Effects of curcumin on crevicular levels of IL-1 b and CCL28 in experimental gingivitis
SJ Pulikkotil,* S Nath †
*Senior Lecturer, Department of Restorative Dentistry, School of Dentistry, International Medical University, Kuala Lumpur, Malaysia.
†Senior Lecturer, Department of Periodontology, Vananchal Dental College and Hospital, Garhwa, Jharkhand, India.
ABSTRACT
Background: Curcumin has anti-inflammatory properties. The aim of this study was to compare interleukin-1b (IL-1b) and chemokine (C-C motif) ligand 28 (CCL28) levels following a topical application of curcumin (CRM), chlorhexidine (CHX) and chlorhexidine-metronidazole (CHX-MTZ) in an experimental gingivitis human model.
Methods:Sixty systemically healthy selected subjects were randomly assigned to one of three topical antigingivitis gels.
Each gel was applied twice daily for 10 minutes as the sole method of oral hygiene for 29 days on the test quadrant only. Modified gingival index (MGI), plaque index (PI), bleeding on probing (BOP) and probing depth (PD) were assessed at baseline, 29 days and 60 days. Estimation of IL-1band CCL28 levels in gingival crevicular fluid was done at baseline and at 29 days.
Results: The increase of IL-1b in the CRM (14.52 16.6 pg/ml) and CHX-MTZ (31.6315.96) groups was signifi- cantly less than that of the CHX group (70.55 38.81). Similar results were also observed for CCL28 (CRM:
8.128.78 pg/ml; CHX-MTZ: 12.81 18.68; CHX: 41.1522.82). All groups had a significant increase in MGI, PI and BOP at 29 days.
Conclusions: The anti-inflammatory potential of topical curcumin was similar to CHX-MTZ but superior to CHX in affecting IL-1band CCL28 levels.
Keywords: CCL28, curcumin/curcuma longa, gingivitis, inflammation, interleukin-1beta.
Abbreviations and acronyms:BOP = bleeding on probing; CHX = chlorhexidine; CHX-MTZ = chlorhexidine-metronidazole; CRM = curcumin; GCF = gingival crevicular fluid; MGI = modified gingivial index; PD = probing depth; PI = plaque index.
(Accepted for publication 17 August 2014.)
INTRODUCTION
Gingivitis is the most prevalent inflammatory periodon- tal disease, affecting more than 80% of the world’s population.1 Pathogenic biofilm around the teeth initi- ate the disease process. The host immuno-inflammatory response leads to pathogenesis and progression of disease from gingivitis to periodontitis in susceptible individuals.2 Excessive production of inflammatory cytokines, especially interleukins (IL) and tumour necrosis factor-a(TNF-a) have been implicated in peri- odontal destruction.2Mechanical plaque control is the most effective method for controlling gingivitis.3How- ever, the perception that the general population does not undertake adequate oral hygiene has led to a search for adjunctive agents to control the effects of biofilm.4 The addition of a chemical agent as an adjunct has been suggested to improve conventional methods of plaque control.4,5Chemical agents can act in two ways:
antimicrobials reduce the microbial burden and host modulation therapeutics modulate the host immuno- inflammatory response.6–8Although bacteria are essen- tial for periodontal disease, most tissue damage is caused by inflammatory mediators and free radicals.2,8 Current preventive and treatment approaches are only partially effective as they mainly focus on biofilm man- agement rather than controlling the host inflammatory mediators responsible for tissue damage.9The potential side effects seen in commonly used topical oral chemo- therapeutics include increasing failure, long-term use and drug resistance.10 There is a need to develop a more effective and efficient preventive and treatment approach for periodontal disease which includes a com- bined approach towards host modulation, inflamma- tion resolution, as well as direct management of bacteria.9 This has led to the screening of several medicinal plants which have equal efficacy and fewer side effects.11
doi: 10.1111/adj.12340
Turmeric has been used for the treatment of many diseases.12 Turmeric (also known as Curcuma longa) is a member of the ginger family, Zingaberaceae.12 This yellow spice contains the polyphenol curcumin in its rhizome. Extensive research on curcumin has demonstrated a wide spectrum of therapeutic effects such as anti-inflammatory, antioxidant, antibacterial, antiviral, antifungal, antitumour, antispasmodic, he- patoprotective and wound healing properties.13–15 The anti-inflammatory properties of curcumin can be useful in the treatment of inflammatory periodontal diseases.14 This effect is mediated through its ability to inhibit cyclooxygenase-2 (COX-2), lipoxygenase (LOX) and inducible nitric oxide synthase (iNOS).14,15 Another mechanism is by modulating the activity of signalling pathways and transcription factors, especially nuclear factor-jB (NF-jB), activat- ing protein-1 (AP-1) and mitogen-activated protein (MAP) kinases.15 In a recent study, curcumin effec- tively inhibited activation of NF-jB in the gingival tissues.14,16 Down-regulation of these pathways leads to suppression of proinflammatory cytokines such as IL-1b, IL-6, IL-12, TNF-a IL-8, matrix metallo- proteinase-2 and matrix metalloproteinase-9.15,16 Curcumin can target both bacteria and host inflam- matory cells and would be highly effective in pre- venting the destruction associated with periodontal diseases.
