Search

Cytosolic mRNA translation is subject to global and mRNA-specific controls. Phosphorylation of translation initiation factor eIF2α anchors a reversible switch that represses translation globally. The stress-responsive GCN2 kinase is the only known kinase for eIF2α inArabidopsis. Here we show that conditions that generate reactive oxygen species (ROS) in the chloroplast, such as dark-light transitions, high light, and the herbicide methyl viologen all rapidly activated the GCN2 kinase, whereas mitochondrial and ER stress did not. In addition, GCN2 activation was light dependent and mitigated by photosynthesis inhibitors and ROS quenchers. Accordingly, seedling growth of multiplegcn2mutant alleles was retarded under conditions of excess light, implicating the GCN2-eIF2α pathway in responses to light and associated ROS. Once activated, the GCN2 kinase preferentially suppressed the ribosome loading of mRNAs for functions such as mitochondrial ATP synthesis, the chloroplast thylakoids, vesicle trafficking, and translation. The transcriptome ofgcn2mutants was sensitized to abiotic stress, including oxidative stress, as well as innate immune responses. Accordingly,gcn2displayed defects in immune priming by the fungal elicitor, chitin. In conclusion, we provide evidence that reactive oxygen species produced by the photosynthetic apparatus help to activate the highly conserved GCN2 kinase, leading to eIF2α phosphorylation and thus affecting the status of the cytosolic protein synthesis apparatus.

Plant activators such as benzothiadiazole (BTH) protect plants against diseases by priming the salicylic acid (SA) signaling pathway. In rice, the transcription factor WRKY45 plays a central role in this process. To investigate the mechanism involved in defense-priming by BTH and the role of WRKY45 in this process, we analyzed the transcripts of biosynthetic genes for diterpenoid phytoalexins (DPs) during the rice–Magnaporthe oryzae interaction. The DP biosynthetic genes were barely upregulated in BTH-treated rice plants, but were induced rapidly after M. oryzae infection in a WRKY45-dependent manner. These results indicate that the DP biosynthetic genes were primed by BTH through WRKY45. Rapid induction of the DP biosynthetic genes was also observed after M. oryzae infection to WRKY45-overexpressing (WRKY45-ox) plants. The changes in gene transcription resulted in accumulation of DPs in WRKY45-ox and BTH-pretreated rice after M. oryzae infection. Previously, we reported that cytokinins (CKs), especially isopentenyladenines, accumulated in M. oryzae-infected rice. Here, we show that DP biosynthetic genes are regulated by the SA/CK synergism in a WRKY45-dependent manner. Together, we propose that CK plays a role in mediating the signal of M. oryzae infection to trigger the induction of DP biosynthetic genes in BTH-primed plants. Electronic supplementary material The online version of this article (doi:10.1007/s11103-014-0221-x) contains supplementary material, which is available to authorized users.

This study is aimed to test whether glucose-free conditions induce the activation of adenosine monophosphate-activated protein kinase (AMPK) and, to investigate association with AMPK expression and cell viability in human dental pulp cells.Human dental pulp cells were initially maintained in culture medium containing glucose and the medium was subsequently changed to glucose-free medium. To evaluate the expression of AMPK, quantitative real-time RT-PCR, Western blot analysis and immunofluorescence were carried out. Cell viability was evaluated by MTT assay.The expression of AMPK mRNA in glucose free conditions was 2.0-2.5 fold higher than the control at 1, 2 and 3 h (P0.01). The expression of phosphorylated-AMPK was characterized by Western blot analysis and by immunofluorescence. Compound C-pre-treated group showed a decline of both AMPK expression and cell viability, while AICAR-pre-treated group showed an increase of AMPK and maintain of cell viability at regular level.AMPK plays an important role on fluctuating in accordance with glucose availability and protects cell viability from glucose free condition in human dental pulp cells.

OsWRKY76 encodes a group IIa WRKY transcription factor of rice. The expression of OsWRKY76 was induced within 48h after inoculation with rice blast fungus (Magnaporthe oryzae), and by wounding, low temperature, benzothiadiazole, and abscisic acid. Green fluorescent protein-fused OsWRKY76 localized to the nuclei in rice epidermal cells. OsWRKY76 showed sequence-specific DNA binding to the W-box element in vitro and exhibited W-box-mediated transcriptional repressor activity in cultured rice cells. Overexpression of OsWRKY76 in rice plants resulted in drastically increased susceptibility to M. oryzae, but improved tolerance to cold stress. Microarray analysis revealed that overexpression of OsWRKY76 suppresses the induction of a specific set of PR genes and of genes involved in phytoalexin synthesis after inoculation with blast fungus, consistent with the observation that the levels of phytoalexins in the transgenic rice plants remained significantly lower than those in non-transformed control plants. Furthermore, overexpression of OsWRKY76 led to the increased expression of abiotic stress-associated genes such as peroxidase and lipid metabolism genes. These results strongly suggest that OsWRKY76 plays dual and opposing roles in blast disease resistance and cold tolerance.

Gap junctions play an important role in differentiation of odontoblasts. Gap junction protein, connexin 43 is expressed in odontoblast. However, the detailed localization in odontoblasts has yet to be fully investigated. We investigated the localization of connexin43 in rat odontoblasts immuno-electron microscopically. The rats were transcardially fixed with 1% paraformaldehyde in 0.1M phosphate buffer, and mandibles were decalcified with 10% ethylene- diamine tetraacetic acid. Pre-embedding method was carried out for immuno-electron micro- scopic analysis. Microscopically, gap junctions were localized between bodies of odontoblasts, and between bodies and processes of odontoblasts. The gap junctions were labeled with gold par- ticles that indicated connexin43. These results suggest that gap junctions between odontoblasts are definitely composed of connexin43 in rats, and our methods used in this study is useful to investigate localization of connexin43 immuno-electron microscopically. Microsc. Res. Tech. 76:988-991, 2013. V C 2013 Wiley Periodicals, Inc.

Hormone crosstalk is pivotal in plant–pathogen interactions. Here, we report on the accumulation of cytokinins (CK) in rice seedlings after infection of blast fungus Magnaporthe oryzae and its potential significance in rice–M. oryzae interaction. Blast infection to rice seedlings increased levels of N6-(Δ2-isopentenyl) adenine (iP), iP riboside (iPR), and iPR 5′-phosphates (iPRP) in leaf blades. Consistent with this, CK signaling was activated around the infection sites, as shown by histochemical staining for β-glucuronidase activity driven by a CK-responsive OsRR6 promoter. Diverse CK species were also detected in the hyphae (mycelium), conidia, and culture filtrates of blast fungus, indicating that M. oryzae is capable of production as well as hyphal secretion of CK. Co-treatment of leaf blades with CK and salicylic acid (SA), but not with either one alone, markedly induced pathogenesis-related genes OsPR1b and probenazole-induced protein 1 (PBZ1). These effects were diminished by RNAi-knockdown of OsNPR1 or WRKY45, the key regulators of the SA signaling pathway in rice, indicating that the effects of CK depend on these two regulators. Taken together, our data imply a coevolutionary rice–M. oryzae interaction, wherein M. oryzae probably elevates rice CK levels for its own benefits such as nutrient translocation. Rice plants, on the other hand, sense it as an infection signal and activate defense reactions through the synergistic action with SA.

