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ORIGINAL ARTICLE
Year : 2021  |  Volume : 39  |  Issue : 2  |  Page : 189-195
 

Association of salivary physicochemical characteristics and peptide levels with dental caries in children


1 Department of Pediatric Dentistry, Faculty of Dentistry, Golestan University of Medical Sciences; Dental Research Center, Golestan University of Medical Sciences, Gorgan, Iran
2 Department of Pediatric Dentistry, School of Dentistry, Shahid Beheshti University of Medical Sciences, Tehran, Iran
3 Department of Medical Immunology, Faculty of Advanced Medical Science Technologies, Golestan University of Medical Sciences, Gorgan, Iran
4 Department of Molecular Medicine, Faculty of Advanced Medical Science Technologies, Golestan University of Medical Sciences, Gorgan, Iran

Date of Submission28-May-2020
Date of Decision03-Sep-2020
Date of Acceptance01-Dec-2020
Date of Web Publication29-Jul-2021

Correspondence Address:
Prof. Ghassem Ansari
Department of Pediatric Dentistry, School of Dentistry, Shahid Beheshti University of Medical Sciences, Tehran
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JISPPD.JISPPD_251_20

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   Abstract 


Aim: The purpose of this investigation was to evaluate the association of physicochemical properties and antimicrobial peptide levels of saliva with caries activity in children. Materials and Methods: The required volume of unstimulated saliva was collected from 41 children aged 3–12 years with no systemic diseases. Caries activity was calculated using DMFS and dmfs records for each participating child. Collected saliva samples were then examined for their flow rate, pH, and buffering capacity. The concentration of three peptides was assessed including LL-37, human neutrophil peptide (HNP) 1–3, and human beta-defensin (HBD)-3 through an enzyme-linked immunosorbent assay. The correlation between caries activity score (CAS) and salivary variables was looked using the linear regression and Spearman's correlation method. The comparison of CAS means between high- and low-value groups of salivary items was performed using independent sample t-test while the association of CAS and salivary parameters in categorical scale was tested by Chi-square test. Results: No statistically significant differences were found between the CAS means at low and high categories of each salivary physicochemical parameter and those of antimicrobial peptides. There was a negative correlation between HNP1–3 and CAS and also between HBD-3 and CAS, but these results were not statistically meaningful. High HNP1–3 concentration was noted in 67% of the low caries rate group and 29% of the high caries rate group, with a statistically significant difference between the low and high caries rate groups (P = 0.019). Conclusion: Salivary inherent factors are not dominant determinants in caries activity. The current results may suggest that α-defensins (HNP1–3) have a protective role against dental caries.


Keywords: Antimicrobial peptides, caries, children, physicochemical characteristics, saliva


How to cite this article:
Ramezani J, Khaligh MR, Ansari G, Yazdani Y, Mohammadi S. Association of salivary physicochemical characteristics and peptide levels with dental caries in children. J Indian Soc Pedod Prev Dent 2021;39:189-95

How to cite this URL:
Ramezani J, Khaligh MR, Ansari G, Yazdani Y, Mohammadi S. Association of salivary physicochemical characteristics and peptide levels with dental caries in children. J Indian Soc Pedod Prev Dent [serial online] 2021 [cited 2022 Aug 11];39:189-95. Available from: http://www.jisppd.com/text.asp?2021/39/2/189/322501





   Introduction Top


Dental caries remains to be one of the most challenging complications to be tackled during early stages of life. Many factors have been proved to be effective in reducing the risks of caries initiation and progression in children. Among these factors, salivary components are known to have a substantial role in mediation and subsequently reduction of the risks involved in caries initiation and progression. Salivary innate protective mechanisms include (1) mechanical cleansing; (2) reducing enamel solubility by calcium phosphate and fluoride ions; (3) buffering and neutralizing potential; and (4) antimicrobial activity using antimicrobial products {Van Nieuw Amerongen, 2004 #40} such as immunoglobulins, lysozyme, lactoperoxidase, lactoferrin, agglutinins, and antimicrobial peptides.[1],[2]

