Effectiveness of Biosurfactant Lipopeptide Adhesive Mucus Paste on the Healing Process of Oral Wounds: A Randomized Controlled Trial

Recurrent aphthous stomatitis is a lesion with a relatively high prevalence. Although this lesion is well-known clinically and histologically, its etiology and treatment are still debatable. Today, many oral specialists believe that this is a multi-cause disease despite the clinical similarity between these lesions. Due to these reasons, in many cases it is not possible to carry out decisive treatment, and the treatment is often symptomatic to reduce pain, reduce burning, and shorten the duration of the disease.¹

One of the goals of new drug delivery systems is to increase the safety and adequacy of the drug molecule with a suitable formulation, leading to better adherence for the patient taking prescribed drugs. One of the new methods of drug delivery is the preparation of an oral adhesive mucosal film, which can prevent the effect of enzymatic degradation of the drug in the digestive system by sticking to the oral mucosa and promoting absorption in this area. This leads to direct access to the blood circulation and can increase the bioavailability and therapeutic effect of the drug.² 

Studies have shown that the oral and sublingual administration of soluble drugs leads to quick absorption due to the conditions of the reticular vessels that are located under the oral mucosa.^3,4 The oral mucosa has a vast blood network that is relatively permeable. One of the advantages of oral adhesive mucus films is that they are in a more permanent environment, which increases the efficacy of the treatment. Biosurfactants are surface-active molecules that are obtained from various plant, animal, and mainly microorganism cells. Based on their chemical structure and microbial origin, biosurfactants are divided into 6 main groups: glycolipids, lipopeptides, lipoproteins, fatty acids, phospholipids, and polymeric surfactants.⁵

In general, biosurfactants have the potential to be used in various industries due to their special abilities, such as reducing surface and interfacial tension. In recent years, biosurfactants—including the subgroup of lipopeptides—have received attention due to their many applications in biomolecules.⁶ Some of their properties include antioxidant,⁷ antimicrobial,⁸ and antitumor activity.⁹ One of the extracted lipopeptide biosurfactants is a lipopeptide complex produced by the bacterium Acinetobacter junii B6, which is isolated from oil-contaminated soils in southern Iran and has been purified using acid precipitation and organic solvent extraction.¹⁰

Previous studies showed that lipopeptide biosurfactant, when compared to other treatments (Cicalfate from Avene gel), had more capability in wound healing and no scarring (wound effect after repair). The results of in vivo, in vitro, and microscopic experiments have all confirmed the power and potential of lipopeptide biosurfactants in healing skin wounds in rats.¹¹ Based on the findings and observations made using fluorescence recovery after photobleaching and DPPH methods, it was determined that biosurfactant lipopeptide has stronger antioxidant properties than the same concentration of standard beta hydroxy acids due to its dual properties (hydrophilic-lipophilic). The active residues in the peptide ring and the hydrocarbon fatty acid chain may be attributed to the increase in free radical scavenging activity. It has been reported that eliminating oxidative and nitrosative stress in wounds using the antioxidant potential of bioactive structures such as lipopeptide biosurfactants can be an important strategy for wound healing.¹¹ Current results showed that biosurfactant lipopeptide, especially at a concentration of 5 mg/mL, was able to reduce oxidative stress by inhibiting free radicals.^12,13

Ulcers of the oral mucosa cause severe and debilitating pain, and their secondary effects cause a change in the sense of taste, sleep disorders, anorexia, weight loss, and a patient’s quality of life. Therefore, the treatment and pain relief of oral lesions helps to improve the patient’s quality of life and treatment tolerance. The use of local formulations of lidocaine and benzocaine (local anesthetics) and diphenhydramine relieves symptoms temporarily, and long-term use of these drugs is associated with side effects. The use of steroidal anti-inflammatory drugs is limited due to their suppression of the immune system, the occurrence of candidiasis, and the effect of other drugs (allopurinol, sucralfate, chlorhexidine, benzydamine, vitamin E, dexpanthenol, etc) has not been proven.^1,2,5 Therefore, the preparation of a special formulation can help in the treatment of inflammation of the oral mucosa by increasing the efficacy and reducing the side effects of the drug. In this study, the formulation of an adhesive mucus gel containing lipopeptide biosurfactant (A baumannii and P aeruginosa) was used to treat oral ulcers.

