Explore The Ability And Mechanism Of Mineralize Type Ⅰ Collagen Induced By Dentin Matrix Protein-1 Biomimetic Analogue PAMAM-PO₃H₂ And PAMAM-COOH

Objectives:Based on the understanding of the classical mineralization theory and the important role of dentine matrix protein-1（DMP-1）plays in the processofmodulatingdentinemineralization,inthisstudy,phosphate-terminated polyamidoamine dendrimer（PAMAM-PO₃H₂）and carboxyl-terminated polyamidoamine dendrimer（PAMAM-COOH）were employed as biomimetic analogues to mimic the dual roles of DMP-1 in regulating amorphous calcium phosphate of dentine mineralization,to determine their potentials for inter-and intra-fibrillar apatite assembly within a two-dimensional collagen model.In this study,the mechanism of intrafibrillar biomimetic mineralization was discussed,and new theoretical strategies could be provided for biomimetic mineralization of dentin.Methods:8 carbon Ni TEM grids with single-layer of reconstituted type I collagen that cross-linked and stabilized with 0.3M EDC/0.06M NHS were selected and phosphorylated with 2.5 wt%PAMAM-PO₃H₂ or hydrolyzed sodium tripolyphosphate（TPP）for 5 min.4 treated grids were immersed in remineralize medium that contained simulated body fluid（SBF）and Portland Cement with the presence of PAMAM-COOH（n=4）or polyacrylic acid（PAA,n=4）,respectively.The rest of PAMAM-PO₃H₂ or TPP-treated grids were immersed in remineralize medium that contained simulated body fluid（n=4）.12grids with reconstituted collagen were divided into three groups randomly,and then immersed in simulated body fluid and Portland Cement with the presence of PAMAM-COOH（n=4）or polyacrylic acid（n=4）or not（n=4）,respectively.After remineralized in the incubator at 37℃for 12、24、48、72h,the mineralized collagen and minerals were examined with transmission electron microscopy（TEM）at 80k V,and the crystallization phases of mineral would be analyed by selected area electron diffracton（SAED）at 200k V.Result:After 12h mineralization,amorphous calcium phosphate（ACP）particles with different shapes and sizes could be seen on the surface of carbon nickel grids of negative control and converted into crystals after 72h,and collagen was not mineralized.Templating analogue groups:In the STPP control,after mineralized of 12h,the nano ACP phase with different sizes and shapes were visible on the collagen surface,and after 24-48h mineralization,the ACP nanophase was transformed to apatite with sharp edges.72h later,irregular crystals deposited on the surfaces of collagen,without any intrafibrous mineralization.Collagen phosphorylated with PAMAM-PO₃H₂ with no PAMAM-COOH stabilized ACP,after the mineralization of 12h,it can be seen that ACP nanoparticles were attached to the surface of collagen,and after 24-48h,the sizes of ACP nanoparticles on the fiber surface were increased and the apatite mineral deposits transformed with sharp edges.After 72h,the mineral deposits on the collagen surface were converted into irregular but with certain orientation.There is no characteristic of fibrous mineralization and the mineral deposits on the surface of the collagen were looser than that in the control group.Sequestration analogue groups:No nano-ACP particles were formed,after12 to 24h of mineralization in the PAA control.But after 24h of mineralization,the diameter of collagen increased,with the electron density decreased and the cross-banding patterns disappeared.ACP nanoparticles could be seen on the surface of the expanded fibers after 48 h.After 72h,the swelling changes of collagen disappeared,the electron density of the collagen increased,but no cross-banding patterns were found.When stabilized ACP with PAMAM-COOH,after 12h of mineralization,a large number of nano ACP particles were formed in the PAMAM-COOH group.After 24h of mineralization,irregular ACP phases were formed locally in collagen.It was found that ACP nano-liquid phases distributed on the surface of collagen 48h later.After mineralized of 72h,the electron density of the fibers was increased and there were not obvious cross-banding patterns.Synergistic induction groups:During the amorphous stage（12-24h）of STPP+PAA control,the cross-banding patterns of the expanded collagen disappeared,and the amorphous ACPs were seen on the surface of the collagen.In the crystalline stage（48-72h）,the cross-banding patterns formed by nano-apatite strips could be observed in some mineralized collagen.The length of the bands was about 44 nm along with the axis of collagen.Type I collagen phosphorylated by PAMAM-PO₃H₂ with the presence of PAMAM-COOH stabilized ACP,in the amorphous stage,the characteristics of cross-banding patterns in the expanded collagen were blurred,and the formation of ACP nanoparticles could be seen on the surfaces of the collagen.In the crystalline stage,collagen was partially dehydration and mineralization,and formation of ACP nanoparticles was observed on the surface of the collagen.After 72h,irregular deposition and mineralization of ACP nanoparticles were observed on the surface of collagen with no cross-banding patterns were found.Conclusion:1.PAMAM-COOH has the ability of stabilizing ACP into nanoprecursors,and PAMAM-PO₃H₂ can induce the phase transition of ACP,and direct nucleation and crystallization to form a loose polycrystalline mineral deposition on the surface of collagen fibers.2.The use of PAMAM-PO₃H₂ as a templating analogue with the presence of PAMAM-COOH to sequester ACP into transient nanophases,which has the ability to induce internal and external mineral deposition of type I collagen fibers in vitro,and accomplish the mineralization of reconstituted type I collagen through the non classical mineralized crystallization pathway,can be used as a potential research strategy for biomimetic remineralization of demineralized dentin collagen fibers.