Cytokine Profile in the Gingival Crevicular Fluid Among Diabetic People: A Case-Control StudyABSTRACT
Objective: To investigate the periodontal conditions and levels of inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), in the gingival crevicular fluid (GCF) of individuals with and without diabetes.
Material and Methods: Seventeen healthy participants and 14 individuals with diabetes were recruited. Various periodontal parameters, including plaque index, gingival index, and probing pocket depth, were assessed by a single, blinded investigator. GCF samples were analyzed for TNF-α, IL-1β, and IL-6 levels by using enzyme-linked immunosorbent assays.
Results: The results showed significant differences between the control and diabetes groups in terms of mean probing depth, mean attachment level, plaque percentage, and percentage of bleeding on probing. The analysis of cytokine levels revealed that the mean ranks for TNF-α and IL-1β were significantly higher in the diabetes group than in the control group, indicating a pro-inflammatory state. However, no significant difference was observed in the levels of IL-6 between the two groups.
Conclusion: Diabetes is associated with increased periodontal inflammation and elevated levels of specific inflammatory cytokines in the GCF. The study offers valuable insights into the interplay between diabetes and periodontal disease, emphasizing the importance of comprehensive dental care and the management of systemic health conditions for individuals with diabetes.
Keywords:
Cytokine; Gingival Crevicular Fluid; Diabetes Mellitus; Periodontitis.
Introduction
Diabetes is a chronic global health challenge with significant public health and economic burdens. In 2021, approximately 8.4 million individuals worldwide were living with type 1 diabetes, including 18% younger than 20 years, 64% aged 20-59 years, and 19% aged 60 years or older [1]. Developing countries account for over 80% of the global diabetes population, with Asian nations alone representing more than 60% [2]. Due to its increasing prevalence and associated complications, diabetes places a substantial strain on healthcare systems worldwide [3]. According to the International Diabetes Federation's (IDF) most recent diabetes atlas, 10.5% of adults aged 20-79 had diabetes in 2021, a figure projected to rise to 12.2% by 2045 [4]. One of the less commonly emphasized yet clinically significant complications of diabetes is periodontitis-a chronic inflammatory disease affecting the supporting structures of the teeth [5]. Several studies have illustrated evidence that type 1 and 2 diabetes increase the risk and severity of periodontitis. This disease involves progressive alveolar bone loss, which can eventually lead to tooth mobility and loss if left untreated [6]. Periodontitis affects 20-50% of the global population and is caused by a dysbiotic bacterial biofilm, triggering host-mediated tissue destruction [7]. It is caused by a specific group of periodontal bacteria that causes bone loss and tooth attachment loss.
The bidirectional relationship between diabetes and periodontitis has been well-documented through epidemiological studies and animal models. Gingival crevicular fluid (GCF), an inflammatory exudate, increases in response to periodontal inflammation and provides a valuable source of biomarkers [8,9]. Non-invasive analysis of GCF cytokines has been widely used to evaluate host immune responses in periodontal disease [10,11]. Hyperglycemia contributes to immunological dysregulation and increased production of pro-inflammatory mediators, including IL-1β, TNF-α, and IL-6 [12,13]. These cytokines drive inflammatory responses that result in cellular and molecular alterations in the periodontium. Patients with periodontitis exhibit significantly higher levels of pro-inflammatory cytokines, such as IL-1β, TNF-α, and IL-6, in GCF compared to healthy individuals [14]. This underscores the bidirectional link between diabetes and periodontal inflammation, making it a condition of concern for both dental professionals and medical practitioners.
Elevated levels of the cytokine interleukin-6 (IL-6) influence the functions of key cell typesincluding leukocytes, osteoclasts, and osteoblasts -thereby affecting tissue turnover. In periodontitis, IL-6 promotes alveolar bone resorption and enhances the activity of matrix metalloproteinases (MMPs), such as MMP-8, contributing to extracellular matrix degradation [15-18]. These cytokines are also implicated in peri-implantitis, suggesting overlapping pathogenic pathways with chronic periodontitis [19]. While dental implants are widely used for tooth replacement, their success in patients with diabetes remains controversial due to impaired bone remodeling and immune function [20,21]. Higher cytokine concentrations in saliva and gingival crevicular fluid (GCF) have been associated with individuals with diabetes and periodontal disorders [11,22,23]. This study aimed to compare clinical periodontal parameters and the levels of key inflammatory cytokines (IL-1β, TNF-α, and IL-6) in gingival crevicular fluid (GCF) between individuals with diabetes mellitus and healthy controls.
