PIEZO1 promotes ATP release from periodontal ligament cells following compression force.

OBJECTIVES: Orthodontic mechanical force on the periodontal ligament induces extracellular adenosine triphosphate (ATP) release. However, mechanosensitive molecules have not been confirmed functionally in periodontal ligament cells. In the present study, we examined the roles of mechanosensitive PIEZO channels in the mechanically stimulated release of ATP in human periodontal ligament fibroblasts (HPdLFs). MATERIALS AND METHODS: To examine PIEZO expression in HPdLFs, we performed reverse transcription-quantitative polymerase chain reaction, fluorescent immunostaining, and Ca2+ imaging. ATP concentrations were measured in culture medium after applications of the PIEZO1 agonist Yoda1 and compression force in a newly developed in vitro weight-loaded cell model (IVWLC) using balance weights and a 48-well plate. The mechanosensitive channel inhibitor GsMTx4 and the ATP-releasing route inhibitors clodronic acid, meclofenamic acid, and probenecid were used. To suppress PIEZO1 expression, short interference RNA (siRNA) treatment of the PIEZO1 gene was performed. RESULTS: PIEZO1 mRNA was expressed more abundantly than PIEZO2 mRNA in HPdLFs. HPdLF cell bodies were immunoreactive to anti-PIEZO1 antibody. Yoda1 increased intracellular Ca2+ and extracellular ATP concentrations in a dose-dependent manner. ATP release was inhibited by GsMTx4 and inhibitors of ATP release routes. In the IVWLC, HPdLFs released ATP in response to compression force but not in response to hypoxic stimulation that was simultaneously applied to cells. Mechanically stimulated ATP release was inhibited by GsMTx4, inhibitors of ATP-releasing routes and siRNA treatment of PIEZO1. CONCLUSIONS: PIEZO1 on the cell membranes of HPdLFs is activated by compression force and then induces ATP release via intracellular Ca2+-dependent exocytosis and ATP-permeable channels.

Discrimination of cellulose microparticles in rats.

Oral perception of food particles is important in mastication and swallowing. However, the mechanism underlying particle perception remains poorly understood because of the lack of suitable experimental systems. We evaluated microparticle perception in rats utilizing insoluble cellulose particles of varying diameters (20-170 µm). The cellulose additives have polycrystalline morphologies and contain smaller crushed particles. The filtrate containing 20 µm particles at a concentration of 1.6% was passed through 3 µm pore-size filter paper, and numerous small particles equivalent to a 0.25 mM soluble solution were observed. In two-bottle preference tests, rats showed no innate preference or avoidance of particles of any size at concentrations ranging from 0.05-1.6%. Next, conditioned preference learning tests employing 8% glucose and fructose solutions were performed. After being repeatedly presented with glucose and fructose solutions containing particles of different sizes (170 and 20 µm particles or 20 µm filtrate) at a concentration of 1.6%, the rats preferred particles in glucose solution even without glucose presentation. Intriguingly, rats preferred the filtrate following repeated presentations of glucose-containing filtrate and water containing fructose. These results suggest that rats can distinguish microparticles in water. The preference learning test is useful for analyzing particle perception mechanisms in mammals.

Correlations of sensations of hardness and springiness of agar and gelatin gels with mechanical properties in human participants.

OBJECTIVES: It is unclear which mechanical properties of foods cause the texture sensation in humans. This study aimed to investigate the relationship between unilateral compression measurements and the sensations of hardness and springiness in gels. METHODS: Three different concentrations of agar and gelatin gels were prepared by the addition of agar (1%, 2%, and 3%) and gelatin (4%, 8%, and 16%) to water or apple juice. In a stress-rupture test, stress-strain curves were obtained by the application of uniaxial compression with a disc plunger at a compression rate of 10 mm/s. The hardness, springiness, and palatability of the gels were evaluated by 12 healthy volunteers using a visual analog scale. RESULTS: The sensation of hardness was positively correlated with the sensation of springiness for the agar and gelatin gels. Palatability decreased as hardness increased for both gels. In terms of mechanical properties, the sensation of hardness was only significantly correlated with the initial elastic modulus, while the sensation of springiness was correlated with the late elastic modulus and other mechanical properties such as fracture strain, time, and stress. CONCLUSIONS: These results suggest that sensations of hardness and springiness are produced in the initial and late stages, respectively, during the food-crushing process using the tongue, palate, and teeth.

