Modulating the oxidative environment during mesenchymal stem cells chondrogenesis with serum increases collagen accumulation in agarose culture.

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.