الفهرس 
AbstractOnly 14 pages are availabe for public view
 |  | from | 314 |  |  |
| | | from | 314 | | |
Abstract
Forensically, wounds specifically traumatic one has great medico-legal importance, they not only indicate the causal agent, but also could determine the vitality; moreover they are a perfect indicator for curtly against human and animals
Medically, thermal wounds represent a major public health issue, they resulting in a marked patient morbidity and mortality annually, also bruises severity ranged from mild to severe; as death may be happen if they are inflicted in a vital organ. Hence it is of great importance to study these types of wounds legally, and trying to provide a highly efficient therapy.
SCs represent the front-line source of regenerative medicine, for reparation of tissues and organs anomalies moreover, they providing efficient, high-quality therapy for skin coverage, minimal risk of hypertrophic scarring, and low-morbidity rate. BM-MSCs are one of the most multi potent progenitor SCs; they could be differentiated into many cell types as the endothelial and epithelial cells, fibroblasts, myofibroblasts, neurons, adipocytes, chondrocytes, and osteoblasts. Besides their powerful ability to regenerate the whole structures of the cutaneous tissue they are also, isolated from other marrow cells easily, and have a high propensity to adhere to the plastic surface of the tissue culture.
In the current study 246 animals were divided into the following seven groups of six animals in each time interval (zero time, 2, 6, 12 and 24 hr, 7 and 14 day in thermal models), and (3, 6, 12, 24, 48, and 72 hr in bruise model). Group1 (Control), animals not exposed to any type of wounds, group 2 (Burn model), The back of the anesthetized animals were shaved and burned with a formerly pre-heated solid aluminum bar in boiling water that maintained in close contact with the animal’s skin for 15 sec on the dorsal proximal area, group 3 (Burn model treated with BM-MSCs), the burned animals were injected (S/C) with BM-MSCs at a dose of 2×106 cells/ mL, group 4 (Scald model): the posterior area of the back of the anesthetized animals was shaved and immersed in water at 100°C for 10 sec which covering 20% TBSA, group 5 (Scald model treated with BM-MSCs), the scalded animals were injected (S/C) with BM-MSCs at a dose of 2×106 cells/ mL, group 6 (Bruise model), the shaved abdomen of the anesthetized rats was lightly pinched by a small pair of Spencer-Well forceps, and group 7 (Bruise model treated with BM-MSCs), the bruised animals were injected with BM-MSCs at a dose of 2×106 cells/ mL S/C.
The clinical course of skin lesions by thermal wounds was evaluated for 14 sequential days with determining the percentage of wound retraction by using a caliper in the tested time intervals. In addition, different colors of bruises were recorded.
At the end of each tested time intervals, animals underwent anesthesia via (ACE) mixture for sacrification, which followed by blood and skin samples collection. The clear non haemolysed supernatant serum was quickly prepared for determining the concentration of VEGF, while formaline- fixed skin specimens were processed and stained with H&E for examining the histopathological lesion, and the immunohistochemical changes. Another part of the skin samples were immediately snap frozen in liquid nitrogen, then preserved under -80oC for analysis the gene expression rate of the wound healing mediators.
The following results have been recorded in each model:-
In burn wound:
Following burning, pale circular area has been noticed grossly then blister was developed at 2hr which become pale at 6hr, then rupturing was noticed at 12 and 24 hr, at 7 days the lesion was covered with reddish stiff crust which decreased in size and still attached to the wound till 14 day. In addition the percentage of the wound contraction rate was decreased significantly at 7 and 14 day post injury.
Histopathologically; there is severe degenerative changes and necrosis of the squamous epithelium lining associated with the absence of the epidermis in certain burn areas that indicates deep second-degree burn at zero time and 2 hr, with developing of oedema and damaging of the hair follicles at 6, 12, and 24 hr. At 7 days, the epidermal layer showed acidophilic scab formation and the granulation tissue was formed in the dermal layer. At 14 day, the epidermal growth with immature differentiation and dermal granulation tissue formation have been evident.
The area percentages of ADAMs10 immunohistochemical expression was decreased at zero time, 6 and 24 hr and increased at 7 and14 day.
On the level of mRNA expression rate of the different molecular markers that incorporated in the healing process, the rate of TGF-β was significantly up-regulated at zero time, 6 hr. and 14 day, and down regulated at 2 hr, the rate of IL-6 was significantly up-regulated at zero time and 14 days, the rate of TNF-α was significatly down regulated at zero time and 2hr, and up-regulated at the remaining intervals, the rate of HSP-90α was dramatically up-regulated, its significantly reduced at zero time and significantly up-regulated at all the remaining intervals, the MMP-9 expression rate was significantly up-regulated at zero time and 6 hr, whist significant down regulated was recorded at 24 hr, 7, and 14 day, the rate of miR-21 was significantly up-regulated at zero time, 6 and 24 hr, and down regulated at 7 and 14 day. Furthermore, the concentration of VEGF has been increased at zero time and 6hr and decreased significantly at 24 hr, 7 and 14 day.
On the other hand, the skin nearly appeared normal following treatment of the burned animals with BM-MSCs, in which the wound assumed pin point appearance, and the percentage of the wound contraction rate was significantly decreased at 14 day.
Moreover, quite an improvement of the epidermal and dermal layers was detected at 6, 12 and 24 hr, epidermal growth with immature differentiation and dermal granulation tissue formation have been evident at 7 days, while at 14 day, the epidermal layer was regularly arranged with thin stratum corneum and the underlying granulation tissue of dermis showed few inflammatory cells and edema, and the area percentage of the immunohistochemical expression of ADAMs 10 was significantly increased at zero time and 6hr, and decreased at 14 day.
