|
|
|
|
|
Back to 2016 Annual Meeting
Skeletal Muscle Regeneration Utilizing Muscle Derived Stem Cell Augmented Scaffolds: In Vitro Optimization and Development of an In Vivo Model
Howard D. Wang, MD, Jose C. Alonso-Escalante, MD, Denver M. Lough, MD, PhD, Amy Quan, MPH, Qiongyu Guo, PhD, Joseph Lopez, MD, MBA, Edward W. Swanson, MD, WP Andrew Lee, MD, Gerald Brandacher, MD, Anand R. Kumar, MD.
Johns Hopkins Hospital, Baltimore, MD, USA.
BACKGROUND: Skeletal muscle loss can result from trauma or oncologic resection, leading to severe cosmetic and functional deficits. Muscle derived stem cells (MDSCs) can be isolated by using a serial preplating technique and have inherent myogenic potential. Since their identification, previous research on MDSCs has mostly focused on ex-vivo gene therapy applications aimed at treating muscular dystrophy, and little is known about the optimal population for skeletal muscle tissue engineering. The aims of this study are to optimize the in vitro conditions for creating an implantable skeletal muscle construct and to develop an in vivo model of volumetric muscle loss (VML) to study muscle regeneration.
METHODS: Murine MDSCs were isolated from transgenic C57BL/6TG(CAG-EGFP) mice expressing green fluorescent protein (GFP) under a beta-actin promoter. Proliferation studies of various preplate populations were performed by utilizing bright-field microscopy to determine time to confluence. Myogenic potential was assessed by immunostaining for myosin-heavy chain (MyHC) to evaluate myotube formation. In vitro skeletal muscle constructs were created using GFP-expressing MDSCs seeded onto collagen scaffolds, and cellular viability and scaffold repopulation were assessed using confocal microscopy. A murine model of volumetric muscle defect was created by excision of a 5mm long and full thickness segment of the rectus femoris muscle. Mini CT images were taken to monitor the volumetric defect. Gross inspection and histologic analysis was performed at 8 weeks after surgery.
RESULTS: Preplates 3, 4 and 5 demonstrated faster expansion rates from time of isolation compared to prelates 1, 2 and 6 (p<0.05). In terms of their myogenic potential, preplates 3, 4, and 5 had comparable fusion indices (2.53±0.51, 3.22±0.80, 3.10±1.46, p=0.316). Confocal imaging of MDSC-seeded construct demonstrated cellular viability at 48 hours and 14 days with progressive concentric scaffold repopulation. All animals (n=6) tolerated the operation, and CT images at 4 and 8 weeks after surgery confirmed persistence of muscle defect. At the study endpoint of 8 weeks, gross inspection of the defect showed evidence of cicatricle healing without muscle regeneration, which was confirmed with histologic analysis.
CONCLUSIONS: The results of this study indicate that preplates 3, 4 and 5 possess high expansion rates and myogenic potentials. Furthermore, MDSCs are capable of successful repopulation of collagen scaffolds in vitro, and a murine model of VML was developed and validated. Future experiments will assess the ability of the MDSC-enriched scaffolds to heal a skeletal muscle defect in vivo.
Back to 2016 Annual Meeting
|
|
