material composition has good mechanical strength to resist gravitational and wind forces, greater rigidity to provide stability, and good fatigue resistance to support the cyclical load that the turbine blades are submitted[38]. Currently, the use of thermosets leads the application in wind turbine blades. However, the blades have been suffering from erosion that causes a significant loss in the aerodynamics efficiency of turbine blades and consequently reduces the wind turbine’s annual performance by between 2 – 25%[39]. Based on this problem, carried out a comparative study of the replacement of thermosets by thermoplastics, such as PP and PU, as they are more ductile and thus, increase the lifetime of wind turbine blades[40,41]. The application of thermoplastics in wind turbine blades has shown great potential, together with many advantages[42,43]. Thermoplastics have more excellent ductility reducing degradation, thus avoiding erosion than thermosets[43]. Thermoplastics have shown excellent aerodynamic performance, and reduced gravity forces may have lowered manufacturing costs. If they need any repair, welding is possible, eliminating the need for adhesive bonds between blade components and increasing the overall strength. Thus, they grow the valuable life of wind turbine blades, 4/12

and in addition, can be recycled after a product has served its useful life[44,45]. Complying with European regulations[46], as the European Union Directive on landfill waste, prohibit the disposal of large parts of composite materials, such as wind turbine blades, in landfills[47]. Given the problems exposed by the use of thermosets and the advantages of thermoplastics, the interest of industry and research in incorporating thermoplastics in the composition of wind turbine blades has been increasing. Research using thermoplastics is still minimal; however, given the existing solutions, a material with properties of high tensile strength, high ductility, and high elongation at break is necessary. As mentioned above, some thermoplastics are already being used, such as PP and PU. However, there are other strong candidates for the composition of wind turbine blades, such as polycarbonate (PC), polyethylene terephthalate (PET), polyamide 69 (PA69), polyamide 11 (PA11), and the PPS[8,10].

4. Processing PPS has relatively easy processability compared to other engineering thermoplastics. Thus, components made of PPS can be molded or extruded, and currently, they are processes widely used industrially[48]. According to Zuo et al.[2], processing by injection is not feasible due to the high fluidity of PPS in the molten state, which requires the use of a closure nozzle and curing with sealed mold. PPS has a high melting temperature (between 270 - 290 °C) requiring high processing temperatures (close to 350 °C), which can often be an obstacle in the use of the material due to the need for unconventional processing equipment, which operates in high temperatures, which often lead to increased costs in the final product[20]. Another problem in PPS processing is the atmosphere in which it is carried out, since the presence of oxygen in contact with the polymer at a high temperature can lead to a degradation of its chains, reducing the properties and useful life of the final part[20,49]. Compression molding is widely used industrially, mainly for PPS composites with some fiber, such as GF and CF[50]. This processing occurs through the lamination of semi-pregs, which are mats with the fiber fabric already impregnated with the polymer matrix. Bruijn and Hattuam[51] used compression molding to make the panel door for a rotorcraft from the recycling of PPS/CF composites. Zhao et al.[52] produced high-performance PPS/CF composites with a high content of reinforcing fabrics (80%) through the hot compression molding. On the other hand, the extrusion process is more used to process the polymer with loads of smaller sizes or obtain polymer blends[53]. To prepare lightweight and highperformance polymer foams with tailored morphologies and excellent properties, Ma et al.[54] used extrusion processing to improve the homogenization of the PPS/PEEK blend. In the study of Lin et al.[55], extrusion processing was used to homogenize the PPS/PA blend with graphene. As with all thermoplastics, a drying process before extrusion processing is always recommended. For PPS, the literature shows that drying must be carried out at a temperature of 120 °C for 3 h[20]. If the PPS has some Polímeros, 32(1), e2022005, 2022

A review on research, application, processing, and recycling of pps based materials carbonaceous load, drying is essential, as water absorption increases in these cases[20,49,53]. Another relatively new process widely used for thermoplastics is three-dimensional (3D) printed through fused deposition modeling (FDM). It can manufacture highperformance components, including parts from PPS-based materials. However, this method involves a complexity associated with selecting its appropriate manufacturing parameters. Many processing parameters can be adjusted to optimize the process to reduce printed parts’ time, cost, quality, and mechanical performance in high-performance components. According to the study carried out by Geng et al.[56], FDM has several challenges due to its inherent crystallization and thermal crosslinking properties of PPS. When the authors verified the degree of crystallinity (Xc) and crosslinking of the PPS, they observed that the thermal history affects the properties of the PPS when printed in three dimensions. Therefore, the authors presented in their study that the accuracy of 3D published PPS samples can be improved using forced-air cooling in the molten deposition modeling. Thus, the balance between mechanical strength and ductility can be improved through changes in heat treatment conditions. El Magri et al.[57] studied the influence of response surface methodology, nozzle temperature, print speed, and layer thickness to optimize output responses, namely Young’s modulus, tensile strength, and Xc through the use of FDM. According to the results obtained by the authors, layer thickness was the most influential printing parameter on Young’s modulus and Xc, as optimal factor levels were reached at nozzle temperature at 338 °C, print speed of 30 mm/s, and layer thickness of 0.17 mm. The authors carried out the reprocessing using various temperatures to eliminate the residual thermal stress generated during the FDM and improve the Xc of the produced parts. The authors found that a temperature of 200 °C for 1 h could improve PPS printed pieces’ thermal, structural, and tensile strengths. Yeole et al.[58] showed that PPS/CF pellets are excellent raw materials for additive manufacturing. The authors showed that the high printing temperatures (between 285 and 400 ºC) did not degrade the CF and did not oxidize the PPS, something that is very worrying for the final properties of the pieces. Furthermore, the authors found that the printed works did not show many voids and that the last properties in all the printing techniques analyzed could be improved by increasing the concentration of CF in the pellets.

5. Machining of PPS/Fiber composites The thermoplastic matrix reinforced fiber leads to higher mechanical strengths, boosting the composite applicability but damaging the machining[59]. In general, reinforced components require a high surface finish and dimensional accuracy only by machining processes affirm that most GF and CF reinforced composites components in the industry are manufactured lacking precision dimension, demanding different finish or semi-finish adjustment machining[60]. Moreover, according to Chen et al.[13], the addition reinforces fibers in the PPS matrix modifies the tribological properties and electrical and thermal conductivity of the PPS blends Polímeros, 32(1), e2022005, 2022

and composites. Therefore, the machinability study should also consider several variables, such as the variation of composition and properties.

5.1 Main characteristics of machining PPS/fiber composites Fiber-reinforced thermoplastic composites are generally manufactured close to the final dimensions, and therefore it is necessary to apply a machining process to meet the dimensional requirements. Carbon fiber-reinforced polymer (CFRP) pieces, used in the aeronautic industry, are assembled by riveting and bolting. To fix components and structure, holes are needed in the CFRP parts, and the most used process for this is conventional drilling[61,62]. High hardness, strength, abrasion resistance and thermal conductivity reduce efficiency, machining quality and accelerate tool wear. Poor machining quality reduces strength against fatigue, compromised structure integrity, and prejudices assembly tolerances[61]. The high abrasiveness of the reinforced constituents, such as CF, make composite materials more difficult to machine than traditional metal materials. Chip formation is also different in machining ductile metals and polymer matrix composites. While machining ductile metals results in curled and continuous chips, the polymer matrix fiber-reinforced composite machining forms crumbled and fragmented chips by fracture of fibers, failure of the matrix, and debonding between the fibers and matrix[63]. Several difficulties are observed in machine PPS composites. One of them is the high strength-to-weight ratio of the PPS matrix, limiting its machinability. Although favoring high-end applications, the correlated anisotropy and the heterogeneity causes serious difficulty in achieving a satisfactory quality of machined components considering surface integrity, dimensional and geometrical tolerances[64]. According to Korugic-karasz and Farugia[65], the anisotropic property of reinforced PPS changes the material removal mechanisms during the different machining phases of the composite structure, and the cutting process favors a specific tendency of damages. Another common factor is the lack of experience in machining fiber reinforced PPS since the knowledge of machining conventional materials cannot be applied to GF and CF reinforced thermoplastics[66]. The most related issue during machining fiber-reinforced polymers matrix is delamination[14,16,67,68]. Delamination is the interlaminar cracking between fiber-reinforcement and polymer matrix, resulting in stiffness and strength loss. Delamination is related to the cutting forces of the machining processes. In drilling, when thrust force exceeds a critical value, pull-out fibers occur in the cutting tool’s entrance, and push-down occurs in the hole’s exit[16,69]. Good machining parameters, tool geometry, and tool material effects cutting forces and are investigated to avoid delamination[15,62,70,71]. In the drilling process, the most used composite material machining for aeronautics and automotive industries, the delamination problem is reduced by high rotations speeds and low feed rates[14,68,70]. 5/12

Montagna, L. S., Kondo, M. Y., Callisaya, E. S., Mello, C., Souza, B. R., Lemes, A. P., Botelho, E. C., Costa, M. L., Alves, M. C. S., Ribeiro, M. V., & Rezende, M. C. According to Iliescu et al.[72], a way to improve drilling is to use diamond-coated tools that can allow from 10 to 12 times longer tool life than carbide tools with three times higher cutting speeds. However, the application of higher cutting speeds can lead to an increase in temperature. According to Sorrentino et al.[73], during drilling, the maximum temperature peak is located close to the drill exit, which can favor material push out and delaminations. Furthermore, the progression of temperature increase is proportional to the composite layers damaged. Nomura et al.[74] observed a strong influence of cutting depth and feed parameters on the burr formation of small holes drilling in PPS for drilling machining. The authors also stated that an increase in feed (from 3 to 12 mm/min) and cutting depth (0.5 to 4.5 mm) decreased the burr formation leading to a better straightness profile of the drilled hole. Basso et al.[75] recommended a drilling strategy between chip thickness and drill cutting lips edge radius to minimize uncut fiber/matrix regions for high feed values in the microdrilling of PPS/CF composites using a 0.6 mm diameter twisted drill. PPS presents better machinability than other highperformance composites[2]. However, factors such as temperature, fiber orientation, and the type of applied lubrication can significantly impact the machining response of fiber-reinforced thermoplastic composites. An important parameter to consider is the temperature since the thermal influence of machining on the polymer matrix must be below a load-critical extent. The process parameters must be adjusted to not exceed the thermoplastic matrix’s glass transition temperature (Tg). An efficient way to evaluate the temperature during machining is to apply thermocouples. However, thermal behavior may vary according to the sensor position. Therefore, regions of non-homogeneity matrix deposition or fiber displacement must be avoided[76]. Another factor to consider is the type and quality of reinforcing fiber added to the thermoplastic. According to Khashaba[77], reinforcement and the cutting speed and tool feed govern the maximum temperature level and heat dissipation during machining. Higher temperatures reduce the matrix stability, leading to stress concentration to matrix smearing, or material loss. The type of applied lubrication during machining can also influence and facilitate the machining of thermoset reinforced composites. The study carried out by Iskandar et al. [78] verified that Minimum Quantity Lubrication (MQL) application for milling reinforced laminates reduced flank wear by 30%.

6. Influence of thermal properties on PPS processing The knowledge of the thermal properties of thermoplastics is essential, as it aims to establish the best processing conditions and know the parameters that influence the final properties of the material. In the study by Batista et al.[79], the influence of the cooling rate on the degree of crystallinity (Xc) of PPS/CF 6/12

composites was verified. The authors evaluate the Xc of 6 laminates, made by hot compression molding, obtained under the same heating parameters (ambient temperature ~25 °C to 315 °C) and with three different cooling rates ((1) cooling natural (or free cooling), (2) slow (1 ºC/min) and (3) fast (10 ºC/min)). According to the authors, DSC analyzes have shown that slower cooling creates higher Xc values (free cooling (61.9 ± 1.9) %, slow (58.5 ± 0.8) %, and fast (51.1 ± 1.0) %, as the polymer crystallite chains will have higher time to create more ordered regions. The authors also observed larger melting peak areas for lower cooling rates because they have higher crystalline content, hindering the melting process. Another point marked was that the slower rate had two endothermic peaks, which probably represents the fusion of crystallites and transcristalinity. This event occurs when fibers influence the crystallization in the interface region between the polymer and the fiber, hindering the growth of spherulites, forcing the longitudinal change of the crystal in the direction perpendicular to the fiber, and generating an increase in binding interfacial between fiber and the polymeric matrix. Therefore, the authors concluded that lower cooling rates favor greater crystallinity in the material and that, at lower cooling rates, CF assists crystallite nucleation. The study carried out by Costa et al.[80] presents an interesting perspective, three PPS/CF laminates with six layers of fabrics were formed by hot compression molding after being subjected to heating by infrared radiation from room temperature to 320 °C, followed by forming at different temperatures (100 °C, 170 °C, and 210°C). The authors reported that 170 ºC was the temperature recommended by the manufacturer. In contrast, the two other temperatures served as a basis for the study to understand material behavior in different situations. The authors verified the crystallinity in the three laminates in three different regions. Batista et al.[79] confirmed these results because the material when taken to conformation, at 320 ºC and coming into contact with a colder mold, its cooling rate is higher. Therefore, its crystallinity will be lower for the three regions studied in each laminate. The results attest to the explanations given, for the three regions of each laminate, the crystallization values obtained are, mold at 100 ºC against the metallic region 14%, against the rubber region 13.3% and median region 20.9%; for mold at 170°C, 18.5%, 20.2%, and 24.2%, mold at 210°C, 21.2%, 20.3%, and 23.1%, respectively, indicating that the cooling rate is inversely proportional to the crystallinity obtained. Taketa et al.[81] also observed similar results in their investigations. Even in a study conducted by Geng et al.[56] that processed PPS through a 3D printer, it has been proven that the cooling rate is also linked to crystallinity. As a comparative option of techniques, in the study carried out by Furushima et al. [82] , another equipment for crystallinity analysis was used, the Fast Scanning Calorimetry (FSC) or Hyper DSC, as the DSC has limitations regarding the scan rate, and thus it is possible to apply a slower temperature scan rate, being able to verify the structural changes of metastable crystals. Furthermore, with the FSC, it is possible to overcome these limitations and investigate the kinetics above without undesired crystallization. Even though it is more profound, Polímeros, 32(1), e2022005, 2022

A review on research, application, processing, and recycling of pps based materials this analysis follows the same line as previous studies regarding the cooling rate of crystallinity. Batista et al.[83], performed tests with the same cooling ranges Costa et al.[80], but with the uniform temperature distribution mold, and they obtained the same conclusions. It is essential to mention the cold crystallization curve, which appears in thermal analyses of thermoplastic polymers before the melting temperature. This event occurs when a high cooling rate is used in the composite manufacturing process. When this same composite is heated above the Tg of the matrix, the polymer chains gain mobility and begin to organize themselves. Therefore, when the cooling occurs quickly, the chains become more disordered, generating a partial amortization in the matrix. This phenomenon can be observed in DSC analysis in the first heating, as a history of insufficient processing. To remove this history, it is necessary to submit the material to the heating and cooling cycle because, after the first heating, the amorphous chains start to move. When cooled with suitable parameters, they begin to reorganize and generate a greater degree of crystallinity, and then in the subsequent warming, this record (cold crystallization) will no longer exist. In the study carried out by Chukov et al.[84], the authors demonstrate these characteristics, presenting DSC analyses with neat and previously unprocessed material, with only one endothermic peak. Two endothermic peaks appear only on the first heating for PPS/CF composites, which were already processed. Soon after the material is cooled to 10 °C/min, second heating is done, and cold crystallization no longer appears, reporting that the material was processed with the best parameters. In the case of the PPS matrix reinforced by GF (PPS/GF), it is possible to verify the exact characteristics of change in the structure of the material as observed with CF reinforcement, mentioned above, as presented in the studies of Wang et al. [85] . These authors give information on the crystallization and fusion behavior of PPS/GF. The survey carried out by Zuo et al.[86] also proposes an analysis of the material, making a comparison between PPS/GF with and without thermal aging. The results point to what was previously described, until a specific time of aging, the crystallinity is increased, after a certain period, the crystallinity starts to decay, the virgin material has 44.2% crystallinity, with 20 h aging 45%, 96 h 55.8% (highest value), 144 h 52.9% and 1080 h 36.6%. Another study related to PPS/GF composites was developed by Zhao et al.[52]. However, the authors used a very high content of GF and the results show the exact characteristics of transcristalinity. Batista et al.[87] experimented to understand the behavior of the crystallinity of PPS/CF composites when exposed to three different types of conditioning (hygrothermal, salt spray, and condensation/ultraviolet) and with three cooling systems (slow, fast). The composites were dried in an oven at 60 ºC, and at each weighing, the samples were dried with a paper towel to remove surface water residue. During hygrothermal conditioning, the samples were exposed to 90% relative humidity at 80 ºC for eight weeks, the salt fog was made in a salt spray chamber, and the samples were exposed to a spray of an aqueous solution of 5 wt% NaCl at 35 ºC for three weeks. UV/condensation conditioning Polímeros, 32(1), e2022005, 2022

was performed in an accelerated weathering tester with ocular solar irradiance control for 900 h, using ASTM G 154 standard[88]. The authors observed that the hygrothermal conditioning showed a 17% increase in crystallinity. The polymer chains could move and reorder to form new crystal structures by remaining at a higher temperature during the process. However, the results showed that the higher the crystalline content, the lower the water penetration, with little difference. In UV conditioning/condensation, a process called chemi-crystallization occurred due to the amorphous chains being broken by UV radiation and gaining mobility to form new crystals. The salt spray conditioning caused salt crystals to start on the sides of the material, indicating that NaCl probably migrated from the inside to the outside of the structure and caused some carbon strands to come out, following this process of salt migration. Samples with higher amorphous contents showed a higher amount of salt. In another study, Batista et al.[89] investigated, through the techniques of immersion in water and ultraviolet radiation (UV) climate chamber, the influence of temperature, humidity, and UV radiation on PPS/CF composites. Hot compression molding produced the composites in a temperature range from 280 ºC to 290 ºC. The artificial photodegradation process was carried out according to ASTM G 154[88], where the samples were submitted to the aging process for periods of 200, 600, and 1200 h. The moisture absorption and diffusion behavior were analyzed with the immersion of the material in water and periodic weighing. The analysis with UV radiation showed an improvement in the compression effort for short periods of exposure, which can be explained by the stiffening caused by crosslinking through the action of UV, temperature, and humidity. For more extended periods of exposure, there was deterioration in mechanical properties, which can be explained by photolysis and photo-oxidation and the embrittlement process caused by extensive crosslinking. DMA and compression tests show an increase in the Tg as the exposure period increases, indicating reduced mobility and narrowing of networks. Since temperature activates the diffusion process, it was found that water absorption increases with temperature. At the same time, Faria et al.[90] evaluated the influence of hygrothermal conditioning on the viscoelastic properties of PPS/CF laminates. Plasticization of the polymer matrix is one of the most pronounced effects of moisture absorption, reducing the glass transition temperature. Then, hygrothermal conditioning was carried out, according to ASTM D 5229/D 5229-04 standard, and the control of the moisture gain of the sample was carried out with weekly weighings. DMA analysis showed that the integrity of the laminates was not affected by moisture absorption. However, the glass transition temperature and dissipation energies increased after conditioning in the climatic chamber. Thus, the crosslinking effect appears caused by the presence of water molecules, which compete with the plasticizing impact in the system, increasing the glass transition temperature and, consequently, expanding its service temperature.

7. Recycling From the growing concern with the environment, the need to reduce CO2 emissions, and the new legislation 7/12

Montagna, L. S., Kondo, M. Y., Callisaya, E. S., Mello, C., Souza, B. R., Lemes, A. P., Botelho, E. C., Costa, M. L., Alves, M. C. S., Ribeiro, M. V., & Rezende, M. C. regarding the disposal of materials, European policies[91,92] of solid waste, which determine the recycling of all plastic components of end-of-life vehicles and the treatment of waste to avoid negative impact to the environment and human health, led industries to increase investments in reuse and recycling of materials[93]. The recycling of thermoplastics is favored over thermosets, mainly due to their greater efficiency in the process. In the presence of temperature, they soften and can be reprocessed in different geometries[33]. The recycling processes can be divided into primary, being carried out only the polymer reprocessing, secondary or mechanical recycling, tertiary or chemical recycling, and quaternary being the energy recycling[94]. The most common process for recycling PPS is the mechanical process, in which the material is milled, homogenized, and reprocessed. Another thermal recycling option, depolymerization, occurs at high temperatures, around 550 °C, from splitting the sulfhydryl groups in the main chain, decomposing mainly into the monomer, benzenethiol. However, thermal recycling can be economically viable with the intention of CF recovery, for example, from PPS/CF composites[93,95-97]. Chemical recycling of PPS is not carried out due to its chemical resistance and insolubility in organic solvents[98]. Figure 4 presents the most viable recycling processes for PPS-based materials. PPS is also widely used for GF and CF reinforcements, mainly in the aerospace and automobile industries. And with the growing use of PPS/CF composites, the volume of waste from the processing steps is also increasing[99]. One of the methodologies related to recycling PPS composites with CF was developed by ThermoPlastic Composites Research Center (TPRC), a consortium of industry and academic members with industrial composites located in the Netherlands. This research center used a methodology based on the mechanical recycling of thermoplastic composites that starts with comminution and then reprocessing. Initially, the PPS/CF composites are ground into large flakes so that the fibers maintain a long length, and then this material is reprocessed. It is carried out by mixing the crushed material with a virgin matrix, followed by melting, which has the same purpose of maintaining the fiber length. The final step consists of reprocessing via hot compression molding.

The study of Bruijn and van Hattum[51] was developed at the TPRC in the Netherlands, using the PPS/CF waste recycling methodology proposed by the TPRC. According to the study, the authors demonstrated a viable and novel recycling route for thermoplastic composites (PPS/CF). They processed an integrally-stiffened access panel door for a rotorcraft, selected for detail design, testing, and current flight testing. One of the studies carried out by Vincent et al.[99] was the mechanical recycling of PPS/CF composites. The authors ground the PPS/CF residues into flakes and used a low-shear blend to homogenize, then extrusion and hot compression molding using a press. After this process, the study aimed to characterize the recycled material heterogeneity and compared it with the commercially available material that is compression molded by long fiber thermoplastics (LFTs) that have been on the market for decades. The authors found that the recycling process was efficient in homogenizing the matrix and fibers. The results were similar to those of LFTs for fiber orientation, percolation, variation of the fiber fraction, and fiber friction. Furthermore, the authors were able to obtain ribbed plates using this methodology. According to the authors, industrial applications of this recycling route will benefit from this similarity, increasing confidence in the combination of material and process. Another process for recycling PPS was developed by Hao Wang et al.[100]. The authors recycled PPS filters used mainly in the thermal energy and metallurgical industries and are replaced frequently, generating a large volume of waste. The process consisted of collecting the filters and removing residues that were adhered to the filter. Afterward, the PPS filters were ground until the formation of a fine powder. Then they were incorporated and homogenized with epoxy resin to act as a flame retardant. The authors found that using this composite (recycled PPS/epoxy resin) as flame retardants resulted in a reduction in CO and CO2 emissions and, consequently, reduced the smoke’s toxicity compared to the burning epoxy/CF composites. Furthermore, the authors observed that the presence of PPS contributed to the formation of a layer of carbon on the surface of the composite, acting like a protective layer, blocking heat. However, if mechanical and thermal recycling of PPS-based materials is not feasible, Li et al.[101] demonstrate a biodegradation

Figure 4. Feasible methods of recycling PPS-based materials. 8/12

Polímeros, 32(1), e2022005, 2022

A review on research, application, processing, and recycling of pps based materials methodology by Pseudomonas sp. for PPS, the most difficult kind of plastic to be degraded due to the excellent physical and chemical stability of this thermoplastic. The study presented by the authors shows the feasibility of biodegradation of PPS beads by Pseudomonas sp, verifying that the process can be carried out in 10 days. According to the authors, the developed method can be used to verify the biodegradation efficiencies of different kinds of plastics within a shorter reaction time, but also provides the possibility to be used for screening and identifying new bacteria strains of various types of plastics.

8. Conclusion PPS-based materials have been used in the aerospace, automotive, and wind energy industries. PPS reinforced with FG or CF-based components presents a high strength-toweight ratio, leading to lighter structures and components with good mechanical resistance. Due to its high melting temperature, the processing temperature is an obstacle in manufacturing PPS-based components. Its process demands equipment that operates in higher temperatures, leading to increased production costs. PPS fiber-reinforced composites are usually produced by compression molding and lamination of semi-pregs and mats with fiber fabric impregnated with the polymer matrix. FDM process has been used recently for printing pieces with PPS filaments. In addition, the PPS processing parameters need to be controlled, as the cooling rate directly affects the material’s crystallinity, where higher cooling rates lead to less crystallinity. Higher polymer crystallinity results in more brittle components, affecting the performance of the PPS-based material parts. The high hardness of the reinforcement materials, high strength, good abrasive resistance, and thermal conductivity make PPS-based composites difficult to machine material. Drilling and milling are the most used machining process, and the major problem is the occurrence of delamination. Delamination occurs when the forces involved in the machining process exceed a critical value, which serves as a point of attention in developing strategies to avoid this phenomenon. The works analyzed exposed strategies to minimize the delamination related to the geometry of cutting tools, different materials for cutting tools, and the adequacy of cutting parameters, mainly the use of high cutting speed and low feeds. One of the significant advantages of applying PPS in the industry is that its residues can be recycled mechanically or thermally. In this way, it is possible to manage and develop technologies that aim at the minimum destination of waste to landfills and incineration, contributing to preserving the environment and natural resources.

9. Acknowledgments and Funding The authors would like to thank the Federal Government Program ‘Rota 2030’ linked to the “Development of Skills for Design and Manufacturing of Tooling for Composite Parts” n° 27194.03.03/2020.01-00 for the financial support; the Lightweight Structures Laboratory from IPT (Instituto de Pesquisas Tecnológicas do Estado de São Paulo) for the coordination, and also the Brazilian Funding Institutions Polímeros, 32(1), e2022005, 2022

FIPT (Fundação de Apoio do IPT), FUNDEP (Fundação de Desenvolvimento da Pesquisa) for the administrative support. The authors are grateful for the National Council for Scientific and Technological Development (CNPq) (303024/2016-8, 305123/2018-1, and 304876/2020-8) and this study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES).

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ISSN 1678-5169 (Online)


Effect of hollow glass microspheres addition on density reduction and mechanical properties of PA6/glass fibers composites Thaysa Rodrigues Mendes Ferreira1 , Matheus de Alencar Lechtman1 , Filipe Lauro Dias2  and Aline Bruna da Silva1*  Laboratório de Polímeros, Departamento de Engenharia de Materiais, Centro Federal de Educação Tecnológica de Minas Gerais – CEFET, Belo Horizonte, MG, Brasil 2 Engenharia de Materiais, Stellantis, Betim, MG, Brasil



Abstract The strategy of combining the traditional reinforcement of glass fibers (GF) with lighter hollow glass microspheres (HGM) can afford to fulfill the need for potential light-weight and high-strength modern materials required in various sectors, such as automotive and aerospace industry applications. This work fabricated composites of PA6/GF/HGM by melting blending in a co-rotating twin-screw extruder, and subsequently, injection molded. The effects of HGM content on the density, morphological and mechanical properties were investigated and the PA6/GF/HGM composites properties were compared to the properties of the traditional PA6/GF (70/30) wt% composite, widely used today in automotive industries. With the increase of HGM amount in the formulations, a reduction of between 3 and 12% in density was achieved with a slight reduction in its mechanical properties, showing that this new strategy can be applied to replace the PA6/GF (70/30) wt% composite, providing a considerable weight reduction for these materials. Keywords: polyamide composites, glass microspheres, glass fibers, density eduction, weight reduction. How to cite: Ferreira, T. R. M., Lechtman, M. A., Dias, F. L., & Silva, A. B. (2022). Effect of hollow glass microspheres addition on density reduction and mechanical properties of PA6/glass fibers composites. Polímeros: Ciência e Tecnologia, 32(1), e2022001. https://doi.org/10.1590/0104-1428.210060

1. Introduction The aerospace and automotive industries are constantly searching for new technologies to improve their performance, especially new materials, to fabricate components lighter, optimize the consumption of fuel, and make the aircraft and vehicles faster and efficient[1]. Throughout history, several advances have been achieved, the replacement of metal parts by plastic material can be highlighted as one of the changes that directly impacted the performance of vehicles and aircraft[2]. Despite the replacement of metal parts by plastic results in a significant reduction in weight, its mechanical properties are usually lower than required by those applications. To enable the replacement of more metal parts by lighter materials polymer composites reinforced with anisotropic fillers, such as glass fiber (GF), and carbon fiber, and or inorganic filler such as talc and calcium carbonate have been produced to be used for those sectors of the industry[3,4]. Considering the reinforcement characteristics, anisotropic fillers are more efficient compared to isotropic fillers[5,6] due to their high surface area ratio. Fiber-reinforced materials have expanded the use of thermoplastics into structural applications, and in this context, GF has long been the reinforcement of choice, since they meet many structural

Polímeros, 32(1), e2022001, 2022

and durability demands of the aerospace and automotive industries[7] because of its high specific strength and high specific modulus of elasticity. Glass fiber-reinforced plastics (GFRP) have thus become a commodity material in the automotive, aerospace, and construction industries[8]. Although, these fillers may result in the increased density of the final composite. Thus, despite the advance in the development already achieved by the GFRPs, the development of new strategies to obtain the composites materials based on polymer and GF are still needed. The strategy of producing a composite combining the traditional reinforcement of GF with a lighter filler can afford to fulfill the need for potential light-weight and high-strength modern materials required in various sectors, such as automotive and aerospace industry applications[9]. This particulate filler can be spheres, blocks, or needle kind. Hollow glass microspheres (HGM), also known as glass bubbles, are an example of sphere inorganic reinforcement applied to obtain lighter composites materials[10,11]. Hollow microspheres, especially glass ones, are used to reduce weight and to impart specific properties to polymeric materials for various applications[12-15]. HGM are spherical thin-walled glass (0.5-2.0 µm) made of outer stiff glass and



Ferreira, T. R. M., Lechtman, M. A., Dias, F. L., & Silva, A. B. inner inert gas, with average diameter between 10-200 µm, which combine very low weight, high resistance to uniform compression, good thermal properties, acoustic insulation and good dielectric properties[16]. The incorporation of HGM to replace the amount of glass fibers face a challenge in the processing of traditional composites, due to the brittle feature of the thin wall glass. Thus, a large amount of HGM fracture during extrusion and injection process, the scientific literature brings as one solution the two-stage processing method, based on twin-screw extrusion[17-20]. The twin-screw extrusion processing is commonly used to improve the distribution and dispersion during the mixing process on the extrusion, and the two stages prevent the breakage of the glass fibers and HGM, what ensure a better efficiency process compared with the traditional single screw method. Nowadays, HGM composites are also an object of study in additive manufacturing, such as 3D printing, to improve flow melting and thermal insulation[21]. Özbay and Serhatlı[22] studied processing and properties of different combinations of HGM filled with polyamide 12 (PA12) matrix, by Selective Laser Sintering (SLS) manufacturing method. As a result, they obtained a 20% of density reduction and a significant rise in the E-modulus with the composition PA12/HGM (80/20). In the automotive industry, the polyamide (PA6) and polyamide 6.6 (PA66) are often used because of their typical hydrogen bonds, due to their polar chemical structure[23], with a short GF reinforcement, commonly 30 wt%. Composites of PA6 or PA66 reinforced with glass fibers ensure great mechanical and thermal properties and can be found in air intake manifolds, rocker covers, radiator end tanks, fuel rails, electrical connectors, engine encapsulation and others[2,24]. In this sense, GF and HGM combination may constitute an excellent solution to combine lower density, dimensional stability, and good mechanical properties. Berman et al.[25] have studied the effects of replacing calcium carbonate (high density filler) with HGM (low density filler) in an unsaturated polyester resin matrix sheet molding compound (SMC) reinforced with short GF (10~15 wt%). The composite was fabricated in SMC manufacturing, lay-up and hot pressing. As a result, they obtained a 12% of density reduction but compromised the mechanical properties. Nevertheless, all values of tensile, flexural and impact properties were higher than the corresponding properties of low and ultra-low-density composites reported in the literature. Thus, the goal of this study was to fabricate a composite based in PA6 reinforced with GF and HGM and to investigate the effects of HGM content on the density, mechanical properties of the composites comparing its properties with the traditional PA6/GF (70/30) wt% composite, widely used today in automotive industries. It’s expected to find a formulation with at least 10% density reduction and maintenance of mechanical properties. In this paper, fundamental results for understanding the relationship between structure and property of both the matrix and the fillers will be discussed in terms of microscopic observations, mechanical properties, and thermal stability. 2/9

2. Materials and Methods 2.1 Materials The injection molding grade polyamide 6 (B30S) with density of 1.14 g/cm3 was provided by LANXESS. Glass fiber was provided by LANXESS, with medium range length of 3 to 4.5 mm, the density of 2.45 g/cm3 and chemical compatibilization with an organosilane. Hollow glass microspheres type S42XHS (3MTM) was provided by 3M with untreated surface, density of 0.42 g/cm3, nominal crush strength = 8,000 psi and size distribution = 20-29 µm.

2.2 Composites preparation The PA6/glass fibers composites and PA6/glass fibers/ hollow glass microsphere composites were produced by melting blending using a Thermo Scientific Haake Rheomex PTW 24 OS co-rotating twin-screw extruder, with a side feeder (L/D = 35). The twin-screw speed and temperature at the die were 200 rpm and 250ºC, respectively. To mixture preparation, the GF and HGM were added in the side feeder, in order to prevent its break. First of all, PA6/GF composites with 30 wt% of glass fibers were prepared and separated as the reference sample. The processing of the PA6/GF/ HGM composites was performed in two stages to prevent the HGM breakage due to abrasive contact with glass fiber and shear stress during mixing [19,20]. In the first stage, only the PA6 and glass fibers were mixed in the extruder and in the second stage, the pellets of the first stage and HGM were mixed. Both fillers were introduced in the side feeder. The twin screw speed and barrel temperature at the die were 200 rpm, 250ºC the same used to the reference sample. The total amount of fillers was kept in 30 wt% for all formulations, changing the HGM content relative to the GF content. Table 1 shows the studied formulations with respective amount of PA6, GF and HGM. After extrusion, tensile specimens were obtained by injection molding, according to ASTM D638, using a Thermo Scientific equipment. The injection molding conditions comprised: barrel temperature profile 260/270/282/280/280ºC, mold temperature 80°C, injection pressure 700 bar and injection speed 25mm/min.

2.3 Filler characterization A calcination experiment was made, based on ISO 3451-1 (method A) to quantitative evaluation of inorganic filler. For this analysis, 3 specimens each formulation were burned using methane gas. After the extinguishment of the flame, only a white residue and black ash were left. They represent the organic load and inorganic filler, respectively. Subsequently, the specimens were placed in a furnace at Table 1. Samples formulation with PA6, GF and HGM content relative. Sample A B C D E

PA6 (wt%) 70.0 70.0 70.0 70.0 70.0

GF (wt%) 30.0 27.0 25.0 22.5 20.0

HGM (wt%) 0.0 3.0 5.0 7.5 10.0

Polímeros, 32(1), e2022001, 2022

Effect of hollow glass microspheres addition on density reduction and mechanical properties of PA6/glass fibers composites 750°C for 30 minutes, to full degradation of the organic load. The amount of inorganic filler in samples was calculated with the Equation 1.

( Masscalcined −

% residue


Masscrucible )



× 100


To evaluate the morphology of the filler after processing a Scanning Electron Microscopy (SEM) Shimadzu SSX-550 Superscan model was used on samples A and C to fibers, and on sample C to HGM. The average L/D of fibers and diameter of HGM after the calcination experiment was measured using the IMAGE Pro-plus 4.5 software. For each sample, 50 examples of GF and HGM was measured.

2.4 Composites characterization To study the PA6/GF and PA6/GF/HGM composites morphology, density and mechanical behavior, the injected specimens were previously placed in an oven for 24 hours at 95ºC. This procedure aims to reduce the humidity of the material, which could affect the results since PA6 is a highly hygroscopic polymer[26]. Scanning electron microscopy (SEM) observations, using a Shimadzu SSX-550 Superscan model, were carried out over the cross-section of cryofractured surface (made with liquid nitrogen). Specimens of all compositions were evaluated and the distribution and adhesion of the fillers were analyzed. The samples densities measured were carried out with a Sartorius analytical balance (2006), according to ISO 1183-1 method A, using Archimedes principle. First, an empty beaker was placed above the plate of the balance, and the balance was tarred. Then the weight of 3 specimens of each composition was record. After that, the beaker was filled with 500 ml of water at 23 ºC and the balance was tarred again. Each specimen, suspended with a copper wire, was submerged in water, and the weight was recorded. The density was calculated following Equation 2. Density water was considered 1.0 g/cm3.  Mass ( sample ) * Densitywater  air Densitysample =   Mass ( sample ) − Mass ( sample ) air submerged 



    


For mechanical characterization, tensile and Izod impact tests were performed. The tensile test was carried out using an Instron universal testing machine, model 4467, according to ISO 527 standard test method (specimen dimension = 80x5x2 mm). The strain rate was set to 50 mm/s, also, an extensometer of 50 mm and a load cell of 30 kN were used. The Izod impact was carried out in a Ceast equipment 6545/000 model, according to ISO 180 standard test method (specimen dimension = 80x10x4 mm), with the pendulum of 2.75 J and preloading of 0.011 J. All mechanical tests were performed at room temperature, and at least, five test specimens were used for testing each formulation.

