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 Table of Contents  
Year : 2021  |  Volume : 33  |  Issue : 2  |  Page : 135-140

Assessment of Clinical Efficacy of Microbiome and Topical Hyaluronic Acid Gel Combination Therapy in Minor Aphthous Ulcer Management – A comparative study

Department of Oral Medicine and Radiology, Tamil Nadu Government Dental College and Hospital, Chennai, Tamil Nadu, India

Date of Submission19-Nov-2020
Date of Decision26-Dec-2020
Date of Acceptance30-Mar-2021
Date of Web Publication23-Jun-2021

Correspondence Address:
Dr. Shilpa Shree Kuduva Ramesh
Department of Oral Medicine and Radiology, Tamil Nadu Government Dental College and Hospital, Frazer Bridge Road, Muthuswamy Salai, Chennai - 600 003, Tamil Nadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jiaomr.jiaomr_236_20

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Aim: This study aimed to determine the clinical efficacy of the combination of microbiomes and 0.2% topical hyaluronic acid gel, alongside topical monotherapy with 0.2% hyaluronic acid gel and steroids, respectively, in minor aphthous ulcer management. Subjects and Methods: A total of 45 patients were divided into three groups: Group A (n = 15) –– a combination of microbiome therapy––pre- and probiotics capsules p. o. and topical 0.2% hyaluronic acid gel; Group B (n = 15)––topical 0.2% hyaluronic acid gel; and Group C (n = 15)––0.1% triamcinolone acetonide oromucosal paste. Numerical rating scale (NRS) for pain, ulcer dimensions measured by Digital Planimetry using android mobile application to determine the degree of ulcer healing (%) and occurrence of new ulcers were recorded on 0, third, and seventh days of treatment. Statistical Analysis Used: One-way ANOVA with post hoc Tukey test among the groups and independent t - test for the degree of ulcer healing between third and seventh days were applied. Results: NRS for pain at day 3 showed a statistically significant pain reduction in Group A compared with Groups B and C (P = 0.04, 0.01, respectively). Though Group A showed the highest grade of closure (93.4 ± 2.5%) and a greater decrease in mean NRS score (0.2 ± 0.42) on the seventh day when compared to Groups B and C, results were not statistically significant (P > 0.05). Conclusion: Microbiome and topical hyaluronic acid gel combination therapy helps in immediate pain relief and faster healing. Hence, it could be an excellent alternative for topical steroid management.

Keywords: Aphthous ulcer, dysbiosis, hyaluronic acid, microbiome, prebiotics, probiotics

How to cite this article:
Kuduva Ramesh SS, Sadaksharam J. Assessment of Clinical Efficacy of Microbiome and Topical Hyaluronic Acid Gel Combination Therapy in Minor Aphthous Ulcer Management – A comparative study. J Indian Acad Oral Med Radiol 2021;33:135-40

How to cite this URL:
Kuduva Ramesh SS, Sadaksharam J. Assessment of Clinical Efficacy of Microbiome and Topical Hyaluronic Acid Gel Combination Therapy in Minor Aphthous Ulcer Management – A comparative study. J Indian Acad Oral Med Radiol [serial online] 2021 [cited 2021 Jul 29];33:135-40. Available from: https://www.jiaomr.in/text.asp?2021/33/2/135/319067

   Introduction Top

Minor aphthous ulcer is the most common form of recurrent aphthous stomatitis (80%) (RAS).[1] One of the current management protocols for RAS includes topical corticosteroids like triamcinolone acetonide gel. It is a potent, intermediate-acting synthetic steroid that acts as an anti-inflammatory agent. Corticosteroids are known to get absorbed systemically even on topical application, that can lead to adverse reactions such as suppression of hypothalamo–hypopituitary axis and central serous retinopathy.[2] Hence, to avoid short-term applications and serious side effects, many other treatments such as cauterization and antibiotics were tried, with unsuccessful results.[3] Oral mucosal dysbiosis, that is, the shift of pathogenic bacteria in place of symbiont bacteria, plays a main role in the initiation of RAS. Pre- and probiotics supplements together called microbiome therapy may act as an ex juvantibus in its management.[4] Hyaluronic acid (HA), a mucopolysaccharide component of the ground substance of many tissues,[5] has been effectively utilized to accelerate wound healing.[6] Within the treatment of RAS, HA as a thick viscous gel and having bioadhesive property remains in contact with the ulcer for a longer time and it quickly decreases the pain and distress caused by the ulcers and essentially has diminished its recurrence.[7] Both Pre- and probiotics and HA when given as partitioned regimens were found to be successful in quicker recuperating in RAS.[7],[8] Therefore, we aimed to compare the clinical efficacy of the combination of microbiome therapy with 0.2% topical HA gel, HA gel as monotherapy, and 0.1% triamcinolone acetonide oromucosal paste (control) within the management of minor aphthous ulcer.

