Penn Dental Medicine Orofacial Stem Cell Research Summit

PENN DENTAL MEDICINE

OROFACIAL STEM CELL RESEARCH

SUMMIT

宾夕法尼亚大学牙医学院 口腔干细胞研究高峰论坛

September 12, 2015 Penn Wharton China Center Beijing, China

SUMMIT

OROFACIAL STEM CELL RESEARCH

Welcome It is with great pleasure to welcome you to the Penn Dental Medicine Orofacial Stem Cell Research Summit. This summit marks the first collaborative research symposium between Penn Dental Medicine and our MOU partner institutions and highlights Penn Dental Medicine’s global engagement in China. The program features some of the most eminent researchers and leaders from around the world in the areas of basic science and clinical delivery. In addition, junior faculty, postdoctoral fellows, graduate students and research investigators gather for a day of exchange and collaboration with colleagues across disciplines. We are especially pleased to celebrate the contribution of these individuals as they represent our future academic leaders in the world of oral health.

We warmly welcome our speakers and academic partners: Songlin Wang, DDS, PhD from Capital Medical University; Zhi Chen, DDS, PhD, Wuhan University, School of Stomatology; Yongsheng Zhou, DDS, PhD, Peking University School of Stomatology; Yan Jin, DDS, PhD, Fourth Military Medical University, School of Stomatology; Xingquan Jiang, Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University; Yi Liu, Capital Medical University School of Stomatology and Yanheng Zhou, Peking University School of Stomatology. We are excited they are joining us.

We hope you will find the Summit a valuable experience as we introduce new initiatives to advance ongoing research, showcase the breadth of Penn Dental Medicine’s continuing strength as a leader in the generation of new knowledge and treatment modalities to prevent oral diseases and promote oral health and cultivate the next generation of academic leaders.

Enjoy the day.

Denis F. Kinane, BDS, PhD Morton Amsterdam Dean

Syngcuk Kim, DDS, PhD, MD (Hon) Louis I. Grossman Professor Department of Endodontics Associate Dean for Global Affairs & Continuing Education

Songtao Shi, DDS, PhD Chair and Professor Department of Anatomy and Cell Biology

SUMMIT

OROFACIAL STEM CELL RESEARCH

尊敬的各位来宾 很荣幸您莅临参加此次宾夕法尼亚大学牙医学院口腔干细胞研究高峰论 坛。这是宾大牙医学院与MOU合作院校间第一次合作举办科研论坛，也彰 显了宾大牙医学院全球协作战略在中国的开展。这次活动特别邀请了来自 各地的基础研究及临床领域的杰出人才和领军者。今天，许多青年学者、 博士后、研究生等在此汇聚一堂，为开展跨学科、院校的交流合作提供契 机。特别感谢所有的与会者对学科的贡献，你们将是未来世界口腔医学领 域的领军者。 热忱欢迎我们的演讲嘉宾和学界同仁：王松灵，DDS，PhD，首都医科大 学；陈智，DDS，PhD，武汉大学口腔医学院；田卫东，DDS，DMD，四 川大学华西口腔医学院；周永胜，DDS，PhD，北京大学口腔医学院；金 岩，DDS，PhD，第四军医大学口腔医学院；蒋欣泉，DDS，PhD，上海交通 大学医学院附属第九人民医院；刘怡，DDS，PhD，首都医科大学；周彦 恒，DDS，PhD，北京大学口腔医学院。很高兴你们应邀而来。 本次论坛我们将阐述新的概念以提升正在开展的研究，并展示宾大牙医学 院作为领军者在多个领域经久不衰的实力，包括创造新知识，发明新疗法 以防治口腔疾病、促进口腔健康，培养新一代学科带头人。期望大家能从 此次论坛获得宝贵的经验。

祝大家愉快！

Denis F. Kinane, BDS, PhD Morton Amsterdam Dean

Syngcuk Kim, DDS, PhD, MD (Hon) Louis I. Grossman Professor Department of Endodontics Associate Dean for Global Affairs & Continuing Education

Songtao Shi, DDS, PhD Chair and Professor Department of Anatomy and Cell Biology

SUMMIT

OROFACIAL STEM CELL RESEARCH

PROGRAM 日程

AM SESSION 8:00 am

Registration and Breakfast 注册、早餐

8:20 am

Welcome Remarks 开幕致辞

8:30 am Mechanisms through which diabetes-enhanced inflammation interferes with bone formation.

DANA GRAVES, DDS, DMSc, Penn Dental Medicine DANA GRAVES, DDS, DMSc, 宾夕法尼亚大学牙医学院 9:00 am Bio-root Based Functional Tooth Regeneration SONGLIN WANG, DDS, PhD, Capital Medical University 王松灵，DDS, PhD, 首都医科大学 9:30 am Molecular regulation of odontoblast differentiation — Transcription factor Klf4 and related epigenetic network ZHI CHEN, DDS, PhD, Wuhan University, School of Stomatology 陈智，DDS, PhD, 武汉大学口腔医学院 10:00 am The epigenetic regulation of osteogenic differentiation of mesenchymal stem cells and its potential usage in bone regeneration or prevention for systemic bone loss YONGSHENG ZHOU, DDS, PhD, Peking University School of Stomatology 周永胜，DDS, PhD, 北京大学口腔医学院 10:30 am - 1:00 pm

4

Poster Session/Lunch 壁报展示／午餐

PENN DENTAL MEDICINE

PM SESSION 1:00 pm Mesenchymal Stem Cell Based Periodontal Regeneration YAN JIN, DDS, PhD, Fourth Military Medical University, School of Stomatology 金岩，DDS, PhD, 第四军医大学口腔医学院 1:30 pm The Use of Stem Cells and Biomaterials for Maxillofacial Bone Regeneration XINGQUAN JIANG, DDS, PhD, Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University 蒋欣泉,DDS, PhD, 上海交通大学第九人民医院 2: 00 pm Hydrogen Sulfide Maintains Mesenchymal Stem Cell Function and Bone Homeostasis via Regulation of Ca2+ Channel Sulfhydration YI LIU, DDS, PhD, Capital Medical University, School of Stomatology 刘怡，DDS, PhD, 首都医科大学口腔医学院 2:30 pm Immunoregulation and activation of PDLSCs under mechanical force stimulation during orthodontic tooth movement YANHENG ZHOU, DDS, PhD, Peking University School of Stomatology 周彦恒，DDS, PhD, 北京大学口腔医学院 3:00 pm Orofacial Mesenchymal Stem Cells SONGTAO SHI, DDS, PhD, Penn Dental Medicine 施松涛，DDS, MS, PhD, 宾夕法尼亚大学牙医学院 3:30 pm

Poster Session/Open 壁报展示

5:30 pm

Depart for Dinner 前往晚宴地点

6:00 pm Dinner and PDM Awards

Dinner location: Hua’s Restaurant at Jingguang Bridge 3/F, Minsheng Baoxian Mansion, No.38 East 3rd Ring North Road, Chaoyang District, Beijing 100020, China

晚宴及颁奖 晚宴地点：花家怡园(京广桥店) 100020朝阳区东三环北路38号院民生保险大厦3楼

OROFACIAL STEM CELL RESEARCH SUMMIT

5

NOTES

6

PENN DENTAL MEDICINE

SPEAKERS

OROFACIAL STEM CELL RESEARCH SUMMIT

7

SPEAKERS

DANA GRAVES, DDS, DMSC Penn Dental Medicine

MECHANISMS THROUGH WHICH DIABETES-ENHANCED INFLAMMATION INTERFERES WITH BONE FORMATION ABSTRACT: Diabetes increases the amount of inflammation in areas of bone formation. In diabetics TNFα (tumor necrosis factor alpha) is increased, which creates a cascade of detrimental downstream effects including activation of several inflammatory, degradative and apoptotic pathways. Increased levels of TNF reduce the number of osteoblasts and mesenchymal stem cells (MSC). MSC numbers are restored to normal levels when TNF is specifically inhibited. The impact of TNF on MSC in diabetic animals is shown by increased MSC proliferation and reduced MSC apoptosis with TNF inhibition. In contrast, inhibition of TNF has no effect on MSCs in normoglycemic conditions. The effect of TNF on MSC is mediated in part by the transcription factor forkhead box-O1 (FOXO1), which regulates expression of cell cycle and apoptotic genes. In addition, TNF stimulates nuclear factor kappa beta (NF-kB), which inhibits the capacity of Wnt and bone morphogenetic protein-2 (BMP-2) to stimulate bone matrix protein expression. This occurs because TNF-activated NF-κB blocks β-catenin and Runx2, transcription factors stimulated by Wnt and BMP that are needed for production of bone. Mutating NF-κB binding sites on osteocalcin (OC) or bone sialoprotein (Bsp) rescues the negative impact of TNF and NF-κB on OC and Bsp expression. This inhibition depends on p65-p50 NF-κB heterodimer formation and deacetylation by HDAC1. The inhibition of RUNX2 and β-catenin is a direct mechanism by NFκB. It does not depend upon indirect mechanisms such as the production of inhibitors (e.g. DKK-1) and provides an explanation for the rapid decrease in new bone formation induced by inflammation. Thus, TNF stimulates FOXO1, which interferes with progression through the cell cycle and promotes apoptosis. TNF also stimulates NF-κB activation that inhibits the binding of β-catenin and Runx2 to nearby consensus response elements. This effects bone formation by inhibiting Wnt and BMP induced transcription of matrix proteins. The importance of the effect of inflammation on periodontal disease was shown in vivo by the capacity of TNF inhibition to overcome the detrimental effects of periodontal disease on osteoblasts in diabetic animals. Thus, these studies reveal two new mechanisms through which diabetes-enhanced inflammation limits bone formation through its effect on MSC and osteoblasts.

ABOUT: Dana Graves completed his DDS training at Columbia University in 1980 and a Certificate in Periodontology and DMSc in Oral Biology from Harvard University in 1984. He was Assistant Professor at the University of Texas Health Science Center from 1985-87 and at Boston University Goldman School of Dental Medicine from 1987-90. He was promoted to Associate Professor 1990-93 and to Professor in 1993. From 2008-2010, Dr. Graves was Chair and Professor of Periodontics at University of Medicine and Dentistry of New Jersey. He arrived at Penn Dental Medicine in 2010 and is currently the Vice Dean for Scholarship and Research, Interim Chair and Professor of Periodontics and Director of the DScD program. He is the Associate Editor for the Journal of Dental Research and sees patients at the Penn Dental Family Practice. Dr. Graves’ research focuses on inflammation, bone, periodontal disease, wound healing, transcription factors and diabetes.

8

PENN DENTAL MEDICINE

Capital Medical University

BIO-ROOT BASED FUNCTIONAL TOOTH REGENERATION ABSTRACT: Tooth loss is devastating and adversely affects most adults in their lives. Currently false tooth is used for tooth loss. Biological restoration using tooth regeneration for tooth loss is a dream of human. As we know, there are two major ways to regenerate tooth. One is based on tissue engineering method, the other is based on the way of development. Tissue engineering methods need scaffold, for example, dental stem cell and HA/TCP based bio-root regeneration and periodontal tissue regeneration. So far, it is still very difficult to regenerate whole tooth using tissue engineering methods. Whole tooth regeneration has been achieved using tooth germ cell reassociation in mice model by development of dental epithelium and mesenchymal cells. The strategy for bio-root regeneration is to use dental pulp stem cells in the root-shaped scaffold to regenerate dentin and to use periodontal ligament stem cells coated with root-shaped scaffold to form periodontal tissues, then repair with artificial crown in a large animal model, miniature pig. Functional bioroot-based tooth regeneration was achieved using either autologous or allogeneic dental stem cells in swine, which may provide a feasible approach for restoring biological root with functional crown for the patients with tooth loss.

Vitamin C treatment promotes mesenchymal stem cell sheet formation and tissue regeneration by elevating telomerase activity, which provides an easy and practical approach for cell-based tissue regeneration. Comparison study between bio-root based tooth and dental implant based tooth showed both could restore functional tooth.

Biomechanical analysis showed no significant difference in compressive strength, modulus of elasticity and twisting force testing between bio-root and normal tooth root, however, 22 fold higher in compressive strength, 5 fold higher in modulus of elasticity, 3 fold higher in twisting force in the dental implant group than those in the bio-root group. Furthermore, element examination revealed that composition of bio-root was similar to natural tooth roots, while dental implants were close to pure titanium. Low successful rate needs further efforts and improvements in bio-root based tooth.

SPEAKERS

SONGLIN WANG, DDS, PHD

ABOUT: Songlin Wang, Professor and Scientific Director, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction; Vice President, Capital Medical University. Dr. Wang got DDS, PhD from Peking University, then works in Capital Medical University since 1989. Dr. Wang visited in Tokyo Medical and Dental University in 1991-1992 and worked in NIDCR, NIH as visiting scientist during 1996-1998. Dr. Wang’s research experience includes clinical, biological and gene therapy studies in salivary glands; tooth development and regeneration. Dr. Wang was honored by AADR/IADR 2007 William J. Gies Award for Biological Research and 2007 Cover of the Year Award. He received two National Science and Technology Advancement Awards of China (second class) as first principle investigator in 2003 and 2010. Now he is President of Chinese Dental Education Association, President of Chinese Society of Oral Biomedicine, Chinese Stomatological Association; editorial broad member of America Journal of Stem Cells, Oral Diseases, and Triple O; Editor-in-Chief for Oral Biomedicine; Associate Editor-in-Chief for Chinese Journal of Dental Research (in English), Chinese Journal of Stomatology, etc. He has authored more than 198 peer reviewed articles (101 in English) including PNAS, Blood, Stem Cells, JDR and 16 review articles, edited 10 books and 16 book chapters.

