Amphiregulin-Releasing Hydrogels for Treating Ischemic Muscle Injuries​

• Caroline LaSalle, Biomedical Engineering, University of Delaware

• Brian Kwee, Biomedical Engineering, University of Delaware

Peripheral artery disease (PAD) causes reduced blood flow, leading to ischemia. Regulatory T-cells (Tregs) are key immune cells that respond to injury sites of ischemic injuries to modulate inflammation. Amphiregulin (AREG), an anti-inflammatory cytokine, can activate Tregs and promote regeneration, making it a promising therapeutic for tissue repair. Biomaterials, such as alginate hydrogels, can locally deliver AREG to the sites of ischemic injuries to promote skeletal muscle repair. Highly oxidized alginate hydrogels were fabricated with a 3:1 ratio of very low viscosity alginate (VLVG) to medium viscosity alginate (MVG), crosslinked with 50 mM calcium, and loaded with varying concentrations of AREG. The hydrogels’ release kinetics were analyzed in vitro over 14 days. Media samples were collected at various timepoints and assessed using an ELISA to determine the hydrogel with the highest release of AREG. Following in vitro analysis, the hydrogel was evaluated in vivo using a murine hindlimb ischemia model with blank and treated groups. Blood perfusion return was monitored using laser speckle perfusion imaging, and necrosis progression was graded visually at various timepoints over 14 days. At the endpoint of the study, tibialis anterior (TA) muscle length was measured and compared between the blank and treated groups. The ELISA confirmed sustained release of AREG for all conditions with increasing oxidation and dosage resulting in a greater release of AREG. In vivo, AREG-treated mice showed no significant increase in blood perfusion return compared to the injured controls. However the AREG-treated mice exhibited reduced necrosis and had a significantly longer TA muscle. These findings suggest that AREG releasing hydrogels have the potential to treat ischemic muscle injuries.