   Subjects and Methods Top

After ethical clearance from Institutional Review Board (TNGDCH IRB approval letter ref. no. 4/IRB/2019 dated August 22, 2019), this study was conducted in the Department of Oral Medicine and Radiology from September 2019 to February 2020. Sample size calculation was done with G*Power v3.1.9.7 software. A total sample size of 18 was required to achieve power (1–β) of 80% with type-I error (α) of 5% and effect size of 0.8 based on the previous study.[9] As ours is a tertiary care center, a total sample size of 45 (power of 98%), with 15 samples each treatment group was chosen.

The inclusion criteria of the study were as follows: (1) both genders with the age range of 18–30 years, (2) patients who were willing to participate in the study, and (3) satisfied diagnostic criteria of RAS minor given by Natah et al.[3] [Table 1].
Table 1: Clinical diagnostic criteria of minor aphthous ulcer[3]

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The exclusion criteria of the study were as follows: (1) patients who were not willing to participate in the study, (2) major herpetiform, RAS and ulcers in the inaccessible areas, (3) RAS associated with other syndromes such as Reiter's, Behcet's, MAGIC (mouth and genital ulcers with inflamed cartilage), PFAPA (periodic fever, Aphthous stomatitis, Pharyngitis, and cervical adenitis), Sweet's; cyclic neutropenia, HIV/AIDS and anemia (4) RAS patients already under topical or systemic management, (5) Medically compromised patients such as Diabetes Mellitus, liver, renal disorders, polyketonuria, (6) pregnant and lactating women, (7) patients allergic to HA, and pre-and probiotics, (8) Smokers.

Detailed recording of patients' histories and clinical intraoral examinations were performed. Investigations, such as serum vitamin B12 level to rule out megaloblastic anemia, serum ferritin level to rule out iron deficiency anemia routine, blood investigations were performed to rule out any other underlying systemic diseases mentioned earlier in the exclusion criteria.

All the participants were informed through written and verbal aspects about the study both in the local and English language and informed consent was obtained. Our study was in accordance with the principles of the Declaration of Helsinki. Study participants were randomly assigned (1:1:1) into three groups using the simple randomization technique.

All patients were given dietary counseling to avoid spicy foods and encouraged a bland diet.

For Group A (n = 15), participants were advised to take each capsule of pre-and probiotic (Bifilac HP Capsule, Tablets Ltd) twice daily with food for a week as a microbiome therapy and educated to apply topical 0.2% HA gel (Gengigel, Milano, EU) thrice daily for a week. For Group B (n = 15), participants were advised to apply topical 0.2% HA gel thrice daily for a week. Participants in Group C (n = 15) were advised to apply topical 0.1% triamcinolone acetonide oromucosal paste (Kenocort, Abbott Healthcare Pvt. Ltd), thrice daily for a week. Both topical 0.2% HA gel (Groups A and B) and 0.1% triamcinolone acetonide paste (Group C) were applied over and around 1 cm of each ulcer by the participants using a cotton applicator tip and advised not to eat/drink for the next 30 mins. Each capsule of Bifilac contains prebiotics-fructooligosaccharides, inulin, and probiotics–lactobacillus sporegens (50 million spores), Bacillus mesentericus (TO-A, 1 million spores), Clostridium butyrcum (TO-A, two million spores), and Streptococcus fecalis (T-110, 30 million spores).