Actin filament assembly in plants is a dynamic process, requiring the activity of more than 75 actin-binding proteins. Central to the regulation of filament assembly and stability is the activity of a conserved family of actin-depolymerizing factors (ADFs), whose primarily function is to regulate the severing and depolymerization of actin filaments. In recent years, the activity of ADF proteins has been linked to a variety of cellular processes, including those associated with response to stress. Herein, a wheat ADF gene, TaADF4, was identified and characterized. TaADF4 encodes a 139-amino-acid protein containing five F-actin-binding sites and two G-actin-binding sites, and interacts with wheat (Triticum aestivum) Actin1 (TaACT1), in planta. Following treatment of wheat, separately, with jasmonic acid, abscisic acid or with the avirulent race, CYR23, of the stripe rust pathogen Puccinia striiformis f. sp. tritici, we observed a rapid induction in accumulation of TaADF4 mRNA. Interestingly, accumulation of TaADF4 mRNA was diminished in response to inoculation with a virulent race, CYR31. Silencing of TaADF4 resulted in enhanced susceptibility to CYR23, demonstrating a role for TaADF4 in defense signaling. Using a pharmacological-based approach, coupled with an analysis of host response to pathogen infection, we observed that treatment of plants with the actin-modifying agent latrunculin B enhanced resistance to CYR23, including increased production of reactive oxygen species and enhancement of localized hypersensitive cell death. Taken together, these data support the hypothesis that TaADF4 positively modulates plant immunity in wheat via the modulation of actin cytoskeletal organization.

SUMMARY Plant ‘activators’, such as benzothiadiazole (BTH), protect plants from various diseases by priming the plant salicylic acid (SA) signalling pathway. We have reported previously that a transcription factor identified in rice, WRKY45 (OsWRKY45), plays a pivotal role in BTH-induced disease resistance by mediating SA signalling. Here, we report further functional characterization of WRKY45. Different plant activators vary in their action points, either downstream (BTH and tiadinil) or upstream (probenazole) of SA. Rice resistance to Magnaporthe grisea, induced by both types of plant activator, was markedly reduced in WRKY45-knockdown (WRKY45-kd) rice, indicating a universal role for WRKY45 in chemical-induced resistance. Fungal invasion into rice cells was blocked at most attempted invasion sites (pre-invasive defence) in WRKY45-overexpressing (WRKY45-ox) rice. Hydrogen peroxide accumulated within the cell wall underneath invading fungus appressoria or between the cell wall and the cytoplasm, implying a possible role for H2O2 in pre-invasive defence. Moreover, a hypersensitive reaction-like reaction was observed in rice cells, in which fungal growth was inhibited after invasion (post-invasive defence). The two levels of defence mechanism appear to correspond to Type I and II nonhost resistances. The leaf blast resistance of WRKY45-ox rice plants was much higher than that of other known blast-resistant varieties. WRKY45-ox plants also showed strong panicle blast resistance. BTH-induced resistance to Xanthomonas oryzae pv. oryzae was compromised in WRKY45-kd rice, whereas WRKY45-ox plants were highly resistant to this pathogen. However, WRKY45-ox plants were susceptible to Rhizoctonia solani. These results indicate the versatility and limitations of the application of this gene.

Background: The purpose of this study was to investigate the differentiation and maturation of osteoblasts on surfaces with different roughnesses. Methods: Blasting a culture dish surface with Al2O3 particles of 25 μm, 50 μm or 180 μm diameter produces mean surface a roughness (Ra) of 0.48 ± 0.07, 1.02 ± 0.14 or 3.10 ± 0.36, respectively. Morphological changes of cells were compared, and the expression of mRNAs of osteopontin and osteocalcin were evaluated. Further, the ratio of alizarin red positive area was calculated. Results: Cells cultured on the 180 μm blasted surface had almost the same morphology as those cultured on the 50 μm blasted surface; however, cellular bridges tended to be much wider and more spread out. The expressions of osteopontin and osteocalcin mRNAs in cells cultured on the 180 μm blasted surface have greater up-regulation than the other cells. Further, the ratio of alizarin red positive area in180 μm blasted surface was significantly higher than those of others. Conclusions: These results suggest that the 180 μm blasted surface, with a surface roughness of approximately 3 μm in width, is the best surface for MC3T3-E1 cells to differentiate and mature in vitro.

Introduction Intracellular Ca 2+ is essential to many signal transduction pathways, and its level is tightly regulated by the Ca 2+ extrusion system in the plasma membrane, which includes the Na + -Ca 2+ exchanger (NCX). Although expression of NCX1 isoforms has been demonstrated in odontoblasts, the detailed properties of NCX remain to be clarified. In this study, we investigated localization and ion-transporting/pharmacologic properties of NCX isoforms in rat odontoblasts. Methods We characterized both the reverse and forward modes of NCX activity in odontoblasts in a dental pulp slice preparation. Ca 2+ influx by reverse NCX activity was measured by fura-2 fluorescence. Ca 2+ efflux by forward NCX activity elicited inward Na + current as measured by perforated-patch clamp recording. For immunohistochemical analysis, cryostat sections of incisors were incubated with antibodies against NCX. Results Immunohistochemical observation revealed localization of NCX1 and NCX3 in the distal membrane of odontoblasts. Inward currents by forward NCX activity showed dependence on external Na + . Fura-2 fluorescence measurement revealed that Ca 2+ influx by reverse NCX activity depended on extracellular Ca 2+ concentration, and that this influx was blocked by NCX inhibitor KB-R7943 in a concentration-dependent manner. However, Ca 2+ influx by NCX showed a slight sensitivity to SEA0400 (a potent NCX1 inhibitor), indicating that expression potencies in odontoblasts were NCX3 > NCX1. Conclusions These results suggest that odontoblasts express NCX1 and NCX3 at the distal membrane, and that these isoforms play an important role in the Ca 2+ extrusion system as well as in the directional Ca 2+ transport pathway from the circulation to the dentin-mineralizing front.

