Influence of Elements on Gene Expression in Human Teeth

Abstract

Several elements (Ca, Fe, Sr, Mn, Mg, P, Zn, Se, B, Pb, Ni, Ti, etc.), classified mainly under three groups namely beneficial, harmless and harmful elements, are measured in human teeth for multiple purposes since they involve in metabolic activities as well as influence gene expression. There are sufficiently available studies reporting roles of the elements in both up and down-regulation of gene expression leading to tooth repair, regeneration, differentiation, biomineralization and demineralization in the dental stem cells. Considering the importance of tooth developmental and protective roles, the association of the elements with gene expression presented in the present review may facilitate for improvement of their selection as one of the criteria for strengthening teeth for a longer life through nutritional sources and dental material formulation.

1. Introduction

1.1 Inorganic and organic composition

Teeth have major parts of enamel, dentin-pulp complex, and cementum. Dentin, the largest tooth portion, contains a majority of 70% minerals by weight, 20% organic content of the matrix and 10% water [1]. The chief constituent of the organic part of dentine is type I collagen with >85% and collagen types III and V are the other remaining contents of it [2, 3]. The dentine phosphoprotein with ~50% of the noncollagenous part is the main composition of the organic matrix, while hydroxyapatite is found as the remaining composition of inorganic matrix [4, 5]. In the case of enamel, less collagen and more noncollagenous protein of 90% amelogenin were found. The inorganic constituent of these hard tissues is composed of biological apatite, Ca₁₀(PO₄)₆(OH)₂. Enamel contains greater inorganic component (~90% prismatic crystals) than dentin (~70%) and cementum (45%) [6]. When the higher inorganic level found is related to higher strength and resistant force in the enamel than in any other hard tissues of body, there are certain means of causing changes in amount of the inorganic constituents linked with both strengthening and weakening of tooth development and structure.

1.2 Element and gene relationship

When essential, non-essential (harmless) and harmful elements reach the dental tissues via blood circulation from natural sources of food, and other man-made sources from environment and dental products used for treatment, the reactions of the tissues rely upon dosage and chemical and biological features of the elements and are markers of specifying the element nature under beneficial, harmful and neutral roles in elucidating actions of gene expression [7, 8]. Since the dental tissues are exposed continuously to multi-elements at varying quantities and with different physiological functions, it is conspicuous to discuss here in details about the consequence of element intake in particular, the interaction between elements and genes. The processes of mineralization and demineralization are important functions of the tissues of teeth and bone and are under the control of several genes identified with varying levels of expression through protein secretion [9]. The diverse genes, levels of expression and related proteins responsible for either strengthening through mineralization and remineralization or debilitating the teeth via demineralization are described well in this review.

1.3 Utilities of dental pulp stem cells

Stem cells are pluripotent cells, having the property of differentiating into various types of cells of the human body [10]. Mesenchymal stem cells (MSCs) that have been developed from various human tissues, peripheral blood and body fluids, are characterized by cellular and molecular markers to understand their specific phenotypes [11]. Dental pulp stem cells (DPSCs) having an MSCs phenotype, can be obtained from different dental tissues viz., human exfoliated deciduous teeth, apical papilla, periodontal ligament (PDL) and dental follicle tissue and can also be developed into induced pluripotent stem cells by incorporation of pluripotency markers [12]. The stem cells isolated from different dental tissues are utilized for in vitro studies about the interaction between elements and gene resulting in gene expression and mineralization. In this chapter, an attempt has been made to explore the relationship of element effects on gene expression in the DPSCs with mineralization, cell differentiation and development of teeth.

2. Influence of genes and mineralization

2.1 Mineralization

The bone, dentin and cementum are the hard connective tissues which under normal biological processes are formed of the collagen fibrils through a scaffold of extremely arranged crystal structures of calcium phosphate [6]. The cells of the hard connective tissues influence various mineralization activities such as crystal structural morphology, composition and localized development and growth. In the bone and the tooth dentin and enamel, the mineral salts of calcium and phosphate ions are fixed through activities of the constituents of the extracellular matrix and a sequence of biocatalysts of enzymes. Mineralization, a lifetime function, occurs through the precipitation of inorganic elements within the matrix of organic components of teeth and bones [13]. Tooth mineralisation processes are implicated with tissues interactions between ectodermal and mesenchymal layers [14] and also with extracellular matrix components and cell-derived microstructures [15]. Knowledge about the mechanism of mineral deposition is essential for prophylaxis and treatments of ill health related to mineralization and also for the renovation and reconstruction of scaffolds.

2.2 Chromosomal defect and mineralization

Alvesalo [16] reported that the Y chromosome regulates the growth of both tooth enamel and dentin, but the X chromosome influences enamel formation only. Hence, various sex chromosome anomalies are associated with variation in tooth crown size and structure of individuals [16] and also with impaired tooth mineralization and defective metabolism of calcium and phosphate [17]. Hypocalcification, an example of defective low calcification was reported in Indonesian individuals with Down syndrome (DS) [18]. To identify chromosome influence on mineralization, Keinan et al. [19] estimated Ca/P ratios in enamel and dentin of exfoliated and extracted lower second primary molars of children with DS, cerebral palsy (CP) and a control group with no adverse medical history. They revealed that the prenatal levels of enamel formed and the mineralization of enamel of the mesial cusps in DS and CP teeth were significantly lower than that of the control group. Further, growth and mineralization of all cusps were found affected in the teeth of DS. It is therefore, opined that determination of Ca/P ratios in the exfoliated deciduous teeth of developmentally challenged children may aid in finding out the inception and severity of growth abnormalities in utero and their retarding effects on later development of teeth [19].

