Abstract
Human mesenchymal stem cells (MSCs) have the capacity for self-renewal, immune evasion and suppression, and differentiation. These characteristics have brought MSCs to the forefront of wound healing research. Chronic wounds have stalled in the inflammatory phase, and thus fail to heal. There is enormous potential for MSCs to promote healing in such stalled wounds by reducing inflammation, promoting collagen deposition and growth factor production. In spite of the high potential of MSCs the actual application has proven difficult, partly due to lack of effective methods to control differentiation and survival of these cells when transplanted into the wound bed. Studies in in vitro culture have indicated the involvement of the extracellular matrix (ECM) in MSC proliferation and attachment. Integrins are the cell surface receptors that recognize motifs within the ECM, allowing for attachment and signaling. Considering the crucial role of integrins in key cellular processes such as proliferation/survival, activation and differentiation, we hypothesized that targeting specific integrins might modulate one or more of these key cellular processes in MSCs. To test this hypothesis, MSCs were characterized from passage 5 through 12 from two donors to determine the base-line expression of key cellular markers, including integrins. Following characterization the synthetic linear peptide LLP2A was used to target integrin α4β1. Integrin α4β1 was targeted due to its involvement in bone homing, migration, and engraftment of MSCs. LLP2A was investigated in classical 2D culture utilizing neutravidin as an anchor for presentation to MSCs. However, the results were inconsistent and lead to a shift to 3D culture model using LXW7, a cyclic peptide specific for integrin αVβ3. For 3D culture beads coated with LXW7, an RGD-like ligand that specifically targets integrin αVβ3, were used in a hanging-drop-like set up, which forced the cells to interact with the peptide. Droplet formation had to be optimized in ultra-low binding tissue culture plates to maximize peptide and cell interaction. Following titration of the bead and cell number the cell aggregates were cultured for ten days and showed increased expression of genes related to smooth muscle differentiation, namely calponin and myosin, and ECM components. These results demonstrated that activation of a single integrin induces change in MSCs in the 3D model, validating the model for future study with alternative peptides. This has therapeutic implications for wound healing as peptide induced integrin activation can be incorporated in tunable hydrogels for delivery into a wound bed.