Department of Bioengineering, Stanford University Schools of Engineering and Medicine, Stanford, California.
Department of Bioengineering and Orthopedic Surgery, Stanford University Schools of Engineering and Medicine, Stanford, California.
Tissue Eng Part A. 2020 Feb;26(3-4):130-139. doi: 10.1089/ten.TEA.2019.0068. Epub 2019 Sep 20.
Rho-associated protein kinase (ROCK) signaling correlates with cell shape, with decreased cell spreading accompanied by decreased ROCK activity. However, how cell shape and ROCK activity impact the chondrogenesis of mesenchymal stem cells (MSCs) remains inconclusive. Here we examine the effects of ROCK inhibition on human MSC chondrogenesis in four different culture models, including three-dimensional (3D) microribbon (μRB) scaffolds, two-dimensional hydrogel (2D-HG) substrates, 3D hydrogels (3D-HGs), and pellet. For each culture model involving biomaterials, four polymers were compared, including gelatin, chondroitin sulfate, hyaluronic acid, and polyethylene glycol. ROCK inhibition decreased MSC chondrogenesis in μRB model, enhanced chondrogenesis in pellet, and had minimal effect in 2D-HG or 3D-HG models. Furthermore, we demonstrate that MSC chondrogenesis cannot be predicted using ROCK signaling alone. While varying biomaterial compositions can impact the amount or phenotype of resulting cartilage, varying biomaterials did not change the chondrogenic response to ROCK inhibition within each culture model. Regardless of culture model or ROCK expression, increased cartilage formation was always accompanied by enhanced N-cadherin expression and production, suggesting that N-cadherin is a robust marker to select culture conditions that promote chondrogenesis. Together, the results from this study may be used to enhance MSC-based cartilage regeneration in different culture models. Impact Statement Here we assessed the effects of Rho-associated protein kinase () inhibition on mesenchymal stem cell (MSC) chondrogenesis in different culture models, including three-dimensional (3D) microribbon scaffolds, two-dimensional hydrogel substrates, 3D hydrogels, and pellet culture. Our results demonstrate that effects of inhibition on MSC chondrogenesis differ substantially depending on culture models. Furthermore, MSC chondrogenesis cannot be predicted using signaling alone. The results from this study fill in a gap of knowledge in the correlation between signaling and MSC chondrogenesis, which may be used to enhance MSC-based cartilage regeneration in different culture models.
Rho 相关蛋白激酶（ROCK）信号与细胞形态相关，细胞铺展减少伴随着 ROCK 活性降低。然而，细胞形态和 ROCK 活性如何影响间充质干细胞（MSCs）的软骨生成仍不清楚。在这里，我们在四种不同的培养模型中研究了 ROCK 抑制对人 MSC 软骨生成的影响，包括三维（3D）微带（μRB）支架、二维水凝胶（2D-HG）底物、3D 水凝胶（3D-HG）和微球。对于涉及生物材料的每种培养模型，我们比较了四种聚合物，包括明胶、硫酸软骨素、透明质酸和聚乙二醇。ROCK 抑制减少了 μRB 模型中的 MSC 软骨生成，增强了微球中的软骨生成，而在 2D-HG 或 3D-HG 模型中影响较小。此外，我们证明仅通过 ROCK 信号不能预测 MSC 软骨生成。虽然不同的生物材料组成会影响产生的软骨的数量或表型，但在每种培养模型中，不同的生物材料并没有改变 ROCK 抑制对软骨生成的反应。无论培养模型或 ROCK 表达如何，增加的软骨形成总是伴随着增强的 N-钙黏蛋白表达和产生，这表明 N-钙黏蛋白是一个强有力的标记，可以选择促进软骨生成的培养条件。总的来说，这项研究的结果可用于增强不同培养模型中基于 MSC 的软骨再生。
