|Year : 2014 | Volume
| Issue : 4 | Page : 456-460
Effect of periodontal therapy on C-reactive protein levels in gingival crevicular fluid of patients with gingivitis and chronic periodontitis: A clinical and biochemical study
Deepika Jayaprakash1, Suchetha Aghanashini2, Ranganatha Rao Vijayendra2, Anirban Chatterjee1, Radhika Mohan Rosh1, Ashit Bharwani3
1 Department of Periodontics, The Oxford Dental College and Hospital, Bengaluru, Karnataka, India
2 Department of Periodontics, DAPMRV Dental College and Hospital, Bengaluru, Karnataka, India
3 Department of Periodontics, K.M. Shah Dental College, Vadodara, Gujarat, India
|Date of Submission||20-Aug-2013|
|Date of Acceptance||27-Jan-2014|
|Date of Web Publication||14-Aug-2014|
#150, First Floor, 15th Main, 35th Cross, Jayanagar, 4th T Block, Bengaluru 560 041, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background and Objectives: C-reactive protein (CRP) is a type I acute phase reactant. A number of studies have reported elevated serum CRP levels in periodontitis subjects, which decrease following periodontal therapy. However, the data of interventional studies on gingival crevicular fluid (GCF) levels of CRP is very scarce. The aim of the present study was to evaluate the effect of periodontal therapy on GCF CRP levels in patients with gingivitis and chronic periodontitis. Materials and Methods: A total of 60 subjects were included in the study with 20 subjects each in following groups: I-Healthy, II-Gingivitis, III-Mild periodontitis based on community periodontal index scores. Periodontal therapy was performed on Group II and Group III patients. GCF was collected from each subject at baseline and 3 months after periodontal therapy. The collected sample was subjected to biochemical analysis to detect CRP levels by using commercially available chemiluminescence immunoassay kit. Results: The present study demonstrated that the periodontitis group had a higher mean CRP level (2.49 ± 0.47 ng/ml) when compared with the Gingivitis group (1.40 ± 0.32 ng/ml) and Healthy group (0.56 ± 0.20 ng/ml). The mean CRP values after periodontal therapy were found to be reduced to 0.44 ± 0.23 ng/ml in Group II and 1.30 ± 0.36 ng/ml in Group III patients. Interpretation and Conclusion: Within the limitations of this study, it can be concluded that GCF CRP level progressively increases from periodontal health to disease. It can also be stated that there is a decrease in GCF CRP levels with periodontal treatment.
Keywords: Chronic periodontitis, C-reactive protein, enzyme immunoassay, gingival crevicular fluid, gingivitis
|How to cite this article:|
Jayaprakash D, Aghanashini S, Vijayendra RR, Chatterjee A, Rosh RM, Bharwani A. Effect of periodontal therapy on C-reactive protein levels in gingival crevicular fluid of patients with gingivitis and chronic periodontitis: A clinical and biochemical study. J Indian Soc Periodontol 2014;18:456-60
|How to cite this URL:|
Jayaprakash D, Aghanashini S, Vijayendra RR, Chatterjee A, Rosh RM, Bharwani A. Effect of periodontal therapy on C-reactive protein levels in gingival crevicular fluid of patients with gingivitis and chronic periodontitis: A clinical and biochemical study. J Indian Soc Periodontol [serial online] 2014 [cited 2019 Dec 10];18:456-60. Available from: http://www.jisponline.com/text.asp?2014/18/4/456/138688
| Introduction|| |
C-reactive protein (CRP) is an acute phase reactant plasma protein produced in response to diverse inflammatory stimuli.  The acute-phase reaction represents an early, non-specific and highly complex reaction of the organism to a variety of injuries such as bacterial, viral or parasitic infection and ischemic necrosis.  Amongst all the acute phase reactants, CRP in particular has been the focus of attention as a key marker of atherosclerosis.  Since the levels of CRP rise earlier than those of other reactants, CRP has been used as an early marker of tissue damage. , CRP is predominantly synthesized by liver hepatocytes and was recognized due to its ability to precipitate with the C-polysaccharide extract of Streptococcus pneumoniae. CRP in plasma is normally present <0.3 mg/dl but may increase dramatically to hundreds of micrograms per milliliter within 72 h of tissue injury. The amount decreases with the subsidence of the disease process and the recovery of the patient.  CRP has been detected in the serum of periodontitis patients and levels were significantly higher than those of non-periodontitis subjects.  Furthermore, there is mounting evidence that effective periodontal therapy can lower serum CRP levels.  A more accurate and non-invasive site for the evaluation of inflammatory biomarkers in subjects with periodontal disease is gingival crevicular fluid (GCF), since it reflects the ongoing events in the periodontal tissues that produce it.  Very few studies regarding CRP levels in GCF of gingivitis patients and effect of periodontal therapy on CRP levels have been reported until date. Hence, this study was undertaken to evaluate the effect of periodontal treatment on CRP levels in GCF in patients with gingivitis and chronic periodontitis.
