A comparative study of the biological and physical properties of viscosity enhanced root repair material (VERRM) AND MTA

A COMPARATIVE STUDY OF THE BIOLOGICAL AND PHYSICAL PROPERTIES OF VISCOSITY ENHANCED ROOT REPAIR MATERIAL (VERRM) AND MTA PALLAVI UPPANGALA NATIONAL UNIVERSITY OF SINGAPORE 2007 A COMPARATIVE STUDY OF THE BIOLOGICAL AND PHYSICAL PROPERTIES OF VISCOSITY ENHANCED ROOT REPAIR MATERIAL (VERRM) AND MTA PALLAVI UPPANGALA (BDS. RGUHS, India) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF ORAL AND MAXILLOFACIAL SURGERY NATIONAL UNIVERSITY OF SINGAPORE 2007 Supervisor A/P Yeo Jin Fei BDS (Singapore), MSc (London), Certificate in Immunology (Distinction) London, MDS (Singapore), FAMS, FDSRCS (Edinburgh), FFOPRCPA (Australia) Head, Dept of Oral and Maxillofacial Surgery Faculty of Dentistry National University of Singapore Co-Supervisor Dr. Chng Hui Kheng B.D.S. (S'pore), DipClinDent (Melb), MDSc (Melb), FAMS (Endodontics) Formerly Asst Prof in Dept of Restorative Dentistry Faculty of Dentistry National University of Singapore DEDICATION To Amma & Appa ACKNOWLEDGEMENTS I would like to thank my Supervisor Associate Professor Yeo Jin Fei, Head, Department of Oral and Maxillofacial Surgery, National University of Singapore for his constant help, guidance and enthusiasm through my candidature. I warmly acknowledge my co-supervisor Dr. Chng Hui Kheng for her help and encouragement. I also acknowledge Prof J. Craig Baumgartner, for his help and guidance with the bacteria leakage project. I would like to thank the staff at Animal Holding Unit, National University of Singapore for their support. I thank Ms. Angeline Han for her help and guidance with the histology work. I would also like to thank Mr. Chan Swee Heng (Lab officer), my colleagues and support staff at the dentistry research labs, DMERI, DSO for their constant help. I also would like to thank my husband Sridhar and my parents for their constant support and encouragement. I would finally like to acknowledge the National University of Singapore for endowing me with the NUS Research Scholarship. i Table of Contents Table of Contents ii Summary iii List of Figures vi List of Abbreviations . ix 1. Introduction . 2. Literature Review 2.1. Portland Cement 2.2. Physical properties of MTA 2.3. Biological Properties of MTA 13 2.4. Comparison of White and Gray MTA 20 2.5. Comparison between MTA and Portland Cement 20 3. Tissue Reaction to Implanted Viscosity Enhanced Root Repair Material 23 3.1. Aim of this study . 23 3.2. Materials and Methods 23 3.3. Results . 26 3.4. Discussion . 46 3.5. Conclusions . 48 4. Comparison of the Root-End sealing ability of Mineral Trioxide Aggregate (MTA) and Viscosity Enhanced Root Repair Material (VERRM) . 49 4.1. Introduction . 49 4.2. Aim of this study . 52 4.3. Materials and Methods 52 4.4. Results . 56 4.5. Discussion . 57 4.6. Conclusions . 59 5. Bibliography . 60 6. Appendix . 77 6.1. Staining Protocols . 77 ii Name: Pallavi Uppangala Degree: Bachelor of Dental Surgery (B.D.S), Rajiv Gandhi University of Health Sciences, Bangalore, India. Department: Oral and Maxillofacial Surgery, Faculty of Dentistry Thesis Title: A comparative study of the biological and physical properties of Viscosity Enhanced Root Repair Material (VERRM) and MTA Summary The emergence of Mineral Trioxide Aggregate (MTA) as a root-end filling material has generated a lot of interest due to its superior sealing ability and biocompatibility. Although MTA possesses superior sealing ability and is less cytotoxic compared to traditional root-end filling materials such as Super-Ethoxy Benzoic Acid (super-EBA) and Intermediate Restorative Material (IRM), it has poor handling characteristics. A novel root-end filling material with similar chemical composition, but improved handling characteristics was recently developed. This material has been tested and was found to fulfill the physical properties requirements for