Differential Effects of the Antioxidants N-Acetylcysteine and Pyrrolidine Dithiocarbamate on Mesenchymal Stem Cell Chondrogenesis
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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 N-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 via 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.
KeywordsChondrogenesis Mesenchymal stem cells Reactive oxygen species Antioxidant N-Acetylcysteine Pyrrolidine dithiocarbamate
Conflict of interest
Suwimol Tangtrongsup declares that she has no conflict of interest. John Kisiday owns stock in Advanced Regenerative Therapies and Regenerative Sciences.
- 1.Amin, A. R., P. E. Di Cesare, P. Vyas, M. Attur, E. Tzeng, T. R. Billiar, S. A. Stuchin, and S. B. Abramson. The expression and regulation of nitric oxide synthase in human osteoarthritis-affected chondrocytes: Evidence for up-regulated neuronal nitric oxide synthase. J. Exp. Med. 182:2097–2102, 1995.CrossRefGoogle Scholar
- 2.Arsalane, K., C. M. Dubois, T. Muanza, R. Begin, F. Boudreau, C. Asselin, and A. M. Cantin. Transforming growth factor-beta1 is a potent inhibitor of glutathione synthesis in the lung epithelial cell line A549: Transcriptional effect on the GSH rate-limiting enzyme gamma-glutamylcysteine synthetase. Am. J. Respir. Cell Mol. Biol. 17:599–607, 1997.CrossRefGoogle Scholar
- 6.Das, K. C., Y. Lewis-Molock, and C. W. White. Activation of Nf-kappa B and elevation of MnSOD gene expression by thiol reducing agents in lung adenocarcinoma (A549) cells. Am. J. Physiol. 269:L588–602, 1995.Google Scholar
- 12.Guadall, A., M. Orriols, J. F. Alcudia, V. Cachofeiro, J. Martinez-Gonzalez, and C. Rodriguez. Hypoxia-induced ROS signaling is required for lox up-regulation in endothelial cells. Front. Biosci. (Elite Ed). 3:955–967, 2011.Google Scholar
- 22.Lepetsos, P., and A. G. Papavassiliou. Ros/oxidative stress signaling in osteoarthritis. Biochim. Biophys. Acta 576–591:2016, 1862.Google Scholar
- 23.Liu, X., Y. Xu, S. Chen, Z. Tan, K. Xiong, Y. Li, Y. Ye, Z. P. Luo, F. He, and Y. Gong. Rescue of proinflammatory cytokine-inhibited chondrogenesis by the antiarthritic effect of melatonin in synovium mesenchymal stem cells via suppression of reactive oxygen species and matrix metalloproteinases. Free Radic. Biol. Med. 68:234–246, 2014.CrossRefGoogle Scholar
- 26.Misra, H. P. Generation of superoxide free radical during the autoxidation of thiols. J. Biol. Chem. 249:2151–2155, 1974.Google Scholar
- 27.Morita, K., T. Miyamoto, N. Fujita, Y. Kubota, K. Ito, K. Takubo, K. Miyamoto, K. Ninomiya, T. Suzuki, R. Iwasaki, M. Yagi, H. Takaishi, Y. Toyama, and T. Suda. Reactive oxygen species induce chondrocyte hypertrophy in endochondral ossification. J. Exp. Med. 204:1613–1623, 2007.CrossRefGoogle Scholar
- 30.Nakagawa, S., Y. Arai, O. Mazda, T. Kishida, K. A. Takahashi, K. Sakao, M. Saito, K. Honjo, J. Imanishi, and T. Kubo. N-Acetylcysteine prevents nitric oxide-induced chondrocyte apoptosis and cartilage degeneration in an experimental model of osteoarthritis. J. Orthop. Res. 28:156–163, 2010.Google Scholar
- 33.Samuni, Y., S. Goldstein, O. M. Dean, and M. Berk. The chemistry and biological activities of N-acetylcysteine. Biochim. Biophys. Acta 4117–4129:2013, 1830.Google Scholar
- 35.Scharf, B., C. C. Clement, S. Yodmuang, A. M. Urbanska, S. O. Suadicani, D. Aphkhazava, M. M. Thi, G. Perino, J. A. Hardin, N. Cobelli, G. Vunjak-Novakovic, and L. Santambrogio. Age-related carbonylation of fibrocartilage structural proteins drives tissue degenerative modification. Chem. Biol. 20:922–934, 2013.CrossRefGoogle Scholar
- 36.Shi, X., S. S. Leonard, S. Wang, and M. Ding. Antioxidant properties of pyrrolidine dithiocarbamate and its protection against Cr(VI)-induced DNA strand breakage. Ann. Clin. Lab. Sci. 30:209–216, 2000.Google Scholar
- 40.Tangtrongsup, S., and J. D. Kisiday. Modulating the oxidative environment during mesenchymal stem cells chondrogenesis with serum increases collagen accumulation in agarose culture. J. Orthop. Res. 36(1):506–514, 2017.Google Scholar
- 41.Tangtrongsup, S., and J. D. Kisiday. Modulating the oxidative environment during mesenchymal stem cells chondrogenesis with serum increases collagen accumulation in agarose culture. J. Orthop. Res. 36:506–514, 2018.Google Scholar
- 44.Wehling, N., G. D. Palmer, C. Pilapil, F. Liu, J. W. Wells, P. E. Muller, C. H. Evans, and R. M. Porter. Interleukin-1beta and tumor necrosis factor alpha inhibit chondrogenesis by human mesenchymal stem cells through NF-kappaB-dependent pathways. Arthritis Rheum. 60:801–812, 2009.CrossRefGoogle Scholar
- 47.Zhang, D. Y., Y. Pan, C. Zhang, B. X. Yan, S. S. Yu, D. L. Wu, M. M. Shi, K. Shi, X. X. Cai, S. S. Zhou, J. B. Wang, J. P. Pan, and L. H. Zhang. Wnt/beta-catenin signaling induces the aging of mesenchymal stem cells through promoting the ROS production. Mol. Cell. Biochem. 374:13–20, 2013.CrossRefGoogle Scholar