**4. Some recent articles "establishing" the vitamin K2 effect on teeth**

locally delivered. However, there is also a possibility that vitamin K2 is absorbed across the mucous membranes of the oral cavity. Quite successfully, one has applied ubiquinone topi-

Vitamin K2's effect on the tooth's outer surface can be seen via its impact on saliva component distribution. This secondary prevention, i.e., the "remineralization success" is mainly relying on whether the saliva composition is altered in order to produce a so-called facilitating "rem-

The famous set of data, collected by Price, when visiting groups of primitive cultures from different parts of the world, should be well known to vitamin K2 enthusiasts. Some groups visited were still primitive as to their alimentation culture and customs, while other groups

Price subsequently analyzed food samples from these groups. And, with little deviation, he registered that the "primitive diets" were high in vitamins A and D, along with factor named "Activator X". This particular ingredient could be retrieved from butter by grass fed animals [5]. He launched the idea that it was a fat soluble nutrient that has now been identified as or

**3.1. The support of a systemic theory of dental caries and K2 as a critical component**

effective. Vitamin K2, however, may serve as a more potent nutrient.

**3.2. What we hope to learn from Dr. Price's discovery**

The "old" theory of dental health, called the 'acid theory,' has been linked to the oral environment as an isolated and unique process, involving the bacteria-acid axis, as it is the only

The systemic version or theory of dental caries acknowledges the effect of refined carbohydrates on oral cavity through the impact of the hypothalamus and the endocrine system. Earlier, free radicals like ROS, typically having been construed as "exhaust energy" from mitochondria. *However, they are now thought of as "critical signals," conveyed to the hypothalamus in order to influence the secretion of hormones, like parotid hormone and insulin. The major task to be undertaken now is: find which nutrients optimally affect the hypothalamus and which would maintain the centrifugal fluid flow through the teeth*. Antioxidants such as EGCG of green tea have proven

The exocrine functions of the salivary glands, i.e., the composition of the saliva, are nutritionally related. As for the prevention of dental caries, optimum nutrition with fat soluble vitamins like K2 may serve as much more significant factor, than the role of traditional dental recommendations which goes like this: *Eat less sugar to minimize the production of bacterial acids* 

cally to subdue periodontal inflammation induced by controlled oxidative stress [51].

ineralization microenvironment."

130 Vitamin K2 - Vital for Health and Wellbeing

linked to menaquinones, vitamin K2 [6].

**3. Evaluation of "the" hypothesis**

cause-effect relationship.

**2.6. Interpretation of "historical" data collected by Price**

and subgroups had adapted to modern civilized diets and ways of living.

It has been reported that vitamin D3 acts synergistically with vitamin K2 to prevent bone loss. A recent study evaluated the impact of vitamin K2 and vitamin D3, as an alimentary supplement in conjunction with scaling and root planning; SRP, as conventional periodontal, on gingival expression of the interleukins IL-1β and IL-10, serum bone-specific alkaline phosphatase (B-ALP), as well as tartrate-resistant acid phosphatase (TRAP, subtype 5b), and the steady state levels of alveolar bone calcium in rats subjected to experimentally provoked periodontitis. Alveolar bone mass in the periodontitis group was markedly larger than the ones in the other experimental groups. No significant differences were seen in the gingival contents of IL-1β and IL-10, blood B-ALP, TRAP-5b, and calcium, nor in alveolar bone mass between the groups receiving SRP and vitamins, and the experimental group receiving SRP alone. Furthermore, vitamin D3 and K2 alone, or combined, failed to affect gingival levels ofIL-1β and IL-10, as well as blood B-ALP and TRAP-5b, or the alveolar bone mass, as compared with traditional periodontal treatment *per se* [52].

