Fluoride is well known for its use in the treatment of dental caries, either systemically or topically. Fluoride intake (such as in drinking water, fluoridated toothpaste, or fluoride supplements) is the cornerstone to preventing dental caries in adults and children. Fluoride prevents dental caries by slowing down the demineralization of enamel, which is caused by the interaction between dental plaque and dental hard tissues. Fluoride may inhibit tooth decay by 40–60% by co-precipitating calcium and phosphate ions and by enhancing the precipitation of fluoridated apatite. Fluoride is also found deposited as calcium fluoride in dental plaque, which helps to prevent dental caries.
1. Fluoride and Epithelial Cells
Epithelial cells function as a barrier to protect organisms from physical, chemical, and microbial damage and to maintain mucosal integrity. Dental epithelial cells can differentiate into various types of cells that can secrete enamel matrix proteins during tooth development [1]. The matrix metalloproteinase-20 (MMP-20) is produced by ameloblasts and is responsible for cleaving other enamel matrix proteins such as amelogenin, ameloblastin, and enamelin [2]. MMP-20 is produced at the secretory stage of dental enamel formation and cleaves proteins that allow the crystals to grow into the space [2]. Ameloblasts also secrete kallikrein-4 (KLK4), which is essential for the breakdown of enamel proteins and enamel crystallization [3]. KLK4 helps crystals to grow and contract, which strengthens the enamel at the last stage of amelogenesis [3].
Recent studies have revealed that different concentrations of fluoride have various effects on epithelial cells.
1.1. High Concentration Fluoride and Epithelial Cells
A high concentration of NaF (millimolar level) induces endoplasmic reticulum stress, apoptosis, and DNA fragmentation and interferes with enamel proteinases [4]. Exposure to a high concentration of fluoride results in enamel hypomineralization, also known as enamel fluorosis [5]. The severity of enamel fluorosis increases depending on the volume of fluoride intake and the duration of fluoride exposure [5]. Fluoride decreases the abilities of proteinases such as MMP-20 and KLK4 that degrade enamel matrix proteins, and as a result, inhibit crystal growth [6]. Alternatively, the intake of fluoride lowers the pH, which inhibits the synthesis and secretion of KLK4 [7]. One study reported that excessive fluoride results in hypomineralization through the reduced expression of Forkhead box O1 (FOXO1) in dental epithelial cells [8]. FOXO1 is reduced after exposure to excessive fluoride, which might affect the expressions of KLK4 and amelotin (AMTN), which ultimately results in enamel fluorosis. One mM fluoride was found to be cytotoxic and induced apoptosis in human gingival epithelial cells. Treatment with 2 mM NaF disturbed the gene network accompanied by endoplasmic reticulum stress [9].
1.2. Low Concentration Fluoride and Epithelial Cells
Studies have shown that fluoride has biphasic effects on epithelial ameloblast-lineage cells at different concentrations. Reduced proliferation was observed at a fluoride concentration higher than 1 mM; however, lower concentrations of fluoride, e.g., around 16 μM, promoted the proliferation of epithelial ameloblast-lineage cells [10].
During gingival wound healing, human epithelial cells cover the wound, a process termed re-epithelialization. The process of wound healing includes epithelial cell proliferation and migration as well as the synthesis and deposition of extracellular matrix components [11]. Extracellular matrix proteins such as fibronectin and laminin-5 are expressed by migrating keratinocytes during wound healing. To understand the characteristics of gingival epithelial cells responding to fluoride, human primary epithelial cells were treated with NaF to characterize their effects on cellular physiology. Cell proliferation peaked at a concentration of 50 μM of NaF, and a higher concentration of NaF (at the millimolar level) reduced proliferation. Cells treated with 50 μM of NaF showed significantly more motility than non-treated cells in vitro. qRT-PCR analyses showed increased mRNA expression levels of fibronectin and laminin-5 in cells treated with 50 μM of NaF [12].
1.3. Fluoride and Epithelial-Mesenchymal Interactions
Epithelial-mesenchymal interactions (EMT) are important for the development of ectodermal organs and for tissue regeneration and wound healing, including cellular events such as cell adhesion, proliferation, differentiation, and maturation [13]. Homeostasis, inflammation, migration and proliferation, and remodeling are the four major processes of wound healing [14]. Fibroblast growth factor 2 (FGF2) and its receptor, fibroblast growth factor receptor 2 (FGFR2), are crucial for proliferation, migration, and protease production in epithelial cells [15]. FGF2 is associated with EMT by inducing mesenchymal characteristics in epithelial cells [16]. Fibroblast growth factor 7 (FGF7) is essential for epithelial morphogenesis [17]. Twist family BHLH transcription factor 1 (Twist1) is a critical mediator for EMT [18]. One study demonstrated that treatment with 50 μM of NaF induces the expression of FGF2 and FGF7, and Twist1 is also upregulated in vivo. It was also found that the untreated group had more persistent wounds in mice [19].
