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ORIGINAL RESEARCH
Year : 2023 | Volume
: 14
| Issue : 1 | Page : 3-6
Assessment of Salivary Interleukin-1β Levels in Patients with Gingivitis and Periodontitis: An Analytical Cross-Sectional Study
Muntadhr A Al-Musawi, Omar H. Ali
Department of Periodontology, College of Dentistry, University of Baghdad, Baghdad, Iraq
Correspondence Address:
Muntadhr A Al-Musawi
Department of Periodontology, College of Dentistry, University of Baghdad, Baghdad
Iraq
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/denthyp.denthyp_148_22
Introduction: The study aimed to analyze the level of salivary Interleukin-1 beta (IL-1β) in subjects with healthy periodontium and patients with gingivitis and periodontitis stage I-III. Methods: A total of 90 individuals participated in the research, separated into five groups, clinically healthy (n = 10), gingivitis (n = 20), stage I periodintitis (n = 20), stage II periodontitis (n = 20), and stage III periodontitis (n = 20). Level of salivary IL-1β was assessed using the enzyme-linked immunosorbent assay. Result: A statically significant difference (P < 0.001) found amongst study groups, further multiple group comparison revealed a statistically significant difference (P < 0.05) when comparing the experimental groups with control and when comparing gingivitis with stage II and III groups. Conclusion: Within the limitations of this cross-sectional study, IL-1β can be considered as a potential salivary biomarkers to differentiate between healthy and unhealthy periodontium.
Keywords: ELISA assay, interleukin-1 beta, gingivitis, periodontal disease, periodontitis, salivary biomarkers
The term “periodontal disease” refers to a class of inflammatory conditions related to bacterial infection, regulated by a combination of numerous host response genes, environmental variables, and lifestyle. The quest for the causal periodontal pathogen(s) remains progressing; however, recent evidence indicates that the inflammatory response plays a key role in altering the periodontal microbiota and accelerating the pathogenesis of periodontitis.[1]
One of the important inflammatory modifiers during the onset and development of periodontal disease is the pro-inflammatory cytokines.[2] Several cytokines are considered to play a significant role in the inflammatory process, like tumor necrosis factor-alpha (TNF-α) and Interleukin 1 (IL-1). IL-1 is an essential inflammatory mediator that promotes the resorption of bone and inhibits its formation. IL-1 induces the production of prostaglandins and the release of proteases as well.[3],[4],[5] Interleukin-1beta (IL-1β) is produced by macrophages, monocytes, lymphocytes, fibroblasts, and epithelial cells.[6],[7],[8] Saliva can be utilized to detect biomarkers specific to the physiological characteristics of periodontal disease. Saliva collection is quick, safe, non-invasive, and easy for patients to accept. Hence, the utilization of saliva in scientific research and clinical practice is growing fast.[9],[10]
We aimed to analyze the level of salivary Interleukin-1 beta (IL-1β) in subjects with healthy periodontium and patients with gingivitis and periodontitis stage I–III.
Material and methodsThe College of Dentistry/University of Baghdad’s ethical committee approved this study on Jan 19th, 2022 (Project number 448/448622). The sample size calculation was based on a previous study by Ebersole et al.,[11] which reported salivary IL-1β levels for healthy individuals 7.24±7.69 (pg/mL) and 90.94±85.22 for patients with periodontitis. With 80% power and a 5% alpha error of probability, the sample size was estimated to be 90 subjects with an allocation ratio of 1:2:2:2:2 (control, gingivitis, periodontitis stage I, II, and III respectively). A total of 90 systemically healthy participants with a minimum of 20 teeth participated voluntarily in this analytical cross-sectinal study after a thorough explanation of the aims and scope of the study and were given informed consent to sign. The subjects were recruited from the College of Dentistry University of Kufa and the specialized dental center in Al Najaf City, from February 2022 to June 2022. They divided into five groups according to the “2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions”[12]: Control group: clinically healthy periodontium (n = 10). Experimental groups: gingivitis group: (n = 20), stage I periodontitis group (n = 20), stage II periodontitis group (n = 20), and stage III periodontitis group (n = 20).
The periodontally healthy group is characterized by “no clinical attachment loss (Probing pocket depth ≤3 mm, Bleeding on probing <10%, no Radiological bone loss).” Gingivitis group is characterized by “intact periodontium (no clinical attachment loss), probing pocket depths ≤3 mm, and bleeding on probing >30% (generalized).” Stage I periodontitis is characterized by “Interproximal bone loss <15% or 1-2 mm.” Stage II periodontitis is characterized by “Interproximal bone loss involving coronal third of the root.” Stage III periodontitis is characterized by “Interproximal bone loss extending to the mid-third of the root.” All periodontitis groups were determined as “generalized (more than 30% of teeth) with unstable condition PPD ≥5mm or PPD at ≥4mm and BOP.”[13]
Participants with systemic disease, or those who had received periodontal treatment or taking antibiotics within 3 months of the study and who used any medication that modifies periodontal parameters values, alcoholics, or smokers, or who receiving orthodontic treatment, and females who are pregnant, or breastfeeding excluded from the study.
