Introduction: Apical periodontitis (AP) is a chronic inflammatory condition resulting from microbial infection of the dental pulp. The host immune response and microbial interactions play a significant role in the disease’s progression. Gingival crevicular fluid (GCF) provides a valuable, non-invasive source for detecting inflammatory biomarkers involved in AP, such as IL-1β, IL-10, and IL-23.

Aim: To evaluate the levels of IL-1β, IL-10, and IL-23 in GCF among individuals with symptomatic apical periodontitis (SAP), asymptomatic apical periodontitis (AAP), and healthy controls, and to assess their potential as diagnostic biomarkers.

Materials and methods: This cross-sectional study included 90 participants aged 20–50 years, divided into three groups: SAP (n=30), AAP (n=30), and healthy controls (n=30). GCF samples were collected using Periostrips, and cytokine levels were measured using enzyme-linked immunosorbent assay (ELISA). Statistical analysis was performed using SPSS version 26, with significance set at p<0.05.

Results: IL-1β, IL-10, and IL-23 levels were significantly elevated in the SAP group compared to the AAP and control groups (p<0.01). IL-10 showed the highest diagnostic accuracy (AUC=0.806), followed by IL-1β (AUC=0.714). IL-23, although significantly elevated in SAP, had lower diagnostic value (AUC=0.636). Strong positive correlations were observed between IL-1β and IL-10, as well as IL-1β and IL-23.

Conclusion: The elevated levels of IL-1β, IL-10, and IL-23 in GCF reflect their involvement in the inflammatory processes of apical periodontitis. IL-10 demonstrated the greatest potential as a diagnostic biomarker. These findings support the clinical utility of GCF cytokine profiling for non-invasive diagnosis and monitoring of AP progression.

apical periodontitis, gingival crevicular fluid, IL-1β, IL-10, IL-23, diagnostic biomarkers

Apical periodontitis commonly results from microbial infection of the dental pulp, an inflammation of the alveolar bone and adjacent tissues at the apex of the tooth.^[1–3] Clinically, distinguishing between symptomatic (SAP) and asymptomatic apical periodontitis (AAP) is crucial, as it directly influences treatment urgency, decision-making, and prognosis. Timely identification of SAP can prevent progression to more severe periapical pathology and patient discomfort.^[1–3]

The body makes an attempt to control the infection and cease its spread when the bacteria cannot be eradicated by the body. This response results in the breakdown of apical tissues and formation of an osteolytic abscess, which is a characteristic feature of chronic apical periodontitis.^[4–6] In AP, there is clinical diversity, and it is classified apically into two types of periodontitis, namely symptomatic apical periodontitis (SAP) and asymptomatic apical periodontitis (AAP), based on the presence or absence of clinical symptoms.^[7] This clinical variability is thought to be due to the immune system fine-tuning of bacterial products with the host’s.^[8] The study has shown that symptomatic apical periodontitis is related to the alterations in microbial flora and richness as well as modulations in the host’s immunological profile.^{[4, 6, 8]}

Gingival crevicular fluid (GCF) is a vital biological fluid that can reveal underlying mechanisms through analysis of its composition and flow rate. Although GCF is primarily used in periodontal research, its proximity to apical tissues and its ability to reflect local inflammatory responses make it a promising diagnostic medium for endodontic inflammatory conditions such as apical periodontitis.^{[9, 10]}

The increased production of cytokines affects the balance of periodontal tissues, and the severity of the periodontal disease depends on the quantity of matrix metalloproteinases (MMPs), which are found in saliva and gingival crevicular fluid.^{[11, 12]}

IL-1β is a key pro-inflammatory cytokine that mediates tissue destruction and bone resorption, processes central to the pathogenesis of apical periodontitis. Its levels are typically elevated in symptomatic lesions, making it a potential marker of acute inflammation.^[13]

IL-10, in contrast, serves as a major anti-inflammatory cytokine that modulates immune responses by inhibiting the synthesis of pro-inflammatory mediators. Its presence may reflect host efforts to counteract excessive tissue damage, especially during early or subclinical phases of AP.^{[14, 15]}

IL-23 is involved in the maintenance and proliferation of Th17 cells, which contribute to chronic inflammation and osteolysis. Its role in sustaining inflammatory responses suggests potential as a biomarker for disease severity and chronicity in apical lesions.^{[14, 15]} Thus, multiple studies have investigated the diagnostic potential of IL-1β, IL-10, and IL-23 in oral fluids as chair-side or point-of-care testing indicators for periodontal and peri-implant diseases.^{[14, 15]}

Recent studies have highlighted the diagnostic potential of IL-1β in GCF collected from teeth affected by apical periodontitis (AAP). Evidence suggests that IL-1β levels in GCF are significantly influenced during the course of AAP.^{[16, 17]}

However, despite evidence of cytokine involvement in apical periodontitis, there remains a lack of comprehensive studies evaluating the diagnostic utility of GCF-derived cytokines, particularly IL-1β, IL-10, and IL-23, for distinguishing between symptomatic and asymptomatic forms of the disease.

