**2. Backing of a new concept**

**1. Introduction**

126 Vitamin K2 - Vital for Health and Wellbeing

(both systemically and locally).

posed of fruits and vegetables, as well as vitamin K2.

only [4].

parotid gland.

on dental health.

The "perfect" and healthy tooth is properly designed to cope with the rough environment in the oral cavity, since virtually cleanses itself in an inside-out manner. Dental caries is supposed to be a result of the phenomenon that a tooth's fluid flow has been halted or even reversed, thus compromising tooth's defense system. It is well known that the local enamel demineralization process, aided by bacterial acid, is vastly stimulated by nutritional conditions, specifically by today's refined carbohydrates (sucrose and corn syrup = free glucose + fructose). Hence, the ensuing process renders the tooth vulnerable, and part of this process begins in the hypothalamus, resulting in alterations of the fluid flow passing through the dentine layer [1]. Not to forget: in this context, nutritional phenomena play very important roles

In the aftermath of acid-induced enamel demineralization of the breakdown of the dentin layer is accomplished by the body's own matrix metalloproteinase (MMP) enzymes [2, 3], a process which follows as a result of an untoward and galloping inflammatory response to an enhanced acid secretion. The present caries process begins as a more or less dormant, but reversible inflammation ("dentinitis"), while developing into a nonreversible dentin phase of caries after a while. This biological phenomenon is very much like reversible versus irreversible pulpitis and the terms gingivitis and periodontitis, while referring to the periodontium

However, there is a consensus that the process of dental caries recognization is multifactorial, as well as systemically based. It may not be sufficient to decrease the detrimental process rendered by the sugar intake with the ensuing enhancement of bacterial number and strains, but also boost the body's defense mechanisms with an antioxidant-rich diet that may be com-

Some research reports document that vitamin K2 can assist in significantly reducing dental caries [5, 6]. A larger body of research, however, is necessary in order to establish the mode, by which this vitamin may augment local defense mechanisms by altering saliva composition, while also systemically, via influencing the hypothalamus, as well as endocrine aspects of the

This concept of systemically delivered impact underscores an important shift in paradigm, from a traditional 'acid theory' explaining the development of dental caries that carries a plethora of implications for the prevention of dental decay in the future. Furthermore, it will necessary to document, via the mechanism of action of vitamin K2, how this small molecule affects gene regulation of the intimate play of osteoblast- and osteoclast-like cells in the organ layers constituting developing and full-frown teeth. The present chapter attempts to create a synthesis of current knowledge and recent research reports and ongoing research projects, with the intention of shedding new light on the impact of K2

#### **2.1. Oral and systemic stress responses with common denominators**

The definition of the stress concept dates back some 60–70 years [7]. It was meant to feature the process of how irritants caused a bodily reaction, and how the body dealt with it. If a stressor was defined as local, e.g. acid-induced enamel demineralization or irritation of the periodontal tissues by plaques, the body might provoke a specific, but local reaction. This would be controlled inflammatory responses that will remain similar throughout the entire body. Hence, they are defined as "local adaptation syndromes" (LAS). In the dentin of a tooth, "dentinitis" represents this local inflammatory response.

A focal reaction is often mild, but resides rather quickly. However, this limited response may develop into a systemic and an exaggerated variant, thus "threatening" the entire body via the endocrine system. The present response is named "GAS" ("general adaption syndrome"), because its "attack" on the organism pretty much resembles the systemic type reaction, even though it represents a local type of stress reaction [7]. The hypothalamus/pituitary/adrenal axis serves as the mechanism in charge of the body's general, untoward reactions.

The essence of the problem resides with the ingestion of refined carbohydrates (i.e., mono and disaccharides in particular) that locally escalate the growth of microorganisms within the oral cavity and their production of acid. This inflammation causes a rapid loss of minerals, such as dental enamel, and it is named "dentinitis" and serves as a local adaptation, being a part of an LAS.

However, subsequent to this local reaction, small-molecular sugar entities (like sucrose, glucose, and fructose) exert a major impact on the body, when absorbed. Blood-sugar spikes are therefore counteracted by emerging dentinal fluid flows through the tooth by coordinated signals emanating from the hypothalamus. This adaptation (GAS) is chiefly endocrine, and affects the entire body. Hence, the present hypothalamic-parotid axis serves as the endocrine axis that is instrumental in maintaining the dental health [8]. The essence of it is the following: local irritation is magnified in the presence of a GAS response [7, 9]. Sugar molecules (mostly monosaccharides), with their marked effect on the whole organism, magnifies the local acid attack by triggering the GAS response. As a consequence, the tooth is rendered more vulnerable to the acid exposure [1], whether the acid is produced from sugar entities (via bacteria) or comes from various carboxylic acids in the diet.

