Donor-dependent regulation of type II and X collagen deposition by early modulation of miR-335-5p and miR-1246 during chondrogenic commitment.

BACKGROUND: Identification of biomarkers to predict the risk of healing delays are of huge clinical interest since 10% of fracture patients progress to delayed or non-union. During endochondral ossification, which takes place in mechanically unstable regions, the bone regenerates through a cartilage intermediate. We previously identified miR-1246, miR-335-5p and miR-193a-5p as fracture-related biomarkers in patient serum, but they appear not to have a functional role in an in vitro model of direct ossification. However, their involvement in other processes related to fracture healing cannot be ruled out and the most common healing process in fracture repair is secondary healing by way of endochondral ossification. Therefore, this study aims to explore the role of miR-1246, miR-335-5p and miR-193a-5p during in vitro endochondral differentiation of human bone marrow-derived mesenchymal stromal cells (BMSCs). METHODS: The activity of miR-1246, miR-335-5p, and miR-193a-5p was transiently inhibited just before pellet formation and the start of chondrogenic differentiation in human BMSCs (n = 5 donors), serving as a model for early endochondral ossification. The effect of miRNA inhibition was assessed by histology (Safranin O/Fast Green), immunohistochemistry (type II and type X collagen), and gene expression analysis by bulk RNA sequencing and RT-qPCR. RESULTS: Inhibition of miR-1246 and miR-335-5p enhanced chondrogenic and hypertrophic differentiation in BMSCs from three out of five donors, while miR-193a-5p inhibition had minimal effect. Donors were categorized as "responders" or "non-responders" based on histological and gene expression profiles. RNA sequencing and RT-qPCR identified differentially expressed genes, including a 1.6 and 1.5-fold upregulation of GDF5 and CCN5 respectively (p < 0.05) and downregulation of SKIL (fold change: 1.3, p = 0.0563) after miR-335-5p inhibition, while the same genes were unaltered by miRNA inhibition in non-responders, suggesting donor-specific responses to miRNA inhibition during early chondrogenesis. CONCLUSIONS: These results suggest that miR-335-5p and miR-1246 have a regulatory effect on endochondral ossification, and genes regulated by miR-335-5p are involved in TGF-β signalling. The function of these miRNAs in human bone formation and repair should be further investigated to validate their potential role as prognostic markers in fracture healing.