2. Fluoride and Bone Marrow Mesenchymal Stem Cells (BMMSCs)
BMMSCs present in the bone marrow is self-renewing precursor cells that have the multipotency to differentiate into osteoblasts, chondroblasts, adipoblasts, and stromal cells [20]. Upon bone injuries, BMMSCs differentiate into osteoblasts and release growth factors during wound healing [21]. Consistent with other studies, fluoride also has dual effects on BMMSCs. A low concentration of NaF (50 or 500 μM) enhanced the proliferation of BMMSCs, while a high concentration of NaF (5 mM) reduced their proliferation. Furthermore, BMMSCs showed elevated motility and migration when compared with the control group. Treatment with 50 μM of NaF upregulated the expression of fibronectin and vimentin, which induced the Runt-related transcription factor (Runx2), induced osteoblast differentiation, and increased the secretion of osteocalcin (OCN) [22]. Treatment with a higher concentration of NaF induced cytotoxicity, DNA damage, and oxidative stress in BMMSCs [23]. Studies have indicated that fluoride-induced cytotoxicity depends on the concentration and the duration of fluoride exposure [24]. High concentrations of NaF (2 mM and above) result in oxidative stress and decreased viability and proliferation via the phosphor-c-Jun N-terminal kinase (JNK) pathway. In dental pulp stem cells, a low concentration of NaF promotes osteo/odontogenic differentiation via the PI3K/Akt pathway [25]. In addition, a low concentration of NAF (0.5 mM) is the optimal concentration to regulate the osteo/odontogenic differentiation of stem cells from apical papilla [26]. When exposed to mouse embryonic stem cells, a high concentration of NaF (over 1 mM) induced ROS and reduced DNA synthesis, which resulted in apoptosis through a JNK-dependent pathway [27].
3. Fluoride and Bone Metabolism
Bone homeostasis is maintained through the bone formation by osteoblasts and bone resorption by osteoclasts [28]. The activity of osteoblasts and osteoclasts is critical for bone maintenance and remodeling. The process is regulated by many factors, such as sex hormones, parathyroid hormones, and calcitonin, as well as various growth factors and cytokines [29]. Osteoblasts and osteoclasts also can control each other’s formation, differentiation, apoptosis through multiple pathways via cytokines, extracellular proteins, and transcription factors [30]. Bone diseases such as osteoporosis are caused by dysfunctional bone remodeling or the disruption of the homeostasis maintained by osteoblasts and osteoclasts [30].
Both positive and negative effects of fluoride on bones and teeth have been well established [31][32]. Fluoride treatment increases the proliferation of osteoblasts and inhibits the function of osteoclasts [33]. Trace amounts of fluoride have been used to treat osteoporosis, vertebral fractures, and bone loss in patients [34], whereas excessive fluoride results in skeletal fluorosis [35]. Long-term exposure to fluoride can result in skeletal and dental fluorosis [36]. Thus, a better understanding of the effects of different concentrations of NaF on bone homeostasis will help to gain more insights into the usage of fluoride.
3.1. Fluoride and Osteoblasts
OCN and Osteopontin (OPN) are non-collagenous proteins secreted by osteoblasts that serve as markers for osteoblast maturation [37]. OCN promotes bone formation and regulates mineralization in the bone matrix, which has a complex regulatory network [38]. OPN is an extracellular matrix protein that has multiple functions associated with bone structuring and destruction in osseous tissues [39]. The transcription factor Runx2 induces the expression of osteoblastic genes including OCN and OPN [40]. Runx2 is expressed at different stages of osteoblasts (pre-osteoblasts, and immature and mature osteoblasts) and is essential for bone formation and osteoblastic differentiation [41]. Osterix (OSX), a zinc-finger containing a transcription factor, is also a downstream target of Runx2 [42]. Together with Runx2, OSX regulates the differentiation of pre-osteoblasts into mature osteoblasts and osteocytes, and the roles of OSX in maintaining bone homeostasis have been well studied [24]. One study showed that NaF treatment affects calcium homeostasis and transcription factor expression [43]. A low concentration of NaF induced Runx2 and OSX while a high concentration of NaF reduced their expression both at the mRNA and protein levels. Treatment of MC3T3-E1 cells with 50 or 500 μM of NaF enhanced their proliferation, alkaline phosphatase (ALP) activity, and extracellular matrix mineralization, as well as their expression of bone-related genes (Runx2, OSX, OCN, and OPN) [44]. Different concentrations of NaF were also found to differentially affect the expression of bone mineralized regulator proteins, such as OCN, OPN, osteonectin (ON), and bone sialoprotein (BSP) in bone marrow stromal cells. A higher concentration of NaF (10−5M) decreased the level of the proteins while a lower concentration (10−7M) increased them [31].
