Physical cues from the cellular microenvironment have a strong influence on the properties and fate specification of mesenchymal stem cells (MSCs) in which factors including the extracellular matrix specificity, presentation and mechanical properties all affect MSC fate via mechanotransductive signalling pathways. This has implications for the development of MSC-based tissue-engineering strategies where the challenge of efficiently directing MSC differentiation into the desired tissue type still remains. For example, although the encapsulation and subsequent differentiation of MSCs in hydrogels has many advantages, the inherent softness of hydrogels means that the mechanical properties are poorly matched to the mechanical cues that drive efficient osteogenesis. We hypothesise that by understanding and manipulating mechanotransductive signalling it will be possible to override the physical signals that MSCs receive from their environment and drive efficient differentiation.
We have previously identified a mechanosensitive miRNA signalling axis in which substrate stiffness alters levels of miR-100-5p and miR-143-3p which then act via the mTOR pathway to regulate MSC osteogenesis. Here we develop and characterise a simple system for in situ delivery of these miRNAs to hydrogel-encapsulated MSCs and test their ability to enhance osteogenic differentiation. Comparing the influence of different transfection agents and hydrogel compositions, we show that it is possible to transfect MSCs with miRNAs in situ and further determine the factors affecting transfection agent release and MSC transfection. We then compare the efficacy of both pre-transfection and in situ transfection on the osteogenic capacity of hydrogel-encapsulated MSCs, showing via increased mineralisation and osteogenic gene expression, that in situ transfected samples actually outperform their pre-transfected counterparts. Overall this system shows the potential to modulate mechanotransduction in MSCs to improve differentiation outcomes whilst developing a system that is simpler and more effective than previous protocols. We therefore believe that this strategy shows significant future promise for MSC-based tissue-engineering.