Altered Rheological Properties of Saliva with Aging in Mouse Sublingual Gland.

Mucin in saliva plays a critical role in the hydration and lubrication of the oral mucosa by retaining water molecules, and its impaired function may be associated with hyposalivation-independent xerostomia. Age-dependent effects on salivary gland function and rheological properties of secreted saliva are not fully understood as aging is a complex and multifactorial process. We aimed to evaluate age-related changes in the rheological properties of saliva and elucidate the underlying mechanism. We performed ex vivo submandibular gland (SMG) and sublingual gland (SLG) perfusion experiments to collect saliva from isolated glands of young (12 wk old) and aged (27 mo old) female C57BL/6J mice and investigate the rheological properties by determining the spinnbarkeit (viscoelasticity). While fluid secretion was comparable in SMG and SLG of both mice, spinnbarkeit showed a significant decrease in SLG saliva of aged mice than that of young mice. There were no significant differences in GalNAc concentration between young and aged SLG saliva. Liquid chromatography/tandem mass spectrometry analysis of SLG saliva revealed that (Hex)1 (HexNAc)1 (NeuAc)1 at m/z 793.31 was the most abundant O-glycan structure in SLG saliva commonly detected in both mice. Lectin staining of salivary gland tissue showed that SLG stained strongly with Maackia amurensis lectin II (MAL II) while Sambucus nigra agglutinin (SNA) stained little, if any, SLG. The messenger RNA expression of St3gal1 that encodes an α-2,3 sialic acid sialyltransferase SIAT4-A showed a decrease in SLG of aged mice, confirmed by a Western blot analysis. Lectin blot analysis in SLG saliva revealed that the relative signal intensity detected by MAL II was significantly lower in aged SLG. Our results suggest that spinnbarkeit decreases in SLG of aging mice due to downregulation of sialic acid linked to α-2,3 sialic acid sialyltransferase expression.

Printable PICN Composite Mechanically Compatible with Human Teeth.

Polymer-infiltrated ceramic network (PICN) composites are mechanically compatible with human enamel, and are therefore promising dental restorative materials. Fabrication technology for PICN composites used in tooth restorative material has been established through computer-aided design/computer-aided manufacturing (CAD/CAM) milling, however, to date, has not been successfully developed using 3-dimensional (3D) printing. This study aimed to develop a 3D-printable PICN composite as a restorative material. The PICN composite was fabricated using a specific method based on 3D printing. A 3D-printable precursor slurry containing a high concentration of silica nanoparticles was produced and 3D-printed using stereolithography (SLA). The 3D-printed object was sintered to obtain a nano-porous object, and subsequently infiltrated and polymerized with resin monomer. Three different fabrication condition combinations were used to produce the 3D-printed PICN composites, which were characterized based on microstructure, mechanical properties, inorganic content, physicochemical properties, and overall shrinkage. The 3D-printed PICN composites were also compared to 2 commercially available CAD/CAM composite blocks, namely a PICN composite and a dispersed-filler composite. The 3D-printed PICN composites exhibited a nano-sized dual-network structure comprising a silica skeleton with infiltrated resin. The 3D-printed PICN composite exhibited a similar Vickers hardness to enamel, and a similar elastic modulus to dentin. The 3D-printed PICN composite exhibited comparable flexural strength (>100 MPa) to the CAD/CAM block, and acceptable water sorption and solubility for practical use. Further, the 3D-printed model-crown underwent isotropic shrinkage during sintering without fatal deformation. Overall, the potential of this 3D-printable PICN composite as a restorative material with similar mechanical properties to human teeth was successfully demonstrated.

Defective NaCl Reabsorption in Salivary Glands of Eda-Null X-LHED Mice.

Mutations in the ectodysplasin A gene ( EDA) cause X-LHED (X-linked hypohidrotic ectodermal dysplasia), the most common human form of ectodermal dysplasia. Defective EDA signaling is linked to hypoplastic development of epithelial tissues, resulting in hypotrichosis, hypodontia, hypohidrosis, and xerostomia. The primary objective of the present study was to better understand the salivary gland dysfunction associated with ectodermal dysplasia using the analogous murine disorder. The salivary flow rate and ion composition of the 3 major salivary glands were determined in adult Eda-deficient Tabby hemizygous male (Ta/Y) and heterozygous female (Ta/X) mice. Submandibular and sublingual glands of Eda-mutant mice were smaller than wild-type littermates, while parotid gland weight was not significantly altered. Fluid secretion by the 3 major salivary glands was essentially unchanged, but the decrease in submandibular gland size was associated with a dramatic loss of ducts in Ta/Y and Ta/X mice. Reabsorption of Na+ and Cl-, previously linked in salivary glands to Scnn1 Na+ channels and Cftr Cl- channels, respectively, was markedly reduced at high flow rates in the ex vivo submandibular glands of Ta/Y mice (~60%) and, to a lesser extent, Ta/X mice (Na+ by 14%). Consistent with decreased Na+ reabsorption in Ta/Y mice, quantitative polymerase chain reaction analysis detected decreased mRNA expression for Scnn1b and Scnn1g, genes encoding the ß and γ subunits, respectively. Moreover, the Na+ channel blocker amiloride significantly inhibited Na+ and Cl- reabsorption by wild-type male submandibular glands to levels comparable to those observed in Ta/Y mice. In summary, fluid secretion was intact in the salivary glands of Eda-deficient mice but displayed marked Na+ and Cl- reabsorption defects that correlated with the loss of duct cells and decreased Scnn1 Na+ channel expression. These results provide a likely mechanism for the elevated NaCl concentration observed in the saliva of affected male and female patients with X-LHED.

