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

Abstract

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