Codelivery of Infusion Decellularized Skeletal Muscle with Minced Muscle Autografts Improved Recovery from Volumetric Muscle Loss Injury in a Rat Model

Abstract

Skeletal muscle is capable of robust self-repair following mild trauma, yet in cases of traumatic volumetric muscle loss (VML), where more than 20% of a muscle's mass is lost, this capacity is overwhelmed. Current autogenic whole muscle transfer techniques are imperfect, which has motivated the exploration of implantable scaffolding strategies. In this study, the use of an allogeneic decellularized skeletal muscle (DSM) scaffold with and without the addition of minced muscle (MM) autograft tissue was explored as a repair strategy using a lower-limb VML injury model (n = 8/sample group). We found that the repair of VML injuries using DSM + MM scaffolds significantly increased recovery of peak contractile force (81 ± 3% of normal contralateral muscle) compared to unrepaired VML controls (62 ± 4%). Similar significant improvements were measured for restoration of muscle mass (88 ± 3%) in response to DSM + MM repair compared to unrepaired VML controls (79 ± 3%). Histological findings revealed a marked decrease in collagen dense repair tissue formation both at and away from the implant site for DSM + MM repaired muscles. The addition of MM to DSM significantly increased MyoD expression, compared to isolated DSM treatment (21-fold increase) and unrepaired VML (37-fold) controls. These findings support the further exploration of both DSM and MM as promising strategies for the repair of VML injury.

FIG. 1.

DSM scaffolds were infusion decellularized using a custom-built infusion bioreactor. During decellularization, SDS solution was delivered using a hypodermic needle (A), which was positioned into the wide mid-belly region of the TA muscle (B). SDS solution was infused using a syringe pump (5 mL/h for 12 h) through each muscle and outflowed to waste collection. The bioreactor was designed to accommodate four side-by-side decellularization units each capable of accommodating a single muscle tissue sample (C). Representative whole TA muscle appearance before and following infusion decellularization treatment (D, E) illustrates the dramatic color change (red to white) following removal of intracellular myoglobin. Infusion prepared DSM scaffolds, when viewed in thin section (scale bar = 100 μm) with hematoxylin and eosin staining, retained the highly aligned architecture of native muscle ECM (F). (arrow = direction of contraction). DSM, decellularized skeletal muscle; SDS, sodium dodecyl sulfate; TA, tibialis anterior; ECM, extracellular matrix. Color images available online at www.liebertpub.com/tea

FIG. 2.

To create the VML injury model, the mid-belly region of the TA muscle in a Fischer 344 rat was visualized through a 1–2 cm incision (A) and ∼20% of the TA muscle (average defect weight = 94 ± 6 mg) was excised using an 8 mm biopsy punch inserted to a depth of 3 mm (B–D). During isolated DSM repair, a single scaffold was cut to size and implanted into the TA defect (E). To prepare each MM autograft, 25% of the previously removed defect plug was minced with a scalpel and scissors and then used to coat the surface of a single DSM scaffold (F). The combined DSM scaffold and MM paste construct were implanted into the TA defect (G). The deep fascia and skin (H) were closed separately using absorbable sutures. VML, volumetric muscle loss; MM, minced muscle. Color images available online at www.liebertpub.com/tea

FIG. 3.

Twelve weeks after treatment, TA peak contractile force was assessed for both unrepaired and DSM repaired groups (n = 8/group). Representative tetanic force responses for normal, unrepaired VML, DSM, and DSM + MM repaired TA muscles (A). Peak tetanic force was normalized to animal weight (N/kg) and computed as a percentage of the untreated contralateral limb for each animal tested (B). Box and whisker plot values shown are the median, first and third quartile, as well as maximum and minimum. The increase in peak contractile force in response to DSM + MM implantation was statically significant compared to both unrepaired VML and isolated DSM repaired samples. (n = 7–8/sample group). ^#Distinguishes significant difference from VML group. p < 0.05; ANOVA with post hoc Tukey's test. ANOVA, analysis of variance. Color images available online at www.liebertpub.com/tea

FIG. 4.

Gross morphology of normal, unrepaired VML, DSM, and DSM + MM repaired whole TA muscles harvested 12 weeks posttreatment (A). TA weight, calculated as the percent normal contralateral muscle, was significantly increased in response to repair with combined DSM + MM implants (B). The EDL muscle mass was similarly increased in response to treatment, suggesting compensatory hypertrophy. Box and whisker plot values shown are the median, first and third quartile, as well as maximum and minimum. (n = 8/sample); ^#distinguishes significant difference from VML group, p < 0.05; ANOVA with post hoc Tukey's test. EDL, extensor digitorum longus. Color images available online at www.liebertpub.com/tea

FIG. 5.

Representative normal (A, E, I), unrepaired VML (B, F, J), DSM (C, G, K), and DSM + MM (D, H, L) repaired histological sections [(E) inset indicates approximate section location]. Sections were stained with either Masson's Trichrome or immunostained for the presence of MHC (red) and collagen type I (green). The defect/repair site (*) for unrepaired VML and isolated DSM repaired samples showed evidence of a thick, collagen enriched, connective tissue repair layer. DSM + MM repair site tissue showed markedly less connective tissue formation than VML or DSM groups. Dotted line highlights the boundary between collagen dense repair tissue and MHC positive muscle tissue. Scale bar = 250 μm unless noted Arrow indicates anterior direction. MHC, myosin heavy chain. Color images available online at www.liebertpub.com/tea

FIG. 6.

Representative normal (A, E), unrepaired VML (B, F), DSM (C, G), and DSM + MM (D, H) tissue sections. Repair site tissue was immunoreactive to antibodies directed against collagen I and III (A–D), as well as laminin and fibronectin (E, F). Scale bar = 100 μm. Arrow indicates anterior direction. Color images available online at www.liebertpub.com/tea

FIG. 7.

Representative DSM (A) and DSM + MM repaired (B) tissue sections immunostained for the presence of collagen type I. The sectional area immunoreactive to collagen type I was calculated using sections prepared from tissue regions located at least 3 mm away from the anterior surface of the muscle. Tissue samples collected from DSM + MM repair samples exhibited a significantly lower immunoreactivity to collagen type I compared to isolated DSM repair samples (C). Scale bar = 100 μm; n = 3/sample group; *distinguishes significant difference from the DSM treatment group; p < 0.05; ANOVA with Tukey's test. Color images available online at www.liebertpub.com/tea

FIG. 8.

RT-PCR results (fold change compared to normal muscle) for the ECM structural proteins Collagen I and III, the ECM regulatory cytokines TGF-β1, MMP2, and TIMP-1, and the myogenic marker MyoD. Values shown are mean + SEM; n = 4/sample group; ^#distinguishes significant difference from both VML and DSM groups; p < 0.05; ANOVA with Tukey's test. SEM, standard error of the mean. Color images available online at www.liebertpub.com/tea

FIG. 9.

Collagen I to MyoD gene expression ratio versus percent normal contractile force (A) for VML, DSM, DSM + MM, and normal TA sample groups. The average expression ratios calculated for VML and DSM samples clustered near an elevated value of 30, while the DSM + MM repair sample ratio was better aligned with normal TA muscle. Average expression ratios (B) for each of the ECM structural and ECM regulatory genes to MyoD. Expression ratio values shown are mean ± SEM; n = 4/sample group. Color images available online at www.liebertpub.com/tea