Correlation Between Salivary Levels of Matrix Metalloproteinases 2,9 and Caries Incidence in An Indian Population

Introduction

Dentin can be described as a very dynamic biological structure that consists of hydroxyapatite and collagen, where the collagen acts as scaffolding for the mineral phase. Approximately 50% of the dentin consists of mineral by volume, 30% is the organic matrix of which, about 90% is type 1 collagen.¹

The dental caries process involves the demineralization of inorganic minerals, mainly hydroxyapatite, by acids produced by oral bacteria. Demineralization occurs when the pH in dental plaque falls below 5.5 within minutes after sugar ingestion until neutralized by salivary buffers. The demineralization is followed by the destruction of the collagenous organic matrix of dentin.²

Bacterial proteases was thought to be responsible for the organic matrix destruction for long. But in vitro studies have shown that they have weak protease activity and were unable to degrade the collagen matrix and they only caused demineralization of the surface dentine.³ Also, previous literature has shown that bacterial collagenases did not resist the acidic fall (pH 4.3) during the demineralization phase of the Ph cycle model. This suggested that this enzyme contribution to dentine matrix degradation may be less important than what was initially thought.⁴

This lead to the breakthrough notion that the matrix metalloproteinases, which are localized in the salivary glands and the dentine can be the culprit in degeneration of the collagen matrix of dentine.^5,6

Matrix metalloproteinases (MMPs) are host proteolytic enzyme which depends on zinc for their activity. They degrade various types of collagen present in extracellular matrix in its pure and denatured from. Currently, 24 MMP genes have been identified in humans, and 26 wellcharacterized members have been reported.^7,8 Human dentinal caries lesion is found to have MMP 8, MMP 9, MMP 2, and MMP 20 are present in human dentinal caries lesions.9 Saliva contains MMP 2, MMP 8, MMP 9 which are from GCF or secreted directly from parotid and sublingual glands.^10,11

MMP activity in saliva can be qualitatively determined using zymography. It determines various MMPs with the help of its molecular weight and degradation of its specific substrates. In this study, we are checking for MMP 2 and MMP 9 which are gelatinases. Hence gelatin zymography is used in the methodology to determine the enzyme activity.¹²

Thus, this study aims to evaluate the activity of MMP2 and MMP 9 in participants with high caries index and compare it with that of caries free participants in the Indian population.

Materials and Methods

Salivary samples were collected from patients who reported to the Department of Conservative Dentistry and Endodontics after obtaining consent from the respective patients. The study duration was two years.Participants within the age group of 18 – 60 were selected who has no active periodontitis, who don't have any systemic disease which can alter the salivary parameters like salivary flow, viscosity, etc.

200 participants were selected and divided into groups A and B, with 100 participants in each group.

GROUP A – participants having DMFT score > 13

GROUP B – Participants having DMFT score 0

Sample collection

All the participants are educated and motivated regarding sample collection. Participants were asked not to brush their teeth, the night before and that day morning of saliva collection. Also, they were refrained from drinking and eating for at least two hours before sample collection. After rinsing the mouth once, the participants were asked not to speak or swallow. Unstimulated saliva which gets pooled on the floor of the mouth was asked to expectorate in the granulated saliva sample collecting vial. 2ml of saliva was collected and stored it in ice bags under -4 degree Celsius and was transported to the lab within two hours. The participants were educated to collect the saliva samples by themselves and individual ice bags and vials were distributed to them to transport the samples to the lab.

Methodology

The pH of both group A and B was determined using a pH meter and recorded. All the samples were centrifuged at 3000 rpm and the supernatant was used for gelatin zymography.

Gelatin Zymography

Zymograms were used for the detection of distinct molecular forms of MMP2 in cell culture media in varying concentrations of bisphosphonates reflecting the proteolytic activation of pro MMP2 from higher molecular form to lower ones. Samples were incubated in Laemmli’s sample buffer for 30 minutes at 22°C, after which 20 µl of the sample was loaded onto each lane of a polyacrylamide (10% sodium dodecyl sulfate) gel containing gelatin. A separate lane was run with molecular weight standards (high and low molecular weight pertained standards from Bio-Rad Lab., Richmond, CA, USA). Electrophoresis was carried out in precooled pool buffer (50 mM Tris; 0.38 M glycine; 0.1% SDS; pH 8.3) at a continuous voltage of 110 V. Gels were washed three times for 10 minutes in 50 mM Tris, buffer, pH 7.5, and supplemented with 2.5% Tween and 0.02% NaN3; then three times for 10 minutes in the same buffer supplemented with 1µM ZnCl2 and 5 mM CaCl2; and overnight at 37°C in 50 mM Tris buffer with 5 mM CaCl2, 1 µM ZnCl2, and 0.02% NaN3, pH 7.5. The reaction was stopped with Coomassie Brilliant Blue R 250 staining followed by destaining in 5% acetic acid and 10% methanol in water. The gelatinases from the sample solubilize their substrate in the gel and therefore were visualized as white bands against the blue-stained surrounding gel, where the substrate is intact and heavily stained.

