The short-term effects of non-surgical periodontal therapy on the circulating levels of interleukin-6 and C-reactive protein in patients with chronic periodontitis.

BACKGROUND: Recent epidemiological studies have shown that periodontal infection is a risk factor for a number of systemic diseases and conditions. In addition to the conventional risk factors, chronic infection and the subsequent generation of a systemic inflammatory response may be associated with this increased risk. AIMS: This study was conducted to determine whether the presence of chronic periodontitis and subsequent non-surgical periodontal therapy could influence the serum levels of interleukin-6 and C-reactive protein (CRP) in patients with severe chronic generalized periodontitis. SETTINGS AND
DESIGN: Participants were selected from subjects who attended the Department of Periodontics and Oral Implantololgy, Government Dental College, Thiruvananthapuram.
MATERIALS AND METHODS: Sera were obtained from 25 patients with periodontitis for baseline examination and reassessment after completion of treatment. As a control, sera were also obtained from 20 subjects without periodontitis. Interleukin-6 was determined by sensitive enzyme-linked immunosorbent assay, and high-sensitivity CRP (hsCRP) was measured using latex turbidometric immunoassay. STATISTICAL ANALYSIS: Data were analyzed using computer software, Statistical Package for Social Sciences (SPSS) version 10.
RESULTS: The level of interleukin-6 and hsCRP in the sera of periodontitis patients was seen to be higher than those of healthy controls. Interleukin-6 level tended to decrease with improvement of the periodontal condition following treatment and approached that of control subjects, and this decline was statistically significant. The hsCRP levels also showed a decreasing trend following periodontal treatment.
CONCLUSIONS: In this study, we were able to show that periodontal disease significantly affects the serum levels of systemic inflammatory markers and that non-surgical periodontal therapy could bring about a decrease in the levels of these inflammatory markers.

INTRODUCTION

Just as periodontal tissues mount an immunoinflammatory response to bacteria and their products, systemic challenge with these agents also induces a major vascular response. This host response may offer explanatory mechanisms for the interaction between periodontal infection and a variety of systemic disorders. Therapeutic oral manipulations or the inappropriate or absence of intervention of progressing periodontitis could have a significant influence on the levels of systemic inflammation.

In this study, we have evaluated the extent of “spill” of proinflammatory cytokines like interleukin (IL)-6 into the systemic circulation, the systemic inflammatory response generated due to severe chronic generalized periodontitis by assessment of IL-6 and C-reactive protein (CRP) levels, and the changes in the systemic levels of cytokine IL-6 and acute phase reactant CRP brought about by non-surgical periodontal therapy.

MATERIALS AND METHODS

Study population

Participants were selected from subjects who attended the Department of Periodontics and Oral Implantololgy, Government Dental College, Thiruvananthapuram. Twenty-five subjects in the age group of 30-60 years with chronic severe (clinical attachment loss ≥5 mm) generalized periodontitis (at least 50% of teeth affected) and who were otherwise healthy were taken under the test group. Twenty subjects who had healthy periodontium were taken as controls. None of the control group subjects had periodontal pockets, loss of attachment, or alveolar bone resorption. Patients with known systemic diseases like cardiovascular diseases, diabetes mellitus, malignancies, pneumonia, severe trauma, or renal transplant, history and/or presence of other infections, those who had taken systemic antibiotic treatment in the preceding 3 months or treatment with any medications known to affect serum inflammatory markers, smokers, obese individuals, and pregnant or lactating females were excluded from the study. All patients gave written informed consent. This study was reviewed and approved by Human Ethical Committee, Medical College, Thiruvananthapuram.

Experimental design

At baseline, patients belonging to the test and control groups were carefully examined regarding their periodontal status and a detailed medical history was taken. Periodontal examinations consisted of oral hygiene assessment by Simplified Oral Hygiene Index (OHS) (Greene and Vermillion), a gingival inflammation record, Gingival Index (GI) (Loe and Silness), probing pocket depth (PPD), and clinical attachment loss measurements. Clinical examination measured four sites per tooth: Mesio-buccal, buccal, disto-buccal, and lingual. Blood samples were collected: 5 ml of blood for estimation of IL-6 and 2 ml for high-sensitivity (hs)-CRP estimation.

Thereafter, test group patients underwent a phase of supra and subgingival scaling and root planing. Pocket irrigation was done with a 1% chlorhexidine gel (three times in 10 min). Oral rinsing with 0.2% chlorhexidine twice a day for 1 min for 14 days was advised. All other necessary dental treatments (extraction of hopeless teeth, restorative treatments) were carried out and comprehensive oral hygiene instructions were given and reinforced at follow-up visits. For the test group, re-examination at 2 weeks and then at 2 months was carried out. At 2 months post-treatment, periodontal examination was repeated and blood samples were collected for re-examination of the levels of IL-6 and CRP.

