This review aims to propose a more consistent interpretation of the concept of matrix vesicle mineralization, and includes current views on biomineralization in general. We first focused on the ultrastructural, cytochemical and biophysical characteristics of matrix vesicles which confirm the assertion that matrix vesicles are the initial sites of bone biomineralization. We offer a scheme describing how cells regulate biomineralization by means of matrix vesicles during bone formation and remodeling and then discuss the subsequential biomineralization, the growth of hydroxyapatite crystals by direct extension, as well as secondary nucleation from previously formed seams of bone and collagen mineralization.

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Mechanical allodynia is defined as pain caused by stimuli that do not normally evoke pain and that are mediated by the activation of low-threshold mechanoreceptive Aβ-fibers. Although mechanical allodynia is a common symptom of the neuropathic and inflammatory pain, the precise mechanisms underlying a mechanical allodynia remained to be determined. A suspected possible interaction between noxious and non-noxious sensory signal transmission pathways may play a key role in the induction of mechanical allodynia. We recently investigated possible mechanisms underlying a mechanical allodynia/neuronal changes in sensitivity at sites remote from temporomandibular joint (TMJ) inflammation by using behavioral, electrophysiological, immunohistochemical and molecular approaches. Both in-vitro and in-vivo studies revealed that under TMJ inflammation, substance P (SP) released from the Aδ-/C-trigeminal ganglion (TRG) neuronal soma (nociceptive) innervating inflamed TMJ via the paracrine mechanism play an important role in the modification of the excitability of Aβ-TRG neurons (non-nociceptive) innervating intact facial skin with up-regulated neurokinin 1 (NK1) receptors. The enhanced excitability of TMJ nociceptive TRG neurons was mainly due to the suppressing A-type voltage-gated potassium currents via a hyperpolarizing shift in the inactivation curve. These results suggest that the paracrine mechanism in the trigeminal ganglia may explain the development of the acute inflammatory mechanical allodynia without sprouting non-nociceptive fibers in the medullary dorsal horn. In other words, either NK1 receptor antagonists or A-type potassium channel openers may be therapeutic agents for the prevention of trigeminal inflammatory mechanical allodynia.

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The revolution in genetics disclosed the types of malformations that occur when expression of a particular gene is lost. In the case of tooth dentin, mutations in the two genes encoding typeIcollagen cause osteogenesis imperfecta, a bone condition that often includes dentin malformations. Besides collagen, there are a number of non-collagenous proteins in dentin. Among the genes encoding the dentin non-collagenous proteins, only mutations in DSPP (dentin sialophosphoprotein) cause inherited dental malformations. DSPP mutations cause dentinogenesis imperfecta typesII and III, and dentin dysplasia typeII. DSPP is the most abundant non-collagenous protein in dentin. DSPP protein is necessary for proper dentin formation, and understanding its structure and function should yield important insights into how dentin forms and biomineralization is controlled. DSPP is expressed and secreted by odontoblasts, the cells that make tooth dentin and that also maintain cell processes extending into the mineralized tissue. Following its secretion, DSPP is cleaved into smaller pieces by multiple extracellular proteases. For the last five years I have devoted myself to characterizing DSPP-derived proteins. DSPP is cleaved by proteases into three main parts: dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP). We have learned that DSP is a proteoglycan that forms covalent dimers, DGP is a phosphorylated glycoprotein, and DPP is a highly phosphorylated intrinsically disordered protein that shows extensive length polymorphisms due to the genetic heterogeneity of its coding region.

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In order to clarify the involvement of primary afferent neurons in neuropathic pain in the trigeminal region, a model involving chronic constriction nerve injury of the infraorbital nerve (ION-CCI) was developed in rats. Mechano-allodynia was observed 3 days after ION-CCI and lasted more than 14 days. At 3, 5, 7, and 14 days after ION-CCI, single unit activities were recorded from the ION. Aβ-, Aδ-, and C-units were identified on the basis of their conduction velocity.
Aδ-units were frequently encountered at the late period after ION-CCI. The highest spontaneous activity was recorded 3 days after ION-CCI during the observation period, and it progressively decreased after that. Mechanically evoked responses of Aδ-units were also highest 3 days after ION-CCI, and gradually decreased thereafter. Patch-clamp recording was performed on middle-to-large sized trigeminal ganglion (TRG) neurons. Patch-clamp recording analysis revealed that I_k (sustained) and I_A (transient) currents in rats with ION-CCI were significantly smaller than those in naïve rats. The hyperpolarization-activated current (I_h) was significantly larger in TRG neurons of rats with ION-CCI compared to that of naïve rats.
These results suggest that I_h, I_k and I_A currents in Aδ-TRG neurons are involved in mechano-allodynia in the trigeminal region following peripheral nerve injury.