2.5 Efficiency metric To compare the performance of the formulations, the percentage of density reduction was divided by the percentage of change in mechanical properties. EDT and EDI refers to Polímeros, 32(1), e2022001, 2022

efficiency density reduction by tensile strength test and by impact test, respectively. Equation 3 and Equation 4 shows how it was calculated. EDT = ∆%density

∆ %Tensile strenght

EDI = ∆ %density

∆ % Absorbed energy

(3) (4)

3. Results and Discussions 3.1 Fillers characterization Table 2 shows the compositions of samples and the amount of inorganic filler after the calcination test. It is noticed a difference between the amount of filler theoretical and after the calcination test. This can be attributed to the low density and small dimensions of HGM, an amount of this filler is lost in the air during the feeding process. Also, some HGM can not pass through the die, getting held back in extruder[20]. The same could happen to the smallest glass fibers. However, there was a homogenous loss of fillers in all samples, around 5% in weight for each one. Thus, this enables a fair comparison of mechanical properties between the samples, considering the same amount of reinforcement for all. Figure 1a shows the SEM images to sample A, PA6 load with 30% of GF, after the calcination of the composite, and Figure 1b shows the aspect ratio distribution of the glass fibers. It was observed that glass fiber (GF) presents a smooth and uniform surface and average aspect ratio of 5.14 ± 3.52 μm. There was a difficulty in determining the beginning and end of the fibers during the measurement, which justified the low L/D value and high standard deviation. Nonetheless, fiber size reduction is expected due to the breakage during the extrusion at samples preparation. Lower aspect ratio results in worse mechanical properties, especially tensile strength and impact absorption[27,28]. Figure 2a shows the SEM images to sample C, PA6 load with 25% of GF and 5% of HGM, after the calcination; through this analysis was possible to evaluate hollow glass microspheres, it has also a smooth surface and the average diameter was between 15 ± 5 μm. It’s noticed a reduction in microspheres diameter due to the breakage during extrusion, owing to your thin brittle glass wall. The major reduction in diameter came to breakage of bigger HGM, which explains a 25% reduction on average diameter. Hu et al.[29] studied the effect of broken HGM in silicon rubber and detected a enhance in mechanical Table 2. Samples formulation with PA6, GF and HGM content relative and inorganic filler after calcination experiment. Sample A B C D E

PA6 (wt%) 70.0 70.0 70.0 70.0 70.0

GF (wt%) 30.0 27.0 25.0 22.5 20.0

HGM (wt%) 0.0 3.0 5.0 7.5 10.0

Calcination (%wt) 24.8 ± 0.1 25.3 ± 0.1 25.9 ± 0.1 25.1 ± 0.2 25.9 ± 0.1


Ferreira, T. R. M., Lechtman, M. A., Dias, F. L., & Silva, A. B.

Figure 1. (a) SEM images of sample A after calcination process; (b) Aspect ratio distribution of sample A.

Figure 2. (a) SEM images of sample C after calcination process; (b) Aspect ratio distribution of glass fibers.

properties due to a bigger contact area between matrix and filler after the HGM break, this observation can explain the enhancement of mechanical properties described in this work in the next section. Fiber average aspect ratio is 5.38 ± 2.19 on sample C. The difference in the glass fiber length between samples A and C is explained by the influence of HGM in attrition increase during extrusion[30]. Again, there was a difficulty in determining the beginning and end of the fibers, which justified the low L/D value and high standard deviation. A reduction in L/D original value was expected due to attrition between flow matrix and fillers during extrusion[19,20]. Also, is well known that lower values of L/D generate worst mechanical properties[28,30]. The average diameter of HGM was measured to analyze the breakage of microspheres during extrusion. A reduction in average diameter was noticed. Scientific papers describe an improvement in tensile and impact strength in composites with broken HGM due to a higher interfacial surface of the filler which leads to more chemical interactions. This improves the efficiency of strain mechanism of composites[29,31].

distribution through to the PA 6 matrix. However, some small holes were observed, probably resulting from the extraction of fibers during the cryogenic fracture (white circle). For sample B, PA6 with 27% GF and 3% HGM, Figure 3b, it was a similar behavior was observed, that is, a uniform fillers distribution through to the PA6 matrix. Although the glass fibers are more adhered to the matrix than the HGM. This can be explained by the fact of GF was chemically treated with an organosilane, improving the interaction fiber-matrix. Due to the non-treated surfaces, HGM showed a weak interfacial adhesion with the matrix. Carvalho et al.[32] studied the influence of interfacial interactions in a polypropylene (PP) /GF extruded composite reinforced with non-treated and aminosilane treated HGM. These authors had describe a difference in the interfacial interaction between matrix-HGM treated and matrix-HGM non-treated. With the treatment, the PP matrix wets almost all HGM, instead of the almost half of non-treated HGM. Kumar et al.[33] concluded the same idea about the surface treatment on HGM after studying the morphology of PP reinforced with bamboo fiber and HGM. Again, SEM images showed the matrix all around the treated surface.

3.2 Composites characterization The micrographs of the surface of composites samples A, B, C, D and E, obtained by SEM, are showed in Figures 3-5. By analyzing reference sample A, PA6 with 30% GF, Figure 3a, it was observed that the glass fibers show uniform 4/9

It can be noted that samples C, D and E, Figures 4 and 5, despite having a larger amount of HGM than samples A and B, do not show agglomerates and maintain good fillers distribution through to the PA6 matrix. These samples also presented holes due to cryogenic fracture. There are more Polímeros, 32(1), e2022001, 2022

Effect of hollow glass microspheres addition on density reduction and mechanical properties of PA6/glass fibers composites

Figure 3. (a) SEM images of sample A after cryogenic fracture; (b) SEM images of sample B after cryogenic fracture (scale = 50µm).

holes coming from the HGM pullout than from fibers. Also, can be noted that those holes have diameters similar to small HGM, about 6 µm. It was expected since a low ratio surface comes with a low adhesion surface between matrix and non-treated HGM[34,35]. Figure 6a show the adhesion failure in sample D (70%PA6/ 22,5%GF/ 7,5%HGM) with a zoom. It’s possible to see an empty space around the filler. On the other hand, as shown in Figure 6b, it can be seen that the PA6 matrix “wets” the glass fibers. Therefore, the fibers have a rough surface, unlike the smooth surface observed after the calcination test; the microspheres remain with the smooth surface. Such behavior can be justified by the presence of an organosilane compatibilizer on the glass fibers surface, which promotes improved chemical interaction between polymer and fiber. The microspheres, however, were not compatibilized. When matrix “wets” the fillers, chemical interactions improve mechanical properties. Although a poor adhesion between the phases leads to premature failure under mechanical stress. The empty space acts as a crack and propagates under loads[36,37]. Samples density obtained according to the Archimedes principle, and the density reduction with the increase of the amount of HGM, in comparison to the PA6/GF with 30 wt% of filler are showed in Table 3. The density test shows a great reduction in the in the density of the composites, in comparison to the reference sample, with the increasing fraction of hollow glass microspheres in the material. In Jang’s study[10], was used polycarbonate Polímeros, 32(1), e2022001, 2022

Figure 4. (a) SEM images of sample C after cryogenic fracture (scale = 50µm); (b) SEM images of sample D after cryogenic fracture (scale = 50µm).

Figure 5. SEM images of sample E after cryogenic fracture (scale = 50 µm).

with density of 1.19 g/cm3 and hollow glass microsphere (density = 0.46 g/cm3). 15 wt% of filler granted a density reduction of 15.2% of the composite. The mechanical properties, i.e. Young’s modulus, tensile strength values, elongation at break, obtained by tensile strength test, and absorbed impact energy, obtained by Izod impact resistance test are shown in Figure 7. The mechanical properties of hybrid composites were reduced with increase of amount of HGM in its composition, in comparison to the reference sample. All samples present the 5/9

Ferreira, T. R. M., Lechtman, M. A., Dias, F. L., & Silva, A. B.

Figure 6. (a) SEM images of HGM after cryogenic fracture (scale = 10µm); (b) SEM images of GF after cryogenic fracture (scale = 10µm).

Figure 7. Mechanical properties of samples: (a) Young’s modulus and tensile strength; (b) Elongation at break and impact absorption.

Table 3. Density test results and analyses of density reduction.

Table 4. Rate reduction of mechanical properties.


Density (g/cm3)


1.32 ± 0.004 1.28 ± 0.003 1.22 ± 0.009 1.20 ± 0.001 1.12 ± 0.024

Density Reduction (%) 3.0 7.6 9.1 15.1

same range of filler after the calcination experiment, so the mechanical properties could be evaluated using the selected mixing method. According to Figure 7, considering the high standard deviations, Young’s modulus was the same to all samples, so it was supposed that hollow glass microspheres do not significantly affect this property. Tensile strength, elongation at break and impact resistance rate reduction of each formulation are summarized in Table 4. Negative values correspond to improvement on the property. According to Table 4, there was a low reduction in the tensile strength, considering the lowest amount of glass fibers in sample E (70%PA/ 20%GF/ 10%HGM), remembering that glass fibers conferees better mechanical properties for these composites[6]. Also, a lower fiber aspect ratio is expected due to the increase of attrition during extrusion with high values of HGM in ternary composites[30]. Can be observed the same range of fiber aspect ratio between binary and ternary composites, so that should not be the reason for 6/9


Tensile Strength (%)

Elongation at break (%)


-12 -3 2 9

-19 23 16 35

Impact Absorption (%) 12 23 30 42

the improvement on mechanical properties. Nevertheless, sample B (70%PA/ 27%GF/ 3%HGM) presented an improvement in properties compared to sample A. This result could be attributed to better distribution and/or dispersion of fillers in the thermoplastic matrix during processing[32]. Other reason that could explain the improvement is better physically interaction that small amount of HGM in ternary composites, like Hu detected in your study. Hu had reported the influence of broken HGM on silicon rubber, with and without intact spheres. Even the broken HGM can be a reinforcement on the composites if there is physical and/ or chemical interaction with the matrix, improved by the higher surface area, although it doesn’t decrease the density. This can explain by broken HGM presenting a higher surface area than an intact microsphere. Doumbia’s[31] study corroborates with Hu’s experiment. Doumbia studies the influence of the various types of hollow microspheres in high-impact Polímeros, 32(1), e2022001, 2022

Effect of hollow glass microspheres addition on density reduction and mechanical properties of PA6/glass fibers composites PP matrix. As broken HGM presents a higher aspect ratio than an intact microsphere, and the only interaction between matrix and HGM is an H bond of hydroxyl group present on the surface of the filler, more surface ensures a higher probability of chemical interaction. Experimentally, it was observed a high elongation at break and high tensile strength compared with the theoretical value. Filler with good physical or chemical interaction with matrix and/ or another reinforcement increases the efficiency of strain mechanism. A small interparticle distance (GF and HGM) decreases substantially mechanical properties due to stress field superimpose[32]. The impact absorption had a relative reduction due to the non-adherence of some hollow glass microspheres with the matrix[38]. However, it could be improved if the hollow glass microsphere were compatibilized with an organosilane, avoiding those failures around the HGM. In your study, Çelebi[39] had compared mechanical properties of pure polypropylene, and the modification after add hollow glass microspheres and HGM silane-modified. The values of tensile strength were reduced, but when the filler is compatibilized, this reduction is smaller. Compared to pure PP, which presented 38MPa of tensile strength, samples with 10% and 20% untreated HGM presented 26 MPa (reduction of 32%) and 21MPa (reduction of 45%), respectively. Although, samples with 10% and 20% of silane modified HGM presented 28 MPa (reduction of 26%) and 23 MPa (reduction of 39%), a difference of 6% compared with value of pure PP. Elastic modulus was increased, at least 20%, for compositions that had modified filler. The rate reduction of tensile strength from PA6/GF/HGM studied in this paper was smaller than the Çelebi’s composites, due to affinity between the materials and fiber reinforcement. However, a treatment on the HGM surface can decrease this rate reduction on tensile strength. To be sure which composition studied is most efficient, Table 5 presents a comparison with rate reduction between density and tensile strength (EDT), also density and energy absorption (EDI). Negative values indicate an increase in mechanical behavior. In terms of absorbed energy, all samples present a near number of reduced density per reduced absorbed energy capacity. Now dealing with tensile strength, the most efficient sample is composition D, once the aim of the study is mass reduction, but if it’s a desire to improve mechanical proprieties and reduce mass, composition C is more efficient. Analyzing the weight reduction of samples, Table 3 shows that a maximum reduction of 15% was achieved (sample E). In all composites studied, a good combination of weight reduction and mechanical properties maintenance was successfully obtained. For example, by replacing five percent of fibers glass with HGM in the composition, it was possible to reduce the composite density in almost 8%, while the mechanical properties were practically the same observed in the reference sample. However, after analyzing the results obtained, it’s possible to have a composition of PA6, glass fiber and HGM that presents the same tensile resistance of PA/GF (70/30), but with a lower density. This composition should be between 5% and 7.5% of hollow glass microspheres. Polímeros, 32(1), e2022001, 2022

Table 5. Comparative of ratio reduction of samples. Sample A B C D E

EDT -0.25 -2.19 3.76 1.75

EDI 0.26 0.33 0.30 0.36

4. Conclusions In this work, a composite of PA6 reinforced with glass fiber (GF) and Hollow glass microspheres (HGM) with light-weight and high-strength was successfully produced. Compared to the traditional PA6/GF (70/30) wt% composite, widely used today in automotive industries, the PA6/GF/HGM (70/22.5/7.5), and PA6/GF/HGM (70/25/5) formulations showed, respectively, a reduction of 9.1 and 7.6% in its density with a slight reduction in its mechanical properties. On the other hand, the PA6/GF/HGM (70/27/03) showed a reduction of 3% in the density and improvement properties compared to the reference sample, which was attributed to better distribution and/or dispersion of fillers in the thermoplastic matrix during processing, besides of the improvement made by broken HGM. The impact absorption reduction observed in the samples, with the increase of the amount of HGM in the composition was attributed to the non-adherence of some hollow glass microspheres in the PA matrix, evidenced in the SEM analysis. However, it could be improved if the hollow glass microsphere were compatibilized with an organosilane, avoiding those failures around the HGM. Thus, was concluded that composites can be used to replace some automotive components, which are currently made by the composite PA6/GF (70/30) wt% composite, providing a considerable weight reduction for these materials.

5. Acknowledgements The authors thank LANXESS for the PA6 supply and the aid with the composite processing.

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Ferreira, T. R. M., Lechtman, M. A., Dias, F. L., & Silva, A. B.















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Effect of hollow glass microspheres addition on density reduction and mechanical properties of PA6/glass fibers composites





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polypropylene-long glass fiber composites. Journal of Thermoplastic Composite Materials, 28(5), 643-655. http:// dx.doi.org/10.1177/0892705713486141. 37. Haverroth, G. E., & Soares, B. G. (2021). Polypropylene and hollow glass microspheres compatibilization via addition of compatibilizing agents. Polymer Composites, 42(9), 4872-4883. http://dx.doi.org/10.1002/pc.26196. 38. Sung, G., & Kim, J. H. (2017). Influence of filler surface characteristics on morphological, physical, acoustic properties of polyurethane composite foams filled with inorganic fillers. Composites Science and Technology, 146, 147-154. http:// dx.doi.org/10.1016/j.compscitech.2017.04.029. 39. Çelebi, H. (2017). Thermal conductivity and tensile properties of hollow glass microsphere/polypropylene composites. Anadolu University Journal of Science and Technology A Applied Sciences and Engineering, 18(3), 746-753. http:// dx.doi.org/10.18038/aubtda.323483. Received: Aug. 13, 2021 Revised: Dec. 03, 2021 Accepted: Dec. 06, 2021


ISSN 1678-5169 (Online)


Incorporation of Aloe vera extract in bacterial nanocellulose membranes Lya Piaia1* , Camila Quinetti Paes Pittella1 , Samara Silva de Souza1 , Fernanda Vieira Berti1  and Luismar Marques Porto1  Departamento de Engenharia Química e Engenharia de Alimentos, Universidade Federal de Santa Catarina, Florianópolis, SC, Brasil



Abstract Nanocellulose (BNC) is a natural polymer produced by bacteria. Its structure has only glucose monomer, it has various properties such as high water holding capacity, unique nanostructure, high crystallinity and high mechanical strength. Pure BNC or in combination with different components can be used for a wide range of applications. Aloe vera is a medicinal plant with polysaccharides in its composition that has a potential for tissue regeneration and repair. The aim of this study was to evaluate the effect of incorporating Aloe vera (A. vera) into BNC membranes produced with three fractions of A. vera extract (BNC-Aloe) on the behavior of epithelial cells. Human fibroblasts and keratinocytes were shown to have increased metabolic activity and proliferation when cultured on BNC-Aloe membranes compared to control. Quantification of collagen biosynthesis was significantly higher in BNC-Aloe membranes. In conclusion, BNC-Aloe membranes are suggested as a material for the purpose of skin tissue repair. Keywords: tissue engineering, nanocellulose, Aloe vera, fibroblasts, keratinocytes. How to cite: Piaia, L., Pittella, C. Q. P., Souza, S. S., Berti, F. V., & Porto, L. M. (2022). Incorporation of Aloe vera extract in bacterial nanocellulose membranes. Polímeros: Ciência e Tecnologia, 32(1), e2022002. https://doi. org/10.1590/0104-1428.210062

1. Introduction The skin tissue is the largest organ in the human body. This organ is considered to be the body’s first defense barrier. Skin tissue performs important functions such as homeostasis, body temperature body temperature and protection against dehydration, in addition to provide support for blood vessels and nerves[1,2]. Extensive and deep damage to the skin and mucous membranes can cause destruction of the dermis and epidermis. The damage of skin can be resolved using human skin grafts, autologous or not. However, this solution is limited by the scarcity of donors and the risk of donors and with the risk of graft rejection[3,4]. The development of temporary and/or permanent and/or permanent replacements for injured tissue arises from this context. One approach to developing functional skin substitutes is to produce to produce three-dimensional biomaterials, which resemble the physiological microenvironment in the presence of the extracellular matrix when cultured with human epidermal cells and human dermal fibroblasts the growth of the artificial tissue[5,6]. The dressings that are being commercialized assist the tissue regeneration[7-9]. As these treatments are costly and often do not induce and often do not induce the effective cure of the lesion, emphasizing the need to invest in the development of new treatments and devices treatments and devices that present a better cost-benefit.

Polímeros, 32(1), e2022002, 2022

In the development of a biomaterial with active substances was used Bacterial Cellulose. It is a natural polymer synthesized by various bacteria, including those from the genus Komagataeibacter, formerly classified in genus Gluconacetobacter[10]. Cellulose-producing bacteria under specific conditions synthesizes a nanofiber network with excellent properties, such as biocompatibility, elasticity, transparency, purity and mechanical stability, which we call bacterial nanocellulose (BNC)[2,11-15]. Along with fractions of Aloe vera (A. vera). The extract of parenchymal tissue of A. vera contains polysaccharides, sugars, minerals, proteins, lipids, phenolic compounds[16]. A. vera has long been considered as a safe functional food material that can be used orally and topically[17]. The extracted material generated three distinct fractions: T, G and F. The T fraction contains all the components present in the parenchymal tissue, synergy with all natural components. A portion of this fraction was centrifuged, producing G fraction containing the components of the parenchymal tissue, except the fibers. F fraction contained only the polysaccharides of the parenchymal tissue of the A. vera[18]. It is a plant that has immune-active properties with therapeutic functions that have been popularly used in a number of applications, such as anti-inflammatory and wound healing purposes[19-21] According to some studies, A. vera interacts with fibroblast growth factors stimulating proliferation and increasing collagen[22,23].



Piaia, L., Pittella, C. Q. P., Souza, S. S., Berti, F. V., & Porto, L. M. The incorporation of other components into the nanocellulose matrix during the synthesis can increase its applications by improving their physicochemical properties[11,12,24-26]. In most cases, considering the main characteristics of BNC, a modification is suitable, since the nanomaterials based on BNC generally present high value-added with great potential of applications. In this perspective BNC membranes incorporated with A. vera extracts were previously developed[18] but the potential application for skin regeneration remains to be evaluated. To widen the applicability of BNC-Aloe membranes, this study presents an in vitro analysis of the behavior of human epithelial cells grown in the porous surface (porous side) of the membranes to provide evidence on the biocompatibility, efficacy of the BNC-Aloe on epithelial tissue repair, in addition to quantifying collagen synthesis.

2. Materials and Methods 2.1 BNC and BNC-Aloe membranes A. vera leaves were used to obtain three different polysaccharide portions following the same procedure previously developed by Godinho[18]. A. vera gel pulp – GP, A. vera gel extract – GE and polysaccharide fraction – PF were used as a supplement of mannitol-based bacterial culture medium[18]. The bacteria Gluconacteobacter hansenii (ATCC 23769) were cultured in mannitol medium containing 60% of GE, GP and GF for 10 days. After 10 days, BNC-Aloe membranes were purified with 0.1 M NaOH for 24 h at 50 °C and finally rinsed with distilled water until reach pH 6.5. BNC-Aloe membranes were sterilized by autoclaving at 121 °C for 20 minutes before using. As a control, G. hansenii were cultured with mannitol-based medium without addition of A. vera to obtain pure BNC membranes, that were purified and sterilized following the same procedure described above. BNC and BNC-Aloe were stored in sterile conditions at room temperature until use.

2.2 Characterization of BNC membranes The porous surface of BNC and BNC-Aloe were used to perform in vitro assays. Scanning Electron Microscopy (SEM) was performed using a JEOL JSM‒6390LV microscope (Jeol, Japan) in order to characterize the surface of BNC and BNC-Aloe. BNC membranes were freeze-dried by lyophilization as described by Berti et al.[27]. After drying, samples were distributed on stubs and then coated with a double gold layer.

2.3 Cell culture Primary human fibroblasts that were extracted of eyelid skin (HDFa)[28] and immortalized keratinocyte cell line (HaCat) (ATCC) were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (Gibco®, USA) supplemented with 10% of fetal bovine serum (Gibco®, USA) and 1% penicillin/ streptomycin (Gibco®, USA). Cell cultures were maintained in a humidified CO2 (5% in air) incubator at 37°C. Primary human fibroblast cells were used in a passage of 5 to 9 to perform all in vitro assays, cultured on petri dishes. Samples 2/8

were supported on glass rings before seeding. The density of cells/samples were in volume of medium for test, before time of cultured the samples were taken and added to new plates to perform the quantitative testes. Cellular metabolic activity - Metabolic activity was determined by mitochondrial activity through MTS [3‐ (4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐ (4‐sulfophenyl)‐2H‐tetrazolium] colorimetric assay using MTS assay kit purchased from Promega Biotecnologia do Brasil Ltda. (São Paulo, Brazil). MTS assay was performed according to the manufacturer’s instructions. HDFa and HaCat cells were seeded on the porous surface of BNC AND BNC-Aloe membranes with 15 mm diameter, in a density of 105 cells/sample. At the end of 1, 3, and 7 days, samples and culture medium were removed, and adhered cells were rinsed with PBS three times. Culture plates were kept in a humidified incubator at 37°C and 5% CO2, protected from light during 2 h for the MTS reaction. Metabolic activity of HDFa and HaCat cells were quantified by Micro ELISA reader (SpectraMaxPlus 384, Molecular Devices, USA) at a wavelength of 490 nm. Cell proliferation - The PicoGreen dsDNA Quantification kit (Molecular Probes, USA) was used to quantify epithelial cells proliferation when cultured on BNC and BNC-Aloe membranes with 15 mm diameter. Epithelial cells were seeded in a density of 105 cells/ sample during 1, 3 and 7 days. To quantify dsDNA of HDFa and HaCat cell, samples were washed with PBS and submersed in 1 mL of ultrapure water for 1 hour in a humidified atmosphere at 37°C and 5% CO2. Thereafter, the plate was removed and stored in a freezer at -80 °C until analysis. Samples were incubated for 2–5 min at room temperature, protected from light and subsequently reacted with Pico Green® following the manufacturer’s protocol. A standard curve was constructed by the quantification of the λDNA provided by the Pico Green® kit. Analysis was performed using Microplate Infinite (model M200 TECAN / LAMEB 1 – UFSC) with excitation filter at 480 nm and emission filter at 530 nm. Cell viability - Live/Dead® Viability/Cytotoxicity kit (Invitrogen, USA) was used to evaluate cell viability. It measures the intracellular esterase activity (calcein) and plasma membrane integrity (ethidium homodimer). HDFa and HaCat cells were seeded on the porous surface of the BNC and BNC-Aloe membranes with 15 mm diameter in a density of 105 cells/sample. A solution of ethidium homodimer and calcein (4:1) was prepared in PBS, and 100 µL of this solution was added on each sample following the manufacturer’s protocol. Afterwards, the culture plate was incubated for 30 minutes at 37°C and 5% CO2 atmosphere. After incubation, samples were mounted on slides and observed using a fluorescence microscope (Eclipse C-L, Nikon, Japan).

2.4 Collagen biosynthesis by fibroblasts (HDFa) HDFa were grown on the surface of BNC and BNC-Aloe membranes with 15 mm diameter for a culturing period of 7 days. Cells were then washed with PBS, fixed with 3.7% formaldehyde for 1 h at 25°C, washed again with PBS and left in a laminar flow hood to dry for 45 min. Samples were then stained with 200 µL of Sirius red solution 0.5 g (Direct Polímeros, 32(1), e2022002, 2022

Incorporation of Aloe vera extract in bacterial nanocellulose membranes Red 80% – Dye content 25%, Sigma, USA) in 500 mL of saturated aqueous picric acid solution for one hour at a temperature of 25°C, protected from light. The supernatant was removed, and samples were washed with 250 µL of 0.01 M hydrochloric acid to remove unfixed dye. Stained collagen fibers were solubilized by adding 150 µL of 0.01 M sodium hydroxide solution and left for one hour. Results of collagen biosynthesis were quantified by Micro ELISA reader (SpectraMaxPlus 384, Molecular Devices, USA) at a wavelength of 535 nm and data were expressed as mean percentage of collagen synthesis. Results were compared with the standard curve of collagen connective tissue of bovine skin, kindly provided by Dr. Durvanei Augusto Maria from Butantan Institute (São Paulo).

2.5 Statistical analysis Statistical analyses were performed using GraphPadPrism® version 5.0 (GraphPad Software Inc., USA). Comparisons between groups were evaluated by one-way analysis of variance (ANOVA), followed by the Bonferroni test. Differences were considered significant when p < 0.05.

3. Results and Discussions In this study we have investigated whether the presence of A. vera fractions incorporated into BNC membranes would stimulate proliferation, metabolic activity, and collagen

synthesis of epithelial cells. To the best of our knowledge this study showed for the first time that BNC membranes incorporated with A. vera could stimulate collagen synthesis of epithelial cells. BNC and BNC-Aloe membranes were successfully synthesized following the previous procedure standardized by our research group[29,30]. BNC and BNC-Aloe membranes were micro structurally characterized by SEM. Figure 1 shows the porous surface of BNC and BNC-Aloe membranes that is corresponding to the surface used to culture epithelial cells. The porous surface of BNC and BNC-Aloe membranes showed similar microstructure after incorporation with A. vera fractions, which resulted in a non-homogeneous surface, as shown on Figure 2. Unlike, BNC-PF membranes showed a thinner fiber network when compared to BNC, BNC-GE and BNC-GP. The porous surface of BNC incorporated with A. vera fractions could determine physical and functional properties of the membranes, proposing a potential biomaterial for tissue engineering applications[25] due their similar microstructure to the extracellular matrix[27,31-33]. Figure 3 shows the metabolic activity profile of epithelial cells cultured on BNC and BNC-Aloe membranes cultured for 1, 3 and 7 days. At the end of 7 days of culture, it is possible to observe an increase of metabolic activity behavior of HDFa cells when cultured on the porous surface of BNC, BNC-GE, BNC-GP, BNC-PF (Figure 2a), with significant

Figure 1. Production steps of the membranes with the different A. vera extracts. Polímeros, 32(1), e2022002, 2022


Piaia, L., Pittella, C. Q. P., Souza, S. S., Berti, F. V., & Porto, L. M.

Figure 2. Micrographs of BNC and BNC-Aloe membranes obtained by SEM. The BNC-Aloe membranes were produced by the addition of 60% of A. vera fractions (BNC-GE, BNC-GP and BNC-PF). BNC (nanocellulose); BNC-GE (nanocellulose/ A. vera gel extract); BNC-GP (nanocellulose/ A. vera gel pulp); BNC-PF (nanocellulose/ polysaccharide fraction). The magnification used to obtain the micrographs was 3000×.

Figure 3. Metabolic activity of epithelial cells cultured on the porous surface of BNC and BNC-Aloe membranes for 7 days. (a) HDFa (primary human fibroblast) cells and (b) HaCat (immortalized keratinocytes cell line). Bars represent the average standard deviation and * represents significant differences at p<0.05 comparing BNC and BNC-Aloe biomembranes.

differences observed in HDFa cells cultured on BNC-PF membrane in comparison to BNC. In the first day, metabolic activity of HDFa cells cultured on BNC-GE, BNC-GP and BNC-PF decreased approximately 4%, 19% and 21%, respectively. However, significant differences were observed only for cells cultured on BNC-GP and BNC-PF. In the third day of culture, metabolic activity of HDFa cells decreased 15% in BNC-GE, 9% in BNC-GP and increased 23% in BNC-PF (considering BNC as 100%). In the last day of culture (7 days), metabolic activity of HDFa decreased 6% in BNC-GE, 2% in BNC-GP and 7% in BNC-PF. The metabolic activity of HaCat cells cultured during 1, 3 and 7 days on BNC and BNC-Aloe membranes are shown in Figure 2b. The metabolic activity profile of HaCaT cells increased with the time when the cells were cultured on the porous surface of BNC-GE and BNC-GP membranes. When HaCaT cells were cultured on BNC, BNC-GE, BNC-GP and BNC-PF it was not possible to observe significant differences after 1 day of culture. Thus, in the third day of culture the metabolic activity of HaCaT significantly increased when cultured in BNC-GE (9%) and BNC-GP (29%) membranes. After 7 days of culture, a significant increase was observed 4/8

in HaCaT cells in BNC-GE (126%), BNC-GP (141%) and BNC-PF (28%) membranes, considering BNC as 100%. The metabolic activity of HaCaT showed a linear increase when cultured on BNC-GP, for 1 day (5%), 3 days (29%) and in 7 days (141%). The incorporation of A. vera fractions into BNC membranes was extremely important to promote significant differences on cell viability and proliferation of epithelial cells. The behaviors of fibroblasts and keratinocytes cells were differently influenced by the composition of A. vera fractions (GP, GE and PF). The metabolic activity and proliferation of HaCat cells increased when they were cultured on BNC-GP and BNC-GE compared to BNC and BNC-PF. On the other hand, after 7 days of culture, the metabolic activity of HDFa cells was similar when compared BNC to BNC-GP. The result obtained by dsDNA quantification is showed in Figure 4. HDFa (Figure 4a) and HaCat (Figure 4b) were cultured on BNC and BNC-Aloe membranes during 1, 3 and 7 days. Significant differences were observed throughout all experimental time when HDFa cells were cultured on BNC compared to BNC-Aloe membranes. In the first day of Polímeros, 32(1), e2022002, 2022

Incorporation of Aloe vera extract in bacterial nanocellulose membranes

Figure 4. Proliferation of epithelial cells cultured on BNC and BNC-Aloe membranes during 1, 3 and 7 days of in vitro culture. The bars represent the average standard deviation and * represents significant differences at p<0.05 comparing BNC and BNC-Aloe membranes. Proliferation of (a) HDFa and (b) HaCat cells cultured on the porous surface of BNC, BNC-GE, BNC-GP, and BNC-PF membranes.

culture, the number of proliferative HDFa cells cultured on BNC-Aloe membranes decreased almost 14% for BNC-GE, 26% for BNC-GP and 31% for BNC-PF. In the third day of culture, this number decreased almost 60%, 56% and 48% for BNC-GE, BNC-GP and BNC-PF, respectively. In the end of 7 days of in vitro culture, HDFa cells increased the proliferative profile when cultured on BNC-Aloe membranes. Unlike, the number of proliferative HDFa cells drastically decreased when they were cultured on BNC during 7 days of culture, (Figure 4a). After 7 days of culture, there were significant differences between the number of proliferative HDFa cells cultured on BNC and BNC-Aloe membranes. HDFa cells were 17% (BNC-GE), 15% (BNC-GP) and 8% (BNC-PF) more proliferative when they were cultured on BNC-Aloe membranes compared to BNC.

into BNC microarchitecture might have influenced the decrease on metabolic activity[27]. Results with HDFa line (Figure 3a and 4a) suggest that the membranes had an increase in the coverage of the BNC fibers by the coating with the polysaccharide fractions. Therefore, they left the fibers wider, decreasing the porosity and the flow of nutrients to the cellular activity as well as to the cellular proliferation by the third day of cultivation[18,34], as well as indicating a directional cellular access to the material[35]. In the work by[36], a lower cellular viability was also observed after three days of cultivation in the samples with higher A. vera concentration.

HaCaT cells were also cultured on BNC and BNC-Aloe membranes to determine if the incorporation of A. vera affects the HaCaT proliferation (Figure 4b). Significant differences in the number of proliferative cells cultured on BNC and BNC-Aloe membranes were observed throughout the total experimental time evaluated, except on the first day of culture when the results observed for BNC-GE were similar to BNC membranes (100%). BNC-GE and BNC-GP increased the number of proliferative HaCaT cells over the 7 days of culture, in 26% and 19% respectively (considering BNC as 100%). In the third day of culture, the number of proliferative HaCat cells increased in 10% and 5% when cultured on BNC-GE and BNC-GP, respectively. On the other hand, the HaCat cells decreased by 28% when cultured on BNC-PF membranes. At the seventh day of experiment, the number of proliferative HaCat cells decreased by 22% when cultured on BNC-PF membranes.

However, the decrease on metabolic activity was not observed in HaCat cells. Interestingly, the metabolic activity and proliferation of keratinocytes increased when they were cultured on BNC-Aloe membranes. HaCat cells remained metabolically active up to 3–7 days of culture on the BNC-GE (9–126%) and BNC-GP (30–141%) membranes. The same was observed on the proliferation of HaCat cells on BNC and BNC-Aloe membranes. An increase of HaCat proliferation in BNC-GE (26%) and BNC-GP (19%) was showed on the seventh day of culture. The increase on metabolic activity and proliferation of HaCat cells seems to be induced by the active compounds present in the BNC-Aloe membranes, as already suggested on the literature[37,38]. Studies with other plants report that natural solutions containing polysaccharides, also present in A. vera, stimulate the proliferation of HaCat cells[39]. Tissue architecture of these membranes closely resembles native human epidermis[40]. Results with HaCat line in BNC-PF (Figure 3b) suggest a lack of synergy of all components when observing cellular activity as well as proliferation.

In addition, the proliferation of HDFa cells was stimulated by BNC-GP membranes in comparison to the BNC membranes without the addition of A. vera. The metabolic activity of HDFa decreased after three days of culture on BNC, BNCGE and BNC-GP. A similar decrease on metabolic activity values were observed when HUVECs were cultured on the porous surface of BNC after 3 days of in vitro culture[27]. According to the referred authors, the cellular adaptation

A complementary qualitative assay was performed to observe the presence of live/dead cells cultured on BNC and BNC-Aloe membranes. Cell viability of epithelial cells cultured on BNC and BNC-Aloe membranes was evaluated over 7 days of culture, as shown on Figure 5. According to the Live/Dead® stain, the green stained dots were related to living cells and the red dots were related to dead cells. Figure 4a shows the behavior of HDFa cells cultured on

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Piaia, L., Pittella, C. Q. P., Souza, S. S., Berti, F. V., & Porto, L. M.

Figure 5. Viability of epithelial cells by fluorescence microscopy using Live/Dead® assay. Live cells are stained in green (calcein) and dead cells are in red (ethidium homodimer). Epithelial cells were cultured on BNC and BNC-Aloe biomembranes over 7 days. (a) HDFa cells and (b) HaCat cells.

BNC and BNC-Aloe membranes. Dead cells were identified after 3 days of HDFa cells cultured on BNC (Figure 5 a-II), BNC-GE (Figure 5 a-V) and BNC-GP (Figure 5a-XI). HDFa cells remained viable when they were cultured on BNC and BNC-Aloe membranes over the entire cultivation period (7 days). Red dots were not observed in Figure 5b, thus viable cells were observed in all experimental times. The incorporation of A. vera fractions into BNC does not cause any cytotoxic effect to HaCat and HDFa cells. This evidence is consistent with other in vitro cytotoxic study involving A. vera/Gellan Gum sponges[41]. The percentage of collagen biosynthesis by HDFa cells cultured on BNC-Aloe membranes for 7 days is shown in Figure 6. As noticed, the concentration of collagen is significantly higher in all BNC-Aloe membranes compared to the control (BNC). The collagen concentration reached 90% of increase on the BNC-GP, 86% on BNC-GE and 70% on BNC-PF membranes, suggesting that BNC-Aloe membranes stimulated the collagen biosynthesis over the entire culture time. The biosynthesis of collagen was stimulated in the presence of fractions of A. vera and the concentration reached 90% of increase in the BNC-GP, 86% in BNC-GE and 70% in BNC-PF membranes. It was reported in the literature that solutions containing acemannan, one of the major components present in A. vera fractions, stimulated the synthesis of collagen[38,42]. The healing of skin lesions in rats using collagen-based membranes containing A. vera gel was also reported by in vivo studies[22]. The histological analysis of the lesions demonstrated the increased synthesis of collagen type I and III, as well as the proliferation of fibroblasts and macrophages in the injured areas after the membrane’s application. These results suggest the potential of BNC-Aloe membranes in the healing process, which 6/8

Figure 6 . Quantification of collagen biosynthesis by fibroblasts cultured on BNC and BNC-Aloe membranes. Data normalized to the BNC control. Bars represent the mean and standard deviation and (* ) represents significant differences at p<0.05.

can stimulate the components present in the extracellular matrix[43]. Therefore, the data indicate that the BNC-Aloe membranes present a favorable environment for cell growth, adhesion and proliferation, as well as stimulating collagen production.

4. Conclusions In this work, we developed membranes with distinct fractions of A. vera polysaccharide extract to investigate the cellular behavior on the porous side of BNC membranes. The presence of A. vera extracts on membranes improved the adaptation of epithelial cells when compared to pure BNC. The use of human dermal and epidermal cells identified a great potential to be applied for wound repair, Polímeros, 32(1), e2022002, 2022

Incorporation of Aloe vera extract in bacterial nanocellulose membranes especially because they may facilitate the healing process by promoting keratinocyte proliferation and collagen synthesis by fibroblasts in vitro. Future in vivo assays will be further conducted in order to confirm our findings.

5. Acknowledgements The authors thank the Brazilian agencies CAPES, CNPq and FINEP for financial support. The authors also thank André Tosello Foundation for the kind donation of Gluconacetobacter hansenii strain; and Cellular and Molecular Immunology Laboratory of Biological Sciences Institute (UFMG) and Antitumor Substances Laboratory UFMG for providing the cells. Electron Microscopy Central Laboratory (LCME, UFSC) is also acknowledged.

