Evaluation of salivary interleukin-6 in children with early childhood caries after treatment.

BACKGROUND: The role of cytokines as a marker in the oral inflammatory process in ECC has not been fully explored before and after full mouth rehabilitation. AIMS: The aim of this study was to assess the level of salivary interleukin-6 (IL-6) in children with ECC and to compare its levels before and after comprehensive full mouth rehabilitation. METHODS AND MATERIALS: Saliva samples were collected from children with ECC prior to dental treatment and 3-month post treatment. The salivary IL-6 levels were analyzed using the ELISA method. The gingival index was also timely recorded. Oral health awareness sessions were conducted for children and their parents at regular intervals during the 3-month study period. STATISTICAL ANALYSIS USED: Wilcoxon Signed Rank test compared the levels of salivary IL-6 while, the paired t test compared the values of gingival index before and after treatment.
RESULTS: The mean level of salivary IL-6 before and 3 months after treatment had reduced and this reduction was statistically significant (P < 0.000). The gingival index scores had also reduced significantly 3-months post treatment (P < 0.002).
CONCLUSIONS: Children with ECC when completely rehabilitated and kept under frequent follow up, which includes reinforcement of oral hygiene measures and maintaining a low caries activity state, the level of inflammation (IL-6) can definitely be minimized and thereby improving the quality of life of affected children.

Introduction

Dental caries is an international public health challenge, especially among young children. It is one of the most common infectious diseases of childhood. Children who are frequently exposed to sugary liquids, breast milk, formula, fruit juices, and other sweet liquids for long periods run at a great risk of suffering from early childhood caries (ECC). The American Academy of Pediatric Dentistry (AAPD) defines ECC as the presence of one or more decayed teeth, missing teeth (resulting from caries), or filled tooth surfaces in any primary tooth in a child of 6 years or younger.[1] Problems encountered by children with ECC are not just pain and infection, also affects speech and communication, eating and dietary nutrition, playing and overall quality of life. There could also be insufficient physical development (in terms of height and weight). Loss of school days is another frequent problem encountered. Another major concern is the cost of treatment. Many children often require multiple comprehensive treatments, frequently requiring hospitalization, and treatments under general anesthesia.[1]

Most children with ECC have a complaint of pain indicating bacterial involvement of the dental pulp. There is a cytokine cascade that induced in response to bacterial infection of the dental pulp which includes interleukin-1 (IL-1), IL-6, IL-8, IL-10, IL-12, and tumor necrosis factor-alpha (TNF-α). Cytokines play a major role in the inflammatory and immune responses, several of them displaying inflammatory and others anti-inflammatory. IL-6 has both proinflammatory and anti-inflammatory properties, and understanding its possible role as a marker for dental caries will prove very useful, especially while dealing with children with ECC. Many studies suggest a strong correlation between IL-6 and dental caries.[23456]

Abdolsamadi et al. found that serum IL-6 could be used as serologic marker for chronic periapical lesions as IL-6 concentrations were significantly higher in patients with chronic periapical lesions.[2] Another study measured and compared the levels of cytokines IL-12, IL-6, IL-8, IL-10, TNF-α, and interferon-gamma (IFN-γ) in pulpal blood from teeth affected with irreversible pulpitis, asymptomatic caries exposure, and those with normal pulps and found that significantly higher levels of IL-6, IL-8, IL-10, TNF-α, and IFN-γ were detected in caries exposed pulp and irreversible pulpitis as compared to normal teeth. Most interestingly, ratios of IL-6/IL-10 and IL-8/IL-10 were significantly higher in irreversible pulpitis when compared to caries-exposed and normal teeth.[3] Garrido et al. in their study on teeth diagnosed with apical periodontitis showed that these teeth when compared to normal healthy teeth stimulated C-reactive protein and IL-6, thereby significantly increasing their levels.[4] A study conducted by Gornowicz et al.[5] was a baseline study comparing the levels of various cytokines in children and adolescents with dental caries and a control group with good oral health. Their study included assessing the levels of salivary IL-6, which was significantly higher in children with dental caries. Varma and Fathima [6] assessed the levels of IL-1β in children with Down syndrome after full mouth rehabilitation and found that the levels of IL-1β significantly decreased after treatment. From the above-mentioned studies, it is clear that there is a strong correlation between levels of these inflammatory mediators and the presence of dental caries, and therefore, children with ECC are also at risk of having higher levels of these cytokines. Hence, the aim of this study was to assess and compare the level of salivary IL-6 in children with ECC before and after full mouth rehabilitation of children with ECC.

