Evaluation of salivary tumor necrosis factor-alpha in patients with the chronic periodontitis: A case-control study.

CONTEXT: Periodontitis is a chronic infectious disease that leads to inflammation of the tissues supporting the teeth, bone loss, attachment loss progressively. In chronic periodontitis for starting the host response and inflammatory reaction, the presence of the infectious agent is necessary. AIMS: One of inflammatory factors is tumor necrosis factor-alpha (TNF-α) that appear to be important in the destruction of periodontal tissues that were examined in this study.
MATERIALS AND METHODS: This study was performed in the laboratory and case-control study. The samples of study collected from 30 individuals with chronic periodontitis and 30 healthy controls that matched for age and sex, together. Unstimulated saliva samples were collected from patients and then TNF-α level were measured by enzyme-linked immunosorbent assay and were compared with the control group. STATISTICAL ANALYSIS USED: In this study for statistical analysis, Mann-Whitney was used.
RESULTS: There were differences in mean salivary concentrations of TNF-α in controls and patients. The average concentration in the case group was 9.1 (pg/ml) and the control group was 8.7 (pg/ml), but there was no significant difference between case and control groups (P > 0.05).
CONCLUSIONS: The results of this analysis showed no significant relationship between two groups TNF-α concentration. This biomarker can not seem to be a good index to evaluate or predict periodontal disease.

INTRODUCTION

Chronic periodontitis is an infectious disease, which leads to inflammation of the tissues supporting the teeth, progressive attachment loss and bone loss. Formation of periodontal pocket is one of the common results in the disease process unless the gingival recession also occurs. In chronic periodontitis, the presence of the infectious agent is necessary to start the host response and subsequent inflammatory reaction.[1]

Periodontal pathogens activate the host immunity-related responses against the bacterial antigens and lead to stimulation a series of cytokines that play an important role in the immune response of patients.[2]

Some recent studies on the course and progression of periodontitis suggest that inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α) cause damage to periodontal tissues.[34] The property and mechanism involved in the process of these cytokines is associated with tissue destruction that stimulated by bone resorption and induction of tissue-degrading proteases. A strong stimulus of proliferation, differentiation and activation of osteoclasts is IL-1.[5] TNF-α has the same effects on osteoclasts, but its intensity and power is less than IL-1.[6] Both IL-1 and TNF-α, induce the production of proteases like matrix metalloproteinase (MMP) from the mesenchymal cells. The MMPs may be related to destruction of connective tissue.[78]

TNF is one of the important of inflammatory factors. Overproduction and uncontrolled function of TNF may lead to pathological conditions such as malnutrition and septic shock. Increased local production of cytokines that are released in the peripheral blood flow can lead to negative changes in immune responses.[910] Studies have proven that two large groups of microorganisms that, namely as Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis are as microorganisms influencing the periodontal diseases, which are capable of stimulating the monocytes and macrophages to produce TNF-α.[111213] The role of bacterial factors in the development process of periodontal diseases and TNF-α secretion in response to bacterial stimulations as a result of polymorphonuclear neutrophils and macrophages activation during body non-specific defense system has been accepted. In some studies, the role of TNF-α in the periodontal destruction has been mentioned; however, in general, the role of this factor as the etiologic factor of periodontitis is still at controversy and there is no consensus of opinion in this relation.[14] In this study, the relationship between generalized moderate chronic periodontitis and the TNF-α biomarker levels in unstimulated saliva of patients and healthy was examined.

MATERIALS AND METHODS

This study was a case-control, which included 60 participants (30 cases and 30 controls) that collected from patients referring to the Periodontology Department of Jundishapur University of Medical Sciences. The case samples diagnosed as generalized moderate chronic periodontitis based on criteria of the American Academy of Periodontology. According these criteria, the patients are classified in the generalized moderate chronic periodontitis group, in which if more than 30% of the mouth areas was involved with 3-4 mm clinical attachment level (CAL) and bone loss determined radiographically.[15] Participants had not a history of using alcohol, tobacco, anti-inflammatory drugs and antibiotics during previous 3 months or periodontal treatments in last 4 months. They had more than 20 teeth, no sustained systemic diseases and the women were not also pregnant or lactating.

The healthy control group had not evidence of CAL or increased periodontal pocket depth (PPD). All participants signed a written consent form approved by the Ethical Committee of Jundishapur University of Medical Sciences. The cases and controls were matched regarding age and sex. All patients would receive periodontal treatment after saliva collection. Clinical parameters were measured with Williams probe (23 W, Hu-Friedy, Chicago, IL, USA) and these parameters consisted of PPD, CAL, plaque index (PI), bleeding on probing (BOP). All positive BOP site divided into all evaluated site and calculated BOP percent. All measurement performed by one calibrated examiner.

To avoid changes in antioxidants, all samples were collected in a given time (11-12 am) in the morning. The participants (cases and controls) washed her/him mouth with water before sampling; then discharge his/her unstimulated saliva in a sterile tube using 5 ml sterile tubes.[16] In order to blind the study, the test tubes were code-labeled after sampling. The samples were immediately centrifuged at 12,000 g for 10 min, in order to remove cells and microparticulate material. The clear supernatant of the centrifuged aliquot transferred to the − 20°C refrigerator in the Department of Immunology, School of Medicine. In this study, the human TNF-α enzyme-linked immunosorbent assay (ELISA) kit (Boster Biological Technology, China) had 1 pg/ml > sensitivity. Regarding specificity, no cross-reaction with other cytokines has been seen in this kit. The concentration of data was analyzed by Kolmograph-Smirnov statistical analysis and its normality was investigated. The analysis indicates the non-normal feature of data (P = 0.02). Therefore, the Mann-Whitney method was used for statistical analysis due to non-normal distribution of data. Comparisons in age, PPD, PI and CAL were made using Student's t-test whereas for BOP and sex data, the χ2 test was used. The correlation among TNF-α levels and clinical parameters were assessed using Spearman rank correlation analysis. The value of P ≤ 0.05 was considered as significance level.

