Effects of Mucoadhessive Paste of Chlorhexidine and Betamethasone on Oral Ulcer Recovery Process in Rats

  Abstract: Introduction. Ulcers are one of the most frequent conditions affecting the oral cavity. The aim of this study was to assess the effects of chlorhexidine and betamethasone on oral mucosa wound healing in rats. Materials and methods. In this study, adult male rats were used in 5 groups (n = 16 per group): bethamethasone (B); chlorhexidine (CHX); betamethasone and chlorhexidine (BC); control (C) no drugs; and blank (BB). A wound measuring 2 mm in diameter was punched into the hard palate of each rat. A mucosal defect measuring 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 the appropriate study material and packed into the wound. The control group was not treated. Histological samples were harvested on post-injury days 2, 4, 6, and 8. Results. Comparison of clinical size of wound showed that groups B and BC had the greatest reduction in wound size on days 4, 6, and 8. A higher count of polymorphonuclear cells was observed on days 2 and 4 in groups BB, CHX, and C. A lower count of mononuclear cells in group BC on days 6 and 8, compared to other groups, was also observed. Groups B and BC showed the highest fibroblast counts at all the intervals, with significant differences between the groups (P < 0.05). The inflammation score was highest at all the intervals in group C. The highest thickness of epithelium was observed in groups B and BC on days 6 and 8, which was significantly different from the other groups (P < 0.05). Conclusion. The results of this study showed the best wound healing processes from clinical and histological aspects in the B and BC groups. Chlorhexidine alone had no significant effect on wound healing and was not an effective drug alone.

Introduction

  Ulcers are one of the most frequent conditions of the oral cavity. A mouth ulcer is characterized by loss or erosion of part of the delicate tissue that lines the oral cavity (ie, the mucous membrane). Although the cause of most mouth ulcers in many cases is not clear, accidental damage is a common cause. 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% in different social and ethnic groups.¹   The most common treatment modality for an oral ulcer is symptomatic treatment. The best medications for this purpose are topical and oral steroids, and antimicrobial agents, such as chlorhexidine.² Chlorhexidine is a bisbiguanid cationic agent with a broad spectrum of antimicrobial activity and a chlorophenyl biguanide that has been used in acetate and, more commonly, the gluoconate salt (which is more soluble) in mouthwashes, gels, and dentifrices for control of plaque and gingivitis.³   Chlorhexidine, which is harmful in high concentrations, is used safely in low concentrations in products such as mouthwash and contact lens solutions. However, numerous scientific papers have reported complications with low-level exposure too.⁴ Previously available only as a prescription in the United States, chlorhexidine is now available over-the-counter, but still prescribed as a wound debridement and cleansing system under the brand name Irrisept.⁵ Also, Chlorhexidine is contraindicated for use on mucous membranes.⁶   In one of the earliest study on the dental applications of chlorhexidine, Schröeder showed a 73% reduction of supragingival calculus and plaque in 3 days.⁶ The efficacy of chlorhexidine mouthwash as an antiplaque/antigingivitis agent is dose-dependent in the range of 0.03% - 0.2%. The volume and frequency of consumption, as well as the concentration, are important in determining the clinical response.3 In a review study, Nashwan found that chlorhexidine might be effective on oral mucosal lesions during chemotherapy in children with cancer.⁷   Corticosteroids, such a betamethasone, are also used in the treatment of oral ulcers. Betamethasone is a potent glucocorticoid steroid with anti-inflammatory and immunosuppressive properties. Unlike other drugs with these effects, betamethasone does not cause water retention. It is applied as a topical cream, ointment, foam, lotion, or gel to treat itching.⁸ A review of literature shows that no studies have been carried out on the effect of chlorhexidine and betamethasone on oral ulcers; therefore, the aim of this study was to assess the effect of chlorhexidine and betamethasone on mucosal wound healing in rats.

