Issuu on Google+


Editorial Boards of JIDMR 2010 Assoc. Prof. Dr. Izzet YAVUZ Editor-in-Chief and General Director Assist. Prof. Dr. Ozkan ADIGUZEL Associate Editor and Director Assoc. Prof. Dr. Refik ULKU Associate Editor for Medicine Prof. Dr. Zulkuf AKDAG, Prof. Dr. Sinerik N. AYRAPETYAN Associate Editor for Biomedical research Assoc. Prof. Dr. Filiz ACUN KAYA, Assist. Prof. Dr. Sabiha Zelal ULKU Associate Editor for Dentistry PhD. Dr. Ediz KALE Language Editor Advisory Board Betul KARGUL (TURKEY) Ferranti WONG (UNITED KINGDOM) Filiz ACUN KAYA (TURKEY) Gauri LELE (INDIA) Gulten UNLU (TURKEY) Jalen Devecioglu KAMA (TURKEY) Moschos A. PAPADOPOULOS (GREECE) Nik Noriah Nik HUSSEIN (MALAYSIA) Sabiha Zelal ULKU (TURKEY) Sadullah KAYA (TURKEY) Editorial Board Xiong-Li YANG (CHINA) Zurab KOMETIANI (GEORGIA) Gajanan Kiran KULKARNI (CANADA) Yuri Limanski (UKRAINE) Gulten UNLU (TURKEY) Nik Noriah Nik HUSSEIN (MALAYSIA) Rafat Ali Siddiqui (PAKISTAN) Jalen Devecioglu KAMA (TURKEY) Ferranti WONG (UNITED KINGDOM) Betul KARGUL (TURKEY) Susumu Terekawa (JAPAN) Moschos A. PAPADOPOULOS (GREECE) Izzet YAVUZ (TURKEY) Halimah AWANG (MALAYSIA) Serdar ERDINE (TURKEY)

Margaret Tzaphlidou (GREECE) Ozkan ADIGUZEL (TURKEY) Muhammad FAHIM (INDIA) Eylem OZDEMIR (TURKEY) Smaragda KAVADIA (GREECE) Sabiha Zelal ULKU (TURKEY) Abdel Fattah BADAWI (EGYPT) Sossani SIDIROPOULOU (GREECE) Mostaphazadeh Amrollah (IRAN) Sadullah KAYA (TURKEY) Medi GANIBEGOVIC (BOSNIA and HERZEGOVINA) Filiz Acun KAYA (TURKEY) Stephen D. SMITH (UNITED STATES OF AMERICA)


Mehmet Zulkuf AKDAG (TURKEY) Sinerik N. AYRAPETYAN (ARMENIA) Süleyman DASDAG (TURKEY) Gauri LELE (INDIA) Ali GUR (TURKEY) Refik ULKU (TURKEY) Shailesh LELE (INDIA) Murat AKKUS (TURKEY) Fadel M. Ali (EGYPT) Betul URREHMAN (UAE) Alpaslan TUZCU (TURKEY) Igor BELYAEV (SWEDEN) Meral ERDİNÇ (TURKEY) Muhammed Mustahsen Urrehman (UAE) Sedat AKDENIZ (TURKEY) KING Nigel MARTYN (HONG KONG SAR, P R CHINA) Muzeyyen YILDIRIM (TURKEY) Christine Bettina STAUDT (SWITZERLAND) M.Sabri Batun (TURKEY) Selahattin TEKES (TURKEY) Claudia DELLAVIA ( ITALY ) Feriha CAGLAYAN (TURKEY) Marco MONTANARI (ITALY)

Aziz YASAN (TURKEY) Filippo BATTELLI (ITALY) Ugur KEKLIKCI (TURKEY) Ali Al-Zaag Iraq Zeki AKKUS (TURKEY) Benik Harutunyan (ARMENIA) Ayca DENIZ IZGI (TURKEY) Neval Berrin ARSERIM (TURKEY) Selahattin KATAR (TURKEY) Guliz Nigar GUNCU (TURKEY) Nurten ERDAL (TURKEY) Feral OZTURK (TURKEY) Serdar ONAT (TURKEY) Selahattin ATMACA (TURKEY) Mehmet Nuri OZBEK (TURKEY) Gonul OLMEZ (TURKEY) Orhan TACAR (TURKEY) Cemil SERT (TURKEY) S. Yavuz SANISOGLU (TURKEY) Ilker ETIKAN (TURKEY) Mahmut METE (TURKEY) Nezahat AKPOLAT (TURKEY) Kursat ER (TURKEY)


Journal of International Dental and Medical Research

ISSN: 1309-100X

TABLE OF CONTENTS DENTISTRY EFFECT OF PULPAL PRESSURE ON MICROTENSILE BOND STRENGTH OF SELF-ETCH SYSTEMS TO DENTIN. APPLICATION OF TWO ADHESIVE LAYERS Montanari Marco, Piana Gabriela Pages 1-5 A COMPARATIVE STUDY OF ORAL HEALTH ATTITUDES AND BEHAVIOR BETWEEN DENTAL AND MEDICAL STUDENTS; THE IMPACT OF DENTAL EDUCATION IN UNITED ARAB EMIRATES Sausan Al Kawas, Kauser Sadia Fakhruddin, Betul Ur Rehman Pages 6-10 INFLUENCE OF CANCELLOUS BONE RIGIDITY ON STRESS DISTRIBUTION IN BONE AROUND DENTAL IMPLANT: A FINITE ELEMENT STUDY Hasanen H. Al-Khafagy Pages 11-14 IN-HOME BLEACHING EFFECT ON COMPRESSIVE STRENGTH VALUES OF SOME DIRECT RESTORATIVE MATERIALS Ali A. Razooki Al- Shekhli Pages 15-18 NEW DIRECTION FOR MEASUREMENT OF MICROLEAKAGE IN CARIOLOGY RESEARCH Izzet Yavuz, Haluk Aydın Pages 19-24 EVALUATING THE FRACTURE STRENGTH OF THREE DIFFERENT PROVISIONAL CROWNS Mustafa Zortuk, Eylem Ozdemir, Suleyman Aguloglu Pages 25-28 INTERDISCIPLINARY APPROACH IN A TREATMENT OF THE PATIENT WITH CLEFT LIP AND PALATE – CASE REPORT Zukanovic Amila, Nakas E, Prohic S Pages 29-33 INTERESTING CASE: AN UNUSUAL LOCATION FOR A LARGE PLEOMORPHIC ADENOMA ARISING IN THE MAXILLA Rezzan Tanrıkulu, Ferhan Yaman, Serhat Atılgan, Nedim Gunes, Belgin Gorgun Pages 34-37 MEDICINE ANTIBIOTIC RESISTANCE AND PLASMID PROFILES OF VIBRIO ISOLATES FROM MUTHUPETTAI MANGROVE ENVIRONMENT, SOUTHEAST COAST OF INDIA P. Manivasagan, S. Ramesh, K. Sivakumar, T. Thangaradjou, S. Vijayalakshmi, T. Balasubramanian Pages 38-44 BIOMEDICAL RESEAERCH FIRST IDENTIFICATION OF A NOVEL PROBIOTIC BACTERIUM STREPTOCOCCUS PHOCAE AND THEIR BENEFICIAL ROLE IN DISEASES CONTROL Paulraj Kanmani, R. Satish Kumar, N. Yuvaraj, K. A. Paari, V. Pattukumar, Venkatesan Arul Pages 45-51

20010 Volume 3 - Number 1


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

Pulpal Pressure on Microtensile Bond Strength Montanari Marco and Piana Gabriela

EFFECT OF PULPAL PRESSURE ON MICROTENSILE BOND STRENGTH OF SELF-ETCH SYSTEMS TO DENTIN. APPLICATION OF TWO ADHESIVE LAYERS Montanari Marco1*, Piana Gabriela2 1. Fellowship DDS PhD; Unit of Dentistry for Disables; Department of Oral Science Alma Mater Studiorum University of Bologna, Italy. 2. Assoc. Prof. MD DDS; Unit of Dentistry for Disables; Department of Oral Science Alma Mater Studiorum University of Bologna.

Abstract The first objective of this study was to examine effect of simulated hydrostatic pulpal pressure (PP) on μTBS of Bond Force (BF) (Tokuyama Corp., Tokyo, Japan), applied in a single or double layer. The second objective was to evaluate dentin location influence (superficial and deep) on μTBS. Flat coronal dentin surfaces of extracted human molars were prepared. Two groups were created with two different conditions of PP. In group A no PP was present (0 cm) during BF application and composite build-up. In group B a PP (20 cm H2O) was present during BF application. Specimens bonded under PP were stored in water at 37C° under 20 cm H2O for 20 min. For both groups BF was applied in single (subgroups A1,B1) or double layer (A2,B2). Flowable resin composite (Estelite Flow Quick Tokuyama Corp., Tokyo, Japan) was used for the build-up. Beams with a 1.0 mm2 area were obtained and stressed to failure using microtensile tester. Application of two layers of BF showed higher μTBS than single application in all tested conditions. PP was responsible for a statistical reduction in μTBS only when Bond Force was applied in single coat. Superficial dentin showed higher values than deep dentin in all conditions but no statistical differences were found. A highly significant correlation was observed between droplets presence and μTBS results. The application of a double layer of BF is a clinical requirement to avoid the reduction in μTBS and prevent any interference of pulpal pressure. (J Int Dent Med Res 2010; 3: (1), pp. 1-5) Keywords: Permeability, water uptake; hydrophilic polymers; dentin bonding systems, microtensile bond strength, water droplets. Received date: 30 October 2009 Introduction The application procedure of a one-step self etch adhesive may take considerably less time compared with their multi-step counterparts, and this may be an important clinical advantage. Unfortunately they exhibit high permeability, resulting in water flow through the adhesive thus, exhibiting a dramatic reduction in bond strength *Corresponding author: Marco Montanari Department of Oral Science Via San Vitale 59, 40125 Bologna, Italy Phone: 0039 051 2088150 Fax: 0039 051 2088111 E-mail: montmarco@virgilio.it

Volume 3 ∙ Number ∙ 1 ∙ 2010

Accept date: 10 December 2009 after water storage1. The outward movement of dentinal fluid under a slight positive pulpal pressure may probably permeate polymerized hydrophilic adhesives2, hindering monomer infiltration into the demineralised collagen matrix contaminating the bonding surface with water. Pulpal pressure has been reported to influence the surface wetness and to affect the bond strength of different types of DBAs3 and it is in relationship with remain dentin thickness (RDT). Periotron device was used to measure the surface humidity of dentin samples, supporting the concept that the dentin surface is wet, especially after smear layer removal and under a physiological pulpal pressure3. For optimal bonding to dentin, the consequence of outward fluid through the dentinal tubules due to Page 1


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

the positive pulpal pressure should be taken into consideration. There have been many bonding studies performed under a simulated hydrostatic pulpal pressure2,4,5,6. However, few in vitro studies have tested the efficacy of a new all-inone adhesive under physiological condition (hydrostatic pulpal pressure) when applied in a single or double layer. The aim of this study was to evaluate the μTBS to deep and superficial dentin bonded with one or two coats of BF bonded and stored under zero and 20 cm H2O hydrostatic PP. The null hypothesis tested was that positive PP does not affect the bond strength of BF to dentin. Materials and Methods Twenty extracted human third molar teeth were stored in 4 °C water for no more than one month. A flat midcoronal dentin disc was prepared by removing the occlusal enamel with a slow-speed diamond saw (Remet, Bologna, Italy) under water cooling. A 180-grit silicon carbide paper was used under running water to create a clinically relevant smear layer on the dentin surface. The root of each tooth was removed below the cement-enamel junction so as to expose the pulp chamber. The pulpal tissue was removed with a small forceps, taking care to avoid touching the pulp chamber walls. A pincertype caliper was used for measurement of the remaining dentin thickness (RDT) that was between 0.9 and 1.5 mm. Each crown segment was attached to a Plexiglas platform (2 ×2 × 0.5 cm) and sealed with cyanocrylate (ROCKET Heavy DVA, Corona, CA, USA). Each platform was penetrated by a short length of 18-gauge stainless steel tube into a center hole created in a piece of Plexiglas. Each Plexiglas-tooth assembly was attached via polyethylene tubing to 20-ml syringe barrel filled with distilled water in order to produce a hydrostatic pressure of 20 cm H2O at the dentin surface to be bonded (Group A). In the control group, the barrel remained empty (Group B). The specimens of the two experimental groups were divided into two subgroups (n = 5) (A1, A2), (B1, B2) according to the number of BF layers applied. To the first subgroup (A1 and B1) a single layer of BF was applied, thinned with a gentle air spray and light cured for 20 s. In the other subgroup (A2 and B2) a first layer of BF was Volume 3 ∙ Number ∙ 1 ∙ 2010

Pulpal Pressure on Microtensile Bond Strength Montanari Marco and Piana Gabriela

applied, thinned with a gentle air spray followed by the application of a second layer, thinned once again with air and finally light cured for 20 s. Finally, a 5 mm-thick resin composite build-up was performed on the resin-bonded dentin surfaces using a light-cured flowable composite (Estelite Flow Quick, Tokuyama Corp., Tokyo, Japan). Each of four 1.5 mm increments was light-cured for 40 s at 600 mW/cm2 using a halogen curing unit (XL-2500, 3M ESPE, St. Paul, MN, USA). The bonded specimens were stored in water at 37°C for 20 min before testing. The simulated hydrostatic pulpal pressure (0 or 20 cm H2O) was maintained during storage. Microtensile Bond Strength Evaluation After a 20 min storage, all samples (A1, A2, B1, B2) were sectioned perpendicular to the adhesive interfaces into 1 mm-thick slabs using the a diamond saw under water cooling. Each slab was subsequently trimmed to produce resindentin beams with a cross-sectional area of 1.0 mm2 (measured with a digital calliper) at the bonded interface. Ten teeth were used for each group and ten to twelve beams were obtained from each tooth. The beams were then attached with cyanoacrylate to a testing jig, and loaded in tension with a universal testing machine (Bisco Inc., Schaumburg, IL, USA) at a crosshead speed of 0.9 mm/min. until failure. Tested specimens were mounted on stubs, sputter coated with gold, and observed with a scanning electron microscope (JSM-5200, JEOL, Tokyo, Japan) for evaluating the failure mode and the morphology the of fracture between dentin and composite build-up. The digitalized SEM images were subjected to quantitative image analysis using a digital slowscan image recording system (SemAfore, JEOL, Sollentuna, Sweden). The microtensile bond strength data was analyzed by using a two-way ANOVA to test the effect of the adhesive system and the experimental condition (simulated pulpal pressure or no pulpal pressure) on bond strength. Results The μTBS results are summarized in Table I. Bond strength was influenced by the number of BF layers applied and by pulpal Page 2


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

pressure during bonding and storage. Pulpal pressure was responsible for a considerable reduction of μTBS, especially when BF was applied in a single layer. The application of two layers of BF showed higher μTBS than the single application in all of the tested conditions. Superficial dentin showed higher bond strength than deep dentin in all tested conditions but no statistical differences were found with respect to deeper dentin.

Pulpal Pressure on Microtensile Bond Strength Montanari Marco and Piana Gabriela

was affected by droplets (c). Droplet dimensions varied from 1 to 20 μ. Figures 1e and 1f show the morphology of the bond surface when BF is applied in two coats in the presence of pulpal pressure. A cohesive failure inside the thickness of the resin composite can be observed (composite side).

Table I. Microtensile bond strengths (means ± standard deviation) of BF bonded to deep and superficial dentin with and without pulpal pressure application. Pulpal pressure was able to considerably reduce microtensile bond strength both for deep and for superficial dentin (Table II) but it had a greater influence in deep dentin.

Table II. Percentage of μTBS change in deep and superficial dentin under pulpal pressure. A highly significant correlation was observed between the presence of droplets inside adhesive layer and μTBS results. SEM micrographs of BF applied in a single layer did not show the presence of voids inside the adhesive thickness (Figure 1a and 1b). Figures 1c and 1d micrographs show representative samples of BF applied with pulpal pressure (dentin side). A mixed fracture pattern was observed. Almost the entire adhesive layer Volume 3 ∙ Number ∙ 1 ∙ 2010

Figure 1. SEM photomicrographs illustrating fractured samples bonded with BF (one layer). a and b micrograph show the morphology of bond surface when BF is applied without pulpal pressure (dentin side). Delamination of the bonding layer were observed. c and d micrographs shows a representative samples of BF applied with pulpal pressure (dentin side). A mixed fracture pattern was observed. Almost the entire adhesive layer was affected by droplets (c). Droplets dimension varying from 1 to 20 μ. e and f show the morphology of the bond surface when BF is applied in two coats in the presence of pulpal pressure. A cohesive failure inside the thickness of the resin composite can be observed (composite side).

Figure 2. Statistical analysis.

Page 3


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

Discussion In vitro simulated pulpal pressure adversely affected bonding of BF to coronal dentin. Therefore, the null hypothesis that positive pulpal pressure does not affect bond strength of BF must be rejected. The all-in-one self-etching adhesive contains hydrophilic and hydrophobic resin monomers with a high solvent content7, 8. The presence of water is essential for providing an ionization medium for self-etching activity9. Prior to photopolymerization, the complete elimination of both water and solvents is needed. The role played by volatile solvents (acetone or ethanol) in promoting water displacement from the dentinal surface is well established10. Water that permeated dentin under a simulated pulpal pressure may have resulted in the lower achieved under pulpal pressure. When a simulated hydrostatic positive pressure is applied to dentin, an outward fluid flow from the dentinal tubules may occur across the smear layer, resulting in “wet bonding” instead of the recommended dry bonding for which these onestep adhesives are ideally designed. In the specimens bonded without simulated pulpal pressure, the water in the adhesive can be evaporated by an air blast. However, specimens bonded under a simulated pulpal pressure may replace that of evaporated water. HEMA-based adhesives are prone to hydrolytic degradation, resulting in reduction of their mechanical properties11. The study demonstrated that the adhesion of BF may be influenced by simulated pulpal pressure only when a single layer of bonding agent has been applied on dentin surface (Figure 2). The resindentin bond achieved with two layers of BF appeared less sensitive to the application of hydrostatic pulpal pressure respect to the application of a single layer. The application of the second layer on the primed dentin may increase hydrophobic layer thickness, prevent the formation of water channels in the adhesive layer after polymerization improving the degree of conversion. Moreover, the second layer may probably fill all the porosities and voids created by the application of first layer and produce a solid layer of resin with a greater and deeper anchorage to the collagen fibers of exposed dentin. In the present study, deep and superficial dentin were divided because of the different Volume 3 ∙ Number ∙ 1 ∙ 2010

Pulpal Pressure on Microtensile Bond Strength Montanari Marco and Piana Gabriela

permeability rate and the different orientation of tubules. In fact it is well known that dentin has far more tubules in the deepest area near the pulp than on the surface, close to enamel junction.12 Conclusions Pulpal pressure is responsible for a significantly reduction in bond strength when adhesives are applied in deep dentin. All-in-one adhesive systems are particularly prone to pulpal pressure. The present study showed that the clinical performance of All-in-one adhesive systems could be improved by the application of a double layer before the polymerization. This particular type of application is important especially in deep dentin in which the effect of pulpal pressure is well detectable. Declaration of Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article. References 1. De Munck J, Van Landuyt K, Peumans M, Poitevin A, Lambrechts P, Braem M, Van Meerbeek B. A critical review of the durability of adhesion to tooth tissue: methods and results. J Dent Res 2005; 84: 118–132. 2. Sauro S, Pashley DH, Montanari M, Chersoni S, Carvalho RM, Toledano M, Osorio R, Tay FR, Prati C. Effect of simulated pulpal pressure on dentin permeability and adhesion of self-etch adhesives. Dent Mater 2007; 23: 705-13. 3. Prati C, Pashley DH. Dentin wetness, permeability and thickness and bond strength of adhesive systems. Am J Dent 1992; 5: 338. 4. Elhabashy A, Swift EJ Jr, Boyer DB, Denehy GE. Effects of dentin permeability and hydration on the bond strengths of dentin bonding systems. Am J Dent 1993; 6: 123-6. 5. Hosaka K, Nakajima M, Monticelli F, Carrilho M, Yamauti M, Aksornmuang J, Nishitani Y, Tay FR, Pashley DH, Tagami J. Influence of hydrostatic pulpal pressure on the microtensile bond strength of all-in-one self-etching adhesives. J Adhes Dent 2007; 5: 437-442. 6. Tao L, Tagami J, Pashley DH. Pulpal pressure and bond strengths of SuperBond and Gluma. Am J Dent 1991; 4: 73-6. 7. Tay FR, Pashley DH. Have dentin adhesives become too hydrophilic? J Can Dent Assoc 2003; 69: 726-31. 8. Van Landuyt KL, De Munck J, Snauwaert J, Coutinho E, Poitevin A, Yoshida Y, Inoue S, Peumans M, Suzuki K, Lambrechts P, Van Meerbeek B. Monomer-solvent phase separation in one-step self-etch adhesives. J Dent Res 2005; 84: 183-8. 9. Schulze KA, Oliveira SA, Wilson RS, Gansky SA, Marshall GW, Marshall SJ. Effect of hydration variability on hybrid layer properties of a self-etching versus an acid-etching system. Biomaterials 2005; 26: 1011-8. 10. Tay FR, Gwinnett JA, Wei SH. Micromorphological spectrum from overdrying to overwetting acid-conditioned dentin in waterfree acetone-based, single-bottle primer/adhesives. Dent Mater 1996; 12: 236-44. 11. Ito S, Hashimoto M, Wadgaonkar B, Svizero N, Carvalho RM,

Page 4


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

Pulpal Pressure on Microtensile Bond Strength Montanari Marco and Piana Gabriela

Yiu C, Rueggeberg FA, Foulger S, Saito T, Nishitani Y, Yoshiyama M, Tay FR, Pashley DH. Effects of resin hydrophilicity on water sorption and changes in modulus of elasticity. Biomaterials 2005; 26: 6449-59. 12. Garberoglio R, Brännström M. Scanning electron microscopic investigation of human dentinal tubules. Arch Oral Biol 1976; 21: 355-62.