This study was conducted as a double-blind randomized clinical trial. Before starting work, this dissertation was approved by the Ethics Committee of the Kerman University of Medical Sciences (ethical code: IR.KMU.REC.1398.165). The study population included 36 individuals who had a history of oral ulcers. Participants were randomly placed into 3 groups, each with 12 patients: positive control group, lipopeptide biosurfactant adhesive mucus group, and base group. In this study, oral ulcer was defined as a wound that recurred at least twice a year and healed within 10 days without leaving a scar. The conditions for inclusion in the study included male and female participants over 18 years of age, wound size less than or equal to 1 cm, and healing of wounds in less than 10 days without medication.

In cases where the wounds were caused by trauma, the sharp edge of a tooth, broken restoration, orthodontic appliances, or partial dentures, the patient was excluded from the study. Also, participants who had a history of known issues, including anemia, unusual skin lesions, recurrent respiratory tract infections, a history of similar wounds in the genital area, swelling and pain in the joints, or eye problems (including blurriness and redness) were excluded from the study. Additional exclusions included women who were pregnant or lactating, persons with a history of immune problems or who received treatment with systemic steroids or other immunosuppressive agents within 1 month of study screening, persons with a known drug sensitivity, persons who had more than 3 days pass since their lesion appeared, persons who used another drug to treat their wound, persons who had used non-steroidal anti-inflammatory drugs or oral antihistamines 1 month before the study, persons who had treated oral ulcers with any medicine within 72 hours of study screening, persons who received treatment with antibiotics within 2 weeks of entering the study, and persons entering any clinical trial during the previous 3 months.

Written informed consent was obtained from all patients; it was explained that they would be placed in 1 of the 3 groups. In addition, they were given explanations of the types of oral ulcers, and pictures were provided so that they could match their oral ulcers with the images. In order to conduct the study, first an intra-oral examination was performed and the characteristics of the wounds were recorded in the relevant form. Patients were randomly assigned to 3 groups: mucoadhesive (group 1), standard treatment (group 2; mouthwash chlorhexidine 0.2%), and base (group 3). The prepared adhesive mucus paste containing biosurfactant lipopeptide (A baumannii and P aeruginosa) and a drug-free blank base (base) after being packaged in a coded container (coded by a person other than the researcher and given to the patients in a double-blind manner) were provided to the patients. Patients were instructed to apply their respective group paste on their wound 4 times a day (after breakfast, lunch, and dinner, and before going to bed). All patients were examined on the initial, second, fourth, and sixth days of treatment and the wound diameter was measured and recorded with a periodontal probe. For patients who had more than 1 ulcer, the area where the ulcer was more common or in the area that was easier to access was considered.

The intensity of the patient’s pain was also measured over 10 days based on the VAS (0 indicating no pain and 10 indicating the most severe pain), and the patient was given the necessary training to record their pain daily. Patients were asked to write down the intensity of pain and burning 1 hour after applying the paste in the morning. The amount of erythema and exudate was measured based on the criteria designed by Greer et al.¹² Additionally, 3 indices—efficacy indices (EI), marked improvement rate (MIR), and improvement rate (IR)—were investigated and calculated.¹²

To evaluate EI, the size of the wound on days 0, 2, 4, and 6 were included in the following formula: 

target day EI = wound size on the target day - wound size on day 0: wound size on day 0 - 100% 

On this basis (1) EI equal to 100% indicated improvement; (2) 70% ≤ EI <100% indicated significant improvement; (3) 30% ≤ EI <70% indicated moderate recovery; and (4) EI <30% indicated no improvement. 

The average of groups 1 and 2 on the second, fourth, and sixth days indicate MIR on the same day, and the average of groups 1, 2, and 3 on the second, fourth, and sixth days indicate IR on the same day.¹² In addition, information including age, sex, number of recurrences of wounds per year, a common site of involvement, duration of wound formation, average number of wounds per period, wound size, and recurrence intervals were recorded. The statistical tests used included a 2-paired sample t test, ANOVA, and Kruskal-Wallis (Wilcoxon signed-rank). Statistical significance was set at a P value of less than .05.