Mesenchymal stem cells have often been used for tissue engineering in regenerative medicine. The present application focused on the features of stem cells obtained from human exfoliated deciduous teeth (SHED) in comparison with dental pulp stem cells (DPSCs) and bone marrow-derived mesenchymal stem cells (BMMSCs). Cells retrieved from various sources displayed MSC characteristics (i.e., fibroblastic morphology and MSC markers). Their growth rate markedly elevated, as compared with that of DPSCs and BMMSCs. Furthermore, it was demonstrated that some 4400 genes altered their expression by a factor of 2.0 or more. A higher gene expression in SHED was witnessed for genes participating in reaction pathways such as "cell proliferation" and "extracellular matrix" [53].

Enhancement of intracellular Ca2+ concentrations is a feature commonly seen during the differentiation period of stem cells. The transient receptor potential melastatin 4 (TRPM4) serves as one of many ion channels controlling the Ca2+ signals in both excitable and nonexcitable cells. Nelson et al. [54] characterized TRPM4 in dental follicle stem cells (DFSCs) of the rat, and defined its impact on Ca2+ mediated signaling in the differentiation process. ShRNA-mediated suppression of TRPM4 decreased the channel activity, resulting in cell proliferation during osteogenesis, with a concomitantly augmented mineralization. Whole genome microarray analysis revealed that a plethora of genes, being associated with both vitamin K2 and SXR = PXR = NR1/2, were affected by TRPM4 during DFSC differentiation. These observations indicate that TRPM4 inhibit osteogenesis. The information provided suggests a link between the Ca2+ signaling pattern and gene expression during the differentiation process, including a recognizable influence of vitamin K2.

Wnt signaling pathways are now heavily linked to bone biology [55]. In the present review, recent advances in how Wnt/Lrp5-mediated signaling modulates osteoblast and osteocyte functioning, introduce new players in the Wnt signaling pathways, proving to play important roles in bone development. Here, emerging areas of Wnt signaling in osteoclastogenesis are discussed, as well as the progress made in translating basic studies to clinical therapeutics and diagnostics centered around inhibiting Wnt pathway antagonists. These are sclerostin, Dkk1, and Sfrp1. In a recent study, (unpublished data) Osmundsen and coworkers have shown that vitamin K2 affects the Wnt system by modulating the expression of DKK1 (a Wnt inhibitor) during the development of teeth in developing molar teeth of the mouse.

mammals, based on analyses of whole genome sequences, and during the course of tooth development and growth, the structure-function relationship of the ECM is altered dynamically. In early phases, the basement membranes (BMs) separate into two cell layers of the dental epithelium and the mesenchyme. These matrix proteins are instrumental in cell functions like adhesion, polarity, as well as differentiation and mineralization of the enamel and

Vitamin K2 and Its Impact on Tooth Epigenetics

http://dx.doi.org/10.5772/66383

133

Interestingly, several of the genes known to be important in tooth development, referred to in the present paragraphs, can be retrieved as SXR and/or vitamin K2-sensitive, or as shown by Osmundsen and coworkers (personal communication), and tabulated underneath in:

**5. Effects of mandibular injection of MK-7 on gene expression in the** 

As an initial experiment, new born (at P1) Balb C mice were given 10 μl intra-mandibular injections of MK-7 (0.2, 2, and 10 mg/kg body-wt., dissolved in corn oil). The control mice

At 24 hours postinjection, the pups were killed and first right-hand side molar tooth germs

Total RNAs were isolated from individual tooth germs and used for analysis of gene expression using deoxyoligonucleotides microarrays and real-time RT-PCR using the RNeasy Mini Kit. The quality of isolated RNA was monitored using the Agilent Bioanalyzer. RNA was

Results from all dosages used suggested that numerous genes exhibited significantly altered expression. At this stage, results from pups given 2mg/kg have been more extensively analyzed. These data results suggested that 281 genes were differentially expressed (p < 0.05), with changes in expression ranging from about 5- (Minpp1, Pdzd2) to about 0.05-fold (Zfp485, Slc2a5). Bioinformatics analysis (using Ingenuity Pathways Analysis) suggested that a major fraction of the observed changes in gene expression was associated with "cell death," the data suggesting a highly significant association to decreased apoptosis. This is likely mediated via altered expression of genes associated with regulation of metabolic substrates being converted to polyamine, retinoic acid-dependent regulation of apoptosis regulation (three genes), and altered osteoclast and osteoblast signaling (six genes). Several genes involved in synthesis of complex carbohydrates e.g., proteoglycans, heparin sulphate of chondroitin

isolated from three separate batches of tooth germs (three tooth germs per batch).

