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IntroductionVolumetric muscle loss (VML) is characterized by permanent tissue impairment resulting from critically-sized muscle loss. We performed time-series transcriptomic and proteomic analyses to reveal key mediators of irreversible pathological remodeling after induction of VML in mice. MethodsThe dynamics of gene and protein expression patterns were analyzed for up to 3 weeks after muscle injury. ResultsRNA Sequencing revealed transcriptional patterns that show rapid upregulation or downregulation shortly after injury, among which a subset of genes failed to return to pre-injury levels within 3 weeks after VML. Time-series analysis revealed gene clusters with sustained upregulation after 3 weeks, including those associated with extracellular matrix remodeling and inflammation, whereas the gene clusters having sustained downregulation were associated with mitochondrial function and metabolism. We further identifiedSPI1andSP1as novel molecular mediators of the pathological remodeling process. DiscussionThis work demonstrates the utility of time-series analysis to reveal dysregulated pathways in the setting of VML. 

Abstract Traumatic muscle injuries associated with volumetric muscle loss (VML) are characterized by muscle loss beyond intrinsic regeneration capacity, leading to permanent functional impairment. Experimental therapies to augment muscle regeneration, such as cell injection, are limited by low cell transplantation capacity, whereas conventional engineered muscle tissue transplants lack geometric customization to conform to the shape of the muscle defect. Here, a facile approach to engineer scaffold‐free high‐density muscle tissues in customizable geometric shapes and sizes with high cell viability and integration potential is developed. Using a facile mold‐based approach to engineer scaffold‐free modular units, transcriptional profiling is performed to uncover the role of pre‐formed cell–cell interactions within scaffold‐free muscle bioconstructs on myogenesis, an the efficacy of muscle bioconstructs in a mouse model of VML is then evaluated. RNA sequencing revealed that pre‐formed cell–cell interactions supported myogenic pathways related to muscle contraction and myofibril assembly, unlike dissociated monodisperse cells. This work further demonstrates the therapeutic efficacy of 3D rectangular solid‐shaped scaffold‐free transplants in improving muscle function and vascular regeneration. Finally, toward clinical translation, the feasibility of this technology to integrate with medical imaging and artificial intelligence‐driven customized bioconstruct design and assembly for intraoperative use is illustrated.