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Chapter 7
Vitamin K2 and its Impact on Tooth Epigenetics
Jan Oxholm Gordeladze, Maria A. Landin,
Gaute Floer Johnsen, Håvard Jostein Haugen and
Harald Osmundsen
Additional information is available at the end of the chapter
http://dx.doi.org/10.5772/66383
© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,
and reproduction in any medium, provided the original work is properly cited.
Vitamin K2 and its Impact on Tooth Epigenetics
Jan Oxholm Gordeladze, Maria A. Landin,
Gaute Floer Johnsen, Håvard Jostein Haugen
and Harald Osmundsen
Additional information is available at the end of the chapter
Abstract
The impact of nutritional signals plays an important role in systemic-based «models» of
dental caries. Present hypotheses now focus both on the oral environment and other organs,
like the nervous system and brain. The tooth is subjected to shear forces, nourishing and
cleansing, and its present “support system” (the hypothalamus/parotid axis) relays endo-
crine signaling to the parotid gland. Sugar consumption enhances hypothalamic oxidative
stress (ROS), reversing dentinal uid ow, thus creating an enhanced vulnerability to the
oral bacterial ora. The acid, produced by the oral bacterial ora, then leads to erosion of the
dentine, and an irreversible loss of dental enamel layers. This aack brings about inam-
matory responses, yielding metalloproteinase-based “dissolution”. However, vitamin K2
(i.e. MK-4/MK-7) may come to the rescue with its antioxidant property, locally (mouth
cavity) or systemically (via the brain), thus sustaining/preserving hormone-induced den-
tinal uid ow (encompassing oxidative stress) and boosting/magnifying bodily inam-
matory responses. However, sugars may also reduce the tooth’s natural defences through
endocrine signaling, thus enhancing acid-supported enamel dentine erosion. Vitamin K2
sustains and improves the salivary buering capacity via its impact on the secretion/ow of
calcium and inorganic phosphates. Interestingly, primitive cultures’ diets (low-sugar and
high-K2 diets) preserve dental health.
Keywords: K2, SXR, endocrine interaction, deiodinases, TH receptors
© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
1. Introduction
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 sup-
posed to be a result of the phenomenon that a tooth’s uid ow 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 condi-
tions, specically by today’s rened 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 uid ow passing through the den-
tine layer [1]. Not to forget: in this context, nutritional phenomena play very important roles
(both systemically and locally).
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 inammatory response to
an enhanced acid secretion. The present caries process begins as a more or less dormant, but
reversible inammation (“dentinitis”), while developing into a nonreversible dentin phase of
caries after a while. This biological phenomenon is very much like reversible versus irrevers-
ible pulpitis and the terms gingivitis and periodontitis, while referring to the periodontium
only [4].
However, there is a consensus that the process of dental caries recognization is multifacto-
rial, as well as systemically based. It may not be sucient 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-
posed of fruits and vegetables, as well as vitamin K2.
Some research reports document that vitamin K2 can assist in signicantly 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 inuencing the hypothalamus, as well as endocrine aspects of the
parotid gland.
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 aects gene regulation of the intimate play of osteoblast- and osteoclast-like
cells in the organ layers constituting developing and full-frown teeth. The present chap-
ter aempts 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
on dental health.
Vitamin K2 - Vital for Health and Wellbeing126
2. Backing of a new concept
2.1. Oral and systemic stress responses with common denominators
The denition 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 dened as local, e.g. acid-induced enamel demineralization or irritation of the
periodontal tissues by plaques, the body might provoke a specic, but local reaction. This
would be controlled inammatory responses that will remain similar throughout the entire
body. Hence, they are dened as “local adaptation syndromes” (LAS). In the dentin of a tooth,
“dentinitis” represents this local inammatory 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 “aack” on the organism prey 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 rened carbohydrates (i.e., mono and
disaccharides in particular) that locally escalate the growth of microorganisms within the oral cav-
ity and their production of acid. This inammation 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 uid ows through the tooth by coordinated signals emanat-
ing from the hypothalamus. This adaptation (GAS) is chiey endocrine, and aects 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 magnied in the
presence of a GAS response [7, 9]. Sugar molecules (mostly monosaccharides), with their marked
eect on the whole organism, magnies the local acid aack 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 uid ow 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 aach to the tooth, where they enhance the
local concentration of acid, leading to the well-known demineralization of the enamel sur-
face. Consequently, the ow of uid 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, inuencing GAS, and eventually aecting the adrenal glands. Finally,
Vitamin K2 and Its Impact on Tooth Epigenetics
http://dx.doi.org/10.5772/66383
127
a normal inammatory 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 neutral-
ize activated MMP’s, when the body has regained its control over the inammatory process.
Antioxidants aid in the control inammation by hampering an ROS activity, thus minimiz-
ing 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 damp-
ening inammation known to cause reversible state, while healing may occur. This process is
called dentinitis [14, 15]. Excessive irritability leads to galloping inammatory processes that
are mainly irreversible in the tooth and recognized in the dentin by the name of caries.