use as a root-end filling material. Earlier studies using a dye leakage test also found the root-end sealing ability of this material to be comparable to MTA. However, there is lack of in vivo studies to ascertain its biocompatibility. The aim of this project is to examine the tissue reactions to Viscosity Enhanced Root Repair Material (VERRM), when implanted in the mandible of guinea pigs and compare the reactions to those induced by MTA and also to test the sealing ability with a bacterial leakage model. iii Fifteen adult male guinea pigs were anesthetized under aseptic conditions, tissue flaps were raised and bony cavities were created in the mandibles of the animals with burs. The materials MTA and VERRM were then implanted in these bony cavities. MTA and VERRM were implanted using Teflon cups as the carrier for the materials. The animals were randomly divided into groups of animals each. Each animal received one implant in the mandible. The animals were euthanized after a period of 80 days and the tissues were processed for histological examination using the Exakt system. Both the materials showed similar tissue reactions and absence of inflammatory reactions suggested that both the materials are biocompatible and there is scope for VERRM to be further developed for clinical use as a root-end filling material. Testing the sealing potential of MTA and VERRM was carried out using a bacterial leakage model. Forty-four extracted single rooted human teeth with single root canals were selected. They were randomly divided into two groups of 18 teeth (among, which teeth in each group were used to test the sterility of the apparatus) to receive the root-end fillings of MTA and VERRM respectively. The remaining teeth were divided into groups of each, to serve as positive and negative controls. After root-canal preparation using the step back technique, root end resections of 3mm were carried out. Root-end cavities were prepared using the ultrasonic technique and root-end fillings were placed. Nail varnish was applied to the external surface of all the teeth except at the apical end, to minimize leakage through the lateral surface. The leakage apparatus consisted of a 2ml micro centrifuge tube with a hole drilled in its cap. Trypticase soy broth was placed in the tube, and the tooth was fitted in the hole, such that 2-3mm of its apical end was immersed in the broth. Trypticase soy broth contaminated with Enterococcus faecalis (a Gram- iv positive bacterium) was introduced into the root canal through the coronal access cavity of the tooth. Bacterial leakage was observed as indicated by the turbidity of the broth. The observation period was 90 days. All the teeth in the positive control group leaked within days. By the end of 1st week, one of the samples out of 16 samples (6.25%) in Group2 (ProRoot MTA) leaked on the 4th day. In the 2nd week, one sample out of the 16 samples (6.25%) in Group1 (VERRM) leaked on the 10th day. In the 3rd week, one sample each in Group1 and Group2 leaked on the 15th and 18th day respectively. There was no leakage in the negative control group throughout the experimental period. After this up to a period of 12 weeks, there was no leakage in any of the samples. There was no significant difference in the leakage between the two materials. Hence, it was concluded that VERRM has the potential to be further developed as a root-end filling material. v List of Figures Figure 1- H &E, Magnification-5x, Gp A. Shows the lateral wall of the Teflon cup (T) surrounded by a thin layer of fibrous connective tissue (C) , free of inflammation, above the bone (B) . 28 Figure - H & E, Magnification 40x, showing the Teflon cup (T), connective tissue (C) and bone (B) 28 Figure - Toluidine blue. Magnification - 5x, Gp A. Deposition of osteoid-like tissue (O), around the Teflon cup (T) indicated by arrow. . 