OROFACIAL STEM CELL RESEARCH SUMMIT

9

SPEAKERS

ZHI CHEN, DDS, PHD

Wuhan University, School of Stomatology MOLECULAR REGULATION OF ODONTOBLAST DIFFERENTIATION - TRANSCRIPTION FACTOR KLF4 AND RELATED EPIGENETIC NETWORK ABSTRACT: Odontoblasts are kind of terminally differentiated cells deriving from dental papilla and play a very important role in dentinogenesis. Dental derived stem cells, which are coming from neural crest, have ability to differentiate into odontoblast, but the mechanism within remained unclear. Previous studies have identified various transcription factors involved in this process, among which Krüppel-like factor 4 (Klf4) was found to have expression in pre-secretory and secretory process of odontoblast differentiation. Despite Klf4 has been known as one of Yamanaka factors, the exact function of Klf4 in odontoblast differentiation still needs to be elucidated.

To investigate the function of Klf4, we obtained transgenic mouse containing inactivated Klf4 in their neural crest cells (Wnt1-Cre; Klf4f/f), and found that the conditional knockout mice had downregulation on Dmp1 and Dsp in odontoblasts of PN0.5 molar germ, thicken predentin layer, sparse alveolar bone, dentin hypoplasia. Further we applied gain- and loss-of-function study in vitro and found that KLF4 could bind to the promoter region of Dmp1 thus promoting odontoblast differentiation.

For epigenetic regulation is an emerging field in developmental biology and cellular biology, we were wondering whether epigenetic factors participate in odontoblast differentiation. By using RNA pull down approach we screened out 11 microRNAs binding to the 3’UTR of Klf4, among which miR-145 and miR-143 were found to build a regulation loop with Klf4 in odontoblast differentiation. Besides of the microRNA level, DNA methylation was also taken into account. We identified 3 CpG islands within the 5’upstream of Klf4, but only one had change of DNA methylation status during odontoblast differentiation, and SP1 could just bind to this only CpG island and promote odontoblast differentiation.

Combine these two parts, we concluded that Klf4 is essential in odontoblasts differentiation and dentinogenesis, and an epigenetic regulatory network including microRNAs and DNA methylation could fine regulate Klf4 thus affecting odontoblast differentiation.

ABOUT: Dr. Zhi Chen got his D.D.S. and Ph.D. from Wuhan University; then he has been working in School & Hospital of Stomatology since 1990. He studied in University Paris Descartes from 1994 to 1995, University of Strasbourg in 2001, University of Missouri Kansas City in 2004 as a visiting scholar. Prof. Chen’s research focus on dental pulp biology, including odontoblast differentiation, dentinogenesis, and pulp regeneration. He is the member of Chinese Stomatological Association; executive member of Chinese Society of Cariology and Endodontics. He is an Associate Editor of Oral Diseases, Chinese Journal of Oral Science Research. He has authored more than 30 peer reviewed articles including JBC, JDR and one patent in dental pulp stem cells.

10

PENN DENTAL MEDICINE

Peking University School of Stomatology

THE EPIGENETIC REGULATION OF OSTEOGENIC DIFFERENTIATION OF MESENCHYMAL STEM CELLS AND ITS POTENTIAL USAGE IN BONE REGENERATION OR PREVENTION FOR SYSTEMIC BONE LOSS

SPEAKERS

YONGSHENG ZHOU, DDS, PHD

ABSTRACT: Human MSCs are a highly attractive source in bone tissue engineering.

It has become increasingly clear that chromatin regulators play an important role in cell fate determination. However, how osteogenic differentiation of hMSCs is controlled by epigenetic mechanisms is not fully understood. Here we report the identification and characterization of Lysine-specific demethylase 1 (LSD1/KDM1A), RBP2, GCN5, SIRT6 and other enzymes which govern the histone modulation, as key regulators in osteogenic differentiation of hMSCs under normal or osteoporotic status. In this lecture, we will demonstrate how these factors epigenetically promote osteogenic differentiation of hMSCs under normal or osteoporotic status. Altogether, our studies defined the functional and biological roles of LSD1, RBP2, GCN5, SIRT6, and extensively explored the effects of its enzymatic activity in osteogenic differentiation of hMSCs. A better understanding of how these factors contributes to osteogenic program associated epigenetic events will provide new insights into the modulation of hMSCs based cell therapy and improve the development of bone tissue engineering.

ABOUT: Dr. Yongsheng Zhou is the Associate Dean of Peking University School of Stomatology (PKUSS), and the professor and chairmanof Department of Prosthodontics. He achieved his DDS degree in PKUSS in 1994 and PhD in the same school in 1998. He accepted postdoctoral training in University of North Carolina Dental Research Center, Department of Prosthodontics for one year. Dr. Zhou is currently a Diplomate of the Chinese Prosthodontic Society. He is the vice-president of Chinese Society for Oral Maxillofacial Rehabilitation, vice-president electof Chinese Prosthodontic Society, a fellow of International College of Dentists, astanding member of Education and Research Committee of International College of Prosthodontists, Council member of Asian Academy of Prosthodontics,vice president of Beijing Prosthodontic Society, and commissioners of Beijing Municipal Stomatological Association, and National Board of Dental Examiners, etc. He is an editor for 6 academic journals in Stomatology. His Researches focus on the usage of bone tissue engineering based on adult stem cells to restore oral bone loss, material surface modification for improving osteogenesis, and digital technology, etc.

OROFACIAL STEM CELL RESEARCH SUMMIT

11

SPEAKERS

YAN JIN, DDS, PHD

Fourth Military Medical University, School of Stomatology MESENCHYMAL STEM CELL BASED PERIODONTAL REGENERATION ABSTRACT: Mesenchymal stem cell (MSC)-mediated tissue regeneration is a promising approach for periodontum regeneration. We postulate that: (1) there is a population of stem cells (a-PDLSC) residing in PDL embedded on the alveolar bone surface, (2) bone marrow (BM) derived MSC are very likely another cell source of physiological repair of periodontal tissues. We investigated if microRNAs and WNT pathway mediate inflammatory cytokines-induced PDLSCs inhibition of osteogenic differentiation potential. We further proposed a possible strategy based on self-assembly approach to enhance reconstruction of periodontium.

Our data suggest that PDLSC may have different characteristics according to different locations. BM-derived cells (BMDC) preferentially migrate into the periodontium and it communicates with periodontal tissues and become tissue-specific mesenchymal progenitor cells to maintain tissue homeostasis. Our work demonstrate that the bone marrow served as source for dental and periodontal mesenchymal tissue and contribute to the maintenance and regeneration, and the resident and migrating cells collectively play crucial roles in. Our data show that it was possible that agents modifying Wnt signaling and miR-17 could be of value to bone regeneration in chronic inflammatory microenvironments by MSCs. Under simulated inflammatory conditions, TNF-α inhibited osteogenic differentiation of PDLSCs more than BMMSCs. Blockage of the canonical Wnt pathway by DKK-1 reconstituted osteogenic differentiation of PDLSCs under inflammtory conditions, whereas activation by Wnt3a increased osteogenic differentiation of BMMSCs. Taken together, our results suggest a diverse regulation of the inhibitory effect of TNF-α in BMMSCs and PDLSCs via modulation of the canonical Wnt pathway.

Our findings show that periodontal tissue regeneration can be achieved with MSC cell aggregate (CA) -engineering approach. We further proposed a possible strategy based on self-assembly approach, which is akin to the physiological phenomenon that occurs during organogenesis, to enhance complete reconstruction of functional complex periodontium-organ systems. The key of complete periodontal regeneration is dependent, to a great extent, on the ability of cells to reconstitute the mineralized tissues of cementum and bone. Our findings show that integrative periodontal tissue regeneration can be achieved with two kinds of stem cell CA-mediated tissue engineering approach.

ABOUT: Graduated from the Fourth Military Medical University in 1985 and awarded the Ph.D. degree in 1991.

He is the professor and director of Tissue Engineering Center of the Fourth Military Medical University (FMMU).

He was offered the position of “Chang Jiang scholars program” Professorship and was awarded National Distinguished Young Scientists Grant of National Natural Science Foundation of China. He is the Chief Scientist of “973 project”. He has published 120 papers in the journals such as Cell Metabolism, Cell Death Diff, Stem Cells, Biomaterials, JBMR, J Control Release, Scientific Report, Mol Therapy and so on.

12

PENN DENTAL MEDICINE

Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University THE USE OF STEM CELLS AND BIOMATERIALS FOR MAXILLOFACIAL BONE REGENERATION ABSTRACT: Dealing with the maxillary and mandibular bone defect is still a large challenging problem for oral and maxillary surgeons and prosthodontists. More importantly, the regeneration of bony defect is the fundament for further implants placement and functional restoration, which are the final prosthetic destination in modern dentistry. Several approaches including autogenous bone grafts, allograft, xenograft and synthetic materials have been explored for bony recovery. However, those means involve in many complications separately.

With the enhancement of living standard and the improvement of medical condition, patients and dentists pay more attention to looking for a safer and more efficient alternative. Tissue engineering is the relatively new and highly promising fields of reconstructive biology. It generally combines three key elements: bioactive scaffolds, stem cells and molecular factors. By combination of those elements, tissue regeneration can often be accomplished. In past ten years, we did many researches on different animal models, such as mandible defect models on rat and canine, vertical alveolar ridge augmentation models on canine, and sinus augmentation models on rabbits, canine and sheep. In addition to investing the regeneration of maxilla and mandible bone defect, we tried to restore the functional deficit in conjunction with oral implant placement. All those works lay a solid foundation for the translational medicine from bench to bed. New attempts are putting on the design of new biomaterials with modifications on structures and compositions to direct the adhesion, proliferation, and differentiation of stem cells. Through repeated attempts, we are trying to shorten the treatment period, to enhance the repair quality and to improve the accuracy of restoration.

SPEAKERS

XINGQUAN JIANG, DDS, PHD

ABOUT: Xinquan Jiang, D.D.S, Ph.D. Professor. He serves now as the director of the department of Prosthodontics, and the director of Oral Bioengineering/Regenerative Medicine Lab, Ninth Peoples Hospital, School of Stomatology, affiliated to Shanghai JiaoTong University. Xinquan Jiang received his Ph.D in 2003 from Shanghai Second Medical University. He got his further training in University of Alberta during 2002~2003 in Canada and University of California, Los Angeles (UCLA) in USA during 2004~2006, and awarded an honorary professorship in Sydney University, Australia (2012~). His research interest focuses on the bone regeneration and dental implantation, and serves in the editorial board of Tissue Eng, Bone Res, IJOS, et al.. In 2005, he won the IADR/Unilever Hatton Award from the international association for dental research. He won the NSFC distinguished young grant, and “Changjiang” Scholar in Chia. Currently, he serves as the regional representative of Asian Academy of Prosthodontics (AAP) and vice president of Chinese Prosthodontic Society (CPS).

OROFACIAL STEM CELL RESEARCH SUMMIT

13

SPEAKERS

YI LIU, DDS, PHD

Capital Medical University, School of Stomatology HYDROGEN SULFIDE MAINTAINS MESENCHYMAL STEM CELL FUNCTION AND BONE HOMEOSTASIS VIA REGULATION OF CA2+ CHANNEL SULFHYDRATION ABSTRACT: Gaseous signaling molecules such as hydrogen sulfide (H2S) are produced endogenously and mediate effects through diverse mechanisms. H2S is one such gasotransmitters that regulates multiple signaling pathways in mammalian cells, and abnormal H2S metabolism has been linked to defects in bone homeostasis. Here, we demonstrate that bone marrow mesenchymal stem cells (BMMSCs) produce H2S in order to regulate their self-renewal and osteogenic differentiation, and H2S deficiency results in defects in BMMSC differentiation. H2S deficiency causes aberrant intracellular Ca2+ influx because of reduced sulfhydration of cysteine residues on multiple Ca2+ TRP channels.

This decreased Ca2+ flux downregulates PKC/Erk-mediated Wnt/b-catenin signaling which controls osteogenic differentiation of BMMSCs. Consistently, H2S-deficient mice display an osteoporotic phenotype that can be rescued by small molecules that release H2S. These results demonstrate that H2S regulates BMMSCs and that restoring H2S levels via nontoxic donors may provide treatments for diseases such as osteoporosis that can arise from H2S deficiencies.

ABOUT: Dr. Liu Yi is a professor and department chair of the Department of Periodontics in Capital Medical University School of Stomatology, China. She is the Standing Committee Member of Chinese Stomatological Association of Periodontal Disease and the Committee Member of the Chinese Stomatological Association of Biomedical. Over the years, Dr. Liu is most noted for her research on mesenchymal stem cell-mediated oral and maxillofacial tissue regeneration and transformation medicine. More recently, she focuse on the mechanism of host immune system controlling tissue regeneration, and investigate the effective immune regulation methods to improve tissue regeneration. Dr. Liu has published more than 30 scientific articles in a variety of scientific journals. Her work has earned her multiple awards including State Science and Technology Advancement Award (second class China, Beijing Science and Technology Advancement Award (First Class China). Dr. Liu earned a DDS degree from West China College of Stomatology, Sichuan University as well as PhD from Capital Medical University School of Stomatology.

