Effects of Locally and Systemically Applied n-3 Fatty Acid on Oral Ulcer Recovery Process in Rats

  Abstract: Introduction. Ulcers are one of the most frequent diseases affecting the oral cavity. The aim of this study was to assess the effects of omega-3 fatty acid (topical and systemic) on oral mucosa wound healing in rats. Methods and materials. In this study, adult male rats in 5 groups (n = 16 per group) were used (L: local, S: systemic, LB: local blank, SB: systemic blank, and C: control.) A wound (2 mm diameter) was punched into the hard palate of each rat. A mucosal defect (2 mm in diameter and 0.2 mm in wall thickness) was made to the depth of the periosteum in the palate with a round stainless steel blade designed for punch biopsy. For topical application, a swab was soaked in L omega-3 or LB and packed into the wound. For systemic treatment, 2 cc of the S omega-3 acid or SB was guided into the stomach by gastric gavage. The control group was not treated. Histological samples were harvested on post-injury days 2, 4, 6, and 8. Results. The highest amount of polymorphonuclear (PMN) cells was observed on days 2 and 4 in the LB group. The control group had the highest inflammation score on days 2 and 4, and the lowest reepithelialization score on days 2, 4, 6, and 8. The thickest epithelium was observed in the L and S groups on days 6 and 8. Conclusion. Omega-3 fatty acid (L and S) increases fibroblast counts and decreases PMN cell counts. Moreover, this compound causes an increase in reepithelialization and epithelial thickness.

Introduction

  Ulcers are one of the most frequent diseases of the oral cavity. A mouth ulcer is characterized by loss or erosion of part of the delicate tissue that lines the inside of the mouth (mucous membrane). Although the cause of mouth ulcers is not clear in many cases, accidental damage is a common cause. Moreover, other factors including certain drugs, chemicals, and infectious diseases such as herpes, thrush, and cancers, can induce oral ulcers. The incidence of oral ulcers has been shown to range from 5% to 50% across different social and ethnic groups.^1,2   The goal of treatment of oral ulcers is to relieve symptoms; therefore, finding suitable drugs with fewer side effects is the goal of many researchers. Steroids (topical and oral) and mouthwashes are usual treatments for oral ulcers; however, systemic absorption of local steroids can have undesirable influences on the immune system and may lead to secondary infections. Moreover, some patients, including children and the elderly, experience the side effects of swallowing mouthwash.⁵ Because of the side effects of chemically synthesized drugs, there is a trend toward the use of more natural treatments.   In recent years, there have been a number of reports in the medical literature regarding the ‘rediscovery’ of n-3 fatty acids as therapeutic agents. N-3 fatty acids, popularly referred to as W-3 fatty acids or omega-3 fatty acids, are types of a series of essential unsaturated fatty acids that cannot be synthesized by the human body but are vital for normal metabolism. Various therapeutic features of omega-3 fatty acids have been reported in clinical studies and animal experiments, where these acids have been used as a dressing for wounds, skin ulcers, and burns. Topical application of omega-3 fatty acids to burn wounds has been found to be effective in controlling infection and producing a clear granulation bed.^3-7   Several clinical trials have been conducted that used omega-3 acids for a variety of wounds. An omega-3 dressing has been used in the treatment of wounds with varied etiology, such as burns, diabetic ulcers, and tropical ulcers.^8-10 The previous data have shown that inflammation, swelling, and pain were quickly reduced; sloughing of necrotic tissue occurred without the need for debridement; dressings could be removed painlessly and without causing damage to tissue regrowth; and healing occurred rapidly with minimal scarring and without the need for grafting. Omega-3 acids were found in almost all cases to be very effective in rapidly clearing infection and promoting healing.¹¹ However, a review of the literature showed that the effect of omega-3 acids on oral ulcers has not been studied. Therefore, the aim of this study is to assess the effects of omega-3 acids (topical and systemic) on mucosal wound healing in rats.

