Correlation between gingival crevicular fluid levels of a disintegrin and metalloproteinase 8 and periodontal disease

Purva Chougule; Avni Raju Pradeep; Patil Rujuta; S Swathika

doi:10.4103/SDJ.SDJ_105_21

ORIGINAL ARTICLE

Year : 2022 | Volume : 6 | Issue : 1 | Page : 18-24

Correlation between gingival crevicular fluid levels of a disintegrin and metalloproteinase 8 and periodontal disease

Purva Chougule, Avni Raju Pradeep, Patil Rujuta, S Swathika
Department of Periodontology, The Oxford Dental College, Bangalore, India

Date of Submission	18-Oct-2021
Date of Decision	30-Nov-2021
Date of Acceptance	15-Dec-2021
Date of Web Publication	21-Feb-2022

Correspondence Address:
Purva Chougule
Department of Periodontology, The Oxford Dental College, Bangalore, House no 2618, Chimgavkar galli, Nipani, Belgaum.,
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/SDJ.SDJ_105_21

Abstract

Background: Periodontal disease is an inflammation caused by host parasite interaction leading to the destruction of tooth supporting structures. During inflammation there is an elevated levels of biochemical signalling molecules. A disintegrin and metalloproteinase 8 (ADAM8) molecule has been implicated in various physiologic functions and pathologies. The expression of ADAM8 is upregulated in inflammatory conditions, and with upregulated expression they play a pivotal role in inflammation, immunity and osteoclastogenesis. Objective: To evaluate ADAM8 levels in gingival crevicular fluid (GCF) collected from healthy individuals and individuals with periodontal disease at baseline and in the periodontal disease group after nonsurgical periodontal therapy. Methods: In total, 48 subjects aged 20–65 years were divided into separate groups: a periodontally healthy group (group 1: males, n = 7, females, n = 9), a chronic gingivitis group (group 2: males, n = 6, females, n = 10), and a chronic periodontitis group (group 3: males, n = 8, females, n = 8). GCF samples were collected from all the groups at baseline and 3 months after nonsurgical periodontal therapy in the chronic gingivitis (group 4) and chronic periodontitis (group 5) cases. Statistical analysis was performed using Shapiro-wilk normality test, One way ANOVA test, Kruskal-Wallis test followed by Mann-Whitney U test, Spearman’s rank correlation test and multiple linear regression analysis was done. The level of significance was determined at P < 0.05. Results: ADAM8 levels in the gingival crevicular fluid was significantly higher in group 3 (26,416.25 ± 7,817.59) than groups 1 and 2 at baseline at P < 0.001. After non-surgical periodontal therapy, ADAM8 levels in the gingival crevicular fluid was significantly reduced for group 2 (13,186.88 ± 3,247.62) and group 3 (18,375.63 ± 3,339.07) at P < 0.001. Conclusion: ADAM8 levels were increased in chronic gingivitis (group 2) and chronic periodontitis group (group 3) and reduced after non-surgical periodontal therapy in groups 2 and 3.

Keywords: A disintegrin and metalloproteinase 8, chronic gingivitis, chronic periodontitis, gingival crevicular fluid

How to cite this article:
Chougule P, Pradeep AR, Rujuta P, Swathika S. Correlation between gingival crevicular fluid levels of a disintegrin and metalloproteinase 8 and periodontal disease. Sci Dent J 2022;6:18-24

How to cite this URL:
Chougule P, Pradeep AR, Rujuta P, Swathika S. Correlation between gingival crevicular fluid levels of a disintegrin and metalloproteinase 8 and periodontal disease. Sci Dent J [serial online] 2022 [cited 2022 Jul 6];6:18-24. Available from: https://www.scidentj.com/text.asp?2022/6/1/18/338001

Background

Periodontitis is an inflammatory disease that leads to the destruction of the periodontal tissues, specifically affecting the periodontal ligament fibers and alveolar bone, ultimately leading to loss of teeth. The primary cause of the disease is microbial plaque that varies in every individual and the degree of causation of the disease also depends on the individual’s inflammatory response.^[1]