14

PENN DENTAL MEDICINE

Peking University School of Stomatology

IMMUNOREGULATION AND ACTIVATION OF PDLSCS UNDER MECHANICAL FORCE STIMULATION DURING ORTHODONTIC TOOTH MOVEMENT ABSTRACT: Orthodontic tooth movement (OTM) induced by mechanical force is governed by local periodontal aseptic inflammation and alveolar bone remodeling.

However, the detailed mechanism of OTM remains unknown. Periodontal ligament (PDL) provides a unique microenvironment during this process. Whether mechanical force could activate periodontal ligament stem cells (PDLSCs), and play a role in modulating the alveolar bone remodeling during OTM needs to be further explored.

SPEAKERS

YANHENG ZHOU, DDS, PHD

Our studies revealed that mechanical force could influence immuno- microenvironment of PDL, which further modulate alveolar bone remodeling during OTM. The number of M1 macrophages increased in PDL after force application. Macrophage depletion decreased OTM distance and the number of osteoclasts, whereas systemic transfusion of M1 macrophages increased them. In addition, OTM distance and the number of osteoclasts in T cell deficiency mice was decreased, whereas systemic infusion of T cells rescued them. Further in vitro experiments showed that mechanical force upregulated the expression of IFN-γ in PDLSC, which may activate M1 macrophges. Moreover, mechanical force activated PDLSCs could promote M1 macrophage polarization. The interaction between PDLSCs and immune cells under force stimulation is under exploration.

We also found that mechanical force-induced Adrb2 expression in PDLSCs contributes to SNS-regulated OTM. Adrb1/2–/– mice showed significantly reduced OTM distance. Moreover, We found that the number of β2adrenergic receptor (Adrb2) positive cells in PDL increased after force application. Further in vitro experiments showed that mechanical force upregulated β2-adrenergic receptor (Adrb2) expression in PDLSCs through increasing the intracellular Ca2+ concentration.

In addition, we found in rat OTM model that collagen fibers in PDL underwent active remodeling, which could regained the pattern similar to the original after stopping the force application. In vitro experiments showed that collagen expression of PDLSCs was suppressed after mechanical force stimulation and could recovered after force withdrawal, indicating that PDLSCs may have a unique memory to modulate PDL extracellular matrix formation under mechanical force stimulation.

ABOUT: Yanheng Zhou, DDS, PhD, Adv Dip Orth. HKU, professor of the orthodontic department, and Director of Center for Craniofacial Stem Cell Research & Regeneration, Peking University School and Hospital of Stomatology, serves as an adjunct professor of Case Western Reserve University School of Dental Medicine.

He is the President of Chinese Orthodontic Society and the vice president of China Association for Medical and Cosmetic Oral Beauty Branch. He is an elected Executive Committee Member of World Federation of Orthodontists (WFO), a Member of International Association for Dental Research (IADR). His main interests are adult orthodontics, combined surgical and orthodontic approach, orthodontic treatment in periodontic patients, microscrew anchorage and the customized lingual orthodontics, as well as invisible orthodontics. His major research areas are mechanism and immunoregulation of orthodontic tooth movement, application of stem cell and bioinspired material in oral tissue defects repair.

OROFACIAL STEM CELL RESEARCH SUMMIT

15

SPEAKERS

SONGTAO SHI, DDS, PHD Penn Dental Medicine

OROFACIAL MESENCHYMAL STEM CELLS ABSTRACT: Mesenchymal stem cells (MSCs) are a population of hierarchical postnatal stem cells with the potential to differentiate into mesodermal lineagederived cells including osteoblasts, chondrocytes, adipocytes, cardiomyocytes, myoblasts and non mesodermal lineage-derived cells such as neural cells. MSCs are a promising cell source for regenerative medicine in terms of forming mineralized tissues to replace damaged and diseased tissues.

The orofacial region contains multiple lineage mesenchymal stem cells, including bone marrow MSCs, dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), Stem cells from Human Exfoliated Deciduous teeth (SHED), Stem Cells from root Apical Papilla (SCAP), and Gingival Stem/Progenitor Cells (GMSC). We identified PDLSCs can be successfully used to treat periodontitis with regeneration of cementum and Sharpey’s fibers in swine and humans. More interestingly, we used orofacial MSCs to regenerate root/periodontal complexes that are capable of supporting artificial porcelain crowns resulting in normal tooth function restoration in swine. Moreover, we generated jaw osteonecrosis model in mouse and employed BMMSC implantation to cure the necrosis, suggesting a novel therapeutic approach to recover jaw necrosis by BMMSC-generated new bone and bone marrow.

In addition to their ability to differentiate into various kinds of cell types, MSCs possess immunomodulatory properties capable of interplaying with several subsets of immune cells, including T cells, B cells, dendritic cells, and nature killer cells, and reducing inflammatory cytokine production. These immunosuppresion functions make MSCs of great interest for clinical applications in treating a variety of human diseases such as SLE and osteoporosis by reconstructing the osteoblastic niche and restoring immune homeostasis.

ABOUT: Songtao Shi, D.D.S., Ph.D., is Professor and Department Chair at the University of Pennsylvania School of Dental Medicine. Dr. Shi received his D.D.S. degree and certificate in Pediatric Dentistry from the Peking University School of Stomatology and Ph.D. in Craniofacial Biology from the University of Southern California.

Prior to joining the faculty at the University of Pennsylvania, he served as a Principal Investigator and Clinical Fellow for nine years at the National Institute of Dental and Craniofacial Research and a professor for more than eight years at the University of Southern California.

His research program focuses on understanding mechanism of mesenchymal stem cell (MSC)-associated diseases, developing new experimental disease models, and exploring feasibility of translating these bench discoveries to clinical therapies. His group and his collaborators were the first to identify dental pulp stem cells, baby tooth stem cells, periodontal ligament stem cells, root apical papilla stem cells, tendon stem cells, gingiva stem cells, sclera MSCs, and benign tumor MSCs from keloid. These novel and landmark discoveries have opened opportunity for scientists to investigate oral tissues derived stem cells and their use for tissue engineering, disease modeling, and clinical treatment.

In translational study, Dr. Shi’s team has used these stem cells to regenerate a variety of tissues, including dentin, pulp, periodontal ligament, tendon, bone, bio-root in preclinical animal models. Dr. Shi and his collaborators were the first to use MSCs to treat systemic lupus erythematosus (SLE), and regenerate periodontal and pulp tissues in patients. Additionally, Dr. Shi and his collaborators were the first to generate bisphosphonate-related osteonecrosis of the jaw-like disease (BRONJ) and keloid disease models in mice and swine.

Dr. Shi, as corresponding author, has published more than 160 peer-reviewed articles in a variety of highimpact scientific journals, of which he served as the corresponding author in Nat Medicine, Cell Stem Cell, Immunity, Lancet, J Clin Invest, Nat Biotechnol, Journal of Experimental Medicine, Proc Natl Acad Sci U S A, Cell Research, Blood, J Bone Miner Res, Stem Cells, and J Dent Res.

16

PENN DENTAL MEDICINE

NOTES

OROFACIAL STEM CELL RESEARCH SUMMIT

17

NOTES

18

PENN DENTAL MEDICINE

ABSTRACTS

OROFACIAL STEM CELL RESEARCH SUMMIT

19

ABSTRACTS

P-1

BREDIGITE CAN IMPROVE THE PLURIPOTENCY OF HUMAN DENTAL PULP CELLS Chen, Lihong1; Hong, Hong1; Wu, Chengtie2; Wei, Xi1 Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Affiliated Stomatological Hospital, Guangdong Province Key Laboratory of Stomatology, Sun Yat-Sen University 2 Biomaterials and Tissue Engineering Research Center, Shanghai Institute of Ceramics, Chinese Academy of Sciences 1

OBJECTIVES: To investigate the effects of silicate bioceramics (bredigite, Ca7MgSi4O16) and traditional bioceramics β-tricalcium phosphate (β-TCP, Ca3(PO4)2) on hDPCs proliferation, senescence, expression of pluripotency-related factors and the property of differentiation induction in vitro. METHODS: Different concentration of bredigite and β-TCP were used to induce hDPCs, growth factor bFGF was taken as a positive control and normal hDPCs served as a blank control. The proliferation activity was detected by CCK8 and flow cytometry cell cycle analysis. The senescence activity was investigated

by SA-β-gal staining. Flow cytometry was used to detect the expression level of Stro-1 of hDPCs at passage 2 and 7. The mRNA levels of Oct4, Sox2 were detected by qPCR. After dentinogenesis induction and adipogenesis induction for 21 days respectively, the expressions of DSPP, DMP-1, PPARγ-2 and LPL were investigated by Western Blot. Alizarin red staining was performed to determine the formation of mineralized nodules, oil red O staining was applied to detect the formation of lipid droplets. RESULTS: CCK8 indicated enhanced proliferation activity in bredigite-induced group and bFGF-induced group after 5-day stimulation (p<0.05). Flow cytometry cell cycle analysis showed that the PI values of bredigite-induced group and bFGF-induced group were up-regulated (p<0.05), whereas β-TCP-induced group showed no significant difference from control (p>0.05). Both control group and β-TCP-induced group were strongly positive for SA-β-gal activity. Bredigiteinduced group and bFGF-induced group showed weaker staining as compared to control group. Flow cytometry revealed that the percentages of Stro1+ cells at passage 2 and 7 were significantly higher in bredigite-induced group and bFGF-induced group than in control group (p<0.05). β-TCP-induced group showed no significant change compared with control group (p>0.05). qPCR results showed increased mRNA levels of Oct4 and Sox2 in bredigite-induced group and bFGF-induced group than in control group (p<0.05). While their expression in β-TCP-induced group were down-regulated (p<0.05). After dentinogenesis induction and adipogenesis induction for 21 days, there were greatly increased amounts of calcium deposition in bredigite-induced group and bFGF-induced group, fewer and smaller mineralized nodules in β-TCP-induced group and control group. The amount of lipid droplets also greatly increased in bredigite-induced group and bFGF-induced group. While the β-TCP-induced group displayed a smaller lipid droplets size, the control group exhibited spindle-shape cells and fewer lipid droplets. Western blot results showed that bredigite-induced group and bFGF-induced group exhibited significantly higher expression of DSPP, DMP-1, PPARγ-2 and LPL than control group (p<0.05), yet there was no significant change in β-TCP-induced group (p>0.05). CONCLUSIONS: Ionic ingredient from bredigite could up-regulate the proliferation activity, delay the progress of cell senescence, and increase the expressions of pluripotency-related genes Stro1, Oct4 and Sox2 of hDPCs. Bredigite also promote the multilineage differentiation potential of hDPCs. The effects of bredigite are similar to bFGF and significantly better than β-TCP. SIGNIFICANCE: Our work may contribute to provide a theoretical basis for the clinical application of bioactive silicate ceramic in repairing and regenerating pulp tissues. It also provides the experimental basis for optimizing the seed cell for oral diseases.

This study was supported by National Natural Science Foundation of China (No.81 371133)

20

PENN DENTAL MEDICINE

TRPM7 SENSES MECHANICAL STIMULATION INDUCING OSTEOGENESIS IN HUMAN BONE MARROW MESENCHYMAL STEM CELLS E, X.1; HuaQiang, Y2; YeHua, G.3; Denghui, D.4; Linhai, H.1; Shiqiang, W.6; Yi, Z.1 Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology College of Life Sciences, Peking University 3 Central Laboratory, Peking University School and Hospital of Stomatology 4 Department of General Dentistry, Peking University School and Hospital of Stomatology 6 College of Life Sciences, Peking University 1

2

ABSTRACTS

P-2

OBJECTIVES: Mesenchymal stem cells (MSCs) are a kind of multipotential stem cells residing in the bone marrow.

Several studies have shown that mechanical stimulation modulates MSC differentiation through mobilization of second messengers, but the mechanism of mechanotransduction remains poorly understood. METHODS: In this study, using fluorescence and laser confocal microcopy as well as patch clamp techniques to evaluated the jaw bone marrow mesenchymal stem cells from the donors. RESULTS: we identified the transient receptor potential melastatin type 7 (TRPM7) channel as the key channel involved in mechanotrasduction in bone marrow MSCs, as TRPM7 knockdown completely abolished pressure induced cytosolic Ca2+ increase and pressure induced osteogenesis. TRPM7 directly sensed membrane tension, independent of cytoplasm and the integrity of cytoskeleton. Ca2+ influx through TRPM7 further triggered Ca2+ release from inositol trisphosphate receptor type 2 (IP3R2) on the endoplasmic reticulum and promoted NFATc1 nuclear localization and osteogenesis. CONCLUSIONS: These results identified a central role of TRPM7 in MSC mechanical stimulation induced osteogenesis. SIGNIFICANCE: This study firstly elucidated MSCs could directly senses the mechanical stimulation by TRPM7 and transfer the mechanical signal to calcium signal and active the downstream pathway.

