基于力学刺激调节的细胞片层培养与叠层组装构建的纤维环组织再生

Replacement of diseased disc using biomimetic tissue prepared through tissue engineering approach is promising for treating intervertebral disc (IVD) degeneration. However, such an approach is largely confined due to the inferior mechanical properties of engineered annulus fibrosus (AF) tissue, which is caused by a combination of problems including homogeneity of cell type, deficient production of matrix, and insufficient inter-layer adhesion. Previously, we have identified AF-derived stem cells (AFSCs) whose differentiation is regulated by the stiffness of substrate. We have also prepared intact cell sheets, in which the cells maintained high activity, using temperature-sensitive hydrogels. In this project, we aim to fabricate elastic silicone substrates which have aligned topography on surface and are coated with a thin layer of temperature-sensitive hydrogel. As the elastic modulus of hydrogel varies, AFSCs may be induced to differentiate into various cell types corresponding to the cells in native AF tissue and subsequently mature into aligned cell sheets. Following this, the cells will be mechanically stretched to promote extracellular matrix (ECM) production. After the cell sheets are carefully harvested by lowering temperature, they will be assembled into an angle-ply structure to form a biomimetic AF tissue, which will further undergo mechanical stimulation in a bioreactor prior to in vivo implantation. By combining cell sheet technology and mechanical regulation methodology, this study will manage to achieve IVD repair and regeneration by simulating the microstructure, biochemical composition, and mechanical characteristics of native AF tissue. Findings from this study will likely provide a profound theoretical basis as well as bench practice toward effective construction of engineered tissues using cell sheet technology.

通过组织工程构建仿生椎间盘进行组织替换在椎间盘退变治疗中极具潜力。但目前仿生纤维环组织存在细胞类型单一、基质表达不足、缺乏层间黏合等问题，导致其力学性能不足，制约仿生椎间盘构建。我们前期已获得分化受基材弹性模量调控的纤维环源干细胞AFSC，并通过温敏性水凝胶构建出具有较高活性的细胞片层。本项目拟在弹性硅胶表面复合温敏性水凝胶薄层，借助其可形变、模量可调及表面含拓扑结构等特性，诱导AFSC分化成对应于纤维环组织不同区域细胞类型的取向细胞片层，并通过力学拉伸促进细胞基质分泌。通过降温无损获取细胞片层后，采用斜交叠层方式构建出仿生纤维环组织，进一步应力刺激下培养后移植体内。本研究通过综合细胞片层技术和力学调控手段，实现对纤维环组织在微观结构、生化组成及力学性能上的高度模拟，以有效应用于椎间盘修复与再生。本研究成果可为利用细胞片层高效构建无支架材料的仿生组织提供理论基础和实践依据。

随着现代社会老龄化加剧，退行性脊柱病变已成为全球性公共健康问题。由椎间盘退变所致住院人数占骨科总数的30%以上，其治疗具有重大经济和社会意义。通过组织工程策略构建仿生椎间盘进行组织替换，在椎间盘退变治疗中颇具潜力，但其进展取决于纤维环这一椎间盘关键结构组件的有效构建。本项目旨在通过综合细胞片层技术和多重力学调控手段，实现对纤维环组织在微观结构、生化组成及力学性能上的模拟，用于椎间盘再生修复。围绕这一目标，我们首先制备了具有不同力学特性和温度敏感性的水凝胶，在其上培养细胞并获得取向细胞片层。自主设计了力学加载细胞培养系统和大鼠尾椎动态牵引系统，在此基础上探究了力学刺激对细胞片层形成及基质代谢的影响，发现适度力学刺激促进细胞增殖和基质分泌，而适度在体力学刺激有利于退变椎间盘修复。另外，适度力学刺激可改善炎性微环境，促进纤维环细胞增殖及合成代谢相关基因的表达和蛋白分泌，这一过程与YAP/NF-κB信号通路以及Cav1/integrin β1/NF-κB信号通路相关。为探究不同因素对细胞分化的协同调控效果，我们制备了具有不同力学特性或微观结构的基材，发现纤维环源干细胞的形态和基质表达显著依赖于基材弹性模量或微观结构，其变化趋势与天然纤维环组织中相关指标的变化趋势一致，并可能与YAP/Cav1信号通路相关。最后，通过斜交层叠组装技术构建仿生纤维环，复合水凝胶人工髓核后，成功构建仿生椎间盘，体内植入实验证明其可维持椎间盘样结构和椎间隙高度，促进椎间盘再生修复。以上研究成果可望为利用细胞片层构建仿生组织以及力学调控在组织再生修复中的应用提供理论基础和实践依据。.项目执行期间，共发表标注项目批准号论文21篇，参编专著5部，获授权专利5项。项目负责人在学术会议上作相关邀请报告17次，其中国际会议7次、国内会议10次。团队成员积极参与国内外学术交流，作口头报告12次。

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