Statistical analysis

All statistical analyses were performed usingSPSS 20.0software, with a pvalue <0.05 regarded as statistically significant.Data from different groups were compared by the Student t -test and fishers exact test.

Results

Mean salivary pH of high caries index individuals are significantly higher than that of low caries index individual (Table 1)

Mean salivary MMP 2 and MMP 9 activity at 280 and 540 nm, for high caries index individuals were significantly higher than that of low caries index individuals. (Table 2)

Discussion

For caries progression the significance of dentine collagen matrix degradation is yet to be unfolded. Previous literature suggests that after demineralization by bacterial acid, dentinal collagen becomes exposed to collagenases in saliva.1 Thus, this supported the view that both demineralization and degradation process of the inorganic and organic part of dentine is inevitable for caries progression.

The Matrix metalloproteinases are activated by variations in pH during caries process. The mean Ph of high caries index was 4.3 ± .02. Previous studies have shown that MMPs will get activated below Ph 4.5 and starts functioning in neutral pH.2 MMPs are kept on check by MMP inhibitors. Aberrant secretion occurs when the balance between MMP and the inhibitors is lost which can result in excessive tissue destruction.^7,13,14

The result of the study affirms the role of MMPs in saliva, specifically MMP 2 and MMP 9 in collagen matrix degradation and thereby caries progression.MMPs are present in dentine, GCF, plaque and saliva. In saliva, it mainly comes from GCF, but studies have shown that salivary glandlike parotid gland directly secrete MMPs.^3,15

In the study, MMP 2 and MMP 9 activity was detected more in high caries index participants than that of caries free participants. Previous researches were done on the role of host MMPs present in dentine collagen.² The participants were selected taking into account the exclusion criteria, participants with periodontal disease, participants under any drug therapy which hinders salivary secretion, participants with any systemic disease which prevents salivary secretions are excluded from the study. Thus, the MMP 2 and MMP 9 detected in high caries index participants were from the salivary source.

MMPs were detected using the method known as zymography. In this method, MMPs can be detected and identified with the help of a specific substrate corresponding to each MMP and their molecular weights. The basic functioning of all zymography’s is the same, except in the substrate which depends on the MMP being detected. As MMP 2 and MMP 9 are gelatinases, the substrate used in the study is gelatin and thus gelatin zymography. In the presence of SDS and non reducing conditions, the proteins are denatured and separated by electrophoresis. The substrate for separation of the protein is held on a gel called polyacrylamide and after the electrophoresis, SDS is washed off. This partially renatures the denatured MMPS and regains its activity. After this, the polyacrylamide gel is stained blue and the MMPs can be vividly viewed as clear bands against the blue background. These bands are then measured with densitometry. The molecular weight of MMPs helps us to distinguish between its active and proenzyme forms. The pronounced advantage of zymography over all other methods is that it separates the MMP-TIMP complex which increases the sensitivity of the test. Thus, gelatin zymography is highly sensitive for MMP 2 and MMP 9.¹²

This is the pioneer study in which salivary MMP 2 and MMP 9 activity has been estimated and compared with the caries experience of the participants. The increased activity of MMP 2 and MMP 9 is because of its aberrant secretion which can be due to some genetic abnormality such as single nucleotide polymorphism.¹⁶

However, gelatin zymography is a qualitative analysis, further advanced studies have to be conducted to ground the correlation between the salivary MMP 2 and MMP 9in caries progression.

Conclusion

Dental caries is the most common oral disease that effects significant number of people. This multifactorial disease progresses with the degradation of dentine collagen matrix. It was thought that bacterial proteases were responsible for dentine matrix degradation which later proved wrong. In the quest to find out the culprit of dentine degradation and caries progression, host proteolytic enzyme, the Matrix Metalloproteinases came into focus.

In our study, it was found that salivary MMP 2 and MMP 9 activity were pronounced in high caries index participants saliva than that of low caries index participants in the Indian population. Further studies has to be undertaken to find out any genetic correlation associated with the pronounced activity of MMP 2 and MMP 9.