The study aimed to compare IL-1β, IL-10, and IL-23 levels in GCF between control and study groups.

This cross-sectional study included ninety individuals aged 20 to 50 years who attended the teaching clinics of the College of Dentistry, Al-Iraqia University. Participants were recruited based on clinical and radiographic diagnoses and were classified into three groups: 30 patients with symptomatic apical periodontitis (SAP), 30 with asymptomatic apical periodontitis (AAP), and 30 healthy controls. The healthy controls were confirmed through both clinical examination and periapical radiographs, ensuring the absence of symptoms, pathology, or periapical radiolucency.

Classification of teeth into symptomatic or asymptomatic apical periodontitis was based on both clinical signs and radiographic findings. Symptomatic AP (SAP) was diagnosed in teeth that presented with spontaneous pain, tenderness to percussion or palpation, and radiographic evidence of periapical radiolucency. Asymptomatic AP (AAP) was identified in teeth with no clinical symptoms but with radiographic evidence of periapical pathology, such as well-defined periapical radiolucency, without sensitivity to percussion or palpation.^[7]

Gingival crevicular fluid sample collection

Sampling was performed from single-rooted and multi-rooted posterior teeth (molars and premolars), including both maxillary and mandibular arches, depending on the location of the diagnosed apical lesion.

To ensure consistency, GCF samples were always collected from the same four standardized sites per participant: mesiobuccal, distobuccal, mesiopalatal, and distopalatal on the selected tooth.

GCF samples were collected from individuals between 9:00 AM and 2:30 PM using Periostrip from four quadrants of each right and left UFP (mesiobuccal, distobuccal, mesiopalatal, and distopalatal). Initially, the tooth surface was softly swabbed with cotton, followed by gentle drying of the teeth using an air syringe aimed occlusally; cotton-wool rolls were used for precise area isolation. A periostrip is inserted into the gingival crevice, 1 mm subgingivally, for 30 sec. Saliva- or blood-contaminated strips were disposed of during this surgery.^[19] To maintain sample integrity, the paper strips were promptly placed in sterile Eppendorf tubes containing 0.5 mL of PBS. The tubes underwent centrifugation at 3000 rpm for a duration of 10 minutes and were subsequently frozen at −20°C until laboratory analysis (Fig. 1).

Figure 1.

Gingival crevicular fluid collection.

Table 1 shows that the mean age of participants in the symptomatic apical periodontitis group was 36.67±6.76 years, the asymptomatic apical periodontitis group was 35.30±7.42 years, and the control group was 35.98±7.07 years. Statistical analysis using one-way ANOVA revealed no significant age difference between the three groups (p=0.757).

The analysis of IL-1β concentrations in the gingival crevicular fluid revealed significant differences among the study groups. The mean ± SD level of IL-1β in the symptomatic apical periodontitis group was 0.06206±0.01779 ng/mL, which was significantly higher than that in both the asymptomatic apical periodontitis group (0.04818±0.01248 ng/mL) and the control group (0.04944±0.01672 ng/mL) (p<0.05) (Table 2).

The comparative analysis of IL-1β levels across the study groups, utilizing the least significant difference (LSD) test, revealed statistically significant differences between certain pairs. The mean difference in IL-1β levels between the SAP group and the AAP group was 0.01388 (p=0.001), and between the SAP group and the control group was 0.01262 (p=0.001). Conversely, no significant difference was detected between the AAP group and the control group (p=0.726) (Table 3).

The analysis of IL-10 levels in GCF demonstrated significant differences across the study groups (Table 4). The symptomatic apical periodontitis group had the highest mean ± SD IL-10 levels (11.17±3.28 pg/mL), followed by the asymptomatic apical periodontitis group (8.47±2.18 pg/mL), and the control group, which had the lowest levels (5.64±4.27 pg/mL). The differences among the groups were statistically significant (p=0.0001).

The findings indicated statistically significant differences in IL-10 pg/mL levels among the study groups, specifically in certain pairings. A statistically significant difference in mean IL-10 pg/mL was observed between the SAP and AAP groups, with a mean difference of 2.70 pg/mL (p<0.05). Similarly, a notable difference was observed between the SAP and control groups, with a mean difference of 5.53 pg/mL (p<0.05). The AAP group exhibited a mean difference of 2.83 pg/mL (p<0.05) from the control group (Table 5).

SAP had the highest mean±SD values of IL-23 (5.018±1.081), followed by AAP (3.083±0.569), and the control group had the lowest mean (1.431±0.586), with statistical analysis indicating a significant difference (p<0.01) (Table 6).

