Modulating mesenchymal stromal cells chondrogenesis through TGF-β1 pre-conditioning during monolayer expansion in pellet culture and in a 3D mechanical load model

Bone marrow–derived mesenchymal stem cells (MSCs) represent a promising source for cell-based regenerative cartilage therapies. However, in vitro chondrogenic differentiation of MSCs varies widely between donors, posing a significant challenge to the clinical translation of these therapies. Here, we explored the potential of pre-conditioning MSCs with TGF-β1 during monolayer expansion in order to enhance chondrogenic differentiation in poorly responsive MSCs and reduce donor-to-donor variability. MSCs from six donors were expanded in monolayer using standard methods. For the final two days of expansion, half of the cells were pre-conditioned with 10 ng/mL TGF-β1, whereas the others were kept in standard expansion medium. MSCs were then detached for 28 days of chondrogenic differentiation in standard pellet culture in chondrogenic medium or in a 3D mechanically induced chondrogenesis model in polyurethane-fibrin scaffolds. After 28 days, samples were collected for gene expression analysis, sulphated glycosaminoglycan (sGAG) assay, DNA quantification and histological evaluations (Safranin-O/Fast green staining). Two days of TGF-β1 pre-conditioning during MSC expansion increased COL2A1 expression after 28 days of pellet culture ( P = 0.0217), but also elevated COL10A1 ( P = 0.0293), leaving the COL2A1/COL10A1 ratio unchanged ( P = 0.8522). There was a trend for an increased COL2A1/COL1A1 ratio and an increased ACAN expression ( P = 0.0837). Pre-conditioning had no effect on sGAGs retained in the pellets or released into the medium over the 28 days but slightly enhanced the Safranin-O staining on histological sections. When chondrogenesis was mechanically induced without exogenous TGF-β1, pre-conditioning had no effect on COL2A1 , COL10A1 , COL1A1 , or ACAN gene expression. Similarly, no differences were observed in sGAG content (retained or released) or in Safranin-O staining. TGF-β1 pre-conditioning enhanced some chondrogenic markers in static pellet culture, but these effects were small and did not translate to the more clinically relevant model of mechanically induced chondrogenesis. Furthermore, TGF-β1 pre-conditioning was associated with increased COL10A1 expression, indicating a concurrent promotion of hypertrophic differentiation.