Estimation of IL-6

Collection of serum for estimation of IL-6

Five milliliters of blood was collected and immediately transferred to the Division of Cancer Research, Regional Cancer Centre, Thiruvananthapuram. Samples were allowed to clot for 1 h at room temperature, centrifuged for 10 min at 1000 rpm, and serum was extracted. Collected serum samples were stored at –70°C before use for assay procedure. IL-6 estimation was done by enzyme-linked immunosorbent assay (ELISA).

Evaluation of high-sensitivity CRP levels in serum

Two milliliters of blood was collected from the cubital fossa by venipuncture using 22-gauge needle and 2-ml syringes at the Advanced Clinical Research Laboratory of Medical College, Thiruvananthapuram. The hsCRP estimation was done by latex turbidometric immunoassay in a fully automated analyzer (Cobas Integra).

Statistical analysis

Data were analyzed using computer software, Statistical Package for Social Sciences (SPSS) version 10. Data were expressed as frequency and percentage, as well as mean and standard deviation. To elucidate the associations and comparisons between different parameters, Chi-square (χ2) test was used as a nonparametric test. Student's paired t test was used to compare different groups. For all statistical evaluations, a two-tailed probability of value <0.05 was considered significant.

RESULTS

Forty-five subjects were taken up for the study: 25 subjects under the test group, i.e., patients with chronic severe generalized periodontitis in the age group of 30-60 years, and 20 subjects under the control group, i.e., patients with healthy periodontium in the age group 30-60 years [Figures 1 and 2, Tables 1 and 2].

Figure 1

Age group distribution in two groups

Figure 2

Gender distribution in two groups

Table 1

Age distribution in cases and controls

Table 2

Gender distribution in cases and controls

Five subjects from the test group could not be reevaluated at follow-up visits, and therefore were not included in the statistical analysis. The subjects under the test group did not report any change of the habits, diet, or lifestyle. They did not give any history of illness or drug administration during the period of study.

Clinical effects of periodontal therapy

The mean OHI score (Greene and Vermillion) of test group subjects at baseline was 3.26 (SD 1.173), whereas the mean OHI score of control group was 0.52 (SD 0.248). Two months after the scaling and root planing phase, the OHI score was lowered to a mean value of 0.920 (SD 0.261) [Figure 3, Tables 3 and 4]. Comparison using Student's paired t test showed statistically significant difference between mean OHI scores of test group and control groups at baseline and between baseline and after therapy scores of test group (P<0.001).

Figure 3

Oralm hygeine index of different groups

Table 3

Comparison of mean OHI scores in test group (pre-treatment) and control group

Table 4

Comparison of mean OHI scores pre- and post-treatment in test group

The test group subjects presented with high levels of gingival inflammation at baseline with a mean value of 2.05 (SD 0.219) (GI of Loe and Silness), whereas the mean GI score of controls was 0.475 (SD 0.177). The mean GI score was significantly lowered after therapy to 0.755 (SD 0.157) [Figure 4, Tables 5 and 6]. Comparison using Student's paired t test showed statistically significant difference between mean GI scores of test group and control group at baseline and between baseline and after therapy scores of test group (P<0.001).

Figure 4

Gingival index of different groups

Table 5

Comparison of mean GI scores in control and test group (pre-treatment)

Table 6

Comparison of mean GI scores pre- and post-treatment in test group

Significant reduction of PPD was achieved after therapy. Baseline mean PPD for the test group was 5.28 mm (SD 0.59), whereas baseline mean PPD for controls was 2.605 mm (SD 0.27). The post-treatment mean PPD was 4.015 mm (SD 0.69) (P<0.001). Reduction of mean PPD achieved for test group subjects was 1.27 mm [Figure 5, Tables 7 and 8]. The mean gain in clinical attachment level after therapy for the test group was 0.88 mm [Figure 6, Tables 9 and 10].

Figure 5

Mean PPD of different groups

Table 7

Comparison of mean PPD in control and test group (pre-treatment)

Table 8

Comparison of mean PPD pre- and post-treatment in test group

Figure 6

Mean CAL of different groups

Table 9

Comparison of mean CAL in control and test group (pre-treatment)

