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Cytokines and their intercellular signals regulate the multipotency of mesenchymal stem cells (MSCs). The present study established the MSC lines SG-2, -3, and -5 from the bone marrow of green fluorescent protein (GFP)-transgenic mice. These cell lines clearly expressed mouse MSC markers Sca-1 and CD44, and SG-2 and -5 cells retained the potential for osteogenic and adipogenic differentiation in the absence of members of the transforming growth factor (TGF)-β superfamily. By contrast, SG-3 cells only retained adipogenic differentiation potential. Analysis of cytokine and cytokine receptor expression in these SG cell lines showed that bone morphogenetic protein (BMP) receptor 1B was most highly expressed in the SG-3 cells, which underwent osteogenesis in response to BMP, while TGF-β receptor II was most highly expressed in SG-3 and -5 cells. However, it was unexpectedly noted that phosphorylation of Smad 2, a major transcription factor, was induced by TGF-β1 in SG-2 cells but not in SG-3 or -5 cells. Furthermore, TGF-β1 clearly induced the expression of Smad-interacting transcription factor CCAAT/enhancer binding protein-β in SG-2 but not in SG-3 or -5 cells. These results demonstrated the establishment of TGF-β-responsive SG-2 MSCs, BMP-responsive SG-3 MSCs and TGF-β/BMP-unresponsive SG-5 MSCs, each of which was able to be traced by GFP fluorescence after transplantation into in vivo experimental models. In conclusion, the present study suggested that these cell lines may be used to explore how the TGF-β superfamily affects the proliferation and differentiation status of MSCs in vivo.

Although the effects of neprilysin (NEP), also called CD10, on the clearance of Alzheimer's disease (AD)-associated amyloid-β (Aβ) have been reported, NEP is not made in the brain, and the mechanism for the transport of NEP to the brain has not been investigated. Our hypothesis is that muscle packages NEP in exosomes in response to a neuromuscular signal and sends it to the brain via retrograde axonal transport. The masseter muscle (MM) and the trigeminal nerve (TGN) are good candidates for this mechanism by virtue of their proximity to the brain. The aim of this study was to trace the NEP protein from the MM, through the TGN, and to the hippocampus (HPC) in muscle contraction models in vitro and in vivo. NEP expression in mouse tissue lysates was analyzed by RT-PCR and Western blot. Four-week-old mice were perfused to remove blood NEP contamination. The MM expressed substantial levels of NEP protein and mRNA. On the other hand, a remarkably high level of NEP protein was measured in the TGN in the absence of mRNA. NEP protein, without the corresponding mRNA, was also detected in the HPC. These results suggested that the MM derived NEP was taken up by the TGN, which in turn permitted NEP access to the central nervous system and within it the HPC. When the MM was induced to contract by electric stimulation in freshly euthanized mice, NEP protein decreased in the MM in a stimulus time-dependent manner, while that in the TGN and the HPC increased sequentially. Furthermore, NIR-labeled exosomes tracked along the same route. Finally, carbachol induced secretion of exosomal NEP in C2C12-derived myotube cells. These results support our hypothesis that MM-derived NEP is transported along the TGN to reach the HPC following electrical or cholinergic stimulation.

Epithelial attachment via the basal lamina on the tooth surface provides an important structural defence mechanism against bacterial invasion in combating periodontal disease. However, when considering dental implants, strong epithelial attachment does not exist throughout the titanium-soft tissue interface, making soft tissues more susceptible to peri-implant disease. This study introduced a novel synthetic peptide (A10) to enhance epithelial attachment. A10 was identified from a bacterial peptide display library and synthesized. A10 and protease-activated receptor 4-activating peptide (PAR4-AP, positive control) were immobilized on commercially pure titanium. The peptide-treated titanium showed high epithelial cell migration ability during incubation in platelet-rich plasma. We confirmed the development of dense and expanded BL (stained by Ln5) with pericellular junctions (stained by ZO1) on the peptide-treated titanium surface. In an adhesion assay of epithelial cells on A10-treated titanium, PAR4-AP-treated titanium, bovine root and non-treated titanium, A10-treated titanium and PAR4-AP-treated titanium showed significantly stronger adhesion than non-treated titanium. PAR4-AP-treated titanium showed significantly higher inflammatory cytokine release than non-treated titanium. There was no significant difference in inflammatory cytokine release between A10-treated and non-treated titanium. These results indicated that A10 could induce the adhesion and migration of epithelial cells with low inflammatory cytokine release. This novel peptide has a potentially useful application that could improve clinical outcomes with titanium implants and abutments by reducing or preventing peri-implant disease.

