种植体表面带电涂层调控成骨细胞粘附、分化的信号通路研究

It has showed that surface charge of material may play a key role on the interactions between the bone tissue and implant. The mechanism is still unclear because it's very hard to exactly measure the polarity and amounts of surface charges on titanium implant in body environment. The unique method has been developed by our team to obtain electrically polarized TiO2 coatings on the surface of titanium implant. The polarity and amounts of surface charges in TiO2 coating can be measured by testing the thermally stimulated depolarization current (TSDC). After electrical polarization treatment, it was found that bonelike apatite-forming ability in SBF was significantly enhanced on the negative-charged coating while the positive-charged coating inhibited the apatite nucleation. The shape of the osteoblast and the arrangement of actin cytoskeleton were totally different between the negative-charged surface and the positive-charged surface.The adhesion and differentiation of MG63 osteoblast were significantly promoted on the negative-charged surface. According to our previous results, we proposed two hypotheses:① surface charge regulates osteoblast adhesion by the signal passway of Ca2+-Protein -Integrin-Vinculin-Rho/ROCK;② surface charge regulates the differentiation of osteoblast by the signal passway of IQGAP1/β-catenin,which is associated with cytoskeletal reorganization. The purpose of the research project is to verify the research hypotheses mentioned above and to further investigate the biological effects of surface charge of TiO2 coating on bone formation,which is very helpful for understanding the interactions between the bone tissue and implant materials.The research protocol will be composed of three parts:① the mechanism study of electrical polarization will be done using surface analysis and the TSDC; ② the mechanism study of surface charge on adhesion and differentiation of osteoblast will be investigated by a series of experiments of cytobiology and molecular biology;③ the animal experiments will be done to further test and verify the research hypotheses in vivo.

我们的研究发现负电TiO2涂层促进了成骨细胞的有效粘附和分化，而正电TiO2涂层起抑制作用，但其分子机制仍不明确。根据前期的研究结果，本课题提出了材料表面电位的识别、导入细胞、调控胞内信号分子变化和调节效应蛋白表达的可能机制：一、表面电位通过钙离子-吸附蛋白-Integrin-Vinculin-ROCK通路影响成骨细胞的有效粘附并诱发细胞骨架的重组；二、细胞骨架的重组通过IQGAP1的表达影响Cadherin/catenin复合物的连接，来改变胞浆内游离β-catenin的浓度并调控β-catenin的核转移，从而激活或抑制β-catenin介导的转录，来调控成骨细胞的分化。本课题拟采用细胞和分子生物学方法进行系统研究，证实该假设，并用体内试验加以验证，从而阐明表面电位影响成骨细胞粘附、分化的具体分子机制，更好的理解细胞与材料间的相互作用，这对骨植入材料表面的优化设计具有重要的指导意义。

本研究发现通过电极化处理可使钛表面TiO2涂层分别带负电或正电，负电表面显著改善了涂层的亲水性，促进了类骨磷灰石的沉积，促进了成骨细胞的有效粘附和分化，而正电TiO2涂层起抑制作用。在获得国科金的资助后，主要对以下几方面内容进行了研究： 研究电极化工艺参数对极化后的TiO2涂层带电量的影响；表面电位是通过什么信号通路影Integrin和actin的表达和排列；电位变化是通过何种机制影响细胞分化的。重要结果和关键数据如下：最佳电极化参数为5000v，300℃；负电表面通过吸附Ca2+，促进了类骨磷灰石的沉积，同时促进了laminin和fibronectin粘附，并通过细胞膜表面的Integrinα1和β3受体结合，促进成骨细胞的粘附和Actin的重新排列；Actin的重排改变了胞浆内游离β-catenin的浓度并调控β-catenin的核转移，通过调控β-catenin介导的转录调控成骨细胞的分化。动物实验也证实负电表面显著促进了钛种植体周围的新骨生成。本课题结果的意义在于揭示了生物材料在于机体的相互作用中，不仅材料的表面形貌、化学组成、亲水性、表面粗糙度等会起到调控作用，而且材料表面的带电特性也起到了重要的调控作用，而且这种调控一定是多因素协同作用的。这对理解细胞与生物材料之间的相互作用，对今后骨植入材料表面的优化设计具有重要的指导意义。本研究虽然揭示了表面电位对成骨的初步调控机制，但其他的协同调控机制仍需进一步的深入研究。