الفهرس 
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Abstract
Trauma to the muscle is a common problem seen by doctors. If the muscle doesn’t become better, there is a high risk for a new problem to evolve. In this case the outcome is worse. A lot of effort is accomplished to find ways to improve and treat these muscles.
Stem cells is a novel way to regenerate these tissues. Cells coming from the marrow of bone are becoming widely tested by doctors around the world.
Lots of work is needed to test these cells and make sure that they can work well.
The present work aimed to study the changes of experimentally-induced gastrocnemius muscle injury in female albino rats, establish an approach for experimental transplantation of bone marrow-derived mesenchymal stem cells in the gastrocnemius muscle of female albino rats and also to explore the possible regenerative potential of transplanted bone marrow-derived mesenchymal stem cells in the repair of gastrocnemius muscle injury
The material and methods of this work included fifty-five healthy adult albino rats, forty-five females, at an average age between 12-15 weeks, weighing from one hundred and eighty to two hundred grams (180 to 200 grams) and ten males aged between 2-3 weeks, weighed thirty to fifty grams (30 to 50 grams). Male rats were used in this study for collecting the bone marrow as a different source of mesenchymal stem cells, and thus to trace the engraftment of these cells by RQ-PCR.
Female rats were assigned into 3 groups;
group I: Control Group: (n=5 adult female albino rats) serving as control without gastrocnemius muscle injury.
group II: Experimental Control Group: (Gastrocnemius muscle injury without BMMSCs local injection) (n=20 adult female albino rats). The purpose of this group was to assess the possibility for spontaneous regeneration. This group was further subdivided into four subgroups:
Subgroup IIa:(n=5 adult female albino rats) sacrificed at the time of gastrocnemius muscle injury.
Subgroup IIb: (n=5 adult female albino rats) sacrificed three days after gastrocnemius muscle injury.
Subgroup IIc: (n=5 adult female albino rats) sacrificed seven days after gastrocnemius muscle injury.
Subgroup IId: (n=5 adult female albino rats) sacrificed fourteen days after gastrocnemius muscle injury.
group III: Experimental Group: (Gastrocnemius muscle injury with BMMSCs local injection) (n=20 adult female albino rats). This group was further subdivided into four subgroups:
Subgroup IIIa: (n=5 adult female albino rats) sacrificed at the time of gastrocnemius muscle injury and stem cell injection.
Subgroup IIIb: (n=5 adult female albino rats) sacrificed three days after gastrocnemius muscle injury and stem cell injection.
Subgroup IIIc: (n=5 adult female albino rats) sacrificed seven days after gastrocnemius muscle injury and stem cell injection.
Subgroup IIId: (n=5 adult female albino rats) sacrificed fourteen days after gastrocnemius muscle injury and stem cell injection.
MSCs were isolated, characterized, cultured and passaged in vitro. All these procedures were conducted in stem cells laboratory in Centre of Excellence for Research in Regenerative Medicine and its Application (CERRMA), Faculty of Medicine, Alexandria University. After third passage, once they became 70-80% confluent, MSCs were injected in gastrocnemius muscle in female rats of group III.
Characterization of MSCs was done by:
1. Daily examination of the morphology of cultured cells using phase contrast inverted microscopy.
2. Colony forming unit-fibroblast (CFU-F) assays.
3. Flow cytometric analysis of the cultured cells’ surface markers.
After the designated time during the study, rats were sacrificed after ether anesthesia. Fresh specimens were taken from the gastrocnemius muscle of all studied groups and prepared for the following studies:
A- Light microscopic study:
a. H&E stain,
b. Gomori’s trichrome stain.
B- Electron microscopic study: Ultrathin sections were studied using transmission electron microscope.
C- RQ-PCR: for detection of Y chromosome.
D- CM-Dil.
Histological examination of group II (Gastrocnemius muscle injury without BMMSCs local injection) showed haemorrhage, vacuolated cells, inflammatory cellular infiltration and fibrosis.
By Gomori’s stain, it showed increase collagen fibres.
Ultrastructure examination confirmed the above findings and also showed disrupted muscle fibres, macrophage with vacuolated cytoplasm and many lysosomes representing the degenerated fibres, sarcolemmal festooning and cytoplasmic depletions.
Examination of rat gastrocnemius group III (Gastrocnemius muscle injury with BMMSCs local injection) showed less degeneration, more regenerating fibres, bridging between the fibres and more internalized nuclei of the muscle fibres.
Lower levels collagen fibres were shown evident by Gomori’s trichrome stain after 14 days.
Electron micrograph of the rat gastrocnemius group III showed macrophages of the M2 type, regenerating muscle fibres, mitochondrial return to more or less normal configuration and nuclear proliferation.
Also CM-Dil was performed on group III and showed shifting of the stem cells from the connective tissue stroma to reside as part of the muscle fibres and thus successful homing of the cells.
Further more evidence of MSCs homing in the injured gastrocnemius was proved by the expression of Y-chromosome (SRY) gene of the injected female rat model. The engraftment of these cells could lead to skeletal muscle regeneration either by their differentiation or through their paracrine effect.
The present results clearly demonstrated the effectiveness of MSCs in the treatment of skeletal muscle injury and the efficiency of MSCs derived from bone marrow in skeletal muscle regeneration and reduction of fibrosis.