Gingival crevicular fluid (GCF) is an exudate whose constituents can be used to assess periodontal dis- ease.17 In our study we used two markers of inflam- matory status, IL-1b and a novel marker CCL28.
IL-1b is important in periodontal tissue destruction and its level is indicative of the activeness of dis- ease.18,19 A site specific increase in IL-1b has been observed in an experimental gingivitis model.20 The chemokine (C-C motif) ligand 28 (CCL28) is a novel marker for the diagnosis of gingivitis and periodonti- tis.21 Previous studies have shown CCL28 is associ- ated with increased epithelial inflammation.22–24 The production of IL-1b increases following contact with a microbial stimulus, which in turn significantly increases the expression of CCL28.21
To our knowledge, there are no studies evaluating the effectiveness of curcumin as a topical chemother- apeutic agent for periodontal disease. Curcumin has a combined anti-inflammatory, antioxidant and anti- bacterial effect.13 We hypothesized that curcumin will be superior in reducing the severity of gingival inflammation compared to popular commercially available topical antibacterials containing chlorhexi- dine and metronidazole. We are not aware of any studies to our knowledge that have assessed anti- inflammatory effects along with clinical changes. The aim of this study was to investigate the anti-inflam- matory effects of curcuma oral gel, chlorhexidine
gel, and a combination gel of chlorhexidine and met- ronidazole by assessing IL-1b and CCL28 levels from GCF and the clinical parameters during experimental gingivitis.
MATERIALS AND METHODS
This study was a randomized, double-blinded, con- trolled, parallel group clinical trial. It was conducted at Chhattisgarh Dental College and Research Institute, Rajnandgaon, India from May 2013 to September 2013. The clinical trial was approved by the Institu- tional Ethics Committee (CDEC/15/2013/01/CP) and registered in the Clinical Trials Registry, India (CTRI/
2013/12/004223). Voluntary informed consent was obtained from the subjects after providing them with detailed information about the trial. By obtaining informed consent and in the conduct of the study, we adhered to the principles of experimentation involving human subjects as outlined in the Declaration of Helsinki.
Sample size
The sample size was calculated for an alpha error of 0.05 and power at 90%. As there were no similar studies, we selected the deviation seen in CCL28 levels as reported by Ertugrul et al.,21 which was greater than in IL-1b levels reported in other stud- ies.18,20On the basis of this data, the sample size was calculated to 17 subjects to enable detection of a difference of 20 ng/ml. However, considering the pos- sibility of drop-out among subjects, the total number of patients was 20 for each group.
Participants
Sixty volunteers aged between 18 to 35 years were recruited. The subjects’ inclusion was based on the following criteria: (1) exhibit good periodontal health with no teeth having a probing depth >3 mm; (2) systemically healthy; (3) possessing at least 20 natural teeth; (4) negative for hypersensitivity to curcumin, chlorhexidine and/or metronidazole. Partic- ipants who used tobacco, who had recently used antibiotics, aspirin or non-steroidal anti-inflammatory drugs (NSAIDs) during the past one month, and pregnant and lactating mothers were excluded from this study.
Test products
Three types of commercially available chemotherapeu- tic preparations were tested in this clinical trial: (1) group I – gel containing curcumin (CRM) (Curenext Oral Gelâ, Abbott Health Care, Mumbai, MH,
India). Each gram contained curcuma longa extract (10 mg); (2) group II – gel containing chlorhexidine (CHX) (Hexigelâ, ICPA Health Products Ltd, Mum- bai, MH, India). Each gram contained chlorhexidine equivalent to chlorhexidine gluconate 1% w/w; (3) group III – gel containing chlorhexidine and metroni- dazole (Metrogyl DG gelâ, JB Chemicals & Pharma- ceuticals Ltd, Mumbai, MH, India). Each gram contained chlorhexidine gluconate solution IP 0.25%
w/w and metronidazole (10 mg).
To ensure blinding, all tubes containing the topical agents were similar and not labelled except for their lot numbers. Neither the examiner (SN) nor the recor- der (RPP) had access to the subject’s treatment code.
All topical agents were packaged in 20 g tubes.
Study design
This was a randomized, double-blinded, controlled, parallel clinical trial. The clinical trial was divided into pre-screening, treatment and post-treatment phases. Figure 1 describes the treatment study design.
Pre-screening phase
The recruited subjects underwent professional scaling and tooth polishing. The importance of maintaining oral hygiene was reinforced by the same operator (SJP). A quadrant for experimentation was randomly selected by lottery method.