Endothelin-1 Elicits TRP-Mediated Pain in an Acid-Induced Oral Ulcer Model.

Oral ulcer is the most common oral disease and leads to pain during meals and speaking, reducing the quality of life of patients. Recent evidence using animal models suggests that oral ulcers induce cyclooxygenase-dependent spontaneous pain and cyclooxygenase-independent mechanical allodynia. Endothelin-1 is upregulated in oral mucosal inflammation, although it has not been shown to induce pain in oral ulcers. In the present study, we investigated the involvement of endothelin-1 signaling with oral ulcer-induced pain using our proprietary assay system in conscious rats. Endothelin-1 was significantly upregulated in oral ulcers experimentally induced by topical acetic acid treatment, while endothelin-1 production was suppressed by antibacterial pretreatment. Spontaneous nociceptive behavior in oral ulcer model rats was inhibited by swab applications of BQ-788 (ETB receptor antagonist), ONO-8711 (prostanoid receptor EP1 antagonist), and HC-030031 (TRPA1 antagonist). Prostaglandin E2 production in the ulcers was suppressed by BQ-788. Mechanical allodynia in the model was inhibited not only by BQ-788 and HC-030031 but also by BQ-123 (ETA receptor antagonist), SB-366791 (TRPV1 antagonist), and RN-1734 (TRPV4 antagonist). In naive rats, submucosal injection of endothelin-1 caused mechanical allodynia that was sensitive to HC-030031 and SB-366791 but not to RN-1734. These results suggest that endothelin-1 production following oral bacterial invasion via ulcerative regions elicits TRPA1-mediated spontaneous pain. This pain likely occurs through an indirect route that involves ETB receptor-accelerated prostanoid production. Endothelin-1 elicits directly TRPA1- and TRPV1-mediated mechanical allodynia via both ETA and ETB receptors on nociceptive fibers. The TRPV4-mediated allodynia component seems to be independent of endothelin signaling. These findings highlight the potential of endothelin signaling blockers as effective analgesic approaches for oral ulcer patients.

Bone Aging by Advanced Glycation End Products: A Multiscale Mechanical Analysis.

The quality and quantity of mandibular bone are essential prerequisites for osseointegrated implants. Only the Hounsfield unit on preoperative computed tomography is currently used as a clinical index. Nevertheless, a considerable mismatch occurs between bone quality and the Hounsfield unit. Loss of bone toughness during aging has been accepted based on empirical evidence, but this concept is unlikely evidence based at the level of mechanical properties. Nonenzymatic bone matrix cross-links associated with advanced glycation end products predominate as a consequence of aging. Thus, loss of tissue integrity could diminish the bone toughening mechanism. Here, we demonstrate an impaired bone toughening mechanism caused by mimicking aging in rabbits on a methionine-rich diet, which enabled an enhanced nonenzymatically cross-linked bone matrix. A 3-point bending test revealed a greater reduction in femoral fracture resistance in rabbits on a methionine-rich diet, despite higher maximum and normalized breaking forces (287.3 N and 88.1%, respectively), than in rabbits on a normal diet (262.2 N and 79.7%, respectively). In situ nanoindentation on mandibular cortical bone obtained from rabbits on a methionine-rich diet did not enable strain rate-dependent stiffening and consequent large-dimensional recovery during rapid loading following constant displacement after a rapid-load indentation test as compared with those in rabbits on a normal diet. Such nanoscale structure-function relationships dictate resistance to cracking propagation at the material level and allow for the overall bone toughening mechanism to operate under large external stressors. The strain-dependent stiffening was likely associated with strain-energy transfer to the superior cross-linked bone matrix network of the normal diet, while the reduction in the enzymatically cross-linked matrix in bone samples from rabbits on a methionine-rich diet likely diminished the intrinsic bone toughening mechanism. The present study also provides a precise protocol for evaluating bone mechanical properties at the material level based on observations from a series of nanoindentation experiments.