Volume 3 ∙ Number ∙ 1 ∙ 2010

Page 5


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

Oral Health Attitudes and Behavior between Students Sausan Al Kawas et al

A COMPARATIVE STUDY OF ORAL HEALTH ATTITUDES AND BEHAVIOR BETWEEN DENTAL AND MEDICAL STUDENTS; THE IMPACT OF DENTAL EDUCATION IN UNITED ARAB EMIRATES* Sausan Al Kawas1**, Kauser Sadia Fakhruddin2, Betul Ur Rehman3 1. Associate Professor Doctor & Head of Oral and Craniofacial Health Sciences Department, College of Dentistry, University of Sharjah, United Arab Emirates. 2. Doctor Lecturer, Oral and Craniofacial Health Sciences Department, College of Dentistry, University of Sharjah, United Arab Emirates. 3. Assistant Professor Doctor, Department of General & Specialist Dental Practice, College of Dentistry, University of Sharjah, United Arab Emirates.

Abstract Background: The aim of this study was to determine the differences in oral health attitudes and behavior between second year dental and medical students studying at the University of Sharjah, United Arab Emirates. Methods: The examination was mainly based upon responses to a questionnaire titled “Hiroshima University – Dental Behavioral Inventory (HU-DBI)”. Higher scores on the HU-DBI indicate better oral health attitudes/behavior. Results: The mean HU-DBI score of the 2nd-year dental students was significantly greater than that of the 2nd-year medical students (9.45 and 6.85, respectively; p<0.001). The percentages of ‘agree’ responses in two HU-DBI questionnaire items were significantly higher among dental students than medical students. They include: “dental visits” and “I am bothered by the color of my gums.” Over 60% of medical students seek dental care only when there is dental pain, compared with 25% of dental students (p<0.01). Also, when students were classified according to gender, significant differences of the mean HU-DBI scores were observed. Conclusion: Present study is the first formal assessment of dental health knowledge and oralhealth related behavior of second year dental and medical students, and there found to be considerable differences related to dental health attitudes/behavior, which indicates the impact of dental health knowledge on student oral health outcomes, such as their positive attitudes toward prevention and personal oral care. (J Int Dent Med Res 2010; 3: (1), pp. 6-10) Keywords: Oral health, knowledge, attitude, behavior, dental education, UAE. Received date: 19 December 2009 Introduction Although dental diseases are not lifethreatening, they are detrimental to the quality of life throughout the life span and can have an *This study was the third winner of poster presentations at the 16th International Dental Congress of Turkish Dental Association & FDI in Istanbul on June 27th 2009. **Corresponding author: Assoc. Prof. Dr., Sausan Al Kawas, (McGill University), FICD College of Dentistry, University of Sharjah P.O. Box: 27272 Sharjah, United Arab Emirates Tel: 00971-50-7929735, Fax: 00971-655585641 E-mail: sausan@sharjha.ac.ae

Volume 2 ∙ Number ∙ 3 ∙ 2009

Accept date: 04 January 2010 impact upon the self-esteem, nutrition and health of an individual. Oral diseases are associated with considerable pain, anxiety, and impaired social functioning 1,2. Oral health information is considered to be an essential prerequisite for health-related behavior. The first step in establishing a habit is to provide relevant information to the patients and to raise their awareness of how to prevent oral diseases3. After going through an undergraduate dental curriculum; dental students are expected to be a role model for oral health behavior. An important task of oral health professionals is to instill in their patients correct oral habits to prevent oral diseases. According to Peker et al. the attitudes and behavior of oral Page 6


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

health providers towards their own oral health reflect their understanding of the importance of preventive dental procedures and of improving the oral health of their target population 4. In the UAE, the oral health system is currently in transition. Our information is limited regarding the knowledge and attitudes about oral diseases and their prevention. Systematic data are needed for public oral healthcare planning. Due to a lack of studies about oral health attitudes and behavior among pre-university and university students in the UAE, this study is of prime importance in this field. The purpose of this study was to assess self-reported oral health attitudes and behavior among a group of dental and medical students at the University Colleges and to compare differences in oral health attitudes between second year dental and medical students at the University of Sharjah, United Arab Emirates.

Oral Health Attitudes and Behavior between Students Sausan Al Kawas et al

was analyzed for frequency distributions. Group comparisons were made using Mann-Whitney Utests for ordinal level data and chi-square tests for categorical data. The significance level was set at p≤0.05. Results One hundred and twenty-six participants from dental (n=63) and medical (n=63) colleges respectively, consented to participate in the study. The mean age of Dental and Medical students was 19.65 (SD=0.53). In both samples, the percentage of female students was higher (n=46, 73% and n=41, 65%, dental and medical, respectively).

Material and Methods The study population consisted of (N=126) students in the second year of medicine and dentistry programs at the University of Sharjah, UAE. The Research Ethics Board of the University of Sharjah approved the project. The students were invited to complete the English versions of a questionnaire titled “Hiroshima University – Dental Behavioral Inventory (HUDBI)” following a lecture. Participation in the study was voluntary and the answers were anonymous. Students were given the option of discontinuing at any time. To ensure anonymity the names were not recorded on the questionnaire. The HU-DBI questionnaire, consists of twenty polar responses (agree-disagree) regarding oral health-related behavior. In addition, two further questions about frequencies of brushing and flossing were included. A total score was calculated based on the response to each item. Higher scores on the HU-DBI indicate better oral health attitudes/behavior. The possible maximum score is 12. Each additional item, i.e. tooth brushing and flossing frequency, has 4 categories (4 times and more, 3 times, twice, and every day for tooth brushing frequency and once a week, once a month, and never, for flossing frequency respectively). The data was entered into SPSS version 14.0. and used for statistical analysis. The data Volume 2 ∙ Number ∙ 3 ∙ 2009

Table 1. Modified English version of HU-DBI Questionnaire survey. Overall, significant differences of the mean HU-DBI scores of dental (9.45±1.48) and medical students (6.85±1.63) were observed. Moreover, in a comparison of the HU-DBI means of medical and dental students by gender, there were statistically significant differences (p<.05) Table 2.

Table 2. Comparison of the HU-DBI Mean scores between school and by gender. Page 7


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

Oral Health Attitudes and Behavior between Students Sausan Al Kawas et al

The HU-DBI questionnaire items and percentage distribution and analysis of “agreed” responses of medical and dental students are shown in Table 3. Statistically significant differences were found for items 1, 4, 7, 10, 11, 15, and 19 between the two sample groups. Only 8% of the dental students were not much worried about visiting a dentist (item 1), compared to 30% of the medical students who were (p<0.01). Moreover, 46% of medical students reported that they “put off going to the dentist until have toothache” compared with 20% of dental students (p<0.01). About one third of the medical students reported that they “have never been taught professionally how to brush” (item 10), whereas 12% of the dental students agreed with this statement (p<0.01). Furthermore, from the medical group only 9% stated “noticing some white sticky deposits on teeth” versus 24% of dental students (p<0.01). According to gender, percentage and analysis of “agreed” responses are shown in Table 3. For both medical and dental students, statistically significant differences were found (p<.05) for items 3, 4, and 12 (I worry about the color of my teeth, I have noticed some white sticky deposits on my teeth, I often check my teeth in a mirror after brushing) respectively, and for items 1, 8 and 10 (I don’t worry much about visiting the dentist, I think my teeth are getting worse despite my daily brushing, and I have never been taught professionally how to brush) respectively.

Table 3. HU-DBI questionnaire and percentage of “agree” response by gender and school. A) D: dental school, M: medical school B) In the calculation of the HU-DBI: (A) = One point is given for each of these agree responses. (D) = One point is given for each of these disagree responses C) *Obtained using the chi-square test; upper left: between gender in D, lower left: between gender in M; right between Dental and Medical, *p<0.05, **p<0.01 , ns= non significant. Volume 2 ∙ Number ∙ 3 ∙ 2009

Table 4. Percentage of tooth brushing per day and flossing frequency by gender and school. *Obtained using Mann-Whitney U-tests, ns= non significant. *p<0.05, **p<0.01.

Page 8


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

In an analysis of oral-health related behavior (tooth brushing and flossing) by gender, medical and dental students showed highly significant differences (p<0.01) Table 4. 64% of female dental students floss every day compared with their male colleagues (25%; p<0.01). However, only about 25% of the female medical students reported that they floss every day, whereas only 14% of the male medical students agreed with this statement and there was found to be no statistical difference between the genders. Discussion Numerous descriptive, clinical, and health services studies make it clear that health behavior in general, and oral health behavior in particular are complex phenomena 5. Several studies have reported that dental health attitudes become more positive and improved with an increasing level of dental education 6-10. The improvement of personal oral health among dental students is linked to their dental education experience, and oral health attitudes and behavior seem to increase significantly in the final years of dental education 11-12. Hence, oral health knowledge is considered to be an essential prerequisite for health-related behavior. Conversely, few studies have suggested that weak association seems to exist between knowledge and behavior 13-14. In the UAE, no data are available on an assessment of the oral self-care level of adolescents. The present study was designed to assess levels of oral health knowledge, beliefs and self-reported behavior among 18-22-year-old medical and dental students, which provides new insights into the impact of curricula on student outcomes, such as their attitudes toward prevention and oral self-care. The second year dental students in the first year of their training program have received knowledge and skills to develop an understanding of the role of preventive dentistry and oral-health care, in the management of common oral health problems, e.g. dental caries, gingivitis, tooth wear etc., though the medical students have never received such information. The mean HU-DBI score of the dental students was high compared to the medical students, which suggested that dental students’ oral self-care levels may have been positively Volume 2 ∙ Number ∙ 3 ∙ 2009

Oral Health Attitudes and Behavior between Students Sausan Al Kawas et al

influenced by the course content related to dental health education. The results of this study were consistent with those reported by Kawamura et al. 15 of a survey, using the HU-DBI, which reported that the mean HU-DBI score of second year dental hygiene students was considerably higher than that of second year nursing students. Though the results were statistically significant, our sample size was relatively small, which was a limitation in our study; a further study with a larger sample population could reveal stronger relations than reported here. In a recent study, gender was a major factor influencing the HU-DBI percentage of agree/disagree responses for items (worry about the color of teeth, noticed some white sticky deposits on teeth and often check my teeth in a mirror after brushing). Female dental students showed a significantly better attitude than their male colleagues. This finding is consistent with previous studies 16-17. Generally, females are more likely to have positive self-care attitudes for internal psychosocial reasons, to improve their appearance and self-esteem. Two findings from the present study are of particular importance. The majority of the medical students reported that they “don’t worry much about visiting the dentist” and “put off going to the dentist until they have toothache”, which is identical to a study from Kuwait 18. These low rates of visiting dental clinics could be due to the UAE medical insurance system, which usually does not cover dental services. It might also be caused by tradition and lack of formal health care at the community level. There may be a previous bad dental experience, or simply the extra time required for frequent visits might not be acceptable. Lack of sufficient knowledge about dentistry can be a factor, as one’s knowledge and positive attitude toward good health care are very important in the preventive cycle 6-10. For that, there is a need for organized intervention, which could lead to improving oral and dental health status through increasing the population’s knowledge, attitudes, and behavior. The results also showed that young people in the UAE are not educated enough when flossing is concerned. Similar data on oral hygiene habits was found in Poland, Kuwait and Switzerland18-20, where respondents had a comprehensive basic dental knowledge and almost everyone used a toothbrush regularly, but Page 9


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

flossing fell short in the majority of cases. Dental health care education in the preuniversity curriculum can have a strong positive influence on oral-health related attitudes and behaviour. These results indicate the need for intervention through oral health education and promotion to alter individuals’ behaviour related to dental health. Additional studies are needed to evaluate information about oral health care information in the pre-university programs in the UAE, which could be an important factor influencing the oral-health-related attitudes and behaviour of the general population. Therefore, more emphasis should be placed on oral health education and basic oral health supplies through school-based and community oral health programs. As oral health means much more than healthy teeth, and is essential for general health2, the dentist’s role should not be limited only to provision of dental treatment. A major role should also be to raise the knowledge and awareness needed to prevent oral diseases. Conclusions This study was the first formal assessment of dental health knowledge and oralhealth related behaviour of medical and dental students. The present study cannot predict the participants’ actual oral health status, which is another limitation of our study. In the second phase of the study, we will assess the epidemiologic relationship between oral health attitudes and behaviour and oral health status of medical and dental students; also their oral and dental health knowledge and oral self care improvement with the increasing level of dental education in the sample population of dental students. Future studies will also include comparison of oral health behavior between dental students in UAE versus other countries in the world. Declaration of Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.

Oral Health Attitudes and Behavior between Students Sausan Al Kawas et al

2. Chen M, Andersen R, Barnes DE, Leclercq M-H, Lyttle CS. Comparing oral health systems: a second international collaborative study. Geneva: World Health Organization, 1997. 3. Levin L, Shenkman A. The relationship between dental caries status and oral health attitudes and behavior in young Israeli adults. Journal of Dental Education 2004; 68: 1185-91. 4. Ilkay P, Alkurt MT. Oral Health Attitudes and Behavior among a Group of Turkish Dental Students. Eur J Dent. 2009; 3:24-31. 5. Petersen PE, Esheng Z. Dental caries and oral health behaviour situation of children, mothers and schoolteachers in Wuhan, People's Republic of China. International Dental Journal. 1998; 48(3):210-16. 6. Al-Wahadni AM, Al-Omiri MK, Kawamura M. Differences in selfreported oral health behaviour between dental students and dental technology/dental hygiene students in Jordan. J Oral Sci. 2004; 24: 191-97. 7. Nusair KB, Alomari Q, Said K. Dental health attitudes and behaviour among dental students in Jordan. Community Dent Health. 2006; 23:147-51. 8. Dagli RJ, Tadakamadla S, Dhanni C, Duraiswamy P, Kulkarni S. Self reported dental health attitude and behavior of dental students in India. J Oral Sci. 2008; 50: 267-72. 9. Kawamura M, Honkala E, Widström E, Komabayashi T. Crosscultural differences of self-reported oral health behavior in Japanese and Finnish dental students. Int Dent J. 2000; 50:46-50. 10. Komabayashi T, Kwan SYL, Hu DY, Kajiwara K, Sasahara H, Kawamura M. A comparative study of oral health attitudes and behavior using Hiroshima University- Dental Behavioral Inventory (HU-DBI) between dental students in Britain and China. J Oral Sci. 2005; 47: 1-7. 11. Cortes FJ, Nevot C, Ramon JM, Cuenca E. The evolution of dental health in dental students at the University of Barcelona J Dent Educ. 2002; 66: 1203-08. 12. Polychronopoulou A, Kawamura M, Athanasouli T. Oral selfcare behavior among dental school students in Greece. J Oral Sci. 2002; 44: 73-8. 13. Almas K, Al-Malik TM, Al-Shehri MA, Skaug N. The knowledge and practices of oral hygiene methods and attendance pattern among school teachers in Riyadh, Saudi Arabia. Saudi Medical Journal. 2003; 24(10):1087-91. 14. Kisumbi BK, Kaimenyi JT, Wakiaga JM. Knowledge on treatment modalities and attitude of Nairobi University students towards dental care. Indian Journal of Dental Research. 1995; 6(4):133-36. 15. Kawamura M, Ikeda-Nakaoka Y, Sasahara H. An assessment of oral self-care level among Japanese dental hygiene students and general nursing students using the Hiroshima UniversityDental Behavioural Inventory (HU-DBI): surveys in 1990/1999. Eur J Dent Educ. 2000:4; 82-8. 16. Al-Omari QD, Hamasha AA. Gender-specific oral health attitudes and behavior among dental students in Jordan. J Contemp Dent Pract. 2005:6; 107-14. 17. Porat D, Kawamura M, Eli I. Effect of professional training on dental health attitudes of Israeli dental students. Refuat Hapeh Vehashinayim. 2001: 18; 51-6. 18. Al-Hussaini R, Al-Kandari M, Hamadi T, Al-Mutawa A, Honkala S, Memon A (2003) Dental health knowledge, attitudes and behaviour among students at the Kuwait University Health Sciences Centre. Med Princ Pract. 2003; 12: 260-65. 19. Oes P, Lutz F. Oral health--the status of knowledge 1985. An analysis of a repeated survey of 761 Swiss recruits. Schweiz Monatsschr Zahnmed. 1989; 99(3):281-91. 20. Kuusela S, Honkala E, Kannas L, Tynjala J. Oral hygiene habits of 11-year-old schoolchildren in 22 European countries and Canada in 1993/1994. Journal of Dental Research. 1997; 76(9):1602-09.

References 1. Kelly M, Steele J, Nuttall N, Bradnock G, Morris J, Nunn J, et al. Adult dental health survey: oral health in the United Kingdom 1998. London: The Stationery Office, 2000.

Volume 2 ∙ Number ∙ 3 ∙ 2009

Page 10


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

Stress Distribution in Bone Around Dental Implant Hasanen H. Al-Khafagy

INFLUENCE OF CANCELLOUS BONE RIGIDITY ON STRESS DISTRIBUTION IN BONE AROUND DENTAL IMPLANT: A FINITE ELEMENT STUDY Hasanen H. Al-Khafagy BDS, MSc, PhD Conservative Department, College of Dentistry, Ajman University of Science And Technology, United Arab Emirates.

Abstract Using the finite element method (FEM), this study sought to investigate how the Young’s modulus of cancellous jaw bones influenced stress distribution in bone surrounding a dental implant. Six models of bone with osseointegrated dental implant, with different modulus of elasticity values of cancellous bone, were used. The stress values of the six models loaded with vertical force were analyzed. The results have shown that the cancellous bone rigidity has a great effect on the stress distribution. Highly rigid cancellous bone results in even stress distribution and reduction in maximum equivalent stress. The influence of bone rigidity may extend even to cortical bone. (J Int Dent Med Res 2010; 3: (1), pp. 11-14 ) Keywords: Stress analysis, implant, bone, finite element. Received date: 29 October 2009 Introduction Finite element analysis is a numerical technique based on the principle of subdividing a structure into a number of finite elements which are interconnected with each other at the nodal points. These nodes are subjected to certain loading conditions, resulting in a behavior of the model similar to that of the structure it represents. The finite element method also allows for the study of stress patterns in two or more dissimilar materials adjacent to each other without affecting their independent behavior1. Finite element models are commonly used in implant dentistry to predict the effect of implant geometry, prosthesis design, and type of loading on the stress and strain distribution in the peri-implant region2,3. The modulus of elasticity (or Young’s modulus) is a measure of the material rigidity; it varies as a function of both the density and microstructure. Accurate values of modulus of elasticity *Corresponding author: Dr Hasanen H. Al-Khafagy. BDS, MSc, PhD Conservative Department, College of Dentistry, Ajman University of Science And Technology. United Arab Emirates Email: dentalhasanen@yahoo.com

Volume 3 ∙ Number ∙ 1 ∙ 2010

Accept date: 19 January 2010 are needed for finite element modeling of the jaws. The modulus of elasticity is measured in MPa4.The actual value of Young’s modulus for any material is normally determined by carrying out a standard tensile test on a specimen of the material5. In the literature, a wide range of elastic properties of bone is reported. The reported values for the Young’s modulus are within a range of 7000 to 30000 MPa for mature cortical bone while the value for the cancellous bone are within a range of 1.1 to 9800 MPa1,4,6,7,8. These variations result from differences in anatomical position9,10, loading direction, methods of storage and testing conditions. Also age seems to be a factor influencing these material properties of bone. For the finite element model, the assignment of accurate values for material properties is an essential step to ensure predictive accuracy. Because of the lack of availability of elastic modulus properties of mandibular cancellous bone, the properties of bone from other parts of the body are frequently used in the finite element models11. The modulus of elasicity has been reported for cortical bone from dentate mandible12. To date, elastic properties have not been reported for cancellous bone in either the dentate or edentulous mandible1. Page 11


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

Material and Methods The two-dimensional finite element model includes a section of mandible at the canine area and dental implant. This modeled section of the mandibule was composed of spongy and cortical bones and was 10 mm in thickness buccolingually, surrounded by 2 mm of cortical bone. Cylindrical implant, 4 mm in diameter and 12 mm length was placed in the mandibule model, and superstructure 4 mm in diameter and 8 mm in height, was also modeled 3. The load which was used in this study was 100 N to the most coronal part of the crown4. An ANSYS program, version 5.4 (Swanson Analysis System, Houston, Pennsylvania), was used to perform the two-dimensional finite element modeling and finite element analysis. The total number of the elements used in the finite element model was 480713. Material property values were assigned in this study and assumptions were made on the basis of previously published data. The materials were homogeneous, isotropic and linearly elastic and the bone/implant interface was assumed to be 100% osseointegrated. The properties of the materials which will be used in this study (Cortical bone, cancellous bone and Titanium) are listed in tables 1 and 2. Six osseointegrated implants with bone models with different value of modulus of elasticity of cancellous bone were used. Stress levels, according to von Mises criteria, were calculated. Von Mises stresses are commonly reported in finite element analysis studies to summarize the overall stress state.

