Abstract
AbstractHydrogels have many properties that emulate biological tissues and are therefore attractive candidates for use in tissue engineering. In particular the encapsulation and subsequent differentiation of mesenchymal stem/stromal cells (MSCs) is a strategy that holds great promise for the repair and regeneration of bone and cartilage. However, MSCs are well-known for their sensitivity to mechanical cues, particularly substrate stiffness, and so the inherent softness of hydrogels is poorly matched to the mechanical cues that drive efficient osteogenesis. This limits the success of bone tissue engineering using MSCs encapsulated in a hydrogel. One approach to overcome this limitation is to harness mechanotransductive signalling pathways and override the signals cells receive from their environment. Previous reports have shown that the mechanosensitive miRNAs, miR-100-5p and miR-143-3p can enhance MSC osteogenesis, but this required a complex multi-step procedure to transfect, encapsulate and differentiate the cells. In this study, we develop and characterise a facile system for in situ transfection of MSCs encapsulated within a light-crosslinkable gelatin-PEG hydrogel. Comparing the influence of different transfection agents and hydrogel compositions, we determine the factors affecting transfection agent release and MSC transfection, showing that it is possible to transfect MSCs with miRNAs in situ. We then compare the efficacy of both pretransfection and in situ transfection on the osteogenic capacity of hydrogel-encapsulated MSCs, demonstrating superior mineralisation and osteogenic gene expression for in situ transfected samples. Our platform therefore demonstrates a simple, one-pot system for delivery of pro-osteogenic miRNAs and in situ transfection that is able to enhance MSC osteogenic potential without the need of multi-step transfection procedures, thus demonstrating significant promise for bone tissue engineering.
Publisher
Cold Spring Harbor Laboratory