| An emerging treatment for peripheral arterial disease is the transplantation of adipose-derived stem/stromal cells (ASCs) by intramuscular (IM) injection to improve limb perfusion by paracrine-mediated angiogenesis and immunomodulation. However, effective clinical translation has been limited due to poor cell retention and survival in ischemic muscle. This thesis focused on the design and in vitro/in vivo evaluation of an injectable hydrogel to improve ASC retention and function following IM delivery.;Initial work developed an injectable, in situ-gelling hydrogel designed specifically to encapsulate ASCs and withstand the mechanical loading of the IM environment. The hydrogel consisted of two polymers: methacrylated glycol chitosan functionalized with a cell-adhesive RGD peptide, selected to support ASC retention and survival, and a triblock copolymer of poly(trimethylene carbonate)-b-poly(ethylene glycol)-b-poly(trimethylene carbonate) diacrylate, designed to improve mechanical resilience. Following in vitro injection and crosslinking within the hydrogel, encapsulated human ASCs demonstrated high viability (>90%) over two weeks under normoxic and hypoxic (2% O2) conditions. The release of angiogenic and chemotactic cytokines from encapsulated ASCs was enhanced under hypoxia, suggesting that the hydrogel can support ASC retention and paracrine function under ischemic conditions.;Subsequent studies investigated the angiogenic and inflammatory responses to the IM injection of allogeneic rat ASCs in the hydrogel, the hydrogel alone, and ASCs in saline using a healthy, immunocompetent rat model. Immunohistochemical analysis over 4 weeks demonstrated that encapsulated ASCs were retained at a higher density and promoted CD68+ macrophage recruitment, as well as M1 (CD68+CCR7+) macrophage infiltration and M2c (CD163+) macrophage polarization at the hydrogel periphery. Coincident with enhanced macrophage infiltration, significantly more blood vessels were observed surrounding and within the hydrogels with ASCs compared to the hydrogels alone.;The therapeutic effects of the IM injection of human ASCs in the hydrogel, the hydrogel alone, ASCs in saline, and saline alone were compared in an immunocompromised NOD/SCID mouse model of hindlimb ischemia. While there were no differences observed in limb perfusion or function at 28 days, immunohistochemical analysis revealed that hydrogel-delivered ASCs were well retained and significantly improved the IM capillary density and enhanced cell proliferation, indicating that the cell delivery approach stimulated localized angiogenesis. |
