BMSC在不同拓扑结构纳米纤维支架中向纤维环细胞定向分化的差异及机制研究

The high recurrence rate of lumbar disc herniation after discectomy is often linked to defects of annulus fibrosus (AF) and its limited repairing potential. It is proposed that tissue-engineering strategy can effectively deal with the damaged AF. As the AF has a complex structure and different cell phenotype, a novel aligned nanoyarn scaffold (AYS) and three-dimensional porous nanofiber scaffold(3-DPS), which mimics the structure of outer and inner AF extracellular matrix individually, have been fabricated. Bone marrow mesenchymal stem cells (BMSC) were seeded onto AYS to differentiated into outer AF cells while differentiated into inner AF cells on 3-DPS, confirmed by the expression of characteristic phenotype markers. The expression pattern of integrin and activation of FAK were also founded different among AYS and 3-DPS. Integrin-FAK signaling participates in cell-scaffold interaction and differentiation process of BMSC. This study aims to investigate the mechanism of differentiation diversity of BMSC inside AYS and 3-DPS through the intervention of integrin-FAK pathway and key transcriptional factor and observe the effect of integrin-FAK pathway regulation in the directed differentiation of BMSC towards AF cells. This study will possibly promote the development of tissue engineering AF research and further repair of AF defects.

腰椎间盘突出术后复发与纤维环缺损无法自行有效修复相关，组织工程纤维环是解决这一难题的重要一步。基于纤维环结构内外层表型差异、不同力学特性，申请者制备了两种不同拓扑结构的取向纳米纱（AYS）和三维多孔纳米纤维支架(3-DPS)，分别模拟外层和内层纤维环。骨髓间充质干细胞(BMSC)在两种支架中表型表达呈现明显差异，在AYS中向外层纤维环分化，在3-DPS中向软骨细胞分化，伴整合素（Integrin）差异表达和黏着斑蛋白激酶（FAK）激活。提示Integrin-FAK通路激活可能是不同支架上BMSC差异分化的原因，将两者复合，可形成仿生化组织工程纤维环。本项目拟通过对不同支架上BMSC分化过程中Integrin、FAK及下游分子表达水平进行检测，验证其与BMSC分化的关系，并通过干预Integrin-FAK及下游分子，观察对BMSC差异分化的影响，阐明其调控机制，最终实现纤维环有效修复。

椎间盘髓核摘除术后出现再突出与纤维环缺损无法生理修复相关。纤维环内部结构复杂，胶原纤维排列高度取向、互相交织，以此缓冲和负载多向机械负荷。在前期研究中，本课题组通过静电纺丝技术构建纳米纤维支架，可以模拟纤维环胶原纤维的机械性能。但这类支架纤维排列无序，并不能模拟纤维环取向排列特点。并且，支架纤维的无序排列无法诱导支架内种子细胞向纤维环细胞定向分化。这些局限性影响了纳米纤维支架在纤维环生理修复中的应用。.为了解决这些问题，本课题组成功构建了两种不同拓扑结构的取向纳米纱（ANS）和三维多孔纳米纤维支架（3-DPS），分别模拟纤维环的外层与内层；然后向支架中接种骨髓间充质干细胞（BMSC），论证纳米纤维支架的拓扑结构（ANS结构与3-DPS）对BMSC向纤维环外层与内层细胞进行定向诱导分化，并通过高通量测序的方法探究不同拓扑结构纳米支架诱导BMSC定向分化的分子机制，探讨转录因子Mkx与蛋白Integrin参与不同拓扑支架内BMSC向纤维环外层与内层分化的调控机制；最后体内评估ANS/3-DPS/BMSC细胞支架复合体在体内修复纤维环的效应。.结果表明：（1）所构建的纳米纤维支架可为骨髓间充质干细胞提供适宜的生长环境，高通量测序结果提示干细胞接种于支架后，与对照组相比有较多差异表达基因，提示支架对干细胞的生物学行为产生了影响，使其在转录层面即出现差异；（3）ANS取向纳米纤维支架可促进骨髓间充质干细胞向外层纤维环细胞分化，Mkx是参与这一过程的调节因子；（3）3-DPS三维多孔纳米纤维支架可促进骨髓间充质干细胞向内层纤维环细胞分化，Integrin β1参与这一过程的调节；（4）取向纳米纤维支架-细胞复合体可有效修复兔腰椎外层纤维环缺损。因此，本项目研究结论为纤维环仿生支架的构建、组织工程学诱导BMSC定向分化与纤维环生理修复提供一定的理论与应用证据，为后续纤维环组织工程支架的临床转化应用奠定基础。