MMP-8, IL-6, and IL-1β: The Most Promising Salivary Biomarkers for Early Diagnosis of Periodontitis

By Maria Ali¹, Yasmeen Taj¹, Farzeen Tanwir², Shaista Bakhat¹, Syed Adeel Ahmed³

Affiliations

1. Department of Pathology, Bahria University Health Sciences Campus, Karachi, Pakistan
2. Department of Periodontology (Dental Section), Bahria University Health Sciences Campus, Karachi, Pakistan
3. Department of Operative Dentistry (Dental Section), Bahria University Health Sciences Campus, Karachi, Pakistan

doi: 10.29271/jcpsp.2025.07.825

abstract
Objective: To evaluate the salivary levels of MMP-8, IL-1β, and IL-6 in patients with periodontitis and healthy controls, and to assess the association between these biomarkers and clinical parameters of periodontal disease.
Study Design: A case-control study.
Place and Duration of the Study: Department of Pathology, Bahria University Health Sciences Campus, Karachi, Pakistan, from February to June 2024.
Methodology: A total of 76 participants were selected using OpenEpi software. Ethical approval was obtained prior to the study. Clinical parameters such as plaque index (PI), calculus index (CI), periodontal probing depth (PPD), clinical attachment loss (CAL), and bleeding on probing (BOP) were measured. Salivary levels of IL-1β, IL-6, and MMP-8 were determined using a Sandwich ELISA kit (BT Lab). Data analysis was performed using IBM SPSS Statistics version 27, and independent t-test / Mann-Whitney U test was used to compare the biomarker levels, with significance set at p <0.05.
Results: The study found significantly higher levels of IL-1β, MMP-8, and IL-6 in periodontitis patients compared to healthy controls. Significant associations were observed between biomarker levels and gender (p = 0.022), age groups (p <0.001), comorbidities (p = 0.012), plaque (p <0.001), CI (p <0.001), BOP (p <0.001), and CAL (p <0.001).
Conclusion: This study demonstrated significant associations between salivary biomarkers (IL-1β, MMP-8, and IL-6) and clinical parameters of periodontitis. These biomarkers show promise as diagnostic tools for the early detection of periodontal disease.

Key Words: Periodontitis, Salivary biomarkers, Saliva, Interleukin 1beta, Interleukin 6, Matrix metalloproteinases 8.

INTRODUCTION

Periodontitis, a chronic infection of the periodontium, is a global concern affecting millions, with dental calculus being a primary contributor.¹ This disease develops due to a combination of microorganisms, poor nutrition, and bacterial plaque retention, with certain populations such as men, African Americans, and the elderly being more at risk.² Traditionally, diagnosing periodontitis relies on clinical indicators such as tooth mobility and probing depth.³ However, the future of diagnosis may lie in the power of biomarkers, offering a non-invasive method for early detection and monitoring.⁴

Key biomarkers such as matrix metalloproteinase-8 (MMP-8), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) emerge as the strong contenders. MMP-8, which correlates with tissue loss, holds significant potential for early diagnosis.⁵

IL-1β and IL-6, both inflammatory cytokines, are elevated in those with periodontal disease and can help gauge the severity of the condition.⁶

National and international studies support the role of these biomarkers in periodontitis diagnosis and management. A study conducted in Pakistan highlighted the effectiveness of MMP-8 and IL-1β levels in assessing disease severity.⁷ Further research, including a clinical study from December 2020 to December 2022, evaluated salivary and GCF biomarkers in 150 patients, reinforcing their role in diagnosing and tracking treatment responses.⁸ A study conducted in Europe shows a link between IL-6 levels in saliva and gingival crevicular fluid to periodontal inflammation.⁹ More recently, Relvas et al. highlighted the importance of IL-1β, IL-6, and MMP-8 as key biomarkers for determining the severity of periodontitis.¹⁰

Traditional diagnostic methods for periodontitis are invasive and often detect the disease only after significant damage has occurred. Salivary biomarkers represent a promising, non-invasive diagnostic approach, enabling earlier intervention before clinical symptoms emerge. The aim of this study was to explore the relationship between MMP-8, IL-1β, and IL-6 levels in periodontitis patients, correlating them with clinical parameters to improve diagnostic and therapeutic strategies.

