الفهرس 
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Abstract
In recent years, exploration of natural compounds capable of promoting tissue repair has gained significant momentum. The practice of regenerative medicine for the treatment of wounds is constantly developing to increase intrinsic regenerative capacity and provide the essential elements for rebuilding damaged tissue. The current experimental study aimed to investigate the efficacy of P25/SWCNTs/Ag and P25/rGO/Ag nanocomposites, separately, on healing of skin wounds in a rabbit animal model and to evaluate their regenerative potential when mixed with PRP in a gelatin matrix.
In the present study, 18 apparently healthy male New Zeeland white rabbits, aged three months and weighing 2-2.25 Kg, provided by faculty of agriculture, Alexandria University were used. The nanomaterials used among the current study included two types; first are the synthetic agents including P25/rGO/Ag nanocomposite (abbreviated as rGO) and P25/SWCNTs/Ag nanocomposite (abbreviated as CNT). These materials were prepared and subjected to characterization and tested for their antioxidant activity using the DPPH radical scavenging activity technique in addition to evaluating the antimicrobial activity, using Staphylococcus, Pseudomonas and Fusarium representing the microbial pathogens. The second nanomaterials used are gelatin (GE) and Platelets rich plasma (PRP) which were prepared and used according to techniques described by Zimmermann et al. (2001); Mazzucco et al. (2009) and Javanmardi et al. (2018).
The rabbits were generally anesthetized by injection of ketamine HCl intramuscularly in a dose rate of 35 mg\kg, preceded by xylazine HCl intramuscularly in a dose rate of 5 mg\kg. Each rabbit was subjected to two square-shaped full- thickness excisional skin wounds on the right and left back-side skin. These wounds were divided into 6 groups (n=9) received these treatments; (GE, as the control group (group 1), PRP/GE (group 2), rGO/GE (group 3), PRP/rGO/GE (group 4), CNT/GE (group 5) and PRP/CNT/GE (group 6). Each wound received a total of 1 ml of the treatment applied topically to cover the whole wound surface area. Each 1 ml (contains a dose of 1 mg nanocomposite) was divided into two separate doses (0.5 ml containing 0.5 mg) applied on day 0 and day 2 of wound conduction. The wounds were followed up until complete healing or till end of the experiment (21 days).
All the experiments and procedures were carried out in the department of surgery, faculty of veterinary medicine, Alexandria University after being approved by the institutional animal use and care committee of faculty of veterinary medicine, Alexandria University (AU-IAUCC), and in accordance with the institutional ethical guidelines for the care and use of laboratory animals.
The wound closure process was assessed macroscopically and microscopically. Wound contraction was determined by calculating wound dimensions at different time points. The wound dimensions (cm) and wound surface area (cm2) were measured in all groups at the same time point through the photographs taken for the wounds on days 3, 7, 10, 15 and 21 of the experiment, where the photographs subjected to analysis using imageJ software. The wound healing rate (%) was calculated according to the this equation: Wound Healing % = [ (Wi – unhealed area) / Wi ] X 100.
Histopathological examination for samples collected on day 3, day 10 and day 21 was considered as an additional evaluation parameter. Statistical analysis for data was carried out and all values were presented as M ± SD.
The collected results showed that the DPPH Radical Scavenging Activity for both used nanocomposites proved positive, indicating that both of them have an antioxidant activity. rGO and CNT showed a promising microbial growth inhibition against Pseudomonas aeruginosa and did not demonstrate any antimicrobial activity, among the tested concentration, against the two other microbial pathogens (Staphylococcus aureus and Fusarium moniliform). Both of the two nanocomposites have a synergistic effect on the action of Gentamicin and Fluconazole against the test microbial pathogens.
Macroscopic examination of wounds revealed that PRP and NPs treated wounds proved free from infection and sepsis compared to the control wounds. The overall results revealed that there was enhanced wound healing outcomes in wounds treated with either nanocomposite with or without conjugation with PRP. Using of CNT loaded in GE minimized the effect of CNT, using of rGO loaded in GE maximized the effect of rGO, conjugating of PRP to the rGO minimized the effect of rGO and conjugating of PRP to the CNT maximized the effect of CNT. GE potentiated the activity of PRP during the first 10-15 days only.
The histopathological findings showed severe scab formation, coagulative necrosis, with hemorrhage beside faint eosinophilic edema and mild to moderate mononuclear macrophage infiltration in control wounds versus the other treated wounds that exhibited fewer lesions, in the first few days. Fibroblastic cell proliferation, neovascularization, and epithelial regeneration were noticed after one week. Complete epidermal regeneration in form of healthy keratin and epidermal layers covering with mature granulation tissue and collagen were noticed at the end of the experiment especially in wounds treated with PRP and CNT.