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 hypo-
thalamic 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 eects of the free radical damage, as shown in the
so-called Asian Paradox, where heavy cigaree 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 antioxi-
dant eects 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 dis-
play 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 cloing, 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 calcications (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/PXR-
vitamin K2 complex may work to enhance the expression of a set of genes that are responsive
Vitamin K2 - Vital for Health and Wellbeing128
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 aract and
retain calcium that is good for bones [31, 32]. The “opposite” process is observed in cardio-
vascular (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 sucient 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 signicantly higher levels of K2 than milk. The higher
levels are obtained from bacterial sources. Nao (which is fermented soy) is, without any
doubt the more potent source of vitamin K2 [30]. Most K2 supplements are cultured from
nao; 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/endo-
crine glands through the hypothalamus. High levels of vitamin K2 are also located in the
brain, heart, and bone [40, 41] which denitely is of signicance 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
Saliva brings nutrients from the outside of the tooth directly to the inside [42]. The uid holds
active ingredients, like minerals and enzymes, as well as buering 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 car-
boxylation related to osteocalcin and matrix GLA proteins for granted, ndings 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-
creas [47] are partially dependent on the presence of vitamin K2.
Furthermore, saliva serves as an important player in the maintenance of proper mineraliza-
tion of the teeth’s enamel. The saliva buers 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 ow. However,
it is yet not known how saliva is connected to vitamin K2, but it has been associated with its
contents of the pH-buering 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
Vitamin K2 and Its Impact on Tooth Epigenetics
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129
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-
cally to subdue periodontal inammation induced by controlled oxidative stress [51].
Vitamin K2’s eect 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-
ineralization 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
dierent 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 lile 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 buer by grass fed animals
[5]. He launched the idea that it was a fat soluble nutrient that has now been identied as or
linked to menaquinones, vitamin K2 [6].
3. Evaluation of “the” hypothesis
3.1. The support of a systemic theory of dental caries and K2 as a critical component
The “old” theory of dental health, called the ‘acid theory,’ has been linked to the oral envi-
ronment as an isolated and unique process, involving the bacteria-acid axis, as it is the only
cause-eect relationship.
The systemic version or theory of dental caries acknowledges the eect of rened carbohy-
drates 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
eective. Vitamin K2, however, may serve as a more potent nutrient.
3.2. What we hope to learn from Dr. Price’s discovery
The exocrine functions of the salivary glands, i.e., the composition of the saliva, are nutri-
tionally related. As for the prevention of dental caries, optimum nutrition with fat soluble
vitamins like K2 may serve as much more signicant factor, than the role of traditional dental
recommendations which goes like this: Eat less sugar to minimize the production of bacterial acids
Vitamin K2 - Vital for Health and Wellbeing130
in the oral cavity. Dental disease will be construed and recognized as an inammation related
degenerative lifestyle disease in line with cardiovascular incidents, as well as with bone brit-
tleness (osteoporosis) and diabetes mellitus.
4. Some recent articles “establishing” the vitamin K2 eect on teeth
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 expres-
sion of the interleukins IL-1β and IL-10, serum bone-specic alkaline phosphatase (B-ALP), as
well as tartrate-resistant acid phosphatase (TRAP, subtype 5b), and the steady state levels of alve-
olar 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 signicant dierences 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 vita-
mins, and the experimental group receiving SRP alone. Furthermore, vitamin D3 and K2 alone, or
combined, failed to aect 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 medi-
cine. The present application focused on the features of stem cells obtained from human exfo-
liated 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., broblastic 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 dieren-
tiation 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 dened its
impact on Ca2+ mediated signaling in the dierentiation 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 aected
by TRPM4 during DFSC dierentiation. These observations indicate that TRPM4 inhibit osteo-
genesis. The information provided suggests a link between the Ca2+ signaling paern and gene
expression during the dierentiation process, including a recognizable inuence 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
Vitamin K2 and Its Impact on Tooth Epigenetics
http://dx.doi.org/10.5772/66383
131
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 aects the Wnt system by modulating the expression of DKK1 (a Wnt inhibitor)
during the development of teeth in developing molar teeth of the mouse.
Another report aims to reveal the biological and physicochemical features of MTA = min-
eral trioxide aggregate, related to its potency in eliciting reparative dentinogenesis. In com-
parison with calcium hydroxide-based materials, MTA is more ecient. 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-contain-
ing uids to precipitate apatite crystals. Furthermore, MTA shows beer 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 participat-
ing in osteogenesis and odontogenesis, is thought to play a vital role underlying the mecha-
nisms that determine the developmental dierences between the root and the crown. During
tooth development, Osx, particularly in odontoblasts and cementoblasts, promote and sustain
their dierentiation as well as and mineralization. Additionally, site-specic roles of Osx in
the formation of tooth root have been established. Hence, Osx is construed as a promoter of
odontoblast and cementoblast dierentiation, 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 mecha-
nism of action remains unclear. Unraveling its modes of action will provide a broader under-
standing of the mechanisms associated with the induced dentinogenesis, as well as helping
to optimize currently available treatment modalities to ensure specic 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 bronectin, BMPs (bone morphogenic proteins)
like BMP-4 and BMP-7, TGFs (transforming growth factors) like TGFβ, IGFs (insulin-like
growth factors), antiinammatory 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
Vitamin K2 - Vital for Health and Wellbeing132
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 dynami-
cally. 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 func-
tions like adhesion, polarity, as well as dierentiation 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.”
5. Eects of mandibular injection of MK-7 on gene expression in the
developing molar tooth
5.1. Methods
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
were injected with vehicle only.
At 24 hours postinjection, the pups were killed and rst right-hand side molar tooth germs
were removed and transferred into RNA-Later solution.