A tooth shield was constructed only for the experi- mental quadrant by taking an alginate impression and pouring it in die stone to obtain a cast. A spacer of 0.5 mm was used over this cast. The tooth shield was made of thermoplastic mouthguard material of 1 mm thickness and then trimmed 2 mm beyond the gingival
margin. Its function was to induce experimental gingi- vitis on the selected treatment site and keep the gel in contact with the gingival margin of the experimental quadrant.
Treatment phase
Two weeks after scaling, participants returned for the recording of clinical data and the collection of GCF as baseline. The experimental quadrant was assessed for periodontal disease. Participants continued in the trial if they had a gingival index and plaque index score of less than 0.5 and absence of bleeding on probing. At baseline the participants were stratified into three groups and distributed randomly according to a computer generated code with 20 participants in each group: in the first group CRM gel; in the second group CHX gel; and in the third group CHX-MTZ gel was applied.
To induce experimental gingivitis, subjects were instructed to wear the provided tooth shield over the selected quadrant during their twice a day brushing and flossing routine for a period of 29 days. Partici- pants self-administered approximately 1 g of gel in the tooth shield before placing over the experimental quad- rant prior to their oral hygiene routine. The shield was left on the quadrant for 10 minutes so as to permit gin- gival contact of the experimental gel as per the manu- facturer’s instruction. The mouth was rinsed following removal of the shield. Participants were instructed not to consume any food or liquid at least one hour after gel application. Thus, the tooth shield prevented any mechanical removal of plaque during brushing and flossing for the purpose of inducing gingivitis in the selected area and allowing the experimental gels to remain in contact during this time.
Fig. 1 Treatment study design.
During the study period participants followed their usual dietary habits and were instructed to avoid any antibacterial rinses (e.g. CHX or phenolic compounds), therapeutic dentifrices (e.g. triclosan or stannous fluo- ride), irrigating devices, chewing gum and mints. All subjects were also given verbal and written instructions about the correct use of oral gel and oral hygiene meth- ods. They were told to report back in case of any use of antibiotics or anti-inflammatory drugs. Patients were recalled after 29 days. During this 29-day period, each patient was given a diary to record the time of application, duration and any adverse effects such as pain, itching, ulceration, discomfort, discolouration and taste disturbance. They were instructed to report back immediately in case of any side effects.
Post-treatment phase
Following the day 29 visit, GCF samples and clinical recordings were made for all participants in the study quadrant by the same examiner (SN). During this per- iod they reinstituted all home care procedures and received professional scaling, tooth polishing and oral hygiene instruction to re-establish periodontal health.
Subjects were recalled again at 35 and 60 days (post- treatment visit) to evaluate for signs of periodontal health. All clinical parameters were re-recorded at 60 days.
Each participant was given four 20 g tubes of the respective experimental gel. To check for compliance, participants were asked to return all used and unused/
partially used tubes after 29 days. This allowed us to measure the amount of gel used by the patients during the treatment period.
Outcome measurements
The single trained and calibrated examiner (SN) con- ducted all clinical examinations. The examiner (SN) and recorder (RPP) were blinded to the gels assigned to each subject.
The primary outcome was assessment of IL-1b and CCL28 levels from GCF at baseline and 29 days fol- lowing treatment for all participants. The secondary outcomes were Plaque Index (PI),25 Modified Gingival Index (MGI),26bleeding on probing (BOP) and probing depth (PD) recorded at baseline, 29 and 60 days on the selected quadrant. PI and MGI were evaluated after air drying the teeth and a score of 0 to 3 was assigned to the four gingival areas of teeth: distofacial, mesiofacial, facial and lingual surface for each tooth. The values of the four sites of each tooth were averaged to determine the score of PI and MGI. BOP was recorded as the overall percentage of sites with bleeding occurring within 15 seconds of probing. PD was measured from the free gingival margin to the base of the pocket at the
selected sites by using a UNC-15 periodontal probe (Hu-Friedy Inc.â, Chicago, Illinois, USA). In addition to this examination, the gingiva and surrounding soft tissue were visually inspected by the same examiner for the presence of any adverse reaction.
Subjective evaluations of participants were done after 29 days using a questionnaire. Subjects were instructed to answer either ‘yes’ or ‘no’ for pain, burning sensation, pruritis/itchiness, dryness of the mouth, taste disturbance, discolouration of teeth and bitter taste in the mouth. Patients were asked to rate the severity of the side effects as mild, moderate or severe if present.
