Journal of Orofacial Sciences

ORIGINAL ARTICLE
Year
: 2022  |  Volume : 14  |  Issue : 2  |  Page : 100--106

Estimation of Matrix Metalloproteinases-9 (MMP-9) Levels in Children with Early Childhood Caries


Ngangbam Reenayai, Manju Raman Nair 
 Department of Pediatric and Preventive dentistry, AB Shetty Memorial Institute of Dental Sciences, NITTE (Deemed to be University), Mangaluru, India

Correspondence Address:
Dr. Manju Raman Nair
Professor and Head of the Department, Department of Pediatric and Preventive Dentistry, AB Shetty Memorial Institute of Dental Sciences, NITTE (Deemed to be University), Deralakatte, Mangaluru, Karnataka 575018
India

Abstract

Introduction: The primal incidence of dental caries is from the childhood. To assess the levels of salivary MMP-9 in children with Early Childhood Caries following caries control. Materials and Methods: 45 children between the age group of 3-5 years were selected for the study and divided into three groups of 15 each. Group A was the control group. The salivary samples were collected before any treatments in ECC (Group B) and S-ECC (Group C), following which caries control measures were done. Following 2 weeks of post-treatment, the saliva samples were collected from experimental Groups and tested for salivary MMP-9, pH, and viscosity levels. Results: The mean value of the salivary MMP-9, pH, and viscosity levels estimated in the control group of children was 7.13 ng/ml (±0.809 mg/ml), 7.06 (±0.338), and 0.41 (±0.072) respectively. The pre-treatment salivary MMPs, pH, and viscosity levels estimated in Group B were 10.95 ng/ml (±1.469 ng/ml), 7.07 (±0.403), and 1.74 (±0.412) respectively, and in Group C these values were 18.55 ng/ml (±9.218ng/ml), 6.92 (±0.542) and 1.28 (±0.123) respectively. The post-treatment salivary MMP-9, pH and viscosity levels estimated in Group B were 7.34 ng/ml (±0.673 ng/ml), 7.24 (±0.394), and 0.42 (±0.208) respectively and in Group C these values were 7.84 ng/ml (±0.647 ng/ml), 7.14 (±0.377) and 0.49 (±0.338), respectively after 2 weeks. Conclusion: The salivary MMP-9 and viscosity levels in groups B and C decreased significantly 2 weeks following caries control. The Salivary MMP-9 level is a good diagnostic and prognostic biomarker for comparing the efficacy of various caries control treatment protocols. comparing the efficacy of various caries control treatment protocols.



How to cite this article:
Reenayai N, Nair MR. Estimation of Matrix Metalloproteinases-9 (MMP-9) Levels in Children with Early Childhood Caries.J Orofac Sci 2022;14:100-106


How to cite this URL:
Reenayai N, Nair MR. Estimation of Matrix Metalloproteinases-9 (MMP-9) Levels in Children with Early Childhood Caries. J Orofac Sci [serial online] 2022 [cited 2023 Jan 30 ];14:100-106
Available from: https://www.jofs.in/text.asp?2022/14/2/100/367448


Full Text



 Introduction



Dental caries is the most common infectious transmissible disease in childhood including in babies.[1] According to the eruption sequence of the teeth, caries begins on the deciduous/upper maxillary incisors[2] and progresses to the first molars, canines, and second molars. Subsurface lesions that show as white-yellow linear (“white spot”) lesions close to the gingival (gum) margin may be the earliest clinical symptoms of caries. Since the primary enamel is relatively thin, these white spot lesions generally cavitate during a short period. The American Association of Pediatric Dentistry has described Early Childhood Caries (ECC) as the presence of one or more caries (cavities or no cavities), missing teeth (due to caries), or presence of restoration deciduous teeth in children ≤71 months of age. If it occurs on the smooth surface of the tooth in children <3 years of age, the disease is classified as Severe Early Childhood Caries (S-ECC).[3]

