生物材料诱导骨生成中破骨细胞的确证及其作用机制研究

It is feasible and practical to apply biomaterials with intrinsic osteoinductivity through the optimization of their composition and structure without the addition of cells or growth factors in the repair of bone defects. At present, there are certain understandings on the physicochemical properties of osteoinductive materials, material-induced osteogenesis is considered as a process similar to intramembranous osteogenesis. However, new evidences imply that material-induced osteogenesis is closer to bone remodeling process. Based on our previous results, the standard materials with osteoinductivity or non-osteoinductivity are implanted into the osteoinductive FVB mouse model for various time points, to identify the time sequence of osteoclastogenesis and osteogenesis by using specific bio-markers. Then, agonists and antagonists of osteoclastogensis are used to further explore the roles of osteoclasts in material-induced bone formation. Moreover in-vitro experiments are applied to verify whether osteoclasts promote the migration and osteogenic differentiation of bone marrow mesenchymal stromal cells by releasing specific factors in the function of osteoinductive materials. The results obtained will be helpful in realizing the role of osteoclasts in material-driven osteoinduction. The outcomes of the current project may lead to not only a creative understanding of the material-induced bone formation and a new theory of osteoinductive materials, but also the finding of the key material properties relevant to smart materials that mimic natural bone and the technology to manufacture such materials.

通过自身组成和结构优化且不外加细胞或生长因子而具备固有骨诱导性的生物材料在骨缺损修复中具有切实可行应用前景。目前对影响材料骨诱导的理化特征和诱导成骨生物学机制有一定认识，一直认为生物材料诱导成骨过程类似于膜内成骨，但近来证据显示生物材料骨诱导过程更可能属于骨改建成骨。本项目将在前期研究基础上,通过骨诱导实验动物FVB小鼠模型，对植入不同时间点的骨诱导和非骨诱导的标准材料样品中相关特异性标志物检测，确认破骨细胞形成与骨形成的时序性；使用破骨细胞促进剂和抑制剂干预，探讨破骨细胞在材料诱导骨生成中的作用；体外实验考察骨诱导材料是否影响破骨细胞释放特定因子促进骨髓间充质干细胞的迁移和骨向分化，回应体内实验结果，探讨破骨细胞形成在材料骨诱导过程中的相关机制。本研究不仅可能引发对材料诱导成骨生物学机制的重新认识，完善材料骨诱导理论，并有望寻找到仿生天然骨的智能人工骨关键材料学特征进一步指导其优化制造。

临床中，颌面部骨缺损修复仍面临挑战。骨诱导性材料是一类可以在不外加细胞和生长因子的情况下在非骨部位诱导新骨形成的人工合成材料，由于其成骨不依赖于骨缺损中宿主骨的骨传导，为颌面部骨缺损的理想修复提供了一种可能。目前，材料诱导成骨的生物学机制仍不明确，限制了材料的优化和临床应用。以往研究认为生物材料诱导成骨过程类似于膜内成骨，但近来证据显示生物材料骨诱导过程更可能属于骨改建成骨。为明确骨诱导的生物学机制，本研究在前期研究基础上，通过一系列体内外实验，确定了破骨细胞形成和参与对材料诱导成骨的关键性作用，并确定了材料诱导成骨的生物学顺序：TCPs植入后，在数小时到数天内巨噬细胞在材料表面定植（在1 d时达到顶峰），数天内极化为M2型巨噬细胞（1周之内），随后破骨细胞（最有可能来自M2巨噬细胞）在数周内生成（主要在第1周到第2周之间），之后骨形成在数周内发生（4周之后）。随后，本项目探索了材料诱导成骨中破骨细胞促进成骨的具体机制，明确了在材料诱导成骨中，破骨细胞分泌因子具有直接的促成骨分化的作用。随后，通过BMM在材料上向破骨细胞诱导5d时TCPb和TCPs上细胞的全转录组测序，将Apoe筛选为材料诱导成骨中偶联破骨与成骨的关键分泌蛋白。本项目进一步补充和完善了骨诱导的生物学机制，为精细调控破骨细胞介导的骨再生奠定了基础。