| Materials and methods|| |
The study was designed as a single-center, longitudinal and interventional study. The study duration was 4 months, in which GCF was collected at baseline and 3 months after periodontal therapy. The research protocol was initially submitted to the Ethical Committee of DAPM R V Dental College, Bangalore, India and the study was conducted in accordance with the Helsinki Declaration of 1975, as revised in 2008. After ethical approval, subjects were selected from the outpatient section of Department of Periodontics, DAPM R V Dental College, Bangalore. All the selected subjects were informed about the nature of the study and signed an informed consent. Duration of the study was from December 2009 to April 2010.
Group sample size was decided by power analysis with 85% power. A total of 60 dentate and systemically healthy subjects (30 males and 30 females) were recruited for the study. Subjects participating in the study were 21-45 years of age.
Age and gender matched subjects without a history of periodontal therapy or previous use of antibiotics or anti-inflammatory medication within the preceding 6 months were included in the study. For Group II, subjects diagnosed with gingivitis, showing clinical signs of inflammation without loss of attachment and with bleeding on probing were selected. For Group III, subjects diagnosed with mild chronic periodontitis, with a community periodontal index (CPI) score of 3 were selected.
Subjects with smoking habit, coronary artery disease, hypertension, metabolic disorders such as diabetes mellitus and with severe infections and other inflammatory conditions like rheumatoid arthritis were excluded.
The subjects were assigned to one of the three groups (20 subjects in each group) based on CPI scores: Group-I (Control): Subjects with clinically healthy periodontium (CPI score of 0), Group-II (Gingivitis group): Subjects with gingivitis (CPI score of 1 or 2) and Group-III (Periodontitis group): Subjects with periodontitis (CPI score of 3). GCF was collected from all the selected subjects at baseline. Group-II subjects underwent periodontal therapy in the form of scaling and Group-III subjects underwent periodontal therapy in the form of scaling and root planning. 3 months after periodontal therapy, GCF was collected from all the subjects. Scaling was done in Group-II subjects using a Universal scaler U15/30, whereas Group-III subjects underwent scaling and root planing using a Universal scaler U15/30 and a Universal curette.
Collection of GCF
The site for collection of GCF was identified during clinical examination based on the highest CPI score in the subject. The crevicular site was dried and isolated with cotton roll and sample of GCF was obtained by placing white color-coded 1-5 μl volumetric and calibrated microcapillary pipettes [Figure 1]. From each test site, a standardized volume was collected using the calibration marks on the micropipettes and by placing the tip of pipette extracrevicularly. The test site that did not express any volume of GCF and micropipettes which were contaminated with blood or saliva were excluded from the study. The collected GCF was immediately transferred into plastic vials containing phosphate buffered saline and the samples were frozed at − 70°C until they were assayed by chemiluminescence immunoassay (CLIA) for CRP levels (sensitivity of the assay - 0.2 μg/ml).
|Figure 1: Collection of gingival crevicular fluid in gingivitis case (pre treatment)|
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The high-sensitivity C-reactive protein (hsCRP) CLIA is based on the principle of a solid phase two-site immunoassay. The assay system utilizes a unique monoclonal antibody directed against a distinct antigenic determinant on the CRP molecule. This mouse monoclonal anti-CRP antibody is used for solid phase immobilization (on the micro titer wells). Another anti-CRP antibody is in the antibody-enzyme (horseradish peroxidase) conjugate solution.
The CRP molecules present in the standard solution or serum are "sandwiched" between the two antibodies. Following the formation of the coated antibody-antigen-antibody-enzyme complex, the unbound antibody-enzyme labels are removed by washing. The horseradish peroxidase activity bound in the wells is then assayed by adding the substrate reagents (4-iodopherol and hydrogen peroxide) and undergoing the chemiluminescent reactions. The intensity of the emitted light from the associated well is proportional to the amount of enzyme present and is directly related to the amount of CRP antigen in the sample. By reference to a series of CRP standards assayed in the same way, the concentration of CRP in the unknown sample is quantified.