Although it is well established that multiple factors, either biological or nonbiological, contribute to an individual’s risk for caries, not many studies evaluated the interactions or correlation between dental carries and host-derived proteases, and in particular matrix metalloproteinases (MMPs) in dentine matrix degradation. MMPs are the Ca/Zn-dependent endopeptidase involved in extracellular matrix degradation[4] and were the first family of proteases implicated in dentin matrix destruction within carious lesions.[5] MMPs generated from the host have been found in saliva, dentine, and Gingival crevicular fluids (GCF). Saliva is a biological fluid that forms the bridge between the formation and susceptibility of caries. There are several functions of saliva such as lubrication, cleaning, digestion, antimicrobial, neutralization of acid and base, demineralization, and remineralization cycle.[6]

MMPs from saliva/dentin are activated by a low pH of 4.5.[7],[8] When there is a decrease in salivary pH, the viscosity of saliva increases. This increase in viscosity is more significant in subjects with high caries activity.[9]

Various studies have been conducted to identify the correlation between MMP-9 and dentinal caries by using dentinal fluid, tooth sections, and genes in human permanent dentition. At present, the knowledge of the correlation between salivary MMP-9, pH, and viscosity with ECC is limited. Hence, based on this background the present study was undertaken.

 Materials and Methods



Saliva samples were collected from 45 children who reported to the Department of Pediatric and Preventive Dentistry. Inclusion criterion was children between 3 and 5 years. While the exclusion criteria were children on medication, with any systemic and metabolic diseases, special health care needs, uncooperative, and parents who were not willing to give their consent. They were further divided into three groups with 15 participants in each group. Group A comprised of carries free, group B of ECC, and group C of S-ECC participants.

A baseline salivary sample was obtained after a thorough dental examination of all three groups was performed using deft index and using World Health Organization. Oral health surveys: basic methods. World Health Organization; 2013. The children in groups B and C were treated according to the requirements for caries control which included advice on oral health education, diet counseling, oral prophylaxis, restorative procedures, topical fluoride and pit and fissure sealants application, and pulp therapy. They were recalled after 2 weeks and salivary samples were collected to analyze the levels of MMP-9, pH, and viscosity. Informed consent was obtained from the parents, and assent was obtained from the children.

Ethical approval for this study (protocol No. ABSM/EC 59/ 2019) was obtained from the Institutional Ethics Committee of AB Shetty Memorial Institute of Dental Sciences, Deralakatte, Mangaluru, India, on October 14, 2019.

Sample collection

The unstimulated whole salivary samples of approximately 1 to 2 mL were collected after 2 hours of consumption of food or water to minimize any possible effect of diurnal variation by draining method[4] and immediately followed by centrifuging at 4000 rpm for 15 minutes at 4°C to diminish salivary residual and viscosity. The supernatants were stored at −70°C[10] and analyzed for MMP-9, pH, and viscosity.

High-sensitivity commercially available human MMP-9 Enzyme-linked immunoassay (ELISA) kit, Sincere Biotech, China was used for determining the levels of MMP-9 in saliva samples of the subjects using a detection range of 0.125 ng/mL to 10 ng/mL. After plate preparation, all of the reagents as well as the standards and samples in the micro-ELISA strip plate were carefully prepared. Following that, 10 µL of samples and 50 µL of standards were collected. The standard and testing wells were filled with Horseradish peroxidase (HRP) conjugate reagent, which was then sealed with a plate sealer and incubated at room temperature for 1 hour. The plate was aspirated and cleaned at least five times.

Each well received 50 mL of chromogen solution A, followed by chromogen B. After properly mixing the two ingredients, they were incubated for 15 minutes at 37°C, away from direct sunlight. Each well was filled with a 50 µL stop solution. The dish was softly tapped to ensure complete mixing. The color shift was noticeable (from blue to yellow). A microplate reader tuned to 450 nm was used to quickly analyze the optical density of each well. The amounts of salivary MMP-9 levels were then measured in ng/mL and calculated. Estimation of salivary pH and viscosity were done by using pH paper strip Ionix, Perfect sales India, Haryana, India Full pH 1-14 and Ostwald, Brand-ABGIL, ABG initiative India, viscometer, respectively.