Materials and Methods

  Local omega-3 fatty acid. An aliquot of pure omega-3 fatty acid (Zahravi Pharmaceutical Co, Tehran, Iran), of concentration 100 mg/kg (0.2% total weight)²¹ was homogenized at 8000 rpm to 25000 rpm with 10% propylene glycol, 3% PEG (300), 10% Tween 80, and therapeutic-grade sesame oil at room temperature for 30 minutes. A local blank was prepared using the same formula, but without the addition of omega-3 acid. These solutions were kept refrigerated.   Systemic omega-3. An aliquot of pure omega-3 acid of concentration 200 mg/kg (0.4% total weight, based on a pilot study) was mixed with 10% to 20% Tween 80. A solution of 1% methylparaben in purified water was prepared at 80°C and then cooled to room temperature. The water phase and fat phase were mixed by homogenization at 800 rpm for 20 minutes. A systemic blank was prepared using the same formula, but without the addition of omega-3 fatty acid. These solutions were kept refrigerated in dark glass vessels.   Animals. Male Wistar rats (200 g to 250 g; 6 weeks to 7 weeks old; n = 80) were purchased from Neuroscience Laboratory Animals Science (Kerman, Iran). Animals were housed under standard conditions (23–25 °C; 12:12 light-dark cycle) and given laboratory food and water ad libitum throughout the study. The experimental protocol was approved by the Research Committee of Kerman University of Medical Sciences prior to commencement of the study.   Animals were divided into 5 groups (n = 16 per group): local (L) omega-3; systemic (S) omega-3; local blank (LB); systemic blank (SB); and control (C). Animals were weighed and then fixed on their backs. A mucosal defect (2 mm in diameter and 0.2 mm in wall thickness) was made to the depth of the periosteum in the palate with a round stainless steel blade designed for punch biopsy. For topical application, a swab was soaked in L omega-3 or LB and packed into the wound. For systemic treatment, 2 cc of the S omega-3 acid or SB was guided into the stomach by gastric gavage. The control group was not treated. Following the procedure, the rats were housed separately with 16 animals per cage.   Wound size was measured with slide callipers (mm), and the soaked swab was reapplied at the same time. Ulcers were assessed clinically every day, immediately prior to application of omega-3. Each animal used for further investigation was weighed daily following the procedure. Weight gain was used as an indirect objective measurement of postoperative pain.¹² On post-injury days 2, 4, 6, and 8, tissues were harvested for histological evaluation. Twenty rats (4 rats from each treatment group) were sacrificed on day 2, with the remaining animals sacrificed on days 4, 6, and 8, respectively.   The animals were euthanized by ether inhalation or an overdose of anesthetic. Wounds were evaluated only once and were not followed over the entire course of healing. Each wound was excised using a 5 mm biopsy punch, maintaining approximately 3 mm of mucosa around the incision. The excised tissue was fixed with 10% formalin and cut at the mid-portion of the wound; each part was then embedded in paraffin. Cross-sections of both portions were thinly sliced (5 µm) and stained with hematoxylin-eosin. Two histological experts used light microscopy to evaluate the pathological changes (eg, granulation tissue formation and reepithelialization in wounds) and comparison with the normal tissue. The length of epithelium regenerated from the wound edge was measured with a micrometer. The mean length of 2 slices was used as the reepithelialization length for each sample.   The ulcer score was graded histopathologically on a 0–5 scale based on the inflammation of tissue (Table 1),¹³ and on a 1–5 scale for the level of reepithelialization (Table 2).¹³ These scores were totaled to provide the condition score of the palatal mucosa.²¹ Residual wound area (µm),¹⁴ epithelium thickness (µm),¹⁵ number of polymorphonuclear (PMN) cells (number/10 HPF),¹⁶ and number of fibroblast cells (number/10 HPF)¹⁶ were also evaluated. To prevent bias in this study, pathologists were blinded.   All values are mean ± SD (standard deviation) of 20 experiments. Statistical significance of differences was evaluated by Student’s t test and one-way ANOVA. When multiple comparisons were performed the significance of differences was evaluated by the modified t test. P < 0.05 was accepted as significant.

