Biophysical cues (especially mechanical cues) embedded in cellular microenvironments show a critical impact on stem cell fate. Despite the capability of traditional hydrogels to mimic the feature of extracellular matrix (ECM) and tune their physicochemical properties via diverse approaches, their relatively large size not only induces biased results, but also hinders high-throughput screening and analysis. In this paper, a microgel model is proposed to recapitulate the role of 3D mechanical microenvironment on stem cell behaviors especially chondrogenesis in vitro. The small diameter of microgels brings the high surface area to volume ratio and then the enlarged diffusion area and shortened diffusion distance of soluble molecules, leading to uniform distribution of nutrients and negligible biochemical gradient inside microgels. To construct ECM-like microenvironment with tunable mechanical strength, three gelatin/hyaluronic acid hybrid microgels with low, medium and high crosslinking densities, i.e., Gel-HA(L), Gel-HA(M) and Gel-HA(H), are fabricated in microfluidic devices by Michael addition reaction between thiolated gelatin (Gel-SH) and ethylsulfated hyaluronic acid (HA-VS) with different substitution degrees of vinyl sulfone groups. Our results show that mouse bone marrow mesenchymal stem cell (BMSC) proliferation, distribution and chondrogenesis are all closely dependent on mechanical microenvironments in microgels. Noteworthily, BMSCs show a clear trend of differentiating into hyaline cartilage in Gel-HA(L) and fibrocartilage in Gel-HA(M) and Gel-HA(H). Whole transcriptome RNA sequencing reveals that mechanical microenvironment of microgels affects BMSC differentiation via TGF-β/Smad signaling pathway, Hippo signaling pathway and Integrin/YAP/TAZ signaling pathway. We believe this microgel model provides a new way to further explore the interaction between cells and 3D microenvironment.

Statement of Significance

In recent years, hydrogels have been frequently used to construct 3D microenvironment for cells. However, their relatively large size not only brings biased experimental results, but also limits high-throughput screening and analysis. Herein we propose a gelatin/hyaluronic acid microgel model to explore the effects of 3D cellular mechanical microenvironment (biophysical cues) on BMSC behaviors especially chondrogenesis, which can minimize the interference of biochemical gradients. Our results reveal that BMSC differentiation into either hyaline cartilage or fibrocartilage can be regulated via tailoring the mechanical properties of microgels. Whole transcriptome RNA sequencing proves that “TGF-β/Smad signaling pathway”, “Hippo signaling pathway” and “Integrins/YAP/ TAZ signaling pathway” are activated or inhibited in this process.

中文翻译：

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工程化细胞机械微环境以调节干细胞软骨形成：来自微凝胶模型的见解。

嵌入细胞微环境中的生物物理线索（尤其是机械线索）显示出对干细胞命运的关键影响。尽管传统水凝胶具有通过多种方法模仿细胞外基质（ECM）的特征并调节其理化特性的能力，但其相对较大的尺寸不仅会导致结果偏倚，而且会阻碍高通量筛选和分析。在本文中，提出了一种微凝胶模型来概括3D机械微环境对干细胞行为特别是体外软骨形成的作用。微凝胶的小直径带来高的表面积与体积之比，然后增大了可溶性分子的扩散面积并缩短了扩散距离，从而导致了营养成分的均匀分布和微凝胶内部微不足道的生化梯度。为了构建具有可调机械强度的类似ECM的微环境，使用三种具有低，中和高交联密度的明胶/透明质酸混合微凝胶，即Gel-HA（L），Gel-HA（M）和Gel-HA（H），在微流控装置中通过硫代明胶（Gel-SH）和具有不同乙烯基取代基取代度的乙基硫酸化透明质酸（HA-VS）之间的迈克尔加成反应制造了这些化合物。我们的结果表明，小鼠骨髓间充质干细胞（BMSC）的增殖，分布和软骨形成均密切依赖于微凝胶中的机械微环境。值得注意的是，BMSCs表现出明显的分化为Gel-HA（L）中的透明软骨和Gel-HA（M）和Gel-HA（H）中的纤维软骨的趋势。全转录组RNA测序表明，微凝胶的机械微环境通过TGF-β/ Smad信号通路，Hippo信号通路和整联蛋白/ YAP / TAZ信号通路影响BMSC的分化。我们相信，这种微凝胶模型为进一步探索细胞与3D微环境之间的相互作用提供了新的途径。

重要声明

近年来，水凝胶经常被用于构建细胞的3D微环境。然而，它们相对较大的尺寸不仅带来有偏差的实验结果，而且限制了高通量的筛选和分析。本文中，我们提出了一种明胶/透明质酸微凝胶模型，以探索3D细胞机械微环境（生物物理线索）对BMSC行为尤其是软骨形成的影响，该行为可以最大程度地降低生物化学梯度的干扰。我们的结果表明，可以通过调整微凝胶的机械性能来调节BMSC向透明软骨或纤维软骨的分化。全转录组RNA测序证明在该过程中“TGF-β/ Smad信号传导途径”，“河马信号传导途径”和“整合素/ YAP / TAZ信号传导途径”被激活或抑制。