In this study it was found that NSAIDs, medication commonly prescribed for children, impaired the synthesis and nuclear translocation of proteins and enzymes important for enamel biomineralization. Those findings shed light on the possibility that those drugs might be involved in the etiology of enamel defects. DEDs occur due to alterations during the period of secretion of the dental matrix in amelogenesis, and its severity will vary according to the period in which there is any disturbance3,4.
MMP-20 is an enzyme present in the extracellular matrix of enamel, being responsible for the degradation of the organic matrix, providing space for the growth of hydroxyapatite crystals and deposition of mineral content10,22. A low expression of MMP-20 in the control group was found. While the groups treated with indomethacin or celecoxib, a selective inhibitor of COX-223, a high expression of MMP-20 was observed. Based on this result, it is hypothesized that MMP-20 deficiency alone might be not responsible for changes in the mineralization quality and cause of enamel defects.
Animals with a mutation on MMP-20 gene have been report to have a greater amount of organic content inside the hydroxyapatite crystals when compared to the group of animals that did not have the MMP-20 mutation24. From this, it was concluded that the absence of MMP-20 in the mineralization process can lead to the accumulation of amelogenin in hydroxyapatite crystals. The presence of these proteins inside the crystals hinders their expansion process, and they end up getting smaller. In another study22, the differences in enamel characteristics of animals with normal expression of MMP-20 in the maturation period and those without MMP-20 were investigated. It was reported that in the group with no enzyme, enamel had a lower hardness, a lower mineral amount and a higher amount of water and protein compared to normal enamel. Accumulation of MMP-20, on the other hand, in pre-ameloblasts has been reported to be responsible for causing enamel defects25.
Runx2 transcription factor is responsible for mediating bone formation. It participates in the differentiation of osteoblasts and in the osteoclastogenesis process around the dental follicle and periodontal ligament26. In an attempt to identify the functions that the transcription factor played in the amelogenesis process, an experiment was carried out with animals in which it was observed that the absence of Runx2 can cause damage such as delay in tooth mineralization and in the expansion of enamel crystals. A delay in the removal of proteins from the enamel extracellular matrix, a decrease in the KLK4 enzyme, which is responsible for protein degradation, and the accumulation of proteins in the matrix during the period of maturation have been reported27.
The present results show that the control group showed high expression of Runx2, different from the group treated with indomethacin and celecoxib, in which there was an inhibition of the transcription factor. Nonetheless, solely indomethacin prevented Runx2 translocation to the nucleus. Mutation in Runx2 cause limitations in enamel formation and bone formation28. It was observed that the mutation in the transcription factor caused alterations in the cell cycle of the dental pulp cells, decreased the cell proliferation rate and its calcification capacity29.
DSP is an important protein for dentin formation, and the deficiency of this protein is related to the occurrence of type III dentinogenesis imperfecta13. Interestingly DSP has been identified in the pre-ameloblast layer as a signaling molecule30, although its role in biomineralization of enamel is widely unknown. Recently, using a mutant DSPP mice, it was found that if DSPP remains retained in the endoplasmic reticulum within the presecretory ameloblasts and causes ER stress25. Because it has been shown that ER stress and its associated unfolded protein response (UPR) is involved in amelogenesis imperfect caused by mutations in the genes that encode enamel matrix proteins31,32,33. Liang et al.25 postulated that the enamel defects observed in DSPP deficient mice may be associated with ER stress and unfolded protein response. The present work sought to explore the presence of DSP on ameloblasts following NSAIDs treatment and found that it is constitutively synthesized in the layer of ameloblasts, contributing to similar evidence mentioned above. In addition to its presence, it was also possible to observe a greater amount in the region of ameloblasts that were in less differentiated stages, thus evidencing the participation of DSP in the earlier stages of enamel matrix secretion. When compared with the groups treated with NSAIDs, it was noticed that there was an inhibition, which was even more evident in the group treated with celecoxib. Despite still not knowing the mechanisms of action of DSP and its functions in amelogenesis, the use of medications can lead to its inhibition and, therefore, potentially damage to the process, causing possible defects in the enamel. Notwithstanding, the accumulation of DSPP in pre-ameloblast would eventually lead to the occurrence of disturbances in the mineralization process such as delay and malformation of dental enamel14. DSPP is an acidic protein, and its accumulation in the endoplasmic reticulum would cause stress. This condition of pathological stress on the endoplasmic reticulum would activate a phenomenon called unfolded protein response, and thus there would be a mutation of the protein, causing enamel defects.
COX-2 is considered pathological enzyme, since its presence has always been associated with inflammatory processes, but one study15 and the present study have identified the presence of COX-2 in physiological tooth enamel formation. COX-2 was expressed in the ameloblastic layer, with a higher expression in the less differentiated ameloblasts, which would be in the initial stage of mineralization. Therefore, because COX-2 appear to be physiological in enamel formation, this study sought to investigate whether medications commonly used in childhood such as NSAIDs would be involved in DEDs. It has been reported that there is a relationship between inhibition of COX-2 and a quantitative reduction in tooth enamel minerals, calcium and phosphorus, caused by the use of ibuprofen. Also, according to the authors, the inhibition of COX-2 activity would lead to a decrease in prostacyclin, which in turn would cause a decrease in blood flow in the dental organ, reducing the arrival of nutrients and entry of ions, and thus impairing the mineralization process15.
It is important to consider that animal models in dental experimental studies are widely used, due to the difficulty of access to developing teeth, which occur during intrauterine life inside the maxillary bones. Mice are an option as an animal model for enamel formation studies, even though they present morphological and numerical differences. It is known that the tooth formation process differs in some points, such as the renewal and growth of dental enamel due to the constant activity of ameloblasts along the incisors6,34. However, studies have shown the presence of important components such as the enzyme MMP-20 and the protein amelogenin in the composition of mice teeth10,35.
Therefore, the results described has showed that indomethacin can inhibit key proteins and enzymes in the dental mineralization process, and might be involved in DED etiology. DSP was also identified in the ameloblast layer, thus being a protein that plays a fundamental role in dentinogenesis, and its role in amelogenesis should be further investigated. It is important to emphasize the importance of carrying out more studies to identify the mechanisms of action that NSAIDs can actually exert to cause DDE if used in the period of tooth formation and mineralization.