Association between alveolar bone loss and serum C-reactive protein levels in aggressive and chronic periodontitis patients

Abstract

Background:

C-reactive protein (CRP) is an acute phase reactant that is produced in response to diverse inflammatory stimuli, and is known predictor of cardiovascular disease risk. Aggressive and chronic periodontitis are two main forms of periodontal disease, which differ mainly in the method of disease progression. This study aims at determining and comparing the relative levels of serum CRP and alveolar bone loss in aggressive and chronic periodontitis patients.

Materials and Methods:

A total of 45 subjects, which were divided into 3 groups diagnosed as having generalized aggressive periodontitis (GAP), chronic generalized periodontitis (CGP) and non-periodontitis controls (NP), were selected for the study. Venous blood samples were collected for quantitative CRP analysis using Turbidimetric immunoassay. Alveolar bone loss (ABL) was measured at proximal sites of posterior teeth on a panoramic radiograph. The relationship between the mean ratio of ABL to root length and serum CRP levels was statistically analyzed using Student unpaired t-test, analysis of variance (ANOVA) and Pearson's correlation coefficient.

Results:

Mean CRP levels were significantly greater in both GAP (7.49±2.31 mg/l) and CGP (4.88±1.80 mg/l) groups as compared to NP (0.68±0.23 mg/l) with P value <0.0001. The mean value of ABL (%) was 31.58 in CGP group and 36.77 in the GAP group, the difference being statistically significant (P=0.0079). Correlation coefficient between CRP and ABL is 0.9310 in CGP, and 0.9252 in GAP, which indicates a positive correlation between both variables.

Conclusion:

Both forms of periodontitis are associated with increased systemic inflammatory response with aggressiveness of disease progression determining the degree of response.

INTRODUCTION

Periodontitis is a destructive inflammatory disease of the supporting tissues of teeth. The host responds to periodontal infections with an array of events involving both innate and adaptive immunity. Although periodontitis is chronic in nature, acute-phase elements are also a part of the innate immunity response seen in periodontitis. The acute-phase reactants receiving the most attention are C-reactive protein (CRP), plasminogen-activator 1 and fibrinogen. CRP in particular has been the focus of attention as a key marker of atherosclerosis, and elevated levels constitute a risk predictor for cardiovascular disease (CVD).[1]

CRP is an acute-phase reactant that is produced in response to diverse inflammatory stimuli, including heat, trauma, infection and hypoxia. It is synthesized by the liver in response to the inflammatory cytokines like Interleukin – 6 (IL-6), Interleukin – 1 (IL-1) and tumor necrosis factor alpha (TNF-α). CRP plays a key role in the innate immune response and is easily measured due to its long plasma half-life (12 to 18 hours). Thus CRP levels provide useful information for the diagnosis, monitoring and therapy of the inflammatory process and associated disease. Importantly, CRP is currently regarded as a biomarker of systemic inflammation.[2]

The international workshop for classification of periodontal diseases and conditions by American Academy of Periodontology (1999) revised the classification of periodontal diseases. Accordingly, periodontal diseases are classified into three main forms - chronic periodontitis, aggressive periodontitis, and periodontitis as the manifestation of systemic disease. Aggressive periodontitis comprises a group of rare (≈1% of population), rapidly progressing form of periodontitis, characterized by severe destruction of periodontal attachment apparatus at an early age. Chronic periodontitis is recognized as most frequently occurring slowly progressing form of periodontitis. The onset of the latter may be at any age with prevalence and severity of disease increasing with age. It may affect a variable number of teeth. Both chronic and aggressive forms of the disease are characterized by alveolar bone destruction. The alveolar bone loss represents the degree of periodontal destruction directly. This bone loss can be readily measured with the help of radiographs.[3]

Recently, it has been shown that patients with periodontitis have elevated CRP levels compared to periodontally healthy individuals.[4–6] Most studies till date have included patients with chronic periodontitis and few investigators have studied CRP levels in subjects with aggressive periodontitis. -However comparison of CRP in these two forms of periodontitis has not been attempted. Also, a correlation between alveolar bone loss and elevated CRP levels seems to be appealing, so as to assess the effect of periodontal disease progression on systemic inflammatory response. Thus, in the present study an attempt is being made to determine the relative levels of serum CRP and alveolar bone loss and compare them in aggressive and chronic periodontitis patients.

MATERIALS AND METHODS

Patient selection and clinical examination

The institutional review board (IRB), B.V.V.S, Bagalkot, Karnataka, India approved the study and an informed consent was obtained from all patients before beginning the study. The subjects were examined from the out patient Department, Department of Periodontics, P.M. Nadagouda Memorial Dental College and Hospital, Bagalkot, Karnataka, India. Following complete medical and dental examination, 45 individuals were selected for the study. Subjects were placed into 3 groups according to the following definitions.

