Dental caries, which is one of the most common diseases in both developing and industrialized countries, has a multifactorial etiology as well as being a chronic and complex disease (1). In a recent systematic analysis evaluating 354 different diseases and injuries, it was reported that 3.5 billion people were affected by oral diseases and the most common of these diseases was caries in permanent teeth. Dental caries in the permanent dentition affected 2.3 billion people globally. Based on these data, it was determined that untreated dental caries was the most common health condition (2).

Secondary caries is a major clinical problem defined as a carious lesion seen on the margins of the existing restoration or caries associated with sealants (3), and progress to the dentin and pulp. This form of caries has been reported in the literature as the most common reason for the replacement of many types of restorations, from the fixed partial prosthesis to fillings, as it alters the dental hard tissue, endangers the lifetime of the tooth, and reduces the longevity of the restoration (4, 5). Since the incidence of dental caries continues to be high among certain groups due to high sugar consumption and change of living conditions, there is a need to determine markers that will help identify individuals with a higher risk of caries (6). For this purpose, genetic studies have been performed in dental caries, as it has been observed that human genetic polymorphisms play a role in the susceptibility of the host to disease (7). Particularly for secondary caries, our data showed that individuals with asthma, high blood pressure, and diabetes were more likely to have secondary caries (8).

One of the genetic factors we identified that appears to impact the progression of a caries lesion into the dentin are members of the matrix metalloproteinases (MMPs) family. We showed that variants in MMP2 and MMP3 were associated of having a deep caries lesion that involved a periapical lesion formation (9). MMP2 is a gelatinase involved in mineralization and dentin degradation, and catalyzes dentine matrix degradation after demineralization (10). MMP3, also called proteoglycanase or stromelysin-1, participates in the mineralization of predentin and is detected in dentin in connection with the odontoblast process. This MMP type also contributes to the disorganization and degradation of the demineralized dentin matrix and the adhesive properties of restorative resins (11, 12). The same MMP2 genetic variant that was associated with deep caries lesions in dentin with periapical lesions was also associated with loss of extensive composite restorations not due to secondary caries (13). Therefore, a caries lesion that begins on unsealed margins of faulty restorations and sealants and progress to the dentin may progress quicker or easier in individuals that have MMP2 expressed differently due to genomic variants.

The hypothesis of the study was that individuals that express variant alleles may have lower MMP activity and secondary caries may develop in a slower pace. On the other hand, individuals with full MMP potential activity due to having wild type alleles may have secondary caries progressing quicker. We tested for statistical evidence of over-representation of alleles of MMP2 and MMP3 in individuals with secondary caries independent of the restorative material. The underlying hypothesis was that genetic variation in these MMP genes will affect the occurrence of secondary caries lesions in treated patients. Carriers of MMP2 or MMP3 fewer common alleles were expected to have secondary caries lesions less frequently. The aim of this study was to test for over-representation of alleles in MMP2 and MMP3 markers to identify statistical evidence that these genes may influence longevity of dental restorative treatments.

Genotype distributions did not deviate from Hardy-Weinberg equilibrium, with the frequency of each genotype within the expected range based on the formula p² + 2pq + q² = 1 (data not shown). The less common allele and homozygotes genotype of MMP2 rs9923304 were less common among individuals with secondary caries. The less common allele of MMP2 rs2287074 was also less frequent among individuals with secondary caries (Table 2).

Dental caries progresses differently into dentin for different people. In some cases, lesions may be devastating to the point that teeth cannot be saved, due to extreme loss of structure. In others, however, the lesions stay confined to dentin without affecting the pulp. Evidence suggests that despite the same environmental conditions and the same time, not everyone will have the same fate regarding dental caries lesions progression (16). One approach that can be used to help elucidate the reasons why these differences occur is to test for evidence of over-representation of alleles, which would suggest a genetic basis for the differences in outcome seen in the clinics. In this study, we expected to see that people without variant alleles of MMP2 or MMP3 markers were more likely to have secondary caries since MMP activity was expected to be in its full potential. The presence of variant alleles was expected to imply that MMP activity was slightly decreased and these carriers were less likely to have secondary caries lesions. The rationale of these assumptions is based on the idea that more hydrolytic degradation of dentin by MMPs activated when pH is lower and expression of MMP2 and MMP3 is at their full potential. The presence of genetic variants (hypomorphic alleles) would decrease MMP activity making it less likely that these carriers of less common alleles to show secondary caries lesions.

