CIRCULATING MIRNA FRACTURE-RELATED BIOMARKERS: MIR-335-5P REGULATES ENDOCHONDRAL DIFFERENTIATION IN HUMAN BONE MARROW MESENCHYMAL STROMAL CELLS VIA REGULATION OF TGF-β SIGNALLING COMPONENTS

Fracture non-union and delayed fracture healing affect up to 10% of fracture patients, leading to significant functional and psychological disability. Identifying biomarkers to predict the risk of healing delays is of immense clinical interest. During endochondral ossification, which occurs in mechanically unstable regions with a higher risk for delayed fracture healing, bone regenerates through a cartilage intermediate. Previous studies identified miR-1246, miR-335-5p, and miR-193a-5p as fracture-related biomarkers, but their functional roles in direct ossification models were not evident. This study aims to validate the role of these miRNAs in the endochondral differentiation of human bone marrow-derived mesenchymal stromal cells (BMSCs). The activity of miR-1246, miR-335-5p, and miR-193a-5p was transiently inhibited before pellet formation and the start of chondrogenic differentiation in human BMSCs from five donors. The effects of miRNA inhibition were assessed through histology, immunohistochemistry, and gene expression analysis via bulk RNA sequencing and RT-qPCR. The transient inhibition of miR-1246 and miR-335-5p during the early stages of human chondrogenesis in a 3D pellet culture model resulted in a donor-dependent increase in cartilage formation, evidenced by increased Safranin-O, type II, and type X collagen staining. RNA sequencing and further RT-qPCR validations revealed that downstream targets of miR-335-5p inhibition include regulators of TGF-β signalling, such as GDF5, CCN5, and SKIL genes. These findings suggest that miR-335-5p has a regulatory effect on endochondral ossification. Its function in human bone formation and repair should be further investigated to validate its potential role as a prognostic marker in fracture healing. The study highlights the importance of miRNA regulation in the early phases of endochondral ossification and its potential implications for improving fracture healing outcomes.