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ORIGINAL ARTICLE
Year : 2018  |  Volume : 36  |  Issue : 2  |  Page : 130-134
 

Salivary interleukin-6 levels in children with early childhood caries and ventricular septal defect


Department of Pedodontics, Amrita School of Dentistry, Kochi, Kerala, India

Date of Web Publication2-Jul-2018

Correspondence Address:
Parvathy Kumaran
Department of Pedodontics, Amrita School of Dentistry, Kochi, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JISPPD.JISPPD_211_17

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   Abstract 


Background: The efficacy of dental treatment on the levels of inflammatory markers in ventricular septal defect (VSD) has not been fully explored. Aims: The aim of this study was to assess the level of salivary interleukin-6 (IL-6) in children with early childhood caries (ECC) and VSD and to compare its levels before and after comprehensive full mouth rehabilitation. Methods: Saliva samples were collected from children with ECC and VSD before dental treatment and 1-month posttreatment. The salivary IL-6 levels were analyzed using the enzyme-linked immunosorbent assay method. Gingival index (GI) was also timely recorded during the study period. Oral health awareness sessions were conducted for children and their parents. The Fisher's Least Significant Difference test was used for intergroup comparison of salivary IL-6 levels, while the paired t-test compared the values of GI before and after treatment. Results: The mean salivary IL-6 levels 1 month after treatment had reduced (P < 0.000). There was a reduction in GI score (P = 0.002). Conclusions: When children having VSD and ECC are dentally rehabilitated and followed up periodically, the level of IL-6 is minimized, hence improving the quality of life of the affected children.


Keywords: Early childhood caries, salivary interleukin-6, ventricular septal defect


How to cite this article:
George T, Kumaran P, Varma R B, Xavier AM, Janardhanan SK, Govinda BS. Salivary interleukin-6 levels in children with early childhood caries and ventricular septal defect. J Indian Soc Pedod Prev Dent 2018;36:130-4

How to cite this URL:
George T, Kumaran P, Varma R B, Xavier AM, Janardhanan SK, Govinda BS. Salivary interleukin-6 levels in children with early childhood caries and ventricular septal defect. J Indian Soc Pedod Prev Dent [serial online] 2018 [cited 2019 Jul 20];36:130-4. Available from: http://www.jisppd.com/text.asp?2018/36/2/130/235681





   Introduction Top


Congenital heart defects (CHDs) are defined as abnormal cardio circulatory structure or function present at birth, although they are often not detected until later in life. CHDs are the most frequent lethal malformation, affecting about 1% of newborns and causing significant morbidity and mortality in infants. These account for 30% of total congenital abnormalities with a birth prevalence of 4–50 per 1000. In a comprehensive study by European Surveillance of Congenital Anomalies that included the live birth registries of 22 countries, prevalence of CHD was 6.5 per 1000 births. Ventricular septal defect (VSD) (33%) is the most predominant individual CHD, followed by atrial septal defect (19%) and tetralogy of Fallot (16.8%). Although the majority of VSD cases were of the isolated type (28.7%), nearly 4.5% were present in combination with a variety of other cardiac defects. ASD and VSD were found together in 6.4% of cases. One-third of VSDs were of the perimembranous type.[1]

The patients with heart diseases are prone to increased caries risk. Risk factors for dental caries may vary over time and are strongly affected by lifestyle and behavioral factors that may expose the individual to risk factors. Poor oral hygiene habits, poor dietary habits, and frequent use of medicines that contain sugar, are acidic, and/or xerogenic are examples of behavioural factors that may lead to an increased caries risk. These sugary foods interact with bacteria mainly Streptococcus mutans on tooth enamel and lead to the initiation of dental caries, which is the most common chronic infectious disease of childhood. These bacteria break down sugars for energy, causing an acidic environment in the mouth and result in demineralization of the enamel. S. mutans can spread from mother to baby during infancy and can inoculate even in predentate infants. Early childhood caries (ECC) can begin early in life, progresses rapidly in those who are at high risk, and often goes untreated.[2]

Most children with ECC have a complaint of pain indicating bacterial involvement of the dental pulp. There is a cytokine cascade that is 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. IL-6 has both pro-inflammatory 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.[3] These pro-inflammatory cytokines play a regulatory role in cardiac remodeling and possess strong cardiac depressant properties. It is possible that a chronic bacterial infection like ECC can induce an upregulation of pro-inflammatory cytokines which may be very much aggravated in patients having an underlying cardiac disease. Many studies suggest a strong correlation between IL-6 with dental caries as well as in VSD.[4] Hence, the aim of this study was to compare and assess the levels of salivary IL-6 in children with ECC and VSD following comprehensive full mouth rehabilitation.


