Non-hypertrophic chondrogenesis of mesenchymal stem cells through mechano-hypoxia programing

Cartilage tissue engineering aims to generate functional replacements to treat cartilage defects from damage and osteoarthritis. Human bone marrow-derived mesenchymal stem cells (hBM-MSC) are a promising cell source for making cartilage, but current differentiation protocols require the supplementation of growth factors like TGF-beta 1 or -beta 3. This can lead to undesirable hypertrophic differentiation of hBM-MSC that progress to bone. We have found previously that exposing engineered human meniscus tissues to physiologically relevant conditions of the knee (mechanical loading and hypoxia; hence, mechano-hypoxia conditioning) increased the gene expression of hyaline cartilage markers, SOX9 and COL2A1, inhibited hypertrophic marker COL10A1, and promoted bulk mechanical property development. Adding further to this protocol, we hypothesize that combined mechano-hypoxia conditioning with TGF-beta 3 growth factor withdrawal will promote stable, non-hypertrophic chondrogenesis of hBM-MSC embedded in an HA-hydrogel. We found that the combined treatment upregulated many cartilage matrix- and development-related markers while suppressing many hypertrophic- and bone development-related markers. Tissue level assessments with biochemical assays, immunofluorescence, and histochemical staining confirmed the gene expression data. Further, mechanical property development in the dynamic compression treatment shows promise toward generating functional engineered cartilage through more optimized and longer culture conditions. In summary, this study introduced a novel protocol to differentiate hBM-MSC into stable, cartilage-forming cells.