Studies on materials and techniques to enhance clinical outcomes for non-vital immature permanent incisors

In this thesis three studies are presented all aimed at enhancing outcomes for the endodontic management of non-vital immature permanent incisors. Study 1. Aim: To study the effect of non-setting calcium hydroxide (NSCH) and mineral trioxide aggregate (MTA) on mechanical properties of dentine. Methodology: Approval was obtained from the Tissue bank of the Leeds Dental Institute for the use of human teeth for this study. Twenty-seven human premolar teeth were sectioned to give a total of 54 dentine slabs. Nine slabs used in each group for each of the two time periods: 3 months and 8 months. A thin layer of NSCH or MTA was applied to the slabs, whilst the control group had no application of a medicament. The total period of the study was eight months. Dentine fracture resistance was measured using nano-indentation and by impact test, after which the slabs were Map Scanned for softening after 3 months and then at 8 months. Results: Impact test showed a significant reduction in the fracture resistance of dentine (p<0.001) after 3 and 8 months of application of MTA and NSCH compared with the control group. MTA application had the worst effect on dentine followed by NSCH and control. Also the map scan showed softer areas in the MTA group than the other groups. Conclusion: There was a significant time dependant reduction in the hardness of dentine with NSCH and MTA application. Study 2 The aim: This study was undertaken to evaluate the most effective intra canal medicament against five bacteria commonly implicated in causation of endodontic infections. Methodology: Five bacteria, namely, Actinomyces odontolyticus (NCTC 9335) Porphyromonas gingivalis (W50), Fusobacterium nucleatum (ACTC 10953), Escherichia coli (NCTC 11954) and Enterococcus faecalis (NCTC 755) were selected for this study. The test materials used to evaluate their effect against the chosen bacteria were calcium hydroxide (CH), mineral trioxide aggregate (MTA) and the recently advocated antibiotics (Minocycline, Metronidazole and Ciprofloxicillin), individually and as a Triple mixture. E. coli and E. faecalis were grown in Brain Heart Infusion (BHI) broth under an atmosphere of 10% CO2 + air and incubated at 37ºC. P gingivalis, F. nucleatum and A. odontolytics were grown in BHI broth supplementd with haemin and menadione at a final concentration of 5 µg/ml and 1 µg/µl respectively, all three bacteria were incubated at 37ºC under anaerobic conditions (80% N2, 10% H2 and 10% CO2) in a Mark III anaerobic Work station (Don Whitley Scientific, Shipley, UK). The antimicrobial activity of test materials were determined in agar diffusion assays. Broth cultures of all five bacteria were adjusted in sterile BHI or BHI supplemented with haemin and menadione to an optical density at 540 nm corresponding to 1 x 106 colony forming units (CFU) per ml. Aliquots (100 µl) of bacterial suspensions were inoculated onto the surfaces of Columbia blood agar plates (pre-reduced for anaerobic bacteria). Wells of 3 mm diameter were cut in each plate using a sterile cork borer and test materials applied using a syringe. Zones of inhibition of bacterial growth were measured using a caliper gauge and the mean of three measurements of each zone was calculated, each experiment was carried out on three different occasions. Results: CH, MTA and Metronidazole were not effective against E. faecalis; whereas Ciprofloxacin and Minocycline showed large inhibition zones with all five types of bacteria used in the present study. When all three antibiotics were used as a mixture, they were found to be effective against all five types of bacteria. Conclusions: In the present study the application of triple antibiotic (TriBioDent) was effective against the five common types of bacteria that cause endodontic lesions. Study 3 Aim: To evaluate the toxicity of antibiotics used for regenerative antibiotic technique, both individually and in combination on pulp cell survival. Methodology: Human dental pulp cells obtained from three different donors from the tissue bank were grown until sub-confluence. These were then incubated for 24 hours with culture media supplemented with different antibiotics (50 mg/ml each) in five groups including: 1- Metronidazole, 2- Ciprofloxacin and 3- Minocycline, 4- Metronidazole + Ciprofloxacin and 5- Metronidazole + Ciprofloxacin + Minocycline. Cells grown in culture medium without any antibiotics were used as a control. The experiments were repeated three times and the cell viability was measured using three methods: Lactate Dehydrogenase (LDH) assay, Trypan blue cell counting and flow cytometery. Results: The three methods used to measure the effect of individual or mixed antibiotics on cell viability showed consistent results. Ciprofloxicillin showed a high cytotoxic when compared to the control group that was significantly different (p<0.00). However a combination of two antibiotics, i.e, Metronidazole and Ciprofloxicillin showed a slightly higher cell survival as compared with a combination of all three antibiotics as currently used for the regenerative endodontic technique. Conclusion: In the present study the application of antibiotics used in the regenerative endodontic technique (double or triple antibiotics) was considered to have no significant effect on the survival of pulp cells. There was no difference between using two or three antibiotics in combination.