Examiner calibration
All measurements were performed by one trained and calibrated periodontist (SN). The repeatability of the examiner for MGI, PI and PD scoring was determined prior to the commencement of the study. Ten subjects were clinically examined and then measurements were reassessed after 60 minutes from the initial examina- tion. The examiner’s repeatability of mean index scores was assessed using the intraclass correlation coefficient. The intraclass coefficients for the mean MGI, PI and PD were 0.89, 0.87 and 0.84
Collection of GCF
The first (baseline) GCF samples were obtained from five selected sites in the test quadrant prior to the first application of the experimental gel. The second GCF samples were collected after 29 days of gel application from the same selected sites. The collection was always done prior to assessment of the clinical param- eters. Before the collection of GCF, supragingival pla- que was carefully removed with gauze. The teeth were isolated with sterile gauzes and the areas around the gingival crevice were gently dried with an air syringe.
GCF samples were obtained from each participant by placing colour coded, 1–5 ll calibrated microcapillary pipettes (Sigma Aldrich Chemicals Company Ltd, St Louis, MO, USA) extracrevicularly. The sites for GCF collection were selected as follows: labial (mid) site of central incisor; mesiobuccal site of first premo- lar and second molar; and mid-buccal and lingual/pal- atal (mid) site of first molar. This ensured a fair representation of sites from the experimental quad- rant. Micropipettes were kept for not more than 5 minutes or until a volume of 1 ll was collected.
The fluid was immediately transferred to a plastic vial and frozen at–70 °C. All quadrants expressed at least 3 ll of GCF. The pooled volume was calculated for each quadrant. Pipettes in sites which did not express any volume of fluid after 5 minutes or those contami- nated with blood/saliva were discarded.
IL-1band CCL28 analysis in GCF
IL-1b and CCL28 levels in pooled GCF samples were measured using a commercially available enzyme- linked quantitative sandwich immunosorbent assay kit (Raybioâ, Human Elisa kits, RayBiotech, Inc., USA), specific for human IL-1b and CCL28. The results were reported as a concentration of IL-1band CCL28 in picogram per millilitre of sample. This assay employs an antibody specific for human IL-1b/CCL28 coated on a 96-well plate. Standards, controls and samples were pipetted into the wells and IL-1b/
CCL28 present in the sample were bound to the wells by the immobilized antibody. The wells were washed and biotinylated antihuman IL-1b/CCL28 antibody was added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin was pipetted to the wells. The wells were again washed, a TMB substrate solution added and the colour developed in proportion to the amount of IL-1b/CCL28 bound.
The Stop Solution changed the colour from blue to yellow, and the intensity of the colour was measured at 450 nm.
Statistical analysis
Data analysis was carried out using SPSS software (Version 16.0, SPSSâ Inc., Chicago, IL, USA). The values of the different parameters are expressed as mean standard deviation. Paired t-tests were used for intragroup comparison. For intergroup comparisons, one-way ANOVA followed by post hoc Tukey’s test was carried out. The mean changes from baseline to 29 days were calculated for all the parameters, and changes from baseline to 60 days were calculated only for the clinical parameter. In each case the level of significance was set at p <0.05. The power of the study was calculated to be of 92%.
RESULTS
Characteristics of the study population
Table 1 summarizes the characteristics of the study population. Four participants did not follow-up in
recall visits and were excluded from the analysis, therefore a total of 56 participants completed the study with 19 subjects in group I, 18 subjects in group II and 19 subjects in group III (Fig. 2). Two sub- jects failed to follow-up for the final visit due to change of residence and the other two withdrew for personal reasons. The mean age was 21.95 3.34, 25.85 5.83 and 20.70 3.38 for groups I, II and III respectively. There were 7 males in groups I and III, and 11 males in group II. There was no significant difference among the treatment groups with respect to age and gender. The GCF volumes increased over the 29-day period with no significant difference in any group. Assessment of gel tube weights indicated no significant difference among groups for the mean quantity of gel used. Examination of soft tissues after 29 days and 60 days revealed no remarkable adverse findings to any of the test products. Curcumin gel application caused a mild yellowish discolouration of the teeth after 29 days, which returned to normal after professional scaling. No other adverse events were reported by any of the participants during the study.
Clinical parameters
In the intragroup comparison (Table 2), there was an increase in mean difference of MGI, PI and BI individ- ually for all three groups from baseline to 29 days, and the difference was significant (p < 0.001). After 60 days there was a gradual decrease in all three parameters and no significant difference from baseline to 60 days in any of the groups. In the case of PD, all values remained similar except for group II (p= 0.01) at 29 days. In the intergroup comparison (Table 3), all groups were similar at baseline (MGI: p= 0.08;
PI: p= 0.48; PD: p = 0.49) and at 60 days (MGI:
p = 0.29; PI: p= 0.43), showing non-significant mean differences for all clinical parameters except for PD at 60 days (p = 0.05). The mean difference from base- line to 29 days was significant for MGI, PI and BI for all three treatment groups (p< 0.001). CRM and CHX-MTZ were similar in MGI and PI scores, and significantly lower than the CHX group at 29 days
Table 1. Characteristics of the study participants
I II III P-value*
Number of subject 19 19 18
Age (meanSD) Age (range)
21.954.68 18–27
20.604.53 17–29
23.254.82 18–36
0.10
Mean volume of GCF at baseline (ll) 3.420.62 3.240.88 3.580.93 0.19
Mean volume of GCF at 29 days (ll) 3.820.89 3.930.99 3.860.75 0.17
Total mean gel mass administered (g) 56.465.97 54.896.75 57.536.93 0.13
I=curcumin group; II=chlorhexidine group; III=chlorhexidine-metronidazole group; GCF=gingival crevicular fluid; SD=standard deviation.
