Correlation between interleukin-1β and matrix metalloproteinase-1 levels in crevicular fluid with a proposed periodontal disease index in children.

BACKGROUND: The crevicular fluid contains biomarkers that allow the identification of periodontal disease, anticipation of its risk, and prediction of its progression.
PURPOSE: The purpose of this study is to correlate interleukin-1β (IL-1β) and matrix metalloproteinase-1 (MMP-1) levels in crevicular fluid with periodontal disease severity in schoolchildren.
METHODS: A cross-sectional study was conducted in 82 schoolchildren between 9 and 12 years. The biofilm percentage, attachment level, gingival recession, dental calculus, and bleeding on probing were measured in the teeth 16, 26, 36, 41, and 46. These five parameters obtained were considered to propose a disease score between 0 and 10 points. In crevicular fluid from the tooth with the highest score, IL-1β and MMP-1 levels were measured.
RESULTS: According to the proposed score, mild gingivitis was identified in 20 schoolchildren, moderate gingivitis in 30, and severe gingivitis in 32. Biofilm percentage, IL-1β, and MMP-1 levels increased as the severity of the disease increase. The mean and 95% confidence interval were 23.2 pg/μl (18.6-27.7), 37.3 pg/μl (27.8-46.9), and 44.6 pg/μl (34.4-58.8) (P = 0.01) for IL-1β and 2.69 mol/min (2.51-2.87), 4.43 mol/min (3.9-4.95), and 5.59 mol/min (4.81-6.38) (P < 0.001) for MMP-1 in each group, respectively. The proposed clinical score correlated with biofilm percentage (r = 0.63), IL-1β (r = 0.50), and MMP-1 (0.45) levels, P < 0.001 in all cases.
CONCLUSIONS: The proposed clinical score for periodontal disease in children correlated with percentage of biofilm, IL-1β, and MMP-1 levels.

INTRODUCTION

Periodontal disease is a chronic inflammatory process induced by microorganisms that progressively destroy dental support tissue and eventually cause tooth loss. In Mexico, it is the second cause of oral disease after caries.[1] In Latin America, the prevalence of gingivitis in children between 6 and 11 years varies between 20 and 73%, and in adolescents, it reaches up to 82.5%.[2]

Periodontal disease in children includes dental plaque-induced gingival disease, chronic periodontitis, aggressive periodontitis, periodontitis as a manifestation of systemic diseases, and necrotizing periodontal diseases.[3] Gingivitis associated with dental plaque is the reversible and nondestructive form of periodontal disease, which can evolve to irreversible and destructive periodontitis characterized by inflammation of periodontal tissues with clinical attachment loss (CAL) and bone support.[4]

In the etiology of periodontal disease, the main bacteria involved are Gram-negative bacteria, mainly because of their ability to colonize the subgingival plaque. They damage the tissues by producing proteases and inducing an inflammatory immune response through interleukin-1 (IL-1), tumor necrosis factor-alpha, interferon-gamma, prostaglandin E2, and matrix metalloproteinase-1 (MMP-1). Systemic and local levels of these cytokines reveal the progression of tissue destruction.[5] IL-1 in its two molecular forms α and ß is an activating factor of osteoclasts.[6]

Elevated levels of IL-1β have been identified in the crevicular fluid of adults with periodontal disease when compared with healthy participants.[7] These levels are related to the periodontal inflammatory process, CAL, and increased probing depth, and they decrease with treatment.[89]

Proteases in the crevicular fluid show the destructive activity of connective tissue, and they increase according to the severity of periodontitis.[10] Active collagenase increases its level in the connective tissue, and it constitutes a marker of the imbalance in the extracellular matrix between metalloproteinases. The main physiological and pathological contribution in the degradation of the collagen in the gingival tissue is attributed to MMP-1, which comes mainly from fibroblasts and is observed to be high in aggressive periodontitis.[11] During remission periods, the tissue inhibitors of metalloproteinases increase.[6]

Different signs of periodontal disease have been used in diagnostic indices either in young or adult patients. In schoolchildren, Silness and Loe plaque and gingival index is the most commonly employed criteria to determine the level of hygiene and the degree of gingival inflammation, without taking into account the status of periodontal tissues that are affected in advanced stages of the disease.[12]

The community periodontal index (CPI) is the most widely used measure in epidemiological surveys.[13] It records the presence of gingival bleeding and inflammation, periodontal pockets, biofilm, dental calculus, and CAL. In children under 20, the index evaluates six teeth to avoid alterations associated with tooth exfoliation and eruption processes. In children under 15 years, it does not take into consideration probing depth, and it only assesses bleeding on probing depth and the presence of supragingival calculus.