Total RNAs were isolated from individual tooth germs and used for analysis of gene expres-
sion 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
isolated from three separate batches of tooth germs (three tooth germs per batch).
6. Results
Results from all dosages used suggested that numerous genes exhibited signicantly altered
expression. At this stage, results from pups given 2mg/kg have been more extensively analyzed.
These data results suggested that 281 genes were dierentially 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 signicant association to decreased apoptosis. This is likely medi-
ated 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
sulphate b, were upregulated.
Vitamin K2 and Its Impact on Tooth Epigenetics
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133
Interestingly, also some genes related to enamel/dentine biosynthesis exhibited dierential
expression (e.g., Amelx, Ambn but not Enam). Microarray results were validated by real-
time RT-PCR. Transcription factor analysis (using Ingenuity Pathways Analysis) suggested
signicant associations to the increased transcriptional activity of Myc (measured as changes
in expression of Hspd1, Ctnnb1, Dkk1, and Psmb8 being in line with the predicted change).
The downregulation of Dkk1 is interesting as denosumab treatment results in reduced Dkk1
level. Further, high Dkk1 levels have been associated with increased bone loss.
The data also suggested highly signicant associations to decreased apoptosis. This is likely medi-
ated via altered signicant associations to polyamine regulation (three genes), altered retinoic,
acid apoptotic regulation (three genes), and altered osteoclast and osteoblast signaling (six genes).
7. Conclusions
The results are based on the measurements from independent biological triplicates and there-
fore suggestive of eects of MK-7 on gene expression during the tooth development. A clear
eect on gene expression was apparent also at a dosage of 0.2 mg/kg body weight. The results
indicate increased transcription of genes involved in development of bone (increased biosyn-
thesis of important carbohydrates) and of enamel/dentin.
Further investigations are, however, required to elucidate these ndings. Such experiments
will likely entail the establishment of a clear dose-response relationship as well as of a time-
course of action. Also, eects of oral administration should be studied.
Summary of information obtained from the microarray analyses
Gene name Description of function: General and bone-related References
Lmcd1 Transcriptional cofactor restricting GATA6 and GATA4 functioning by inhibiting
DNA-binding:
Gata6 and Gata4 are transcription factors shown to be involved in TGFβ- and
estrogen-mediated regulation of gene expression in osteoblasts, and thus bone
mineralization to hamper the development of brileness.
Int J Biochem
Cell Biol. 2013
Mar;45(3):696–705.
J Bone Miner Res. 2014
Dec;29(12):2676-87.
J Cell Physiol. 2013
Jul;228(7):1594–600.
Dmxl2 Protein involved in many functions including participation in signal transduction
pathways, such as Notch signaling:
NOTCH signaling in BMSCs (bone marrow stromal/stem cells) is required
for fracture repair performed by mature osteoblastic cells.
J Biol Chem. 2010 Nov
5;285(45):34757–64.
J Clin Invest. 2016 Mar
7. pii: 80672.
Abcb4 The membrane-associated protein encoded by this gene is a member of the
superfamily of ATP-binding cassette (ABC) transporters. ABC proteins transport
various molecules across extra- and intra-cellular membranes. The gene is heavily
involved in a plethora of liver functions:
In experimental animals, i.e., Abcb4 (-/-) mice with hepatic osteodystrophy,
serum RANKL and TGFβ-levels were augmented, resulting in an excess bone
resorption rate, probably due to a dysregulation of genes like osteoprotegerin,
osteocalcin, and osteopontin, as well as vitamin D metabolism.
Bone. 2013
Aug;55(2):501–11.
doi: 10.1016/j.
bone.2013.03.012.
Vitamin K2 - Vital for Health and Wellbeing134
Gene name Description of function: General and bone-related References
Slc12a6 This gene is a member of the K-Cl cotransporter (KCC) family. K-Cl cotransporters
are integral membrane proteins that lower intracellular chloride concentrations
below the electrochemical equilibrium potential:
Human osteoblasts express functional K-Cl cotransporters in their cell
membrane that seems to be able to induce activation of volume-sensitive
channels by KCl, necessary for normal osteoblast membrane currents, and
thus secretory functions.
Am J Physiol Cell
Physiol. 2003
Jul;285(1):C22–30.
Mta1 Transcriptional coregulator that can act as both a transcriptional corepressor and
coactivator, and stimulates the expression of WNT1 by inhibiting the expression of
its transcriptional corepressor, SIX3:
Hypoxia-induced MTA1 (via HIF-1α) stimulates growth (and inhibits
dierentiation) of osteoblastic (MC3T3) cells which is deemed important in
the process of fracture healing.
Eur J Med Res. 2015
Feb 3;20:10.
Pou3f3 This gene encodes a POU-domain containing protein that functions as a
transcription factor. The encoded protein recognizes an octamer sequence in the
DNA of target genes. This protein may play a role in development of the nervous
system:
However, it was recently shown that this gene normally upregulates the
genes of the Dlx-familiy (Dlx1, 2, 5, 6), as well as downstream genes like
Gbx2, is involved in the paerning of the mammalian jaw.
Development. 2008
Sep;135(17):2905–16.