*ANOVAp-value was calculated.
when compared individually. BOP was significantly less in the CRM group when individually compared to the other two groups.
Cytokine levels
Table 4 summarizes the intragroup and Table 5 the intergroup comparison of cytokine levels from base- line to 29 days. There was an increase in IL-1b levels from baseline to 29 days for all three groups and the intra mean difference was significant (I: 14.52 16.0, p =0.002; II: 70.55 38.81 p< 0.001; III: 31.63 15.96, <0.001). A similar increase in CCL28 was seen with a significant difference for groups II and III only (I: 8.12 18.78, p= 0.10; II: 41.1522.82, p <0.001; III: 12.81 18.68, p = 0.02) from base- line. An intergroup comparison of IL-1b and CCL28 levels found all groups were similar at baseline (IL-1b:
p =0.92, CCL28: p = 0.56) but showed significant changes after 29 days (p < 0.001) (Table 5). No dif- ference was seen between group I and III (IL-1b:
p =0.18; CCL28: p= 0.80), whereas significant difference was seen when group II was compared with group I (IL-1b: p < 0.001; CCL28: p< 0.001) and III (IL-1b: p <0.001; CCL28: p <0.001) at 29-day levels.
DISCUSSION
The purpose of this randomized, double-blinded, con- trolled clinical trial was to assess the effect of curcu- min on IL-1b and CCL28 levels in GCF in a human experimental gingivitis model. Gingivitis is inflamma- tion of the gingival tissues in response to the presence of dental plaque. Regular removal of plaque accumu- lation can prevent as well as resolve gingivitis.27 Mechanical plaque removal is the superior treatment for gingival inflammation while topical application of chemotherapeutic agents has an adjunct role in reduc- ing the severity of the disease.27 Hence in our study we expected a reduction in the severity of gingival inflammation rather than a complete prevention or resolution of gingivitis through the use of any topical antigingivitis products. Our results also showed that use of the three experimental gels did not prevent the onset or development of experimental gingivitis when evaluated clinically. However, there was a difference in the cytokine levels assessed which will be discussed further.
Two popular topical antibacterials, CHX and CHX-MTZ combination gels, were selected for com- parison. CHX is a broad spectrum antiseptic having both bactericidal and fungicidal action against Gram- Enrolment
Allocation
Follow-up
Analysis
Analysed (n =19) Analysed (n =19)
Assessed for eligibility (n = 75)
Excluded (n = 15)
Not meeting inclusion criteria (n = 8) Declined to participate (n = 4) Other reasons (n = 3)
Randomized (n = 60)
Allocated to curcumin gel group intervention (n = 20)
Lost to follow-up (due to change of residence) (n = 1)
Lost to follow-up (due to personal reason) (n = 1)
Lost to follow-up (1 subject changed residence, other withdrew due to
personal reason) (n = 2) Allocated to chlorhexidine gel group
(n = 20)
Allocated to chlorhexidine-metronidazole gel group
(n = 20) Received allocated intervention (n = 20) Received allocated intervention (n = 20)
Analysed (n =18) Fig. 2 CONSORT studyflow chart of various phases of the clinical trial.
