| Mesenchymal stem cells (MSCs) have the potential to revolutionize regenerative medicine. Ultimately, when combined with a photopolymerizable, injectable polyethylene glycol) (PEG)-based hydrogel scaffold, MSCs can be delivered to bone defects throughout the body in a minimally invasive manner. Advantages of this technique include complete spatial and temporal control over the polymerization process and the facile exchange of nutrients and wastes between encapsulated cells and their surrounding environment.; In this research, we first characterized the osteogenic differentiation of human MSCs (hMSCs) in two-dimensional culture and gained insights into how the scaffold might be best designed for the three-dimensional photoencapsulation of hMSCs. Unfortunately, when hMSCs were photoencapsulated in PEG hydrogels, cell viability was low. Viability was dramatically improved when either the well-known cell-adhesive RGD peptide sequence or a phosphate-containing, photoreactive charged molecule was incorporated into the hydrogel network. Both of these approaches led to improved viability up to at least 4 weeks in culture. Furthermore, phosphate-containing PEG hydrogels led to a mineralized region similar in composition and structure to biological apatites.; Next, the osteogenic differentiation of hMSCs in these cell-permissive hydrogels was investigated with respect to the expression of various osteogenic genes. While differentiation could be achieved by adding the osteogenic factor dexamethasone in media surrounding gel-encapsulated hMSCs, our goal was to develop an osteogenic PEG-based scaffold that would release dexamethasone to the encapsulated cells in a sustained and local manner. In this way, dexamethasone would not be needed in the surrounding media.; To synthesize an osteogenic scaffold, dexamethasone was covalently linked to a mono-methacrylated PEG molecule through a hydrolytically-degradable bond. When co-polymerized with the photocrosslinkable PEG, this molecule was covalently incorporated into the hydrogel network. Over time, these pendent dexamethasone molecules were released into the surrounding gel environment. This led to osteogenic differentiation of encapsulated hMSCs, as measured by increased gene expression of the important osteogenic transcription factor core binding factor alpha I.; Finally, the interactions of hMSCs with and the limitations of degradable PEG-based scaffolds were investigated and discussed. The studies presented herein represent the founding studies for using hMSCs in photopolymerizable PEG scaffolds for bone regeneration. |