Objective To investigate the effects of wearing complete dentures on pharyngeal shape for swallowing in edentulous older people. Background In the absence of complete dentures, edentulous older people often lose the occlusal support necessary to position the mandible, which leads to an anterosuperior shift of the mandible during swallowing. This may result in pharyngeal shape changes effecting swallowing function in older people. However, the details of this phenomenon are currently unclear. Materials and methods Participants were 17 older edentulous volunteers. Cone-beam computed tomography imaging was performed with the participant in the seated position and wearing (i) both maxillary and mandibular dentures, (ii) maxillary dentures only and (iii) no dentures. During imaging, participants were instructed to keep their mouth closed to the mandibular position determined in advance during swallowing for each denture-wearing condition. The volume, height and average cross-sectional area of the velopharynx and oropharynx were measured, and the positions of the epiglottis and mandible were recorded. Results While the vertical height of the oral cavity and pharynx significantly decreased, the volume and average cross-sectional area of the oropharynx significantly increased when dentures were not worn (p

The key objective of mastication is to form a food bolus suitable for smooth swallowing. However, chewing is usually performed without a conscious effort. Poor bolus formation can cause pharyngeal residue and suffocation in elderly individuals with reduced swallowing function. Therefore, chewing with a conscious effort may help the bolus to more easily pass the pharynx. This study aimed to clarify the impact of mastication with a conscious effort on the feeding sequence. Subjects included 25 dentulous volunteers who were informed and provided written consent. Lateral videofluoroscopy was performed during the feeding of solid agar jelly under two conditions: chewing naturally in their usual manner (without volition) and chewing with a conscious effort (with volition). Temporal evaluation was performed for mastication, stage II transport (STII), swallow onset and oropharyngeal transit time. Moreover, bolus volume at swallow onset and subjective evaluation of swallowing easiness were measured. Volitional chewing with a conscious effort lengthened the duration of the chewing sequence before and after STII and delayed the swallow onset despite the fact that the bolus volume in the vallecula and hypopharynx (HYP) had significantly increased. Furthermore, with volition, the bolus transit time from swallow onset in the oral cavity, upper oropharynx and HYP was reduced, and subjective evaluation of swallowing easiness demonstrated significant improvement. These results suggest that volitional chewing with a conscious effort can alter bolus transport and swallowing, resulting in easier swallowing.

This position paper proposes a definition and naming standard for removable partial dentures (RPDs) using thermoplastic resin, and presents a guideline for clinical application. A panel of 14 experts having broad experience with clinical application of RPDs using thermoplastic resin was selected from members of the Japan Prosthodontic Society. At a meeting of the panel, “non-metal clasp denture” was referred as the generic name of RPDs with retentive elements (resin clasps) made of thermoplastic resin. The panel classified non-metal clasp dentures into two types: one with a flexible structure that lacks a metal framework and the other having a rigid structure that includes a metal framework. According to current prosthetic principles, flexible non-metal clasp dentures are not recommended as definitive dentures, except for limited cases such as patients with a metal allergy. Rigid non-metal clasp dentures are recommended in cases where patients will not accept metal clasps for esthetic reasons. Non-metal clasp dentures should follow the same design principles as conventional RPDs using metal clasps.

