Immunomodulatory capacity of the local mesenchymal stem cells transplantation after severe skeletal muscle injury in female rats

Cell therapy technique with stem cells is a very attractive strategy for the treatment of muscle disorders.The objective of this study was to investigate the mechanism of local transplantation of mesenchymal stem cells (MSCs) which could contribute to skeletal muscle healing.Female rats were divided into three equal groups as the following: group 1, the negative control group (untreated group), group 2, sham-treated group, rats with muscle injuries involving volumetric muscle loss (VML) of adductor brevis muscle and injected locally with phosphate-buffered saline (PBS) 0.5 ml without stem cells after 7 d of muscle injury, group 3, treated group, rats with VML and injected locally (intramuscular) with 1.5 × 106 bone marrow MSCs suspended in PBS 0.5 ml (1) after 7 d of muscle tissue injury. All animals were sacrificed after 4 weeks of stem cell transplantation.In vitro culture the morphology of MSCs reached confluence and appeared as long spindle in shape on 9-14 d. Most of the cells did not express the hematopoietic cell marker, CD34 and CD45 but expressed MSCs marker CD44, CD90 and CD105. The remarkable increase of proliferating cell nuclear antigen positive nucleus was recorded in MSCs group as compared to PBS group. After 28 d of injection, administration of only PBS into the site of muscle injury caused up-regulation in the levels of interleukins IL-1β, IL-6, tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β1), interferon alpha (IFN-α) and down-regulate the level of IL-10 in muscular tissue comparing to the untreated control. Bone marrow MSCs + PBS injected at the site of muscle injury significantly down-regulate the inflammatory cytokines levels IL-1β and IL-6 and TNF-α, TGF-β1 and IFN-α and up-regulate the level of IL-10. Collagen concentrations in the injured skeletal muscle estimated by enzyme-linked immuno sorbent assay and stained with Masson trichrome stain were increased with PBS group and decreased after transplantation of bone marrow MSCs in the site of injury. Muscle sections stained with H&E showed a higher number of centronucleated regenerating myofibers in the stem-cell-treated group than in the (PBS) and untreated control group. Microvasculature of skeletal muscle was decreased as demonstrated by immunostaining technique for CD34 in PBS group from untreated control. The MSCs group showed angiogenesis and marked increase of skeletal muscle microvasculature than PBS group.MSCs can modify the local immunological responses and improve muscle regeneration by suppressing of inflammatory cytokines, activating of the anti-inflammatory cytokine, restoration of muscle fibers and angiogenesis. By means of increase in TGF-β production in response to muscle injury prevent the repair of injured fibers and increase connective tissue production (collagen fibers), thus propagating skeletal muscle weakness and fibrosis whereas MSCs + PBS injected at the site of muscle injury significantly down-regulate (TGF-β1) and hence the level of collagen (fibrosis or scar areas). MSCs are able to block the fibrotic signaling cascade by declining TGF-β1 and scar areas in the injured muscle.