الفهرس 
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Abstract
Maggot debridement therapy (MDT) is one of the ‘old’ techniques in wound healing. It is basically a therapeutic wound myiasis. The most commonly used fly in MDT is Lucilia sericata (L.sericata). Owing to the unavailability of an effective alternative to L.sericata, researches tried the repositioning of the ordinary house fly, Musca domestica (M. domestica) as a substitute of L.sericata.
One of the major obstacles in the utilization of MDT is the poor acceptance by both patients and health care professionals. Recent researches are focused on extraction of certain molecules from these larvae which can stimulate wound healing.
Chitin and chitosan are capable of accelerating the healing process and the repair of different tissues, facilitating contraction of wounds and regulating secretion of the inflammatory mediators. Besides, several studies suggested the presence of an antibacterial substance and proteolytic enzymes in the alimentary tract of maggots, thus having great role in the mechanism of MDT for being capable of bacterial digestion and promotion of wound healing.
Wound healing is a dynamic and complex process that begins at the time of wounding and involves the replacement of destroyed tissue by living one. The orchestra of skin healing involves inflammation, re-epithelialization, angiogenesis, granulation tissue formation, and deposition of interstitial matrix, besides other events carried out by different types of cells, such as keratinocytes, fibroblasts, and inflammatory and endothelial cells
Immunocompromised patients are more susceptible to bacterial, fungal, and viral infections than those with healthy immune systems especially, in wounded areas. Deficient immune systems likely cause decrease in cellular proliferation, neovascularization, and in matrix production, thus, a defect in normal wound healing.
Research in the field throws light recently on different substances derived from natural sources especially insects and insect derivatives to show their effects on wound healing. Moreover, as several maggot extracts have a role by one way or another in the success of maggot therapy, the present study is designed to assess the role of M.domestica larvae-derived chitosan and gut extracts on wound healing in immunocompetent and immunosuppressed experimental mice.
In the present work, cultures of M. domestica adults were maintained at the laboratory of Zoology Department, Faculty of Science, University of Alexandria. Adults were identified in the lab depending on the shape of the posterior spiracles of the third instar larvae developed during rearing of the adults. Fresh beef liver was used for oviposition. Then, part of liver containing eggs was used for cycle maintenance and the rest was used for chitosan and larval gut extraction. Chitosan was prepared from the third instar larvae by deproteinization, demineralization and deacetylation. For gut extraction, egg masses were sterilized, then incubated till hatching. The third instar larvae were dissected, and the gut contents were extracted into petri dish, then pooled into aliquots and stored at -20 ̊C till used.
For evaluating the efficacy of the extracted substances, one hundred and eighty Swiss strain Albino mice, four to six weeks old, were used. They were divided into two main groups, group I: control group, wounded non-treated (60 mice) and group II: experimental group, wounded treated mice (120 mice). Animals of group I and II were equally subdivided into 2 subgroups, a: immunocompetent and b: immunosuppressed. Each subgroup in experimental animals was further classified into chitosan treated and gut-treated (30 mice each). Mice were wounded on their backs midway between shoulders (one centimeter diameter). The tested substances were applied daily, then six mice from each subgroup in the control and experimental groups were sacrificed on the third, seventh, fourteenth, twenty first and twenty eighth days post-wounding or till complete closure of the wound after being clinically observed for wound healing.
The efficacy of the tested material was assessed through naked eye examination of the wound to detect the signs of healing and inflammation. Wound diameter was also measured to determine wound contractures. The quality of wound healing and remodeling process was assessed by histopathological examination of skin sections taken from the wounded areas at different durations using light microscope equipped with camera.
Hematoxylin &Eosin (H&E) stained sections were done for examining the epithelial cells in the epidermis and the dermis. The epidermal thickness was measured using a PathPic image analysis software developed at the institute of medical research.
Trichrome stained skin sections were also performed to evaluate the collagen deposition in the wounded areas. The mean collagen fraction was measured using the ImageJ software (NIH) to compare the amount of collagen in the healed wound between the treated and untreated groups.
Immunohistochemical study was performed to assess the maturation, differentiation and proliferation of epithelial cells of the neo-epidermis and neo-dermis after wound healing. This was done by using Primary antibody (Cytokeratin Cocktail AE1/AE3) to detect the deposition of keratin which is a marker denoting the maturation of keratinocytes.
Bacteriological study was performed by using quantitative culture technique at different time interval, till the time of complete healing by naked eye to detect the antibacterial effect of the tested material.
The obtained chitosan was of high molecular weight (2,000,000 MW) and high degree of deacetylation (85%DD).
The best results in wound contraction were revealed in immunocompetent chitosan treated subgroup (IIa1) that showed significant reduction in the mean wound size [(0.80±0.14) and (0.38±0.15)] on the 3rd and 7th day post wounding respectively in comparison to subgroup Ia that had a mean wound size of (1.20±0.26) and (0.87±0.24) respectively. The reduction in wound diameter was significant only on the seventh day post wounding to the side of competent treated subgroup (IIa1) (0.38±0.15) over immunosuppressed one (IIb1) (0.73±0.12). On the other hand, wound size reduction was not significant in gut treated subgroups on the 3rd and 7th days post wounding compared to their corresponding untreated subgroups in both immunocompetent and immunosuppressed mice. In addition, there was significant reduction in wound diameter on the 7th days post wounding between immunocompetent treated subgroups [(IIa1) and (IIa2) with a mean wound size of (0.38±0.15) and (0.78±0.08) respectively].
