基于纤维膜/细胞层构建梯度修复材料及其调控骨-软骨再生机制研究

Osteochondral defect is a common disease with high incidence rate，while its repairing remains a big challenge in clinical therapy. Current strategies applied in clinical therapy are mainly focusing on alleviating the patients’ pain but not curing the disease. Osteochondral defects cannot be adequately self-repaired due to the presence of the sophisticated hierarchical structure and the lack of blood supply in cartilage. Therefore, osteochondral tissue engineering is envisioned promising in treating this disease, however, there is still a long way to go. The main obstacle comes from the understanding and the reconstruction of the bone-cartilage interface, although knowledge about cartilage and bone tissues has been established after so many years of investigation.To this end, the present project intends to carry out studies on how to induce the formation of bone-cartilage interface, i.e. calcified cartilage, and how to construct osteochondral repairing implant with biomimetic gradient design. The proposed strategy in this project is basing on electrospun fibrous mesh and cell sheet technology. Briefly, bone marrow mesenchymal stromal cells (BMSCs) are cultured on electrospun fibrous meshes to form cell sheets, The BMSCs sheets are induced into chondrocyte, hypertrophic chondrocyte or osteoblast-like cells by controlling the features of fibrous meshes such as composition and fiber arrangement, etc. To investigate the formation of calcified cartilage, several strategies are proposed to try by using the mixtures of chondrogenic and osteogenic differentiation media, decellularized matrix or BMSC-affinity peptide modification. Finally, the osteochondral repairing implant is built by paper-stacking different fibrous mesh/BMSC sheet complexes to mimic the natural arthrosis with gradient design. The positive hypothesis is the osteochondral defect able to be satisfactorily regenerated with the multilayered constructs. Finally, the correlation between several key factors, such as preparation of fibrous mesh, culture and differentiation of BMSCs sheet, as well as reconstruction of osteochondral defect, is expected to be clarified as a guidance for relative studies in osteochondral tissue engineering.

关节骨-软骨损伤发生率高，但其修复治疗仍是整形外科面临的一项艰巨挑战，针对目前临床修复方法还是以缓解病痛为主而非治愈的现状，组织工程是有希望实现骨-软骨缺损修复的技术。虽然在骨和软骨组织工程这两个相对独立的领域，已对各自生理结构、生物学和力学特性建立了较为清晰的认识，但在骨-软骨组织工程，其最大挑战在于对骨-软骨界面结构和两者间结合的认识，及如何诱导界面层再生。本项目拟重点探索钙化软骨中间层的形成机制，基于静电纺丝纤维膜孵育骨髓间充质干细胞层（BMSCs），依据软骨、钙化软骨、骨组织分化的个异性需求，利用纤维膜特性、成软骨/成骨诱导分化液混合、脱细胞外基质和BMSCs募集多肽等多种手段定向调控BMSCs分化，最后采用多层堆叠技术构建具有仿生结构的骨-软骨梯度修复体，达到实现骨-软骨缺损的再生修复目标，建立纤维膜材料——BMSCs细胞层——定向诱导分化——骨-软骨修复效果间的相互关联机制。

本项目是以关节骨-软骨损伤的再生修复治疗为最终目标，针对制约骨-软骨组织一体化修复最大的挑战，即对骨-软骨界面结构和两者之间结合的认识，以及如何诱导界面层的再生，重点开展了针对钙化软骨中间层形成机制的研究。采用的主要策略是基于细胞层技术和静电纺丝纤维膜技术，在纤维膜上孵育BMSCs细胞层，依据软骨层、钙化软骨层、骨层组织再生的个异性需求，利用纤维膜特性、生物活性成分添加和成软骨/成骨诱导分化液混合、脱细胞外基质和BMSCs募集多肽修饰等多种手段，来定向调控BMSCs向成软骨、成钙化软骨和成骨方向分化，最后采用多层堆叠技术构建具有梯度结构的仿生三维骨-软骨修复材料，完成了加速骨-软骨缺损再生修复的研究目标，并形成了对材料—细胞层定向诱导分化—骨-软骨修复效果之间相互关联机制的系统认识。.项目开展的具体研究内容和策略包括：1）分别适用于软骨区/钙化软骨区/骨区支架的静电纺丝纤维膜制备研究与体外表征；2）纤维膜的梯度矿化、BMSC亲和多肽E7负载和三维梯度支架的制备与体外表征；3）骨-软骨再生修复体内评价（策略1）—— 软骨诱导分化液+成骨诱导分化液混合诱导BMSC细胞层定向预分化；4）骨-软骨再生修复体内评价（策略2）—— 策略1结合纤维膜组成（促成软骨、促成骨）调控BMSC细胞层的定向预分化；5）骨-软骨再生修复体内评价（策略3）—— 由预诱导分化细胞层获得的脱细胞外基质促钙化软骨生成研究；6）骨-软骨再生修复体内评价（策略4）—— E7多肽负载的三维梯度仿生支架体内募集细胞和定向分化。