For detecting the early periodontal disease activity most of the studies have focused on evaluating the biomarker levels such as interleukin 1beta (IL-1β), tumor necrosis factor alpha.^[2] Similarly, a disintegrin and metalloproteinase 8 molecule has been thought to play an important role as osteoclast stimulating factor.^[3] Seals and Courtneidge, have reported that proteins belonging to zinc protease superfamily consist of an integrin receptor binding disintegrin domain and therefore referred as a disintegrin and metalloproteinase.^[4] The study also states their physiologic role in muscle development, cell migration, cytokine and growth factor shedding and fertilization and, also in pathologies such as inflammation and cancer.^[4]

In terms of chemical structure ADAM8 consists of 824 amino acid, a carboxyl- terminal transmembrane domain, and potential extracellular adhesion and protein domains.^[5] It has been shown to hydrolyze myelin basic protein and a variety of peptide substrates.^[5]

ADAM8 role has been studied in various systemic diseases like non-small cell lung cancer cells where it is a potential target for malignancy that acts through signal transducer and activator of transcription 3 signaling pathway.^[6]

It is also involved in the invasion of tumor cell in the central nervous system and allergen induced acute airway inflammation by promoting dendritic cells recruitment and C-C motif chemokine ligand 11, C-C Motif chemokine Ligand 22 production.^[7],[8] Furthermore, ADAM8 contributes to alveolar bone resorption by enhancing the formation of mature multinucleated osteoclast.^[9] Thus, ADAM8 may play a role in the pathogenesis of periodontal disease.

A recent study showed that ADAM8 levels were reduced after non-surgical periodontal therapy in patients with chronic periodontitis implying that ADAM8 reflect inflammatory and bone-resorbing activities in periodontal pocket.^[10]

To date, no study has been carried out on ADAM8 levels in GCF of healthy, chronic gingivitis, and chronic periodontitis subjects before and after non-surgical periodontal therapy in the Indian population.

To explore the possibility of using ADAM8 as a predictive marker of periodontal disease, this study investigated the ADAM8 levels in GCF of Indian subjects with clinically healthy periodontium, chronic gingivitis, and chronic periodontitis at baseline and after non-surgical periodontal therapy in latter two groups.

Materials and Methods

Study group

A total of 48 systemically healthy individuals of age 20–65 years who attended out patient department were recruited. Chronic gingivitis and periodontitis were diagnosed based on the criteria of American Academy of Periodontology classification of periodontal disease 1999.^[11] The 48 individuals were divided into 5 groups, 16 individuals in each group. A periodontally healthy group (group 1: males, n = 7, females, n = 9), a chronic gingivitis group (group 2: males, n = 6, females, n = 10), and a chronic periodontitis group (group 3: males, n = 8, females, n = 8). GCF samples were collected from all the groups at baseline and 3 months after nonsurgical periodontal therapy in the chronic gingivitis (group 4) and chronic periodontitis (group 5) cases. The clinical parameters to be measure for each group are as follows, Group 1: individuals with healthy periodontium, gingival index≤0, probing pocket depth≤3 mm and clinical attachment level≤0 (n = 16), Group 2: individuals with chronic gingivitis where GI≥1, PPD≤3 mm and CAL≤0 (n = 16), Group 3: individuals with chronic periodontitis where GI≥1, PPD≥5 mm and CAL≥3 mm with radiographic evidence of bone loss. Group 4: Chronic gingivitis subjects after non-surgical periodontal therapy, Group 5: Chronic periodontitis subjects after non-surgical periodontal therapy. All individuals had ≥20 teeth present. None of the participants included in the study had received periodontal therapy within the preceding 6 months. The exclusion criteria were as follows: individuals with aggressive periodontitis, under medication, with the history of any underlying systemic disease, smoking and tobacco chewing habits and pregnant, lactating women were excluded from the study.