Oral Diseases (2010) 16, 263–268 Objective: The aim of this study was to investigate the proliferation, migration and death of periodontal ligament (PDL) cells after tooth replantation. Materials and methods: Maxillary first molars were extracted from 4-week-old male (n = 28) Sprague–Dawley rats and immediately replanted, after which, proliferation, migration and death of PDL cells were investigated. Results: At 3 days after tooth replantation, many proliferative cell nuclear antigen (PCNA)-positive PDL cells were observed on the alveolar bone side, but fewer on the root side. However, while a gradual decrease was observed in number of PCNA-positive PDL cells on the alveolar bone side until 7 days, an increase was seen on the root side. At 3 weeks, cells labeled with PKH26 (fluorescent dye into plasma membrane) were located in the middle of the PDL space. However, these PKH26-labeled cells did not spread to the surface of the cementum or the alveolar bone. TUNEL-positive cells were observed on both the bone and root sides at 3 days. Number of apoptotic cells increased until 7 days on the bone sides, but decreased on root sides. Conclusion: These results suggest that both cell proliferation and apoptosis occur in different patterns and at different times to maintain regular spacing of the PDL after tooth replantation.

Although the central role of ameloblasts in synthesis and resorption of enamel matrix proteins during amelogenesis is well documented, the Ca2+-transport/extrusion mechanism remains to be fully elucidated. To clarify Ca2+-transport in rat ameloblasts, we investigated expression and localization of Na+-Ca2+ exchanger (NCX) isoforms and the functional characteristics of their ion transporting/pharmacological properties. RT-PCR and immunohistochemical analyses revealed expression of NCX1 and NCX3 in ameloblasts, localized in the apical membrane. In patch-clamp recordings, Ca2+ efflux by Na+-Ca2+ exchange showed dependence on external Na+. Ca2+ influx by Na+-Ca2+ exchange, measured by fura-2 fluorescence, showed dependence on extracellular Ca2+ concentration, and it was blocked by NCX inhibitors KB-R7943, SEA0400, and SN-6. These results showed significant expression of NCX1 and NCX3 in ameloblasts, indicating their involvement in the directional Ca2+ extrusion pathway from cells to the enamel mineralizing front. Keywords:: transporter, enamel, mineralization, channel, SLC8 gene family

Mechanical stress such as occlusal and orthodontic loading has been suggested to induce a homeostatic and regenerative response in periodontal ligament (PDL), but the underlying mechanism remains to be clarified. The purpose of this study was to investigate expression of mRNAs encoding proteins involved in osteogenesis and homeostasis by PDL cells following application of tensile stress and characterize the relationship between such expression and the regenerative and homeostatic functions of the PDL. PDL cells were obtained from rats and stretched by 9% or 18% at a frequency of 6 cycles/min for 12 hr to 5 days in a FX-4000Ttrade; culture system. After stretching, expression of mRNAs encoding collagen type I (Col-I), alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP-2), bone morphogenetic protein-4 (BMP-4), heat shock protein 70 (HSP70) and basic fibroblast growth factor (bFGF) was investigated. The highest levels of Col-I, ALP and BMP-2 mRNA expression occurred at 12 hr, while those of BMP-4 and HSP70 occurred at 1 day and 5 days, respectively. Expression levels of Col-I, ALP, BMP-2, BMP-4 and HSP70 increased magnitude-dependently with stretching force in the stretching groups. In contrast, expression of bFGF mRNA showed statistically significant reduction in both stretching groups, with the largest reduction seen in the 9% stretching group (p0.01). These results suggest that stretching of PDL cells provokes significant increases in expression of factors promoting osteogenic differentiation and HSP70, which protects PDL cells undergoing mechanical stress and contributes to maintenance of PDL homeostasis. However, expression of bFGF was restrained. Reduced expression of bFGF mRNA suggested that there was an optimum magnitude of stretching force for increasing expression.

Background and Objective: The 4-META/MMA-TBB [4-(2-methacryloxyethyl)trimellitic anhydride/methyl methacrylate-tributylborane] resin is widely used as a dental adhesive. It has also been applied in the dressing of gingival wound surfaces following periodontal surgery. However, its effect on the regeneration and/or cell attachment of the oral epithelium remains to be clarified. To evaluate the effect of the resin applied as a wound dressing, we investigated expression of laminin 5, integrin β4 and cytokeratin 14 in regenerating oral epithelium treated with this resin following gingivectomy from the viewpoint of cell attachment and differentiation. Material and Methods: The resin was applied to the entire wound surface in rats after gingival surgery, and regenerating epithelium was examined immediately and at 1, 3, 5, 7 and 14 days later. The resin was removed 2 weeks after application in some animals and tissue further examined at 1, 3, 5 and 7 days later. Results: Regenerating epithelium under the resin was not keratinized, but became keratinized immediately after removal of the resin. Laminin 5 and integrin β4 were immunolocalized in the basal lamina, the internal basal lamina, in marginal cells of the regenerating epithelium and at the resin–regenerating epithelium interface. Cytokeratin 14 localized in the regenerating epithelium underneath the resin, as well as in healthy and regenerated junctional epithelial cells. Conclusion: These results suggest that this resin covers the wound surface and that the regenerating epithelium biologically adheres to the resin during the initial process of its regeneration.

Fatty acids and their derivatives play important signaling roles in plant defense responses. It has been shown that suppressing a gene for stearoyl acyl carrier protein fatty-acid desaturase (SACPD) enhances the resistance of Arabidopsis (SSI2) and soybean to multiple pathogens. In this study, we present functional analyses of a rice homolog of SSI2 (OsSSI2) in disease resistance of rice plants. A transposon insertion mutation (Osssi2-Tos17) and RNAi-mediated knockdown of OsSSI2 (OsSSI2-kd) reduced the oleic acid (18:1) level and increased that of stearic acid (18:0), indicating that OsSSI2 is responsible for fatty-acid desaturase activity. These plants displayed spontaneous lesion formation in leaf blades, retarded growth, slight increase in endogenous free salicylic acid (SA) levels, and SA/benzothiadiazole (BTH)-specific inducible genes, including WRKY45, a key regulator of SA/BTH-induced resistance, in rice. Moreover, the OsSSI2-kd plants showed markedly enhanced resistance to the blast fungus Magnaporthe grisea and leaf-blight bacteria Xanthomonas oryzae pv. oryzae. These results suggest that OsSSI2 is involved in the negative regulation of defense responses in rice, as are its Arabidopsis and soybean counterparts. Microarray analyses identified 406 genes that were differentially expressed (≥2-fold) in OsSSI2-kd rice plants compared with wild-type rice and, of these, approximately 39% were BTH responsive. Taken together, our results suggest that induction of SA-responsive genes, including WRKY45, is likely responsible for enhanced disease resistance in OsSSI2-kd rice plants.

Many morphological and developmental studies have demonstrated the characteristics of tight junctions (TJs) between odontoblasts. However, detailed localization of TJ-associated proteins in odontoblasts and their functions has not yet been clarified. To elucidate the relationship between the establishment of TJ structures and the differentiation of odontoblasts during early dentinogenesis, we studied the expression and localization of constituent proteins of TJs (claudin-1, occludin, ZO-1 and ZO-2) between odontoblasts in rat lower incisors using Western blotting, immunofluorescence and immunoelectron microscopy. When the expression of claudin-1 increases at the distal portion of mature odontoblasts, the TJs form complex networks of strands, and odontoblasts differentiated by developing distal membrane domains and by secreting specific molecules for mineralization. We conclude that the TJs of odontoblasts may play an important role in the differentiation of odontoblasts in rat lower incisors during early dentinogenesis.

