Release of pro-inflammatory cytokines from muscle and bone causes tenocyte death in a novel rotator cuff in vitro explant culture model

Purpose: Tendinopathy is a significant clinical problem thought to be associated with altered mechanical loading. Explant culture models allow researchers to alter mechanical loading in a controlled in vitro environment while maintaining tenocytes in their native matrix. However, current models do not accurately represent commonly injured tendons, ignoring contributions of associated musculature and bone, as well as regional collagen structure. This study details the characterization of amouse rotator cuff explant culture model, including bone, tendon, and muscle (BTM). Materials and Methods: Following harvest, BTM explants were maintained in stress-deprived culture for one week and tendon was then assessed for changes in cell viability, metabolism, matrix structure and content. Results: Matrix turnover occurred throughout culture as manifested in both gene expression and biosynthesis, but this did not translate to net changes in total collagen or sulfated glycosaminoglycan content. Furthermore, tendon structure was not significantly altered throughout culture. However, we found significant cell death in BTM tendons after 3 days in culture, which we hypothesize is cytokine-induced. Using a targeted multiplex assay, we found high levels of pro-inflammatory cytokines released to the culture medium from muscle and bone, levels that did cause cell deathin tendon-alone controls. Conclusions: Overall, this model presents an innovative approach to understandingrotator cuff injury and tenocyte mechanobiology in a clinically-relevant tendon structure. Our model can be a powerful tool to investigate how mechanical and biological stimuli can alter normal tendon health and lead to tendon degeneration, and may provide a testbed for therapeutics for tendon repair.

研究目的：肌腱病（Tendinopathy）是一类严重的临床病症，目前认为其与机械负荷改变密切相关。外植体培养（explant culture）模型可使研究者在可控的体外环境中调控机械负荷，同时保留腱细胞（tenocytes）处于天然细胞外基质（native matrix）中。然而，现有模型无法准确模拟临床常见的受损肌腱，忽略了相关肌肉、骨骼以及区域胶原结构的贡献。本研究详细表征了一种包含骨骼、肌腱与肌肉（BTM）的小鼠肩袖（rotator cuff）外植体培养模型。

材料与方法：取材完成后，BTM外植体在无应力培养条件中维持一周，随后对肌腱开展细胞活力、代谢水平、基质结构与成分变化的检测。

结果：培养过程中基质重塑贯穿始终，这一现象在基因表达与生物合成层面均有体现，但并未导致总胶原或硫酸化糖胺聚糖（sulfated glycosaminoglycan）含量出现净变化。此外，培养全程肌腱结构未发生显著改变。不过，我们发现培养3天后的BTM肌腱出现显著细胞死亡，推测其由细胞因子介导。通过靶向多重检测法（targeted multiplex assay），我们检测到肌肉与骨骼向培养基中释放了高水平的促炎细胞因子（pro-inflammatory cytokines），该浓度足以在单独培养的肌腱对照组中引发细胞死亡。

结论：总体而言，本模型为探究临床相关肌腱结构中的肩袖损伤与腱细胞力学生物学提供了一种创新方法。本模型可作为有力工具，用于研究机械与生物学刺激如何改变肌腱正常健康状态并引发肌腱退变，同时可为肌腱修复治疗手段提供测试平台。