P-3

NANOTOPOGRAPGY OF MINERALZED COLLAGEN DETERMINES PERIODONTAL LIGAMENT STEM CELL FATE Fu, Yu1; Liu, Shuai1; Liu, Xiao-Mo1; Sun, Yue1; Chang, Da-Tong1; Wang, Gao-Nan1; Liu, Yan1; Zhou, Yan-Heng1 Center for Craniofacial Stem Cell Research and Regeneration, Department of Orthodontics, Peking University School, Hospital of Stomatology

1

OBJECTIVES: The interplay between scaffolds and cells is of critical importance to the function of engineered tissues. This study examined the effects of nanotopography of mineralized collagen scaffolds on human periodontal ligament stem cell (hPDLSC) behaviors. METHODS: Intrafibrillarly-mineralized collagen (IMC) and extrafibrillarly-mineralized collagen (EMC) scaffolds were fabricated through a biomimetic crystallization method and a conventional crystallization method respectively.

The nanotopography of these two scaffolds was observed by scanning electron microscopy (SEM) and transmission electron microscope (TEM). Micro-Fourier transform infrared spectroscopy (micro-FTIR) was employed to measure the spatial distribution of different chemical groups and mineralization level of different scaffolds at micro-level. Cell morphology and adhesion by immunofluorescence staining and SEM, proliferation by CCK-8 assay, mineralization by Alizarin red staining and the mRNA expression of differentiation-related genes of hPDLSCs on different scaffolds were evaluated. RESULTS: These two mineralized collagen scaffolds exhibited different nanotopography. The IMC scaffold exhibited highly smoothed surface morphology with filamentous substructure parallel to the fibrils, whilst flower-like apatites deposited around the surface of the fibrils in the EMC scaffold. From the micro-FTIR mapping test, the IMC scaffold showed more uniform distribution of mineral and matrix, and higher mineralization level than the EMC scaffold. The hPDLSCs seeded on the IMC scaffold exhibited extended pseudopodia with polyagonal and spindle shape, improved cell adhesion and proliferation. mRNA levels of major osteogenic differentiation genes including osteopotin, alpha-1 type I collagen and bone morphogenetic protein-2 were highly expressed in the IMC scaffold. CONCLUSIONS: IMC fabricated through the biomimetic intrafibrillar mineralization method could promote the adhesion, actin aggregation, proliferation and osteogenic differentiation of hPDLSCs, compared with EMC made with conventional crystallization method. SIGNIFICANCE: The IMC scaffold with a similar nanotopography to natural mineralized collagen improved cell spreading, adhesion, proliferation and osteogenic differentiation, providing a promising scaffold in the periodontal tissue regeneration.

OROFACIAL STEM CELL RESEARCH SUMMIT

21

ABSTRACTS

P-4

PDLSCs PROMOTE M1 MACROPHAGE POLARIZATION DURING ORTHODONTIC TOOTH MOVEMENT He, Danqing1; Kou, Xiaoxing1; Yang, Ruili1; Liu, Dawei1; Zhou, Yanheng1 Department of Orthodontics,Peking University School and Hospital of Stomatology

1

OBJECTIVES: This study examined: 1) whether and how M1 macrophage polarization involves in mechanical force-induced orthodontic tooth movement (OTM); 2) whether periodontal ligament stem cells (PDLSCs) could promote M1 macrophage polarization during OTM. METHODS: Orthodontic nickel–titanium springs were applied to the upper first molars of rats or mice to induce OTM. Micro-CT was used to analyze the OTM distances. The number of osteoclasts was detected by Tartrate-resistant acid phosphatase (TRAP) staining. The expressions of M1 markers CD68, tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS) was detected by immunohistochemistry and immunofluorescence staining. The role of M1 macrophage polarization during OTM was confirmed by the experiments of monocyte/macrophage depletion, systemic transfusion of M1 macrophages and systemic injection of recombinant TNF-α or TNF-α inhibitor. In addition, static compressive force was applied to primary cultured human PDLSCs, and the expression of interferon (IFN)-γ was detected by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Supernatant of force-loaded PDLSCs was added to THP-1 derived macrophages and the expressions of M1 or M2 markers were detected using real-time polymerase chain reaction and western blot. RESULTS: M1-like macrophage polarization and expression of M1 cytokine TNF-α increased during OTM after force application. Monocyte/macrophage depletion in mice decreased the distance of OTM and the number of TRAP+ osteoclasts, CD68+ macrophages and the expressions of TNF-α and iNOS, whereas systemic transfusion of M1 macrophages in mice increased them. Moreover, injection of rTNF-α enhanced OTM distance, whereas blockage of TNF-α by etanercept injection repressed it. When static compressive force was applied to the PDLSCs, the levels of IFN-γ mRNA expression and IFN-γ secretion in the culture supernatant were upregulated. After incubation with the supernatants from force-treated PDLSCs, the mRNA expression of M1 markers TNF-α, IL-1β, and IL-6 were upregulated in THP-1-derived macrophages. Western blot analysis showed that the protein expression of TNF-α in THP-1-derived macrophages was upregulated after incubation with the supernatant from force-treated PDLCs, whereas no changes were observed in the expression of arginase-1. CONCLUSIONS: These data suggest that M1-like macrophage polarization promotes alveolar bone resorption and consequent OTM after mechanical force application. Force-loaded PDLSCs could promote M1 macrophage polarization during OTM. SIGNIFICANCE: These data are the first time to showed that force-loaded PDLSCs could promote M1 macrophage polarization during OTM, which provide new insights into novel modalities to accelerate OTM. /

P-5

HEMATOPOIETIC STEM CELLS IN THE CRANIOFACIAL BONE Jiang, Nan1; Chen, Mo2; Ding, Lei2; Mao, Jeremy2; Zhou, Yanheng1 Peking University School of Stomatology Columbia University

1

2

OBJECTIVES: Appendicular bone marrow is known to harbor two distinct stem-cell populations: hematopoietic stem cells (HSCs) and mesenchymal stem/stromal cells (MSCs). One of the fundamental hypotheses in stem cell biology is that MSCs, along with osteoblasts, form niches for HSCs in bone marrow. Whether craniofacial bones harbor HSCs is obscure. In this study, we harvested mandibular bone marrow cells to identify putative hematopoietic stem cells (HSCs) from craniofacial bones. METHODS: Adult C57BL/6 mice (8 wks old) were scarified to harvest bone-marrow contents from mandibles and two appendicular bones, the femur and tibia, by repeated aspirations. Following established HSC isolation protocols, we subjected mandibular bone-marrow aspirates to centrifuging @ 400 g/20 min to form a density gradient, using femoral and tibial marrow aspirates as a positive control. Mononucleated cells were aspirated from a thin cell-rich layer and then subjected to Lin- selection by magnetic-activated cell sorting (MACS). Lin- cells were then selected with CD34-, Sca1+ and c-kit+ antibodies, and further sorted with MoFlo, and additional characterization. Adult mice were administered a minimum lethal dose of radiation with a Cesium 137 GammaCell 40 Irradiator (MDS) to deliver two doses of 540rad (1,080rad in total) at least 2h apart. Mononucleated cells were freshly harvested from donor matched long bones and mandibles before transplantation. C57BL/6-SJL (CD45.1) mice were used as the recipient. Cells were injected into the retro-orbital venous sinus of anaesthetized mice to test the long-term competitive reconstitution probability. RESULTS: A total of 1.5% of Lincells from mandibular bone marrow were CD34-/low/c-Kit+ /Sca1+ cells, or otherwise known as CD34-KSL cells, which are typically regarded as hematopoietic stem cells (HSCs) in the literature. We further established single cell colony assays from

22

PENN DENTAL MEDICINE

P-6

ABSTRACTS

CD34-KSL cells. Mandibular CD34-KSL cells or putative HSCs showed self-renewal over the tested 14 days, which is typical HSC self-renewal time lines prior to spontaneous differentiation. SLAM family receptors can further distinguish functionally distinct subpopulations of HSCs and multipotent progenitors. Lineage-/lowSca-1+c-kit+ (LSK) cells are subdivided into MPPs (CD150-CD48+ LSK), HPC (CD48+ LSK) and HSCs (CD150+CD48+ LSK). Besides CD150 and CD48), CD229 and CD244 further divided the mouse bone marrow LSK cells into seven functionally subpopulation. Mandibular HSCs and MPPs obtained the similar frequency as the long bone HSCs. Less HPC-1 (CD150-CD48+ LSK) were detected in the mandible (0.090±0.011) than long bone (0.144±0.009), while more HPC-2 (CD150+CD48+ LSK) were seen in the mandible (0.013±0.001) than long bone (0.009±0.001). When we further looked at the HSCs subpopulation, obviously more CD229- subpopulation and less CD229+ population in the mandibular HSCs (n=3-5, three independent experiments).

CONCLUSIONS: The mandibular bone harbors putative hematopoietic stem cells (HSCs) that are distinctive from donormatched mesenchymal stem/stromal cells. We discovered and characterized CD34-/low, Sca-1 and c-kit double positive cells in the mandibles of postnatal mice. Mandibular CD34-KSL cells showed similar proliferation rates to donor-matched HSCs, and differentiated into all hematopoietic cell types with robust capacity towards the myeloid lineage, but were severely deficient of the ability to differentiate into the lymphoid lineage. SLAM family markers testified that mandibular bone marrow cells expressed significantly fewer CD229+ fractions in the HSCs and multipotent hematopoietic progenitors (MPPs) subpopulations. Mandibular HSCs showed similar capacity to restore all tested hematopoietic lineages in the irradiated bone marrow in vivo as HSCs isolated from donor-matched tibia/femur. SIGNIFICANCE: Here, we first characterized neural crest derived bone marrow harbors hematopoietic stem cells that are consistent with appendicular bone in the aspects of stemness and frequency. Interestingly, a lymphoid lineage deficiency was observed in the mandibular bone hematopoiesis, which may be related with the niche cells and molecules.

TNF-α INHIBITS FOXO1 TO AGGRAVATE OXIDATIVE DAMAGE IN OSTEOPOROTIC BMMSCS Liao, L1; Su, X2; Deng, Z3; Jin, Y1 State Key Laboratory of Military Stomatology, Center for Tissue Engineering, School of Stomatology, Fourth Military Medical University 2 Department of Orthodontics, Stomatology Hospital, Jiaotong University College of Medicine 3 Department of Otolaryngology, Xijing Hospital, Fourth Military Medical University 1

OBJECTIVES: Decline of antioxidant defense after estrogen deficiency leads to oxidative damage in bone marrow-derived mesenchymal stem cells (BMMSCs), resulting a defect of bone formation in osteoporosis. Forkhead box O1 (FOXO1) protein is crucial for defending physiological oxidative damage in bone. This study aimed to explore whether FOXO1 is involved in oxidative damage during osteoporosis and identify the molecular mechanism. METHODS: To address these questions, ovariectomized (OVX) mice were used as animal models of osteoporosis. Flow cytometry, fluorescent microscope and Realtime-time RT-PCR were used to detect ROS levels and antioxidants expression in BMMSCs isolated from OVX and sham surgery mice. Immunohistochemistry and Western blotting were performed to detect FOXO1 protein accumulation in bone and BMMSCs. The function of TNF-α on FOXO1 expression was determined by in vitro and in vivo gain- and loss-of-function assay. MicroRNA (miRNA)-mediated mechanism was determined by in silico analysis, luciferase reporter assay and functional validation. RESULTS: We found that FOXO1 protein accumulation was decreased in BMMSCs of OVX mice. The decrease of FOXO1 resulted in the suppression of manganese superoxide dismutase (SOD2) and catalase (CAT) expression and accumulation of reactive oxygen species (ROS), inhibiting the osteogenic differentiation of BMMSCs. The decline of FOXO1 protein was caused by TNF-α accumulated after estrogen deficiency. Mechanistically, TNF-α activated NF-κB pathway to promote microRNA-705 expression, which function as a repressor of FOXO1 through posttranscriptional regulation. Inhibition of NF-κB pathway or knockdown of miR-705 largely prevented the decline of FOXO1mediated antioxidant defense caused by TNF-α, and ameliorated the oxidative damage in osteoporotic BMMSCs. Moreover, the accumulated ROS further activated NF-κB pathway with TNF-α, which formed a feed-forward loop to persistently inhibiting FOXO1 protein accumulation in BMMSCs. CONCLUSIONS: Taken together, our study revealed that the decline of FOXO1 is an important etiology factor of osteoporosis, and unclosed that TNF-α decreased FOXO1-mediated antioxidant defense through post-transcriptional regulation by miR-705. SIGNIFICANCE: These findings revealed a novel mechanism of TNF-α in oxidative damage regulation, suggesting a close correlation between inflammation and oxidative stress in stem cell dysfunction during degenerative bone diseases. Since oxidative damage is caused by FOXO1 dysfunction, FOXO1 is a potential target for recovery of mesenchymal stem cell dysfunction and treatment of degenerative bone diseases.

