Carboxyl Modified Polyamide-Amine/N Ano-hydroxyapatite Induced Dentin Remineralizationin Acid Environment

Objective:1.To prepare Carboxyl modified polyamide-amine/nanohydroxyapatite?PAMAM-COOH/n-HAP?compositeand explore the optimum raw material quality ratio.2.To observe the effect of PAMAM-COOH/n-HAPcomposite in inducing biomimetic remineralization of demineralized dentin in acid environment,and to provide a new theoretical reference for the application of a new remineralization agent in clinical treatment of it.3.To investigate the toxicity of PAMAM-COOH/n-HAPcompositetodental pulp cells and evaluate its feasibility as a new type of remineralization agent.Methods:1.Prepared different mass ratios?m?PAMAM-COOH?:m?n-HAP??of PAMAM-COOH/n-HAPcomposite.The chemical information was measured by fourier transform infrared spectroscopy,the surface morphology and particle size were observed by transmission electron microscope,and the amount of PAMAM-COOH bound to nano-hydroxyapatite?n-HAP?was detected by thermogravimetric analysis.2.Preparated demineralized dentin discs and randomly divided into8groups?n=15?:?1?⁴were the CaP?-?groups:?1?Control group,dentins withdeionizedwater;?2?PAMAMCOOH/n-HAPgroup,dentinswith PAMAM-COOH/n-HAPcompositepaste;?3?PAMAM-COOH group,dentins with PAMAM-COOH;?4?n-HAPgroup,dentinswithn-HAPpaste.After corresponding treatment,?1?⁴groups were directly remineralized in acid environment daily.?5?⁸were the CaP?+?groups:?5?Control+CaP group,dentins withdeionized water;?6?PAMAM-COOH/n-HAP+CaP group,dentins with PAMAM-COOH/n-HAP paste;?7?PAMAM-COOH+CaPgroup,dentins with PAMAM-COOH;?8?n-HAP+CaP group,dentins with n-HAP paste.After corresponding treatment,?5?⁸groups were treated with calcium and phosphorus solution?CaP?and remineralized in acid environment daily.After 2weeks ofcontinuous remineralization,the ability of dentins remineralization were evaluated by scanning electron microscope?SEM?and X-ray diffraction?XRD?.3.Human dental pulp cells were cultured in vitro.The fourth passage dental pulp cells were used.PAMAM-COOH/n-HAP composites were extracted in common cell culture medium without serum for 1 day,3 days and 7 days,respectively.The supernatant was taken and divided into experimental groups for 1 day,3 days and 7 days.Positive control group and negative control group were also set up.Positive control group:0.64%phenol was added to the normal cell culture medium.Negative control group:common cell culture medium.The relative growth rate?RGR?of dental pulp cells was detected by CCK-8 after cultured dental pulp cells in CO₂ cell incubator for 1 day,3 days and 5 days,in order to evaluate the cytotoxicity of the composites.Results:1.PAMAM-COOH/n-HAP composites with different mass ratios were successfully prepared,and the thicknesses of PAMAM-COOH coating on PAMAM-COOH/n-HAP were different.The thickness of PAMAM-COOH coating was?26.648±1.035?nm,when the mass ratio was 1:4.It was significantly higher than that of other mass ratios?P<0.05?.When the mass ratio was 1:2,the thickness was smallest,which was only?6.620±0.501?nm.2.In PAMAM-COOH/n-HAP group treated with CaP solution,the optimal effect of remineralization was obtained.The dentindiscs surface were almost completely covered by the new mineralized layer,and the sealing depth of dentin tubules on the longitudinal section was up to 15.5?m³9.3?m.However,in PAMAM-COOH/n-HAP group without CaP solution,most of the dentin tubules were completely closed with a closed depth of 8.8?m⁸.3?m.But there were still a few dentin tubules in a semi-closed state.However,the remineralization effect of the other control groups were weak.3.The A value increased gradually with the prolongation of culture time in the extraction group for 1 day,3 days and 7 days,and the difference was statistically significant?P<0.05?.There was no significant difference in A value between each extraction group and negative control group at the same time point?P>0.05?,but there was significant difference between each extraction group and positive control group?P<0.05?.The RGR of each experimental group was above 90%in 1,3 and 5 days after dental pulp cell culture,which met the cytotoxicity requirements of national medical biomaterials.In the positive control group,the RGR of the cells was less than 24%,and the cytotoxicity of the cells in the positive control group was higher.Conclusion:1.In this study,when the PAMAM-COOH/n-HAP composite was synthesized,the optimum mass ratio of PAMAM-COOH and n-HAP was 1:4.2.This study shows thatPAMAM-COOH/n-HAP compositescould induce demineralized dentin remineralization in acid environment,but the effect of demineralized dentin was more obvious with the aid of exogenous CaP solution.3.The results of this study show thatPAMAM-COOH/n-HAP composites was below grade I,which met the cytotoxicity requirements of national medical biomaterials and had the potential to be used in clinical remineralization therapy.However,due to the limitations of experimental conditions and time,a large number of experiments still need to be verified in the later stage.