2.3 Loss of amelogenin gene and mineralization

Amelogenin proteins are essential for the normal development of enamel and are products of ameloblast cells and the primary composition of mineralizing organic matrix of enamel, that becomes mineralized with a hydroxyapatite phase to become the mature enamel [20, 21, 22]. The gene loci for human amelogenin, the major protein component of the organic matrix in enamel are on both the X and Y chromosomes [23]. Particularly, the amelogenin genes (AMGX and AMGY) that are identified respectively on the X and Y chromosomes, take part in a prominent role at the time of dental enamel development [24] and exert biologic functions as signaling molecules through cell-surface receptors [25]. When one X-chromosome is absent or malfunctioning in Turner syndrome (TS), expression of the X-chromosome was investigated for mineral incorporation during amelogenesis and indirectly during dentinogenesis. Primary tooth enamel and dentin from girls with TS were analyzed with the use of X-ray microanalysis, microradiography and rule induction analysis and compared with that of healthy girls [26]. High levels of calcium and phosphorus, and low levels of carbon, were found in both TS enamel and dentin; a lower degree of mineralization found in TS enamel is ascribed to low values of carbon. Thus, it is evident that the absence of amelogenin gene (AMGX) expression of the X-chromosome has affected on formation of dental hard tissue.

2.4 Exon4 amelogenins transcription and enamel biomineralization

Amelogenins are proteins formed by alternative splicing of the amelogenin gene and are essential for tooth enamel formation [27, 28]. In a study, Stahl et al. [29] demonstrated the spatiotemporal position of amelogenins, a derivative from transcripts having exon4 (AMG+4) in the enamel matrix and the relative binding of recombinant AMG+4 to hydroxyapatite. Besides, they illustrated that secretion of AMG+4 proteins into the enamel matrix occurred at an early maturation stage and that binding of recombinant AMG+4 to hydroxyapatite was more as compared to binding of recombinant AMG-4. The spatiotemporal site of amelogenins containing exon4 peptide, and their functional variation in hydroxyapatite binding, indicated that the inimitable features of amelogenins having exon4 was responsible for an explicit increase of biomineralization connected to stabilization of early-formed hydroxyapatite at the maturation stage [29].

2.5 Genes and tooth development

Genes above 300 are found in regulating various stages of tooth development [30]. Main genes associated with tooth development include homeobox, MSX, DLX, PAX group and their mutation causes tooth deformity and development disorders [31]. Ogawa et al. [32] and Kapadia et al. [33] showed that Pax9 controls Msx1 expression and acts with Msx1 at the protein level to activate Msx1 and Bmp4 expression at the time of tooth development. Expression of KROX-26 gene was identified in the epithelial tissues of the developing tooth during the early bud and cap stages of the growing fetus [34]. Further, in human beings, the mutation of MSX, DLX genes [35] and AXIN2 [36] are related to certain syndromes (tooth agenesis and tricho-dento-osseous (TDO) syndrome) and hypodontia or oligodontia. To establish relationships between the syndromes of TDO and the expression patterns of MSX-2, DLX-5, and DLX-7 homeogenes in dentition, Davideau et al. [37] investigated the variation in expression of these three genes and compared their activities in orofacial samples of 7.5–9 week old human embryos. They reported two different patterns of gene expression in different tissues; (1) a stronger gene (MSX-2) expression in the progenitor cells of human orofacial skeletal structures namely bones of mandible and maxilla, Meckel’s cartilage, and tooth germs and (2) other two gene expression (DLX-5, DLX-7) only in vestibular lamina and vestibular part of dental epithelium. When the expression patterns of the three genes (MSX-2, DLX-5, and DLX-7) were correlated to the various human tooth types of early development stages, it was discerned that there existed expression of the homeogene in spatially ordered sequences along the vestibular/lingual axis of dental epithelial tissues and different organizing centers involved in the control of human tooth morphogenesis [37].

3. Influences of elements

3.1 Calcium

Insufficient dietary calcium and a reduction in the calcium: phosphorous ratio may promote bone reabsorption, risk of osteoporosis and periodontal disease and increased calcium intake is associated with alleviation of suffering from inflammatory processes, tooth mobility in patients of gingivitis with haemorrhaging [38]. Hence, enough calcium intake is suggested for patients suffering from periodontal disease and to help in the prevention of osteoporosis. Calcium is a key component of the mineralized enamel matrix, but may also have a role in ameloblast cell differentiation. In a study, Chen et al. [39] used human ameloblast lineage cells to determine the effect of calcium on cell function. Primary human ameloblast lineage cells were isolated from human fetal tooth buds and were treated with calcium ranging from 0.05 mM to 1.8 mM. Enhancing calcium levels resulted in notably decreased proliferation of ameloblast cells. The pretreatment of calcium at of 0.1 mM and 0.3 mM concentrations to the cultures caused respectively overexpression of amelogenin, amelotin and type I collagen and formation of a mineralized matrix. Thus, Chen et al. [39], by culturing in vitro human ameloblast lineage cells, showed that the addition of calcium at 0.1 mM and 0.3 mM, induced cell differentiation and upregulation of amelogenin type I collagen and amelotin.

Cementoblasts are tooth root lining cells that are essential for formation of cementum on the root surface and for creating a functional periodontal ligament [40]. Cementoblasts share phenotypical features with osteoblasts. When the increased concentration of extracellular Ca²⁺ is linked to stimulating proliferation and differentiation of osteoblasts, the influence of extracellular Ca²⁺ signaling in cementogenesis has been reported by Kanaya et al. [41]. Using reverse transcription polymerase chain reaction (RT-PCR), they found that enhanced levels of extracellular Ca²⁺ increased fibroblast growth factor (FGF)-2 gene expression.