Statistical analysis

The gathered information was entered into an MS Office Excel spreadsheet (v 2019, Microsoft Redmond Campus, Redmond, WA). The data were statistically analyzed using the Statistical Package for Social Sciences (SPSS IBM Corporation, Chicago v 26.0, IBM). Descriptive statistics such as frequencies and percentages for categorical data, as well as the mean and standard deviation for numerical data, have been shown. Intergroup comparisons (more than two groups) were performed using one-way Analysis of variance (ANOVA). For intragroup, the paired t test was used to compare groups within the same group (up to two observations). Based on a 5% level of significance, 80% power, and effect size of 0.5, the total sample required for three groups was 45, that is, 15 participants in each group.

 Results



Levels of MMP-9, pH, and viscosity for baseline (or pretreatment) salivary samples for all three groups and posttreatment salivary samples for groups B and C after 2 weeks were analyzed.

Pretreatment and posttreatment

For pretreatment, mean value of salivary MMP-9 levels estimated were 7.13 ng/mL (±0.809 ng/mL), 10.95 ng/mL (±1.469 ng/mL,) and 18.55 ng/mL (±9.218 ng/mL) for groups A, B, and C, respectively. Salivary pH levels were found to be 7.06 (±0.338), 7.07 (±0.403), and 6.92 (±0.542), respectively. And salivary viscosity levels estimated were 0.41 (±0.072), 1.74 (±0.412), and 1.28 (±0.123), respectively [Table 1].{Table 1}

Posttreatment salivary samples were analyzed in groups B. The MMP-9, pH, and viscosity levels estimated in group B were 7.34 ng/mL (±0.673 ng/mL), 7.24 (±0.394), and 0.42 (±0.208), respectively. While for group C, MMP-9, pH, and viscosity levels estimated were 7.84 ng/mL (±0.647 ng/mL), 7.14 (±0.377), and 0.49 (±0.338), respectively [Table 2].{Table 2}

Intragroup and intergroup comparison

Intragroup comparison

In pretreatment and posttreatment intragroup comparison of MMP-9, pH, and viscosity in group B, the mean salivary MMP-9 levels before and after intervention were 10.96 and 7.34, respectively. These differences were statistically highly significant with higher values before treatment. The mean salivary pH levels before and after the intervention were 7.03 and 7.24, respectively. Even though there was a slight increase in pH after the treatment, it was not statistically significant. The mean salivary viscosity levels before and after intervention were 1.78 and 0.42, respectively. These differences were statistically highly significant with higher values before treatment [Table 3].{Table 3}

In group C, the mean salivary MMP-9 levels before and after intervention were 20.32 and 7.84, respectively. These differences were statistically highly significant with higher values before treatment. The mean salivary pH levels before and after the intervention were 6.93 and 7.14, respectively. Even though there was a slight increase in pH after the treatment, it was not statistically significant. The mean salivary viscosity levels before and after the intervention were 1.29 and 0.50, respectively. These differences were statistically highly significant with higher values before treatment [Table 4].{Table 4}

Intergroup comparison

Intergroup comparison of pretreatment means salivary MMP-9 among the three groups showed the highest value in group C as compared to groups A and B which was statistically highly significant. Intergroup comparison of mean pH among the three groups showed no significant difference. For intergroup comparison of mean viscosity, the highest value was shown in group B as compared to groups A and C which was statistically highly significant [Figure 1].{Figure 1}

In the posttreatment intergroup comparison of MMP-9, pH, and viscosity between groups B and C (t test), there was no statistically significant difference in the values between the two groups (P > 0.05) after treatment [Figure 2].{Figure 2}

