Effects of Melatonin on Primary Oral Wound Healing in Rats

This study aimed to investigate the effects of melatonin on primary wound healing of the oral mucosa in rats. Thirty healthy male albino rats were used in this study, almost at the same weight, age, and circumstance. All animals were submitted to operation in the buccal mucosal region. Each animal was given a mixture of xylazine and Ketamine hydrochloride Intraperitoneal (I.P.). After about 5 minutes, anesthetic integrity was checked by testing rats' reflexes. After complete anesthesia, each animal was laid down on the dorsal recumbency, via intraoral approach, a full-thickness soft tissue incision 0.5cm length in the right buccal mucosa. The incision was closed by a single simple interrupted stitch using a 5/0 silk suture to heal by primary intention. The animals were randomly divided into two main groups: control group (n=15) and melatonin (n=15)group, which receives melatonin (10 mg/kg/daily) solution orally, then each main group were subdivided into three subgroups (each group 5 rats ) according to the time of sacrifice 3 rd ,7 th and 9 th days after surgery respectively. Biopsies were taken from the site of operation and examined by three histopathologists. The statistical analysis showed significant differences in the inflammatory response between the melatonin-treated and control group at all periods of healing. There were significant differences in granulation tissue formation between the melatonin-treated and control groups at the 7 th and 9 th day healing times. Also, a significant difference existed in reepithelialization between the melatonin-treated and control groups at the 9 th day periods of healing. This study concluded that the melatonin appeared good healing accelerator of oral soft tissue healing in rats . ـــــــــــــــــــــــــــــــــــــــــ


INTRODUCTION
The oral mucosa, a specialized moist tissue lining the oral cavity, extends from the skin mucosal junction (vermillion border) of the lips anteriority to the oropharynx posteriorly (Bahadur, 2021). the oral mucosa plays a critical role as a barrier against exogenous substances, pathogens, and mechanical stresses (Liu et al., 2010).
The wound is defined as primarily damaged or disruption of skin/mucosa contributed to the loss of its microstructure stability and undergoes a complex wound healing process (Salleh and Fauzi, 2021). The primary goals of wound management are rapid wound closure, a functional and aesthetically satisfactory scar (Kujath and Michelsen, 2008). According to the physiology of wound healing, wounds can be divided into two types: acute and chronic (Martin and Nunan, 2015). Wound healing can also be divided into three categories, depending on the mechanism and timing of closure: primary intention, secondary intention, and tertiary intention (Prasetyono, 2009). Overall, wound healing is accomplished through three overlapping but distinct biological processes: (hemostasis), inflammation, proliferation, and remodeling (Almadani et al., 2021).
The use of natural products and antioxidants substances, such as melatonin, has attracted a great deal of attention (Ozler et al., 2010). Melatonin (N-acetyl-5-methoxytryptamine) is an endogenous neurohormone with well-reported anti-inflammatory and antioxidant properties (García-Bernal et al., 2021); melatonin was discovered and isolated from bovine pineal in 1958 by Turkish scientist Aaron Lerner (Lerner et al., 1958). melatonin is a natural methoxyindole first described as a pineal hormone and later present in most mammalian and non-mammalian cells (Pandi et al., 2017). Exogenous melatonin's present in dietary meats such as chicken, lamb, beef, pork but also in fish, eggs, and colostrum, melatonin regulates a variety of physiological functions, such as circadian rhythm and circadian clock genes, reproduction, modulation of mood, sexual behavior or the immune system, and regulation of body temperature and blood pressure (Anadón et al., 2021).
Melatonin exerts antioxidant, immunomodulatory, pro-apoptotic, antiproliferative, and antiangiogenic effects (Najafi et al., 2019). Therapeutic effects of melatonin have been reported in several disorders such as certain tumors, cardiovascular diseases, or psychiatric disorders. Indeed, oncostatin effects of melatonin have been reported in several tumors (breast cancer, ovarian and endometrial carcinoma, human uveal melanoma, prostate cancer, hepatomas, and intestinal tumors) (Gurunathan et al., 2021). Many research shows that endogenously produced and exogenously-applied melatonin is beneficial to the oral cavity in several conditions such as periodontitis, mucositis, cancers. Furthermore, melatonin has been observed to enhance osseointegration and bone regeneration (Najeeb et al., 2016). So, this study aimed to investigate the effects of melatonin on primary wound healing of the oral mucosa in rats.

Modeling Experiment:
This research will include thirty healthy male albino rats of similar age (3 months) and weight (250-300 gm). Rats were chosen from animal maintenance situated in the Veterinary Medicine/University of Mosul (Iraq). Plastic cages were used to keep the animals with a 12-hours light\12 hours dark period at a temperature (22±3˚C) and access to the daily diet and tap water ad-libitum. This research was carried out following international veterinary science protocols on the use of animals. All measurements were taken with the permission of the Mosul University ethical committee to avoid animal cruelty.

