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Skeletal Muscle Regeneration in a Rat Model with Extracellular Matrix and Adipose Derived Stem Cells
Tiffany Sarrafian, DVM1, Sue Bodine, PhD1, Susan Stover, DVM, PhD1, Kevin Grayson, DVM, PhD2, Hakan Orbay, MD, PhD1, David E. Sahar, MD1, Daren Danielson, MD2.
1University of California, Davis, Davis, CA, USA, 2Clinical Investigation Facility, David Grant USAF Medical Center, Fairfield, CA, USA.
PURPOSE: This study aims to address the important clinical need for repair of volumetric muscle loss by employing an extracellular matrix (ECM; decellularized porcine small intestinal submucosa) in a rat gastrocnemius muscle regeneration model. We hypothesize that ECM, either as a sheet or emulsification, will stimulate a muscle regenerative response in a rat skeletal muscle volumetric loss model, and the combination will provide synergistic benefit. Muscle healing will be further enhanced when the matrix is seeded with rat syngeneic adipose derived stem cells (ASCs).
METHODS: A 7mm full thickness volumetric muscle loss defect was surgically created under anesthesia in the medial head of the rat gastrocnemius muscle, utilizing a specially designed muscle biopsy clamp. Study groups include using a 4-ply sheet of ECM and an emulsified matrix suspension, with or without ASCs seeded onto the matrix to repair the muscle defect (Figure 1). In group I muscle was repaired with ECM only. In group II the ECM was sutured to bridge the muscle gap, with emulsification injected into the gap (0.2 ml). In group III ECM was seeded with ASCs, prior to repair, with a seeding density of 300,000 cells / cm2. In group IV the defect was repaired with emulsified matrix only. Defects in the control group were left unrepaired. Rats were sacrificed at 3 and 6 months for histologic evaluation and immunohistochemistry to evaluate muscle healing.
RESULTS: The animal model described here was shown to be reproducible and the specially designed muscle biopsy clamp helped to standardize the size of muscle defects. ASCs attached to the ECM scaffolds after 3 days of incubation and were visualized with DiI labeling. ECM sheet and emulsified matrix were successfully implanted to bridge the gap in study animals. No visible wound related complications, gait deficits or pain were observed in animals post-operatively. At 3 and 6 months post-operatively, there was histologic and immunohistochemical (myosin and desmin positivity) evidence of angiogenesis and new striated muscle formation (Figure 1). A minimal degree of inflammation was observed in the regenerated tissue within the ECM tube along with a compensatory increase in soleus muscle mass in response to defect creation.
CONCLUSION: Herein, we present a novel model for evaluation of muscle regeneration. ECM shows promise to regenerate skeletal muscle, opening a therapeutic avenue for patients with devastating traumatic injury and irreparable volumetric muscle loss. We anticipate that future study will reveal more ingrowth and functionality of repaired muscle at later time points.
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