Controlled Release of Amphiregulin from Alginate Hydrogels for Treating Ischemic Muscle Injuries

Introduction: Ischemia, the loss of blood flow, as a result of injury or disease affects millions of people per year. Ischemia in skeletal muscle, in particular, can lead to muscle degeneration, muscle denervation, and tissue necrosis. Amphiregulin (AREG), a cytokine that can stimulate the growth of muscle stem cells, blood vessel cells, and motor neurons, may be a potent therapeutic for treating ischemic muscle injuries. Furthermore, AREG can activate regulatory T-cells (Tregs), which are anti-inflammatory immune cells that can also promote muscle regeneration. As a treatment of ischemic muscle injuries, we engineered an injectable, oxidized (degradable by hydrolysis), calcium crosslinked alginate hydrogel that provides sustained release of AREG.

Materials and

Methods: To engineer our hydrogel, we utilized alginate of very low viscosity (VLVG) and medium viscosity (MVG), oxidized at 1%, in a ratio of 75:25 VLVG:MVG. We loaded the hydrogel with 2 µg of AREG and crosslinked the hydrogel with calcium sulfate dihydrate. We tested the release of AREG from the alginate hydrogel with 25 mM calcium crosslinking, 50 mM calcium crosslinking, and 50 mM calcium crosslinking with the addition of 0.1 mg/mL Laponite-RD. We tested the injectability of the hydrogel through a 25-gauge needle at 25 mM calcium crosslinking and 50 mM calcium crosslinking for in vivo delivery. We surgically induced hindlimb ischemia in female BALB/c mice by ligating the femoral artery, vein, and nerve. 24 hours post-surgery, we injected 50 µL of alginate hydrogel with or without 2 µg AREG into the thigh and calf muscles of the ischemic limb . We performed laser speckle blood perfusion imaging at day 0,3,7 and 14 post-surgery and quantified the blood perfusion ratio of the ischemic and non-ischemic foot. We qualitatively determined the progression of necrosis in the toes of the ischemic limb over 14 days. On day 14, we measured the in-situ contractile force of the tibialis anterior (TA) muscle upon direct electrical stimulation of the muscle or the sciatic nerve innervating the TA muscle. Ischemic muscles were excised and fixed in 4% PFA, weighed.

Results, Conclusions, and Discussions: Incorporation of AREG into the alginate hydrogel resulted in moderate burst release followed by sustained, linear release over 28 days. The incorporation of 0.1 mg/mL Laponite-RD into the alginate hydrogels reduced the burst release of AREG. The release of AREG over 28 days was not significantly different between the 25 mM crosslinked hydrogel and the 50 mM crosslinked hydrogel. The 25mM calcium crosslinking concentration was chosen for in vivo experiments due to ease of injection through a 25-gauge needle. Injection of the alginate hydrogel loaded with 2 µg of AREG into the ischemic limb of mice resulted in no significant enhancement in blood perfusion return to the ischemic foot over 14 days relative to the blank alginate control. Compared to the blank alginate control, the AREG treated mice had less progression of necrosis in their ischemic limb over 14 days. Upon direct muscle stimulation, ischemic TA muscles demonstrated no significant difference in twitch or maximum tetanic force when treated with AREG or blank hydrogels. When stimulating the sciatic nerve, the TA muscles demonstrated significantly higher twitch when treated with AREG hydrogels compared to blank hydrogels, but showed no significant differences in terms of tetanic force. This suggests that AREG delivery may enhance innervation of the ischemic TA muscle. In conclusion, this study yielded an injectable alginate hydrogel to deliver AREG in vivo for the treatment of hindlimb ischemia in mice. Although delivery of AREG from alginate hydrogel in vivo did not increase blood perfusion of the ischemic limb, the AREG hydrogel slowed the progression of necrosis. AREG delivery did not significantly influence the maximum tetanic force of the TA muscle. However, the enhancement in twitch force with nerve stimulation indicates potential for improved innervation. Future experiments will engineer an alginate hydrogel with more rapid release of AREG from the hydrogel, as less than half of the AREG dose comes out after 28 days Future studies will further analyze the skeletal muscle, blood vessel, and motor neuron regeneration through histology and evaluate immune cells recruited to the ischemic muscle site with flow cytometry.

Acknowledgements (Optional): This was supported by the Delaware Center for Musculoskeletal Research from the National Institute of Health’s National Institute of General Medical Sciences under grant number P20GM139760, as well as by the University of Delaware Biomedical Engineering Department start up funds.