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35. Petersen, A., Princ, A., Korus, G., Ellinghaus, A., Leemhuis, H., Herrera, A., Klaumünzer, A., Schreivogel, S., Woloszyk, A., Schmidt-Bleek, K., Geissler, S., Heschel, I., & Duda, G. N. (2018). A biomaterial with a channel-like pore architecture induces endochondral healing of bone defects. Nature Communications, 9(1), 4430. http://dx.doi.org/10.1038/s41467-018-06504-7. PMid:30361486. 36. Carter, P., Rahman, S. M., & Bhattarai, N. (2016). Facile fabrication of Aloe vera containing PCL nanofibers for barrier membrane application. Journal of Biomaterials Science. Polymer Edition, 27(7), 692-708. http://dx.doi.org/10.1080/ 09205063.2016.1152857. PMid:26878323. 37. McAnalley, B. H., Carpenter, R. H., & McDaniel, H. R. (1995). US 5468737A. USA. Retrieved in 2021, December 20, from http://www.google.com/patents/US5468737. 38. Atiba, A., Nishimura, M., Kakinuma, S., Hiraoka, T., Goryo, M., Shimada, Y., Ueno, H., & Uzuka, Y. (2011). Aloe vera oral administration accelerates acute radiation-delayed wound healing by stimulating transforming growth factor-β and fibroblast growth factor production. American Journal of Surgery, 201(6), 809-818. http://dx.doi.org/10.1016/j. amjsurg.2010.06.017. PMid:21396624. 39. Deters, A., Dauer, A., Schnetz, E., Fartasch, M., & Hensel, A. (2001). High molecular compounds (polysaccharides and proanthocyanidins) from Hamamelis virginiana bark: influence on human skin keratinocyte proliferation and differentiation and influence on irritated skin. Phytochemistry, 58(6), 949958. http://dx.doi.org/10.1016/S0031-9422(01)00361-2. PMid:11684194. 40. Bell, E., Parenteau, N., Gay, R., Nolte, C., Kemp, P., Bilbo, P., Ekstein, B., & Johnson, E. (1991). The living skin equivalent: its manufacture, its organotypic properties and its responses to irritants. Toxicology In Vitro, 5(5-6), 591-596. http://dx.doi. org/10.1016/0887-2333(91)90099-Y. PMid:20732083. 41. Silva, S. S., Oliveira, M. B., Mano, J. F., & Reis, R. L. (2014). Bio-inspired Aloe vera sponges for biomedical applications. Carbohydrate Polymers, 112, 264-270. http://dx.doi.org/10.1016/j. carbpol.2014.05.042. PMid:25129743. 42. Jettanacheawchankit, S., Sasithanasate, S., Sangvanich, P., Banlunara, W., & Thunyakitpisal, P. (2009). Acemannan stimulates gingival fibroblast proliferation; expressions of keratinocyte growth factor-1, vascular endothelial growth factor, and type I collagen; and wound healing. Journal of Pharmacological Sciences, 109(4), 525-531. http://dx.doi. org/10.1254/jphs.08204FP. PMid:19372635. 43. Boonyagul, S., Banlunara, W., Sangvanich, P., & Thunyakitpisal, P. (2014). Effect of acemannan, an extracted polysaccharide from Aloe vera, on BMSCs proliferation, differentiation, extracellular matrix synthesis, mineralization, and bone formation in a tooth extraction model. Odontology, 102(2), 310-317. http://dx.doi. org/10.1007/s10266-012-0101-2. PMid:23315202. Received: Sep. 10, 2021 Revised: Nov. 23, 2021 Accepted: Dec. 20, 2021

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ISSN 1678-5169 (Online)


Scaffold based on castor oil as an osteoconductive matrix in bone repair: biocompatibility analysis Fabianne Soares Lima1 , Luis Felipe Matos2 , Isnayra Kerolaynne Pacheco2 , Fernando Reis3 , João Victor Frazão Câmara4* , Josué Junior Araujo Pierote5 , José Milton Matos3 , Alessandra Ribeiro6 , Walter Moura2  and Ana Cristina Fialho2  Departamento de Biomateriais e Biologia Oral, Universidade de São Paulo, São Paulo, SP, Brasil Departamento de Patologia e Clínica Odontológica, Universidade Federal do Piauí, Teresina, PI, Brasil 3 Departamento de Química, Centro de Ciências da Natureza, Universidade Federal do Piauí, Teresina, PI, Brasil 4 Departamento de Ciências Biológicas, Faculdade de Odontologia de Bauru, Universidade de São Paulo, Bauru, SP, Brasil 5 Faculdade de Odontologia de Piracicaba, Universidade Estadual de Campinas, Piracicaba, SP, Brasil 6 Centro de Biotecnologia e Química Fina, Universidade Católica Portuguesa, Porto, Portugal 1



Abstract To analyze the biocompatibility of the scaffold produced from a natural polymer derived from castor oil through hemolytic activity and antimicrobial activity, to enable the clinical application. Three in vitro tests were performed: Hemolytic activity test - Polymer partially dissolved in contact with blood agar; Hemolytic activity test in sheep’s blood - Polymer extract with red blood cells solution; Antimicrobial activity test - Solid polymer in direct contact with E. Coli and S. Aureus. For hemolytic tests, none of the samples showed hemolysis. Negative hemolytic activity is a good indicator, as the maintenance of the blood clot in the area of the lesion is essential for the formation of new tissue. For the antimicrobial activity test, no significant activity was observed against the bacteria used. The polymer is not toxic to red blood cells, being viable for clinical application as a matrix for tissue regeneration. Keywords: bone matrix, materials testing, tissue scaffolds. How to cite: Lima, F. S., Matos, L. F., Pacheco, I. K., Reis, F. Câmara, J. V. F., Pierote, J. J. A., Matos, J. M., Ribeiro, A., Moura. W., & Fialho, A. C. (2022). Scaffold based on castor oil as an osteoconductive matrix in bone repair: biocompatibility analysis. Polímeros: Ciência e Tecnologia, 32(1), e2022003. https://doi.org/10.1590/0104-1428.210018

1. Introduction There is a vast amount of surgical procedures performed in an attempt to repair bone tissue damaged by disease or trauma. The field of tissue engineering research aims to develop biological substitutes that restore, maintain or improve the function of damaged tissue by combining body cells with biomaterials. Scaffolds, commonly produced from polymeric biomaterials, provide structural support for cell binding and subsequent tissue development[1]. Scaffolds produced from various biomaterials are used in the field in an attempt to regenerate different tissues and organs in the body. Regardless of the type of fabric, a number of considerations are important when designing or determining the suitability of scaffold for use in tissue engineering, this generally requires that the devices be equivalent in performance, biocompatibility, safety, stability and sterility to previously approved devices[2]. The characteristics that biomaterials must have are: a) biocompatibility: the material must be non-toxic, not promote an acute or chronic inflammation reaction, have a

Polímeros, 32(1), e2022003, 2022

low tissue reactivity, that is, do not promote host rejection; b) bioabsorption: the material must have degradability that will accompany the formation of a new tissue; c) porosity: the material must have a pore density of around 75% with average sizes of 200 to 400 mm in diameter, to favor protein adhesion, in addition to increasing the collagen formation; d) chemotaxis: the material must attract mesenchymal cells and provide means of cell adhesion, facilitating cell proliferation and differentiation; e) angiogenesis: the material must promote vascularization, being hydrophilic, to absorb blood fluid and reinforce the initial coagulation after implantation; f) low cost: the material cannot exceed the value of the autograft, having abundant constituent materials and efficient sterilization[3-5]. Most tests performed on new scaffold devices follow the protocols of the International Organization for Standardization - ISO 10993, for the Biological Assessment of Medical Devices. Bearing in mind that biocompatibility is an important property for human use



Lima, F. S., Matos, L. F., Pacheco, I. K., Reis, F., Câmara, J. V. F., Pierote, J. J. A., Matos, J. M., Ribeiro, A., Moura, W., & Fialho, A. C. of biomaterials, the need to conduct in vitro studies of cellular behavior at the interface with these materials is evident. The in vitro tests are fast, cost-effective, do not involve ethical problems and simulate the performance of the material in the body. Biocompatible biomaterials should not have a toxic or harmful effect on biological systems[6]. The object of study of this research was a scaffold produced from a natural polymer derived from castor oil, it is an innovative biomaterial produced at the Federal University of Piauí. This study aims to analyze the biocompatibility of the castor oil scaffold, with the main tests of toxic activity against red blood cells and antimicrobial activity, to enable the clinical application of this biomaterial as an osteoconductive matrix in the repair of bone injuries.

2. Materials and Methods The production and characterization of the material were carried out at the Materials Physics Laboratory of the Federal University of Piauí (FISMAT-UFPI). The biocompatibility experiments were carried out at the Interdisciplinary Laboratory of Advanced Materials at the Federal University of Piauí (LIMAV/UFPI).

2.1 Production and characterization The scaffolds were found from castor oil monoglyceride and characterized as described in previous studies[7-9]. The pure Castor oil was commercially acquired. Also, the reagents used in the production of monoacylglycerides (MAG) and its polymer (CPU) were glycerol (C3H8O3, Impex), lithium hydroxide (LiOH, Vetec), and Hexamethylene Diisocyanate (HDI) (C8H12N2O2, Sigma-Aldrich) for polymerization[7]. Glycerol was added to the castor oil in a heating bath at 140ºC. After 10 minutes of preparation, lithium hydroxide (0.05% w/w) was added and kept under stirring for 5 hours. To form the polymer in scaffolding format, the initial temperature of 80ºC was preserved. After this process, polyethylene glycol (2.5g) was added to the monoglyceride (5g) and stirred until complete dissolution. Hexamethylene diisocyanate (HDI) was added at a ratio of 1:4.5 (MAG:HDI), still under composition until completion of polymerization. During the temperature, it was possible to observe the formation of the spongy material, remaining 12 hours at the initial temperature until the end of the process[7,8]. Characterization was performed by Fourier Transform Infrared Spectroscopy (FTIS) and thermal analysis. To confirm the presence of the materials in the sample, spectroscopy was performed in a Thermo Fisher Scientific Nicolet iS5 apparatus, with a purge pump and a wavelength between 800 cm-1 and 4000 cm-1, 128 accumulated cans, 4 cm-1 resolution, in attenuated total reflection. To verify the stability and thermal decomposition as a function of mass loss, the thermal analyzer TGA-51H, Shimadzu, standardized with a heating rate of 10 ◦C min-1 in a nitrogen atmosphere, up to a temperature of 600 ◦C and a sample mass of approximately 7 mg[7-9]. 2/5

2.2 Hemolytic activity test on blood agar Blood agar culture medium was used in 90 mm diameter Petri dishes for the test. Dilutions of the polymer were prepared in two different solvents (methanol and ethanol). In a 1: 1 ratio, 1.3 mg of the fragmented polymer was collected and added to 1.3 ml of solvent. Using the common concentration formula: 𝐶=

mass of solute (1,3mg )

volume of solution (1,3ml )

, 1.3 ml of solution in volume

was obtained, at a concentration of 1 mg / ml, being stirred in a solution shaker (Model AP56 – Phoenix Luferco)[10]. After obtaining the two solutions, a dosing pipette with disposable tips, fixed at 40 μl, was used to soak sterile filter paper discs number 1 with a diameter of 7 mm. The solutions were divided into two groups, one with methanol solvent and the other with ethanol solvent. Each group was divided into two subgroups: Control group: Discs impregnated only with the solvent (Methanol or ethanol) and Experimental group: Discs impregnated with the solvent and polymer solution. For each group (Control and experimental), three discs were used, in a triplicate test[11]. After the natural evaporation of volatile solvents, the Petri dishes were opened in an oven, next to the Bunsen burner flame, to avoid contamination of the medium. The discs were inserted into the Petri dish using a toothless Adson forceps, and then incubated at 35 ° C for 24 hours. The analysis of hemolytic activity was performed macroscopically after the incubation time determined[10,11].

2.3 Blood compatibility evaluation The hemolytic activity test in sheep’s blood was performed as described by Grilo[12] and Hou et al.[13] with some adaptations. This analysis consists of a colorimetric assay to measure the release of cyanomethemoglobin caused by hemolytic activity through spectrophotometry. Defibrinated sheep blood (Newprov®, Paraná, Brazil) was used to produce a 2% w/v suspension of red blood cells in the following steps: 1) Blood centrifuge at 4.000 rpm, for 15 minutes at 4ºC; 2) Removal of supernatant plasma with a micropipette; 3) Three successive washes with saline at 4ºC; 4) Weighing the red blood cell pellet; 5) Addition of saline to obtain a suspension at 2% w/v according to the formula: [%] =

pellet ( g )

solution volume ( ml )


To obtain an extracting solution, the biomaterial was weighed and mixed with saline solution to a concentration of 2mg/ml and taken to incubation at 37º for 1 hour. Then, 800μl of the extraction solution was mixed with 200μl of the 2% red blood cell solution in tubes. For the negative control, saline solution was used, for the positive control, distilled water. The tubes were slightly agitated and taken to incubation at 37°C for 1 hour, the test was performed in triplicate. After the incubation time, the tubes were centrifuged at 3.000 rpm for 10 minutes, the supernatant liquid was collected with a micropipette and taken for analysis in the DU® 800 UV/Visible Spectrophotometer at 545nm (Beckman Coulter, California, EUA). After Polímeros, 32(1), e2022003, 2022

Scaffold based on castor oil as an osteoconductive matrix in bone repair: biocompatibility analysis reading, the percentage of hemolysis was obtained using

3. Results and Discussions

impregnated disc. As expected, the negative controls did not show a hemolytic halo. This result indicates that the castor polymer is not toxic against red blood cells. The hemolytic test in defibrinated sheep blood confirmed the result obtained in the test with the blood agar culture medium. An average of the absorbance obtained was performed and the formula was applied to obtain the hemolysis percentage (Figure 1). According to the standard of hemolysis assay, samples with percentage hemolysis between 0-2% are classified as non-hemolytic[14]. The scaffold showed hemolytic activity below 1%, being considered a non-hemolytic material, this is an important factor for its application in bone tissue. For the antimicrobial activity test, no significant activity was observed against the bacteria used, Escherichia coli and Staphylococcus aureus. All vials of bacteria in contact with scaffold had turbidity similar to the control vial, for both bacteria. After adding the mixtures in MHA culture medium, the presence of bacteria in the flasks was confirmed. Thus, the biomaterial has no antimicrobial activity against the bacteria under study. The significant development of biomaterials has represented a powerful therapeutic tool in surgical activities, especially in the correction of critical bone defects. However, despite the proven benefits, its use requires careful clinical and ethical care from the professional in the analysis of the risks and benefits that each biomaterial may present[15]. The performance of in vitro tests is extremely important for the viability of biomaterials in the health sciences, toxicity tests can predict whether a material presents any type of damage to the cells[16]. The hemolytic activity test seeks to understand whether the biomaterial in which it is intended to be used in living organisms has toxic activity on erythrocytes, since the free hemoglobin molecule in plasma due to red cell lysis can cause elevation of plasma hemoglobin, inducing deleterious effects mainly in kidneys (Nephrotoxicity) and the cardiovascular system (vasomotor effect)[11]. Hemocompatibility is one of the main criteria that limit the clinical applicability of blood contact biomaterials. Adverse interactions between newly developed materials and blood

The spectra obtained by Fourirer transform infrared spectroscopy were compatible with what has already been reported in the literature, confirming the standardization of the scaffolds production. In the thermal analysis, the biomaterial also behaved in a predicted way, showing thermal degradation in three different stages, starting at 180ºC, going through 280ºC and ending at 380ºC with the greatest loss of mass (57%), according to previous studies[7-9]. The blood agar culture medium is widely used in hemolytic activity tests. This culture medium is a mixture of defibrinated sheep blood with a 1:20 base, red in color and PH 6.8 +/- 2. Being a medium rich in healthy erythrocytes, it contributes to the identification of hemolysis halos caused by toxic substances. The 2% red blood cell solution allows the material under study to come into direct contact with blood cells[10]. For the hemolytic diffusion test on blood agar, the partially dissolved samples did not show hemolytic ability, that is, none of the samples presented a hemolytic halo around the

Figure 1. Percentage of hemolysis in the Positive control (Distilled water) and Scaffold group.

AB - AS the formula: %H = AW - AS *100

Where AB, the absorbance of the tube with bioadhesive; AS, negative control absorbance (Saline solution) and AA, positive control absorbance (Distilled water).

2.4 Antimicrobial activity test The antimicrobial activity test was performed as described by NCCLS[14] with some necessary adaptations due to the properties of the scaffold under study. Strains of Escherichia coli (ATCC® 25922 ™) and Staphylococcus aureus (ATCC® 25293™) were used to perform the test. The strains were inoculated in liquid culture medium of Mueller Hinton in broth (MHB) and incubated at 37ºC until a turbidity corresponding to 0.5 of the McFarland scale (0.5 x 108 Colony Forming Units - UFC) was obtained, in Then, the mixture was diluted until a mixture was obtained at 0.5 x 105 CFU. After obtaining the bacteria, 0.5 ml of the mixture of culture medium + bacteria was inoculated in each of the 8 vials, 4 for Escherichia coli and 4 for Staphylococcus aureus, distributed as follows: 1) Experimental Groups: Flasks 1 and 5: 4.5 ml of MHB culture medium + 1mg of polymer, Flasks 2 and 6: 4.5 ml of MHB culture medium + 10 mg of polymer and Flasks 3 and 7: 4.5 ml of MHB culture medium + 100 mg of polymer; 2) Control Groups: Flasks 4 and 8: 4.5 ml of MHB culture medium (positive control). The tubes were fitted into an adapted device and incubated (Nova Ética 430-RDBP) with constant agitation and a temperature of 37ºC for 24 hours. After the determined time, the flasks were analyzed according to turbidity, using the control groups as a reference. To confirm the results obtained, 100 μl of the content of each flask was transferred to Petri dishes with MHA culture medium using a dosing pipette. The plates were incubated in an SPLabor SP-200 oven at 37ºC for 24 hours for analysis of bacterial development.

Polímeros, 32(1), e2022003, 2022


Lima, F. S., Matos, L. F., Pacheco, I. K., Reis, F., Câmara, J. V. F., Pierote, J. J. A., Matos, J. M., Ribeiro, A., Moura, W., & Fialho, A. C. must be extensively analyzed to prevent the activation and destruction of blood components[12]. The negative result for the hemolytic activity test is relevant for the clinical application of the scaffold, since the maintenance of the blood clot in the area of the lesion is fundamental for the healing and formation of new tissue, as it contains elements that are essential to the bone regeneration process[17-19]. The scaffold under study was developed from the natural polymer of castor oil. Despite the high toxicity of castor seeds, castor oil is not toxic, since ricin, a toxic protein in seeds, is not soluble in lipids, the entire toxic component being restricted to pie[20-22]. That is, even though it originated from a toxic seed, the castor polymer scaffold did not present toxicity against blood cells. For the negative result of antimicrobial activity, there is no implication in the clinical failure of the biomaterial, as this property would be desirable to avoid bacterial infections in the area to be repaired. However, in addition to the possibility of systemic antibiotic therapy, some studies have explored the hypothesis of using biodegradable antimicrobials impregnated in scaffolds to prevent infection and support new bone growth without contamination, avoiding several problems during healing[23-26]. The main function of the scaffold in the bone defect is to provide mechanical resistance to the lesion site and serve as an osteoconductive matrix, the antimicrobial activity would be an additional feature for the success of the therapy, but it is not essential, since the infection can be fought with association of scaffold with other materials and drug therapy[27,28].

4. Conclusions With hemolytic activity tests, it was observed that the polymer does not present toxicity to blood cells, being, therefore, viable for clinical application as a matrix for bone tissue regeneration. The absence of antimicrobial activity observed during the tests does not compromise the clinical use of the material, since antibiotic therapy allows the control of bone infections. In vivo biocompatibility tests are required to confirm the biomaterial’s biocompatibility.

5. References 1. Perier-Metz, C., Duda, G. N., & Checa, S. (2020). Mechanobiological computer model of scaffold-supported bone regeneration: effect of bone graft and scaffold structure on large bone defect tissue patterning. Frontiers in Bioengineering and Biotechnology, 8, 585799. http://dx.doi.org/10.3389/ fbioe.2020.585799. PMid:33262976. 2. Jeong, H., Gwak, S., Seo, K. D., Lee, S., Yun, J., Cho, Y., & Lee, S. (2020). Fabrication of three-dimensional composite scaffold for simultaneous alveolar bone regeneration in dental implant installation. International Journal of Molecular Sciences, 21(5), 1863. http://dx.doi.org/10.3390/ijms21051863. PMid:32182824. 3. Fernandes, H. R., Gaddam, A., Rebelo, A., Brazete, D., Stan, G. E., & Ferreira, J. M. F. (2018). Bioactive glasses and glassceramics for healthcare applications in bone regeneration and tissue engineering. Materials, 11(12), 2530. http://dx.doi. org/10.3390/ma11122530. PMid:30545136. 4/5

4. Sergi, R., Bellucci, D., & Cannillo, V. (2020). A review of bioactive glass/natural polymer composites: state of the art. Materials, 13(23), 5560. http://dx.doi.org/10.3390/ma13235560. PMid:33291305. 5. Taale, M., Schütt, F., Zheng, K., Mishra, Y. K., Boccaccini, A. R., Adelung, R., & Selhuber-Unkel, C. (2018). Bioactive carbon-based hybrid 3d scaffolds for osteoblast growth. ACS Applied Materials & Interfaces, 10(50), 43874-43886. http:// dx.doi.org/10.1021/acsami.8b13631. PMid:30395704. 6. Keong, L. C., & Halim, A. S. (2009). In vitro models in biocompatibility assessment for 6 biomedical-grade chitosan derivatives in wound management. International Journal of Molecular Sciences, 10(3), 1300-1313. http://dx.doi.org/10.3390/ ijms10031300. PMid:19399250. 7. Moura, F. N., No., Fialho, A. C. V., Moura, W. L., Rosa, A. G. F., Matos, J. M. E., Reis, F. S., Mendes, M. T. A., & Sales, E. S. D. (2019). Castor polyurethane used as osteosynthesis plates: microstructural and thermal analysis. Polímeros: Ciência e Tecnologia, 29(2), e2019029. http://dx.doi.org/10.1590/01041428.02418. 8. Pacheco, I. K. C., Reis, F. S., Carvalho, C. E. S., Matos, J. M. E., Argolo, N. M., No., Baeta, S. A. F., Silva, K. R., Dantas, H. V., Sousa, F. B., & Fialho, A. C. V. (2021). Development of castor polyurethane scaffold (Ricinus communis L.) and its effect with stem cells for bone repair in an osteoporosis model. Biomedical Materials, 16(6), 065006. http://dx.doi. org/10.1088/1748-605X/ac1f9e. PMid:34416741. 9. Morais, J. P. P., Pacheco, I. K. C., Maia, A. L. M., Fo., Ferreira, D. C. L., Viana, F. J. C., Reis, F. S., Matos, J. M. E., Rizzo, M. S., & Fialho, A. C. V. (2021). Polyurethane derived from castor oil monoacylglyceride (Ricinus communis) for bone defects reconstruction: characterization and in vivo testing. Journal of Materials Science: Materials in Medicine, 32(4), 39. http:// dx.doi.org/10.1007/s10856-021-06511-z. PMid:33792773. 10. Merck. (1990). Cultivation measurement manual. Darmstadt: Merck KGaA. 11. Kalegari, M., Miguel, M. D., Dias, J. F. G., Lordello, A. L. L., Lima, C. O., Miyazaki, C. M. S., Zanin, S. M. W., Verdam, M. C. S., & Miguel, O. G. (2011). Phytochemical constituents and preliminary toxicity evaluation of leaves from Rourea induta Planch. (Connaraceae). Brazilian Journal of Pharmaceutical Sciences, 47(3), 635-642. http://dx.doi.org/10.1590/S198482502011000300023. 12. Grilo, K. T. M. (2016). Determinação da concentração de hemoglobina livre em concentrados de hemácias pela espectrofotometria direta: método de Harboe (Dissertação de Mestrado). Universidade de São Paulo, Ribeirão Preto, Brasil. 13. Hou, Y., Wang, X., Yang, J., Zhu, R., Zhang, Z., & Li, Y. (2018). Development and biocompatibility evaluation of biodegradable bacterial cellulose as a novel peripheral nerve scaffold. Journal of Biomedical Materials Research. Part A, 106(5), 1288-1298. http://dx.doi.org/10.1002/jbm.a.36330. PMid:29316233. 14. Clinical and Laboratory Standards Institute – CLSI. (2005). Performance standards for antimicrobial susceptibility testing: fifteenth informational supplement CLSI/NCCLS document M100-S15. USA: Clinical and Laboratory Standards Institute. 15. Shah, F. A., Thomsen, P., & Palmquist, A. (2018). A review of the impact of implant biomaterials on osteocytes. Journal of Dental Research, 97(9), 977-986. http://dx.doi. org/10.1177/0022034518778033. PMid:29863948. 16. Weber, M., Steinle, H., Golombek, S., Hann, L., Schlensak, C., Wendel, H. P., & Avci-Adali, M. (2018). Blood-contacting biomaterials: in vitro evaluation of the hemocompatibility. Frontiers in Bioengineering and Biotechnology, 6, 99. http:// dx.doi.org/10.3389/fbioe.2018.00099. PMid:30062094. Polímeros, 32(1), e2022003, 2022

Scaffold based on castor oil as an osteoconductive matrix in bone repair: biocompatibility analysis 17. Zhu, T., Cui, Y., Zhang, M., Zhao, D., Liu, G., & Ding, J. (2020). Engineered threedimensional scaffolds for enhanced bone regeneration in osteonecrosis. Bioactive Materials, 5(3), 584-601. http://dx.doi.org/10.1016/j.bioactmat.2020.04.008. PMid:32405574. 18. Kawai, T., Shanjani, Y., Fazeli, S., Behn, A. W., Okuzu, Y., Goodman, S. B., & Yang, Y. P. (2018). Customized, degradable, functionally graded scaffold for potential treatment of early stage osteonecrosis of the femoral head. Journal of Orthopaedic Research, 36(3), 1002-1011. http://dx.doi.org/10.1002/jor.23673. PMid:28782831. 19. Wang, G., Li, Y., Sun, T., Wang, C., Qiao, L., Wang, Y., Dong, K., Yuan, T., Chen, J., Chen, G., & Sun, S. (2019). BMSC affinity peptide-functionalized β-tricalcium phosphate scaffolds promoting repair of osteonecrosis of the femoral head. Journal of Orthopaedic Surgery and Research, 14(1), 204. http://dx.doi. org/10.1186/s13018-019-1243-5. PMid:31272458. 20. Tan, A. C. W., Polo-Cambronell, B. J., Provaggi, E., ArdilaSuárez, C., Ramirez-Caballero, G. E., Baldovino-Medrano, V. G., & Kalaskar, D. M. (2018). Design and development of low cost polyurethane biopolymer based on castor oil and glycerol for biomedical applications. Biopolymers, 109(2), e23078. http://dx.doi.org/10.1002/bip.23078. PMid:29159831. 21. Patel, V. R., Dumancas, G. G., Viswanath, L. C. K., Maples, R., & Subong, B. J. J. (2016). Castor oil: properties, uses, and optimization of processing parameters in commercial production. Lipid Insights, 9, 1-12. http://dx.doi.org/10.4137/ LPI.S40233. PMid:27656091. 22. Oryan, A., Alidadi, S., Moshiri, A., & Maffulli, N. (2014). Bone regenerative medicine: classic options, novel strategies, and future directions. Journal of Orthopaedic Surgery and Research, 9(1), 18. http://dx.doi.org/10.1186/1749-799X-9-18. PMid:24628910. 23. McLaren, J. S., White, L. J., Cox, H. C., Ashraf, W., Rahman, C. V., Blunn, G. W., Goodship, A. E., Quirk, R. A., Shakesheff,

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K. M., Bayston, R., & Scammell, B. E. (2014). A biodegradable antibiotic-impregnated scaffold to prevent osteomyelitis in a contaminated in vivo bone defect model. European Cells & Materials, 27, 332-349. http://dx.doi.org/10.22203/eCM. v027a24. PMid:24908426. Yang, K., Han, Q., Chen, B., Zheng, Y., Zhang, K., Li, Q., & Wang, J. (2018). Antimicrobial hydrogels: promising materials for medical application. International Journal of Nanomedicine, 13, 2217-2263. http://dx.doi.org/10.2147/IJN. S154748. PMid:29695904. Kamaruzzaman, N. F., Tan, L. P., Hamdan, R. H., Choong, S. S., Wong, W. K., Gibson, A. J., Chivu, A., & Pina, M. F. (2019). Antimicrobial polymers: the potential replacement of existing antibiotics? International Journal of Molecular Sciences, 20(11), 2747. http://dx.doi.org/10.3390/ijms20112747. PMid:31167476. Pramanik, S., Ataollahi, F., Pingguan-Murphy, B., Oshkour, A. A., & Osman, N. A. A. (2015). In vitro study of surface modified poly(ethylene glycol)-impregnated sintered bovine bone scaffolds on human fibroblast cells. Scientific Reports, 5(1), 9806. http://dx.doi.org/10.1038/srep09806. PMid:25950377. Turnbull, G., Clarke, J., Picard, F., Riches, P., Jia, L., Han, F., Li, B., & Shu, W. (2017). 3D bioactive composite scaffolds for bone tissue engineering. Bioactive Materials, 3(3), 278314. http://dx.doi.org/10.1016/j.bioactmat.2017.10.001. PMid:29744467. Qasim, M., Chae, D. S., & Lee, N. Y. (2019). Advancements and frontiers in nano-based 3D and 4D scaffolds for bone and cartilage tissue engineering. International Journal of Nanomedicine, 14, 4333-4351. http://dx.doi.org/10.2147/IJN. S209431. PMid:31354264. Received: Feb. 19, 2021 Revised: Dec. 19, 2021 Accepted: Dec. 27, 2021


ISSN 1678-5169 (Online)


CO2 adsorption by cryogels produced from poultry litter wastes Lídia Kunz Lazzari1* , Daniele Perondi2 , Ademir José Zattera2  and Ruth Marlene Campomanes Santana1  1 Programa de Pós-graduação em Engenharia de Minas, Metalurgia e Materiais, Laboratório de Polímeros, Departamento de Engenharias, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil 2 Programa de Pós-graduação em Engenharia de Processos e Tecnologias, Laboratório de Polímeros, Centro de Ciências Exatas e Tecnologias, Universidade de Caxias do Sul, Caxias do Sul, RS, Brasil


Abstract Poultry litter waste (PLW) is the main by-product generated by the Brazilian poultry industry. A sustainable approach for reusing this waste is the production of biochar to be further used aiming CO2 adsorption. In this work, biochars were produced by varying the N2 flow along the pyrolysis process of 150 (PLW-150) and 1000 (PLW-1000) mL min-1. PLW and biochars were characterized for their morphology, porosity, specific surface area, and CO2 adsorption capacity. From the biochars, carbon cryogels (CC) were produced aiming their use as CO2 adsorbents. The results of the cryogel adsorption test showed a CO2 adsorption capacity of 13.1±2.9 and 33.8±3.3 mg g-1 for the CC-PLW.150 and CC-PLW.1000 cryogels, respectively. Therefore, reusing this residue for cryogels production and its use in the CO2 adsorption signifies an attractive perspective to minimize the environmental damage caused by CO2 emissions. Keywords: biochar, carbon cryogels, CO2 adsorption, porous materials, poultry litter wastes. How to cite: Lazzari, L. K., Perondi, D., Zattera, A. J., & Santana, R. M. C. (2022). CO2 adsorption by cryogels produced from poultry litter wastes. Polímeros: Ciência e Tecnologia, 32(1), e2022004. https://doi.org/10.1590/0104-1428.210075

1. Introduction According to Brazilian Agricultural Research Corporation (EMBRAPA), Brazil had a 3% increase in national chicken production in 2019, holding third place in the world ranking. Considering other poultry species in addition to chickens rearing, there is an excessive waste generation, mostly made up of poultry litter. Poultry litter waste is the material spread out on the aviaries’ floor to provide bedding for the birds. This material is usually composed of wood chips, sawdust, wheat, straw, peanut husks, rice husks, among others, and cellulose as its main component. Using this residue as fertilizer seems to be economically attractive, since it represents an internal resource in the crop, as it contains a great concentration of nutrients. Nevertheless, there are great environmental restrictions regarding its application, which may cause environmental damage, such as antibiotics leaching and the spread of pathogens towards ecosystems. A sustainable approach for these wastes’ reuse is thermochemical conversion since the by-products generated have greater economic value and can also be converted into energy[1]. Pyrolysis is known as the thermal degradation process of organic matter in the total or partial absence of oxygen, in solid (char), liquid, and gaseous compounds. The solid portion is rich in carbon and consists of a

Polímeros, 32(1), e2022004, 2022

large part of the inorganic compounds present in the biomass. Maximum char yields and moderate amounts of tar might be achieved with low heating rates and long residence times[1,2]. Cryogels are three-dimensionally structured solid materials with high porosity, which provides them excellent physical and thermal properties. They are extensively studied in thermal and acoustic insulation areas, adsorption of organic fluids, dyes, and gases[3]. Cellulose-derived cryogels present the advantage of being derived from plant biomass, without the need for synthetic modifications[4]. The main contribution to CO2 emissions into the atmosphere is the use of fossil fuels[5]. Recently, great efforts have been made to reduce the problems generated by CO2 emissions, within technologies for capturing, storing, and using carbon. The most developed techniques currently used are liquid amine absorption and membrane separation. Nevertheless, these technologies display limitations, such as equipment corrosion, high energy consumption, and harmful products generation. Due to the high levels of CO2 emission, it is necessary to develop efficient low-cost technologies in CO2 separation and purification from flue gases[6,7]. Porous solid materials such as zeolites, activated carbons, and polymer-based aerogels can minimize the aforementioned issues[8,9]. Among these materials, polymeric



Lazzari, L. K., Perondi, D., Zattera, A. J., & Santana, R. M. C. natural-based aerogels, especially those derived from cellulose, are considered the most promising candidates for CO2 adsorption, as they are materials with threedimensional networks, with low densities, high specific surface areas, thermal stability, and mechanics, and good sorption properties[8,10]. Therefore, the goal of the present work was to produce carbon aerogels, from the pyrolysis of poultry litter waste, for use as a CO2 adsorbent. Additionally, the scientific contribution of this work, when compared to other works that report CO2 adsorption in biochars, is its incorporation into the cryogel, which presents a different structure from the powder, facilitating its use. It can also be highlighted that the reuse of agro-industrial waste as a carbon source is being proposed.

2. Materials and Methods 2.1 Materials Poultry litter waste (PLW) was used as biomass. The sample was collected in an aviary in the city of Antônio Prado – Brazil. It consists of wood chips and sawdust. Polyethylene glycol (PEG) and carboxymethyl cellulose (CMC) reagents were purchased from Sigma-Aldrich S.A. and used as received.

2.2 Poultry litter waste (PLW)

2.3 Carbon Cryogels (CC) 2.3.1 Carbon Cryogels (CC) production For cryogels production, 7 g of biochar, 90 mL of deionized water, 2.5 g of PEG and 1.0 g of CMC were used. PEG was previously solubilized in water at 60°C for 30 minutes with constant agitation. Afterward, the CMC was added and stirred till the system homogenization. Finally, the biochar was manually mixed. The formed gel was placed into cylindrical molds and then frozen at -80°C for 24 hours and afterward dried in a Liobrás lyophilizer – model LioTop L101 (Brazil) for approximately 72 hours. The cryogels were named CC-PLW.150 and CC-PLW.1000 about the nitrogen flow used, 150 and 1000 mL min-1 respectively. 2.3.2 Cabon Cryogels (CC) characterization The CO2 adsorption capacity of the cryogels was determined in a Netzsch STA 449 F3 Jupiter® thermobalance. Approximately 10 mg of cryogel was used for each run. The test started with a flow of 50 mL min-1 of N2 and a temperature of 120ºC for 60 minutes, to eliminate possible volatile compounds present in the biochar. Then, the temperature was decreased to 25°C and the CO2 replaced the N2 (flow of 50 mL min-1). The experiment was maintained for 30 minutes with CO2 flow. The values corresponding to the CO2 adsorption mass were collected every 1.5 seconds and the result was expressed in mg of CO2 per g of cryogels. To evaluate the adsorption cycles, the procedure was repeated five times.

2.2.1 Poultry litter waste pyrolysis Biomass pyrolysis experiments were firstly performed. Approximately 100 g of poultry litter waste was placed into a bench reactor. The cylindrical quartz reactor operated under a nitrogen gas (N2) atmosphere using two different flow rates: 150 (PLW.150) and 1000 (PLW.1000) mL min1 . The oven temperature was then increased at a speed of 5 °C min1 until reaching 800 °C. The reactor was kept at this temperature for 60 minutes (holding time) and then cooled, still with a N2 flow, to room temperature. The total cooling time was approximately 10 h. After pyrolysis, the biochar formed was then macerated to be later used in powder form. 2.2.2 Biochar characterization The poultry litter waste was characterized regarding its morphology through field emission scanning electron microscopy, using a FEG Mira 3 - Tescan equipment (Czech Republic); thermogravimetry using a TGA-50 - Shimadzu® thermobalance (Japan) at a heating rate of 10 ºC min-1 over a temperature range from 23 to 800 ºC in a N2 atmosphere. Biochars were characterized for their specific mass; specific surface area by the Brunauer, Emmet, and Teller (BET) method using the Quantachrome Instruments equipment (model 1200e), managing the nitrogen adsorption/desorption process at -196 ºC. The samples went through a degassing process conducted under vacuum and at 380ºC, for a period of 20 h; ultimate analysis following the following technical standards: ASTM D5373/02 (carbon, hydrogen and nitrogen) and ASTM D4239-14e2 (sulfur), using ELEMENTAR instruments, Vario Macro model. 2/7

3 Results and Discussion 3.1 Raw-material and biochars characterization The poultry litter waste, received in the form of sawdust, was pyrolyzed as described above and was used as the carbon source to produce the carbon cryogels. PLW presents the chemical composition of 37.38%wt of cellulose, 7.34%wt of hemicellulose and 20.23%wt of lignin. Figure 1 shows the photographs and micrographs of the poultry litter waste before and after pyrolysis. As it can be seen in the photographs, the PLW.150 and PLW.1000 samples present different size particles, and the N2 flow did not modify their characteristics visually. By electron microscopy images, it is possible to verify the appearance of cavities in biochars. The fibrous structure of the material remained, even after the fibers were broken and weakened during the pyrolysis process. During the material devolatilization process, there is a gradual release of different volatile compounds as the temperature increases at a low heating rate. As a result, cracks occur on the surface of the fibers, which cause them to break. The carbonized particles formed by PLW devolatilization contain complex pore structures. The EDS spectra (Figure 2) showed that in biochars there was an increase in carbon, and consequently, a decrease in the oxygen level due to the pyrolysis process. This behavior is corroborated by the results found in the ultimate analysis presented in Table 1, where there was an increase above 30% in the amount of carbon in biochars concerning PLW. The biochars yield was similar, around 44% to the raw materials mass was converted into biochar. The main Polímeros, 32(1), e2022004, 2022

CO2 adsorption by cryogels produced from poultry litter wastes

Figure 1. Photographies; (a) PLW; (d) PLW.150 and (g) PLW.1000. Micrographs: (b) PLW; (e) PLW.150 and (h) PLW.1000.