Table 10

Comparison of mean CAL pre- and post-treatment in test group

Changes in circulating levels of IL-6 and hsCRP

The mean IL-6 level of patients under the test group was 13.85 pg/ml (SD 7.72), whereas the mean value of control group subjects was 6.12 pg/ml (SD 4.29). The mean IL-6 value after therapy was 5.99 pg/ml (SD 5.51) [Figure 7, Tables 11 and 12]. Student's paired t test showed statistically significant difference between mean IL-6 levels of test group and control group (P<0.001) and the mean serum IL-6 values before and after therapy (P<0.001). The difference between the mean of pre-treatment and post-treatment values was 7.43 pg/ml. The mean value of serum hsCRP level in the test group subjects was 0.575 mg/dl (SD 0.215), whereas the mean value of control group was 0.240 mg/dl (SD 0.05) (P<0.001) [Figure 8, Tables 13 and 14]. The difference in the mean hsCRP values between the test group subjects and control group was 0.34 mg/dl. The mean hsCRP values after therapy was 0.47 mg/dl (SD 0.113) compared to pre-treatment mean value of 0.575 mg/dl (P>0.05). The reduction of hsCRP levels obtained was 0.10 mg/dl and was not statistically significant.

Figure 7

Mean serum interleukin-6 of different groups

Table 11

Comparison of mean serum IL-6 in test group (pre-treatment) and control group

Table 12

Comparison of mean serum IL-6 pre- and post-treatment in test group

Figure 8

Mean serum Hs CRP og different groups

Table 13

Comparison of mean serum hsCRP in test group (pre-treatment) and control group

Table 14

Comparison of mean serum hsCRP pre- and post-treatment in test group

DISCUSSION

This study was done to assess the systemic inflammatory burden caused by chronic periodontitis in an otherwise “healthy” individual and also to evaluate if periodontal therapy could bring about a change in the serological markers of systemic inflammation.

Subjects with severe chronic generalized periodontitis (1999 International Workshop for a Classification of Periodontal Diseases and Conditions) with clinical attachment loss 5 mm and more than 50% of the sites involved were included under the test group. These levels of extent and severity of disease were chosen to increase the probability of detection of a systemic burden from the local periodontal infection.

The mean OHI score, mean GI score, and PPD were significantly higher for patients in the test group than in the control group. Mean clinical attachment loss of the test group was 5.835 mm, whereas there was no attachment loss in controls. After non-surgical periodontal therapy, there was a significant reduction of the OHI score, GI score, and PPD, and a gain of attachment. Baseline value of IL-6 was 13.85 pg/ml for the test group subjects with severe generalized periodontitis, compared to the control group mean value of 6.12 pg/ml (P<0.001). Two months after non-surgical periodontal therapy, the serum IL-6 level was reduced to a mean value of 5.99 pg/ml (P<0.001). These results correlate well with the findings by Loos et al.[1] who examined the levels of IL-6 and CRP in the peripheral blood of periodontitis patients. They found that CRP, IL-6, and neutrophil levels were elevated in periodontitis patients. Loos[2] found IL-6 to increase in a dose-dependent manner. In general, patients with chronic severe generalised periodontitis had much higher levels and were much more often positive for IL-6 than the controls, because IL-6 could not be measured in many controls .

Sothern et al.[3] conducted two studies which examined the circadian characteristics of IL-6 in the blood and urine samples of clinically healthy men. They found that average IL-6 values ranged from 1.66 to 5.38 pg/ml, with the lowest to highest values within 24 h ranging from 1.20 to 7.58 pg/ml between subjects. IL-6 has been demonstrated to increase the expression of adhesion molecules in human umbilical vein endothelial cells at 10 pg/ml according to Watson et al.[4] Ridker et al.[5] reported that in apparently healthy men, elevated levels of IL-6 are associated with increased risk of future myocardial infarction However, the biological effect of these low levels of IL-6 and the role of periodontal infection on the systemic levels of IL-6 remained to be clarified.

The hsCRP value of the test group at baseline was 0.575 mg/dl compared to the control group mean value of 0.240 mg/dl (P<0.001). These results are similar to those obtained by Ebersole,[6] Noack,[7] Slade,[8] Craig,[9] and Waranuch Pitiphat,[10] who reported high mean CRP level among people with extensive periodontal destruction. The elevated values of IL-6 and hsCRP in the test group subjects indicate that severe, generalized periodontitis in these otherwise healthy individuals contributed to their systemic inflammatory burden. These results, however, do not allow for generalization to periodontal patients suffering from less severe and/or more localized forms of disease. Data will have to be confirmed and expanded in larger trials if we are to better understand what proportion of the 10-15% of subjects suffering from severe periodontitis has increased systemic inflammation as a result of this chronic infection (Papapanou).[11]

Mean IL-6 value was reduced from 13.85 pg/ml pre-treatment to 5.99 pg/ml after therapy. Student's paired t test showed statistically significant difference between pre-treatment and post-treatment values (P<0.001). These results are in accordance with those obtained by D’Aiuto et al.,[1213] Elter et al.,[14] Loos et al.,[15] and Tonetti et al.[16] in their studies. All test group subjects had an elevated value prior to therapy and all the patients in the test group showed a decrease in the levels after therapy. This points to the fact that the increase in IL-6 values in these patients could be attributed to the severe generalized periodontitis they suffered from. The rapid decline in the levels of IL-6 could be due to its short plasma half-life.

