Direct Neurotization of Decellularized Muscle Matrix Leads to Enhanced Muscle Regeneration and Neural Tissue Ingrowth
Mimi Wu Young, MD1, Hari Iyer, BS2, Seok Hong, PhD1, Robert Galiano, MD1.
1Northwestern Feinberg School of Medicine, Chicago, IL, USA, 2McGill University School of Medicine, Montreal, QC, Canada.
Traumatic soft tissue injuries to the face and extremities with associated volumetric muscle loss can result in devastating functional deficits, and there are currently no available treatments to stimulate the repair or regeneration of functional skeletal muscle in patients with these debilitating injuries. Decellularized extracellular matrix has been a promising candidate for induction of skeletal myocyte regeneration. However, functionalization of the regenerated tissue requires motor nerve innervation and the presence of motor end plates to properly contract. Nerve-specific growth factors have also been demonstrated to enhance muscle regeneration. In this study, we examined the myogenic and neurogenic effects through direct neurotization of decellularized muscle extracellular matrix. METHODS:
External oblique muscles were harvested from adult Sprague-Dawley rats and underwent a negative pressure assisted decellularization process to generate decellularized muscle matrix. Bilateral full-thickness, 1 cm latissimus dorsi defects were surgically created in 12 rats. 8 defects were left as is, 8 defects were implanted with decellularized matrix alone, and 8 defects were implanted with decellularized matrix then neurotized. Peripheral motor nerves innervating the panniculus carnosus were microsugically dissected free and transected distally to allow transposition onto the ipsilateral decellularized matrix implant. Neurotization was performed on both the deep and superficial surfaces of the matrix with 3-5 nerves total implanted based on anatomic feasibility. Rats were sacrificed and tissue harvest was carried out on post-operative days 90 and 180. Tissue samples were assessed through gross appearance, immunohistochemistry, and western blot analysis for myogenesis, inflammation, neovascularity, and neural ingrowth within the explanted matrices and defects alone. RESULTS:
At the time of tissue harvest, both the neurotized and non-neurotized matrices appeared well integrated within the surrounding native muscle as compared to the defect alone, which healed with a thin layer of fibrous connective tissue. However, neurotized decellularized muscle matrix demonstrated greater infiltration by mature myocytes compared to non-neurotized control implants based on myosin heavy chain analysis. CD-31 staining revealed similar levels of microvascular networks within the matrix of neurotized and non-neurotized matrices, both greater than defect alone. Cholinergic neural tissue ingrowth was only observed in neurotized muscle matrix. Direct stimulation of the matrix implants with a peripheral nerve stimulator demonstrated contraction of both the neurotized and non-neurotized matrices, however a difference could not be grossly appreciated. CONCLUSION:
Direct neurotization of decellularized muscle matrix in a model of volumetric muscle loss leads to more robust myogenesis and neurogenesis within the implant as compared to non-neurotized matrix and defect alone. Further studies are needed to evaluate functional differences in more detail.
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