The limited ability of articular cartilage to self-repair has motivated the development of tissue engineering strategies that aim to harness the regenerative potential of mesenchymal stem/marrow stromal cells (MSCs). Understanding how environmental factors regulate the phenotype of MSCs will be central to unlocking their regenerative potential. The biophysical environment is known to regulate the phenotype of stem cells, with factors such as substrate stiffness and externally applied mechanical loads known to regulate chondrogenesis of MSCs. In particular, hydrostatic pressure (HP) has been shown to play a key role in the development and maintenance of articular cartilage. Using a collagen-alginate interpenetrating network (IPN) hydrogel as a model system to tune matrix stiffness, this study sought to investigate how HP and substrate stiffness interact to regulate chondrogenesis of MSCs. If applied during early chondrogenesis in soft IPN hydrogels, HP was found to downregulate the expression of ACAN, COL2, CDH2 and COLX, but to increase the expression of the osteogenic factors RUNX2 and COL1. This correlated with a reduction in SMAD 2/3, HDAC4 nuclear localization and the expression of NCAD. It was also associated with a reduction in cell volume, an increase in the average distance between MSCs in the hydrogels and a decrease in their tendency to form aggregates. In contrast, the delayed application of HP to MSCs grown in soft hydrogels was associated with increased cellular volume and aggregation and the maintenance of a chondrogenic phenotype. Together these findings demonstrate how tailoring the stiffness and the timing of HP exposure can be leveraged to regulate chondrogenesis of MSCs and opens alternative avenues for developmentally inspired strategies for cartilage tissue regeneration.

中文翻译：

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静水压力调节骨髓源性基质细胞的体积、聚集和软骨分化

关节软骨自我修复能力有限，促使组织工程策略的发展，旨在利用间充质干/骨髓基质细胞 (MSC) 的再生潜力。了解环境因素如何调节 MSCs 的表型将是释放其再生潜力的核心。已知生物物理环境可调节干细胞的表型，已知基质硬度和外部施加的机械载荷等因素可调节 MSC 的软骨形成。特别是，静水压力 (HP) 已被证明在关节软骨的发育和维持中起着关键作用。使用胶原-藻酸盐互穿网络 (IPN) 水凝胶作为模型系统来调整基质刚度，本研究旨在研究 HP 和基质硬度如何相互作用以调节 MSCs 的软骨形成。如果在软 IPN 水凝胶的早期软骨形成过程中应用，发现 HP 会下调 ACAN、COL2、CDH2 和 COLX 的表达，但会增加成骨因子 RUNX2 和 COL1 的表达。这与 SMAD 2/3、HDAC4 核定位和 NCAD 表达的减少相关。它还与细胞体积减少、水凝胶中 MSC 之间的平均距离增加以及它们形成聚集体的趋势降低有关。相比之下，HP 延迟应用于软水凝胶中生长的 MSCs 与细胞体积和聚集的增加以及软骨形成表型的维持有关。