Background and Objective: It is known that epithelial islands are embedded in the cementum during tooth root formation, but details of this process remain unknown. The purpose of this study was to investigate the dynamic characteristics of Malassez's epithelial rest cells in the cementum during tooth root formation in pigs in vivo. Material and Methods: The first molars of 6-mo-old pigs were used in this study. Specimens were decalcified before being embedded in paraffin. Paraffin sections were investigated using TdT-mediated dUTP-biotin nick end labeling (TUNEL), immunohistochemical, and ultrastructural techniques. Results: Malassez's epithelial rest cells were located close to the root surface at the apical one-third of the periodontal ligament, and epithelial clusters surrounded by distinct lamina cementia were sometimes observed in the cementum. TUNEL-positive cells were detected only in the cementum. Malassez's epithelial rest cells in the periodontal ligament were completely surrounded by basement membranes, but epithelial clusters in the cementum were only intermittently surrounded by such membranes. Cytokeratin-positive cells in the superstratum of the cementum were directly connected by cementocytes and by desmosome-like structures. However, organelles were scarce in the cytokeratin-positive cells in the substratum of the cementum, and the matrix of the cementum was deposited in the cells. Conclusion: These results suggest that the majority of the fragmented Hertwig's root sheath remains in the periodontal ligament and that some cells, which are connected to cementoblasts, are embedded in the cementum and progress to apoptosis.

Previous reports have shown the expression of several mechanosensitive ionic channels on the plasma membrane in odontoblasts, which are the cells responsible for dentin formation. The membrane characteristics of odontoblasts imply that they could play critical roles in the mechano-transduction of fluid displacement within dentinal tubules into the electrical cell signals, to carry dentin sensation to the central nervous system. However, the direct ionic mechanism underlying such a dentin nociceptive function remains unclear. In the present study, we investigated the expression of the transient receptor potential vanilloid subfamily member 1 (TRPV1) channel--which essentially contributes to the detection of pain sensation--in rat odontoblasts by immunohistochemical and nystatin perforated patch-clamp techniques. Immunohistochemical observation showed the localization of TRPV1-immunoreactions on the distal regions of odontoblast membranes. In the patch-clamp experiments, we observed capsaicin-induced inward currents that were inhibited by capsazepine, a TRPV1 channel antagonist. Our results indicate a significant expression of TRPV1 channels in odontoblasts, suggesting that odontoblasts may directly respond to noxious stimuli such as a thermal-heat stimulus, and point to the necessity for a reconsideration of the cellular mechanisms of dentin sensation based on the transmembrane ionic signals in odontoblasts.

The purpose of the current study was to evaluate the bone formation when beta-tricalcium phosphate (TCP) was implanted in bone defects of rat femurs. beta-TCP granules were applied to defects created in the femurs of 65 male rats who were sacrificed 3, 7, 10, 14 or 30 days later. Bone tissues were embedded in paraffin, serial sections were cut and then stained with hematoxylin-eosin. Histomorphometric analyses were also conducted. Furthermore, total mRNAs were extracted, homogenized, and reverse transcribed, after which quantitative PCR assays were conducted with a LightCycler using the double-stranded DNA dye Syber Green I with primers for either rat osteopontin or osteocalcin. Tissues in defects without beta-TCP were used as controls. The amount of newly formed bone tissue in the beta-TCP implanted group was significantly greater in both the side areas and the central area of defects than in the control group. Expressions of osteopontin and osteocalcin mRNAs of cells in the defects of the experimental group were up-regulated compared with the control group at all time periods. Taken together, these results prove that beta-TCP is an appropriate material for osteoconduction and promotes bone formation in bone defects.

Cementoblastoma is a relatively rare type of odontogenic tumor. This study investigated the features of cementoblastoma tumor cells in three cases using immunohistochemistry. Immunopositive reactivity for Ki-67 was recognized in the nuclei of spindle-shaped cells that were located in the fibrous connective tissue among the cementum-like hard tissue. Those cells were mainly discernible at the periphery of the tumors. Polygonal cementoblast-like cells with large nuclei surrounding the hard tissue were immunopositive for osteopontin and osteocalcin but not for Ki-67, which suggests that these cells may be more differentiated cells that have the potential to form hard tissue.

Property modification of an implant surface is known to influence the behavior of cells surrounding implant material. This study examined the osteogenic cell behavior on protein immobilized surface in vitro. Following plasma surface modification, fibronectin or bone morphogenetic protein-2 (BMP-2) was immobilized. The number of cells adhering to fibronectin-immobilized surface increased after 1 and 2 h of incubation compared with non-immobilized surface. Alkaline phosphatase activity and osteocalcin mRNA expression of the osteogenic cells on the BMP-2 immobilized surface was greater than that on the non-immobilized surface. This study demonstrated that protein can be immobilized to a polystyrene surface after treatment with O2 plasma and that the osteogenic cells surrounding a biomaterial can be controlled by the immobilization of protein to the biomaterial.

The purpose of this study was to investigate the behavior of rat bone marrow cells (RBM) growing on surfaces with different pore sizes. RBM behavior on Millipore filters (MF-Millipore membrane filter) made from cellulose mixed esters with 5 different pore surfaces (0.45 microm, 1.2 microm, 3.0 microm, 5.0 microm and 8.0 microm) were compared in terms of morphological changes on the different pore sizes. Furthermore, the expressions of osteopontin and osteocalcin mRNAs were investigated. On the 1.2 microm and 3.0 microm pore surfaces, RBM attached to the substrate well, but cells on the 5.0 microm and 8.0 microm pore surfaces invaded deeply into the pores. Higher levels of both osteopontin and osteocalcin mRNA expression were always observed in cells cultured on the 1.2 microm filter. These results suggest that the 1.2 microm Millipore filter pore size is the most suitable for inducing RBM to differentiate into an osteoblastic phenotype among these surfaces and is probably related to production of the ECM but not to the phenomenon of cell spreading.

The purpose of this study was to investigate pulp cell responses during hypoxia and reoxygenation. Pulp tissues obtained from beagle dogs were cultured. In the control group, pulp cells were incubated in normoxic conditions (20% O2) for 1-4 d. In the hypoxia group, pulp cells were incubated under hypoxic conditions (2% O2) for 1-4 d. In the reoxygenation group, pulp cells were first incubated under hypoxic conditions for 24 h, and were then incubated in normoxic conditions (20% O2) for one to three additional days. Cell viability, MTT (3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay, cellular proliferation, and alkaline phosphatase (ALPase) activity were determined. Expression of heat shock protein 70 (HSP70) and vascular endothelial growth factor (VEGF) was analysed by Western blotting. Hypoxia inducible factor-1alpha (HIF-1alpha) in pulp cells was analysed by reverse transcriptase polymerase chain reaction (RT-PCR). The cell growth rate and ALPase activity were significantly higher in the hypoxia group than in the control group. After reoxygenation, cellular proliferation and ALPase activity decreased to the level of the control group while HSP70 expression increased. Hypoxia inducible factor-1alpha expression was detected in pulp cells, and VEGF expression (which is regulated by HIF-1alpha) increased under hypoxic conditions. These results suggest that dynamic responses to hypoxia and reoxygenation occur in pulp cells.

