Neurotization Of A Tissue Engineered Muscle Repair Construct, Potential For Improved Functional Outcomes
Kristen Knapp, B.S., Juliana Amaral-Passipieri, Ph.D., Angela Pineros-Fernandez, M.D., George T. Christ, Ph.D., Patrick S. Cottler, Ph.D., Christopher A. Campbell, M.D..
University of Virginia, Charlottesville, VA, USA.
Background: The traumatic loss of a large volume of skeletal muscle and associated functional debilitation is a significant clinical problem and currently available treatments to restore the functional deficit of volumetric muscle loss (VML) remains the transfer of pedicled or innervated free muscle flaps into the defect in conjunction with physical therapy. This approach leads to significant donor site morbidity by sacrificing existing neuro-muscular flaps instead of promoting regeneration of lost skeletal muscle. Engineered tissue constructs hold tremendous potential to achieve the muscle bulk and function needed for recovery. However, such approaches have been limited to the implantation of engineered muscle grafts with myocyte regeneration limited to the interface of the host skeletal muscle in animal models. Innervation and vascularization of regenerated tissue remains a significant hurdle in creating functional repairs of VML.
Methods: Using a small animal model of neurotization, we investigated the ability of maintaining viability of a large donor motor nerve within a tissue engineered muscle repair (TEMR) construct that has previously shown the ability to promote functional regeneration in a skeletal muscle defect. TEMR constructs (muscle-derived myoblasts seeded on a bladder acellular matrix (BAM) ) were fabricated, preconditioned with uniaxial mechanical strain and anchored to the gracilis muscles of Lewis rats. At the time of placement, the TEMR constructs were folded around the dissected and isolated femoral vascular pedicle. Additionally, the motor branch of the femoral nerve was sharply divided and the proximal stump was embedded into the construct. Animals were recovered and observed for 12 weeks post injury when the constructs were carefully excised, fixed and stained with hemotoxin and eosin (H&E) and markers for neural and vascular tissue.
Results: The animals recovered fully from the procedure without a noticeable gait deficit. Histological and immunohistological analysis showed that the femoral nerve placed within the TEMR maintained its original diameter with organized NF200+ filaments and axons, in addition to neural filaments throughout the constructs. These filaments were closely associated with support cells. Additionally, the samples exhibited regions of skeletal muscle within the constructs. A robust cellular integration was seen, including the presence of regenerative M2 macrophages throughout the constructs, confirmed through macrophage staining targeting CD68 and CD163. There was also evidence that the vascular pedicle maintained patency and the construct was able to integrate around it with a robust microcirculation throughout the tissue. This was confirmed through CD31 (endothelial cell marker) and alpha smooth muscle actin (αSMA) staining.
Conclusions: This is the first study to evaluate the potential of neurotization of a TEMR construct as a means to promote more complete integration and functional recovery from VML. By demonstrating that a motor nerve used to neurotize the construct was able to maintain its size and architect along with newly forming neural and muscle tissue, provides the foundation for full neurotization of the TEMR through embedding the terminal end of a nerve, including the motor end plates. Subsequently, placing the neurotized TEMR into a volumetric muscle defect will provide the regenerative signaling needed for improved functional outcomes.
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