METHODOLOGY

Seventy-six healthy and chronic periodontitis patients were selected by consecutive sampling technique from the Dental OPD of the Periodontology Department, affiliated with the Bahria University Health Sciences Campus, Karachi, Pakistan, from February to June 2024. To determine the appropriate sample size, the open-source online EPI software was utilised, ensuring that the sample size was sufficient to obtain statistically significant results. These participants were divided into two groups: A control group and a case group, with each group consisting of thirty-eight individuals. Ethical approval for the study was obtained from the Institutional Review Board (IRB) of Bahria University Health Sciences Campus, and written informed consent was obtained from all participants.

The inclusion criteria for participation in the study were as follows: Participants were between 20 and 60 years of age, had at least sixteen natural teeth, and had not undergone any medical treatment during the last three months prior to examination and sampling.

The control group consisted of individuals who met the following conditions: No systemic health conditions, no sites with probing depth (PD) >3 mm or clinical attachment loss (CAL) >3 mm, with ≤10% sites exhibiting bleeding on probing (BOP), and no radiographic evidence of alveolar bone loss.

The case group included individuals with the following characteristics: Presence of at least sixteen natural teeth, a minimum of 35% of sites with CAL >3 mm, PD ≥3 mm, and presence of ≥40% sites with BOP. Smokers, pregnant women, or individuals who had undergone post-radio and chemotherapy treatments were excluded from the study.

Each participant underwent a comprehensive periodontal examination and a review of their medical and dental history. All permanent teeth were measured using a CPITN probe. Various clinical parameters, including PI, CI, BOP, PD, and CAL, were measured at six sites on all teeth, excluding third molars.

For saliva collection, participants were instructed to refrain from eating, drinking, chewing gum, or cleaning their teeth for at least 30 minutes prior to sample collection. First, participants gargled with tap water and then expectorated the entire saliva into a small sterile cup for up to five minutes using the drooling technique. A saliva sample of 2 to 5 ml was transferred to a polypropylene tube for analysis.

Salivary analysis was performed using the Invitrogen Sandwich ELISA kit, which is equipped with a pre-coated plate. All measurements were conducted in triplicate. Data analysis was conducted using IBM SPSS Statistics version 27. Normality was checked by Shapiro-Wilk’s test. For quantitative variables, the mean and standard deviation were calculated, and those variables that did not follow the assumption of normality were expressed as median and interquartile range.

For qualitative variables, frequency and percentage were reported. The comparison of means for IL-1BETA, IL-6, and MMP-8 across study groups was performed using an independent t-test / Mann-Whitney U test as appropriate. Chi-square/ Fisher’s exact test was applied to determine the association between qualitative variables. ROC analysis was performed to check the performance of biomarkers. A p-value of less than 0.05 was considered statistically significant.

RESULTS

The study involved 76 participants, with a mean age of 24.5 years for controls (IQR = 12.25) and 45.0 years for cases (IQR = 22.25). In the case group, 50% were over 45 years, while 94.7% of controls were in this age range. The case group had a higher percentage of males (60.5%) compared to the control group (34.2%). Clinically, 71.1% of controls and 68.4% of cases had thin plaque. The median PD and CAL were 1.00 mm (IQR = 1.00) and 1.00 mm (IQR = 1.00) for controls, whereas for cases, they were 5.00 mm (IQR = 2.00) and 7.00 mm (IQR = 2.00), respectively. Significant associations were found between study groups and gender (p = 0.022), age (p <0.001), comorbidities (p = 0.012), plaque (p <0.001), calculus (p <0.001), BOP (p <0.001), and CAL (p <0.001), Table I.

In terms of inflammatory markers, the case group had higher levels: IL-1β (4.95 ± 1.26 pg/mL), IL-6 (4.68 ± 1.41 pg/mL), and MMP-8 (Median = 3.96 pg/mL, IQR = 1.22 pg/mL) compared to the control group, which had lower levels: IL-1β (3.90 ± 1.01 pg/mL), IL-6 (3.78 ± 1.52 pg/mL), and MMP-8 (Median = 3.53 pg/mL, IQR = 1.07 pg/mL).

Table II shows that a significant mean difference was found for IL-1β for males (p = 0.007), females (p = 0.010), for older patients aged >45 (p = 0.151), comorbid (p = 0.021), and non- comorbid (p = 0.001). It also shows a significant mean IL-6 difference between patients with no comorbid conditions (p = 0.014) and for individuals ≤45 years (p = 0.004).

Figure   1: Receiver  operating  curve.

Table I: Association of demographic and clinical parameters according to study groups (n = 64).