Figure 2. EDS: (a) PLW; (b) PLW.150 and (c) PLW.1000.

variation was in the bio-oil formation, where the increase in nitrogen flow (from 150 to 1000 mL min-1) increased the liquid fraction yield (from 25.75 to 32.64% wt). This might be due to the shorter residence time of the vapors into the reactor. Longer times promote cracking reactions and secondary char formation[3,11]. Polímeros, 32(1), e2022004, 2022

The (inert) gas flow rate in pyrolysis processes plays an important role in the formation of products. With the increase in the inert gas flow, there is a significant reduction in the residence time of the pyrolysis vapors in the bed, and, consequently, a reduction in the occurrence of secondary reactions. The increase in bio-oil yield of 25.75%wt. (experiment conducted 3/7

Lazzari, L. K., Perondi, D., Zattera, A. J., & Santana, R. M. C. Table 1. Yield, ultimate analysis and specific surface area of poultry litter waste and biochars. PLW 2.35 ± 0.09 30.72 ± 1.37 5.36 ± 0.20 0.19 ± 0.02 61.39 ± 1.49 -

Biochar (%wt.) Oil (%wt.) Gases (%wt.) N (%wt.) C (%wt.) H (%wt.) S (%wt.) O* (%wt.) Specific surface area (m2 g-1) Total pore volume (cm3 g-1)

PLW.150 43.26 25.75 30.99 1.15 ± 0.02 44.08 ± 0.28 1.29 ± 0.02 0.35 ± 0.02 53.14 ± 0.31 161.896 0.102

PLW.1000 44.07 32.64 23.29 1.04 ± 0.08 40.00 ± 2.28 1.21 ± 0.08 0.45 ± 0.02 57.30 ± 2.46 141.800 0.089

*Calculated by difference.

Table 2. Pore volume of carbon cryogels.

CC.PLW-1000 CC.PLW-150

Mesopore (cc g-1)

Micropore (cc g-1)

0.02348 0.031

0.06547 0.07119

with a flow of 150 mL/min) to 32.64%wt (experiment conducted with a flow of 1000 mL/min), is associated with the shorter residence time of pyrolysis vapors in the gas phase in the experiment with higher flow, minimizing the secondary cracking reactions. Long residence times of pyrolysis vapors allow volatile species formed during primary pyrolysis to undergo secondary decomposition reactions in the gas phase to form non-condensable gases (CO/CO2/ CH4). The highest content of non-condensable gases was observed for the experiment carried out with the lowest inert gas flow (30.99%wt), that is, longer residence time of the pyrolysis vapors, when compared to the experiment carried out with a flow of 1000 mL/ min, which presented 23.29%wt. of non-condensable gases. The carbon, nitrogen, hydrogen, sulfur, and oxygen yields, obtained by ultimate analysis, are shown in Table 1 and it is possible to verify that the carbon content increases considerably when compared the biochar with the raw material. This takes place because during the pyrolysis there is a decrease in the hydrogen and oxygen concentrations, due to dehydration, decarboxylation and condensation reactions. The PLW carbon concentrations was 30.72%wt. and 44.08 and 40.00%wt for PLW-150 and PLW-1000 biochars, respectively. Regarding to the total pore volume and the specific surface area, the increase in the inert gas flow caused the reduction in total pore volume and, consequently, in the specific surface area. According to Mortari et al.[12] fast release of volatiles produces substantial internal overpressure and coalescence of the smaller pores, which also justifies the smaller volume of micropores and larger volume of mesopores for the experiment with higher flow. The specific surface area found for both biochars is representative of cellulosebased biochars, such as the values found by Zazycki et al.[13] for 93 m2 g-1 pecan nutshell and by Yu et al.[14] for hinoki cypress of 143 m2 g-1. The adsorbent-used materials are classified based on their pore size, microporous (pore diameter < 20 Å); mesoporous 4/7

Total Volume (mesopore+micropore) (cc g-1) 0.08895 0.10219

% micropores (micropore volume/volume total*100) 73.6 69.7

(20 Å < pore diameter < 500 Å), and macroporous (mean diameter > 500 Å). Microporous materials are extensively applied for gases and vapors adsorption[15]. Table 2 presents the volumes of micropores and mesopores of biochars, as well as the percentage of micropores calculated considering the total pore volume. The micropores fraction in biochars is 69.8% and 73.6% (to the total pore volume in each biochar) for PLW.1000 and PLW.150, respectively. Due to their high micropores fraction, biochars present great potential to be applied in CO2 adsorption, and therefore used for the cryogels production. Figure 3a displays the poultry litter waste thermogravimetry before and after pyrolysis process. The total poultry litter mass lost was 67%. The results show two mass loss events, the first between 19 and 200ºC, caused by the initial of hemicellulose thermal degradation phenomenon, related to the low molecular weight components evaporation, including moisture. The second mass loss event starts at 200ºC and it might be associated to several degradation phenomena of the PLW different components, mainly cellulose and lignin, which are the major components present in this organic matter[1,16]. Biochars behaved as expected, where there was only one mass loss event related to the sample moisture. Due to the pyrolysis process having been carried out at 800ºC, the organic matter present in the PLW suffered degradation, causing the produced biochars to be considered thermally stable materials. Figure 3b shows the weight loss curves of cryogels, where it is possible to note two mass-loss events. The first, referring to the degradation of CMC molecules starting at 250ºC and the second event referring to the degradation of PEG, starting at 350ºC, as can be seen in Figure S.1 – Supplementary Material. Due to the addition of these two components, had an increase in the initial degradation temperature of cryogels (T = 220°C) over PLW (T = 177°C). The CC-PLW.150 cryogel showed a residual mass of 37%, whereas the CC-PLW.1000 of 34%. Polímeros, 32(1), e2022004, 2022

CO2 adsorption by cryogels produced from poultry litter wastes

Figure 3. Thermogravimetry: (a) poultry litter waste (PLW) before and after pyrolysis with Nitrogen rate of 150 (PLW.150) and 1000 (PLW.1000) mL.min-1 and (b) cryogels CC-PLW.1000 and CC-PLW.150.

Figure 4. CO2 adsorption capability of the CC-PLW.150 and CC-PLW.1000 cryogels: (a) 30 min and (b) for 5 cycles.

3.2 Carbon Cryogels (CC) Table 3 shows cryogels CC-PLW.150 and CC-PLW.1000 photographs beyond the apparent density of both. As it is seen, they present the same physical appearance and threedimensional structure besides having an apparent density very close due to their same amount of solid particles. The cryogels CO2 adsorption capability was tested at 25ºC and atmospheric pressure. The cyogels behavior is shown in Figure 4. As seen in Figure 4a CO2 adsorption capacity was stable for up to 5 cycles. This shows the cryogels effectiveness does not drop after the desorption process, which is an important feature in selecting adsorbents. The previously described behavior was also found by Leung et al.[7] for cellulose aerogels synthesized from old corrugated containers. The maximum CO2 adsorption capacity (Figure 4b) was 13.1±2.9 and 33.8±3.3 mg g-1 to CC.PLW.150 cryogels and CC-PLW.1000 respectively. This CO2 adso The different micropore volumes of biochars used to produce cryogels, seen in Table 2, are the possible inferences for the difference in adsorption capacity between Polímeros, 32(1), e2022004, 2022

Table 3. Specific mass of carbon cryogels. Photography


Apparent density (g cm-3)


0.118 ± 0.014


0.121 ± 0.016

the materials. According to Zhang et al.[17], the large CO2 capture capacity is exclusively due to their high volume of narrow micropores and not to the high surface areas or pore volumes, neither to the presence of heteroatoms. The biochar microporosity plays a fundamental role in the adsorption capacity of CO2 and is the main subject for the increase in the CO2 adsorption capacity in the CC-PLW.1000 cryogel. Table 4 shows the CO2 adsorption capacity of aerogels produced with different raw materials[8]. summarized a series of CO2 adsorption capacity data for different aerogels, and 5/7

Lazzari, L. K., Perondi, D., Zattera, A. J., & Santana, R. M. C. Table 4. CO2 adsorption capacity of aerogels produced with different raw materials. Aerogels Amino functionalised Silica-Aerogels[9] Chitosan grafted graphene oxide aerogel[10] Carbon nanotube/PVA aerogels[16]

CO2 adsorption capacity (mg g-1) 23.01 11.31 145.2

highlighted that silica aerogels had an average of 23.03 - 347.60 mg g-1 at 1 bar; carbon aerogels synthesized by different organic precursors have CO2 adsorption capacity in the range of 14.52 - 651.20 mg g, and hybrid aerogels have a capacity in the range of 49.28 - 293.04 mg g. Several factors influence the adsorption of CO2, including the texture and chemical properties of the surface of the adsorbents, whether or not chemical modifications are carried out. Furthermore, temperature and partial pressure of CO2 are important parameters regarding the adsorption kinetics that strongly influence the CO2 rate.

633 33 59


8. 9.

4. Conclusions Carbon cryogels were synthesized with biochars produced from the pyrolysis of poultry litter waste and evaluated for their performance for CO2 adsorption. The major presence of micropores into the biochar porous structure provided the cryogels great CO2 adsorption capacity, as well as stability in their adsorption capacity for 5 cycles. These results demonstrate that cryogels are promising candidates for CO2 adsorption at room temperature.


5. Acknowledgements


The authors would like to thank the Federal University of Rio Grande do Sul (UFRGS), University of Caxias do Sul (UCS) and the National Council for Scientific and Technological Development (CNPq).



6. References 1. Perondi, D., Poletto, P., Restelatto, D., Manera, C., Silva, J. P., Junges, J., Collazzo, G. C., Dettmer, A., Godinho, M., & Vilela, A. C. F. (2017). Steam gasification of poultry litter biochar for bio-syngas production. Process Safety and Environmental Protection, 109, 478-488. http://dx.doi. org/10.1016/j.psep.2017.04.029. 2. Basu, P. (2010). Biomass gasification and pyrolysus: pratical design ans theory. USA: Academic Press. 3. Hüsing, N., & Schubert, U. (1998). Aerogels-airy materials: chemistry, structure, and properties. Angewandte Chemie International Edition, 37(1-2), 22-45. http://dx.doi.org/10.1002/ (SICI)1521-3773(19980202)37:1/2<22::AID-ANIE22>3.0.CO;2-I. PMid:29710971. 4. Onay, O., & Kockar, O. M. (2003). Slow, fast and flash pyrolysis of repeseed. Renewable Energy, 28(15), 2417-2433. http:// dx.doi.org/10.1016/S0960-1481(03)00137-X. 5. Miao, Y., Luo, H., Pudukudy, M., Zhi, Y., Zhao, W., Shan, S., Jia, Q., & Ni, Y. (2020). CO2 capture performance and characterization of cellulose aerogels synthesized from old corrugated containers. Carbohydrate Polymers, 227, 115380. http://dx.doi.org/10.1016/j.carbpol.2019.115380. PMid:31590848. 6. Khalilpour, R., Mumford, K., Zhai, H., Abbas, A., Stevens, G., & Rubin, E. S. (2015). Membrane-based carbon capture 6/7

BET surface área (m2 g-1)





from flue gas: a review. Journal of Cleaner Production, 103, 286-300. http://dx.doi.org/10.1016/j.jclepro.2014.10.050. Leung, D. Y. C., Caramanna, G., & Maroto-Valer, M. M. (2014). An overreview of current status of carbon dioxide capture and storage technologies. Renewable & Sustainable Energy Reviews, 39, 426-443. http://dx.doi.org/10.1016/j. rser.2014.07.093. Maleki, H. (2016). Recent advances in aerogels for environmental remediation applications: a review. Chemical Engineering Journal, 300, 98-118. http://dx.doi.org/10.1016/j.cej.2016.04.098. Zhao, S., Malfait, W. J., Guerrero-Alburquerque, N., Koebel, M. M., & Nystrom, G. (2018). Biopolymer aerogels and foams: chemistry, properties, and applications. Angewandte Chemie International Edition, 57(26), 7580-7608. http://dx.doi. org/10.1002/anie.201709014. PMid:29316086. Dassanayake, R. S., Gunathilake, C., Dassanayake, A. C., Abidi, N., & Jaroniec, M. (2015). Amidoxime-functionalized nanocrystalline cellulose-mesoporous silica composites for carbon dioxide sorption at ambient and elevated temperatures. Journal of Materials Chemistry. A, Materials for Energy and Sustainability, 5(16), 7462-7473. http://dx.doi.org/10.1039/ C7TA01038A. Aegerter, M. A., Leventis, N., & Koebel, M. M. (2011). Aerogels handbook. Switzerland: Springer. http://dx.doi. org/10.1007/978-1-4419-7589-8. Mortari, D. A., Perondi, D., Rossi, G. B., Bonato, J. L., Godinho, M., & Pereira, F. M. (2021). The influence of water-soluble inorganic matter on combustion of grape pomace and its chars produced by slow and fast pyrolysis. Fuel, 284, 118880. http:// dx.doi.org/10.1016/j.fuel.2020.118880. Zazycki, M. A., Godinho, M., Perondi, D., Foletto, E. L., Collazzo, G. C., & Dotto, G. L. (2018). New biochar from pecan nutshells as na alternative adsorbent for removing reactive red 141 from aqueous solutions. Journal of Cleaner Production, 171, 57-65. http://dx.doi.org/10.1016/j.jclepro.2017.10.007. Yu, S., Park, J., Kim, M., Ryu, C., & Park, J. (2019). Characterization of biochar and byproducts from slow pyrolysis of hinoki cypress. Bioresource Technology Reports, 6, 217-222. http://dx.doi.org/10.1016/j.biteb.2019.03.009. Wu, F., Tseng, R., & Hu, C. (2005). Comparisons of pore properties and adsorption performance of KOH-activated and steam-activated carbons. Microporous and Mesoporous Materials, 80(1-3), 95-106. http://dx.doi.org/10.1016/j. micromeso.2004.12.005. Baniasadi, M., Tugnoli, A., Conti, R., Torri, C., Fabbri, D., & Cozzani, V. (2016). Waste to energy valorization of poultry litter by slow pyrolysis. Renewable Energy, 90, 458-468. http:// dx.doi.org/10.1016/j.renene.2016.01.018. Zhang, X., Li, W., & Lu, A. (2015). Designed porous carbon materials for efficient CO2 adsorption and separation. New Carbon Materials, 30(6), 481-501. http://dx.doi.org/10.1016/ S1872-5805(15)60203-7. Received: Oct. 13, 2021 Revised: Dec. 28, 2021 Accepted: Dec. 29, 2021 Polímeros, 32(1), e2022004, 2022

CO2 adsorption by cryogels produced from poultry litter wastes

Supplementary Material Supplementary material accompanies this paper. Figure S.1 – TGA of PEG and CMC. This material is available as part of the online article from https://www.scielo.br/j/po

Polímeros, 32(1), e2022004, 2022


ISSN 1678-5169 (Online)


Cellulose nanocrystals into Poly(ethyl methacrylate) used for dental application Andressa Leite1 , Hamille Viotto1 , Thais Nunes1 , Daniel Pasquini2  and Ana Pero1*  Laboratório de Prótese, Departamento de Materiais Odontológicos e Prótese, Faculdade de Odontologia de Araraquara, Universidade Estadual Paulista “Júlio de Mesquita Filho” – UNESP, São Paulo, SP, Brasil 2 Laboratório de Reciclagem de Polímeros, Instituto de Química, Universidade Federal de Uberlândia – UFU, Campus Santa Mônica, Uberlândia, MG, Brasil



Abstract Cellulose nanocrystals (CNCs) can improve the mechanical properties of dental resins. However, there is a deficiency of information about the behavior of physical properties of resins after this addition. The purpose was to evaluate the characterization and physical properties of hard chairside reline material modified with CNCs (0.25%, 0.5%, 0.75% or, 1.0%). Addition of CNCs at 0.5%, 0.75% and 1% increased Vickers hardness; 0.75% decrease surface free energy; 0.75% and 1% showed similar to control on the surface roughness. The simple and straightforward approach of adding CNCs, a renewable material, provides good potential for its future practical application as it has shown promise with increasing hardness. It means that the incorporation of CNCs into this denture reline resin could improve the abrasion resistance of this material, which is desirable in the long term. Keywords: reline, acrylic resin, physical properties, nanocrystal cellulose. How to cite: Leite, A., Viotto, H., Nunes, T., Pasquini, D., & Pero, A. (2022). Cellulose nanocrystals into Poly(ethyl methacrylate) used for dental application. Polímeros: Ciência e Tecnologia, 32(1), e2022006. https://doi.org/10.1590/01041428.20210066

1. Introduction Hard chairside reline for relining dental prostheses is commonly used in dentistry directly in the mouth to regain the adaptation of the denture base to the residual ridges as a consequence of constant bone resorption that provides to an improper fit and a deficiency of support for the denture base[1]. Chairside or direct relining presents as advantages, simpler, faster, less expensive, and more practical than indirect use of a laboratory setting[2]. In addition, the relined denture must exhibit satisfactory strength to prevent fracture during function[3]. However, there is always the presence of residual monomer in the polymeric material due the conversion of monomer into polymer is not complete during the polymerization reaction[4]. This has a potential impact and this residual monomer is extensively identified as a plasticizer that affects the mechanical properties[5]. A possible alternative to solve this problem could be to incorporated reinforcing material[6]. In this respect, with the natural resources in evidence and the advent of nanotechnology, the organic fillers such as nanocelluloses (nano-structured cellulosic materials) have emerged due to its inherent characteristics and appear to have quickly drawn the attention of both academia and industry[7,8]. Cellulose nanocrystals (CNCs) or cellulose whiskers obtained from cellulose are arranged in crystallized structures obtained through acid hydrolysis into nano-sized needle-

Polímeros, 32(1), e2022006, 2022

like crystallites that supplies potential for reinforcing mechanical properties of polymer materials due to their great mechanical strength and elastic modulus, besides renewability, sustainability, abundance, low cost, lower density and biocompatibility[9]. CNCs have been used in the matrixes for collagen scaffolds, silk fibroin membranes and grafted onto the matrix of Poly(methyl methacrylate) PMMA for applications of packaging, flexible screens and optically transparent films[10-12]. The improvement of mechanical properties of composites of PMMA filled with cellulose has been reported, achieving promising results[13-15]. For dental application, the incorporation of CNCs in dental composites and glass ionomer cements has shown an improvement in the properties of these materials[16-18]. Previous studies have been demonstrated the strengthen of conventional PMMA denture base resins after the incorporation of cellulose fibers[19] microfibers[20], nanofibers[21] and microcrystalline cellulose[22], which may be promising materials to be used as active fillers. A recent study demonstrated that pure cellulose nanofiber could be used as a good substitute as a “petroleum-free” for the conventional PMMA denture base resin[23]. In contrast, Chen et al.[10] observed that the incorporation of CNCs modified by silver nanoparticles (AgNPs) did not improve the flexural strength of a 3D-printable PMMA denture base resin. Thus, little information is found



Leite, A., Viotto, H., Nunes, T., Pasquini, D., & Pero, A. in the literature concerning the incorporation of cellulose nanocrystals into denture base resins, especially that used for denture relines. In this study, based on the literature reports where it was assumed the CNCs could be used as an active filler of PMMA to be used for dentures, CNCs was incorporated to a hard chairside reline resin essentially composed by Poly(ethyl methacrylate) presuming to improve their properties. The objectives of this investigation were to characterize one hard chairside reline material to denture base containing CNCs and to evaluate the physical properties. The null hypothesis of this research was that the incorporation of CNCs would not modify these properties of the hard chairside reline resin.

2. Materials and Methods The hard chairside reline resin tested was Soft Confort Dura (Poly(ethyl methacrylate), phtalate ester, ethyl alcohol; Dencril, Pirassununga, Brazil), a powder/ liquid resin. A control group (0% CNCs, unmodified acrylic resin) and four experimental groups of acrylic resins with CNCs in different ratios were prepared: 0.25%, 0.5%, 0.75%, and 1%. These concentrations were chosen in accordance with a previous study[24], considering that higher levels of CNCs beyond 1% did not allow to obtain the proper consistency of the mixture of powder and liquid.

2.1 Synthesis of CNCs The isolation of the CNCs from bleached Kraft Eucalyptus wood pulp (Suzano Papel e Celulose S.A, São Paulo, SP, Brazil) in the form of sheets was obtained through acid hydrolysis, as previously described[15,25]. The sheets was triturated with the aid of a blender until the resulting material appeared to resemble cotton, centrifuged twice for 10 minutes at 10,000 rpm using a refrigerated centrifuge (Centrifuge 580 R; Eppendorf, Hamburg, Germany) at 10°C to remove any excess acid and the concentrated phase was then dialyzed against distilled water to neutral pH for removal of the excess sulfuric acid, salts and soluble sugars[25]. Then the material was treated with a probe ultrasound (UP100HP; Hielscher, Teltow, Germany) for 15 min (pulse used: 7s on and 2s off) and the resulting suspension was reserved under refrigeration at 4°C[25].

2.2 Solvent changes A solvent change was performed, in decreasing order of polarity, of the CNCs that were initially in water for ethanol, acetone and finally the liquid used in the polymerization of the resin according to the manufacturer. Solvent changes were carried out at 7000 rpm at 10°C for 10 minutes twice with each solvent using a centrifuge (Centrifuge 580 R; Eppendorf, Hamburg, Germany). After the last liquid centrifugation, the CNCs were dispersed using a probe ultrasound (UP100H; Hielscher, Teltow, Germany) for 5 minutes (pulse used: 7s on and 2s off).

2.3 Chemical characterization For the chemical characterization [Fourier Transform Infrared Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC), Scanning Electron Microscopy (SEM)], two glass 2/7

slides (26 × 76 × 1mm) were invested in high-viscosity silicone (Zetalabor; Zhermack S.p.A., Badia Polesine, Italy), further flasks supported by dental stone (Herodent; Vigodent S.A. Ind. Com., Rio de Janeiro, RJ, Brazil)[26]. After the dental stone had set, the glass slides were removed from the flasks, replaced by mixtures of acrylic resin and CNCs and packed under 0.5kgf pressure, for 30 min until complete polymerization[26]. After polymerisation, excess was removed by polishing. The specimens were used 30 minutes after its making, simulating the minimum time for realization an immediate reline the clinic[26]. Infrared spectroscopy analyses were performed by the KBr pellet method[27]. FTIR spectra of samples (n=1) were obtained in a spectrometer (IR prestige-21; Shimadzu, Kyoto, Japan). Differential scanning calorimetry thermograms of the samples (n=1) were recorded between room temperature (~25°C) and 250°C with a DSC Q20 instrument (TA Instruments, New Castle, USA), operating at a scanning rate of 10°C min-1 in a nitrogen atmosphere with N2 flow of 50 mL.min.-1 For the experiments, approximately 5mg of the samples were encapsulated in aluminum sample pans. A scanning electron microscope (Vega 3 SBH; Tescan, Kohoutovice, Czech Republic) was used to analyze the morphology of the fractured samples in liquid nitrogen (n=1), at an accelerating voltage of 20 kV[27,28]. After drying, the samples were sputtered with gold coating of 3 nm thickness in an argon atmosphere at 20 mA for 2 min. For the physical evaluation [Surface free energy (SFE), surface roughness (SR), Vickers hardness (VH)], discs (15 mm diameter × 3 mm thickness) were produced using stainless steel mold placed on an acetate sheet and a glass slab technique[29]. After polymerization, excess flash of each disc was removed with a bur (Max‐Cut; Densply Malleifer, Tulsa, OK, USA).

2.4 Physical evaluation SFE was obtained through a goniometer (200; Raméhart Instrument Co., Succasunna, NJ, USA) and two wet agents: water and diiodomethane[30]. The test was performed twice for each agent on ten discs of each group. The Young– Laplace equation was used for right and left contact angles measured for each wet agent[31]. For the SFE calculation, a software (DROPimage Standard; Ramé-hart Instrument Co., Succasunna, NJ, USA) was used[31]. A profilometer (SJ 400; Mytutoyo Corp, Kanagawa, Japan) with 0,01μm resolution, interval (cutoff length) of 0.8 mm, transverse length 2.4 mm, stylus speed 0.5 mm/second, and the radius of the active tip of 5µm was used for measured SR, mean value Ra (μm), of each specimen (n = 10)[22]. A microhardness tester (Micromet 2100; Buehler, Lake Bluff, Illinois, USA) with a Vickers diamond was utilized to measure the VH of the specimens from each group (n = 10)[32]. Two measurements were made for each sample for 10 seconds with a force of 50 g.

2.5 Statistical analysis Statistical analysis was performed using ANOVA/ Welch and the Games-Howell test for post-hoc comparisons for normal and heteroscedastic data of VH. Normal and homoscedastic data of SFE, and SR were evaluated by oneway ANOVA and the Bonferroni post-hoc test. All analyses Polímeros, 32(1), e2022006, 2022

Cellulose nanocrystals into Poly(ethyl methacrylate) used for dental application were performed at α=0.05, using the with the PAWS Statics software (v. 19, SPSS Inc). For the FTIR, DSC and SEM descriptive analyses were performed.

3. Results and Discussions According to the results of this study, SC had the highest mean hardness values in 0.5, 0.75% and 1% (Figure 1). The favorable results of the present investigation may have been attributed to the formation of a highly crystalline CNCs structure or to the reduction voids and distances between CNCs within the hard liners, compared to the macroscale structures[33,34]. In this study, we used CNCs that are rigid and dense nanoparticles with nanometric dimensions and acicular shape, where lengths are around 200 nanometers and thicknesses around 5 nanometers. Due to the rigid and dense structure of these CNCs, we obtained a stronger material in the hardness tests than when using commercial fibers, because these fibers are flexible and hollow, and would result in loss of stiffness in the material with its insertion in the polymeric matrix. Hardness is a physical property that has been utilized to predict the wear resistance and to the resistance to plastic deformation of dental materials, that is, it higher hardness increases the longevity of the denture because it reduces the risk of fracture and also reduces abrasion, preserving aesthetics and biofilm formation difficult[35,36]. In addition, it is used as an indirect method of evaluating polymerization depth and self-curing, that is, to evaluate the degree of conversion of monomer to polymer during polymerization[35,37,38]. As the molecular weight of straight-chain alkyl groups increases, the hardness continues to decrease. Thus, poly (methyl methacrylate) is the hardest resin of polymethacrylate esters, then the isobutyl, and finally the n-butyl[39]. That is, denture base resins have a greater hardness when compared to hard chairside resins. For this reason, the increase in hardness provided by the addition of the CNCs proved to be favorable. Nanofillers are recognized to be promising fillers for resins owing to their high specific surface area and high surface free energy, that improve the bending strength and fracture toughness of the resin effectively[10]. Studies have evaluated the effect of adding nanoparticles on the mechanical properties of acrylic resins[10,40] since denture fractures are one of the deficiencies of current denture base materials[40]. It has been observed that 56% of dentures fractures were accidental[40]. These fractures usually occur due to the flexural stress generated by chewing, so high resistance to flexural strength is required[41]. Data shows monomer residues ratios are higher auto-polymerized resins and they may adversely affect the mechanical properties and they are associated low wear resistance by a plasticizing effect, which decreases interchain forces so that deformation happens more swimmingly under load throughout flexural strength tests[42-44]. Moreover, adverse effects of certain foods and beverages can affect the mechanical properties consequently the longevity of the dentures relined with certain hard liners[45]. Previous reports showed improvement of mechanical properties by adding modified CNCs by silver nanoparticles in dental resin[10]. Polímeros, 32(1), e2022006, 2022

FTIR analyses demonstrated that there was no change in the structure of the hard chairside resin after the addition of CNCs, probably due to the small amount of this component (Figure 2). In contrast, Silvério et al. (2021)[24] observed typical peaks and bands related to the structure of the cellulose in the FTIR spectra obtained from a denture resin after the incorporation of CNCs using the same concentrations (0.25%, 0.5%, 0.75%, 1%). The FTIR technique uses a very small amount of sample, with the fraction used containing 1% or less of CNC. This makes the detection of CNC difficult, as it is a technique in which the peaks are proportional to the concentration of the constituents of the analyzed sample. A little variation was observed in the DSC thermograms of hard chairside resin increasing the Tg values when the CNCs was added suggesting that nanocrystals are affecting the molecular organization of the polymer, which consequently interfered in the Tg of the materials (Figure 3). These results are in accordance with previous studies, in which the CNC presence in the polymer enhanced the Tg values. Voronova et al. and Qin et al. explained that it might be due to the macromolecular nanoconfinement provided by the CNC surfaces[46,47].

Figure 1. Effect of CNC incorporation into SC specimens on Vickers hardness, according to the group. Different capital letters indicate significant differences among groups. (ANOVA/Welch and Games-Howell test for post-hoc comparisons, p=.000 and p=.000, respectively).

Figure 2. Infrared spectrum of CNC incorporation into SC specimens. 3/7

Leite, A., Viotto, H., Nunes, T., Pasquini, D., & Pero, A. The SEM image showed a change in the structure of the resin; however, it was not possible to observe the distribution of the CNCs in the polymer matrix (Figure 4). It can be seen that in the images presented using nanocrystals, it is difficult to identify their presence because they are in a nanometric scale. Thus, it could be hypothesized that CNCs are perfectly compatible and dispersed in the polymeric matrix. In contrast, if commercial fibers were used, their presence in the matrix would be displayed, and the presence of two phases, the fiber phase and the matrix phase, would be very evident. SR and SFE values of the SC resin were shown in Table 1. The results showed that the incorporation of CNC

Figure 3. DSC thermograms of CNC incorporation into SC specimens.

did not affect the roughness for groups 0.5 and 0.75% of SC, however for other groups there was an increase. This property is directly related to microorganism accumulation once microorganisms adhere to the surface of hard liners, colonize these abiotic structures and can lead to the development of oral pathologies[48]. Thus, to facilitate the hygiene of dentures, the smoother the prosthetic surface the better[48]. The SFE determines the interaction between cohesion and adhesion forces and the wetting capacity of a solid[49]. The clinical significance of this physical property is related to its surface wetting capability, where high SFE is desired when adhesion is required, but undesirable if plaque resistance is required[50]. In the present study, the addition of CNCs to SC (0.75%) was also favorable for the variable SFE where a decrease was noted. Studies have been conducted to verify the association between SFE and Candida spp. adhesion and it was observed that the surface of polymeric materials with high SFE may induce greater adhesion and proliferation of Candida albicans[48]. The attraction forces for liquid (or microorganism) with the same chemical composition could be explain for a high SFE, that is understood as a high number of active ions on surfaces[51]. Nanometric materials, such as CNCs, diamond, carbon and metal oxide, have been incorporated into acrylic resins for reinforcement purposes[10,52]. The use of CNCs stands out because it is a material from renewable and easily found sources. Complementary studies with microbiological and cytotoxic tests should be carried out before applying this material in clinical research.

Figure 4. Surface morphologies of the samples obtained from SEM of CNC incorporation into SC specimens.; original magnification ×200, ×5000, and ×2000. 4/7

Polímeros, 32(1), e2022006, 2022

Cellulose nanocrystals into Poly(ethyl methacrylate) used for dental application Table 1. Mean results and standard deviation for the flexural strength, surface roughness and surface free energy assessment according to different CNC concentrations for SC. Title Control 0.25% 0.5% 0.75% 1% Control

Surface Roughness (µm) 2.23 (1.05) A 3.56 (0.47) B 3.18 (1.19) AB 3.14 (0.80) AB 3.65 (1.17) B 2.23 (1.05) A

P .019

Surface Free Energy (erg cm-2) 40.76 (3.01) A 43.89 (2.41) AB 44.48 (4.34) AB 40.38 (2.94) B 41.85 (3.01) AB 40.76 (3.01) A

P .019

Means (SD) followed by the same uppercase in columns are significantly similar (P>.05).

Promising results were obtained when the CNCs was associated with a commercial resin with increased surface hardness and decreased SFE of the material, which was not harmful to the other physical properties evaluated here. CNCs within the hard liners can be used to customize temporary or long-term use of dentures with better physical properties when compared to conventional hard chairside resins. It means that the incorporation of CNCs into this denture base resin could improve the abrasion resistance of this material, which is desirable in the long term since the denture base is constantly subjected to brushing[53]. Further studies are required to assess other proprieties of CNCs within hard liners and characterize conditions of maintenance in long-term use, for a better clinical application in dentistry. In addition, the evaluation of the antimicrobial activity and biocompatibility are recommended before these findings can be applied in a clinical environment. The present study has the potential to present as an alternative the improvement of hard chairside reline resins produced from residual vegetable biomass and should be considered promising in future studies, which may provide technological, economic and environmental benefits since they can promote the increase of the useful life of the complete dentures through the use of a renewable material.







4. Conclusions The simple and straightforward approach of adding CNCs, a renewable material, provides good potential for its future practical application as it has shown promise with increasing Vickers hardness. It means that the incorporation of CNCs into this denture reline resin could improve the abrasion resistance of this material, which is desirable in the long term.

5. Acknowledgements



Study supported by FAPESP (São Paulo Research Foundation), grant no. 2017/26512-9.

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ISSN 1678-5169 (Online)


Ecological structure: production of organic impregnation material from mussel shell and combustion Hüseyin Tan1* , Murat Şirin2  and Hasan Baltaş2  Department of Materials and Material Processing Technologies, University of Recep Tayyip Erdoğan, Rize, Turkey 2 Department of Physics, University of Recep Tayyip Erdoğan, Rize, Turkey



Abstract In the research, sea mussel shell (Chamelea gallina) powders were impregnated on the samples of Eastern spruce (Picea orientalis (L.) Link.) and Anatolian chestnut (Castanea sativa Mill.) by dipping method at different concentrations (1%, 5%, 10%, 15%). To investigate the level of use in the wood industry and especially its effects against fire; adhesion, thermogravimetric analysis (TGA), limiting oxygen index (LOI) test measurements were carried out. According to the TGA results, while the residue quantity in the spruce wood sample was the highest at 5%, the residue amount in the chestnut wood sample was the highest at 15%. With increasing amounts of mussel shell powder, the limiting oxygen index values in both wood species samples increased. As a result, it was discovered that impregnating wood samples with mussel shell powder improved the wood’s fire resistance. Keywords: ecosystem, thermogravimetric TGA analysis, LOI analysis, mussel shell, human/environmental health. How to cite: Tan, H., Şirin, M., & Baltaş, H. (2022). Ecological structure: production of organic impregnation material from mussel shell and combustion. Polímeros: Ciência e Tecnologia, 32(1), e2022007. https://doi.org/10.1590/01041428.20210110

1. Introduction The ecosystem’s health, the acquisition and development of new organic preservatives/top surface treatment materials in the wood industry, and the creation of novel impregnation technologies are all critical for humanity’s future. Wood can provide valuable services in a variety of sectors when used properly. However, Unfavorable usage environments and fire, can easily cause the wood to deteriorate. As a result, fire retardant treatment of wood is vital process. The proper application of fire retardant chemicals protects the wood from burning and, as a result, increases the wood’s service life. The use of fire retardant chemicals does not guarantee that the wood will be fully non-flammable. However, they can make it harder for the wood to ignite and delay the quick spread of fire once it has begun[1,2]. The transport of moisture away from the wood is influenced by permeabiliy or moisture ingress into the wood under the charred layer affects its movement. The intensity of the fire has an impact on the rate of carbonization. The rate of carbonization lowers as the temperature to which the wood is exposed rises[3]. Pabeliña et al.[4] It has been reported that the pyrolysis of cellulose and its interaction with oxygen causes wood combustion and the process of pyrolysis starts with a rise in temperature. Wood is an essential raw material that has been utilized by humans in a variety of industries since humankind existence. Without the use of chemicals, wood can be made to be durable with some precautions. Chemical operations

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are also necessary due to the diversity and continuity of risks. Impregnated wood has a significant role in the construction industry due to its economics, aesthetics, and appearance, in addition to its resistance to environmental factors. It has been reported that synthetic/chemical materials in the living environment pose major risks to human and environmental health, thus the creation of organic wood preservatives has become a requirement[5,6]. In the field of impregnation, new approaches are being developed. To provide fluid fluidity and increase retention rate depending on the impregnation process; drying, steaming, engraving, and vacuum pressure application are among the techniques employed in addition to biological, chemical, mechanical, and physical processes. The effectiveness of fire retardant chemicals on wood has been studied in various academic researches. Because of their environmental friendliness, low cost, and fire retardant efficacy, nitrogen and phosphorus-containing compounds are known to be essential criteria in the usage of fire retardant chemicals[7]. The wood was impregnated with boric acid, a borax combination, and a variety of natural tanning chemicals; and it is resulted that natural tanning materials produce positive results in terms of combustion parameters[8]. Carbonization occurs between 500 and 800 °C, and wood components disintegrate into hemicellulose, cellulose, and lignin, respectively[9]. Stone water has been shown to be effective in preventing fire effects in the wood sector in a variety of experiments[10,11].



Tan, H., Şirinc M., & Baltaş H. In this respect, natural mussel shells are grinded and prepared in different concentrations, and chestnut and spruce woods are impregnated by dipping method; physical properties were determined by performing adhesion level/ thermogravimetric (TGA) analysis and Limit Oxygen Index (LOI) tests. In terms of environmental health, it is aimed that this natural preservative will be applied in the wood industry as an impregnation substance and a fire retarder.

2. Materials and Methods 2.1 Material Within the study’s scope, chestnut (Castanea sativa Mill.) and spruce (Picea orientalis (L.) Link) woods were preferred. As a natural impregnation medium, a solution made from mussel shell was employed. The mussel shell samples were pulverized in a ring mill and then passed through a 63 micron screen to guarantee particle size uniformity before being used to make the solution. From the sieved mussel powder samples, 100 mL of 1 percent, 3 percent, 5 percent, 10 percent, and 15 percent solution were prepared using weights of 1, 3, 5, 10, and 15 g, respectively. Weighed samples were placed in a 200 mL beaker on a magnetic stirrer sequentially. To dissolve the mussel powder, the beaker was filled with 15-20 mL ultrapure 32 percent HCl and 10 mL distilled distilled water. The shell samples were dissolved at 500 rpm and 200 oC temperatures. Then, the solution obtained from the dissolved shell samples was completed to a final volume of 100 mL with the help of distilled distilled water.

certain temperature rise rate is precisely detected. The thermal deterioration of polymeric materials and the characterization of kinetic events happening during the degradation are the two primary applications of this approach[13]. TGA analysis was carried out according to ASTM E1131-08[14] that has conditions; approximately 10 mg of wood flour passing through 40 mesh, not passing through 60 mesh, 50 mL/min, under nitrogen gas at a flow rate of 57, 10°C/min, with a temperature rise rate of 25 °C to 600 °C. The % weight loss in the sample at the highest temperature point, the time period with the maximum amount of instant weight loss, and the rapid pyrolysis temperature point values were all analyzed as a consequence of the experiment. In addition, the temperature-dependent weight loss curves were graphically obtained.