29 Figure - Toluidine blue. Magnification - 40x, Gp A. Higher magnification of the area in the dash-box (Figure - 3), showing osteoid-like tissue (O) and bone (B) . 29 Figure - Toluidine blue. Magnification - 40x, Gp A. Higher magnification of the area in the solid-box (figure – 3) showing osteoid-like tissue (O) and lateral wall of Teflon cup (T). 30 Figure - VK with VG. Magnification - 5x, Gp A. Osteoid-like tissue (O) next to VERRM (V) indicated by arrow . 30 Figure - VK with VG. Magnification - 40x, Gp A. Higher magnification of the area in the dash-box (Figure – 6) showing osteoid-like tissue (O) and VERRM (V) 31 Figure - VK with VG. Magnification - 40x, Gp A. Higher magnification of the area in the solid-box (Figure – 6) showing osteoid-like tissue (O) and bone (B). 31 Figure - H & E. Magnification - 5x, Gp B. Normal healing of bone (B) with a thin layer of connective tissue (C) free of inflammation around the Teflon cup (T) 32 Figure 10 - H & E. Magnification - 40x, Gp B. Higher magnification of the area in the dash-box (Figure- 9) showing the lateral wall of the Teflon cup (T) and a thin layer of fibrous connective tissue (C) free of inflammation. . 32 Figure 11 - H & E. Magnification - 40x, Gp B. Higher magnification of the area in the solid-box (Figure- 9) showing bone (B) and a thin layer of fibrous connective tissue (C). . 33 Figure 12 - Toluidine blue. Magnification - 5x, Gp B. Normal healing of bone (B) around Teflon cup (T). 33 Figure 13 - Toluidine blue. 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Rinse for 10 mins in running tap water to dye blue 6. Stain for mins in Eosin 7. Differentiate in 80% alcohol 8. Differentiate in 96% alcohol 9. Differentiate in 100% alcohol 10. Dip quickly once in xylene 11. Dry completely before you coverslip 12. Coverslip in Technovit 7200 77 Appendix Results: • Nuclei Blue-black • Cytoplasm-Varying shades of pink • Muscle fibers -Deep pinky red • Fibrin- Deep pink • Red blood cells- Orange/red. 78 Appendix Toluidine blue staining 1. Sections to rinsed in hydrogen peroxide for minutes 2. Toluidine blue/EDTA hour 3. Wash in distilled water Blot dry (37°C overnight or 60°C hours) 4. Rinse in methyl cyclohexane 5. Mount in Technovit 7200 Results: • Nuclei- Blue • Mineralized bone- Light purple • Osteoid- Colourless - pale blue • Mineralised front -Light blue • Reversal line-Dark blue/purple. To prepare Toluidine blue solution • Toluidine blue-1g • Diaminoethanetetra - acetic acid disodium salt-5g • Distilled water-100ml. 79 Appendix Von Kossa with Van Gieson: 1. Sections have to be rinsed in hydrogen peroxide for minutes. 2. Place sections into a clean small glass beaker of 1% silver nitrate (filter solution) and expose to UV light for 20 minutes. 3. Wash in distilled water minutes. 4. Fix in 1% solution thiosulphate for 30 seconds. 5. Repeat step 3. 6. Counterstain in Van Gieson (Unna's variant) saturated with picric acid for 15 minutes. 7. Rinse in distilled water. 8. Blot dry. 9. Rinse in alcohol. 10. Wash in methyl cyclohexane. 11. Mount in Technovit 7200. Results: • Mineralized bone- Black • Osteiod/Collagen- Red • Marrow/Muscle- Yellow 80 Appendix To prepareVan Gieson (Unna Variant): • Acid fuchsin 2.5g • Nitric acid (conc) 5ml • Glycerine 100ml • Distilled water 900ml • Picric acid to saturation. 81 Appendix 82 [...]... ettringite as the main hydration products Fridland and Rosado (2003) and (2005) found that MTA was capable of maintaining its high pH over a long duration of time and calcium was the main salt released when MTA was mixed with water It was shown by Holland et al (199 9a) , and Holland et al (2001b), that the mode of action of MTA was similar to Calcium hydroxide The basis for the biologic properties of MTA was... better sealing ability than amalgam, super-EBA and IRM Al-Hezaimi et al (2005b) found that MTA provided a better sealing ability against leakage of human saliva than vertically condensed guttapercha and sealer In a study of leakage using endotoxin by Tang et al (2002), it