This study proposes the use of specific criteria for the diagnosis of periodontal health in schoolchildren with mixed dentition and evaluates its correlation with pro-inflammatory cytokines in crevicular fluid.

MATERIALS AND METHODS

Participants

The study population included 82 schoolchildren from the rural community of San Juan de Otates in Leon, Guanajuato, and 44 girls (53.7%) and 38 boys (46.3%) aged 9–12 years with mixed dentition were examined. We did not include children with fixed or removable appliances that had undergone antibiotic and/or anti-inflammatory treatment in the previous 15 days and cases with premature loss of permanent first molars. Samples of the crevicular fluid that had been contaminated with blood were excluded from the study.

The sample size was calculated on the basis of a simple correlation between the clinical score of gingival disease and IL-1β levels, with an expected correlation coefficient of 0.6, and values of α = 0.05, 1β = 0.8, constant k = 6.2, and one-tailed hypothesis testing. Following this statistical approach, the sample size comprised 70 participants. Considering a probable loss of 15%, the sample size was increased to 82 students.

The parents of the participating schoolchildren were informed about the purpose of the study and its risks and benefits. They signed an informed consent to authorize their participation. The study was reviewed and approved by the research and ethics committee of the university ID number 2017-12.

Clinical procedure

Clinical examination and collection of crevicular fluid were performed by two dentists previously trained and standardized in the corresponding procedures,[14] by an expert in pediatric dentistry and periodontics. Intra- and interrater reliability was assessed using Cohen's kappa coefficient. The results showed a level of reliability >0.8.

Periodontal examinations took place in a dental chair equipped with adequate lighting. The teeth 3, 14, 19, 25, and 30 were examined in occlusion with their antagonist.

The biofilm percentage was determined using a revealing tablet (Viarden, Mexico) and the results were interpreted according to the Silness and Loe index.[12] These data were coded as 0 = incipient presence of biofilm ranging from 1% to 25%; 1 = moderate presence ranging from 26% to 50%; and 2 = severe >50%.

The clinical attachment level was determined through the probing depth measurement, and gingival recession was measured with a Michigan periodontal probe (Hu-Friedy, Chicago, US). For the probing depth, we obtained four sites per tooth in the mesial, medial, distal buccal, and palatal or lingual areas measured in millimeters.[15] The deepest site was coded as 0 = 1–3 mm, code 1 = 4–5 mm, and 2>5 mm. When X-rays taken to validate bone levels ascertained that pockets of 5 mm or greater were actually pseudo pockets, the probing depth was subtracted to the biological thickness because there was no loss of clinical insertion.

In case of gingival recession, it was measured in millimeters using the cementoenamel junction as the cervical reference to the most apical position of the gingival margin. The gingival recession with the largest measure of probing depth was added to CAL. The result was coded as follows: 0 = 1–3 mm; 1 = 4–5 mm; and 2≥6 mm.

Supragingival and/or subgingival dental calculus was identified using an explorer and a #5 mirror according to standardized procedure.[16] The absence or presence of dental calculus was coded as follows: 0 = absence; 1 = supragingival calculus; 2 = subgingival calculus, and 3 = supragingival and subgingival calculus.

Bleeding on probing was coded as 0 = absence and 1 = presence.

The clinical score proposed can obtain a score of 0–10 points considering the 5 sections of evaluation, with higher sum scores reflecting increased periodontal disease. The tooth with the highest score was classified into three groups: 0 = absence of gingivitis; 1–3 points = mild or early biofilm-induced gingivitis; 4–6 points = moderate biofilm-induced gingivitis; and 7–10 points = severe biofilm-induced gingivitis.