The statistical analysis was carried out using analysis of variance, fisher exact test, paired proportion test and Student's t-test.
| Results|| |
The study was carried out on a total of 60 systemically healthy subjects with normal gingiva (Group-I), gingivitis (Group-II) and periodontitis (Group-III). All the subjects finished the study; there were no drop-outs. CRP was assessed in all the subjects before therapy and in Groups II and III after periodontal therapy. The subjects included in the study had a mean age of 38.05 ± 10.65 years.
The selected subjects were divided into above mentioned three groups based on the CPI score. The mean CPI score was found to be 0 in Group-I, 1.45 ± 0.51 in Group II and 3.00 ± 0.00 in Group III before periodontal therapy [Table 1].
|Table 1: CPI score in sample population and their percentage before periodontal therapy using paired proportion test |
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The mean CRP values at baseline were found to be 0.56 ± 0.20 ng/ml in Group I; 1.40 ± 0.32 ng/ml in Group II and 2.49 ± 0.47 ng/ml in Group III patients. The values reduced to 0.44 ± 0.23 ng/ml in Group II and 1.30 ± 0.36 ng/ml in Group III patients post-therapy.
Inter-group comparison of baseline CRP values among the three groups using ANOVA test revealed a statistical significant difference (P < 0.001). CRP levels were significantly higher in Group-II and Group-III when compared with control group [Table 2].
|Table 2: Mean GCF CRP values (ng/ml) before therapy using analysis of variance |
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Fisher Exact test revealed a significant difference (P < 0.001) in CRP values among the three groups: 0.84 between Group I and II; 1.93 between Groups I and III and 1.09 between Groups II and III [Table 3].
|Table 3: Fisher exact test for comparison of GCF CRP values (ng/ml) before treatment |
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Comparison of pre-treatment and post-treatment CRP levels was carried out using a Student's t-test. It was observed that there was a significant decrease (P < 0.001) in CRP values post-therapy in both Group-II (1.40 ± 0.32-0.44 ± 0.23) and Group-III (2.49 ± 0.47-1.30 ± 0.36). An additional observation made was that the post-therapy values reached normal levels in Group-II but remained marginally higher than Group-II in Group-III [Table 4] and [Figure 2].
|Table 4: Evaluation of CRP values (ng/ml) pre- and post-therapy using student's t test |
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|Figure 2: Graph comparing C-reactive protein levels between Group II and III before and after periodontal therapy|
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The CPI scores were observed to be reduced after periodontal therapy in both Group II and Group III. In Group II, 0% subjects were observed with score 2, 30% with score 1 and 70% with score 0. In Group III, 0% subjects were observed with score 3, 5% with score 2, 12% with score 1 and 3% with score 0 [Table 5].
|Table 5: Evaluation of CPI, before and after treatment (using paired proportion test) |
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| Discussion|| |
While most studies of periodontitis have emphasized the local nature of periodontitis, , it appears that systemic manifestations of this disease are also detected through the production of CRP and other acute-phase proteins and pro-coagulant mediators.  As a response to the presence of bacteria and bacterial products, such as lipopolysaccharides, cell-mediated inflammation is triggered and a number of proinflammatory cytokines (tumor necrosis factor [TNF], interleukin [IL]-1 and IL-8) are synthesized. Systemic inflammation primed by periodontal infection and the release of lipopolysaccharides into the periphery activates both inflammatory cells and endothelial cells and cytokines are carried to the liver where they induce the production of acute-phase proteins such as CRP. The reason for the interest in plasma/serum levels of CRP in periodontitis is due to the epidemiological research indicating association of periodontitis with CVD  and that it is an exceptionally stable analyte in plasma and immunoassays for it are robust, well standardized, reproducible and readily available. 
GCF is proved to be a more specific sample for analysis of periodontal disease activity. GCF can be easily and non-invasively collected and contains products of the host, the plaque and their interactions. Analysis of GCF in our study showed elevated CRP levels in gingivitis and periodontitis patients when compared with control group.
The elevated levels of serum CRP in gingivitis and periodontitis patients have been observed in numerous large scale cross-sectional studies, ,,,,,, the third National Health and Nutrition Examination Survey longitudinal study , and meta-analysis by Paraskevas et al.