Table 7 shows the comparison of IL-23 levels among the study groups—including the control group, SAP, and the asymptomatic group—using post-hoc ANOVA least significant difference.

The findings indicated extremely significant differences (p=0.0001) in IL-23 levels between the asymptomatic and control groups. In a similar manner, a comparison between the symptomatic and control groups revealed significant differences (p=0.0001). Furthermore, a significant difference was seen between the symptomatic and asymptomatic groups (p=0.0001).

A strong positive significant correlation was found between IL-1β, IL-10, and IL-23 levels in SAP and AAP patients (Table 8).

The analysis of the diagnostic performance of IL-1β and IL-10 levels in distinguishing between study groups using the receiver operating characteristic (ROC) curve revealed the following:

• The IL-1β area under the curve (AUC) of 0.714 indicated reasonable diagnostic accuracy in distinguishing SAP from controls. AUC was statistically significant with a p-value of 0.000 and a 95% CI of 0.615 to 0.812.
• IL-10 demonstrated a higher diagnostic accuracy, with an AUC of 0.806, which was also statistically significant (p-value=0.000). The 95% CI for IL-10 was 0.729 to 0.883.
• IL-23 had the lowest AUC at 0.636, suggesting limited diagnostic utility compared to IL-1β and IL-10. However, it was still statistically significant (p=0.026) (Table 9) (Fig. 2).

This study investigated the levels of IL-1β, IL-10, and IL-23 in gingival crevicular fluid among patients with symptomatic apical periodontitis, asymptomatic apical periodontitis, and healthy controls. The findings showed that all three cytokines were significantly elevated in the SAP group compared to the AAP and control groups. Among them, IL-10 exhibited the highest diagnostic accuracy, followed by IL-1β, while IL-23 showed a weaker but still significant association with symptomatic inflammation.

Figure 2.

ROC curves for examined GCF biomarkers.

The current study revealed that the levels of IL-1β, IL-10, and IL-23 were significantly elevated in the SAP group compared to the AAP and control groups. Among these, IL-10 demonstrated the highest diagnostic accuracy.

Gingival crevicular fluid (GCF) serves as a valuable non-invasive source for detecting inflammatory biomarkers associated with periodontal and periapical diseases. It offers a localized and sensitive assessment of disease progression, being less affected by external factors than saliva. This advantage supports its suitability as a diagnostic tool.‌^{[21, 22]} GCF may be a superior diagnostic choice for assessing mediators of health and disease, given its simple and non-invasive collection compared to blood, along with its sensitivity, convenience, and potential for repeated sampling.^[23] This research investigated inflammatory biomarkers in GCF, which are difficult to obtain and have some practical constraints compared to saliva. Nonetheless, GCF offers information into the localized characteristics of the illness and is unaffected by salivary flow.^[24] Salivary samples were inappropriate for this investigation, as all analyzed teeth were from the same patient.^[25]

The relationship between GCF flow and outcomes in apical periodontitis is an intriguing topic in dental research. GCF, a serum-derived fluid found in the gingival sulcus, can be influenced by various factors, including inflammation and tissue health.^[21] In the context of apical periodontitis, GCF flow may reflect the status of inflammation.

Without convincing evidence and data from well-designed studies on GCF analysis techniques and after determining their pros and cons, it was difficult to say which technique was the best. This is especially true for biomarker detection and quantification accuracy, efficiency, feasibility, cost, and time. In clinical investigations, most researchers employed ELISA, perhaps because of its simplicity. In this study, ELISA technique was selected for its high sensitivity, specificity, and reproducibility in detecting cytokine levels, allowing accurate quantification necessary for diagnostic biomarker development.^[26] The latter feature is critical for determining cut-off concentrations of the chosen biomarkers, which facilitates their conversion into a chairside clinical tool.

IL-1β is a key pro-inflammatory cytokine involved in the destruction of connective tissue and bone resorption in apical periodontitis. In this study, significantly elevated IL-1β levels were observed in the SAP group compared to the AAP and control groups, reinforcing its role in acute inflammation and symptomatic disease. These results align with previous studies suggesting that IL-1β plays a central role in neutrophil recruitment and matrix degradation during active periapical lesions.^{[27, 28]}

Interestingly, IL-1β levels did not differ significantly between AAP and control groups, suggesting its upregulation may be more reflective of acute inflammatory responses rather than subclinical or chronic presentations.