Material and Methods
Study Design, Ethical Clearance and Recruitment
In this case-control study, a rigorous process was followed to ensure patient participation and collect relevant data. Before the study commenced, all patients involved were required to provide written informed consent after receiving a thorough explanation of the study design and protocol. The study was approved by the Ethics Committee of Prince Sultan Military College of Health Sciences (IRB-2018-CLS-003).
The initial aim was to recruit 30 participants per group, resulting in a total of 17 healthy controls and 13 individuals with diabetes mellitus being ultimately enrolled due to constraints in recruitment and resource availability. The inclusion criteria for the two groups, healthy individuals and patients diagnosed with either type 1 or type 2 diabetes mellitus between 18 and 60 years old, consisted of not receiving any periodontal treatment that could affect the outcomes within the last six months for diabetes, with a history of diabetes in the previous two years.
Additionally, Participants must not have used antibiotics or anti-inflammatory drugs within three months before the study. The study excluded participants who were pregnant, breastfeeding, or smokers. Individuals with hormonal disorders, those taking hormonal agents, and those with infections such as HIV, hepatitis, or tuberculosis were also excluded. Additionally, individuals with systemic diseases, such as osteoporosis or immunological disorders, or a history of oncological treatment were not eligible to participate. HbA1c was performed for all participants, which is a blood test used to measure the average blood sugar (glucose) levels over the past two to three months. An HbA1c level of less than 6% is considered non-diabetic.
Clinical Data Collection
To assess the periodontal condition of the patients, various parameters were recorded from specific teeth known as Ramfjord teeth. These teeth included the maxillary right first molar, maxillary right lateral incisor, maxillary left first premolar, mandibular left first molar, mandibular left lateral incisor, and mandibular right first premolar. Calibrated periodontal probes, specifically the University of North Carolina - 15 (UNC-15) Probe, were used to measure the periodontal parameters. The collected data were then recorded on a periodontal chart for each patient. Notably, a single-blinded investigator evaluated the parameters to minimize bias.
Furthermore, the Plaque Index (PI), Plaque accumulation was assessed using the Plaque Control Record described by O'Leary [24]. This index evaluates plaque presence on the mesial, distal, facial, and lingual surfaces of each tooth. A disclosing solution was applied to all exposed tooth surfaces, and after rinsing, each surface was examined using a probe to detect plaque at the dentogingival junction. Only plaque was present at this junction, and each surface was scored dichotomously as either "yes" or "no." The plaque index was calculated by dividing the number of surfaces with plaque by the total number of available surfaces (number of teeth × 4), and multiplying the result by 100 to express the score as a percentage [24]. The Gingival Index (GI) was also assessed; Bleeding was evaluated using a periodontal probe in accordance with the method described by Ainamo and Bay [25]. The presence or absence of bleeding was recorded alongside probing depth measurements taken from all surfaces of each tooth [25]. Lastly, Probing Pocket Depth (PPD) was measured on all teeth using a UNC-15 periodontal probe, by determining the distance from the gingival margin to the base of the periodontal pocket, following the method described by Armitage [26].
Before collecting GCF samples, the supragingival plaque was carefully removed, and the collection sites were isolated and dried. Sterile strips were then placed at the gingival crevice region to obtain GCF samples from the mesiobuccal root. The strips were left in place for 30 seconds to minimize mechanical irritation, and any contaminated strips were discarded. Following collection, the strips were stored at -80 ΰC in sterile test tubes for the ELISA test.
Cytokines Level Measurement
The GCF samples underwent analysis for TNF-α, IL-1β, and IL-6 using commercially available enzyme-linked immunosorbent assays (ELISA) provided by the MOLEQULE-ON ELISA Kit (MOLEQULE-ON, Auckland, New Zealand). The studies were conducted following the protocol provided by the manufacturer. Each sample determination was performed twice to ensure accuracy and reliability. The results were determined by employing the standard curves generated during each assay. The cytokine concentrations were adjusted for GCF volume and expressed as picograms per milliliter (pg/ml). The overall quantity of cytokines in the GCF was expressed in picograms (pg).
Statistical Analysis
The results were statistically analyzed using SPSS Statistics 26 software (IBM Corp., Armonk, NY, USA). The values of demographic data and clinical characteristics are presented as means and standard deviations. The Independent Samples t-test was used to test the statistical differences between the means of two groups (diabetes and control). A p-value of 0.05 or less was considered statistically significant. On the other hand, the cytokine levels in GCF were expressed as mean ranks, and the significance of differences between groups was assessed using the Mann-Whitney test, as the data were not normally distributed. A p-value of 0.05 or less was considered statistically significant. To evaluate the relationship between cytokine levels and periodontal parameters, a Spearman's rank correlation analysis was performed, as the data were not normally distributed.
