Macromolecular crowding for chondrogenic phenotype maintenance and stem cell differentiation
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Chondrocyte-based tissue engineering therapies require in vitro cell expansion, which is associated with loss of phenotype, decrease in synthesis of collagen type II and increase in synthesis of collagen type I. Another major obstacle in clinical translation of chondrocyte-based therapies is the lack of extracellular matrix (ECM) in the cartilage substitutes. Macromolecular crowding (MMC) is a biophysical phenomenon, based on the excluded volume effect, known to dramatically increase tissue-specific ECM deposition during in vitro culture and to modulate cell phenotype and stem cell differentiation. The effect of MMC in chondrogenesis has not been well investigated as yet. Herein, we hypothesized that MMC will increase hyaline ECM deposition in chondrocytes and favour chondrogenic, as opposed to osteogenic and adipogenic, differentiation of bone marrow mesenchymal stem cells (BMSCs). In early passage (3) human chondrocytes, MMC increased, albeit not significantly, both collagen type I and collagen type II deposition. In late passage (7) chondrocytes, re-differentiated using a commercial medium, MMC significantly increased collagen type I deposition, whilst almost no collagen type II was detected. These data suggested that the commercial medium used was not able to correctly restore cell phenotype and that the optimal medium for expansion of adult chondrocytes is still elusive. MMC was then assessed in the pre-condition and/or differentiation media of human BMSCs. A moderate increase in collagen type II and chondroitin sulphate, a lower increase in collagen type I and collagen type X and an unaffected Sox-9 expression were detected, suggesting that carrageenan, the highly sulphated crowder used, enhanced chondrogenesis. Osteogenesis was also enhanced, as evidenced by increased mineralisation, collagen type I deposition and osteopontin expression. Adipogenesis was not affected. Collectively, these data provide further knowledge on the use of MMC for the modulation of chondrogenic phenotype maintenance and stem cell differentiation.