“一步法”植入功能性水凝胶支架诱导内源性干细胞募集和差异性分化在体修复半月板缺损

Till nowadays we fail to construct a bionic tissue-engineered meniscus (TEM) with structural and functional heterogeneity to effectively repair the meniscus defect, which will lead to the presence of osteoarthritis prematurely. Recruitment of seed cells, regulation of regenerated tissues, and remodeling of structure and function in the microenvironment are major obstacles to TEM construction. Previously, the applicant confirmed that Mg2 + possesses the potential to promote mesenchymal stem cell (MSCs) recruitment and meniscal repair. Scaffold pore structure, growth factor (TGFβ3 and CTGF) and mechanical microenvironment affect the phenotypic differentiation of MSCs and the function of regenerated tissues. In this study, the "one-step" implantation system was proposed, including three components: first, the high mechanical strength and high adaptability hydrogel scaffold is adapted as the carrier; secnond, the Mg2 + interface modification is applied to play a role of recruiting endogenous MSCs; third, the induction of differential differentiation of stem cells in the in-vivo micro-environment through the different scaffold pore sizes and time-area differences of releasing growth factor. The best TEM construction programs will be selected in vitro, and implanted in vivo to observe the outcome. The aim of this study is to elucidate the mechanism of Mg2 + in the recruitment of endogenous stem cells, and the effect of scaffold pore size and double stimuli of growth factors and in vivo stress in the differential differentiation of MSC, and finally to construct a heterogeneous TEM, to provide a noval approach for the repair of meniscus defects in clinic.

目前组织工程半月板（TEM）不具有结构和功能异质性，这种非仿生组织不能有效修复半月板缺损，致骨性关节炎过早发生。种子细胞的募集、再生组织的调控和微环境下结构功能的重塑，是TEM构建的主要障碍。申请人前期证实：Mg2+在促进间充质干细胞（MSCs）募集及半月板修复方面具备潜能；支架孔径结构、生长因子（TGFβ3和CTGF）和力学微环境均影响MSCs的表型分化和再生组织的功能。本研究拟构建“一步法”植入体系，以高力学强度高顺应性水凝胶支架为载体，界面修饰Mg2+发挥其募集内源性MSCs作用，再通过支架孔径及释放生长因子时间区域差异，诱导在体力学微环境下干细胞差异性分化，体外优选TEM构建方案，实验动物兔体内植入观察其转归。本研究旨在阐明Mg2+募集内源性干细胞，及支架孔径差异、生长因子-在体应力双刺激在诱导MSC差异性分化的作用和机制，并构建异质性TEM，为临床半月板缺损修复提供新方案。

目前组织工程半月板（TEM）不具有结构和功能异质性，这种非仿生组织不能有效修复半月板缺损，致骨性关节炎过早发生。种子细胞的募集、再生组织的调控和微环境下结构功能的重塑，是TEM构建的主要障碍。本研究：1 通过体外体内实验，阐明镁具有募集内源性干细胞，发挥促进半月板再生修复的作用；2 通过构建体外应力刺激模式，证明了生长因子（TGFβ3和 CTGF）和力学微环境均影响MSCs的表型分化和再生组织的功能；3 基于以上研究，我们设计了含镁的水凝胶（壳聚糖）体系，从6周体内试验结果看，含镁水凝胶植入有促进半月板再生作用，但是由于早期其炎症反应比较明显，并未能解释镁是否可以定向趋化诱导在体干细胞作用于损伤处发挥作用，与我们初始设想存在一定差距，无法达到精准调控的要求。基于此，我们进一步探索可以精准控制干细胞靶向募集的新方式，即适配体技术。其中转铁蛋白受体核酸适配体transferrin receptor （CD71）与MSC干细胞结合好，目前“一步法”适配体-水凝胶体系的体外筛选及体内实验已完成，从初步分析看对干细胞的募集及促进再生修复发挥较好的作用。目前共发表论文 5篇，包括在 SCI 收录杂志发表论文5 篇，最高影响因子17.16，目前申请发明专利1项（申请号：202110037269.7；申请公布日：2021.4.9）。通过该课题，培养1名博士研究生和1名硕士研究生，同时申请人基于此课题，申请获得国家自然科学基金优秀青年科学基金项目（82022046），相关研究内容在国际软骨修复协会ICRS2019年会发言并荣获ON基金会奖励。基于此项目，阐明镁募集内源性干细胞，及生长因子-应力双刺激在诱导MSC差异性分化的作用和机制，并构建异质性TEM，为临床半月板缺损修复提供新方案。