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

#### **5.1. Methods**

roles in bone development. Here, emerging areas of Wnt signaling in osteoclastogenesis are discussed, as well as the progress made in translating basic studies to clinical therapeutics and diagnostics centered around inhibiting Wnt pathway antagonists. These are sclerostin, Dkk1, and Sfrp1. In a recent study, (unpublished data) Osmundsen and coworkers have shown that vitamin K2 affects the Wnt system by modulating the expression of DKK1 (a Wnt inhibitor)

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

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

nism of action remains unclear. Unraveling its modes of action will provide a broader understanding of the mechanisms associated with the induced dentinogenesis, as well as helping to optimize currently available treatment modalities to ensure specific regenerative processes of tooth preservation. A compilation of articles on "mechanisms of dentinogenesis involving calcium hydroxide" is featured in this paper, and recommendations related to dentinogenic

release of biologically active molecules, like fibronectin, BMPs (bone morphogenic proteins) like BMP-4 and BMP-7, TGFs (transforming growth factors) like TGFβ, IGFs (insulin-like growth factors), antiinflammatory interleukins, alkaline phosphatase (ALP), and others [60]. It is well known that a plethora of these factors are encoded by genes that are sensitive to the impact of vitamin K2 (via the transcription factor SXR = PXR = NR1/2), as well as the vitamin

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

has been extensively used in the dentistry; however, its mecha-

range from direct irritating action by the material to induction of

to a feasible way of promoting/sustaining Osx expression experimentally [59].

during the development of teeth in developing molar teeth of the mouse.

maintains structural stability, however [56].

132 Vitamin K2 - Vital for Health and Wellbeing

Calcium hydroxide Ca(OH)2

mechanisms of Ca(OH)2

A (RXR) and vitamin D (VDR) receptors [61].

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 first 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 expression using deoxyoligonucleotides microarrays and real-time RT-PCR using the RNeasy Mini Kit. The quality of isolated RNA was monitored using the Agilent Bioanalyzer. RNA was isolated from three separate batches of tooth germs (three tooth germs per batch).
