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

Abstract

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.

Expansion of Chondrocytes for Cartilage Tissue Engineering: A Review of Chondrocyte Dedifferentiation and Redifferentiation as a Function of Growth in Expansion Culture

“…Insulin was found to be a critical factor for generating histological evidence of redifferentiation [47]. Similar results have been reported for bone marrow MSCs as withholding insulin or ITS from chondrogenic medium greatly suppressed chondrogenesis [48,49]. As demonstrated for immature bovine chondrocytes, cartilage matrix synthesis by primary chondrocytes can be stimulated by insulin [50].…”

Culture Conditions that Support Expansion and Chondrogenesis of Middle-Aged Rat Mesenchymal Stem Cells

“…17 Recently, we reported that supplementing defined chondrogenic medium containing ITS+ with FBS moderately enhanced collagen accumulation in young adult equine MSC cultures. 19 For middle-aged rat MSCs, the effect of serum was more pronounced as GAG and hydroxyproline accumulation, and cell survival improved over defined medium. With serum-supplementation of middleaged rat cultures, the accumulation of GAG (~2 µg/mg w/w) was approximately similar to young adult equine MSCs that were cultured in the same manner, while hydroxyproline accumulation in rat cultures (~0.18 µg/mg w/w) was approximately 50% of equine cultures.…”

“…With serum-supplementation of middleaged rat cultures, the accumulation of GAG (~2 µg/mg w/w) was approximately similar to young adult equine MSCs that were cultured in the same manner, while hydroxyproline accumulation in rat cultures (~0.18 µg/mg w/w) was approximately 50% of equine cultures. 19 While differences in variables such as seeding density, scaffold, or duration of chondrogenic cultures limits the extent to which comparisons to additional studies can be considered, it appears possible that with serum-supplementation the chondrogenic potential of adult rat MSCs may be comparable to MSCs from other species.…”

“…In the absence of ITS, serum supplementation has demonstrated a poor capacity to support MSC chondrogenesis. 17,19,20 Therefore, in middle-aged MSC cultures it is likely that serum did not improve chondrogenesis per se, but functioned to prevent cell death and improve collagen accumulation of differentiated cells. The large (70%) difference in cell viability between defined and serum-supplemented conditions was unexpected as MSCs have generally proven to tolerate serum-free chondrogenic culture, although loss of viability of MSCs in agarose and defined chondrogenic medium has been reported for cells from numerous species, 18,[21][22][23] and has been attributed to apoptosis.…”

“…18 It is possible that the relatively low viability for middle-aged rat MSCs in defined medium was due to apoptosis that was induced by serum withdrawal. 24 Furthermore, a possible contributing factor to apoptosis is elevated levels of intracellular reactive oxygen species, 25 which have been reported to increase substantially with induction of MSC chondrogenesis, 19,26 and can be lowered with serum supplementation. 19 In contrast to middle-aged rats, MSCs from 6-week-old rats experienced robust chondrogenesis in defined medium, with cell viabilities that were not significantly different between defined and serum-supplemented cultures.…”