| Background:Periodontal disease, trauma and dental caries caused by tooth loss, and inconvenience to patients with chewing function [1]. Preparation of hydroxyapatite coating in the surface of the implant titanium have been attention, but the HA-coated existence of the degradation and flaking will result in the loss of stability of the implant-bone interface, affecting the long-term effects[2]. Complexes of calcium and phosphorus compounds of type I collagen closer to the organizational structure of the physiology of bone, because calcium phosphate ion deposition of collagen, which is conducive to the stability of the calcium phosphate; subsequent induction of osteoblast, collagen for calcium phosphate to hydroxyapatite conversion into a nuclear environment; calcium phosphate enhance the stability of the collagen, so that collagen continue to maintain the osteoinductive activity after all the calcium phosphate into apatite phase[3,4]. Calcium phosphate can be degraded and converted to hydroxyapatite which is similar to human teeth and bone composition under physiological conditions, inducingof osteoblast differentiation. Type I collagen can provide a good environment for the early osteoblast adhesion, proliferation, differentiation.The complexes of CaP/COL-I accord with the human physiology Bone Tissue[6,7]. CaP/COL-I biomimetic coating in the environment of the titanium implant simulats biological mineralization, and promotes early bone healing, and finally achieves to a stable and long-term osseointegration of dental implants and human bone tissue. In recent years, the reseach of the calcium phosphate/collagen composite biomimetic coating less, and its special biological properties provide good biological activity and osteoinductive potential, which expecting to create a new kind of chemical coating for dental implants[4,5].Objective:The complex of calcium phosphate/the type I collagen induced osteoblast osteogenesis is to determine a key factor in the early osseointegration. This experiment attempt to understand the reaction mechanism of CaP/COL-I composite coatings on osteoblast, by observing the MC3T3-E1osteoblast adhesion, proliferation and differentiation changes.Methods:1. Material groups:Titanium sample,calcium phosphate coating of titanium sample,(porous type) calcium phosphate/collagen type I titanium sample,(compact type) calcium phosphate/collagen type I titanium sample.2. MTS proliferation detection:each group of materials and MC3T3-E1osteoblast cells were cultured in24-well plates detect osteoblast adhesion and proliferation in culture Id,2d,3d,5d.3. SEM obserbations:Each group of materials and MC3T3-E1osteoblast cells were cultured in24-well plates,24h,72h, gathering and preparing samples, scanning electron microscopy of osteoblasts on the surface of the growth, morphology and adhesion.4. Mechanism of osteogenesis:each group of materials and MC3T3-E1osteoblast cells were cultured in24-well plates, alkaline phosphatase (ALP) protein expression levels in cells by Western blot analysis, to understand the material to induce changes in the bone cell function; through real-timequantitative PCR detection of ALP, osteocalcin (OC), COL-I mRNA in the expression level of understanding of osteoblast differentiation, osteoblast gene expression changes in the level.Results:1. MTS proliferation detection:osteoblast were cultured after Id,2d,3d,5d, the growth of the MTS test values indicate cells on the surface of the material growth and proliferation in each group of materials over time. Compared to different groups of materials, the proliferation of osteoblasts were significantly different (p<0.05) indicating that the (compact type) calcium phosphate/I collagen titanium sample cell proliferation can obviously promote the proliferation of osteoblast.2. SEM observation:electronic microscopy revealed the bone cells in the normal adhesion of different materials cultured for24h,72hours, growth, proliferation, cell spreading, but in the (compact type) calcium phosphate/collagen type I titanium sample group stretched out pseudopodia, anchorthe material surface, growth is better than the other groups.3. Western blot: osteoblast cultured for1d,3d,7d, Western blot detection value indicates that the ALP protein expression inceases over time, and the expression level of (compact type) calcium phosphat e/collagen type I titanium sample group is higher.4. qRT-PCR:osteoblasts cultured for4d,7d,14d, real-time quantitative PCR detection value indicates that the expression of ALP, OC and COL-ImRNA increases over time, but in the (compact type) calcium phosphate/type I collagen titanium sample group is the highest.Conclusion:(Compact type) calcium phosphate/collagen type I titanium specimen is conducive to the adhesion, proliferation and differentiation of MC3T3-E1osteoblasts. Studies have shown that the (compact type) calcium phosphate/type I collagen compound scaffold for biomineralization environment has a good induction of osteogenic activity and biodegradability, bionic, also have a synergistic effect to promote bone regeneration. |
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