Effect Of Electric-assisted Technique On Dentin Bonding&Preliminary Study On Prevalence Of Enamel Defects Of Human Third Molar

PART ONE Effect of electric current-assisted technique on dentin bonding CAPTER ONE Setting up electric current-assisted deviceObjective:We tried to generate the electric current-assisted device according to the requirements of cells experiments in vitro and dentin bonding.Materials and methods:An electric current generating device was set up and modified according to the descriptions by other investigators. Current intensities of0μA,10μA,20μA,50μA,60μA,70μA,90μA were added to our device (Fig.1A). Modifications in electric circuit were also made to simplify the operations in cellular experiments.Results:We generated the electric current-assisted device. It had been proven that the device met requirements of dentin bonding and cells experiments in vitro. The stimulation of direct currents were stable and accurate.CAPTER TWO Effect of electric-current assisted technique on the proliferation and differentiation of human dental pulp cellsExp.1Effects of electric-current assisted technique on proliferation of human dental pulp cellsObjective:To study the effects of different intensities of direct-current stimulation on proliferation of human dental pulp cells in vitro.Materials and methods:Human dental pulp cells were harvested and serially passaged. Cells at passage3were used in this study. HDPCs were seeded in6wells plates at a density of2×104cells/cm2and cultured for24hours. Electrical stimulation is applied through two pure titanium (Ti) electrodes (30mm×10mm×0.5mm) placed on opposite sides of each well. HDPCs were stimulated for30s. For experimental groups, with the device on, the current intensity for electrical stimulation was kept constant at10,20,50,60,70and90μA, respectively. While for the control group, HDPCs were stimulated with the electric device off. Cell morphology was observed under the fluorescent microscope, and cell proliferation was analysed by MTT assay.Results:The morphology of HDPCs in the experimental groups were similar to the control cells. the cell density in the20μA,50μA,60μA and70μA groups was not visibly changed, it was increased in the10μA group and decreased in the90μA group. MTT assay showed no significant difference in the relative cell viability in the groups of20μA,50μA,60μA and70μA(P>0.05). the cell viability was affected when the cells were stimulated by90μA (P<0.05). However, the cell proliferation was significantly improved in the10μA group (P<0.05).Exp.2Effects of electric-current assisted technique on differentiation of human dental pulp cellsObjective:To study the effects of different intensities of direct-current stimulation on differentiation of human dental pulp cells in vitro.Materials and methods:Human dental pulp cells were harvested and serially passaged. Cells at passage3were used in this study. HDPCs were seeded in6wells plates at a density of2×104cells/cm2and cultured for24hours. Electrical stimulation is applied through two pure titanium (Ti) electrodes (30mm×10mm×0.5mm) placed on opposite sides of each well. HDPCs were stimulated for30min. For experimental groups, with the device on, the current intensity for electrical stimulation was kept constant at30,60and90μA, respectively. While for the control group, HDPCs were stimulated with the electric device off. After the stimulation, we evaluate the differentiation of HDPCs by RT-PCR, ALP dye and alizarin red dye.Results:The real-time PCR results showed that relative Gene expression were different for ALP an OCN stimulated with different intensities of direct currents. And it has the potential to promote remineralization when stimulated by60μA. However, we still need further experiments to prove it. The results of ALP dye and alizarin red dye indicated that the formation and numbers of Nodules of calcification showed no significant differences compared to the control.CAPTER THREE Effect of electric-current assisted technique on dentin bondingExp.1Effects of electric-current assisted technique on μTBS and failure modes analysisObjective:To study effects of different intensities of direct current on μTBS and failure modes of debonded specimens.Materials and methods:Thirty-five freshly extracted intact human third molars were ground and bonded with Clearfil S3Bond with use of electric-current assisted equipment. Specimens were stored in37℃water for24hours and then sectioned into beams by slow speed diamond saw. Specimens were randomly divided into seven groups:the control group,10μA,20μA, 50μA,60μA,70μA and90μA. The microtensile bond strengths were performed. After the tests, scanning electron microscopy (SEM) were used to evaluate the failure modes of resin-dentin bonding.Results:One-factor variance analysis indicated that, compared to the control group, the10μA,20μA group showed no significant difference (P>0.05), while50μA,60μA,70μA and90μA group were significantly increased (P<0.05). The predominant fracture pattern of all debonded specimens was adhesive failure. Less common type was mixed failure. While cohesive failures in dentine were very rare and no cohesive failures in composite were found in all the fractured specimens. Fracture mode analysis did not indicate any significant statistical differences between different experimental and control groups or within the groups (P>0.05).Exp.2Effects of electric-current assisted technique on interfacial nanoleakageObjective:To study effects of different intensities of direct current on interfacial nanoleakage.Materials and methods:Thirty-five freshly extracted intact human third molars were ground and bonded with Clearfil S3Bond with use of electric-current assisted device. Specimens were stored in37℃water for24hours and then sectioned into beams by slow speed diamond saw. Specimens were randomly divided into seven groups:the control group,10μA,20μA,50μA,60μA,70μA and90μA. Interfacial nanoleakage was evaluated by scanning electron microscopy.Results:Compared with the control group, clusters of silver deposits still could be identified in the10μA and20μA experimental groups. Whereas, only sparse or even scarce silver deposition was detected in the60μA,70μA and90μA groups.Conclusions:1. Compared with the control group, the μTBS was significantly increased when the adhesive was bonded to specimens with electric-assisted device using current intensities of50μA,60μA,70μA and90. In particular, the highest μTBS was achieved with60μA and70μA. No significant difference was found in10μA and20μA groups.2. The interfacial nanoleakage images were in concordance with the results of μTBS. Only sparse or even scarce silver deposition was detected in the60μA and70μA. 3. The cell viability was affected when the cells were stimulated by90μA. The cell proliferation was significantly improved in the10μA group. The relative cell viability was not affected in the groups of20μA,50μA,60μA and70μA.4. Compared to the control group, the differentiation of HDPCs was restrained by direct current stimulation of90μA. While the stimulation of30μA and60μA didn’t promote differentiation of HDPCs. PART TWO Preliminary study on prevalence of Enamel Defects of Human Third MolarObjectives:The purpose of this study was to measure the prevalence of enamel defects in a dental school patient population through direct observation, photographic examination, observation of cross-sections, and polarized light microscopy of histological sections in routinely extracted third molars.Methods:A cross-sectional study examined100subjects. Participants completed a questionnaire designed to identify and quantify psychosocial childhood stressors experienced from approximately8-12years of age, the time of third molar enamel formation, then donated their extracted third molars. Enamel defects were identified and analyzed using plain eyesight;2.5x magnification loupes; macro photography; and transmitted light microscopy with polarization of hard tissue sections. Teeth were classified on the type of enamel defects using a modification of the Witkop classification.Results:The prevalence of enamel defects was high. Approximately two thirds of teeth had pits or valley defects, spots, bands, or demineralization; more had snow capping. However, linear enamel hypoplasias and some subtypes of the above defects were rare. Most spots or bands were white. Many subjects had experienced harsh non-nutrative childhood environments.Conclusions:Enamel defects were extremely common; demineralization and caries were extremely common; linear enamel hypoplasias and valley defects were rare; teeth with single pits were rarer than those with multiple pits. Polarized light microscopy of histological sections distinguished between defects which had indistinguishable surface appearances. Most defects were white, but brown and translucent defects were also seen. Snow capping can be considered to be normal in this third molar sample; some other white spots and bands had the same histological features as snow capping. All translucent spots and some other spots and bands exhibited a histological pattern consistent with a developmental origin.