Stress Distribution in Bone Around Dental Implant Hasanen H. Al-Khafagy

Results Numerical equivalent stress values were determined by the von Misses failure theory at the cortical and cancellous bones. The results of the stress are illustrated in the figures 1 and 2. The stress in the first model doesn’t exceed 21.1 MPa while it reaches 84.02 MPa in the sixth model. The stresses in all the six models are higher in the cortical bone than in the cancellous bone. The stress in the first model, with high modulus of elasticity which is about ½ of cortical bone, has shown even stress distribution. While the models with lower values of modulus of elasticity of the spongy bone stress distribution has appeared with uneven distribution. The stress also concentrated on the cortical bone, especially in the sixth model. 90 80 70 60 50 MPa 40 30 20 10 0

Minimum Maximum

Model Model Model Model Model Model 6 5 4 3 2 1

Figure 1. The maximum equivalent stress of six models with dental implant and different values of modulus of elasticity (in MPa).

Table 1. The properties of the cortical bone and titanium.

Table 2. The properties of the cancellous bone of the six models. Volume 3 ∙ Number ∙ 1 ∙ 2010

Figure 2a. Page 12


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

Stress Distribution in Bone Around Dental Implant Hasanen H. Al-Khafagy

Figure 2b.

Figure 2e.

Figure 2c.

Figure 2f. Figure 2a, b, c, d, e, f. Stress contour of the six models with dental implant at the cortical and cancellous bone and different value of modulus of elasticity where used. Modulus of elasticity of cancellous bone (A): 6850 MPa, (B): 2740 MPa, (C): 1370 MPa, (D): 685 MPa, (E): 457 MPa, (F): 274 MPa. Discussion

Figure 2d.

Volume 3 ∙ Number ∙ 1 ∙ 2010

Several factors appear to affect biomechanical failures of implant-supported prostheses. Resultant stresses, which are the product of occlusal loading, when excessive, are detrimental to the stability and longevity of the implant. Therefore it is important to consider the bone rigidity which result in the lowest levels of stress concentration and produce even Page 13


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

distribution of these stresses. In 1992, Meijer, used the finite element method where the model of the mandible was either consisting of only a cortical bone or of a cancellous bone. By his model, he concluded that with an increase of modullous of elasticity the principal stresses in the bone around dental implants will become more extreme14. But his model of the mandible showed many shortages of bone representation. The results that were obtained by the twodimensional finite element method analysis suggest that the modulus of elasticity of the cancellous bone play a key role in good stress distribution. When a load is applied to the super structure, the results stress transfers to the bone surrounding the implant. When there is a small difference in the values of the modulus of elasticity of both cortical and cancellous bone, the stress will be distributed evenly. Since a dense bone has the ability to bear the stress which is applied on it, while in the case of great difference, (model number six), the stress will be concentrated on the cortical bone. The relationship between the values of modullous of elasticity of cancellous bone and the maximum equivalent stress is linear. As the values of elasticity decrease, the stress in the bone around the implant increases. Conclusions

Stress Distribution in Bone Around Dental Implant Hasanen H. Al-Khafagy

3. Akca K. Cehreli MC and Iplikcioglu H. A comparison of three dimensional finite element stress analysis with in vitro strain gauge measurements on dental implants. International J Prosthod 2002; 15, 2,115-121. 4. O’Mahony AM, Williams JL, Katz JO, Spencer P. Anisotropic elastic properties of cancellous bone from a human edentulous mandible. Clin Oral Impl Res 2000.11:415-21. 5. Hearn EJ. Mechanics of Materials. Volume (1) Second Edition, Pregamon Press. 1985. 369-375. 6. Papavasilliou G, Kamposiora P, Bayne SC, Felton DA. Three dimensional finite element analysis of stress-distribution around single tooth implant as a function of bony support, prosthesis type, and loading during function. J Prosth Dent 1996; 76, 6,633-40. 7. Farah JW, Craig RG, Meroueh KA. Finite element analysis of mandibular model. J Oral Rehabil 1988; 15,615-24. 8. Petrie CS, Williams JL. Shape optimization of dental implant designs under oblique using the ρ-version finite element method. J mechanics in medicine and biology. 2002; 2, 3&4; 339-345. 9. Seong WJ, Kim UK, Swift JQ, Heo YC, Hodges JS, Ko CC. Elastic properties and apparent density of human edentulous maxilla and mandible. Int J Oral Maxillofac Surg. 2009 Oct;38(10):1088-93. Epub 2009 Jul 31. 10. Seong WJ, Kim UK, Swift JQ, Hodges JS and Ko CC. Correlation between physical properties of jawbone and dental implant initial stability. J Prosthet Dent. 2009 May;101(5):303-318. 11. Guan H, van Staden R, Loo YC, Johnson N, Ivanovski S, Meredith N.Influence of bone and dental implant parameters on stress distribution in the mandible: a finite element study.Int J Maxillofac Implants.2009 Sep-Oct;24(5):866-876. 12. O’Mahony AM, Williams JL and Spencer P. Anisotropic elasticity of cortical and cancellous bone in the posterior mandible increases peri-implant stress and strain under oblique loading. Clin Oral Impl Res.2001; 12:648-657. 13. Al-Khafagy HH. A three dimensional finite element analysis of the effect of cortical bone thickness on the stress distribution around single0unite osseointegrated implant. International Dentistry SA.2006; 8, 5. 14. Meijer HJA. A biomechanical study on bone around dental implants in an edentulous mandible – a finite element analysis and a design for a radiographic analysis. [Met een samenvatting in het Nederland].1992, 91-103.

In conclusion, it was confirmed that von Misses equivalent stress was sensitive to the Young’s modulus of cancellous jaw bones. Rigid cancellous bone results in even stress distribution and reduction in maximum equivalent stress especially at the cortical bone around dental osseointegrated implants. Declaration of Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article. References 1. Tuncelli B, Poyrazoglu E, Koyoglu AM, Tezcan S. Comparison of load transfer by implant abutment of various diameters. Eur J Prosth Rest Dent 1997; 5-2:79-83. 2. Assunção WG, Barão VA, Tabata LF, Gomes EA, Delben JA, dos Santos PH. Biomechanics studies in dentistry: bioengineering applied in oral implantology. J Craniofac Surg. 2009 Jul;20 (4) : 1173-7.

Volume 3 ∙ Number ∙ 1 ∙ 2010

Page 14


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

Home Bleaching Effect Ali A. Razooki Al- Shekhli

IN-HOME BLEACHING EFFECT ON COMPRESSIVE STRENGTH VALUES OF SOME DIRECT RESTORATIVE MATERIALS Ali A. Razooki Al- Shekhli1* 1. B.D.S.,MSc, Ph.D.; Assistant Prof., Faculty of Dentistry, Ajman University of Science & technology, UAE.

Abstract Patients commonly have restorations in posterior teeth, made of resin-based composite, amalgam or glass ionomer or another material. Questions remain concerning the need for replacement of posterior restorations after bleaching. It's not clear if the bleaching agents exert some effect on the restorative materials that could harm the quality and longevity of these restorations. The aim of this study is to evaluate the effect of in home bleaching material on the compressive strength of different direct restorative materials. Three types of direct restorative materials: (two types of light activated composites, TG fine glass (Technical & General Ltd, Germany); Cavex (Quadrant Universal LC, CE 0197; Germany)) and one type of amalgam restorative material; World-Cap (Ivoclar vivadent FL-9494 schaan/Liechtenstein, Sweden) were used in this study. Forty resin composite specimens and 20 amalgam specimens were prepared using a circular nickel-chromium split mold with 3 mm in inner diameter and 6 mm in height. The twenty samples of each restorative material being selected were divided into 2 groups: ten tested before treatment with home bleaching tooth whitening system and the other ten, tested after treatment with home bleaching system (WHITE smile HOME BLEACHING, 35% carbamide peroxide, Germany) for 8 hours: (4X2 hours). Specimens were placed into a dark bottle containing distilled water at 37°C for 7 days before testing procedure. Compressive testing was performed in a Universal Testing Machine at a crosshead speed of 0.5 mm/min. The data was calculated in MPa and data were analyzed by one way ANOVA at 0.05 level of significance. All direct restorative materials being tested, exhibited statistically insignificant differences (P≥0.05) in compressive strength values between the two groups (before and after bleaching) except for TG direct restorative composite, which exhibited statistically significant differences (P<0.05) in compressive strength values between the two groups (before and after bleaching). All the restorative materials being tested exhibited lower compressive strength values after bleaching in comparison with their values before bleaching. In-Home bleaching material should not be used when TG light activated composite restorations. (J Int Dent Med Res 2010; 3: (1), pp. 15-18 ) Keywords: Composite resins; compressive strength; amalgam; in-home bleaching. . Received date: 29 December 2009

Accept date: 23 March 2010 Introduction

*Corresponding author: Ali A. Razooki Al- Shekhli B.D.S.,MSc, Ph.D. Assistant Prof., Faculty of Dentistry, Ajman University of Science & technology, UAE. E-mail: alirazooki@yahoo.com

Volume 3 ∙ Number ∙ 1 ∙ 2010

Esthetic dentistry, particularly tooth whitening, is one of the most rapidly growing areas in dentistry, and vital tooth bleaching is a popular treatment modality1, 2. Many techniques for vital tooth bleaching are available: in-office and at home bleaching systems. The latest one is called also night guard vital Bleaching or NGVB. NGVB is an esthetic procedure where the patient, at-home, uses Page 15


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

custom-fitted prostheses to apply a solution to lighten vital teeth. The American Dental Association’s (ADA) accepted standard for the procedure recognizes the use of a 10% carbamide peroxide or CP material. The CP concentration, however, may vary from 10-35%. Restorative filling materials used in dentistry require long-term durability in the oral cavity3. In order to find out the performance of restorative materials against masticatory forces, it is required to determine the compressive strength values of the restorative materials. Several investigators have studied the effects of home bleaching on oral tissues and restorative materials4, 5. At-home as well as in-office bleaching agents have a softening effect on some tooth colored restorative material, and the patient must be aware before using them6. Material and Methods Three types of direct restorative materials (two types of light activated composites, TG fine glass (Technical & General Ltd, Germany) and Cavex (Quadrant Universal LC, CE 0197; Germany) and one type of amalgam, World-Cap) were used in this study. Forty resin composite specimens were prepared (20 for each type of composites being tested) by incremental (three increments) insertion of composite into a circular nickelchromium split mold with 3 mm in inner diameter and 6 mm in height and cured using Blue phase C5 (Ivoclar, Vivadent) LCU for 40 seconds for each 2 mm increment of composite thickness. For the preparation of the 20 amalgam specimens, the freshly triturated World-Cap amalgam (Non-gamma-2, spherical 40% silver alloy amalgam in self-activating capsule, Ivoclar vivadent FL-9494 schaan/Liechtenstein, Sweden) was introduced into the mold and condensed incrementally into the mold until it became over filled then by simple movement of a glass slide over the excess amalgam, the top surface of the specimen became flat. After 24 hours, the two halves of the mold were separated and the amalgam cylindrical specimen was removed. Twenty samples were prepared for each restorative material being tested, ten tested before treatment with home bleaching tooth whitening system and the other ten tested after treatment with home bleaching system (WHITE Volume 3 ∙ Number ∙ 1 ∙ 2010

Home Bleaching Effect Ali A. Razooki Al- Shekhli

smile HOME BLEACHING, 35% carbamide peroxide, Weinheimer strabe 6. 69488 birkenau. Germany) for 8 hours: (4X2 hors). Specimens were placed into a dark bottle containing distilled water at 37°C for 7 days before testing procedure. Specimens were positioned vertically on the testing machine base and subjected to compressive load until failure. Compressive testing was performed in a Universal Testing Machine at a crosshead speed of 0.5 mm/min. The compressive strength (CS) was calculated by dividing the failure load (F) by the cross-sectional area, i.e.: CS = F/πR2, where R is the radius of the cross-section of the specimen. Mean compressive strength values were expressed in MPa and data were analyzed by one way ANOVA at the 0.05 level of significance. Results Mean compressive strength values, standard deviations of amalgam, Cavex and TG composites in MPa before and after bleaching are presented in table 1.

Table 1. Mean compressive strength values and standard deviations of tested restorative materials before and after bleaching in MPa. For amalgam restoration, statistical analysis of data by using one-way analysis of variance (ANOVA) revealed that, there was statistically insignificant difference ≥0.05) (P in compressive strength values between the two groups before and after bleaching as shown in Table (2). Source Factor Error Total

DF 1 18 19

SS MS F P 2142 2142 1.80 0.197 21464 1192 23607 Table 2. ANOVA for amalgam before and after bleaching. For Cavex composite restoration, statistical analysis of data by using one-way analysis of variance (ANOVA) revealed that, Page 16


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

there was statistically insignificant difference (P≥0.05) in compressive strength values between the two groups before and after bleaching as shown in Table (3). Source Factor Error Total

DF 1 18 19

SS 45 51091 51136

MS F 45 0.02 2838

P 0.901

Table 3. ANOVA for Cavex composite before and after bleaching. For TG composite restoration, statistical analysis of data by using one-way analysis of variance (ANOVA) revealed that, there was statistically significant difference (P<0.05) in compressive strength values between the two groups before and after bleaching as shown in Table (4).

Home Bleaching Effect Ali A. Razooki Al- Shekhli

number of studies done on their effects on restorative materials1, 10. TG composite is the only direct restorative showed a significant difference reduction in compressive strength values (Table 4) after the exposure to the at home bleaching process compared to other direct restorative materials being tested in this study (World-Cap amalgam and Cavex composite were insignificantly affected by bleaching material (Tables 2&3). This could be contributed to the type, size, and volume fraction of the filler particles and the degree to which the filler is bonded to the resin matrix. Filler-matrix interactions appear to have a great effect on increasing the resistance of composite resin11.

Source Factor Error Total

DF SS MS F P 1 17523 17523 6.68 0.019 18 47192 2622 19 64715 Table 4. ANOVA for TG composite before and after bleaching. Discussion In order to find out the performance of restorative materials against masticatory forces, it is required to determine the compressive strength values of the restorative materials. Bleaching agents affect lightening of discolored tooth structure through decomposition of peroxide into free radicals. The free radicals break down large pigmented molecules included in reflecting a specific wavelength of light and are responsible for the color stain in enamel, into smaller less pigmented molecules through oxidation and reduction7, 8. In addition to the oxidizing agent used in the at-home bleaching procedure an additive called carbopol (carboxy polymethelene) may be added to thicken the gel that improves adherence to the tooth surface and prolongs the release of oxygen. This additive keeps the gel contained within the tray better and slows the chemical reaction9. The effect of the active agents of bleaching solutions has not been adequately investigated since there have been a limited Volume 3 ∙ Number ∙ 1 ∙ 2010

Table 5. Composition of the materials being tested (manufacturer's data).

Figure 1. Mean compressive strength values for restorative materials before and after bleaching.

Page 17


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

Carbamide peroxide (at-home bleaching) breaks into urea and hydrogen peroxide. Hydrogen peroxide in turn breaks down into free radicals, which eventually combine to form molecular oxygen and water. Some aspect of this chemical process may accelerate the hydrolytic degradation of tooth colored restorative materials12. Since both Cavex and TG composites used in this study had an approximately the same filler percentage by weight (72% and 74% respectively) (Table 5) , the main cause behind this difference in compressive strength values before and after bleaching is in the resin matrix. Cavex composite contained only one type of resin matrix (Bis-GMA without diluents monomers) (Table 5) while TG composite contained three types of resin matrixes (Bis-GMA, TEGDMA and UDMA with diluents monomers) and the incorporation of TEGDMA diluents monomer in TG composite showed less resistance to the bleaching material than the principal monomers (Bis-GMA and UDMA) in spite of its high mean compressive strength value before bleaching in comparison with Cavex composite (Figure 1).

Home Bleaching Effect Ali A. Razooki Al- Shekhli

3- Okada K, Tosaki S, Hirota K, et al: surface hardness change of restorative filling materials stored in saliva. Dent- Mat.2001; 17: 34-39. 4- Lenhard M. Assessing tooth color change after repeated bleaching in vitro with a 10 percent carbamide peroxide gel. J Am Dent Assoc 1996;127: 1618 - 1624. 5- McCaslin AJ, Haywood VB, Potter BJ, Dickinson GL and Russell CM. Assessing dentin color changes from nightguard vital bleaching. J Am Dent Assoc 1999; 130: 1485 - 1490. 6- Taher NM. The Effect of Bleaching Agents on the Surface Hardness of Tooth Colored Restorative Materials. The Journal of Contemporary Dental Practice, , 2005 Volume 6, No. 2, May 15:1-8. 7- Oltu ü, Gürgan S: Effect of three concentrations of carbamide peroxide on the structure of enamel. J- Oral Rehabil. 2000; 27: 332-340. 8- Flaitz CM, Hicks MJ: Effects of carbamide peroxide whitening agents on enamel surfaces and caries-like lesion formation: An SEM and polarized light microscopic in vitro study. J- of Dent- Child (ASDC) July-August 1996; 249-256. 9- Gutmann MS, Gutmann JL: Some current perspectives on tooth bleaching and management of tooth stains. Dent- news 2001; 8: 19-24. 10- Baily SJ, Swift EJ: Effect of home bleaching products on composite resin. Quint- Int.1992; 23: 489-494. 11- Manhart J, Kunzelmann KH, chen HY, Hickel R: Mechanical properties and wear behavior of light-cured packable composite resins. Dent-Mat.l 2000; 16: 33-40. 12- Baily SJ, Swift EJ: Effect of home bleaching products on composite resin. Quint- Int.1992; 23: 489-494.

Conclusions 1. All the restorative materials being tested exhibited lower compressive strength values after bleaching in comparison with their values before bleaching. 2. All the materials being tested showed insignificant differences in compressive strength values before and after bleaching except for TG composite which showed significant differences in compressive strength values before and after bleaching. Declaration of Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article. References 1- Nathoo SA, Chmielewski MB, Kirkup RE: Effect of colgate platinum professional tooth whitening system on micro hardness of enamel, Dentin, and composite resins. Comp- contEduc- Dent .1994; 15: 627-630. 2- Luk K, Tam L, Hubert M: Effect of light energy on peroxide tooth bleaching. J- Am-Dent-Assoc (JADA) .2004; 135: 194 -201.

Volume 3 ∙ Number ∙ 1 ∙ 2010

Page 18


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

New Direction for Measurement of Microleakage Izzet Yavuz and Haluk Aydın

NEW DIRECTION FOR MEASUREMENT OF MICROLEAKAGE IN CARIOLOGY RESEARCH Izzet Yavuz1, Haluk Aydın2* 1. Associate Professor MsC, PhD, DDs, Faculty of Dentistry, Department of Pediatrics, Dicle University. Diyarbakir / Turkey. 2. Associate Professor MsC, PhD, Faculty of Science, Department of Chemistry, Dicle University. Diyarbakir / Turkey.

