Rho GTPases对骨诱导材料介导破骨细胞分化成熟的作用和机制研究

The study on the mechanism of osteoinductive materials is very important for the optimization of materials, which is the basis for its wide application. Previous studies demonstrated the osteoinductive materials can promote the osteoclastogenic differentiation of monocyte in vivo, and the microstructures on the materials believed to be the most important contributing factor, by affecting the arrangement of cytoskeleton and focal adhesions, finally determine the differentiation of cells. However, as one of the most important factors in cytoskeleton dynamics, whether the osteoclastogenic differentiation of monocyte on the microstructures controlled by Rho GTPases has not been clarified. In this study, two types of tricalcium phosphate ceramic disc with submicron or micron topography would be used, the murine RAW264.7 macrophages would be cultured on the surface of each discs for up to 5 d in the presence of osteoclast differentiation factor RANKL. TRAP staining, and Fluorescent staining for F-actin, vinculin and Hoechst would be used to evaluate the osteoclast differentiation of cells, meanwhile the qRT- PCR would be used to evaluate the markers for osteoclastogenesis, after 21 day of cell culture the resorption lacuna formation would be evaluate by SEM. Finally, the Rac1 and Rac1 inhibitors would be used to clarify whether the osteoinductive materials induced osteoclastogenesis could be regulated by Rho GTPases. This study would help us clarify how the osteoinductive material affect the osteoclast differentiation and further elucidate the biological mechanisms of osteoinduction, which provide the basis for further optimization the osteoinductive materials.

骨诱导性生物材料在骨缺损修复中具有切实可行的应用前景，对材料的优化及相关机制研究是其广泛应用的基础。前期研究表明骨诱导性材料植入体内后可促进破骨细胞分化；而材料的微结构可能在此过程中通过对细胞粘附及细胞骨架的调节而影响破骨细胞的分化成熟；Rho GTPases作为细胞骨架的关键调控因子，其是否在此过程中发挥作用尚不清楚。本研究通过对反应条件的控制形成具有骨诱导性的磷酸三钙陶瓷材料，与小鼠前破骨细胞株RAW264.7复合培养，利用TRAP染色、细胞骨架蛋白和粘附相关蛋白的荧光染色、吸收陷窝的扫描电镜分析以及qRT-PCR系统地评价破骨细胞在骨诱导材料中分化成熟的动态变化；通过引入Rac1及其抑制剂探索骨诱导材料对破骨细胞分化成熟的作用是否受到Rho GTPases的调控，该研究有助于完善骨诱导相关生物学机制，为进一步优化材料奠定基础。

骨诱导性生物材料在骨缺损修复中具有切实可行的应用前景，对材料的优化及相关机制研究是其广泛应用的基础。前期研究表明骨诱导性材料植入体内后周围可见破骨细胞形成，然而破骨细胞在其中的具体作用及机制却不清楚。材料植入体内后，各种细胞间的信息交流至关重要，前期研究发现，破骨细胞可通过分泌细胞外因子或直接接触促进成骨细胞的分化，其中外泌体在细胞间通讯中可能具有关键作用，然而目前关于破骨细胞来源的外泌体在骨诱导性材料诱导成骨中作用尚不清楚。因此，本研究旨在探究骨诱导性材料表面对破骨细胞分化的影响和在材料刺激下破骨细胞来源外泌体对干细胞分化的影响。结合前期研究结果，通过对反应条件的控制制备了具有骨诱导性的磷酸三钙陶瓷材料，并通过小鼠皮下异位成骨模型证实了材料的骨诱导性。体外实验中将小鼠前破骨细胞株RAW264.7分别在材料和孔板表面培养，利用TRAP染色、TRAP活性定量、细胞骨架蛋白的荧光染色、扫描电镜系统评价了破骨细胞的分化，发现材料表面破骨细胞大小和核的数量虽然较少，TRAP活性却更高；随后获得破骨细胞的条件培养基后提取外泌体，并通过western blot，透射电镜和动态光散射法进行鉴定后将外泌体分别与干细胞共培养，通过荧光染色发现外泌体可以被干细胞吞噬，证实了破骨细胞来源外泌体是破骨细胞-干细胞间信息交换的媒介之一；接着，我们通过碱性磷酸酶定量，ELISA和RT-qPCR对不同来源外泌体处理过的骨髓间充质干细胞进行了成骨向分化的检测，发现相比与孔板表面培养的破骨细胞来源的外泌体，材料刺激下破骨细胞来源的外泌体可促进干细胞的成骨向分化。因此本研究进一步证明了破骨细胞在骨诱导性材料促进成骨中的关键作用，并首次探索了材料刺激下破骨细胞来源外泌体的潜在作用，进一步完善了骨诱导相关生物学机制，为骨诱导性材料的进一步优化和临床转化提供了理论基础。