微结构一致而基质刚度不同骨组织工程支架的构建及其促骨修复机制

To improve the repair and reconstruction quality of massive bone defect is a research hotspot and difficulty in the field of bone repair. Matrix stiffness plays a vital role in regulation of biological behavior of stem cells and the effects have obvious difference between two-dimensional (2D) and three-dimensional (3D) conditions. 3D acellular bone scaffold has been successfully obtained by our group. This proposal intends to construct 3D scaffolds with different stiffness but same microstructure by differentially demineralizing acellular bone scaffolds, in which the protein active factors will be inactivated by guanidine hydrochloride. Rat bone marrow mesenchymal stem cells (MSCs) will be used as the seeding cells. The effects of 3D matrix stiffness on MSCs adhesion, proliferation, osteogenic differentiation, stem cell recruitment and micro-angiogenesis will be investigated in cell experiments and rat subcutaneous implantation tests. Further, a rabbit femur defect model will be established to evaluate the constructed 3D scaffolds in vivo. The matrix stiffness that can promote the repair of bone defect will be expected to be obtained in this proposal by aforementioned experiments. The influence of stromal cell-derived factor-1α (SDF-1α) on this process will also be studied. The mechanobiology mechanism of 3D matrix stiffness on the biological behavior of MSCs will be further explored. An in-depth understanding of the difference between 2D and 3D culture conditions will also be gotten. The accomplishment of this proposed research project might present new ideas for seeking efficient methods for bone repair and enriching research methods of matrix mechanics.

提高大块骨修复重建质量是骨修复领域的研究热点和难点，基质刚度对干细胞生物学行为有重要的调控作用，且三维与二维培养有明显差异。申请人所在课题组前期已成功获得脱细胞骨，本项目拟对脱细胞骨进行不同程度脱钙处理，通过盐酸胍灭活消除支架内蛋白活性因子的影响，构建一种新型微结构一致而基质刚度不同的三维支架，以大鼠骨髓间充质干细胞(MSCs)为种子细胞，通过细胞实验和大鼠皮下植入实验考察三维基质刚度对MSCs粘附、增殖、成骨分化以及干细胞招募和微血管生成的作用；进一步利用兔股骨缺损模型在体评价所构建三维支架，以期获得促进骨修复的基质刚度，研究基质细胞衍生因子-1α (SDF-1α)对三维基质刚度调控作用的影响，探索三维基质刚度调控MSCs生物学行为的力学生物学机制，深入理解其与二维培养的差异。本项目的完成可为探寻高效骨修复方法和丰富基质力学研究方法提供新思路。

提高大块骨修复重建质量是骨修复领域的研究热点和难点，基质刚度对干细胞生物学行为有重要的调控作用，且三维(three-dimensional, 3D)与二维(two-dimensional, 2D)培养有明显差异，然而现有的关于基质力学在支架材料构建方面的研究主要集中在2D或者准3D条件下，而且大部分3D支架的制备方法在改变基质刚度的同时也改变了其3D微结构，难以考察基质力学单一因素对干细胞生物学行为的影响。本项目通过对3D脱细胞骨支架进行不同时长脱钙，构建了一种微结构一致而基质刚度不同的新型3D脱钙骨支架材料。离体考察了不同基质刚度脱钙骨支架对骨髓间充质干细胞(mesenchymal stem cells, MSCs) 粘附、增殖、成骨分化以及干细胞招募和微血管生成的影响；通过皮下埋植实验考察了不同基质刚度脱钙骨支架对细胞浸润、胶原纤维沉积以及内源性细胞成骨分化的作用；进一步利用兔股骨缺损模型在体评价所构建三维支架。研究证实所构建支架微结构一致，弹性模量分别为66.06±27.83 MPa (高刚度组)、26.90±13.16 MPa (中刚度组)和0.67±0.14 MPa (低刚度组)，具有良好的孔径尺寸和孔隙率，并且保留了天然骨的细胞外基质(extracellular matrix, ECM)成分。不同基质刚度支架均有利于MSCs的粘附、生长和成骨分化，低刚度组支架在皮下有利于细胞浸润、胶原纤维沉积以及内源性细胞成骨分化，可有效促进兔股骨髁骨缺损修复。本项目的完成可为探寻高效骨修复方法和丰富基质力学研究方法提供新思路，有助于进一步理解3D基质刚度在骨修复中的作用及其机制。