2.5 Limit oxygen index test (LOI) All The oxygen index test is applied to assess the flammability of combustible materials. This value represents the quantity of oxygen that must be present in the atmosphere for combustion to proceed; it also determines how the combustion event occurs, as well as the amount of carbon in post-combustion residues and decomposable organic compounds ASTM 2863-09[15]. For the LOI test, 1%, 3%, 5% and 10% impregnated wood material groups were contructed.

3. Results and Discussions 3.1 Solution properties

2.2 Preparation of experimental samples For TG Analysis, both wood types were taken to represent the entire main mass, that is, the entire material from which the sample was taken, the materials that remained on the 40 and 60 mesh (250 1847 micron) sieves were ground in a Willey mill and placed in jars[9]. For LOI analysis, wood samples were prepared as 100x10x10 mm.

2.3 Impregnation process

The solution properties are given in Table 1. Before and after impregnation, the pH/density did not change. It has been reported in the literature that the acidic/ basic structure has a positive/negative effect on the anatomical and technological properties of the wood.

3.2 Retention (%) % Retention results are presented in Table 2.

The immersion (medium-term immersion) approach is preferred for impregnation. Ten test samples were processed for each TGA and LOI test. The completely dried samples were kept in the solution for 12 hours and re-weighed, and the measurement was carried out again at 103±2 °C after 24 hours[12].

Chestnut wood has the highest retention value (1.88%) and spruce wood has the lowest retention value (0.22%).

2.4 Thermogravimetric analysis (TGA)

Table 3 shows the findings of a thermogravimetric (TGA) examination of chestnut wood, whereas Figure 1, 2, 3 shows the corresponding graphics.

Thermogravimetric analysis is an analysis method in which the weight loss of polymeric or other samples at a

3.3 Thermogravimetric (TGA) analysis 3.3.1 Chestnut wood thermogravimetric (TGA) analysis results

Table 1. Solution properties. Concentration and Material 1% 5% 10% 15%

Mussel shell


Temperature (ºC)



pH BI 1,38 1,08 0,88 1,00

AI 1,38 1,08 0,88 1,00

Density (g/ml) BI AI 0,988 0,988 1,006 1,006 1,010 1,010 0,999 0,999

BI: Before impregnation; AI: After impregnation.


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Ecological structure: production of organic impregnation material from mussel shell and combustion When the table and graph were analyzed, it was discovered that a peak at 80°C was generated by the elimination of moisture from the material, and that the primary degradation peak was about 335-342°C. The thermal resistance of the control sample is generated by the degradation of lower hemicelluloses and some extractives; we can conclude that the thermal resistance of this peak value is formed by the degradation of lower hemicelluloses and some extractives. With the amount of additive supplied to the material, the peak height reduced and the decomposition temperature of the readily pyrolysis sample components decreased. The turning point temperature increased from 335°C to 340°C when the amount of additive was raised in the sample compared to the control sample; this could be owing to the amount of additive added.

Table 2. % Retention and duncan test results. Wood type Concentration Chestnut wood %1 %5 %10 % 15 Spruce Wood %1 %5 %10 %15

% Retention 0.89 1.61 1.88 1.70 0.22 0.31 0.88 0.64


HG: Homegeneity groups.

3.3.2 Spruce Wood Thermogravimetric (TGA) analysis results Table 4 shows the results of the thermogravimetric (TGA) study of spruce wood, with accompanying visuals in Figures 4, 5, 6. According to the results obtained; with the increased amount of chemicals, the turning point temperature was 388.95 °C, and even with the addition of 1% chemical, the turning point temperature reached 377.63 °C. However, as can be seen in the table and graph, it is seen that rates above 5% do not have a positive effect. It can be concluded that the most suitable ratios for spruce samples are 1% and 5%. The amount of residues at 550°C rose as expected with the addition of the chemical. It’s worth noting that impregnating spruce wood is anatomically challenging. When compared to the literature, it has been discovered that identical results have been found we can say that this is due to the anatomical structure of the wood, wood moisture, wood dimensions and wood type.

3.4 LOI analysis Table 5 shows the results of the limit oxygen index test (LOI), and Figure 7 shows the corresponding graphic. In comparison to the control samples, the limiting oxygen index percentages increased as the solution % increased in both wood types. The results demonstrate that the highest LOI percentage (26%) of the 5 percent and 10% values of chestnut wood samples was identified; the results show

Table 3. Results of thermogravimetric analysis (TGA) of Chestnut Wood. Concentration Control %1 %5 %10 %15

Initial Temperature (°C) 304.00 309.45 305.91 306.15 302.81

Turning point (°C) 335.18 338.64 341.91 342.34 340.61

Final (oC) Temperature 350.15 351.25 354.03 356.59 358.80

Delta Y (%) 58.41 58.00 55.84 54.66 53.31

Residual amount at 550 °C (%) 25.18 25.50 27.11 26.16 27.01

Figure 1. Chestnut wood TGA spectra. Polímeros, 32(1), e2022007, 2022


Tan, H., Şirinc M., & Baltaş H.

Figure 2. TGA spectrum derivatives of chestnut wood.

Figure 3. TGA Weight loss derivatives of chestnut wood.

Figure 4. Spruce wood TGA spectra (%).

Figure 5. Spruce wood TGA spectra (%). Table 4. Spruce wood thermogravimetric (TGA) analysis results.



Initial Temperature (°C)

Turning point (°C)

(oC) Final Temperature

Delta Y (%)

Residual amount 550 °C (%)































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Ecological structure: production of organic impregnation material from mussel shell and combustion

Figure 6. Spruce wood weight loss derivative.

Figure 7. Chestnut and spruce wood samples LOI values. Table 5. Spruce and chestnut wood LOI results. Wood type Spruce wood

chestnut wood

Solvent Concentration Kontrol %1 %3 %5 %10 Kontrol %1 %3 %5 %10

Limiting Oxygen index (%) 24.00 24.00 24.25 24.50 25.00 24.75 25.00 25.50 26.00 26.00

parallelism with retention values. It has been resulted that increasing the amount of substance penetrating the wood material raises the wood’s fire resistance as well as the oxygen percentage, which is required by another method.

4. Conclusions The mussel shells (CaCO3), which are found inert in nature and can be used sparingly (poultry feed, etc.), are pulverized and treated with HCl acid, then, the impregnation material is formed by concentration with water at appropriate percentages in the research. Polímeros, 32(1), e2022007, 2022

Örs and Keskin[16] Wood’s resistance to acids at low concentrations is higher than its resistance to bases. This is due to the fact that cellulose and lignin are acid resistant, whereas hemicellulose and lignin are strong base resistant. 2% solutions of HCl and NaOH and other acids and bases do not cause any significant deterioration in wood at room temperature. However, the degradation rate increases as the concentration rises and the temperature and duration of action rise. Many tree species’ resistance drops by 50-75 percent at a concentration of 10% and a temperature of +50 °C. In this respect, bases are more destructive. Coniferous woods are more resistant to chemicals than leafy woods because they contain less hemicellulose. HCl, when applied at low rates, appears to be a favorable choice in terms of wood’s acid resistance, according to the research. Thermogravimetric analysis (TGA) and oxygen limit index (LOI) analyses were performed after impregnation of the produced impregnation material on spruce and chestnut woods. It was determined that the LOI analysis result percentages increased in the same period in parallel with the concentrations of the impregnation material. Furthermore, based on the findings of TGA analysis, it was found that impregnation material with a 5% solution would enough, particularly for spruce wood and the highest result was obtained with a 10% solution on chestnut wood. In both wood types, the amount of residue increased in parallel with the amount of solution. Based on these findings, the ideal proportions for spruce and chestnut are 5% solution combination for 5/8

Tan, H., Şirinc M., & Baltaş H. spruce and 10% solution mixture for chestnut, taking into account time, labor, and cost. Hirata et al.[17] The thermal breakdown of cellulose, hemicellulose, and lignin in wood occurs at various temperatures, according to thermogravimetric analysis (TGA) research. Uysal[18] When wood is heated from room temperature to 100 °C, essentially no chemical reactions occur, and only the moisture and essential oils evaporate in the wood. It has been reported that when the temperature is significantly raised, such as to 200 °C, chemical bonds in wood weaken and degrade as a result of dehydration processes, and above 250 °C, all wood components are exposed to thermal deterioration. Rowell and Dietenberger[19] The majority of the carbohydrate components were destroyed between 300 and 375 °C, while lignin remained. Hill[20] Ranking of wood components for thermal resistance were given as at low temperatures; hemicellulose < lignin < cellulose, while at high temperatures; hemicellulose < cellulose < lignin. It has been reported that the thermal degradation of hemicelluloses starts at 180-200 °C, and the thermal degradation of cellulose starts at 210-220 °C and reaches the highest level at 270-280 °C and is completed between 300 °C and 340 °C. Thermal degradation was shown to occur between 294-388 °C for spruce wood and 302-342 °C for chestnut wood in the research. The hemicellulose and lignin content of spruce wood is higher than chestnut wood, this situation plays an active role in thermal deterioration at high temperature values. Goldstein[21] Methanol, acetic acid, furan, and furfural are released during rapid pyrolysis when hemicellulose is degraded, levoglucosan (1,6-anhydro-beta-D glucopyranose) is released when cellulose is degraded, and phenols are released when lignin is degraded. Rapid pyrolysis produces aromatic moieties such as xylenols, guaiacols, cresols, and catasols. It was determined that charcoal remained after carbonization between 400 and 500 °C and the removal of combustible gases. Rowell et al.[22] It has been discovered that the rate of lignin coal generation during burning is higher than that of cellulose and hemicellulose, indicating that lignin has a more heat-resistant structure and a high char formation rate. It is concluded that it reduces the generation of flammable gas, hence preventing further thermal deterioration of the wood. Akgün[23] The cell wall does not contain extractive chemicals; instead, they are present in the cell gaps. Coniferous woods typically contain high levels of resinous compounds (oil and resin), resulting in high ether extract rates. The amount of this resinous material in spruce is typically less than 1%, although the resin rate in pines has been found to be between 2 and 6 percent. Although the impregnation process is particularly tough in the study, the low amount of resin in spruce wood is one of the reasons why it is preferred over coniferous plants. Peker and Atılgan[24] It is resulted that; when wood is ignited, it has been discovered that the rate of flame propagation on its surface is substantially governed by the wood’s heat conduction and heat capacity, with an inverse relationship between wood density and flame propagation speed. Wang et al.[25] The accelerated decomposition is 6/8

is owing to the fact that fire retardant bonds like P-O-C (Phosphorus-Carbon) are substantially less stable than C-C bonds in the control sample, and they decompose around 180 oC. Basak et al.[26] This early breakdown reduces dangerous flammable gases generation while also promoting the formation of carbonized coal. Tutuş et al.[9] The thermal degradation of wood and wood components happens between 300 and 500 °C, according to research. The number of residues at 550 °C increased in parallel with the amount of retention in our study in both tree species following TG analysis. Kartal and Imamura[5] Since the impregnation material that we use has not been tested before, the most effective mixture can be determined by preparing mixtures at certain percentages with other tried (boron, borax, etc.) Even without the use of various chemicals, wood can become durable with some precautions. Nonetheless, chemical activities are required due to the variety and continuity of dangers. Kartal[6] With its economy, aesthetics, and appearance, as well as its resistance to factors, impregnated wood (biotic/ abiotic, etc.) plays a significant role in the building industry. According to Özdemir[27], the usage of synthetic/chemical materials in his environment creates major hazards to human and environmental health, the creation of organic wood preservatives is necessary. Finding and developing wood organic preservatives is extremely significant in this study. In the field of impregnation, new approaches are being developed. To ensure fluid fluidity and boost the retention (retention) rate, methods such as drying, heating, vacuum, and pressure application are utilized in addition to biological, chemical, mechanical, and physical processes, depending on the impregnation procedure. The effectiveness of fire retardant chemicals on wood has been studied in a number of scholarly research. Environmental friendliness, low cost and fire retardant performance are important parameters in the use of nitrogen and phosphorus-containing chemicals as fire retardant chemicals[28]. In line with the literature, it is critical for the industry to provide ecologically acceptable mussel shells, which we have tested in combustion experiments and shown to be effective as a fire retardant, and which are plentiful in nature and also was not completely evaluated before our research. Peker and Atılgan[29] impregnated with waste tea plant extract, Iroko wood had the lowest percent retention rate (1.58 percent), beech wood had the highest percent retention rate (6.75 percent), iroko had the lowest overall retention at (31,27 kg /m3), and they determined that the highest total retention value in beech wood (100.65 kg/m3). Flynn[30] The differences in retention rates may have been caused by the wood species, the anatomical structures of the trees, thus the physical properties, the impregnation process and the solution and it was determined that many factors related to the anatomical structure such as heartwood, sapwood, spring wood, summer wood, density, sapwood, tracheid and resin affect the permeability. Both TGA and LOI data indicate favorable results in terms of combustion retardation as the quantity of retention rises, according to the research. When the variations in retention are compared to spruce and chestnut trees, as stated in the literature, spruce wood impregnation is difficult due to its anatomical structure. Polímeros, 32(1), e2022007, 2022

Ecological structure: production of organic impregnation material from mussel shell and combustion Özdemir et al.[31] Polymer chemical structures have a considerable impact on LOI values. LOI values can be determined by the number of oxidizable atomic or molecular groups of polymers. The greater the hydrogen-to-carbon ratio, which impacts the materials’ flammability, the more flammable they are. A high LOI rating for any substance suggests that it will be difficult to ignite in the atmosphere. As a result, materials with a LOI value of less than 25% may readily burn in air, whereas those with a LOI value of more than 25% have reported that they are self-extinguishing in air. In our research, we discovered that, as the solution percentages increase, the LOI values in both tree species increased. As a result, the amount of CO2 emitted into the atmosphere by CaCO3 during burning leaves the environment without oxygen. Therefore, the LOI values increased. Göker and Ayrılmış[32] when wood is subjected to high temperatures, its structure deteriorates, forming an insulating layer (the carbonized section) that prevents oxygen from entering the burning material and slows further degradation. The cross-sectional dimensions of structural wood pieces determine their load-bearing capability. Thefore, the carbonization rate of the cross-section is a significant determinant for the resistance of structural wood parts, among other things. It prevents the rapid chemical degradation of the material and the diffusion of oxygen from the charred surface into the interior. The fire resistance of structural wood elements is intimately connected to the rate of carbonization of wood and wood-based materials. The carbonization and pyrolysis (heat decomposition) layer impregnated results can be seen in the TG analysis tables of spruce and chestnut wood samples in our experiments. Compared to the control groups, the initial temperatures decreased, that is, it charred earlier and formed the pyrolysis layer, therefore, as mentioned in the literature, it is possible to prevent oxygen from entering the part whose structure is deteriorated. As a consequence, the LOI results also support this. In other words, TG Analysis increased the LOI (Limit Oxygen Index) value due to the fact that the decrease in the initial temperatures, this causes early carbonization and pyrolysis, and the lack of oxygen in the environment. Çavdar[33] investigated the effects of copper-based and water-based wood preservatives on LOI values on fir wood. CbWPs and new generation wood preservatives have a partial fire retardant effect on wood other than CCB. It was concluded that concentrations of CCB treatment greater than 3% may have a potential fire retardant. Based on our LOI results, it is an example of the literature that refers to the increase in LOI values as the solution percentage increases. In general, it is known that the mechanical and physical resistance of wood decreases with impregnation. In the percentages of this combination that we developed in another investigation, it is suggested that several mechanical and physical resistance capabilities should be assessed in the same wood species. The physical and mechanical resistance properties of the boards, particularly the adhesion resistance, can be investigated by using the same impregnation material for wooden boards and coatings, in addition to TGA and LOI tests on wooden boards, because we know that impregnation materials have a negative effect on adhesion resistance. After impregnation, the surface gloss and surface treatments of the wood material can be examined. The wood Polímeros, 32(1), e2022007, 2022

samples we utilized in our studies did not change color after being impregnated. Therefore, they can be utilized in the construction of wood materials (wooden door and window joinery, roofing materials, building materials, toy industry, playgrounds, collective living spaces, wooden structures, insulation boards, furniture) especially for indoors. In nano-technology with different methods other than forest products; in industries such as the textile industry, impregnated products can be considered for also military textile products, especially with fire protection. As a result, a raw material (mussel shell), which has never been tried before and is abundant in nature, and it was applied for the first time as imperagnated material, a solution was prepared (as a result of melting the ground mussel shell powders with HCl) and its effect on the burning property of wood was evaluated; TGA analysis and LOI analysis showed positive results as flame retardant. When compared to any interior wood preservative on the market, the raw material utilized is far more cost-effective. Furthermore, delivering a raw material that has never been analyzed and left to the ecology as inert in nature will contribute significantly to the country’s economy.

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Tan, H., Şirinc M., & Baltaş H. 9. Tutuş, A., Kurt, R., Alma, M. H., & Meriç, H. (2010). Chemıcal analysis of scotch pine wood and its thermal properties. In III Ulusal Karadeniz Ormancılık Kongresi (pp. 1845-1851). Artvin: Artvin Çoruh Üniversitesi Orman Fakültesi. 10. Can, A., Özlüsoylu, İ., Grzeskowıak, W., & Sözen, E. (2017). Improvement of fire performance of impregnated wood with copper based chemicals. In 28th International Conference on Wood Science and Technology, ICWST 2017: Implementation of Wood Science in Woodworking Sector (pp. 21-27). Croatia: Faculty of Forestry, University of Zagreb. Retrieved in 2021, January 12, from https://www.researchgate.net/publication/321803829 11. Çakal, S. (2018). The effect of impregnation/ diffusion times on some technological properties of oriental spruce (Picea orientalis (L.) Link) wood (Master’s Thesis). Artvin Coruh University, Artvin. 12. Var, A. (1994). The effect of the use of natural resin (Colophan) on the water-repellent properties of wood material (Master’s Thesis). Karadeniz Technical University, Trabzon. 13. Le Van, S. L. (1989). Thermal degredation. In Schniewind, A.P. (Ed.), Concise encyclopedia of wood and wood-based materials (pp. 271-273), New York: Pergamon Press. 14. American Society for Testing and Materials – ASTM. (2014). ASTM E1131-08: standard test method for compositional analysis by thermogravimetry. USA: ASTM International. 15. American Society for Testing and Materials – ASTM. (2006). ASTM D2863-06: standard test method for measuring the minimum oxygen concentration to support candle-like combustion of plastics (Oxygen Index). USA: ASTM Internatıonal. 16. Örs, Y., & Keskin, H. (2001). Wood material information. Ankara: Nobel Academic Publishing. 17. Hirata, T., Kawamoto, S., & Nishimoto, T. (1991). Thermogravimetry of wood treated with water-insoluble retardants and a proposal for development of fire-retardant wood materials. Fire and Materials, 15(1), 27-36. http://dx.doi. org/10.1002/fam.810150106. 18. Uysal, B. (1997). The effects of various chemicals on the fire resistance of wood material (PhD Thesis). Gazi University, Ankara. 19. Rowell, R. M., & Dietenberger, M. A. (2012). Thermal properties, combustion, and fire retardancy of wood. In R. M. Rowell (Ed.), Handbook of wood chemistry and wood composites (pp. 121-151). USA: CRC Press. http://dx.doi. org/10.1201/b12487-11 20. Hill, C. A. S. (2006). Wood modification: chemical, thermal and other processes. UK: John Wiley & Sons Ltd. http://dx.doi. org/10.1002/0470021748 21. Goldstein, I. S. (1973). Degradation and protection of wood from thermal attack. In D. D. Nicholas (Ed.), Wood deterioration and its prevention by preservative treatment (pp. 307-339). USA: Syracuse University Press. 22. Rowell, R. M., Susott, R. A., DeGroot, W. F., & Shafizadeh, F. (1984). Bonding fire retardants to wood. I. Thermal behavior of chemical bonding agents. Wood and Fiber Science, 16(2), 214-223. Retrieved in 2021, January 12, from https://agris.fao. org/agris-search/search.do?recordID=US8630248


23. Akgün, H. C. (2005). Chemical composition of Anatolian chestnut wood and its suitability for paper making (Master’s Thesis). Zonguldak Karaelmas Unıversity, Bartın. 24. Peker, H., & Atılgan, A. (2015). Wood as a natural energy source: combustion properties and protection methods. Afyon Kocatepe University Journal of Science and Engineering Sciences, 15, 022201. Retrieved in 2021, January 12, from https://dergipark.org.tr/tr/download/article-file/205867 25. Wang, X., Hu, Y., Song, L., Xing, W., & Lu, H. (2010). Thermal degradation behaviors of epoxy resin/POSS hybrids and phosphorus-silicon synergism of flame retardancy. Journal of Polymer Science. Part B, Polymer Physics, 48(6), 693-705. http://dx.doi.org/10.1002/polb.21939. 26. Basak, S., Samanta, K. K., Chattopadhyay, S. K., & Narkar, R. (2015). Thermally stable cellulosic paper made using banana pseudostem sap, a wasted by-product. Cellulose, 22(4), 27672776. http://dx.doi.org/10.1007/s10570-015-0662-7. 27. Özdemir, B. (2020). Esgin (Rheum ribes L.) plant (Antioxidant/ Antibacterial) extract on the improvability and technological properties of wood (Master’s Thesis). Artvin Coruh University, Artvin. 28. Özkan, E. O. (2018). The performance of fire retardant treated wood in outdoor conditions (Doctoral Dissertation). Kastamonu Üniversitesi, Kastamonu. Retrieved in 2021, January 12, from http:// earsiv.kastamonu.edu.tr:8080/xmlui/bitstream/handle/123456789/803/ Osman%20Emre%20%c3%96zkan.pdf?sequence=1&isAllowed=y 29. Peker, H., & Atılgan, A. (2015). Using various mordant-water solvent varnish of waste tea extract dye on wood and effects of dynamic bending strength. Selcuk-Technic Journal, 644-651. Retrieved in 2021, January 12, from http://sutod.selcuk.edu. tr/sutod/article/view/263 30. Flynn, K. A. (1995). A review of the permeability, fluid flow, and anatomy of spruce (Picea SPP.). Wood and Fiber Science, 27(3), 278-284. Retrieved in 2021, January 12, from https:// wfs.swst.org/index.php/wfs/article/view/1659 31. Özdemir, F., Tutuş, A., & Bal, B. (2013). The effect of fire retardants on the thermal conductivity and limit oxygen index of high-density fiberboard. Turkish Journal of Forestry, 14(2), 121-126. Retrieved in 2021, January 12, from https://dergipark. org.tr/en/pub/tjf/issue/20901/22447 32. Göker, Y., & Ayrılmış, N. (2002). Performance characteristics and thermal degredation of wood and wood based products in fire. Istanbul Üniversitesi Orman Fakültesi Dergisi, 54(2), 1-22. Retrieved in 2021, January 12, from https:// arastirmax.com/en/publication/istanbul-universitesi-ormanfakultesi-dergisi/53/1-2/yanginda-odun-odun-esasli-urunlerinperformans-karakteristikleri-termal-degredasyonu/arid/ bdaed57e-ed0c-4d56-8157 33. Cavdar, A. D. (2014). Effect of various wood preservatives on limiting oxygen index levels of fir wood. Measurement, 50, 279-284. http://dx.doi.org/10.1016/j.measurement.2014.01.009. Received: Jan. 12, 2022 Revised: Feb. 20, 2022 Accepted: Mar. 08, 2022

Polímeros, 32(1), e2022007, 2022

ISSN 1678-5169 (Online)


Fatigue damage propagation and creep behavior on sisal/epoxy composites Mateus da Silva Batista1* , Linconl Araujo Teixeira1 , Alisson de Souza Louly1 , Sayra Oliveira Silva1  and Sandra Maria da Luz1  Laboratório de Tecnologias em Biomassa, Departamento de Engenharia Mecânica, Universidade de Brasília – UnB, Brasília, DF, Brasil



Abstract The lack of knowledge about the behavior under creep and fatigue limits the use of polymeric composites reinforced with natural fibers. Thus, this work assessed the behavior of epoxy composites reinforced with sisal fibers under tensile, fatigue, and creep tests. Also, thermogravimetry and scanning electron microscopy assessed the sodium hydroxide (NaOH) treatment efficiency in sisal fibers. Further, differential scanning calorimetry determined the degree of cure of the composites, and stereomicroscopy allowed the evaluation of the surface’s fracture. As a result, the tensile strength of the composite was 1.4 times the value of neat epoxy resin after 100,000 cycles in the fatigue test. Moreover, when loaded with 20% of the maximum tensile strength, it is estimated that the composite resists 200,000 h without rupturing by creep. To conclude, the efficient adhesion between sisal fibers and epoxy obtained by NaOH treatment allowed good mechanical behavior to the epoxy composite. Keywords: natural fibers, NaOH treatment, mechanical properties, thermal behavior. How to cite: Batista, M. S., Teixeira, L. A., Louly, A. S., Silva, S. O., & Luz, S. M. (2022). Fatigue damage propagation and creep behavior on sisal/epoxy composites. Polímeros: Ciência e Tecnologia, 32(1), e2022008. https:// doi.org/10.1590/0104-1428.20210093

1. Introduction Although polymer matrix composites reinforced with carbon, glass, or Kevlar fibers have excellent mechanical properties associated with low weight, the use of these synthetic fibers has adverse effects on the environment since their production requires a large amount of energy[1]. Polymeric composites reinforced with natural fibers from plants are attractive alternatives. There is a wide variety of plants used as reinforcement in polymeric matrices, for example, sisal, jute, palm, curauá, and others. The fibers used as reinforcement come from different plants parts such as the trunk, leaf, fruit branches, and stem[2]. And even if the adhesion between natural fibers and polymers is low, it can be improved with chemical treatments, removing fiber amorphous and nonpolar components and impurities that repel polymers[3]. The treatment with sodium hydroxide (NaOH) is simple and widely used. Palm fruit branch fibers show about 180% greater tensile strength when treated with a NaOH solution, reaching about 290 MPa[2]. In epoxy composites, natural curauá fibers treated with NaOH improve tensile, flexural, and impact strength by 24%, 44%, and 47%, respectively, comparing untreated samples[4]. This sums up the potential of natural fibers for composite applications. Recently, several studies have reported the mechanical performance of polymeric composites reinforced with natural fibers, but almost all reports use only tensile,

Polímeros, 32(1), e2022008, 2022

flexural, and impact tests. However, more detailed studies on the behavior of polymeric composites reinforced with natural fibers under cyclic loads or quasi-static loads are still required for structural applications. Since composites reinforced with natural fibers are applied for non-structural purposes, such as panels, engine covers, and internal parts of cars and aircraft[5]. Fatigue is the leading cause of failure for failures in aircraft components[6], vehicle storage tanks[7], machine and rail bearings[8], among other engineering applications subjected to cyclic loads. Furthermore, the variability in fatigue properties of the same type of composite due to differences in composition and structure properties requires an understanding of the fatigue mechanism in these materials[9]. Experimental studies are still needed to understand damage propagation and failure modes in composites, and in this sense, the analysis of material stiffness during fatigue tests is used[10]. However, the material’s stiffness, which should be reduced with increasing damage to the composites, increases in some cases[11]. Therefore, fatigue tests controlled by strain instead of stress have a consistent behavior with the stiffness of composites, which is reduced until the material breaks[12]. Furthermore, the study of the creep behavior of polymeric composites is also crucial for safe structural design because it presents a time-dependent degradation in stiffness due to strains which can lead to material breakage[13]. The effects



Batista, M. S., Teixeira, L. A., Louly, A. S., Silva, S. O., & Luz, S. M. of creep are long-term, and research aims to predict the strength of polymer composites through accelerated testing. However, in general, the tests use temperature variation to accelerate the creep effect on the material, which makes it challenging to obtain results[14]. Therefore, the stepped isostress method (SSM) is a form of life prediction in which the temperature is constant, and what varies is the stress, which is systematically applied to the material[15]. SSM effectively tests amorphous or crystalline polymers and reduces test time by 70 times to predict material life[16]. Although there are reports of epoxy composites reinforced with synthetic fibers under fatigue and creep, few works have studied the performance of composites reinforced with natural fibers. However, natural fibers are promising as reinforcement. For example, hybrid composites of epoxy resin reinforced with fiberglass and kenaf (natural fibers) have fatigue life close to that of material reinforced with only fiberglass[17]. For creep, the stepped isostress method has not yet been reported for composites reinforced with natural fibers. Still, it is promising in predicting the life of polymer composites reinforced with fibers[18]. Therefore, this innovative work intends to promote natural fibers, especially sisal fibers, and provide valuable and new information about the mechanical performance of sisal fibers/epoxy composites under tension-tension fatigue controlled by strain and creep loading tests using the stepped isostress method.

2. Materials and Methods 2.1 Materials

on the fibers with a roller. The prepreg was kept at room temperature until it reached stage B, also known as the gel stage, and then kept under refrigeration at -18ºC. Laminated composites were prepared with three unidirectional prepreg layers. The layers were placed in a hydraulic press under a load of 0.5 tons for 1 h. Then, the material was cured at room temperature for 24 h. The postcure was carried out in the oven at 60ºC for 12 h. A neat epoxy resin plate was prepared for a comparison with the composite materials.

2.3 Thermal characterization Untreated and NaOH-treated sisal fibers, neat uncured epoxy, untreated, and NaOH-treated sisal 15 wt.%/epoxy prepregs were analyzed in a simultaneous TGA-DSC thermal analyzer (SDT Q600, TA Instruments, USA). The samples weighing 10 ± 1 mg were deposited on an alumina pan. The analysis was from 20 to 600°C with a heating rate of 5°C/min under a nitrogen atmosphere at a 50 mL/min flow rate. TGA was used to assess the surface treatment effect on sisal fibers, and DSC to calculate the degree of cure in the composites, α, determined by Equation 1: α = 1−

∆H p



where ∆Hp is the partial enthalpy (integral area of the prepregs cure peaks), and ∆Ht is the total enthalpy of the cure reaction (integral area of the uncured epoxy resin cure peak).

2.4 Microscopic analysis

Sisal fibers were supplied by Sisalsul Industry and Commerce LTD (Bahia, Brazil) and treated using sodium hydroxide (NaOH) 97% pure (Greentec) aqueous solution. The polymer matrix is constituted by epoxy resin AR 260, Di-Glycidyl Ether of Bisphenol A (DGEBA), and hardener agent AH 260, Triethylenetetramine (TETA), both supplied by E-composites Commerce of Composite Materials LTD (Rio de Janeiro, Brazil).

The surface morphology of the treated and untreated sisal fibers was analyzed by scanning electron microscopy (SEM) (TM-4000Plus, Hitachi, Japan), using 15 kV voltage and 400 × magnification. After the rupture in tensile tests, the fracture region of neat epoxy and NaOH-sisal 15 wt.%/epoxy composite specimens were analyzed under a stereomicroscope (DFC-700T, Leica, Germany). The investigated samples were tested before fatigue tests, and 31.5 x magnification and incident light were used.

2.2 Prepregs and laminated composites obtaining

2.5 Mechanical characterization

A superficial treatment was carried out for the sisal fibers soaking them in a 5% (w/v) sodium hydroxide (NaOH) solution with a ratio of 10:1 (solution: fiber). Then, the mix (fibers and sodium hydroxide solution) was kept at 80°C for 2 h under constant stirring. This condition followed the best result from a recent study developed by Teixeira et al.[19]. After, the fibers were washed with distilled water until pH 7. Finally, the fibers were dried at room temperature (~ 25°C) for 96 h and subsequently in an oven (32 °C) for 24 h[20]. The epoxy prepregs were obtained with 15 wt.% of fibers by the hand lay-up method[21]. This fiber content was chosen based on a previous work, where the authors studied the influence of cure agent, treatment, and fiber content by using statistical analysis[22]. Sisal fibers were placed unidirectionally in a steel mold of 200 x 220 mm. The epoxy system was prepared by mixing the resin and hardener in a ratio of 100:21 (epoxy resin: hardener agent), as recommended by the supplier. Then, the resin was spread 2/9

2.5.1 Tension-tension fatigue Tension-tension fatigue tests were carried out according to the American Society for Testing and Materials Standard (ASTM), ASTM D3479[23]. The test was performed on a universal testing machine (8801, Instron, USA) with 100 kN load cell. The test specimen dimensions were 140 × 25 × 3 mm. Tabs of epoxy reinforced with glass fibers of 40 x 25 mm were attached during the composites manufacturing. The fatigue tests were carried out with a frequency of 5 Hz, strain ratio R = 0.1, and controlled by strain. The cyclic load was applied in a sinusoidal waveform with three maximum strain levels (εmax), 0.14, 0.10, and 0.02%. For each strain level, one NaOH-sisal 15 wt.%/epoxy composite specimen and two neat epoxy specimens were analyzed. The pressure on the grips was 15 bar. The evolution of the dynamic stiffness of the materials during the fatigue tests, Ef, is calculated by the slope of the Polímeros, 32(1), e2022008, 2022

Fatigue damage propagation and creep behavior on sisal/epoxy composites secant that links the lowest and the highest peaks in the stress-strain hysteresis loop of each loading cycle. Then it is normalized by the dynamic stiffness of the first cycle, E. The damage propagation during fatigue tests, D, was calculated by D = 1 – (Ef/E)[24], where Ef and E are the same mentioned in dynamic stiffness. Dynamic stiffness values ​​and accumulated damage were plotted as a function of loading cycles. The x-axis, N/Nf, presented the number of cycles, N, divided by the maximum number of loading cycles applied to the materials, Nf.

at a staggering rate: 20% (12.98 MPa), 25% (16.23 MPa), 30% (19.46 MPa), 35% (22.72 MPa), and 40% (19.48 MPa) of the ultimate tensile strength of the specimen. After the first stress level was defined, the tension was maintained on the material for 3 h for each tension step. With these measurements, the strain vs time curve was made. From the curve from the experimental data of strain vs time, three shifts in the curve were made: the vertical, rescaling, and horizontal. For the vertical shift, each step of the initial curve is adjusted by Equation 2:

2.5.2 Tensile tests

= ε

Tensile tests were performed according to ASTM D3039[25], in two different steps: 1) Tensile before fatigue tests: To measure the maximum tensile strength and determine the maximum stress levels used in creep tests. Five specimens of NaOH-sisal 15 wt.%/epoxy composites specimens (200 × 20 × 3 mm) were tested before fatigue tests using a test machine (Electropuls E10000, Instron, USA) equipped with 10 kN load cell at a speed 2 mm/min. 2) Tensile after fatigue tests: To determine the remaining tensile strength and properties on epoxy composites reinforced with sisal fibers and neat epoxy samples that reached the run-out on fatigue tests, defined as 100,000 cycles. For each strain level in fatigue tests, one NaOH-sisal 15 wt.%/epoxy composite specimen and two neat epoxy specimens were tested. The specimen size was 140 × 25 × 3 mm, manufactured with tabs, and the tests were performed using a test machine (8801, Instron, USA) equipped with a 100 kN load cell at a speed of 0.3 mm/min. In these tests, the presence of tabs influenced the premature rupture of the material, so the execution speed was lower than that of the previous tensile test, used to determine the stress levels of the creep test. 2.5.3 Creep Creep tests were performed using a test machine (ElectroPuls E10000, Instron, USA) equipped with a 10 kN load cell and mechanical grips. The NaOH-sisal 15 wt.%/ epoxy composite rectangular specimens (200 × 20 × 3 mm) were manufactured without tabs and tested before fatigue tests. The creep test was performed according to the Stepped Isostress Method (SSM)[15]. Different loads are applied to a specimen during the same test at 25oC. The loads were changed

Ci * ( t − t0i ) n


where 𝜀 is the strain, 𝑡 is the time, 𝐶𝑖 and 𝑛 are materials constants, 𝑡0𝑖 is the point where the i-th fitting curve crosses the abscissa. Second, the rescaling shift, each step of the curve is discounted by 𝑡0𝑖. This shifts each step to the left. Finally, in horizontal shift, the curve points are divided by a displacement1factor, as obtained by Equation 3: C as =  0  Ci

n  


where C0 is the coefficient CI of the first level of strain. The creep curves are plotted with these strains vs the log time, estimating the strain after long periods.

3. Results and Discussion 3.1 Effect of chemical treatment on sisal fibers’ morphology The structural morphology of sisal fibers for untreated and NaOH-treated sisal fibers can be seen in SEM images in Figure 1. The chemical treatment removed part of the hemicellulose layers on the surface of sisal fibers, presented in Figure 1a, facilitating the adhesion between the fibers and the polymeric matrix. Thus, bundles of cellulose fibrils, a component with a high percentage of crystallinity and mechanical resistance, are exposed, as shown in Figure 1b. The adhesion between the reinforcement and the polymeric matrix could improve due to the significant surface roughness of fibers and the effective contact area[26,27]. In previous work, the treatment conditions applied to natural fibers

Figure 1. Scanning electron microscopy (SEM) for untreated (a) and NaOH-treated sisal fibers (b). Polímeros, 32(1), e2022008, 2022


Batista, M. S., Teixeira, L. A., Louly, A. S., Silva, S. O., & Luz, S. M. (5% solution, for 2 h at 80°C) improved its crystallinity. Also, the hemicellulose was removed without losses in cellulose content[19].