positive and Gram-negative strains and is considered the gold standard for chemical plaque control.4–6 CHX has been recognized by the pharmaceutical industry as the positive control for evaluating the effi- cacy of alternative antiplaque agents.28 MTZ is effec- tive against Gram-positive and Gram-negative obligate anaerobe.6,10 CHX and MTZ have demon- strated anti-inflammatory properties although the improvement in gingivitis is due to their antibiofilm properties.29 The anti-inflammatory action of CHX and MTZ is mainly due to their inhibitory action on oral biofilm. However, the use of topical chemothera- peutics has certain disadvantages. Side effects of CHX are brown discolouration of teeth, taste perturbation, oral mucosal ulceration, paraesthesia, parotid swelling and enhanced supragingival calculus formation,10 while MTZ can cause resistance against antibiotics.6 Due to the drawbacks of the synthetic agents, the rel- atively safe nature of herbal extracts has led to their use in various fields as an alternative.11
Curcumin is a naturally occurring phytochemical known to have antibacterial and anti-inflammatory properties.13,15It is a major constituent of the popular Indian yellow spice turmeric derived from the rhi- zomes of Curcuma spp. Curcumin has a number of applications. It has been shown to prevent cancer in the oral cavity, colon, skin, lung, liver, duodenum and breasts.13,14 It is effective against gastric ulcers, arthritis, inflammatory bowel disease and Crohn’s dis- ease.13,14 Curcumin has the potential to treat inflam- matory disease, especially periodontal disease.14,30–32
In our study we chose an experimental gingivitis model of 29 days similar to previous studies to com- pare the efficacy of three commercially available anti- gingivitis topical agents.33,34 The original model proposed by Loe et al.35 was of 21 days but we pre- ferred the 29-day model because there is heightened host inflammatory mediator response to plaque build- up at this time point.33,34 Instead of the full mouth model, we restricted it to the selected quadrant to increase patient compliance and reliability, similar to Paquetteet al.34and Saxton and van der Ouderaa.36In this model, gingival health is first established followed by abandonment of all oral hygiene procedures for a period of 29 days to induce gingivitis. According to Deinzeret al.,37the experimental gingivitis model may not be identical to chronic gingivitis in a natural setting but it is preferable to study the anti-inflammatory effects of topical gel in a well-controlled environment.
The three groups of subjects were similar in age, male–female ratio, average volume of GCF at baseline and at 29 days. Researchers have shown an increase in GCF volume after 21 days of no oral hygiene, but no such change was seen in our study.38 The interven- tions might have controlled an increase. Smokers were excluded from this study due to the depressive effects Table2.Intragroupcomparisonandmeanchangefrombaselineofclinicalparameteratdifferenttimepointsforallgroups ParameterGroupBaseline29days60daysBaseline–29daysBaseline–60days DifferencePvalueDifferencePvalue MGII II III
0.410.20 0.310.13 0.290.18 0.860.27 1.590.49 1.010.31 0.360.34 0.390.18 0.260.24 0.450.31 1.290.55 0.680.38
<0.001* <0.001* <0.001*
(-)0.050.32 0.090.25 (-)0.030.18
0.50 0.16 0.48 PII II III
0.110.11 0.130.13 0.150.11 0.520.14 0.980.29 0.530.17 0.230.23 0.140.17 0.180.13 0.420.16 0.850.37 0.360.21
<0.001* <0.001* <0.001**
0.120.27 0.010.12 0.020.10
0.05*0.70 0.30 BOPI II III
0.00.0 0.00.0 0.00.0 0.260.19 0.680.15 0.500.13 0.00.0 0.00.0 0.00.0 0.260.19 0.680.15 0.500.13
<0.001* <0.001* <0.001*
0.00.0 0.00.0 0.00.0 PDI II III
2.090.48 2.090.39 2.260.57 2.380.53 2.310.49 2.420.54 2.230.52 2.030.31 2.420.54 0.290.67 0.220.32 0.160.74
0.07 0.01* 0.37
0.140.67 (-)0.060.25 0.160.74 0.37 0.28 0.34 *Statisticallysignificant.I=curcumingroup;II=chlorhexidinegroup;III=chlorhexidine-metronidazolegroup;MGI=modifiedgingivalindex;PI=plaqueindex;BOP=bleedingonprob- ing;PD=probingdepth.
of tobacco on gingival inflammation.34 In our study we did not use any negative or placebo controls as there is evidence that experimental gingivitis would lead to gingival inflammation and an increase in inflammatory markers.33 There is also a possibility of
cross-over contamination of the intervention agent on the contralateral side if used as a control as in split mouth design.39 An adequate amount of gel was used in all groups and usage was not dissimilar. We used a tooth shield so that the experimental agent would Table 3. Intergroup comparison of clinical parameter at various follow-up visits from baseline (p-values only)
Time points Parameter I/II/III‡ I/II I/III II/III
Baseline MGI 0.08
PI 0.48
BOP 1
PD 0.49
29 days MGI <0.001* <0.001* 0.43 <0.001*
PI <0.001* <0.001* 0.99 <0.001*
BOP <0.001* <0.001* 0.003* <0.001*
PD 0.71 NC NC NC
60 days MGI 0.29
PI 0.43
BOP 1
PD 0.05
Difference Baseline–29 days MGI <0.001* <0.001* 0.13 <0.001*
PI <0.001* <0.001* 0.85 <0.001*
BOP <0.001* <0.001* 0.007* <0.001*
PD 0.81 NC NC NC
Baseline–60 days MGI 0.23
PI 0.15
BOP 1
PD 0.47
Onlyp-values are given here as the individual groups’ MGI, PI, BOP and PD values are given in Table 2.
‡To compare groups I, II and III, ANOVAp-value was calculated. Pairwise comparison and p-value was done only when comparison by ANOVA was significant.
