“区域”干细胞定向诱导活性去细胞骨腱界面书页支架制备及其再生效应研究

Bone-tendon interface (BTI) plays a vital role in transmitting and buffering stress during exercise, and characteristic bone-fibrocartilage-tendon structure is difficult to regenerate after BTI injury. Consequently, the regenerated BTI cannot effectively disperse stress, and is prone to re-injury. In the previous study, the applicant constructed a book-shaped acellular fibrocartilage scaffold with biomimetic property in morphological structure, which was determined to be able to promote the regeneration of BTI, but it lacks anti-stretching properties and region-dependent differentiation inducibility for stem cell, thus cannot promote the regeneration of bone-fibrocartilage-tendon structure effectively and simultaneously. To solve these problems, this project intends to prepare a book-shaped acellular bone-tendon scaffold (BABS) with morphological structure bionic, anti-stretching property and region-dependent differentiation inducibility for stem cell. Firstly, the BTI tissue was cut into book shape from the tendon end to bony end and parallel to the myotility direction, and then decellularized to acquire BABS. After that, collagen affinity peptide-BMP-2 (CAP-BMP-2), collagen affinity peptide-TGF-β3 (CAP-TGF-β3) and collagen affinity peptide-GDF-7 (CAP-GDF-7) were innovatively synthesized to respectively bind to the bone, fibrocartilage, and tendon regions of the BABS, so as to make it have the region-dependent differentiation inducibility for stem cell and sustained growth factor releasing ability. In-vitro studies are designed to determine its region-dependent differentiation inducibility for stem cell and sustained growth factor releasing ability. Meanwhile, the molecular mechanisms of stem cell differentiation will also be investigated. In addition, a new graft will be constructed by sandwiching BMSCs-sheets into the book page of this novel scaffold, and the regeneration effect of the graft was evaluated in-vivo. This project may provide a new treatment strategy for the regeneration of BTI injuries.

骨腱界面在运动中发挥着传递和缓冲应力的作用，其损伤后特征性骨-软骨-肌腱结构再生困难，因而不能有效分散应力，容易再撕裂。申请人前期构建了形态结构仿生的“书页状”去细胞纤维软骨支架。该支架虽有骨腱再生效应，但缺乏抗牵拉特性及“区域”干细胞诱导活性，无法同时再生骨-软骨-肌腱结构。为此，本项目拟沿着应力方向切割骨腱界面组织，脱细胞制备具有形态结构仿生及抗牵拉特性的去细胞骨腱界面书页支架。创新合成胶原结合特性的BMP-2、TGF-β3和GDF-7，分别靶向结合支架的骨、纤维软骨、肌腱区域，使其具有“区域”干细胞定向诱导活性及生长因子缓释能力，从而制备具有形态结构仿生、抗牵拉力、“区域”干细胞定向诱导活性的去细胞骨腱界面书页支架。体外研究明确该支架的区域干细胞定向诱导活性及作用分子机制。将BMSCs片包夹于支架书页间隙构建新型移植物，体内评估其骨腱再生效应。本项目实施将为骨腱再生提供新的治疗策略。

骨腱界面是机体肌腱/韧带与骨相连接移行的部位，发挥着传导和缓冲从肌腱/韧带到骨的机械应力的作用。典型的骨腱界面由肌腱/韧带、未钙化的纤维软骨层、钙化的纤维软骨层和骨四层梯度渐变的组织构成。在机体运动过程中，骨腱界面处于机械应力集中部位，因此容易遭受损伤。骨腱界面损伤修复发生在机体最软的组织（肌腱/韧带）和最硬的组织（骨）之间；同时损伤过程中或者手术治疗后骨腱界面结合部位纤维软骨层结构常常被破坏，由于该部位的血供差、再生能力弱，使得骨腱界面损伤修复过程十分缓慢而困难。如何有效促进梯度渐变的骨腱界面组织再生，从而加速骨腱界面损伤后的快速而优质的愈合，成为了运动医学研究领域极富挑战性的难题。.当今组织工程技术的发展为骨腱界面再生提供了新的思路。为此，本项目采用组织工程研究思路，进行了如下工作：1）采用犬冈下肌肌腱止点骨腱界组织，部分脱钙后，最大化保留抗牵拉力学性能的情况下切割为“书形”，创新搭建负压吸引装置，优化去细胞方案，制备出保留胶原、蛋白聚糖、抗牵拉力学性能的高仿生骨腱界面支架；2）合成具备胶原结合特性的BMP-2、胶原结合特性的TGF-β3 、胶原结合特性的GDF-7，区域化粘附于高仿生骨腱界面支架的骨、纤维软骨、肌腱区域，强化支架的“区域化”成骨、成软骨、成肌腱诱导效应；3）提取犬尿源干细胞，制备细胞片，包夹于诱导效应强化的高仿生骨腱界面支架构建新型组织工程骨腱界面移植物，在犬冈下肌肌腱止点损伤模型证实该组织工程移植物可有效地促进骨腱界面再生，进而加速骨腱界面损伤后生物力学性能恢复，为制定骨腱界面损伤修复新治疗策略提供科学依据。.项目实施以来，以第一作者或通讯作者（含共同）发表SCI论文9篇，以第一申请人授权国家发明专利3项。论文发表于Biomaterials、Bioactive Materials、eLife、Am J Sports Med、J Orthop Res、Stem Cells Int、Ann Transl Med杂志。已经授权与本项目直接相关国家发明专利4项（第一申请人3项）。联合培养博士生2名（已毕业），硕士生1名（在读）。