Chen Ziming, Chen Peilin, Ruan Rui, Chen Lianzhi, Yuan Jun, Wood David, Wang Tao, Zheng Ming Hao
Centre of Orthopaedic Translational Research, Medical School, University of Western Australia.
Centre of Orthopaedic Translational Research, Medical School, University of Western Australia;
J Vis Exp. 2020 Aug 1(162). doi: 10.3791/61278.
Tendinopathy is a common chronic tendon disease relating to inflammation and degeneration in an orthopaedic area. With high morbidity, limited self-repairing capacity and, most importantly, no definitive treatments, tendinopathy still influences patients' life quality negatively. Tendon-derived stem cells (TDSCs), as primary precursor cells of tendon cells, play an essential role in both the development of tendinopathy, and functional and structural restoration after tendinopathy. Thus, a method that can in vitro mimic the in vivo differentiation of TDSCs into tendon cells would be useful. Here, the present protocol describes a method based on a three-dimensional (3D) uniaxial stretching system to stimulate the TDSCs to differentiate into tendon-like tissues. There are seven stages of the present protocol: isolation of mice TDSCs, culture and expansion of mice TDSCs, preparation of stimulation culture medium for cell sheet formation, cell sheet formation by culturing in stimulation medium, preparation of 3D tendon stem cell construct, assembly of the uniaxial-stretching mechanical stimulation complex, and evaluation of the mechanical stimulated in vitro tendon-like tissue. The effectiveness was demonstrated by histology. The entire procedure takes less than 3 weeks. To promote extracellular matrix deposition, 4.4 mg/mL ascorbic acid was used in the stimulation culture medium. A separated chamber with a linear motor provides accurate mechanical loading and is portable and easily adjusted, which is applied for the bioreactor. The loading regime in the present protocol was 6% strain, 0.25 Hz, 8 h, followed by 16 h rest for 6 days. This protocol could mimic cell differentiation in the tendon, which is helpful for the investigation of the pathological process of tendinopathy. Moreover, the tendon-like tissue is potentially used to promote tendon healing in tendon injury as an engineered autologous graft. To sum up, the present protocol is simple, economic, reproducible and valid.
肌腱病是一种常见的慢性肌腱疾病，与骨科领域的炎症和退变有关。由于发病率高、自我修复能力有限，且最重要的是缺乏明确的治疗方法，肌腱病仍然对患者的生活质量产生负面影响。肌腱源性干细胞（TDSCs）作为肌腱细胞的主要前体细胞，在肌腱病的发生发展以及肌腱病后的功能和结构恢复中都起着至关重要的作用。因此，一种能够在体外模拟TDSCs向肌腱细胞体内分化的方法将是有用的。在此，本方案描述了一种基于三维（3D）单轴拉伸系统来刺激TDSCs分化为肌腱样组织的方法。本方案有七个阶段：小鼠TDSCs的分离、小鼠TDSCs的培养与扩增、用于细胞片形成的刺激培养基的制备、在刺激培养基中培养形成细胞片、3D肌腱干细胞构建体的制备、单轴拉伸机械刺激复合体的组装以及体外机械刺激的肌腱样组织的评估。通过组织学证明了其有效性。整个过程耗时不到3周。为促进细胞外基质沉积，在刺激培养基中使用了4.4 mg/mL的抗坏血酸。带有线性电机的分隔腔室提供精确的机械加载，且便携且易于调节，适用于生物反应器。本方案中的加载方案为6%应变、0.25 Hz、8小时，然后休息16小时，持续6天。该方案可以模拟肌腱中的细胞分化，这有助于研究肌腱病的病理过程。此外，肌腱样组织有可能作为工程化自体移植物用于促进肌腱损伤中的肌腱愈合。综上所述，本方案简单、经济、可重复且有效。
