Highly Porous Foam Scaffolds Imprinted with human iPSCs Regenerate Skeletal Muscle and Improve Function Following Volumetric Muscle Loss

PURPOSE: Volumetric muscle loss (VML) is a composite loss of skeletal muscle tissue (greater than 20%) that heals with minimal muscle regeneration, substantial fibrosis, and subsequent functional deficits (1,2). Standard treatment, involving free functional muscle transfer and physical therapy, cannot restore full muscle function following VML (3). Tissue engineered scaffolds, 3D structural templates that mimic native extracellular matrix, are promising to enhance functional muscle formation and recovery (4,5). Bioprinted 3D scaffolds, engineered using scaffolding material and stem cells, can replicate skeletal muscle architecture with control over cellular alignment and differentiation. METHODS: The present study evaluates a highly porous, 3D-bioprinted scaffold containing engineered muscle fibers developed from human induced pluripotent stem cell-derived myogenic precursor cells (hiPSC-MPCs), derived from a serum-free differentiation protocol of a hiPSC cell line, for the treatment of VML in a murine model. Human induced pluripotent stem cells (hiPSCs) exhibit excellent proliferation potential and are capable of being differentiated into myogenic (precursor) cells and regenerate skeletal muscle. The gelatin scaffold was subjected to high rates of stirring to increase porosity. These muscle tissue implants were then integrated and implanted into a VML injury model, created through a 4-mm punch biopsy to a mouse gastrocnemius muscle. RESULTS: At four weeks post-VML injury to the gastrocnemius, gross imaging of the muscle showed migration of host cells into the construct and identified newly regenerated muscle tissue in the hiPSC-MPC muscle group compared to the acellular GelMA group. At eight weeks post-VML injury, histological analysis confirmed significant 2.5-fold increase (p<0.0001) in de novo muscle regeneration, measured by the number of myofibers identified with centrally located nuclei per high power field, in the hiPSC-MPC group compared to the untreated VML group. At four weeks post-VML injury, the hiPSC-MPC group also showed significantly reduced % area fibrosis (<3.58% area) in gross imaging compared to the acellular GelMA group (p<0.0001) and untreated VML group (p<0.05). Functional strength assessment of ankle plantarflexion of isolated gastrocnemius demonstrated a significant increase in both in situ twitch (p<0.033) and tetanic (p<0.033) force in the hiPSC-MPC group compared to the acellular GelMA group and VML untreated group. CONCLUSIONS: This study pioneers a combination of bioengineering and stem cell technologies to develop a treatment for VML. Taken together, these results demonstrate successful graft-host integration and de novo muscle formation upon in vivo implantation of hiPSC-MPC-derived muscle scaffold in a mouse gastrocnemius model of VML injury. This work is an important step toward the next generation of VML regenerative therapies. References: 1. Corona BT, Rivera JC, Greising SM. Inflammatory and Physiological Consequences of Debridement of Fibrous Tissue after Volumetric Muscle Loss Injury. Clinical and Translational Science. 2018;11(2):208-217. doi:10.1111/cts.12519 2. Corona BT, Rivera JC, Owens JG, Wenke JC, Rathbone CR. Volumetric muscle loss leads to permanent disability following extremity trauma. J Rehabil Res Dev. 2015;52(7):785-92. doi:10.1682/jrrd.2014.07.0165 3. Garg K, Ward CL, Hurtgen BJ, et al. Volumetric muscle loss: persistent functional deficits beyond frank loss of tissue. J Orthop Res. Jan 2015;33(1):40-6. doi:10.1002/jor.22730 4. Panayi AC, Smit L, Hays N, et al. A porous collagen-GAG scaffold promotes muscle regeneration following volumetric muscle loss injury. Wound Repair and Regeneration. 2020;28(1):61-74. doi:10.1111/wrr.12768 5. Porzionato A, Sfriso MM, Pontini A, et al. Decellularized Human Skeletal Muscle as Biologic Scaffold for Reconstructive Surgery. Int J Mol Sci. Jul 1 2015;16(7):14808-31. doi:10.3390/ijms160714808