Study On Zinc Or Copper-doped Calcium Silicon Based Root Canal Sealer

Chapter1 Preparation and physicochemical properties of the zinc or copper-doped calcium-silicon-based root canal sealersPurpose:The efficacy of root canal treatment was affected by choice of root canal sealers.The ideal root canal sealers must have specific characteristics,including biocompatibility,biological activity,adequate root canal sealing ability,proper setting time and degradation properties which were suitable for clinical practice.In this experiment,zinc or copper-doped calcium-silicon-based root canal sealers were prepared,and the physical and chemical properties of the materials were evaluated.Method:Five different kinds of ?-dicalcium silicate materials,including zinc-doped dicalcium silicate(5 mol%or 10 mol%),copper-doped dicalcium silicate(5 mol%or 10 mol%)and pure dicalcium silicate were prepared by a modified sol-gel method.The phase composition of the materials was confirmed by XRD and ICP analysis.The particle size distribution of the powders was measured by a nanometer particle size analyser.The surface morphology of the materials was observed by SEM.The setting time was measured by the Vicat instrument,and the compressive strength was measured by a universal testing machine.Degradation of the materials was performed in Tris buffer solution.By the characterization of the above physical and chemical properties,the preparation method of zinc-doped or copper-doped dicalcium silicate material was optimised.Results:In this study,five kinds of dicalcium silicate powders were prepared successfully by the modified sol-gel method.XRD and ICP tests found that the main components of the five materials were p-dicalcium silicate and the actual molar ratios of the four elements Ca,Zn,Cu and Si were consistent with the theoretical synthesis values.The nanometer particle size analyzer measured that all five samples were nano-sized powders,and the particle size distribution ranged from 200 nm to 700 nm.Vicat test showed that the initial setting and final setting time of Zn/Cu-doped dicalcium silicate was longer than pure dicalcium silicate.The Zn-doped dicalcium silicate was solidified for a more extended period than Cu-doped dicalcium silicate.Alternatively,10%Cu-doped dicalcium silicate has a longer setting time than 5%Zn or 5%Cu-doped dicalcium silicate.The universal testing machine found that the compressive strength of the five samples increased with the curing time,but the compressive strength of the Zn-doped dicalcium silicate was lower than other materials at the same curing time.The compressive strength of low molar ratio Zn/Cu-doped dicalcium silicate is higher than high molar ratio Zn/Cu-doped dicalcium silicate at the same curing time.Degradation studies found that the weight loss of the five samples after soaking for three days was about 10%.After four weeks,the weight loss of the five samples was between 26%and 42%,and the weight loss of zinc-doped dicalcium silicate was the least.The pH in the Tris buffer solution increased rapidly within two weeks,and the liquid became alkaline very quickly.After two weeks,the pH value was still rising,but the development was slowed down.The pH values of the dicalcium silicates powers did not differ considerably from each other at the same time.Conclusion:In this experiment,zinc or copper-doped dicalcium silicate biomaterials were successfully prepared,and zinc or copper doping optimized the physicochemical properties of dicalcium silicate,making it a candidate material for the ideal root canal sealer.Chapter 2 Evaluation of in vitro biological properties of the zinc or copper-doped calcium-silicon-based root canal sealersPurpose:To evaluate the in vitro biocompatibility,bioactivity,root canal sealing ability and antibacterial activity of the zinc or copper-doped calcium-silicon-based root canal sealers,and to provide a scientific basis for preparing bioactive root canal sealing materials.Method:In vitro bioactivity and biocompatibility of zinc(5 mol%or 10 mol%)-doped,copper(5 mol%or 10 mol%)-doped and pure beta-dicalcium silicate was assessed by soaking the materials in simulated body fluid solutions and MC3T3-E1 osteoblasts cultures for various time periods,respectively.The apical sealing ability was measured using dye penetration method.The antibacterial experiment was conducted using the agar diffusion method against E.faecalis and P.gingivalis.Result:The release of Ca2+ from the five kinds of dicalcium silicate samples in the SBF solution increased rapidly within 14 days and slowed down after 14 days.The release of Ca2+ from the zinc or copper-doped dicalcium silicate group was significantly higher than the pure dicalcium silicate group at the same time throughout the study period.The release of Si ions from the five kinds of dicalcium silicate increased significantly in 14 days and slowed down after 14 days.The release of Si ions from the zinc-doped dicalcium