| The main purpose of surface modification of dental implant is to shorten the time of osseointegration, and as to reach immediately or early loading. From the aspect of biomimetics, this investigation focused on the detailed methodology research of designing and constructing a biomimetic ECM bioactive coating on pure commercial titanium implant surface. Based on this, biological evaluation of different coatings was undertaken.We produced a basal surface by the following treatments:first grinding and polishing, then acid-etching with a mixture of H2SO4/H2O2. Previous studies indicated it is a basal surface with plenty of hydroxy group and owns quite good biological activity. The interaction of hydrogen bonds, between hydroxy group and polyelectrolyte with lots of amino group, provides the feasibility of the following self-assembly. Then four biomacromolecules with good osteoinductive, collagen typeâ… , chitosan, hyaluronic acid and polyglutamic acid were choosed. and four polyelectrolyte solutions with disparity charge were prepared according to their different isoelectric point. Four different coatings were prepared on pure titanium basal surface with layer by layer technique and were sent for characterization. SEM analysis demonstrated that surface morphology of the coatings was changed compared with that of basal surface, and were also covered with different granules in nano size. The results of AFM examination also proved almost the same tendancy. XPS analysis showed that the element nitrogen content of the coating surface was dramatically increased with deposition number. On the other hand, the results of quartz crystal microbalance measurement indicated that the thickness of coating was also increased with approximately linearly with the deposition number. Finally, contact angle measurement showed that surface wettability of the coating was dramatically altered along the deposition number. Taken together, we can draw a conclusion that organic biomacromolecules we applied have been successfully constructed on the titanium surface. After that, we cultured pre-osteoblasts (MC3T3-E1) on different coatings in order to evaluate cell attachment, cell proliferation and cell differentiation. The biological evaluation results suggested that the coating based on collagen type I and hyaluronic acid promote cell growth at mostly.Next, GRGDSPC polypeptide sequence was designed and synthesized, and was then tointroduced to hyaluronic acid. Finally, collagen/hyaluronic acid PEM was constructed on titanium surface by LBL technique. Owing to the high activity of sulfhydryl group from aminothiopropionic acid reside of the polypeptide; the sulfhydryl group can change into disulfide bond with cross linkage reaction under moderate condition, which makes the collagen/hyaluronic acid PEM more stable. Thus, the RGD sequence was introduced to the PEM and the PEM was cross linked at the same time. The in vitro degradation experiment showed, the stability of collagen/hyaluronic acid PEM, which was cross linked, was significantly promoted. Furthermore, the degradation rate of this coating was largely elevated in GSH fluid, which suggested the degradation rate of this coating could be accelerated with the action of in vivo glutathion. In vitro biological evaluation demonstrated, compared with collagen/hyaluronic acid PEM, the RGD functionalized PEM significantly promoted the attachmen, proliferation, differentiation and calcification of preosteoblasts. Still on this base, we plan to introduce growth factors to this coating. BMP-2 has been extensively explored because of its strong osteoinductivity, for the same reason BMP-2 was choosed in this experiment. So BMP-2 was dissolved in collagen solution and thus positively charged, and was introduced to the coating by LBL technique, then cross linkage reaction was also carried out. Degradation test manifestated BMP-2 bioactive molecules could be slowly released from the treated coating, which also shared a higher stability. Biological evaluation results suggested that the released BMP-2 molecules do not loss any activity. Compared with RGD functionalized COL/HA PEM, this bioactive coating significantly improved the differentiation and function expression of preosteoblasts. Similar to the unique structure and function of ECM, that coating serves not only as a passive scaffold for preosteoblasts, but RGD sequence and several growth factors slowly released from this coating can interact with presosteoblasts via integrins or other specific cell surface receptors, thus directly and indirectly promoting growth, migration and differentiation of these cells.Moreover, bFGF can also be incorporated into the RGD functionalized COL/HA PEM successfully with the same strategy. However, recently reseach has verified that bFGF can not only stimulate vascularization, but also promote osteogenesis. The constructed coating also shares several advantages such as stability and drug delivery. This bioactive coating also shares some character of biomimetics, and significantly increased the differentiation and calcification of preosteoblasts than RGD functionalized COL/HA PEM.Finally, we try to introduce both BMP-2 and bFGF to the coating at the same time, and in vitro degradation test also indicated that BMP-2 and bFGF could be slowly released from the coating respectively, without any intervention. Compared with all the coatings mentioned above, this special coating which incorporated with both BMP-2 and bFGF could significantly promote cell attachment, cell proliferation and cell differentiation, however, there exists conspicuous synergistic effect between BMP-2 and bFGF. This newly biomimetic ECM bioactive coating on dental surface, which was modified with organic macromolecules, could encourage attachment, proliferation and differentiation of MC3T3-E1 cells, and might facilitate early bone integration to dental implant surface.Obviously, from this investigation, we can get a conclution that biomimetic ECM bioactive coating could be constructed on dental implant surface successfully by LBL technique, and this coating remarkably enhance preosteoblasts attachment, proliferation and differentiation, and might facilitate better integration with bone.
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