Assessment of local Interleukin 1β and CCL28 levels and clinical effects of curcumin gel in an experimental gingivitis model. A randomized double blind controlled clinical trial.

Gingivitis is one of the most frequently occurring periodontal diseases affecting human. In India more than 80% of the population regardless of the age, sex, and race are affected by gingivitis. Indian epidemiologic studies have shown high prevalence of gingivitis ranging from 71% -94%. Pathogenic bacteria organized in a biofilm around the teeth initiate the disease process; the host inflammatory response to these bacteria plays a central role in the pathogenesis and progression of the disease from gingivitis to periodontitis. Prospective studies including clinical trials indicate that controlling inflammation remains a principal strategy for preventing and managing periodontal disease. Similarly, evidence from humans and animal models demonstrate that anti-inflammatory agents or host modulatory agents used as adjunct can improve periodontal status.

Since ages turmeric has been used for medicinal purpose for the treatment of many diseases in India and other parts of the world as a form of herbal medicine. Turmeric, otherwise known as Circuma longa, is a member of the ginger family, Zingaberaceae. Extensive research on curcumin have demonstrated a wide spectrum of therapeutic effects such as anti-inflammatory, antibacterial, antiviral, antifungal, antitumour, antispasmodic, and hepatoprotective. The most important property of curcumin is anti-inflammatory property that can be put to oral use for the treatment of gingivitis. The anti-inflammatory effect of curcumin is most likely mediated through its ability to inhibit cyclooxygenase-2 (COX-2), lipoxygenase (LOX), and inducible nitric oxide synthase (iNOS). COX-2, LOX, and iNOS are important enzymes that mediate inflammatory processes. It is also a potent inhibitor of pro-inflammatory cytokines like Interleukin 1 beta (IL 1β), IL-6, IL-12, Tumor necrosis factor alpha (TNF-α) and Interleukin 8 (IL 8).

There are no studies at present evaluating the levels of IL1β and CCL28, in gingival crevicular fluid (GCF) after the use of curcumin based gel, chlorhexidine gel and metronidazole gel in patients with gingivitis.

Aim:

The present study is done with the following aims and objective:

1. To evaluate clinically curcuma oral gel, chlorhexidine gel, and metronidazole gel in experimental gingivitis.

2. To evaluate the levels of IL1β and CCL28 in Gingival Crevicular Fluid (GCF) before and after using curcuma oral gel, chlorhexidine gel and metronidazole gel in experimental gingivitis.

Hypothesis:

ï‚· The use of curcuma oral gel topically will lead to clinical reduction of gingival inflammation by 20% more than metronidazole gel and chlorhexidine gel.

 The use of curcuma oral gel topically will lead to reduction of IL1β and CCL28 levels in the GCF by 20% more than metronidazole gel and chlorhexidine gel.

Participants:

The volunteers for this study will be selected from the patients visiting the outpatient department of periodontology, Chhattisgarh Dental College and Research Institute, Rajnandgaon. 60 volunteers will be recruited for this study with age ranging from 18 to 35 years.

Inclusion Criteria:

i. Systemically healthy individuals

ii. Possessing at atleast 20 natural teeth.

iii. All the posterior teeth in upper right quadrant excluding the third molar should be present and exhibit periodontal health or gingivitis.

Exclusion criteria:

i. There should be no teeth with a probing depth > 3mm.

ii. Participants with any tobacco use

iii. Recent use of antibiotics, aspirin or non-steroidal anti-inflammatory drugs (NSAIDs) during past one month.

iv. If they necrotizing periodontal conditions, gross oral pathology, severe unrestored dental caries or clinically significant laboratory abnormalities.

v. Pregnant and lactating mothers

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Tooth shield Construction:

Alginate impression of the experimental teeth will be a taken and poured in die stone to obtain casts. A tooth shield will be constructed of 1mm thick thermoplastic mouth-guard material over this cast. The tooth shield will be trimmed 2 mm beyond the gingival margin to prevent oral hygiene measures so as to induce experimental gingivitis and will be used to keep the gel in contact with the gingival margin of the experimental teeth.

Study design:

This will be double blind randomized controlled clinical trial. The clinical trial will be divided into, prescreening, screening and treatment and post treatment phase.

