肌腱干细胞脱细胞基质诱导BMSCs归巢及腱向分化的实验研究

Our previous studies have demonstrated that decellularized tendon extracellular matrix (ECM) is a promising substrate to confer bioactivity and direct stem cell fate through the presentation of a native and biophysical and biochemical relevant microenvironment cues. Further experiments showed that the decellularized matrix components of tendon-derived stem cells(TDSCs) in the decellularized tendon ECM microenvironment contained the growth factors involved in the regulation of stem cell homing and the critical proteoglycans of directing stem cell tenogenic differentation fate. However, it remains an unanswered question whether the decellularized matrices of TDSCs possess the capacity to induce the homing and tenogenic differentiation of stem cell. It is hypothesized that TDSCs in the decellularized tendon ECM microenvironment can secrete pontent bioactive molecules of directing stem-cell fate, which can endow the novel bioscaffold with the inductive capacity and induce the homing and tenogenic differentiation of BMSCs. In this project, a new idea that using the decellularized matrix of TDSCs and decellularized bovine tendon sheet (DBTS) substrate to develop a novel bioscaffold with inductive capacity is raised to testify our hypothesis. Then, the inductive capacity of the novel bioscaffold endowed by the decellularized matrix of TDSCs in vitro and in vivo are determined from gene and protein expression level based on bone-marrow mesenchymal stem cells (BMSCs) model. This project aims to develop a novel bioscaffold with inductive capacity, and provide a new idea for the design of tendon tissue engineering scaffolds.

我们前期研究证明：脱细胞肌腱胞外基质(ECM)可通过其天然的生理及生化微环境决定干细胞分化命运。进一步实验发现肌腱干细胞(TDSCs)在脱细胞肌腱ECM微环境中分泌的基质组分中含有可调控干细胞归巢的生长因子及决定腱向分化的重要蛋白聚糖等，但TDSCs的脱细胞基质能否诱导干细胞归巢及腱向分化尚不清楚。因此我们推测TDSCs在脱细胞肌腱ECM微环境中可以分泌调控干细胞命运的生物活性因子并发挥功能，利用TDSCs脱细胞基质赋予支架材料诱导性功能，可诱导BMSCs归巢及腱向分化。本项目拟利用TDSCs脱细胞基质复合脱细胞小牛肌腱压片(DBTS)模板构建新型生物支架，以BMSCs为细胞模型，通过体外、体内实验，从基因和蛋白水平上研究证实TDSCs的脱细胞基质赋予新型生物支架可诱导BMSCs归巢及腱向分化的能力，旨在探索一种具有组织诱导性的新型生物支架材料，为肌腱组织工程的支架材料设计提供一种新思路。

由于各种创伤或疾病导致的肌腱损伤是一种常见的临床问题，因其修复困难长期以来一直威胁着人类健康并明显降低了病人的生活质量。组织工程肌腱的发展曾为解决这一难题带来希望，然而利用传统的组织工程方法构建组织工程化肌腱因其技术“瓶颈”难以用于损伤肌腱的修复与再生。研究组织诱导性生物材料为组织工程肌腱的发展开辟了崭新的思路。在前期研究中，我们成功地研制了脱细胞肌腱切片(DTSs)支架，并证明脱细胞肌腱胞外基质(ECM)可通过其天然的生理及生化微环境决定干细胞分化命运。在此基础上，本项目利用肌腱组织特异性干细胞-TDSCs脱细胞基质(TECM)复合DTSs模板构建新型生物支架，并证实TDSCs在DTSs模板提供的脱细胞肌腱ECM微环境中能够分泌调控干细胞命运的生物活性因子，包括调控干细胞归巢的趋化因子(SDF-1/MCP-1)和生长因子(TGF-β/IGF-1/ VEGF)以及决定腱向分化的重要蛋白聚糖(Bgn/Fmod)，在体外具有诱导BMSCs增殖、迁移及腱向分化的能力，且发现利用TDSCs脱细胞基质(TECM)构建的新型生物支架对BMSCs迁移能力的提升可能依赖于SDF-1/CXCR4/PI3K/AKT信号通路的激活。该项目为肌腱组织工程的支架材料设计提供一种新策略，对于进一步研究具有组织诱导性的新型生物支架材料具有重要意义。