#### **2.2. How the tooth develops caries**

The tooth is fed by the alimentary elements delivered by fluid flow through the dentin which may be halted or reversed, when impacted by a systemic stressor, like excessive sugar intake [10, 11]. This allows bacteria in the oral cavity to attach to the tooth, where they enhance the local concentration of acid, leading to the well-known demineralization of the enamel surface. Consequently, the flow of fluid through the tooth is enhanced by parotid hormone [8]. The secretion of parotid hormone, as well as the secretion of insulin [12, 13] is regulated by the hypothalamus, influencing GAS, and eventually affecting the adrenal glands. Finally, a normal inflammatory reaction (LAS) occurs with a corresponding enhanced metabolism, i.e., (1) an increase in reactive oxygen species (ROS) production, and (2) activation of MMP's (e.g., collagenase). Normally, tissue inhibitors of metalloproteinases (TIMP's) serve to neutralize activated MMP's, when the body has regained its control over the inflammatory process. Antioxidants aid in the control inflammation by hampering an ROS activity, thus minimizing the necessity for stimulating MMP activation. It is known that optimal nutrition exerts an important role in this process. Temporary challenges are followingly managed by the dampening inflammation known to cause reversible state, while healing may occur. This process is called dentinitis [14, 15]. Excessive irritability leads to galloping inflammatory processes that are mainly irreversible in the tooth and recognized in the dentin by the name of caries.

to the presence of vitamin K2 (ref). Some examples of these processes are osteocalcin, located in bones and teeth, and matrix GLA protein expressed in cardiovascular (i.e., soft) tissues. Both of these protein structures require the vitamins A and D, as well as vitamin K2 for their production [30, ref]. The carboxylation of osteocalcin by vitamin K2 allows it to attract and retain calcium that is good for bones [31, 32]. The "opposite" process is observed in cardiovascular (i.e., soft) tissues, since matrix GLA proteins allow for calcium to be deposited in arteries, when uncarboxylated, but shed or blocks the "entry" of calcium, when carboxylated

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Dietary K2 is processed in the liver and released into the circulation via high and low density lipoproteins that make them readily available for uptake into extrahepatic tissues [36–38]. Fermented foods such as cheese have significantly higher levels of K2 than milk. The higher levels are obtained from bacterial sources. Natto (which is fermented soy) is, without any doubt the more potent source of vitamin K2 [30]. Most K2 supplements are cultured from natto; however, synthetic products with unsurpassed bioavailability and stability are now to

Menaquinones are taken up and stored in several tissues throughout the body. Some of the highest concentrations to be found are in the pancreas and the salivary glands [39]. Hence, it can be construed that there exists a close relationship between both of these exocrine/endocrine glands through the hypothalamus. High levels of vitamin K2 are also located in the brain, heart, and bone [40, 41] which definitely is of significance for many disease states, including dental caries, which has been shown to be associated with oxidative stress [6, 24].

Saliva brings nutrients from the outside of the tooth directly to the inside [42]. The fluid holds active ingredients, like minerals and enzymes, as well as buffering agents. The free cytosolic calcium levels sustain the more important or critical role in the signaling potential of the salivary glands' contents [43–45]. Taken calcium's dependency on vitamin K2–assisted carboxylation related to osteocalcin and matrix GLA proteins for granted, findings to come may reveal that vitamin K2 exerts an impact on salivary signaling and composition and activation potency. Since long, one has known that insulin [46] and the exocrine secretions of the pan-

Furthermore, saliva serves as an important player in the maintenance of proper mineralization of the teeth's enamel. The saliva buffers demineralization seen with acid-induced mineral dissolution, and it delivers building blocks for remineralization "on request." The optimal pH for tooth health is variable [48], and is more related to saliva composition and flow. However, it is yet not known how saliva is connected to vitamin K2, but it has been associated with its contents of the pH-buffering inorganic phosphate, decreasing the counts of lactobacilli in the

Some types of cheese have been asserted to display anticariogenic properties [49], and this is due to its contents of fermented bacteria that produce higher amount of vitamin K2 [30, 50]. This favorable feature would serve as a source of systemically located vitamin K2, rather than

creas [47] are partially dependent on the presence of vitamin K2.

with the assistance of sufficient vitamin K2 [33–35].

be available in the market.

