Marcellan, A. | Search bioRxiv

bioRxiv2024

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 Youngs 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. HighlightsO_LIStiff and non-porous dense collagen matrices synthesized by 3D printing recapitulate the key characteristics of fibrosis observed in muscular dystrophies C_LIO_LIFibrotic matrices negatively impact myoblast proliferation and differentiation into mature myocytes C_LIO_LIFibrotic matrices impede the 3D organotypic organization of myocytes C_LIO_LIThis 3D pathological model of skeletal muscle offers a valuable tool for investigating how persistent fibrosis influences the progression of muscular dystrophy C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/630059v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@c2403corg.highwire.dtl.DTLVardef@1074147org.highwire.dtl.DTLVardef@14eb055org.highwire.dtl.DTLVardef@11ac419_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Anisotropic dense collagen hydrogels possessing two ranges of porosity to create the adequate microenvironment for muscle bundles: a step towards skeletal muscle modeling

Despite the crucial role of the extracellular matrix (ECM) in the organotypic organization and function of skeletal muscles, most 3D models do not mimic its specific characteristics, namely its biochemical composition, stiffness, anisotropy, and porosity. Here, a novel 3D in vitro model of muscle extracellular matrix was developed to differentiate myogenic cells (C2C12 line) into myotubes and reproduce their natural cell/cell and cell/matrix interactions. An anisotropic hydrogel mimicking the perimysium was obtained thanks to unidirectional 3D printing of dense collagen with aligned collagen fibrils. The space between the different layers was tuned to generate an intrinsic porosity (100 {micro}m) suitable for nutrient and oxygen diffusion. By modulating the gelling conditions, the mechanical properties of the construct reached those measured in the physiological muscle ECM. The addition of large channels (600 {micro}m) by molding permitted to create a second range of porosity suitable for cell colonization without altering the physical properties of the hydrogel. C2C12 cells embedded in Matrigel(R), seeded within the channels, organized in 3D, and differentiated into multinucleated mature myotubes. This organization reproduced the global muscular bundles, i.e., the endomysium encompassing myotubes. These results show that porous and anisotropic dense collagen hydrogels colonized with myoblasts are promising biomaterials to model skeletal muscle. Table of contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=39 SRC="FIGDIR/small/496716v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@bae7e8org.highwire.dtl.DTLVardef@cf453corg.highwire.dtl.DTLVardef@1ca69a2org.highwire.dtl.DTLVardef@1dd13cd_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA novel extracellular matrix-like hydrogel increases the physiological relevance of the skeletal muscle model. C_LIO_LIPorous and anisotropic dense collagen hydrogels mimic the muscle ECM physical properties. C_LIO_LIUnidirectional printing of dense collagen creates a porous and anisotropic scaffold in a single step. C_LIO_LIAnisotropic dense collagen hydrogels promote C2C12 differentiation into myotubes and their 3D organization. C_LI

bioengineering↗