3.1.1 Calcium sensing receptor gene

Mathias et al. [42] reported that Ca²⁺ may regulate tooth formation and the Ca²⁺-sensing receptor (CaSR) that is expressed in bone and cartilage has indicated a mechanism by which extracellular Ca²⁺ can regulate cell function. They identified CaSR protein and messenger ribonucleic acide (mRNA) in an immortalized ameloblast-like cell line (PABSo-E) and the expression of CaSR in cultured ameloblasts. In PABSo-E cells, increasing extracellular Ca²⁺ in the medium from 0 (baseline) to 2.5 mM or 5.0 mM resulted in an increase in intracellular Ca²⁺ above baseline to 534 +/− 69 mM and 838 +/− 86 mM, respectively. They revealed that enhancement of Ca²⁺ concentration in the medium could induce the intracellular Ca²⁺ signal transduction pathway and that the CaSR is expressed in developing teeth and may provide a mechanism by which these cells can react to changes in extracellular Ca²⁺ to regulate cell function and eventually, tooth formation. Further, Spurr [43] indicated several sites of expression and functions for the CaSR gene, which includes a role in tooth development and fluid regulation.

3.1.2 Pathway of tricalcium silicate induced gene expression for biomineralization

Calcium-silicate cement is used as a liner and a dentin substitute base under definitive restorative materials [44]. Hence, Du et al. [45] aimed to study tricalcium silicate (C₃S) driven pathway of extracellular signal-regulated kinase 1/2 (ERK1/2) and its role in influencing proliferation and biomineralization occurring in human dental pulp cells (hDPCs) in vitro. They cultured hDPCs in a medium containing C₃S for comparing with controls without C₃S and for measuring biomineralization, cell viability and phosphorylated ERK1/2. The ERK1/2 inhibitor U0126 was used to assess the role of this pathway on stage of the cell cycle and mineralization-dependent gene expressions of hDPCs by using flow cytometry and RT-PCR, respectively. It was observed that C₃S extracts promoted (P < 0.05) biomineralization and viability of hDPCs. When hDPCs were cultured in the medium of C₃S extracts, phosphorylated ERK1/2 was noticed within half an hour time. Furthermore, proliferation and the expression of mineralization-dependent genes, including collagen type I, dentin sialophosphoprotein, osteopontin, and osteocalcin were found decreased (P < 0.05) due to inhibition of the ERK1/2 pathway by inhibitor U0126 [45]. In conclusion, C₃S stimulated the proliferation and biomineralization of hDPCs in vitro, through the ERK1/2 pathway.

3.2 Roles of calcium of MTA

Mineral trioxide aggregate (MTA), a therapeutic, endodontic repair substance, is associated with activities of tissue calcification even though its mode of action needs further clarification [46]. In order to observe calcium release, calcification activity, calcium-sensing receptor (CaSR) gene expression and bone morphogenetic protein-2 (BMP-2), and BMP-2 receptor protein and gene expression, two populations of human periodontal ligament cells (HPLCs) that were donated by two patients, were cultured in the presence or absence of MTA discs and/or CaCl₂ [47]. It was found out that within 2 weeks MTA released a considerable concentration of calcium (4 mmol/L) into culture media. HPLCs innately exhibited gene expression encoding for the receptors of CaSR and BMP-2. Supplementation of exogenous CaCl₂ in the media effected expression of CaSR gene, calcification and BMP-2 synthesis during the whole HPLC cultures, while supplementation of magnesium chloride yielded no impact on mineralization and gene expression. Maeda et al. [47] concluded that HPLCs cocultured directly with MTA up-regulated BMP2 expression and calcification.

3.3 Calcium and phosphorus

Several culture systems with human dental pulp cells are employed to find out the mechanisms involved in dentin formation through promotion of differentiation of dental pulp (DP) cells into odontoblasts [48, 49]. When explants from human teeth were cultured in Eagle’s basal medium supplemented with 10% or 15% fetal calf serum, with or without beta-glycerophosphate (beta GP), Couble et al. [48] reported that addition of beta GP to the culture medium induced odontoblast features in the cultured pulp cells. Further, they showed through splendid structural evaluation of the cultured pulp cell that the presence of typical intracellular organelles was manifested in the body of odontoblast and afterward there appeared an area of mineralization in type I collagen rich matrix. The presence of calcium and phosphorus was evident from X-ray microanalysis while the apatite crystal structure of the mineral was confirmed through electron diffraction pattern. Finally, two patterns of expression were identified, viz., elevated expression of alpha 1(1) collagen mRNAs in all polarized cells and dentin sialoprotein gene expression in mineralizing areas including their association with calcium and phosphorus [48].

3.4 Calcium, magnesium and enamel formation genes

A genetic component in caries susceptibility is related to variation in enamel formation genes [50]. The trends of tooth demineralization and remineralization in a group of subjects are related to chosen five genes (ENAM (enamelin), MMP20 (matrix metalloproteinase 20), TUFT (tuftelin), TFIP (tuftelin-interacting protein), and AMBN (amelobalstin)). In a study, Halusic et al. [51] exposed primary baseline teeth (20 h) to an artificial caries solution as well as remineralizing solution to measure Ca and Mg concentrations in the biopsies of three categories of baseline, carious, and fluoridated teeth and to compare these tooth categories with allele and genotype frequencies for calcium and magnesium levels. Halusic et al. [51] pointed out that calcium content was higher than magnesium levels in each sample and there existed associations of genetic variation of only two genes, ENAM and AMBN with mineral concentrations. As a result, it is substantiated that there exits obvious association among influencing roles of enamel formation genes, tooth levels of calcium and magnesium and the caries development.

3.5 Selenium from dental material for stronger teeth

Selenium, an essential trace element is a constituent of antioxidant enzymes [52] and can replace sulphur in bonds of collagen resulting in stronger Se-collagen bond than a sulphur bond. Since collagen is the most important component of the organic matrix of the tooth and Se-collagen bond is stronger, the beneficial role of selenium is evident from stronger teeth of children and adults [53]. Dental filling materials are one of selenium sources of body intake and its additional benefits were tested in the experimental tooth samples of 60 subjects who were treated with endodontic dressing in the following four groups: selenium (Se), calcium hydroxide, calcium hyrdoxide + selenium and controls without Se (n = 15) [54]. With use of RT-PCR, expression of the prokaryotic 16S ribosomal RNA and microbial growth were evaluated before cleaning and shaping procedures and after 15 days of treatment of the groups with or without filling materials. The finding of the evaluation indicated that selenium use from the source of tooth material filling was significantly effective in reducing the microbial growth by decreasing the IFN-γ mRNA expression for healthy strong teeth [54].