Variables	Study groups n (%)		p-value
	Controls	Cases
Gender
Male	13 (34.2)	23 (60.5)	0.022*
Female	25 (65.8)	15 (39.5)
Age groups
≤45 years	36 (94.7)	19 (50)	<0.001*
>45 years	2 (5.3)	19 (50)
Any comorbidity
Yes	2 (5.3)	10 (26.3)	0.012*
No	36 (94.7)	28 (73.7)
Plaque
No plaque	10 (26.3)	0 (0)	<0.001*
Thin plaque	27 (71.1)	26 (68.4)
Moderate plaque	1 (2.6)	12 (31.6)
Calculus
No calculus present	26 (68.4)	0 (0)	<0.001*
Grade I	12 (31.6)	17 (44.7)
Grade II	0 (0)	20 (52.6)
Grade III	0 (0)	1 (2.6)
BOP
Normal gingival	25 (65.8)	5 (13.2)	<0.001*
No bleeding / mild inflammation	13 (34.2)	18 (47.4)
Bleeding on probing / moderate Inflammation	0 (0)	15 (39.5)
CAL
No clinical attachment loss	17 (44.7)	0 (0)	<0.001*
Stage I	20 (52.6)	0 (0)
Stage II	1 (2.6)	0 (0)
Stage III	0 (0)	38 (100)
Chi-square / Fisher’s exact test was applied. p-value of ≤0.05 was considered significant. *Significant at 0.05 levels.

Table II: Mean comparison of IL-1β according to study groups (n = 76).

Variables	IL-1β (pg/mL) Mean ± Std. Dev			IL-6 (pg/mL) Mean ± Std. Dev			MMP-8 (pg/mL) Median (IQR)
	Controls	Cases	p-value	Controls	Cases	p-value	Controls	Cases	p-value
Overall (n = 76)	3.90 ± 1.01	4.95 ± 1.26	<0.001*	3.78 ± 1.52	4.68 ± 1.41	0.009*	3.53 (1.07)	3.96 (1.22)	0.006*
Male  (n = 36)	3.99 ± 0.78	5.00 ± 1.32	0.007*	3.90 ± 1.10	4.70 ± 1.40	0.086	3.72 (2.17)	3.95 (0.81)	0.172
Female (n = 40)	3.86 ± 1.13	4.89 ± 1.21	0.010*	3.72 ± 1.71	4.65 ± 1.47	0.090	3.47 (1.27)	4.00 (1.60)	0.036*
≤45 years (n = 55)	3.89 ± 1.04	4.63 ± 1.36	0.030*	3.81 ± 1.55	5.12 ± 1.50	0.004*	3.53 (1.05)	4.16 (2.13)	0.016*
>45 years (n = 21)	4.08 ± 0.37	5.28 ± 1.10	0.151	3.24 ± 0.98	4.24 ± 1.20	0.274	3.81 (NA)	3.90 (0.71)	0.719
With comorbid  (n = 12)	4.01 ± 0.14	5.08 ± 1.22	0.021*	4.78 ± 1.47	4.70 ± 1.43	0.947	4.03 (NA)	4.15 (1.41)	1.000
Without comorbid  (n = 64)	3.90 ± 1.04	4.91 ± 1.30	0.001*	3.72 ± 1.52	4.67 ± 1.43	0.014*	3.51 (1.05)	3.96 (1.55)	0.005*
Mann-Whitney U test was applied.  p-value of ≤0.05 was considered as significant.  *Significant at 0.05 levels.

A noteworthy difference in MMP-8 was seen among female patients (p = 0.036), patients aged 45 years or older (p = 0.016), and patients without comorbid conditions (p = 0.005).

IL-1β emerged as the most reliable biomarker for periodontitis detection, demonstrating the highest AUC (0.720) along with a strong balance of sensitivity (51.75%) and specificity (86.8%). IL-6 and MMP-8 also displayed notable discriminatory capabilities but were less effective than IL-1β. IL-6 exhibited moderate sensitivity (63.2%) and specificity (68.4%), while MMP-8 had the highest sensitivity (71.1%) but lower specificity (55.3%). In summary, IL-1β provided the most balanced diagnostic performance, followed by MMP-8 and IL-6, as illustrated in Figure 1.

