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Description
Skeletal muscle exhibits a highly organized, anisotropic architecture, where fascicles - bundles of aligned myofibers - are embedded within a connective tissue matrix. Such structural organization is the key for skeletal muscle achieving uniaxial contractions, mechanical stability, and functional performance in vivo.
The presented engineered skeletal muscle (eSM) platform aims the development of biomimetic fascicles through the co-culture of myoblasts and fibroblasts, integrated via a novel aqueous two-phase system (ATPS)-based hydrogel interface formulation. Highly anisotropic in vitro skeletal muscle fascicles were biofabricated using a stable ATPS biomaterial ink for the shell region and a fibrin-based bioink for the core, via rotary wet spinning (RoWS)1,2. The ATPS composition was specifically designed to entrap thermoresponsive hydrogel droplets (THD) within a fast-crosslinkable alginate matrix during fiber formation, allowing THD sacrificial removal under incubation conditions. Upon incubation, the THDs gradually dissolve, creating a porous shell network. This porosity improves nutrient diffusion and metabolic waste removal, and more importantly, it enhances biochemical crosstalk between the external environment and the encapsulated myogenic cells in the fiber core.This porous infrastructure not only facilitates mass transport but also establishes a biologically active interface that supports signal exchange and cellular communication.
The physiologically relevant biocomposition of fascicles was developed by introducing a co-culture system. Myoblasts were embedded within the fibrin-based fiber core, while fibroblasts were seeded onto the porous outer surface of the fibers. This design mimics the native distribution of myofibers encased in a collagen-rich extracellular matrix produced by fibroblasts. The interplay between myoblasts and fibroblasts promotes both myogenic differentiation and matrix remodeling, thereby recapitulating the organization and mechanical support observed in native muscle. Immunofluorescence and proteomic analysis of myogenic markers and extracellular matrix proteins was used to evaluate fascicle maturation and validate the biomimetic co-culture strategy.
Our robust and tunable strategy for the development of highly anisotropic in vitro skeletal muscle models emphasizes biochemical integration, architectural fidelity, and functional maturation. By leveraging ATPS-enabled porosity and co-culture dynamics, our eSM model advances current paradigms in skeletal muscle biofabrication, with implications for biorobotics, cultured meat development, disease modeling, drug screening, and regenerative therapies.
1Reggio et al. (2025), 3D Rotary Wet-Spinning (RoWS) Biofabrication Directly Affects Proteomic Signature and Myogenic Maturation in Muscle Pericyte–Derived Human Myo-Substitute. Aggregate, 6: e727.
2 Celikkin et al (2023), Combining rotary wet-spinning biofabrication and electro-mechanical stimulation for the in vitro production of functional myo-substitutes, Biofabrication, 15 045012
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