 Discussion



MMPs have various roles in dental caries, pulpal, and periapical inflammation. Normally, MMPs are secreted as inactive proenzymes and need to be activated for degrading the dentin organic matrix component. When pH drops (becomes acidic), the MMPs are activated from pre-form only for a short duration. The activated MMPs are functional only at neutral pH. The acidic pH does not remain for long since the buffering mechanism of the salivary system causes neutralization of the acidic pH, thereby restricting the degradation of the dentine matrix component by the activated MMPs.[11],[12] A study done by Wang et. al.[13] demonstrated that MMP-9 along with MMP-20 genes can be the markers for white spot lesions. A study proved that MMP-9 and MMP-20 genes are involved in white spot lesion and ECC development.[14]

The main predisposing factor for initiating ECC is the physical properties like pH and viscosity. The low pH of saliva creates a conducive environment for the development of ECC. Saliva does the function of flushing sugary substances and any other food products in the oral cavity and the viscosity of the saliva depends on the amount of food inside the mouth.[15],[16] Thicker the viscosity, lesser the clearance of food from the mouth which then decreases the pH level. This consequently increases the demineralization of tooth structure.[17] Saliva has been demonstrated to be a convenient, noninvasive medium for the detection and diagnosis of various health conditions in children.[18] Because of this, saliva has been considered for the estimation of MMP-9, pH, and viscosity levels in this study.

In the study, the mean salivary MMP-9 level was 7.13 ng/mL in these children. The normal salivary MMP-9 levels range between 1 and 10 ng/mL, which directly reflects the presence of inactive proenzyme in the matrix of dentine.[19] The optimum pH in these children ranged from 6.77 to 7.39 and the salivary viscosity was clear with a mean of 0.41.[20]

In groups B and C (intervention groups), salivary samples were estimated after a gap of 2 weeks, considering the time required for the carries control protocol, stabilization of oral biofilm following restorative procedures,[9] for the inflammatory reactions to subside, and the duration of time for setting in of new routines in oral hygiene practices. This is per observations made by Van et al.,[11] Marsh,[21] Van Der Hoeven et al.,[22] and Winnier et al.[23] here.

A study was conducted to estimate the pretreatment and posttreatment levels of salivary MMP-9, pH, and viscosity in ECC children (group B). In these children, the mean pretreatment salivary MMP-9 levels were 10.95 ng/mL. This could be due to the activation of MMP-9 which initiates demineralization of dentine either by degrading the collagen or by dissolving in the dentinal fluid which in turn mixes with the saliva.[7] Here, the pretreatment mean salivary pH of ECC children was 7.07. Because the breakdown of enamel begins when the pH goes below the critical pH level, the values found were insufficient to trigger the demineralization of the tooth’s inorganic substance.[24] As a result, other variables such as microflora, diet, and food retention may have overwhelmed the function of saliva pH in the development of caries. The viscosity of the saliva was found to be more viscous and thicker in consistency.[25],[26]

In group B, the mean posttreatment salivary MMP-9 levels were observed as 7.34 ng/mL, which is within the normal range of 1 to 10 ng/mL. This could be because of the inactivation of MMP-9[27] as well as the endogenous activity of MMPs in dentin after the treatment.[28] The posttreatment mean salivary pH of ECC children was 7.24.[29],[30] The posttreatment mean viscosity of the saliva was 0.49, which is suggestive of watery consistency.[21],[31]

Further, the pretreatment and posttreatment levels of salivary MMP-9, pH, and viscosity in S-ECC children (group C) were estimated. The pretreatment mean salivary MMP-9 level was 18.55 ng/mL, which is above the normal range. This could be due to the involvement of pulpal inflammation resulting in inflammatory cell aggregation and the production of inflammatory cytokines (C-reactive protein (CRP), Interleukin-1 (IL-1), and Interleukin-6 (IL-6)),[32],[33] polymorphonuclear leukocytes, and neutrophils, as well as their enzymes elastase and lactoferrin. The pretreatment mean salivary pH level of group C was 6.92. Children with S-ECC were found to have thick and ropy saliva with a mean value of 1.28. The high viscosity of saliva in these children could be the reason for the high prevalence of carious activity occurring in this group as it reduces the clearance of bacteria from the oral cavity.[34]