Regarding the mRNA expression rates, the rate of TGF-β was significantly up-regulated at 12 hr and down regulated at 24 hr, 7 and 14 days, the rate of IL-6 was significantly reduced at zero time, 2, 6 and 12 hr, and 14 day, the TNF-α expression rate was remarkably elevated at zero time, 2 and 6hr, and reduced at 14 day, the rate of HSP-90α of was significantly up-regulated at 6 and 24 hr, and 7and 14 day, the MMP-9 expression rate was significantly down regulated at 6 and 24 hr, 7 and 14 day, the rate of miR-21was significantly up-regulated at zero time, and down regulated significantly at the remaining time intervals. The concentration of VEGF was significantly decreased at 6 hr and 7 days.
In scald wound:
Immersing of the animal’s back in boiling water causing slight cutaneous redness and blistering for 2hr after exposure, which become pale with time with developing of ruptured blisters at 6, 12 and 24hr, a thin crust or thick crust in some animals was observed after 7 days from the immersing which sloughed and the underlying granulation tissue was appeared at 14 day, and the percentage of the wound contraction rate was significantly decreased at 14 day.
Histopathologically; severe necrosis of the epidermal and dermal layers was reported indicating the full thickness injury, with presence of severe degenerative changes and necrosis of the hair follicles, sweat, and sebaceous glands, and coagulative acidophilic fused collagen fibers. The immunohistochemical expression of ADAMs10 was progressively increased in all-time intervals until 7 days and decreased at 14 day.
On the level of mRNA expression rate, the rate of TGF-β was remarkably down regulated at zero time and 2hr and elevated at 24 hr and 14 day, the expression rate of IL-6 was up-regulated at zero time, 2 and 12 hr, and 7 days and down regulated at 24 hr and 14 day, while the rate was down regulated significantly at 6hr, the rate of TNF-α was significantly down regulated at zero time and 6 hr and elevated at 12 and 24 hr 7 and 14 day, the rate of HSP-90α was significantly down regulated at zero time and 6hr, while at 24 hr, 7 and 14 day the rate was significantly up-regulated, The rate of MMP-9 rate was down regulated significantly at 7 and 14 day, the rate of miR-21 was significantly up-regulated at zero time, 6 & 24 hr and down regulated at 7 and 14 day. The VEGF concentration was increased significantly at 24 hr and decreased at zero time and 14 day.
Therapy of the scalded animals with BM-MSCs causing developing of intensive bluish coloration at 12 and 24 hr, formation of thick stiff crust which detected at 7 days and continued until the end of the experiment, and increasing the percentage of wound contraction significantly at 12 hr, while no improvements were recorded in the damaged tissue but the inflammatory cell infiltration was increased and the expression of ADAMs 10 was increased in all-time intervals.
Concerning the mRNA expression rates, the rate of TGF-β was significantly down regulated at zero time, 2, 6, and 24 hr and up-regulated at 12 hr, 7and 14 day, the rates of IL-6 and TNF-α were significantly down regulated, the HSP-90α expression rate was significantly up-regulated at 6and 24 hr, 7 and 14 day, the rate of MMP-9 was significantly reduced at 6 and 12hr 7, and 14 day, the miR-21 expression rate was down regulated at 6 and 24 hr, 7 and 14 day, and significant decreasing of theVEGF concentration at 24 hr.
In bruise wound:
The sequences of color changes were appeared as follow, the red color was developed at 3hr, and its intensity was reduced with time and become slight red at 6 and 12hr, slight bluish coloration was appeared after 24hr, and the yellow coloration was appeared at 48hr and become prominent at 72hr.
Histopathologically; degenerative changes of the squamous epithelium lining, presence of leuckocytic infiltrations mainly neutrophils in the dermal layer associated with congested blood vessels were reported at 3, 6 and 12 hr. At 24 hr, the neutrophilic infiltration was extended to the muscular layer. At 48 and 72 hr, the leuckocytic infiltration of the dermal layer was reduced.
For mRNA expression rates, a significant down regulation of TGF-β was reported at 3 hr, while the up-regulation was noted at 72 hr, the rate of IL-6 was down regulated at 3, 12, 48, and 72hr and up-regulated at 6 and 24 hr, the rate of TNF-α was down regulated throughout the all-time intervals, the expression rate of HSP-90α was significantly down regulated at 3hr, then up-regulated significantly until 72 hr, the expression rate of MMP-9 and miR-21 were significantly up-regulated at 3, 6, and 24 hr and down regulated at 48 & 72 hr, significant up-regulation of the of apoptotic rate at 3 and 6 hr, and down regulation at 72 hr, significant increasing of the VEGF concentration at 3and 48 hr and decreasing at 6, 24, and 72 hr.
Contrary, S/C injection of the lightly pinched skin with BM-MSCs resulting in reducing the color intensity especially at 72hr, marked increasing in the neutrophilic infiltration in the muscular layer at 24 hr, whilst, at 48 and 72 hr, minimal leuckocytic infiltration in the dermal layer could be found.
Significant up-regulation of TGF-β mRNA expression rate at 3, 6, 12 and 48 hr and down regulation at 72hr, a significant down regulation in the IL-6 expression rate at 6 & 24 hr, and up-regulation at 48 and 72 hr, significant up-regulation of TNF-α and HSP-90α, significant down regulation of MMP-9 and miR-21 expression rate, significant decreasing in the VEGF concentration at 3, 48, and 72 hr, and significant reduction in the rate of apoptosis.