Department of Materials Science and Engineering, Monash University, Clayton, Victoria 3800, Australia; ARC Training Centre for Cell and Tissue Engineering Technologies, Monash University, Clayton, Victoria 3800, Australia.
Department of Materials Science and Engineering, Monash University, Clayton, Victoria 3800, Australia.
Acta Biomater. 2023 Sep 15;168:144-158. doi: 10.1016/j.actbio.2023.06.041. Epub 2023 Jul 7.
Mesenchymal stromal cells (MSCs) have significant therapeutic potential due to their ability to differentiate into musculoskeletal lineages suitable for tissue-engineering, as well as the immunomodulatory and pro-regenerative effects of the paracrine factors that these cells secrete. Cues from the extracellular environment, including physical stimuli such as substrate stiffness, are strong drivers of MSC differentiation, but their effects upon MSC paracrine activity are not well understood. This study, therefore sought to determine the impact of substrate stiffness on the paracrine activity of MSCs, analysing both effects on MSC fate and their effect on T-cell and macrophage activity and angiogenesis. The data show that conditioned medium (CM) from MSCs cultured on 0.2 kPa (soft) and 100 kPa (stiff) polyacrylamide hydrogels have differing effects on MSC proliferation and differentiation, with stiff CM promoting proliferation whilst soft CM promoted differentiation. There were also differences in the effects upon macrophage phagocytosis and angiogenesis, with the most beneficial effects from soft CM. Analysis of the media composition identified differences in the levels of proteins including IL-6, OPG, and TIMP-2. Using recombinant proteins and blocking antibodies, we confirmed a role for OPG in modulating MSC proliferation with a complex combination of factors involved in the regulation of MSC differentiation. Together the data confirm that the physical microenvironment has an important influence on the MSC secretome and that this can alter the differentiation and regenerative potential of the cells. These findings can be used to tailor the culture environment for manufacturing potent MSCs for specific clinical applications or to inform the design of biomaterials that enable the retention of MSC activity after delivery into the body. STATEMENT OF SIGNIFICANCE: • MSCs cultured on 100 kPa matrices produce a secretome that boosts MSC proliferation • MSCs cultured on 0.2 kPa matrices produce a secretome that promotes MSC osteogenesis and adipogenesis, as well as angiogenesis and macrophage phagocytosis • IL-6 secretion is elevated in MSCs on 0.2 kPa substrates • OPG, TIMP-2, MCP-1, and sTNFR1 secretion are elevated in MSCs on 100 kPa substrates.
间充质基质细胞 (MSCs) 具有显著的治疗潜力，因为它们能够分化为适合组织工程的肌肉骨骼谱系，以及这些细胞分泌的旁分泌因子的免疫调节和促再生作用。细胞外环境中的信号，包括基质硬度等物理刺激，是 MSC 分化的主要驱动力，但它们对 MSC 旁分泌活性的影响尚不清楚。因此，本研究旨在确定基质硬度对 MSC 旁分泌活性的影响，分析其对 MSC 命运的影响及其对 T 细胞和巨噬细胞活性和血管生成的影响。数据表明，在 0.2 kPa（软）和 100 kPa（硬）聚丙烯酰胺水凝胶上培养的 MSC 的条件培养基 (CM) 对 MSC 增殖和分化有不同的影响，硬 CM 促进增殖，而软 CM 促进分化。巨噬细胞吞噬作用和血管生成也存在差异，软 CM 的效果最好。对培养基成分的分析表明，包括 IL-6、OPG 和 TIMP-2 在内的蛋白质水平存在差异。使用重组蛋白和阻断抗体，我们证实了 OPG 在调节 MSC 增殖中的作用，涉及调节 MSC 分化的多种因素。数据共同证实，物理微环境对 MSC 分泌组有重要影响，这可以改变细胞的分化和再生潜力。这些发现可用于调整培养环境，以制造用于特定临床应用的有效 MSC，或为设计能够在输送到体内后保留 MSC 活性的生物材料提供信息。 研究意义： • 在 100 kPa 基质上培养的 MSC 产生一种促进 MSC 增殖的分泌组 • 在 0.2 kPa 基质上培养的 MSC 产生一种促进 MSC 成骨和成脂、血管生成和巨噬细胞吞噬作用的分泌组 • 在 0.2 kPa 基质上培养的 MSC 中 IL-6 分泌增加 • 在 100 kPa 基质上培养的 MSC 中 OPG、TIMP-2、MCP-1 和 sTNFR1 分泌增加。
