The present study aimed to investigate possible changes that the absence of pro-inflammatory receptor TNFR1 could cause in amelogenesis and biomineralization of dental enamel. It was found that the absence of the receptor can impact amelogenesis, leading to microscopic changes in enamel surface and in mineral content. Also, the synthesis of proteins and enzymes that are crucial for amelogenesis was affected.
The results have shown that even though there were no visual differences in the dental enamel, at a microscopic level, it was possible to observe a change in the conformation of the enamel rods of knockout mice. Scanning electron microscopy analysis in deciduous lower molars with hypomineralization revealed that teeth also had structural changes in the enamel surface and in the mineral content (21). The samples had a lower content of calcium, phosphorus, oxygen and carbon. The changes described by the authors are similar to the changes of hypermineralization in permanent molars (6, 22). In addition, a recent study has investigated the possible effects in the enamel of rodents that have had a mutation in the gene KDF1. This gene is associated with the development of dental enamel in humans and animals. It was described as the presence of structural abnormalities in enamel rods, which had a large area of cracks. Analysis of volume and enamel layer density showed a significant difference between KDF1 deficient and wild type mice (23). The present study found that the absence of the TNFR1 receptor did not influence the volume or density of the enamel layer, although it was possible to observe a decrease in microhardness and in the amount of calcium and phosphorus. Also, there was an increase of carbon in enamel composition. Usually, teeth affected with DED have had a lower mineral content and a reduction in the microhardness (24, 25). In addition, studies have shown that it is common to find a higher protein content in the organic enamel matrix of those teeth (6, 22).
MMP-20 is an enzyme that plays a role during the final stage of the secretory phase and during the maturation phase of amelogenesis. The main role of MMP-20 is to remove proteins that are present in enamel organic matrix, allowing the mineral incorporation and extension of hydroxyapatite crystals (26; 27, 28). A study that investigated the effects of the absence of MMP-20 in the teeth of rodents revealed that those teeth had a low mineral content because MMP-20 has a role in the removal of proteins such as amelogenin and guide the length growth and thickness of hydroxyapatite crystals (28). However, the present study analyzed the gelatinolytic activity in TNFR1-/- animals and it was observed the storage of the enzyme through the ameloblast layer with a high expression during the maturation stage of amelogenesis. It is hypothesized that the overexpression of MMP-20 can be associated with enamel malformations. The presence of the enzyme in a higher quantitate could cause the disarrangement of the ameloblast layer and lead to a gap between the ameloblasts cells and the enamel layer (29).
Runx2 transcription factor has a fundamental role in the different stages of osteogenesis, and gene mutations are associated with the occurrence of cleidocranial dysplasia, a condition that causes clavicle hypoplasia (30; 31). Absence of Runx2 during amelogenesis results in a high protein content such as ameloblastin, amelogenin and enamelin in organic matrix (16). Runx2 overexpression can also be associated with enamel mineralization changes during amelogenesis causing a reduced expression of KLK4, a protease responsible for protein removal (32). In the present results, Runx2 transcription factor has had a high expression in knockout animals for TNFR1 receptor, showing that the absence of the receptor can impact enamel maturation.
Studies have shown that in most cases where there are molecular changes in the signaling of enamel formation, there will be a failure in the protein removal of enamel organic matrix, which leads to a reduction of mineral content and an increase of proteins (28, 32, 33). Amelogenin is the main protein in the enamel organic matrix during amelogenesis, also serving as a scaffold that will guide the mineralization of hydroxyapatite crystals (27). On the other hand, ameloblastin is the second most abundant protein in the enamel organic matrix, and it has an important role in the differentiation process of ameloblasts cells (34, 35). In addition, it is assumed that ameloblastin can have a role in cell adhesion. The absence of this protein during the secretory stage could lead to cell polarity loss of ameloblasts (36). Although these proteins are essential for enamel formation, amelogenin and ameloblastin should not be present in the final enamel composition (18, 27). Failures during the protein removal process in the maturation stage can lead to protein remaining in dental enamel, which can affect the growth and mineral incorporation of hydroxyapatite crystals (15). Ameloblastin had a higher expression in the knockout animals when compared to the wild type animals, this result shows the protein accumulation in enamel layer, which can be associated with low microhardness of the enamel surface of these animals.
During amelogenesis there are other molecules that play a crucial role and are essential for this process. Some inflammatory cytokines such as COX-2, TGF-β and TNF-α and/or their receptors were already described during tooth and enamel formation (8, 11, 12, 19, 20). COX-2 is an enzyme known for the participation during inflammatory processes, and its induction is mediated by pro-inflammatory cytokines such as interleukins and TNF-α (37). Recently, the physiological expression of COX-2 has been reported in the ameloblast layer, and when COX-2 inhibitor drugs have been used, the enamel has shown lower mineral content and changes in the expression of enzymes and proteins, essential for amelogenesis (20).
TNF-α is a pleiotropic inflammatory mediator responsible for the proliferation and differentiation process. This mediator has an important role in immune response during an inflammatory process (38, 39). In dental development, members of the TNF-α superfamily act as a signaling pathway through the EDAR receptor. EDAR receptor is activated by specific intracellular proteins that play a role in dental morphogenesis (40) and enamel formation (12). The present study demonstrated that TNFR1, another receptor for TNF-α, can regulate enamel biomineralization, once the absence of this receptor has resulted in a lower mineral content, a decrease microhardness of enamel surface and modifications of the signaling of amelogenesis. It is important to consider the presence and mechanisms of action of inflammatory cytokines during enamel formation in the search for etiological factors of DED. The expression of inflammatory cytokines during the first years of life, a period in which the occurrence of childhood illness is common, could alter the expression of these cytokines and be associated with changes in the biomineralization of tooth enamel.
The absence of proinflammatory TNFR1 receptor can directly impact the biomineralization of dental enamel once there have been changes in expression of the protein and enzymes that are essential for the correct mineralization process, and may be associated with the occurrence of enamel defects.