Results
Demographic Data and the Clinical Characteristics
Table 1 summarizes the key characteristics of the study, comparing the control group (17 participants) and the diabetes group (14 participants). The table includes data on age, HbA1c levels, mean probing depth, mean attachment level, plaque percentage, and bleeding on probing (BoP) percentage. Regarding age, the control group had a mean age of 33 years (SD = 13), while the diabetes group had a mean age of 42 years (SD = 15). There was no significant difference between the groups in terms of mean age. For HbA1c levels, the control group had a mean of 5.2 ± 0.49, whereas the diabetes group had a significantly higher mean HbA1c level of 9.17 ± 1.62 (p=0.0001).
In terms of clinical periodontal parameters, the control group had a mean probing depth of 2.22 mm, while the diabetes group had a slightly higher mean probing depth of 2.87 mm. The mean attachment level in the control group was -2.24 mm, compared to -3.14 mm in the diabetes group, indicating greater attachment loss in the latter group.
The plaque percentage was also significantly higher in the diabetes group (42.50%) compared to the control group (18.29%). Similarly, the rate of individuals experiencing bleeding on probing was higher in the diabetes group (45.36%) compared to the control group (12.58%). These results demonstrate significant differences between the two groups in terms of mean HbA1c, probing depth, attachment level, plaque percentage, and bleeding on probing. However, no significant difference was found between the groups in terms of mean age.
The analysis results are summarized in Table 2, which presents the mean ranks for three cytokines: TNF-α, IL-1β, and IL-6. For TNF-α, the control group (n = 17) had a mean rank of 9.50, whereas the diabetes group (n = 14) had a significantly higher mean rank of 23.89. Similarly, for IL-1β, the control group had a mean rank of 9.71, while the diabetes group had a mean rank of 23.64. For IL-6, the control group showed a mean rank of 14.41, while the diabetes group had a mean rank of 17.93.
The Mann-Whitney U test was used to compare the cytokine levels between the two groups. The test results yielded U values of 8.500 for TNF-α, 12 for IL-1β, and 92 for IL-6. Statistical significance was assessed using two-tailed tests, with the Asymptotic Significance (p-values) reported for each comparison. The p-values for TNF-α and IL-1β were both < 0.001, indicating a significant difference between the control and diabetes groups. However, for IL-6, the p-value was 0.283, suggesting no significant difference between the two groups.
Correlation analysis revealed that tumor necrosis factor-alpha (TNF-α) showed strong and statistically significant positive correlations with all periodontal clinical parameters and HbA1c levels. Specifically, TNF-α was significantly correlated with HbA1c (r = 0.733, p<0.001), mean probing depth (r = 0.699, p<0.001), mean attachment level (r = 0.738, p<0.001), plaque percentage (r = 0.566, p<0.001), and bleeding on probing percentage (r = 0.608, p<0.001). Interleukin-1 beta (IL-1β) also demonstrated significant positive correlations with all measured parameters, including HbA1c (r = 0.670, p < 0.001), mean probing depth (r = 0.521, p<0.001), mean attachment level (r = 0.590, p<0.001), plaque percentage (r = 0.604, p<0.001), and bleeding on probing percentage (r = 0.596, p<0.001). In contrast, interleukin-6 (IL-6) did not show significant correlations with clinical parameters and HbA1c.
Discussion
This study aimed to compare the prevalence of periodontitis between healthy individuals and those with diabetes by measuring the levels of TNF-α, IL-1β, and IL-6 cytokines in gingival crevicular fluid (GCF). As expected, the HbA1c levels were significantly higher in the diabetes group compared to the control group, aligning with previous research [6] that has shown elevated HbA1c levels in individuals with diabetes due to increased blood glucose levels. However, the effect of age on HbA1c was not evaluated in the current study. While some studies have reported a correlation between age and HbA1c fluctuations, with younger individuals often showing higher HbA1c levels than older adults [2], this was not a focus of our analysis. It is noteworthy that the diabetes patients demonstrated average attachment levels and probing depths that were both greater than those of the control group in the present study. The latter findings of this study align with those reported before, which demonstrated that increased probing depths were associated with tooth attachments and bone loss induced by diabetes in diabetes patients [8]. Plaque formation around the tooth, observed in diabetes patients with greater probing depths, may be associated with the loss of alveolar bone surrounding the tooth. Consistent with the results reported before, which reported relatively higher rates of Plaque formation in diabetes patients due to the role of glucose in feeding harmful bacteria in the mouth, it was established from the findings of this study that diabetes patients exhibited a higher Plaque percentage compared to the control/healthy group of participants [8,9]. However, another study found that immune responses and the presence of inflammation caused by diabetes could also be a defining factor in the increased plaque formation in periodontitis cases of diabetic patients [18].