Ethics

The study protocol was approved by the Ethics committee (258/2017–18). Written informed consent was obtained from all the individuals before clinical periodontal examination, GCF sampling and non-surgical periodontal therapy for group 4 and 5. The study was conducted in accordance with the Helsinki Declaration of 1975, as revised in 2000.

Clinical measurements

Subjects were selected for each group after detailed case history recording and radiographic examination. All the clinical measurements were carried out by a single operator using mouth mirror and william’s graduated periodontal probe. The clinical parameters recorded were plaque index, gingival index, probing pocket depth, clinical attachment level and bleeding on probing. PI and GI measurement were performed according to silness and loe for PI and loe and silness for GI at four sites per tooth (mesial, distal, buccal and lingual/palatal) with william’s graduated periodontal probe.^[12],[13] PPD measurement were performed at six sites per tooth (mesiobuccal, distobuccal, midbuccal, mesiolingual/palatal, distolingual/palatal, midlingual/palatal). CAL was measured from the cento enamel junction to the base of the sulcus/pocket. BOP was measured as presence or absence of bleeding on gentle probing of the gingival tissue.^[14]

Gingival crevicular fluid collection

The GCF samples were collected following day after the clinical parameters were recorded to avoid contamination of GCF with blood. The site selected for GCF collection was air dried and isolated with cotton rolls, supragingival plaque was removed without touching the marginal gingiva and GCF was collected by placing 5 μl microcapillary pipette commercially available as Drummon – Microcap by Drummond scientific company from Unites states of America. A standardized volume of 3 μl was collected from the test site. Only one site per subject was selected as a sampling site in chronic gingivitis and periodontitis group, whereas for healthy group to ensure the collection of an adequate amount of GCF, multiple sites without inflammation were sampled. GCF samples were stored at −80°C till the assay procedure was carried out.

Non-surgical periodontal treatment

The subjects included in the chronic gingivitis (group 2) and chronic periodontitis (group 3) group received initial periodontal therapy consisting of full mouth supragingival and subgingival scaling and root planing with gracey curettes and ultrasonic scaler under topical anaesthesia as required, on the same day of GCF collection. The subjects were advised about oral hygiene maintenance and recalled after 3 months. Following non-surgical periodontal therapy, at the end of 3 months the GCF samples were collected from the same sites for group 2 and 3. After the collection of all the samples they were subjected to ELISA for the estimation of ADAM 8 levels.

Measurement of ADAM8 levels

The ELISA kit was obtained from Biocodon USA. In the blank wells of microtiter plate 100μlof standard diluent was added. In the sample wells 50 μl of streptavidin-horseradish proxidase were added. Then 40 μl of undilute sample, 10 μl of ADAM8 antibody and 50 μl of streptavidin – horseradish peroxidase were added. Then the assay plate was sealed using plate sealer, gently shaken and incubated the plate at 37°C for 60 minutes. After the incubation the solution was discarded completely and plate was tapped on paper towel. 1X washing buffer solution was added in each well and plate was incubated for 20–30 seconds at 22°C. Then the solution was discarded and the plate was tapped on paper towel to drain the reagents out of wells and the above procedure of washing buffer solution was repeated 5 times. After the last wash any remaining buffer was removed by aspirating or decanting plate. Then 50 μl of chromogen reagent A was added to each well followed by addition of 50 μl of chromogen reagent B to each well and gentle shaking was done to mix both the reagents and incubated for 10 minutes at 37°C in dark. After incubation 50 μl of stop solution was added to each well to stop the reaction. The absorbance of each well was read immediately on the ELISA reader using 450 nm wavelength. The concentration of ADAM8 in tested sample was estimated using standard curve.

Statistical analysis

Statistical analysis was performed using Shapiro-Wilk normality test to verify the normality of the data. Since all the variables were not normally distributed, the data was analysed using non-parametric test. One-way ANOVA was done to compare the mean of the groups in order to determine the statistical evidence. Differences between the groups were tested using Kruskal-Wallis test followed by Mann-Whitney U test. To determine the strength of association between clinical parameters and ADAM8 levels, Spearman’s rank correlation test was done. Then the multiple linear regression analysis was done to explain the relationship between two or more variables.