Medication:
Melatonin (Green Field Nutrition, USA) solution was prepared by mixing(10 mg/kg) of melatonin powder with (1ml) of normal saline with continuous mixing until a homogenous solution was formed (Dos et al., 2020).

Experimental Groups and Procedures:
All rats received a mixture of general anesthesia ketamine hydrochloride (100 mg /kg) and xylazine (8 mg /kg) as a sedative, analgesic, and muscle relaxant drug intraperitoneally (Heng et al., 2020). Complete anesthesia was obtained within 5 minutes; this kept the animal anesthetized for about 50 minutes.Then each animal was laid down on the dorsal side on the surgical board, via intraoral approach, a full-thickness soft tissue incision 0.5cm length in the right buccal mucosa then the incision was closed by a single simple interrupted stitch using (5/0) silk suture to heal by primary intention (Tang et al., 2015). After the surgical procedure had been completed then randomly selected animals were divided into two main experimental groups as the following:  Control group (n=15): no treatment was received.  Melatonin group(n=15): which received melatonin (10 mg/kg/daily) solution orally. Then Each main group subdivided according to the time of sacrifice as a following:(Tas et al.,2015) G1: Three days after surgery, the animals were sacrificed. G2: Seven days after surgery, the animals were sacrificed. G3: Nine days after surgery, the animals were sacrificed.

Histological Observation:
Oral mucosa specimens were preserved in 10% neutral buffer formalin solution then washed with distal water to remove the excess of formalin. After that, it was processed with ethanol and xylol, then embedded and marked in paraffin wax blocks and lastly freeze for 24 hours. These blocks were sliced into slices using a microtome; each slice is between 4-5 µm in thick and stained with Hematoxylin and eosin stain (H&E), then examined under a light microscope (Canciani et al., 2021).

Criteria of histopathological examination:
1.Criteria of inflammatory response parameter (scoring):  Score 1: Nil No inflammatory cells seen in the field of operation.  Score 2: Mild When inflammatory cells present in few numbers, less than ½ of the field.  Score 3: Moderate Inflammatory cells could be seen in more than ½ of the field.  Score 4: Severe or abundant when inflammatory cells present in huge numbers, more than ¾ of the field. 2. Criteria for the amount of granulation tissue formation (Scoring) (Sultana et al., 2009; Gupta and Kumar, 2015).  Score 1: Absent of granulation tissue formation in the wound.  Score 2: Quantity of granulation tissue formation in the wound gap is scanty.  Score 3: Amount of granulation tissue formation is moderate in tissues.
 Score 4: Total amount of granulation tissue formation in the wound is profound. 3. Criteria of re-epithelization parameters (Scoring) (Al-Saffar et al., 2020)  Score 0: Reepithelialization at the edge of the wound.  Score 1: Reepithelialization covering less than half of the wound.  Score 2: Reepithelialization covering more than half of the wound.  Score 3: Reepithelialization covering the entire wound, irregular thickness.  Score 4: Reepithelialization covering the entire wound, normal thickness.

Statistical Analysis:
The data were expressed as mean ± standard deviation (S.D.), the variation between two experimental sets was statistically evaluated using the Mann-Whitney U test. Statistical significance was accepted for (p-value ≤ *0.05).

RESULTS
On day 3 of an induced wound, the mean value of inflammatory cells (3.8±0.44) in the control group is the highest, while the mean value of inflammatory cells (2.8±0.44) in melatonin groups and the mean value of granulation tissue is (1.6±0.54), and the mean value of reepithelialization ( 0.2 ±0.44) of control group while the mean value of granulation tissue (2.4±0.98), and the mean value of reepithelialization (0.4±0.89) which is the highest value of melatonin group. The p-value of inflammation was found to be less than 0.05, indicating that the two groups are significantly different. Still, there is no significant difference between the two groups in granulation tissue formation and reepithelialization ( Figs. 1 & 2 ). Fig. 1: Mean rank differences between variables treated systemically on the 3 rd day of oral mucosa wound healing for inflammation, granulation tissue formation, and reepithelialization. Post-wounding on day seven, the group treated with melatonin remained the lowest in inflammatory cells infiltration and the highest in granulation tissue and reepithelialization wound healing score. The mean value of inflammatory cells infiltration (3.4±0.54) of the control group and the mean value of granulation tissue (2.2 ±0.44) and reepithelialization (0.8±0.83), while the mean value of inflammatory cells infiltrates (2.2±0.44), granulation tissue (3.4±0.54), and reepithelialization (1.8±0.44) of melatonin group. A *p-value of the wound scores was discovered to be ≤0.05 that indicates a significant variation between the two groups in inflammation and granulation tissue formation, while the (p-value ) of reepithelialization was found to be more than 0.05 which indicates no significant difference between the two groups ( Figs. 3  & 4 ).  Post-wounding at day ninth, the melatonin group remained the lowest in inflammatory cells infiltration and the highest in granulation tissue and reepithelialization wound healing score. The mean value of inflammatory cell infiltration (2.8±0.44 )of the control group and the mean value of granulation tissue (2.6± 0.54)and reepithelialization (1.4±0.54), while the mean value of inflammatory cell infiltrates (1.6±0.89), granulation tissue (3.6±0.83)and reepithelialization (3.2±0.83) of the group treated with melatonin that showed there was a significant difference in inflammation, granulation tissue formation, and reepithelialization (Figs. 5 & 6).

