Muscle-specific acellular ECM fibers made with anchored cell sheet engineering support regeneration in rat models of volumetric muscle loss.

Volumetric muscle loss (VML), a condition affecting millions due to trauma, represents a critical unmet need in regenerative medicine, with no established standard of care. This study introduces a de novo therapeutic strategy using tissue-specific skeletal muscle acellular extracellular matrix (aECM) fibers fabricated using scaffold-free Anchored Cell Sheet Engineering technique. These engineered fibers replicate the native ECM composition and microarchitecture of skeletal muscle, incorporating essential structural and basement membrane proteins. In a rat VML model, aECM demonstrated promising regenerative capacity compared to commercial porcine-derived small intestine submucosa (SIS) ECM. Over an 8-week period, compared to contralateral muscle, aECM preserved muscle volume and weight, regulated inflammatory and fibrotic responses, and promoted vascularization. In contrast, SIS was rapidly degraded by week 4 and associated with fibrosis. Force recovery in aECM was lower at the 8-week time point (77 % compared to 91 % in control), but histological and immunohistochemical analyses revealed newly formed, dispersed muscle fibers exclusively in aECM treatment. Importantly, muscle weight was preserved only when aECM was used, resulting in similar normalized force-to-weight across all groups (87 % in aECM vs. 88 % in SIS). The histological analyses further demonstrated ongoing tissue remodeling, indicative of sustained regeneration, in contrast to the premature fibrotic healing observed in the other groups. An innovative quantitative image analysis workflow enabled assessment of spatial tissue heterogeneity through histology and immunohistochemistry images, setting a new standard for regeneration analysis. These findings establish engineered tissue-specific aECM as a transformative approach for VML treatment, laying the groundwork for translation to clinical applications. STATEMENT OF SIGNIFICANCE: The current study introduces a transformative approach to treating volumetric muscle loss (VML) through the development of tissue-specific acellular extracellular matrix (aECM) fibers engineered using a scaffold-free biofabrication platform uniquely suited for recreation of such aECM components. The engineered fibers represent a significant advancement over current commercial options by recreating native ECM composition and microarchitecture while eliminating complications associated with xenogenic materials. Through comprehensive in vivo evaluation in a rat model, it is demonstrated that these engineered fibers maintain muscle mass and promote controlled tissue regeneration, addressing key limitations of existing treatments. The scaffold-free biofabrication of tissue-specific aECM provides a new paradigm for biomaterial design in regenerative medicine.