الفهرس 
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Abstract
Skeletal muscle injuries are common injuries occurring in frequent situations as sports, accidents and military combat. Muscle healing following major traumatic injuries requires very long duration, and is usually incomplete. Moreover, the injured muscle tissue is replaced by non-contractile fibrous scar, which persists and leads to diminished muscle capabilities and so a decline in quality of life.
Recently, regenerative medicine and stem cell therapy are showing promising results in treatment of uncorrectable end stage muscle diseases. They are new tools by which damaged tissue can be compensated with fully functioning healthy tissue.
The aim of our work was to compare the long-term effect of using BM-MSCs versus ADSCs in regeneration and re- innervation of experimentally induced skeletal muscle laceration injury.
Five weaned male albino rats weighing 70 -80gm and of average ages around one month, were used for preparation of MSCs. In these rats, the bone marrow and adipose tissue were obtained for separation of BM-MSCs and ADSCs respectively.
The experiment was conducted on forty-five adult female albino Wister rats weighing 150-200g. They were randomly divided into five groups. group I (negative control group): included five rats that were left without any interference. group II (Positive control group-Sham): included ten rats. A skin incision was done over the right gluteal muscle, followed by IM injection of 1 ml PBS then the skin was sutured. The skin was incised and injection of 1 ml PBS in the right gluteal muscle was done then the skin was sutured. group III (Untreated Laceration group): included ten rats in which laceration injury was done in the right gluteal muscle and then suturing was done. Rats were left for spontaneous healing. group IV (Laceration treated with BM-MSCs group): included ten rats in which laceration injury was done as in group III, but with concomitant local injection 10⁶ BM-MSCs suspended in 1 ml PBS in the muscle then suturing was done. group V (Laceration treated with ADSCs group): included ten rats in which laceration injury was done then rats received 10⁶ ADSCs suspended in 1 ml PBS in the muscle at site of injury then suturing was done.
Muscle injury was performed at about 0.5 cm depth in the muscle using scissors at middle of the gluteal region then, we injected BM-MSCs in the edges of the lacerated muscle in group IV and ADSCs in group V. After the procedure, a suture was taken between the ends of the muscles then the skin was sutured. Rats from each group were subdivided into two subgroups; subgroup “a” in which the animals were sacrificed after ten days of the operation and subgroup “b” in which the animals were sacrificed after eight weeks of the operation.
At the end of the experiment, animals in all subgroups were sacrificed and longitudinal muscle biopsies were obtained with the area of laceration in the middle. Histological examination was done to study muscle regeneration (using H&E and Mallory`s Trichrome stains). Immune-histochemical examination was also done for the expression of Neuro-Filament light chain protein “NFL” to study muscle re-innervation. Morphometric study was done to detect transverse diameter of muscle fibers, number of myotubes, and area percentage of collagen fibers and expression of NFL. Statistical analysis was also performed.
After ten days from the laceration (subgroup IIIa), histological examination of the gluteal muscle showed the site of the injury filled with granulation tissue and many inflammatory cells as lymphocytes and macrophages and few neutrophils. These cells were seen extending into the interstitium between adjacent muscle fibers. Necrotic fragments of the injured muscle fibers and dilated congested blood vessels were noticed. At the side of the injury, few amounts of granulation tissue could be seen and the muscle fibers appeared disorganized and running in different directions. The interstitium of the muscle showed groups of fat cells. Few degenerated muscle fibers were seen with wide diameter, cytoplasmic vacuolations and disrupted striations. Many cells with vesicular nuclei and prominent nucleoli -most probably activated satellite cells- were seen. Furthermore, immature regenerating myotubes with indistinct cross striations and chain of central vesicular nuclei were seen. Immune-histochemical examination revealed negative expression of NFL. This indicates failure of muscle re-innervation.