A total of 34 patients were included in the study. There were 11 patients in the mucoadhesive group, 12 patients in the positive control group, and 11 in the base group; 2 patients left the study. The study included 19 female and 15 male individuals. The participants were between 20 and 41 years (average age, 25.72 ± 3.08 years). Statistically, there was no significant difference observed among the studied population (Table 1), and demographic characteristics including age and sex were not significant between the 3 groups (P > .05). 

Although the average wound pain scores of the 3 groups were not significantly different at the beginning of the study (P > .05), a significant difference was found in the mucoadhesive group and the positive control in the continuation of the study (days 2, 4, 6) (P < .05).

With the passage of time, the pain score decreased in the mucoadhesive group and the positive control group, which was statistically significantly different from the base group (P < .05). Although the positive control group experienced more pain reduction, there was no significant difference compared with the mucoadhesive group (Table 2, Figure 1).

On the second treatment day, the efficacy index of the positive control group was higher than that of the mucoadhesive group and the base group (P = .04). There was also a significant difference between the mucoadhesive and the positive control groups and the base group (P = .001). The positive control group had a significantly higher recovery rate compared to the mucoadhesive and base groups, and this recovery rate was significant in the positive control and base groups (P = .001; Table 3). On the fourth day, the efficacy index of the positive control group was found to be higher than the mucoadhesive and the base groups, and this group had a higher recovery rate. The efficacy index and recovery rate of the mucoadhesive group were significantly higher than the base group (P > .05). On the sixth day, more patients recovered, and the positive control group showed a significant improvement compared to the base group, which was statistically significant (P < .05). A difference between the mucoadhesive group and the positive control group was observed, but it was not statistically significant (P > .05).

There was no significant difference in the average size of the wounds between the 3 groups at the beginning of the study (P > .05), but a significant difference was observed on days 4 and 6 (P < .05). The average wound size in the positive control group on days 4 and 6 (Table 4, Figure 2) was statistically less than the mucoadhesive and base group (P < .05), while there was no significant difference between the mucoadhesive and base groups (P > .05). On the second day, the efficacy index of the positive control group was much greater than that of the base group (P < .05) but there was no significant difference between the other groups (P > .05), although the recovery rate was similar in all groups (P < .05). On the fourth day, the efficacy index of the positive control group was much higher than the other groups (P < .05) and the recovery rate was significant (P < .05). There was no significant difference between the mucoadhesive and base groups in terms of efficacy index and recovery rate (P > .05). On the sixth day, compared to the mucoadhesive and base groups, the positive control group maintained a significantly higher effect (P < .05). Although the improvement rate was similar in all groups, the specific improvement rate in the positive control group was statistically higher than in other groups (P < .05) and there was a significant difference between the mucoadhesive and base groups (Table 4). All patients tolerated these drugs, and no complications were reported during the study.

Wound healing is an important biological process that involves tissue repair and regeneration. A wound is described as a disorder or damage to the structure and function of tissue that results in damage to the internal tissue barriers.¹³ Healing is a complex process initiated in response to injury that restores the function and integrity of damaged tissues.¹⁴ Wound healing involves cell-cell as well as cell-matrix interactions, which accept the process in 3 overlapping steps. Those steps are inflammation (0–3 days), cell proliferation (3–12 days), and regeneration (3–6 months).¹⁵ Platelet aggregation during homeostasis releases various soluble mediators in the healing process.¹⁶ Hemostasis is established by a slightly pre-inflammatory state characterized by vasodilatation, increased capillary permeability, complement activation, and polymorphonuclear and macrophage movement to the wound site within 3 days. Moist wounds are less susceptible to infection than dry wounds, given that periwound moisture stimulates tissue regeneration and esthetics, reduces pain, and prevents microbial invasion.¹⁷ The use of natural compounds and polymers is a leading development for tissue regeneration, and a wide array of bioactive metabolites have been described as having potential for wound healing applications.¹⁸

Recurrent aphthous stomatitis is a complex disease that has spread worldwide with high prevalence, the exact cause of which is still unknown. The wide age range, recurrent character of the disease, and decrease in the quality of life of patients have led to much research on the cause and treatment of this condition, and various treatment methods with local and systemic agents have been evaluated in different countries for several years.¹⁹ 

The results of the current study showed that both mucoadhesive and routine treatment reduced the pain and wound size on the second to sixth days, and these effects were significant (P < .05) compared to the base group. Although routine treatment includes chlorhexidine mouthwash, the chlorhexidine mouthwash showed better treatment effects compared to mucoadhesive; however, there was no significant relationship between routine treatment and mucus glue. 