OROFACIAL STEM CELL RESEARCH SUMMIT

23

ABSTRACTS

P-7

RESVERATROL REVERSES IMPAIRED REGENERATION POTENTIAL OF PERIODONTAL LIGAMENT STEM CELLS Liu, N1; Zhao, P1; Hu, C1; Sui, B1; Zhou, C1; Jin, Y1 Research and Development Center for Tissue Engineering, School of Stomatology, The Fourth Military Medical University

1

OBJECTIVES: Adult stem cells isolated from human periodontal ligaments (PDLs) can be as a potential autologous cell source for clinical use in periodontal tissues regeneration. Periodontal ligament stem cells derived from periodontitis (PPDLSCs) can be a potential candidates because their easily obtainment. However, their low pluripotent comparing with normal periodontal ligament stem cells (NPDLSCs) restrict their use. Though it was well known that resveratrol could be as an anti-aging and anti-inflammation small compound, we still did not know whether resveratrol could be benefited for PDLSCs. So the aims of this study were to improve the pluripotential capacity and regenerative potential of the PPDLSCs by treating with resveratrol. METHODS: PDL tissues were obtained from surgically extracted normal and inflamed molars respectively. All PDLSCs were characterized by colony forming unit assay, cell surface marker and their osteogenic/adipogenic differentiation potential. The biological effects of resveratrol as an anti-inflammation activator were then investigated and quantified for statistical analyses, including alkaline phosphatase (ALP) activity, gene expression and western blotting.

RESULTS: It was found that resveratrol induced a significant and continuous stimulation of osteogenesis in human PPDLSCs. Furthermore, resveratrol improve the regenerative ability by increasing ALP activity and decreasing the inflamed level of PPDLSCs. The in vivo tissue regeneration potential was assessed by in vivo ectopic transplantation model in nude mice and in situ transplantation model in rat. CONCLUSIONS: Resveratrol is a promising candidate for PPDLSCs based periodontal tissues regeneration therapies, enabling us to expand autologous pathological cell resources for clinical use.

SIGNIFICANCE: Our study support a possible method regenerates periodontal tissues in patients with periodontitis using autologous PDLSCs.

P-8

MSC TRANSPLANTATION DURABLY RESCUES RECIPIENT LUPUS MSCS VIA EPIGENETIC MODIFICATIONS Liu, S1; Liu, D1; Chen, C1; Hamamura, K2; Yang, R1; Liu, Y1; Jin, Y3; Shi, S1 Department of Anatomy and Cell Biology, School of Dental Medicine, University of Pennsylvania Center for Craniofacial Molecular Biology, University of Southern California 3 Research and Development Center for Tissue Engineering, School of Stomatology, The Fourth Military Medical University 1

2

OBJECTIVES: Mesenchymal stem cell transplantation (MSCT) has been widely used to treat a variety of human diseases.

It is very interesting that in most cases, MSCT is just performed once, however, the therapeutic effects generated by the onetime MSCT could last for a long term. These evidences indicate that MSCT generate durable therapeutic effects and generate a â&#x20AC;&#x153;therapeutic memory.â&#x20AC;? The mechanisms underlying these phenomena have not been explored yet. In this study, we aim to explore the molecular regulation mechanisms underlying the durable therapeutic effects generated by MSCT. METHODS: In Fas-deficient-MRL/lpr systemic lupus erythematosus (SLE) mice model, we will determine the MSCT-generated durable therapeutic effects on osteoporotic disease phenotype and osteogenic differentiation capacities of the recipient BMMSCs. Then, we will explore the role of epigenetic modifications, especially DNA methylation, in the durable therapeutic effects.

Finally, we will dissect the mechanisms underlying donor mesenchymal stem cells (MSCs) regulate the epigenetic status of the recipient bone marrow mesenchymal stem cells (BMMSCs). RESULTS: Here we show that MSCT rescues recipient BMMSC function in SLE mice via rescuing their hypomethylated DNA profile. Mechanistically, we show that elevated intracellular levels of miR-29b, caused by Fas-deficient-mediated failure of releasing, downregulate expression of DNA methyltransferase 1 (Dnmt1) in MRL/lpr BMMSCs. This results in hypomethylation of Notch1 promoter and activation of Notch signaling, in turn leading to reduced osteogenic differentiation. Furthermore, we show that exosomes secreted due to MSCT reduce intracellular levels of miR-29b in recipient MRL/lpr BMMSCs, and that exosome-derived Fas is reused by these BMMSCs. Finally, the reduced intercellular miR-29b levels result in recovery of Dnmt1-mediated Notch1 promoter hypomethylation and thereby

24

PENN DENTAL MEDICINE

P-9

ABSTRACTS

improve MRL/lpr BMMSC function. CONCLUSIONS: Collectively our findings unravel the means by which MSCT rescues recipient MRL/lpr BMMSCs through reuse of donor exosome-provided Fas to regulate the miR-29b/Dnmt1/Notch cascade, and also highlight the potential of MSCT-mediated epigenetic regulation as a therapeutic approach to rescue BMMSC function. SIGNIFICANCE: We reveal the mechanisms underlying the durable therapeutic effects generated by MSCT, indicating that stem cell therapy can durably regulate the functions of the recipient cells through epigenetic modifications.

Thus, this study provides new therapeutic method for reversing the abnormal epigenetic status during disease development.

Meanwhile, we find that, the components of donor cells can be reused by recipient cells for rescuing their own functions, indicating that the interactions between donor cells and recipient cells play an important role in MSCT. Therefore, this study provides further theory basis for clinical applications of stem cell therapy.

NANOTOPOGRAPHY OF MINERALIZED COLLAGEN DETERMINES NANOSTRUCTURE OF REGENERATED BONE Liu, Shuai1; Fu, Yu1; Sun, Yue1; Chang, Da-Tong1; Liu, Yan1; Zhou, Yan-Heng1 Peking University, School of Stomatology

1

OBJECTIVES: Traditional artificial scaffolds designed to support cell and bone regeneration have aimed on a macroscopic level to match the properties of natural bone without recreating the nanoscale detail observed in natural bone. In this study, we aimed to investigate the effect of nanotopography of mineralized collagen scaffolds on the nanostructure of regenerated bone. METHODS: Intrafibrillar mineralized collagen scaffold (IMC) and extrafibrillar mineralized collagen scaffold (EMC) were fabricated by a biomimetic bottom-up mineralization approach and a conventional top-down mineralization approach separately. The nanostructure and elemental analysis of representative scaffolds was performed on non-osmicated, unstained thin sections prepared by ultramicrotomy, using a Tecnai G2 scanning-transmission electron microscopy (STEM) at 200 kV. Rat bone marrow stromal cells were seeded on different scaffolds to investigate the effects of their nanostructure on cell behaviors. For in vivo experiments, different scaffolds without seeded cells were randomly implanted in the critical size defects in rat mandibles, while the control group had no implants (N = 6). Eight weeks after implantation, Micro-CT, histological staining and TEM analyses were used to evaluate the quantity and quality of the newly-formed bone. RESULTS: The IMC scaffold exhibited bone-like hierarchy with mineral platelets arranged in a parallel staggered pattern in a collagen fibril, while spherulitic clusters composed of randomly oriented minerals deposited around a collagen fibril in the EMC. In the IMC, periodically-repeated band regions of the collagen fibril exhibited increased HAADF intensity possessed relatively high concentrations of Ca (indicative of nanoapatite) and significantly reduced concentrations of C (indicative of gap zones between collagen molecules) by STEM. Compared with the EMC, the IMC improved the initial cell adhesion and proliferation, and calcification of bone marrow mesenchymal stem cells. In vivo assessment in rat critical-size mandibular defects showed that the defects were almost entirely filled with fibrous bone structures, with unidirectionally-aligned collagen fibrils similar to natural bone in the IMC scaffold. Limited bone formation mainly restricted to the defect margins with randomly-arranged collagen fibrils in the EMC group, and no obvious bone formation in the control group. The undegraded IMC scaffold integrated with the regenerated bone due to its nanostructural similarity to natural bone. Furthermore, TEM of undecalcified regenerated tissues showed that collagen fibril arrays were parallel-aligned with mineral platelets arranged in a parallel staggered pattern in the IMC group; while needle-like minerals randomly arranged round collagen fibrils in the EMC group.

Immunohistochemistry displayed intensive expressions of osteogenesis- and angiogenesis-related genes such as TGF-β1 and VEGF in the IMC group. CONCLUSIONS: The IMC scaffold reproduces the nanoscale staggered pattern in natural bone and exhibits excellent osteoinductivity and osteoconductivity. The nanotopography of mineralized collagen scaffolds determines the nanostructure of the regenerated bone. SIGNIFICANCE: Our results reveal that the IMC scaffold can not only serve as a support for ingrowth of host cells, but also participate directly in bone formation like a natural extracelluar matrix. This challenges the traditional concept of tissue engineering concept that scaffolds can only act as temporary templates.

OROFACIAL STEM CELL RESEARCH SUMMIT

25

ABSTRACTS

P-10

STAGGERED NANOSTRUCTURE CONTROLS MECHANICAL AND BIOLOGICAL PERFORMANCE OF MINERALIZED COLLAGEN Liu, Yan1; Liu, Shuai1; Wang, Dong-Xue1; Fu, Yu1; Zhou, Yan-Heng1 Center for Craniofacial Stem Cell Research and Regeneration, Department of Orthodontics, Peking University School, Hospital of Stomatology 1

OBJECTIVES: Nature bone and bone-like tissues exhibit a generic staggered nanostructure, yet its role remains unclear.

In this study, we aim to test the effect of staggered pattern of nanohydroxyapatites on the mechanical and biological performance of mineralized collagen. METHODS: Mineralized collagen with staggered pattern (SMC) and with continuous strands (CMC) were fabricated separately by a polyacrylic acid (PAA) mediated mineralization method and a poly-L-aspartic acid (PASP) as a mineralization-directing agent. The nanotopography of different mineralized collagen was observed by scanning electron microscopy (SEM) and transmission electron microscope (TEM). Atomic force microscopy (AFM) was employed to measure the nanomechanical properties of different mineralized collagen. To illustrate the mechanism of SMC formation, the Gibbs free energy in each mineralization phase is calculated. The effect of staggered nanostructure on cell morphology by immunofluorescence staining, proliferation by CCK-8 assay and differentation by ALP activity were evaluated.

Furthermore, a critical size defect in Sprague-Dawley rat mandibular bone was created to evaluate the bone regeneration potential of different collagen scaffolds. Micro-CT, histological staining, SEM and TEM were applied to evaluate the quantity and quality of new bone. RESULTS: The SMC exhibited a periodic banding pattern (ca. 67 nm), with nanohydroxyapatites arranged in a parallel staggered pattern in collagen; while the CMC displayed as clusters of short filaments, which contained mineral strands destroying the banding pattern of nature collagen. It was demonstrated by nanomechanical testing that the Young's modulus of single fibril in the SMC also features periodic distribution, with 8.1 ± 0.5 GPa in the gap regions and 6.7 ± 0.5 GPa in the overlap regions. While the modulus of single fibril in the CMC showed uniform distribution with the modulus of 6.27 ± 0.58 GPa. These nanomechanical properties resulted in few cracks in the SMC scaffolds and collapse of the CMC scaffold, when a vertical force with 8N was applied to the scaffolds. Thermodynamic calculation demonstrated that the Gibbs free energy for PAA-Ca is between Gap-Ca and Overlap-Ca, resulting in periodic deposition of nanohydroxyapatite crystals in the SMC. While the free energy of PASP-Ca is much higher than that of Gap-Ca as well as Overlap-Ca, resulting in unselectivity of mineralization whether in the gap or overlap regions in the CMC. In vitro cell biology showed that the hierarchical, staggered arrangement of nanohydroxyapatites within a collagen fibril determined the osteogenic-specific cell fate of bone marrow mesenchymal stem cells. Moreover, in vivo assessment showed that the SMC improved bone regeneration with nanostructure similar to natural bone. CONCLUSIONS: The staggered arrangement of nanohydroxyapatites in a collagen fibril controls the mechanical and biological performance of mineralized collagen. SIGNIFICANCE: These findings reveal that production of hierarchical nanostructure with a staggered fashion is critical in material science.

P-11

ZOLIDRONIC ACID-LOADED GELATIN NANOPARTICALES FOR ALVEOLAR BONE RESTITUTION Ni, Shilei1; Su, Hongchen1 School of Stomatology, Jilin University

1

OBJECTIVES: The resorption of the rest ridge after tooth extraction, which brings difficulties to the design of denture restoration is very common in dental clinical treatment. Zoledronic acid (ZA) is the most potent bisphosphonate which is widely used to inhibit bone resorption, whereas it also brings severe side effects while systemically administrated. The purpose of this work is to prepare ZA compound drug and study the efficiency of local slow release of ZA in the tooth extraction socket(TES) and find out the drug's efficiency on the restitution of alveolar bone. METHODS: Gelatin is chosen as a ZA carrier because of its biodegradability and biocompatibility in physiological environments. 500mg gelatin is dissolved with 10ml deionized and double-distilled water, and then acetone is added as polycoagulant. The suspension is discarded and sediment derived is dissolved again together with ZA. The PH value is adjusted to the isoelectric point of the mixture. Then,

26

PENN DENTAL MEDICINE

P-12

ABSTRACTS

acetone is added again to prepare ZA-loaded gelatin nanoparticles and glutaraldehyde follows for cross linking. The surface potential, size, morphology, drug loading and encapsulation efficiency of the nanoparticles are measured respectively for qualitation of the nanoparticles. Wistar rats (6 weeks, 180g-200g) are chosen as the experimental animals. One incisor of the Wistar rat is extracted to set up an animal model. As three experimental groups, nanoparticles contain 3μg, 6μg and 12μg ZA respectively are inserted into the TESs, whereas same weight of blank gelatin nanoparticles are inserted into TESs of three control groups. The animals are sacrificed after 15d, 30d and 60d. The relative height of rest ridge, bone mineral density, and histological section are measured to see the condition of bone restitution in the TES. RESULTS: 50nm-450nm ZA-loaded gelatin nanoparticles with an average size of 163 nm were prepared in this work. Through SEM, we found the nanoparticles are regular round and relatively disperse well. The drug loading and encapsulation efficiency were 6.85% and 60.52% respectively.