Prescreening Phase: During the prescreening examination participants are assessed for inclusion and exclusion criteria. Also during this phase patients will be brought to gingival health with mechanical scaling, tooth polishing and oral hygiene instruction. Additionally alginate impressions will be taken of the selected quadrant for preparation of tooth shield. The importance of oral hygiene will be strongly reinforced.

Screening and treatment phase: The participants return after 7 days, for screening phase. The periodontal status is assessed for the experimental quadrant and the participants are continued for the study if they had a gingival index of less than 0.5 and absence of bleeding on probing. The participants will be randomly distributed by lottery method into three treatment groups, with 20 participants in each group: 1) Application of curcuma gel; 2) application of metronidazole gel 2) application of chlorhexidine gel. For this group of three, a quadrant for the experimentation will be randomly selected. This will ensure that same quadrant is being evaluated in the three who were randomly assigned.

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To induce experimental gingivitis subjects will be instructed to abstain from the twice a day brushing and flossing in the selected quadrant for a period of 29 days by wearing the tooth shield. Subjects will be asked to wear their tooth shields over the selected quadrant during daily brushing procedures for a period of 29 days. Following brushing, the participants are instructed to fill the respective gel in tooth shield which then is used as application trays. They are instructed to keep the tray in place over the experimental quadrant for 10 minutes and then followed by rinsing of the mouth. This is done twice daily following brushing in the non experimental quadrants. They will be instructed to avoid any anti-bacterial rinses (e.g. chlorhexidine or phenolic compounds), therapeutic dentifrices (e.g. triclosan or 0.454% stannous fluoride) or irrigating devices. Subjects will be instructed to use a dispensed fluoride dentifrice and dental floss for plaque control in non-study quadrants (maxillary left, mandibular left and mandibular right).

Blinding: All the tubes will have plain white covering labeled only with the lot numbers to ensure proper blinding of the product from the patient and the examiner. The gels will be packaged in masked encoded tube. This is done to ensure blinding so that neither the examiner nor the participant knows the contents of the gel (double blinding).

Post treatment phase: Following the day29 visit, subjects will enter a post-treatment period. During this period they will reinstitute all home care procedures and receive professional scaling, tooth polishing and oral hygiene instruction to re-establish periodontal health. Subjects will be recalled at 6 weeks and 8 weeks (post-treatment visit) to evaluate signs of periodontal health.

Compliance: Each patient will be given 50g tube containing gel. To check for the compliance participants will be asked to return the tubes after 29 days and see the remaining amount of gel left in the tube.

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Outcome Measurements:

The clinician who was blinded to the gels assigned to the subjects, will conduct all the examination and scoring.

Primary Outcome:

Gingival crevicular fluid (GCF) will be collected with microcapillary pipettes inserted into the gingival sulcus. The assessment of IL1 β/ ccl28 levels will be done using enzyme linked immunosorbent assay (ELISA) at induction baseline and 29 day recall following treatment for all participants.

Secondary outcome:

The following index will be recorded with a graduated periodontal probe (UNC 15 HuFriedy®) at baseline and after 29 day recall for the entire participants. Efficacy parameters will be collected around maxillary right molars and premolars only, and at six periodontal sites per tooth (i.e. mesiobuccal, buccal, distobuccal, mesiolingual, lingual, distolingual).

1. Plaque index (Silness and Loe)

2. Modified Gingival Index (Loe and Sillness Index)

3. Bleeding on probing

Also subjective evaluations of the participants will be done using a questionnaire. Subjects will be requested to mention yes/no for pain, burning sensation, pruritis/ itichiness, dryness of the mouth, taste disturbance, discolouration of the teeth, feeling bitter taste in the mouth. The patients will be asked to rate the severity of the side effects as none, mild, moderate or severe.

Statistical Analysis:

Student’s t-test will be used to evaluate statistical differences between the weight of thethree gels on days 0 and 29( α=0.05). Mann-Whitney test will be performed to evaluate statistical differences between control and test groups on days 0 and 29 (α=0.01) for non-parametric

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data. In each group, the mean scores of GI, BOP and PI will be compared between baseline and the end of the trial by the Wilcoxon test (α=0.01). Results will be presented as mean and standard deviation.

Utilization of research:

Curcumin is an age old product of Indian herbal medicine that has been used for many purposes. One of the important properties of Curcumin is to inhibit the inflammatory response mediated by pro inflammatory cytokines such as IL1 β, TNFα and IL8. It can be brought into periodontal use for the prevention and treatment of gingivitis and mild periodontitis.: