组合干细胞ECM协同构建新型支架募集内源性干细胞促进肌腱再生的研究

Bone-marrow mesenchymal stem cells(BMSCs) were demonstrated to secrete some chemokines, which can regulate the recruitment of stem cells. Our previous studies have demonstrated that the tendon-derived stem cells(TDSCs) in the decellularized tendon slices (DTSs) scaffold can secrete potent bioactive factors for directing tenogenic differentiation fate of the stem cells. However, whether the combined stem cells (including BMSCs and TDSCs) in the DTSs scaffold act synergistically to promote the secretion of bioactive molecules of regulating the stem cell recruitment and tenogenic differentiation. Hence, it is hypothesized that the synergistic effect of extracellular matrix (ECM) of the combined stem cells (BMSCs/TDSCs) can endow the novel scaffold with the bio-inductive capacity and achieve tendon regeneration through the induction of the recruitment and tenogenic differentiation of the endogenous stem cells. In this project, a new idea that using the combined stem cells (BMSCs/TDSCs) ECM modified the DTSs scaffold to develop a novel scaffold with bio-inductive capacity is raised to testify our hypothesis. The novel scaffold will be developed and characterized in vitro, and then the bio-inductive capacity of novel scaffold that inducing the recruitment and tenogenic differentiation of endogenous stem cells will be tested by the Achilles tendon defect in a rat model. This project aims to provide a new idea for the scaffolds design for tendon regeneration.

有研究证实：骨髓间充质干细胞(BMSCs)可分泌调控干细胞募集的趋化因子。我们的前期研究发现：肌腱干细胞(TDSCs)在脱细胞肌腱片(DTSs)支架中可分泌促进干细胞腱向分化的生物活性因子。然而，组合干细胞(BMSCs/TDSCs)在DTSs支架中能否协同促进分泌调控干细胞募集及腱向分化的生物活性因子并发挥功能尚不清楚。因此，我们推测利用组合干细胞(BMSCs/TDSCs)ECM的协同效应赋予新型支架生物诱导活性，可通过诱导内源性干细胞募集和腱向分化实现肌腱再生。本项目拟利用组合干细胞(BMSCs/TDSCs)ECM修饰DTSs支架协同构建新型支架，通过跟腱缺损模型证明该新型支架可通过诱导内源性干细胞募集和腱向分化实现肌腱再生，为探索具有肌腱再生功能的支架材料设计提供崭新的思路。

干细胞胞外基质修饰的支架具有独特的生物功能，为研制组织诱导性生物支架提供了新的策略。已有研究报道骨髓间充质干细胞（BMSCs）可分泌调控干细胞募集的趋化因子。我们前期研究发现：肌腱干细胞（TDSCs）可分泌促进干细胞腱向分化的活性因子。为此，本研究探索组合干细胞（BMSCs/TDSCs）在脱细胞支架中能否协同促进分泌调控干细胞募集和腱向分化的生物活性因子并发挥生物功能。本项目利用组合干细胞（BMSCs/TDSCs）在脱细胞肌腱支架上进行复合培养，待细胞汇合成致密细胞片层后行脱细胞处理，成功制备了覆盖组合干细胞胞外基质（ECM）修饰的新型支架。对该支架的形态学和生化组分分析证实，组合干细胞ECM修饰构建的新型支架表面覆盖了干细胞ECM，其组分中含有更多纤调蛋白聚糖（FMOD）等生化因子，但类胰岛素生长因子-1（IGF-1）和基质细胞衍生因子-1的含量与单纯TDSCs的ECM修饰的支架并无明显差异。更多的生化因子谱系的表征有待蛋白组学的结果进行揭示。将TDSCs的ECM修饰脱细胞脱钙骨基质支架构建新型支架，支架中含有生物活性因子（SDF-1、BGN、FMOD等）比修饰前高1倍以上；更重要的是，将TDSCs的ECM修饰脱细胞肌腱支架，发现修饰后TGF-beta1、VEGF、SDF-1比修饰前高3倍以上。体外实验证实干细胞ECM修饰后的新型支架具有促进BMSCs增殖、迁移和腱向分化的能力；将上述新型支架植入体内修复跟腱缺损和肩袖肌腱撕裂，证明该支架具有募集内源性干细胞促进肌腱再生和腱骨界面愈合的功能。本项目研究结果证明了采用组合干细胞ECM修饰的支架可能赋予新型支架更多生物功能，为进一步探索肌腱缺损修复的支架设计和肌腱再生机制提供了重要的实验依据。