The compound drug dissolved well in the TES in 15 days. In the experimental group, bone trabaculars in the TES were found more and bigger than that of the control group through histology section. They grew towards the center of the TES. In the experimental group which 6μg ZA was administrated, the TES was full of massive and reticular bone trabaculars after 30 days whereas in the control group, the bone trabaculars were thinner and less significantly. According to statistical analysis, the bone mineral density in the TESs and height of rest ridge were significantly higher than that of the control group after 60 days. CONCLUSIONS: We found an ideal method to prepare ZA-loaded gelatin nanoparticles. The drug we prepared could promote the restitution and inhibit the resorption of Wistar rats™ alveolar bone by slow release. SIGNIFICANCE: With further research, as a new form of drugs, ZA-loaded gelatin nanoparticles can be used in clinical to prohibit the resorption of alveolar bone in periodontitis. And also, it can be used to promote the restitution of alveolar bone after tooth extraction.

BONE MARROW DERIVED CELLS CAN DIFFERENTIATED INTO OCS OR FBGCS Quan, JJ1 Guanghua Dental Hospital, Sun Yat-sen University

1

OBJECTIVES: This study was to analyze the phenotype and characteristics of multinucleated osteoclasts (OCs) or foreign body giant cells (FBGCs), derived from the bone marrow. METHODS: Humerus and tibia were obtained from six-week old C57/BL6 mice, and bone marrow cells were flushed with media and then filtered through a 600 μM cell strainer, which was utilized for the differentiation of OCs and FBGCs. Cells were seeded in 150 mm x 25 mm dishes at a density of 1.5x104/cm2 with 30 ng/mL of MCSF. After 2 days, adherent cells were separated and plated in 96 and 6-well plates at a density of 1.5x104/cm2. OCs were generated with MCSF (30 ng/mL) and RANKL (35 ng/mL) to media. FBGCs were generated with GMCSF (50 ng/mL) and IL4 (50 ng/mL) to media. After a continuous culture for 10 days, all cells were fixed with 10% formalin, one part of cells was TRAP stained, the other part was stained by DAPI and phalloidin. To measure the bone resorption activity of OCs and FBGCs, cells were plated on dentin slices. Total RNA were extracted from cultured cells on each day from Day 0 to Day 4, and converted into cDNA using reverse transcriptase. Quantitative gene analysis of TRAP, MMP9, CTSK, RANK, NFATc1, CCL2, CCL3, CCL4, CCL5, and CCL9 was performed by using SYBR Green qPCR Supermix in a Real-time PCR system. RESULTS: Apart from osteoblasts (OBs), bone marrow derived cells could differentiate into OCs or FBGCs under various treatments. TRAP staining and immunofluorescence have showed typical multinucleated and F-actin ring for OCs, while FBGCs displayed giant cell type with multinuclear but not obvious F-actin ring.

Bone report assay found typical bone resorption pits for OCs, while FBGCs could not. qPCR analyzed expression of TRAP, MMP9 and CTSK were highly increased in OCs, while CTSK, MMP9 were not expressed in FBGCs. RANK and NFATc1 was expressed by OCs significantly more than FBGCs. For inflammation specific chemokines, FBGCs expressed CCL2, CCL3, CCL4 and CCl5 significantly more than OCs. CCL9 was found to be highly induced by FBGCs on day 2 compared to OCs, while on day 3 the difference was not significant, on day 4 CCL9 was expressed more by FBGCs than OCs. CONCLUSIONS: Since OCs and FBGCs are normally detected in periodontitis and apical periodontitis, these bone marrow derived cells have played important roles in the progression of inflammatory bone destruction. SIGNIFICANCE: Chemokines expressed from OCs and FBGCs may have positive feedback to enlarge the inflammatory response. How to control the balance between differentiation of bone form and bone resorb would be useful to inhibit the inflammatory bone destruction.

OROFACIAL STEM CELL RESEARCH SUMMIT

27

ABSTRACTS

P-13

THE EFFECT OF TET1 ON THE PROLIFERATION AND ODONTOGENIC DIFFERENTIATION OF HUMAN DENTAL PULP CELLS Rao, Lijia, Li, Qimeng, Yi, Baicheng, Xu, Qiong* Guanghua School of Stomatology & Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University) *Corresponding author OBJECTIVES: Human dental pulp cells (hDPCs) possess the capacity to differentiate into odontoblast-like cells and generate reparative dentin in response to exogenous stimuli or injury. Ten-eleven translocation 1 (TET1) is a novel DNA methyldioxygenase that plays an important role in the promotion of DNA demethylation and transcriptional regulation in several cell lines. However, the role of TET1 in the biological functions of hDPCs is unknown. The aim of this study was to investigate the role of TET1 in the proliferation and odontogenic differentiation of hDPCs. METHODS: A recombinant shRNA lentiviral vector was used to knockdown TET1 expression in hDPCs. To identify the effect of TET1 knockdown on the proliferation and odontogenic differentiation potential of hDPCs, the CCK8 assay was used to measure cell proliferation.

Alkaline phosphatase (ALP) activity was measured, and alizarin red staining was used to assess the formation of mineralized nodules. The expression levels of the odontogenic differentiation markers DSPP and DMP1 were also analyzed. RESULTS: Following TET1 knockdown in hDPCs, TET1 was significantly down-regulated at both the mRNA and protein levels.

Proliferation of the hDPCs was suppressed in the TET1 knockdown groups. ALP activity, the formation of mineralized nodules, and the expression levels of DSPP and DMP1 were all reduced in TET1-knockdown hDPCs undergoing odontogenic differentiation. CONCLUSIONS: TET1 knockdown can prevent the proliferation and odontogenic differentiation of hDPCs, suggesting that TET1 may play an important role in dental pulp repair and regeneration. SIGNIFICANCE: Our results may elucidate the role of TET1-dependent DNA demethylation in the regulation of the differentiation potential of hDPCs\ This study is supported by National Natural Science Foundation of China (81570971)

P-14

BMMSC INFUSION-BASED THERAPY AGAINST OSTEOPOROSIS ARE IMPAIRED BY RECIPIENT HYPERGLYCEMIA Sui, B1; Hu, C1; Zhang, X1; Chen, Y1; He, T1; Li, M2; Zhao, X2; Jin, Y1 Research and Development Center for Tissue Engineering, School of Stomatology, The Fourth Military Medical University State Key Laboratory of Military Stomatology, School of Stomatology, The Fourth Military Medical University

1

2

OBJECTIVES: Bone marrow mesenchymal stem cells (BMMSCs) are potent immunomodulators that have demonstrated therapeutic benefits for autoimmune- or inflammation- related bone loss based on systemic infusion.

The immunomodulation of BMMSCs occurs via induction by proinflammatory cytokines. However, whether the immunomodulatory and therapeutic potential of BMMSCs for bone loss are maintained under complicated recipient conditions such as type 1 diabetes (T1D) is unknown. We aimed to explore the clinical utility of BMMSCs in T1D-induced osteoporosis and the underlying mechanisms. METHODS: The BMMSCs used in our study were identified by colony formation, multilineage differentiation and surface markers. 1x106 syngeneic BMMSCs were infused via caudal vein into C57 mice 1 w after ovariectomy (OVX), orchidectomy (ORX) or first streptozotocin (STZ) injection, or into 12 w-old senescence-accelerated mice-prone 6 (SAMP6) mice. 4 w post infusion, the bone mass were evaluated by microCT, and the bone formation and the bone resorption rates were evaluated by calcein and TRAP staining, respectively. 6 h post infusion, percentages of CD3+T cells in mononuclear cells from recipient peripheral blood, bone marrow and spleen were determined with flow cytometry. 4 w post infusion, concentrations of proinflammatory cytokines tumor necrosis factor-alpha (TNFα), interferon-gamma (IFN-γ) and interleukin-1beta (IL-1β) were detected by ELISA. Both 24 h and 4 w post infusion, migration and engraftment of BMMSCs in bone marrow were determined with GFP-labeling and immunofluorescent staining. The levels of blood glucose were detected each week through the study. T-cell apoptotic percentages with or without co-culture with BMMSCs were determined by flow cytometry using Annexin V FITC-PI double staining, where BMMSCs were treated with either 5.55 mM or 25 mM glucose, or either PBS or 200 μg/ml AGE-BSA in vitro. The secretion of TNF-α and IFN-γ into conditional media were detected by ELISA. RESULTS: Infusion of BMMSCs prevented bone loss induced by OVX and restored the balance between bone formation and bone resorption. However in T1D mice, therapeutic effects of BMMSCs were diminished, failed to prevent the development of osteoporosis with an inhibited bone formation and an elevated bone resorption. The migration and engraftment of BMMSCs in bone marrow were not significantly different in OVX and T1D mice, both at 24 h and 4 w post infusion. On the other side, the immunomodulation of BMMSCs against T cells occurred

28

PENN DENTAL MEDICINE

P-15

EFFECTS AND MECHANISM OF SIRT6 ON OSTEOGENIC ABILITY OF RAT BONE MESENCHYMAL STEM CELLS

ABSTRACTS

in OVX mice but was impaired in T1D mice, although T1D mice also developed up-regulated proinflammatory cytokines.

Mechanistically, hyperglycemia inhibited the immunosuppressive effects of BMMSCs on T-cell survival through advanced glycation end products (AGEs). Furthermore, the immunomodulatory and therapeutic potential of BMMSCs were maintained under euglycemia in ORX-induced and age-related osteoporosis. CONCLUSIONS: Mesenchymal stem cell infusion-based therapeutic effects on bone loss were diminished by recipient hyperglycemia through AGEs inhibiting immunomodulation of BMMSCs. SIGNIFICANCE: We first report that hyperglycemia impairs the immunomodulatory and therapeutic potential of infused BMMSCs in osteoporosis. These findings suggest a potential challenge for the clinical utility of BMMSCs in treating autoimmune and skeletal diseases.

Sun, Hualing1; Wu, Yanru1; Fu, Dongjie1; Liu, Yinchen1; Huang, Cui1 The State Key Laboratory Breeding Base of Basic Science of Stomatology and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan

1

OBJECTIVES: To evaluate the effect of a aging-related gene SIRT6, a NAD-dependent deacetylase on osteogenesis of rat bone marrow mesenchymal stem cells (rBMSCs). METHODS: SIRT6 cDNA was cloned into lentiviral vector. rBMSCs with enhanced or reduced SIRT6 function were developed. Their osteogenic abilities were examined by qRT-PCR, ALP staining, Alizarin staining, and Von kossa staining. The effect of SIRT6 knockdown on NK-κb signaling pathway was evaluated by a dual-luciferase reporter assay system. Additionally, the bone formation ability of SIRT6 overexpression rBMSCs combined with collagen/chitosan/ hydroxyapatite scaffold (CCHS) was investigated in rat calvarial defect in vivo. RESULTS: SIRT6 knockdown significantly reduced the mRNA levels of several key osteogenic markers in vitro, including alkaline phosphatase (ALP), Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN), while overexpression of SIRT6 enhanced their expression. Additionally, SIRT6 knockdown activated nuclear factor-κB (NF-κB) transcriptional activity and upregulated the expression of acetyl-NF-κB p65 (Lys310). The decreased osteogenic differentiation ability of rBMSCs could be partially rescued by the addition of NF-κB inhibitor BAY 11-7082. Furthermore, SIRT6 overexpression in rBMSCs combined with the use of CCHS could significantly boost new bone formation in rat cranial critical-sized defects, as determined by microcomputed tomography and histological examination. CONCLUSIONS: SIRT6 is mainly located in the nuclei of rBMSCs and plays an essential role in their normal osteogenic differentiation, partly by suppressing NF-κB signaling. SIGNIFICANCE: It is important to find SIRT6 agonist, which may be used to treat aging-related disease, such as osteoporosis.

