The advent of bioprinting has enabled the creation of precise three‐dimensional (3D) cell cultures suitable for biomimetic in vitro models. In this study, we developed a novel protocol for 3D printing methacrylated collagen (ColMa, or PhotoCol®) combined with tendon stem/progenitor cells (hTSPCs) derived from human tendon explants. Although pure ColMa has not previously been proposed as a printable hydrogel, this paper outlines a robust and highly reproducible pipeline for bioprinting this material. Indeed, we successfully fabricated a 3D bioengineered scaffold and cultured it for 21 days under perfusion conditions with medium supplemented with growth/differentiation factor‐5 (GDF‐5). This bioprinting pipeline and the culture conditions created an exceptionally favorable 3D environment, enabling the cells to proliferate, exhibit tenogenic behaviors, and produce a new collagen type I matrix, thereby remodeling the surrounding environment. Indeed, over the 21‐day culture period under perfusion condition, tenomodulin expression showed a significant upregulation on day 7, with a 2.3‐fold increase, compared to days 14 and 21. Collagen type I gene expression was upregulated nearly 10‐fold by day 14. This trend was further confirmed by western blot analysis, which revealed a statistically significant difference in tenomodulin expression between day 21 and both day 7 and day 14. For type I collagen, significant differences were observed between day 0 and day 21, as well as between day 0 and day 14, with a p‐value of 0.01. These results indicate a progressive over‐expression of type I collagen, reflecting cell differentiation towards a proper tenogenic phenotype. Cytokines, such as IL‐8 and IL‐6, levels peaked at 8566 and 7636 pg/mL, respectively, on day 7, before decreasing to 54 and 46 pg/mL by day 21. Overall, the data suggest that the novel ColMa bioprinting protocol effectively provided a conducive environment for the growth and proper differentiation of hTSPCs, showcasing its potential for studying cell behavior and tenogenic differentiation.

中文翻译：

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基于 ColMA 的生物打印 3D 支架可用于研究人类肌腱干细胞中的肌腱发生事件


生物打印的出现使创建适用于仿生体外模型的精确三维 （3D） 细胞培养物成为可能。在这项研究中，我们开发了一种用于 3D 打印甲基丙烯酸酯胶原蛋白（ColMa 或 PhotoCol®）与源自人类肌腱外植体的肌腱干细胞/祖细胞 （hTSPCs） 相结合的新方案。尽管以前从未提出过纯 ColMa 作为可打印水凝胶，但本文概述了一种用于生物打印这种材料的稳健且高度可重复的管道。事实上，我们成功地制造了 3D 生物工程支架，并在灌注条件下用补充生长/分化因子-5 （GDF-5） 的培养基培养了 21 天。这种生物打印管道和培养条件创造了一个非常有利的 3D 环境，使细胞能够增殖，表现出肌腱形成行为，并产生新的 I 型胶原蛋白基质，从而重塑周围环境。事实上，在灌注条件下的 21 天培养期内，肌腱调节蛋白表达在第 7 天显示显着上调，与第 14 天和第 21 天相比增加了 2.3 倍。到第 14 天，I 型胶原基因表达上调了近 10 倍。western blot 分析进一步证实了这一趋势，该分析揭示了第 21 天与第 7 天和第 14 天之间肌腱调节蛋白表达的统计学显着差异。对于 I 型胶原蛋白，在第 0 天和第 21 天之间以及第 0 天和第 14 天之间观察到显着差异，p 值为 0.01。这些结果表明 I 型胶原蛋白进行性过表达，反映了细胞向适当的肌腱发生表型分化。 细胞因子（如 IL-8 和 IL-6）水平在第 7 天分别达到 8566 和 7636 pg/mL 的峰值，然后在第 21 天降至 54 和 46 pg/mL。总体而言，数据表明，新型 ColMa 生物打印方案有效地为 hTSPCs 的生长和适当分化提供了有利的环境，展示了其在研究细胞行为和肌腱发生分化方面的潜力。