Effect of cisplatin on oral ulcer-induced nociception in rats.

OBJECTIVE: The aim of this study is to investigate effects of cisplatin preadministration on oral ulcerative mucositis-induced nociception by using an experimental model of rats. DESIGN: After two rounds of cisplatin administration, oral ulcers developed with topical acetic acid treatment in rats. Spontaneous mouth rubbing behavior was observed as spontaneous nociceptive behavior in a plastic cage. Head-withdrawal behavior was observed as mechanical allodynia by using von Frey test in the oral mucosa of conscious rats. Bacterial invasion and inflammatory cell infiltration into oral ulcerative region and systemic leukocyte phagocytic activity were assessed. RESULTS: Following cisplatin preadministration, oral ulcerative mucositis-induced spontaneous nociceptive behavior was not observed in the model. The preadministration enhanced leukocyte phagocytic activity, leading to reduce bacterial invasion and inflammatory cell infiltration in the oral ulcerative region. In contrast, oral ulcerative mucositis-induced mechanical allodynia was induced. The exaggerated mechanical allodynia in the oral ulcerative region was largely inhibited by topical treatment with the antioxidative drug, ɑ-lipoic acid, or the blocker of N-formyl peptide receptor 1, N-t-butoxycarbonyl-methionyl-leucyl-phenylalanine. CONCLUSIONS: These results suggest that cisplatin preadministration suppresses spontaneous nociception in oral ulcerative region, due to antiinflammatory effects by enhancement of leukocyte phagocytic activity, but exaggerates mechanical allodynia due to oxidative stress with N-formyl peptide receptor 1 activation. The suppression of spontaneous nociception is one of the advantages of cisplatin treatment for head and neck cancer patients although the exaggerated allodynia is a serious symptom.

Deletion of epithelial cell-specific p130Cas impairs the maturation stage of amelogenesis.

Amelogenesis consists of secretory, transition, maturation, and post-maturation stages, and the morphological changes of ameloblasts at each stage are closely related to their function. p130 Crk-associated substrate (Cas) is a scaffold protein that modulates essential cellular processes, including cell adhesion, cytoskeletal changes, and polarization. The expression of p130Cas was observed from the secretory stage to the maturation stage in ameloblasts. Epithelial cell-specific p130Cas-deficient (p130CasΔepi-) mice exhibited enamel hypomineralization with chalk-like white mandibular incisors in young mice and attrition in aged mouse molars. A micro-computed tomography analysis and Vickers micro-hardness testing showed thinner enamel, lower enamel mineral density and hardness in p130CasΔepi- mice in comparison to p130Casflox/flox mice. Scanning electron microscopy, and an energy dispersive X-ray spectroscopy analysis indicated the disturbance of the enamel rod structure and lower Ca and P contents in p130CasΔepi- mice, respectively. The disorganized arrangement of ameloblasts, especially in the maturation stage, was observed in p130CasΔepi- mice. Furthermore, expression levels of enamel matrix proteins, such as amelogenin and ameloblastin in the secretory stage, and functional markers, such as alkaline phosphatase and iron accumulation, and Na+/Ca2++K+-exchanger in the maturation stage were reduced in p130CasΔepi- mice. These findings suggest that p130Cas plays important roles in amelogenesis (197 words).

The effect of flavor on the oral perception and palatability of viscosity in healthy human subjects.

OBJECTIVES: Thickeners are frequently used in various foods, including ice cream and sauces, to impart viscosity. Generally, viscous foods have some flavor (smell and taste). In this study, we examined the effects of flavor on the oral perception and palatability of viscosity in humans. METHODS: Viscous fluids were prepared by adding the commercial thickener Tsururinko® (0.5 and 3.0%) to water and apple juice, which were used as the control and flavor fluids, respectively. The viscosity and palatability perception of the test fluids were evaluated in nine healthy volunteers using a visual analog scale. In the other seven volunteers, fluid viscosities were measured before and after spitting following retention in the mouth for 5 s to investigate the dilution of viscous fluids by flavor-stimulated saliva. RESULTS: With 1.5% Tsururinko®, there was no difference between the physical viscosity of water and apple juice, but the perceived viscosity of apple juice was significantly lower than that of water. With 3.0% Tsururinko®, the viscosity of apple juice was significantly higher than that of water, but the perceived viscosities did not differ significantly. The addition of Tsururinko® reduced palatability in water in a dose-dependent manner. Apple juice suppressed this Tsururinko®-induced reduction. The reduction in viscosity after spitting was significantly larger in apple juice than in water. CONCLUSION: Our results suggest that a favorable flavor reduces the perception of oral viscosity, which is due to mixing with stimulated saliva, and suppresses the unpalatability of thickeners.

