Biomimetic Remineralization Of Human Dentin Type Ⅰ Collagen Fibrils Induced By Carboxylated Polyamidoamine/Amorphous Calcium Phosphate Nanocomposite

ObjectiveThis study aims to synthesize a carboxylated polyamidoamine/ amorphous calcium phosphate nanocomposite,evaluate the biomimetic remineralization ability of type I collagen fibrils in vitro,and demonstrate the intrafibrillar remineralization of collagen fibrils.Herein,we develop a new strategy for the synthesis of biomimetic remineralized collagen fibrils.Hope to provide an experimental reference for future studies.Moreover,an experimental basis for improving the resin-dentin bonding interface,as well as prevention and cure of dental caries,is provided.MethodsThe PAMAM-COOH/ACP was prepared using the solution coprecipitation method and characterized using TEM,FTIR,TGA,and XRD.Herein,41 samples of demineralized human dentin type I collagen fibrils were prepared and randomly divided into 4 non-fluorescent groups(n=4): Blank group(A),ACP group(B),ACP+PAMAM-COOH group(C),and PAMAM-COOH/ACP group(D)and 3 fluorescent experimental groups(n=4): fluorescent ACP group(E),fluorescent ACP+PAMAM-COOH group(F),and fluorescent PAMAM-COOH/ACP group(G).After demineralization,they were incubated in HEPES equilibrium solution with or without 0.33wt% mineralized materials for30 days.In non-fluorescent groups,the remineralization of the demineralized collagen fibrils were observed using TEM.In fluorescent groups,the remineralization of the demineralized collagen fibrils were observed using CLSM.In all the groups,another samples were demineralized with 1 wt%chitosan to remove the extrafibrillar minerals and were observed using TEM or CLSM.Results1.TEM images showed that PAMAM-COOH/ACP comprised a double-layered circular particle with a diameter of 22.03 ± 3.85 nm.FTIR and XRD analysis confirmed that the composite of ACP and PAMAM-COOH is an amorphous phase,which is necessary for the remineralization of the collagen fibrils.TGA showed that the proportion of PAMAM-COOH in PAMAM-COOH/ACP was 10.09%.2.After 30 days of remineralization,which was induced using the PAMAM-COOH/ACP in human dentin type I collagen fibrils,the intrafibrillar remineralization of the collagen fibrils was observed,and intrafibrillar mineral deposits in the collagen fibrils was also observed after treatment with chitosan.3.When fluorescent PAMAM-COOH/ACP was used to induce the remineralization of the demineralized collagen fibrils layer,dense new crystals were observed,and the morphology and density of the new minerals remained unchanged after treatment with chitosan.This confirmed the formation of intrafibrillar minerals in the collagen fibrils layer.The results showed that PAMAM-COOH/ACP induced a remineralization depth of 92.50 ± 12.25 μm,which was retained up to 77.50 ± 8.86 μm after treatment with chitosan.The amount of intrafibrillar mineralized products in the collagen fibrils was 83.78%.Conclusion1.The PAMAM-COOH/ACP was successfully prepared using the solution coprecipitation method and it comprised double-layer circular particle with a diameter of 22.03 ± 3.85 nm.2.PAMAM-COOH/ACP can induce the biomimetic remineralization of completely demineralized human dentin type I collagen fibrils,especially the formation of intrafibrillar minerals.3.The method of verifying the intrafibrillar mineralization of collagen fibrils combined with fluorescent labeling technology and the extrafibrillar demineralization of collagen fibrils can be used to verify the existence of intrafibirillar mineralization of collagen fibrils.