Background: The association of total salivary antioxidant capacity (TAC) to severe early childhood caries (S-ECC) has been studied in the past. It is not clear if TAC is an indicator of the inflammatory response to the lesion or a marker of the disease.
Aim: This study aimed to measure the salivary TAC levels in children with early childhood caries before and after dental treatment and compare the results to caries free children.
Methodology: Salivary samples were obtained from 20 children in their fifth year of life diagnosed S-ECC and 20 age and gender matched controls. Complete dental rehabilitation under general anesthesia was performed for the 20 children with S-ECC, and follow up salivary samples were obtained one week and three months post operatively. The TAC was measured using a commercially available Oxygen Radical Absorbance Antioxidant Assay measurement kit (Zen-Bio ORAC™, AMS Biotechnology, Abington UK). The data was analyzed using SPSS version 20 utilizing descriptive analysis and chi-square test, and the level of significance for all tests was set at p<0.05. Results: The mean TAC for the control group was significantly lower than the TAC for the experimental group prior to treatment (p=0.003). Post treatment there was significant reduction in TAC of children with S-ECC and no significant differences were observed between the test and control groups at either one week(p=0.076) or three month recall(p=0.096). There was a significant reduction the TAC of children post treatment when compared to their pretreatment values (p<0.001) Conclusion: Dental treatment significantly reduces the TAC in children with S-ECC suggesting that TAC is a marker of the lesion rather than the disease. Keywords: Severe Early Childhood Caries, Dental Treatment, Total Antioxidant Capacity Introduction The American Academy of Pediatric Dentistry defines severe early childhood caries (S-ECC) as the presence of any smooth surface carious lesion in a child under three years of age, in the ages three to five, it is defined as 1 or more cavitated, missing (due to caries), or filled smooth surfaces in primary maxillary anterior teeth or a decayed, missing, or filled score of ?4 (age 3), ?5 (age 4), or ?6 (age 5).1 S-ECC has been recognized as a serious public health issue in children2. The etiology of dental caries in children is multifactorial, however it has been shown that severe dental caries results in a conventional inflammatory immune response from the body3. Battino et al proposed that, saliva is a heterogeneous fluid that is made up of small organic compounds and molecules, electrolytes, glycoproteins, and proteins. They state that saliva guards against the free radical-mediated oxidative stress4. Since saliva plays an important role in the defense of the oral cavity it natural that saliva's physiochemical properties are linked to caries. It has been shown that markers of antioxidant capacity such as Nitric Oxide (NO) as well as total antioxidant capacity (TAC) are increased in children with early childhood caries3, 5-7. While it is clear that increased TAC is a response to inflammation, it is still debated if the increase in TAC is due to the inflammatory reaction to the bacteria in the lesion or the overall reaction of the body to the disease itself8-10. There is evidence to suggest that both the microbial profile and the immune response of children with S-ECC differs from that of caries free children 11-13. In recent years there has been a tendency to look in to the systemic nature of dental caries, with researchers showing that individual susceptibility to dental caries may be a result of systemic factors such as immune response and oxidative stress14. This has led to several authors to look into the possibility of TAC as a biomarker for dental caries and periodontal disease in children 7, 13, 15, 16. While it is accepted that TAC levels in children are higher than controls, it is not clear if this increase is a marker of the disease or the lesion. The aim of this study was to evaluate the impact of complete dental rehabilitation of children with S-ECC on the total antioxidant capacity of their saliva and to compare their pre and post treatment levels of TAC to age and gender matched caries free children. Methodology Ethical Approval: Ethical approval for this study was obtained from the Institutional Review Board (IRB) of the Riyadh Colleges of Dentistry and Pharmacy, Riyadh Saudi Arabia (FPGRP/ /43435005/126). Informed consent was obtained from the parents of all children prior to examination and assent of the child was obtained verbally in the presence of the parent before proceeding with examination and saliva collection. Sample Power Calculation: Sample power was calculated using the G-Power sample power calculator (Universtat Kiel, Kiel, Germany) Sample Selection and collection of Saliva: A total of 30 children in their fifth year of life diagnosed with severe early childhood caries and scheduled for dental treatment under general anesthesia were included in the test group. Children with long term chronic illnesses and those on medications for chronic illness were excluded from the study. Saliva was collected from the children prior to dental treatment using the passive drool method17. The saliva samples were stored in plastic vials with caps (Plastico, SPS Industries, Jeddah, Saudi Arabia) which were transported in an ice box (Polarbag™, SGS , Fujian, China) to a deep freezer (TSX40086D, Thermofisher Scientific, Waltham, MA, USA) where they were stored at -800C