Modulation of miRNA-16-5p in adipose-derived stem cells in a tendon mimetic microenvironment results in a differential regulation of tenogenic and fibrosis markers

Tendons heal poorly after injury, and the molecular mechanisms governing tendon healing remain incompletely understood. Among the regulatory molecules involved in tissue repair, microRNAs have emerged as important modulators of gene expression and represent attractive candidates for directing tendon regeneration. In this study, we investigated the in situ delivery of miRNA-16-5p to modulate tendon-associated gene expression in encapsulated human adipose-derived stem cells (hASCs) using anisotropic gelatin methacryloyl (GelMA) hydrogels as tendon-mimetic culture platform. For this, hASCs were encapsulated in the GelMA hydrogels containing miRNA-16-5p mimics or inhibitors formulated as transfection complexes. Hydrogel retention of the miRNA complexes, transfection efficiency, SMAD3 modulation, and the expression of tendon-associated genes were evaluated over 21 days of culture. The hydrogels retained the miRNA complexes during the initial culture period and supported in situ transfection of the encapsulated hASCs. Delivery of miRNA-16-5p mimics resulted in sustained downregulation of SMAD3 expression for up to 14 days and was associated with increased expression of TNMD , TNC , and DCN , together with reduced COL3A1 expression, compared with the miRNA-16-5p inhibitor group. Together, these findings show that, modulation of miRNA-16-5p influences tendon-associated gene expression of the hASCs cultured within a tendon-mimetic microenvironment, and provide a foundation for further studies investigating the role of this miRNA in tendon biology.