Carbon foams are suitable for scaffolds used in tissue engineering that have a porosity ratio of more than 90% with a three-dimensional honeycomb structure. Titanium films were deposited isotropically using an electron cyclotron resonance (ECR) sputtering system on 5 mm-thick carbon foams. SEM observation and electron probe microanalysis revealed that all the surfaces of the carbon foam in the center of the specimens were coated with an approximately 5 μm-thick titanium film, indicating that the ECR sputtering method enables titanium coating into both the surrounding and deep regions of carbon foams. The flexural strength and elastic modulus of the coated specimens increased by approximately 1.5 and 2.5 times, respectively, than those of the uncoated specimens. Cell culture tests with mouse fibroblasts (L-929) and rat bone marrow osteoblast-like cells demonstrated the presence of cells not only on the surface but also inside the foam. No toxic effect on both cells on the titanium-coated carbon foams was apparent. A small amount of extracellular matrices appeared on the foam struts, but those matrices did not grow enough to occupy the spaces between them. These results indicate that carbon foams coated with titanium are useful for skeletons used for tissue engineering.

The purpose of this study was to investigate the immunohistochemical and the ultrastructural features of the implant circumference epithelium of the beagle dog using various types of antibodies. The peri-implant epithelium was at an acute-angle from the gingival epithelium and was arranged in parallel to the implant surface. With immunohistochemical staining, the peri-implant epithelium was strongly positive for KL-1, and weakly positive for CK4, CK8 and CK19. These positive reactions for keratins and also for PCNA and BM-1 were similar to those seen in the oral mucosa. In the peri-implant epithelium, a plentitude of microvilli were observed at the periphery of cells at the implant sites, and bacteria were observed between the implant and the peri-implant epithelium without the formation of half desmosomes. There were many lipid-like vacuoles or lysosome-like granules. The intercellular space was wider than the junctional epithelium, and random migrations of large numbers of neutrophils could be seen. Taken together, the peri-implant epithelium is similar to that seen in the oral mucosa, and it is structurally different from the junctional epithelium.

Because dental implants contact many different tissues, the implant material must have optimum surface compatibility with the host bone tissue, subepithelial connective tissue, and epithelial tissue. In addition, dental implant surfaces exposed to the oral cavity must remain plaque-free. Such materials can be created under well-controlled conditions by modifying the surfaces of metals that contact those tissues. “Tissue-compatible implants,” which are compatible with all host tissues, must integrate with bone tissue, easily form hemidesmosomes, and prevent bacterial adhesion. This research was aimed at developing such tissue-compatible implants by modifying titanium surfaces using a dry process for closely adhering to the titanium substrate and ensuring good wear resistance. The process includes ion beam dynamic mixing (thin calcium phosphates), ion implantation (Ca+, N+, F+), titania spraying, ion plating (TiN, alumina), and ion beam mixing (Ag, Sn, Zn, Pt) with Ar+. At the bone tissue/implant interface, a thin calcium phosphate coating and rapid heating with infrared radiation were effective in controlling the dissolution without cracking the coating. This thin calcium phosphate coating may directly promote osteogenisis, but it may also enable immobilization of functional proteins or drugs. At the oral fluid/implant interface, an alumina coating and F+ implantation were responsible for inhibiting the adhesion of microbial plaque. In conclusion, dry-process surface modification is useful in controlling the physicochemical nature of surfaces, including the surface energy and the surface electrical charge, and in developing tissue-compatible implants.

Since dental implants are used in contact with many different tissues, it is necessary to have optimum surface compatibility with the host bone tissues and soft tissues. Furthermore, dental implant surfaces exposed to the oral cavity must remain plaque-free. Such materials can be created under well-controlled conditions by modifying the surfaces of metals that contact those tissues. “Tissue-compatible implants,” which are compatible with all host tissues, must integrate with bone tissue, easily form hemidesmosomes, and prevent bacterial adhesion. This research was aimed at developing such tissue-compatible implants by modifying titanium surfaces using a dry process for closely adhering to the titanium substrate and ensuring good wear resistance. The process includes ion beam dynamic mixing (thin calcium phosphates), ion implantation, titania spraying, ion plating and ion beam mixing. At the bone tissue/implant interface, a thin calcium phosphate coating and rapid heating with infrared radiation was effective in controlling the dissolution without cracking the coating. This thin calcium phosphate coating may directly promote osteogenisis, but also enable immobilization of functional proteins or drugs such as bisphosphonate for dug delivery system. At the oral fluid/implant interface, an alumina coating and F + -implantation were responsible for inhibiting the adhesion of microbial plaque. In conclusion, dry-process surface modification is useful in controlling the physicochemical nature of surfaces, including the surface energy and the surface electrical charge, and in developing tissue-compatible implants.

In the present study, we investigated the localization of amelogenin in odontogenic tumors, using an anti-amelogenin polyclonal antibody. In order to make the antibody, antisera against an amelogenin fraction obtained from the enamel matrix of unerupted bovine tooth was raised in rabbits. By Western blot analysis, a main band of 25 kDa and six minor bands (6.8, 12, 18, 20, 23, and 27 kDa) were detected under nonreducing conditions. Immunoreactivity for the amelogenin was observed in ameloblasts and in the immature enamel matrix of 4-day-old rats. In odontogenic tumors, positive reactions for amelogenin were localized in limited areas in adenomatoid odontogenic tumor, calcifying odontogenic cyst, primary intraosseous carcinoma and odontoma. The strongest immunoreactions were shown in enamel matrices in odontomas. Small mineralized foci in epithelial nests showed positive reactions, and a few reactions were observed in epithelium adjacent to the mineralized foci. In calcifying odontogenic cysts, some ghost cells in the lining epithelium were strongly stained. The results indicate that the present antibody for amelogenin is useful for the determination of odontogenic tumors, especially in those in which small mineralized foci are present in the epithelilal nests.

We have examined the expression and localization of connexin 43 (CX43) in rat incisor odontoblasts using reverse transcriptase polymerase chain reaction, in situ hybridization and immunohistochemistry. The CX43 gene was expressed in odontoblasts, and levels of gene expression increased throughout the course of development. In contrast, CX43 was down-regulated at an incisal segment. In situ hybridization analysis showed no positive signal for CX43 RNA in the cytoplasm of differentiating dental papilla cells, but faint positive signals for CX43 RNA were observed in early pre-odontoblasts. Those signals were more intense in young and in old odontoblasts, but were less in short odontoblasts. CX43 could not be detected in differentiating dental papilla cells or in early pre-odontoblasts by immunohistochemical localization, but a positive reaction was found in the late pre-odontoblast stage where predentin had been produced. The positivity gradually increased during odontoblast maturation, and was highest in the layer of old odontoblasts. These results indicate that odontoblasts that secrete actively dentin matrix components are tightly in contact with each other by gap junctions as suggested by the intense CX43.

