Wagner John R, Taguchi Takashi, Cho Jane Y, Charavaryamath Chandrashekhar, Griffon Dominique J
College of Veterinary Medicine, Western University of Health Sciences.
Applied Medical.
J Vis Exp. 2018 Mar 30(133):56810. doi: 10.3791/56810.
Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across species, combined with the limited healing properties of this tissue, have prompted the search for cellular therapies and propelled the development of experimental models to study their efficacy. Umbilical cord matrix-derived mesenchymal stem cells (UCM-MSC) are appealing candidates because they are abundant, easy to collect, circumvent the ethical concerns and risk of teratoma formation, yet resemble primitive embryonic stem cells more closely than adult tissue-derived MSCs. Significant interest has focused on chitosan as a strategy to enhance the properties of MSCs through spheroid formation. This paper details techniques to isolate UCM-MSCs, prepare spheroids on chitosan film, and analyze the effect of spheroid formation on surface marker expression. Consequently, creation of a bilateral patellar tendon injury model in rats is described for in vivo implantation of UCM-MSC spheroids formed on chitosan film. No complication was observed in the study with respect to morbidity, stress rising effects, or tissue infection. The total functional score of the operated rats at 7 days was lower than that of normal rats, but returned to normal within 28 days after surgery. Histological scores of tissue-healing confirmed the presence of a clot in treated defects evaluated at 7 days, absence of foreign body reaction, and progressing healing at 28 days. This bilateral patella tendon defect model controls inter-individual variation via creation of an internal control in each rat, was associated with acceptable morbidity, and allowed detection of differences between untreated tendons and treatments.
再生医学为传统治疗方法难以应对的疾病提供了新的选择。肌腱病在各个物种中的患病率和发病率，以及该组织有限的愈合能力，促使人们寻求细胞疗法，并推动了用于研究其疗效的实验模型的发展。脐带基质间充质干细胞（UCM-MSC）是颇具吸引力的候选细胞，因为它们数量丰富、易于收集，避免了伦理问题和畸胎瘤形成的风险，并且与原始胚胎干细胞的相似性高于成体组织来源的间充质干细胞。壳聚糖作为一种通过形成球体来增强间充质干细胞特性的策略受到了广泛关注。本文详细介绍了分离UCM-MSC、在壳聚糖膜上制备球体以及分析球体形成对表面标志物表达影响的技术。此外，还描述了在大鼠中建立双侧髌腱损伤模型以用于体内植入壳聚糖膜上形成的UCM-MSC球体。在该研究中，未观察到与发病率、应激增加效应或组织感染相关的并发症。手术大鼠在7天时的总功能评分低于正常大鼠，但在术后28天内恢复正常。组织愈合的组织学评分证实，在7天时评估的治疗缺损处存在血凝块，无异物反应，并且在28天时愈合进展良好。这种双侧髌腱缺损模型通过在每只大鼠中建立内部对照来控制个体间差异，具有可接受的发病率，并能够检测未治疗肌腱与治疗之间的差异。
