Cell-based therapy is needed to overcome the lacking intrinsic ability of cartilage to heal. Generating cartilage tissue from human bone marrow-derived stromal cells (MSC) is limited by up-regulation of COL10, ALP and other hypertrophy markers in vitro and calcifying cartilage at heterotopic sites in vivo. MSC hypertrophic differentiation reflects endochondral ossification, unable to maintain a stable hyaline stage, as observed by redifferentiation of articular chondrocytes (AC). Several transcription factors (TF), are held responsible for hypertrophic development. SOX9, the master regulator of chondrogenesis is also, alongside MEF2C, regulating hypertrophic chondrocyte maturation and COL10 expression. RUNX2/3 are terminal markers driving chondrocyte hypertrophy, and skeletogenesis. However, so far regulation of these key fate determining TFs has not been studied thoroughly on mRNA and protein level through chondrogenesis of human MSC. To fill this gap in knowledge, we aim to uncover regulation of SOX9, RUNX2/3, MEF2C and other TFs related to hypertrophy during MSC chondrogenesis in vitro and in comparison to the gold standard AC redifferentiation. Expression of SOX9, RUNX2/3 and MEF2C was compared before and during 6-week chondrogenic re-/differentiation of human MSC and AC on mRNA level via qRT-PCR and protein level via Western-Blotting. Chondrogenesis was evaluated by histology at d42 and expression of chondrogenic markers like COL2. Hypertrophic development was characterized by ALP activity and expression of hypertrophic markers like COL10.Introduction
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