Mechanically Robust Injectable Hydrogel Scaffolds for the Intramuscular Delivery of Adipose-Derived Stem/Stromal Cells

Abstract

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.