Management of sleep-time masticatory muscle activity using stabilisation splints affects psychological stress.

To treat sleep bruxism (SB), symptomatic therapy using stabilisation splints (SS) is frequently used. However, their effects on psychological stress and sleep quality have not yet been examined fully. The objective of this study was to clarify the effects of SS use on psychological stress and sleep quality. The subjects (11 men, 12 women) were healthy volunteers. A crossover design was used. Sleep measurements were performed for three consecutive days or longer without (baseline) or with an SS or palatal splint (PS), and data for the final day were evaluated. We measured masseter muscle activity during sleep using portable electromyography to evaluate SB. Furthermore, to compare psychological stress before and after sleep, assessments were made based on STAI-JYZ and the measurement of salivary chromogranin A. To compare each parameter among the three groups (baseline, SS and PS), Friedman's and Dunn's tests were used. From the results of the baseline measurements, eight subjects were identified as high group and 15 as low group. Among the high group, a marked decrease in the number of bruxism events per hour and an increase in the difference in the total STAI Y-1 scores were observed in the SS group compared with those at baseline (P < 0·05). No significant difference was observed in sleep stages. SS use may be effective in reducing the number of SB events, while it may increase psychological stress levels, and SS use did not apparently influence sleep stages.

DNA amplification using phi29 DNA polymerase validates gene polymorphism analysis from buccal mucosa samples.

Venous blood is currently the most common source of DNA for gene polymorphism screening; however, blood sampling is invasive and difficult to perform in general dental treatment. Buccal mucosa samples provide an alternative source of DNA, but it is frequently difficult to effectively amplify the DNA owing to the small amounts of sample material obtained. This study was performed to establish a method for performing total genomic DNA amplification from buccal mucosa samples using phi29 DNA polymerase. Total genomic DNA was isolated from buccal mucosa samples obtained from healthy subjects and was amplified using phi29 DNA polymerase. To determine the suitability of the extracted DNA for genotyping, polymerase chain reaction and restriction fragment length polymorphism analyses were performed for the IL-1 gene polymorphism. Genotyping of the IL-1 polymorphism was successful using the amplified DNA from a buccal mucosa, but genotyping was unsuccessful using the unamplified control because of low DNA purity. The method of extracting DNA from a buccal mucosa is painless, simple, minimally invasive, and rapid. Genomic DNA from a buccal mucosa can be amplified by phi29 DNA polymerase in sufficient quantity and quality to conduct gene polymorphism analyses.

The roles played by the D2 and D3 domains of recombinant human thrombomodulin in its function.

Thrombomodulin (TM) is composed of five domains. We investigated the roles of the sixth epidermal growth factor (EGF)-like structure (E6) in the second domain (D2) and of an O-glycosylation site rich domain (D3) in the function of TM in more detail using deletion analysis. Two soluble mutants of TM, TMD123 and TMD12, and three deletion mutants lacking respectively 6, 16, and 38 C-terminal residues of the E6 portion, TMD12 delta 6, TMD12 delta 16, and TMD12 delta 38, were expressed in COS cells and purified. The results of protein C-activating cofactor assay showed that TMD12 delta 6, TMD12 delta 16, and TMD12 delta 38, which lack the C-terminal region, had remarkably weak cofactor activities in comparison with TMD123 (9.1, 1.4, and 1.1% of TMD123 activity, respectively). Similar findings were obtained for anticoagulant activity. These findings indicate that the last loop structure in E6 is required for full activity of recombinant human TM. We also determined in vivo stabilities of TMD12, TMD123, and TMD12 delta 6 in a pharmacokinetic study in rats. TMD12 and TMD12 delta 6, which lack the D3 domain, exhibited increased clearance (about twice that of D123). This finding suggested that the D3 domain of TM plays an important role in stabilizing TM in vivo.

A novel type of life cycle "delayed homothallism" in Saccharomyces cerevisiae wy2 showed slow interconversion of mating-type.

Saccharomyces cerevisiae wy2 segregated to 2 mater and 2 non-mater in relation to mating ability. The non-mater segregants behaved as the normal type of homothallic life cycle. On the other hand, the mater segregants gradually formed spores during successive subcultures, indicating that slow interconversion of mating-type happened to occur during subcultures. We termed this novel type of life cycle "delayed homothallism". The results of complementation tests with standard ho strains and introduction of a wild type HO gene showed that delayed homothallism was caused by a defective HO gene. The amino acid sequence deduced from the nucleotide sequence of the wy2 HO gene differed from the wild type HO gene in three amino acid residues. In the carboxy terminus of HO protein, there are three repeats of cysteine and histidine that are postulated to play a role in binding of HO protein to DNA. However, wy2 HO protein lacked one such repeat at residues Cys470-His475, where His was replaced by Leu.