P-16

miR-21 MODULATES THE IMMUNOREGULATORY FUNCTION OF MESENCHYMAL STEM CELLS Wu, Tingting1; Liu, Yi1; Fan, Zhipeng1; Xu, Junji1; Wang, Jinsong2; Zhang, Chunmei2; Chen, Wanjun3; Wang , Songlin2 Capital Medical University School of Stomatology Molecular Laboratory for Gene Therapy and Tooth Regeneration 3 National Institute of Dental and Craniofacial Research, National Institutes of Health 1

2

OBJECTIVE: Bone marrow mesenchymal stem cells (BMMSCs) are multipotent adult stem cells. microRNAs (miRNAs) have been indicatedto be closely involved in theosteogenesis in BMMSCs, but whethermiRNAs regulate the immunoregulatory function of mesenchymal stem cells is incompletely understood. The purpose of this study is to investigate effect of miR-21 on the immunoregulatory function of BMMSCs and its' mechanism. METHODS: miRNA expression was overexpressed or knockdown by transfection with miRNA mimics or inhibitor into MSCs; The protein expression levels of TGF-β1, IL-6, and IL-17 were detected by CBA; After preparing BMMSCs from miR-21-/-mice, the differentiation of Tregs and the expression levels of TGF-β1 were detected when these cells were co-cultured with CD4+T cells; miR-21 target was predicted in the database PicTar`miRTar`TargetScans; miR-21 target was confirmed by the Luiciferase assay, and the function of miR-21 was confirmed by Western blot assay; Knockdown of PTEN expression was conducted by transfection with PTEN siRNA; Western blot assay was conducted to detect the down stream pathway. The expression levels of TGF-β1

OROFACIAL STEM CELL RESEARCH SUMMIT

29

ABSTRACTS

was determined after using special pathway inhibitor LY294002 to block AKT pathway. Colitis was induced in C57BL/6 mice during a 9-day time course with DSS-feeding on the second day. Then BMMSCs were transferred into mice with experimental colitis. Body weight changes %, colitis score, proportion of Tregs in mesenteric lymph nodes, and Hematoxylineosin staining of the anus were showed in each groups of mice. RESULTS: Here, we demonstrate that miR-21 negatively regulates the paracrine activity of immunoregulatory cytokine TGF-β1 in BMMSCs. Over-expression of miR-21 in BMMSCs resulted in decreased TGF-β1 expression, whereas knockdown of miR-21 increased TGF-β1 expression. BMMSCs from miR-21-/- mice enhanced their immunosuppressive function compared to those from wild-type (WT) mice. Anti-TGF-β1 eliminated the effect of miR-21 on the immunoregulatory functions of BMMSCs. Mechanistically, bioinformatic analysis identified potential targets of miR-21 in the 3’-untranslated region (3’UTR) of the phosphatase and tensin homolog deleted on chromosome ten (PTEN). miR-21 inhibited TGF-β1 expression by suppressing PTEN translation, as knock-down of PTEN using siRNA partially abolished the inhibitory effect of miR-21 on TGF-β1. Furthermore, after targeting PTEN, miR21 inhibited TGF-β1 expression via promoting activation of AKT, and then following promoting activation of the NF-κB pathways in BMMSCs. Importantly, adoptive transfer of miR-21-/--BMMSCs into mice with experimental colitis could more effectively ameliorate both the clinical and histopathological severity of colonic inflammation. This therapeutic effect was abolished when anti-TGF-β1 antibody was con-injected with miR-21-/--BMMSC in the colitis mice. CONCLUSIONS: We have uncovered a previously unrecognized mechanism that may interfere the immunoregulatory function of BMMSCs through miR-21 regulation of TGF-β1 activity. SIGNIFICANCE: This study for the first time uncovered a previously unrecognized mechanism that miR-21 controls immunoregulatory function of BMMSCs through inhibition of TGF-β1 activity. Our findings provide new insights into understanding the regulatory role of miRNAs in the process of MSCs, which may have implications in developing a new therapy for autoimmunity and other inflammatory diseases.

P-17

WHOLE TOOTH REGENERATION IN THE JAWBONE OF SWINE BASED ON CELL RE-ASSOCIATED TECHNIQUE Wu, Zhifang 1; Wang , Fu2; Wang, Songlin3 Molecular Laboratory for Gene Therapy & Tooth Regeneration, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, School of Stomatology, Capital Medical University 2 Department of Oral Basic Science, College of Stomatology, Dalian Medical University 3 Molecular Laboratory for Gene Therapy & Tooth Regeneration, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, School of Stomatology, Capital Medical University, Department of Biochemistry and Molecular Biology 1

OBJECTIVES: Whether the strategy of epithelial-mesenchymal interaction-based whole-tooth regeneration can be applied to humans remains elusive. An essential pre-clinical study in large animal is required. Here, we developed feasible methods for whole tooth regeneration in minipigs based on the tooth developmental biology. METHODS: Single epithelial and mesenchymal cells were dissociated from cap staged tooth germ of the deciduous second incisor, and then reconstituted into re-associated tooth germs. The re-associated and intact tooth germs were cultured in an improved high-throughput suspension culture system loaded with Aspirin, and then transplanted into the jawbone of host miniature pigs pretreated with BMMSC infusion. RESULTS: Images from computer tomography showed a calcified tissue with tooth-like shape in transplantation area. Histological analysis of the regenerated explant showed it contained organized enamel, dentin, cementum, periodontal membrane and pulp tissues similar with the natural tooth structure. CONCLUSIONS: Taken together, we developed a practical method for whole tooth regeneration with the swine tooth germ cell-based scaffold-free approach. SIGNIFICANCE: This study will offer a reference for studying regeneration of human tooth in situ.

30

PENN DENTAL MEDICINE

RE-OSSEOINTEGRATION FOLLOWING REGENERATIVE THERAPY OF TISSUE-ENGINEERED BONE IN PERI-IMPLANTITIS Xu, LY1; Wen, J1; Li, GL1; W, X1; Jiang, XQ1 Department of Prosthodontics, Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University; Oral Bioengineering and Regenerative Medicine Lab, Shanghai Key Laboratory of Stomatology, Ninth People’s Hospital, School of Medicine, Shanghai

1

OBJECTIVES: This study was designed to see whether the adoption of tissue-engineered bone complex of adipose-derived stem cells (ASCs) and bone morphogenetic protein-2 (BMP-2) gene delivery would work efficiently in the correction of experimental peri-implantitis. METHODS: All premolars in both side of mandibular were removed from six beagle canines three months before implant placement. Typical peri-implantitis were then induced by three month ligature placement. After the implementation of identical anti-bacterial and mechanical debridement therapy, the shaped peri-implant defect were stuffed with four groups of constructs, as A: beta tricalcium phosphate (β-TCP); B: β-TCP with adipose-derived stem cells (ASCs); C: β-TCP with enhanced green fluorescent protein gene transduced ASCs (AdGFP-ASCs); and D: β-TCP with bone morphogenetic protein-2 gene-modified ASCs (AdBMP-2-ASCs). Systematic radiographic, micro-CT, and histomorphometrical assessments were performed. RESULTS: After six months of healing, more bone formation and reosseointegration was found around the implant of groups B and C than group A. And group D further promoted the new bone height and re-osseointegration percentage. Moreover, sequential fluorescence labeling tells that group D exhibited the quickest and strongest bone formation on the cleaned implant surface during the entire observation period as compared to the other three groups. CONCLUSIONS: These data demonstrated that tissue engineered bone of ASCs, BMP-2 gene delivery, and β-TCP could exert powerful therapeutic effect on peri-implantitis as expected, which may suggest a feasible way to maintain the stability and masticatory function of dental implant. SIGNIFICANCE: Re-osseointegration is difficult to realize by current therapies. Here we demonstrated the positive regenerative effect of gene modified stem cell in the correction of intractable bone defect.

P-19

ABSTRACTS

P-18

USING SILICA NANOPARTICLES ENCAPSULATING ASPIRIN/ BMP-2 TO PROMOTE BONE FORMATION Xu, Xiaowei1; Zhang, Kai2; Zhao, Liang1; Wang, Dandan1; Bu, Wenhuan1 College and Hospital of Stomatology, Jilin University College of Chemistry, Jilin University

1

2

OBJECTIVES: Regeneration of large bone defects is still challenging for the clinician, since there is little achievement on the regulation of bone material and bone defect niche. In this study, the bone formation ability of silica nanoparticles (SNs) encapsulating aspirin/BMP-2 gene is studied in vitro and in vivo to reveal their potential in bone regeneration. Materials & METHODS: The carbon dots (CDs) were prepared by pyrolysis of aspirin and SNs were synthesized by an ethyl ether emulsion approach which covalently grafted PEI on the surface. Drug delivery efficiency and gene binding capability were evaluated by UV-vis spectroscopy measurement and agarose gel electrophoresis. In vitro anti-inflammatory effect of aspirin and CDs was compared by quantitative real-time polymerase chain reaction using RAW264.7 cell line. The bone formation ability of SNs encapsulating aspirin/BMP-2 gene was evaluated by Micro-CT after 12 weeks. RESULTS: The size of CDs and SNs observed by transmission electron microscopy were 3~5nm and 100~200nm, respectively. Drug loading efficiency of SNs could reach 21.08% and higher gene expression of plasmid DNA was obtained at a weight ratio of 30:1. Antiinflammatory effect of CDs was more obvious than that of aspirin by measuring pro-inflammatory cytokines (IL-1β, TNF-α and MMP-9) under the stimulation of LPS. Furthermore, we demonstrated that bone formation area of SNs encapsulating aspirin/BMP-2 gene group could reach 69.8±3.5%, which was significantly higher than that of the control group (26.4±1.6%) (P<0.01), SNs/aspirin group (36.9±1.4%) (P<0.01) and SNs/BMP-2 group (50.9±1.7%) (P<0.01). CONCLUSIONS: Our results demonstrate that SNs encapsulating aspirin/BMP-2 could efficiently deliver the drug aspirin, functionally express BMP-2 plasmid in vivo, and effectively possess bone regeneration ability compared to SNs encapsulating BMP-2 plasmid only. SIGNIFICANCE: SNs encapsulating aspirin/BMP-2 gene could efficiently deliver the drug aspirin and functionally express BMP-2 plasmid in vivo, suggesting their potential applications in bone regeneration.

OROFACIAL STEM CELL RESEARCH SUMMIT

31

ABSTRACTS

P-20

Tcf12 SUPRESSES MESENCHYMAL STEM CELLS OSTEOGENIC DIFFERENTIATION Yi, Siqi1; Zhang, Yufeng1 Wuhan University, Stomatological Hospital

1

OBJECTIVES: In studies described here, we documented the effect of Tcf12 on differentiation and mineralization of mouse mesenchymal stem cells. METHODS: In order to investigate the functional role of Tcf12 in differentiation of mMSCs, lentivirus system was employed during the inducing differentiation period. RESULTS: We observe that downregulation of Tcf12 significantly enhanced mRNA level of the vital regulator of mMSCs differentiation, including Alp, Ocn, Bsp, while overexpression of Tcf12 decreased their expression. What's more, downregulation of Tcf12 enhanced alkaline phosphatase (ALP) activity and boosted mineralization nodules formation. Unexpectedly, we observe knockdown the expression of Tcf12 can significantly promote the phosphorylation of Erk1/2 and p-smad1/5. CONCLUSIONS: these data supported knockdown of Tcf12 can promote the differentiation of mMSCs, partially by promoting Erk signaling and BMP signaling.

SIGNIFICANCE: the present study reveals the functional role of Tcf12 during the osteogenic differentiation period.

P-21

FUNCTION OF SLC7A11 IN OSTEOGENIC DIFFERENTIATION OF HASCS Zhang, Ping1 Peking University School, Hospital of Stomatology

1

The cystine-glutamate transporter SLC7A11, also known as xCT, has been identified to be a critical regulator of tumor migration and invasion, and some central nervous system disease. Generally, the function of this glutamate transporter remains largely unclear. In the present study, we detected that both the mRNA and protein level of SLC7A11 were significantly decreased during the osteogenic differentiation of human adipose-derived stem cells(hASCs). In order to investigate the potential role of SLC7A11 during the osteogenic commitment, we next established SLC7A11 stable knock down cells. After osteogenic induction, we find that the down-regulation of SLC7A11 led to increased osteogenic differentiation examined by ALP staining and quantification. Moreover, the extracellular matrix mineralization was also augmented in SLC7A11 knockdown cells at 2 weeks after osteogenic induction. In addition, knockdown of SLC7A11 resulted in significantly increased mRNA expression levels of RUNX2 and BGLAP in contrast to the control cells. Most importantly, the expression of SLC7A11 was increased in the bone tissue sections of ovariectomized mice as determined by immunohistochemistry staining. Collectively, these data suggested an unrevealed role of SLC7A11 in regulating osteogenic commitment of hASCs.

