Aspiration-assisted intra-operative 3D bioprinting of scaffold-free modular units for skeletal muscle tissue regeneration

Traumatic muscle injury associated with volumetric muscle loss results in reversible muscle regeneration and function. Bottlenecks to conventional tissue engineering strategies to augment muscle regeneration are limited by low cell transplantation capacity by conventional injection of dissociated cells, the lack of customization of engineered tissues based on muscle defect geometry, as well as the feasibility of intra-operative use. Here, we developed a facile approach to engineer modular units of scaffold-free high-density muscle tissues in customizable geometric shapes and sizes with integration potential. For the first time, we describe aspiration-assisted intra-operative bioprinting of scaffold-free modular units for preclinical treatment of volumetric muscle loss. We demonstrated the ability of 3D modular muscle units to be bioprinted to the site of the muscle defect, leading to improved muscle, vascular, and neuromuscular regeneration in a mouse model of volumetric muscle loss. Transcriptional profiling revealed that the scaffold-free muscle units enable pre-formed cell-cell interactions that support myogenesis, unlike dissociated cells that lack cell-cell interactions. This technology of intraoperative modular muscle unit implantation is promising for clinical use for precise bioprinting of scalable tissues from geometrically tunable modular units. RNA Sequencing was performed on modular muscle tissue units composed of either murine C2C12 myoblasts or primary human muscle cells. For the murine muscle tissue units formed from C2C12 myoblasts, the experimental groups consisted of muscle units formed from cells with pre-differentiation treatment as were denoted as follows: Group I: C2C12 myoblasts were used to fabricate tissue strands on day 0 without prior differentiation and then maintained in the mold in differentiation media for 5 days. Group II: C2C12 cells were subjected to 2 days of pre-differentiation on tissue culture plastic before 3D tissue fabrication for an additional 3 days. Group III: C2C12 cells were subjected to 4 days of pre-differentiation on tissue culture plastic for 4 days before 3D tissue fabrication for an additional 1 day (n=4-5 per group). To study the molecular effects by which pre-formed cellular interactions present within the modular muscle units may prime muscle myogenesis after implantation, RNA Sequencing was performed on human muscle units composed of primary human muscle cells cultured within rectangular solid-shaped molds for 2 days in differentiation media prior to RNA isolation. As a basis for comparing to pre-formed inter-cellular interactions within the modular unit in the setting of VML, an additional group consisted of primary human muscle cells cultured for 2 days in differentiation media were suspended in ultralow adhesion dishes, where there was negligible cell-cell interaction (n=3 per group).