Stablishment Of Epicallocatechin-3-gallate-encapsulated Nanohydroxyapatite/mesoporous Silica Biomaterial And Its Application On Dentin Hypersensitivity

Part I Synthesis of nanohydroxyapatite/mesoporous silica nanoparticle(nHAp@MSN)biocomposite and its application on dentinal tubule occlusion in vitroObjectives:To fabricate a nHAp@MSN biocomposite and investigate its effectiveness on in vitro dentinal tubule occlusion,acid-resistant stability,and biocompatibility.Materials and methods:As the principal ingredients,calcium nitrate,diammonium hydrogen phosphate,mesoporous silica nanoparticle were used to synthesize nHAp@MSN.It was characterized by XRD,FTIR,N2 adsorption-desorption,FESEM,and TEM.The in vitro cytotoxicity towards human dental pulp stem cells(hDPSCs)was detected by CCK-8 test.Thirty-two intact caries-free human third molars were collected,preparing dentin disc specimens with the thickness of 1 mm ±0.1 mm.After wet and graded ground,the specimens were immersed in 1%(w/v)citric acid solution for 20 s to simulate a sensitive tooth model.According to different treatments on dentin surfaces,all dentin specimens were randomly divided into four groups(n = 8 each):Group 1,no treatment(control);Group 2,NovaMin-containing paste-treated for 15 s × 2;Group 3,MSN slurry-treated for 15 s × 2;Group 4,nHAp@MSN slurry-treated for 15 s × 2.Then,four discs were randomly selected from each group and treated with 6%(w/v)citric acid solution for 1 min to test the acid-resistant stability after different desensitizing treatments.The dentinal tubule occlusion of each specimen was inspected by FESEM.Results:Characterization results revealed that the nHAp@MSN biocomposite was successfully synthesized,and nHAp was loaded on MSN,it induced low cytotoxicity to hPDSCs.nHAp@MSN could effectively occlude the dentinal tubules,and tightly combined with tubular inwall.After citric acid attack,the majority of tubule orifices were still occluded,and a membrane-like layer that covered the dentin surface was created.Conclusions:The application of nHAp@MSN biocomposite could effectively block the dentinal tubules,provide certain acid-resistant stability,and possessed favorable in vitro biocompatibility,suggesting great potential for treating dentin hypersensitivity.Part II Establishment of EGCG-encapsulated nanohydroxyapatite/mesoporous silica nanoparticle(EGCG@nHAp@MSN)biomaterialObjectives:To fabricate a EGCG@nHAp@MSN biomaterial and investigate its in vitro release profiles of EGCG,Ca2+,PO43-,and biocompatibility.Materials and methods:Forty milligram of EGCG was dissolved into 20 mL of absolute ethanol under darkness at room temperature,followed by adding 100 mg of nHAp@MSN.After magnetic stirring for 2 h and shaking for 72 h,the mixture was centrifuged for 15 min to retrieve precipitate.The precipitate was triple-washed by ethanol,filtered,and vacuum-dried to obtain a white powdery product.Morphology and ultrastructural features were examined by TEM,thermal stability of nanoparticles and encapsulating capacity of EGCG were determined by TGA.The in vitro cytotoxicity towards human dental pulp stem cells(hDPSCs)was detected by MTT test.A hundred milligram of EGCG@nHAp@MSN was added into 10 mL of TBS solution at room temperature protected from light,and kept shaking at 37?.At each designated time interval(0.5,1,2,4,8,12,24,48,72,96 h),the release of EGCG and Ca2+ and PO43-were measured and analyzed by an UV-Vis spectrophotometer and an ICP-AES,respectively.Results:Characterization results revealed that EGCG was successfully encapsulated into the inside pores of MSN with the encapsulating capacity of approximately 11.29%,and the EGCG@nHAp@MSN biomaterial was successfully synthesized,it induced low cytotoxicity to hPDSCs.The release of EGCG,Ca2+,PO43-from EGCG@nHAp@MSN displayed a two-step pattern with an initial burst release and a relatively slow sustained release over 96 hours.Conclusion:EGCG@nHAp@MSN possessed favorable in vitro biocompatibility,and was able to slowly sustained release EGCG,Ca2+,PO43-in TBS solution.Part III In vitro study on dentin hypersensitivity treatment by EGCG@nHAp@MSN and its application on adhesive restorationObjectives:To evaluate dentinal tubule occlusion ability of EGCG@nHAp@MSN and its effect on dentin permeability in vitro,and the application on adhesive restoration.Materials and methods:Thirty-two intact caries-free human third molars were collected,preparing dentin disc specimens with the thickness of 1 mm ± 0.1 mm.After wet and graded ground,the specimens were immersed in 0.5 M EDTA solution for 2 min to simulate a sensitive tooth model.All dentin specimens were randomly