RESULTS

There were 18 males (60%) and 12 females (40%) in the controls group and the mean age of participants in this group was as 36.3 ± 5.2 years. There were 16 males (53%) and 14 females (47%) in the case groups and the mean age of participants in this group was as 37.7 ± 4.3 years. For the case and control group examined 460 and 488 teeth and 1840 and 1950 sites respectively. There were not observed statistically significant differences in age or sex between groups. Table 1 summarizes demographic data and clinical parameters of all participants that evaluated in this study.

Table 1

Demographic data and clinical parameters of case (chronic periodontitis) and control (healthy patients)

The TNF-α was detected in all saliva sample of both case and control groups the average concentrations of TNF-α in the experimental groups were calculated as 9.1 (pg/ml) in the case and 8.7 (pg/ml) in controls groups. Despite the numerical difference in the medians obtained from the case and control groups, the results of statistical analysis suggest no significant relationship between two groups of the case and control groups (P = 0.806). Significantly were not observed between clinical parameter (PPD [P = 0.6], BOP [P = 0.78], CAL [P = 0.8]) and TNF-α concentration. There was no significant difference between men and women in groups of experiments (control and patient) in terms of mean concentrations of TNF-α [Table 2].

Table 2

Evaluation TNF-α concentrations between two groups

DISCUSSION

This study was performed as experimentally case-study method. The use range of this kit covers all concentrations related to this cytokine in serum, saliva, body fluids, lyzed tissues and cell culture media. After data analysis, no significant relationship was seen between the presence of TNF-α and generalized moderate chronic periodontitis (P = 0.05).

In a study, Teles et al. examined the salivary levels of different cytokines, including TNF-α by ELISA method in patients with chronic periodontitis and people with healthy periodontium. No significant statistical relationship was found between different groups after laboratory experiments. This study suggested that the salivary levels of studied cytokines can not be an appropriate factor to differentiate the periodontal disease from healthy periodontium that its results regarding lack of significant differences are similar to the current research.[17]

In China, Geng et al. assessed the salivary activity rates of TNF-α and IL-6 in patients with chronic periodontitis using ELISA test; at the end of the study, they stated that the levels of these two cytokines were higher in patients with chronic periodontitis than the healthy individuals. It was suggested in this study that TNF-α and IL-6 level can be used as biomarkers to diagnose chronic periodontitis. Similar to our research, ELISA test and unstimulated saliva samples were used in this study. However, the difference in results can be caused by differences in patient selection method compared with our research since the samples were selected from patients with chronic periodontitis including all types of mild, moderate, severe, generalized and localized types in this study, but in our study, only the generalized moderate chronic was selected.[18]

In a study in Finland, Gursoy et al. examined and evaluated salivary concentrations of IL-1-β, elastase, lactate dehydrogenase, TNF-α and IL-6 and the presence of different pathogens in periodontitis. The results indicated that there was no relationship between the concentrations of TNF-α and periodontal disease, which are similar to our study results.[19]

Based on Rai et al. research conducted in United States, in which salivary levels of TNF-α were studied in patients with periodontitis, it was found salivary levels of TNF-α were significantly higher in patients with periodontitis than the control group (P < 0.001). It was suggested that the salivary TNF-α analysis can be used as a useful diagnostic tool and as a prognostic biomarker.[20] The different age groups may be possible causes of difference result that may be mentioned. In this study, the age of samples were 30-45 years and in Rai's study were 17-30 years. Another reason of such difference could be the lower number of samples in Rai's study that included 10 controls and 25 patients. Differences in sampling and evaluation methods can be other possible causes. Patients in Rai's study included all types of periodontitis and no separation had been done. Due to the small size of the samples, this could have led to reduced sensitivity in samples assessment.

In Ng et al. study in the United States conducted aiming at evaluation of potential salivary biomarkers in correlated with bone resorption. Various salivary factors, including TNF-α were reviewed and evaluated. In this study, unlike the current study, the stimulated saliva samples were examined that no significant correlation was observed in levels of salivary TNF-α between case and control groups. Despite using different sample collection methods, similar results to our study findings were obtained in this study.[21]

Based on Slotwinska et al. study in Poland, which was performed to examine the salivary levels of the antagonist receptor of IL-1 and soluble TNF receptor Type I (sTNFR I) in chronic periodontal disease, no significant differences in the levels of salivary sTNFR I were seen in two groups that are similar to our study findings and consistent with them.[22]

According to all collected information and reviewed studies, it seems that the major difference in the final results of the studies was due and related to the methods used and precision in selecting the patients; in none of the studies reviewed, a certain subgroup of the periodontal disease had been considered and the presence of periodontitis had been studied in general. In this study, generalized chronic periodontitis was studied that was more common among the referring patients. Since generalized moderate chronic periodontitis is the most common type of chronic periodontitis, this study was performed on these patients.

CONCLUSION

Although, no relationship was found in this study between periodontal diseases and the TNF-α biomarker, it is suggested to design further studies with more sample size on other different groups of periodontal diseases to assess this biomarker and other ones and also evaluated in gingival crevicular fluid and compare with saliva in different groups.