Materials and Methods

Twenty-two children diagnosed with ECC were included in this study. The inclusion criteria were children between 3–6 years of age and those with Type II ECC by Wayne's criteria [7] (labio-lingual carious lesions on maxillary incisors with/without involvement of molars and unaffected mandibular anterior). The exclusion criteria were Type III severe ECC, Type I ECC, presence of any abscess, sinus, or fistula, medically compromised children, and those with special health care needs. The study was carried out in the Department of Pedodontics and Preventive dentistry of our institute after obtaining approval from the Institutional Ethical Committee. Informed consent was also obtained from each parent prior to the commencement of the study.

An intraoral examination was carried out prior to the commencement of the study. Saliva samples were collected before the dental rehabilitation initiated. The gingival index (GI) was also recorded prior to dental treatment. Patients were asked to refrain from eating 60 min before saliva sample collection, and they were then asked to rinse their mouths 20–30 min before sample collection. Saliva was collected in sterile Eppendorf tubes using disposable plastic pipettes. Samples were then immediately stored at a low temperature of 4°C and transported to a laboratory within 2 h. The saliva samples were then centrifuged at 1500 g (3000 rpm) for 15 min to remove mucosal and other constituents. The clear supernatant was then transferred to another Eppendorf tube, was labeled and arranged in an Eppendorf tube rack, and stored at −80°C ultra-cold storage unit until subjected to enzyme-linked immune sorbent assay (ELISA) tests. Salivary IL-6 assessment was done using the Salimetrics ELISA kit (Salimetrics LLC, State College, PA, USA), specific for salivary IL-6 in the Department of Biochemistry [Figures 1a, 1b, 2a, 2b, 3a and 3b].

Figure 1

(a) Diluting saliva samples with IL-6 sample diluent. (b) Plates placed on mini-rotator shaker which mixes constantly

Figure 2

(a) Blue color obtained after adding 100 μL Tetramethylbenzidine solution. (b) Yellow color obtained after adding stop solution

Figure 3

(a and b) Optical density is read on a standard plate reader at 450 nm

Based on examination, history, and investigations, a treatment plan was formulated to be carried out as a chairside procedure. General anesthesia was preferred in cases where the child was not cooperative. Treatments carried out were oral prophylaxis, restorations, pit and fissure sealants, fluoride application, pulp therapies, and extractions. Pulp therapies were followed by placement of stainless steel crowns. Extractions were followed by placement of appropriate space maintainers depending upon the case. Follow-up appointments included reinforcement of oral hygiene measures, and oral prophylaxis was done if required. Oral hygiene measures were also shown to parents and guardians to assist the child efficiently. Children were also trained to brush. Restorations and other treatments were re-assessed for intactness at the recall visits.

The second salivary sample was collected 3 months after the completion of comprehensive dental treatments in a similar manner as explained earlier, for IL-6 estimation. Oral health education sessions were also conducted for both child and parent to ensure and reinforce good oral hygiene measures. The GI scores were again recorded at the end of 3 months and compared.

Results

The mean values of the levels of salivary IL-6 of the pretreatment (sample A) and 3 months posttreatment (sample B) were 100.46 ± 112.47 and 21.46 ± 35.80, respectively [Table 1]. Wilcoxon signed-rank test compared the pretreatment values with 3 months posttreatment values. The reduction in levels of salivary IL-6 was found statistically significant (P < 0.000). This clearly indicated postdental rehabilitation of children with ECC, and there is a statistically significant reduction in salivary IL-6 levels.

Table 1

Mean value and standard deviation of salivary interleukin-6 and the P value obtained

The mean GI values of both sample A (prior to treatment) and sample B (3 months posttreatment) were 0.68 ± 0.22 and 0.55 ± 0.21, respectively [Table 2]. The paired t-test compared the pre- and post-treatment GI values (P < 0.002), and the values were statistically significant.

Table 2

Mean value and standard deviation of gingival index and the P value obtained

Discussion

IL-6 is known to have a strong correlation with severity and extent of carious lesions in the oral cavity, and therefore, minimizing its levels will help in improving the inflammatory condition in the oral cavity. This was a pioneering approach to assess the levels of salivary IL-6 in children with ECC and to evaluate the difference in levels of salivary IL-6 after full mouth dental rehabilitation.

This study included 22 healthy children diagnosed with Type II ECC, excluded to be from any systemic disease, medically compromised condition, etc., These exclusion criteria were decided as such because many studies done by various authors have shown that levels of IL-6 may be increased in many systemic conditions.[89] Studies done by Erta et al.[8] suggest that IL-6 has a key role in the central nervous system and its expression may be enhanced in multiple sclerosis, psychiatric disorders such as depression, autism, and schizophrenia. Diepold et al.[9] showed that pediatric oncology patients who have higher levels of IL-6 showed risk of developing septicemia.