Histological examination of H&E stained sections revealed an epidermal creeping starting early on the 3rd day post wounding in the immunocompetent treated subgroups (IIa1) and (IIa2). This epidermal tongue was more formed in gut treated subgroup, while was completely absent in subgroup Ia. Hair follicles and sebaceous glands were encountered on the 14th day post wounding in subgroups (IIa1) and (IIa2), however, they were delayed till day 21 and 28 post wounding in subgroup Ia. On the other hand, highly vacuolated epidermis extended behind the edge of the wound with many pyknotic nuclei detected in subgroup Ib with less vacuolation in subgroup IIb1 as a result of the immunosuppressive drug. Surprisingly, early improvement was observed in subgroup IIb2 that showed creeping epidermal tongue on the 3rd day post-wounding. Hair follicles and sebaceous glands were encountered on day 14 post wounding in subgroup IIb2. However, this was delayed till day 28 post wounding in subgroup IIb1. Moreover, subgroup Ib showed no skin appendages till the end of experiment. It is worth mentioning that complete re-epithelialization was seen seven days post wounding in both immunocompetent and immunosuppressed treated subgroups compared to their corresponding control in which re-epithelialization was delayed till day 14 post wounding.
The results revealed significant reduction in the epidermal thickness on day 14 post wounding (time of wound closure) in subgroup IIa1 (99.37±40.09) compared to the subgroup Ia (191.55±39.61). This reduction was also significant between subgroup IIa2 (57.80±14.26) and subgroup Ia. As regards the immunosuppressed mice, the only significant reduction was observed in subgroup IIb2 (130.07±30.14) when compared with its corresponding control one (Ib) (263.64±89.55). There was also significant reduction between chitosan treated subgroups (IIa1 &IIb1), but, this reduction was not significant between the two gut treated subgroups (IIa2& IIb2). However, gut treatment in the immunosuppressed condition was superior to chitosan treatment that showed significant reduction in epidermal thickness.
The best results in collagen deposition were detected in gut treated subgroups. Significant increase in the mean collagen fraction was detected in subgroup IIa2 (226.99±8.55) compared to subgroup Ia that showed a mean collagen fraction of (184.53±12.96). On the other hand, in immunosuppressed gut treated mice, there was a significant increase in the mean collagen fraction in subgroup IIb2 and its corresponding control (Ib) [the mean collagen fraction was (196.99±10.28) and (118.97±1.57) respectively]. The only significant increase in the mean collagen fraction in chitosan treated mice was detected in subgroup IIb1 (188.42±16.29) when compared with its immunosuppressed control subgroup Ib (118.97±1.57). There was no significant effect on collagen deposition in comparing treated competent and suppressed subgroups and between gut and chitosan treated subgroups.
Immunohistochemical results in the present study showed that subgroups IIa1&IIa2 gave the best maturation detected by deep brown colour of keratin however in subgroup IIa2 dense positive reaction (deep brown color) of the thickened epidermis detected as early as seven days post wounding. The same result was also detected in immunosuppressed mice in subgroup IIb2 on the 14th day post wounding. However, in chitosan treated immunosuppressed mice, they showed lighter reaction (brown colour) till the end of the experiment. Other groups showed weak reaction detected as scanty brown colour denoting improper keratin deposition.
Microbiological study revealed the presence of Staphylococcal colonies in opened wounds at deferent intervals. The difference in colonial numbers was assessed. The results revealed significant increase in colonial numbers in subgroup Ib (18.36×104 ±24.37) compared to subgroup Ia (1.0×104 ±1.67) on the 7th day post wounding. While in immunosuppressed treated mice, there was significant reduction in colonial numbers on the 3rd and 7th day post-wounding as compared to subgroup Ib. This reduction was not significant between the immunocompetent subgroups.
The marvelous effect of gut extracts could be a new light in the field of wound healing. Mature and intense collagen deposition was observed in mice treated with our new extract. Additionally, it had antibacterial effect evident in reducing the colonial count, thus protecting against wound infection that can be a major threat against healing especially in immunosuppressed mice.
On the other hand, chitosan was superior in wound closure acceleration. Its effect was mainly evident in immunocompetent subgroup. Furthermore, the antibacterial effect of chitosan was evident by colonial number reduction in competent and suppressed subgroups. The delay in hair growth in chitosan treated subgroups besides, the poor epidermal remodeling at the time of wound closure could be a defect in tissue repair that we were looking for, especially in immunosuppressed subgroup. The benefits of chitosan should be outweighed with its drawbacks on considering it for wound healing during immunosuppression. This should enhance further investigation interpreting the effect of possible combination therapy including the two extracts.
Our results opened a new field using M.domestica larval extracts as a relevant and reliable substitute to that of the famous L.sericata larvae in the field of maggot therapy.