The primary outcome measure was pain relief and was recorded using ten graded Numerical Rating Scale (NRS) (0––nil, 1 to 3––mild, 4 to 6––moderate, 7 to 9––severe, >10––worst, unimaginable) and secondary outcome measures were degree of ulcer healing and occurrence of new ulcer. To calculate the degree of ulcer healing, we measured the diameter and area of a single large ulcer in that particular episode, irrespective of the site using ON 2D-CameraMeasure version 7.0 (PotatotreeSoft, 2013), a two-dimensional digital planimetry android mobile application [Figure 1]. NRS for pain, diameter, and area of the largest ulcer were recorded at three-time points: baseline (day 0), third and seventh day of the treatment. Based on the ulcer area and diameter, the degree of ulcer healing (%) was calculated on the third and seventh days.
Figure 1: (a) Minor aphthous ulcer with an erythematous halo; (b) Digital planimetric measurement of the ulcer

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Statistical analysis was performed using Statistical Package for Social Sciences (SPSS) software program, version 25.0 (IBM). One-way ANOVA with post hoc Tukey test was performed among three groups and independent t- tests for comparing the degree of ulcer healing at third and seventh days. Paired t -test was performed for intragroup comparison of outcome on day 3 and day 7. The level of statistically significant difference (α) was set at P < 0.05.

   Results Top

A total of 45 patients were enrolled in the study and completed the treatment and follow-up. The mean age of the participants was 23 ± 3.7 years with the range of 18–30 years. Differences in baseline ulcer measurements such as NRS of pain, ulcer diameter, and area between the groups were statistically insignificant (P > 0.05) [Table 2]. Patients were followed up continuously to check the treatment adherence and diet. None of the patients developed an anaphylactic reaction to the gels and microbiome therapy.
Table 2: One-way ANOVA for baseline parameters

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No new ulcers appeared on day 7 in Group A patients. Mean and standard deviation of NRS, ulcer diameter, area, and degree of ulcer healing are tabulated in [Table 3].
Table 3: Intergroup and Intragroup comparisons with Mean±SD of NRS score, ulcer diameter, ulcer area, degree of ulcer healing at day 3 and 7

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The intragroup comparison of treatment outcome at third and seventh days was highly significant (P < 0.001). One-way ANOVA with post hoc Tukey test showed no statistically significant differences in pain NRS [Figure 2]a, ulcer diameter (P = 0.35, 0.67) [Figure 2]b, area (P = 0.14, 0.68) [Figure 2]c and degree of ulcer healing (P = 0.11,0.72) [Figure 2]d between the study groups (A, B) and conventional treatment group C (P > 0.05) at day 3 and day 7. Pain relief on the third day alone was statistically significant (P < 0.05), on comparing group A vs. group B (P = 0.04) and Group A vs. Group C (P = 0.01). It concludes that Group A participants had faster pain relief than B [Table 4].
Table 4: Post hoc Tukey test from One way ANOVA for intergroup comparison of NRS score, ulcer diameter, ulcer area, and independent t test for degree of ulcer healing at third day and seventh day

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Figure 2: Line graph showing reduction in (a) pain intensity, (b) ulcer diameter, (c) ulcer area in Group A, B & C at 0, third and seventh day measured using Digital Planimetry and (d) degree of ulcer healing among groups on day 3 and day 7

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   Discussion Top

RAS is a common multifactorial inflammatory disorder with an overall predominance rate somewhere in the range of 5% and 66% in the age group of 10-40 years.[10] In our study, the mean age of participants was 23 ± 3.7 years which was similar to previous studies.[7],[8] Ulcers are usually round or oval, regular, shallow floor covered by pseudomembrane with an erythematous halo, predominantly manifest in the non-keratinized mucosa.[1] Almost all the RAS patients in our study reported pain on the scale of mean NRS – 8.5 out of 10, which significantly reduced the patient's quality of life, as it caused constant painful/burning sensation which interfered with daily activities like speaking, eating, or even drinking as reported in the literature.[11] RAS can be classified into three different types: Minor, Major, and Herpetiform ulcers.[12] Among them, Minor RAS is the most frequently observed one and measures below 1 cm in diameter that typically heal without scarirng.[1]

Several factors like host and genetic factors, hormonal imbalance, stress, vitamin deficiency have been implicated in the etiologies of RAS. However, all these factors affect the disease progression by influencing the composition of microbiota residing on oral mucosae, which in turn causes immunomodulation. This is called Microbial Dysbiosis in which there is a potential shift of pathobiont microbial community in the place of symbiont ones.[13],[14]

Many bacterial species have been identified in abundance in RAS patients compared to healthy controls like L-form of Streptococcus sanguinis 2A,[15] Bacteroidales,[16] Neisseria, Veillonella, Rothia dentocariosa, Streptococcus mitis;[17] Acinetobacter johnsonii[18] showing the association between dysbiosis and RAS. Also, an autoimmune reaction is suggested against oral epithelial peptides due to cross-reaction of some 65-kDa heat shock proteins produced by Streptococcus species, leading to mucosal damage.[18]