IL-10 levels were significantly higher in SAP than in AAP and control groups. Periodontitis involves the breakdown of periodontal connective tissues, making IL-10 therapeutically important in controlling MMP enzyme activity. Tissue injury, fibrosis, and ECM degradation may arise from MMP activity regulation imbalances, indicating disease progression.^[29] Thus, the lack of AP explains this study’s lower IL-10 levels, which are consistent with previous data.^{[12, 24]}

Other longitudinal studies^[30] showed lower IL-10 levels in progressing periodontitis active areas and showed that MMPs over their tissue inhibitor are responsible for tissue damage. Interestingly, IL-10 was elevated in AAP compared to controls, which may reflect the subclinical nature of inflammation in asymptomatic cases. This emphasizes the importance of IL-10 as a sensitive marker even in early or less severe disease.^[24] The suggested approach may be used for the short-term follow-up of endodontically treated teeth with healed periodontal lesions to verify the efficacy of the intervention and observe the progression of periapical lesions. IL-10 expression was marginally upregulated under the same circumstances.^{[12, 31]}

The findings of this study showed that IL-23 levels were significantly elevated in the SAP group compared to both the AAP and control groups. This suggests a strong association between IL-23 and active inflammation in symptomatic apical periodontitis. IL-23 is a pro-inflammatory cytokine that contributes to the survival and proliferation of Th17 cells, which are involved in chronic inflammation and bone resorption.^[32]

The elevated levels in SAP patients support the hypothesis that IL-23 plays a crucial role in sustaining inflammation and tissue damage in the apical region.^[33] Additionally, the significant difference in IL-23 levels between the SAP and AAP groups indicates that IL-23 could potentially serve as a biomarker for symptom severity in apical periodontitis.

While IL-23 showed lower diagnostic accuracy (AUC=0.636) compared to IL-10 and IL-1β, its role in promoting chronic inflammatory processes still highlights its value in understanding disease mechanisms.

Several previous studies support the notion that IL-23 is involved in the pathogenesis of inflammatory oral diseases, particularly in the activation and maintenance of the Th17 immune response, as a study by Liu et al. demonstrated increased expression of IL-23 in periapical lesions, particularly those with intense inflammatory cell infiltration, suggesting a key role in maintaining chronic inflammation in apical periodontitis.^[34] Similarly, Veloso et al. found a skewing toward the M1 macrophage profile in symptomatic apical periodontitis.^[35]

On the contrary, Kaymaz and Beikler observed no significant IL-23 expression in GCF samples of asymptomatic apical periodontitis patients, emphasizing that IL-23 might not always reflect disease activity, especially in lesions with low cellularity.^[32]

Saccon et al. reported that IL-23 expression did not correlate with the severity of bone resorption in apical granulomas, suggesting its contribution may be secondary or context-dependent.^[36]

The findings of this study highlight the clinical relevance of measuring cytokine levels in gingival crevicular fluid (GCF) as a non-invasive method for differentiating between symptomatic and asymptomatic apical periodontitis. Given their proximity to the site of inflammation, GCF biomarkers such as IL-10 and IL-1β could support more precise diagnosis, reduce unnecessary interventions, and improve treatment planning. Moreover, their high diagnostic accuracy suggests potential for integration into chairside diagnostic tools that enable rapid, real-time assessment of endodontic inflammation, in line with current trends in personalized and minimally invasive dentistry.

This study has several limitations. First, the cross-sectional design makes it difficult to establish causal relationships or track cytokine levels over time. Second, the sample size, while statistically adequate, was confined to a single center, which may affect generalizability. Third, the study focused on a limited panel of cytokines; other important inflammatory mediators such as TNF-α, IL-6, or aMMP-8 were not included. Additionally, while care was taken to standardize GCF collection, factors such as individual variation in gingival inflammation or fluid volume could still influence cytokine concentrations.

Future studies should include longitudinal designs to observe cytokine fluctuations during treatment and healing phases.

The present study demonstrated the levels of IL-1β, IL-10, and IL-23 in gingival crevicular fluid were significantly elevated in symptomatic apical periodontitis compared to asymptomatic cases and healthy controls. Each cytokine reflects different aspects of the host immune response: IL-1β is a hallmark of active inflammation, IL-10 is indicative of regulatory anti-inflammatory activity, and IL-23 is associated with chronic immune activation. Among these, IL-10 demonstrated the highest diagnostic accuracy, suggesting its potential as a promising non-invasive biomarker for assessing disease severity. These findings support the clinical use of GCF-based biomarker profiling to assist clinicians in early diagnosis, case classification, and potentially in monitoring treatment response over time.

The authors declare no conflict of interest.

No external funding was received.

Zeena Tariq Abdulhadi: conceptualization, methodology, data collection, formal analysis, and writing – original draft preparation; Shareef Radhi Jawad: review and editing; Thuraya K. Alwandawi: review and editing; Zainab Y. Muhsin: review and editing. All authors read and approved the final version of the manuscript.

The research protocol was approved by the scientific committee at Al-Iraqia University College of Dentistry. All patients received detailed information about the study’s objectives and provided informed consent.