3.6 Strontium

Strontium is recognized as a most recent version of anti-osteoporotic agent that causes immediately anti-catabolic and anabolic effects on bone cells [55]. Römer et al. [56] employed strontium in vitro to explore its application to promote bone marker transcription and hydroxyapatite formation on isolated Runx2 (Runt-related transcription factor 2) osteoblasts samples of subjects with a disease of cleidocranial dysplasia. This ailment is caused owing to insufficient gene dosage and heterozygous mutations of Runx2 which is an essential transcription factor 2 for maturing of osteoblast and transcription of osteogenic genes. This genetic deficiency is attributed to supernumerary teeth, aplasia or hypoplasia of clavicles, symptoms of hypophosphatasia (HPP) and patent fontanelles. In an investigation, Römer et al. [56] aimed to examine strontium influence on the formation of hydroxyapatite, the cell proliferation of strontium-treated Runx2 osteoblasts and gene expression of bone marker proteins. The results of their study manifested improved hydroxyapatite formation in the extracellular matrix and gene expression of bone marker proteins in strontium-treated Runx2 osteoblasts. A water soluble tetrazolium salts-1 cell proliferation assay with strontium-treated Runx2 osteoblasts indicated that cell proliferation and growth were promoted by strontium. As a consequence of strontium inducing effects, enhanced mineralization of the extracellular matrix was recognized in the strontium-treated Runx2(+/−)-osteoblasts.

When strontium forms a significant component of dental restorative materials and is widely used in toothpastes, Huang et al. [57] mentioned that low dose Sr (between 0.1 and 2.5 mM) induced proliferation and alkaline phosphatase (ALP) activity, collagen formation and mineralization of human dental pulp stem cells (hDPSC) in vitro. With the use of quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blotting and immunocytochemistry techniques in hDPSCs, strontium was found regulating gene expression and the protein secretion of the odontogenic markers (dentine sialophosphoprotein), dentine matrix protein 1, calcium sensing receptor, the downstream pathway of MAPK and ERK signaling pathway. Strontium specifically in the bioavailable form from bioglass (BG) appeared regulating metabolic and alkaline phosphate activities in hDPSCs. Henceforth, it is viewed that the element, strontium at definite concentrations considerably gives rise to odontogenic differentiation, proliferation and mineralization of hDPSCs in vitro through calcium-sensing receptor resembling to the pathway of osteoblast differentiation [57]. It is suggested based on these findings that Sr treatment of hDPSCs could be a promising therapeutic agent in dental applications and that Sr from a substituted BG could be used more effectively in biomaterials designed for dental applications.

3.7 Strontium phosphate

In another study, Su et al. [58] found out that strontium phosphate had an impact on the osteogenic differentiation of SHEDs (stem cells from human exfoliated deciduous teeth); especially, the action of the phosphate compound was linked to improved osteogenic differentiation of SHEDs along with elevated expression of the osteoblast-related genes. Two modes of activities were reported on chitosan scaffolds containing strontium, namely (1) reduced proliferation of SHEDs and (2) notably increased activities of type-I collagen expression, alkaline phosphatase role, and calcium deposition. Therefore, it is proposed that strontium has an important function in tooth remodeling because it can simulate tooth formation and decrease resorption.

3.8 Strontium ranelate

Tian et al. [59] in research, produced strontium ranelate-loaded chitosan film on titanium surfaces with five values of strontium ranelate (SR) (0, 2, 20, 40, and 80 mmol/L of the strontium ion [Sr²⁺]) to find Sr²⁺ effects of bone healing. The low levels of 2 mmol/L or 20 mmol/L of SR loaded onto the chitosan film caused improved cell responses of primary oestoblasts (POBs) with obvious proliferation, alkaline phosphatase (ALP) activity, and expression of bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (Runx2), ALP, and osteocalcin, whereas SR at great values of 40 mmol/L or 80 mmol/L suppressed the growth of POBs. The conclusive finding is that the SR-imbibed chitosan film on a titanium exterior surface supports proliferation and differentiation of osteoblasts in a concentration-dependent way and the subsequent recommendation is that strontium and titanium have definite utilities as safe implant materials of dental treatment [59].

Osteoporosis that is caused due to intensified bone loss, is associated with periodontitis and the two have the general causal factors of bone resorption [60]. One of the strategies commonly practiced to deal with the illness of periodontitis is that when SR is used, strontium ions released from SR, involves in an explicit influence on arresting activation of osteoclast and inducing differentiation of osteoblast. Jia et al. [61] studied the processes of periodontal regeneration promoted by strontium and elucidated that the epigenetic mechanism of splicing factor, heterogeneous nuclear ribonucleoprotein L (hnRNPL) promoted osteogenesis processing of periodontal ligament stem cells (PDLSCs) that were activated by strontium chloride. When SET domain containing 2 is an enzyme that in humans is encoded by the SETD2 gene and hnRNPL has osteogenesis promotion, there are chances of utilizing strontium, hnRNPL and SET domain containing 2 for curing periodontitis patients concurrently ailing from osteoporosis [61].