until the time of analysis. Saliva was also collected one week after completion of the treatment. A total of 20 children completed the three month recall appointment when a new salivary sample was collected. The children who completed the recall examination were cross matched for age and gender with control caries free children (Fig 1). Salivary Analysis: The collected saliva was analyzed for total antioxidant capacity using a using a commercially available Oxygen Radical Absorbance Antioxidant Assay measurement kit (Zen-Bio ORAC™, AMS Biotechnology, Abington UK) and analyzed using a mutli detection microplate reader (FLx800™, BioTek® US, Winooski VT, USA). Statistical Analyses: A Shapiro-Wilk test showed that there was significant skew in the sample (p=0.012) thus non-parametric tests were used for the analysis. The Mann-Whitney U test was used to compare mean TAC between the test and control group at baseline and between the test and control group at the three month follow up visit. The same test was also used to compare TAC levels between boys and girls. The Wilcoxon Sign Rank Test was used to compare between the pretreatment and post treatment salivary TAC in the children with S-ECC. Results The final sample comprised of 20 children with S-ECC (8 boys and 12 girls) and 20 age and gender matched controls. The mean age of the sample was 5.14 years (SD+/-0.85 years) with no significant age difference between the boys and the girls in the sample (t=1.529, p=120). No significant differences were observed between the mean TAC levels of boys and girls at base line (U= 184, p=0.098 ) , one week or at the three month follow up visit When the baseline TAC of the children was compared to that of control children it was observed that the baseline TAC of children with S-ECC was significantly higher than those of the age matched controls (p=0.003). One week after complete dental treatment under general anesthesia, the children in the S-ECC group still had a higher TAC than controls but the differences were not statistically significant. Three months after treatment the S-ECC group actually had a lower mean TAC level than the control group, although the differences were not statistically significant (Table 1). When the progression of TAC levels were compared using the Wilcoxon sign rank test, it was observed that significant reductions were observed in the salivary TAC levels of children from baseline to one week and from one week to three months (Table 2). Discussion The role of antioxidants, and salivary total antioxidant capacity (TAC), in severe early childhood caries is a topic that has received much attention in literature 6, 18-21. The focus of this literature has been the comparison between caries free children and those with severe early childhood caries (S-ECC) 18-20. The aim of this study was to look into the impact of dental treatment on the TAC of children with S-ECC and compare them to caries free children. The children with S-ECC in our study had significantly greater salivary TAC before treatment than caries free controls which in keeping with most previous studies on the topic 7, 18-20, 22. Salivary TAC has been proposed by some authors to be a biomarker for dental caries 6, 21, 23. Some have even gone so far as to suggest that anti-oxidant rich preparations may help prevent or treat dental caries24, 25. However, there are others who suggest that the elevated anitoxidants are a marker of the inflammatory response to dental caries or faulty dental restorations 26-28. Complete dental rehabilitation of the child under general anesthesia routinely performed in children with multiple carious lesions, in whom behavioral problems prevent the treatment of the lesions in the dental chair2. The rationale for selecting this group of children in this study was not only the fact that they suffered from S-ECC but also that the treatment would ensure complete removal of dental caries as a source of inflammation. This methodology has been previously used to study the impact of dental treatment on salivary cortisol levels, but has never been used in the assessment of salivary TAC29. The significant reduction of salivary TAC post-operatively suggests that increases in TAC in our study sample were related to the untreated dental caries. The lack of significant difference in salivary TAC between the control group and the samples taken at one week and three months post-operatively seem to suggest that rather than a biomarker for the disease, TAC may simply be a measure of inflammatory changes. The results of this study have to be viewed keeping in mind certain limitations. The relatively small sample size make it difficult to generalize the results. The follow up period in this study was limited to three months in order to avoid the possible confounding effects of secondary dental caries or new carious lesions. However, long term effects of dental treatment and the role of TAC in predicting the recurrence of dental caries cannot be assessed with the present design and are beyond the scope of the present study. Conclusions Within the limitations of this study we can conclude that the increase of salivary TAC observed in patients with S-ECC appears to be due to the inflammatory effects of the lesions. The increased TAC disappears after the removal of the lesions and this reduction is maintained one month postoperatively. Long term studies into the effects of dental treatment on salivary TAC are recommended to further understand the relationship between salivary TAC and S-ECC.