Microbiome therapy reverses the oral mucosal dysbiosis to eubiosis, that is, conversion of pathogenic to symbiont microbiota community similar to cariogenic bacteria and periodontal pathogens by modulating host immune response.[19]

The possible mechanism of actions of microbiome therapy are bacteriocin and hydrogen peroxide production; co-aggregation and growth inhibition of pathogenic bacteria; competitive antagonism on adhesion and nutrition; Immunomodulation by modulating the cytokines like IL-10, TNF-α, IL-8, and Toll-like receptors also may be a contributing factor.[20],[21] Its metabolites activate the aryl hydrocarbon receptor, hereby enhancing the synthesis of the tight junction proteins and increases the production of ZO-1 to strengthen the epithelial barrier.[4] Our study showed significant pain relief at the end of the third day in Group A patients receiving both microbiome therapy and topical HA gel, which could be attributed to their epithelial barrier formation. Similar results were shown by Trincheiri et al.[8] However, recent studies by Dugourd et al.[22] and Pederson et al.,[23] showed no statistically significant results on the comparison of the probiotics over placebo in the treatment of RAS.

According to Cheng et al.,[24] the combination of probiotics and steroids or anesthetic antiseptic gel is more efficacious than its monotherapy in the management of RAS patients. But here, we attempted to utilize the epithelial barrier-inducing property of both probiotics, and HA and studied its efficacy when comparing it with monotherapy using conventional steroids. Thence, it forms the first kind of its study in the literature to the best of our knowledge.

HA is a linear polymer of glucuronic acid N-acetylglucosamine disaccharide glycosaminoglycan with anti-inflammatory, anti-edematous effect and also HA is implicated in initiation and modulation of the inflammatory responses, angiogenesis necessary for local collagen synthesis. It induces re-epithelization through basal keratinocyte proliferation.[25]

In our study, Group B patients who were treated with HA alone, showed statistically significant pain relief and ulcer healing when comparing the baseline and post-treatment values and only 8 patients showed new ulcers during treatment. Similar results were shown by Nolan et al.,[26] in which only a few ulcers were recorded on day 5 of the investigation than those treated with placebo. In another study by Lee JH et al.,[7] the maximum ulcer size was reduced by 62% on the fourth day in HA group which was a little greater than our study results where only 58.9% of healing was recorded on the third day. Dalassendri et al.[27] compared the two different formulations of the HA gel, and mouth rinse in the treatment of RAS and showed both the barrier formulations were effective in the reduction of ulcer size and pain intensity on the third day. But HA-based oral gel determined better results both in terms of the number of oral lesions and in terms of lesion sizes when compared with chlorhexidine mouth rinses.[28]

0.1% Triamcinolone acetonide is a potent topical corticosteroid (Class II) and intermediate-acting glucocorticoid. It suppresses the pain, shortens the aphthae healing time, and is most widely used in the management of RAS.[29] Hence, we compared our study groups with the steroid group.

Digital planimetry has been used in the measurement of trophic ulcers; however, this is the first study to use digital planimetry in the measurement of oral ulcer dimensions. We used an Android smartphone application for digital planimetry and hence we were able to calculate the area of the ulcer effectively, rapidly, and at a low cost.

Group A showed nil occurrence of new ulcer at the end of the seventh day and also there is no risk of an accidental overdose in both HA and microbiome therapy. Hence it can be safely recommended to individuals who may not comply with instructions. Additionally, younger patients (<20 years old) in all three groups showed rapid healing and pain relief than elder patients (20–30 years) which may be due to greater healing capacity in the younger age. No variation was found among males and females.

Limitations and future prospects

The smaller sample size, limited follow-up is the major drawback of the study. Only the single largest ulcer during the particular episode was assessed for evaluation. Ulcers in the inaccessible areas were excluded in the study which was the other prime limitation. Future implications could be studying its effects in different age groups, patient's quality of life, the site-specific impact of the drug, ELISA assessment of the microbiome.

   Conclusion Top

Altogether results of this study showed the immediate pain control of topical HA and microbiome combination therapy in the management of RAS. Hence, this could be an excellent alternative for topical steroids in the management of RAS.

Declaration of patient consent

Patient(s)/study participants were informed about the nature of the study and their consent were obtained to use the relevant clinical details and images for the scientific publication purpose without revealing the identity.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2]

  [Table 1], [Table 2], [Table 3], [Table 4]


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