"Summary of information obtained from the microarray analyses."

were removed and transferred into RNA-Later solution.

dentin matrices [62, 63].

**developing molar tooth**

were injected with vehicle only.

sulphate b, were upregulated.

**5.1. Methods**

**6. Results**

Another report aims to reveal the biological and physicochemical features of MTA = mineral trioxide aggregate, related to its potency in eliciting reparative dentinogenesis. In comparison with calcium hydroxide-based materials, MTA is more efficient. It has been asserted that the action of MTA is associated with natural wound healing processes of exposed pulps, even though MTA may also stimulate matrix formation and its mineralization *in vitro*. Physicochemical analyses have shown that MTA may also interact with phosphate-containing fluids to precipitate apatite crystals. Furthermore, MTA shows better sealing ability and maintains structural stability, however [56].

Congenital diseases of tooth roots (e.g., developmental abnormalities of short and thin roots) may lead to tooth loss [57]. Recently, studies have shown that Osterix (Osx), serving as an important transcriptional factor, along with Runx1, Runx2, SP1, and SP3 [58], all participating in osteogenesis and odontogenesis, is thought to play a vital role underlying the mechanisms that determine the developmental differences between the root and the crown. During tooth development, Osx, particularly in odontoblasts and cementoblasts, promote and sustain their differentiation as well as and mineralization. Additionally, site-specific roles of Osx in the formation of tooth root have been established. Hence, Osx is construed as a promoter of odontoblast and cementoblast differentiation, as well as a factor determining root elongation. Research featuring mechanistic properties of teeth delineates a regulatory network involving Osx expression which is controllable via either BMP-signaling or Runx2-expression, pointing to a feasible way of promoting/sustaining Osx expression experimentally [59].

Calcium hydroxide Ca(OH)2 has been extensively used in the dentistry; however, its mechanism of action remains unclear. Unraveling its modes of action will provide a broader understanding of the mechanisms associated with the induced dentinogenesis, as well as helping to optimize currently available treatment modalities to ensure specific regenerative processes of tooth preservation. A compilation of articles on "mechanisms of dentinogenesis involving calcium hydroxide" is featured in this paper, and recommendations related to dentinogenic mechanisms of Ca(OH)2 range from direct irritating action by the material to induction of release of biologically active molecules, like fibronectin, BMPs (bone morphogenic proteins) like BMP-4 and BMP-7, TGFs (transforming growth factors) like TGFβ, IGFs (insulin-like growth factors), antiinflammatory interleukins, alkaline phosphatase (ALP), and others [60]. It is well known that a plethora of these factors are encoded by genes that are sensitive to the impact of vitamin K2 (via the transcription factor SXR = PXR = NR1/2), as well as the vitamin A (RXR) and vitamin D (VDR) receptors [61].

The extracellular matrix (ECM) provides physical support for various tissues. However, it also contributes to the development of same, their homeostasis, and prevention of disease. More than some 200–300 ECM molecules are listed as comprising the "core matrisome" in mammals, based on analyses of whole genome sequences, and during the course of tooth development and growth, the structure-function relationship of the ECM is altered dynamically. In early phases, the basement membranes (BMs) separate into two cell layers of the dental epithelium and the mesenchyme. These matrix proteins are instrumental in cell functions like adhesion, polarity, as well as differentiation and mineralization of the enamel and dentin matrices [62, 63].

Interestingly, several of the genes known to be important in tooth development, referred to in the present paragraphs, can be retrieved as SXR and/or vitamin K2-sensitive, or as shown by Osmundsen and coworkers (personal communication), and tabulated underneath in: "Summary of information obtained from the microarray analyses."