T-test analysis between the control/healthy group and the diabetes patient group showed significant differences in terms of bleeding on probing, with diabetes patients experiencing higher bleeding rates. These findings align with those published by Manjushree et al. [5], who attributed bleeding to attachment loss around the tooth caused by inflammation resulting from high glucose levels circulating in the mouth region. It is imperative that diabetes results in higher glucose levels, which decreases the immune responses in the areas around the tooth, thereby causing higher bacterial build-up, plaque formation, and bone and attachment loss, ultimately exacerbated by periodontitis diseases.
ELISA was used to determine the concentrations of the cytokines IL-1β, TNF-α, and IL-6. The findings revealed that the diabetes group exhibited elevated IL-1β, TNF-α, and IL-6 cytokines compared to the control and healthy groups. A t-test analysis yielded a P-value of 0.0001, indicating statistically significant differences between the two groups. The results of this research align with those of prior investigations, which have similarly observed that individuals with diabetes exhibit elevated concentrations of cytokines, such as IL-1β, TNF-α, and IL-6 [9, 10, 13, 15, 16, 27]. Nevertheless, this study documented significant fluctuations in the concentrations of these cytokines among individuals with diabetes, mirroring findings from other research [10,11,13] that observed IL-6 and IL-5 to be significantly more prevalent than TNF-κ, and that the duration of diabetes influenced these concentrations [14]. IL-1β and TNF-α are pro-inflammatory cytokines that contribute to periodontal tissue destruction by promoting inflammatory responses and matrix degradation. High blood glucose levels in diabetic individuals can lead to enhanced production of these cytokines, which then exacerbate the progression of periodontal disease.
On the other hand, the lack of a significant difference in IL-6 levels between the two groups may be explained by the multifaceted role of IL-6 in inflammation. IL-6 is involved in both pro-inflammatory and anti-inflammatory pathways. While it is elevated in the bloodstream of diabetic individuals, its localized effects in gingival tissues may not be as pronounced as those of IL-1β and TNF-α. This complexity suggests that other factors, such as the duration of diabetes, overall glycemic control, and the presence of comorbidities, may influence IL-6 expression in periodontal tissues. In general, it was apparent that diabetes patients exhibited the presence of cytokines that are strongly associated with inflammation [11,12]. This association heightens the risk of periodontal disease and complicates treatment, indicating that diabetes patients require specialized periodontal disease treatment.
This study has several limitations that should be taken into account when interpreting the findings. First, the final sample size was relatively small (n = 31), comprising 17 healthy participants and 14 individuals with diabetes, which falls short of the originally intended 60 participants. This limited sample reduces the statistical power of the study and may have hindered the detection of significant differences between groups. Additionally, the cohort may not adequately represent the broader diabetic population, particularly individuals with type 1 or type 2 diabetes who present with multiple complications, poor glycemic control, irregular clinical follow-up, or advanced periodontal disease. These exclusions limit the generalizability of the findings.
Furthermore, due to the exclusive use of gingival crevicular fluid (GCF) sampling for periodontal assessment, it was not feasible to compare specific periodontal sites (i.e., healthy vs. diseased) within participants. The absence of radiographic evaluation also meant that the 2017 World Workshop classification system for periodontitis, which requires both clinical and radiographic data, could not be applied. Lastly, the cross-sectional nature of the study precludes any conclusions about causality between diabetes and periodontal inflammation. Future studies with larger, more diverse samples and longitudinal designs are necessary to validate and expand upon these preliminary findings.
The findings of this research suggest that diabetes has a significant influence on the progression and management of periodontitis. Specifically, biomarkers, including cytokines, indicate that patients with diabetes have a compromised immune system, which increases their susceptibility to periodontal disease. Therefore, when treating diabetes patients, particular care must be taken to reduce the risk of developing the disease.
Conclusion
Patients diagnosed with diabetes exhibited elevated concentrations of IL-1β and TNF-α, cytokines, compared to the control group, as indicated by the findings of this study. The increased incidence of periodontitis among individuals with diabetes may be attributed to the compromised immune system and inflammation in the periodontal tissues, which contribute to the deterioration of tooth attachment and the surrounding bone tissue. Diabetes patients should be treated with exceptional care for periodontitis, with blood sugar control being of the utmost importance. Further research is needed to compare participants with varying degrees of periodontal inflammation and poorly managed versus well-controlled diabetes to gain a more comprehensive understanding of site-specific periodontal destruction in this population.
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Financial Support
None.
Acknowledgments
We are grateful to the study participants and the study physicians, nurses, and other staff who contributed to the collection of data for this study.
Data Availability
The data used to support the findings of this study can be made available upon request to the corresponding author.
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Edited by
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Academic Editor:
Alessandro Leite Cavalcanti