Dio2 This gene belongs to the iodothyronine deiodinase family. It activates thyroid
hormone by converting the prohormone thyroxine (T4) by deiodination to bioactive
3,3',5-triiodothyronine (T3). It is highly expressed in the thyroid, but is known to be
expressed many other peripheral tissues:
Cold exposure (in Misty mice) compensates for BAT (brown adipose
tissue) dysfunction by increasing the expression of Acadl, Pgc1a, Dio2,
and other thermogenic genes, by altering the expression of osseous Runx2
and Rankl.
Genes upregulated by BMP-7 showed a strong enrichment for established
osteogenic marker genes, and several others (MFI2, HAS3, ADAMTS9,
HEY1, DIO2 and FGFR3) in osteoblasts. Furthermore, for DIO2 seems to
impact osteoblastic dierentiation.
Outer ring deiodination (ORD) activity was seen in bone extracts of whole
skeleton, bone marrow, and MC3T3-E1 osteoblasts. [1,25(OH)2VD]-treatment
induced D2 activity, while estradiol, PTH, forskolin, leptin, TNFα, TGFβ,
and dexamethasone did not.
J Bone Miner Res. 2013
Sep;28(9):1885-97. doi:
10.1002/jbmr.1943.
Bone. 2009
Jul;45(1):27–41.
doi: 10.1016/j.
bone.2009.03.656. Epub
2009 Mar 21.
Endocrinology. 2005
Jan;146(1):195–200.
Epub 2004 Oct 7
Camk4 Camk4 is a member of he serine/threonine protein kinase (PK) family, and the Ca2+
calmodulin-dependent protein kinase subfamily. It serves as a multifunctional
serine-threonine protein kinase, and has been implicated in transcriptional control of
lymphocytes, neurons, as well as male germ cells:
Silencing of CaMK1β obliterates the proliferation ability of osteoblasts, as
well as expression of c-Fos. However, this does not inuence the skeleton
markersRunx2, Osterix, and/or Osteocalcin.
CaMKs activate pathways mediated by CREB and NFATc1. Inhibition of
CaMKs obliterates CREB phosphorylation, lowering c-Fos, and NFATc1
expression, and thus osteoclastogenesis activated by NF-κB ligand
(RANKL).
Finally, BMP-receptor signaling in stem cells from human exfoliated
deciduous (SHED) teeth enhances the expression of genes like BMP-4,
Runx2, as well as DSPP.
Bone. 2008
Oct;43(4):700–7.
Nat Med. 2006
Dec;12(12):1410–6.
Epub 2006 Nov 26
J Endod. 2011
Dec;37(12):1647–52.
doi: 10.1016/j.
joen.2011.08.023. Epub
2011 Oct 6.
Vitamin K2 and Its Impact on Tooth Epigenetics
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135
Gene name Description of function: General and bone-related References
Ppp2r2b This gene encodes the family of phosphatase 2 regulatory B subunits. Protein
phosphatase 2 functions as one of four main Ser/Thr phosphatases, and is involved in
the inhibitory control system of cellular division:
Loss of estrogen during menopause causes changes in the female body.
Hence, it is expected that HRT-associated gene expression is due to the
changes in the DNA methylation prole (DMP). Of the DMP genes, ACBA1,
CCL5, FASLG, PPP2R2B, and UHRF1 were dierentially expressed, all of
which are associated with HRT or estrogenic regulation. All genes were also
associated with bone mineral content (BMC), while ABCA1, FASLG, and
UHRF1 were also associated with body adiposity.
Bahl A1, Pöllänen
E, Ismail K, Sipilä S,
Mikkola TM, Berglund
E, Lindqvist CM,
Syvänen AC, Rantanen
T, Kaprio J, Kovanen V,
Ollikainen M.
Stard4 Cholesterol homeostasis is regulated by sterol regulatory element (SRE)-binding
proteins (SREBPs) and by liver X receptors (LXRAs). When sterols are depleted,
LXRs are inactive and SREBPs bind promoter SREs and activate genes involved
in cholesterol turnover. The protein STAR is involved in this process, and it is
homologous to a family of proteins STARD4:
Estrogen-containing hormone replacement therapy (HRT) leads to a relief
of typical menopausal symptoms, benets bone and muscle health, and is
associated with tissue-specic gene expression proles. Hence, It is plausible
that part of the HRT-associated gene expression is due to changes in the
DNA methylation prole.
The gene expression patterns of white blood cells (WBCs) and their
associations with body composition, including muscle and bone
measures of monozygotic (MZ) female twin pairs discordant for
HRT were assessed. Of genes with differentially methylated regions
(DMRs), five (ACBA1, CCL5, FASLG, PPP2R2B, and UHRF1) were
also differentially expressed. These have been associated with HRT
or estrogenic regulation, but were also associated with bone mineral
content (BMC). Additionally, ABCA1, FASLG, and UHRF1 were also
related to the body adiposity.
Bahl A1, Pöllänen
E2, Ismail K1, Sipilä
S2, Mikkola TM2,
Berglund E3, Lindqvist
CM3, Syvänen AC3,
Rantanen T2, Kaprio J1,
Kovanen V2, Ollikainen
M1.