The development of new osteoconductive bone substitute materials is expected in medicine. In this study, we attempted to produce new hydroxylapatite (HAP)/collagen (Col) composites using two HAP particles of different sizes and porcine type I collagen. The two HAP particles were either nano-sized (40 nm in average diameter; n-HAP) or had macro-pore sizes of 0.5–1.0 mm in length with fully interconnected pores (m-HAP). The aim of this study was to investigate the effects of apatite particle size in two HAP/Col composites on the osteogenic differentiation profile in osteoblast-like cells (SaOS-2). We created a collagen control sponge (Col) and two HAP/Col composite sponges (n-HAP/Col and m-HAP/Col) using freeze-drying and dehydrothermal cross-linking techniques, and then punched out samples of 6 mm in diameter and 1 mm in height. The SaOS-2 cells were cultured on three test materials for 1, 2, 3 and 4 weeks. Total RNA was extracted from the cultured cells and the expression of osteogenic differentiation-related genes was evaluated by reverse transcription PCR (RT-PCR) using primer sets of alkaline phosphatase (ALP), type 1 collagen (COL1), bone sialoprotein (BSP) and osteocalcin precursor [bone gamma-carboxyglutamate (gla) protein (BGLAP)] genes, as well as the β-actin gene. The cells were also cultured on Col, n-HAP/Col and m-HAP/Col specimens for 1 and 4 weeks, and were then observed under a scanning electron microscope (SEM). The experimental results were as follows: RT-PCR indicated that osteogenic differentiation, particularly the gene expression of BSP, was most accelerated when the cells were cultured on n-HAP/Col specimens, followed by m-HAP/Col, whilst the weakest accelaeration was observed when the cells were cultured on Col specimens. As shown by the SEM images, the SaOS-2 cells were fibroblastic when cultured on Col specimens for up to 4 weeks; they were fibroblastic when cultured on n-HAP/Col specimens for 1 week, but appeared as spheroids, while actively phagocytizing n-HAP particles at 4 weeks; however, they appeared as deformed fibroblasts when cultured on m-HAP/Col specimens, detached from the particles. Despite limited experimental results, our study suggests that n-HAP/Col may be employed as a new osteoconductive bone substitute material.

Palatal coverage is often required for elderly edentulous patients with complete dentures. The purpose of this study was to clarify impacts of palatal coverage on bolus formation and subsequent adaptive changes. Subjects were 18 healthy young dentulous adults who wore 1·5-mm-thick palatal plates. Subjects were asked to feed 12 g of bicoloured rice as usual, and the bolus formation by mastication and swallowing in the pharynx was observed using a nasal videoendoscopy. The bolus formation index (BFI), number of mastication strokes until swallowing, visual analogue scale about swallowing easiness and masticatory performance using colour-changeable gum were measured under three conditions: before placement of the palatal plate (day 0), immediately after placement (day 1) and after 7 days of wearing the plate (day 7). BFI and visual analogue scale on day 1 were significantly lower than those on day 0, but those on day 7 significantly recovered to the level of day 0. The number of mastication strokes did not change from day 0 to day 1, however, that on day 7 was significantly higher. Masticatory performance on days 1 and 7 was significantly lower than that on day 0. Although palatal coverage inhibits bolus formation during feeding, subjects increased the number of mastication strokes until swallowing threshold as they adapted to palatal coverage over time. This adaptive change was due to compensate for the lowered masticatory performance to achieve bolus formation for comfortable swallowing.

Recently, we identified the scrapie responsive gene 1 (SCRG1) secreted from mesenchymal stem cells (MSCs) and its receptor bone marrow stromal cell antigen 1 (BST1) as positive regulators of stem cell qualities such as self-renewal, migration abilities, and osteogenic differentiation potential. Here, we examined the effect of the paracrine activity of SCRG1 in macrophages. The mouse macrophage-like cell line Raw264.7 expressed BST1/β1 or BST1/β2 integrin as possible SCRG1 receptors. Unexpectedly, recombinant SCRG1 did not enhance cell proliferation, migration, or adhesion in these macrophages. However, further examination of the effect of SCRG1 in Raw264.7 cells did reveal a potent anti-inflammatory effect whereby SCRG1 suppressed LPS-induced CCL22 production. SCRG1 also induced the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in these cells and, moreover, a mitogen-activated protein kinase (MAPK)/ERK kinase inhibitor U0126 significantly suppressed the effect of SCRG1 on LPS-induced chemokine CCL22 production. Taken together, these data indicate that SCRG1 signals through the MAPK pathway and suppresses the LPS signaling pathway. CCL22 is generally known to be chemotactic for monocytes, dendritic cells, natural killer cells and chronically activated T lymphocytes, suggesting that MSC-derived SCRG1 may block infiltration of these cells. A mechanism is proposed by which MSCs play their immunosuppressive role through suppressing chemokine expression in monocyte/macrophage lineage cells.