We originally reported associations between MMP2 and MMP3, and individuals with deep caries lesions in dentin that were accompanied by periapical pathology (9). The MMP2 marker rs9923304, that was associated with periapical pathology of teeth with deep carious lesions in the original work, was also associated with failure of extensive composite restorations that were not due to secondary caries (13). In this report, we showed that markers in MMP2, including rs9923304 and rs2287074, were also associated with secondary caries, independent of the type of restoration. These data, in sum, provide evidence that MMP2 contributes to failed outcomes resulting from restorative treatment and/or pulp involvement. The association of these two particular MMP2 variants, rs9923304 and rs2287074, is due to the increased number of wild type homozygotes among individuals with failed restorations. Since it is expected that MMP2 acts by degrading components of the extracellular matrix and non-matrix proteins (mainly type IV collagen), the result fits perfectly with our hypothesis that individuals that show a higher MMP2 activity may be prone to more failures of extensive restorations. These individuals with higher MMP2 activity would carry zero copies of the variant allele, and individuals carrying zero copies are presumed to have less MMP2 activity and be less prone to secondary caries. It is possible that these markers are the ones providing direct information on MMP2 activity and could be considered as genomic markers for future clinical management.

The pattern of MMP2 expression in dentin suggests that treating the dentin to minimize the effects of metalloproteinases action may be useful. Determining an individual's MMP2 rs9923304 variant genomic status may be a useful tool in proactively implementing dentin crosslinking to prevent restoration failure. Future clinical research should be designed to incorporate genomic analysis in the evaluation of dentin crosslinking outcomes to determine the profiles of patients who will benefit most from these approaches. Dental practitioners would find value in understanding the genetic contribution to dental caries for at least two reasons: First, they would be able to explain to patients that some forms of decay are more strongly associated with inherited risk. This would help explain for both the patient and dentist why persons with similar behavioral risks (e.g., tooth brushing frequency or dietary habits) have different caries rates, thus avoiding the “blaming the victim” that sometimes accompanies preventive health messages in dentistry. Second, future technological developments may make it possible to someday identify patients who are genetically at higher risk for caries. These patients then could be monitored more closely and provided with more aggressive prevention programs (16).

We and others (7, 17) have shown that genes involved in enamel formation (tuftelin, amelogenin, enamelin, kallikrein 4, ameloblastin, MMP20) are associated with dental caries. Although most of the existing data are on primary lesions or models of dental caries initiation in enamel, it is not possible to exclude the potential effect of these genes in the occurrence of secondary caries lesions.

The use of a registry and existing data may be seen as a limitation, since no reassessments of the cases studied could be proposed and possible recording errors may exist. Information used was recorded by different professionals over the years and analysis per type of restoration was not performed.

In conclusion, MMP2 rs9923304 and rs2287074 are associated with failure of dental restorative treatments and have the potential to be used for determination of risks impacting longevity of dental treatments.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

The studies involving human participants were reviewed and approved by University of Pittsburgh Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.

LF and MB generated data extraction and organization. KD processed samples and supported recruitment. AM supported recruitment and obtaining data. AV designed the project, created all infra-structure, supervised all aspects of the project, and wrote the manuscript. All authors contributed to the article and approved the submitted version.

Data for this project was obtained from the Dental Registry and DNA Repository project, which is supported by the University of Pittsburgh School of Dental Medicine. MB was supported by the Fulbright Postdoctoral program (FY-2020-TR-PD-02).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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