Results

[Table 1] represents the mean age and gender of the study population. The Shapiro-wilk test for normality and Chi square test was done that showed no statistically significant difference between the mean age and the gender distribution among the groups at P = 0.31. However, there was more of female predominance seen in all the groups.

Table 1: Age and gender distribution

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One-way ANOVA test was carried out to determine the mean values of all the clinical parameters recorded [Table 2]. Plaque index was highest for group 3(1.42 ± 0.13) compared to group 2(1.29 ± 0.15) and group1(0.41 ± 0.11) and is statistically significant at P < 0.001 [Table 2]. Similarly, the gingival index, probing pocket depth were highest for group 3 (1.42 ± 0.17 and 6.44 ± 0.89) than group 2(1.24 ± 0.17 and 2.69 ± 0.48) and group 1(0.40 ± 0.09 and 2.25 ± 0.45) and this finding was statistically significant at P < 0.001. The clinical attachment level was also highest for group 3 (6.88 ± 0.96) than group 2(0.00) and was statistically significant at P < 0.001. The mean values of the clinical parameters decreased in groups 4 and 5 after nonsurgical periodontal therapy, with the PI decreasing significantly in the two groups (0.42 ± 0.11 and 0.44 ± 0.12) respectively; P < 0.001). The mean gingival index also decreased significantly in group 4 (0.56 ± 0.11) and group 5 (0.40 ± 0.15) which was statistically significant at P < 0.001. The PPD and CAL decreased significantly in group 5 (chronic periodontitis) after non-surgical periodontal therapy.

Table 2: Comaprison of mean values of different study parameters using One-way ANOVA test

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The mean GCF ADAM8 levels were determined by Kruskal Wallis test followed by Mann Whitney post hoc analysis that is represented in [Table 3]. The test results demonstrated mean GCF ADAM 8 levels were significantly higher for group 3 (26,416.25 ± 7,817.59) than group 2 and group 1 at P < 0.001. The GCF ADAM8 level for the post treatment group that is group 4 and 5 were reduced that is (13,186.88 ± 3,247.62) and (18,375.63 ± 3,339.07) at P < 0.001.

Table 3: Comparison of mean GCF ADAM8 levels between different groups

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Spearman’s correlation test was conducted to determine the relationship between GCF ADAM8 levels and the periodontal parameters measured. As shown in [Table 4], the results revealed a strong positive correlation between GCF ADAM8 levels and the PI (rho value: 0.65, P < 0.001). Similarly, rho value for GI was 0.71 at P < 0.001 which also shows strong positive correlation with GCF ADAM8 levels. Similarly, the rho value for PPD was 0.52 and for CAL was 0.50 at P < 0.001 and which shows a moderate positive correlation with GCF ADAM8 levels. Further stepwise multiple linear regression analysis for GCF ADAM8 levels was done in overall samples. The test results demonstrated that coefficient value for gingival index was 5785.68 at P = 0.009 and for CAL it was 661.51 at P = 0.03. This indicates that for every 1 mm increase in CAL and every 1 score increase in GI there will be 5785.68 and 661.5 increase in GCF ADAM 8 levels and the r² which is the predicting potential of particular model and here it is for GCF ADAM8 levels is 0.40. Thus, GI and CAL are the predictors in overall samples [Table 5].

Table 4: Spearman’s correlation test to establish relationship between GCF ADAM8 levels and periodontal parameters

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Table 5: Multiple linear regression analysis for GCF-ADAM8 in overall samples

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Discussion

Periodontitis is caused by abnormal host immune responses that are initiated by polymicrobial infections from anaerobic periodontal pathogens.^[1] The degree of severity of periodontitis depends on various factors such as genetic and environmental factor. Uncontrolled host immune responses and failure to resolve inflammation can lead to overproduction of cytokines, chemokines and matrix metalloproteinases proteins (MMPs).^[15]

