Our understanding of tendon biology continues to evolve, thus leading to opportunities for developing novel, evidence-based effective therapies for the treatment of tendon disorders. Implementing the knowledge of tendon stem/progenitor cells (TSPCs) and assessing their potential in enhancing tendon repair could fill an important gap in this regard. We described different molecular and phenotypic profiles of TSPCs modulated by culture density, as well as their multipotency and secretory activities. Moreover, in the same experimental setting, we evaluated for different responses to inflammatory stimuli mediated by TNFα and IFNγ. We also preliminarily investigated their immunomodulatory activity and their role in regulating degradation of substance P. Our findings indicated that TSPCs cultured at low density (LD) exhibited cobblestone morphology and a reduced propensity to differentiate. A distinctive immunophenotypic profile was also observed with high secretory and promising immunomodulatory responses when primed with TNFα and IFNγ. In contrast, TSPCs cultured at high density (HD) showed a more elongated fibroblast-like morphology, a greater adipogenic differentiation potential, and a higher expression of tendon-related genes with respect to LD. Finally, HD TSPCs showed immunomodulatory potential when primed with TNFα and IFNγ, which was slightly lower than that shown by LD. A shift from low to high culture density during TSPC expansion demonstrated intermediate features confirming the cellular adaptability of TSPCs. Taken together, these experiments allowed us to identify relevant differences in TSPCs based on culture conditions. This ability of TSPCs to acquire distinguished morphology, phenotype, gene expression profile, and functional response advances our current understanding of tendons at a cellular level and suggests responsivity to cues in their in situ microenvironment.

我们对肌腱生物学的理解不断发展，从而为开发新的、基于证据的有效疗法来治疗肌腱疾病带来了机会。实施肌腱干/祖细胞 (TSPC) 的知识并评估它们在增强肌腱修复方面的潜力可以填补这方面的重要空白。我们描述了受培养密度调节的 TSPCs 的不同分子和表型谱，以及它们的多能性和分泌活动。此外，在相同的实验环境中，我们评估了对 TNFα 和 IFNγ 介导的炎症刺激的不同反应。我们还初步研究了它们的免疫调节活性及其在调节 P 物质降解中的作用。我们的研究结果表明，低密度 (LD) 培养的 TSPC 表现出鹅卵石形态和降低的分化倾向。当用 TNFα 和 IFNγ 引发时，还观察到具有高分泌和有希望的免疫调节反应的独特免疫表型特征。相比之下，高密度 (HD) 培养的 TSPC 显示出更细长的成纤维细胞样形态、更大的脂肪形成分化潜力以及与 LD 相关的肌腱相关基因的更高表达。最后，HD TSPCs 在用 TNFα 和 IFNγ 引发时显示出免疫调节潜力，略低于 LD。TSPC 扩增过程中从低培养密度到高培养密度的转变证明了中间特征，证实了 TSPC 的细胞适应性。综合起来，这些实验使我们能够根据培养条件确定 TSPC 的相关差异。TSPCs 获得独特的形态、表型、基因表达谱和功能反应的能力促进了我们目前在细胞水平上对肌腱的理解，并表明对肌腱中的线索的反应性原位微环境。