Various cellular mechanisms, including the Mitogen-activated protein kinase (MAPK) pathway, are proposed to function in bone formation during fluoride treatment [45]. It is well established that a low concentration of NaF (micromolar levels) regulates tyrosine kinase and ALP activity to increase osteoblast proliferation [46][47]. Fluoride treatment of osteoblasts is also biphasic in that low concentrations promote proliferation, whereas higher concentrations result in signs of weakened osteoblast activity [48]. Long periods of fluoride exposure reduce the expression of bcl-2 family proteins, which promotes apoptosis in osteoblastic cells [49]. Fluoride induces mitochondrial respiratory chain complex abnormal expressions, which in turn cause oxidative stress and result in apoptosis [50]. High concentrations of NaF also regulate the proliferation and cell cycle of the p16 gene methylation during the development of skeletal fluorosis [51]. The schematic diagram shown inFigure 1summarizes the functions of NaF in osteoblasts.
Figure 1.A schematic diagram of the biphasic functions of fluoride in osteoblasts. A low concentration of NaF stimulates the expression of fibronectin, vimentin, Runx2, and OSX to promote the expression of osteoblastic genes (OCN and OPN). A high concentration of NaF inhibits Runx2 and induces oxidative stress in osteoblasts.
3.2. Fluoride and Osteoclasts
Osteoprotegerin (OPG) is a soluble cytokine receptor of the tumor necrosis factor (TNF) receptor family that binds to the OPG ligand [52]. The receptor activator of nuclear factor kappa-B ligand (RANKL) is also a member of the TNF superfamily, which binds to RANK on target cells. OPG/RANKL/RANK is a key signaling pathway in bone metabolism. The overexpression of OPG alters osteoclast differentiation, and recombinant OPG impedes ovariectomy-induced bone loss in rats [53]. Recombinant OPG binds to OPG ligand on BMMSCs, thereby inhibiting osteoclast differentiation [54]. RANKL is an essential cytokine that regulates osteoclastogenesis and bone resorption [55]. In response to RANKL activation, the nuclear factor of activated T cells 1 (NFATc1) regulates the terminal differentiation of osteoclasts, and NFATc1-deficient embryonic stem cells were unable to differentiate into osteoclasts [56]. NFATc1 participates in the transcription of the genes involved in heart/valve septum formation, angiogenesis, T cell proliferation, and osteoclast formation. NFATc1 binds to the promoter of genes associated with bone resorption, including cathepsin K and matrix metalloproteinase 9 (MMP-9), thus inducing their gene expression [57].
Low concentrations of NaF (micromolar level) have little influence on the viability of BMMSCs and significantly downregulate both mRNA and the protein expression levels of NFATc1 in rat osteoclasts, which result in a reduction in the levels of cathepsin K and the attenuation of bone destruction [58]. Treatment with 0.5 mM to 1 mM of NaF inhibits the activity of osteoclasts in vitro [59]. Studies suggest that fluoride can act on matrix proteinases such as Metalloproteinases-2 and -9, thus inhibiting matrix degradation [60][61]. Low concentrations of fluoride can possibly regulate osteoclasts via the B lymphocyte-induced maturation protein-1 (Blimp1)/B cell lymphoma 6 (Bcl6) axis which is a critical signaling pathway that regulates osteoclast differentiation and bone homeostasis [62]. On the other hand, the stimulation of NaF exhibits a U-shaped curve in a dose-dependent manner [63]. High concentrations of NaF induce RANKL and decrease OPG, thus increasing osteocyte-driven osteoclastogenesis via the RANK-JNK-NFATc1 signaling pathway [64]. A recent study found that osteoclasts demonstrate the most sensitivity to high concentrations of NaF with respect to other bone cell types and that fluoride exposure induces apoptosis via the Transforming growth factor (TGF)-β signaling pathway [65]. A schematic diagram of the functions of NaF in osteoclasts is shown inFigure 2.
Figure 2. A schematic diagram of the biphasic functions of fluoride in osteoclasts. A low concentration of NaF inhibits the expression of RANKL, NFATc1, MMP9, and Cathepsin K but induces OPG to bind RANKL. A low concentration of NaF is possibly associated with Blimp1/Bcl6 to repress the expression of osteoclastic genes (MMP9 and Cathepsin K). A high concentration of NaF decreases the expression of OPG while inducing RANKL to bind RANK. Induced NFATc1 translocate into the nucleus to promote osteoclastogenesis.