*Statistically significant.
I = curcumin group; II=chlorhexidine group; III=chlorhexidine-metronidazole group; MGI=modified gingival index; PI=plaque index;
BOP=bleeding on probing; PD=probing depth; NC=not calculable as all values were similar.
Table 4. Intragroup comparison of cytokines levels from baseline to 29 days
Cytokine Group Baseline 29 days Difference P-value
IL-1b I
II III
24.716.84 24.937.27 25.646.20
39.2715.71 95.5441.67 57.2415.42
14.5216.0 70.5538.81 31.6315.96
0.002*
<0.001*
<0.001*
CCL28 I
II III
76.489.65 81.6415.26 78.1316.23
84.5916.94 122.7819.30 90.9 415.90
8.1218.78 41.1522.82 12.8118.68
0.10
<0.001*
0.02*
*Statistically significant.
I=curcumin group; II=chlorhexidine group; III=chlorhexidine-metronidazole group.
Table 5. Intergroup group comparison of cytokine levels from baseline to 29 days (p-values only)
Time points Parameter I/II/III‡ I/II I/III II/III
Baseline IL-1b 0.92
CCL28 0.56
29 days IL-1b <0.001* <0.001* 0.17 <0.001*
CCL28 <0.001* <0.001* 0.57 <0.001*
Difference Baseline–29 IL-1b <0.001* <0.001* 0.18 <0.001*
CCL28 <0.001* <0.001* 0.80 <0.001*
Onlyp-values are given here as the individual groups’ cytokine levels are given in Table 4.
‡To compare groups I, II and III, ANOVAp-value was calculated. Pairwise comparison and p-value was done only when comparison by ANOVA was significant.
*Statistically significant.
I=curcumin group; II=chlorhexidine group; III=chlorhexidine-metronidazole group.
come into contact with the gingiva undisturbed for 10 minutes as per the manufacturers’ instructions for the individual gels. We could not locate any other studies using a tooth shield for application of test gels during initiation of experimental gingivitis. No adverse effects including pain, hypersensitivity reac- tions or ulceration were observed in or reported by the participants. Teeth staining was observed in the CRM group at 29 days, but this cleared after resum- ing mechanical plaque control.
Plaque, BOP and gingival inflammation scores increased progressively from baseline to 29 days, simi- lar to other studies with or without the use of topical agents in experimental gingivitis.33,35,40 Refraining from mechanical plaque control resulted in increased plaque accumulation, leading to gingival inflammation.
This confirmed the superiority and effectiveness of complete mechanical plaque removal. All three experi- mental gels could not prevent development of gingivitis as well as lead to any resolution of established gingivi- tis. However at 29 days, the CRM group was more effective in reducing the severity of clinical signs of experimental gingivitis. Inflammatory signs of BOP scored significantly less in the CRM than the other two groups. MGI was similar in the CRM and CHX-MTZ group, while it was significantly higher for the CHX group. This is reflected in the high plaque scores for CHX. Thus at 29 days, we found that CRM and CHX-MTZ were more efffective in controlling plaque and gingival inflammation compared to the gold stan- dard CHX. Pradeepet al.10also reported the effective- ness of the CHX-MTZ combination gel in the control of gingivitis compared to the individual agents. Similar to our result, Waghmare et al.30 and Suhag et al.31 demonstrated the superior anti-inflammatory proper- ties of curcumin mouthwash compared to CHX. CHX and MTZ have well-characterized antimicrobial effects and potential anti-inflammatory properties.29 The antiplaque effect of CHX-MTZ may be explained by CHX properties acting on the cell walls of the microor- ganism and changing their surface structures, whereas the cytototoxic metabolites of MTZ directly interact with bacterial DNA, resulting in cell death.10,29 All clinical scores at 60 days were similar to baseline lev- els, which again reiterates the excellent results achieved with good mechanical plaque control.
GCF is a transudate that accumulates bacterial and host metabolic products. Therefore, it is an ideal diag- nostic tool for analysing the inflammatory mediator.
To assess the anti-inflammatory effect of the test product, we used two markers: IL-1b and CCL28.
IL-b is a reliable marker associated with the presence and severity of plaque induced gingival inflamma- tion.41,42 IL-1b is associated with bone resorption exhibiting increased levels in diseased rather than healthy sites.41,42 Studies have shown that CCL28
levels were higher in samples from a group of subjects with inflammation compared to a healthy control group.22,23 Ertugrul et al.21 found a significant increase (1.5 times) of IL-1b and CCL28 levels in GCF in naturally occurring gingivitis subjects com- pared to periodontal healthy subjects.
