Abstract The objective of this study was to evaluate serum C-reactive protein (CRP) and gingival crevicular fluid (GCF) osteoprotegerin (OPG) and neutrophil elastase as biomarkers for periodontal disease diagnosis in patients with Type 2 diabetes mellitus (T2DM) compared with non-diabetic periodontitis patients and healthy controls. This case-control study included 90 participants aged 20–60 years, divided into three groups: healthy controls (n = 30), periodontitis with T2DM (n = 30), and periodontitis without diabetes (n = 30). Periodontal parameters (probing depth, clinical attachment loss, bleeding on probing, and plaque index) were recorded. Serum CRP was measured using a Cobas 411 analyzer, while GCF OPG and neutrophil elastase were assessed by ELISA. Data were analyzed using ANOVA with significance different (P < 0.05). Periodontal parameters were significantly higher in both periodontitis groups compared to controls. Serum CRP levels were highest in controls and significantly lower in periodontitis groups, with the lowest levels observed in non-diabetic periodontitis. GCF OPG levels were highest in controls and significantly reduced in disease groups; however, patients with T2DM showed intermediate OPG levels, significantly higher than those without diabetes. Neutrophil elastase levels were markedly elevated in both periodontitis groups, with the highest levels in diabetic periodontitis. A strong positive correlation between CRP and OPG was observed in the non-diabetic periodontitis group.

Keywords: Type 2 diabetes mellitus, Periodontitis, Gingival crevicular fluid (GCF), C-reactive protein (CRP), Osteoprotegerin (OPG), Neutrophil elastase

Citation: Mohammed, H.A. and Talib, E.Q. 2026. Assessment of serum CRP and gingival crevicular fluid OPG and neutrophil elastase as biomarkers for periodontal disease in type 2 diabetes mellitus. Natural and Life Sciences Communications. 25(3): e2026066.

Graphical Abstract:

INTRODUCTION

Periodontal disease is one of the most common chronic inflammatory disorders affecting humans, characterized by the destruction of the tooth-supporting tissues including the periodontal ligament and alveolar bone (Ray, 2023). The prevalence of periodontitis is particularly high among adults, and its negative effects extend beyond oral health, affecting quality of life and systemic health (Agnese et al., 2025). Among the systemic conditions influencing periodontal disease, Type 2 Diabetes Mellitus (T2DM) is of special significance due to its widespread prevalence and intricate interaction with periodontal inflammation (Mohamed et al., 2015; Păunică et al., 2023).

T2DM is a metabolic disorder defined by insulin resistance and relative insulin deficiency, leading to chronic hyperglycaemia (Zhao et al., 2023). It is well-established that individuals with T2DM are at an increased risk of developing periodontitis, with evidence indicating susceptibility rates two to three times higher than those observed in non-diabetic individuals (Zhao et al., 2023). The degree and duration of hyperglycaemia play a critical role in determining the severity and progression of periodontal disease, and poor glycaemic control is associated with more severe periodontal breakdown, greater clinical attachment loss, and increased tooth loss (Graves et al., 2020; Mirzaei et al., 2021). Importantly, this relationship is bidirectional: not only does T2DM exacerbate periodontal inflammation and tissue destruction, but active periodontitis can also negatively impact glycaemic control, complicating diabetes management (Preshaw et al., 2012).

The underlying mechanisms linking T2DM and periodontitis involve a combination of altered immune response, increased inflammatory mediator production, enhanced oxidative stress, and shifts in oral microbiota composition (Zhao et al., 2023). Hyperglycaemia in T2DM leads to the formation of advanced glycation end-products (AGEs), which interact with their receptors (RAGE) in periodontal tissues, thereby provoking pro-inflammatory cytokine release, upregulating the RANKL/OPG system, and promoting alveolar bone resorption (Preshaw et al., 2012; Zhao et al., 2023). Diabetic patients also exhibit impaired neutrophil function and reduced tissue repair capabilities, making them more susceptible to persistent and severe periodontal infection (Graves et al., 2020).

Given the complex interplay between diabetes and periodontitis, biomarkers capable of reflecting local immune and systemic inflammatory status are of growing importance for early diagnosis, prognosis, and therapeutic monitoring (Cafiero et al., 2021; Hajishengallis and Chavakis, 2021). Serum C-reactive protein (CRP) serves as a systemic marker of inflammation, while gingival crevicular fluid (GCF) biomarkers such as osteoprotegerin (OPG) and neutrophil elastase provide insights into bone remodelling and tissue breakdown at the periodontal site (Zhao et al., 2023). The measurement of these markers in T2DM patients with periodontal disease could help unravel disease activity, forecast risk, and guide intervention (Graves et al., 2020).