Tyk2 This gene encodes the Janus protein kinases (JAKs). These proteins associate with
and activate cytokine receptors with ensuing phosphorylation (activation) of receptor
subunits. It is also a component of the interferon signaling pathways. It may
therefore play a role in anti-viral immunity:
Interleukin-23 (IL-23) belonging to the IL-6/IL-12 family that plays a key
role in autoimmune and inammatory disorders. IL-23 binding to dendritic
cells, macrophages and monocytes triggers the activation of Jak2 and Tyk2
which in turn phosphorylates STAT1, STAT3, STAT4, and STAT5 as well as
induce formation of STAT3-STAT4 heterodimers. IL-23 is essential for the
survival and/or expansion of inammatory Th17 cells which, when activated
by IL-23, sustain osteoclasto-genesis via the production of IL-17 (stimulator
of the NF-kappa B) of mesenchymal cells. As a group, the IL-17 - IL-23 “axis”
includes Th17 cells that play a major role in the development and maintenance
of autoimmune arthritic inammation.
Scand J Immunol. 2010
Mar;71(3):134-45.
doi: 10.1111/j.1365–
3083.2009.02361.x.
Pdcd5 This gene encodes a protein that is upregulated during apoptosis. The encoded
protein is a regulator of K(lysine) acetyltransferase-5, involved in transcription,
DNA damage response and the cell cycle control, by blocking its degradation:
The programmed cell death gene (PDCD5) was overexpressed in an
osteosarcoma (OS) cell line, MG-63. The results indicate that PDCD5 can induce
apoptosis and G(2) phase arrest in MG-63 cells. Furthermore PDCD5 expression
in established xenografted tumors was associated with a decrease in tumor size
and weight. Furthermore, it was found that the Ras/Raf/MEK/ERK signaling
pathway was hampered, leading to the inhibition of cyclin B and CDK1, and to
the activation of caspases 3 and 9, respectively. These results are consistent with
the G(2) phase arrest observed.
Cell Signal. 2012
Aug;24(8):1713-
21. doi: 10.1016/j.
cellsig.2012.04.011.
Epub 2012 Apr 25.
Vitamin K2 - Vital for Health and Wellbeing136
Gene name Description of function: General and bone-related References
Psenen Presenilins are required for intramembranous processing of transmembrane proteins,
such as the Notch proteins. Signaling by Notch receptors mediates a wide range of
developmental cell fates.
Titanium implant surfaces with modified topographies improve osteogenic properties
in vivo. The activation of signaling stem cell pathways (such as TGFβ/BMP, Wnt,
FGF, Hedgehog, and Notch) was characterized subsequent to incubations (24 and 72
h) with BCs to SLA and modSLA surfaces in the absence of osteogenic cell culture
supplements.
Key regulatory genes belonging to the TGFβ/BMP (TGFBRs, BMPRs, ACVRs,
SMADs, Wnts, FZD1, FZDs, LRP5, NFATCs, PYGO2, LEF1) and Notch species
(including PSENEN) pathways were upregulated on the modified surfaces. These
data correlate with an increased expression of osteogenic markers (e.g. BSP and
osteocalcin, as well as BMP2 and BMP6.
These finding indicate that activation of proosteogenic cell signaling pathways by
modSLA and SLA surfaces leads to enhanced osteogenic
Clin Oral
Implants Res. 2014
Apr;25(4):475–86. doi:
10.1111/clr.12178.
Epub 2013 Apr 21.
Rhob RHOB (Ras Homolog Family Member B) is a Protein Coding gene. Diseases
associated with RHOB include oculo auricular syndrome and sertoli cell-only
syndrome. Among its related pathways are Signaling by GPCR and Developmental
Biology. GO annotations related to this gene include GTP binding and GDP
binding. An important paralog of this gene is RHOA.
Defects (mild affection to complete destruction) in the sealing zone were observed
in the OPG-deficient animals. Resorption lacunae were not detected, indicating the
loss of osteoclast-mediated bone resorption activity. Treatment with OPG resulted
in a significant decrease in the expression of a cluster of instrumental genes (like for
instance) Rho guanine nucleotide exchange factors (RhoGEFs), RhoGTPases, ROCK1
and ROCK2. This resulted in damage to or destruction of the sealing zone, thus
inhibiting osteoclast-mediated bone resorption.
Int J Mol Med. 2014
Sep;34(3):856–62.
doi: 10.3892/
ijmm.2014.1846. Epub
2014 Jul 10.
Rs1 The present gene encodes an extracellular protein serving an important
organizational role in the retina. The protein encoded is assembled and secreted
as a homooligomeric complex. Mutations in the present gene are lead to X-linked
retinoschisis with ensuing severe loss in vision.
G-protein-coupled receptors (GPCRs) are key regulators of skeletal homeostasis and
important in fracture healing. It was earlier shown that blockade of G(i) signaling in
maturing osteoblasts enhanced cortical and trabecular bone formation and prevented
age-related bone loss in female mice. Furthermore, activation of G(s) signaling
induced massive trabecular bone formation, but a concomitant cortical bone loss.
Here, “labile” tibial fractures, where endogenous G(i) signaling are blocked by PTX,
or G(s) signaling activated by Rs1, were achieved.
Inhibition of endogenous G(i) activity gave a smaller callus, but enhanced net bone
formation in both mice, irrespective of age.
PTX treatment lowered the expression of Dkk1 and upregulated Lef1 mRNA
upon fracture healing, indicating endogenous G(i) signaling in maintaining
Dkk1 expression, while suppressing Wnt signaling. On the contrary, mice
with activated Gs signaling demonstrated an increase in the initial callus size
with enhanced callus bone production. These results indicate that G(i) blockade and
G(s) activation are important for proper fracture healing.