After the subjects were selected, unstimulated whole saliva samples were collected by passive drooling from all participants into plastic containers (CNWTC, Jangsu, China) between 9 and 11 a.m.[14] Participants were asked to avoid eating, drinking, chewing gum, and practicing any oral hygiene activities for at least 1 hour before sample collection. The samples were placed in an ice box (Wanmei, Foshan, China) immediately, followed by centrifuging (80-1 Electric Centrifuge, Shanghai, China) at 4000 rpm for 15 minutes, and the clear supernatants were stored in Eppendrof tubes (CNWTC/OEM, Jiangsu, China) inside a deep freezer at (−80 °C) (Angelantoni Life Science, Perugia, Italy) until further analysis.[15]
A single calibrated examiner measured all clinical periodontal parameters for all participants. The following items were assessed: (1) bleeding on probing (BOP), which was noted within 30 seconds of probing. (2) Probing pocket depth (PPD), which was assessed “from the gingival margin to the base of the pocket.” (3) Clinical attachment level (CAL), which was assessed “from the cementoenamel junction to the base of the pocket.” These parameters were assessed with the aid of a periodontal probe (UNC 15 probe, New York, NY, USA) at six sites for each tooth. The plaque index (PI) was assessed with the aid of disclosing agent[16] (Sunstar, Etoy, Switzerland) at four sites for each tooth. The periodontal parameters (PPD, and CAL) for five subjects were used for the determination of inter- and intra-examiner calibrations.
Laboratory procedure
Human specific Enzyme-Linked Immunosorbent Assay (ELISA) kits (Bioassay Technology Laboratory, Shanghai, China) were utilized to determine salivary IL-1β levels as per the manufacturer’s instructions. Salivary samples were thawed and labeled prior to the procedure. Kits’ sensitivity values were 10.07 pg/mL, with a detection range of 20–6000 pg/mL. The optical density was measured at 450 nm. The operator who performed the procedures was blinded to the tested groups.
Statistical analysis
Data analyses were performed using one-way analysis of variance (ANOVA) and the Pearson correlation coefficient. For the assessment of intra- and inter-examiner agreement, the interclass correlation coefficient (ICC) was used. R software (R Foundation for Statistical Computing, Vienna, Austria) was utilized to analyze the data.
ResultsNinety participants were included in this study (mean age = 36.87 ± 9.46, range 22–58, 60% male) (Supplementary Figure 1).[17] Intra- and inter-examiner agreements were 0.955, 0.935 for clinical attachment loss (CAL), respectively, and 0.954, 0.909 for probing pocket depth (PPD), respectively.
Analysis of levels of IL-1β showed a statically significant difference (P < 0.001) amongst study groups, further multiple group comparison revealed a statistically significant difference (P < 0.05) when comparing the experimental groups with the control and when comparing gingivitis with stage II and III groups [Figure 1].
Figure 1 Box and whisker plot showed the spread and centers of the levels of IL1-β (pg/mL) among different study groups.A moderate positive correlation was found between levels of IL1-β, PI, and BOP among periodontitis, gingivitis, and control groups (P < 0.05). Week positive correlation was found between levels of IL1-β, CAL, and PPD (P ≥ 0.05) [Figure 2].
Figure 2 Heatmap showed correlations between levels of IL1-β and clinical periodontal parameters. Discussion IL-1 β is an essential pro-inflammatory cytokine in tissue injury.[18] Many studies found that IL-1β is detectable in lower amounts in healthy periodontium compared to unhealthy periodontium (gingivitis and periodontitis).[11],[19],[20] In this study, assessment of IL-1 β levels, revealed a progressive increase from healthy to periodontitis groups and theoretically showed the potential to differentiate between healthy and unhealthy periodontium. The result is consistent with many studies.[15],[21],[22],[23] At the same time, disagree with Teles et al.,[24] who found no statistically significant change between a healthy periodontium and periodontal disease groups.
Regarding periodontitis stages, the level of IL-1β increased from stage I to stage II, then slightly decreased in stage III, with no statistically significant difference between different stages, which disagrees with Dikilitas et al.,[25] except for stage II and III, which is consistent with the study result. All periodontitis stages showed a statistically significant difference with the healthy control group, which is consistent with Tan et al.[20]
When comparing IL-1β levels of gingivitis and periodontitis stages, stages II and III showed a statistically significant difference, while stage I showed no significance, which is in harmony with Dikilitas et al.,[25] and disagrees with Tan et al.,[20] who found a significant difference between stage I and gingivitis.
As for the transition from gingivitis to periodontitis, no significant difference was found between gingivitis and stage I periodontitis, which brings to light the importance of IL-1β as an inflammatory mediator (gingivitis) and as a stimulus for initiating extracellular matrix and bone resorption (periodontitis)[15] which is consistent with the result of Dikilitas et al.,[25] and disagrees with Tan et al.[20]
IL-1β was positively correlated with BOP in all experimental groups; this might be because IL-1β is a pro-inflammatory cytokine that rises with inflammation, and increasing periodontal inflammation causes vasodilation, which leads to a higher BOP. This result agrees with Tan et al.[20] and Balaji et al.[26]
Limitations of the study
The usage of saliva does not reflect the specific site of active disease. Readers must be aware of the inherent limitations of cross-sectional studies, which cannot make a causal inference. We did not follow individuals over time and selected a sample of participants from a large and heterogeneous study population.
Financial support and sponsorship
Nil.
Conflicts of interest
The authors report no conflicts of interest.
References
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