Supplementary Material for: Chondroitin Sulfate Microparticles Modulate Transforming Growth Factor-β₁-Induced Chondrogenesis of Human Mesenchymal Stem Cell Spheroids

Mesenchymal stem cells (MSCs) have been previously explored as a part of cell-based therapies for the repair of damaged cartilage. Current MSC chondrogenic differentiation strategies employ large pellets; however, we have developed a technique to form small MSC aggregates (500-1,000 cells) that can reduce transport barriers while maintaining a multicellular structure analogous to cartilaginous condensations. The objective of this study was to examine the effects of incorporating chondroitin sulfate methacrylate (CSMA) microparticles (MPs) within small MSC spheroids cultured in the presence of transforming growth factor (TGF)-β₁ on chondrogenesis. Spheroids with MPs induced earlier increases in collagen II and aggrecan gene expression (chondrogenic markers) than spheroids without MPs, although no large differences in immunostaining for these matrix molecules were observed by day 21 between these groups. Collagen I and X were also detected in the extracellular matrix (ECM) of all spheroids by immunostaining. Interestingly, histology revealed that CSMA MPs clustered together near the center of the MSC spheroids and induced circumferential alignment of cells and ECM around the material core. This study demonstrates the use of CSMA materials to further examine the effects of matrix molecules on MSC phenotype as well as potentially direct differentiation in a more spatially controlled manner that better mimics the architecture of specific musculoskeletal tissues.

间充质干细胞（Mesenchymal stem cells, MSCs）此前已被作为基于细胞疗法的组成部分，用于受损软骨的修复。当前的间充质干细胞软骨分化策略多采用大体积细胞微团；本研究开发了一种可形成小型间充质干细胞聚集体（500~1000个细胞）的技术，该技术能够在维持类似软骨原基的多细胞结构的同时，降低物质运输屏障。本研究的目的是探究在添加转化生长因子-β1（transforming growth factor, TGF-β1）的培养体系中，于小型间充质干细胞球状体内部掺入甲基丙烯酸软骨素硫酸盐（chondroitin sulfate methacrylate, CSMA）微粒（microparticles, MPs）对软骨形成的影响。相较于未掺入微粒的球状体，掺入微粒的球状体可更早上调II型胶原与聚集蛋白聚糖这两种软骨分化标志物的基因表达；尽管在培养至第21天时，两组球状体的上述基质分子免疫染色结果未出现显著差异。所有球状体的细胞外基质（extracellular matrix, ECM）中均通过免疫染色检测到I型胶原与X型胶原。值得注意的是，组织学分析显示CSMA微粒会在间充质干细胞球状体的中心区域聚集，并诱导细胞与细胞外基质围绕材料核心形成周向排列。本研究证实，利用CSMA材料可进一步探究基质分子对间充质干细胞表型的影响，同时也有望以空间调控更精准的方式引导细胞分化，更逼真地模拟特定肌肉骨骼组织的结构特征。