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Abstract
There are two main types of stem cells; HSCs and MSCs. The main source of the MSCs is the BM. BM-MSCs have the capacity to migrate to remote tissues and play a critical role in wound repair and tissue regeneration. So the aim of the present study was to isolate and culture BM-MSCs from adult male albino rats and also to evaluate their benefit in wound healing.
In the current study, thirty five adult male albino rats of average weight 150 grams were used. They were divided into three groups.
In group I: BM was isolated and cultured. In this group, also skin specimens were obtained from the mid back as a control skin specimen.
In group II: a standardized 1.5 cm2 full-thickness wound was done on the mid back. The wound was injected intradermally by 0.5ml of PBS at 6 different sites. Rats of group II were subdivided into 2 subgroups. In subgroup IIA, the skin wound specimens were examined on day 3 and those of subgroup IIb were examined on day 7.
In group III: a wound was performed as in group II and then treated with BM-MSCs suspended in 0.5 ml PBS and was injected by the same method as in group II. Group III rats were subdivided into two subgroups and examined as in group II.
In the current study, MSCs were isolated from the BM and were cultured in complete media. The cultured cells were incubated in a humidified incubator containing 5% CO2 at 370C. The cultured cells were examined every day after culture by the inverted microscope. Removal of the supernatant and changing the media were done on day five and day eight of culture. Some culture dishes were stained by Giemsa-stain and others were examined immuno-histochemically for detection of CD44 positive cells on day 9 after culture.
In the present study, the cultured cells examined on day one were rounded in shape. On day two, some cells became adherent. On day three, the examined BM-MSCs appeared as sporadic spindle and star shaped cells, with multiple processes and central vesicular nucleus. Some cultured cells showed different mitotic figures. On day five, the attached cells appeared as spindle or star or large flat shaped cells with granular cytoplasm. Some of them appeared binucleated. On day eight, colonies of adherent cells with showed different shapes. On day nine the adherent cells appeared confluent and most of them were seen as dense population of spindle shaped fibroblast like cells. Immuno-histochemical staining using CD44 antibodies on day nine of culture, showed BM-MSCs with positive brownish immuno-reaction.
The skin specimens in each group were examined histologically using Hx&E, Mallory’s Trichrome and Aldehyde fuchsin stains. Immuno-histochemical staining was performed for detection of positive CD44 cells. Morphometric study was done and confirmed with statistical analysis.
Untreated wounds examined on day 3 and 7 showed discontinuity between the two ends of the wound which was occupied by granulation tissue. The dermis appeared densely cellular with mononuclear cellular infiltration and was highly vascular. The collagen and elastic fibers were arranged in irregular pattern and showed a significant increase on day 7 in contrast to day 3. In immuno-histochemical examination by CD44 antibodies, there were positive brownish cells in the granulation tissue and epidermal cells on day 3. On day 7 they appeared in the epidermis and in the dermis in hair follicles and sebaceous glands. The total count of CD 44 positive cells showed a significant decrease on day7 in contrast to day 3.
The wounds treated by BM-MSCs were examined on day 3 and 7 and showed healing of the wound edges. Epidermal cells were arranged in better organization in contrast to untreated wounds. The total epidermal thickness showed significant increase in the treated wounds in contrast to untreated wounds. Dermis showed mononuclear cellular infiltration with neovascularization. There was apparent increase in number of hair follicles and sebaceous glands. The amount of collagen fibers and elastic fibers in dermis showed significant increase in treated wounds in contrast to untreated wounds, and were better organized compared to untreated wounds. The total count of CD44 positive brownish cells showed significant increase in treated wound in contrast to untreated wound.
It is concluded that, local administration of BM-MSCs is effective in promoting wound healing. The findings of the current study indicate that BM-MSCs cell therapy may provide a powerful technique to augment healing of clinically problematic wounds.