3.9 Iron role in cytodifferentiation of human periodontal ligament cells

The periodontal ligament (PDL) is essential in maintaining homeostasis of tooth and periodontal tissue [62] and iron overload or deficiency can have adverse impacts on alveolar bone density. In a study, the requirement of iron levels for the cytodifferentiation of PDL cells was reported by Hou et al. [63]. After supplementing in a culture medium of human PDL cells with 10–50 μm ammonium ferric citrate or 5 μm deferoxamine (an iron chelator) during differentiation, the status of intracellular iron was measured by determining the level of expression of ferritin RNA and protein; in addition, the differentiation and function of PDL cells were assessed by estimating osteoblast differentiation gene markers and the capability of formation of mineralized nodules in the culture. The results of the study indicated that the accumulation of iron caused increased regulation of light and heavy chain ferritin protein. Concomitantly, inhibition of osteoblast differentiation gene markers and mineralized nodule formation appeared in the culture medium. Deficiency of iron occurred during PDL cell differentiation led to decreased three activities namely (1) downregulation of both the light and heavy chain ferritin proteins, (2) diminished alkaline phosphatase activity and (3) poor mineralized nodule formation. Thus, it is concluded that iron is critical for the normal cell differentiation of human PDL cells [63].

3.10 Potassium hydrogen phosphate in mineralization

Stem cells from dental apical papilla (SCAPs) can be induced to differentiate along both osteoblast and odontoblast lineages [64] and effect of KH2PO4 was studied on differentiation efficiency in SCAPs by Wang et al. [65]. Stem cells that were isolated from apical papillae of immature third molars were exposed to two kinds of mineralization-inducing media, MM1 and MM2, with two different concentrations of KH₂PO₄. The levels of proliferation and osteo/odontogenic differentiation of SCAPs were correlated between MM1 and MM2 treatments. Investigation with cell counting and flow cytometry revealed that the proliferative potential of SCAPs was greater in MM2 containing 1.8 mM additional KH₂PO₄ than in MM1. Similarly, the SCAPs were much better in MM2 medium than in MM1 for various higher activities such as osteo/odontogenesis, alkaline phosphatase activity, calcium deposition and expression of osteo/odontoblast-specific genes/proteins (e.g., runt-related transcription factor 2, and osteocalcin). When KH₂PO₄ 1.8 mM was added into the media, there were positive effects of notably increased cell proliferation, better differentiation capacity of SCAPs along osteo/odontogenic cell lineages and enhanced mineralization, in comparison to control media lacking additional KH₂PO₄ [65].

3.11 Effect of phosphate

Mutations in the gene ALPL in hypophosphatasia (HPP) decreased activities of tissue nonspecific alkaline phosphatase and the consequent rise in pyrophosphate (PP(i)) caused bone and tooth mineralization malfunction by affecting calcium-phosphate (P(i)) precipitation [66]. To find out mechanisms involved in HPP-associated pulp/dentin phenotypes, Rodrigues et al. [67] cultured primary pulp cells from hypophosphatasia (HPP) subjects to assay alkaline phosphatase (ALP) activity, mineralization, and gene expression for comparison with cells from healthy controls. Exogenous P(i) was provided to the correct P(i)/PP(i) ratio in cell culture. The results of the culture studies demonstrated that HPP cells showed remarkably lower ALP activity (by 50%) and mineral nodule formation (by 60%) than the activities of controls. The affected expression of PP(i) regulatory genes was found in the HPP pulp cells, including a decrease in the progressive ankylosis gene (ANKH) and high ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). P(i) supplementation in the culture restored a remedial activity for the mineralization and moderately rescued a few gene expressions, even though the cells had already changed messenger RNA contents for PP(i)-connected genes. Rodrigues et al. [67] opined that low mineralization and disrupted odontoblast profile caused in pulp cells under HPP conditions, were the first steps of the molecular mechanisms for dentin phenotypes observed in HPP.

3.12 Aluminum oxide particles

Periodontal ligament stem cells (PDLSCs) are capable of regenerating the periodontal tissues including alveolar bone tissue [68]. Further, PDLSCs being considered as the stem cells are associated with the osseointegration of titanium implants, immediately after an implant is fixed in the fresh gap of the extracted socket. Therefore, Heo et al. [69] cultured PDLSCs on three sorts of titanium surface textures ((1) smooth machined, (2) blasted with 75 and 125 μm Al₂O₃ particles, and (3) anodized) to check their proliferation and gene expressions of osteocalcin, osteopontin, type I collagen, and GAPDH. From these experiments, Heo et al. [69] proposed three deliberations namely, (1) elevated proliferation of PDLSCs on the rough surface blasted with 75 μm Al₂O₃ particles, (2) increased osteocalcin expression on the Al₂O₃ particle treated-surface regardless of its particle size and generally lowered type I collagen expression with time in 6 days culture. In conclusion, titanium surface coating has inducing effects of Al₂O₃ due to greater proliferation of PDLSCs in the culture and higher expression of osteocalcin.

3.13 Phosphate of Mg, Sr and Zn and chloride of Mg

Divalent Mg, Sr and Zn contribute significantly to bone remodeling through osteoinductivity because of their roles in inducing bone formation and reducing bone resorption [70, 71]. So, Huang et al. [72] experimented to examine the impacts of a few divalent metal phosphates on osteogenic differentiation from human exfoliated deciduous teeth by steadily releasing the divalent metal ions from the scaffold into the culture medium and repetitively activating osteoblastic differentiation. They reported that osteoblastic differentiation was conspicuously higher in the SHEDs cultured within chitosan scaffolds containing the phosphates of divalent metals than in the cells cultured without the phosphates of metals. The observed metal influence was attributed to elevated action of alkaline phosphatase and also the bone-related gene expression of collagen type I, Runx2, osteopontin, osteocalcin, VEGF, and Ang-1, which were evident from the technique of RT-PCR and immunocytochemistry staining of bone-related protein [72]. A noteworthy enrichment of deposited minerals, especially the phosphates of Mg and Zn, which were discerned on the scaffolds after 21 days of culture, was apparent from a calcium-content assay. Similarly, MgCl₂ at 10 mM concentration increased odontogenic differentiation in human dental pulp stem cells by promoting ERK/BMP2/Smads signaling through enrichment of intracellular magnesium [73]. It is, therefore, concluded and recommended that except barium phosphate, other metal (Sr, Mg and Zn) phosphates and MgCl₂ which are found effectively promoting SHED cell differentiation and osteoblastic cell maturation, have significant efficacy and immense utility in tissue engineering and bone repair.