Antimicrobial peptides are essential components of natural immunity, participating in first-line protective reactions.[3],[4] Antimicrobial peptides are divided by amino acid composition and three-dimensional structure into three main families of α-helical peptides without cysteine (the cathelicidins), peptides with three disulfide bonds (the α- and β-defensins), and peptides with an unusually high proportion of specific amino acids, like histatins.[4],[5],[6] More recent findings suggest that defensins and cathelicidins have an antibacterial activity in the oral cavity while histatins are primarily antifungal agents.[6],[7]

Human beta-defensins (HBDs) are widely detected in oral tissues and in the gingival epithelium.[8],[9],[10] HBD-1 and HBD-2 are found in salivary glands, their ducts, and saliva.[11],[12] HBD-3 is a relatively new discovered member of the host-defense peptide family which has recently attracted more attention. This molecule is produced either inherently or by stimulation. This peptide exhibits antibacterial activities toward Gram-negative and Gram-positive bacteria while being a chemoattractant.[13] Alpha-defensins or Human Neutrophil Peptides (HNP1-3) that are detected in neutrophils and play a role in nonoxidative microbial death which is also present in gingival crevicular fluid.[14],[15] Human cathelicidin peptide LL-37 is found in neutrophils and inflamed epithelium as well as in saliva. Both the mRNA and protein for cathelicidin peptides have been centralized to the salivary glands, especially in acinar cells of the submandibular gland and palatine minor glands. They are also seen in the lingual epithelium and palatal mucosa of mice while in submandibular duct cells of humans.[16],[17] The defensins and cathelicidins have broad antimicrobial activity against both Gram-negative and Gram-positive bacteria and even Candida albicans. They have also been tested positive in laboratory investigation on oral microorganisms such as Streptococcus mutans, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans. Therefore, both the defensins and LL-37 could have a role in protecting the tooth structure from caries as well as oral mucosa.[18],[19],[20]

It is critical to keep the pH of the oral cavity at about 6.7 in order to maintain the tooth structure integrity. In normal condition, saliva is saturated and the ion activity products are equal to the solubility product of hydroxyapatite, and therefore, no demineralization or remineralization will occur.[21],[22] The critical pH value is referred to as a fixed value of 5.5, used as the threshold for determining demineralization point of teeth. The primary determinants of critical pH are the total calcium and phosphate concentrations in saliva. As flow-dependent variations happen in total calcium and phosphate concentrations of saliva, critical pH may be changed by up to one pH unit from the mean with different flow rates. Unstimulated saliva has a lower critical pH than stimulated saliva because of a higher total phosphate concentration in unstimulated one. Different individuals may have different critical pH values due to interindividual variations in saliva total calcium and phosphate concentrations. Thus, the critical saliva pH is not static, but more a dynamic variable, which varies around a mean pH value of 5.5.[22],[23]

There are three buffering systems in human saliva which include phosphate, bicarbonate, and protein buffer systems. The carbonic acid/bicarbonate is the main buffer in stimulated saliva. The equilibrium for bicarbonate buffer system is CO2 + H2O ↔ H2CO3 ↔ HCO3 + H + where CO2 is carbon dioxide, H2CO3 is carbonic acid, and HCO3 is bicarbonate. When food enters the mouth, two notable changes occur as drop in pH and rise in bicarbonate concentration. Increased bicarbonate concentration removes the additional amounts of H + produced by bacteria; thus, the above equilibrium shifts to the left to produce more carbonic acid. However, the oral cavity concentration of carbonic acid is constant, and the excess carbonic acid must be removed. An increase in carbonic acid concentration shifts the equilibrium further to the left, producing more CO2. Extra CO2 diffuses out from the mouth due to the higher CO2 salivary partial pressure than that of the atmosphere. When the concentration of carbonic acid falls, more bicarbonate ions bind to hydrogen to form carbonic acid, establishing a new equilibrium. With this mechanism, excess H+ produced within plaque is neutralized and removed, reducing the risk of tooth erosion.[22],[24],[25]