Materials and Method

  This study was approved by the ethics committee of Kerman University of Medical Sciences (Kerman, Iran) (No.K.90.68). Pure powders of chlorhexidine and betamethasone were purchased. Oral mucoadhesive paste was prepared from sodium carboxylmethyl cellulose, pectin, and gelatin in plasty base. Plasty base gel is made by the quick cooling of a warm mixture of 5% polyethylene with low density in liquid paraffin. To this end, polyethylene was poured into a beaker with liquid paraffin with a temperature of 80°C at a 2:1 weight ratio of polyethylene:paraffin. This preparation was mixed at 130°C until the gel achieved the desired viscosity, and warm paraffin was mixed in. After 1 hour of mixing, the pure mixture was placed inside aluminum foil containers to cool completely to yield plasty base gel (formula: gelatin [16.6%], pectin [16.6%], sodium carboxymethyl cellulose [16.6%], and plasty base to reach 100%).   Preparation of the mucoadhesive paste containing a drug involved passing the powder of the drug (chlorhexidine and betamethasone) through a 200-mesh sieve, gently adding it to the plasty base in the primary stage, and mixing continuously. After preparing the mucoadhesive paste, this material was kept in a refrigerator until used for the purpose of the study.^9-11   Eighty male Wistar rats, weighing 200 g –250 g and between 6–7 weeks of age, were purchased from the Razi Institute, Kermanshah, Iran. The animals were housed under standard conditions (23°C -25°C; 12 hours of light-dark cycles) and given laboratory food and water ad libitum throughout the study. Animals were divided into 5 groups (n = 16 per group): betamethasone (B); chlorhexidine (CHX); betamethasone and chlorhexidine (BC); control (C); and blank (BB). The animals were anesthetized with a ketamine dose of 60 mg/kg and xylanzine (Rompun®, Bayer Parsian AG, Tehran, Iran) dose of 7.5 mg/kg as a muscle relaxant on the first day. A mucosal defect measuring 2 mm in diameter and 0.2 mm in wall thickness was produced to the depth of the periosteum on the palate with a round stainless steel blade designed for punch biopsy. After generating the wound, different kinds of mucoadhesive pastes containing drugs were placed on the wounds of 48 animals. In addition, 16 animals received mucoadhesive paste without any drug, and 16 remaining animals were considered the control group, such that no material was used for remedy of the wounds. On the second day, before placing any material on wounds, 4 rats were euthanized, by ether inhalation and an overdose of anesthetic agent from each of the 5 groups; each wound was excised using a 5-mm biopsy punch, maintaining approximately 3 mm of intact mucosa around the incision. The excised tissue was fixed with 10% formalin and referred to the laboratory for histological evaluation. The same manner was repeated up to the 8th day of study such that 20 animals were sacrificed at day 2, 4, 6, and 8.   Each animal used for further investigation was weighed daily following the procedure. Weight loss was used as an indirect objective measurement of postoperative pain.¹² Evaluation of wounds was carried out in all the animals during the study period of 8 days before represcription of mucoadhesive. An individual who was not aware of the animal groupings measured depth and width of ulcers using slide calipers.   Samples, cut at the mid-portion of the wound, were sent to the laboratory where each part was then embedded in paraffin. Cross-sections of both portions were thinly sliced (5 µm) and stained with hematoxylin-eosin. Light microscopy was used to evaluate the pathological changes (eg, granulation tissue formation and reepithelialization in wounds) and comparisons were carried out with the normal tissue by 2 histological experts. The length of epithelium regenerated from the wound edge was measured with a micrometer.   Each ulcer was scored histopathologically on a 0-5 scale based on the inflammation of tissue (Table 1), 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. Residual wound area (µm),¹⁴ epithelium thickness (µm),¹⁵ the number of polymorphonuclear cells (PMNs) (number/10 per high power field [HPF]), and the number of fibroblasts (number/10 HPF) were also evaluated.16 Statistical significance was defined at P < 0.05 using ANOVA. Significant treatment means were subjected to 2-way ANOVA and post-hoc analysis (Bonferroni correction) using SPSS 18.

Results

  In this study, 5 groups were evaluated, consisting of: bethamethasone (B); chlorhexidine (CHX); betamethasone and chlorhexidine (BC); control (C); and blank (BB). Comparison of clinical wound size showed groups B and BC had the greatest reduction in wound size on days 4, 6, and 8, which was significantly different from the other groups (P < 0.05). (Graph 1) (Figure 1).   The highest thickness of epithelium was observed in groups B and BC on days 6 and 8, which was significantly different from the other groups (P < 0.05). In addition, group C had the lowest thickness of epithelium, which was significantly different from the other groups at all intervals (P < 0.05) (Figure 2). In relation to the residual wound area, groups B and BC showed the lowest values among all the treatment groups at all the intervals (P < 0.05).   Furthermore, clinical examination of the wounds in groups B and BC showed gradual healing over time in all the groups. Slow wound healing was observed on postoperative days 3 and 5. Macroscopically, bone was covered with a serofibrinous layer. Margins of the wound became irregular and started to migrate towards the center of the wound. The area of fibrin covering the base of the wound decreased rapidly from day 3 to day 5. On day 5, the defect was largely covered with epithelium. At the end of the experiment (day 8), most of the defects were healed with minimal central depression.   The inflammation score was highest at all the intervals in group C, and a significant difference was observed between the groups (P < 0.05); groups B and BC showed the lowest values on days 2, 4, 6, and 8, with a significant difference between the groups (P < 0.05) (Figure 3).   There were also significant differences between reepithelialization results. Groups B and BC showed the highest values on days 6 and 8 (P < 0.05). Group C showed the lowest values at all the intervals (Figure 4).   According to 2-way ANOVA, a higher count of PMNs was observed on days 2 and 4 in groups BB, CHX, and C, with significant differences from groups B and BC (P < 0.05). (Figure 5).   This study showed a lower mononuclear cell counts in group BC on days 6 and 8 compared to the other groups, which was statistically significant; the counts were lower in groups BB and CB on day 2; however, the difference was not significant (P < 0.05) (Figure 6). The average cell counts in groups CHX and BB was lower on day 4 compared to other groups, with a significant difference from group B (P < 0.05).   Groups B and BC showed the highest values of fibroblast counts at all the intervals, and a significant difference was observed between the groups (P < 0.05) (Figure 7). Clinical examination of the wounds (Figure 8) showed gradual healing over time in BC, CHX, and B groups. Figure 9 shows the healing of the excisional wounds in the B, CHX, BC, C, and BB groups at day 8 based on histological presentation.

