Hydrogen peroxide: a potent inducer of differentiation of human adipose-derived stem cells into chondrocytes

Common protocols for chondrogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) are generally expensive and time-consuming and, so far, have not successfully recreated pure chondrocytes. We hypothesise that a low level of H₂O₂ may induce differentiation of ADSCs into chondrocytes in a shorter incubation time and relatively lower cost. Therefore, this study aimed to comparatively investigate the effectiveness of H₂O₂-containing or free medium in the induction of ADSCs to chondrocytes. ADSCs were isolated from the lipoaspirate of four healthy females and evaluated by immunophenotyping for their CD90, CD73, CD44, CD34, and CD45 cell surface markers. Chondrogenic differentiation was carried out using differentiation medium in the presence or absence of 10 and 50 µM H₂O₂ in normal and three-dimensional culture system. The intracellular contents of reactive oxygen species (ROS) were detected by flow cytometry and fluorescence microscopy. The hydroxyproline, was assessed as marker of collagen and the glycosaminoglycans (GAGs) content was both qualitatively detected and quantitatively determined. Real-time PCR was performed to determine the gene expression level of aggrecan (ACAN), type-II collagen, and transcription factor Sox9. H₂O₂-treated cells showed pre-chondrocyte morphology on day 1 and chondrocyte pellets were formed on day 14. H₂O₂-treated cells induced greater pellet sizes and showed significantly higher content of GAGs and hydroxyproline level compared with untreated cells. The gene expression levels of ACAN, collagen type-II, and Sox9 were markedly upregulated by H₂O₂. Our findings showed for the first time that H₂O₂-containing differentiation medium is potentially more effective than H₂O₂-free differentiation medium in the induction of chondrogensis of ADSCs.