Aging significantly alters cellular mechanics and mitochondrial physiology, with chronic low-grade inflammation (inflammaging). However, its role in skeletal muscle atrophy and fibrosis is poorly understood. This study addressed the unresolved mechanism using a 2.5D coculture model of RAW264.7 macrophages and C2C12 myoblasts, exposed to lipopolysaccharide (LPS, a fibrosis inducer), with a focus on myogenesis, fibrogenesis, cellular stiffness, and mitochondrial metabolism. Paracrine signals from LPS-stimulated macrophages decreased myogenic markers MyHC and MyoG, increased fibrosis markers, and elevated fibrotic cell stiffness. Mitochondrial metabolism was disrupted, indicated by lowered maximal respiration and increased proton leak, demonstrating impaired energy production. To explore the alleviation of muscle atrophy and promote regeneration, a biomaterial-based therapeutic approach involving the use of pirfenidone (PFD, pulmonary antifibrotic drug)-loaded hydrogels composed of silk fibroin and agarose was investigated. Treatment reduced fibrotic stiffness by ∼40%, increased myotube formation by 33%, improved mitochondrial function, and restored mitochondrial structure, with a 20% increase in maximal respiration and a 50% decrease in proton leak in the seahorse assay. Sustained release of PFD from tissue-mimicking hydrogels effectively suppressed the expression of fibrotic markers such as α-SMA and COL1 while simultaneously increasing the expression of myogenic genes. RNA transcriptomics further corroborated the upregulation of myogenic pathways and the downregulation of fibrogenic signaling. This study highlights the potential of PFD-loaded hydrogels as a novel therapeutic strategy to target inflammation-induced muscle fibrosis and promote skeletal muscle regeneration, demonstrating prevention of fibrotic progression in the established inflammation-induced reversal of established fibrosis in vitro, with promising translational potential for treating sarcopenia. • A macrophage-myoblast coculture model was developed to recapitulate inflammaging-driven fibrosis in skeletal muscle in vitro • Pro-inflammatory macrophages induced myoblast-to-myofibroblast phenotypic transition, a key step in fibrosis progression • Inflammaging disrupts cellular bioenergetics and alters the mechanical microenvironment • We investigated a natural polymer–based, tissue-mimicking hydrogel for sustained drug delivery that restores mitochondrial function and reduces fibrotic stiffness • Therapeutic intervention prevented progression in the established disease model and enhanced the myogenic differentiation pathway • This biomaterial strategy targets both bioenergetic and biophysical drivers of fibrosis, with translational potential for regenerative medicine
Gopinath et al. (Sun,) studied this question.