Tangtrongsup Suwimol, Kisiday John D
Department of Clinical Sciences, Orthopaedic Research Center, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, 300W. Drake Road, Fort Collins, CO 80523 USA.
Cell Mol Bioeng. 2019 Jan 18;12(2):153-163. doi: 10.1007/s12195-019-00566-3. eCollection 2019 Apr.
Mesenchymal stem cell (MSC) chondrogenesis is associated with increases in intracellular reactive oxygen species (ROS), which may result in oxidative stress that is detrimental to cartilage regeneration. This study evaluated the ability of the antioxidants -acetylcysteine (NAC) or pyrrolidine dithiocarbamate (PDTC) to reduce intracellular ROS, and their effect on MSC chondrogenesis and maturation of cartilage-like extracellular matrix.
Equine bone marrow MSCs were cultured in serum-supplemented chondrogenic medium with or without NAC or PDTC. ROS was quantified in monolayer after 8 and 72 h of culture. MSCs were seeded into agarose, cultured for 15 days, and analyzed for viable cell density, glycosaminoglycan (GAG) and hydroxyproline accumulation, and collagen gene expression. PDTC cultures were evaluated for oxidative damage by protein carbonylation, and mechanical properties compressive testing.
NAC significantly lowered levels of ROS after 8 but not 72 h, and suppressed GAG accumulation (70%). In secondary experiments using serum-free medium, NAC significantly increased levels of ROS at 72 h, and lowered cell viability and extracellular matrix accumulation. PDTC significantly reduced levels of ROS (~ 30%) and protein carbonylation (27%), and enhanced GAG accumulation (20%). However, the compressive modulus for PDTC-treated samples was significantly lower (40%) than controls. Gene expression was largely unaffected by the antioxidants.
NAC demonstrated a limited ability to reduce intracellular ROS in chondrogenic culture, and generally suppressed accumulation of extracellular matrix. Conversely, PDTC was an effective antioxidant that enhanced GAG accumulation, although the concomitant reduction in compressive properties is a significant limitation for cartilage repair.
间充质干细胞（MSC）向软骨细胞分化与细胞内活性氧（ROS）水平升高有关，这可能导致氧化应激，对软骨再生产生不利影响。本研究评估了抗氧化剂——N-乙酰半胱氨酸（NAC）或吡咯烷二硫代氨基甲酸盐（PDTC）降低细胞内ROS的能力，及其对MSC软骨形成和类软骨细胞外基质成熟的影响。
将马骨髓间充质干细胞在添加血清的软骨形成培养基中培养，培养基中添加或不添加NAC或PDTC。培养8小时和72小时后，对单层细胞中的ROS进行定量分析。将间充质干细胞接种到琼脂糖中，培养15天，分析活细胞密度、糖胺聚糖（GAG）和羟脯氨酸积累情况以及胶原蛋白基因表达。通过蛋白质羰基化和机械性能（压缩试验）评估PDTC培养物的氧化损伤情况。
NAC在培养8小时后显著降低了ROS水平，但在72小时后未降低，并且抑制了GAG积累（70%）。在使用无血清培养基的二次实验中，NAC在72小时时显著增加了ROS水平，并降低了细胞活力和细胞外基质积累。PDTC显著降低了ROS水平（约30%）和蛋白质羰基化水平（27%），并增强了GAG积累（20%）。然而，PDTC处理样品的压缩模量比对照组显著降低（40%）。基因表达在很大程度上不受抗氧化剂的影响。
NAC在软骨形成培养中降低细胞内ROS的能力有限，并且通常会抑制细胞外基质的积累。相反，PDTC是一种有效的抗氧化剂，可增强GAG积累，尽管其压缩性能的同时降低对软骨修复是一个重大限制。