Results

  The average weights of the animals in the 5 treatment groups (L, S, LB, SB, and C) at the beginning of the study were 210 g, 204 g, 208 g, 218 g, and 202 g, respectively, and after 4 days with oral ulcers, weights decreased to 201 g, 200 g, 200 g, 208 g, and 198 g, respectively. There were no significant differences between groups in the reduction of weight (P > 0.05) (Table 3).   On the basis of one-way ANOVA analysis, a higher score of PMN cells was observed on days 2 and 4 in the LB group (P < 0.05), and a lower score was observed in the L and S groups on all days (P < 0.05) (Table 4). Fibroblast counts in the control group were the lowest on all days. The L and S groups showed the highest values on all days, and a significant difference was observed between the groups (Table 5). The inflammation score was highest on days 2 and 4 in the C group (P < 0.05), and the L and S groups showed the lowest inflammation score values on days 2, 6, and 8 (P < 0.05) (Table 6).   There was a significant difference among the reepithelialization results. The S and L groups showed the highest values on days 6 and 8 (P < 0.05). The C group showed the lowest values on all days (Table 7). The highest thickness of epithelium was observed in the L and S groups on days 6 and 8, which was significantly different from the other groups (P < 0.05) (Table 8). In the case of the residual wound area, the S and L groups showed the lowest values among all treatment groups on all days (P < 0.05) (Table 9). Differences between the clinical wound size reduction of the L and S groups on days 4, 6, and 8 versus the other groups were significant (P < 0.05).   Finally, this study showed the L group, compared to the S group, had higher fibroblast cells, higher reepithelizalization scores, higher thickness scores, a lower PMN cell count, lower inflammation scores, and a smaller residual wound area.   Figure 1 shows the gradual healing of the excisional wounds in the L omega-3 group at days 2, 4, 6, and 8.

Discussion

  Oral ulcers are among the most common manifestations of oral diseases, and result from vesiculobullous disease, aphthae, herpetic lesion, mucositis, drug eruption, allergic and contact stomatitis, chemical burns, trauma, and other conditions. Interaction with food may cause oral ulcers to be painful, thereby affecting mouth function.¹ Thus, the treatment and management of oral ulcers is very important. Although several medical and palliative therapies are available, because of the importance and prevalence of oral ulcers, application of a new therapy that is less expensive, easy to use, and readily available would be helpful. The findings of this study indicate omega-3 fatty acids (L and S) promote the healing process of oral ulcers. The authors’ results showed wound size reduction, reepithelialization, and granulation tissue formation were significantly higher, and inflammatory cell infiltration was significantly lower, in groups treated with L and S omega-3. Thus, omega-3 was clinically and histologically effective in the healing of oral ulcers.   Although omega-3 fatty acids have been known to be essential for normal growth and health since the 1930s, awareness of their health benefits has increased dramatically since the 1990s.¹⁷ These benefits were discovered in the 1970s by researchers studying the Greenland Inuit tribe, who consumed large amounts of fat from seafood, but displayed virtually no cardiovascular disease. The high level of omega-3 fatty acids consumed by the Inuit reduced the amount of triglycerides in the body, consequently reducing heart rate, blood pressure, and atherosclerosis.¹⁸   A number of investigations have demonstrated that a diet supplemented with fish oil enriched in omega-3 fatty acids has profound beneficial health effects against various conditions,¹⁹ including cardiovascular diseases, respiratory diseases, diabetes, depression, cancers, inflammatory disease, and immunological renal disorders.²⁰ Dietary omega-3 fatty acids have shown a significant protective effect against gastric ulcerations by inhibition of offensive mucosal factors and augmentation of defensive mucosal factors.²¹   Fatty acids are one of the main components in the structure of leukotriene B4 and prostaglandin E2 (PGE2), which have an important role in a number of inflammatory processes including fever, increased vascular permeability, proliferation of PMN cells and natural killer cells, and inhibition of the production of interferon-a.⁵   In several reports, the rapid healing observed with an omega-3 dressing is noted. Wounds treated with omega-3 show less edema, less granular and mononuclear cell (MNC) infiltration, less necrosis, and better wound contractions. Omega-3 acids play a role in the prevention of inflammation.^4-6   In addition to its use as a wound-healing agent, omega-3 has been used as an alternative treatment for clinical conditions ranging from gastrointestinal problems to ophthalmologic disorders. Although research studies have widely been performed on the effect of honey on skin wounds, burn ulcers, and intestinal mucosa,^4-10 there is no evidence about the effects of omega-3 on the healing of oral ulcers.   Thies et al²² and Kang et al²³ showed omega-3 fatty acids play an important role in the regulation of inflammation through multiple pathways. Omega-3 fatty acids inhibit the formation of pro-inflammatory eicosanoids while inducing the formation of several potent anti-inflammatory lipid mediators. Together, these actions directly or indirectly suppress the activity of nuclear transcription factors, such as NF kappa B, and reduce the production of pro-inflammatory enzymes and cytokines, including COX-2, tumor necrosis factor (TNF)-a, and interleukin (IL)-1b.^22,23   In this study, the fibroblast count in the L and S groups on all days of the study showed the highest values, and a significant difference was observed between the results. The effects of omega-3 fatty acids on the in vitro healing response of medial collateral ligament (MCL) fibroblasts were investigated by Hankenson et al.²⁴ This research shows that omega-3 fatty acids positively affect the healing characteristics of MCL cells, and therefore may represent a possible non-invasive treatment to improve ligament healing. Additionally, these results show MCL fibroblasts produce PGE2, IL-6, and TNF-a, and that IL-6 production is related to MCL collagen synthesis.   Omega-3, in addition to its antibacterial activity, also may clear infection by stimulating the activity of leukocytes. Cell culture studies have shown that omega-3 stimulates proliferation of lymphocytes and monocytes to release cytokines, which are activators of the immune response to infection.^6,7,25-27   The author’s results showed that reepithelialization occurred more quickly in the L and S groups than in the other groups. Moreover, the greatest value of epithelial thickness was observed in the L and S groups on days 6 and 8, which was significantly different from the other groups. Terkelesn et al⁸ showed topical application of a 25% cod liver oil ointment significantly accelerated both the epithelial and vascular component of healing compared to saline.⁸ Other studies have shown omega-3 fatty acids to play a role in reepithelialization of experimental skin, and in intestinal and stomach wounds in rat, rabbit, and dog models.⁹ In this study, the residual wound area in the S and L groups showed the lowest values among all groups on all days of the study, which is in agreement with the findings of other researchers.^{25, 28-31}   Two groups using topical and systemic administration of omega-3 showed significant differences in mean inflammation (less inflammation in group L), mean reepithelialization (reepithelialization higher in group L), and average thickness of the epithelium (more in group L). These differences could be caused by delayed effects of the S omega-3 form circulating in the blood versus the topical from of omega-3 placed directly in the wound.