The enamel knot-like structure is eternally maintained in the apical bud of postnatal mouse incisors.

OBJECTIVES: The boundary where inner and outer enamel epithelia meet is referred to as the cervical loop (CL) in molar tooth germs. In contrast, rodent incisors are continuously growing: the labial side of the teeth is covered with enamel (crown-analog), and the lingual side is covered with cementum (root-analog). These results in the appearance of CL in the frontal sections apart from the apical end. However, many researchers have used the term "labial CL" to indicate the apical end of incisors. DESIGN: This study investigated the gene expression patterns for the enamel knot signaling center in tooth morphogenesis to clarify the difference between "labial CL" and molar CL. We examined the three-dimensional expression patterns of markers for the enamel knot including fibroblast growth factor 4 (Fgf4), sonic hedgehog (Shh), Msx2, and P21 in frontal sections of murine mandibular incisors. RESULTS: Serial frontal sections from the apical end of the postnatal incisor clearly demonstrated the existence of enamel knot-like structures composed of supra-inner enamel epithelium and stellate reticulum in the "labial CL". This structure includes the expression of all examined markers for enamel knots such as Fgf4, Shh, Msx2, and P21. CONCLUSIONS: The molar tooth germ-like structure is maintained indefinitely in the "labial CL". Therefore, the "labial CL" is not equivalent to the molar CL. The existence of an EK-like structure in the apical end of incisors implies that the usage of "labial CL" may be inappropriate for indicating this region. The "apical bud" could be used as an alternative term.

The distinct distributions of immunocompetent cells in rat dentin pulp after pulpotomy.

Pulpotomy involves the removal of the coronal portion of pulp, including the diseased tissue, with the intent of maintaining the vitality of the remaining pulpal tissue via a therapeutic dressing. Once odontoblasts suffer injuries, the differentiation of mesenchymal cells is induced from the precursor cell population in the dental pulp, and these cells are recruited to the injured site to differentiate into odontoblasts. However, the involvement of immunocompetent cells during pulpal regeneration remains unclear. Thus, the purpose of this study was to investigate the properties of macrophages that infiltrated wound healing sites in rats between 1 and 28 days after pulpotomy (dap). During the inflammatory phase, ED1(+) (CD68(+) ) macrophages significantly increased throughout root pulp, especially apical to the demarcation zone, and this population persisted until 3 dap before decreasing gradually until 28 dap. OX6(+) macrophages expressing class II MHC also increased in the apical pulp at 1 dap and declined thereafter. However, OX6(+) cells appeared prior to dentin bridge formation at 3 dap and appeared again apical to the dentin bridge during the healing stage at 14 dap. The shift from ED1(+) cells in the inflammation phase to OX6(+) cells during dentin bridge formation might contribute to wound healing.

The novel IκB kinase ß inhibitor IMD-0560 prevents bone invasion by oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) cells display significantly augmented nuclear factor-κB (NF-κB) activity, and inhibiting this activity suppresses malignant tumor characteristics. Thus, we evaluated the effect of IMD-0560, a novel inhibitor of IκB kinase (IKK) ß that is under assessment in a clinical trial of rheumatoid arthritis, on bone invasion by the mouse OSCC cell line SCCVII. We examined the inhibitory effects of IMD-0560 on NF-κB activity and tumor invasion using human OSCC cell lines and SCCVII cells in vitro. Using a mouse model of jaw bone invasion by SCCVII cells, we assessed the inhibitory effect of IMD-0560 on jaw bone invasion, tumor growth, and matrix degradation in vivo. IMD-0560 suppressed the nuclear translocation of NF-κB and the degradation of IκBα in OSCC cells. IMD-0560 also inhibited invasion by suppressing matrix metalloproteinase-9 (MMP-9) production in OSCC cells. IMD-0560 protected against zygoma and mandible destruction by SCCVII cells, reduced the number of osteoclasts by inhibiting receptor activator of NF-κB ligand (RANKL) expression in osteoblastic cells and SCCVII cells, increased SCCVII cell death and suppressed cell proliferation and MMP-9 production in SCCVII cells. Based on these results, IMD-0560 may represent a new therapeutic agent for bone invasion by OSCC cells.