3.14 Ca²⁺, Gd³⁺, Sr²⁺, or Al³⁺

Kanaya et al. [41] showed that treatment with trivalent/divalent inorganic ions, Ca²⁺, Gd³⁺, Sr²⁺ and Al³⁺ except Mg²⁺ in cementoblasts (tooth root lining cells) resulted in a dose-dependent elevation of fibroblast growth factor (FGF-2) mRNA levels in a cAMP-dependent fashion. Because of this finding, it is presumed that a mechanism of extracellular Ca²⁺-sensing exists in the cementoblasts and its regulation results in FGF-2 activation in a cAMP/PKA dependent manner. The knowledge of the pathway leading to primary genes’ expression for modulating the regeneration of oral tissues will guide in making use of these inorganic elements and designing regenerative therapies in dentistry. In support, the regenerative capacity of dental pulp stem cells was found higher due to higher osteocalcin and the Runx2 gene expression caused by the scaffolds containing Sr₂ and Ce₃ [74].

3.15 Manganese effects on Streptococcus mutans virulence gene expression

Comparison of trace metals in drinking water with that of tooth enamel has led to prediction of a caries-influencing role of manganese (Mn) [75]. Manganese, an essential element, is required for the expression of mutant Streptococci sobrinus’s virulence factors, glucan-binding lectin (GBL) whose functional analogue is a glucan-binding protein (Gbps/GbpC) that is found contributing to biofilm architecture and virulence. Therefore, Arirachakaran et al. [76] explored the effects of Mn on the transcription of genes encoding Streptococcus mutans (S. mutans) Gbps, including GbpC, along with other critical S. mutans virulence genes for understanding Mn role in mutant induced tooth caries. With the use of northern and western blots and RT-PCR techniques, they aimed to find the differences of Mn impacts on the selected Gbp genes under conditions of planktonic and biofilm cultures of S. mutans in media with 50 μM Mn and without Mn. Their findings showed different forms of Mn effects namely, (1) increased expression of GbpC and gtfB, (2) decreased expression of wapA, in both planktonic and biofilm cultures, (3) decreased expression of GbpA and GbpD only in biofilms and (4) increased expression of gtfC only in planktonic cultures [76]. Thus, it is delineated that Mn availability affects the expression of multiple S. mutans genes involved in adhesion and biofilm formation.

3.16 Boron and molecular mechanism of gene expression

Boron is an essential micronutrient participating in metabolism and a few boron derivatives are found promoting the growth of bone and teeth in vivo [77]. So, Taşlı et al. [78] studied molecular mechanism of bone formation, while evaluating cell differentiation and toxicity of sodium pentaborate pentahydrate (NaB) at various concentrations. Further, they assessed odontogenic, osteogenic differentiation and biomineralization of human tooth germ stem cells (hTGSCs) by measuring the levels of mRNA expression, odontogenic and osteogenic protein expression, alkaline phosphatase (ALP) activity, mineralization, and calcium deposits. In comparison to control, the hTGSCs exposed to NaB showed the uppermost ALP activity and expression of osteo- and odontogenic-related genes and proteins. Since NaB has the functional role of promoting in vitro odontogenic and osteogenic differentiation, it is viewed as a promising compound for the development of new scaffold systems in both bone and tooth tissue engineering [78].

3.17 Lead

Lead (Pb²⁺) exposure continues to be a significant public health problem and teeth have been recognized as a useful long-term record of lead (Pb²⁺) uptake [79, 80]. Thaweboon et al. [81] cultured dental pulp cells (DP cells) from the teeth of young patients (aged 17–24 years) and treated them with lead glutamate to examine in vitro effects of lead on DP cells. The results of their study denoted that in serum-free conditions lead at all three concentrations (4.5 × 10⁻⁵ M, 4.5 × 10⁻⁶ M, and 4.5 × 10⁻⁷ M) caused radically enhanced proliferation of DP cells and only one lead concentration of 4.5 × 10⁻⁵ M and in 2% fetal bovine serum caused increasing cell proliferation. But the significantly decreased levels of protein, procollagen type I, and osteocalcin secretion observed are indicative of the affected state of DP cells and toxic effects of lead.

In another in vitro study, stem cells obtained from primary and secondary teeth, and periodontal ligament were exposed to five concentrations of lead nitrate (160, 80, 40, 20, and 10 μM) for 24 hours to find out its adverse impacts on the proliferation, differentiation, and gene expression in these cell lines [82]. The findings of the study revealed damaging lead effects viz., (1) altered morphology and adhesion of the cells in a concentration-dependent fashion, (2) a severe downregulation of osteogenesis and ectoderm and endoderm markers, demonstrating an irregular and untimely differentiation trail and (3) a regular expression of key markers associated with stemness (Oct 4, Rex 1) and DNA repair enzyme markers. Abdullah et al. [82] conclusively corroborated the harmful lead effects of modified differentiation and expression of the stem cells.

3.18 Titanium

Titanium is also another element finding its way from dental alloy into the oral cavity to cause various effects. Peri-implant granulation tissue fibroblasts (PIGFs) were exposed to TiO₂ particles, Porphyromonas gingivalis and a mixture of TiO₂ particles and P. gingivalis to determine gene expression and protein production of pro-inflammatory mediators by PIGFs with the use of the techniques of PCR and enzyme-linked immunoassay (ELISA) [83]. It was observed that at high concentration TiO₂ was toxic to PIGFs and at sub-toxic level, it promoted high gene expression of tumour necrosis factor A (TNF-A) and enhanced protein production of TNF-α, interleukin (IL)-6 and IL-8. Both TiO₂ particles and P. gingivalis caused higher effects than P. gingivalis alone. In another investigation, Wang et al. [84] observed that after treatment with submicron particles of titanium, human mesenchymal stem cells showed reduced levels of several activities namely, bone sialoprotein (BSP) gene expression, collagen type I and BSP production, cellular proliferation and viability and matrix mineralization. Further, Salvi et al. [85] revealed that incorporation of titanium dental implants into hard and soft tissue resulted in a multifarious biological activities viz., osseointegration linked to inflammation and a raise of gene expression for osteogenesis, angiogenesis and neurogenesis of wound healing.