It was previously asserted that Rs1 constitutively activated Gs-coupled GPCR,
under the control of the 2.3 kb Col I promoter, enhancing the steady state level
mineral mass in trabecular bone of femurs. In this article, it was further concluded
that Gs-signaling in OBs on enhanced intramembranous bone formation in
calvariae of Col1(2.3)/Rs1 mice. Rs1 calvariae displayed a dramatic increase in bone
volume with partial loss of cortical structure. Gene expression analysis of calvarial
OBs showed that genes were affected by Rs1 signaling, featuring processes like:
(a) differentiation, (b) synthesis of cytokine/growth factors, (c) angiogenesis, (d)
coagulation, as well as (e) energy metabolism.
J Bone Miner Res. 2015
Oct;30(10):1896–904.
doi: 10.1002/jbmr.2540.
Epub 2015 May 14
Exp Cell Res. 2015
May 1;333(2):289–302.
doi: 10.1016/j.
yexcr.2015.02.009.
Epub 2015 Feb 20.
Vitamin K2 and Its Impact on Tooth Epigenetics
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137
8. Emulation of the interaction between genes and microRNA species
known to be instrumental in the development of the osteoblastic/
odontoblastic phenotype by Mir@nt@n
The bioinformatics program Mir@nt@n, developed by Le Bechek et al. [64], was used to arrive
at high stringency interactions between microRNA species known to be instrumental in the
development and stability of osteoblastic and odontoblastic cells from stem cells. The dental =
osteoblastic/dentinoblastic genes (being signicantly regulated by Vitamin K2 in the present
study) described in the present array were fed into the program along with other genes and
microRNA-species known to be instrumental in the development and stability (both posi-
tively and negatively) of the osteoblastic and/or odontoblastic phenotype:
Gene name Description of function: General and bone-related References
Trpm5 This gene encodes a member of the transient receptor potential (TRP) protein family.
This protein plays a role in taste transduction. It is activated by lower concentrations
of intracellular Ca2+, and inhibited by higher concentrations.
Elevation of intracellular Ca2+ is commonly observed during stem cell differentiation
(e.g., osteoblastogenesis), but cease after process completion. These findings suggest
an inhibitory role for TRPM4 (Ca2+ ion channel) on osteogenesis while it appears to
be required for adipogenesis. The data provide a link between
the Ca2+ signaling pattern and gene expression during stem cell differentiation.
Stem Cells. 2013
Jan;31(1):167–77. doi:
10.1002/stem.1264.
Amelx This gene encodes a member of the amelogenin family of extracellular matrix
proteins. Amelogenins are involved in biomineralization during tooth enamel
development.
Research on enamel matrix proteins (EMPs) is centered on the understanding of their
role in enamel biomineralization, as well as of their bioactivity for tissue engineering.
It was shown that mRNA expression of AMELX and AMBN in mandibular
alveolar and basal bones RNA-positive for AMELX. Furthermore, AMELX and
AMBN mRNA levels varied according to: 1) ontogenic stage, and 2) tissue-type. In
conclusion, it was asserted AMELX and AMBN may function as growth factor-like
molecules in jaws, where they might play a role in bone physiology via autocrine/
paracrine pathways, and especially during adaptation of stress-induced remodeling.
Thymosin beta 4 is associated with RUNX2 expression through the Smad and Akt
signaling pathways in mouse dental epithelial cells.
Thymosin β4 (Tβ4) is associated with the initiation and development of the tooth
germ, via enhancement of RUNX2. The transcription factor regulates the expression
of genes involved in odontogenesis, like amelogenin, X-linked (Amelx), ameloblastin
(Ambn), as well as enamelin (Enam).
It appeared that the mDE6 mouse epithelial cell line expressed Runx2, Amelx, Ambn
and Enam, and yielded calcified matrices upon the induction of calcification.
PLoS One. 2014 Jun
16;9(6):e99626. doi:
10.1371/journal.
pone.0099626.
eCollection 2014.
Int J Mol Med. 2015
May;35(5):1169–78.
doi: 10.3892/
ijmm.2015.2118. Epub
2015 Mar 2.
DKK1 The present gene encodes a member of the dickkopf protein family. It is secreted,
including two cysteine rich regions, and it parttakes in embrygenesis due to its
inhibition of Wnt-ediated signaling. Enhanced DKK1 levels in bone marrow and
blood correlates with bone osteolysis in patients suffering from multiple myeloma.
In this article, the authors review advances and discrepancies in how Wnt/Lrp5
signaling regulates osteoblasts and osteocytes, and describe new players in Wnt
signaling pathways exerting important roles in bone development, i.e., Wnt
signaling in osteoclastogenesis, inhibition of Wnt pathway antagonists, such as
sclerostin, Dkk 1, and Sfrp1.
Gene. 2012 Jan
15;492(1):1–18.
doi: 10.1016/j.
gene.2011.10.044. Epub
2011 Nov 3. Monroe
DG1, McGee-Lawrence
ME, Oursler MJ,
Westendorf JJ.