With mounting evidence supporting the strong association between T2DM and periodontitis, as well as the utility of specific immune biomarkers in disease detection, research in this area is pivotal to improve integrated management strategies. Early identification of periodontal inflammation in T2DM patients may not only support oral health but also improve overall glycaemic control and reduce systemic complications (Zhao et al., 2023).

Aim of this study

To evaluate the levels of C-Reactive Protein (CRP) in serum, and Osteoprotegerin (OPG) and Neutrophil Elastase in Gingival Crevicular Fluid (GCF) as biomarkers for periodontal disease diagnosis in Type 2 Diabetes Mellitus.

MATERIALS AND METHODS

Study design and subjects

In this case–control study was conducted over a period between November 2024 to April 2025. A total of 90 Participants were recruited attending the College of Dentistry clinics using a convenience sampling method. Eligible subjects were screened according to predefined inclusion and exclusion criteria and were then allocated into three groups: 30 healthy controls, 30 periodontitis without diabetes, and 30 periodontitis with Type 2 Diabetes Mellitus.

This case–control observational study was reported in accordance with the STROBE guidelines (Supplementary file 1).

All participants provided informed consent prior to enrollment.

Sample size calculation

Using G power 3.1.9.7 by Franz-Faul at Universität Kiel, Germany, the sample size was determined with a study power of 0.97%, a two-sided alpha error probability of 0.05, and a large effect size of 0.80 for three groups. This resulted in a sample size of 82 subjects, increased by 10% to account for error, totaling 90 subjects.

Ethics approval

The study protocol was reviewed and approved by the Research Ethics Committee of the Al-Iraqia University/ College of Dental/Teaching Hospital (Approval No. 102, November, 2024). Written informed consent was obtained from all participants before enrollment.

Inclusion criteria

The following criteria were required for participation:

1. Aged between 20 and 60 years.

2. Good general health, with the exception of a T2DM diagnosis for participants in Group 2.

3. For Groups 2 and 3, a diagnosis of periodontitis based on the criteria established by the American Academy of Periodontology (AAP).

Exclusion criteria

Individuals were excluded from the study if they met any of the following conditions:

1. Had used antibiotics or anti-inflammatory drugs within the past 3 months.

2. Were current smokers or users of tobacco in any form.

3. Were pregnant or lactating.

4. Had a history of systemic diseases known to affect periodontal health (e.g., autoimmune diseases).

5. Had undergone periodontal treatment in the 6 months preceding the study.

Regrading healthy control group, included systemically healthy individuals with clinically healthy periodontium, defined by probing pocket depth (PPD) ≤3 mm, absence of clinical attachment loss (CAL), no bleeding on probing, and no radiographic evidence of alveolar bone loss. Participants were required to have at least 20 natural teeth. Individuals were excluded if they had Type 2 Diabetes Mellitus or any systemic inflammatory disease, history of periodontal therapy within the previous six months, use of antibiotics, anti-inflammatory or immunosuppressive medications within the past three months, smoking habit, pregnancy or lactation, or any other oral inflammatory condition that could influence biomarker levels.

Periodontal diagnosis

Patients were diagnosed with periodontitis according to the 2017 World Workshop Classification of Periodontal and Peri-Implant Diseases and Conditions, developed jointly by the American Academy of Periodontology and the European Federation of Periodontology.

Oral examination

A comprehensive oral examination was performed for all participants to assess their periodontal status. The following clinical parameters were recorded:

•   Probing Depth (PD): Measured in millimeters from the gingival margin to the base of the periodontal pocket (Berglundh et al., 2018).

•   Clinical Attachment Loss (CAL): Measured in millimeters from the cementoenamel junction to the base of the periodontal pocket (Berglundh et al., 2018).

•   Bleeding on Probing (BOP): Recorded as present (1) or absent (0) within 30 seconds after probing (Renvert et al., 2018).

•   Plaque Index (PI): Assessed to evaluate oral hygiene status (Silness and Löe, 1964; Lindhe and Meyle, 2008).

Periodontal diagnosis, staging, and grading

Sample collection

Blood Sample:

A 5 mL venous blood sample was drawn from each participant in the morning between 8:00 AM and 10:00 AM after an overnight fasting period of approximately 8–12 hours. The blood was centrifuged to separate the serum, which was then aliquoted and stored at -20°C until analysis (Talib and Taha, 2024).

The blood sample was used for two purposes

Approximately 2 mL of blood was collected in EDTA tubes for the measurement of glycated hemoglobin (HbA1c), using whole blood samples. The remaining blood, about 3 ml, was collected in plain tubes, allowed to clot, and then centrifuged to separate the serum. The serum was aliquoted and stored at −20°C until analysis.