Wearing dentures and dysphagia are common in older individuals; however, it is still unknown how dentures affect oral and pharyngeal swallowing. The purpose of this study was to reveal the effects of wearing and removing dentures on oropharyngeal movements during pharyngeal swallowing in the feeding sequence of solid food. Participants were 25 edentulous volunteers (nine men, 16 women; mean age 76·2 years) who wore complete dentures. The test food was minced agar jelly containing barium sulphate. Subjects were instructed to feed and swallow the test food with or without dentures during observation using videofluorography. We quantitatively evaluated the range, distance and duration of oropharyngeal movements during pharyngeal swallowing. When dentures were absent, the range of mandible and hyoid movements were significantly expanded in the anterosuperior direction, and the range of laryngeal movement was significantly expanded in the anterior direction. Additionally, the posterior pharyngeal wall contraction and upper oesophageal sphincter opening significantly increased. In addition, the distances of the mandible, hyoid and laryngeal movements and the mandibular duration were significantly extended when dentures were absent. No significant differences were observed in the duration of movements of other organs between wearing and removing dentures. The hyoid bone, larynx, posterior pharyngeal wall and upper oesophageal sphincter do not change their duration of movements when dentures were removed but, rather, expand their range of movement. This might be a spatial change of oropharyngeal movement to avoid temporal changes in pharyngeal swallowing when dentures were absent in edentulous older individuals.

Background It is important to visualize the phenomenon which occurs in actual clinical cases to decide the timing of implant placement subsequently after sinus graft. Although several clinical cases of bone augmentation using xenograft have been evaluated, the number of those reports which have described bone remodeling by alloplasty are not sufficient. Findings In the present report, to investigate bone remodeling histologically after maxillary sinus augmentation with porous hydroxyapatite alloplast, bone cores from the sinus floor of three female nonsmoking patients (aged 64–73 years) were collected 6 months after sinus lift surgery, embedded in methyl methacrylate resin, and prepared by conventional methods. Bone architecture and graft residues were evaluated by micro-computed tomography of the same specimens. Hematoxylin–eosin and Villanueva–Goldner staining revealed mature osteoblasts and multinucleated osteoclasts on the grafted sinus floor and surface of residual hydroxyapatite particles. The particulate interspace was partially filled with osteoid and calcified tissue and showed active vascularization. Conclusion The results suggested that bone regeneration and angiogenesis within and between porous hydroxyapatite particles were sufficiently found after 6 month histologically in the grafted sinus floor. Electronic supplementary material The online version of this article (doi:10.1186/s40064-016-1885-2) contains supplementary material, which is available to authorized users.

An impression technique called optical impression using intraoral scanner has attracted attention in digital dentistry. This study aimed to evaluate the accuracy of the optical impression, comparing a virtual model reproduced by an intraoral scanner to a working cast made by conventional silicone impression technique. Two implants were placed on a master model. Working casts made of plaster were fabricated from the master model by silicone impression. The distance between the ball abutments and the angulation between the healing abutments of 5 mm and 7 mm height at master model were measured using Computer Numerical Control Coordinate Measuring Machine (CNCCMM) as control. Working casts were then measured using CNCCMM, and virtual models via stereo lithography data of master model were measured by a three-dimensional analyzing software. The distance between ball abutments of the master model was 9634.9 ± 1.2 μm. The mean values of trueness of the Lava COS and working casts were 64.5 μm and 22.5 μm, respectively, greater than that of control. The mean of precision values of the Lava COS and working casts were 15.6 μm and 13.5 μm, respectively. In the case of a 5-mm-height healing abutment, mean angulation error of the Lava COS was greater than that of the working cast, resulting in significant differences in trueness and precision. However, in the case of a 7-mm-height abutment, mean angulation errors of the Lava COS and the working cast were not significantly different in trueness and precision. Therefore, distance errors of the optical impression were slightly greater than those of conventional impression. Moreover, the trueness and precision of angulation error could be improved in the optical impression using longer healing abutments. In the near future, the development of information technology could enable improvement in the accuracy of the optical impression with intraoral scanners.

Date Y, Yokoyama Y, Kondo H, Kuroda S, Ohya K, Ota MS, Iseki S, Kasugai S. Restricted expression of chromatin remodeling associated factor Chd3 during tooth root development. J Periodont Res 2012; 47: 180–187. © 2011 John Wiley & Sons A/S Background and Objective: The tooth root is one of the critical parts to maintain tooth function; however, the molecular mechanisms of root development remain unknown. We aimed to identify specific factors for root morphogenesis using a newly developed experimental system. Material and Methods: Tentative cementoblasts and periodontal ligament cells from mouse mandibular molars were isolated using laser capture microdissection. More than 500 cementoblasts and periodontal ligament cells were separately captured. After RNA extraction and amplification, mRNA expression in isolated cementoblasts was compared with that of periodontal ligament cells by cDNA microarray analysis. Then, putative cementoblast-specific genes were subjected to in situ hybridization analysis to confirm the results in mouse mandible. Results: Approximately 2000 genes were differentially expressed between these tissues. Among those genes, zinc finger helicase (ZFH), also termed chromodomain-helicase-DNA-binding protein 3 (Chd3), was one of the highly expressed transcripts in tentative cementoblasts. In situ hybridization revealed that ZFH/Chd3 was strongly expressed in Hertwig’s epithelial root sheath rather than in cementum. Moreover, its expression disappeared when root formation was advanced in the first molar. In contrast, Chd3 was continuously expressed in dental epithelial cells of the cervical loop, in which root extension is never terminated. Conclusion: These results suggest that ZFH/Chd3 might play an important role in tooth root development and subsequent cementogenesis.

