الفهرس 
REVIEW OF LITERATUREOnly 14 pages are availabe for public view
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Abstract
The integrity of skin is essential to maintain the physiological homeostasis of the human body. Skin wounds cause loss of skin’s integrity which results in physiologic imbalance. Skin wounds also have serious psychological impacts due to the disfigurement caused by scarring which can be impossible to prevent and difficult to treat.
Exosomes are effective in treating various diseases and do not have the drawbacks of MSCs. Therefore, the attention of researchers was directed towards cell free therapy in treating skin injuries.
This histological study was carried out to study the potential role of exosomes derived from MSCs on healing of cutaneous wound in adult male albino rats.
Forty-two adult male albino Wistar rats with average weight of 200 grams were used in this study. In addition, five young weaned male albino rats weighing 70-80 grams were used for the isolation of MSCs. The adult rats were randomly divided into three groups, 14 rats each: group I: served as a control group, and it was further subdivided into two subgroups, subgroup Ia and subgroup Ib in which rats were sacrificed after 14 days and 21 days from the beginning of the experiment, respectively. group II: served as spontaneous wound healing group in which full thickness skin wound was generated on the back of the rats. This group was further subdivided into two subgroups, subgroup IIa and subgroup IIb in which rats were sacrificed after 14 days and 21 days from the beginning of the experiment, respectively. group III: served as treated skin wound with exosomes, in which full thickness skin wound was generated on the back of the rats and on the second day after surgery, rats were given single intravenous injection of 1ml PBS in the tail vein containing purified exosome concentrate collected from the conditioned medium of one million MSCs. This group was further subdivided into two subgroups, subgroup IIIa and subgroup IIIb in which rats were sacrificed after 14 days and 21 days from the beginning of the experiment, respectively.
Exosomes were characterized using transmission electron microscope.
Rats in groups II and III were monitored for nociceptive behavior via using the Rat Grimace Scale (RGS). Skin wounds in groups II and III were grossly observed on days 7, 14, and 21 with measuring the wound’s length and width. Skin specimens were examined histologically via Hematoxylin and eosin (H&E) stain, Masson’s trichrome stain, Periodic acid-Schiff (PAS) stain as well as immunohistochemical analysis for detection of proliferating cell nuclear antigen (PCNA) reaction.
In the current study, rats in group III showed nociceptive behavior for shorter duration than the rats in group II. Regarding the wound size, group III showed significant decrease in wound size when compared to group II on days 7, 14, and 21.
On day 14, H&E-stained sections of subgroup IIa showed the beginning of the re-epithelialization in the form of flattened cells with flat nuclei and acidophilic cytoplasm covering the wound site, except for small areas that were not covered by epidermis. Also, separation between the newly formed epidermis and the new dermis was frequently seen. In contrast, in subgroup IIIa, the wound edge was covered with a thick layer of stratified squamous keratinized epithelium which appeared migrating towards the wound’s center. In addition, a significant increase in the mean epidermal thickness at edge and center of the wound and the mean dermal thickness was noticed compared to subgroup IIa.
Masson’s trichrome stained sections of subgroup IIa showed the presence of few collagen fibers with high amount of interfibrillar matrix. While in subgroup IIIa, the newly formed collagen at the wound edge showed better organization and consisted of thin collagen fibers and thick, wavy collagen bundles deeper in the dermis, forming the papillary and reticular layers of the dermis respectively. While the dermis at the center of the wound showed disorganized collagen with few thick collagen bundles in-between the fine collagen fibers. In addition, a significant increase in the mean area percentage of collagen fibers was noticed compared to subgroup IIa.
PAS-stained sections of subgroup IIa showed inapparent basement membrane underneath the regenerated epidermis while in subgroup IIIa, the newly formed basement membrane was well-defined with few areas of ill-defined basement membrane.
Immunohistochemical examination of PCNA in subgroup IIa showed a significant decrease in number of epidermal PCNA positive nuclei compared to control group. Mild positive brownish PCNA reaction was noticed in the nuclei of some of the flattened cells in the newly formed epidermis as well as few PCNA positive nuclei in the papillary dermis. While in subgroup IIIa, a significant increase in number of epidermal PCNA positive nuclei was noticed compared to subgroup IIa. PCNA positive nuclei were seen in all the layers of the newly formed epidermis and in most cells of the papillary dermis.
On day 21, H&E-stained sections of subgroup IIb showed undifferentiated layers of keratinocytes in the newly formed epidermis covering the wound site, with separation of the most superficial layers. While in subgroup IIIb, the wound area was covered with a thin layer of stratified squamous keratinized epithelium, differentiated into stratum basale, spinosum, granulosum, and corneum. In addition, a significant decrease in the mean epidermal thickness at edge and center of the wound and a significant increase in the mean dermal thickness, were noticed compared to subgroup IIb.
Masson’s trichrome stained sections of subgroup IIb showed a significant increase in collagen fibers compared to subgroup IIa, but it was still significantly decreased compared to control group and subgroup IIIb. The papillary layer of dermis contained thin, fragmented, faintly stained, irregularly arranged, and widely separated collagen fibers. The reticular layer contained relatively thicker, but still disorganized, and widely separated collagen bundles. In contrast, subgroup IIIb showed better organization of the newly formed collagen fibers that appeared similar to control group.
PAS-stained sections of subgroup IIb showed ill-defined basement membrane while subgroup IIIb showed a well-defined basement membrane underneath the newly formed epidermis.
Immunohistochemical examination of PCNA in subgroup IIb showed a significant increase in the number of epidermal PCNA positive nuclei compared to subgroup IIIb. PCNA positive nuclei were seen in the basal layer of the epidermis as well as in the papillary dermis. While in subgroup IIIb, positive nuclear PCNA immune reaction was seen in many cells of the stratum basale and stratum spinosum of the epidermis, the outer root sheath of hair follicles, and in the papillary dermis.
Concerning the recovery of skin appendages, subgroup IIIa showed downgrowth of the epidermis without the formation of new hair follicles while in subgroup IIIb, newly formed hair follicles and sebaceous glands were seen. No skin appendages could be observed in the other subgroups.
Such findings revealed that group III showed faster re-epithelialization, the formation of well-defined basement membrane, recovery of skin appendages, better organization of collagen, as well as increasing the proliferation of epidermal and dermal cells in comparison with group II.
Hence, we concluded that intravenous injection of MSCs-derived exosomes was effective in healing skin wounds via accelerating the re-epithelialization, and the proliferation of epidermal and dermal cells, enhancing collagen fibers’ organization as well as promoting the recovery of skin appendages.