Similar studies in GCF are scarce. The elevated levels of GCF CRP found in Group II and Group III of our study is in accordance to a study by Pradeep et al. in 2010 which showed that the levels increased proportionately with the increase in severity of the disease and positively correlated with the clinical parameters.  The findings of our study are also in accordance to an extensive literature review conducted by Pradeep et al. in 2012 which demonstrated sufficiently large quantities of hsCRP levels in GCF of periodontitis patients. 
The periodontal literature has documented different time intervals for reevaluation after periodontal therapy. These time intervals range from 2 weeks to 6 months.  Based on the rate of healing, it has been reported that 3 months post treatment is a suitable interval for the primary evaluation of initial non-surgical therapy, even in areas with preliminary deep lesions. Beyond this time interval, the subgingival microbial repopulation occurs in the absence of improved plaque control. 
Recent studies have investigated the potential role of periodontal therapy on systemic inflammation, but the results are somewhat conflicting.  Patients treated by non-surgical mechanical periodontal therapy showed a significant increase in plasma CRP, TNF-α and IL-6 levels immediately after the intervention, indicating a systemic acute-phase response, apparently due to a massive bacterial inoculation in conjunction with instrumentation  followed by a steady decrease. The meta-analysis conducted by Ioannidou et al.  did not show any statistically significant reduction in serum CRP following periodontal therapy whereas systematic review by Paraskevas et al.  showed modest evidence of lowered serum CRP.
There are very few studies which correlate periodontal disease and GCF CRP levels and even fewer which study the effect of intervention on the CRP levels. Studies by Pradeep et al. have shown increased GCF levels of CRP in obese and non-obese patients with chronic periodontitis.  Another study by the same group of authors showed a decrease in both serum and GCF CRP levels following short term non-surgical periodontal therapy in type 2 diabetes mellitus patients. 
Also recent research has reported local production of CRP in gingiva itself, which could justify the increased levels of GCF CRP levels in cases of inflammation of the periodontal tissues. 
Megson et al. have deduced that the CRP detected in the GCF is of systemic origin and cannot be produced locally due to the absence of CRP messenger ribonucleic acid (mRNA) in periodontal tissues. They also stated that genetic susceptibility to inflammation may also modify CRP levels in response to periodontal destruction or systemic disease thus explaining the dissimilar correlation of CRP levels found in healthy and diseased groups.  The authors stated that the elevated CRP levels were not due to local production but due to systemic or periodontal inflammation.
Contradictory to this study, Lu and Jin have detected CRP protein and mRNA in cultured gingival epithelia hypothesizing that gingiva per se produces CRP locally and contributes to systemic inflammation too.  Though, the functions of the CRP produced by gingival epithelium and their mechanism of production is unclear. The study further showed no much significant difference in CRP values of healthy and periodontitis patients which was attributed to genetic and other acquired factors.
As with our study, these two studies too have not correlated the GCF CRP levels with that of the serum which makes it difficult to arrive at a definite conclusion about the source of GCF CRP.
Since CRP is a non-specific protein and its level increases in response to any trauma or infection, the interventional nature of our study gives a better understanding of its role in periodontitis since its values have been shown to reduce after periodontal therapy. GCF levels of CRP is considered as a substitute source to assess systemic inflammation.  However some authors  have demonstrated no correlation between serum and GCF hsCRP values. Hence, further studies are needed to correlate levels of CRP in the GCF with that of serum before GCF could be considered to be suitable as a source for the non-invasive assessment of systemic inflammation in both periodontitis and non-periodontitis patients. 
| Conclusion|| |
Collectively, these data suggest that GCF in the active phase of periodontal disease possesses a greater amount of CRP when compared with periodontally healthy and gingivitis sites. As the severity of inflammation increases, there is a significant increase in the CRP levels as indicated by higher CRP levels in periodontitis compared with gingivitis suggesting that there is a direct relationship between CRP levels in GCF and periodontal destruction. With the subsidence of disease process after periodontal therapy, the levels of CRP are reduced. The values almost reach the normal levels in gingivitis cases suggesting that CRP might play a role in the inflammatory process. Since the levels of CRP have been shown to reduce after periodontal therapy, it may be used as a potential biochemical marker for assessment of periodontal disease activity in gingivitis and periodontitis.
| ACKNOWLEDGeMENT|| |
We extend our gratitude to Dr. Kishore Bhat, Prof and Head, Department of Microbiology, Maratha Mandal Dental College, Belgaum and Dr. Asawari Sant of Aabha diagnostics, Belgaum for guiding and conducting the chemiluminescence assay respectively.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]