The aim of this study was to elucidate the distribution and morphological changes of tight junctions during secretion in parotid gland acinar cells. Localization of tight junction-associated polypeptide ZO-1, and of tight junction transmembrane protein Occludin, was examined in rat parotid gland by immunofluorescence and immunogold labelling of ultrathin sections. Adult male Sprague-Dawley rats were intraperitoneally injected with IPR and, after 10 and 30 minutes, parotid glands were extirpated. In control specimens, positive immunoreaction for ZO-1 and Occludin was observed on the adluminal side between adjacent cells in the form of narrow elongated profiles corresponding to intercellular canaliculi. After IPR injection, canaliculi became dilated and fluorescence was no longer seen as a continuous line but appeared as an aggregation of separate bright particles. ZO-1 was more widely distributed and was recognized in other areas of the cytoplasm as well. Concurrently, omega-shaped concavities, marked by actin fluorescence, appeared along the intercellular canaliculi. We concluded that, during exocytosis, the selective permeability barrier to the paracellular pathway, based on tight junctions, becomes more leaky, owing to segregation of Occludin caused by intracellular ZO-1 distributional changes associated with actin filaments.

The purpose of this study was to investigate nerve regeneration and proliferative activity in amputated pulp tissue after the application of 4-META/MMA-TBB adhesive resin (4-META resin). Calcium hydroxide was used as a control material. At 3 days, fibroblast-like cells were positive for proliferating cell nuclear antigen (PCNA) in both 4-META resin- and calcium hydroxide-treated groups and were located mainly within 0.5 mm from the cut surface. Only a few fragmented neurofilament protein (NFP)-positive nerve fibers were observed in this area. At 7 and 14 days, the number of PCNA-positive cells had gradually decreased and regenerated NFP-positive nerve fibers were observed close to the cut surface of the pulp in both groups. At 21 days in the experimental group, several PCNA-positive cells were still found in the area 0.5 mm from the cut surface, and NFP-positive nerve fibers were detected about 0.15-;0.2 mm from the cut surface. In contrast, a dentin bridge was produced under the necrotic layer at 21 days in the control group. PCNA-positive cells were not found underneath the dentin bridge, but NFP-positive nerve fibers had regenerated close to it. These results suggest that although cell differentiation and nerve regeneration are delayed, wound healing occurred even after the application of 4-META resin to exposed pulp surface the same as calcium hydroxide application.

The role played by the internal basal lamina (IBL) and hemidesmosomes between an implant and the peri-implant epithelium (PIE) in the adherence of the epithelium to the implant is controversial. This study used rat maxilla implantation models to clarify the ultrastructure of the PIE-implant interface.Ti-6Al-4V implants were inserted either immediately or 2 weeks after the extraction of the upper left first molar of 6- or 4-week-old rats, respectively. The junctional epithelium (JE) of the upper right molars in the same animals was used as a control. Four weeks after implantation, the animals were sacrificed to prepare specimens for light and immunoelectron microscopy.Under light microscopy, the PIE appeared to attach to the implant surface. Ultrastructurally, IBL, consisting of the lamina densa and lamina lucida, and hemidesmosomes were formed only in the lower region, and rarely in the middle region, of the PIE-implant interface. In control teeth, the IBL and hemidesmosomes formed throughout the dento-JE interface. Laminin-1 was found in the IBL and also in the vesicles and vacuoles of the PIE and JE cells. Statistical analysis showed that there was also a significant difference in the amount of IBL between the PIE-implant and dento-JE interfaces.PIE attached to the implant via hemidesmosomes and IBL in the lower region of the PIE-implant interface. Although PIE cells may secrete laminin-1, which contributes to epidermal cell adhesion, the PIE which attaches to implants only in the lower region of the interface is considered to be the poorly adhered epithelium.

We investigated the distribution and localization of connexin 26, 32, and 43 in rat parotid glands. Immunofluorescence microscopy revealed the presence of spots reacting for connexin 26 and 32 between endpiece cells. A few spots indicating connexin 43 occasionally were observed in the parotid glands. Connexin 43-positive spots were localized around intercalated ducts. No positive spots for these connexins were detected between ductal cells. Double immunofluorescence microscopy revealed that connexin 26-reactive sites could be observed within the same spots as connexin 32. Double immuno-electron microscopy confirmed that connexin 26 and 32 were co-localized in the same gap junction. These results suggest that connexin 26 and 32 are associated with secretory function and permeability between endpiece cells and that connexin 43 is associated with contraction of the myoepithelial cells that surround intercalated ducts in the rat parotid glands.

A rare case of clear cell odontogenic carcinoma was investigated using histochemical and immunohistochemical methods. The tumor occurred in the anterior mandible of a 69-year-old Japanese man. Histologically, the tumor was composed mostly of large clear cells and squamous cells. Columnar-shaped cells with basophilic nuclei polarized away from the basement membrane were observed at the periphery of the tumor foci. The tumor cells had aggressively invaded muscle and perineural tissues. The tumor cells were positive for PAS staining. Immunohistochemically, tumor cells reacted positively to keratin, cytokeratin19, epithelial membrane antigen, and S-100 protein. The tumor was diagnosed as a clear cell odontogenic carcinoma. Its characteristics are discussed in term of its histopathological, histochemical and immunohistochemical features.

To investigate the ultrastructure of gap junctions and functional differences between connexin (gap junction structural protein) 32 and 43 in the rat submandibular and sublingual glands, tracer and freeze-fracture methods were carried out, and the expression of both connexins was examined. In both glands, western blot analysis with anti-connexin 32 and 43 antibodies revealed bands of about 27kD and 43kD, respectively. Immunofluorescence showed the presence of connexin32-positive spots between acinar cells in both glands. In contrast, connexin43-positive spots were observed at the periphery of the acinar structures in either gland. Positive spots for both connexins could not be detected between ductal cells in both glands. By immunocytochemistry, connexin32 was found on the gap junctional membranes of acinar cells and connexin43 on the gap junctional membranes of myoepithelial cells. It is surmised that connexin32 of the gap junction is related to the secretory function of acinar cells and that connexin43 is associated with the contraction of the myoepithelial cells.

The purpose of this study was to establish the criteria for oral epithelial dysplasia (OED). A total of fifty-six biopsies of white lesions in the buccal mucosa and gingiva were classified into Type I, II and III, and their relationship to expression of PCNA or p53 and keratinization patterns were examined. The appearance of PCNA positive cells coincided with severe atypia in the basal layer. p53 expression revealed focal, diffuse and basal layer patterns. The focal pattern was found in only Type I; both the diffuse and basal layer patterns were seen in Type II. All the cases of Type III showed the basal layer pattern. It is suggested that p53 expression of the basal layer pattern is useful for estimating the malignant potency. Parakeratosis demonstrating a tendency toward severe atypism and general disturbance is an important malignant sign.