   Methods Top


Eighteen children diagnosed with ECC and VSD who visited the Department of Pedodontics and Preventive Dentistry and Pediatric Cardiology of our sister medical institute formed a part of this study. Children between 3 and 6 years of age and those with Type II ECC by Wayne's criteria (labiolingual carious lesions on maxillary incisors with/without involvement of molars and unaffected mandibular anterior) were included in this study.[5] However, children suffering from other common congenital cardiac conditions such as coarctation of aorta, transposition of the great arteries, patent ductus arteriosus, tetralogy of Fallot, and pulmonary atresia were excluded from this study due to comorbid health concerns. The Institutional Ethical Committee granted clearance and an informed consent was obtained from parents before the initiation of the study.

Before the commencement of the study, an intraoral examination that included decayed, extracted, filled, decayed, missing, filled (dmf), and gingival index (GI) were recorded. Patients were asked to refrain from eating 60 min before and rinse their mouths 20–30 min prior the saliva 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, labeled, and arranged in racks stored at −80°C ultracold temperature until subjected to the enzyme-linked immunosorbent assay (ELISA) tests. Salivary IL-6 assessment was done using the SINCERE™ IL-6 kit (Sincere Biotech Co., Ltd., Beijing, China) specific for salivary IL-6 in the Department of Biochemistry [Figure 1], [Figure 2]a, [Figure 2]b, [Figure 3]a, [Figure 3]b and [Figure 4]a, [Figure 4]b.
Figure 1: Study flowchart

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Figure 2: (a) Diluting saliva samples with IL-6 sample diluent. (b) Plates placed on mini-rotator shaker which mixes constantly

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Figure 3: (a) Blue color obtained after adding 100 μL tetramethylbenzidine solution. (b) Yellow color obtained after adding stop solution

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Figure 4: (a and b) Optical density read on a standard plate reader at 450 nm

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Based on the oral examination and findings, a tailor-made treatment plan was formulated for each patient. After the comprehensive dental rehabilitation, the children were timely followed up for a period of 1 month. During the follow-up, the children and parents were instructed and demonstrated of the brushing techniques and other oral hygiene measures. Dental restorations were re-assessed for intactness at recall visits.

The posttreatment salivary samples were collected 1 week and 1 month after the completion of comprehensive dental treatments in a similar manner for IL-6 estimation. The indices were finally recorded at the end of 1 month and compared. The Fisher's Least Significant Difference (LSD) test was used for intergroup comparison of salivary IL-6 levels, while the paired t-test compared the values of GI before and after treatment.


   Results Top


The mean of the pretreatment levels of salivary IL-6 was 492.94 ± 114.49 (sample A) which decreased to 415.46 ± 86.60 (sample B) after 1 week and then to 343.46 ± 61.75 (sample C) after 1 month. Fisher's LSD test used for the intergroup comparison of salivary IL-6 levels showed a high statistical significance (P < 0.000). This clearly indicated the effectiveness of dental rehabilitation of children with ECC and VSD [Table 1].
Table 1: Mean value and standard deviation obtained for salivary interleukin-6 levels

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The paired t-test compared the pre- and post-treatment GI values. The mean pretreatment GI was 0.68 ± 0.22 (sample A) which reduced to 0.55 ± 0.21 (sample B) after 1 month. This reduction in the values was found to statistically significant (P < 0.002) [Table 2].
Table 2: Mean value, standard deviation, and the P value obtained for gingival index

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   Discussion Top


It is known that children with very complex CHDs frequently require regular long-term medication, but the knowledge of oral health effects caused by long-term medication in medically compromised children is sparse. It has been noticed that most of these children do not practice proper oral hygiene measures and dental care for these children are often neglected by the parents or caregivers. Furthermore, the patients with CHD are indulged more frequently in sweet snack foods due to their chronic illness.[6] These sugary foods interact with bacteria mainly S. mutans on tooth enamel and lead to the initiation of dental caries, which is the most common chronic infectious disease of childhood. These bacteria break down sugars for energy, causing an acidic environment in the mouth and result in demineralization of the enamel. S. mutans can spread from mother to baby during infancy and can inoculate even in predentate infants. ECC can begin early in life, progresses rapidly in those who are at high risk, and often goes untreated. Its consequences can affect the immediate and long-term quality of life of the child and family and can have significant social and economic consequences beyond the immediate family as well.[2]

Several studies showed that children with congenital heart disease had significantly more caries in their primary teeth [7] and the dental ages of the children with a congenital heart disease were significantly lower.[8] Ajami et al.[9] evaluated the rate of oral S. mutans and the rate of caries in children suffering from heart disease and concluded that the rate of S. mutans in children with congenital heart disease was significantly higher than the rates in children with acquired heart disease and healthy control subjects.

