Caries and Count: Exploring Salivary Biomarkers in Children With Down Syndrome - A Prospective Study

INTRODUCTION

The most prevalent genetic condition, Down syndrome, results from the presence of an extra copy of chromosome 21 (trisomy 21), causing intellectual disability with a varying degree of decline in cognitive function. Patients with Down syndrome present with characteristic phenotypic features and are at a higher risk for haematological disorders, cardiovascular abnormalities, immunological impairment, and age-related neural degenerative diseases. The affected individuals have accelerated cell aging and neurological disorders due to a buildup of excessive oxidative stress from impaired metabolism of reactive oxygen species (ROS). A healthy human body possesses an antioxidant defence, which protects both enzymatic and non-enzymatic metabolic pathways. One such mechanism involves superoxide dismutase (SOD-1), an enzyme encoded on chromosome 21 (21q22.1),^[1] which catalyses the conversion of superoxide anions into hydrogen peroxide and oxygen. Because of the gene dosage effect, which raises ROS levels and can cause oxidative stress, SOD-1 activity is increased in people with Down syndrome.^[2,3]

Children with Down syndrome have a high incidence of dental caries,^[4] which emphasises the role of early diagnosis in these children. This can be achieved through testing of salivary biomarkers, which may reduce the need for future invasive treatment modalities. The matrix metalloproteinase enzymes, such as collagenase, are stimulated by bacterial toxins during the carious process. This results in the breakdown of the collagen matrix in dentin and the start of an inflammatory carious process. With any form of inflammatory process, lipid peroxidation reaction is stimulated, which results in the production of malondialdehyde (MDA),^[5] a byproduct found in saliva, which is a marker of oxidative stress. This forms an important etiologic factor in dental caries.

MDA is a derivative of lipid peroxidation, which alters the function and structure of host cells via the following mechanism. The hydroxyl radical that is generated sets off a series of events that result in the peroxidation and membrane disruption of lipids. These lipid radicals can permeate through membranes, changing their composition and properties, and impairing cell homeostasis.^[6]

Saliva is the first line of defence against caries process, by imparting essential protective qualities- like the natural flushing action, buffer capacity, pH, and flow characteristics. The bacterial acids are subjected to saliva, and the buffering capacity is based on the bicarbonate system, carbonic acid, and phosphate. In children with active caries, this quality is reduced.^[7] The acids cause an overall reduction in the pH of saliva, which contributes to the growth of acidogenic and aciduric bacteria, making the environment unfavourable for the protective species. As a result, the oral microbiota alters, which favours cariogenic bacteria and further causes a reduction in the salivary pH.^[8] The viscoelastic nature of saliva, provided by glycoproteins and mucins, allows the removal of debris, bacterial aggregates. However, elevated salivary viscosity has been noted in individuals with increased caries risk.^[9]

To date, no research has explored the association between dental caries in children with Down syndrome, oxidative damage, and ROS. The present study aims to assess salivary parameters, buffering capacity, pH, viscosity, and MDA levels in children with Down syndrome and caries, and to evaluate the changes in these parameters before and after dental treatment.

MATERIAL AND METHODS

This study comprised 15 Down syndrome, aged 6 to 14 years-of both genders, who reported to a department of a teaching dental hospital. Informed consent in English and the native language was obtained from the parents before the commencement of the study. Children with three or more active carious lesions (ICDAS-II Code 3-6) and who, after receiving consent from their parents, were willing to take part in the study were enrolled.

The children included in the study underwent a thorough dental examination on a dental chair using a mouth mirror, blunt explorer, and active carious lesions were ascertained via the International Caries Detection and Assessment System-II (ICDAS-II Code 3-6).^[10]

Every patient’s oral findings were documented, and a profile was compiled for convenience in following up with each patient until the study’s conclusion.

Unstimulated salivary samples were obtained to get the baseline values for evaluating the salivary MDA, pH, viscosity, and buffer capacity before caries control.

Treatment of dental caries was accomplished using the following interventions: ICDAS-II Code 3-5 was used to identify active carious lesions that displayed localised enamel destruction and an underlying dark shadow from dentine,^[10] were excavated such that the nidus of inflammation was removed, and they were restored with GC Fuji Gold Label Type IX Glass Ionomer Cement.