• Non -periodontitis controls (NP): This group comprised of 15 patients with no evidence of attachment loss

• Generalized aggressive periodontitis (GAP): A total of 15 patients with probing depth of ≥5 mm and/or clinical attachment loss on 8 or more teeth, at least 3 of which were not first molars and incisors and demonstrated age of onset under 30 years

• Chronic generalized periodontitis patients (CGP): A total of 15 patients with probing depth of ≥5 mm and/or clinical attachment loss in more than 8 teeth were classified as generalized chronic periodontitis.

Dental examination consisted of oral hygiene index simplified, gingival index, probing depth and clinical attachment loss measurements using William's graduated periodontal probe and an Orthopantomogram. All the selected subjects were systemically healthy and none had received periodontal treatment and antibiotic therapy for at least 6 months prior to sampling and recording. Active smokers and subjects with systemic diseases like diabetes, arthritis, CVD etc were not considered. Also pregnant and lactating females were excluded from the study.

Quantitative determination of CRP

Serum levels of CRP were quantified using Turbidimetric immunoassay according to the manufacturer's instructions. Kit used was “TURBILYTE-CRP” (Tulip diagnostics, Goa, India) which had detection limit of 0.5 mg/l and measuring range of 0-22 mg/l.

Examination of alveolar bone loss

Panoramic radiographs were taken using an OPG machine (Gendax, Orthoralix 9200) with standard exposure specifications of 74 kvp and 10 mA for 12 seconds. Alveolar bone loss was measured at proximal sites of posterior teeth on a OPG taking cemento-enamel junction as reference point. Only posterior teeth were selected because of possibility of overlapping in anterior teeth. The distal sites of second molars were excluded to eliminate the influence of the third molar, which was frequently impacted. A maximum of 28 sites and minimum of 10 sites were measured in each subject. The length from cemento-enamel junction CEJ to the alveolar bone adjacent to the root surface and the length from the cemento-enamel junction to the root apex was measured. The former was divided by the latter for each site and the mean value of the ratio in percentage for all sites was designated the mean alveolar bone loss (M) of the subject.

Statistical analysis

Data are presented as mean, standard deviation and 95% confidence interval of the mean difference. Comparison of clinical and biochemical parameters between the groups was carried out by using ANOVA-one way (Analysis of Variance). Pairwise comparison was done by using Student's unpaired t-test. Correlation between variables carried out by using Pearson's correlation coefficient ‘r′, and all levels of significance were set at P<0.05.

RESULTS

The mean CRP (mg/l) levels for the GAP, CGP and NP groups, as seen in Table 1, were 7.49±2.31, 4.88±1.80 and 0.68±0.23 respectively. CRP values of the three patient groups were significantly different from each other, with CRP levels in the GAP group greater than those in the CGP, which were in turn greater than those in the NP subjects. Furthermore, comparison of three groups (CGP, GAP, and NP) with respect to CRP values by one-way ANOVA showed statistical significance (P<0.0001), as seen in Table 2. Also, pair wise comparison of all three groups with respect to CRP values by student's unpaired t-test [Table 3] illustrated statistical significance. The mean value of alveolar bone loss (ABL) (%) was found to be 31.58 in CGP group and 36.77 in GAP group. The difference was found to be statistically significant with P value of 0.0079 by using student's unpaired t-test [Table 4]. The Correlation coefficient between CRP and ABL is 0.9310 and 0.9252 in CGP and GAP, respectively [Tables 5 and 6], thus indicative of a positive correlation.

Table 1.

Mean and standard eeviation of C reactive protein *(mg/l) values according to three groups (CGP, GAP, NP) in the study

Table 2.

Comparison of three groups in the study (CGP, GAP, NP) with respect to C reactive protein values by one-way analysis of variance

Table 3.

Pair wise comparison of three groups (CGP, GAP, NP) with respect to C reactive protein values by student's unpaired t-test

Table 4.

Comparison of CGP and GAP groups with respect to ABL (%)* values by student#x0027;s unpaired t-test

Table 5.

Correlation coefficient between CRP and ABL levels in CGP group

Table 6.