was found that MTA allowed less leakage than amalgam, super-EBA and IRM Micro leakage 10 Literature Review assessment of MTA using a fluid... less inflammation as compared to amalgam and also the presence of a fibrous capsule adjacent to MTA was noted In another study conducted by Torabinejad et al (1997), it was seen that in monkeys, MTA demonstrated less inflammation as compared to amalgam and also encouraged the growth of cementum Shabahang and Torabinejad (2000) in a clinical study on patients showed that when MTA was placed as the root- end... Torabinejad and Chivian (1999), described the various uses of MTA in vital pulp therapy, repair of root perforations, and as a root- end filling material Schwartz et al (1999) reported that MTA was successful in the treatment of cases such as vertical root fracture, apexification, perforation repair and repair of a resorptive defect In a study conducted by Arens and Torabinejad (1996), MTA, when used as a. .. MTA was compared to amalgam, super-EBA and ZOE, it was found that MTA had some 12 Literature Review antibacterial effect against some of the facultative anaerobes but no effect on the strict anaerobes In a study conducted by Al-Hezaimi et al (200 6a) , it was found that white MTA had less antibacterial action than gray MTA 2.2.4 Antifungal effect of MTA In a study conducted by Al-Nazhan and Al-Judai (2003)... placed in chopped meat carbohydrate broth and leakage observed It was found that there was no significant difference in the ability of the three materials to prevent leakage Nakata et al (1998) evaluated the ability of MTA and amalgam to seal furcal perforations in extracted human molars using an anaerobic bacterial leakage model Fusobacterium nucleatum was used in this study and it was concluded that... with minor additives of other oxides to enhance its physical and chemical properties (Schwartz et al 1999) According to United States patent for MTA (Torabinejad et al 199 8a) , the principal component of MTA is Portland Cement There are 2 kinds of MTA available: one is Gray MTA and the other is White MTA The main difference between the two is the lack of the aluminoferrite phase in the White MTA, 3 Introduction... et al 1995) Daoudi and Saunders (2002) compared MTA and Vitrebond for the repair of furcations They found that furcations repaired with MTA leaked less than those with Vitrebond Hardy et al 15 Literature Review (2004) found that MTA and One-Up Bond had similar sealing capabilities Lee et al (1993) determined that MTA had a better sealing ability than amalgam and IRM when used as a lateral furcation repair. .. selection of a suitable root- end filling material The aim of a root- end filling material is to provide an air-tight seal to prevent the movement of materials such as bacteria and their byproducts from the root canal to the periradicular tissues (Gutmann & Regan 2004) The requirements of an ideal root end filling material are: 1 Introduction • should be capable of sealing all the borders of the prepared cavity... caused a less toxic reaction Torabinejad et al (1995c) and (1998b) studied the biocompatibility of MTA, IRM, and super-EBA by implanting them in the mandible and tibia of guinea pigs After anesthetizing the guinea pigs, tissue flaps were raised and bony cavities drilled in the mandible and tibia The materials were implanted in these cavities using Teflon cups The animals were euthanized after 80 days and . India. Department: Oral and Maxillofacial Surgery, Faculty of Dentistry Thesis Title: A comparative study of the biological and physical properties of Viscosity Enhanced Root Repair Material (VERRM). A COMPARATIVE STUDY OF THE BIOLOGICAL AND PHYSICAL PROPERTIES OF VISCOSITY ENHANCED ROOT REPAIR MATERIAL (VERRM) AND MTA PALLAVI UPPANGALA . NATIONAL UNIVERSITY OF SINGAPORE 2007 A COMPARATIVE STUDY OF THE BIOLOGICAL AND PHYSICAL PROPERTIES OF VISCOSITY ENHANCED ROOT REPAIR MATERIAL (VERRM) AND MTA