Conclusion

  Omega-3 (L and S) increased the MNC and fibroblast counts and decreased the PMN cell count. Moreover, this compound increased reepithelialization and epithelium thickness. The findings of the present study indicate omega-3 increases the formation of granulation tissue, epithelialization, and fibroblast action, and reduces the residual wound area and clinical wound size. Therefore, the use of omega-3 fatty acids is effective, safe, and practical, and could be an alternative treatment for oral ulcers.

Acknowledgement

  This study was supported by Kerman University of Medical Sciences. The authors would like to thank the Research Deputy for their financial support.

References

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Albina JE, Gladden P, Walsh WR. Detrimental effects of an omega-3 fatty acid-enriched diet on wound healing. J Parenter Enteral Nutr. 1993;17:519-521. 30. Matsuba S, Itoh M, Joh T, Takeyama H, et al. Effect of dietary linoleate/alpha-linoleate balance on experimentally induced gastric injury in rats. Prostaglandins Leukot Essent Fatty Acids. 1998;59:317-323. 31. Gercek A, Yildirim O, Konya D, et al. Effects of parenteral fish-oil emulsion (Omegaven) on cutaneous wound healing in rats treated with dexamethasone. JPEN J Parenter Enteral Nutr. 2007;31:161-166. Maryam Alsadat Hashemipour, DDS, MSc is from the Department of Oral Medicine, Kerman Oral and Dental Disease Research Center, Kerman University of Medical Sciences, Kerman, Iran. Amir Reza Ghasemi, DDS is from the Kerman Oral and Dental Disease Research Center, Kerman University of Medical Sciences, Kerman, Iran. Mahdi Ansari Dogaheh, PhD is from the Department of Pharmaceutics, Kerman University of Medical Sciences, Kerman, Iran. Molok Torabi, DDS, MSc is from the Department of Oral Pathology, Kerman Oral and Dental Disease Research Center, Kerman University of Medical Sciences, Kerman, Iran. Address correspondence to: Maryam Alsadat Hashemipour Kerman Oral and Dental Diseases Research Center Qusar Boulevard Kerman, Iran m_hashemipoor@kmu.ac.ir