Abstract The present study introduces the feasibility of a different new direction to determining the microleakage volume associated with dental restorations and the relative marginal adaptation deficiency of teeth in in vitro conditions. Our method utilizes the molecular adsorption characteristics of Methylene Blue. It was concluded that investigations showed the microleakage volume measurement method looking as a valuable new technique for the in vitro study of microleakage dynamics around dental restorations. (J Int Dent Med Res 2010; 3: (1), pp. 19-24 ) Keywords: Microleakage, marginal adaptation, volume measuremnt, new tecnique, dye adsorption test. Received date: 15 September 2009 Introduction Main principle in restorative dentistry states that the transition between the restorative material and the dental hard tissue must be continuous to increase the survival probability of the restoration1. One of the most important problems of restorative dentistry today is the failure of restorative materials to completely bond to enamel and dentin, causing microleakage. Microleakage has been defined as the passage of ions, molecules, fluids or bacteria between a cavity wall and the applied restorative material. Microleakage has been reported as the cause of hypersensitivity of restored teeth, discoloration at the margins of cavities and restorations, recurrent caries, pulp inflammation and failure of endodontic treatment2-4. It is also affected by a number of other factors: for example, the operative techniques used, including the size of the cavity, the angle at *Corresponding author: Assoc.Prof. Dr. Haluk AYDIN, Faculty of Science, Department of Chemistry, Dicle University. 21280 Diyarbakir / Turkey. E-mail: halukaydin@dicle.edu.tr

Volume 3 ∙ Number ∙ 1 ∙ 2010

Accept date: 09 December 2009 which the enamel prisms and dentin tubules are cut depending on their location, the method used to condition dental hard tissues1. Microleakage is determined today by many in vitro techniques with or without thermal cycling, such as staining; scanning electron microscope; bacterial activity; decay; air pressure; chemical agents; markers; neutron activation analysis; radioisotope; ionization; autoradiography, reversible radioactive adsorption and electrochemical technique2-9. The significant differences between these models and materials suggest that an ideal method for the determination of microleakage has not yet been established. The aim of some researchers is to develop an in vitro model to replicate microleakage at a tooth/restoration interface3,1012 . Laboratory tests and clinical trials must also be added to the process of evaluating whether the microleakage measurement methods fulfill the objectives of evidence-based dentistry. It is only possible to obtain sufficient evidence in the natural sciences if proven and correct evaluation methods are applied and if those methods are subjected to the process of validation. If the test method confirms by examination and the provision of objective evidence that the particular requirement for a Page 19


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

specific intended use can be consistently fulfilled, the method can be called “validated” and the results are “internally valid” 1. The purpose of present study was to introduce valuable an in vitro model to determine the microleakage volume in in vitro marginal adaptation research studies.

New Direction for Measurement of Microleakage Izzet Yavuz and Haluk Aydın

of MB. Theoretically, the volume measurement method was created and applied as described below. The MB molecule is made up of an acid combined with an organic base. Its molecular weight is (MA=319.868g.mol-1) and a single piece of the absorbed covers an area of (σ)=120 A0 2 on the surface16. (Figure 1).

Materials and Methods Primary human teeth were obtained from the Department of Maxillo-Facial Surgery of Dental Faculty, they were selected by Binocular Stereo Microscope (Olympus Co., Japan) as without caries and cracked for this study. The teeth species were used for microleakage volume study. Before the cavity preparation and restorative procedure, all teeth were cleaned. Class V cavity prepared on the buccal surface of each tooth. The cavities had a mesio-distal width of 3mm, an occluso-cervical length of 2mm and a depth of 1.5 mm. The teeth were restored in the following way: The type 2 light-hardening powder and liquid glass ionomer cement (Variglass VLC, Dentsply, USA) were prepared in accordance with the manufacturer’s instructions, applied to the bottom of the preparations and polymerized. (Astralis3, Vivadent, Australia). Following polymerization, all preparations were filled with a high copper amalgam (Cavex Avalloy, Cavex Co., Holland) and 24 hours later finishing and polishing were performed. The specimens were subjected to thermo cycling between 5oC ±4oC and 55oC ±4oC for 500 cycles (Guidance on substrate selection, storage, handling, and execution of bond strength tests according to the ISO Technical Specification 11405103). After thermo cycling, the surface of the teeth, up to approximately 1.5 mm to the restoration, were coated with a layer of nail varnish, melted utility wax and a second layer of nail varnish3,10,12-15. The methylene Blue(MB) solution was prepared to a concentration of MB 4.75 g/l. A stock solution was prepared using a buffer of H2PO4- / HPO4-2 (phosphate / biphosphate) with a pH of 6.98 and 24 hours did storage the specimens in the MB solution. Each individual sample was quantitatively measured for volume of the marginal gaps using the chemical molecular characteristic properties Volume 3 ∙ Number ∙ 1 ∙ 2010

Figure 1. The methylene blue.

molecular

structure

of

Adsorption is the accumulation of dissolved molecules over the surface of a solid matter, the dissolved molecules could be atoms or ions of matter present in any solution of a gas, vapour or liquid phase. In the phase, which allows the accumulation to occur between surface, is known as the adsorbent (the teeth), the matter which accumulates, is known as the adsorbate (MB) 16-18. Adsorption, in the liquid form, is usually measured using an indirect method. After the experiment, the teeth were dissolved in a 50% solution of nitric acid, the MB that filled the microleakage gaps dissolves into the solution and it’s the MB concentration is determined. To draw the calibration graph, a part of the MB solution was taken and determined to have a wavelength of 664 nm in a spectrophotometer λmax (maximum absorption wavelength). Some of the MB stock was taken and diluted (10 different concentrations were prepared using 100 milliliters of distilled water in each beaker to dilute the 2% MB, ranging from 0 mL added to 180 mL added in 20 mL increments ) to form a series of solutions of varying concentration. These varying concentrations were measured for their absorption wavelengths. These measurements were then used to construct the calibration graph (Figure 2). Page 20


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

New Direction for Measurement of Microleakage Izzet Yavuz and Haluk Aydın

0,9

0,8

0,7

0,6

A

0,5

0,4

0,3

0,2

0,1

0 0

0,001

0,002

0,003

0,004

0,005

0,006

C(g/L)

Figure 2. The calibration curve of Methylene Blue (A; Absorbance, C; Concentration). A=E C (A: Absorbance, E:molar absorption coefficient, C: concentration) The molar absorption coefficient was determined to be 170.57 dm3.cm-1.g-1 . After the calibration graph was drawn, the concentrations were calculated using the absorption values. In the absorption experiments of our study, the teeth were placed in joje balloons along with 100ml of MB solution. The primary teeth were subjected to MB dye penetration at 37oC for a 24-hour period, to allow dye penetration into any possible existing gaps between the tooth substance and the restorative material3,8,10. Afterwards, the tooth tissue around the restoration surfaces was removed in a block by making cuts 1.5 mm around the restored area (Figures 3,4).

Figure 4. Teeth specimens around the restoration surfaces were removed in a block by making cuts 1.5 mm (MagnificationX10). These blocks were then dissolved separately in 50% nitric acid (Figure 5) and the amount of MB absorption was calculated using the previously created calibration graph. These values were converted to volume values (V=m/d) and the individual tooth volume values are reported in Table 1.

Figure 5. Removed blocks were then dissolved separately in 50% nitric acid. Results

Figure 3. Appearance of cutting model of tooth tissue 1.5 mm around the restoration surfaces was marked. Volume 3 ∙ Number ∙ 1 ∙ 2010

Dye penetration for group calculated according to the present method. Measurement values of the volume measurements of the group and the MB molecular counts equivalent to median values are shown in Table 1. In the present study we didn’t use control group due to that study introducing a way for microleakage research. Different specimens Page 21


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

groups, could be compare and evaluate statistically for evaluation of microleakage volume using One Way ANOVA Test and statistically significant differences could be revealed.

Table 1. Measurement values of the volume measurements of the group as median values human teeth which restored by Amalgam. Discussion The purpose of microleakage tests is to evaluate the seal of restorations placed in extracted teeth and to give a prognosis about the clinical performance with regard to the occurrence of postoperative sensitivity and/or secondary caries19. The in vitro microleakage phenomenon and the adaptation of filling materials into the cavity walls under clinical and laboratory conditions constituted the focal points of researchers for many years and a variety of methods have been used to research this4,6,7,10,19Volume 3 ∙ Number ∙ 1 ∙ 2010

New Direction for Measurement of Microleakage Izzet Yavuz and Haluk Aydın

22

. Some of these laboratory models have been successfully used to in order to determine microleakage, but they are not quantitative methods. Also comparison of the marginal adaptation results using Due to its ease and simplicity, the most frequently used method is the measurement of the microleakage of a specific dye after sectioning teeth that have been restored1. The second most frequent method is the quantitative marginal analysis of replicas of restored teeth with the scanning electron microscope (SEM) and an appropriate software for length measurements, is used less often, because it is more time consuming, and complex1,19 but SEM analysis, is semi quantitative method within samples allocated from the study groups. In fact, in the studies of dye penetration, the dentin staining was observed to be more different than the actual gaps between cavity walls and restoration materials. This resulted in the use of a dye with a particle diameter equal to the bacterial size or smaller by researchers (around 2µm) 3. Dye penetration is a diffusion phenomena and the consequences are that the results are not obtained immediately, they are semiquantitative, and the defect is evaluated on a section (two-dimensional evaluation) 23. For microleakage studies a popular dye is Methylene Blue 19,24. In present study, a 2.00% solution of the MB molecule was used (one MB molecule=1.2nm2=120 A0 2 ) since the particle size is less than that of the bacterial one. MB molecules were used because the also dissolve as monomer and bimer in an aqueous environment in which the pH is adjusted to 6.98 with a phosphate and biphosphate buffer 3,17. Another important issue in microleakage studies arises from the scoring systems. Since the evaluation in those studies largely depends on the observer’s interpretation, the leakage scoring is at best a semi-measurable method3. Various studies performed show that the dye leakages in different sections taken at different places of the restorations may show significant differences6,23. For this reason, the accuracy of a leakage study based on a single section made from a tooth may be negligible. As of today, there are no quantitative methods applicable and valuable for the Page 22


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

microleakage determination; we have above indicated the amount of microleakage through quantification. In the stereo microscopic studies, the method is based on the interpretation of the leakage of dye on the cavity wall and is defined as a semi-quantitative approach where the leakage is calculated solely at the surface where the section is made3,6,7. In that method, the researcher’s observation and interpretation do not come into play in the determination of microleakage volume quantity and all surfaces where a leakage occurs between tooth/restoration materials is quantitatively measured by a chemist. The volumetric measurement method leads to the immediate behavior of the whole interface and not only the sealing ability of the margin (volumetric evaluation). The groups can be comparing as a statistically for microleakage volume measurements using the One Way Anowa Test. Conclusions It was also concluded in that preliminary and our previous investigations25, 26 show that the method of measuring the microleakage volume can be best a valuable tool for the in vitro study of microleakage dynamics around dental restorations, and this method can be use as a new technique for the determination of microleakage volume. The use of a measurement of volume to detect microleakage in magrin of restoration appears to be effective, although it is not suitable for simulating in vivo clinical circumstances. It is necessarily more further work to establish the true scope of the model, but this preliminary investigation shows promise and its supported with previous experimental study. Declaration of Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article. References 1. Siegward D. Heintzea, Systematic Reviews: I. The Correlation Between Laboratory Tests on Marginal Quality and Bond Strength. II. The Correlation Between Marginal Quality and Clinical Outcome J Adhes Dent 2007; 9: 77-106.

Volume 3 ∙ Number ∙ 1 ∙ 2010

New Direction for Measurement of Microleakage Izzet Yavuz and Haluk Aydın

2. Taylor, M.J; Lynch, E. Microleakage. J. Dent. February 1992, vol. 20, p. 3-10. 3. Yavuz, I; Aydin H. New Method For Measurement of Surface Areas of Microleakage at The Primary Teeth By Biomolecule Characteristics of Methilene Blue. Biotechnol. & Biotechnol. Eq. 2005, vol. 19, p. 181-187. 4. Tjan, A. and Tan, D. Microleakage at gingival margins of class V composite resin restorations rebonded with various low-viscosity resin systems. Quintessence Int. July 1991, vol. 22, p. 565-573. 5. Pradelle-Plassea N, Wengerb F, Picarda B, Colona P.Evaluation of microleakage of composite resin restorations by an electrochemical technique: the impedance methodology Dental Materials (2004) 20, 425–434. 6. Yavuz, I and Atakul, F. Evaluation of microleaking in different in vitro restorations of cow permanent teeth. Balk J Stom. March 2001, vol. 5, p. 42-45. 7. Yavuz, I and Atakul, F. The comparison of amount of leaking occurred in different sections of in vitro prepared restorations in cow permanent teeth. Balk J Stom. July 2001, vol. 5, p. 104-107. 8. Yavuz, I; Atakul, F. The comparison of amount of microleaking for in vitro restorations in permanent teeth of human, dog and cow. Balk J Stom. July 2000, vol. 4, p. 103-106. 9. Sano, H et al. Nanoleakage: Leakage within the hybrid layer. Operative Dent. January-February 1995, vol. 20, p. 18-25. 10. Yavuz, I ; Aydin, H and Kaya, S. New method for measurement of microleakage. T Klin J Dent Sci. July 2003, vol. 9, p. 79-85. 11. Matharu, S et al. A new in vitro model for the study of microbial microleakage around dental restorations: A preliminary qualitative evaluation. Int End J. October 2001, vol. 34, p. 547-553. 12. Iwamı, Y; Yamamoto, H; Ebısu, S. A new electrical method for detecting marginal leakage of in vitro resin restorations. J Dent. May 2000, vol. 28, p. 241-247. 13. Olmez, A; Oztas, N and Bodur, Haluk. The effect of flowable resin composite on microleakage and internal voids in class II composite restorations. Oper Dent. November-December 2004, vol. 29, p. 713-719. 14. Derhami, K; Coli, P and Brannstrom, M. Microleakage in class 2 composite resin restorations. Oper Dent. May-June. 1995, vol. 20, p. 100-105. 15. Gungor, H C; Turgut, M D; Attar, N and Altay, N. Microleakage evaluation of a flowable polyacid-modified resin composite used as fissure sealant on air-abraded Permanent teeth. Oper Dent. 2003, vol. 28, p. 267-273. 16. Aydin, H and Tez, Z. Comparative determination of the adsorption capacities and possible fields of usage of active carbons prepared from the shells of walnut and hazelnut through some commercial active carbons. Turk J Chem. 1996, vol. 16, p. 51-58. 17. Nelsen, R J.; Wolcoltt, RB and Paffenbarger, GC. Fluid exchange at the margins of dental restorations. J Am Dent Assoc. March 1952, vol. 44, p. 228-295. 18. Davies, RG. The use of benzene adsorption isotherms in evaluating the surface and pore characteristics of activated carbons. Chemistry and Industry. February 1952, vol. 23, p. 160-165. 19. Roulet JF, Reich T, Blunck U, Noack M. Quantitative margin analysis in the scanning electron microscope. Scanning Microsc 1989;3:147-58. 20. Turgut, M D.; Tekcicek, M and Olmez, S. Clinical evaluation of a polyacid-modified resin composite under different conditioning methods in primary teeth. Oper Dent. September-October 2004, vol. 29, p. 515-523. 21. Ersin, N K. and Eronat, N. The comparison of a dentin adhesive with calcium hydroxide as a pulp-capping agent on the exposed pulps of human and sheep teeth.Quintessence Int. 2005, vol. 36, p. 271-280. 22. Kelsey, WP; Latta, MA; Meara, JD; Barkmeier, WW; Hammesfahr, PD and Jefferies, SR. An accelerated in vitro model for adhesive testing. Am J Dent. October 2004, vol. 17, p. 323-326. 23. Prati C, Tao L, Simpson M, Pashley DH. Permeability and microleakage of class II resin composite restorations. J Dent 1994;22:49-56. 24. Raskin A, D'Hoore W, Gonthier S, Degrange M, Dejou J. Reliability of in vitro microleakage tests: a literature review. J Adhes

Page 23


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

New Direction for Measurement of Microleakage Izzet Yavuz and Haluk Aydın

Dent 2001;3:295-308. 25. Yavuz, I., Aydın H., Ulku R., Kaya S. ve Tümen C., “A New Method: Measurement of Microleakage Volume Using Human, Dog and Bovine Permanent Teeth,” Electronic Journal of Biotechnology, 9(1), 8-17 (2006). 26. Yavuz, I.,. Aydın A.H, Ulku R., Dulgergil T.C. ve Akdag M.Z., “New Technique: Measurement of Microleakage Volume in The Marginal Gaps of The Dental Restorations,” Biotechnol. & Biotechnol. Eq, 19 (3), 184-191 (2005).

Volume 3 ∙ Number ∙ 1 ∙ 2010

Page 24


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

The Fracture Strength of Different Materials Mustafa ZORTUK et al

EVALUATING THE FRACTURE STRENGTH OF THREE DIFFERENT PROVISIONAL CROWNS Mustafa Zortuk1, Eylem Ozdemir2*, Suleyman Aguloglu2 1. Assist. Prof.Dr. Department of Prosthetic Dentistry, Faculty of Dentistry, Erciyes University, Kayseri- Turkey. 2. Assist. Prof.Dr. Department of Prosthetic Dentistry, Faculty of Dentistry, Dicle University, Diyarbakir- Turkey.

Abstract With the advent of new provisional crown materials, it has become imperative to evaluate their strength in order to select the appropriate crown material. This study examined the fracture resistance of three different provisional materials. To simulate oral conditions, the specimens were fabricated in brass molds, ensuring their similarity to premolars. The 33 fabricated specimens were divided into three groups (n=11) and stored at 37°C in artificial saliva (1.6 g NaHCO3, 0.4 g NaH2PO4∙H2O, and 0.1 g CaCl2∙H2O per L H2O). After conditioning, the fracture resistance was assessed using a universal testing machine (Testometric). Analysis of variance and Student’s t-test were used for statistical analysis. The study concluded that the mechanical properties of provisional restorative materials are strongly influenced by their proportions of methyl methacrylate and bisphenol A glycidyl methacrylate. (J Int Dent Med Res 2010; 3: (1), pp. 25-28 ) Keywords: Fracture strength, provisional crowns, dental materials. Received date: 18 August 2009 Introduction Today, provisional crowns represent an important stage in fixed prosthesis treatment. Provisional prostheses are intended to protect the support teeth and restore function, phonation, esthetic appearance, and tissue compatibility during preparations for permanent restoration1,2. Provisional crown and bridge materials must be esthetically acceptable, must resist functional loads, and must possess adequate stability and biocompatibility. The resistance of a material assumes greater importance in the presence of parafunctional habits or if a long-term prosthesis is planned, especially if the patient needs to wear a provisional crown for a long time1-3. Various resins are used to fabricate provisional restorations; these include ethyl methacrylate, methyl methacrylate, and bis-acryl *Corresponding author: Assist. Prof. Dr. Eylem Ozdemir Department of Prosthetic Dentistry, Faculty of Dentistry, Dicle University, 21280 – Diyarbakır / Turkey. Tlf: +90 412 2488101/3415; Fax: +90 412 2488100 E-mail: dteylem@yahoo.com

Volume 3 ∙ Number ∙ 1 ∙ 2010

Accept date: 22 December 2009 composites. Ethyl methacrylate has poor esthetics, despite being resistant to abrasion. Methyl methacrylate and bis-acryl composites are superior to ethyl methacrylate in both respects4. Provisional crown materials have been evaluated in terms of marginal aperture, polymerization shrinkage, color stability, temperature increase, and fracture resistance. The specimens used for resistance tests were generally prepared as disks, rods, or bars, which were subjected to three-point bending tests5. However, researchers have reported the need to test specimens with close resemblance to crown morphology, emphasizing that data obtained otherwise might be unrealistic6,7. This study analyzed the fracture resistance of different provisional crown materials in vitro using specimens prepared in premolar form. Material and Methods Three provisional crown materials were selected for use (Table 1). Thirty brass dies with the dimensions of premolars were prepared for crown fabrication. The conic specimens had a crown height of 7 mm, a crown width of 8 mm, Page 25


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

and a planned shoulder width of 1.5 mm in all directions. The labial, palatinal, and axial surfaces were inclined at a 6°-angle of approach.

The Fracture Strength of Different Materials Mustafa ZORTUK et al

program. As the groups were independent and each contained fewer than 30 specimens, Student’s t-test was used for statistical analysis.

