Recombinant proteins and cytokines are under broad preclinical and clinical investigation to promote angiogenesis, but their success is limited by ineffective delivery, lack of long-term stability and excessive cost. Mesenchymal stem/stromal cells (MSC) secrete bioactive trophic factors, and thus, may provide an effective alternative to address these challenges. Glycine-Histidine-Lysine (GHK) is a peptide fragment of osteonectin, a matricellular protein with reported proangiogenic potential. We examined the capacity of GHK to up-regulate secretion of proangiogenic factors from human MSC in culture and when covalently coupled to alginate hydrogels. GHK had no apparent cytotoxic effects on MSC in culture over a wide range of concentrations. We detected a dose-dependent increase in vascular endothelial growth factor (VEGF) concentration in media conditioned by GHK-treated MSC, which increased endothelial cell proliferation, migration and tubule formation. We covalently coupled GHK to alginate using carbodiimide chemistry, and human MSC were entrapped in alginate hydrogels to assess VEGF secretion. Similar to monolayer culture, MSC responded to GHK-modified gels by secreting increased concentrations of VEGF and basic fibroblast growth factor compared to unmodified gels. The pre-treatment of MSC with antibodies to α6 and β1 integrins prior to entrapment in GHK-modified gels abrogated VEGF secretion, suggesting that the proangiogenic response of MSC was integrin-mediated. These data demonstrate that the proangiogenic potential of MSC can be significantly increased by the presentation of GHK with a biodegradable carrier, therefore increasing their clinical potential when used for tissue repair.

The efficacy of cell-based therapies as an alternative to autologous bone grafts requires biomaterials to localize cells at the defect and drive osteogenic differentiation. Hydrogels are ideal cell delivery vehicles that can provide instructional cues via their composition or mechanical properties but commonly lack osteoconductive components that nucleate mineral. To address this challenge, we entrapped mesenchymal stromal cells (MSCs) in a composite hydrogel based on two naturally-derived polymers (alginate and hyaluronate) containing biomineralized polymeric microspheres. Mechanical properties of the hydrogels were dependent upon composition. The presentation of the adhesive tripeptide Arginine-Glycine-Aspartic Acid (RGD) from both polymers induced greater osteogenic differentiation of ovine MSCs in vitro compared to gels formed of RGD-alginate or RGD-alginate/hyaluronate alone. We then evaluated the capacity of this construct to stimulate bone healing when transplanting autologous, culture-expanded MSCs into a surgical induced, critical-sized ovine iliac crest bone defect. At 12 weeks post-implantation, defects treated with MSCs transplanted in composite gels exhibited significant increases in blood vessel density, osteoid formation, and bone formation compared to acellular gels or untreated defects. These findings demonstrate the capacity of osteoconductive hydrogels to promote bone formation with autologous MSCs in a large animal bone defect model and provide a promising vehicle for cell-based therapies of bone healing.