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AMP-activated protein kinase (AMPK) acts as a fuel gauge during musclar exercise and stress-induced energy crisis. The function of the masseter muscle changes during postnatal development, along with an alteration in the type of muscle fibers. We investigated expressions of AMPK subunit isoforms in the masseter muscle by quantitative real-time RT-PCR and Western blotting, and compared them with those in the tibialis anterior muscle in 2-, 4-, and 9-week-old mice. The most abundantly expressed isoform of the catalytic subunit was α2, and those of the regulatory subunits were β1, β2, and γ1 in both types of muscle; mRNA expression of all the main isoforms in the masseter muscle was higher than that in the tibialis anterior muscle. Expression of α protein increased with development in both muscle types. Messenger RNA expression of regulatory subunit isoforms β1 and γ1 in the masseter muscle rose, together with an increase in the amounts of their corresponding proteins. On the other hand, mRNA expression of these isoforms in the tibialis anterior muscle decreased with development, while their corresponding proteins increased at a similar rate to that of the development of the α subunit. This altered expression of AMPK subunit isoforms in each muscle during postnatal development may be related to the changing function of each type of muscle.

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Apert syndrome (AS) is an autosomal dominant disorder characterized by craniosynostosis and bony syndactyly from a gain-of-function mutation of the fibroblast growth factor receptor 2 gene (FGFR2) (Ser252Trp and Pro253Arg mutations). However, the effects of FGFR2 mutation on oral conditions are not fully understood. To elucidate the effects of AS-type FGFR2 mutation on periodontal disease, we examined the production of matrix metalloproteinases (MMPs) and inflammatory cytokines. Gingival keratinocytes were obtained from a normal subject and an AS patient with Ser252Trp mutation. Cells were treated with lipopolysaccharide (LPS), and the production of MMPs and inflammatory cytokines was examined. No difference in MMP-2 production and MMP-2 activation was observed between normal and AS keratinocytes treated with LPS. LPS-treated normal keratinocytes produced interleukin (IL)-6 and IL-8 in a dose-dependent manner. However, LPS-treated AS keratinocytes produced only very low levels. LPS-induced cytokine production was not significantly decreased in normal keratinocytes when cells were treated with LPS and FGF-7. A mitogen-activated protein kinase kinase (MAPKK/MEK) inhibitor, PD98059, had no effect on LPS-induced cytokine production in AS keratinocytes. These results suggest that AS-type FGFR2 mutation causes the hypoproduction of inflammatory cytokines independent of the MEK/ERK pathway, and that AS-type FGFR2 mutation is an inhibitory factor for periodontal disease.

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Enterococcus faecium, a lactic acid bacterium, is a normal bowel commensal inhabitant that is rarely found in the oral cavity. We investigated whether E. faecium functions as a probiotic strain inhibiting biofilm formation by Streptococcus mutans, which is an etiological agent for dental caries, using a flow cell system. Cell suspensions were cultured in flow cell systems coated with salivary components in tryptic soy broth including 0.25% sucrose without dextrose. The resultant biofilm formation was stained using a LIVE/DEAD^®BacLight^TM Viability Kit, and examined using confocal laser scanning microscopy. E. faecium showed cell density-dependent inhibition of biofilm formation in dual species culture with S. mutans in flow cell cultures at ratios of 100: 1 and 10: 1. Biofilm formation with increased numbers of voids and hollows was observed at the base of the culture using a confocal microscope. In contrast, increasing the ratio of L. casei or L. salivarius, other lactic acid bacteria, to S. mutans did not affect biofilm formation. In addition, a sonic extract sample of E. faecium was sub-purified by salting out and gel filtration, and its inhibitory effects on S. mutans biofilm were similarly observed in the same assays. Together, our results suggest that E. faecium possesses an inhibitory substance and functions as a probiotic bacterial inhibitor of streptococcal biofilm formation. Further, more they provide important information regarding bacterial communication and diversity, as well as for potential therapies and materials for the prevention of biofilm development in the oral cavity.

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The purpose of this study was to elucidate changes in serum biochemical markers related to bone turnover after orthognathic surgery in skeletal Class III patients. The subjects consisted of 16 healthy, adult men aged 22.1 to 40.3 years, one week before surgery. Samples of venous blood (5-6 mL) were taken at 10: 00 a. m.-12: 00 a. m.. The timing of each sample collection was: before-surg, one week before surgery; 3-d, three days after surgery; 10-d, ten days after surgery; 1-m, one month after surgery; 3-m, 3 months after surgery; and 6-m, 6 months after surgery. Serum osteocalcin, bone alkaline phosphatase (BAP), and tartrate-resistant acid phosphatase (TRAP) were measured at each stage. Additionally, serum C-reactive protein (CRP) and other serum components were measured as general examinations of bone metabolism and for health checks. Results: 1. Among all values of serum components during the observation period, only CRP at 3-d was higher than the standard value for Japanese adults. 2. Serum osteocalcin (p<0.001) and TRAP (p<0.01) showed a significant decrease at 3-d versus before-surg, and recovered after 10-d. Serum BAP showed significant decreases at 3-d (p<0.001) and 10-d (p<0.01) versus before-surg and recovered after 1-m. 3. Serum CRP increased significantly at 3-d (p<0.001) versus before-surg beyond the normal range, but returned to normal levels after 10-d. Conclusion: Each early fall at 3-d in serum osteocalcin, BAP, and TRAP seemed to reflect the suppression of bone metabolism due to the stress of orthognathic surgery.

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