In group C, the salivary MMP-9 level was 7.84 ng/mL. This could be due to zinc contained in zinc oxide eugenol and Ethylenediamine tetraacetic acid (EDTA) inhibiting the MMP-9 activity by competing with the tissue inhibitory metalloproteinases.[35],[36] As this group was associated with an increased pulpal inflammation, caries controlled the decline in the levels of salivary MMP-9 which subsequently inhibit the neutrophils, interleukins, and C-reactive protein.[37] A decline in the MMP-9 level also hampered the degradation of dentine collagen. The posttreatment mean salivary pH and viscosity level of group C were 7.14 and 0.49, respectively.

Intragroup comparison of pretreatment and posttreatment values of MMP-9 and viscosity in both groups B and C were found to be statistically highly significant with higher values before treatment (P < 0.001). This could be attributed to an increase in awareness and knowledge of the parents following diet counseling and oral hygiene instructions.[38] The posttreatment of mean pH for both groups was higher than the pretreatment value, however, it was not statistically significant (P > 0.05). The result was following the study done by Ravikumar et al.,[27] Basma et al.,[24] Muchandi et al.,[39] and Animireddy et al.[40] The drop in pH may have been neutralized by the salivary buffer systems.

Intergroup comparison of pretreatment MMP-9 levels between groups A, B, and C showed a highly statistically significant difference between the three groups, with the highest levels of MMP-9 seen in group C. This is under the studies by Antunes et al.,[41] Ying et al.,[42] Wang and Qin,[19] and Ballal et al.[25] This could again be attributed to the increasing number of teeth involved and the inflammatory state in group C.[43] There was no statistically significant difference in pretreatment pH values between the three groups. A high statistical difference was found in pretreatment viscosity between the three groups with the highest value in group B. This may be due to the small sample size and improper techniques during saliva collection or diet such as dietary products which can affect the viscosity of saliva.[44]

Intergroup comparison of posttreatment MMP-9, pH, and viscosity levels between groups B and C showed no statistically significant difference between them. From these results, we may conclude that decreased levels of MMP-9, pH, and viscosity following oral rehabilitation are independent of pretreatment caries and inflammatory status.

In the present study, the dropout rate in groups B and C were 13.3% and 20%, respectively. The prevalence of dropouts in our study is consistent with prior research, which could be due to parental avoidance behavior and/or lack of awareness about the gravity of the consequences of dental caries on a child’s overall health and well-being which persists even today among the general population in the country.

The MMP-9 levels have decreased posttreatment in both groups B and C, indicating that undergoing caries control treatment helps to reduce their inflammatory status as well as inhibits the demineralization of the dentinal organic matrix. The diagnostic and prognostic significance of ECC has not been explored to date. Thus, from the present study, we can conclude that salivary MMP-9 holds a true potential to be a future prognostic and diagnostic inflammatory marker as well as an indicator of the level of degradation of the organic dentin matrix.

 Conclusion



In caries-free children, the salivary MMP-9, pH, and viscosity levels were estimated to be 7.13 ng/mL, 7.06, and 0.41, respectively.

Intragroup comparison of the salivary MMP-9 and viscosity levels in children with ECC before and after the caries control protocol showed a statistically highly significant difference (P < 0.001) but no statistically significant difference in pH values.

Intragroup comparison of the salivary MMP-9 and viscosity levels in children with S-ECC before and after the caries control protocol showed a statistically highly significant difference (P < 0.001) but no statistically significant difference in pH values.

Intergroup comparison of pretreatment salivary MMP-9 and viscosity were statistically highly significant (P < 0.001) between the control and study groups but no significant difference in pH level.

Intergroup comparison of posttreatment salivary MMP-9, pH, and viscosity showed no significant difference between the control and study groups.

Financial support and sponsorship

Nil.

Conflict of interest

There are no conflicts of interest.

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