Table 1. Provisional crown materials tested. To obtain provisional crowns with the same dimensions and shape, a brass mold was used (Figure 2). A socket to hold the die was prepared in the brass mold. With the die inserted in the mold, a negative space, 9.5 mm in diameter and 8.5 mm deep, resulted. The 30 dies were divided into three separate (totally 33 samples) groups, and three different provisional crown materials were used (Figure 1). The provisional crown materials were prepared according to the manufacturers’ recommendations and were poured into the space formed inside the mold. The mold was vibrated to eliminate air, and excess material was removed. Polymerization proceeded to completion under a fixed pressure of 2.5 kg. Subsequently, excess material was removed using stone grinders. The specimens were matched to a crucible, and abraded surfaces were rubbed with 600-grit sandpaper to obtain smooth surfaces. The crown-shaped provisional crown specimens were cemented (Proviscell; Septodont, France) onto the brass molds following the manufacturer’s instructions. Before fracture testing, the specimens were kept for 14 days at 37°C in artificial saliva (1 L doubledistilled H2O, 1.6 g NaHCO3, 0.4 g NaH2PO4∙H2O, and 0.1 g CaCl2). The prepared specimens were placed in a measurement device (Testometric; Testometric, UK) for fracture testing. The ends of the fracturing apparatus were hemispherical, and the base of the crown was marked so that the fracture test could be performed at the same point on each crown. During the test, the force was loaded at an approach speed of 0.5 mm/min. The data were recorded digitally (Figure 3) and were analyzed using the SPSS 10 statistical Volume 3 ∙ Number ∙ 1 ∙ 2010

Figure 1. Brass dies with premolar dimensions were prepared with the aim of crown fabrication.

Figure 1. The use of brass mold, in order to obtain provisional crowns with all the same dimensions and shape. Results The fracture resistances of the three different provisional crown materials are presented in Figure 4. The provisional crown material Temdent had the highest fracture resistance (581.9 N), followed by Tempofit duomix (403.7 N), and Protemp III (304 N). The difference between groups was significant (p < 0.05) based on one-way analysis of variance (ANOVA). The results of two-way Student’s ttests are presented in (Table 2).

Page 26


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

The Fracture Strength of Different Materials Mustafa ZORTUK et al

Discussion

Figure3. The specimens prepared were consecutively placed in a test measurement device (Testometric, Testometric Co., UK) for fracture testing.

Table 2. Statistical results (p<0.005).

Figure 4. Fracture resistances among the groups. Volume 3 ∙ Number ∙ 1 ∙ 2010

The fracture resistances of three different provisional crown materials were examined in the laboratory8,9. We used brass molds to produce standard specimens of provisional prostheses prepared in a crown shape. In addition to the preparation of standard specimens and the establishment of appropriate storage conditions, the loading speed selected during testing is also thought to influence fracture resistance. A variety of loading speeds have been used in studies of the fracture resistance of provisional restorations. Resistance has been shown to increase with increased loading speed, owing to the lack of time for cracks to grow. Thus, fast loading speeds can produce faulty data, and the loading speed should be relatively slow10-12. In light of these studies, we used a loading speed of 0.5 mm/min and applied axial (vertical) loading, considering the intraoral forces on the premolar crown-shaped specimens. The average values for resistance to fracture obtained in our study were 581.9 N for Temdent (methyl methacrylate), 403.7 N for Tempofit (bis-acryl composite), and 304 N for Protemp (bis-acryl composite). The differences between groups were significant (p < 0.05), and we believe that these differences arise from differences in the chemical structures of the materials9,13-15. Provisional crowns are fabricated from materials containing bisphenol A glycidyl methacrylate (BIS-GMA) and methyl methacrylate, with each material exhibiting unique physical and chemical properties. When combined in different provisional materials, the monomers display differences in properties such as exothermic heat of polymerization and shrinkage resistance4,9. Haselton et al. examined the shrinkage resistance of various provisional crown materials, obtaining the lowest shrinkage resistance with methyl methacrylate and the highest with bisacryl13. Osman et al. reported that provisional crown material containing methyl methacrylate had higher resistance to shrinkage compared with composite-based provisional crown material8. In contrast, after testing methyl methacrylate and composite-based provisional crown materials, Wang et al. reported no significant difference between them11. Ireland et al. investigated the shrinkage resistance of four Page 27


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

provisional restoration materials and reported that bis-acryl had the highest shrinkage resistance14. In contrast to our study, those studies applied flexural testing. It is thought that methyl methacrylate is not resistant to flexural stress because it consists of linear, mono-functional, low-molecular-weight molecules, whereas materials containing BIS-GMA comprise multidirectional, flexible chains that provide high flexural resistance8,12,13. With advanced monomer systems using bis-acryl, it is necessary to establish a balance between high mechanical resistance and limited elasticity. Methyl methacrylate increases fracture resistance, while BIS-GMA provides flexural resistance. Studies have shown that materials containing both are able tolerate brief deformation and resist high stress13,14,16. In our study of provisional crown materials with different structural properties, Temdent, which contains only methyl methacrylate, was the most resistant to pressure-induced fracture. Protemp, which contains BIS-GMA, was the least resistant, probably because BIS-GMA, although resistant to flexural forces, is not resistant to pressure forces. Researchers obtained similar findings in a study of three different resins and bis-acryl materials9,12,13. Materials containing methyl methacrylate have serious disadvantages such as high heat emission during polymerization, shrinkage, and high residual monomer levels. Compared with the mono-functional acrylate, bis-acryl materials have the advantage of relatively low heat emission during polymerization8,9,12,13,16. Conclusions We found only a few studies of provisional crown restorations in our review of the literature. Our study evaluated the resistance properties of three materials. However, a material with good mechanical properties may have other less desirable features (e.g., polishing, difficult manipulation, and esthetic appearance). As no provisional crown material can meet all requirements under all circumstances, clinicians must evaluate several characteristics such as esthetic appearance, ease of application, and cost when selecting products. The requirements will differ for a single provisional crown implanted in the anterior region, emphasizing the Volume 3 ∙ Number ∙ 1 ∙ 2010

The Fracture Strength of Different Materials Mustafa ZORTUK et al

importance of selecting a provisional crown material appropriate to each patient based on the particular features the material. Declaration of Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article. References 1. Haselton DR, Diaz-Arnold AM, Dawson DV. Effect of storage solution on surface roughness of provisional crown and fixed partial denture materials. J Prosthodont 2004;13(4):227-32. 2. Young HM, Smith CT, Morton D. Comparative in vitro evaluation of two provisional restorative materials. J Prosthet Dent 2001; 85(2):129-32. 3. Shillingburg HT, Hobo S, Whitsett LD, Jacobi R, Brackett SE. Fundamentals of fixed prosthodontics. 3rd ed. Chichago: Quintessence Publishing; 1997. p. 225-56. 4. Ulker M, Ulker HE, Zortuk M, Bulbul M, Tuncdemir AR, Bilgin MS. Effects of current provisional restoration materials on the viability of fibroblasts. Eur J Dent. 2009; 3(2):114-9. 5. Rosentritt M, Behr M, Lang R, Handel G. Flexural properties of prosthetic provisional polymers. Eur J Prosthodont Restor Dent. 2004; 12(2):75-9. 6. Yoshinari M., Derand T. Fracture strength of all-ceramic crowns. Int J Prosthodont. 1994;7: 329-338. 7. Pröbster L. Survival rate of in-ceram restorations. Int J Prosthodont. 1993; 6: 259-263. 8. Osman YI, Owen CP. Flexural strength of provisional restorative materials. J Prosthet Dent 1993; 70(1): 94-6. 9. Kim SH, Watts DC. Polymerization shrinkage-strain kinetics of temporary crown and bridge materials. Dent Mater 2004; 20(1): 8895. 10. Pfeiffer P, Grube L. In vitro resistance of reinforced interim fixed partial dentures. J Prosthet Dent 2003; 89(2): 170-4. 11. Wang RL, Moore BK, Goodacre CJ, Swartz ML, Andres CJ. A comparison of resins for fabricating provisional fixed restorations. Int J Prostodont 1989; 2(2): 173-84. 12. Hernandez EP, Oshida Y, Platt JA, Andres CJ, Barco MT, Brown DT. Mechanical properties of four methylmethacrylate-based resins for provisional fixed restorations. Biomed Mater Eng. 2004; 14(1): 107–22. 13. Haselton DR, Diaz-Arnold AM, Vargas MA. Flexural strength of provisional crown and fixed partial denture resins. J Prosthet Dent 2002; 87(2): 225-8. 14. Ireland MF, Dixon DL, Breeding LC, Ramp MH. In vitro mechanical property comparison of four resins used for fabrication of provisional fixed restorations. J Prosthet Dent 1998; 80(2): 15862. 15. Chan D, Giannini M, Goes MF. Provisional anterior tooth replacement using nonimpregnated fiber and fiber-reinforced composite resin materials. A clinical report. J Prosthet Dent 2006;95(5):344-8. 16. Akova T, Ozkomur A, Uysal H. Effect of food-simulating liquids on the mechanical properties of provisional restorative materials. Dent Mater 2006; 22(12): 1130-4.

Page 28


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

Cleft Lip and Palate: case report Zukanović Amila et al

INTERDISCIPLINARY APPROACH IN A TREATMENT OF THE PATIENT WITH CLEFT LIP AND PALATE – CASE REPORT Zukanovic Amila1*, Nakas E2, Prohic S3 1. Department of Preventive and pediatric dentistry, Faculty of Dentistry, University of Sarajevo, Bosnia and Herzegovina. 2. Department of Orthodontics, Faculty of Dentistry, University of Sarajevo, Bosnia and Herzegovina. 3. Department of Oral surgery, Faculty of Dentistry, University of Sarajevo, Bosnia and Herzegovina.

Abstract Background: Therapy of patients with cleft lip and palate does not comprise only surgical closure of the cleft – it requires multidisciplinary approach in a dental treatment, with the aim to achieve aesthetically and functionally optimal results. Case report: 12-years-old patient, referred to the orthodontic clinic with surgically closed unilateral cleft lip and palate. After taking diagnostic records, the patient was scheduled for orthodontic treatment. Before banding, crown lengthening on first upper molar has been performed due to a short clinical crown. Length of active orthodontic treatment was 30 months, after which retainer has been placed, and patient scheduled for crown lengthening on upper left incisor and canine. Multiple tooth restorations have been performed on upper anterior region using adhesive, build - up technique (teeth 13, 21, 22, 23). Dental photography editing with image editing software enabled information about possible aesthetic solution for our patient. Follow up: Clinical evaluation of composite restorations has been performed using USPHS criteria. Functionality, pleasing aesthetic results and satisfied patient are outcome of 4 years long comprehensive dental treatment. (J Int Dent Med Res 2010; 3: (1), pp. 29-33 ) Keywords: Cleft lip, palate. Received date: 14 December 2009 Introduction Cleft lip and palate (CLP) is a congenital malformation characterized by morphologic changes and deficiencies of soft and hard tissues in oral and maxillofacial region. The prevalence of cleft lip and palate among population has been estimated between 1:500 – 1:2500 live births1,2. This congenital deformity shows a multifactorial and complex etiology, where genetic and environmental factors cause abnormal facial development during gestation.

*Corresponding author: Zukanovic Amila, DDS, PhD Department of preventive and pediatric dentistry Faculty of Dentistry, University of Sarajevo Bolnička 4a 71 000 Sarajevo Bosnia and Herzegovina E-mail: migulin@lsinter.net

Volume 3 ∙ Number ∙ 1 ∙ 2010

Accept date: 08 February 2010 Cleft lip and palate is usually associated with different dental anomalies. Several authors report crown and root malformations, delay in tooth development/dental maturity, anomalies in tooth structure and size, variation in the number of teeth (oligodontia)3-6. Aesthetic and functional disturbances are often associated with cleft lip and palate that require early medical, surgical and dental interventions, which start early after birth and continues in various stages until maturity7. Patients with cleft often born with some missing teeth, where the lateral incisor in the line of cleft is usually absent, that can create additional functional and aesthetic problems. Treatment plan should be made individually, made by a team of dental specialists, including maxillofacial and oral surgeons, orthodontists, pediatric dentists, prosthodontists in order to perform treatment for these patients with the best result.

Page 29


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

Children with cleft lip and/or palate require a multidisciplinary approach in which different specialist are involved. The team concept remains the key to success in the care of these patients8. Due to that, some parents seek care for children in cleft palate or craniofacial treatment centers, if exists. No matter cleft can produce variety of oral problems; a child with a cleft lip/palate requires the same regular preventive and restorative care as the child without a cleft. Case Report In 2002, 12-years-old female patient, referred to the orthodontic clinic after surgical correction of unilateral clef lip and palate. After taking diagnostic records, orthopantomography and cephalometric analysis, patient was scheduled for comprehensive dental treatment with following diagnosis: status post op. cheilognathopalatoshisis, with consequent pseudoprogenia and progenia vera, hypodontia of upper right incisors (teeth 12, 11), rotation of upper premolars and palatinal position of tooth 25.

Figure 1. 1a. Plaster models of the patient before the therapy with mobile functional appliance. 1b. Plaster models of the patient before the therapy with fixed orthodontic treatment. 1c. Plaster models of the patient after completing of active orthodontic treatment. Orthodontic treatment was planned in two phases. In first phase (2002-2003) mobile functional appliances have been used, in order to Volume 3 ∙ Number ∙ 1 ∙ 2010

Cleft Lip and Palate: case report Zukanović Amila et al

achieve somewhat better position of bone segments to provide underlying framework for soft tissue. Second phase begin in 2004 when full fixed orthodontic treatment begun, with use of Roth 0.22 bracket system, transpalatal arch as additional anchorage due to palate cleft, and band on upper molar teeth. Patient’s mother rejected orthognatic surgery after completing of orthodontic treatment. Before banding, crown lengthening on first upper molar has been performed due to a short crown. Length of treatment was 20 months - retainer had been placed after, and patient scheduled for crown lengthening on upper left incisor and canine (Figure 1,2).

Figure 2. 2a. Orthopantomography before any orthodontic treatment started. 2b. Orthopantomography during fixed orthodontic treatment. Surgical crown lengthening has been proposed to improve restorative procedures and preventing periodontal injuries in teeth with structurally inadequate clinical crowns or exposing tooth structure in the presence of deep, subgingival pathologies which may hamper the Page 30


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

access

for proper restorative measures. Periodontal surgical procedures consisting of gingival flaps and osseous recontouring are indicated for crown lengthening of several contiguous teeth in the esthetic zone; both in cases where restorations are required and in cases where no restorations are planned, such as in patients with excessive gingival smile due to altered passive eruption9,10. Subepithelial connective tissue graft is one of the most used periodontal plastic surgery procedure in cases that already show recession defects (Miller class I and II). In our case crown elongation, as a surgical procedure on central incisor, showed satisfying clinical result, without any signs of recession. In 2007, after orthodontic and surgical crown lengthening treatment, patient had been scheduled for conservative treatment of pediatric dentist in order to find satisfying optimal aesthetical solution for the patient (Figure 3).

Cleft Lip and Palate: case report Zukanović Amila et al

Multiple tooth restorations have been performed on upper anterior region (teeth 13, 21, 22, 23), in four dental visits. Canine teeth were reshaped in lateral incisors, tooth 22 in a central left incisor and tooth 21 in a right central incisor. In all teeth, the enamel surface was etched with 37% phosphoric acid for 30 seconds (Eco - Etch®), due to additional micro retention, no matter that self-etching system was planned to be use after. On already prepared enamel surface, self – etching system AdheSe® (Ivoclar Vivadent) has been additionally used. Build-up technique (dental composite bonding) for teeth restoration has been performed using light – curing ARTEMIS® composite (Ivoclar Vivadent) (Figure 4,5).

Figure 4. Direct composite restorations of teeth 21 and 22.

Figure 3. Face of the patient and dental status after finishing orthodontic treatment, before starting multiple tooth restorations. Possible prosthodontic and conservative treatments had been discussed in order to decide if the better approach was veneers, crowns or composite build-up technique. Respecting the patient’s age, patient started with conservative teeth restoration, using direct restorative approach. Teeth morphology had been modified with adhesive techniques in order to achieve satisfying functional and aesthetic goals (position, shape and color). Volume 3 ∙ Number ∙ 1 ∙ 2010

Figure 5. Direct composite restorations of teeth 13 and 23. Page 31


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

During dental visits, while tooth restorations using build up technique have been performed, digital dental photography was taken with non-professional digital camera (Nicon Coolpix P50, 8.1 megapixels). Dental photography editing, with image editing software Adobe photoshop 6.0, enabled information about possible aesthetic solution for our patient regarding teeth morphology and size variations. Using this software, the optimal esthetic has been achieved with saving the time spent on dental chair for the patient and dentist as well. Follow up: Clinical evaluation of composite restorations has been performed using modified USPHS (United States Public Health Service) criteria11. Discussion Orthodontic treatment of patient with CLP should be performed in phases. The recommendation is to avoid continuous active treatment in early childhood, because these patients are often seeking some more medical procedure (ENT, speech therapist, maxillo-facial or plastic surgeons, etc). Our treatment was performed in two phases. First phase was 1 year long and second one - 20 months. Due to previously performed operation, mother refused orthognatic surgery as a part of treatment plan, so we discuss the alternatives, and the plan was to align the teeth and achievement as much contact points as possible for good occlusion and then restoration of the teeth. After completing of permanent dentition, fixed orthodontic treatment started in 2004, and finished in 2007. Due to the fact that patients with CLP usually have different dental anomalies, beside surgical and orthodontic treatment, very often these patients require comprehensive aesthetic dental treatment. In older patients, anomalies in size, shape and number of teeth can be solved using different prosthodontic solutions. But, regarding the age of the patient, one of the possible solutions was teeth reshaping using adhesive technique. This technique is time consuming and requires specific personal skill of the therapist. But on the other hand, it is noninvasive, repeatable, and comfortable for the patient. Using light – curing ARTEMIS® composite, with wide range of shades and various degrees of translucency, we were able to Volume 3 ∙ Number ∙ 1 ∙ 2010

Cleft Lip and Palate: case report Zukanović Amila et al

match the colors until we found satisfying aesthetic solution. Multiple tooth restorations have been performed on teeth 13, 21, 22, 23. Even after orthognatic treatment, patient stayed with disharmonic dental arches. Very high positioned central incisor in the oral vestibulum, near to the line of the cleft, with mesio-distal inclination, followed with wide diastema, limited our therapy. During teeth restorations with dental adhesive systems, digital photography was taken in order to find the best aesthetic appearance between two dental visits. Image editing software (Adobe photoshop 6.0) enabled information about possible treatment for our patient regarding teeth morphology and size variations. Treatment’s plan was done respecting total face and dental appearance (Figure 6).

Figure 6. Possible solutions in teeth remodelation using image editing software.

As it can be seen, we intentionally reshaped teeth on the way that certain disharmony still exists. Incisal edge of frontal teeth is not “perfectly” aligned intentionally – incisal edge of remodelated tooth 22 is longer comparing with tooth 21. The reason why we choose it on that way is the line of cleft lip which is on the right side of the face. With tooth 22 reshaped in central incisor, one millimeter longer than tooth 21 on the left patient’s side, we succeed in delusion of “optically decreasing” lip cleft, while patient smiles. No matter the direct restoration technique is demanding in the sense of personals skills of a therapist and time Page 32


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

consuming for the dentist and patient too, the outcome of this treatment seems to be very satisfying (Figure 7). Clinical evaluation of composite restorations in the term of anatomic form, color match, marginal discoloration and adaptation, retention, secondary caries and sensitivity, has been performed using modified USPHS (United States Public Health Service) criteria at 3, 6, 12 and 24 months interval.

Cleft Lip and Palate: case report Zukanović Amila et al

2. Ohyama T. Prosthodontic considerations for patients with cleft lip and palate. Int Dent J 1986;36:140-5. 3. Pöyry M, Nyström M, Ranta R. Tooth development in children with cleft lip and palate: a longitudinal study from birth to adolescence The European Journal of Orthodontics 1989;11(2):125-130. 4. Normastura, A.R. and Nizam Abdullah, and Abdul Rani, S. and Lin, N. Dental Anomalies And Facial Profile Abnormality Of The Non-Syndromic Cleft Lip And Palate Children In Kelantan. Malaysian Journal of Medical Sciences 2004;11(2): 41-51. 5. Cabete HF, Gomide MR, Costa B. Evaluation of primary dentition in cleft lip and palate children with and without natal/neonatal teeth Cleft Palate Craniofac J. 2000;37(4):406-9. 6. Shapira Y,Lubit E,Kuftinec MM,DStom.Congenitally missing second premolars in cleft lip and cleft palate children. Am J Orthod Dentofacial Orthop 1999;115:396-400. 7. Saunders ID, Geary L, Fleming P, Gregg TA. A simplified feeding appliance for the infant with a cleft lip and palate. Quintessence Int 1989;20:907-10. 8. Mese A, Ozdemir E. Removable Partial Denture in a Cleft LIP and Palate Patient. J Korean Med Sci 2008;23:924-7. 9. Pontoriero R, Carnevale G. Surgical Crown Lengthening: A 12Month Clinical Wound Healing Study. Journal of Periodontology 2001;72(7): 841-848. 10. Camargo PM, Melnick PR, Camargo LM. Clinical crown lengthening in the esthetic zone. J Calif Dent Assoc 2007;35(7):487-98. 11. Cvar JF, Ryge G: Criteria for the clinical evaluation of dental restorative materials. US Government Printing Office, San Francisco. USPHS Publication No. 190-244, 1991.

