Long-term effectiveness of local BM-MSCs for skeletal muscle regeneration: a proof of concept obtained on a pig model of severe radiation burn

Abstract

Background: Medical management of the severe musculocutaneous radiation syndrome involves surgical intervention with debridement of necrotic tissue. Even when skin excision is replaced by specific plastic surgery, treatment of the muscle radiation injury nonetheless remains difficult, for it involves a massive muscle defect in an unpredictable environment, subject to inflammatory waves weeks to months after irradiation, which delay healing and predispose the patient to the development of fibrous scar tissue. In this study, we investigated the long-term effect of local injections of bone marrow-derived mesenchymal stromal cells (BM-MSCs), combined with plastic surgery, to treat muscle necrosis in a large animal model.

Methods: Three months after irradiation to the rump, minipigs were treated by excision of necrotic muscle tissue, vascularized flap surgery, and four injections with or without local autologous BM-MSCs, performed weekly. The quality of the muscle wound healing was examined 1 year post-surgery.

Results: The skeletal muscle surgery without MSC treatment led to permanent deposition of collagen 1 and 3, decreased myofiber diameter, failed muscle fiber regeneration, a reduced number of capillaries, and the accumulation of high calcium and fat. In animals treated by surgery and MSC injections, these indicators were substantially better and demonstrated established regeneration. MSC therapy acts at several levels by stimulating growth factors such as VEGF, which is involved in angiogenesis and satellite cell pool maintenance, and creating a macrophage M1/M2 balance.

Conclusion: Thus, cell therapy using BM-MSCs is an effective and safe way to improve recovery of irradiation-induced skeletal muscle damage without signs of long-term degeneration.

Keywords: BM-MSC; Irradiation; Muscle; Pig; Regeneration.

Fig. 1

Development of a pig model of musculocutaneous radiation lesion. a Experimental design. b Photographs at surgery for fibrosis/necrosis. The skin and muscular necrosis and underlying tissues up to healthy muscle were excised, and all deep fibrosis was removed until bleeding and muscle contraction occurred. A pedicled fasciocutaneous perforator flap was elevated and closed at the site with absorbable suture. c Histological characteristics of the skeletal muscle after hematoxylin and eosin staining. d Alizarin-stained transverse sections of nonirradiated skeletal muscle (control group), irradiated skeletal muscle on the day of surgery (Irr D100 group), and 12 months after flap surgery with (flap-MSC group) and without (flap group) MSC treatment. The flap group showed large areas of fat cell infiltration, which is absent in the groups with MSC treatment. Scale bar 100 μm

Fig. 2

Effect of MSC treatment on deposition of extracellular matrix (ECM) components. a Representative whole-muscle cross-sections stained with Picro Sirius red to identify scar tissue (dark red) and Collagen-3 immunostaining of nonirradiated skeletal muscle (control group), irradiated skeletal muscle on the day of surgery (Irr D100 group), and 12 months after flap surgery with (flap-MSC group) and without (flap group) MSC treatment showed a net reduction of Col3 staining with the MSC treatment. Scale bar 100 μm. b Real-time-PCR of Col3a and Col1a. The flap-MSC group exhibited a net reduction of Col3a and Col1a expression compared with the irradiated and flap groups. Data are expressed relative to control skeletal muscle and normalized to GAPDH. Results are expressed as means ± SEM. P values were calculated by ANOVA with Bonferroni correction, *P < 0.001; compared with nonirradiated skeletal muscle; ^#P < 0.05; ^##P < 0.01; ^###P < 0.001compared with irradiated-untreated controls

Fig. 3

Effect of MSCs on myofiber regeneration. a Quantification of muscle fiber diameter, densities (calculated by normalizing the total number of fibers to section area), and number of regenerated fibers (identified by their central nuclei). b Immunostaining of the slow- and fast-twitch myofibers. c Percentage of each fiber type in nonirradiated skeletal muscle (control group), irradiated skeletal muscle on the day of surgery (Irr D100 group), and 12 months after flap surgery with (flap-MSC group) and without (flap group) MSC treatment. Scale bar 100 μm. d Real-time-PCR of fast myosin heavy chain 2 and slow myosin heavy chain 7. Data are expressed relative to control skeletal muscle and normalized to GAPDH. Results are expressed as means ± SEM. P values were calculated by ANOVA with Bonferroni correction, *P < 0.05; **P < 0.01; ***P < 0.001 compared with nonirradiated skeletal muscle; ^#P < 0.05; ^##P < 0.01; ^###P < 0.001 compared with irradiated-untreated controls

Fig. 4

MSC injections accelerated vascular restoration after flap surgery. a Representative immunostaining of Van Willebrand factor. b Real-time expression of angiogenic factors VEGF and eNOS. Scale bar 50 μm. Results are expressed as means ± SEM. P values were calculated by ANOVA with Bonferroni correction, *P < 0.05; **P < 0.01 compared with nonirradiated controls; ^#P < 0.05 compared with irradiated controls; ^#P < 0.05 compared with irradiated-untreated controls

Fig. 5

MSC controlled the repair process. a Modulation of CD34⁺ cells by MSC treatment. Representative CD34 immunostaining (better illustrated in the magnified view in the box) and real-time expression. b S100β representative immunostaining of nonirradiated skeletal muscle (control group), irradiated skeletal muscle on the day of surgery (Irr D100 group), and 12 months after flap surgery with (flap-MSC group) and without (flap group) MSC treatment. Scale bar 100 μm. Data are expressed relative to control skeletal muscle and normalized to GAPDH. Results are expressed as means ± SEM. P values were calculated by ANOVA with Bonferroni correction, *P < 0.05; **P < 0.001 compared with nonirradiated skeletal muscle; ^#P < 0.001 compared with irradiated-untreated controls

Fig. 6

MSC treatment modulated macrophage infiltration and phenotype. a Representative immunostaining for calprotectin-positive macrophages. b Anti-calprotectin staining and anti-Arg-1 staining on muscle section of nonirradiated skeletal muscle (control group), irradiated skeletal muscle on the day of surgery (Irr D100 group), and 12 months after flap surgery with (flap-MSC group) and without (flap group) MSC treatment. c Real-time-PCR of iNOS, Arg1, and iNOS/Arg-1 mRNA level ratio as an index of M1/M2 activity balance. d Real-time-PCR of CD163 gene related to M2 polarization. Data are expressed relative to control skeletal muscle and normalized to GAPDH. Results are expressed as means ± SEM. P values were calculated by ANOVA with Bonferroni correction, *P < 0.05; **P < 0.01; ***P < 0.001 compared with nonirradiated skeletal muscle; ^#P < 0.05; ^##P < 0.001 compared with irradiated-untreated controls

Fig. 7

Course of gene expression from blood monocytes differentiated in different culture conditions from pigs before irradiation (C), on the day of surgery (Irr), and 2, 3, 4, 6, and 12 months after flap surgery with or without BM-MSC treatment. Macrophages were generated from blood monocytes in the presence of M-CSF. Real-time-PCR a in an unstimulated condition, b in an M1 condition after stimulation with LPS and IFN-γ, and c in an M2a condition, after stimulation with IL-4. Data are expressed relative to control skeletal muscle and normalized to GAPDH