The phosphate buffer system acts by HPO42−ion through binding to a hydrogen ion to form H2PO4-ion. This acid-base pair has a pKa value ranging from 6.8 to 7.2, with maximum buffering capacity that is relatively close to the salivary pH range of 6–8. However, its effectiveness is low due to insufficient concentrations of phosphate in the oral cavity. Phosphate buffer is believed to be moderately efficient in unstimulated saliva.[22]

Saliva proteins can act as buffers when the pH varies in relation to their isoelectric points. Below their isoelectric point, proteins can absorb protons, while above that, they can release protons. Many salivary proteins have their isoelectric point between pH 5 and 9 which are considered as good buffers at alkaline and especially at acidic pH values. Moreover, some of the saliva proteins increase the viscosity of saliva when the pH decreases and thereby protect the teeth against acid load by formation of a physical barrier.[22],[25]

The purpose of this investigation was, therefore, to determine the salivary flow rate, pH, buffering capacity, and concentration of LL-37, HNP1–3, and HBD-3 peptides as well as evaluation of their association with dental caries experience in children.


   Materials and Methods Top


This in vitro study was approved by the Ethics Committee of the Golestan University of Medical Sciences (IR.GOUMS.REC.1394.117). Informed consent forms were collected, signed by the parents or guardians of the children prior to sample collection. Forty-one patients were included aged 3–12 years from those referred to the Pediatric Dentistry Department at Golestan University of Medical Sciences, Gorgan, Iran.

Inclusion criteria were as follows: (1) medically healthy children; (2) no tooth structure abnormality; (3) children with primary or mixed dentition; (4) free from conditions that affect salivation such as asthma, diabetes, surgery, radiotherapy, and medication; and (5) should not be under orthodontic treatment. Exclusion criteria were children who did not show enough cooperation for examination or saliva evaluation and all children with interfering systemic diseases.

Daily exposures to sugar-containing snacks or beverages as well as brushing activity per day were recorded. A full-mouth caries assessment was performed using visual/tactile criteria, supplemented by standard radiographic views as needed. DMFS and dmfs were recorded for each case, and finally, caries activity score (CAS) was determined as the sum of decayed, missed, and filled surface (DMFS + dmfs) in each patient.

Unstimulated saliva was collected through simple spitting into the specific laboratory container provided. All specimens were collected between 9 am and 1 pm. Children were asked not to eat, drink, or brush their teeth for at least 1 h before the test, however, they were advised to rinse the mouth with still water before sample collection. Sampling was performed while the child was sitting in an upright relaxed chair position under natural light. They were asked to spit in a clean plastic container, and the collected saliva was registered under their coded mark in 5 min time. Spitting was continued until 2 ml of saliva was collected.

The salivary flow rate was calculated by dividing the amount of collected saliva to the duration of the collection (5 min). The pH and buffering capacity were evaluated using GC Saliva Check (Tokyo, Japan) based on the manufacturer's instructions [Figure 1].
Figure 1: Evaluation of salivary pH and buffering capacity with GC Saliva Check kit. (a) The color of the test strip shows pH 6.4. (b) The buffer test strip shows green, green/blue, and red/blue color, based on conversion table buffering capacity is 8

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Saliva samples were centrifuged at 10,000 g for 5 min. Cleared unfractionated saliva was frozen at − 80°C for later analysis. LL-37, HNP1–3, and HBD-3 concentrations were evaluated by an enzyme-linked immunosorbent assay (ELISA) as well as quantitative sandwich ELISA technique using a commercially available analysis kit, specific for these peptides (OmniKine BD-3 Human ELISA Kit and Elabscience Human HNP1–3 and LL-37 ELISA Kit, USA) [Figure 2].
Figure 2: Evaluation of peptide concentration with enzyme-linked immunosorbent assay kit. Wells contain human neutrophil peptide 1–3 in the absorbance reader for calculating the concentration

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Descriptive statistics were used to express centering and variability values along with comparison of the means between genders were done with Independent sample t-test Mann-Whitney U test. The correlation between CAS and salivary variables and also between salivary variables and age was performed using the linear regression, Spearman's, and Pearson correlation tests. The comparison of means of CAS between high- and low-value groups of salivary items was performed with the independent sample t-test. Chi-square test was used to evaluate the association between CAS and salivary parameters in categorical scale. All analyses were performed with SPSS v19.0 (IBM Corp, Armonk, NY, USA). The significance level of all statistical tests was predetermined at P < 0.05.