divided into two groups(n = 16 each);Group 1,no treatment(control);Group 2,dentin surfaces were polished by EGCG@nHAp@MSN slurry for 30 s × 2.Then,the specimens in each group were randomly divided into two subgroups(n = 8 each):Subgroup 1,specimens were immersed in 6%(w/v)citric acid solution for 1 min to test the acid-resistant stability;Subgroup 2,specimens were mechanically brushed for 3 min to test the abrasion-resistant stability.All specimens were utilized to measure the dentin permeability and then compared statistically,the dentinal tubule occlusion was observed by FESEM.Additional twenty human third molars were collected and then sectioned to expose middle dentin,and wet-ground by 600-grit silicon carbon,simulating a sensitive tooth model and randomly dividing all teeth into two groups(n=10 each)according to the above mentioned treatment procedures.The teeth in each group were then randomly divided into two subgroups(n = 5 each):Etch-and-rinse adhesive Single Bond 2 or self-etch adhesive G-Bong were employed to conduct adhesive restoration,respectively,and the micro-tensile bond strength(MTBS)in each group was tested and compared statistically.Results:The dentin permeability in EGCG@nHAp@MSN-treated group was significantly lower than the permeability before treatment or the control group,the dentinal tubules were completely occluded and the intratubular nanoparticles were tightly combined with the tubular inwall.After citric acid challenge,although the dentin permeability in EGCG@nHAp@MSN-treated group was higher than the permeability before acid challenge,it was significantly lower than the control group,the majority of the tubule orifices were still blocked with certain blocking depth and the tubular inwall combination.After mechanical brushing challenge,the dentin permeability in EGCG@nHAp@MSN-treated group was not changed compared to the permeability before brushing challenge,it was significantly lower than the control group,all dentinal tubules were remained sealed and the sealing depth was almost maintained.MTBS result showed that there was no significant difference between the control group and the EGCG@nHAp@MSN-treated group regardless of Single Bond 2 or G-Bond adhesive systems.Conclusions:The application of EGCG@nHAp@MSN biomaterial could effectively occlude the dentinal tubules,decrease dentin permeability,provide excellent acid-resistant and abrasion-resistant stability,and did not affect the dentin bond strength of etch-and-rinse or self-etch adhesives,implying that EGCG@nHAp@MSN is of great potential to treat dentin hypersensitivity,prevent the occurrence of post-operative dentin hypersensitivity after adhesive restoration,and improve the dentin bond strength.Part IV Study on inhibitory effect of EGCG@nHAp@MSN on Streptococcus mutans biofilm formationObjectives:To evaluate the inhibitory effect of EGCG@nHAp@MSN on Streptococcus mutans(S.mutans)biofilm formation in vitro.Materials and methods:Twenty intact caries-free human third molars were collected,preparing dentin disc specimens with the thickness of 1 mm ± 0.1 mm.After wet-ground by 600-grit silicon carbon,the specimens were immersed in 0.5 M EDTA solution for 2 min to simulate a sensitive tooth model.All dentin specimens were disinfected under UV light and randomly divided into two groups(n = 10 each):Group 1,no treatment(control);Group 2,dentin surfaces were polished by EGCG@nHAp@MSN slurry for 30 s x 2.Then,all specimens were placed in a 24-well plate with the treated surface upwards,each well was added by 1 mL of S.mutans inoculation medium containing BHI broth and 1%sucrose,and incubated at 37 °C under anaerobic condition for 24 h for biofilm formation.After that,the specimens were triple-washed with sterile PBS to remove non-adherent bacteria,CLSM,MTT assay,CFU counts,and FESEM were utilized to inspect and analyze the inhibitory effect on S.mutans biofilm formation after different treatments.Results:Results of CLSM,MTT assay,and CFU counts revealed that,the biomass of S mutans biofilm,the metabolic activity of S.mutans,and the viable S.mutans colonies in EGCG@nHAp@MSN-treated group were significantly fewer than the control group.FESEM observation showed that nearly all of the dentinal tubules in the EGCG@nHAp@MSN-treated group were occluded,and the S.mutans biofilm formed on dentin surfaces was significantly fewer than control group.Conclusion:The application of EGCG@nHAp@MSN biomaterial on sensitive dentin surface could effectively occlude the dentinal tubules,and inhibit the formation and growth of S.mutans biofilm,indicating the potential importance of EGCG@nHAp@MSN on dental caries prevention.