Correlation coefficient between CRP and ABL levels in GAP group

DISCUSSION

Periodontal disease is a chronic inflammatory destruction of tissues surrounding the teeth caused by specific anaerobic pathogens contained in dental plaque organized on the tooth surface. Two major types of periodontal diseases are recognized: Chronic periodontitis, which has a major prevalence in adults with a slow pattern of progression and Aggressive periodontitis, a less common type generally affecting young patients with rapid periodontal destruction that may result in tooth loss with aesthetic, functional and psychological problems. CRP is an acute phase reactant synthesized by the liver in response to the inflammatory cytokines, IL-6, IL-1, and TNF-α. Circulating CRP levels are a marker of systemic inflammation and are associated with periodontal disease.[7] Also of significance are the results of prospective longitudinal trial that indicate that this marker appears to be a useful predictor for future cardiovascular events in a variety of population.[8]

The present study evaluated CRP levels and ABL% in Chronic and Aggressive Periodontitis patients. Also comparison was made between CRP levels and ABL% amongst Chronic and Aggressive Periodontitis patients. All the subjects included in the study were systemically healthy and were excluded for all the other risk factors for periodontitis. The mean CRP (mg/l) levels for CGP was 4.88±1.8 mg/l, for GAP it was 7.49±2.31 mg/l as compared to NP with 0.68±0.23 mg/l. The pair wise comparison of three groups with respect to CRP values showed statistical significance with (P=0.0018) for Chronic Periodontitis patients and for Aggressive Periodontitis patients, (P<0.0001); for Chronic Periodontitis and control group; and, (P<0.0001) for Aggressive Periodontitis patient and control group. These results were similar to study by Loos et al.,[5] which concluded that CRP levels in Chronic Periodontitis patients are higher than non-periodontits controls. Similarly, study by T.N. Salzberg et al.,[2] concluded that CRP levels were elevated in Aggressive Periodontitis as compared to non-periodontitis controls. Also, they reported that CRP levels in chronic group were intermediate between non-periodontitis subjects and Aggressive group, which was also seen in the present study.

Periodontitis is a mixed infection of supporting tissues of the teeth; and, dental plaque harbors many putative pathogens. Once the disease sets in, the subgingival tissues become inflamed and ulcerated. This provides a pathway for bacteria and bacterial products such as lipopolysaccharide (LPS) as well as locally produced pro-inflammatory cytokines to enter the systemic circulation. Subsequently, the systemically dispersed bacteria and LPS, as well as cytokines from periodontal lesion, may stimulate hepatocytes to produce elevated CRP levels.[5] In the present study, alveolar bone loss (ABL) was calculated using panoramic radiographs OPG. Although panoramic radiographs are enlarged by about 20%, our use of the ratio of ABL to root length should eliminate any effect of their enlargement. This was in accordance with study done by Saito et al.[3] Here we found a statistically significant difference with respect to ABL% between chronic group (31.5±4.78) and Aggressive group (36.77±5.13) with P=0.0079.

The correlation co-efficient between CRP and ABL% showed statistically significant results’ in both the CGP and GAP groups, and as the ABL% increased the CRP levels were also seen to be elevated. These findings were similar to those reported by Saito et al.,[3] and confirmed the dose-dependent relationship between ABL and CRP elevation in chronic periodontitis. ABL% depicts the severity of periodontal destruction caused by periodontal pathogens and their products. These pathogens not only cause local inflammation and tissue destruction but are also involved in systemic inflammation and immune responses. Thus as the severity of periodontal disease increases, the systemic component of inflammation also increases. This may be seen as increased production of CRP due to activation of cascade of inflammatory cytokine production in presence of bacteria and bacterial products.[6] Also, the amount of bone loss in GAP was significantly higher as compared to CGP thus portraying the aggressive nature of the disease. The CRP levels in GAP were much higher compared to CGP as well as NP. This indicates that rapid alveolar bone destruction in GAP in presence of cytokines must be adding to the systemic inflammatory burden.

The results of the present study confirm the outcomes of recent investigations, which reported an elevation of CRP levels in periodontitis patients. Also, we assessed the ABL and CRP levels in chronic and aggressive periodontitis patients and compared them in both the groups. CRP levels were significantly higher in both groups as compared to controls, with levels in chronic periodontitis group lying in between those of controls and aggressive periodontitis group. ABL in aggressive periodontitis patients was significantly higher than chronic periodontitis patients, which was due to the rapid rate of disease progression in aggressive periodontitis. The ABL was significantly associated with CRP levels in both the groups. Though ABL is not always related to current inflammation in periodontium, it represents the degree of periodontal destruction directly. These findings point to an association between severity of periodontal destruction recorded as ABL% and systemic inflammatory response, as measured by serum CRP levels. Lately, many studies investigating the effect of periodontal treatment on serum C-reactive protein levels reported a reduction in CRP levels following periodontal therapy.[9–12] Recently, CRP has been proved to be the strongest and most significant predictor of future cardiovascular events.[6] The results of the present study suggest that elevated CRP levels in periodontitis patients can possibly contribute at least in part to the increased risk for cardiovascular disease. Thus, there is a complementary need to recognize that apparently systemically healthy individuals may be having extensive periodontal disease, which might be adding to the systemic inflammation. Also, this evidence based association between oral infection in general and periodontal infection in particular can be used effectively in emphasizing the need for optimum oral health in the population at large. Hence, periodontal disease assessment and its treatment should be instituted in current health programmes to improve overall health status of an individual.