3.2 Thermal characterization Figures 2a and b show the TG and DTG curves for samples of untreated and NaOH-treated sisal fibers, respectively. The degradation steps are indicated in the TG curves. The first step, from 30-105 °C for untreated and 30150 °C for treated fibers, is associated with loss of moisture absorbed by the fibers (about 5% weight loss). In treated fibers, the range of temperature is higher, as the treatment increases its moisture absorption[28]. In the second step, the degradation of fiber components begins at 160°C for the untreated fiber and refers to the degradation of hemicellulose

(about 20% weight loss) followed by cellulose degradation (about 40% loss weight) in the third step. The untreated fibers showed two degradation peaks at 280 °C and 340 °C (DTG curves), corresponding to hemicellulose and cellulose degradation, respectively[29]. The treated fibers, however, have a single cellulose degradation stage (about 60% weight loss), which starts at 230 °C, indicating an increase in the thermal stability of the fibers by 50 °C. The increase in the thermal stability of the fibers happens because the alkali treatment with NaOH removes hemicellulose, which has low thermal stability[30]. In previous work, the components losses are followed by chemical characterization and Fourier transform infrared spectroscopy, confirming the effects of the treatments on hemicellulose and cellulose thermal degradation. The degradation steps (TG curves), for untreated (fourth step) and treated

Figure 2. TG and DTG curves for untreated sisal fibers (a) and NaOH-treated sisal fibers (b); DSC curves for neat uncured epoxy, untreated-sisal 15 wt.%/epoxy, and NaOH-sisal 15 wt.%/epoxy (c). 4/9

Polímeros, 32(1), e2022008, 2022

Fatigue damage propagation and creep behavior on sisal/epoxy composites fibers (third step) are linked to lignin degradation (about 12% weight loss)[29]. A residue of 23% in weight of the carbonized fibers was observed. In our previous work with the same epoxy and fiber content, 15 wt.%, also presented high thermal stability[31]. Through the DSC analyzes, it is possible to observe the exothermic peaks of the prepregs. Figure 2c shows the DSC curves for sisal (NaOH treated and untreated) 15 wt.%/epoxy prepregs compared to uncured neat epoxy resin. The first exothermic peak, around 40 °C, is related to the glass transition temperature of epoxy and agrees with what is reported in the literature[32]. The exothermic peaks around 100°C are related to the curing reactions of the crosslinks in the epoxy resin[21]. Enthalpy is the energy released by the system in the formation of crosslinks, calculated by the area under the peak curing temperature in DSC curves delimited by the baselines (dashed lines). The degree of cure of prepregs is inverse to the enthalpy value. The higher degree of cure is related to the more significant crosslinking number in the epoxy resin matrix and the elevated mechanical resistance of the composite[33]. The high crosslink content in epoxy resins reduces the strain rate of the material, increases its recovery capacity, and affects the fatigue strength of epoxy resin-based materials[34]. The degree of cure of NaOH-sisal/epoxy prepreg was 51.55%, 1.7 times higher than that of untreated sisal/epoxy

prepregs (30.36%). The presence of hydroxyl groups, OH, increases the number of cross-links established between the resin and the curing agent[35]. The chemical treatment exposes the cellulose chains of the natural fibers and their OH groups, hence the higher concentration of cross-links in the treated sisal-reinforced prepreg. In addition, the treatment on sisal fibers allows a higher crosslinked interphase due to the intermediate module between reinforcement and polymer[36]. Undoubtedly, it influenced the better fatigue and tensile strength for NaOH-sisal 15 wt.%/epoxy composites compared to neat epoxy, as discussed in the following section.

3.3 Laminated composites characterization Figures 3a and 3b shows the stiffness loss of neat epoxy and NaOH-sisal 15 wt.%/epoxy composite during the fatigue tests, respectively. For all materials, a dynamic stiffness loss during the fatigue tests is observed. This behavior is as expected, presenting the stiffness reduction[37]. When comparing the materials, at 0.14% of maximum strain, the stiffness loss was lower, about 3% for the composite. However, the composite was more resistant to fatigue than neat epoxy because the fibers transfer the applied load and provide a barrier preventing crack propagation in composites[38]. The stiffness is reduced because only part of the energy released in the loading is recovered[39].

Figure 3. Normalized dynamic stiffness during fatigue tests for (a) neat epoxy; and (b) NaOH-sisal 15 wt.%/epoxy; and damage propagation during fatigue tests for (c) neat epoxy; and (d) NaOH-sisal 15 wt.%/epoxy. Polímeros, 32(1), e2022008, 2022


Batista, M. S., Teixeira, L. A., Louly, A. S., Silva, S. O., & Luz, S. M. Figures 3c and 3d show the accumulated damage during fatigue tests of neat epoxy and NaOH-sisal 15 wt.%/epoxy composite, respectively. The presented damage curve is expected because damage to epoxy composites is inversely proportional to degradation in stiffness[40]. This is also reflected in a consistent damage curve representation. Strain control prevents the false increase in dynamic stiffness by residual strains observed for some epoxy composites reinforced with natural fibers in fatigue tests[12]. After the run-out in fatigue tests, the specimens were tested under tensile. The properties of neat epoxy and NaOH-sisal 15 wt.%/epoxy composite are shown in Table 1, where εmax is the maximum strain applied during fatigue tests cycles. The addition of treated sisal fibers to the epoxy resin increased the tensile strength by about 40%, achieving 47 MPa against 34 MPa of neat epoxy. The found values are as expected for tensile strength of epoxy composites reinforced with aligned sisal fibers, 51.5 ± 5.6 MPa[41], and superior to epoxy composites reinforced with jute fabrics, 40 MPa of tensile strength[42]. In general, composites reinforced with unidirectional fibers have better tensile properties than those reinforced with woven fabrics due to the undulations in the yarn which form the fabric structure[43]. In addition, the NaOH-sisal 15 wt.%/epoxy composite presented superior tensile strength comparing epoxy composites reinforced with 10 wt.% of untreated sisal fibers, by 50%[44]. In addition, the tested specimens broke at the base of tabs. Silva et al.[21], reported a 40% higher tensile strength

for NaOH-treated sisal fibers (15 wt.%) epoxy composites in samples manufactured without tabs and not subjected to cycles, showing the negative influence of these conditions on the tensile properties of the material. The presence of tabs can cause the premature rupture of the specimens due to the increase in the concentration of stresses caused by geometric discontinuities among the parts[45]. The stress level used in creep tests was also determined by testing NaOH-sisal 15 wt.%/epoxy under tensile. The samples presented 64.86 ± 12.20 MPa on tensile strength and 5.04 ± 0.49 GPa on Young’s Modulus, as shown in Table 1. This tensile strength is similar to the value given for epoxy composites reinforced with glass fibers[46]. Stereomicroscopy was used to analyze the morphology of surface fracture of the neat epoxy and NaOH-sisal 15 wt.%/ epoxy composites materials after the tensile tests, as shown in Figure 4. The specimens were manufactured without tabs and tested before fatigue tests. The micrograph of neat epoxy resin, Figure 4a, shows the cracks propagation without material flow and the absence of voids and defects in the epoxy resin. This region is flat, not reflective, has riverbed patterns, and demonstrates its brittle characteristics[47]. The river lines with a smooth surface are due to the excellent bonding of the epoxy resin[48]. Figure 4b shows the NaOH-sisal 15 wt.%/epoxy composite, and it is possible to observe the fiber rupture. Compared with neat epoxy resin, the best mechanical performance is linked to the efficient adhesion between sisal fiber and epoxy

Table 1. Tensile properties for neat epoxy and NaOH-sisal 15 wt%/epoxy composite specimens. Sample

εmax (%)


NaOH-sisal 15 wt.%/epoxy* Neat epoxy Neat epoxy Neat epoxy NaOH-sisal 15 wt.%/epoxy NaOH-sisal 15 wt.%/epoxy NaOH-sisal 15 wt.%/epoxy

0.02 0.10 0.14 0.02 0.10 0.14

64.9 ± 12.2 31.8 ± 2.3 35.4 ± 0.6 35.1 ± 11.9 51.0 48.9 46.2

Max. Elongation (%) 1.7 ± 0.4 1.1 ± 0.1 1.3 ± 0.1 1.3 ± 0.5 1.9 1.9 1.8

Young’s Modulus (GPa) 5.0 ± 0.5 2.9 ± 0.1 2.8 ± 0.2 2.7 ± 0.1 3.0 3.1 3.1

* NaOH-sisal/epoxy specimens manufactured without tabs and tested before the fatigue tests.

Figure 4. Stereomicroscopy of a fractured surface of the specimens after the tensile tests for (a) neat epoxy; and (b) NaOH-sisal 15 wt.%/ epoxy. 6/9

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Fatigue damage propagation and creep behavior on sisal/epoxy composites

Figure 5. Creep results for (a) Strain vs time curve for NaOH-sisal 15 wt.%/epoxy composite; Vertical shift (b), and rescaling shift for NaOH-sisal 15 wt.%/epoxy composite (c); and Stepped Isostress Method (SSM) curve for NaOH-sisal 15 wt.%/epoxy composite (d).

resin. In addition, voids are observed, defects caused by the release of volatiles and trapping of the gases generated in the resin curing reactions during the manufacturing process. The presence of voids in the epoxy resin reduces its tensile properties, but resin degassing with an ultrasonic bath, as was done in this work, improves its tensile strength[49]. Also, in recent literature, alkali-pretreatment jute fibers (natural fibers) reduce the number of epoxy composites voids and increase their tensile strength[50]. Figure 5a shows the strain vs time curve obtained for NaOH-sisal/epoxy composites after the creep test. It is possible to observe that strain increases with each step’s time (20, 25, 30, 35, and 40% of UTS). Figures 5b and 5c show the vertical and rescaling shifts, respectively. Finally, Figure 5d shows the Stepped Isostress Method (SSM) curve and, according to this, it is valid to state that with 20% UTS the material resists for 200,000 hours without undergoing a creep break. This is due to the insertion of natural fibers in the composite, as the incorporation of fibers can successfully reduce creep since the tension transmitted to the matrix can be transferred to the fibers before the material breaks[51].

15wt.%/epoxy composites present the best strength under fatigue and tensile compared to neat epoxy. This behavior can be explained by adding unidirectional sisal fibers with high tensile strength when the load is applied on its longitudinal axis. Due to the NaOH treatment of the sisal fibers, the adhesion between the fiber and the epoxy matrix was also efficient in distributing loads applied to the composite. The more significant crosslinks number was observed for the NaOH-sisal 15 wt.%/epoxy compared to the neat epoxy. The higher crosslink content also contributed to the higher fatigue and tensile strength of the material. These characteristics were also significant in the resistance of the composite against creep.

4. Conclusions

6. References

In conclusion, the NaOH treatment removed hemicellulose and increased its thermal stability. Also, the NaOH-sisal

1. Fitzgerald, A., Proud, W., Kandemir, A., Murphy, R. J., Jesson, D. A., Trask, R. S., Hamerton, I., & Longana, M. L. (2021).

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5. Acknowledgements This work was supported by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), DPG/ UnB (Decanato de Pós Graduação/University of Brasília), FAPDF (Fundação de Apoio à Pesquisa do Distrito Federal) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico).


Batista, M. S., Teixeira, L. A., Louly, A. S., Silva, S. O., & Luz, S. M.




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ISSN 1678-5169 (Online)


C ss-NMR Singular value decomposition and fitting for sorghum proteins conformation elucidation


Tatiana Santana Ribeiro1* , Juliana Aparecida Scramin2 , José Avelino Santos Rodrigues3, Rubens Bernardes Filho4 , Luiz Alberto Colnago4  and Lucimara Aparecida Forato4  Departamento de Ciências da Natureza, Matemática e Educação, Universidade Federal de São Carlos – UFSCar, Araras, SP, Brasil 2 Programa de Pós-graduação Inter Unidades em Biotecnologia, Universidade Federal de São Carlos – UFSCar, São Carlos, SP, Brasil 3 Embrapa Milho e Sorgo, Sete Lagoas, MG, Brasil 4 Embrapa Instrumentação, São Carlos, SP, Brasil



Abstract Kafirins, water-insoluble proteins from Sweet Sorghum BR501 grains, have been an alternative to prepare edible coatings for food due to their hydrophobic character. In this work, the secondary structures (SS) content of reduced (SSr) and unreduced (SSu) kafirins were determined by 13C solid-state-NMR spectroscopy using areas of carbonyl peak. The SS elucidate by fitting the signal with the Lorentzian function shows 56% and 59% of α-helix and 40% and 12% of β-sheet structures for SSr and SSu, respectively. The SS also were elucidated by a Singular value decomposition- SVD method shows 55% and 49% of α-helix and 12% and 8% of β-sheet structure for SSr and SSu, respectively. Since SVD does not depend on the operator and has higher correlation coefficients for α-helix (0.96%) and β-sheet (0.91%), it is a reliable method to quantify the SS of insoluble proteins using the 13C NMR signal. Keywords: kafirin, secondary structures proteins, sweet sorghum, insoluble protein, solid-state 13C NMR spectroscopy. How to cite: Ribeiro, T. S., Scramin, J. A., Rodrigues, J. A. S., Bernardes Filho, R., Colnago, L. A., & Forato, L. A. (2022). 13C ss-NMR Singular value decomposition and fitting for sorghum proteins conformation elucidation. Polímeros: Ciência e Tecnologia, 32(1), e2022009. https://doi.org/10.1590/0104-1428.20210082

1. Introduction The sweet sorghum has a sucrose-rich stem (like sugar cane). It can be used for ethanol production with advantages such as fast-growing and wide adaptability to different environments[1-3]. The grain is the by-product when sweet sorghum is used[4]. Although the grains can be used to feed livestock, it is preferable to find more technological applications that could increase the value of this by-product. Some food industries already use zeins, proteins extracted from the endosperm of corn kernels, to produce edible films and coatings. Kafirins are proteins extracted from sweet sorghum and can also be a good strategy for making these films and/or other technological applications[5-7]. Kafirins are a group of proteins that respond for 68% to 73% of the total protein contents of the sorghum grain and between 77% and 82% of the endosperm. Kafirins are classified according to their solubility and kDa in dodecylsulfate polyacrylamide gel (SDS/PAGE). The electrophoresis analysis shows four kafirin bands known as α-, γ-, β- and δ-. The most abundant ones are the α-kafirins with values between 23 and 25 kDa, representing 66% to 84% of the total kafirins. The α-kafirins, in the same way as α-zeins, are insoluble in water and soluble in aqueous alcohol solutions of 70% ethanol (w/w). γ-kafirins are soluble in water after

Polímeros, 32(1), e2022009, 2022

disulfide bonds reduction and present the monomer and dimer bands at 28 kDa and 49 kDa, respectively. The γ-kafirins represent 9% to 21% of the total kafirins. The β-kafirins represent 7% to 13% of the total kafirins, and there is some controversy about their kDa values. Some authors assigned the bands at 15, 17, and 18 kDa to these fractions, and others declare that it is only one band from 18 kDa to 19 kDa. β-kafirins are soluble in aqueous alcohol solutions after disulfide bonds reduction process[8]. The δ-kafirins possibly comprise less than 1% of the protein content of sorghum grain when in its mature stage[8,9]. Some authors studied the kafirins’ conformation, vital information to understand their secondary structure. Gao et al. [10] suggest that the secondary structure of these proteins is quite relevant to obtaining a good film formation. Wu et al. [11] analyzed kafirins in 60% t-butanol solution by optical rotatory dispersion and circular dichroism (CD). The authors extracted kafirins from grain sorghum hybrids cultivars OK612, RS626, TE77, and Funk G-766, and they found 40% of α-helix content. Duodu et al.[12] analyzed the sorghum protein bodies (PB) by ATR-FTIR and concluded that the kafirins in the PB have 54%, 55%, 58%, and 59% of α-helix for condensed-tannin-free sorghum P851171, P850029,



Ribeiro, T. S., Scramin, J. A., Rodrigues, J. A. S., Bernardes Filho, R., Colnago, L. A., & Forato, L. A. KAT 369, and NK 283 mutants respectively. Gao et al.[10] analyzed kafirins from a mixture of two tannin-free cultivars (PANNAR 202 and 606) extracted with different solvents and drying methods using ATR-FTIR. They observed that kafirins extracted with 60% t-butyl alcohol + 0.5% DTT and freeze-dried had FTIR peak intensity ratio of helix/ intermolecular β-sheet (1650 cm-1/1620 cm-1) of 1.39. Using 70% ethanol, 0.5% sodium metabisulfite, and 0.35% sodium hydroxide, they noticed a 1.10 ratio, and then using 70% ethanol plus 0.5% sodium metabisulfite as the solvent, they observed a 1.00 ratio. However, during all preparations, the ratio was 0.90 when samples were heated and dried at 40 °C. They concluded that the best conditions for film formation occurred when there were more α-helix contents since the intermolecular β-sheet can induce protein aggregation. Wang et al.[13] extracted kafirins from distillers dried grains with soluble (DDGS) using three different extraction procedures based on HCl/ethanol, acetic acid, and sodium hydroxide-ethanol. In all preparations, using the FTIR technique with samples prepared as KBr pellets, it was noticed that the α-helix is the predominant SS with a small portion of β-sheet. Xiao et al.[14] extracted kafirins using 60% t-butanol and ultrasonication, and their SS were evaluated by ATR -FTIR of kafirin powder. The authors used high-resolution methods to calculate the amount of SS as second derivative and Gaussian fitting and found 49% of α-helix, 24% of β-sheet, and 27% of β-turns. They also analyzed the SS kafirins in solution by Circular Dichroism (CD). They found 68.4% of α-helix for kafirins dissolved in 85% ethanol: 57.9% in 60% t-butanol and 53.1% in 65% isopropanol. The authors concluded that these differences are due to different polarities of the solvents and that the lower polarity is related to the higher helical content. Dianda et al.[15] prepared kafirins from pre-heated sorghum flour (70°C) using 70% ethanol at 68°C added by sodium metabisulfite and acetic acid in a water bath (70 °C) as described by Olivera et al.[16]. By processing ATR-FTIR with Gaussian fitting of kafirin powder, Dianda et al.[15] found 43.88%, 23.53%, and 19.30% for α-helix, β-sheet, and β-turns, respectively. They also evaluated the SS kafirins in ethanol at 70%, 80%, 85% and 90% and at 50%, 60%, 70% and 80% t-butanol using CD. In ethanol solutions, α-helix proportion varied from 40.47 to 47.12% and β-sheet from 8.76 to 15.12%. In t-butanol solutions, α-helix proportion varied from 26.89 to 41.72% and β-sheet from 16.54 to 11.626%. Compared to Xiao et al. [14] , these authors concluded that the differences are due to the different calculation methods for the solvent polarities. Dianda et al.[15] also predicted a theoretical value of about 66% of α-helix based on amino acid sequences analysis of the kafirins. High-resolution solid-state 13C NMR studies using crosspolarization magic angle spinning (CPMAS) sequence have been developed to determinate insoluble proteins. It uses 13 C chemical shift of carbonyl (C=O) peaks to determine α-helix and β-sheet conformation, usually observed between 172 ppm and 176 ppm, respectively[17]. However, 13C solid-state NMR shows problems as the broad linewidth leads to low resolution, overlapped peaks, and inaccurate determination of secondary structures. The fitting methods have been used for analyzing them, though it depends 2/6

on operators such as the number of peaks, specific line shape, and other parameters that are set by the operator[17]. In this research, SS kafirins were quantified using a pattern recognition method based on singular value decomposition (SVD) applied in areas of carbonyl peaks by solid-state 13C NMR spectra. The Lorentz fitting method was also used for comparison. The SVD method does not depend on analyst information, such as function fitting and peak numbers and positions, leading to 0.96% and 0.90% correlation coefficient for α-helix and β-sheet, respectively[17].

2. Materials and Methods 2.1 Protein extraction Kafirins extraction was adapted according to the literature[18] using the Sweet Sorghum grains (BR501-white) provided by Embrapa Maize and Sorghum, Minas Gerais, Brazil. Reduced protein fractions were extracted: Sorghum grains were ground and defatted with hexane in a Soxhlet apparatus for 24-hour. Defatted flour was mixed with NaCl 1,25 mol/L solution and maintained by agitation for 3 hours to solubilize the albumins and globulins. After vacuum filtration, a 100 mmol/L sodium bisulfite aqueous solution was added to the flour for 2 hours, and then the residue was added to 70% ethanol (w/w) for 24 hours under agitation (at room temperature). The solvent was evaporated, and the obtained kafirins were lyophilized. The unreduced fractions were extracted with this mentioned methodology without only the step of adding sodium bisulfite.

2.2 SDS/PAGE The SDS/PAGE was a 15% polyacrylamide gel, and the gel was stained with Coomassie Blue dye. The standard molecular weight employed was Benchmark Protein Ladder Cat. Nº. 10747-012 from Invitrogen.

2.3 13C solid-state Nuclear Magnetic Resonance (13C ss-NMR) The solid-state 13C NMR spectra were obtained with a Bruker Advance III HD 400 MHz spectrometer equipped with a solid-state MAS probe, with two channels configured for 1H and 13C frequencies of 400 MHz and 100.5 MHz, respectively. 13C cross-polarization magic angle spinning (CP/MAS) was the sequence used for the analysis. The operational conditions were: 90 1 H pulse length of 2.3 us, contact time of 2 ms, a spectral width of 50 kHz, and a recycle delay of 3 s and 4096 scans[17]. Spectra were filtered by an exponentially decaying function with 20 Hz of line broadening. Samples were packed in a zirconia rotor of 5 mm and rotated at the magic angle at 10 kHz. External hexamethylbenzene standard chemical shift (at 17.3 ppm) was used as reference. The carbonyl peak area in the 13C NMR spectra for reduced and unreduced kafirins has been used to calculate the SS with CP/MAS 13C NMR pulse sequence[19,20]. The region from 180 to 160 ppm was obtained with a total of 859 data points from each ss-13C NMR spectrum, and its second derivative was used to find the three individual peaks: at 172 ppm that were assigned to β-sheet, 174 ppm to unordered ones, and 176 ppm to the α-helix structure. Polímeros, 32(1), e2022009, 2022

C ss-NMR Singular value decomposition and fitting for sorghum proteins conformation elucidation


These three peaks were used in the Lorentzian multiple peak fitting, and the area corresponded to each SS proportion as already indicated. The Lorentzian multiple peaks fitting Adjusted R- Square was 0.994 and 0.992 for unreduced and reduced kafirins, respectively, and both fittings reached the chi-square tolerance of 1x10-9. The calibration matrix to the SVD method was published elsewhere[17]. Equation 1 shows the SVD method correlation with each NMR protein spectrum (R) and protein secondary structure concentration (F) by a calibration matrix (X). The F and R matrices with a medium square error are ± 1.5 for a-helix and ±1.8 for β sheet structures prediction were used by Andrade et al.[17]. The F matrix (15x4) consists of 15 proteins with four secondary structures proteins proportions obtained by X-ray crystallographic data[21]. The R matrix comprises the 13C NMR spectra (carbonyl area) of each protein in the F matrix. (1)

F = XR

The SVD method reduces the rank of the R matrix, making it consistent with the information, and calculates the generalized inverse as shown in Equation 2: R = USV T and R −1 = VS −1U T


where: S: diagonal matrix with singular values on the diagonal and zeros out of the diagonal; U: is an orthogonal matrix of eigenvectors on the matrix rows; V: is an orthogonal matrix of coefficients. The X matrix can be calculated as shown in Equation 3:


X = F VS −1U T



It is possible to calculate the SS of a protein that was not determined (SS unknown protein) by its NMR spectrum (N) multiplied by the X matrix as shown in Equation 4: SS unknown = XN


3. Results and Discussion 3.1 Characterization by gel electrophoresis containing polyacrylamide sodium dodecyl sulfate (SDS/PAGE) As described in the literature, kafirins are extracted with different solvents and reducing agents[1-15]. We extracted the kafirins using the reduced and unreduced forms to compare the SS obtained in this work with that already published by other authors and therefore evaluate the SS calculation methods used here. Reduced kafirin fraction showed four bands (Figure 1, second well): β-kafirin at 17 kDa and other three bands at 21, 25, and 26 kDa were assigned to α2-, α1- and γ-kafirins, respectively. This result is in good agreement with the description of Byaruhanga et al.[22]. Unreduced kafirins fraction (Figure 1, first well) showed a more intense band at 17 kDa attributed to β-kafirin and a weak one at 25-26 kDa to the α+γ-kafirins agglomerate. El Nour et al.[23] extracted kafirin with 60% t-butanol without reducing agent, and they also found the presence of the β-kafirin and weak bands for Polímeros, 32(1), e2022009, 2022

Figure 1. Unreduced kafirin fraction (first well) and reduced kafirin fraction (second well) characterized by gel electrophoresis containing polyacrylamide sodium dodecyl sulfate (SDS/PAGE).

α2-, α1- and γ-kafirins. They concluded that in unreduced conditions, β-kafirin is extracted in the monomeric form.

3.2 SS analysis using solid-state 13C NMR spectroscopy Figures 2A and 2B show the NMR spectra of unreduced and reduced kafirins fractions, respectively. The NMR spectra show typical protein signals: from 172 ppm to 176 ppm due to carbonyl peaks, from 140 ppm to 100 ppm due to amino acids with aromatic side chains; from 70 ppm to 45 ppm to α carbons, and from 45 ppm to 15 ppm to the aliphatic amino acid side chains[19,21,24]. The signals at 73 ppm were assigned to starch in unreduced kafirin (2A), which are stronger than in the reduced kafirins (2B). Figure 2 spectra also showed an intense peak at 130 ppm. This peak has been assigned to unsaturated fatty acids, obtained using the single-pulse technique (Figure 3) used to detect the presence of mobile molecules[25,26]. Figure 3 shows the unreduced kafirin spectrum by single-pulse sequence, similar for both reduced and unreduced kafirins. The carboxyl signal at 173 ppm was attributed to unsaturated fatty acids. The peak at 130 ppm was attributed to the double bond carbons. The intense peaks from 10 ppm to 40 ppm were assigned to the methyl and methylene carbons. Similar fatty acid contents have been noted in zeins extract[25,27], indicating that kafirins may also be fatty acid-binding proteins. The carboxyl peak at 173 ppm can be overlapped with other carbonyl signals from the aminoacids. SVD method has the advantage over Lorentzian fitting because it does not need the assignment of peaks to calculate SS % proteins. The area of carbonyl peak was calculated by Equation 3 (section 2.3) using the 13C NMR of the 15 proteins (R matrix) as well as the generalized inverse of F (F-1) calculated by the SVD method[17]. Figure 4 shows the expansion of carbonyl signals of unreduced kafirins (A) and reduced kafirins (B). The Lorentzian 3/6

Ribeiro, T. S., Scramin, J. A., Rodrigues, J. A. S., Bernardes Filho, R., Colnago, L. A., & Forato, L. A.

Figure 2. Solid-state 13C NMR (CPMAS) spectra for (A) Kafirins from sweet sorghum extracted without the reducing agent; (B) Kafirins from sweet sorghum extracted with the reducing agent. Table 1. α-helix and β-sheet kafirins solid structures (SS %) elucidated by CP/MAS 13C NMR spectroscopy. These kafirins were extracted with sodium bisulfite (reduced form) and without sodium bisulfite (unreduced form). Lorentzian fitting and SVD method were used for SS % calculation. Applied method Lorentzian fitting (unreduced kafirin) Lorentzian fitting (reduced kafirin) SVD (unreduced kafirin) SVD (reduced kafirin)

Figure 3. The single pulse ss- 13C NMR spectrum of unreduced kafirins from sweet sorghum.

fitting presents three signals, at 176 ppm, 174 ppm, and 172 ppm, attributed to the α-helix, unordered, and β-sheet structures, respectively[19,20]. The results of SS calculated for kafirins in this method and with the Lorentzian fitting and SVD method are in table 1. Spectrum 4A (unreduced kafirins) peaks areas show 59% of α-helix, 12% of β-sheet, and 29% of unordered structures. The carbonyl signals of reduced kafirins (Figure 4B) show 56% of α-helix, 40% of β-sheet, and 4% of unordered structures. As a result, both extracted kafirins offer a high content of α-helix, regardless of the reducing agent utilized. The high content of α-helix and β-sheet is confirmed through the signals of α-carbon. Signals at 56 ppm and 59 ppm are typical of the α-helix, and signals at 53 ppm are typical of the β-sheet[19,20]. This fitting method depends on the following factors: the number of peaks and respective positions and the line shape function used in the fitting process, among other factors[28,29]. The SS was also calculated by the SVD method. This method multiplied the carbonyl area of kafirins ss-13C NMR spectra by a calibration matrix [17], as shown by Equation 3. 4/6

α-helix (SS %) 59 56 49 55

β-sheet (SS %) 12 40 8 13

The SVD method also observed high helical content for reduced and unreduced kafirins, 55% and 49%, respectively, and 13% and 8% for β-sheet, respectively. The difference in the SS values may occur due to the different electrophoretic patterns, given that β-, α1-, α2- and γ-kafirin fractions were observed in the reduced kafirins, while in unreduced kafirins an intense signal for β-kafirin was observed, which may occur as a monomer[23], and a weak signal in the range of α1-, α2- and γ-kafirin that was named as α+γ- agglomerate. The difference in absolute values of SS kafirins used to calculate Lorentzian fitting must be attributed because this fitting method depends on the operator, necessary to choose the number of peaks and positions assigned to each carbonyl area. The SVD method does not need the process described above, which is dependent of the operator. The αand β- secondary structures content of reduced (SSr) and unreduced (SSu) kafirins has a high correlation coefficient for reduced and unreduced kafirins by the SVD method applied in CP/MAS 13 C NMR. Table 1 shows the result of SS % calculate to reduced and unreduced kafirins using Lorentzian fitting and SVD methods. It is noteworthy that the CP/MAS 13C NMR is not a quantitative method. Its signal depends on cross-polarization (CP) efficiency and spin-lattice relaxation in the rotating frame [17,30]. Despite that, when the carbons have similar chemical environments, such as the Polímeros, 32(1), e2022009, 2022

C ss-NMR Singular value decomposition and fitting for sorghum proteins conformation elucidation


Figure 4. Expansion of CP/MAS 13C NMR spectra carbonyl area. A) Kafirins from sweet sorghum extracted without the reducing agent. B) Kafirins from sweet sorghum extracted with the reducing agent. Solid Line: Signal; Dashed line: Total Lorentzian fitting; Dotted lines; Individual Lorentzian fitting peaks (Adjusted R- Square=0.994 for A and 0.992 for B)) and the chi-square tolerance of 1x10-9 was reached.

carbonyl proteins, these two parameters are alike, giving quantitative results. The high content of α-helix structure in kafirins from sweet sorghum grains agrees with the data published by Gao et al.[10], Wu et al.[11], and Duodu et al.[12], who used another sorghum cultivar. According to these authors, the study of SS kafirins was quite relevant for obtaining good films to apply on foods once a higher proportion of β-sheet structures could induce protein agglomeration and reduce their solubility.

4. Conclusion We applied the Lorentz fitting and a SVD method in carbonyl peaks area by CP/MAS 13C NMR of unreduced and reduced kafirins spectra to verify which mathematical method is more reliable to calculate their %SS. Kafirins reduced fraction extract from sweet sorghum grains are composed of four protein fractions assigned to β-, α1-, α2- and γ-kafirins and the unreduced by β-kafirins in the monomeric form and an α+γ agglomerate. Higher proportions of α-helix structure and smaller β-sheet structure were identified in the reduced and unreduced kafirins, confirmed by other spectroscopy techniques. Thus, high-resolution 13C solidstate NMR spectroscopy is promising to elucidate secondary structures of insoluble proteins. The main difference was in the absolute values for SS proportions since the SVD method does not depend on the operator, and it has high coefficient correlations for α-helix and β-sheet prediction[17]. Our research indicates that the SVD method is more reliable than the Lorentzian fitting to calculate the % SS of proteins by CP/MAS 13C NMR spectroscopy.

5. Acknowledgements We acknowledge CNPq (process 562470/2010-7) and CAPES for the assistance received and Rede Agronano/ Embrapa. Polímeros, 32(1), e2022009, 2022

6. References 1. Chen, H., Tian, X., Yu, Q., Hu, W., Chen, J., & Zhou, L. (2021). Sweet sorghum stalks extract has antimicrobial activity. Industrial Crops and Products, 170, 113746. http://dx.doi. org/10.1016/j.indcrop.2021.113746. 2. Zegada-Lizarazu, W., & Monti, A. (2012). Are we ready to cultivate sweet sorghum as a bioenergy feedstock? A review on field management practices. Biomass and Bioenergy, 40, 1-12. http://dx.doi.org/10.1016/j.biombioe.2012.01.048. 3. Ratnavathi, C. V., Suresh, K., Kumar, B. S. V., Pallavi, M., Komala, V. V., & Seetharama, N. (2010). Study on genotypic variation for ethanol production from sweet sorghum juice. Biomass and Bioenergy, 34(7), 947-952. http://dx.doi. org/10.1016/j.biombioe.2010.02.002. 4. Shah, U., Dwivedi, D., Hackett, M., Al-Salami, H., Utikar, R. P., Blanchard, C., Gani, A., Rowles, M. R., & Johnson, S. K. (2021). Physicochemical characterization of kafirins extracted from sorghum grain and dried distillers grain with solubles related to their biomaterial functionality. Scientific Reports, 11(1), 15204. http://dx.doi.org/10.1038/s41598-021-94718-z. PMid:34312467. 5. Buchner, S., Kinnear, M., Crouch, I. J., Taylor, J., & Minnaar, A. (2011). Effect of the kafirin protein coating on sensory quality and shelf life of ‘Pacham Triumph’ pears during ripening. Journal of the Science of Food and Agriculture, 91(15), 28142820. http://dx.doi.org/10.1002/jsfa.4526. PMid:21725981. 6. Gao, C., Stading, M., Wellner, N., Parker, M. L., Noel, T. R., Mills, E. N. C., & Belton, P. S. (2006). Plasticization of a protein-based film by glycerol: a spectroscopic, mechanical and thermal study. Journal of Agricultural and Food Chemistry, 54(13), 4611-4616. http://dx.doi.org/10.1021/jf060611w. PMid:16787005. 7. Wu, S., Myers, D. J., & Johnson, L. A. (1997). Factors affecting yield and composition of zein extracted from commercial corn gluten meal. Cereal Chemistry, 74(3), 258-263. http://dx.doi. org/10.1094/CCHEM.1997.74.3.258. 8. Belton, P. S., Delgadillo, I., Halford, N. G., & Shewry, P. R. (2006). Kafirin structure and functionality. Journal of Cereal Science, 44(3), 272-286. http://dx.doi.org/10.1016/j. jcs.2006.05.004. 5/6

Ribeiro, T. S., Scramin, J. A., Rodrigues, J. A. S., Bernardes Filho, R., Colnago, L. A., & Forato, L. A. 9. Laidlaw, H. K. C., Mace, E. S., Williams, S. B., Sakrewski, K., Mudge, A. M., Prentis, P. J., Jordan, D. R., & Godwin, I. D. (2010). Allelic variation of the β-, γ- and δ-kafirin genes in diverse sorghum genotypes. Theoretical and Applied Genetics, 121(7), 1227-1237. http://dx.doi.org/10.1007/s00122-0101383-9. PMid:20563549. 10. Gao, C., Taylor, J., Wellner, N., Byaruhanga, Y. B., Parker, M. L., Mills, E. N. C., & Belton, P. S. (2005). Effect of preparation conditions on protein secondary structure and biofilm formation of kafirin. Journal of Agricultural and Food Chemistry, 53(2), 306-312. http://dx.doi.org/10.1021/jf0492666. PMid:15656666. 11. Wu, Y. V., Cluskey, J. E., & Jones, R. W. (1971). Sorghum prolamins: their optical rotatory dispersion, circular dichroism, and infrared spectra. Journal of Agricultural and Food Chemistry, 19(6), 1139-1143. http://dx.doi.org/10.1021/jf60178a008. 12. Duodu, K. G., Tang, H., Grant, A., Wellner, N., Belton, P. S., & Taylor, J. R. N. (2001). FTIR and solid state 13C NMR spectroscopy of proteins of wet cooked and popped sorghum and maize. Journal of Cereal Science, 33(3), 261-269. http:// dx.doi.org/10.1006/jcrs.2000.0352. 13. Wang, Y., Tilley, M., Bean, S., Sun, X. S., & Wang, D. (2009). Comparison of methods for extracting kafirin proteins from distillers dried grains with solubles. Journal of Agricultural and Food Chemistry, 57(18), 8366-8372. http://dx.doi.org/10.1021/ jf901713w. PMid:19754169. 14. Xiao, J., Li, Y., Li, J., Gonzalez, A. P., Xia, Q., & Huang, Q. (2015). Structure, morphology, and assembly behavior of kafirin. Journal of Agricultural and Food Chemistry, 63(1), 216-224. http://dx.doi.org/10.1021/jf504674z. PMid:25510968. 15. Dianda, N., Rouf, T. B., Bonilla, J. C., Hedrick, V., & Kokini, J. (2019). Effect of solvent polarity on the secondary structure, surface, and mechanical properties of biodegradable kafirin films. Journal of Cereal Science, 90, 102856. http://dx.doi. org/10.1016/j.jcs.2019.102856. 16. Olivera, N., Rouf, T. B., Bonilla, J. C., Carriazo, J. G., Dianda, N., & Kokini, J. L. (2019). Effect of LAPONITE addition on the mechanical, barrier and surface properties of novel biodegradable kafirin nanocomposite films. Journal of Food Engineering, 245, 24-32. http://dx.doi.org/10.1016/j. jfoodeng.2018.10.002. 17. Andrade, F. D., Forato, L. A., Bernardes, R., Fo., & Colnago, L. A. (2016). Quantification of protein secondary structure by 13 C solid-state NMR. Analytical and Bioanalytical Chemistry, 408(14), 3875-3879. http://dx.doi.org/10.1007/s00216-0169484-1. PMid:27068694. 18. Park, S.-H., & Bean, S. B. (2003). Investigation and optimization of the factors influencing sorghum protein extraction. Journal of Agricultural and Food Chemistry, 51(24), 7050-7054. http:// dx.doi.org/10.1021/jf034533d. PMid:14611170. 19. Kricheldorf, H. R., & Müller, D. (1984). Secondary structure of peptides 16th. Characterization of proteins by means of 13C NMR CPMAS spectroscopy. Colloid & Polymer Science, 262(11), 856-861. http://dx.doi.org/10.1007/BF01452215.