**2.5. How dental tissue is nourished**

oral cavity [5].

#### **2.3. A systemic approach and the impact of oxidation control**

The systemic "angle" of caries may be construed as a link to diabetes mellitus. When enhanced blood glucose is registered in the hypothalamus, the production of free radicals like "reactive oxygen species (ROS)" is enhanced. These molecules serve as a warning signal for the hypothalamic gland to down-regulate the secretion of parotid hormone, while simultaneously upregulating the insulin secretion. Antioxidant loading has since long been known to manage the glucose-induced free radical storm sweeping the hypothalamic gland [16].

Antioxidants counteract the detrimental effects of the free radical damage, as shown in the so-called Asian Paradox, where heavy cigarette smoking is paralleled with reduced rates of coronary disease and cancer amongst consumers of green tea [17–19] that is famous for its antioxidant properties. However, along with the fact that systemically administered antioxidant effects of green tea reduce the incident of dental caries, vitamin K2 may prove to be an even more potent antioxidant [20–24].

#### **2.4. A summary of the features of vitamin K2**

Vitamin K2 is known as menaquinones, while vitamin K1 is phylloquinone. The quinones display oxygen-containing ring structures that render them suitable for the transport of electrons [25–27]. K2 was added to the vitamin K category of molecules, since it may be produced in the body from K1 [28]. K1 is deemed essential to blood clotting, and accordingly, our body has "invented" modes to recycle K1 for repeated use. Hence, it has rendered itself less dependent on a constant dietary intake of K1.

Vitamin K2 takes several forms that are linked to the structure of their side chains (e.g., MK4 and MK7). While MK4 is the form produced by our bodies produced from K1, supplemental MK4 is entirely synthetic. MK7 is a (more) biologically active form that entertains a longer half-life. Therefore, it is often the preferred or recommended supplement [29], especially in the treatment of osteoporosis and untoward soft tissue calcifications (ref).

Vitamin K2 is bounded to the transcription factor SXR/PXR (ref) and may serve as a cofactor of vitamin K dependent carboxylases. This enzyme, when associating with vitamin K2, will change the structure of proteins by the process of gamma-carboxylation, or the SXR/PXRvitamin K2 complex may work to enhance the expression of a set of genes that are responsive to the presence of vitamin K2 (ref). Some examples of these processes are osteocalcin, located in bones and teeth, and matrix GLA protein expressed in cardiovascular (i.e., soft) tissues. Both of these protein structures require the vitamins A and D, as well as vitamin K2 for their production [30, ref]. The carboxylation of osteocalcin by vitamin K2 allows it to attract and retain calcium that is good for bones [31, 32]. The "opposite" process is observed in cardiovascular (i.e., soft) tissues, since matrix GLA proteins allow for calcium to be deposited in arteries, when uncarboxylated, but shed or blocks the "entry" of calcium, when carboxylated with the assistance of sufficient vitamin K2 [33–35].

Dietary K2 is processed in the liver and released into the circulation via high and low density lipoproteins that make them readily available for uptake into extrahepatic tissues [36–38]. Fermented foods such as cheese have significantly higher levels of K2 than milk. The higher levels are obtained from bacterial sources. Natto (which is fermented soy) is, without any doubt the more potent source of vitamin K2 [30]. Most K2 supplements are cultured from natto; however, synthetic products with unsurpassed bioavailability and stability are now to be available in the market.

Menaquinones are taken up and stored in several tissues throughout the body. Some of the highest concentrations to be found are in the pancreas and the salivary glands [39]. Hence, it can be construed that there exists a close relationship between both of these exocrine/endocrine glands through the hypothalamus. High levels of vitamin K2 are also located in the brain, heart, and bone [40, 41] which definitely is of significance for many disease states, including dental caries, which has been shown to be associated with oxidative stress [6, 24].

#### **2.5. How dental tissue is nourished**

a normal inflammatory reaction (LAS) occurs with a corresponding enhanced metabolism, i.e., (1) an increase in reactive oxygen species (ROS) production, and (2) activation of MMP's (e.g., collagenase). Normally, tissue inhibitors of metalloproteinases (TIMP's) serve to neutralize activated MMP's, when the body has regained its control over the inflammatory process. Antioxidants aid in the control inflammation by hampering an ROS activity, thus minimizing the necessity for stimulating MMP activation. It is known that optimal nutrition exerts an important role in this process. Temporary challenges are followingly managed by the dampening inflammation known to cause reversible state, while healing may occur. This process is called dentinitis [14, 15]. Excessive irritability leads to galloping inflammatory processes that

are mainly irreversible in the tooth and recognized in the dentin by the name of caries.

the glucose-induced free radical storm sweeping the hypothalamic gland [16].

