PDF(Pubmed)
Abstract：
Adipose-derived stem cells (ADSCs) hold promise for tendon repair, even if their tenogenic plasticity and underlying mechanisms remain only partially understood, particularly in cells derived from the ovine animal model. This study aimed to characterize oADSCs during in vitro expansion to validate their phenotypic properties pre-transplantation. Moreover, their tenogenic potential was assessed using two in vitro-validated approaches: (1) teno-inductive conditioned media (CM) derived from a co-culture between ovine amniotic stem cells and fetal tendon explants, and (2) short- (48 h) and long-term (14 days) seeding on highly aligned PLGA (ha-PLGA) electrospun scaffold. Our findings indicate that oADSCs can be expanded without senescence and can maintain the expression of stemness (Sox2, Oct4, Nanog) and mesenchymal (CD29, CD166, CD44, CD90) markers while remaining negative for hematopoietic (CD31, CD45) and MHC-II antigens. Of note, oADSCs\' tendon differentiation potential greatly depended on the in vitro strategy. oADSCs exposed to CM significantly upregulated tendon-related genes (COL1, TNMD, THBS4) but failed to accumulate TNMD protein at 14 days of culture. Conversely, oADSCs seeded on ha-PLGA fleeces quickly upregulated the tendon-related genes (48 h) and in 14 days accumulated high levels of the TNMD protein into the cytoplasm of ADSCs, displaying a tenocyte-like morphology. This mechano-sensing cellular response involved a complete SOX9 downregulation accompanied by YAP activation, highlighting the efficacy of biophysical stimuli in promoting tenogenic differentiation. These findings underscore oADSCs\' long-term self-renewal and tendon differentiative potential, thus opening their use in a preclinical setting to develop innovative stem cell-based and tissue engineering protocols for tendon regeneration, applied to the veterinary field.
摘要：
脂肪来源的干细胞（ADSC）有望修复肌腱，即使它们的张力可塑性和潜在机制只得到了部分理解，特别是在来自绵羊动物模型的细胞中。本研究旨在表征体外扩增过程中的oADSC，以验证其移植前的表型特性。此外，使用两种体外验证的方法评估了它们的肌腱潜能：（1）来自绵羊羊膜干细胞和胎儿肌腱外植体之间的共培养的诱导条件培养基（CM），和(2)在高度对齐的PLGA(ha-PLGA)电纺支架上短期(48小时)和长期(14天)接种。我们的发现表明，oADSC可以扩增而不衰老，并且可以维持干细胞（Sox2，Oct4，Nanog）和间充质（CD29，CD166，CD44，CD90）标志物的表达，同时对造血（CD31，CD45）和MHC-II抗原保持阴性。值得注意的是，oADSCs的肌腱分化潜力很大程度上取决于体外策略。暴露于CM的oADSCs显著上调肌腱相关基因(COL1，TNMD，THBS4），但在培养14天时未能积累TNMD蛋白。相反，接种在ha-PLGA羊毛上的oADSCs迅速上调肌腱相关基因（48小时），并在14天内将高水平的TNMD蛋白积累到ADSCs的细胞质中，显示肌腱细胞样形态。这种机械感应细胞反应涉及完全的SOX9下调，伴随着YAP激活，强调生物物理刺激在促进肌腱分化中的功效。这些发现强调了oADSC的长期自我更新和肌腱分化潜能，从而打开它们在临床前环境中的使用，以开发创新的基于干细胞和组织工程的肌腱再生方案，适用于兽医领域。