Discussion

  Oral ulcers are common conditions of the oral cavity, involving 12% - 20% of the population.¹⁷ Many agents have been investigated in the quest for an agent that promotes oral wound healing and reduces postoperative complications.¹⁸ Wound healing is a complex procedure that involves a series of controlled events,^19,20 during which epithelial cells begin to express extracellular receptors not usually present in the resting epithelium. Fibroblasts are found with a special form in healing granulation tissue.²¹   In this investigation, the effects of chlorhexidine and betamethasone separately and combined were assessed on the palatal excisional wound in rats. Rats were used as a model representing a reproducible wound that could be followed clinically and histologically. The main concept in using chlorhexidine to promote wound healing is that chlorhexidine reduces the bacterial load of the wound and has been recommended as a treatment for oral ulcers.^22,23 Invasive wound infection interferes with the normal wound healing process as the body’s defense against infection disrupts granulation through activation of inflammatory cells and mediators, and injures the surrounding normal tissues by a variety of interactions.^24,25   It is important to note that studies on chlorhexidine have shown considerable contradictions on its effect on wound healing. A number of in vitro studies have shown that chlorhexidine negatively affects fibroblast and keratinocyte proliferation in a concentration- and time-dependent manner.^21,26,27 However, only some in vivo studies^28,29 have indicated a negative effect of chlorhexidine on wound healing, while a considerable number of studies have shown the value of chlorhexidine rinses after various oral surgical procedures and a positive postoperative effect of chlorhexidine on wound healing.^19,30-32   The contradiction between in vivo and in vitro results might be explained by different cellular, molecular, and environmental interactions affecting the tissue. For instance, in the oral cavity, chlorhexidine binds mostly to bacteria, and an additional amount of the applied chlorhexidine is precipitated by serum proteins.^33,34 Therefore, the remaining amount of chlorhexidine molecules available to bind to and harm host cells in the wound is significantly reduced.¹⁹   The results of this investigation showed that the combination of betamethasone and chlorhexidine had a significant effect on wound healing. The number of PMNs at all intervals in groups B and BC was less than that in other groups, and also the number of mononuclear cells (MNCs) and fibroblast cells in group BC was the lowest compared to other groups, consistent with the results of other investigations.^35,36   This study showed that inflammation was highest at all study intervals in the control group compared to other groups. In addition, groups B and BC had the lowest inflammation score compared to other groups at all intervals. Glucocorticoids, like betamethasone, are potent inhibitors of the inflammatory response; by their action on gene regulation, they induce the production of lipocortin, which is an inhibitory protein of phospholipase A2. The resultant inhibition leads to a decrease in the production of prostaglandins and leukotrienes. Glucocorticoids also suppress the synthesis of cyclooxygenase. The final result of these 2 actions is an inhibition of neutrophil, eosinophil, and monocyte chemotaxis.³ However, using chlorhexidine, or adding this drug to betamethasone, had no significant effect on increasing this effect.   The reepithelialization rate was highest in groups B and BC on days 6 and 8, which seems to be predictable because the intensity and extension of inflammation inhibit the epithelialization; in other words, the groups with more severe inflammation had lower regeneration.   In this study, no significant differences were exhibited in groups BB and C. On some days the BB group showed weaker signs of healing, which might be attributed to local wound irritation.

Conclusion

  The results of this study showed that the best wound healing processes from clinical and histological aspects were achieved in the betamethasone (B) and betamethasone-chlorhexidine (BC) groups. The benefit of glucocorticoids applied topically for oral ulcers is greatest when the period of contact with the tissue is maximal and maintained in the oral cavity for a long time, such as the case with adhesive pastes or mucoadhesives. Also, use of chlorhexidine alone had no significant effect on wound healing and other criteria; therefore, the authors concluded it is not effective when used alone.   Pectin and gelatin are the 2 main materials used in the production of mucoadhesive paste in many articles,^37-41 although there is no justification for the inclusion of pectin and gelatin in the experimental formulae. These foreign proteins have been shown over many years to both create allergies and impede the wound healing process. Therefore, the authors recommend other studies be conducted in the field with different mucoadhesive compounds; although, in this study, no increase in cell numbers of eosinophils and basophils or allergic reaction in microscopic examination were reported.

Acknowledgement

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

References

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Application of pectin in oral drug delivery. Expert Opin Drug Deliv. 2011;8(8):1009-1023 The authors are from the Department of Oral Medicine, Kerman Oral and Dental Disease Research Center, Kerman University of Medical Sciences, Kerman, Iran. Address correspondence to: Maryam Alsadat Hashemipour Kerman Oral and Dental Disease Research Center Kerman University of Medical Sciences Qusar Boulevard Kerman, Iran m_s_hashemipour@yahoo.com