Figure 7. Status praesens on the first control check-up. Conclusions Functionality, pleasing aesthetic results and satisfied patient are outcome of a comprehensive dental treatment. Declaration of Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article. References 1. Slayton RL, Williams L, Murray J, Wheeler JJ, Lidral AC, Nishimura CJ. Genetic Association Studies of Cleft Lip and/or Palate with Hypodontia Outside the Cleft Region. Cleft PalateCraniofac J 2003;4:274-9.

Volume 3 ∙ Number ∙ 1 ∙ 2010

Page 33


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

Pleomorphic adenoma Rezzan TANRIKULU et al

INTERESTING CASE: AN UNUSUAL LOCATION FOR A LARGE PLEOMORPHIC ADENOMA ARISING IN THE MAXILLA Rezzan Tanrikulu1, Ferhan Yaman2*, Serhat Atilgan2, Nedim Gunes3, Belgin Gorgun4 1. Assoc.Prof.Dr. University of Dicle Faculty of Dentistry Department of Oral and Maxillo Facial Surgery Diyarbakir / TURKEY. 2. Assist.Prof.Dr. University of Dicle Faculty of Dentistry Department of Oral and Maxillo Facial Surgery Diyarbakir / TURKEY. 3. DDS, MsC. University of Dicle Faculty of Dentistry Department of Oral and Maxillo Facial Surgery Diyarbakir / TURKEY. 4. Prof.Dr. University of Dicle Faculty of Dentistry Department of Oral and Maxillo Facial Surgery Diyarbakir / TURKEY.

Abstract Pleomorphic adenoma is the most common benign tumor of the major salivary glands, especially of the parotid gland. It is much less common in the minor salivary glands of the oral cavity, and it rarely occurs in others sites in the head and neck (intraosseous, lacrimal gland, chest). A 51-year-old woman was referred to our department complaining of painless swelling of the left maxillary vestibular sulcus, which had been felt pain with the pressure. CT scan revealed a well defined expansile mass arising from pterygoid plates, infratemporal space to palatinal bone and arcus zygoma. Transoral biopsy specimen showed pleomorphic adenoma. Patient was treated by surgical excision. Follow up goes on in postoperative period. Our study’s aim is to present the pleomorophic adenoma’s case which is extended up large propotion in an unusual location. Our case is evaluated by in terms of diagnose and treatment result. Furthermore in the light of literature reviews we discuss the pleomorphic adenoma’s incidence, location, and recurrence rates. We believe that our presentation is very interesting in point of view rare location and expansive mass. (J Int Dent Med Res 2010; 3: (1), pp. 34-37 ) Keywords: Pleomorphic adenoma, maxilla, salivary gland. Received date: 03 February 2010 Introduction This neoplasm was originally called benign mixed tumor in 1866, and its classic microscopic description was given in 1874. A name change to “pleomorphic adenoma” was first suggested in 1948, and considerable controversy has followed. The decision rests on the origin of the myoepithelial cell. The cellular component of a pleomorphic adenoma consists of epidermoid cells and myoepithelial cells. If the two cells have independent origins, the proper *Corresponding author: Assist. Prof. Dr. Ferhan YAMAN University of Dicle Faculty of Dentistry Department of Oral and Maxillo Facial Surgery 21280 Diyarbakir / TURKEY E-mail: dtferhan@dicle.edu.tr

Volume 3 ∙ Number ∙ 1 ∙ 2010

Accept date: 09 April 2010 name is “mixed tumor”. If the two cells have a common origin, the proper name is “pleomorphic adenoma”. Evidence favors the latter. Pleomorphic adenomas, or benign mixed salivary gland tumors, are the most common of all salivary gland neoplasms. They usually present as a unilateral, painless, slow-growing mass in the parotid gland. However, when they originate in the minor salivary glands, they mostly occur in the hard and soft palate. The palate has the highest concentration of minor salivary glands in the upper aerodigestive tract, and it is the most common site for benign and malignant minor salivary gland tumors1,2. Pleomorphic adenoma appears as a painless firm mass and, in most cases, does not cause ulceration of the overlying mucosa. Generally it is mobile, except when it occurs in the hard palate. Intraoral mixed tumors, especially those noted within the palate, lack a well-defined capsule. Lesions of the palate Page 34


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

frequently involve periosteum or bone. Approximately 25% of benign mixed tumors undergo malignant transformation. And also the clinical presentation of a pleomorphic adenoma arising from the hard palate is typically a firm or rubbery submucosal mass without ulceration or surrounding inflammation. Histology characteristically demonstrates myoepithelial, epithelial, and stromal components. The exact etiology for this tumor is debated, but the current theory is that pleomorphic adenomas originate from intercalated and myoepithelial cells1,3. Mixed tumors range in size from a few millimetres to several centimetres in diameter and are capable of reaching giant proportions in the major salivary glands, especially the parotid. The tumor is typically lobulated and enclosed within a connective tissue pseudocapsule that varies in thickness. In areas where the capsule is deficient, neoplastic tissue may lie in direct contact with adjacent salivary tissue and may contribute to recurrences4,5. Microscopically, mixed tumors demonstrate a wide spectrum of histologic features. The pleomorphic patterns and the variable ratios of ductal to myoepithelial cells are responsible for the synonym pleomorphic adenoma4,6. The treatment of choice is surgical excision. Lesions of the palate or gingiva often involve or about periosteum or bone, making complete removal difficult unless some bone is removed. Other oral benign mixed tumors can be more easily excised, preferably including tissue beyond the pseudocapsule4. Since the majority of minor salivary gland neoplasm’s arising in the palate is malignant, patient evaluation should include a detailed history and physical exam, especially focusing on signs of cranial nerve involvement. As with other salivary gland tumors, fine needle aspiration (FNA) biopsy should be performed as an adjunct to diagnosis prior to definitive surgical treatment. Computed tomography (CT) or magnetic resonance imaging (MRI) should be considered when assessing for presence of bony erosion or soft tissue and nerve involvement. Ultimately, complete surgical excision will provide the definitive diagnosis and treatment for this noteworthy salivary gland neoplasm1,7.

Pleomorphic adenoma Rezzan TANRIKULU et al

Case Report A 51-year-old woman was referred to our department complaining of painless swelling of the left maxillary vestibular sulcus, which had been felt pain with the pressure. No anomalous (pathologic) feature was observed by extra-oral examination. On the other hand a mass was detected in the left maxillary sulcus by bidigital palpation on the intra-oral examination. Except for the mass there was no pathologic feature on the case. Axial and coronal Computerized Tomography (CT) scan revealed a well defined expansile mass arising from pterygoid plates, infratemporal space to palatinal bone and arcus zygoma and also the mass’ size was 5x4 cm on CT scan (Figure 1).

Figure 1. Preoperative CT of tumoral mass. Incisional biopsy was performed under local anesthesia and the specimen was hardy consistency and grey- white colored and was sent for histopathological examination with prediagnosed as chondroma, neurofibroma, and pleomorphic adenoma (Figure 2).

Figure 2. Macroscopic appearance of tumour. Volume 3 ∙ Number ∙ 1 ∙ 2010

Page 35


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

Histopathologic examination revealed the typical mixed tumor has a biphasic appearance resulting from the initimate admixture of epithelium and stroma. Most of the epitelhial component is of a glandular nature, but foci of squamous metaplasia are common, accompanied by keratinized epithelial plugs in the lumen (Figure 3).

Pleomorphic adenoma Rezzan TANRIKULU et al

The patient was operated under general anesthesia and incision was made horizontally to prevent any damage to Stenon’s duct and orificis of duct. After the incision submucosal tissues dissected and also dissections was made to reach whole tumor from tuber maxilla to zygomatic bone and masseteric region. Then the whole tumor was excised. After the macroscopic view tumor was hardy consistency, fibroticchondroid in form, grey-white in color. Operation region was saturated primarily after hemorrhagic control. Then postoperative axial CT showed that whole tumor was excised (Figure 5).

Figure 3. Histological features of specimen (HE 20 X 10). The stroma has fibro-collagen appearance, containing vascular tissue and mononuclear infectious cells. Minor infiltration of the tumor margins is seen although the majority of the tumor is well circumscribed and surrounded by a zone of thin fibrous connective tissue (Figure 4). Therefore the mass reported as Pleomorphic Adenoma.

Figure 5. Postoperative axial assessment of lesion. In the postoperative period, due to spreading to masseteric region the mechanictherapy was started to prevent myositis ossificians and trismus due to scar formation. Such a clinical situation was not developing on the follow up. There are no recurrence for 4 months period and follow up goes on. Discussion

Figure 4. Histological features of specimen (HE 20X 10).

Volume 3 ∙ Number ∙ 1 ∙ 2010

Approximately 750 minor salivary glands are found in the oral cavity, nasal cavity, paranasal sinuses and pharynx8. The majority of these glands are found at the junction of the hard and soft palate, which is the common site for minor salivary glands tumors. In the palate, 50% of these tumors are malignant, whereas 85% to 90% are malignant in the tongue, and only 20% are malignant in the upper lip6. Page 36


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

CT scanning is the best for bony involvement in palatal lesions, and MRI is better to display soft tissue invasion or perineural spread1. Biopsy should be undertaken at the center of the tumor and should include overlying mucosa3,6. The most common benign tumor is pleomorphic adenoma; malignant tumors are divided into low-grade and high-grade lesions6. Pleomorphic adenoma is treated by local excision with 0.5 to 1 cm margins. Enucleation will lead to recurrence1,2,9. Except in the case of larger neglected tumors, reconstruction may be by primary or secondary healing or the use of local flaps2,9. Bony excision is usually not required, as pleomorphic adenoma dose not invade bone, although it may cause pressure resorbtion6. There is small risk of recurrence, as well as a small (5%) risk of malignant transformation to a carcinoma- ex- pleomorphic adenoma10. About 25% of pleomorphic adenomas may present malignant evaluation if not treated2. Said et al reported the case of a 77year-old woman who presented with a six year history of pleomorphic adenoma with multiple recurrences and myoepitelial carcinoma ex pleomorphic adenoma of salivary glands3.

Pleomorphic adenoma Rezzan TANRIKULU et al

4. Regezi JA, Sciubba JJ, Jordan RCK. Oral Pathology Clinical Pathologic Correlations. Saunders Company, 2003. 5. Piekarski J, Nejc D, Szymczak W, Wronski K, Jeziorski A. Results of extracapsular dissection of pleomorphic adenoma of parotid gland. J Oral Maxillofac Surg. 2004; 62(10): 1198-202. 6. Ord RA. Salivary gland disease in ed: Fonseca RJ. Oral and Maxillofacial Surgery vol. 5. W.B. Saunders Company, 2000: 273-94. 7. El-Nagar AK, et al. Pleomorphic adenoma. The American Journal of Surgical Pathology 1998; 22(6): 772. 8. Ord RA: Management of intra-oral salivary gland tumors. Oral Maxillofac Surg Clin North Am 1994; 6: 499-522. 9. Pogrel MA. The management of salivary gland tumors of the palate. J Oral Maxillofac Surg. 1994; 52: 454-9. 10. Schow SR, Miloro M. Diagnosis and management of salivary gland disorders in ed: Peterson LJ. Contemporary Oral and Maxillofacial Surgery. Mosby Company 1998.

Conclusions In our case we decided to perform, with the patient under general anesthesia, a radical removal of the tumor within the surrounding soft tissue with 5-mm margins. Although the pleomorphic adenoma is a benign tumor, enucleation alone is not advisable because of the high rate of recurrence and dissemination. About 25% of pleomorphic adenomas may present malignant evolution if not treated. Declaration of Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article. References 1. Lowry TR, Heichel DJ. Pleomorphic adenoma of the hard palate. Otolaryngol Head Neck Surg. 2004; 131(5): 793. 2. Clauser L, Mandrioli S, Dallera V, Sarti E, Galie M, Cavazzini L. Pleomorphic adenoma of the palate. J Craniofac Surg. 2004; 15(6): 1026-9. 3. Said S, Campana J. Myoepithelial carcinoma ex pleomorphic adenoma of salivary glands: Aproblematic diagnosis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2005; 99(2): 196-201.

Volume 3 ∙ Number ∙ 1 ∙ 2010

Page 37


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

Antibiotic resistance and plasmid profiles of vibrio P. Manivasagan et al

ANTIBIOTIC RESISTANCE AND PLASMID PROFILES OF VIBRIO ISOLATES FROM MUTHUPETTAI MANGROVE ENVIRONMENT, SOUTHEAST COAST OF INDIA P. Manivasagan1*, S. Ramesh1, K. Sivakumar2, T. Thangaradjou3, S. Vijayalakshmi4, T. Balasubramanian6 1. Research Scholar, CAS in Marine Biology, Annamalai University, Tamil Nadu, INDIA. 2. Senior Lecturer, CAS in Marine Biology, Annamalai University, Tamil Nadu, INDIA. 3. Lecturer, CAS in Marine Biology, Annamalai University, Tamil Nadu, INDIA. 4. Research Associate, CAS in Marine Biology, Annamalai University, Tamil Nadu, INDIA. 5. Professer & Director, CAS in Marine Biology, Annamalai University, Tamil Nadu, INDIA..

Abstract A total of 209 pathogenic vibrios strains were isolated from Muthupettai mangrove environment, Southeast coast of India. All strains were identified to be 8 species (Vibrio cholera, Vibrio harveyi, Vibrio mimicus, Vibrio splendidus, Vibrio aestuarianus, Vibrio vulnificus, Vibrio parahaemolyticus and Vibrio metschnikovii) from all stations. The levels of resistance of bacteria to various antibiotics differed considerably. Among these 3 species (13 strains) multiple antibiotic resistance bacteria were identified from all isolates such as Vibrio cholerae (5 strains), Vibrio parahaemolyticus (5 strains) and Vibrio vulnificus (3 strains). The all strains were also able to resistance concentration of antibiotics up to 150µg/ml. The isolated strains were screened for plasmid DNA by agarose gel electrophoresis and tested for susceptibility to 10 antibiotics by the agar dilution method. 13 strains belonging to 3 species have been found to Muthupettai mangroves 1 – 3 plasmids, with sizes ranging from 11 – 112kb. (J Int Dent Med Res 2010; 3: (1), pp. 38-44 ) Keywords: Vibrio spp., muthupettai, mangroves, multiple antibiotic resistance, plasmid profiling. Received date: 23 October 2009 Introduction Mangrove wetlands along the coastal zone act as barrier against cyclones, protect coastal erosion and provide good nursery ground for number of commercially important aquatic organisms 1. Bacteria of the genus Vibrio are commonly found in coastal, estuarine and mangroves waters. Some Vibrio strains are pathogenic and can cause vibriosis, a serious infectious disease in both wild and cultured finfish *Corresponding author: P. Manivasagan Centre of Advance Study in Marine Biology Annamalai University, Parangipettai - 608 502. Tamil Nadu, INDIA E-mail: manimaribtech@gmail.com

Volume 3 ∙ Number ∙ 1 ∙ 2010

Accept date: 04 February 2010 and shellfish2. In recent years, vibriosis has become one of the most important bacterial diseases in maricultured organisms, affecting a large number of species of finfish and shellfish3, 4. Antibiotics and other chemotherapeutic agents are commonly used in aquaculture farms either as feed additives or immersion baths to achieve either prophylaxis or therapy. However, extensive use of these drugs has resulted in an increase of drug-resistant bacteria as well as Rplasmids5, 6. Furthermore, many species of halophilic vibrios have become recognized as potential human pathogens causing serious gastroenteritis or severe wound infection upon exposure to contaminated seafood and/or seawater7. Elucidation of the antimicrobial susceptibilities of potential pathogenic vibrios will be important for prophylaxis and treatment of vibrio infections in human beings and in cultured marine organisms. Diseases are, perhaps, the major cause of losses in the aquaculture industry, among Page 38


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

them; vibriosis is one of the most frequent diseases affecting fishes, molluscs and crustaceans8. Plasmids have been found in vibrios9,10, and in some cases, their involvement in resistance to many antibiotics has been proven10,11. To our knowledge, plasmid presence, profiling, or their relationship with antibiotic resistance, have not been reported from bacterial strains isolated from mangrove in the Muthupettai. The aim of this study was to investigate the presence of plasmids and their relationship with antibiotic resistance in strains isolated from Muthupettai mangrove environment in Southeast coast of India. Material and Methods STUDY AREA: Muthupettai mangroves (Lat. 10o 25’N; Long. 79o 39’E) situated 400km south of Chennai lies along the south east coast of India. It has total area of 6800ha in which the water spread area covers approximately 2720 ha. It has two specialized habitats were noted viz. mangroves and lagoon (Figure 1).

Figure 1. Map of Muthupettai mangrove environment showing different locations. Many tributaries of the river Cauvery delta such as Paminiyar, Koraiyar, Kilaithangiyar, Kandankurichanar and Marakkakoraiyar flow through Muthupettai and nearby villages and form a lagoon before they reaches the sea, Bay of Bengal. Avicennia marina is the dominant mangrove species in Muthupettai and accounts for nearly 95% of the vegetative cover. The sampling areas for present study viz.,1, Aquaculture discharge area; 2. Sethuguda; 3. Lagoon; 4. Sellimunai; 5. Sea mouth region and 6. Open sea. Volume 3 ∙ Number ∙ 1 ∙ 2010

Antibiotic resistance and plasmid profiles of vibrio P. Manivasagan et al

A sediment samples at six stations were collected during April (2006) to March (2007) and transported on ice to the laboratory and processed within 18-24h. Selective isolation and purification of Vibrio species Sediment samples (1 g) were directly spread on Thiosulphate Citrate Bile Salts Sucrose (TCBS) agar plates (HiMedia Laboratories Pvt. Ltd, Mumbai) and incubated at 30 oC for about 12 h. Distinctive yellow and green colonies on agar plates were picked and streaked on new TCBS agar plates several times to purify bacterial isolates until pure cultures of isolates were obtained. A total of 209 bacterial isolates were randomly selected from Muthupettai mangroves. Only thirteen (MAR) strains were highly selective from each site for screening for the presence of plasmids 12. Identification of Vibrio species Morphological and biochemical properties of the bacteria were investigated according to Bergey’s manual of determinative bacteriology 13. Determination of antibiotic resistance Antibiotic resistance of bacteria was determined by the single disc diffusion method with the use of Mueller-Hinton agar, according to the Kirby-Bauer method14. Bacteria were multiplied on agar slants (ZB) at 20oC. After 72h they were washed off the slants with 5 cm3 of sterile buffered water and adjusted to a turbidity of 4 on the Mac Farland scale, which corresponds to 109 bacterial cells per 1 cm3. Subsequently, 0.2 cm3 of bacterial suspension prepared and introduced into steriled MuellerHinton medium cooled to 40oC. After mixing, the sample was poured onto Petri dishes and dried in a drier at 37oC for 1h. Paper discs impregnated with an antibiotic were than applied to the surface of the seeded medium. The blotting paper discs (13mm) were manufactured by HIMEDIA. The dishes were kept at 4 o C for 1h in order to allow antibiotic diffusion from the discs into the agar medium. The dishes were then incubated at 27 o C for 24h. Bacteria were classified as antibiotic resistant according to the manufacturer instructions. The following ten clinical antibiotics, with their concentrations given in parentheses were used in antibiograms: Ampicillin (AM, 10µg), Chloramphenicol (CP, 30µg), Gentamycin (GE, 10µg), Kanamycin (KM, 30µg), Nalidixic acid (NA, 30µg), Novobiocin (NB, 30µg), Penicillin (PL, 10µg), Rifampicin (RF, 10µg), Streptomycin (SM, Page 39


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

30µg) and Tetracycline (TE, 30µg). The results were used to calculate the antibiotic resistance index (ARI) for bacteria15. Time course for growth of the bacterial isolates Exponentially grown cultures of the test organisms were inoculated into treated (30, 60, 90, 120 and 150 µg/ml of antibiotics) and untreated liquid culture medium and incubated at 28oC for different time intervals. A control was also run simultaneously. The growth was determined turbidometrically at different time intervals by measuring the optical density (OD) at 540 nm in a Spectronic-20 spectrophotometer16. Extraction of plasmid Plasmid DNA of bacterial isolates was extracted using alkaline lysis method as described by Sambrook et al., (1989). QIAprep spin miniprep kit (Valencia, CA, USA) was also applied to confirm the plasmid extraction result by alkaline lysis method. The plasmid DNAs were loaded onto 0.7% horizontal agarose gels for separation and viewing. Gels were run at 5 Vper cm, strained in ethidium bromide, destained in water and photographed on a UV transilluminator 17.

