Bochon Karolina, Zielniok Katarzyna, Gawlak Maciej, Zawada Katarzyna, Zarychta-Wiśniewska Weronika, Siennicka Katarzyna, Struzik Sławomir, Pączek Leszek, Burdzińska Anna
Department of Immunology, Transplantology and Internal Diseases, Medical University of Warsaw, Warsaw, Poland.
Department of Pharmacodynamics and Pathophysiology, Centre for Preclinical Research and Technology, Medical University of Warsaw, Warsaw, Poland.
Int J Stem Cells. 2021 Feb 28;14(1):33-46. doi: 10.15283/ijsc20023.
Despite significant improvement in the treatment of tendon injuries, the full tissue recovery is often not possible because of its limited ability to auto-repair. The transplantation of mesenchymal stromal cells (MSCs) is considered as a novel approach in the treatment of tendinopathies. The question about the optimal culture conditions remains open. In this study we aimed to investigate if serum reduction, L-ascorbic acid supplementation or a combination of both factors can induce tenogenic differentiation of human adipose-derived MSCs (ASCs).
Human ASCs from 3 healthy donors were used in the study. The tested conditions were: 0.5 mM of ascorbic acid 2-phosphate (AA-2P), reduced serum content (2% FBS) or combination of these two factors. The combination of AA-2P and 2% FBS was the only experimental condition that caused a significant increase of the expression of all analyzed genes related to tenogenesis () in comparison to the untreated control (evaluated by RT-PCR, 5 day of experiment). Moreover, this treatment significantly increased the synthesis of SCLERAXIS, MOHAWK, COLLAGEN_1, COLLAGEN_3 proteins at the same time point (evaluated by Western blot method). Double immunocytochemical staining revealed that AA-2P significantly increased the extracellular deposition of both types of collagens. Semi-quantitative Electron Spin Resonance analysis of ascorbyl free radical revealed that AA-2P do not induce harmful transition metals-driven redox reactions in cell culture media.
Obtained results justify the use of reduced content of serum with the addition of 0.5 mM of AA-2P in tenogenic inducing media.
尽管肌腱损伤的治疗取得了显著进展，但由于其自身修复能力有限，往往无法实现完全的组织恢复。间充质基质细胞（MSCs）移植被认为是治疗肌腱病的一种新方法。关于最佳培养条件的问题仍未解决。在本研究中，我们旨在探讨血清减少、添加L-抗坏血酸或这两种因素的组合是否能诱导人脂肪来源的间充质干细胞（ASCs）向肌腱细胞分化。
本研究使用了来自3名健康供体的人ASCs。测试条件为：0.5 mM的抗坏血酸2-磷酸酯（AA-2P）、降低血清含量（2%胎牛血清）或这两种因素的组合。与未处理的对照组相比（通过RT-PCR在实验第5天评估），AA-2P和2%胎牛血清的组合是唯一能使所有分析的与肌腱形成相关基因的表达显著增加的实验条件。此外，在同一时间点，这种处理显著增加了硬骨素、莫霍克蛋白、胶原蛋白1、胶原蛋白3的合成（通过蛋白质印迹法评估）。双重免疫细胞化学染色显示，AA-2P显著增加了两种胶原蛋白的细胞外沉积。抗坏血酸自由基的半定量电子自旋共振分析表明，AA-2P不会在细胞培养基中诱导有害的过渡金属驱动的氧化还原反应。
所得结果证明在肌腱诱导培养基中使用降低的血清含量并添加0.5 mM的AA-2P是合理的。