For carious lesions with extensive distinct cavities in dentine and involving pulp, identified by ICDAS-II Code 6, pulp therapy was performed for them, followed by which interim restorations were given using GC Fuji Gold Label Type IX Glass Ionomer Cement. Oral health education and motivation were provided by giving oral hygiene instructions, and diet counselling was done. After two weeks post caries control, the patients from both groups were called back, and saliva samples were collected again.^[11]

Saliva check buffer kit

To determine the pH and buffering capacity of saliva, the “Saliva Check Buffer Testing Mat” (GC Dental Products Corp., Kasugai City, Aichi, Japan) was utilised.^[7]

Estimation of salivary pH utilising a “Saliva Check Buffer Testing Mat”

The enclosed pH strip was taken, and one end was immersed in the collected saliva sample for 10 seconds. Subsequently, the colour of the strip was checked. When the paper was still wet, the test strip’s colour was compared. The pH value was monitored, and the findings were documented. The results were determined by the dental saliva pH indicator, as given in the kit [Figure 1].

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Figure 1:

Estimation of saliva pH.

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Estimation of salivary buffering ability by utilising “Saliva Check Buffer Testing Mat” [Figure 2].

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Figure 2:

Saliva check buffer testing mat.

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After opening the buffer test foil pack, saliva was collected from the cup using a pipette. Each of the three test pads received one drop of saliva, and any excess was drained onto a tissue. After 2 minutes, the colour of each pad was compared with the table below, and the 3 scores were added to record the results [Figure 3].

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Figure 3:

Estimation of buffering capacity of saliva.

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RESULTS

Application of the Wilcoxon signed rank test showed a statistically significant difference (p<0.01) in salivary MDA levels between the time intervals, with higher values recorded in the pre-treatment group compared to the post-treatment group. The test also indicated no statistically significant difference (p>0.05) in salivary pH values between the time intervals, although slightly higher values were observed in the pre-treatment group. A statistically significant difference (p < 0.01) was found in salivary viscosity, with higher values in the pre-treatment group compared to the post-treatment group. Similarly, salivary buffering capacity values showed a statistically significant difference (p<0.01) between the time intervals, with higher values noted in the pre-treatment group compared to the post-treatment group.

DISCUSSION

Saliva serves as the primary line of defence against harmful free radicals. The imbalance between the formation of free radicals and counteracting antioxidant activity is known as oxidative stress.^[5] These free radicals are extremely reactive and possess a very short half-life. The compounds are generated from the interaction of ROS/RNX and cellular macromolecules, mediated via the iron-catalysed Haber-Weiss reaction, and are preferentially used as biomarkers of oxidative damage. Oxidative stress is evaluated using markers such as lipid peroxidation byproducts, oxidised proteins, and DNA oxidation and fragmentation derivatives. MDA is synthesised from fatty acids via the lipid peroxidation process.^[6] Oxidative stress increases levels of lipid peroxidation, which plays a major role in the pathogenesis of various inflammatory diseases. Apart from MDA, salivary properties, such as pH, viscosity, and buffer capacity, also act as salivary biomarkers to identify and detect various diseases.

The defence system of saliva includes oral clearance, buffering action, inherent innate immunity, and potential for remineralisation. Thus, the balance between protective and pathogenic factors in saliva and plaque, along with the interaction between cariogenic and non-cariogenic microbiota, plays a key role in determining the progression of dental caries.^[7] Also, among the other protective factors, salivary viscosity plays an important role. Fresh mixed human saliva is a viscoelastic fluid.^[13]

In children with Down syndrome, mouth breathing is common due to narrower nasal passages and a large tongue. This increases the viscoelasticity of saliva. Glycoproteins play a crucial role in imparting saliva its viscoelastic properties, forming a lubricating layer that facilitates smooth movement of oral tissues. Assessing salivary viscosity is highly significant, as Higher viscosity has been associated with an increased risk of dental caries.^[14] Buffering capacity is another physical property of saliva that serves as an important biomarker for the initial diagnosis of dental caries.^[7]