C-reactive protein (CRP), lipid profile parameters including total cholesterol (Chol), triglycerides (TG), very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and uric acid (U.A.) were measured in serum using the Cobas e 411 analyzer.

Gingival crevicular fluid (GCF) collection:

The GCF collection site was isolated using cotton rolls and gently air-dried to prevent contamination. Using a calibrated microcapillary pipette, a standardized volume of 2 μL of GCF was collected from the deepest periodontal pocket in participants with periodontitis (Ali and Taha, 2024) (Groups 2 and 3) or from a healthy interproximal site in control participants (Group 1). The GCF samples were immediately transferred to Eppendorf tubes and stored at -80°C until further analysis by Enzyme-linked Immunosorbent Assay (ELISA).

Enzyme-linked immunosorbent assay (ELISA)

Kits and assay procedure:

The levels of the biomarkers in the collected samples were quantified using commercially available ELISA kits according to the manufacturers' protocols.

Osteoprotegerin (OPG) and neutrophil elastase:

The concentrations of OPG (Cat: ELK1155/ ELK Biotechnology/USA) and Neutrophil Elastase (Cat: ELK1076// ELK Biotechnology/USA) in the GCF samples were determined using specific, commercially available ELISA kits. The assays involved adding samples and standards to antibody-coated wells, followed by a series of incubations and washes. The reaction was developed with a substrate, and the intensity of the color formed was measured spectrophotometrically, with the concentration being proportional to the color intensity.

Statistical analysis

Data were analyzed using SPSS version 26 and Excel 2019. Normality was tested using the Shapiro-Wilks test. Comparisons between groups and genders were conducted using ANOVA test and LSD, Games-Howell, Chi-square, with significance set at P < 0.05.

RESULTS

Table 1 shows the sex distribution across the three study groups. There was not-significant difference in gender distribution between groups (P > 0.05), indicating that sex was balanced among the control, periodontitis with diabetes, and periodontitis without diabetes groups.

Table 1. Demographic distribution of participants by sex and study group.

Age differed significantly between groups (P < 0.0001). The periodontitis without diabetes group had the highest mean age (years), followed by the periodontitis with diabetes group and the control group. As demonstrated in Table 2.

Table 2. Age distribution across study groups.

All periodontal parameters (PD, BOP, PI, CAL) in Table 3 showed significant differences between groups (P < 0.05). Periodontitis groups (with and without diabetes) had higher probing depth, bleeding on probing, plaque index, and clinical attachment loss compared to the control group.

Table 3. Level of periodontal clinical parameters (PD, BOP, PI and CAL) among study groups.

HbA1c, cholesterol, triglycerides, and VLDL levels were significant different between groups (P < 0.05). The periodontitis with diabetes group had the highest mean HbA1c, while non-significant differences were found in LDL, HDL and uric acid levels. (Table 4).

Table 4. Levele of biochemical biomarkers (HbA1c, liped profile and uric acid) among study groups.

CRP were increased levels in the control group and significantly decreased in the periodontitis groups, with the lowest levels observed in the PD without diabetes group.

Regarding OPG levels were highest in the control group and progressively decreased in the periodontitis groups, with the lowest levels in the PD without diabetes group and increased in PD with diabetes group.

Neutrophil elastase levels were elevated significant in both periodontitis groups compared to controls, with the highest levels in the PD with diabetes group and PD without diabetes group. As shown in Table 5.

Table 5. Mean levels of serum CRP and GCF immune biomarkers (OPG and neutrophil elastase) in control, periodontitis without diabetes, and periodontitis with type 2 diabetes mellitus groups.

In Table 6, CRP was significantly lower in the PD without diabetes group compared to both the control (P < 0.05) and PD with diabetes groups (P = 0.010). Not significant difference was found between control and PD with diabetes (P = 0.355).

OPG was significantly lower in periodontitis groups, with the largest decrease seen in the PD without diabetes group compared to controls (P < 0.05). The PD with diabetes group also showed a significant reduction compared to controls (P = 0.0001), and was significantly higher than the PD without diabetes group (P = 0.000). (Table 6)

Neutrophil elastase was significantly elevated in both periodontitis groups compared to controls, with the greatest increase in the PD with diabetes group (P < 0.05). The PD with diabetes group also showed significantly higher levels than the PD without diabetes group P = 0.001). (Table 6).

Table 6. Post hoc multiple comparisons of CRP, OPG, and neutrophil elastase levels between study groups.