The present study was designed to examine the bone-titanium interface of the titanium-coated or titanium rod implants inserted for 28 days in the tibiae of 6-week-old rats. Basically, there were hardly differences in the interfaces between the titanium-coated and titanium rod implants. Light microscopically, titanium layer appeared to make direct contact with the mature bone or poorly mineralized layer, and one or a few layers of slender cells were localized contacting the titanium. Ultrastructurally, titanium came in direct contact with either the bone, the poorly mineralized layer consisting of delicate fibril-like structures or the slender cell layer through a thin amorphous zone (20-40nm). This amorphous zone was positive for ruthenium red which has a high affinity for proteoglycans. Part of the interfacial slender cells had abundant rough-ER and glycogen granules as osteoblasts, while some slender cells had well-developed rough-ER but no glycogen, and often endocytosed the titanium fragments, as shown in the fibroblasts.These findings suggest that an amorphous zone consisting of proteoglycans is thus essential for attachment between the slender cell layer, poorly mineralized layer or mature bone and titanium. Furthermore, slender cells close to the implant may also be considered to function as either osteoblasts for bone production adjacent to the implant or as scavengers for the removal of interfacial debris at the bone-titanium interface.

本研究はマラッセの上皮遺残由来の上皮細胞 (MER) と歯根膜線維芽細胞 (PLF) を混合培養することにより, 上皮-間葉の相互関係を検索することである。MERは敷石状に配列するKeratin弱陽性細胞で, PLFは紡錘形のVimentin弱陽性細胞であった。PLFは日数の経過とともに著明に増殖し, その増殖率は級数的であったが, MERの増殖率は緩やかであった。MERをPLF上で培養した群では日数の経過とともに, PLF上でMERの敷石状細胞集団が形成されその領域を増加させていく像が観察された。PLFをMER上で培養した群では集族するPLFの細胞集団とその辺縁から放射状に増殖する細胞が観察されるようになった。しかしその増殖は極めて弱いものであった。PLFおよびMERを混合培養した群では, 2日例でPLFの細胞が主体で, 散在性にMERが観察されたが日数の経過とともにPLFの占有率をしのぐまで増殖していた。以上の所見よりマラッセの上皮遺残由来の細胞は線維芽細胞に比べると緩慢ではあるが徐々に増殖を開始し, 線維性組織の上を覆い, 角化重層扁平上皮の構造をとるようになることが示唆された。

ビーグル成犬に歯槽骨-歯根窩洞を形成した後に再生する歯根膜組織の細胞密度, 血管分布, 増殖細胞核抗原陽性率およびマラッセの上皮遺残の分布と密度を検索した。術後1カ月例で切断部には, 周囲歯槽骨より伸びる細い骨組織が侵入しており, 切断された歯牙との間に歯根膜様組織が再生し, 歯牙の象牙質切断面には上下壁ともにセメント質が沈着していた。細胞成分はセメント質領域で最も多く, 血管は中央部領域で最も高い値を示していた。PCNAに陽性を示す細胞は中央領域に最も多く, これらの結果は対照群とほぼ同様であった。しかし再生歯根膜内には, マラッセの上皮遺残は観察できなかった。2カ月例では部分的に再生し侵入した骨組織と歯牙とが強直を示す例が観察された。以上再生歯根膜内にはマラッセの上皮遺残が観察されずに強直が観察されたことより, マラッセの上皮遺残が歯根膜空隙の恒常性維持に大きく寄与している可能性があると思われた。

Epithelial cell rests of Malassez (ERM) are involved in the maintenance and homeostasis of the periodontal ligament. The objective of this study was to investigate the effect of mechanical stretching on cell growth, cell death and differentiation in the ERM. Cultured porcine ERM were stretched for 24 hr in cycles of 18% elongation for 1 sec followed by 1 sec relaxation. The numbers of cells and TUNEL-positive cells were then counted. The expression of mRNAs encoding gap junction protein α1 (Gja1), ameloblastin, bone morphogenetic protein 2 (BMP2), bone morphogenetic protein 4 (BMP4) and noggin were evaluated using quantitative real-time PCR. The number of cells in the stretching group was approximately 1.3-fold higher than that in the non-stretching controls at 24 hr (p

ビーグル成犬の前臼歯歯根膜内をセメント質領域, 中央領域および歯槽骨領域に三等分し細胞密度および分布について, PKK-1とPCNAを抗体とした免疫組織化学的検索を行った。細胞密度は, セメント質領域で他の部位に比べ有意差をもって多く観察された。血管の占有面積率では, 歯槽骨領域が他の部位に比べ有意差をもって高い値を示していた。増殖細胞の指標となるPCNAに陽性を示す細胞は, 中央領域で他の部位に比べ有意差をもって高い値を示し, 中でも血管周囲に位置したものが多かった。マラッセ上皮遺残はセメント質領域のみにみられ, 占有面積率で約2.32%を占めていた。以上の結果より, セメント質側の細胞の代謝回転は歯槽骨側のそれに比べ非常に活発であることが示唆された。また歯根膜内の芽細胞の供給は血管周囲の細胞から行われ, マラッセの上皮遺残は歯根膜腔の恒常性を調節する可能性が示唆された。

Plant hormones play pivotal signaling roles in plant–pathogen interactions. Here, we report characterization of an antagonistic interaction of abscisic acid (ABA) with salicylic acid (SA) signaling pathways in the rice–Magnaporthe grisea interaction. Exogenous application of ABA drastically compromised the rice resistance to both compatible and incompatible M. grisea strains, indicating that ABA negatively regulates both basal and resistance gene–mediated blast resistance. ABA markedly suppressed the transcriptional upregulation of WRKY45 and OsNPR1, the two key components of the SA signaling pathway in rice, induced by SA or benzothiadiazole or by blast infection. Overexpression of OsNPR1 or WRKY45 largely negated the enhancement of blast susceptibility by ABA, suggesting that ABA acts upstream of WRKY45 and OsNPR1 in the rice SA pathway. ABA-responsive genes were induced during blast infection in a pattern reciprocal to those of WRKY45 and OsPR1b in the compatible rice–blast interaction but only marginally in the incompatible one. These results suggest that the balance of SA and ABA signaling is an important determinant for the outcome of the rice–M. grisea interaction. ABA was detected in hyphae and conidia of M. grisea as well as in culture media, implying that blast-fungus-derived ABA could play a role in triggering ABA signaling at host infection sites.