   Results Top


From the total population, 27 were female and 14 were male. All participants were between 5 and 11 years of age. Based on their race, 73% were Persian and 27% Torkman and Sistani. Thirty-six subjects were at mixed dentition stage and 5 were at their primary dentition stage. Majority of the cases (38 children) were at high risk of caries development while only 3 were scored at moderate risk for caries. The descriptive results and distribution of variables by sex are presented in [Table 1].
Table 1: Descriptive data and distribution of variables by sex

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No significant correlation with age was noted for the CAS, flow rate, pH, buffering capacity, and salivary protein concentration [Table 2].
Table 2: Correlation evaluations of caries activity score, flow rate, pH, buffering capacity, and protein concentration with age

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No significant correlation with CAS was seen for the flow rate, pH, buffering capacity, and salivary protein concentration [Table 3].
Table 3: Correlation evaluations between salivary parameters and caries activity score

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Simple linear regression showed that CAS was not predictable based on pH, flow rate, buffering capacity, and LL-37 concentration [Table 4].
Table 4: Simple linear regression for predicting caries activity score based on pH, flow rate, buffering capacity, and LL-37 concentration

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The comparison of CAS means between low- and high-value groups of salivary items is presented in [Table 5].
Table 5: Comparison of the means of caries activity score between high- and low-value groups of salivary parameters

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The association between low or high salivary parameters and low (CAS ≤ 22) or high (CAS > 22) caries activity was established [Table 6].
Table 6: Associations between caries activity score categories and salivary item groups

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   Discussion Top


Potential caries prevention is well supported by the presence of certain salivary proteins, and investigations on their effects and their mechanisms in reducing caries risk have long been going on. The result of this investigation was supportive to such hypothesis with certain limitations on variations of the salivary components. The result of this investigation was supportive to such hypothesis with certain limitations on variations of the salivary components.

Participants of this investigation were matched for their caries risk based on the standard caries-risk assessment tool.[26],[27] The frequency of sugar-containing snack or beverage consumption between meals was more than three times per day. They reported to have had no daily brushing regimen with fluoridated toothpaste and had more than one interproximal lesion. The total of decayed, missed, and filled surface (DMFS + dmfs) was calculated in each patient, using a new numerical scale called CAS, along with the nominal scale of high or low caries activity.

No statistically significant correlation was found between CAS and salivary physicochemical parameters. The correlation between buffering capacity and CAS was negative, with no statistically significant correlation.

The differences of the mean were not significant in CAS between low and high categories of each salivary physicochemical parameter. There was no significant association between high or low CAS groups and high or low physicochemical parameters.

Animireddy et al.[1] stated that salivary pH had been significantly higher in caries-free group compared to low caries and nursing caries groups. However, there was no statistically significant difference between low caries and nursing caries groups. Prabhakar et al.[28] demonstrated that pH, buffering capacity, and salivary flow rate were not significantly different between caries-free and caries-active groups in 7–14-year-old children. Thaweboon et al.[29] showed that pH and salivary flow rate were not statistically different between caries-free and rampant caries cases of 5–10 year-olds.

Tayab et al.[30] reported that pH, buffering capacity, and salivary flow rate of the caries-active group as being significantly lower than the caries-free group. Kaur et al.[31] showed that the salivary flow rate is normal in 90% of caries-free and 33% of caries-active cases in 4-6 year-olds. Sufficient pH in 100% of caries-free children and 30% of caries-active children was reported, with the differences being statistically significant.

These controversies along with a lack of significant association between physicochemical characteristics of saliva and CAS in the current investigation could be an indication of more powerful environmental factors compared to salivary inherent factors. It appears that the ideal physicochemical characteristics are not adequate to overcome undesirable factors in high caries risk groups, where the environmental parameters are strong.