Collection and analysis of crevicular fluid samples

The samples were obtained from the tooth with the highest clinical score. Any presence of dental calculus was previously removed using a Gracey curette (Hu-Friedy, Chicago, US) without touching gingival margin. The tooth was previously isolated with cotton rolls and air-dried for 15 s. PerioPaper (Oralflo, NJ, US) strips were then placed into the gingival sulcus for 2 min to absorb the crevicular fluid and stored at −70°C until their analysis. The IL-1β and MMP-1 were measured using a commercial ELISA test according to the manufacturer's protocol (Enzo Life Sciences, Inc., Farmingdale, NY, US).

Data analysis

Quantitative variables were summarized in means and 95% confidence intervals. The normality of the data was evaluated by the Kolmogorov–Smirnov test. The differences between the three groups were established using the Kruskal–Wallis hypothesis test and the Bonferroni post hoc test was applied. The correlation was determined using Spearman's test. P < 0.05 was considered as statistically significant. The analysis was performed with the Statistical Package for the Social Sciences version 19.

RESULTS

Three groups were established according to their clinical score: mild biofilm-induced gingivitis n = 20 (24.4%); moderate biofilm-induced gingivitis n = 30 (36.6%), and severe gingivitis n = 32 (39%).

We found that biofilm percentage, IL-1β, and MMP-1 levels increased as the severity of the disease increase. Biofilm was lower in mild degree than in moderate and severe gingivitis. IL-1β levels were only different between mild and severe gingivitis, while MMP-1 levels were lower in mild than in moderate and severe gingivitis degree as well as lower in moderate than in severe gingivitis [Table 1].

Table 1

Comparison of biofilm percentage, matrix metalloproteinase-1, and interleukin-1β levels in crevicular fluid according to a new proposed gingivitis score in children

In Figures 1–3, we can observe that the clinical score of periodontal disease was positively correlated with the biofilm percentage (r = 0.63, P ≤ 0.001), IL-1β (r = 0.50; P < 0.001), and MMP-1 levels in crevicular fluid (r = 0.45, P < 0.001), respectively.

Figure 1

Correlation between clinical score for gingivitis in children and interleukin-1β levels in crevicular fluid

Figure 3

Correlation between clinical score for gingivitis in children and biofilm percentage

Correlation between clinical score for gingivitis in children and matrix metalloproteinase-1 levels in crevicular fluid

DISCUSSION

Periodontal disease is common in children; however, it is usually limited to the gingiva and does not cause CAL or resorption of the alveolar bone. Literature has reported that the destructive phase of periodontal disease initiates around puberty and is known as aggressive periodontitis.[17] Primary dentition and early permanent dentition may also present with chronic periodontitis. Although not as common as in the adult population, it does happen. The worse periodontal conditions in primary dentition have been identified using the simplified periodontal index.[18] The lack of identification of periodontitis may be due to the fact that its existence is not considered in primary dentition and that its consequence, which is teeth loss is natural and not due to periodontal disease.[3] The clinical criteria employed to diagnose periodontitis such as probing depth, clinical attachment level, or bleeding on probing have limited the assessment of the clinical status in children. The ideal method should consider not only the presence but also the severity of the disease and it should be able to predict its progress as well. This study proposed and used a diagnostic method that considered five parameters obtained from different validated indexes: biofilm percentage, attachment level, gingival recession, dental calculus, and bleeding on probing.[1319] This yields a score according to the severity of periodontal damage in schoolchildren with mixed dentition. The results showed that all the students exhibited some degree of periodontal disease, ranging from mild to severe gingivitis, possibly because the sample was obtained from a population with low socioeconomic level, with little or no knowledge about oral hygiene, limited access to health services, malnutrition, and poor oral hygiene.[20] Using the current proposed index, the category of severe gingivitis was identified in 39% of the schoolchildren, which is in agreement with another study in which severe gingivitis was reported in 32% schoolchildren. Cases with severe gingivitis were associated with poor oral hygiene characterized by high levels of biofilm with a high bacteria content, which are the main trigger of the periodontal inflammatory response and the subsequent development of the disease.[21] The prevalence of periodontal pathogens is higher in the mixed dentition than in the primary dentition stage and increases with the number of permanent teeth, which confirms that the infection by periodontal pathogens progresses with the development of dentition, especially during the transition from primary to mixed dentition.[22] The schoolchildren's high clinical scores predominantly resulted from the presence of dental calculus, which is the mineralized form of chronic biofilm that acts as a gingival irritation factor. It in initial stages appears as gingivitis and subsequently as periodontitis increasing bone resorption and risk for tooth loss.[23]