Ten days after administration of BM-MSCs to the lacerated area (subgroup IVa) showed filling the center of laceration injury by granulation tissue. Granulation tissue was less fibrous but more cellular as compared to the untreated laceration injury (subgroup IIIa). It contained mononuclear inflammatory cells as macrophages and lymphocytes. Few dead neutrophils were detected surrounding deeply stained necrotic muscle fragments. Small blood vessels which might indicate angiogenesis and congested blood vessels were also detected together with extravasated RBCs. Large accumulations of fat cells were seen adjacent to the muscle fibers at the edge .At the side of injury, apoptotic muscle fibers showed deep acidophilic cytoplasm and pyknotic nuclei. Degenerated muscle fibers and groups of cells with vesicular nuclei and prominent nucleoli were frequently seen. These cells were most probably activated satellite cells or BM-MSCs. Immature regenerating myotubes were noticed. The mean number of myotubes was significantly higher than in untreated laceration (subgroup IIIa). The mean area percentage of collagen fibers showed a significant increase compared to (subgroup IIIa) (p <.05). Immune-histochemical examination revealed positive expression of NFL indicating the start of re-innervation.
On the other hand, ten days after administration of ADSCs to the lacerated area (subgroup Va) showed filling the center of laceration injury with granulation tissue that extended in-between muscle fibers at the side of injury. It was highly cellular and less fibrous than in the untreated laceration injury. The granulation tissue contained inflammatory cells as lymphocytes and macrophages. Few neutrophils were also observed. Deeply stained necrotic muscle fragments were also observed. At the side of the injury, lesser amount of granulation tissue and inflammatory cells were seen in the interstitium. Aggregations of fat cells, and congested blood vessels were seen. Few degenerated muscle fibers were also detected. Cells with vesicular nuclei and prominent nucleoli were frequently noticed. These cells were most probably activated satellite cells or ADSCs. Immature regenerating myotubes were seen. Their mean number showed significant increase compared to untreated laceration injury (subgroup IIIa) and laceration treated with BM-MSCs (subgroup IVa) (p <0.05). Also, a significant decrease in the mean area percentage of collagen fibers was noticed compared to subgroup IIIa and subgroup IVa (p <0.05). Immune-histochemical examination revealed positive expression of NFL indicating the start of re-innervation with no significant difference in the mean area percentage compared to subgroup IVa (p <0.05).
After eight weeks from the laceration, examination of untreated laceration group (subgroup IIIb) showed replacement of the granulation tissue by high amount of fibrous tissue at the site of injury. Fibrous tissue extended between disorganized muscle fibers. Some muscle fibers appeared pale indicating immaturity. Large accumulations of fat cell were seen within the fibrous tissue. Immune-histochemical examination revealed negative expression of NFL which indicates failure of re-innervation.
Eight weeks after administration of BM-MSCS to the lacerated area (subgroup IVb) showed filling the site of injury with new muscle fibers. The muscle fibers were of two types. Mature muscle fibers with dark acidophilic sarcoplasm and well-defined cross striations. Also, immature regenerating muscle fibers with pale acidophilic sarcoplasm and absent cross striations were noticed. Some muscle fibers were seen in the remodeling stage. A significant decrease in the mean area percentage of collagen fibers was noticed compared to untreated laceration group (IIIb) (p <0.05). Immune-histochemical examination revealed positive expression of NFL. This indicates successful re-innervation of gluteal muscle.
Furthermore, eight weeks after administration of ADSCs to the lacerated area (subgroup Vb) showed filling the site of injury with new muscle fibers. The muscle fibers were of two types. Mature muscle fibers with dark acidophilic sarcoplasm and well-defined cross striations. Also, immature regenerating muscle fibers with pale acidophilic sarcoplasm and absent cross striations were seen. Some muscle fibers were seen in the remodeling stage. Fat cells were seen adjacent to newly regenerated muscle fibers. There was a significant decrease in the mean area percentage of collagen fibers compared to untreated laceration group (subgroup IIIb) and laceration treated with BM-MSCs group (subgroup IVb) (p <0.05). Immune-histochemical examination revealed successful re-innervation as indicated by the positive expression of NFL with a non-significant difference compared to subgroup IVb (p >0.05).