Hierarchical dual-conductive networks enable high-cell-density spheroid bioprinting for volumetric muscle loss repair.

Abstract

Volumetric muscle loss (VML) remains a significant clinical challenge, as the intrinsic regenerative capacity of skeletal muscle is insufficient for restoring large tissue defects, often leading to extensive fibrotic scarring. While 3D bioprinting enables precise control over tissue architecture, challenges remain in high-cell-density printing and in maintaining cell viability during extrusion and photocrosslinking. Herein, we report a dual-conductive, cell spheroid-laden, bioprinted hydrogel construct for in situ VML repair. Specifically, myogenically differentiated adipose-derived stem cells (M-ADSCs) were assembled with carbon nanotube-loaded short fibers (F_C) into cell spheroids (F_C-CS). These spheroids were then encapsulated within a hydrogel matrix containing additional F_C, yielding the final F_C-CS@Gel-F_C. This spheroid-based printing strategy effectively shields M-ADSCs from nozzle-induced shear stress during high-cell-density bioprinting, while the integrated F_C establishes a dual-conductive network. This network functions as an internal micro-scaffold within spheroids while forming conductive bridges between them in hydrogels, thereby enhancing electrical signal transmission, promoting cytoskeletal proteins expression, and facilitating myotube fusion. In vivo evaluation in a VML rat model demonstrates that F_C-CS@Gel-F_C significantly suppresses fibrotic scarring and accelerates regeneration of densely packed, aligned myofibers, leading to marked recovery in motor function and endurance. Transcriptomic analysis reveals extensive reprogramming of the cellular microenvironment, characterized by upregulation of myogenesis, angiogenesis, and extracellular matrix remodeling. This study presents a synergistic strategy that integrates high-cell-density bioprinting of lineage-specific spheroid microtissues with dual-conductive hydrogel networks to overcome current biofabrication limitations and enhance functional muscle restoration. STATEMENT OF SIGNIFICANCE: Volumetric muscle loss (VML) represents a persistent clinical challenge due to the limited regenerative capacity of skeletal muscle, often leading to irreversible functional impairment and fibrotic scarring. While 3D bioprinting has emerged as a powerful platform for engineering complex tissues, the field faces two critical bottlenecks: (i) achieving therapeutic cell densities without sacrificing viability, and (ii) recreating the native electrical microenvironment essential for functional muscle regeneration. In this study, we address these challenges through a synergistic strategy that integrates high-cell-density spheroid bioprinting (overcoming cell viability bottlenecks) with hierarchically structured dual-conductive hydrogel networks (mimic native muscle microenvironment). This strategy holds great promise for skeletal muscle engineering and could serve as a reference for developing constructs aimed at other electrically excitable tissues.