In the periodontitis without diabetes group, there was a strong positive correlation between CRP and OPG (P <0.05), HbA1c and OPG, HbA1c and CRP. Non significant correlations were found between biomarkers in the periodontitis with diabetes group. As shown in Table 7.

Table 7. Correlation Analysis between immune biomarkers and glycemic status in periodontitis groups with and without type 2 diabetes mellitus.

Note: r= Pearson correlation, NS: Non-Significant.

DISCUSSION

The present study evaluated serum C-reactive protein (CRP) and gingival crevicular fluid (GCF) levels of osteoprotegerin (OPG) and neutrophil elastase as potential biomarkers for periodontal disease in type 2 diabetes mellitus (T2DM) and non-diabetic individuals.

Serum CRP is a well-established marker of systemic inflammatory burden and is known to be elevated in various chronic inflammatory conditions including periodontitis and diabetes. However, in our study, CRP levels were unexpectedly highest in healthy controls and decreased with periodontal disease presence, particularly in non-diabetic periodontitis. CRP production is primarily hepatic but can increase locally in inflamed periodontal tissues and spill into circulation.

The finding that CRP levels were highest in the healthy control group and decreased in periodontitis patients, particularly in the non-diabetic group, appears contrary to the conventional understanding that CRP increases with inflammatory burden. However, several factors may explain this observation. First, CRP is a systemic acute-phase protein synthesized by the liver and primarily reflects systemic inflammation rather than localized periodontal inflammation. Periodontitis is a chronic low-grade inflammatory condition, and localized periodontal inflammation may not consistently induce marked systemic CRP elevation, especially in otherwise healthy individuals. Moreover, CRP levels can be influenced by multiple confounding factors including subclinical infections, obesity, lifestyle habits, and metabolic variations, which may not be fully captured during screening.

Previous studies have reported heterogeneous findings regarding CRP levels in periodontal disease. Some investigations demonstrated elevated CRP in periodontitis patients, supporting a systemic inflammatory link.

Study by (Mohan et al., 2014) reports generally describe high level CRP in periodontitis, reflecting systemic inflammatory stimulation by periodontal infections, with higher CRP in T2DM patients with periodontitis compared to healthy controls (Mohan et al., 2014).

Another study by Jayaprakash et al. (2014) show CRP reductions after periodontal therapy, which supports that CRP can reflect disease inflammatory burden rather than being disease-specific (Jayaprakash et al., 2014). The lower CRP in periodontitis in your cohort may reflect variations in systemic inflammatory set points, sample differences, or compensatory immune responses in chronic periodontitis, rather than acute systemic inflammation.

However, Baser et al. (2014) show non significant difference in CRP between disease severity categories or between serum and GCF CRP, indicating that CRP responses can be variable and may not directly reflect periodontal severity across all populations (Baser et al., 2014).

Rapone et al. (2021) show CRP reductions after periodontal therapy only when glycemic control improves, indicating that CRP may reflect a combined systemic inflammatory burden rather than solely periodontal inflammation (Rapone et al., 2021).

Osteoprotegerin (OPG) is a decoy receptor for RANKL and a key regulator of osteoclast activity and bone resorption. In healthy periodontal conditions, OPG helps preserve alveolar bone by neutralizing RANKL’s pro-osteoclastic effects. In line with Hassan et al. (2015), the current results show higher OPG in healthy controls and lower in periodontitis, consistent with enhanced bone resorption in diseased states.

The partial increase in OPG among periodontitis patients with diabetes suggests a complex interplay between local bone metabolism regulators and systemic metabolic factors that may modify the host response, potentially as a compensatory attempt to limit bone loss (Hassan et al., 2015).

Consistent with Mogi et al. (2004) GCF OPG is generally reduced in periodontal disease relative to healthy controls, reflecting a shift toward alveolar bone destruction (Mogi et al., 2004).

In contrast, Senkal et al. (2024) who found a non-significant differences in serum OPG between periodontitis and healthy subjects, indicating that local changes in GCF might be more sensitive to periodontal status than systemic levels (Senkal et al., 2024).

Actually, the greater reduction in OPG in PD without diabetes compared to PD with diabetes, and the intermediate level in the PD with diabetes group, may reflect a complex interaction between diabetic metabolic factors and local periodontal inflammatory pathways. Diabetes is known to affect bone metabolism and bone-related cytokine expression, which can alter the RANKL/OPG balance. These findings align with study by Xu et al. (2015) demonstrated that serum OPG levels were lower in patients with T2DM and chronic periodontitis compared to periodontitis alone and healthy controls, implying that diabetes exacerbates disruption in bone regulation (Xu et al., 2016).