P-22

Foxc2 POTENTIATES BMP2-INDUCED OSTEO-/ODONTOGENIC DIFFERENTIATION OF HUMAN SCAP Zhang, Wen; Ling, Junqi Department of Operative Dentistry and Endodontics, Guanghua School and Hospital of Stomatology & Guangdong Province Key Laboratory of Stomatology, Sun Yat-sen University OBJECTIVES: Stem cells from apical papilla (SCAP) own powerful osteo-/odontogenic differentiation capability, which are regarded as the major cell source for root dentin development. Bone morphogenetic protein 2 (BMP2) and Foxc2 play a pivot role in modulating osteo-/odontogenic differentiation of SCAP and dentin formation. However, the synergy effects of BMP2 and Foxc2 on osteo-/odontogenic differentiation of SCAP are still unclear. Current study aimed to investigate proliferation and osteo-/odontogenic differentiation characters of BMP2 and Foxc2 gene co-transfected SCAP (SCAP-BMP2-Foxc2) in vitro. 2) METHODS: Basic characters of the isolated SCAP were identified by multipotential differentiation induction and flowcytometry. Lentiviral vector mediated gene transfection was applied on SCAP to construct blank vector transfected SCAP (SCAP-GFP), BMP2 gene transfected SCAP (SCAP-BMP2), Foxc2 gene transfected SCAP (SCAP-Foxc2) and SCAP-BMP2-Foxc2. Cell Counting Kit 8 was used to analyze the proliferation character of the four group cells. The

32

PENN DENTAL MEDICINE

P-23

ABSTRACTS

expression of osteo-/odontogenic genes and protein of the cells were evaluated by real-time PCR and Western blot. The mineralized nodules formed by the four group cells were visualized by ALP and Alizarin Red staining. RESULTS: Among four group cells, SCAP-Foxc2 demonstrated superior proliferation status, and SCAP-BMP2-Foxc2 showed inferior proliferation during eight days observation. SCAP-BMP2-Foxc2 obtained significantly better expression of ALP, OCN, DSPP, DMP1 and DSP than the other three groups at most of the time points. Furthermore, SCAP-BMP2-Foxc2 exhibited significantly more ALP positive mineralized nodules than other groups after sixteen days culture in vitro. CONCLUSIONS: Lentiviral vector mediated BMP2 and Foxc2 gene co-transfection was more effective in activating the osteo-/odontogenic differentiation potential of human SCAP than a single gene transfection. SIGNIFICANCE: Foxc2 potentiates BMP2-induced osteo-/ odontogenic differentiation of human stem cells from apical papilla in vitro.

VASCULARIZATION STRATEGY FOR STEM CELL-BASED TISSUE REGENERATION Zhang, Wenjie1; Li, Guanglong1; Jiang, Xinquan1 Department of Prosthodontics, Ninth Peopleâ&#x20AC;&#x2122;s Hospital Affiliated to Shanghai Jiao Tong University, School of Medicine

1

OBJECTIVES: Stem cell-based tissue engineering shows great advantages in promoting tissue regeneration. But there is still a major obstacle in maintaining the survival of the seeded cells within a large scaffold for critically-sized tissue regeneration due to low degree of vascularization. A special design by co-culture of Human umbilical vein endothelial cells (HUVECs) with arrayed hollow channels modified silk scaffolds was applied in this study in an attempt to facilitate fast vascularization to enhance large stem cell-based tissue regeneration. METHODS: Large cylindrical silk scaffolds with arrays of linear hollow channels were fabricated. HUVECs were pre-seeded in the scaffolds and incubated for 7 days in vitro. MTT assay and VEGF ELISA assay were applied to assess the biological activities of HUVECs during in vitro culture. CM-Dil labeled HUVECs were was used to trace capillary-like tubes formation initially after implanting subcutaneously in nude mice. And microfil perfusion was applied to test the function of the newly formed capillary vessels. We also sequentially detected the process of vascularization using a Laser Doppler-based perfusion meter. Finally, we evaluated the effect of this vascularization strategy as stem cells carrier using in vivo bioluminescence imaging method. RESULTS: In vitro, hollow channels improve HUVECs survival, VEGF secretion and prevascularization. Three days after implantation in vivo, those Dil-labeled cells still formed typical capillary-like tubes. At 7 days after implantation, most of the newly-formed vessels were filled with red cells and microfil. Hollow channels and pre-seeded HUVECs synergistically promoted scaffold rapid vascularization. And for stem cells carrying, the intensity of light emission in 254C/HUVECs group was largest in all four groups. CONCLUSIONS: In this study, we successfully constructed a new strategy by combining 254 Îźm hollow channels and prevascularization with HUVECs to promote in vivo rapid vasularization and integration of large-scale silk scaffolds, which further enhanced the survival of transplanted stem cells. SIGNIFICANCE: The prevascularization strategy provides an option for the use of stem cells with enhanced survival for the regeneration of large tissues and complex organs by overcoming mass transfer limitations via improved scaffold designs.

P-24

THE FUNCTION OF ALK2 IN TOOTH DEVELOPMENT Zhang, Xue1; Liu, Qi Lin1 Jilin University

1

As is known to all, ALK2 is the receptor of BMP signaling, and have multiple functions in vivo. However, the function of ALK2 in tooth development is little known. In our experiment, we use the osterix-cre;ALK2-floxed mouse model to study the possible function of ALK2 in tooth development. Now,our previous micro-CT and HE staining has shown us that the conditional knockout of ALK2 can lead to dentin dysplasia and even bone-like dentin in the lingual part of the mandibular incisor.Apparentlly,dentin dysplasia means thinnner dentin and broader pre-dentin in ALK2 knockout mouse.All of this hint us ALK2, as a receptor of BMPs,is important in tooth development.

OROFACIAL STEM CELL RESEARCH SUMMIT

33

ABSTRACTS

P-25

THE LOSS OF ODONTOGENIC POTENTIAL IN CULTURED EMBRYONIC DENTAL MESENCHYMAL CELLS FROM MICE Zheng, Yunfei1,2,3; Cai, Jinglei3; Hutchins, Andrew Paul3; Jia, Lingfei2; Liu, Pengfei3; Yang, Dandan3; Chen, Shubin3; Ge, Lihong4; Pei, Duanqing3; and Wei, Shicheng1,2 Department of Oral and Maxillofacial Surgery, Laboratory of Interdisciplinary Studies, Peking University School and Hospital of Stomatology, Beijing 100081, P.R. China 2 Central Laboratory, Peking University School and Hospital of Stomatology, Beijing 100081, P.R. China 3 Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, P.R. China 4 Department of Pediatric Dentistry, Peking University School and Hospital of Stomatology, Beijing 100081, P.R. China 1

OBJECTIVES: Tooth regeneration is a very promising potential use of cell-based regeneration. Mouse dental mesenchymal cells from E14.5 tooth germs possess vigorous tooth forming potential (odontogenic potential). Efficient culturing of dental mesenchymal cells not only ameliorates the laborious experimental procedures of acquiring cells from embryonic tissues, but also provides insight into the derivation of cells with odontogenic potential from pluripotent stem cells. However, the impact of in vitro culture on the odontogenic potential of dental mesenchymal cells remains unknown. Thus, we undertook to test the odontogenic potential of cultured mouse dental mesenchymal cells. METHODS: Dental mesenchymal cells from E14.5 ICR mouse lower molars were isolated and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. To examine the odontogenic potential of dental mesenchymal cells, freshly isolated and cultured dental mesenchymal cells were harvested and recombined with freshly isolated E14.5 dental epithelium prior to subrenal transplantation in adult male ICR mice. We also performed RNA sequencing to explore the changes in dental mesenchymal cells as the cells adapt to culture. RESULTS: We found that the tooth-forming potential in dental mesenchymal cells was maintained for only 1 day and was completely impaired afterwards, indicative of suboptimal culture medium and/or in vitro differentiation. Dental mesenchymal cells cultured for 1 day gave rise to tooth structures with a ratio of 60%, but cells cultured for 48 h failed to regenerate a tooth. RNA sequencing revealed massive changes in their transcriptome even after just 12 hours in cell culture. The transcriptome of cultured dental mesenchymal cells correlated well with each other, but separated from that of freshly isolated dental mesenchymal cells. Amongst these gene expression changes we noticed a significant loss of marker genes for the dental mesenchyme, particularly the loss of Msx1, Pax9, Lhx6, and Pdgfrα. This loss of dental mesenchymespecific markers correlated strongly with the ability to form tooth. CONCLUSIONS: The odontogenic potential of dental mesenchymal cells is lost after 1 day in culture owing to the loss of dental mesenchyme-specific genes including Msx1, Pax9, Lhx6, and Pdgfrα. SIGNIFICANCE: Our work revealed the characteristics and behavior of dental mesenchymal cells in culture and suggested the need for efficient culturing approach of odontogenic dental mesenchymal cells.

P-26

AUTOPHAGY REGULATES PRE-ODONTOBLAST DIFFERENTIATION VIA SUPPRESSING NF-kB IN INFLAMMATORY ENVIRONMENT Zhi, Chen1; Zhang, Lu1; Pei, Fei1 1

School and Hospital of Stomatology, Wuhan University

OBJECTIVES: To explore the role of autophagy in pre-odontoblasts differentiation in inflammatory environment in cells in vitro and caries teeth in vivo. METHODS: To explore the capability of odontoblasts differentiation, mDPC6T (preodontoblast cell line) were cultured with complete medium (Ctrl), mineralized induced medium (MM) and mineralized induced medium added LPS (MM+LPS). The mineralized nodules were stained with Alizarin red S after cells cultured for 14 days. Proteins were collected for ALP activity and western blot analysis. Monodansylpentane (MDH) and mRFPEGFP-LC3 plasmid were used to detect autophagosome and autophagy flux. To confirm the role of autophagy in the upregulation capacity of differentiation in LPS treated pre-odontoblasts, autophagy was blocked with CQ, and enhanced with

34

PENN DENTAL MEDICINE

P-27

ABSTRACTS

rapamycin. BAY 11-7082 was used to inhibit the activation of NF-kB. To further investage the mechanism, Atg5 siRNA was transiently transfected to cells to inhibit autophagy. The distribution of LC3 and p-NF-kB was detected with double immunofluorescense in pre-odontoblasts and caries teeth with reactionary dentin. RESULTS: The differentiation capacity of pre-odontoblast was enhanced with LPS stimulation, which was proved with increased mineralized nodules and ALP activity. The expression of DSP, DMP1 and OSX was up-regulated during pre-odontoblasts differentiation in MM+LPS. Autophagy markers LC3, Atg5, Beclin1 and TFE3 were also increased time dependently. The autophagosome was detected with monodansylpentane (MDH) staining, and autophagy flux was observed to be increased with mRFP-EGFP-LC3 transfection. These suggest autophagy was induced in pre-odontoblasts cultured with MM+LPS. Mineralized nodules were decreased when inhibit autophagy with CQ, while they increased with rapamycin treatment. The expression of DSP, DMP1 and OSX was consistent with Alizarin red staining RESULTS. Autophagy was induced and essential to regulate the differentiation of pre-odontoblast in inflammatory environment. The expression of p-NF-kB was found negatively related with autophagy, meanwhile it inhibited the differentiation of pre-odontoblasts. The colocalization of p-NF-kB and LC3 can be detected in cells cultured with MM+LPS. The nucleus translocation of p-NF-kB was increased when autophagy was inhibited with Atg5 siRNA in vitro. The colocalization of p-NF-ÎşB and LC3 in odontoblasts and sub-odontoblastic layer was confirmed further in caries teeth with reactionary dentin. CONCLUSIONS: Autophagy regulates the differentiation of odontoblast via suppressing the activation of NF-kB in inflammatory environment. SIGNIFICANCE: It showed a new insight into the mechanisms of dentin-pulp complex defense in inflammation invasion, and the knowledge might be useful for pulp regeneration in inflammatory environment.

PERIODONTAL LIGAMENT STEM CELLS PLAY DISTINCT ROLES IN PERIODONTIUM REGENERATION Zhu, B1; Hu, C1; Zhang, L1; Ding, Y2; Jin, Y1 Research and Development Center for Tissue Engineering, School of Stomatology, The Fourth Military Medical University Department of Orthodontic, Fourth Military Medical University

1

2

OBJECTIVES: Periodontitis is a highly prevalent chronic inflammatory bone disease. It destructs periodontium seriously, then leads to alveolar bone absorption and teeth missing. Periodontal ligament stem cells (PDLSCs) are proved to have the potential to form cementum/PDL-like tissue in vivo. Previously, we found that the osteogenic differentiation of PDLSCs was inhibited significantly in inflammation microenvironment. However, how to rescue their function are largely unknown.

METHODS: Culture PDLSCs of normal (N-PDLSCs) and periodontitis patients (P-PDLSCs) respectively and human bone marrow-derived mesenchymal stem cells (BMMSCs). Compare their proliferation via FCM and EDU analysis, multidifferentiation capacity through oil-red, alizarin red staining, RT-PCR, western blotting and immunohistochemistry. Transfect miR-17 into PDLSCs and testify the function changed. Construct PDLSCs/BMMSCs aggregates system, and observe their effect on periodontium regeneration. RESULTS: P-PDLSCs manifested decreased osteogenic differentiation and increased proliferation ability. MiR-17 regulated the function of N-PDLSCs and P-PDLSCs via targeting different genes under different environment. The differentiation capacity was more distinct in BMMSCs than in PDLSCs, and BMMSCs showed stronger resistance to inflammation cytokine compared to PDLSCs. PDLSCs/BMMSCs aggregates could repair the periodontium defect successfully. CONCLUSIONS: Our RESULTS proved that recovering the function of endogenous PDLSCs and using exogenous PDLSCs/BMMSCs complex aggregates could rescue the regeneration of periodontium. SIGNIFICANCE: Our results may provide therapeutic METHODS to solve the periodontitis problem.

OROFACIAL STEM CELL RESEARCH SUMMIT

35

NOTES

36

PENN DENTAL MEDICINE

NOTES

OROFACIAL STEM CELL RESEARCH SUMMIT

37

NOTES

38

PENN DENTAL MEDICINE

THANK YOU THIS RESEARCH SUMMIT IS IN PART MADE POSSIBLE THROUGH

Global Education Fund