Global DNA methylation was determined in 21 subjects with peri-implantitis and 24 subjects with healthy implants with use of immunohistochemical measurement of 5-methylcytosine (5mC) in peri-implant crevicular fluid samples and related to titanium levels analysed with inductively coupled plasma mass spectrometry (ICP-MS) in submucosal plaque samples [86]. The levels of 5mC were notably greater in peri-implantitis samples than the healthy implant samples (P = 0.002). Therefore, it is opined that there is a relationship between peri-implantitis and epigenetic alterations in the peri-implant tissues and that methylation may be affected by titanium dissolution products. In conclusion, it viewed that because of corrosion and deposition from implants, titanium toxicity may pose at gene level several tooth weakening effects namely, inflammation, allergy, bone loss, failure of osseointegration and dental implants [87].

3.19 Nickel

Nickel becomes a source from alloys of dental application and causes intraoral metal contact allergy [88, 89], inflammatory response [90, 91] and cytotoxicity [92] that are associated with the expression of different genes described as follows. X-ray fluorescence microscopy and spectrometry for measurement of metal level released from the alloy, histochemical analysis, RT-PCR and western blotting for the expression level of HLA-DR were employed to compare between gingival tissues collected from the subjects with allergy and affected by alloy restoration and normal gingival tissue samples [88]. The allergic groups showed significantly higher levels of protein and gene expression of human leukocyte antigen DR (HLA-DR) than (P < 0.01) control group without any metal exposure [88]. In allergic patients metals of alloys are responsible for inducing HLA-DR which is one of the major histocompatibility complex class II antigens and associated with antigen presentation to CD₄⁺ T lymphocytes [93].

In a study reported by Li et al. [91], impacts of nickel(ii) on the expression of inflammatory cytokine, receptor genes and nuclear factor-kappa B (NFκB)-related genes were determined by using qRT-PCR and PCR-based arrays in the human THP1 monocytic cell line pre-exposed to Ni(ii) for 72 h. Both downregulation of 10 inflammatory genes and up-regulation of IL8 and seven NFκB-related genes’ expression were found induced by Ni(ii) only in the pre-exposed group. Wylie et al. [90] treated H400 oral keratinocytes with two Ni-based dental alloys (Matchmate and Dsign10) and NiCl₂ (1–40 μg/mL Ni²⁺) and found that exposure to increasing concentrations of NiCl₂ decreased cell growth and morphology and increased all cytokine transcripts at 1 day. On day 6, IL-1beta and IL-8 transcripts were negatively affected, whereas granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-11 enhanced with Ni²⁺ dose. Further, Messer and Lucas [92] indicated that Ni ions released from alloys affected several cellular functions and their cytotoxic effects relied on the factors namely ion chemistry, ion valence, and dose-time dependence. In conclusion, it is viewed that there exist the main concerns about biocompatibility of harmful nickel ions released from the alloys into dental and surrounding tissues [92].

3.20 Mineralization and genes

The human TUFT1 gene is expressed at the time of development and mineralization of enamel through coding for tuftelin, a glycoprotein identified in enamel [94, 95]. Halusic et al. [51] reported that among the selected genes, ENAM, MMP20, TUFT, TFIP, and AMBN, only two genes EAM and AMBN were found associated with calcium mineralization, whereas Jeremias et al. [96] reported a significant association of 3 genes (TFIP11, ENAM, and AMELX) with molar-incisor hypomineralization.

Pang et al. [50] have used a Streptococcus mutans biofilm model to verify if variants in genes are connected with mineral loss in dental enamel. In the samples of saliva and enamels from 213 individuals, they carried out DNA extraction and analyses of 16 single nucleotide polymorphisms in the saliva and also analyses of physical and chemical properties, mineral loss and the lesion depth of the demineralized enamel samples under cariogenic challenge for comparison between different genotypes at each single nucleotide polymorphism. Their findings pointed out the genes associated with minerals both at higher levels [Mn in GG genotype of AMBN (rs7694409), Ca in GT genotype of MMP20 (rs1612069), Ca/P ratio in GG genotype of AMBN (rs13115627)] and also at lower levels [Mn in CT genotype of TFIP11 (rs2097470) and P in GG genotype of AMBN (rs13115627), AG genotype of ENAM (rs12640848) and AA genotype of MMP20 (rs2292730)]. Subsequently, the lower mineral levels were connected with the mineral loss, depth loss and the genes (TFIP11, TUFT1, MMP20, and ENAM). They suggested that genetic variations in the genes of TFIP11, TUFT1, MMP20, and ENAM influenced enamel demineralization in a Streptococcus mutans biofilm model and the possibilities of weakening the tooth structure due to demineralizing action of certain genes [50].

4. Summary and conclusions

Elements are found influencing mineralization, cell differentiation and development of teeth. Differences in the effects of elements are associated with ionic and compound forms and dose. Their influence is through gene expression. Table 1 shown the various elements’ influence on gene expression associated with the mineralization, cell differentiation and tooth development. Prominently four kinds of gene expressions were reported, namely up regulation by B, Ca, Sr, Fe, K, P, MgCl₂, Divalent ions (Mg, Zn, etc.), down-regulation by tricalcium silicate and lead, no expression by barium phosphate and adverse gene expression by lead nitrate. Broadly the effects could be positive, neutral and negative correspondingly related to their types as essential, non-essential and harmful elements which are to be considered for their multiple sources from diet, environment and dental materials and implants. Besides other properties and utilities, essential elements (Ca, Sr, Se, Cr, Fe, K, P etc.) are included and harmful elements (Pb) are excluded from oral intake and in the composition of dental products formulated and fabricated for multiple requirements in dentistry, whereas Ni and Ti use is continued since their effects of allergy and inflammation are very rare and possibilities of their substitutes are explored.