Saliva is considered as the gold standard in biochemical assays and analysis.[5],[610],[11121314] Similarly, saliva was used as the diagnostic fluid for IL-6 estimation in this study. Although GCF is considered as the main source of periodontitis associated cytokines, it only reflects periodontal inflammation at each specific site sampled and these agents eventually enter into saliva and mix with salivary IL-6 as suggested by Gursoy et al.[15] Gursoy et al.[15] and Lima et al.[16] have stated that saliva, unlike GCF, is an easily and noninvasively collected specimen which can be used for the detection of immune components. Seyberth et al.[17] demonstrated that saliva sampling is a very effective technique in children due the following reasons such as it is both noninvasive and painless; minimal patient discomfort when compared to venous blood sampling; repeated samples are easy to obtain, and good correlation has been found with blood concentrations of various parameters.

Prior to saliva collection, a general physical examination and a thorough oral examination were carried out. Patients were asked to refrain from eating 60 min before saliva sample collection and were asked to rinse their mouths 20–30 min before sample collection so as to clear the oral cavity of any debris, similar to the methodology used by Gornowicz et al.[5] Saliva was collected using a pipette and Eppendorf tube as suggested by Cousins.[18] Polythene pipettes do not traumatize the patient, even if the patient bites onto the pipette, no harm is done, it can be easily cleaned, and it has longer life than ordinary bulb Pasteur pipette.[18] After collection of saliva, the samples were centrifuged at 1500 × g (3000 rpm).[5192021] Centrifuging removes mucins and other particulate matter which may interfere with antibody binding, ultimately leading to falsely elevated results.[192021] The saliva supernatant was separated and stored at low temperature in an “ultra-cool temperature storage unit” at −80°C so as to prevent microbial growth and avoid degeneration of cytokines.[15161718192021]

The posttreatment saliva samples were collected and handled in a fashion similar to the pretreatment samples. In our study, the samples were subjected ELISA analysis to measure the levels of IL-6 in saliva using Salimetrics™ IL-6 kit. This kit is a sandwich immunoassay, specifically designed for the measurement of salivary IL-6. ELISA has been used previously in numerous studies for determining concentration of IL-6.[510111213]

Based on the clinical examination and investigations, a tailor-made treatment plan was formulated for each patient. Dental treatment was carried out either as a chairside procedure or under general anesthesia, depending on patient compliance. According to the pediatric oral health policy by AAPD in 2012,[22] conducting comprehensive dental rehabilitation under general anesthesia for children with ECC has many additional benefits to children as well as their families. Some of them are improvement in the quality of life of child and parent, facilitating dental access for very young patients, improved positive oral health behaviors and oral health outcomes. In this study, 6 out of 22 children required treatment under general anesthesia.

After the full mouth comprehensive treatments were completed, the children were followed up for 3 months as performed in other studies.[623242526] During those 3 months, children and respective parents were instructed and demonstrated the brushing techniques and other oral hygiene instructions. The GI (Loe and Silness, 1967) was recorded first before the treatment commenced and 3 months after completion of full mouth rehabilitation to evaluate the difference in oral health. The GI has been used by many authors to assess the oral health of children as in studies conducted by Gopinath et al.[27] and Al-Haddad et al.[28] The mean pretreatment GI was 0.68 ± 0.22 which reduced to 0.55 ± 0.21 after 3 months.

The mean salivary IL-6 as estimated in this study was 100.46 ± 112.47 which reduced to 21.46 ± 35.80 after 3 months posttreatment. This may be attributed to the complete rehabilitation performed. In a similar study, there was a reduction in IL-1β levels in children with Down Syndrome after full mouth dental rehabilitation, but the reduction was not statistically significant because of delayed response or the defective neutrophil chemotaxis and prolonged T-cell activation seen in individuals with Downs syndrome.[6]

This was a pioneer effort to evaluate the relation between salivary IL-6 and ECC and also to analyze the effect of dental rehabilitation on reducing it in the oral cavity. Further studies with larger sample size and longer follow-up period may help to conclusively establish the efficacy of dental treatments in minimizing the levels of these inflammatory markers.

Conclusion

Children with ECC were found to have higher levels of salivary IL-6. This study also showed that after full mouth rehabilitation significantly contributed to reducing salivary IL-6 levels.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.