With the sustained biofilm challenge for 29 days, it was observed that all three groups showed a signifi- cant increase in GCF IL-1b levels over their respective baseline values. The increase was reflective of the induced gingival inflammation due to the accumula- tion of plaque. A similar relationship between GCF cytokines and clinical gingival inflammation has been reported previously.38,42 The application of any topi- cal gel was not effective in reducing IL-1b levels to baseline levels in our study. At 29 days, IL-1b levels in GCF were significantly lower in the CRM and CHX-MTZ groups compared to the CHX group.
Groups using CRM gel and CHX-MTZ gel showed reduced IL-1b levels during the development of exper- imental gingivitis compared to CHX gel. This again corroborates our clinical findings. Similar results were observed with respect to CCL28 wherein the CRM and CHX-MTZ gel were similar in having signifi- cantly lower levels compared to CHX. In addition, we observed that curcumin prevented any significant increase of CCL28 levels over baseline at 29 days.
Statistically better results obtained by the topical application of curcumin can be attributed to its anti- inflammatory, antioxidant and antibacterial properties, which might have resolved inflammation in an earlier stage, reflected by lower cytokine levels and reduced clinical inflammation when compared to CHX. Curcu- min has been shown to be effective when evaluated as a treatment for periodontal disease.14,30–32 Curcumin acts similarly to aspirin and aspirin-like anti-inflamma- tory drugs in diminishing inflammatory mediators of arachidonic acid metabolism, thus inhibiting prosta- glandins and leukotreines.15 Curcumin use reduces inflammatory mediator levels of IL-6, IL-1b, TNF-a, MMP-2 and MMP-9 by modulation of signalling path- ways and transcription factors, especially NF-jB, AP-1 and MAPKinase.14,15 Curcumin causes shrinkage of tissues by reducing inflammatory oedema and vascular engorgement of connective tissues.43 It promotes migration of fibroblasts in the wound bed and enhances wound healing by an increase in fibronectin and transforming growth factor btranscription.43 The antioxidant activity of curcumin is comparable to vita- mins C and E.13 Rao et al.44 demonstrated the scav- enging effect of curcumin on superoxide radicals, hydroxyl radicals and lipid per oxidation. Curcumin suppressed the growth of Prevotella intermedia, a major periodontal pathogen.16 Therefore, due to the diverse range of actions, curcumin treated sites showed faster resolution of inflammatory signs.
Curcumin is a naturally occurring molecule and a major limiting factor is its low solubility in water (i.e.
0.0004 mg/ml at pH 7.3) and low bioavailability.
This has been attributed to its poor absorption, high rate of metabolism and rapid elimination from the body.45 A conventional oral formulation of curcumin resulted in a very low availability of curcumin in the blood circulation to achieve therapeutic effects.46 Various types of nanoparticle (NPs), such as polymer NPs, polymeric micelles, liposome/phospholipid, nano-/microemulsions, nanogels, solid lipid NPs, poly- mer conjugates, self-assemblies, are suitable for the delivery of an active form of curcumin.46 Shahani et al.47showed that curcumin formulation in the form of microparticles had a sustained release for nearly a month. Nanoformulations of curcumin vastly enhance aqueous solubility and thus bioavailability in animals as well as humans, evidenced by a higher release pro- file of nanocurcumin over curcumin suspension.45 Toxicity of curcumin was reduced by nanoformula- tion while increasing therapeutic efficacy.48
Some limitations were identified in our study.
Even though compliance was checked, it is difficult to tell if the at-home products were used correctly.
A cross-over design with a suitable control for cross- contamination would eliminate any bias of the variable host response. The absence of a negative con- trol did not allow us to measure the magnitude of change for the individual groups.
CONCLUSIONS
CRM and CHX-MTZ topical application were supe- rior to CHX in affecting the development of gingival inflammation in human experimental gingivitis. CRM and CHX-MTZ were similar in clinical inflammation and cytokine levels of IL-1b and CCL28. However, a mild superiority can be claimed for curcumin over CHX-MTZ. CRM prevented any increase of CCL28 levels as well as having the least BOP scores. The evi- dence from this study suggests that curcumin has potential in the treatment of inflammatory periodontal disease as an adjunct to good mechanical plaque con- trol. Improved formulations employing recent advances in nanotechnology would help realize the true antibacterial and anti-inflammatory potential of curcumin. Topical, sustained and systemic delivery formulations of curcumin need to be explored. Fur- ther studies are required to ascertain the effect of curcumin on periodontitis patients.
ACKNOWLEDGEMENTS
The materials and facilities used in the study were funded by Abbott Healthcare Pvt. Ltd, Mumbai, MH, India. The authors would like to thank Mr Manoj Naik
(Abbott India), Mr Manoj Prabhu (Abbott India), Mr Tushar Fegade (Abbott India), Dr RM Zade and Dr Ritu Prabha Patel for their support during the study.
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Address for correspondence:
Dr Shaju J Pulikkotil International Medical University School of Dentistry Kuala Lumpur 57000 Malaysia Email: [email protected]