Vitamin K2 - Vital for Health and Wellbeing138
Lmcd1, Dmxl2, Abcb4, Slc12a6, Mta1, Pou3f3, Dio2, Camk4, Ppp2r2b, Stard4, Tyk2, Pdcd5,
Psenen, Rhob, Rs1, Trpm5, Amelx, DKK1, SP1, SP3, SP7, Runx2, Runx1, NR1/2, ADRB3, Foxc2,
PGC1α, PPARA, PPARG, Dio2, UCP1, Adipoq, LEP, BETA3AR/ADRB3R/B3AR, hsa-mir-155,
and c/EBPB.
Hsa-mir-196a, hsa-mir-16, hsa-mir-455, hsa-mir-339, hsa-mir-125b, hsa-mir-328, hsa-mir-16, hsa-
mir-149, hsa-mir-125b, hsa-mir-760, hsa-mir-133, hsa-mir-29, hsa-mir-27, hsa-mir-23, hsa-mir-320,
hsa-mir-26b, hsa-mir-21, hsa-mir-302, hsa-mir-132, and hsa-mir-223.
9. Major ndings
The genes, signicantly modulated (directly or indirectly) by vitamin K2, are presented in
Table 1.
Of major interest here, from a regulatory point of view, and as a minimal “cluster” of nec-
essary and sucient genes, are probably the following species: RUNX1, RUNX2, SP1, SP3,
and DIO2, along with the microRNA-species 149, 328, 339, and 760 (see Figure 1). It is
well known that the osteoblast and odontoblast phenotypes are “determined” and “stabi-
lized” by the RUNX- and SP-families of transcription factors (upregulated), as well as the
Table 1. “Dental” genes aected directly or indirectly by exposure to vitamin K2 (MK-7).
Vitamin K2 and Its Impact on Tooth Epigenetics
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139
microRNA-species 149, 328, and 339 (downregulated). Recently, it was shown [58] that mir-
760 is involved in the eect of vitamin K2, since it associates with the transcription factor
NR1/2 = SXR = PXR [65].
Using high/maximal stringency emulations rendered by the bioinformatics program Mir@
nt@n [64], it was quite interesting to nd that the gene DIO2 (deiodinase2) that encodes the
enzyme transforming T4 to T3 in peripheral tissues, was associated with has-mir-760, also
found to exert an impact on the levels of Runx2, as well as being involved in the steady state
of SP1 and SP3, transcription factors upstream of the Runx species deemed to be markers of
the osteoblast/odontoblast phenotype (see Figure 2). It therefore does not come as a surprise
that bone tissue is heavily dependent on DIO2 activation to function properly, i.e., replen-
ishing “lost” osteoblasts from precursor cell, as well as proper functioning of dierentiated
osteoblasts/dentinoblasts to maintain bone/dentine mass at a stable level [66]. It may though
come as a surprise to many that, in fact, vitamin K2 serves a rather prominent role in this
process.
Finally, when applying low-stringency criteria to the Mir@nt@n-emulation process, a
larger and less rigid network of mutual interactions was obtained (see Figure 3). From
the interactions predicted, one may hypothesize the following: It is not trivial to ingest a
dose that is too small to see the broad spectrum of benecial eects of vitamin K2 on osteo-
blasts/odontoblasts. Furthermore, the dose should be titrated to ensure proper levels and
characteristics and amounts of bodily beige versus white adipocytes (confer the postulated
Figure 1. Interactions between transcription factors, “functional” genes, and microRNA species as emulated in the
bioinformatics program Mir@nt@n.
Vitamin K2 - Vital for Health and Wellbeing140
Figure 3. Extended interaction scheme (low stringency emulation) of transcription factors, microRNA species, and
dierentiation-related genes in tooth germs from the rat.
Figure 2. Interactions (high/maximal stringency emulation) of transcription factors, microRNA species, and dierentiation /
function-related genes in tooth germs from the rat.
Vitamin K2 and Its Impact on Tooth Epigenetics
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impact of vitamin K on mir-760 on SP1 and mir-149 with reciprocal regulatory loops), and
the mir-760 “junction” between RUNX1, SP1, SP3, and PPARG versus DIO2, ADIPOQ,
and RUNX2, which are all part of a mutually interacting network regulated by vitamin K2
odontoblasts/osteoblasts.
Finally, it should be emphasized that vitamin K2 (MK-7) upregulates Amelx and DKK1 in
tooth germs, the former is instrumental in the building and maintenance of tooth enamel,
and thus their resilience toward enamel erosion; the laer, DKK1 (dickopf1 WNT signaling
pathway inhibitor 1), takes part in the modulation of osteoclast-related bone degradation, and
in this context, the healthy transition between osteoclast-induced resorption and renewal of
bone tissue with microcracks [67].
10. Pertinent question: is the dental lling material toxic to the living
tooth? Contemplations on the making of live and articial teeth
Monomers from methacrylate based dental materials both prior to and post polymerization
have demonstrated adverse eects both in vitro and in vivo in terms of cytotoxicity [68], muta-
genicity/genotoxicity [69–72], negative eects on fertility [73], xenoestrogenicity [74–76], and
allergy induction [77]. The degree of cytoxicity will vary from the type assay used, materials
tested, time intervals for testing, and cell types tested [78].
It is most pertinent to perform in vitro cytotoxicity testing on cells from cell types and tissues
relevant to the area of in vivo placement of dental materials [79]. Recent studies on elution
of monomers available in both dental composites and methacrylate- and epoxy-based root
canal sealers looked at reactions in submandibular salivary gland acinar cells for the evalua-
tion of cytotoxicity, cell proliferation, and apoptosis [69, 80]. The ndings in such studies are
of great interest and importance, but the authors of one of these studies [80] stated that their
model would have been more realistic had they utilized human primary cells from direct
target tissue. Tissues that often share the closest proximity to dental llings are certainly not
salivary glands but rather gingiva, mucosa, and, in particular, pulp tissues [79].