silicate group was much lower than copper-doped and pure dicalcium silicate group.The Zn or Cu ions from the five groups released rapidly in the SBF solution within seven days and released slowly after seven days.The release of Zn or Cu ions from the more Zn or Cu doping dicalcium silicate group was significantly more extensive than that of other groups.A large number of interlocking needle-like nanoparticles appeared on the surface of all samples after soaking in SBF for three days.The diameter of the particles was about 50 nm,and the length was about 200-300 nm.After soaking for seven days,many needle-like particles became coarse,and the gap between them was denser than before.Finally,the surface of the samples covered with these particles.Root canals filled with the 5%Zn doping dicalcium silicate have shown lowest microleakage when compared to other samples according to dye penetration depth.There was no significant difference between the zinc-doped and the copper-doped dicalcium silicate group.Dye penetration in the pure dicalcium silicate group showed the maximum penetration depth.The antibacterial activity of the five kinds of dicalcium silicate against P.gingivalis showed that Cu-doped dicalcium silicate group>Zn-doped dicalcium silicate group>pure dicalcium silicate group.With the increase of the content of Cu or Zn doping,the antibacterial effect of dicalcium silicate also enhanced.The 10%copper or 10%zinc doping dicalcium silicate has the most potent antibacterial impact against E.faecalis,following the 5%copper or 5%zinc dicalcium silicate.The pure dicalcium silicate exhibited the worst antibacterial activity.After 72 hours,the osteoblasts were naturally observed to proliferate in the 3.125?100 mg mL-1,and the relative proliferation rate was nearly six times than that of the negative control group(p<0.05).After 120 hours,the rate of osteoblasts was almost six times in 3.125-25 mg mL-1(p<0.05),while the rate of cell proliferation is nearly double than that of the negative control group in 100 mg mL-1.The relative proliferation rate of 10%zinc or 5%copper-doped dicalcium silicate group was higher than that of other groups at 72h and 120h,respectively(p<0.05).After 24 hours,osteoblasts were observed to contact with the surface of all samples and form into a single layer.They had a small number of filopodia extending and adhering to the surface of the materials.After 120 hours,osteoblasts were observed to spread on the surface of samples and to have a large number of plasma membranes stretching out and adhering firmly to the materials.Conclusion:Zn-doped or Cu-doped dicalcium silicate showed excellent biocompatibility,and optimised bioactivity also exhibited superior anti-microleakage ability and potent antibacterial effect.It was expected to be a promising biomaterial as root canal sealing substitute.Chapter 3 Biological properties of the zinc or copper-doped calcium-silicon-based root canal sealing materials in vivoPurpose:Repair and regeneration of alveolar bone defects caused by chronic apical periodontitis is still a challenge in the medical field.In this study,a rabbit model of the mandibular alveolar bone defect was established,then the zinc or copper-doped calcium-silica-based bioactive materials were implanted in the bone defect area to reveal the mechanism of alveolar bone repair and regeneration.Method:Standardized bilateral alveolar bone defects were created in the mandibular body of twenty adult New Zealand white rabbits.These defects were randomly filled with an equal amount of Zn-doped(5%or 10%)dicalcium silicate,Cu-doped(5%or 10%)dicalcium silicate or the pure dicalcium silicate.After 4 and 16 weeks,the animals were sacrificed,followed by analyzed bone regeneration in the bone defect area by gross anatomy,X-ray,Micro-CT,HE staining and Masson trichrome staining observation.Result:The relative bone volume(BV/TV),trabecular thickness(Tb.Th),and trabecular bone number(Tb.N)of the Zn-doped or Cu-doped dicalcium silicate group were significantly higher than the pure dicalcium silicate group.On the contrary,the surface area to volume ratio(BS/BV)and the trabecular bone separation(Tb.Sp)were significantly higher in the pure dicalcium silicate group.The results of three-dimensional reconstruction showed that trabecular bone was thicker and more number,the interstitial space is small,the porosity is low,the bone is dense,all of which suggesting that the formation of new bone is more significant in the Zn-doped or Cu-doped dicalcium silicate group than in the pure dicalcium silicate group.HE staining and Masson staining indicated high expression of collagen and fibre in the Zn-doped or Cu-doped dicalcium silicate group.Gross anatomy and X-ray observation further supported the above results.Conclusion:Zn-doped or Cu-doped dicalcium silicate exhibited good biocompatibilityand osteoconductivity,which resulted in promoting the repair and reconstruction of mandibular alveolar bone defects.