DISCUSSION

This study explored the role of MMP-8, IL-1β, and IL-6 as biomarkers for periodontitis, aiming to identify significant differences in their levels between periodontitis patients and healthy controls. The present study assessed the salivary levels of IL-1β, IL-6, and MMP-8 in both periodontally healthy individuals and chronic periodontitis patients. The demo-graphic analysis revealed significant differences in age and gender between the periodontitis and healthy groups (p <0.001 and p = 0.022, respectively). A study conducted in Pakistan found that 82% population above 60 years of age had periodontitis. Regarding gender, it is more prevalent in men than women, with approximately 57% of men being affected compared to 39% of women.¹¹ The most frequently associated comorbid conditions were diabetes mellitus (20%) and hypertension (40%). The p-value was significant (p = 0.012). Hypertension can amplify the activation of immune cells, including monocytes, macrophages, and lymphocytes, while periodontopathogens exacerbate inflammation by activating Th1 and Th17 lymphocytes. This interaction creates a bidirectional relationship where inflammatory responses contribute to the development and progression of hypertension.¹² Studies have shown that poorly managed diabetes increases the incidence of periodontitis by 86% compared to those with or without properly managed diabetes.^13,14 The present study found significantly higher levels of IL-1β, IL-6, and MMP-8 in the case group. Notably, MMP-8 levels were significantly higher in females, individuals aged 45 years or older, and those without comorbid conditions, suggesting its potential as a marker in these subgroups. MMP-8 is closely linked to disease severity, CAL, BOP, and PPD.¹⁵ Elevated MMP-8 levels in females may be influenced by hormonal fluctuations, while in individuals aged 45 years and older, age-related immune changes may exacerbate inflammation. However, some studies report inconsistent correlations between MMP-8 and CAL.¹⁶ The present study also found significant differences in IL-1β levels for both male (p = 0.007) and female (p = 0.010) periodontitis patients, with elevated IL-1β correlating strongly with PPD, BOP, and CAL. IL-1β plays a critical role in collagen and extracellular matrix degradation, driving inflammation and tissue destruction in periodontitis.¹⁷

In the present study, the authors found a significant difference in mean IL-6 levels among patients without comorbid conditions (p = 0.014) and those aged 45 years or younger (p = 0.004). IL-6 shows promise as a reliable salivary biomarker for early periodontitis detection, with previous research indicating elevated IL-6 levels in periodontitis patients compared to healthy individuals.^18,19 IL-6 contributes to the inflammatory response, promoting tissue-destructive enzymes and mediators, leading to greater CAL and deeper periodontal pockets, key indicators of disease severity.²⁰ The present study also assessed the diagnostic performance of these biomarkers. IL-1β had the highest AUC (0.720), indicating its strongest potential for periodontitis. Recent studies have shown that IL-1β has a significant discrimi-natory capacity, with a sensitivity of 88.24% and a specificity of 62.5% for diagnosing periodontitis. Future recommendations for the use of MMP-8, IL-1β, and IL-6 in diagnosing periodontitis include developing standardised biomarker panels to enhance diagnostic accuracy. Advancements in point-of-care testing may facilitate rapid chairside devices for real-time measurement, enabling immediate clinical decisions. Continuous monitoring could become standard for managing chronic periodontitis, allowing for treatment adjustments based on disease activity. Furthermore, targeting these biomarkers therapeutically could lead to adjunctive treatments aimed at modulating their levels.

CONCLUSION

This study revealed significant associations between these biomarkers and factors such as age, gender, and comorbi-dities. The future of salivary biomarkers for periodontal disease is promising, with advancements in molecular biology and diagnostic technologies. Key developments include the discovery of novel biomarkers through omics and microbiome research, personalised risk assessment and treatment monitoring, and the integration of portable diagnostic devices for accurate analysis. Salivary diagnostics may also help monitor chronic conditions and explore links between periodontal and systemic diseases.

There were some variations in the findings of research regarding the presence of cytokines in periodontitis. This could be due to study’s protocols or the sample size of the studied groups.

ETHICAL  APPROVAL:
Ethical approval was taken from the Institutional Review Board of Bahria University Health Sciences Campus (BUHS-IRB # 037/24, Date: 03-01-2024).

PATIENTS’  CONSENT:
Written informed consent was obtained from all participants.

COMPETING  INTEREST:
The authors declared no conflict of interest.

AUTHORS’  CONTRIBUTION:
MA, FT: Contribution to the conception and design of the work.
YT, SB: Drafting of the work and revision of the manuscript critically for important intellectual content.
SAA: Data collection, interpretation of the data, and drafting of the manuscript.
All authors approved the final version of the manuscript to be published.

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