Isolation and propagation of muscle-derived mesenchymal cells and assessment of their regenerative potential in skeletal muscle injuries in mice
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Date
2019-08
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G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand)
Abstract
The present study was conducted on 24 clinically healthy Swiss albino mice (8-10 weeks old) of either sex, weighing 25-35 gm. The animals were divided into three groups viz. group A, B and C, having 8 animals in each. In all the groups, the gastrocnemius muscle was injured by surgical resection of the muscle. After creating muscle injury, PBS was injected in the mice of group A (control group) on day 1 and 5. In the mice of group B, MDSCs were injected in the injured muscle on day 1 and 5. While in the mice of group C, MDSCs were injected in the injured muscle on day 5 and 10. Muscle-derived mesenchymal cells were isolated by modified preplate method and cultured in proliferation media and incubated at 37 °C in a humidified 5% CO2 incubator. When the cells reached 70-80 % confluency, they were harvested using 0.25% trypsin-EDTA and cells were implanted at the site of injury in animals of group B and C. The regenerative potential of MDSCs for repair of the injured muscle was assessed by the evaluation of physical parameters, physiological parameters, wound condition, biochemical parameters,
histopathological studies and free wire hanging test at different time intervals. Also, gene expression profiling of mVEGF was done for the evaluation of muscle regeneration via angiogenesis on day 7 and 15 after creation of muscle injury wound. On the basis of parameters observed in the present study, early and better healing of injured muscle was revealed in the mice of group B. There was early reduction in
swelling, exudation, warmth and pain at the repaired site in the mice of group B. The level of enzymes CK, LDH, AST and AST was decreased significantly in the mice of group B. Also, the score of free wire hanging test was highest in the mice of group B as compared to the mice of group A and C indicating complete regeneration of the injured muscle. Histopathological observations revealed the presence of well organized, polygonal shaped regenerated myofibers having peripherally arranged nucleus in the mice of group B. Gene expression analysis showed upregulation of the gene mVEGF in group B as compared to control group. Results concluded that muscle-derived mesenchymal cells can be isolated by modified preplate method, cultured in-vitro and could be used without any scaffold for regeneration of injured skeletal muscle. Therefore, the current study confirmed that muscle-derived mesenchymal cells may be used for early, better and complete muscle
regeneration in clinical cases of muscle injuries.
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