Tangtrongsup Suwimol, Kisiday John D
Department of Clinical Sciences, Orthopaedic Research Center, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins 80523, Colorado.
J Orthop Res. 2018 Jan;36(1):506-514. doi: 10.1002/jor.23618. Epub 2017 Jun 26.
Chondrogenesis of mesenchymal stem cells (MSCs) is induced in culture conditions that have been associated with oxidative stress, although the extent to which the oxidative environment affects differentiation and extracellular matrix (ECM) accumulation is not known. The objectives of this study were to evaluate the oxidative environment during MSCs chondrogenesis in conventional serum-free medium, and the effect of serum-supplementation on intracellular reactive oxygen species (ROS) and chondrogenesis. Young adult equine MSCs were seeded into agarose and cultured in chondrogenic medium, with or without 5% fetal bovine serum (FBS), for up to 15 days. Samples were evaluated for intracellular ROS, the antioxidant glutathione, ECM and gene expression measures of chondrogenesis, and carbonylation as an indicator of oxidative damage. Intracellular ROS increased with time in culture, and was lower in medium supplemented with FBS. Glutathione decreased ∼12-fold during early chondrogenesis (p < 0.0001), and was not affected by FBS (p = 0.25). After 15 days of culture, FBS supplementation increased hydroxyproline accumulation ∼80% (p = 0.0002); otherwise, measures of chondrogenesis were largely unaffected. Protein carbonylation in chondrogenic MSCs cultures was not significantly different between serum-free and FBS cultures (p = 0.72). Supplementation with adult equine serum increased hydroxyproline accumulation by 45% over serum-free culture (p = 0.0006). In conclusion, this study characterized changes in the oxidative environment during MSC chondrogenesis, and suggested that lowering ROS may be an effective approach to increase collagen accumulation. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:506-514, 2018.
间充质干细胞（MSCs）的软骨形成是在与氧化应激相关的培养条件下诱导产生的，尽管氧化环境对分化和细胞外基质（ECM）积累的影响程度尚不清楚。本研究的目的是评估在传统无血清培养基中MSCs软骨形成过程中的氧化环境，以及血清补充对细胞内活性氧（ROS）和软骨形成的影响。将年轻成年马的MSCs接种到琼脂糖中，并在软骨形成培养基中培养，添加或不添加5%胎牛血清（FBS），培养长达15天。对样本进行细胞内ROS、抗氧化剂谷胱甘肽、ECM以及软骨形成的基因表达测量，并用羰基化作为氧化损伤的指标进行评估。细胞内ROS在培养过程中随时间增加，在添加FBS的培养基中较低。谷胱甘肽在软骨形成早期下降了约12倍（p < 0.0001），且不受FBS影响（p = 0.25）。培养15天后，添加FBS使羟脯氨酸积累增加了约80%（p = 0.0002）；否则，软骨形成的测量指标基本不受影响。无血清培养和FBS培养的软骨形成MSCs培养物中的蛋白质羰基化没有显著差异（p = 0.72）。添加成年马血清比无血清培养使羟脯氨酸积累增加了45%（p = 0.0006）。总之，本研究描述了MSCs软骨形成过程中氧化环境的变化，并表明降低ROS可能是增加胶原蛋白积累的有效方法。© 2017骨研究学会。由Wiley Periodicals, Inc.出版。《矫形外科研究杂志》36:506 - 514, 2018。