The severity of the clinical expression and the inflammatory response induced by bacteria can be explained by the genetic predisposition of the host, i.e., its genome and the epigenetic events that induce the immune response manifested in the profile of the cytokines that cause bone and tissue destruction.[624] Pro-inflammatory cytokines by different mechanisms collectively contribute to periodontal soft-tissue destruction and their levels are a useful and sensitive diagnostic marker of periodontal destruction.[25] The clinical score obtained through the diagnostic proposal used in mixed dentition schoolchildren was positively and significantly correlated with the levels of cytokines IL-1β and MMP-1 in crevicular fluid, which conveys the degree and progression of periodontal disease.[26] Schoolchildren with higher clinical scores have higher levels of IL-1β in crevicular fluid. In adults, it has been shown to be elevated in saliva and crevicular fluid according to the severity of periodontal disease,[727] with the CAL,[8] and with the amount of bone loss.[28] We did not assess bone loss, which is difficult to identify in early stages, but it is important to consider that clinical parameters correlate with a biomarker that explains the pathophysiology of bone resorption.

We also identified a correlation between the levels of the MMP-1 biomarker and the severity of periodontal tissue disease, which shows the imbalance between MMPs and their tissue inhibitor, actively participating in the progression of inflammatory stages of gingivitis and periodontal disease. MMPs are the most important participants in the destruction of periodontal tissue associated with periodontitis due to their role in the pathological degradation of the extracellular matrix in periodontal tissues.[29] In patients with aggressive periodontitis and chronic periodontitis, elevated levels of MMP-1 in crevicular fluid have been reported.[30] MMP-1 levels have been reported to increase up to 50-fold in individuals who develop the disease, and up to 9 months before, they exhibit radiological bone loss. A similar behavior can be observed in the levels of IL-1β.[31] Both inflammatory biomarkers increase the risk of developing periodontal disease up to 45 times.[32] Therefore, the use of the proposed periodontal evaluation in schoolchildren with mixed dentition and its confirmation by the inflammatory biomarkers of periodontal disease validate the proposed methodology as a diagnostic method in the early stages during the mixed dentition stage.[2733]

The identification of the presence of destructive periodontal disease with CAL, resorption of bone is possible before puberty and possibly with a more common frequency than is believed. Searching for it intentionally will allow earlier diagnosis, facilitate adequate treatments, and therefore improve the prognosis since periodontal disease causes 30%–35% of the teeth loss and is related to the presence or severity of systemic diseases in adulthood.[34]

We do not know how exfoliation behaves according to the severity of the periodontal disease or if it could have repercussions on the growth and development of the orthognathic system. One of the limitations of this study is that it was a cross-sectional study and periodontal disease should be considered in a longitudinal manner.

Future studies should consider not only the amount of biofilm but also the type of microbiome and the inflammatory response associated with the patient's genome, in order to understand the expression of the severity and progress of the disease in the early stages of life, as a response to different therapeutic schemes. In addition, we did not consider the systemic effects generated by the immune response during the periodontal infectious process that begins in childhood during temporary dentition and continues with final dentition.

Bone loss from chronic periodontitis can be seen in adolescents and even in children with primary dentition. The age at which the patient seeks treatment does not reflect the onset of the disease. It is a disease that has progressed without treatment and can be treated and reversed.

Although our proposed clinical score is related to IL-1ß and MMP-1 levels in crevicular fluid, it needs future validation in a bigger population. Furthermore, this requires periodontal evaluation in children, using periodontal probes and radiological assessment of alveolar bone.

CONCLUSIONS

The proposed clinical score correlates with the inflammatory biomarkers that are part of the pathophysiology of periodontal disease. We suggest this new diagnostic tool to compare the various clinical and biochemical parameters for periodontal disease. It could diagnose the disease in the early stages and implement more appropriate, timely dental care and treatment that prevent, stop, or reverse periodontal damage and its consequences.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.