Fibrotic-like collagen matrices as innovative 3D in vitro models for investigating the impact of pathological ECM on muscle regeneration in muscular dystrophies

Muscular dystrophies are characterized by impaired skeletal muscle contraction due to genetic mutations. Beyond the disruption of muscle function, the extracellular matrix (ECM) surrounding muscle fibers becomes fibrotic, further impeding tissue repair and function. In this study, we designed both healthy and fibrotic matrices to examine the impact of persistent fibrosis on muscle cell behavior. Fibrotic matrices were synthesized by 3D printing of dense collagen solutions in air, followed by slow gelation to yield non-porous, isotropic hydrogels. These matrices were subsequently crosslinked using EDC/NHS chemistry, resulting in a Young’s modulus of approximately 50 kPa. The behavior of C2C12 myoblasts cultured within the fibrotic matrices was compared to cells grown in healthy matrices. Our results showed that the fibrotic matrix had a detrimental effect on cell behavior. Myoblasts were unable to differentiate into mature myotubes, exhibited poor alignment, and suffered from hypoxia. Furthermore, these cells failed to proliferate, secreted inflammatory cytokines, and were unable to remodel their ECM. Using matrices possessing a single characteristic of the fibrotic matrix (i.e stiff or non porous), the predominant factor for each feature of the cell phenotype was determined. These findings underscore the detrimental effects of a fibrotic persistent ECM on muscle homeostasis. The development of these two distinct 3D muscle models, one representing healthy muscle and the other fibrotic, offers a valuable tool for investigating the pathophysiology of muscular dystrophy.