Myogenesis and Inflammatory Response of Fibro-Adipogenic Progenitors are Regulated by Hydrogel Biophysical Properties

Stem cell-based interventions to treat volumetric muscle loss (VML) and other skeletal muscle disorders offer promising avenues for muscle repair. Fibro-adipogenic progenitors (FAPs) support satellite cell function, yet under dysregulated conditions, FAPs can contribute to pathological adipogenesis and fibrosis. FAPs are highly responsive to biophysical cues of their environment that can alter cell fate, and their responsiveness to such cues can be readily interrogated using hydrogels. However, current approaches to study FAPs are often limited by the tunability of underlying substrates. Gelatin hydrogels exhibit favorable cell compatibility but are relatively elastic. In contrast, alginate hydrogels possess more physiologically relevant viscoelastic properties but lack essential cell-adhesive ligands. To overcome these limitations, we blended alginate into horseradish peroxidase-catalyzed gelatin hydrogels (cGA), forming a viscoelastic interpenetrating polymer network (IPN) structure termed ciGA, through covalent cross-linking of gelatin and ionic crosslinking of alginate. These hydrogels mimic the native mechanical properties of muscle, providing enhanced stiffness, tunable gelation, and improved viscoelasticity. ciGA hydrogels supported FAP viability, proliferation, and spreading while preserving mitochondrial dynamics, enhancing metabolic activity, and potentiating pro-myogenic paracrine functions. While elastic hydrogels promoted myotube formation and M1-like macrophage polarization, FAPs entrapped in ciGA did not induce macrophage polarization, suggesting their suitability for applications following the inflammatory phase. These findings highlight viscoelastic hydrogels as promising biomaterials for muscle repair, capable of supporting key stromal cell functions and modulating regenerative signaling via biophysical cues without compromising immunogenicity.