We investigated cell response, including cell proliferation and expression of heat stress protein and bcl-2, to clarify the influence of low-level [gallium-aluminum-arsenide (Ga-Al-As) diode] laser irradiation on Par-C10 cells derived from the acinar cells of rat parotid glands. Furthermore, we also investigated amylase release and cell death from irradiation in acinar cells from rat parotid glands. The number of Par-C10 cells in the laser-irradiated groups was higher than that in the non-irradiated group at days 5 and 7, and the difference was statistically significant (P < 0.01). Greater expression of heat shock protein (HSP)25 and bcl-2 was seen on days 1 and 3 in the irradiated group. Assay of the released amylase showed no significant difference statistically between the irradiated group and the non-irradiated group. Trypan blue exclusion assay revealed that there was no difference in the ratio of dead to live cells between the irradiated and the non-irradiated groups. These results suggest that low-level laser irradiation promotes cell proliferation and expression of anti-apoptosis proteins in Par-C10 cells, but it does not significantly affect amylase secretion and does not induce rapid cell death in isolated acinar cells from rat parotid glands.

The present study investigated the expression and distribution of osteopontin in the mouse major salivary glands. The level of osteopontin expression in the mouse submandibular gland was higher (12.7-fold) than that in parotid and sublingual glands at the mRNA level. By Western blot analysis, intense positive bands were seen at the predicted molecular mass (about 55 kDa) in all the major salivary glands, while an approximately 30 kDa band of osteopontin was detected only in the submandibular gland. Indirect immunofluorescent and immuno-electron microscopy analyses demonstrated the localization of osteopontin in the luminal (apical) membranes of acinar cells in all the salivary glands. Osteopontin was also localized at the lumen of acini in the submandibular gland. These results suggest that the expression of osteopontin in the submandibular gland is different from that in the parotid and sublingual glands and that osteopontin may be degraded in the mouse submandibular gland.

Simvastatin acid (SVA) has been reported to stimulate bone formation with increased expression of BMP-2. Therefore, immobilization of SVA onto dental implants is expected to promote osteogenesis at the bone tissue/implant interface. The aim of this study was to evaluate the immobilization behavior of SVA onto titanium (Ti), O(2)-plasma treated titanium (Ti + O(2)), thin-film coatings of hexamethyldisiloxane (HMDSO), and O(2)-plasma treated HMDSO (HMDSO + O(2)) by using the quartz crystal microbalance-dissipation (QCM-D) technique. HMDSO surfaces were activated by the introduction of an OH group and/or O(2)-functional groups by O(2)-plasma treatment. In contrast, titanium surfaces showed no appreciable compositional changes by O(2)-plasma treatment. The QCM-D technique enabled evaluation even at the adsorption behavior of a substance with a low molecular weight such as simvastatin. The largest amount of SVA was adsorbed on O(2)-plasma treated HMDSO surfaces compared to untreated titanium, HMDSO-coated titanium, and O(2)-plasma treated titanium. These findings suggested that the adsorption of SVA was enhanced on more hydrophilic surfaces concomitant with the presence of an OH group and/or O(2)-functional group resulting from the O(2)-plasma treatment, and that an organic film of HMDSO followed by O(2)-plasma treatment is a promising method for the adsorption of SVA in dental implant systems.

To examine the influence of cholinergic and beta-adrenergic agents on paracellular transport, we applied confocal microscopy and freeze-fracture to the isolated, perfused submandibular gland of the rat. By confocal microscopy, perfusion of lucifer yellow through an arterial catheter, revealed a bright fluorescence in the basolateral spaces of acini, but not in the intercellular canaliculi. However, addition of isoproterenol on carbachol stimulation, induced lucifer yellow fluorescence in intercellular canaliculi. This finding indicates that isoproterenol is capable of opening the paracellular route. The tight junction strands surrounding intercellular canaliculi were visualized using freeze replicas. Fixation was carried out both by vascular perfusion with Karnovsky's solution and by metal contact rapid freezing with liquid helium. In the chemically-fixed specimens, the strand particles of tight junctions formed 2-5 lines at the P-face along most of the apical portion at rest. With carbachol/isoproterenol stimulation, the strand particles rearranged with free ends and terminal loops. In the rapidly frozen specimens, the strand particles were arranged more irregularly even in the resting state. The meshwork of strands became more disheveled and interrupted during carbachol/ isoproterenol stimulation. The present findings led us to conclude that: 1) the beta-adrenergic agent, isoproterenol, can open the paracellular transport. 2) in the rapidly frozen specimen, the tight junction strand particles are arranged roughly and become disheveled and interrupted during stimulation by carbachol/isoproterenol. These findings may be related to rearrangement of subcellular structures, especially of the actin filament network.

This study evaluated the behavior of osteoblast-like cells on multigrooved surfaces consisting of a combination of microgrooves and macrogrooves. A polystyrene substrate was fabricated with multigrooves with 90-degree, V-shaped microgrooves with a 2-μm pitch cut on trapezoidal macrogrooves, which had a 50-μm ridge width, a 50-μm wall width, a 50-μm bottom width, and 25-μm depth. Smooth polystyrene substrates were also prepared as controls. Rat bone marrow cells were cultured as osteoblast-like cells on the substrates for morphological evaluation using a scanning electron microscope, and for biochemical evaluation using the quantitative reverse transcriptase-polymerase chain reaction technique for osteopontin and osteocalcin mRNA expression. After 8 days of incubation, the osteoblast-like cells were aligned parallel to the surface grooves on the multigrooved substrates. After 16 days of incubation, a dense mineralized extracellular matrix (ECM) was produced along the multigrooves. The ECM on the multigrooved surface appeared oriented more in the direction of the grooves than on the smooth surface, and trapezoid-shaped macrogrooves of the ECM were cast upside down. Although there were not significant differences, the osteopontin and osteocalcin mRNA expressions of the osteoblast-like cells on the multigrooved surfaces tended to be higher than on smooth surfaces. These results suggest that multigrooves could be used to control the orientation of mineralized ECM as well as of cells, and also to enhance the production of mineralized ECM. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 68A: 227–234, 2004

We investigated the expression and distribution of osteopontin in mouse salivary glands. Western blot analysis showed intense positive bands at the predicted molecular mass (about 60 kDa) in mouse parotid and sublingual glands. However, a cross-reacted band around 30 kDa was strongly detected in submandibular glands. Indirect immunofluorescent analysis showed that osteopontin was localized at the luminal (apical) membranes of the acinar cells in parotid and sublingual glands. However, it was not detected in acinar cells of submandibular glands. No expression was found in ductal cells of any glands. We also examined the expression of matrix metalloproteinase (MMP)-3 and -7. In parotid gland, MMP-3 was observed at 57 kDa, indicating a latent form, but MMP-7 was not detected. In contrast, MMP-7 definitely was observed at 28 kDa area in submandibular gland, whereas MMP-3 was not detected. These results suggest that osteopontin localizes at luminal sites of acinar cells and may be associated with saliva secretion in mouse salivary gland. It is also suggested that osteopontin may be cleaved by MMP-7 in mouse submandibular gland.