**9. Major findings**

**8. Emulation of the interaction between genes and microRNA species known to be instrumental in the development of the osteoblastic/**

**Gene name Description of function:** *General* **and bone-related References**

*This protein plays a role in taste transduction. It is activated by lower concentrations* 

*Stem Cells. 2013 Jan;31(1):167–77. doi: 10.1002/stem.1264.*

*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.*

*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.*

*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* 

*the Ca2+ signaling pattern and gene expression during stem cell differentiation*.

*proteins. Amelogenins are involved in biomineralization during tooth enamel* 

*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* 

*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* 

*It appeared that the mDE6 mouse epithelial cell line expressed Runx2, Amelx, Ambn and Enam, and yielded calcified matrices upon the induction of calcification*.

*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* 

**Trpm5** *This gene encodes a member of the transient receptor potential (TRP) protein family.* 

*of intracellular Ca2+*, *and inhibited by higher concentrations*.

*be required for adipogenesis. The data provide a link between*

**Amelx** *This gene encodes a member of the amelogenin family of extracellular matrix* 

*signaling pathways in mouse dental epithelial cells.*

**DKK1** *The present gene encodes a member of the dickkopf protein family. It is secreted,* 

*(Ambn), as well as enamelin (Enam)*.

*development*.

138 Vitamin K2 - Vital for Health and Wellbeing

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 significantly 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:

**odontoblastic phenotype by Mir@nt@n**

*sclerostin, Dkk 1, and Sfrp1*.

The genes, significantly 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 necessary and sufficient 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 "stabilized" by the RUNX- and SP-families of transcription factors (upregulated), as well as the


**Table 1.** "Dental" genes affected directly or indirectly by exposure to vitamin K2 (MK-7).

**Figure 1.** Interactions between transcription factors, "functional" genes, and microRNA species as emulated in the bioinformatics program Mir@nt@n.

microRNA-species 149, 328, and 339 (downregulated). Recently, it was shown [58] that mir-760 is involved in the effect 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 find 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., replenishing "lost" osteoblasts from precursor cell, as well as proper functioning of differentiated 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 beneficial effects of vitamin K2 on osteoblasts/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 3.** Extended interaction scheme (low stringency emulation) of transcription factors, microRNA species, and

**Figure 2.** Interactions (high/maximal stringency emulation) of transcription factors, microRNA species, and differentiation /

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differentiation-related genes in tooth germs from the rat.

function-related genes in tooth germs from the rat.

**Figure 2.** Interactions (high/maximal stringency emulation) of transcription factors, microRNA species, and differentiation / function-related genes in tooth germs from the rat.

microRNA-species 149, 328, and 339 (downregulated). Recently, it was shown [58] that mir-760 is involved in the effect of vitamin K2, since it associates with the transcription factor

**Figure 1.** Interactions between transcription factors, "functional" genes, and microRNA species as emulated in the

Using high/maximal stringency emulations rendered by the bioinformatics program Mir@ nt@n [64], it was quite interesting to find 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., replenishing "lost" osteoblasts from precursor cell, as well as proper functioning of differentiated 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

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 beneficial effects of vitamin K2 on osteoblasts/odontoblasts. Furthermore, the dose should be titrated to ensure proper levels and characteristics and amounts of bodily beige versus white adipocytes (confer the postulated

NR1/2 = SXR = PXR [65].

bioinformatics program Mir@nt@n.

140 Vitamin K2 - Vital for Health and Wellbeing

process.

**Figure 3.** Extended interaction scheme (low stringency emulation) of transcription factors, microRNA species, and differentiation-related genes in tooth germs from the rat.

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.

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 define human stem cells: (1) adherence to plastic, (2) specific surface antigen expression, and (3) multipotent differentiation potential [83]. The cells to be used in this project fulfil all three criteria [84], and were isolated in accordance with a published procedure described by Sorrentino

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Dental specimens were obtained from extraction after signed informed consent, and the pulp was exposed by cutting the tooth, while maintaining sterile conditions: The enamel of the tooth crown is partially cut, following the sagittal plane, applying a diamond bur. Thereafter, the cut is completed using a piezoelectric ultrasound scalpel to avoid overheating of the tissue. The pulp is then treated collagenase and dispase for 1 hour at 37°C, and then incubated

Phenotyping of the DPSCs yielded a CD-profile very much like the one seen for bone marrow 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).

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 affected tooth. The authors list different techniques, ranging from stem cell therapy, the use of growth factors, pulp implants, implant of 3D cell printed in hydrogels, injectable scaffolds, bioactive materials, the use of co-enzymes, and root canal revascularization. However, despite alleged advantages of the subject approaches, they also suffer major disadvantages like low cell survival, lack of de novo production of pulp, necrosis of reinfected pulp, and lack of vascularity,

By determining the cut point of toxicity (i.e., cell death/enhanced apoptosis and lack of proper differentiation induced by the leakage of monomers of endodontic filling materials), it is possible to develop new filling materials without an acute and long term detrimental effect on DPSCs. Hence, the development of a test battery to check the monomers that may diffuse into

et al. [85].

**11. Characterization of the DPSCs**

in a bioSpherix chamber under normoxic conditions [85].

and requirement for precise root canal fitting.

**12. Tissue engineering using stem cells: can it be avoided?**

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 latter, 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].