The systemic "angle" of caries may be construed as a link to diabetes mellitus. When enhanced blood glucose is registered in the hypothalamus, the production of free radicals like "reactive oxygen species (ROS)" is enhanced. These molecules serve as a warning signal for the hypothalamic gland to down-regulate the secretion of parotid hormone, while simultaneously upregulating the insulin secretion. Antioxidant loading has since long been known to manage

Antioxidants counteract the detrimental effects of the free radical damage, as shown in the so-called Asian Paradox, where heavy cigarette smoking is paralleled with reduced rates of coronary disease and cancer amongst consumers of green tea [17–19] that is famous for its antioxidant properties. However, along with the fact that systemically administered antioxidant effects of green tea reduce the incident of dental caries, vitamin K2 may prove to be an

Vitamin K2 is known as menaquinones, while vitamin K1 is phylloquinone. The quinones display oxygen-containing ring structures that render them suitable for the transport of electrons [25–27]. K2 was added to the vitamin K category of molecules, since it may be produced in the body from K1 [28]. K1 is deemed essential to blood clotting, and accordingly, our body has "invented" modes to recycle K1 for repeated use. Hence, it has rendered itself less dependent

Vitamin K2 takes several forms that are linked to the structure of their side chains (e.g., MK4 and MK7). While MK4 is the form produced by our bodies produced from K1, supplemental MK4 is entirely synthetic. MK7 is a (more) biologically active form that entertains a longer half-life. Therefore, it is often the preferred or recommended supplement [29], especially in

Vitamin K2 is bounded to the transcription factor SXR/PXR (ref) and may serve as a cofactor of vitamin K dependent carboxylases. This enzyme, when associating with vitamin K2, will change the structure of proteins by the process of gamma-carboxylation, or the SXR/PXRvitamin K2 complex may work to enhance the expression of a set of genes that are responsive

the treatment of osteoporosis and untoward soft tissue calcifications (ref).

**2.3. A systemic approach and the impact of oxidation control**

even more potent antioxidant [20–24].

128 Vitamin K2 - Vital for Health and Wellbeing

on a constant dietary intake of K1.

**2.4. A summary of the features of vitamin K2**

Saliva brings nutrients from the outside of the tooth directly to the inside [42]. The fluid holds active ingredients, like minerals and enzymes, as well as buffering agents. The free cytosolic calcium levels sustain the more important or critical role in the signaling potential of the salivary glands' contents [43–45]. Taken calcium's dependency on vitamin K2–assisted carboxylation related to osteocalcin and matrix GLA proteins for granted, findings to come may reveal that vitamin K2 exerts an impact on salivary signaling and composition and activation potency. Since long, one has known that insulin [46] and the exocrine secretions of the pancreas [47] are partially dependent on the presence of vitamin K2.

Furthermore, saliva serves as an important player in the maintenance of proper mineralization of the teeth's enamel. The saliva buffers demineralization seen with acid-induced mineral dissolution, and it delivers building blocks for remineralization "on request." The optimal pH for tooth health is variable [48], and is more related to saliva composition and flow. However, it is yet not known how saliva is connected to vitamin K2, but it has been associated with its contents of the pH-buffering inorganic phosphate, decreasing the counts of lactobacilli in the oral cavity [5].

Some types of cheese have been asserted to display anticariogenic properties [49], and this is due to its contents of fermented bacteria that produce higher amount of vitamin K2 [30, 50]. This favorable feature would serve as a source of systemically located vitamin K2, rather than 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 topically to subdue periodontal inflammation induced by controlled oxidative stress [51].

*in the oral cavity*. Dental disease will be construed and recognized as an inflammation related degenerative lifestyle disease in line with cardiovascular incidents, as well as with bone brit-

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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,

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

or the alveolar bone mass, as compared with traditional periodontal treatment *per se* [52].

such as "cell proliferation" and "extracellular matrix" [53].

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

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

tleness (osteoporosis) and diabetes mellitus.

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 "remineralization microenvironment."

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

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

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 linked to menaquinones, vitamin K2 [6].