Neutrophil elastase is a proteolytic enzyme released by activated neutrophils, capable of degrading extracellular matrix proteins and contributing to periodontal tissue destruction. The observed elevated elastase in periodontitis groups, with the highest levels in T2DM associated periodontitis, supports its role as an indicator of heightened local inflammatory activity and neutrophil infiltration at disease sites (Alpagot et al., 2001). Previous research by Smith et al. (1995) shown that GCF neutrophil elastase is higher in periodontitis compared to health, validating its association with active periodontal tissue breakdown and inflammation.

Excessive neutrophil elastase activity contributes to connective tissue breakdown and may further amplify inflammatory cascades by promoting pro-inflammatory cytokine release within the periodontal microenvironment (Tseng et al., 2022).

According to Alpagot et al. (2001), not all populations show the same magnitude of change, potentially due to differences in periodontal severity or metabolic control, suggesting that neutrophil elastase may be influenced by both local disease activity and systemic conditions like diabetes (Alpagot et al., 2001).

In the present study, a strong positive correlation was observed between serum CRP and GCF OPG in the periodontitis without diabetes group (r = 0.799, P = 0.000), indicating that as systemic inflammatory burden increases, there is a concomitant increase in local bone remodeling regulatory activity marked by OPG. This relationship suggests a linked host response in which systemic inflammation, reflected by CRP, may stimulate compensatory mechanisms in local bone protective pathways, even though absolute OPG levels were reduced in periodontitis. CRP is an acute-phase reactant that increases in response to pro-inflammatory cytokines and has been widely documented to reflect chronic low-grade inflammation associated with periodontal disease and other systemic conditions. Indeed, Paraskevas et al. (2007) show elevated serum CRP levels in periodontitis patients compared with controls, indicating a systemic inflammatory component of periodontal disease (Paraskevas et al., 2008).

Although much of the literature emphasizes CRP as a marker of systemic inflammation, its association with osteoprotegerin underscores interconnected inflammatory and bone metabolic pathways in periodontal pathology. OPG acts as a decoy receptor for RANKL to inhibit osteoclastogenesis and protect against alveolar bone loss; reductions in OPG have been consistently observed in periodontitis, reflecting dysregulated local bone metabolism (Xu et al., 2016).

Although several previous studies have evaluated individual biomarkers such as neutrophil elastase, osteoprotegerin (OPG), and C-reactive protein (CRP) in periodontal disease, the present study provides additional value by simultaneously assessing systemic and local immune biomarkers in patients with periodontitis with and without Type 2 Diabetes Mellitus. Unlike earlier investigations that focused on single-marker analysis, this study explored the combined diagnostic relevance of serum CRP and gingival crevicular fluid OPG and neutrophil elastase within the same patient population.

LIMITATIONS

The relatively small sample size, although statistically calculated, may limit the generalizability of the findings to larger populations.

The study was conducted over a limited time period, which may not fully reflect long-term variations in immune biomarker levels.

Occasional delays and limited availability of laboratory kits may have influenced the timing of sample analysis, although standardized procedures were followed to minimize potential bias.

CONCLUSION

This study highlights the distinct roles of systemic and local biomarkers in the pathology of periodontitis associated with Type 2 Diabetes Mellitus. Neutrophil elastase in gingival crevicular fluid emerged as a sensitive marker for local periodontal destruction, with significantly elevated levels in diabetic patients reflecting heightened neutrophil activity and tissue breakdown exacerbated by hyperglycaemia.

In contrast, the reduction of Osteoprotegerin (OPG) in periodontitis groups confirms the dysregulation of the RANKL/OPG system favoring bone resorption in diseased states. Interestingly, the intermediate decrease of OPG in diabetic periodontitis patients compared to non-diabetic patients suggests a complex metabolic interference that may modulate local bone protective mechanisms. Furthermore, the unexpected inverse relationship observed with serum CRP challenges its utility as a linear marker for periodontal severity in this specific population, suggesting it may reflect a broader, multifactorial systemic inflammatory burden. Future research should focus on longitudinal multicenter studies with larger sample sizes to confirm the predictive value of these biomarkers over time.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the College of Dentistry, Al-Iraqia University, Baghdad, Iraq.

AUTHOR CONTRIBUTIONS

Haneen Ahmed Mohammed: Data Curation (Equal), Formal Analysis (Equal), Validation (Equal), Visualization (Equal), Writing – Review & Editing (Equal); Ehab Qasim Talib: Conceptualization (Lead), Data Curation (Lead), Software (Lead), Validation (Lead), Writing – Original Draft (Lead).

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

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