IL-6 is known to have a strong correlation with severity and extent of carious lesions in the oral cavity, as well as an inflammatory marker in VSD.[3],[10] Qing et al.[4] detected the presence of pro-inflammatory cytokines such as TNF-α, IL-1-beta, IL-6, and the anti-inflammatory cytokine IL-1 in the myocardium of patients with VSD. Studies done by McQuinn et al.[10] found that, in children following VSD repair, inflammatory cytokine induction occurs including IL-2, 4, 6, 8, and 10, which is related to the emergence of a specific matrix metalloproteinase (MMP) profile. Moreover, this specific cytokine and MMP profile were associated with ischemic time (cross-clamp time), inotropic requirements, postoperative fluid balance, and blood product utilization. These studies clearly indicate the significance of IL-6 in patients with VSD. Therefore, minimizing its levels will help in improving the inflammatory condition in the oral cavity, thereby improving the general health. This study was a pioneering approach to assess the levels of salivary IL-6 in children with ECC and VSD and to evaluate the difference in levels of salivary IL-6 after full mouth dental rehabilitation.

This study included 18 children diagnosed with Type II ECC with VSD. Literature shows evidence of increased IL-6 levels in many systemic conditions, thus warranting us to set the exclusion criteria as children with systemic diseases, medically compromised conditions, mentally and physically disabled children, and other cardiac problems.[3],[11],[12] Studies by Chia et al.[13] found that S. mutans antigens exerted immune modulatory effects on human cells of different origins and might contribute to the development of immunopathological reactions in cardiac patients by stimulating the production of pro-inflammatory cytokines such as IL-1, IL-6, IL-8, and TNF-α.

In the present study, saliva was used as the diagnostic fluid as it is both noninvasive and painless, causes minimal patient discomfort when compared to venous blood sampling, easy to obtain repeated samples, and provides good correlation with blood concentrations of various parameters.[3],[14],[15],[16],[17],[18],[19]

Before 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 as to clear the oral cavity of any debris.[20] A similar methodology was used by Menon et al.[3] and Gornowicz et al.[17] Saliva was collected using a pipette and Eppendorf tube. The advantages of using polythene pipette for saliva collection are that it does not harm the patient, can be easily cleaned, and has a longer life than ordinary bulb Pasteur pipette. After collection of saliva, the samples were taken to the laboratory for centrifugation within 2 h. Samples were subjected to centrifugation at 1500 ×g (at 3000 rpm). Abdul-Wahab and AWD Hamad et al.[21] and Sexton et al.[22] have shown that centrifuging removes mucins and other particulate matter which may interfere with antibody binding, ultimately leading to falsely elevated results. The saliva supernatant was separated and stored under low temperature in an “ultracool” temperature storage unit at −80°C so as to prevent microbial growth and avoid degeneration of cytokines.[3],[17],[22]

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 Sincere™ IL-6 kit. This kit is a sandwich immunoassay, specifically designed for the measurement of salivary IL-6. ELISA has also been used previously in numerous studies for determining the concentration of IL-6.[3],[15],[17],[18],[19]

The GI has been used by many authors to assess the oral health of children.[3],[23],[24],[25] The GI (Loe and Silness, 1967) and the dmfs index were recorded first before the treatment commenced and 1 month after completion of full mouth rehabilitation to evaluate the difference in oral health. The mean pretreatment GI was 0.68 ± 0.22 which reduced to 0.55 ± 0.21 after 1 month.

The mean pretreatment salivary IL-6 level was 492.94 ± 114.49 in this study which was decreased to 415.46 ± 86.60 after week and then to 343.46 ± 61.75 after 1 month. This may be attributed to the completion of oral rehabilitation in these children. Frequent follow-up appointments to reinforce good oral hygiene habits would have also contributed to the reduced IL-6 levels.


   Conclusion Top


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. This study proves that if children with ECC and VSD are completely rehabilitated and kept under frequent follow-up, which includes the reinforcement of oral hygiene measures and maintaining a low caries activity state, the level of inflammation can definitely be minimized and thereby improving the quality of life of the affected children. 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.

Acknowledgment

The authors extend their gratitude to Prof. (Dr) Krishnakumar, Head of Pediatric Cardiology and Prof.(Dr) Sajitha Krishnan, Department of Biochemistry, for their contribution and wholehearted support toward this clinical research. We are grateful to Department of Nanosciences and Nanomedicine for their valuable guidance and help rendered.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
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