(J-374) Bioprinted nanopatterned skeletal muscle constructs for treatment of volumetric muscle loss

Introduction::
Volumetric muscle loss (VML) refers to the substantial loss of skeletal muscle tissue, typically due to traumatic injury, surgical resection, or disease. VML can lead to persistent functional deficits, such as weakness, impaired mobility, and chronic pain, significantly impacting an individual's quality of life. Despite the substantial impact of VML, limited treatment options are available that can effectively restore muscle volume and function.









VML pathophysiology involves a complex interplay of inflammation, fibrosis, and impaired muscle regeneration. Currently, treatment strategies for individuals with VML include surgical approaches, such as muscle flaps and grafts, as well as physical therapy and rehabilitation. However, these approaches have limited efficacy in restoring muscle volume and function, particularly in cases of severe VML. Approaching the chronic issue via the avenue of tissue engineering offers the promise of addressing the significant unmet clinical need in VML through the addition of supporting scaffolds and cellular intervention. Here, we build on our laboratory's previous work by engineering 3D printing-assisted endothelialized skeletal muscle composed of muscle myoblasts co-cultured with vascular endothelial cells on parallel-aligned nanofibrillar collagen scaffolds.




Materials and Methods::
To generate the patterned collagen scaffolds, a 3D printer was modified using a blunt-tip needle (22G) and syringe set at a 30° angle attached to a pump, which allowed for consistent extrusion and high velocity sheering of concentrated, acidic rat tail collagen type-I (30mg/ml) into a concentrated buffer solution (10x PBS, pH 7.4). Randomly oriented scaffolds were made through a lower velocity, omitting the sheering stressors necessary to achieve the nanopatterned effect. Once the fibrillogenesis was completed, the scaffolds were washed with 1x PBS and sterilized for cell culture in a 20-minute 90% ethanol bath. Human dermal microvascular endothelial cells were seeded at a density of 150k/scaffold and allowed to attach overnight before seeding the human skeletal myoblasts at the same density. The cell-seeded scaffolds were implanted into a VML murine model the following day, with the experimental endpoint at day 21. Histological analyses were performed on the explanted tissues to determine myofiber regeneration capacity as well as determine if an increase of vascularization was seen in the engineered tissue and surrounding areas.




Results, Conclusions, and Discussions::
The 3D printing of collagen scaffolds provides a consistent and easily scalable procedure that can be used to make various types of muscle constructs. Histological analysis demonstrated that the aligned nanopatterned scaffolds led to a larger area of muscle regeneration, compared to unaligned scaffolds. Additionally, the aligned scaffolds showed a higher number of overall myofibers and a significant increase in total vasculature and perfusion seen throughout the area of regeneration. By shifting away from the transplantation of acellular scaffolds, our lab demonstrates the ability to produce aligned skeletal muscle constructs which assist the damaged native tissue in stimulating muscle regeneration.




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