In an in vitro study using a mouse model conducted by Zouari et al,²⁰ the authors found that a topical application of a gel (based on Bacillus subtilis SPB1 biosurfactant) to the wound site every 2 days caused the percentage of wound closure to increase over a period of 13 days when compared to untreated and Cicaflora-treated groups. The wound healing effect of gel based on SPB1 biosurfactant was confirmed by histologic study. 

Farias et al²¹ showed that mouthwashes prepared from 3 biosurfactants produced by P aeruginosa UCP 0992 (PB), Bacillus cereus UCP 1615 (BB), and Candida bombicola URM 3718 (CB) constitute a safe, effective, natural alternative to commercially available mouthwashes for the control of oral microorganisms. The combination of CB and PB biosurfactants with chitosan showed an additive effect on most of the tested microorganisms. The toxicity of the mouthwashes was significantly lower than commercial mouthwashes. In the research conducted by Resende et al,²² toothpaste containing biosurfactant and fungal chitosan with sodium fluoride was evaluated for cytotoxicity, antimicrobial effect, and inhibition potential against the biofilm formed by Streptococcus mutans. Biosurfactants produced by PB, BB, and CB were tested. All materials had minimum inhibitory concentration for S mutans. The combination of CB and PB with chitosan had an enhanced effect against S mutans, while BB combined with chitosan had an indifferent effect. The toothpaste was non-toxic. All formulations inhibited the cell viability of S mutans in the biofilm, with similar results compared to the tested commercial toothpaste. The current results show that the proposed formulations are promising compared to commercial toothpaste.

Microbial-derived biosurfactants are amphipathic molecules composed of both hydrophobic and hydrophilic domains that allow them to assemble at the interface of immiscible liquids such as water and oil.²³ Surface-active metabolites reduce surface tension and interfacial tension in different material phases such as gas, liquid, and solid.²⁴ Such properties lead to potential applications of biosurfactants in the cosmetic (anti-cellulite products), pharmaceutical (nano-sized drug delivery systems), food (emulsifier and stabilizer additives), and petrochemical (microbial-enhanced oil recovery process) industries.^25,26 According to the origin of biosurfactants and the nature of their chemical structure, they are divided into 5 main groups: lipopeptides, lipoproteins, glycolipids, phospholipids, and polymers.²⁷ The lower toxicity, higher biodegradability, mild production conditions, and environmental compatibility of biosurfactants, as well as their high selectivity and stable activity at extreme pH, salinity, and temperature, make them a valid alternative to their petroleum-derived chemical synthesis counterparts.²⁸ Despite these advantages, the commercial success of biosurfactant applications has not been fully achieved due to the difficulty of establishing efficient and economical development of bioprocesses and downstream production technologies.²⁹ Therefore, optimizing biosurfactant production conditions and developing cost-effective recovery methods are among the most important approaches to maximizing biosurfactant production.²⁷

Biosurfactant producers can facilitate the adsorption of hydrocarbons through the formation and release of surface-active molecules owing to their abundance in oil-contaminated soils.³⁰ As such, various reports on the isolation of Bacillus and Pseudomonas species as biosurfactant producers from oil-contaminated areas are available.^24,31 However, such ability has rarely been described in the genus Acinetobacter. For example, Zou et al²³ isolated Acinetobacter baylyi from oil-contaminated soil and used it for the microbial enhanced oil recovery process. In a recent study by Jimoh et al,³⁰ biosurfactant produced from Acinetobacter spp was optimized using the response surface methodology design, which achieved a final increase of 57.5% in biosurfactant production. Reports have shown that microbial lipopeptide surfactants, which are surface-active compounds produced extracellularly or as part of the cell membrane by several bacterial and fungal species, have unique properties that are potentially useful for the cosmetic industry; surface properties include maintaining anti-wrinkle and moisturizing activities on human skin.^31,32