Collagen containing bone morphogenetic protein-2 (BMP-2) expression vector, which is called “gene-activated matrix,” promotes bone regeneration when transplanted to the bone defect. We speculated that hydroxyapatite fiber (HF) would be an ideal matrix for “gene-activated matrix” especially for bone regeneration, because it is oseteoconductive and has high affinity to DNA. The purpose of this study is to clarify whether HF containing BMP-2 expression vector induces ectopic bone formation. We prepared HF containing 0, 10, 50, and 100 μg BMP-2 expression vector. Wistar male rats (8 weeks) were used and each rat received two HF implants in the left and right dorsal muscle. The rats were sacrificed 4, 8, and 12 weeks after the operation, and implants were analyzed radiographically by softex, dual-energy X-ray absorptiometry, and they were histologically examined. At 4 weeks, HF containing 50 or 100 μg BMP-2 expression vector showed high bone mineral contents and large radiopaque volume compared to the other implants. At 8 and 12 weeks, HF containing 50 μg BMP-2 expression vector exerted the highest values in the radiographic analyses. Bonelike tissue was histologically observed in HF containing 50 and 100 μg BMP-2 expression vector groups but not detected in the other implants. The present results suggest that HF is potential as a matrix for “gene-activated matrix” for bone tissue engineering. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2009

The detailed features of cementum and PDL remain unknown and specific markers for cemento- blasts and PDL cells have not been identified. Moreover, the molecular mechanism of periodontal tissue development, homeostasis and regeneration is also obscure. Since cementum or PDL tissue in previous studies was usually obtained by manual curettage, it has been difficult to isolate pure cementum or PDL. In the current study, laser capture microdissection(LCM)of undecalcified frozen sections of murine mandible was employed for precise tissue isolation and to obtain intact RNA from cementoblasts and PDL cells of the murine mandible. Over 500 tentative cementoblasts and PDL cells were individually captured using an infra- red laser. A bioanalyzer detected peaks of 18S and 28S rRNA, suggesting that the quality of RNA was sufficient. Subsequently, mRNA expression in cementoblasts was compared with that in PDL cells by GeneChip analysis, showing that approximately 2,000 genes were differently expressed between those tissues. Serial cementoblast-positive and PDL-negative genes were subjected to real-time RT-PCR analy- sis to support GeneChip results, using mandible and femur RNA samples. Several genes were expressed at higher levels in the mandible than in the femur, suggesting that several genes found in this study might be candidates for cementum-specific markers. In this study, a novel experimental system was established to isolate target tissues from undecalcified frozen sections at the cell unit level and to obtain intact RNA. Our methodologies could help to guide further investigation of mineralized tissues and to explore tissue-specific factors.

Periosteum has been demonstrated to contain mesenchymal progenitor cells differentiating to osteoblasts, and both bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) may play important roles in cell-based approaches to bone regeneration. The purpose of this study was to evaluate the feasibility and efficacy of BMP-2 and/or VEGF on periosteal cell differentiation to osteoblasts in vitro and ectopic bone formation in vivo. Human periosteum-derived cells were transfected with BMP-2, VEGF, BMP-2 + VEGF, or vehicle as a control by non-viral gene transfer and then cultured and implanted to nude mice intramuscularly. Real-time polymerase chain reaction analysis of the culture revealed that transgenes for BMP-2 and BMP-2 + VEGF induced more mRNA expression of alkaline phosphatase, collagen type I, and osteocalcin than VEGF and vehicle treatments; additionally, alizarin red S staining, alkaline phosphatase staining, and alkaline phosphatase activity were significantly higher in the BMP-2 + VEGF transgene than in the other versions. After implantation, ectopic bone was observed at 4 weeks and greatly increased at 8 weeks in all groups. In particular, the combination of BMP-2 and VEGF formed significantly more bone at 4 weeks, and VEGF transfection resulted in more blood vessels relative to the conditions without VEGF. Thus, VEGF might enhance BMP2-induced bone formation through modulation of angiogenesis. Keywords:: tissue engineering, periosteal cell, bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), gene transfer