20. Wishart, D. S., & Sykes, B. D. (1994). Chemical shifts as a tool for structure determination. Methods in Enzymology, 239, 363-392. http://dx.doi.org/10.1016/S0076-6879(94)39014-2. PMid:7830591. 21. Bicudo, T. C., Forato, L. A., Batista, L. A. R., & Colnago, L. A. (2005). Study of the conformation of γ-zeins in purified maize protein bodies by FTIR and NMR spectroscopy. Analytical and Bioanalytical Chemistry, 383(2), 291-296. http://dx.doi. org/10.1007/s00216-005-0003-z. PMid:16132146. 22. Byaruhanga, Y. B., Emmambux, M. N., Belton, P. S., Wellner, N., Ng, K. G., & Taylor, J. R. N. (2006). Alteration of kafirin and kafirin film structure by heating with microwave energy and tannin complexation. Journal of Agricultural and Food Chemistry, 54(12), 4198-4207. http://dx.doi.org/10.1021/ jf052942z. PMid:16756347. 23. El Nour, I. N. A., Peruffo, A. D., & Curioni, A. (1998). Characterization of sorghum kafirins in relation to their crosslinking behavior. Journal of Cereal Science, 28(2), 197-207. http://dx.doi.org/10.1006/jcrs.1998.0185. 24. Kumashiro, K. K., Kurano, T. L., Niemczura, W. P., Martino, M., & Tamburro, A. M. (2003). 13C CPMAS NMR Studies of the Elastin-Like Polypeptide (LGGVG)n. Biopolymers, 70(2), 221-226. http://dx.doi.org/10.1002/bip.10470. PMid:14517910. 25. Forato, L. A., Colnago, L. A., Garratt, R. C., & Lopes, M. A. (2000). Identification of free fatty acids in maize protein bodies and purified alpha zeins by 13C and 1H nuclear magnetic resonance. Biochimica et Biophysica Acta, 1543(1), 106-114. http://dx.doi.org/10.1016/S0167-4838(00)00190-4. PMid:11087946. 26. Bardet, M., Foray, M. F., Bourguignon, J., & Krajewski, P. (2001). Investigation of seeds with high-resolution solid-state 13 C NMR. Magnetic Resonance in Chemistry, 39(12), 733-738. http://dx.doi.org/10.1002/mrc.958. 27. Forato, L. A., Yushmanov, V. E., & Colnago, L. A. (2004). Interaction of two prolamins with 1-13C Oleic Acid by 13 C NMR. Biochemistry, 43(22), 7121-7126. http://dx.doi. org/10.1021/bi035562k. PMid:15170349. 28. Forato, L. A., Bernardes-Filho, R., & Colnago, L. A. (1998). Protein structure in KBr pellets by infrared spectroscopy. Analytical Biochemistry, 259(1), 136-141. http://dx.doi. org/10.1006/abio.1998.2599. PMid:9606154. 29. Hegland, M., & Anderssen, R. S. (2005). Resolution enhancement of spectra using. Differentiation. Inverse Problems, 21(3), 915-934. http://dx.doi.org/10.1088/0266-5611/21/3/008. 30. Kolodziejski, W., & Klinowski, J. (2002). Kinetics of crosspolarization in solid-state NMR: a guide for chemists. Chemical Reviews, 102(3), 613-628. http://dx.doi.org/10.1021/cr000060n. PMid:11890752. Received: Oct. 14, 2021 Revised: Mar. 07, 2022 Accepted: Apr. 11, 2022

Polímeros, 32(1), e2022009, 2022

ISSN 1678-5169 (Online)


Determination leaching of boron from Oriental beech wood coated with polyurethane/polyurea (PUU) hybrid and epoxy (EPR) resins Çaglar Altay1 , Hilmi Toker2 , Mustafa Kucuktuvek3* , Mehmet Yeniocak2 , İlknur Babahan Bircan4  and Ergun Baysal2  Department of Interior Design, Aydın Vocational School, Aydın Adnan Menderes University, Aydın, Turkey 2 Department of Wood Science and Technology, Faculty of Technology, Muğla Sıtkı Koçman University, Muğla, Turkey 3 Department of Interior Architecture, Faculty of Architecture, İskenderun Teknik University, İskenderun, Hatay, Turkey 4 Department of Chemistry, Faculty of Arts and Sciences, Aydın Adnan Menderes University, Aydın, Turkey



Abstract In this study, the leaching performance of boron compounds from Oriental beech (Fagus orientalis L.) wood coated with polyurethane/polyurea (PUU) hybrid resin and epoxy resin (EPR) was investigated. Leaching of boron test and SEM (Scanning Electron Microscopy) analyzes were applied to the prepared test specimens. According to the leaching of boron test results, specimens coated with polyurethane/polyurea (PUU) hybrid resin gave the most positive result against boron leaching. It was found that boric acid (BA) impregnated and epoxy resin (EPR) coated Oriental beech wood showed a smoother surface than other treatment groups. Keywords: boron compounds, epoxy resin, leaching of boron, Oriental beech, polyurethane/polyurea hybrid resin. How to cite: Altay, Ç., Toker, H., Kucuktuvek, M., Yeniocak, M., Bircan, İ. B., & Baysal, E. (2022). Determination leaching of boron from Oriental beech wood coated with polyurethane/polyurea (PUU) hybrid and epoxy (EPR) resins. Polímeros: Ciência e Tecnologia, 32(1), e20220010. https://doi.org/10.1590/0104-1428.20210099

1. Introduction Wood material has been among the most important building materials since ancient times due to its nature, structural features, and easy shaping. Among the most prominent features of wood being preferred in architecture; being light, resistant to physical and mechanical effects, low heat and sound insulation, aesthetic appearance, easy workability, ensuring color and design integrity, not having harmful effects on human health and the environment, being suitable for indoor and outdoor use. For these reasons, wood is preferred building materials[1-3]. There is increasing interest in using renewable natural resources in architecture. The main reason for this is the negative effects of industrial activities on the environment in recent years. Wood is an important natural material that is preferred in furniture production as well as in the building and construction industry. Wood is a desired building material in terms of its physical and mechanical properties. However, due to its organic structure, its chemical composition is cellulose, hemicellulose, lignin, resins, etc. consists of substances, it can decompose when exposed to environmental effects such as water, light, heat, fungi, bacteria, insects. Some inorganic materials are used to protect the wood. These substances are derivatives of copper, boron, and zinc. It also performs impregnation or coating

Polímeros, 32(1), e2022010, 2022

processes by using organic compounds such as polymers, vegetable oils, essential oils, phenols, triazoles, benzoic acid derivatives, waxes, resins[4-9]. Structural changes in wood occur in connection with various mechanical and physical properties of wood. The permeability of wood should be improved in order to increase the amount of retention and to make a good impregnation process. The amount of retention can be increased by pre-treating wood biologically, chemically, physically, or mechanically. Wood that is not impregnated or coated begins to deteriorate chemically. The mechanical properties of chemically degraded wood generally continue to deteriorate. For example, alkali can cause cell collapse and increase wood density. The mechanical properties of wood, which is damaged by acids, are partially reduced not only in the wet state but also in the dry state of wood[10]. As concerns about the quality and durability of buildings increase, solutions are needed to increase the likelihood that structures can maintain their physical and mechanical integrity. It is even more important to preserve the wooden material in historical buildings[11]. Notable among the most observed anomalies in wooden parts of buildings are those caused by xylotrophic organisms such as rot fungi and subterranean termites, as well as dry wood termites and



Altay, Ç., Toker, H., Kucuktuvek, M., Yeniocak, M., Bircan, İ. B., & Baysal, E woodworms. The first two of these anomalies occur in timber with high moisture content and the second two occur in dry timber. Keeping timber in a good preservation condition and curing infection or infestation requires the use of chemical compounds. These chemical protection methods include impregnation, varnish coating, and paints[12]. One of the good ways to protect forests and ensure sustainability is the treating of wood. The use of treated wood in buildings also helps homeowners save money[13,14]. Until the early 1990s, the use of active substances such as pentachlorophenol, copper, tin or lindane, and CCA (chromated copper arsenate) compounds applied in organic solvents (LOSP) were common as wood preservatives[12-14]. Some of these formulations can be very effective at prolonging the life of the wood. However, the health hazard to workers and the risk of environmental impact on soil and landscape must be seriously considered[13,14]. Therefore, severe restrictions have been placed on the use of many of the above-mentioned constituents in the last few decades in Europe[15] and, for instance, in the USA[16] where CCA has been phased out from all new housing uses from the beginning of 2004[16]. With the increase in industrialization and technological developments, functional material options that are environmentally friendly, non-toxic, resistant to fire and flame, and widely used in architecture are also increasing. Wood material meets most of these characteristics. However, wood material, as mentioned above, is adversely affected by the damages in the outdoor environment due to its organic structure[17]. These problems can be partially overcome by modification or impregnation of the wood[18]. The use of boron compounds instead of using impregnation materials that have harmful effects on the environment can be an important step for a solution to the problems. Boron compounds protect the wood material against fungi, fire, and pests. However, it is necessary to increase the retention amount of boron minerals and to fix the boron compounds by decreasing the leaching amount. Attempts have been made to improve the leaching performance of boron compounds by using water repellents, monomers, and polymer systems[19]. Lloyd et al.[20] reported that the addition of polyols to borate solutions highly decreased boron leaching from wood by borate/polyole chalate complexion. Yalinkilic[21] investigated leaching characteristics of borates from sugi wood impregnated with boric acid-vinyl monomer combination treatment. Results indicated that sugi wood impregnated with boric acid ready lost its boron content in early leaching cycles, whereas vinyl monomer treatment resulted in five times lesser boron release into the water. Baysal and Yalinkilic[22] studied a natural wood polymer composite that was obtained under the catalytic effect of borates by using furfuryl alcohol. Furfuryl alcohol and borates were mixed at different ratios before treatment. When borates alone are used, they were leached from wood after cyclic leaching periods. However, this was not encountered with the mixture of furfuryl alcohol and borates. Baysal et al.[23] studied boron leaching from Douglas wood impregnated with borates and secondary some water repellent chemicals. Results indicated that secondary treatments of wood with the water repellent chemicals following borate impregnation reduced the leaching of chemicals from wood in water[24]. Among the construction materials which are used in architecture, wood holds a special place because of its 2/9

impressive range of attractive properties, including low thermal extension, low density, and high enough mechanical strength. However, wood material has a leaching issue after impregnation with boron compounds. This study was carried out to measure the leaching of boron from Oriental beech (Fagus orientalis L.) wood, which is widely preferred in the architecture and furniture industry. In this context, experimental specimens impregnated with boron minerals, which have fire retardant properties, and coated with polyurethane/polyurea (PUU) hybrid resin and epoxy resin (EPR) were used.

2. Materials and Methods 2.1 Materials Oriental beech (Fagus orientalis L.) wood was collected from Muğla province in Turkey. Wood specimens were prepared in 20 mm × 20 mm × 20 mm (radial, tangential, and longitudinal) dimensions. H3BO3 (Boric acid; CAS: 10043-35-3) and Na2B4O7.10H2O (Borax; CAS 1303-96-4) were purchased from Merck Chemicals company and were used without any purification. Epoxy (sikafloor®-156) and polyurethane/polyurea hybrid resin (sikalastic®-851 R) resins were purchased from Sika company in Turkey. The microstructures of the specimens were obtained by using a Jeol JSM 7600F scanning electron microscope (SEM).

2.2 Impregnation process 3% aqueous solutions of boric acid (BA), borax (BX), and, a mixture of boric acid and borax (1:1; weight:weight) (BA+BX) were prepared to impregnate the Oriental beech wood specimens. The impregnation of the Oriental beech wood specimens was carried out according to ASTM D 1413-07[25]. In this study, the specimens were prepared for the impregnation process were first subjected to pre-vacuum for 30 minutes and then to the impregnation process under 1.01 bar pressure for 30 minutes in the impregnation device. Then the borates retention of Oriental beech was calculated from the following Equation 1: R=


G.C X 103 kg / m3 V



where: G = T2 -T1;

T2 = Specimen weight after impregnation (gr);

T1 = Specimen weight before impregnation (gr); V = Specimen volume (cm3);

C = Solution concentration (%).

2.3 Coating process In this study, epoxy and polyurethane\polyurea hybrid resins were used for the surface coatings. Firstly, the impregnated test specimens were primed with epoxy resin (Sikafloor®-156) to prepare EPR coatings, afterwards, they were coated with polyurethane/polyurea hybrid resin (Sikalastic®-851 R) to obtain the PUU coatings. PUU coatings should be noticed that they were primed with epoxy resin (Sikafloor®-156) before coating with polyurea/polyurethane Polímeros, 32(1), e2022010, 2022

Determination leaching of boron from Oriental beech wood coated with polyurethane/polyurea (PUU) hybrid and epoxy (EPR) resins hybrid resin (Sikalastic®-851 R). Coating processes are depicted in Figure 1. 2.3.1 Preparation of epoxy coatings (EPR) Sikafloor®-156 was used to prepare epoxy resin (EPR) coatings. It is composed of two parts, an epoxy part (A) and a hardener part (B). Sikafloor®-156 resin has some advantages such as low viscosity and being solvent-free[26]. The mixtures were prepared by mixing the epoxy and hardener with a 3:1 ratio of epoxy to hardener as follows the manufacturer’s instructions. Then they were applied to the wood specimens which are impregnated with boron compounds (Figure 1). The possible cure reaction between an epoxy part and a hardener part is given in Figure 2. The hardeners are known as the chemicals which are converted epoxy resin to thermosets, have usually bear active hydrogen attached to an electronegative atom such as N, O, or S. The curing reaction is a ring-opening reaction between the oxirane ring and a nucleophile (Figure 2). The ring-opening reaction occurs via nucleophilic attack by the hardener to the oxirane ring, then a second reaction follows until the remaining active hydrogens attached to the hardeners are fully reacted[27, 28]. 2.3.2 Preparation of polyurea/polyurethane coatings (PUU) Sikalastic®-851 R was used for PUU coatings. PUU coated by using Sikalastic®-851 R resin were made after test specimens were primed with epoxy resin (Sikafloor®-156). Sikalastic®-851 R is known as a two-component, elastic, crack-bridging, rapid-curing modified polyurethane/ polyurea hybrid resin. Component A consists of isocyanate derivative and component B consists of polyol/amine

derivative[28,29]. The coatings were professionally made by the manufacturer firm. Two layers were applied to the floor using special polyurea coating machines (GAMA G-30 H) at the consumption of 1.7-2.2 kg per m2 and the second layer application was started within a maximum of 6 hours after the first layer application. A possible reaction, between polyurea/polyurethane and epoxy moieties which forms an epoxy-urea bond, and displays the new interfacial chemical reaction between Sikafloor®-156 and Sikalastic®-851 R, is illustrated in Figure 3[28,29].

2.4 Leaching test procedure Wood specimens impregnated with boron compounds then coated with epoxy and polyurea/polyurethane hybrid resins were subjected to leaching of boron test. A leaching test was performed regarding the Yeniocak and Kahveci[30] study. First, the maximum wavelengths of the impregnation materials were determined by UV spectrophotometer to be a reference point for absorbance in evaluating the leaching results in the study. Three wood specimens from the same group were placed in the erlenmeyer with 250 ml of distilled water. The leaching properties of boron were determined by taking the erlenmeyer in a shaking water bath at 120 rpm shaking speed at different time intervals, different pH, and different temperatures. The tests were carried out at 10 °C, 22 °C, 40 °C temperature, in pH 7. The leaching of boron was measured at 5, 15, 30, 45, 60, 75, 90, and 120 minutes and the amount of substance (absorbance) transferred to the leaching water was measured in the UV spectrophotometer device, regarded maximum wavelengths values. Biochrom Libra S70 model, UV-Visible Spectrophotometer was used in

Figure 1. Preparation of epoxy coatings (EPR) and polyurea/polyurethane coatings (PUU). Polímeros, 32(1), e2022010, 2022


Altay, Ç., Toker, H., Kucuktuvek, M., Yeniocak, M., Bircan, İ. B., & Baysal, E Table 1. Maximum wavelength values of solutions.

Figure 2. Possible curing reaction between epoxy part and hardener part for EPR coatings.

Impregnation group BA BX BX+BA

Max. wavelength (nm) 201 195 192

BA: Boric acid; BX: Borax.

Table 2. Retention values of boron compunds. Impregnation group

Figure 3. The reaction between epoxy and polyurea/polyurethane moieties.

the tests. The UV/VIS Spectrophotometer had a dual-beam in the wavelength range of 190-1100nm.

2.5 SEM (scanning electron microscope) analyzes Specimens impregnated with boron compounds and coated with EPR and PU (control, EPR, BA+EPR, BX+EPR, and ((BX+BA)+EPR) were coated with gold for surface morphology. The specimens were then examined with a scanning electron microscope (Jeol JSM 7600F). In this study, SEM analyzes could not be performed on PUU-coated specimens due to imaging problems of the scanning electron microscope device.

3. Results and Discussions 3.1 Leaching test The maximum wavelengths of solutions are given in Table 1. Maximum wavelengths were measured in nanometers in a UV spectrophotometer device, taking pure water as a reference. The maximum wavelength indicates the maximum amount of substance that can pass into the pure water during leaching. These obtained data were used as a reference in determining the amount of substance transferred to the water in the leaching test. Retention is a factor in determining the total amount of solution added to the wood material in the impregnation process. Table 2 shows the retention values of boron compounds. The highest retention value was obtained as 17.23 kg/m3 in BA+PUU applied group, while the minimum retention value was measured as 14.29 kg/m3 in the ((BX+BA) +PUU) applied group. Results of Oriental beech wood specimens coated with PUU and EPR and leaching of boron compounds under conditions of pH: 7, 10 °C, 22 °C, and 40 °C temperatures and 120 rpm in shaking speed were given in Table 3 in the unit of absorbance (abs). In Table 3, after a temperature of 10 ˚C and 120 minutes, there was no leaching of boron observed in the group covered by PUU. On the other hand, low leaching of boron was observed in specimens impregnated with BX, BA, and BX+BA and coated with EPR. Here, minimum leaching of boron rate of 0.38 abs was observed in the wood specimens impregnated with BX+BA and coated with EPR, while the 4/9


Retention (kg/m3) Mean 17.23 15.53 17.08 16.52 14.29 15.30

Note: PUU: Polyurethane/Polyurea hybrid resin (Sikalastic®-851 R); EPR: Epoxy resin (Sikafloor®-156); BA: Boric acid; BX: Borax.

maximum 0.67 abs was measured in the wood specimens impregnated with BA and coated with EPR. After a temperature of 22 ˚C and 120 minutes, no leaching of boron in all groups was covered by PUU. Besides, low leaching of boron was observed in wood specimens impregnated with BX, BA, and BX+BA and coated with EPR. While the minimum leaching of boron with 0.92 abs was observed in the wood specimens impregnated with BA and coated with EPR, maximum leaching of boron was measured in wood specimens impregnated with BX + BA and coated with EPR as 1.18 abs. After a temperature of 40 °C and 120 minutes, no leaching of boron was observed in all experimental specimens impregnated and coated with PUU at 40 °C temperature, 120 rpm shaking speed, and after 120 minutes. Low leaching of boron was observed in wood specimens impregnated with BX, BA, and BX+BA and coated with EPR. The minimum leaching of boron rate was observed in wood specimens impregnated with BA and coated with EPR as 0.48 abs and maximum leaching of boron was measured in wood specimens impregnated with BX and coated with EPR as 1.27 abs. In this study; by applying polyurethane (PUU) and epoxy (EPR) resins to wood specimens subjected to the impregnation process, they were subjected to leaching of boron tests and it was aimed to prevent the water from physically dissolving and losing the preservative material by reaching the impregnation materials. When the results obtained were evaluated in general, it was observed that leaching of boron was completely prevented in all groups in which PUU was applied. On the other hand, it was observed that particular proportions of leaching of boron occurred in the epoxy resin applied groups. The factor of temperature plays an important role in the leaching of boron performance. Leaching of boron was observed at least at 10 °C, while the maximum leaching of boron was monitored at 40 °C (except for the BA+EPR group). The temperature rise may cause the water to overcome the resin layer physically bonded to the wood surface, causing the preservative to dissolve. In this case, it can be said that the groups coated with PUU are more resistant to the temperature increase than the EPR applied group. Polímeros, 32(1), e2022010, 2022

Determination leaching of boron from Oriental beech wood coated with polyurethane/polyurea (PUU) hybrid and epoxy (EPR) resins 3.2 Scanning electron microscope (SEM) analysis The SEM images of untreated (control) wood specimens and coated wood specimens of Oriental beech (EPR, BA+EPR, BX+EPR, and (BX+BA) +EPR) are given in Figure 4 (Magnification are 50, 100, 250 and 500 times and bar is 100 μm). In this study, SEM analyzes could not be performed on specimens coated with PUU, due to problems in taking images in the scanning electron microscope device.

When the SEM images of the control specimen in Figure 4 are examined, a macroscopically smooth surface can be seen, even if there is a microscopically rough surface at x500 magnification. When the SEM images of epoxy-coated specimens are examined in Figure 5, layered and rough surfaces can be seen easily. Since epoxy resin was cured with its hardener on the wood surface as was illustrated in Figure 2, it provides layered surfaces. While pure epoxy resin exhibited smooth

Table 3. Leaching of boron compounds from Oriental beech wood coated with PUU and EPR. Leaching temperatures Leaching at 10 °C temperature

Leaching at 22 °C temperature

Leaching at 40 °C temperature


5 min 0.21 0.23 0.01 0.38 0.17 0.82 0.57 0.01 0.49

15 min 0.23 0.29 0.04 0.04 0.14 1.05 0.72 0.09 0.82

30 min 0.26 0.36 0.11 0.49 0.34 1.18 1.02 0.17 1.05

Leaching time 45 min 60 min 0.30 0.35 0.44 0.50 0.18 0.05 0.60 0.71 0.48 0.59 1.11 1.14 1.11 1.26 0.24 0.30 0.98 1.09

90 min 0.41 0.59 0.30 0.86 0.73 1.14 1.26 0.38 1.00

120 min 0.48 0.67 0.38 1.02 0.92 1.18 1.27 0.48 0.99

Note: PUU: Polyurethane/Polyurea hybrid resin (Sikalastic®-851 R); EPR: Epoxy resin (Sikafloor®-156); BA: Boric acid; BX: Borax.

Figure 4. Magnified SEM images of untreated wood specimens. (a) 50x, bar 100 μm; (b) 100x, bar 100 μm; (c) 250x, bar 100 μm; (d) 500x, bar 100 μm. Polímeros, 32(1), e2022010, 2022


Altay, Ç., Toker, H., Kucuktuvek, M., Yeniocak, M., Bircan, İ. B., & Baysal, E surfaces at 50x magnification in the literature, EPR exhibited rough surfaces in this study. It is expected that EPR reduces surface roughness. The low viscosity of the EPR or the insufficient amount of application to the surface may be the main reasons for the rough surfaces in the Figure 5. The SEM images impregnated with BX and coated with EPR displayed that the specimens covered the micropores and

cell walls (Figure 6). It was observed for BX impregnated and EPR coated wood specimens had more layered, hollow, and rough surfaces compared to the impregnated with BA and coated with EPR (Figure 7). The reason could be that the presence of empty cells and these empty cells create aspiration of these passages after the polymerization or drying process of the specimens[31]. Besides this, the cause

Figure 5. Magnified SEM images of epoxy coated wood specimens (EPR). (a) 50x, bar 100 μm; (b) 100x, bar 100 μm; (c) 250x, bar 100 μm; (d) 500x, bar 100 μm.

Figure 6. Magnified SEM images of impregnated with borax and coated with epoxy resin wood specimens (BX+EPR). (a) 50x, bar 100 μm; (b) 100x, bar 100 μm; (c) 250x, bar 100 μm; (d) 500x, bar 100 μm. 6/9

Polímeros, 32(1), e2022010, 2022

Determination leaching of boron from Oriental beech wood coated with polyurethane/polyurea (PUU) hybrid and epoxy (EPR) resins of the bulky distribution of borax in some places within the wood cells could be the other reason. When the SEM images of impregnated with BA+BX and coated with EPR are examined (Figure 8), It can be seen on the pictures that the mixture of BA+BX covers the micropores and cell walls. Especially at x50 magnification, it is seen that some cell voids are formed as a result of impregnation with BA+BX on the wood surface. It can be said that this is caused by the bulky distribution of boric acid and borax at some points.

It can also be seen that at x250 magnification the borax and boric acid polymer chains interact and form an interface. While the layered and rough part could be borax in the upper part of the image, the polymer with a flatter and smoother appearance in the lower part could be boric acid. There is no clear phase separation between them. On the other hand, SEM photographs show some broken layers. These broken layers are indicative of penetration or copolymerization in

Figure 7. Magnified SEM images of impregnated with boric acid and coated with epoxy resin wood specimens (BA+EPR). (a) 50x, bar 100 μm; (b) 100x, bar 100 μm; (c) 250x, bar 100 μm; (d) 500x, bar 100 μm.

Figure 8. Magnified SEM images of impregnated with boric+borax mixture and coated with epoxy resin wood specimens ((BA+BX)+EPR). (a) 50x, bar 100 μm; (b) 100x, bar 100 μm; (c) 250x, bar 100 μm; (d) 500x, bar 100 μm.

Polímeros, 32(1), e2022010, 2022


Altay, Ç., Toker, H., Kucuktuvek, M., Yeniocak, M., Bircan, İ. B., & Baysal, E the cell walls. This could be due to the preservatives in the cell wall interacting with the hydroxyl groups.

4. Conclusions In this study, Oriental beech wood was impregnated with boron compounds and then was coated with polyurethane/ polyurea (PUU) hybrid resin and epoxy resin (EPR). According to the leaching of boron test results, the coating with polyurethane/polyurea (PUU) hybrid resin gave the most positive results against leaching of boron after impregnation at 10 °C, 22 °C, and 40 °C. Results showed that temperature plays an important role in the leaching of boron characteristics. The lower temperature values resulted in lower leaching of boron rates. When the SEM images of boron compounds impregnated and EPR coated Oriental beech wood are examined, it is seen that the impregnation material covers the cell spaces and cell walls. SEM results showed that the micropores on the BA impregnated Oriental beech wood specimens are less than the other treatment groups.

5. Acknowledgements This article study was taken from some results of the Ph.D. thesis of Çağlar ALTAY, who is studying in Woodworking Industrial Engineering at Muğla Sıtkı Koçman University Institute of Science. This study is supported by the Scientific Research Project BAP20/099/01/2.

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ISSN 1678-5169 (Online)


Cross-link density measurement of nitrile rubber vulcanizates using dynamic shear testa Gustavo Ninho Campos1* , Ana Carolina Ribeiro Coimbra1, Arianne Aparecida da Silva1 , Elisson Brum Dutra da Rocha1 , Felipe Nunes Linhares2 , Cristina Russi Guimarães Furtado1  and Ana Maria Furtado de Sousa1  Laboratório de Processamento de Polímeros II, Instituto de Química, Universidade do Estado do Rio de Janeiro – UERJ, Rio de Janeiro, RJ, Brasil 2 Centro de Engenharia, Modelagem, e Ciências Sociais Aplicadas, Universidade Federal do ABC – UFABC, Santo André, SP, Brasil a This paper has been partially presented at the 16th Brazilian Polymer Congress, held on-line, 24-28/Oct/2021



Abstract Cross-link density impacts most mechanical properties of rubber, therefore it is necessary to have a method to measure it. The most widely used method is via equilibrium swelling, however, it is time consuming and uses organic solvents. Dynamic Shear Test (DST) can be used to calculate both chemical and physical cross-links in rubber compounds in shorter times than by swelling equilibrium method, and without the use of solvents. In this work, equilibrium swelling using toluene and acetone was used to validate the dynamic shear tests for different nitrile rubber (NBR) compounds. The DST had a good correlation with the swelling equilibrium method using acetone, with a correlation coefficient of ~0,91, validating the use of DST. Moreover, the use of the Modified Guth-Gold equation (instead of Guth-Gold Equation with Medalia correction) also allowed to deduct the effect of carbon black on the cross-link density from the DST. Keywords: nitrile rubber, cross-link density, dynamic shear test, swelling equilibrium, solubility parameter. How to cite: Campos, G. N., Coimbra, A. C. R., Silva, A. A., Rocha, E. B. D., Linhares, F. N., Furtado, C. R. G., & Sousa, A. M. F. (2022). Cross-link density measurement of nitrile rubber vulcanizates using dynamic shear test. Polímeros: Ciência e Tecnologia, 32(1), e2022011.

1. Introduction Rubber compounds are designed as a complex mixture of components that includes vulcanization (curing) systems, reinforcement fillers, protective systems, and process aids. The choice of the vulcanization system is of extreme importance in the manufacturing and final properties of rubber products. The three-dimensional network formed during the vulcanization process is responsible for the high elasticity behavior and the reversible deformability of rubber materials, besides, it also affects their mechanical and thermal properties[1,2]. Therefore, it is necessary to have suitable techniques for evaluating the cross-linking density (CLD). Although there are several techniques in the literature that measure the CLD, it is unquestionable that Equilibrium swelling is the most used method[2,3]. The Equilibrium swelling method is based on the Flory−Rehner theory of swollen networks. It quantifies the cross-link density, μ (mol.g-1), which is proportional to the inverse of the average molecular weight between cross-links (μ ∝ ½ Mc, g.mol-1)[2,4,5]. Although it is widely used, the Equilibrium swelling method uses organic solvents, and it is a laborious and time-consuming technique. Furthermore, the results will directly depend on the solvent used.

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Lee et al.[6] presented a fast method for assessing the CLD from rheological properties of natural rubber (NR) and styrene butadiene rubber (SBR), using the Rubber Process Analyzer (RPA 2000). This dynamic shear test (DST) method allows the measurement of physical crosslink density, which is related to the chain’s entanglements, and the measurement of total crosslink density, which also includes the contribution from chemical cross-links formed during the vulcanization process. The chemical cross-links can be calculated as the difference between total and physical crosslink densities[6-9]. Silva et al[8] compared the crosslink density values of epoxidized natural rubber filled with hydrotalcite measured by equilibrium swelling and DST methods. The authors reported a good correlation between both methods and highlighted that DST is a fast and efficient alternative, with the advantage of not using any organic solvent[8]. Ünügül and Karaagaç[9], employed the dynamic shear test to study the effect of reactive silane on the vulcanization of the chlorinated polyethylene (CPE) and chloroprene rubber (CR) compounds. The authors reported that CPE showed a noticeable increase in physical



Campos, G. N., Coimbra A. C. R., Silva, A. A., Rocha E. B. D., Linhares F. N., Furtado C. R. G., & Sousa A. M. F. cross-link density and that amino silane compounds exhibit significantly higher chemical and total cross-link densities. Despite its potential and its use in different rubbers, there are few reports in literature of the dynamic shear test and even less of this method applied to nitrile rubber. A more detailed understanding of the relationship of the DST with other, more conventionally used methods such as equilibrium swelling, will prove useful to attest the validity of the DST and to better understand rubber cross-link density. Aiming at validating the applicability of the dynamic shear test (DST) in nitrile rubber, unfilled and carbon black filled NBR compounds were vulcanized at different times to generate different degrees of vulcanization, and their cross-link density was measured by equilibrium swelling (using both toluene and acetone) and DST.

2. Materials and Methods 2.1 Materials Three different grades of poly(acrylonitrile-cobutadiene), also known as nitrile rubber or NBR, with different acrylonitrile (ACN) content were kindly donated by Nitriflex S/A Indústria e Comércio: N726 (28% of ACN), N615 (33% of ACN), and N206 (45% of ACN). Moreover, carbon black (N330), zinc oxide (ZnO), stearic acid, and n-tert-butyl-2-benzothiazolesulfenamide (TBBS) were used as received.

2.2 Rubber compounding and samples preparation Unfilled (gum) and carbon black filled rubber formulations were designed according to Table 1, using ASTM D3187 as reference. The experimental coding used was AA%/BB, in which AA is the CAN content (28, 33, and 45%) and BB is the amount of carbon black. The compounds were prepared in an open mixing mill (Luxor, model BML 150) following the procedure described in ASTM D3187.

Rheometric curves were determined based on ASTM D5289 using the Rubber Process Analyzer (RPA 2000, Alpha Technologies). The test was performed at 160°C, oscillation amplitude of ± 0.5° arc, and frequency of 1.67 Hz. For each rubber compound, four different vulcanization times, tx, (ranged from t20 to t90, Table 2) were established from the torque versus time curves with the purpose of producing different cross-link densities. “tx” is the time needed to achieve a “x” % of vulcanization. The same specimen from DST was submitted to equilibrium swelling test method to compare the two methods.

2.3 Cross-link density characterization by Dynamic Shear Test (DST) The dynamic shear test to calculate the compounds’ CLD was conducted on an RPA 2000 (Alpha Technologies). The DST protocol was divided in four steps, following the parameters defined by Lee and Coran as to avoid sample shrinkage and degradation[6]. In the first step, the test specimen was preconditioned during 2 min at 100ºC, 0.2º of strain, and 0.5-Hz frequency. The second step consisted of measuring the elastic modulus at 5.0 Hz frequency (G’5Hz), at 100ºC of temperature and 0.25º of strain. In the third step, the test specimen was vulcanized at 160ºC using the respective vulcanization times (Table 2). In the fourth step, the temperature was reduced to 100°C, and then the elastic modulus was measured at 0.5 Hz frequency (named as G’0.5Hz), at 100ºC of temperature and 0.25º of strain. At the end, the test specimen was removed from RPA and reserved for testing in the equilibrium swelling test. For the unfilled rubber compounds, the physical [μ]P, total [μ]T, and chemical [μ]C cross-link densities were calculated using Equations 1, 2, and 3, respectively, where R is the gas constant (8.314 J.K-1mol-1) and T is the absolute temperature in Kelvin (K)[6,7]. [ µ ]P = ( G '5 Hz ) / ( 2 RT )


Table 1. Unfilled and filled nitrile rubber (NBR) formulations. Amounts in part per hundred parts of rubber (phr). Component NBR with 28% of acrylonitrile NBR with 33% of acrylonitrile NBR with 45% of acrylonitrile ZnO Stearic acid Sulphur TBBS1 N3302

28%/00 100 3 1 1.5 0.7 -

33%/00 100 3 1 1.5 0.7 -

45%/00 100 3 1 1.5 0.7 -

33%/40 100 3 1 1.5 0.7 40

1 – n-tert-butyl-2-benzothiazolesulfenamide; 2 – carbon black N330.

Table 2. The curing times of the compounds and their percentage of vulcanization*. 28%/00 t23 t55 t83 t90

33%/00 6 min 7 min 9 min 11 min

*Based on time of t(x) related to the


t20 t63 t78 t90

45%/00 7 min 9 min 11 min 15 min

 x  M ( x ) =ML + ( MH − ML ) *  ,  100 

t20 t60 t70 t90

33%/40 5 min 7 min 11 min 20 min

t25 t53 t78 t90

4 min 5 min 7 min 11 min

where x is the percentage of vulcanization (20 to 90%).

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Cross-link density measurement of nitrile rubber vulcanizates using dynamic shear test [ µ ]T = ( G '0.5 Hz ) / ( 2 RT )


[ µ= ]C [ µ ]T − [ µ ]P


It is important to highlight that Equations 1, 2, and 3 were developed for unfilled rubber. Therefore, it is necessary to deduct the filler’s contribution in the G’5Hz and G’0.5Hz values for filled rubber compounds, i.e., it is necessary to estimate the modulus values of the respective “gum-state”. Therefore, it was used the Guth-Gold Equation with Medalia correction[6,7] (Equation 4) and the modified Guth–Gold equation[10] (Equation 5) developed for carbon black–filled rubbers[5]. G ' filled = G ' unfilled (1 + 2.5Ø + 14.1Ø 2 )


G ' filled = G ' unfilled (1 + 2.5Ø + 14.1Ø ² + 0.20( S )3 Ø 3 )


Wherein G ' filled is the elastic modulus of filled samples, G ' unfilled is the elastic modulus without filler contribution, Ø is the volume fraction of the filler, and S is the BET nitrogen surface area (NSA) of carbon black. According to literature[10], Equation 4 is recommended when the particles are dispersed from each other in a rubber matrix, behaving almost independently, while Equation 5 is applied to systems where particles or aggregates are connected to each other forming a network structure.

2.4 Cross-link Density characterization by Equilibrium swelling test The equilibrium swelling test was assessed using toluene and acetone. Each test specimen from the DST test was cut in four pieces, being two tested with toluene and the other two with acetone. The test protocol consisted in weighing the test specimen in air and solvent to calculate their initial mass and density (according to Archimedes’ principle). Then, each test specimen was swollen in the solvent until the system reached equilibrium. After this time, the test specimen was removed from the solvent and weighed. Lastly, the solvent inside the swollen test specimen was removed and the sample reweighted thereafter. The crosslink density was calculated by the equation developed by Flory-Rehner, shown in Equation 6. Wherein μ is cross-link density (mol.cm-3), ν r is the volume fraction of rubber in the swollen sample determined by Equation 7, V0 is the molar volume of the solvent (toluene: 106.83 cm3.mol-1 and acetone: 73.7 cm3.mol-1) and χ is the Flory–Huggins interaction parameter for the solvent and the elastomer.  − ln 1 −ν +ν + χ *ν 2   ( r) r r      1    3 ν r  V0 * ν r − 2       

   µ=

 M 1 − M 1* f f    ρc   Vr =  M 1 − M 1* f f   M 2 − M 3   +      ρc ρs    

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Wherein M1, M2, and M3 are, respectively, the initial, the swollen, and the dried sample masses; ƒ is the filler ƒ fraction in volume; ρ c is the sample density, and ρ s is the solvent density. The interaction parameters ( χ ) of NBR/Acetone and NBR/toluene were calculated by using the Hildebrand model (Equation 8)[11,12], wherein δsol ((cal/cm3)0.5) is the solubility parameter of solvent (δtoluene = 8.90; δacetone = 9.88) and δrub ((cal/cm3)0.5) is the solubility parameter of rubber (δNBR28% = 9.35; δNBR33% = 9.57; δNBR45% = 10.19). The χ values are shown in Table 3. V χ= 0.35 + 0 (δ sol − δ rub ) ²



2.5 Statistical analysis Pearson product-moment correlation coefficient was used to assess the relationship between DST and equilibrium swelling methods. The Pearson correlation coefficients range between -1 and +1 and measure the strength of the linear relationship among the variables[13]. As the correlation coefficients gets closer to +1 or -1, the more correlated the datasets will be, with a positive trend (+1) or negative trend (-1). When the correlation coefficient is close to zero, its linear relationship is poor. The p-value was used to evaluate the statistical significance of the correlation coefficient. The Pearson product-moment correlation coefficient was preferred instead of the conventional coefficient of determination of a linear fit, R2, because it is a more accurate way to describe the strength of the linear relationship rather than R2 because we are not evaluating the strength of a linear model. The cross-link data were processed using the statistical software STATGRAPHICS Centurium 18 with 95,0% of confidence level. This analysis was conducted for the groups of variables: [μ]C, [μ]T, μTol, and μAcet, using all data from unfilled NBR.

3. Results and Discussions 3.1 Cross-link density characterization by Dynamic Shear Test Figure 1 shows the values of the physical ([μ]P), chemical ([μ]C) and total ([μ]T = [μ]C + [μ]P) cross-link densities measured from DST test for unfilled NBR. The vulcanization times (from t20 to t90) did not affect the values of [μ]P for each of NBR sample. This behavior was expected, since the [μ]P is mainly associated with the presence of physical entanglements for unfilled rubber. Moreover, the physical entanglements ([μ]P) vary according to the type of NBR, increasing from 28% to 45% of acrylonitrile content. Table 3. Calculated rubber-solvent interaction parameter ( χ ) values based on Equation 8.


Solvent Toluene Acetone

NBR28% 0.3760 0.3945

NBR33% 0.4253 0.3627

NBR45% 0.6482 0.3618


Campos, G. N., Coimbra A. C. R., Silva, A. A., Rocha E. B. D., Linhares F. N., Furtado C. R. G., & Sousa A. M. F.