Lipopeptide biosurfactants are safely introduced into skin products as long as they have low cytotoxicity against human cells.¹⁸ Interestingly, surfactin, a lipopeptide biosurfactant produced by the genus Bacillus, has received much attention in the industry due to its multifunctionality.³³ Surfactin is a biosurfactant with exceptional surface properties that reduces the surface tension of water from 72 to 27.9 mN-1.^32,34 Surfactins are exceptionally biocompatible due to their low cytotoxicity to mammalian cells and minor irritation to human skin.³⁵ A number of companies have used surfactin derivatives in skin formulations as well as in cleansing cosmetics.¹⁸ In addition to using them as cleansing and anti-wrinkle agents, they have been used to stimulate the production of collagen and elastin because of their anti-free radical and moisturizing properties. Lipopeptide biosurfactants have strong antimicrobial activity and have shown potential against multidrug-resistant bacteria or fungi.¹⁸ During a screening program for biosurfactant-producing strains, B subtilis SPB1 (HQ392822) was isolated³⁶ and the crude lipopeptide biosurfactant mixture produced by B subtilis SPB1 was tested for its inhibitory activity against 11 bacterial and 8 fungal strains. It demonstrated significant antimicrobial activity against microorganisms with multidrug-resistance profiles.³⁷ The in vivo toxicity of SPB1 crude lipopeptide biosurfactant was evaluated in male rats in a previous study. The results showed that the daily consumption of SPB1 biosurfactant did not result in mortality in any dose. Also, during the 28-day treatment period, no change in the animals’ behavior was observed.³⁸

In a 2011 review by Nashwan³⁹ on 5 interventional articles (randomized controlled trials) from 1980 to 2010 that investigated the effect of chlorhexidine mouthwash in children undergoing chemotherapy, 4 articles reported that chlorhexidine has a preventive effect on the development and severity of oral mucositis, while the fifth study showed no beneficial effect. It was concluded that chlorhexidine may have a role in reducing oral mucosa damage during chemotherapy in children with cancer and this effect is probably through the reduction of microbial flora and oral plaque.³⁹ In the case of chlorhexidine, it is important to know that the results of studies on its efficacy in wound healing are contradictory. Many in vitro studies have shown that chlorhexidine has a negative effect on the proliferation of fibroblasts and keratinocytes, which depends on the concentration and duration of its use.⁴⁰ But only a limited number of in vivo studies show its negative effect, and a large number of these studies demonstrate the beneficial effect of chlorhexidine mouthwash after various oral surgery procedures on wound healing.⁴⁰ The difference between the findings of the studies^40,41 may be justified by the interaction of molecular and environmental processes affecting the tissue in vivo compared to in vitro. For example, in the oral cavity, chlorhexidine often binds to bacteria and any excess is precipitated by serum proteins,^19,41 so the excess amount of chlorhexidine molecules that bind to host cells and inflict damage is reduced.⁴¹

In a study by Alsadat Hashmipour et al,⁴¹ the authors showed betamethasone alone and in combination with chlorhexidine has anti-inflammatory effects due to glucocorticoids (like betamethasone) being strong inhibitors of inflammatory responses and due to their effect on gene regulation. They cause lipocortin production, which is an inhibitor of the phospholipase A2 protein. This inhibition reduces the production of prostaglandins and leukotrienes, a function that is especially important in macrophages, monocytes, endothelial cells, and fibroblasts. Glucocorticoids also suppress COX synthesis.^40,42 The final result of these 2 functions is to inhibit the chemotaxis of eosinophils, neutrophils, and monocytes. It also inhibits the production of monocytes and the synthesis of cytokines in monocytes and macrophages and, as a result, reduces inflammation.⁴² Merchant et al⁴³ showed that betamethasone was effective in reducing symptoms and shortening recovery time, but not in preventing relapse.

The patients included did not adhere to all study protocols. As a result, the patients were not properly treated before inclusion in the study or during the study.

The results of this study showed that the use of mucoadhesive compared to mucoadhesive without biosurfactant lipopeptide treatment reduces pain and wound size. However, the mucoadhesive had less effect in reducing pain and wound size than the routine treatment. Therefore, further studies should be conducted on the effects of the biosurfactant lipopeptide adhesive mucus paste on oral ulcers.