Qom University of Medical Sciences Journal

Introduction
Burn-related injury is the fourth most common cause of injury in the world, accounting for 5-12% of hospitalized cases. Finding new therapies in combination with antibiotics is necessary to treat these injuries. Herbal antimicrobial agents have high potential in the treatment of infectious diseases. Their simultaneous use with antibiotics may reduce the side effects of antibiotics. Regarding to the important role of Staphylococcus aureus in burn wound infection, the present study aims to investigate of the effect of Polycaprolactone (PCL) scaffold with Myrtus communis extract on the healing of burn wound infections induced by Methicillin-resistant Staphylococcus aureus (MRSA) in rats.
Methods
 In this quasi-experimental study, the methanolic extract of the Myrtus communis was obtained using Maceration method. The synthesis of silver nanoparticles was done using silver salt solution, green method, and the methanolic extract of Myrtus communis. The nanofiber scaffolds of the Myrtus communis extract was prepared by electrophoresis from PCL polymer. Totally, 42 male Wistar rats weighing 200-250 g were randomly divided into seven groups of 6. Rats were kept in separate cages with adequate water and food.
After anesthesia by intraperitoneal injection of ketamine (50 mg/kg) and xylazine (50 mg/kg) intraperitoneally, second-degree burns were induced on the back of rats. After 24 hours and confirmation of second-degree burns, 0.1 ml of MRSA suspension with SCCmec type 3 was injected subcutaneously into the wound area in several directions. After 24 hours, rats were treated daily with scaffolding for 14 days with PCL alone, PCL+Myrtus communis extract 1%, PCL+Myrtus communis 0.5%, PCL+silver nanoparticles, and PCL+Myrtus communis extract + silver nanoparticles. For evaluation of wounds and infection on days 1, 5, 7, 9, 12 and 14, direct culture was prepared from the infected wound site by using sterile swabs.
To measure the level of burn on the first, seventh and 14th days of burn induction, the radius of the wound was measured with a ruler and the area of the wound was calculated. After taking picture of the wound site and killing the rats, to compare the amount and quality of wound shrinkage and wound healing, the entire skin of the affected area and some of the surrounding healthy skin was deeply removed. After washing with physiological serum, a part of the tissue was kept in the Eppendorf microtube for biochemical tests (malondialdehyde level, nitrite oxide level, and total antioxidant capacity). The remaining part was kept in 10% formalin solution for 24 hours and then transferred to the new 5% formalin solution in the second 24 hours. Wound histopathological changes were observed by Nikon light microscope. Malondialdehyde level was measured based on a reaction with Thiobarbituric Acid (TBA). Nitric oxide level was measured suing Griess reaction based on microplastic method. The Total Antioxidant Capacity (TAC) was measured by the ferric reducing/antioxidant power test. Scanning Electron Microscopy (SEM) test was used to evaluate the presence of extracts and nanoparticles on nanofibers, and contact angle test was used to evaluate the hydrophilicity of scaffolds. GRAPH PAD software was used for statistical analysis and the data were reported using mean and standard deviation. Data were statistically analyzed by one-way ANOVA and Tukey’ test after determining the normality of data distribution. The significance level of the tests was set at 0.05.
Results
The results showed that treatment with PCL+Myrtus communis extract and PCL+Myrtus communis extract+silver nanoparticles in comparison with other groups resulted in a significant increase in wound healing. SEM analysis showed the presence of Myrtus communis extract and silver nanoparticles on the prepared nanofibers. These particles had a diameter of about 636 nm.
In bacteriological studies on the 12th and 14th days, PCL+Myrtus communis extracts and PCL+silver nanoparticle groyps stopped the growth of bacteria better than PCL alone, and a significant difference was observed.
The results of wound area assessment on the seventh and 14th days, showed that the PCL+Myrtus communis extract significantly reduced the wound area compared to other study groups and helped the wound healing. In histological studies, the results showed that the PCL+Myrtus communis extract and PCL+Myrtus communis extract+silver nanoparticles had the best effect on the skin wound healing speed compared to other groups. The thickness of epidermis and dermis at the end of the 14th day in these two groups was significantly increased compared to positive control and PCL alone groups.

The results of biochemical tests to investigate the inhibitory effects of oxidative stress showed that the burn increased the level of malondialdehyde and nitric oxide and reduced TAC in rats. On the fourteenth day, in the group received treatment by PCL+Myrtus communis extract significantly reduced malondialdehyde and nitric oxide levels and increased TAC compared to other groups.
Discussion
Prevention of infections caused by MRSA is essential due to its potential pathogenicity and increasing resistance to antimicrobial drugs. For this reason, researchers have made great efforts to find suitable antibiotic compounds and assess the effects of herbal nanoparticles against this bacterium. Herbal extracts and silver metal in nano-state in combination with nanofiber scaffolds increase the hydrophilicity and antimicrobial activity of nanofibers. Although many antibacterial and antifungal activities of herbal extracts have been reported, their effectiveness against antibiotic-resistant bacteria is very low. The results of this study in vitro revealed that the Myrtus communis extract and silver nanoparticles play an important role in reducing the time required for complete burn wound healing and also in preventing the progression of burn wound infections by modifying the properties of PCL.
Ethical Considerations
Compliance with ethical guidelines
This plan has been approved by Yasuj University of Medical Sciences ethics committee (Ethics Code: IR.YUMS.REC.1399.083).
Funding
This study was carried out with the financial support of Yasouj University of Medical Sciences.
Authors contributions
All authors contributed equally in preparing all parts of the research.
Conflicts of interest
The authors declared no conflict of interest.
Acknowledgements
The authors appreciate and thank the officials of the microbiology laboratory of the Faculty of Medicine and the Medicinal Plants Research Center of Yasouj University of Medical Sciences.