A murine model of volumetric muscle loss and a regenerative medicine approach for tissue replacement

Abstract

Volumetric muscle loss (VML) resulting from traumatic accidents, tumor ablation, or degenerative disease is associated with limited treatment options and high morbidity. The lack of a reliable and reproducible animal model of VML has hindered the development of effective therapeutic strategies. The present study describes a critical-sized excisional defect within the mouse quadriceps muscle that results in an irrecoverable volumetric defect. This model of VML was used to evaluate the efficacy of a surgically placed inductive biologic scaffold material composed of porcine small intestinal submucosa-extracellular matrix (SIS-ECM). The targeted placement of an SIS-ECM scaffold within the defect was associated with constructive tissue remodeling including the formation of site-appropriate skeletal muscle tissue. The present study provides a reproducible animal model with which to study VML and shows the therapeutic potential of a bioscaffold-based regenerative medicine approach to VML.

Figures

FIG. 1.

Induction of VML injury. The exposed quadriceps muscle compartment after skin incision and blunt dissection of the surrounding fascia (A). Volumetric defect consisting of a 4×3 mm full-thickness resection of the tensor fasciae latae quadriceps muscle (B). Treated defect filled with a size-matched piece of vacuum-pressed SIS-ECM (C). Single-layer SIS-ECM sheet overlay sutured to adjacent native muscle before dermal closure (D). VML, volumetric muscle loss; SIS, small intestinal submucosa; ECM, extracellular matrix. Color images available online at www.liebertpub.com/tea

FIG. 2.

VML injury is of critical size. Histological analysis of uninjured (A) and injured (B) muscle at 56 days postsurgery. After 56 days low-power magnification (left panels) reveals an obvious volumetric defect (dotted line). Higher magnification (right panels) shows the defect partially remodeled with a collagenous connective tissue consistent with scar formation ({) and some adipose (*). No signs of new skeletal muscle formation are seen in untreated injured muscle. The black boxes on the left represent the area of the high-power images on the right (scale bar=1 mm). Color images available online at www.liebertpub.com/tea

FIG. 3.

Defect site at 7 days postsurgery. Low magnification (left panels) shows the defect site (dotted lines). Untreated defects (A) show a VML injury characterized by necrotic skeletal muscles (arrows), while treated defects (B) are filled with the SIS-ECM scaffold. High magnification (right panels) shows a robust mononuclear cell infiltrate in both untreated and treated defects. The black boxes on the left represent the area of the high magnification images on the right (scale bar=1 mm). Color images available online at www.liebertpub.com/tea

FIG. 4.

Defect site at 14 days postsurgery. Low magnification (left panels) shows the defect site (dotted lines). Untreated defects are filling with granulation tissue, while the ECM scaffold is becoming infiltrated with cells. The black boxes on the left represent the area of the high magnification images on the right. (#, host derived neomatrix; *, scaffold) (scale bar=1 mm). Color images available online at www.liebertpub.com/tea

FIG. 5.

Defect site at 28 days postsurgery. Low magnification (left panels) shows the defect site (dotted lines). High magnification (right panels) shows predominantly spindle-shaped cells populating the untreated defect site (A), while treated defects are comprised of cells with varying morphologies (B). The black boxes on the left represent the area of the high-magnification images on the right (scale bar=1 mm). Color images available online at www.liebertpub.com/tea

FIG. 6.

Vascularity of treated versus untreated VML defects. CD31 staining (green) of endothelial cells at 7, 14, 28, and 56 days after VML in untreated defects (A) or defects treated with an SIS-ECM scaffold (B). CD31 immunopositive blood vessels were counted per 400× field of view (C). Three fields per surgical site were examined at the interface with underlying host tissue (*p<0.01) (scale bar=1 mm). (Error bars=standard deviation). Color images available online at www.liebertpub.com/tea

FIG. 7.

Evidence of innervation within treated versus untreated VML defects. Representative images of Gap-43+ (green) neurons at 7, 14, 28, and 56 days after VML in untreated defects (A) or defects treated with an SIS-ECM scaffold (B) (scale bar=1 mm). Color images available online at www.liebertpub.com/tea

FIG. 8.

Site-appropriate remodeling by ECM scaffolds. Defect sites treated with SIS-ECM (dotted line) maintain robust high cellularity after 56 days (A). Immunolabeling shows desmin+ cells (green staining) populating the area of ECM implantation along the interface of the underlying native muscle [(B), left panel]. Desmin+-striated skeletal muscle cells were also observed throughout the SIS-ECM scaffold [(B), right panel]. The black box on the left represents the area of the high magnification image on the right. Color images available online at www.liebertpub.com/tea