Figure 1. Physical ([μ]P), chemical ([μ]C) and Total ([μ]T = ([μ]C + [μ]P) cross-link densities (.10-5 mol.cm-3) of unfilled rubber with acrylonitrile content of (a) 28%, (b) 33% and (c) 45%, cured at times related to specific vulcanization percentages.

Figure 2. Physical ([μ]P), chemical ([μ]C) and Total ([μ]T = ([μ]C + [μ]P) cross-link densities (.10-5 mol.cm-3) of filled rubber 33%/40 vulcanized at crescent times and deducting the filler content using Guth-Gold Equation with Medalia correction (Equation 4) and the modified Guth–Gold equation (Equation 5).

Regarding the chemical crosslink density ([μ] C), as expected, there is an increase in [μ] C values as the vulcanization time increases, and at t90 vulcanization time the three NBR compounds presented similar [μ] C values (~ 6.10 -5 mol.cm-3). This behavior is reasonable since the same vulcanization system (Table 1) was used for all NBR compounds. Therefore, the difference of [μ] T values at t 90 (28%/00 > 33%/00 > 45%/00) is directly related to the different physical crosslinks of each sample. Figure 2 shows the values of [μ]P, [μ]C, and [μ]T crosslink densities for the 33%/40 compound, whose calculation was done considering two conditions: (i) “filled-state”, i.e., with the contribution of carbon black in the result, and (ii) “gum-state”, in which the carbon black contribution was discounted using Guth-Gold Equation with Medalia correction[6,7] (Equation 4) and the modified Guth–Gold equation[10] (Equation 5). 4/6

A noticeable increase in [μ]P values of 33%/40 is observed for the “filled-state” compared to unfilled NBR (33%/00, Figure 1b). A similar behavior was observed with carbon black filled natural rubber (NR) compounds[14] using 1 H-NMR method. Higher values of physical crosslink density for carbon black filled NR was observed than to the unfilled one. This is because carbon black restricts the rubber chains’ mobility, besides the inherent rubber chains’ entanglements. The comparison between the results of 33%/40 (Figure 2) and 33%/00 (Figure 1b) shows that the correction of modulus made with modified Guth–Gold Equation (Equation 5) produced values of [μ]P “gum-state” close to the unfilled [μ]P of 33%/00. Therefore, this result indicates that modified Guth–Gold equation (Equation 5) was more effective deducting the filler’s contribution from moduli values. Regarding the higher values observed of values of [μ]P “gum-state” using Equation 4, the Guth-Gold Equation with Medalia correction only accounts for the amplification of the modulus caused by rigid particles that do not deform; but does not consider the amplification of the modulus caused by the interaction of rigid particles, as in the formation of a network structure. Fukahori et al.[10] indicates that the modulus increase caused by carbon black network is better described using the modified Guth–Gold equation (Equation 5). Furthermore, as expected, the chemical crosslink ([μ]C) values of “filled-state” are higher than “gum-state” ones (Equation 4 and 5). Comparing the 33%/40 [μ]C “gumstate” from Guth-Gold equation to the 33%/00 [μ]C, one can infer that carbon black affected negatively in the chemical cross-links. However, there is no major consensus in the literature on carbon black/cross-link density effect. Some studies[15] suggest carbon black increases the formation of cross-links, whereas others[16,17] indicates carbon black does not affect the cross-link density.

3.2 Comparison of cross-link densities between Equilibrium swelling and DST Figure 3(a), (b) and (c) shows the cross-link densities values of unfilled NBR measured from equilibrium swelling method using two different solvents: toluene (μTol) and acetone (μAcet). Regardless of the solvent type, all crosslink densities increased with the vulcanization time. Similar values were observed when the compounds were vulcanized at respective t90, with a small tendency of a lower Polímeros, 32(1), e2022011, 2022

Cross-link density measurement of nitrile rubber vulcanizates using dynamic shear test

Figure 3. Cross-link densities (10-4 mol.cm-3) of unfilled NBR, (a) 28%/00, (b) 33%/00 and (c) 45%/00, and filled NBR, (d) 33%/40, cured at different times determined from Equilibrium swelling data.

value for 45%/00. The cross-link density for the different NBR samples were similar, as expected, because the same vulcanization system was employed. The same trend was also observed on the results from DST. Figure 4a shows the scatterplot matrix with a scatterplot for each pair of the variables [μ]C, [μ]T, μTol, and μAcet (using data from unfilled NBR) plotted against each other. Figure 4b shows the Pearson product-moment correlation coefficient plot, corr-plot, which consists of cells with the correlation coefficient of each pair of variables, as well as the p-value of each correlation coefficient in parenthesis. This was done to assess the relationship between DST and equilibrium swelling methods: as the correlation coefficient gets closer to +1 or -1 (straight line in the scatterplot), the correlation between variables gets stronger[13]. It is important that the cross-link densities obtained with any method are correlated, because in principle they are measuring the same property. The scatterplot and the correlation plot of the variables [μ]C and [μ]T were not shown because they are linearly dependent by definition ([μ]T = [μ]C+[μ]P). As shown in Figure 4b, there are strong positive linear correlation (color from orange to red) for all pairs of variables, except for “[μ]T X μTol”. Regarding the equilibrium swelling with toluene and acetone, μAcet produced higher correlation than μTol, being the strongest correlation found for “[μ]C x μAcet”. These findings are interesting for showing an agreement between the techniques and showing the importance of solvent type. It has been shown that “good” solvents, which have a better interaction with the rubber and subsequent lower interaction parameters, give more accurate results of cross-link density when using the Hildebrand equation than “bad” solvents[2]. As the interaction parameters of NBR/ acetone are lower than the NBR/toluene ones (Table 3), the use of acetone is more appropriate for NBR samples. As for the DST, the use of [μ]C produced higher correlation than [μ]T with the equilibrium swelling with acetone, with correlation coefficients of approximately 0.91 for the pairs “[μ]C X μAcet” against 0.80 for “[μ]T X μAcet”. This shows that best variable from DST to compare the cross-link density with the equilibrium swelling is the [μ]C and confirms the validity of using the dynamic shear method. Figure 3(d) and (b) shows the cross-link density comparison between 33%/40 (filled rubber) and 33%/00 measured by equilibrium swelling data. The test performed with toluene indicated that there was no appreciable difference in cross-link densities between 33%/40 and 33%/00, while the test with acetone resulted in Polímeros, 32(1), e2022011, 2022

Figure 4. Correlation of variables [μ]C, [μ]T, μTol, and μAcet by (a) Scatterplot matrix, with each variable plotted against each other; (b) Correlation plot, with each cell with the corresponding pair correlation coefficient and the p-value in parenthesis. (*) correlation between “[μ]C X [μ]T” was not considered because they are linearly dependent by definition ([μ]T = [μ]C+[μ]P).

higher values of CLD for 33%/40. Literature shows that the calculated CLD from carbon black filled rubber is higher than unfilled, given that the immobilized rubber next to the filler acts as a cross-link (bound rubber)[16-20].

4. Conclusions The cross-link density of the three grades of unfilled NBR was successfully determined using both the Dynamic Shear Test and the Equilibrium swelling method. Based on the results obtained, we could conclude: 1) Both dynamic shear test (DST) and the equilibrium swelling method have good correlation (correlation coefficient of ~ 0.91) for NBR, when using the chemical cross-link density, [μ]C, for DST and when swelling acetone instead of toluene as solvent; 5/6

Campos, G. N., Coimbra A. C. R., Silva, A. A., Rocha E. B. D., Linhares F. N., Furtado C. R. G., & Sousa A. M. F. 2) Acetone as solvent for equilibrium swelling method for NBR compounds gives better results than toluene as solvent when using the Hildebrand solubility parameter to calculate the interaction parameter; 3) Vulcanization times did not affect the physical crosslinks of the NBR compounds; 4) Modified Guth-Gold equation gives better results deducting carbon black effect on cross-link density than Guth-Gold Equation with Medalia correction; 5) Dynamic shear test method is a reliable, solventless method for calculating cross-link densities for both filled and unfilled rubber compounds, and it conducted faster than equilibrium swelling method.

5. Acknowledgments The authors thank Nitriflex for donating the raw materials and for the use of its facilities. This study was supported by Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro – FAPERJ [E-26/200.905/2020-Bolsa: Scholarship received by Ana Carolina R. Coimbra and E-26/200.289/2021: PhD Scholarship received by Gustavo N. Campos], Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPQ [PQ-2:309461/2021-9], and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) [Financing code 001, and the PhD Scholarship received by Arianne A. Silva].

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8. Silva, V. M., Nunes, R. C. R., & Sousa, A. M. F. (2017). Epoxidized natural rubber and hydrotalcite compounds: rheological and thermal characterization. Polímeros: Ciência e Tecnologia, 27(3), 208-212. http://dx.doi.org/10.1590/01041428.03416. 9. Ünügül, T., & Karaağaç, B. (2021). Vulcanization of chlorinated polyethylene / chloroprene rubber compounds at lower temperatures in the presence of reactive silanes. Journal of Applied Polymer Science, 138(23), 50544. http://dx.doi. org/10.1002/app.50544. 10. Fukahori, Y., Hon, A. A., Jha, V., & Busfield, J. J. C. (2013). Modified guth-gold equation for carbon black-filled rubbers. Rubber Chemistry and Technology, 86(2), 218-232. http:// dx.doi.org/10.5254/rct.13.87995. 11. Lee, J.-Y., Park, N., Lim, S., Ahn, B., Kim, W., Moon, H., Paik, H.-J., & Kim, W. (2017). Influence of the silanes on the crosslink density and crosslink structure of silica-filled solution styrene butadiene rubber compounds. Composite Interfaces, 24(7), 711-727. http://dx.doi.org/10.1080/09276 440.2017.1267524. 12. Kim, D. Y., Park, J. W., Lee, D. Y., & Seo, K. H. (2020). Correlation between the crosslink characteristics and mechanical properties of natural rubber compound via accelerators and reinforcement. Polymers, 12(9), 2020. http://dx.doi.org/10.3390/ polym12092020. PMid:32899685. 13. Xu, Q., Majlingova, A., Zachar, M., Jin, C., & Jiang, Y. (2012). Correlation analysis of cone calorimetry test data assessment of the procedure with tests of different polymers. Journal of Thermal Analysis and Calorimetry, 110(1), 65-70. http://dx.doi. org/10.1007/s10973-011-2059-7. 14. Fei, Z., Long, C., Qingyan, P., & Shugao, Z. (2012). Influence of carbon black on crosslink density of natural rubber. Journal of Macromolecular Science, Part B: Physics, 51(6), 1208-1217. http://dx.doi.org/10.1080/00222348.2012.664494. 15. Robertson, C. G., & Hardman, N. J. (2021). Nature of carbon black reinforcement of rubber: perspective on the original polymer nanocomposite. Polymers, 13(4), 538. http://dx.doi. org/10.3390/polym13040538. PMid:33673094. 16. Yadollahi, S., Ramezani, M., Razzaghi-Kashani, M., & Bahramian, A.-R. (2018). Nonlinear viscoelastic dissipation in vulcanizates containing carbon black and silanized silica hybrid fillers. Rubber Chemistry and Technology, 91(3), 537547. http://dx.doi.org/10.5254/rct.18.82611. 17. Koenig, J. L. (2000). Spectroscopic characterization of the molecular structure of elastomeric networks. Rubber Chemistry and Technology, 73(3), 385-404. http://dx.doi. org/10.5254/1.3547598. 18. Negri, R. B. P., Silva, A. H. M. F. T., Sousa, A. M. F., Silva, A. L. N., & Rocha, E. B. D. (2021). Improved mechanical and rheological behavior of nitrile rubber reinforced with multi-walled carbon nanotubes and carbon black dual-filler system. Materials Today Communications, 26, 101884. http:// dx.doi.org/10.1016/j.mtcomm.2020.101884. 19. Rocha, E. B. D., Batista, M. R., Linhares, F. N., Silva, A. L. N., Delpech, M. C., Sousa, A. M. F., & Furtado, C. R. G. (2019). Cyclic uniaxial stress-strain test and rheological behavior of carbon black/metakaolin dual-filler system used in nitrile rubber compounds. Polymer Testing, 77, 105906. http://dx.doi. org/10.1016/j.polymertesting.2019.105906. 20. Fan, R., Zhang, Y., Huang, C., Zhang, Y., Fan, Y., & Sun, K. (2001). Effect of crosslink structures on dynamic mechanical properties of natural rubber vulcanizates under different aging conditions. Journal of Applied Polymer Science, 81(3), 710718. http://dx.doi.org/10.1002/app.1488. Received: Mar. 24, 2022 Revised: May 16, 2022 Accepted: May 22, 2022 Polímeros, 32(1), e2022011, 2022

ISSN 1678-5169 (Online)


Effect of non-thermal argon plasma on the shear strength of adhesive systems Isabella de Almeida Guimarães Passos1 , Juliana das Neves Marques1 , João Victor Frazão Câmara2* , Renata Antoun Simão3 , Maíra do Prado1  and Gisele Damiana da Silveira Pereira1  Departamento de Clínica Odontológica, Faculdade de Odontologia, Universidade Federal do Rio de Janeiro – UFRJ, Rio de Janeiro, RJ, Brasil 2 Clinic of Operative Dentistry, Periodontology and Preventive Dentistry, Saarland University Hospital, Homburg, Saar, Germany 3 Departamento de Engenharia Metalúrgica e de Materiais, Centro de Tecnologia, Universidade Federal do Rio de Janeiro – UFRJ, Rio de Janeiro, RJ, Brasil 1


Abstract This study evaluated the influence of non-thermal argon plasma applied to dentin on the shear strength of two adhesive systems. Ninety tooth fragments were embedded in epoxy resin and distributed into experimental groups (n=15): G1 and G4 - adhesive systems applied according to the manufacturers’ instructions; G2 and G5 - dentin treated with nonthermal argon plasma for 30 seconds before hybridization; G3 and G6 - dentin treated with non-thermal argon plasma for 30 seconds after hybridization. Cylinders were made with composite resin in the adhesive area, and the specimens were submitted to the shear strength test. Higher values were observed when applying the plasma treatment after hybridization (G1: 26.51 MPa, G2: 29.22 MPa, G3: 30.27 MPa, G4: 22.66 MPa, G5: 28.33 MPa, G6: 29.32 MPa). The treatment with non-thermal argon plasma significantly increased the shear strength values regardless of the application time. Keywords: argon plasma, non-thermal plasma, dentin bonding agents, dentin, shear strength. How to cite: Passos, I. A. G., Marques, J. N., Câmara, J. V. F., Simão, R. A., Prado, M., & Pereira, G. D. S. (2022). Effect of non-thermal argon plasma on the shear strength of adhesive systems. Polímeros: Ciência e Tecnologia, 32(1), e2022012. https://doi.org/10.1590/0104-1428.20220019

1. Introduction Several factors inherent to the physicochemical structure of adhesives and their intrinsic properties interfere with the formation of the hybrid dentin layer[1-4]. The morphological, physiological, and pathological heterogeneity of dentin, the moisture needed to maintain the expanded collagen network, its low surface energy, and the limited degree of conversion of resin monomers are among the main obstacles to achieving uniform adhesion[5]. Moreover, the hydrolytic degradation of both components of the hybrid layer - collagen matrix and composite resin - seriously compromises the long-term adhesive interface integrity and the bond strength durability, causing postoperative sensitivity, bacterial microleakage, and secondary caries[4,5]. Non-thermal plasmas are considered the fourth state of matter, comprising partially ionized gases with different concentrations of highly reactive low molecular weight particles, including electronically excited atoms, molecules, ionic species, and free radicals applied at temperatures close to body temperature, which allows using them in vivo[6,7]. Plasma has been used in the surface engineering industry for improving biomaterial adhesion by depositing thin films[8]. When used correctly, the non-thermal plasma modifies the physical and chemical properties of surfaces, maintaining

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the interior characteristics of the material[9]. Recent studies have proposed using plasma technology in Dentistry for different purposes, including dental caries treatment, sterilization, biofilm elimination, root canal disinfection, and tooth whitening, among others[6,10,11]. Furthermore, plasma has been used to improve adhesion to the dental substrate because treatments with this gas increase the contact surface area of collagen fibers and their hydrophilicity[6,10], allowing a higher interaction with the adhesive and increasing bond strength[6,9]. Plasma also induces and increases the degree of conversion of resin monomers. The high complexity of the dentin tissue and the factors that influence bond durability require scientific evaluations of bond strength and surface treatments. Thus, this study aimed to evaluate the influence of non-thermal argon plasma applied to deep dentin, before and after hybridization, on the shear strength of two adhesive systems: a conventional three-step system and a self-etching single-step system. The following null hypotheses were tested: 1 - The composition and application technique of adhesive systems do not affect adhesive strength; 2 - The application of nonthermal argon plasma does not affect the shear strength of ​​ adhesive systems; 3 - The application of non-thermal argon



Passos, I. A. G., Marques, J. N., Câmara, J. V. F., Simão R. A., Prado, M., & Pereira, G. D. S. plasma before the adhesive systems does not interfere with adhesion; 4 - The application of non-thermal argon plasma on hybridized dentin does not interfere with the shear strength of adhesive systems.

2. Materials and Methods 2.1 Ethical aspects The research project was submitted to and approved by the Research Ethics Committee of the University Hospital Clementino Fraga Filho (HUCFF) of the Federal University of Rio de Janeiro - UFRJ (RJ, Brazil), with approval number 79803517.6.0000.5257.

2.2 Materials Two adhesive systems were used: a conventional waterbased multi-bottle system (Adper Scotchbond Multi-Purpose, 3M do Brasil, Sumaré, SP, Brazil) and a single-bottle selfetching ethanol-based system (Single Bond Universal, 3M do Brasil, Sumaré, SP, Brazil). The study also used an Opallis nanohybrid restorative composite in A3 color (FGM LTDA, Joinville, SC, Brazil), Condac 37% phosphoric acid conditioner (FGM LTDA, Joinville, SC, Brazil), and non-thermal plasma from Argon gas (White Martins, Rio de Janeiro, RJ, Brazil). Table 1 describes the brands and compositions of the materials selected.

2.3 Experimental groups The samples were divided into six experimental groups with 15 repetitions each, as follows: Group 1 – SBMPSP: after acid etching, the Adper Scotchbond Multi-Purpose adhesive system was applied to the dentin surface; Group 2 – SBMPPA: after acid etching, argon plasma was applied for 30 seconds to the dentin surface, followed by the application of Adper Scotchbond adhesive; Group 3 - SBMPPD: after acid etching, Adper Scotchbond Multi-Purpose adhesive was applied, followed by the application of argon plasma for 30 seconds; Group 4 – SBUSP: the Single Bond Universal adhesive

system was applied to the dentin surface; Group 5 – SBUPA: argon plasma was applied for 30 seconds to the dentin surface, followed by the application of the Single Bond Universal adhesive system; Group 6 – SBUPD: the Single Bond Universal adhesive system was applied to the dentin surface, followed by the application of argon plasma for 30 seconds.

2.4 Preparation of dentin samples The study used 45 human third molars impacted and freshly extracted by therapeutic indication. The teeth were collected in a private office after patients had signed the donation terms and the informed consent form. The teeth were stored for up to 30 days in a 0.1% thymol solution (UFRJ-CCMN, Department of Biochemistry, Rio de Janeiro, RJ, Brazil) at a pH of 7 for disinfection, and a temperature of 37ºC in an oven (Quimis Scientific Apparatus, São Paulo, SP, Brazil) until starting the external surface cleaning with # 13/14 Gracey periodontal curettes (Hu-Friedy do Brasil, Rio de Janeiro, RJ, Brazil) to remove periodontal tissue residues. Subsequently, the teeth were submitted to prophylaxis with a pumice stone (SS-White, Rio de Janeiro, RJ, Brazil) and water, using Robinson brushes (KG Sorensen, Barueri, SP, Brazil) mounted in a counterangle at low rotation (Kavo do Brazil, Joinville, SC, Brazil). The coronal portions of the 45 upper or lower third molars were separated from their roots 1 mm below the cemento-enamel junction with a double-sided diamond disc (KG Sorensen, Barueri, SP, Brazil) at low rotation and under abundant water cooling/air. Then, the fragments obtained were sectioned in two parts mesiodistally from the occlusal surface with the same process, producing 90 tooth fragments. The enamel surface of each fragment was sanded and fixed on a glass plate aided by adhesive tape to facilitate their inclusion in the epoxy resin (Redecenter Materials Plásticos e Acessórios LTDA, São Paulo, SP, Brazil) poured into PVC tube rings with 21 mm in internal diameter and

Table 1. Brands and composition of materials. Material Composition Condac 37% 37% phosphoric acid Adper Scotchbond Primer: Aqueous solution of 2-hydroxyethyl methacrylate Multi-Purpose (HEMA) and a copolymer. Adhesive: Bisphenol-a-glycidyl methacrylate (Bis-GMA), 2-hydroxyethyl methacrylate (HEMA), and camphorquinone solution. Single Bond Universal Bisphenol-a-diglycidyl ether dimethacrylate (Bis-GMA), 2-hydroxyethyl methacrylate, water, 1,10-decanediol methacrylate phosphate, acrylic, itaconic acid copolymer, camphorquinone, N,N-dimethylbenzoin, 2-dimethyl monoethyl methacrylate, and methyl ethyl ketone. Opallis Monomeric matrix: Bis (GMA), Bis (EMA), UDMA, and TEGDMA. Fillers: Barium-aluminosilicate glass, silanized barium-aluminosilicate glass, and silicon dioxide nanoparticles. Photoinitiator: camphorquinone, accelerators, stabilizers, and pigments. The composite particles range from 40 nm to 3.0 microns with an average particle size of 0.5 microns, total filler content by weight from 78.5% to 79.8%, and volume from 57% to 58% of inorganic filler. Non-thermal plasma Argon gas.


Manufacturer Classification FGM LTDA, Joinville, SC, Brazil Acid conditioner 3M do Brasil, Sumaré, SP, Brazil Conventional 3-step adhesive system

3M do Brasil, Sumaré, SP, Brazil Self-etching single-step adhesive system

FGM LTDA, Joinville, SC, Brazil Nanohybrid resin

White Martins, Rio de Janeiro, Propagation gas nonRJ, Brazil thermal plasma

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Effect of non-thermal argon plasma on the shear strength of adhesive systems 10 mm in height. These tubes were also fixed on the glass plate to centralize the tooth fragments[12]. After polymerization, the epoxy resin blocks (Redecenter Materials Plásticos e Acessórios LTDA, São Paulo, SP, Brazil) were removed from the PVC cylinders and the buccal, palatal, and lingual surfaces were abraded with a #180 silicon carbide sandpaper (3M do Brasil, Sumaré, SP, Brazil) in a water-cooled rotary electric polisher (Aropol 2V, Arotec Indústria e Comércio, Cotia, SP, Brazil) to expose a flat area in the deep dentin. The control distance from the dentin surface to the pulp chamber was based on the protocol by dos Santos et al.[13]. Finishing was performed similarly with a #600 silicon carbide sandpaper. Then, the flat surfaces (3D) were washed with distilled water spraying for 15 seconds. The samples were identified so they could be distributed by drawing among the experimental groups. The specimens were stored in containers with distilled water and maintained in an oven at 37ºC while awaiting the preparation of specimens for the mechanical test.

2.5 Non-thermal plasma application The non-thermal plasma treatment was performed with a glass reactor, which consisted of a glass tube of 5 cm in diameter and 30 cm in length, evacuated by a mechanical pump to pressures below 2 Pascals (Pa). The gas was allowed to fill the reactor up to 10 Pa of pressure[6]. The non-thermal plasma was produced inside the glass cylinder under vacuum by the action of a magnetic field induced by the current passing through an electric coil surrounding the cylinder. The dentinal surfaces were treated with argon gas at 60 watts (w) for 30 seconds[6]. At the end of the process, the radio frequency was turned off before exposing the samples to air. In groups 2 and 5, this procedure was performed before applying the adhesive systems under study. In groups 3 and 6, this procedure was performed after applying and polymerizing the adhesive systems.

height was placed in the adhesive area to facilitate the insertion of the Opallis A3 color nanohybrid restorative composite (FGM LTDA, Joinville, SC, Brazil). The composite was inserted in 2-mm increments aided by a Suprafil spatula and cured for 20 seconds according to the manufacturer’s instructions with the Radii-cal curing apparatus (SDI Brasil Indústria e Comércio LTDA, São Paulo, SP, Brazil) at a power of 1200 mw/cm2. After 24 hours, the specimens were subjected to the mechanical shear strength test.

2.7 Mechanical shear strength test The sequence of specimen fractures was randomly performed after a draw. The tests were performed in an INSTRON 33R5567 universal testing machine (Instron, Canton, Massachusetts, USA) with a 200-kg load cell adjusted for the speed of 0.5 mm/min. The load was applied with a chisel with a 0.5-mm wide active tip positioned flush with the base of the composite cylinder, as close as possible to the adhesive interface. The load required for fracturing each specimen was expressed in Newton (N), and the results were transformed into Megapascal (MPa) with the formula: P=F/A and submitted to the statistical analysis.

2.8 Statistical analysis Statistical analyses were performed with the R Project 3.4.2 (R Foundation for Statistical Computing, Vienna, Austria) and IBM SPSS 22 (IBM Corporation, Armonk-NY, United States) software. The Shapiro-Wilks test assessed data normality. Considering the deviation from normality, non-parametric tests were used to assess differences among the groups. The Mann-Whitney test was used for comparing two groups. The Kruskal-Wallis test was performed for analyzing more than two groups and, if there was a significant difference among the groups in the two-by-two analysis (post-hoc), the Dunn test was applied. A 5% significance level was used.

2.6 Adhesive system application and restorative procedures

3. Results and Discussion

For the Adper Scotchbond Multi-Purpose adhesive system: acid application for 15 seconds, washing with water for 30 seconds, drying, primer application, mild air spraying for 5 seconds, adhesive application, and light-curing for 10 seconds. For the Single Bond Universal adhesive system: adhesive layer application for 15 seconds, mild air spraying for 5 seconds, new adhesive layer application, air drying for 5 seconds, and light-curing for 10 seconds. After polymerizing the adhesive system, a rubber matrix with a central perforation of 3 mm in diameter and 5 mm in

Table 2 presents the mean values and standard deviations of all groups and the maximum and minimum values of each group. The highest median (30.27 ± 0.54) was recorded for the group that received argon plasma after the application of the Adper Scotchbond Multi-Purpose (SMPPD) adhesive system, and the lowest median (22.66 ± 1.84) was found in the group that received the Single Bond Universal adhesive system without plasma (SBUSP). Table 3 compares the values obtained between the control groups (which did not receive argon plasma) and

Table 2. Mean, median, standard deviation, minimum/maximum value, and the number of samples for each group tested. Group SMPSP SMPPA SMPPD SBUSP SBUAP SBUPD Total

Mean 26.74 29.25 30.20 22.46 28.20 29.61 27.74

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Median 26.51 29.22 30.27 22.66 28.33 29.32 28.79

Standard deviation 0.88 0.44 0.54 1.84 0.78 0.91 2.80

Minimum 25.27 28.19 29.46 19.71 26.55 28.13 19.71

Maximum 28.30 30.07 31.00 26.51 29.22 31.14 31.14


Passos, I. A. G., Marques, J. N., Câmara, J. V. F., Simão R. A., Prado, M., & Pereira, G. D. S. the groups that received plasma. According to the MannWhitney test (p<0.05), the groups that received plasma had higher resistance values (29.30 MPa) than the control groups (25.54 MPa). Table 4 shows the results of the two adhesive systems without plasma application (control group). The Adper Scotchbond Multi-Purpose group showed bond strength values significantly higher than the Single Bond Universal group, whose medians were 26.51 MPa and 22.66 MPa, respectively. Different letters indicate significant differences between the groups (Mann-Whitney, p <0.05). n = 90. Table 5 shows the results of the influence of plasma application before or after the adhesive system. The results of the Kruskal-Wallis test (p<0.05) rejected the null hypothesis that the groups have equal strength values. In both adhesive systems, bond strength values were higher when applying plasma after the adhesive system. Table 6 shows the comparison of the three treatments tested for the Adper Scotchbond Multi-Purpose adhesive system. The results of the Kruskal-Wallis test (p<0.05) rejected the null hypothesis that the groups have equal strength values. The two-by-two comparison with the Dunn test showed higher values for Scotchbond with plasma after > plasma before > no plasma. Table 7 shows the comparison of the three treatments tested for the Single Bond Universal adhesive system. The results of the Kruskal-Wallis test (p<0.05) rejected the null hypothesis that the groups have different strength values. The two-by-two comparison with the Dunn test showed the highest values in the group that received plasma after applying the adhesive system (SBUPD), followed by groups SBUPA and SBUSP. Forming a dense, homogeneous, and uniform hybrid layer, regardless of thickness, is crucial for strong and lasting adhesion. Thus, the adhesive must completely infiltrate the demineralized dentin, otherwise, porosities or nanometric defects are formed within the hybridized area, facilitating the infiltration of oral fluids and bacterial enzymes that degrade the restoration over time and decrease bond strength and durability[14]. Several factors inherent to the physicochemical structure of the adhesive, the application techniques, and the intrinsic properties of adhesives can affect this diffusion. Considering the tendency to use simpler and faster systems that are less susceptible to operator errors and attempting to eliminate the inconveniences of the total acid etching technique caused by acid washing and substrate drying, self-etching systems were developed with increasing concentrations of acidic monomers in their composition[15]. The low pH values of these systems allow them to partially diffuse through the dentin slurry, reaching and superficially demineralizing the underlying intact dentin until dissolution products buffer its acidity[15]. Acidic monomers decalcify the adhesive and simultaneously open channels that facilitate the diffusion of resin monomers within the dentin, forming a thin hybridized complex. However, there are no discrepancies between the depths of monomer demineralization and infiltration, providing satisfactory initial bond strength values[16,17], as observed in this study for the Single Bond Universal system in the control group. 4/7

Considering the more uniform interdiffusion zone, it was expected that the adhesion values of the self-etching system evaluated would be the highest among the control groups. However, the results obtained for the previously acid-etched Scotchbond Multi-Purpose conventional threestep system were statistically higher than those of the Single Bond Universal system. This may have occurred because the SBMP system is less sensitive to substrate moisture variations due to the presence of water in its composition, which can rehydrate the collapsed collagen, recover its original Table 3. Values, ​​in MPa, of control groups and groups that received plasma. Group With plasma Without plasma Total

Median 29.30 A 25.54 B 28.79

Standard deviation 1.00 2.60 2.80

Table 4. Shear strength values, in MPa, of the two adhesive systems without plasma application. Group SBMPSP SBUSP Total

Median 26.51 A 22.66 B 25.54

Standard deviation 0.88 1.84 2.60

Different letters indicate significant differences between the groups (Mann-Whitney, p <0.05). n = 30.

Table 5. Shear strength values, in MPa, of the two adhesive systems with plasma application. Group SMPPA SMPPD SBUPA SBUPD Total

Median 29.22 B 30.27 A 28.33 C 29.32 B 29.30

Standard deviation 0.44 0.54 0.78 0.91 1.00

Different letters indicate significant differences between the groups (Mann-Whitney, p <0.05). n = 60.

Table 6. Comparison of shear strength values, in MPa, for the three treatments with the Adper Scotchbond Multi-Purpose adhesive system. Group ASMPSP ASMPPA ASMPPD Total

Median 26.51 C 29.22 B 30.27 A 29.22

Standard deviation 0.88 0.44 0.54 1.61

Different letters indicate significant differences between the groups (Dunn test, p <0.05). n = 45.

Table 7. Comparison of shear strength values, in MPa, for the three treatments with the Single Bond Universal adhesive system. Group SBUSP SBUPA SBUPD Total

Median 22.66 C 28.33 B 29.32 A 28.29

Standard deviation 1.84 0.78 0.91 3.36

Different letters indicate significant differences between the groups (Dunn test, p <0.05). n = 45.

Polímeros, 32(1), e2022012, 2022

Effect of non-thermal argon plasma on the shear strength of adhesive systems framework in dry dentin situations after acid washing, and present hydrophilic monomers with a high affinity for the substrate under higher moisture conditions[12]. Although this system does not have a simultaneous hybridization to dentin demineralization, it is less sensitive to errors caused by the application technique. Therefore, reliable adhesion values can be obtained in different substrate moisture conditions, among other characteristics, which qualifies this system as the gold standard for dental adhesion[12]. Additionally, the narrow thickness of the interdiffusion zone formed by the Single Bond Universal system results in a hybrid layer with a high elastic modulus, reducing its flexibility and ability to absorb the stresses generated when polymerizing the restorative composite, forming more cracks that propagate fractures in the joint area during the mechanical test. In turn, the SBMP system uses a more viscous adhesive composed of Bis-GMA and HEMA after the primer, which allows forming a more elastic layer with a higher potential for absorbing the forces generated during polymerization shrinkage[18]. It is also worth noting that the potentially acidic medium promoted by the self-etching primer makes it hard to convert resin monomers into polymers[11], and incorporating the smear layer in the hybrid complex of self-etching systems causes a weak bond adherence[17]. These findings reject the null hypothesis that the composition of adhesive systems does not influence dentin bond values, considering the values of the SBMP system were higher than those of the SBU system in the three variables studied. There have been several attempts to create a defectfree hybrid layer. Several authors have proposed different types of dentin pretreatments to increase the durability of adhesive restorations. Dentin deproteinization, collagen cross-linking agents, and laser irradiation are examples of poorly consolidated attempts to improve adherence[19,20]. Recent efforts have been focused on developing a technique to electrically or chemically modify the dentin surface. Methods have been used to increase the permeability and wettability of this substrate and facilitate the penetration and absorption of adhesive agents[21]. Thus, the non-thermal plasma application technique has attracted considerable interest and has been extensively used to modify the surfaces of biomaterials[22]. This study used non-thermal argon plasma because it is inexpensive compared to other noble gases, and the application temperature is close to the human body temperature, (lower than 40°C at the time of application), which allows using it in vivo. Non-thermal argon plasma also presented favorable adhesive strength results in the articles evaluated[6,10]. The time of 30 seconds was chosen due to its clinical application feasibility. Studies have suggested that non-thermal plasma can increase interfacial bond strength by increasing the surface contact area with the collagen fibers and their hydrophilicity, allowing a higher interaction with the adhesive and penetration into the substrate. This reduces defects and voids at the interface, forms longer resin extensions, and increases the conversion of resin monomers into polymers[23-25], corroborating the findings of this research, in which bond strength values were significantly higher for both adhesive systems when treating the dentin substrate with plasma. Polímeros, 32(1), e2022012, 2022

Therefore, the null hypothesis that the treatment with argon plasma does not interfere with the adhesive strength of the systems used was rejected. This behavior was verified for both adhesive systems when the plasma was applied before the adhesives to dentin. As dentin is a substrate rich in organic matter, the contact angle between this substrate and a liquid is higher than that of enamel, impairing the adhesive process[10,26]. The results obtained with non-thermal argon plasma indicate that this gas is can make the dental surface more hydrophilic, considerably reducing the dentin contact angle, and increasing wettability and consequent penetration of the adhesive system[10]. This occurs because plasma breaks down and removes the protein content of hydrocarbons from the dentin surface. This is associated with the stiffening effect on the hybrid layer, inhibiting the enzymatic activity of matrix metalloproteinase (MMP). Another possible explanation is that breaking interfibrillar bonds, such as hydrocarbon bonds, can induce structural changes in the exposed collagen fibers, preventing the collagen networks from collapsing, protecting them, and inhibiting MMP enzymes, thus improving bond durability. Therefore, reducing the amount of organic matter increases the amount of mineral content, mainly calcium and phosphate - the main components of hydroxyapatite. This reduces the surface contact angle, favoring the close contact of the adhesive and its penetration[27-29]. This behavior becomes more significant when using self-etching adhesive systems because of the absence of a pre-etching step, therefore, not removing the smear layer from this surface. The presence of the dentin slurry reduces the surface energy of the substrate, making it less receptive to adhesion. It also works as a physical barrier to monomer penetration, further impaired by the increased thickness of this debris layer, reducing primer acidity and hindering monomer diffusion in the underlying dentin[30]. Applying plasma directly to the dentin without pre-etching in the groups that received the SBU system may have changed the composition of the dentin slurry, increasing the surface energy of the substrate and favoring bonding[30], which was translated by the significant difference in the numerical results obtained for the adhesive. The strong chemical and physical bonds promoted by plasma increase mechanical strength values, as verified in this study, in which shear strength values were significantly higher for the groups with previous plasma applications. The null hypothesis that applying non-thermal argon plasma before the adhesive system does not influence the shear strength values of the adhesives evaluated was rejected. Despite the increase in bond strength values with the prior dentin treatment with argon plasma, these values were significantly lower than those obtained with the application of the gas after substrate hybridization in both adhesive systems evaluated. This possibly occurred due to using deep dentin as the substrate for adhesion, considering that morphological, structural, and compositional differences occur at different depths of the dentin substrate[22,31,32]. Some studies[33,34] report that deep dentin has a lower mineral content and amount of collagen fibers caused by the increase in density, width, and area occupied by dentinal tubules, which may have impaired plasma performance when applied before the adhesive. 5/7

Passos, I. A. G., Marques, J. N., Câmara, J. V. F., Simão R. A., Prado, M., & Pereira, G. D. S. The benefits of this treatment are closely related to the existing amount of tissue, especially collagen fibers[32-34]. However, the higher dentin depth did not influence the values obtained with plasma application after the hybrid layer formation, as they were the highest adhesion values for both systems evaluated. This occurred because in vitro adhesion is not affected by increased moisture in the deep substrate, which can cause adhesive system dilution, phase separation, and reduction of the degree of monomeric conversion[12] – the main action mechanism of plasma on the hybridized tooth surface[33]. The null hypothesis that applying non-thermal argon plasma gas after hybridization does not influence the bond strength values of the systems evaluated was rejected. The acidic monomers in these systems react with the initiating amines, reducing their concentration and polymerization reaction, which can negatively affect adhesion[10,11]. However, applying plasma after the adhesive may have minimized this problem due to the additional polymerization, as seen in the values of the Single Bond Universal system in this experimental condition. Also, self-etching systems consist of a certain amount of water, as in the single-bottle system evaluated in this study, which contains 20%. Water is essential for ionizing acid monomers, but it can be an interference factor, reducing the photopolymerization of adhesives by diluting its components. However, the water contained in acidic adhesives does not have a deleterious effect when using plasma, considering that an appropriate amount of water can facilitate the injection of free radicals into the plasma and increase the propagation of the monomeric chain, thus increasing conversion[30-32].



8. 9.




4. Conclusions Applying non-thermal argon plasma for 30 seconds improved the bond strength of deep dentin. Non-thermal argon plasma applied to deep dentin before the adhesives significantly increased the bond strength of both adhesive systems, which ensured higher wettability and adhesion.

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