In this study, salivary MDA levels in children with Down syndrome and dental caries showed a highly statistically significant difference (p<0.01) between the two time intervals (2 weeks). This can be attributed to the multifactorial nature of dental caries. The inflammatory response in dentin due to caries is mainly due to oxidative stress, which can cause destruction of dental hard tissues, hence initiating the caries process.^[5,15]

In the test group comprising children with Down syndrome and caries, salivary pH values showed no statistically significant difference. Previous investigations have also reported similar outcomes, demonstrating no significant variation in salivary pH between groups. This can be attributed to the buffering capacity of saliva, which counteracts the fall in pH and serves as a key protective mechanism against dental caries.^[16]

Studies investigating the relationship between lipid peroxidation and dental caries have reported no significant differences in salivary MDA levels between healthy children with or without Early Childhood Caries. Likewise, research assessing the association between DMFT scores and salivary MDA levels in individuals with caries also found no statistically significant variations in MDA levels across the examined groups.^[17]

The present research examined the extent of oxidative stress that decreased significantly in patients following rehabilitation. It may be due to increased production of salivary antioxidant enzymes post-interventions. Salivary peroxidase facilitates the oxidation of the thiocyanate ion, producing reactive compounds that suppress the growth and metabolic activity of various microorganisms, thus slowing down caries progression and inflammation post-treatment. This aligns with the findings of studies, which investigated inflammatory biomarkers and demonstrated that increased levels of interleukin-6 in cases of early childhood caries significantly decreased following treatment.^[18]

Similarly, earlier studies assessed Interleukin-1β levels in children with Down syndrome after comprehensive dental rehabilitation and reported a significant decrease in interleukin-1β following treatment.^[19] These studies collectively highlight a strong association between elevated inflammatory mediators and the incidence of dental caries in special needs children, with their levels decreasing after appropriate rehabilitation.

This study revealed a highly statistically significant difference in the test group for salivary viscosity values (p < 0.01), with higher viscosity recorded prior to treatment compared to post-treatment of carious lesions. Similar observations have been reported previously; the relative viscosity of saliva in children with active caries was significantly greater compared to those without caries. Increased salivary viscosity diminishes the natural cleansing capacity of saliva, thereby predisposing to a greater incidence of dental caries.^[13]

The buffering capacity of children with Down syndrome has been reported to be higher, consistent with the present study, where intergroup comparisons also demonstrated higher values in this group, although the difference was statistically nonsignificant. This finding has been attributed to elevated phosphorus levels in their saliva. Other studies have similarly reported an increased buffering ability in individuals with Down syndrome compared to control groups.^[20] In contrast, some investigations have found no significant differences in salivary buffering ability between the groups, while suggesting that the relatively decreased occurrence of dental caries in individuals with Down syndrome may be associated with elevated salivary bicarbonate levels.^[21]

A highly statistically significant increase in buffering capacity was observed in the group with Down syndrome and dental caries following intervention (p<0.01). This can be explained by the role of the major salivary buffering systems, bicarbonates, phosphates, and proteins, which act synergistically to neutralise the pH reduction caused by bacterial activity in the oral cavity. The enhanced buffering action helps in stabilising oral pH, thereby contributing to the slowing of caries progression after treatment.^[7]

These findings align with previous studies that reported a slight decrease in salivary buffering capacity among caries-active children compared to caries-free children, though the difference was not statistically significant.^[22,23] In contrast, some studies have shown that the buffering ability in children with early childhood caries was statistically higher than in caries-free children, while others reported comparable results with no significant differences.^[24]

CONCLUSION

The levels of MDA, viscosity, and buffer capacity significantly decreased post-treatment of carious lesions in children with Down syndrome. This suggests that caries control treatment is effective in reducing oxidative stress in individuals affected by caries. With various rehabilitative interventions, it helped in minimising the levels of oxidative stress among these children, thus bringing down the severity of their disease. Therefore, evaluation of these biomarkers in children with Down syndrome helps in accurate caries risk evaluation and improved treatment efficacy, which will improve the overall oral health status of these children.