Meniscus fibrochondrocytes (MFCs) experience simultaneous hypoxia and mechanical loading in the knee joint. Experimental conditions based on these aspects of the native MFC environment may have promising applications in human meniscus tissue engineering. We hypothesized that in vitro "mechano-hypoxia conditioning" with mechanical loading such as dynamic compression (DC) and cyclic hydrostatic pressure (CHP) would enhance development of human meniscus fibrocartilage extracellular matrix in vitro. MFCs from inner human meniscus surgical discards were pre-cultured on porous type I collagen scaffolds with TGF-β3 supplementation to form baseline tissues with newly formed matrix that were used in a series of experiments. First, baseline tissues were treated with DC or CHP under hypoxia (HYP, 3% O2) for 5 days. DC was the more effective load regime in inducing gene expression changes, and combined HYP/DC enhanced gene expression of fibrocartilage precursors. The individual treatments of DC and HYP regulated thousands of genes, such as chondrogenic markers SOX5/6, in an overwhelmingly additive rather than synergistic manner. Similar baseline tissues were then treated with a short course of DC (5 vs 60 min, 10-20% vs 30-40% strain) with different pre-culture duration (3 vs 6 weeks). The longer course of loading (60 min) had diminishing returns in regulating mechano-sensitive and inflammatory genes such as c-FOS and PTGS2, suggesting that as few as 5 min of DC was adequate. There was a dose-effect in gene regulation by higher DC strains, whereas outcomes were inconsistent for different MFC donors in pre-culture durations. A final set of baseline tissues was then cultured for 3 weeks with mechano-hypoxia conditioning to assess mechanical and protein-level outcomes. There were 1.8-5.1-fold gains in the dynamic modulus relative to baseline in HYP/DC, but matrix outcomes were equal or inferior to static controls. Long-term mechano-hypoxia conditioning was effective in suppressing hypertrophic markers (e.g., COL10A1 10-fold suppression vs static/normoxia). Taken together, these results indicate that appropriately applied mechano-hypoxia conditioning can support meniscus fibrocartilage development in vitro and may be useful as a strategy for developing non-hypertrophic articular cartilage using mesenchymal stem cells.

中文翻译：

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工程人体半月板的机械缺氧调节。

半月板纤维软骨细胞 (MFC) 在膝关节中同时经历缺氧和机械负荷。基于天然 MFC 环境这些方面的实验条件可能在人类半月板组织工程中具有广阔的应用前景。我们假设体外“机械缺氧调节”具有机械负荷，如动态压缩 (DC) 和循环静水压 (CHP) 将增强体外人半月板纤维软骨细胞外基质的发育。来自人体内半月板手术丢弃物的 MFC 在补充 TGF-β3 的多孔 I 型胶原支架上预培养，以形成具有新形成的基质的基线组织，用于一系列实验。首先，在缺氧（HYP，3% O2​​）下用 DC 或 CHP 处理基线组织 5 天。DC 是诱导基因表达变化的更有效负载方案，并且组合 HYP/DC 增强了纤维软骨前体的基因表达。DC 和 HYP 的单独治疗以压倒性的相加方式而非协同方式调节了数千个基因，例如软骨形成标记 SOX5/6。然后用具有不同预培养持续时间（3 对 6 周）的短期 DC（5 对 60 分钟，10-20% 对 30-40% 应变）处理类似的基线组织。较长的加载过程（60 分钟）在调节机械敏感和炎症基因（如 c-FOS 和 PTGS2）方面的收益递减，这表明仅 5 分钟的 DC 就足够了。较高 DC 菌株的基因调控存在剂量效应，而不同 MFC 供体在预培养期间的结果不一致。然后将最后一组基线组织在机械缺氧条件下培养 3 周，以评估机械和蛋白质水平的结果。在 HYP/DC 中，动态模量相对于基线增加了 1.8-5.1 倍，但基质结果等于或低于静态对照。长期机械缺氧调节可有效抑制肥大标志物（例如，COL10A1 10 倍抑制与静态/常氧）。综上所述，这些结果表明，适当应用的机械缺氧调节可以支持体外半月板纤维软骨的发育，并且可用作使用间充质干细胞开发非肥大关节软骨的策略。在 HYP/DC 中，动态模量相对于基线增加了 1 倍，但基质结果等于或低于静态对照。长期机械缺氧调节可有效抑制肥大标志物（例如，COL10A1 10 倍抑制与静态/常氧）。综上所述，这些结果表明，适当应用的机械缺氧调节可以支持体外半月板纤维软骨的发育，并且可用作使用间充质干细胞开发非肥大关节软骨的策略。在 HYP/DC 中，动态模量相对于基线增加了 1 倍，但基质结果等于或低于静态对照。长期机械缺氧调节可有效抑制肥大标志物（例如，COL10A1 10 倍抑制与静态/常氧）。综上所述，这些结果表明，适当应用的机械缺氧调节可以支持体外半月板纤维软骨的发育，并且可用作使用间充质干细胞开发非肥大关节软骨的策略。