DISCUSSION
Wound healing is a complex process consisting of multi-steps that begins with the hemostasis and then inflammation and continue to granulate tissue formation, angiogenesis, reepithelialization, and wound contraction (Amirian et al., 2021). The first step in wound healing starts with the progression of the inflammatory reaction and the initial stage of the proliferation, with the granulation tissue progression (Broughton et al., 2006). this study showed that in contrast to the control group, the melatonin treated group reported a decrease in the inflammatory response and an increase in collagen deposition and this is an agreement with Soybir et al., (2003) that show melatonin has a positive effect on wound healing and also melatonin increases the angiogenesis which accelerates wound healing process.
The inflammatory phase is responsible for extensive response to microbial іnfectіon and removing the cellular debris from tissue (Politis et al., 2016;Li et al., 2021). It's also liable for the production of oxygen-free radicals. Tissue growth factors trigger the proliferative phase of the healing procedure and the new connective tissue matrix (Lin et al., 2012). Furthermore, persistent inflammation can cause wound healing to be delayed, leading to irregular wound repair or tissue fibrosis (Smith and Martínez 2018). The current study's hіstopathologіcal fіndіng sіgnіfіcant dіfference іn wound healіng process between the control group and melatonin-treated groups, this action may be due to anti-inflammatory and antioxidant effect of melatonin (Nabavi et al., 2019), melatonin interacts with Tumor necrosis factor(TNF), Interleukin-2 (IL-2), and interleukin-4 (IL-4).
Many studies have demonstrated that the level of TNF is significantly lower in high melatonin levels, thus indicating a feedback mechanism between the pineal gland and TNF released from macrophages (Soybİr et al., 2003). Besides that, many studies show that melatonin significantly suppressed the proinflammatory factors IL-1β and TNF-α and reduced the relative gene expression of IL-1β, TNF-α, and iNOS, so melatonin significantly promoted the healing of diabetic wounds by inhibiting inflammation, thereby further facilitating angiogenesis and collagen synthesis in vivo (Liu et al., 2020).
Also, the histopathological findings of the present research showed an acceleration in the healing of the wound in the treated group with melatonin and this is in agreement with Pugazhenthi et al., 2008 that shows melatonin treatment significantly improved the quality of scarring, both in terms of maturity and orientation of collagen fibers.
Evidence shows that many factors can cause cellular damage such as tissue neutrophil accumulation, the formation of the high level of reactive oxygen species (ROS) while undergoing reperfusion and high levels of calcium, and the activation of a high level of epithelial apoptosis are all important factors in the etiology of different forms of ischemia reperfusion injury in organ dysfunction and tissue damage (Chen  et al., 2018), Melatonin treatment significantly decreased inducible Nitric oxide synthases (iNOS) activity during the acute inflammatory phase but significantly increased iNOS activity during the resolving phase (Pugazhenthi, et al., 2008).
Many studies show that melatonin has a powerful antioxidant effect, melatonin can scavenge the free radicals and also melatonin increases the levels of the antioxidant enzyme such as catalase (CAT), glutathione peroxidase (GSH-px), and Superoxide dismutase (SOD) also melatonin treatment decrease the level of Malondialdehyde (MAD) (Başak et al., 2003). Furthermore melatonin plays an important role as a potent radical scavenger of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Its antioxidant properties are reported to be even more effective than vitamins C and E (Pieri et al.,1994).
In this research, there was an acceleration in the wound healing process and this is an agreement with Ozler et al., 2010 that shown wound healing was prolonged in experimental animals deprived of melatonin through pinealectomy, so melatonin exerts positive effects on wound healing, whether it is administered topically or systemically (Ozler et al., 2010). Furthermore, the previous study shows that melatonin has antimicrobial activity, accelerating wound healing of burns and other skin lesions (Soriano et al., 2020).

CONCLUSION
The data of this study revealed that melatonin has anti-inflammatory effects and melatonin increases granulation tissue formation, so melatonin accelerates wound-healing of the primary oral mucosal wound.