Previous research revealed elevated GCF levels of metalloproteinases belonging to the metzicin superfamily, including those of ADAM17 and ADAM8, in periodontitis patients, pointing to the importance of these two ADAMs in the pathogensis of periodontitis.^[15] A study done by Khongkhunthian et al. have demonstrated increased levels of ADAM8 in GCF of chronic gingvitis and periodontitis patients as compared to healthy individuals.^[3]

In the present study, which included individuals aged 20–65 years, there was no significant difference in GCF ADAM8 levels according to age. Although there was a female predominance, there was no difference in GCF ADAM8 levels which is in accordance with Khongkhunthian et al. study.^[15] The results of the present study demonstrated that the mean GCF ADAM8 levels was least in healthy group (group1) as compared to chronic gingivitis (group2) and chronic periodontitis group (group3) (1,6081.25 ± 3,095.40, 21,183.75 ± 5,000.43, 26,416.25 ± 7,817.59 respectively). The GCF ADAM8 levels increased according to the severity of periodontal disease and this can be attributed to the fact that ADAM8 has a role to play in inflammation, immunity and in the formation of mature mutinucleated osteoclast.^[3],[9] It is likely that raised GCF ADAM8 levels in chronic periodontitis and gingivtis patients in the prsent study were the result of inflammatory cells (neutrophils, macrophages) infiltration, as well as from inflammed epithelial cells within gingival tissues of chronic periodontitis as found previously in a study done by Aung et al.^[15] They observed increased expression of ADAM8 in chronic periodontitis tissues and within gingival epithelium consistent with an upregulation of ADAM8 expression in Fusobacterium nucleatum stimulated human gingival epithelial cells suggesting a role of ADAM8 in innate immunity of periodontal tissue.^[15]

In several pathological and inflammatory-related conditions, elevated levels of soluble ADAM8 have been detected in serum (e.g., myocardial infarctions), bronchoalveolar lavage (e.g., rheumatoid arthritis, and amniotic fluid (e.g., preterm deliveries).^{[16],[17],[18],[19]} A number of studies demonstrated ADAM8 to be a novel serologic marker of lung cancer and is associated with increased invasiveness in primary brain tumors, pancreatic cancer, head and neck squamous cell carcinoma and an unfavourable prognosis of prostrate cancer.^{[20],[21],[22],[23],[24]} In the present study the clinical parameters correlated positively with GCF ADAM8 levels in the gingivitis and periodontitis group. The chronic gingivitis and periodontitis patients after non-surgical periodontal therapy the mean levels of ADAM8 in GCF reduced from 21,183.75 ± 5.000.43 to 13,186.88 ± 3,247.62 for the chronic gingivitis and 26,416.25 ± 7,817.59 to 18,375.63 ± 3,339.07 for chronic periodontitis group which were significant and also correlated positively with clinical parameters measured. This finding was in accordance with the study done by Nimcharoen et al, who reported significant reduction of ADAM8 levels in GCF were found at sites with moderate periodontitis and severe periodontitis after non-surgical periodontal therapy but no significant reduction of ADAM8 levels in the GCF.^[10] In the present study we found significant reduction of ADAM8 levels in the GCF in both gingivitis and periodontitis group after the treatment and these levels were positively correlated with the clinical parameters measured.This could be attributed to expression of ADAM8 in various types of immune cells including monocytes/macrophages, neutrophils, eosinophils, dendritic cells, and B lymphocytes suggesting its role in innate immunity. The possible limitation of the study could be that, it was not gender matched with more number of females compared to male which can act as confounding factor. The study consisted of small sample size.

Conclusion

Within the limitation of the present study, it can be postulated that increased level of ADAM8 can be detected in GCF of gingivitis and periodontitis patients and greater the extent of periodontal destruction higher the levels of ADAM8 in GCF. However further longitudinal prospective multicentered studies involving larger population should be carried out to validate the findings of the present study and to better understand the role of ADAM8 in pathogenesis of periodontal disease.

Acknowledgments

The authors are grateful to laboratory technician for carrying out ELISA procedure. All the patients are highly appreciated for their co-operation and sincere involvement in the study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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