The pulp is a loose connective tissue within a nonresilient capsule of dentin and enamel.
Pulpal inammation is considered a protective mechanism and can either be of an acute or
chronic nature. Acute and chronic responses are related to the “magnitude and duration of
the insult [81].” Inammation will inevitably cause vasodilation, increased vessel permeabil-
ity which in turn will result in relatively large changes in tissue pressure [82]. Bacterial infec-
tion is the most common reason for pulpal inammation, but any insult or stimuli will most
probably result in a response. It is an established fact that many of the constituents in dental
adhesive resin are cytotoxic [81], and the dierence in cytoxicity varies among commercial
materials commonly used by public dentists in Norway [unpublished results in a report to the
Norwegian National Directorate of Health].
This project aims at elucidating the cellular eects of “leachables” (residual monomers) from
dental lling materials exerted on dental pulp stem cells (DPSCs) ex vivo. It is important to
Vitamin K2 - Vital for Health and Wellbeing142
ensure that the cells used in the study are, indeed, stem cells. The International Society for
Cellular Therapy has released a position statement wherein they list three criteria to dene
human stem cells: (1) adherence to plastic, (2) specic surface antigen expression, and (3) mul-
tipotent dierentiation potential [83]. The cells to be used in this project full all three criteria
[84], and were isolated in accordance with a published procedure described by Sorrentino
et al. [85].
11. Characterization of the DPSCs
Dental specimens were obtained from extraction after signed informed consent, and the pulp
was exposed by cuing the tooth, while maintaining sterile conditions: The enamel of the
tooth crown is partially cut, following the sagial plane, applying a diamond bur. Thereafter,
the cut is completed using a piezoelectric ultrasound scalpel to avoid overheating of the tis-
sue. The pulp is then treated collagenase and dispase for 1 hour at 37°C, and then incubated
in a bioSpherix chamber under normoxic conditions [85].
Phenotyping of the DPSCs yielded a CD-prole very much like the one seen for bone mar-
row mesenchymal stem cells (BM-MSCs) with an approximately identical percentage of cells
expressing CD10 (CALLA), CD 13 (Aminopeptidase N), CD29 (β1-integrin), CD44 (H-CAM,
Pgp-1), CD49acd (VLA-1,3,4 = α1,3,4-integrin), CD54 (I-CAM-1), CDw90 (THY-1), CD105
(Endoglin, TGFβ-R), CD140b (PDGF-Rb), CD146 (M-CAM), CD147 (Neurothelin/basigin),
CD166 (Alcam, CD6-ligand), and also comparable amounts of GD2 (Neural ganglioside).
12. Tissue engineering using stem cells: can it be avoided?
It has been asserted that tissue engineering might be the future of endodontics [86]. It is stated
in the abstract that pulpal regeneration after tooth injury is not easily accomplished, since the
infected pulp is required for tooth extraction or root canal therapy. It is further asserted that
an ideal form of therapy might consist of regenerative approaches where diseased or necrotic
pulp tissues are removed and replaced with healthy pulp tissue to revitalize the aected
tooth. The authors list dierent techniques, ranging from stem cell therapy, the use of growth
factors, pulp implants, implant of 3D cell printed in hydrogels, injectable scaolds, bioactive
materials, the use of co-enzymes, and root canal revascularization. However, despite alleged
advantages of the subject approaches, they also suer major disadvantages like low cell sur-
vival, lack of de novo production of pulp, necrosis of reinfected pulp, and lack of vascularity,
and requirement for precise root canal ing.
By determining the cut point of toxicity (i.e., cell death/enhanced apoptosis and lack of proper
dierentiation induced by the leakage of monomers of endodontic lling materials), it is pos-
sible to develop new lling materials without an acute and long term detrimental eect on
DPSCs. Hence, the development of a test baery to check the monomers that may diuse into
Vitamin K2 and Its Impact on Tooth Epigenetics
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143
the root canal, for cytotoxicity and ability to aain proper and functional cell phenotypes (i.e.,
odontoblasts, neural laice, and endothelial cells constituting blood vessels) seems manda-
tory. The present project description aims to dene such a test baery using highly sophisti-
cated techniques like proteomics (including phosphoproteomics) and mass cytometry.
The advantage of using such techniques resides with the fact that extremely few cells may
be used in complex arrays of incubation conditions, while still yielding reliable results. The
technology described in the present project outline, may also enable the denition of a mini-
mal and sucient array of variables, which precisely describes a robust test baery to be
implemented as a gold standard to be adopted in the development of endodontal biomaterial
llings in the future.
Author details
Jan Oxholm Gordeladze1*, Maria A. Landin2, Gaute Floer Johnsen3, Håvard Jostein Haugen3
and Harald Osmundsen2
*Address all correspondence to: j.o.gordeladze@medisin.uio.no
1 Department of Molecular Medicine, Section for Biochemistry, Institute of Basic Medical
Science, University of Oslo, Oslo, Norway
2 Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo, Norway
3 Institute of Clinical Odontology, Faculty of Odontology, University of Oslo, Norway
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