Based on the results achieved from the collected data, the concentration of LL-37, HBD-3, and HNP1–3 was between 0–75.10, 0–4890, and 0–277 ng/ml, respectively. Tao et al.[20] reported these values at 120–12,000, 0–6210, and 60–10,500, respectively. Phattarataratip et al.[32] reported 3.93–71.02, 0.15–11.56, and 548.63–5231.06 in order for LL-37, HBD-3, and HNP1–3, respectively. Davidopolou et al.[33] found LL-37 concentration to be between 0.22 and 275 ng/ml.

An extensive variation was noted in these peptides concentration between individuals and different populations. One explanation could be the differences in genetic and racial backgrounds. Several earlier studies have revealed the differences in salivary protein concentration between people with various racial backgrounds.[34],[35],[36],[37] However, more studies are required to confirm ethnic influences or genetic polymorphisms on salivary protein expressions.

A negative correlation was noted between HNP1–3 and CAS and also between HBD-3 and CAS, with nonstatistical significance. There were no significant differences in CAS means between low and high salivary antimicrobial peptide values. High HNP1–3 concentration was noted in 67% of the low caries rate group and 29% of the high caries rate group, which was statistically significant.

The role of HNP1–3 in caries susceptibility was also reported by Tao et al.[20] who indicated that HNP1–3 was significantly higher in caries-free children than in those with evidences of caries. Ribeiro et al.[38] showed that HBD-3 and HNP1–3 could desirably decrease caries risk, but LL-37 could not. Davidopolou et al.[33] looked at caries-free children and those with low-to-moderate caries activity and reported that they tend to have significantly higher levels of LL-37 saliva concentrations than those with high caries activity.

Phattarataratip et al.[32] reported no statistically significant difference between the two caries activity groups (caries-active and caries-free groups) on salivary levels of HNP1–3, LL-37, HBD-3, and HBD-2. It appears that peptide concentration is somehow related to salivary flow rate, and caries-active subjects may have lower salivary flow rates than caries-free subjects that could result in more caries activity. They also stated that caries-active subjects may have higher concentrations of specific protein components that facilitate dental caries initiation.[32] The role of specific salivary proteins has been documented in promoting the adhesion of bacteria onto the oral surfaces through the formation of adherent biofilms and pellicles.[39],[40]

In contrast to the current study, Davidopolou et al.[33] reported a significant correlation between peptide concentration and age. They stated that the concentration of the peptide increases with age and reaches a plateau in late adolescence. A greater age range was used for the current investigation including those in puberty; thus, a higher protein concentration which is age correlated could be expected.

The current study results revealed that the mean LL-37 concentration in girls was found to be higher than boys who participated, although the difference was not statistically significant. The same findings were reported earlier by Davidopolou et al.,[33] where it was suggested that such higher concentration could be due to sexual differences in immunocompetency. This sexual dimorphism has been observed in immune function, with the females being more immunocompetent than males.[41] Srivastava et al.[42] reported differences in gene expression related to age and gender in the human parotid gland.

A potential protective effect is acknowledged for α-defensins (HNP1–3) on caries prevention. These peptides are present in submandibular glands, which are the major sources of unstimulated saliva. They are also seen in neutrophils that migrate into the oral cavity within the gingival crevicular fluid. It has been estimated that 30,000 neutrophils/min enter the oral cavity through this route and the junctional epithelium surrounding the teeth.[43] Therefore, α-defensins could have extensive antimicrobial effects implemented through saliva.

It appears that salivary inherent factors could have a determinant role in caries development, but environmental factors have strong adverse effects in this process that overshadow the role of salivary factors.


   Conclusion Top


  1. α-defensins (HNP1–3) have a protective role against dental caries
  2. These peptides could be used for clinical evaluation of caries risk
  3. Environmental factors such as oral hygiene and diet have a greater role in preventing dental caries than salivary inherent factors.


Acknowledgment

The authors would like to express their appreciation to the staff of the Pediatric Dentistry, School of Dentistry also staff members of Molecular Medicine, School of Advanced Medical Science, Golestan University of Medical Sciences, for their valuable cooperation and support.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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    Abstract
   Introduction
    Materials and Me...
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  2005 - Journal of Indian Society of Pedodontics and Preventive Dentistry | Published by Wolters Kluwer - Medknow 
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