SN	Element/compound	Stem cells used	Method used	Gene identified	Nature of gene expression & [reference]
1	Ca^2+, Gd^3+, Sr^2+, Al^3+	Cementoblast	RT-PCR	Fibroblast growth factor (FGF)-2 gene	Increase in FGF-2 expression [41]
2	Ca from MTA	Human periodontal ligament cells	In vitro & gene expression techniques	Calcium-sensing receptor (CaSR) gene	Increase in gene expression [47]
3	Ca	Human ameloblast lineage cells	Techniques of PCR, Immunoassay, phase contrast microscope	AMELX, COL1A1, AMTN	Upregulated expression of amelogenin, type I collagen & amelotin [39]
4	Se	Intracanal dental cells	RT-PCR	IFN-γ mRNA expression	Down regulated IFN-γ expression for anti-inflammation [54]
5	Sr	Runx2(+/−) osteoblasts	RNA isolation, RT-PCR, fluorescence staining & quantification, cell growth analysis & WST-1 cell proliferation test	Runx2	Improved mineralisation of the extracellular matrix [56]
6	Sr	Human dental pulp stem cells	qRT-PCR, western blotting and immune-cytochemistry techniques	(DSPP) and dentine matrix protein 1 (DMP-1) genes	Proliferation, odontogenic differentiation and mineralisation of hDPSCs [57]
7	Strontium phosphate	Stem cells from human exfoliated deciduous teeth	Osteogenic differentiation, RT-PCR, alkaline phosphatase assay, calcium quantification, perfusion dynamic culture	Osteoblast-related gene	Up-regulated expression for enhanced cellular differentiation [58]
8	Strontium ranelate	Primary oestoblasts	X-ray diffraction, scanning electron microscopy, Fourier transform infrared spectroscopy	Osteoblastic gene	Promotes osteoblast proliferation and differentiation [59]
9	Strontium ranelate	Periodontal ligament stem cells	Immune-histochemistry, western blotting, RNA extraction, RT-qPCR	Heterogeneous nuclear ribonucleo-protein L-gene	Preventing osteoclast & promoting osteoblast differentiation [61]
10	Iron	Human periodontal ligament cells	Techniques of measurements of ferritin RNA and protein, gene expression	Osteoblast differentiation gene	Fe is critical for normal differentiation of human PDL cells [63]
11	Potassium hydrogen phosphate	Stem cells from the dental apical papilla	Flow cytometry, alkaline phosphatase assay, alizarin red staining, TR-PCR, western blot, immune-cytochemistry	Osteo/odontoblast-specific genes	Upregulated gene expression for enhanced cell growth & improved differentiation [65]
12	P	Primary pulp cells from hypophosphatasia (HPP) subjects	qRT-PCR, von Kossa assay, alizarin red S staining, ALP activity assay	Odontoblast marker genes	P addition enhanced mineralization & rescued some of gene expression [67]
13	Divalent Mg, Sr, Zn & P	Stem cells from human exfoliated deciduous teeth	RT-PCR, immune-cytochemistry, ALP activity assay	Gene of collagen type-I, Runx2, osteopontin, osteocalcin, VEGF, and Ang-1	High activity of alkaline phosphatase, & high osteoblastic cell maturation & differentiation [72]
14	Boron	Human tooth germ stem cells	RT-PCR, immune-cytochemistry, ALP activity assay	Osteo- and odontogenic related genes	Increased alkaline phosphatase) activity, osteo- & odontogenic differentiation & biomineralization [78]
15	Aluminum oxide particles	Periodontal ligament adult stem cells	RT-PCR, immune-cytochemistry, enzyme activity assay	Genes of osteocalcin, osteopontin, type I collagen & GAPDH	Increased osteocalcin & decreased type I collagen expression [69]
16	MgCl2	Human periodontal ligament cells	RT-PCR	ERK/BMP2/Snads signaling genes	Increased odontogenic differentiation [73]
17	Mg^2+	Shed human exfoliated deciduous teeth	RT-PCR, western blot	Genes of collagen type 1, Runx2, osteopontin, osteocalcin, VEGF, and Ang-1,	Elevated gene expression and mineral deposit [72]
18	Barium phosphate	Stem cells from human exfoliated deciduous teeth	RT-PCR, western blot	Genes of collagen type 1, Runx2, osteopontin, osteocalcin, VEGF, and Ang-1,	No effect [73]
19	Tricalcium silicate	Human dental pulp cells	RT-PCR, ALP activity analysis, alizarin red S staining	Mineralization-dependent genes	Decrease of gene expression [45]
20	Lead	Human dental pulp fibroblasts cells of patients	RT-PCR, ALP activity analysis, western blot	Genes of procollagen type I, and osteocalcin	Decreased protein, procollagen type I, and osteocalcin productions [81]
21	Lead nitrate	Stem cells of deciduous & permanent teeth, periodontal ligament & bone marrow	RT-PCR, MTT & LDH assay, immune-phenotyping assay	Genes of Oct 4, Rex 1 and DNA repair enzyme	Alteration in the differentiation and gene expression in the cells [82]
22	Titanium	Peri-implant granulation tissue fibroblasts	PCR & ELISA	TNF-α, IL-6 & IL-8	High gene expression for causing peri-implantitis [83]
23	Nickel	Human THP1 monocytic cells	qRT-PCR	10 inflammatory genes, IL-8 & 7 NFκB-related genes	Up regulation of IL-8 & 7 NFκB-related genes for inflammation [91]

Table 1.

Effects of elements on the genes expression, differentiation and mineralization in the different dental stem cells.

Acknowledgments

The author thanks the Principal, Regional Institute of Education, Mysore and the Director, National Council of Educational Research and Training, New Delhi, India for their moral support and encouragement.