Antibiotic resistance and plasmid profiles of vibrio P. Manivasagan et al

mangroves environment (percentages derived from the pooled date of all stations). Over 15% of the bacterial microflora was resistant to clinically used antibiotics such as kanamycin, nalidixic acid, novobiocin and pencillin; less than 6% of the isolates were resistant to gentamycin and streptomycin. Differences in the level of antibiotics resistance between the Vibrio spp. isolated from different station of the mangroves environment were determined (Table. 1).

Results Vibrio spp. isolated from different stations of the Muthupettai mangroves environment were subjected to analysis for resistance to ten widely used antibiotics. Our observations were carried out in the month of April to March during the years 2006-2007. Vibrio spp. isolated from the Muthupettai mangrove environment are characterized by large differences in the level of resistance to studied antibiotics (Figure 2).

Percentage of strains

30 25 20 15 10 5 0 AM CP

GE KM NA NB

PL

RF SM

TE

Antibiotics

Figure 2. Resistance to different antibiotic among bacteria isolated from Muthupettai Volume 3 ∙ Number ∙ 1 ∙ 2010

Table 1. Resistance to the antibiotics of bacteria isolated from different stations (in %).

Most of the antibiotic resistant bacteria were found in the aquaculture pond discharge area of the mangrove environment (Station 1) (ARI 0.16) and the most sensitive bacteria were isolated from open sea (Station 6) (ARI 0.11). At all studied station, most of the bacteria were resistant to kanamycin, nalidixic acid, novobiocin and pencillin, and most sensitive to gentamycin and streptomycin. Figure 3 presents the results of the study of antibiotic resistance in yellow and green Vibrio spp. isolated from the Muthupettai mangroves environment. Generally no differences between yellow and green bacteria were noted, more resistant kanamycin, nalidixic acid, novobiocin and pencillin and most sensitive to gentamycin and streptomycin.

Page 40


Percentage of strains

Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

30 25 20 15 10 5 0 AM

CP

GE KM NA

NB

PL

RF

SM

TE

Antibiotics Green colonies

Yellow colonies

Antibiotic resistance and plasmid profiles of vibrio P. Manivasagan et al

parahaemolyticus (5 strains) and Vibrio vulnificus (3 strains) were tested for their resistance to the all antibiotics. The Vibrio cholerae strains resistance concentration up to 150µg/ml of 8 antibiotics, remaining antibiotics 120 µg/ml of SM and TE. The Vibrio parahaemolyticus strains resistance concentration up to 150µg/ml of 9 antibiotics, 120µg/ml of SM. The Vibrio vulnificus resistance concentration up to 150µg/ml of CP, KM, NA, NB and PL, 120 µg/ml AM, GE, RF, SM and TE.

Figure 3. Differential resistance of yellow and green bacteria to studied antibiotics.

Percentage of strains

Chosen strains were analyzed for multiple antibiotic resistance (MAR) (Figure 4). About 1115% of the studied bacteria were resistant to KM and NA. 3-10% of the studied bacteria showed an AM, CP, NB and PL MAR pattern (i.e. resistance to KM and NA of the 10 antibiotics tested). 0.5-2% of studied bacteria showed a SM and TE MAR. 16 14 12 10 8 6 4 2 0 AM CP

GE KM NA NB

PL

RF SM

TE

Antibiotics

Table 2. Antibiotic resistance of Vibrio species identified from mangrove environment.

Figure 4. Multiple antibiotic resistance bacterial strains inhabiting Muthupettai mangrove environment. A total of 209 strains were isolated based on colony morphology, gram-negative, motile by means of polar flagella, oxidase-positive, fermented glucose, yellow and green color from antibiotic resistance of TCBS selective agar plates for biochemical identification (Table 2). The majority of the isolated strains from station 1 were identified. The lowest number of isolates was identified from station 6. Highly resistance of the 13 (MAR) strains isolates were classified and identified as 3 species using MIC of resistance bacteria and plasmid profile. All the thirteen multiple antibiotic resistance of bacterial strains (Table. 3) isolates of to Vibrio cholerae (5 strains), Vibrio Volume 3 ∙ Number ∙ 1 ∙ 2010

Table 4. Plasmid profiles of the 13 Vibrio species isolates. Page 41


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

Thirteen strains belonging to Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus have been found to Muthupettai mangroves 1 – 3 plasmids, with sizes ranging from 11 – 112kb (Table. 4). Ten strains were found to contain 1 plasmid, two strains contained 2 plasmids and 1 strain (strain no 80) contained 3 plasmids of different molecular weights. The antimicrobial resistant patterns of these strains which Muthupettai mangroves one or more plasmids were very similar, almost all of them were resistant to all antibiotics. Discussion Vibrio species occur widely in aquatic environments and are part of the normal flora of coastal seawater. They also exist as normal flora in fish and shellfish but also been recognized as opportunistic pathogens in many marine animals2. In the present study, cultivable antibioticresistant Vibrio species were widespread in the Muthupettai mangrove environment. Generally no differences between yellow and green bacteria were noted, most resistant to kanamycin, nalidixic acid, novobiocin and pencillin, and most sensitive to gentamycin and streptomycin. Similar results were obtained by 18 in southern Baltic Sea. This is not surprising since the intrinsic resistance of many marine bacteria to antibiotics is well documented19- 21. According to 22, 23, such a high level of antibiotic resistance in marine bacteria might result from terrestrial bacteria with antibiotic resistant plasmids entering the seawater; this fact may be responsible for the observed prevalence of resistance genes in the marine environment. Bacteria occurring in many water basins show multiple antibiotic resistance (MAR), as has been reported by 18, 23-29. Regarding MAR Vibrio species of Muthupettai mangroves, majority of the bacteria were resistant to KM and NA. That means that they are perfectly capable of detoxicating those antibacterial substances. The percentage of MAR was higher than those reported by 18, 19, 23, 27-31. As pointed out by32, differences in the percentage of bacteria resistant to various antibiotics may reflects the history of antibiotic application and hence serve as its indicator. In the present work, all the 209 strains of Vibrio species featuring antibiotic resistance in the sample collected at different stations, which Volume 3 ∙ Number ∙ 1 ∙ 2010

Antibiotic resistance and plasmid profiles of vibrio P. Manivasagan et al

are affected by monsoon season heavy fresh water inflow, agricultural discharges, shrimp effluent pollution with indicators of sewage pollution and this result suggests that perhaps other anthropogenic sources of pollution are present and influencing the microbial communities at all sites. The majority of the isolated strains from station 1 were identified, which are affected by aquaculture pond discharge water in mangrove environment. Similar results were observed by23. The lowest number of isolates was identified at station 6. In the station, fresh water inflow, pollution sources and aquaculture effluent were low in marine environment. Highly resistance of the 13 (MAR) strains isolates were classified and identified as 3 species using MIC of resistance bacteria and plasmid profile. The present study has shown that 3 Vibrio species isolates of to Vibrio cholerae (5 strains), Vibrio parahaemolyticus (5 strains) and Vibrio vulnificus (3 strains) were tested for their resistance to the all antibiotics. Growth pattern of 3 Vibrio species isolates in broth at different time intervals was studied. Growth of the isolates at the lowest concentrations (30µg/ml) was comparable to that of control. However, the growth declined at (120µg/ml) and dropped more sharply at 150µg/ml. Vibrio parahaemolyticus resistance of high concentration up to 150µg/ml of 9 antibiotics. It is well known that plasmid is one of the most important mediators facilitating the fast spreading of antibiotic resistance among bacteria33. In order to examine if there is any plasmid involved in antibiotic resistance profile mentioned above, plasmid extraction with alkaline lysis and QIAgen miniprep kit were also applied in this study. From the results of the plasmid extraction experiment, bacteria gave large plasmids (1 - 3 plasmids per strain) with molecular weights ranging from 11 – 112kb. The similar results were reported by12, 16, 17, 34, 35. However, in the present study, a large number of strains were devoid of plasmids but were resistant to all antibiotics an observations which indicates that resistance to these antibiotics is chromosomal. However, the presence of plasmids in these isolates seemed to increase their antibiotic resistance. According to26, adaptive responses of bacterial communities to several antibiotics observed in the present investigation may have Page 42


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

possible implications for the public health. Public health risk is further stressed by the occurrence of a high frequency (77%) of strains that are typically resistant to more than one antibiotic. Result obtained from this study indicates that antibiotics are a significant selection factor and probably play an important role in regulating the composition of bacterial communities in mangroves environments. Hence, further studies on establishing the role of antibiotic substances in controlling mangroves sediment bacterial populations are needed. Conclusions In view of these studies, it is evident that the Vibrio strains isolated from Muthupettai mangroves sediment were able to grow in the presence of antibiotics. This property of antibiotic resistance in these bacteria may be important in the decontamination of mangrove sediment polluted by the antibiotics. This is the few report, where a comprehensive study on the plasmids present in Vibrio species isolated. Resistance to antibiotics is widespread in Vibrio species and their relationship with transferable plasmids should be further studied. Declaration of Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article. References 1. Janaki-Raman D, Jonathan MP, Srinivasalu S, Armstrong-Altrin JS, Mohan SP, Ram-Mohan V. Trace metal enrichments in core sediments in Muthupet mangroves, SE coast of India: Application of acid leachable technique. Environmental Pollution 2007;145:245-257. 2. Austin B, Austin DA. (eds) Bacterial Fish Pathogens, 2nd edn., 1993;pp. 265-307. Ellis Horwood, Chichester. 3. Woo NYS, Ling JLM, Lo KM. Pathogenic Vibrio spp. in the sea bream, Sparus sarba. Journal of Sun Yatsen University Supp 1995; 3, 192-193. 4. Wu HB, Pan JP. Studies on the pathogenic bacteria of the vibriosis of Seriola dumerili in marine cage culture. Journal of Fisheries China 1997; 21, 171-174. 5. Son R, Rusul G, Sahilah AM, Zainuri A, Raha AR, Salmah I. Antibiotic resistance and plasmid profile of Aeromonas hydrophila isolates from cultured fish, Tilapia (Tilapia mossambica). Letters in Applied Microbiology 1997;24, 479-482. 6. Saitanu K, Chongthaleong A, Endo M, Umeda T, Takami K, Aoki T, Kitao T. Antimicrobial susceptibilities and detection of transferable R-plasmids from Aeromonas hydrophila in Thailand. Asian Fishery Science 1994;7, 41-46. 7. French GL, Woo ML, Hui YW, Chan KY. Antimicrobial susceptibilities of halophilic vibrios. Journal of Antimicrobial Chemotherapy 1989; 24 183-194.

Volume 3 ∙ Number ∙ 1 ∙ 2010

Antibiotic resistance and plasmid profiles of vibrio P. Manivasagan et al

8. Rheinheimer G. Microbial ecology of a brackish water environment. In: Ecological Studies, vol. 25. Springer-Verlag, Berlin, Heidelberg, New York 1977; p. 291. 9. Hada HS, Sizemore RK. Plasmids in marine Vibrio spp.: incidence and determination of potential functions using numerical taxonomic methods. In: Vibrios in the Environment (Colwell, R.R., Ed.), 1984 pp. 183-194. Wiley, New York. 10. Toranzo AE, Barja JL, Colwell RR, Hetrick FM. Characterization of plasmids in bacterial fish pathogen. Infect. Immun. 1983; 39, 184-192. 11. Zhao J, Kim EH, Kobayashi T, Aoki T. Drug resistance of Vibrio anguillarum isolated from ayu between 1989 and 1991. Nippon Suisan Gakkaishi/Bull. Jpn. Soc. Sci. Fish. 1992; 58: 1523-1527. 12. Zhang R, Wang Y, Gu JD. Identification of environmental plasmid-bearing Vibrio species isolated from polluted and pristine marine reserves of Hong Kong, and resistance to antibiotics and mercury. Antonic van Leeuwenhoek. 2006; 89:307-315. 13. Holt GH, Kreig NR, Sneath PHA, Staley JT, Williams ST. Bergey’s Manual of Determinative Bacteriology. 1997; Williams & Wilkins (Ed.), Baltimore, MD. 14. Arvanitodou M, Tsakris A, Constantindis TC, Katsouyannopulus VC. Transferable antibiotic resistance among Salmonella strains isolated from surface water. Water Research 1997; 37, 1112–1116. 15. Jones JG, Gardner S, Simon BM, Pickup RW. Factors affecting the measurement of antibiotic resistance in bacteria isolated from lake water. Journal of Applied Bacteriology 1986; 60, 455– 462. 16. Shafiani S, Malik A. Tolerance of pesticides and antibiotic resistance in bacteria isolated from wasterwaterirrigated soil. World journal of Microbiology & Biotechnology, 2003; 19:897901. 17. Wang Y, Leung PC, Qian PY, Gu JD. Antibiotic resistance and plasmid profile of environmental isolates of Vibrio species from Mai Po Nature Reserve, Hong Kong. Ecotoxicology 2006;15:371-378. 18. Zbigniew Jan Mudryk. Occurrence and distribution antibiotic resistance of hetrotrophic bacteria isolated from a marine beach. Marine Pollution Bulletin 2005; 50:80-86. 19. Nair S, Chandramohan D, Bharathi L.. Differential sensitivity of pigmented and non-pigmented marine bacteria to metals and antibiotics. Water Research 1992; 26, 431–434. 20. Sabry SA, Ghozian HA, Abou-Zeid D. Metal tolerance and antibiotic resistance patterns of a bacterial population isolated from seawater. Journal of Applied Bacteriology 1997; 82, 245– 252. 21. Mudryk Z, Skorczewski P.. Antibiotic resistance in marine neustonic and planktonic bacteria isolated from the Gdan´sk Deep. Oceanologia 1998;40, 125–136. 22. Chandrasekarn S, Venkatesh B, Laithakumari D.. Transfer and expression of a multiple antibiotic resistance plasmid in marine bacteria. Current Microbiology 1998; 37, 347–351. 23. Tendencia EA, LD de la Pena. Antibiotic resistance of bacteria from shrimp ponds. Aquaculture, 2001; 195:193-204. 24. De Vincente A, Aviles M, Codina JC, Borrego JJ, Romero P. Resistance to Antibiotics and heavy metals of Pseudomonas aeruginosa isolated from natural water. Journal of Applied Bacteriology 1990; 68, 625–632. 25. Davis J. Another look at antibiotic resistance. Journal of General Microbiology 1992;138, 1553–1559. 26. Qureshi AA, Qureshi MA. Multiple antibiotic resistant fecal coliforms in raw sewage. Water, Air and Soil Pollutions 1992; 61, 47–56. 27. Mudryk Z. Antibiotic resistance among bacteria inhabiting surface and subsurface water layers in estuarine lake Gardno. Polish Journal of Environmental Studies 2002; 11, 401–406. 28. Lobova TI, Maksimova EY, Popova LY, Pechurkin NS. Geographical and seasonal distribution of multiple antibiotic resistance of heterotrophic bacteria of Lake Shira. Aquatic Ecology, 2002; 36:299-307. 29. Lobova TI, Bakhatov YV, Salamatina OV, Popova LY. Multiple antibiotic resistance of heterotrophic bacteria in the littoral zone

Page 43


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

30.

31.

32.

33. 34.

35.

Antibiotic resistance and plasmid profiles of vibrio P. Manivasagan et al

of Lake Shira as an indicator of human impact on the ecosystem. Microbiological Research 2006; Article in press. Nemi M, Sibakov M, Niemela S. Antibiotic resistance among different species of fecal coliforms isolated from water samples. Applied and Environmental Microbiology 1983; 45, 79–83. Herwig RP, Gray JP, Weston DP. Antibacterial resistant bacteria in surficial sediments near salmon net-cage farms in Puget Sound, Washington. Aquaculture 1997; 149, 263–283. Hsu CH, Hwang SC, Liu JK. Succession of bacterial drug resistance as an indicator of antibiotic application in aquaculture. Journal of Fisheries Society of Taiwan 1992; 19, 55–64. Dale JW, Park S. Molecular genetics of bacteria. 4th ed. John Wiley & Sons Inc., Chichester, UK 2004. Jun Li, Jun Yie, Rita W T F, Julia ML, Ling, Huaishu Xu, Norman YSW. Antibiotic Resistance and Plasmid Profiles of Vibrio Isolates from Cultured Silver Sea Bream, Sparus sarba. Marine Pollution Bulletin, 1999; 39:245-249. Molina-Aja A, Garc-Gasca A, Abreu-Grobois A, Bolan-Mejia C, Roque A, Gomez-Gil B. Plasmid profiling and antibiotic resistance of Vibrio strains isolated from cultured penaeid shrimp. FEMS Microbiology Letters 2002 ; 213: 7-12.

Resistance of bacteria Vibrio cholerae

Vibrio parahaemolyticus

Vibrio vulnificus

MIC (µg/ml) 30 60 90 120 150 30 60 90 120 150 30 60 90 120 150

Antibiotics AM 2(5.0) 1(2.5) 1(2.5) 1(2.5) 1(2.5) 1(2.5) 1(2.5) 2(5.0) 1(5.0) 2(10) -

CP 1(2.5) 2(5.0) 1(2.5) 1(2.5) 1(2.5) 2(5.0) 1(2.5) 1(2.5) 2(10) 1(5.0)

GE 1(2.5) 2(5.0) 1(2.5) 1(2.5) 2(5.0) 1(2.5) 1(2.5) 1(2.5) 2(10) 1(5.0) -

KM 1(2.5) 1(2.5) 1(2.5) 2(5.0) 1(2.5) 1(2.5) 2(5.0) 1(2.5) 1(5.0) 2(10)

NA 1(2.5) 1(2.5) 2(5.0) 1(2.5) 1(2.5) 1(2.5) 1(2.5) 2(5.0) 1(5.0) 2(10)

NB 2(5.0) 1(2.5) 1(2.5) 1(2.5) 1(2.5) 2(5.0) 1(2.5) 1(2.5) 2(10) 1(5.0)

PL 1(2.5) 2(5.0) 1(2.5) 1(2.5) 1(2.5) 2(5.0) 1(2.5) 1(2.5) 2(10) 1(5.0)

RF 1(2.5) 2(5.0) 1(2.5) 1(2.5) 1(2.5) 1(2.5) 2(5.0) 1(2.5) 1(5.0) 2(10) -

SM 2(5.0) 1(2.5) 1(2.5) 1(2.5) 1(2.5) 1(2.5) 2(5.0) 1(2.5) 2(10) 1(5.0) -

Table 3. Multiple antibiotic resistance of 3 Vibrio species isolates. Values in parentheses indicates the percentage of the otal isolates Total number of Vibrio cholerae isolates = 5 Total number of Vibrio parahaemolyticus isolates = 5 Total number of Vibrio vulnificus isolates = 3 Not detected = -

Volume 3 ∙ Number ∙ 1 ∙ 2010

Page 44

TE 2(5.0) 1(2.5) 1(2.5) 1(2.5) 1(2.5) 1(2.5) 2(5.0) 1(2.5) 2(10) 1(5.0) -


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

First identification of a novel probiotic Paulraj Kanmani et al

FIRST IDENTIFICATION OF A NOVEL PROBIOTIC BACTERIUM STREPTOCOCCUS PHOCAE AND IT’S BENEFICIAL ROLE IN DISEASES CONTROL Paulraj Kanmani1, R. Satish Kumar1, N. Yuvaraj1, K. A. Paari1, V. Pattukumar1, Venkatesan Arul2* 1. PhD scholars, Department of Biotechnology, School of Life Sciences, Pondicherry University, Pondicherry-605014, INDIA. 2. Dr. Reader, Department of Biotechnology, School of Life Sciences, Pondicherry University, Pondicherry-605014, INDIA.

Abstract The strain PI80 which showed promising antimicrobial effect on different shrimp and fish pathogens was isolated from gut of shrimp Penaeus indicus. Based on 16Sr RNA gene sequence similarities and phenotypic characterization, strain PI80 was identified as a member of the genus Streptococcus. The G+C content of the partial DNA sequence was 53 %. Streptococcus phocae PI80 exhibited resistance against methicillin, kanamycin, neomycin and amikacin. Hemolytic and phenotypic results showed the differentiation of strain PI80 from other species within the genus Streptococcus. S. phocae PI80 showed broad spectrum of antagonistic activity against Grampositive and Gram-negative pathogens by producing its own antimicrobial compound like bacteriocin. Moreover the culture growth parameters revealed that the PI80 having potential to grow at high temperature, pH and NaCl concentration. (Journal of International Dental and Medical Research 2010; 3: (1), pp. 45-51 ) Keywords: S. phocae, penaeus indicus, probiotic, bacteriocin.

