Articular cartilage is one of the most important weight-bearing components in human body, thus the chondrogenesis of stem cells is reactive to many intracellular and extracellular mechanical signals. As a unique physical cue, matrix stiffness plays an integral role in commitment of stem cell fate. However, when examining the downstream effects of matrix stiffness, most studies used different soluble factors to assist physical inducing process, which may mask the chondrogenic effects of matrix stiffness. Here we fabricated polyacrylamide (PAAm) hydrogels with gradient stiffness to unravel the role of matrix stiffness in chondrogenic process of mesenchymal stem cells (MSCs), with or without TGF-β3 as induction factor. The results showed that with micromass culture mimicking relatively high cell density in vivo, the chondrogenic differentiation of MSCs can be promoted by soft substrates (about 0.5 kPa) independently with assembled cytoskeleton. Further analysis indicated that addition of TGF-β3 generally increased expression level of cartilage-related markers and masked the stiffness-derived expression pattern of hypertrophic markers. These results demonstrate how mechanical cues experienced in developmental context regulate commitment of stem cell fate and have significant impact on the design of tissue regeneration materials.

关节软骨是人体最重要的负重成分之一，因此干细胞的软骨形成对许多细胞内和细胞外的机械信号都有反应。作为一个独特的物理线索，基质刚度在干细胞命运的承诺中起着不可或缺的作用。然而，在检查基质刚度的下游效应时，大多数研究使用不同的可溶性因子来辅助物理诱导过程，这可能掩盖了基质刚度的软骨形成效应。在这里，我们制造了具有梯度刚度的聚丙烯酰胺 (PAAm) 水凝胶，以揭示基质刚度在间充质干细胞 (MSCs) 软骨形成过程中的作用，有或没有 TGF-β3 作为诱导因子。结果表明，通过模拟体内相对高细胞密度的微量培养，软底物（约 0.5 kPa）与组装的细胞骨架独立地促进 MSCs 的成软骨分化。进一步的分析表明，添加 TGF-β3 通常会增加软骨相关标志物的表达水平，并掩盖肥大标志物的刚度衍生表达模式。这些结果证明了在发育背景下经历的机械线索如何调节干细胞命运的承诺，并对组织再生材料的设计产生重大影响。