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

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。

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.