Received date: 30 December 2009 Introduction The genus Streptococcus consists of a phenotypically diverse group of catalase-negative, Gram-positive, cocci shaped bacterium. Many novel species from human and animal sources have been described in this genus1. Though more than 60 species has been reported in the genus, only few species have been isolated from marine animals. Microbial strains isolated from marine mammals are Streptococcus phocae2-4, Streptococcus halichoeri5, Streptococcus dysgalactiae subsp dysgalactiae4 and Streptococcus marimammalium6.

*Corresponding author: Dr. Venkatesan. Arul, Reader, Department of Biotechnology, School of Life Sciences, Pondicherry University, Pondicherry-605014, INDIA Phone: 091-413-2654429, Fax: 091-413-2655212 E-mail: varul18@gmail.com

Volume 3 ∙ Number ∙ 1 ∙ 2010

Accept date: 09 March 2010 Streptococcus phocae was first isolated from seals2 later in fur seals3. This organism was suspected to be associated with respiratory infection, starvation both in adults and offsprings of fur seals. Vossen et al. (2004)4 isolated beta haemolytic units of S. phocae from harbor seals (Phoca vitulina) and Grey seals (Halichoerus grypus). Gibello et al. (2005)7 first identified S. phocae in Atlantic salmon Salmo salar. All these strains were beta hemolytic Streptococci. Though S. phocae was isolated from diseased fish it was not proved, whether the disease was caused by the microorganism. Skaar et al. (1994)2; Henton et al. (1999)3; Vossen et al. (2004)4; Gibello et al. (2005)7 reported that hemolytic Streptococcus phocae isolated from different species of seals and fish. Till to date no one reported non hemolytic Streptococcus phocae isolated from shrimp. Here, first time we have reported a non hemolytic probiotic S. phocae isolated from gut of shrimp Penaeus indicus. The present study was carried out to identify and characterize a novel non-hemolytic Streptococcal strain isolated from Indian white Page 45


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

prawn Penaeus indicus. S. phocae has been used as a probiotic to culture the fish Cyprinus carpio and post larvae of Penaeus monodon and to restrain the following fish pathogen such as Aeromonas hydrophila, Vibrio parahaemolyticus and V. harveyi 8. Isolation and characterization of a heat stable bacteriocin (9.2kDa) from S. phocae and its usage as biopreservative in food preservation and probiotics in shrimp farming is in progress.

First identification of a novel probiotic Paulraj Kanmani et al

extension at 72oC (30 Sec) and final extension at 72oC (10 min). Amplification was carried out in Eppendorf Thermocycler, programmed for 30 cycles. The amplified DNA fragment was separated on a 1% agarose gel and purified using Quick PCR purification spin columns. The purified fragment was directly sequenced in MACROGEN, Korea. These sequences were then identified by homology search using BLAST program and sequences were submitted in Genbank.

Material and Methods Isolation of strain PI80 Under aseptic conditions, gut was removed from Penaeus indicus shrimp caught in the wild, homogenized with 0.8% (w/v) NaCl, plated on lactobacillus MRS agar plates (Himedia, MUMBAI, INDIA) and incubated at 37°C for 16 h. Separate bacterial colonies were picked and grown on fresh MRS agar plates incubated at 37°C for 16 h. Over top of these plates, 10 ml of soft agar containing indicator strains (105 CFUml1 ) were poured. After incubation for 16 h at 37°C, the plates were examined for antagonistic activity. Biochemical characterization of strain PI80 The biochemical characterization such as Gram staining, motility, starch hydrolysis, nitrate reduction, oxidase, catalase, indole, H2S production and carbohydrate profile tests were analyzed for bacterial strain PI80. Polymerase Chain Reaction for 16SrRNA gene amplification The total genomic DNA was isolated from Streptococcus phocae PI80 and used as template DNA for gene amplification. The 16SrRNA gene sequence was amplified using 16SrRNA gene specific primers (Forward primer 5’- AGA GTT TGA TCC TGG CTC AG-‘3 and Reverse primer 5’- ACG GCT ACC TTG TTA CGA CTT-‘3 ). The reaction mix was carried out totally in 25µl containing water (7.5µl), Taq buffer (3µl), dNTP (2.5µl), both forward and reverse primers (2µl), Taq polymerase (5µl) and template DNA (5µl). All chemicals were procured from GENEI (BANGALORE, INDIA). The reaction conditions were performed with initial denaturation temperature at 95oC (4min), denaturation temperature at 95oC (30 Sec), annealing temperature at 55oC (30 Sec), Volume 3 ∙ Number ∙ 1 ∙ 2010

Methods for analyzing the probiotic properties 1. Cell adherence and acid stability S. phocae PI80 (5 x1010) was administered orally to the animal model and the cells attached to mucous layer were calculated as colony forming unit (CFU ml-1). Moreover, acid tolerance was tested in vitro in gastric system model as described by Mouecoucou et al. (2004)9; Duc et al. (2004)10 with some modifications. The total cell viability was recorded after exposure of strain PI80 to the above explained condition and was compared with control. 2. Assay of antibiotic susceptibility and hemolytic properties The susceptibility of the strain S. phocae PI80 against Penicillin, Methicillin, Tetracyclin, Oxytetracyclin, Erythromycin, Ampicillin, Amoxycillin, Kanamycin, Gentamycin, Vancomycin, Neomycin, Amikacin, Chloramphenical, Nitrofurantoin and cefpodoxin were tested in Lactobacillus MRS agar medium. All antibiotics were purchased in HIMEDIA (MUMBAI, INDIA). Moreover, their hemolytic property was analyzed in sheep and human blood agar plates. 3. Assay of bacteriocin activity Bacteriocin activity was estimated using the agar well diffusion method 11 and expressed in arbitrary units (AUml-1), calculated as ab × 100, where “a” represents the dilution factor, and “b” represents the last dilution that produce an inhibition zone of 2 mm in diameter. One arbitrary unit (AU) of bacteriocin activity was defined as the reciprocal of the highest two fold dilution showing a clear zone of inhibition of the indicator strains and its activity is expressed per ml after multiplication by 100. More than five pathogenic organism such as Vibrio Page 46


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

parahaemolyticus, V. vulnificus, V. anguilarum, V. fischeri, A. hydrophila and Listeria monocytogenes 657 was used for the assay of bacteriocin activity. Effect of probiotic S. phocae treatment on control of Vibrio harveyi Black tiger shrimps (Penaeus monodon) were procured from a commercial hatchery. After 40 days of acclimation period, shrimps were separated in triplicates (30 animals each) in to 500 L-capacity fiber tank. The control group was fed with regular diet for entire trial period and one group was fed with probiotic S. phocae PI80 and the third group was fed with probiotic S. phocae PI80 after Vibrio infection. V. harveyi (MTCC 3435) were used as disease causing agent in the experiment. These strains were grown in TSA broth (Himedia, MUMBAI, INDIA) at 30oC for 16hr. At end of 15 days feeding trial, treatment and control group were exposed to V. harveyi at 106 CFUml-1 for 20 – 30 min. Infection was ensured by inoculating the diseased shrimp on TCBS agar (Himedia, MUMBAI, INDIA) and verified by biochemical tests.

First identification of a novel probiotic Paulraj Kanmani et al

was selected for in vivo study by maximum zone of inhibition against fish and shrimp pathogens such as A. salmonicida, Vibrio anguillarum, V. fischeri, V. vulnificus and V. parahaemoliticus which classifies it as a novel ‘antagonistic probiotic bacteria’ for shrimp larval rearing systems in hatcheries and farms. This was confirmed by testing the strain in common carp Cyprinus carpio and shrimp Penaeus monodon culture systems. LAB isolates were characterized on the basis of morphological, physiological and biochemical tests by Bergey’s Manual of Systematic Bacteriology14. The biochemical characterization is commonly used technique to differentiate one bacterial strain to others7. In respect of biochemical identification, isolate belongs to the group of cocci, non motile, gram positive, oxidase and catalase negative (Table 1).

Effect of growth parameters for S. phocae PI80 The effect of temperature, pH and salinity on the growth of S. phocae in MRS broth was determined by inoculation of early log phase culture and incubation at different temperatures (25°C, 30°C, 35°C, 40°C and 45°C), pH (5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0) and NaCl (0%, 0.5%, 1%, 2% and 3%) for 48hrs. The samples were collected at every 6 hr intervals and absorbance at 600nm was read. Fresh MRS broth was taken as blank. Experiments were carried out in triplicates. Results and Discussion Identification and characterization of strain PI80 A total of 100 numbers of lactic acid bacteria were isolated from gut of shrimp (Penaeus indicus-PI) in MRS agar plates. These isolates were screened for antagonistic activity against fish pathogen Aeromonas hydrophila by cross streaking 12 and agar well diffusion method13. Finally, a single pinpoint isolate (PI80) Volume 3 ∙ Number ∙ 1 ∙ 2010

Table 1. Biochemical characterization of newly isolated strain of S. phocae PI80. Page 47


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

The Lab isolate utilizes the sugar of glucose, lactose, xylose, rhamnose and sucrose. The strain PI80 was non hemolytic in human blood-agar plates where as α hemolytic in sheep blood agar plates. The 16S rRNA gene sequences are sufficient enough for the identification of bacterial isolates15 and identification of bacterial isolate in molecular level was attempted by sequencing the 16S rRNA gene of streptococcal sp 16. The identified sequences of our strain consisted of 847 base pairs and the BLAST program was used to compare the sequences with those of the gram positive cocci, oxidase and catalase negative species available in the Genbank database. The 16S rRNA gene sequence analysis revealed that PI80 displayed the highest sequence similarity (98.0%) with Streptococcus phocae. (GenBank accession number EU117220).

First identification of a novel probiotic Paulraj Kanmani et al

also tested for the culture isolate. The alpha and non hemolytic activities were observed in sheep and human blood agar plate. These results clearly differentiated that our isolate isn’t similar to previously reported culture2-4,7.

Probiotic characteristic properties 1. Mucous layer attachment and acid tolerant in invitro gastric system The attachment of cells in mucous layer was analyzed after two days of cells administration. The concentration of cells was observed to be same and was compared with control group (data not shown). For analyzing the acid resistance properties, S. phocae PI80 was introduced into the artificially formulated gastric environment. The equal bacterial cell viability was recorded after exposure of S. phocae PI80 in gastric conditions. 2. Antibiotic susceptibility and hemolytic test Antibiotic susceptibility test was performed for S. phocae PI80 against fifteen antibiotics. Among the antibiotic, only six antibiotics such as, erythromycin amoxicillin, nitrofurantoin, chloramphenicol, ampicillin, penicillin, oxytetracyclin, and tetracycline highly inhibited (20-37mm zone) the growth of culture PI80. Moreover, the isolate was less susceptible (10-19mm of zone of inhibition) to cefpodoxin and vancomycin. As well as, gentamicin also inhibited moderately about 6-7mm zone of inhibition. But no zone of inhibition was observed in antibiotics amikacin and methicillin, kanamycin and neomycin (Table 2). The property of hemolytic activity was Volume 3 ∙ Number ∙ 1 ∙ 2010

Table 2. Antibiotic susceptibility test of newly isolated strain of S. phocae PI80. 3. Bacteriocin activity The isolate PI80 produce bacteriocin, which exhibited broad spectrum of inhibitory activity against closely related gram positive and gram negative bacterial strains. All indicator strains and their bacteriocin activity were listed in Figure 1. Among the pathogenic strains used V. anguillarum and Listeria monocytogenes 657 showed higher bacteriocin activity by the probiotic streptococcus. Agar well diffusion method was used to detect bacteriocin activity. Previously Skaar et al. (1994) 2, Henton et al. (1999) 3, Vossen et al. (2004) 4, Gibello et al. (2005)7 reported that S. phocae didn’t have any capability to produce the any antimicrobial compounds. Where as, Kabuki et al. (2007)17 reported that Streptococcus thermophilus SBT1277 produce heat stable bacteriocin within the Streptococcus sp. Moreover, Satish kumar and Arul (2009)18 reported the high molecular weight of bacteriocin was produced by S. phocae PI80 and its antilisterial activity.

Page 48


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

First identification of a novel probiotic Paulraj Kanmani et al

The mortality rate (25 %) was observed in control group. Similarly Balcazar et al. (2007)19 observed 17-22% mortality in shrimp exposed to V. parahaemolyticus and 33% in shrimp not treated with probiotics. Results of this study reinforce the view that probiotic is a effective addition to disease restrain phenomenon in aquaculture system20. Pseudomonas fluorescens AH2 was able to reduce the mortality of rainbow trout infected with V. anguillarum and 47% of mortality was observed in control group inoculated with pathogenic strain 21. In conclusion, the probiotic S. phocae PI80 was ability to restrain the mortality of shrimp while also improving the shrimp survival rate (100%). Figure 1. Effect of bacteriocin activity (AUml-1) produced by probiotic S. phocae PI80. Effect of probiotic S. phocae PI80 on survival of P. monodon To investigate whether probiotic strain S. phocae are able to defend the shrimp P. monodon against vibriosis infection, shrimp were infected with V. harveyi. Supplementation of S. phocae (10 ml) showed 23 % mortality in shrimp exposed to Vibrio harveyi but not treated with probiotic whereas 0 % mortality was observed in shrimp exposed to Vibrio harveyi and treated with probiotic S. phocae PI80 (Figure 2).

Figure 2. Accumulated mortality of Penaeus monodon infected with V. harveyi for 30 min with and without probiotic treatment. Volume 3 ∙ Number ∙ 1 ∙ 2010

Effect of temperature, pH and NaCl on growth of S. phocae PI80 The effect of temperature, pH and NaCl on growth of S. phocae PI80 was investigated in MRS broth. The log phase reached at very early stage in all culture parameters especially in pH 5.0. Among the growth parameters, increased growth (1.391± 0.073, 1.341± 0.028 and 1.351± 0.057) rate was observed at end of the incubation period at temperature 40oC, pH 7.5 and 0% NaCl (Figure 3, 4 and 5).

Figure 3. Growth profile of S. phocae PI80 in MRS broth at different temperature (25oC, 30oC, 35oC, 40oC and 45oC)

Page 49


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

First identification of a novel probiotic Paulraj Kanmani et al

shrimp intestine and it was proved by the production of bacteriocin at different Na Cl concentration. The higher amount bacteriocin profile was observed in 0.5-1% (Figure 6). Our results correspond to the report observed by kabuki et al. (2007)17 in S. thermophilus SBT1277.

Figure 4. Growth of S. phocae PI80 in MRS broth at different pH (5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0).

Figure 6. Tricine SDS -PAGE showing the effect of salinity on bacteriocin production by S. phocae PI80. Lane 1- MWM and Lane2 – Lane 6 shows the order of increasing concentration salinities (0, 0.5, 1, 2, and 3%). Conclusions

Figure 5. Growth of S. phocae PI80 in MRS broth containing different concentration of NaCl (0%, 0.5%, 1%, 2% and 3%). Moreover, the log phase growth reached within 4hrs of incubation period. So, temperature 40oC, pH 7.5 and NaCl 0% are found to be optimum for only growth of S. phocae PI80. Also these results revealed that the probiotic bacterium S. phocae PI80 have the capacity to grow in higher temperature, pH and Na Cl. Because of this culture was isolated from marine Volume 3 ∙ Number ∙ 1 ∙ 2010

The phenotypic and genotypic characterization revealed that strain PI80 was Streptococcus phocae and it has got ability to restrain gram positive and gram negative bacterial pathogenic strains by producing its antimicrobial compounds like bacteriocin. S. phocae PI80 survive well in higher environmental factors like temperature, pH and salinity. Hence, we conclude that S. phocae PI80 is able to displace bacterial pathogens by competitive process in the shrimp hatchery and it act as a better replacement than administering antibiotics and is now gaining acceptance for control of pathogens in aquaculture. Acknowledgements This work was supported by a grant from Department of Biotechnology, New Delhi. Declaration of Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article. Page 50


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

First identification of a novel probiotic Paulraj Kanmani et al

References 1. Facklam R. What happened to the streptococci: overview of taxonomic and nomenclatural changes. Clin. Microbiol. Rev 2002; 15:613 - 630. 2. Skaar I, Gaustad P, Tonjum T, Holm B, Stenwig H. Streptococcus phocae sp. nov., a new species isolated from clinical specimens from seals. Int. J. Syst. Bacteriol 1994; 44:646 - 650. 3. Henton MM, Zapke O, Basson PA. Streptococcus phocae infections associated with starvation in cape fur seals. J. South Afr .Veter. Asso 1999; 70:98 - 99. 4. Vossen A, Abdulmawjood A, Lammler C, Wei R, Siebert U. Identification and molecular characterizaction of beta-hemolytic streptococci isolated from harbor seals (Phoca vitulina) and grey seals (Halichoerus grypus) of the German North and Baltic Seas. J. Clin Microbiol 2004; 42:469 - 473. 5. Lawson PA, Foster G, Falsen E, Davison N, Collins MD. Streptococcus halichoeri sp. nov., isolated from grey seals (Halichoerus grypus). Int. J. Syst. Evol. Microbiol 2004; 54:1753 -1756. 6. Lawson PA, Foster G, Falsen E, Davison N, Collins MD. Streptococcus marimammalium sp. nov., isolated from seals. Int. J. Syst. Evol. Microbiol 2005; 55:271-274. 7. Gibello A, Mata AI, Blanco MM, Casamayor A, Domı´nguez L, Ferna´ndez-Garayzabal JF. First Identification of Streptococcus phocae Isolated from Atlantic Salmon (Salmo salar). J. Clin. Microbiol 2005; 43(1):526 -527. 8. Gopalakannan A. Studies on the control of Aeromonas hydrophila infection and Cyprinus carpio and Tilapia mossambicus by immunostimulants and probiotics, Ph.D. Thesis, Department of Biotechnology, Pondicherry University, Pondicherry, India, 2006. 9. Mooecoucou J, Villaume C, Sanchez C, Mejean I, β-lactoglobulin /polysaccharide interaction during invitro gastric and pancreatic hydrolysis assessed in dialysis bags of different molecular weight cut-offs. Biochimi. Biophy. Acta 2004; 1670:105-112. 10. Duc LH, Hong HA, Barbosa TM, Henriques AO, Cutting SM. Characterization of Bacillus probiotics available for human use. Appl. Environ Microbiol 2004; 70:2161-2171. 11. Todorov SD, Dicks LMT. Lactobacillus plantarum isolated from molasses produces bacteriocins active against gram negative bacteria. Enz. Microbi. Technol 2005; 36:318-326. 12. Kekessy DA, Piquet JD. New method for detecting bacteriocin production. Appl Microbiol 1970; 20:282-283. 13. Lyon WJ, Glatz BA. Isolation and purification of propionicin PLG-1, a bacteriocin produced by a strain of Propionibacterium theoni. Appl. Environ. Mcrobiol 1993; 59:83-88. 14. Holt JG, Krieg NR, Sneath PHA, Williams ST, Bergey’s manual of determinative bacteriology, The Williams & Wilkins Co. Baltimore, Md. 9th eds.1994; p. 527-558. 15. Kolbert CP, Persing DH. Ribosomal DNA sequencing as a tool for identification of bacterial pathogens. Cur. Opin. Microbiol 1999; 2:299 - 305. 16. Vela AI, Goyache J, Tarradas C, Luque I, Mateos A, Moreno MA, Borge C, Perea JA, Domı´nguez L, Fernandez-Garayzabal JF. Analysis of genetic diversity of Streptococcus suis clinical isolates from pigs in spain by pulsed-field gel electrophoresis. J.Clin Microbiol 2003; 41:2498-2502. 17. Kabuki T, Uenishi H, Watanabe Y, Seto Y, Nakajima H. Characterization of bacteriocin, Thermophilin 1277, produced by Streptococcus thermophilus SBT1277. J. Appl. Microbiol 2007; 102:971-980. 18. Satish kumar R, Arul V. Purification and characterization of Phocaecin PI80 an antilisterial bacteriaocin produced by Streptococcus phocae PI80 isolated from the gut of Indian White Shrimp (Peneaus indicus) J. Microbiol. Biotechnol 2009; 19(11): 1393-1400.

Volume 3 ∙ Number ∙ 1 ∙ 2010

19. Balcazar JL, Luna TR, Cunninggham DP. Effect of the addition of four potential probiotic starins on the survival of pacific white shrimp (Litopenaeus vannamei) following immersion challenge with Vibrio parahaemolyticus. J. Invert. Pathol 2007; 96:147150. 20. Balcazar JL, Decamp O, Vendrell D, de Blas I, Ruiz-ZarZuuela I. Health and nutritional properties of probiotics in fish and shelfish. Microb. Ecol. Health Dis 2006; 18:65-70. 21. Gram L, Melchiorsen J, Spanggaard B, Huber I, Nielsen T. Inhibition of Vibrio anguillarum by Pseudomonas fluorescens strain AH2, a possible probiotic treatment of fish. Appl. Envion. Microbiol 1999; 65:969-973.

Page 51


Journal of International Dental and Medical Research