The hsCRP value reduced from 0.575 mg/dl pre-treatment to 0.470 mg/dl post-treatment. There was a trend toward lowering of hsCRP levels after therapy. However, Student's paired t test did not show statistically significant difference between pre-treatment and post-treatment mean values. The reduction in hsCRP level after therapy was 0.11 mg/dl. Although all patients in the test group had an elevated hsCRP value of above 0.3 mg/dl, only half of the subjects showed a slight decrease in hsCRP 2 months after therapy. These results are similar to those reported by Anna Dongari Bagtzoglou et al.,[17] who, in their meta-analysis, do not support the hypothesis that periodontal treatment can reduce systemic CRP levels. The data presented here are for patients following a single course of periodontal treatment, and hence some residual diseased sites remained, and there may have been insufficient time for biochemical changes to be established after disease reduction. These results could also mean that non-surgical periodontal therapy alone is not able to bring down the chronic inflammatory stimulus in these patients. D’Aiuto et al. found that periodontal therapy results in lowering of serum hsCRP values. It should be noted that the difference in CRP values was obtained by D’Aiuto et al. only after 6 months in their study. Mattila et al.[18] also reported reduction of hsCRP after therapy. They reported that periodontitis seems to increase CRP only in some individuals, presumably the ones reacting to it with a systemic inflammatory reaction, and that periodontal treatment decreases CRP levels in these individuals, and it may thus decrease their risk of coronary heart disease.

D’Aiuto et al. conducted a pilot study on 94 subjects and assessed serum CRP and IL-6 levels at baseline and at 2 and 6 months following non-surgical periodontal therapy. They found significant reductions in CRP and IL-6 serum levels, along with improvement in all clinical periodontal parameters with therapy. Tonetti et al. report from their study that CRP and IL-6 levels did decrease 6 months after therapy in both treatment groups, standard periodontal therapy and intensive periodontal therapy, but Tonetti and colleagues suggest that CRP and other markers might not adequately reflect the relevant inflammatory pathways or that the long-term improvements were independent of the systemic inflammatory response. Ide, et al.[19] concluded from their studies that treatment does not produce statistically significant changes in the levels of any of the systemic markers, IL-6 and CRP. Kazuhisa Yamazaki et al.[20] report from a study on Japanese that there was no statistically significant difference in IL-6 and hsCRP before and after therapy. They report that this lack of statistical significance may reflect the various contributions made by periodontal disease to the total burden of inflammation in different patients and the relatively small numbers of patients.

Recent studies have investigated different avenues of therapy for combating periodontal inflammation and perhaps subsequent systemic inflammation. In a study by Tipton et al.,[21] the role of cyclooxygenase-2 (COX-2) inhibitors in periodontal therapy was examined. Gingival fibroblasts were cultured in a medium with IL-1β, with or without COX-2 inhibitors. Results showed that COX-2 inhibitors caused dose-dependent decreases in prostaglandin E2 (PGE2) and IL-6 production by IL-1β-stimulated gingival fibroblast and may be useful in controlling inflammation in these patients. Thus, a link is shown between systemic therapy and treatment of periodontitis. Takashiba et al.[22] discussed a possible role for anti-cytokine therapy, such as anti-IL-6 antibodies, in combating periodontal inflammation. If a direct relationship is found among the serum levels of IL-6 and other inflammatory cytokines and periodontitis on a systemic level, then systemic therapy such as vaccinations may be possible and warranted.

A limitation of this study is the small sample size. Five patients were lost to follow-ups. Larger sample size is needed to account for correction for lost follow-up. Although we utilized an hsCRP assay, and excluded current smokers, obese individuals, and, as far as possible, subjects with any other infections or inflammatory conditions, we did not assess the effects hypertension, education, or cholesterol, and it is possible that these confounders may have influenced our findings. Results of this study point to the importance of diagnosing cases of severe periodontal destruction, and the need for proper health education and prompt periodontal therapy for the cases identified.

CONCLUSION

Severe generalized periodontitis seems to contribute to systemic inflammation. Periodontal therapy can cause reductions in the serum levels of proinflammatory cytokines like IL-6. The level of the acute phase reactant CRP did not show significant reduction 2 months after non-surgical periodontal therapy.

Although the current therapies used to manage periodontitis may be adequate to simultaneously manage systemic sequelae, no periodontal treatment protocols are available that are specifically designed to improve systemic status. Further studies in this regard could lead to the emergence of novel therapeutic measures.