Purpose We examined safety issues related to the presence of various metallic dental materials in magnetic resonance (MR) imaging at 7 tesla. Methods A 7T MR imaging scanner was used to examine 18 kinds of materials, including 8 metals used in dental restorations, 6 osseointegrated dental implants, 2 abutments for dental implants, and 2 magnetic attachment keepers. We assessed translational attraction forces between the static magnetic field and materials via deflection angles read on a tailor-made instrument and compared with those at 3T. Heating effects from radiofrequency during image acquisitions using 6 different sequences were examined by measuring associated temperature changes in agarose-gel phantoms with a fiber-optic thermometer. Results Deflection angles of the metallic dental materials were significantly larger at 7T than 3T. Among full metal crowns (FMCs), deflection angles were 18.0° for cobalt-chromium (Co-Cr) alloys, 13.5° for nickel-chromium (Ni-Cr) alloys, and 0° for other materials. Deflection angles of the dental implants and abutments were minimal, ranging from 5.0 to 6.5°, whereas the magnetic attachment keepers were strongly attracted to the field, having deflection angles of 90° or more. Increases in temperature of the FMCs were significant but less than 1°C in every sequence. The dental implant of 50-mm length showed significant but mild temperature increases (up to 1.5°C) when compared with other dental implants and abutments, particularly on sequences with high specific absorption rate values. Conclusion Although most metallic dental materials showed no apparent translational attraction or heating at 7T, substantial attraction forces on the magnetic attachment keepers suggested potential risks to patients and research participants undergoing MR imaging examinations.

Gene-activated matrix (GAM) is a matrix, such as collagen-containing plasmid vector, that encodes a protein to stimulate tissue regeneration. In the original GAM system, gene transfer efficiency was extremely low. We have recently reported that modifying GAM with calcium-phosphate precipitates (CaP) enhances the efficiency of gene transfer. The purpose of this study was to evaluate the effects of our modified GAM on tissue regeneration. We prepared critical size segmental bone defects in rat tibiae and transplanted GAM consisting of bovine atelocollagen and expression plasmid vector (bmp2), which encodes human BMP2, with or without CaP. The tibiae were later examined radiographically, histologically, and mechanically. Implantation of bmp2-CaP-collagen at 12 microg bmp2 bridged the bone defect at 4 weeks, and the strength of the bone was comparable to that of an intact tibia at 6 weeks. Implantation of bmp2-collagen at the same dose of bmp2 bridged the defect to a smaller extent. Neither collagen alone nor vacant vector-CaP-collagen bridged the defect. These results indicate that our modified GAM with CaP has the potential to be effective in tissue regeneration at lower plasmid DNA doses than used in previous studies.

A cell line of murine osteogenic progenitor cells, Kusa, was established from femoral bone marrow stromal cells with other types of mesenchymal progenitor cells. We characterized two sublines of Kusa (Kusa-A1 and Kusa-O) from several aspects, including the use of an expression profiling system, a cDNA microarray. The original Kusa subline (Kusa-A1) had high alkaline phosphatase activity and high accumulation of calcium deposits in a condi- tion inducing mineralization, with ascorbic acid and � - glycerophosphate. Kusa-O, a low osteogenic subline of Kusa, had high alkaline phosphatase activity but slow accu- mulation of calcium deposits even in the inducing condition. These two Kusa sublines differed in the expression of the osteogenic marker genes, osteocalcin and osteopontin, dur- ing mineralization. A type of cDNA microarray revealed marked downregulation of gene expression in the inducing condition in both Kusa-A1 and Kusa-O. Another type of high-throughput microarray was performed to examine the difference in gene expression patterns between Kusa-A1 and Kusa-O. By this analysis, periostin, which would be involved in a stage of osteogenesis, was low in Kusa-A1. On the contrary, Myocyte enhancer factor 2C (MEF2C), a myogenic transcriptional factor, was high in Kusa-A1, although no expression of any other myogenic genes was shown.

In vivo gene transfer with plasmid vector has been applied experimentally and clinically; however, the low level of gene transfer efficiency with plasmid vector is a problem. We speculated that the combination of calcium phosphate precipitate (CaP) and plasmid vector could solve this problem because CaP stabilizes plasmid DNA. In the present study, we used a plasmid exression vector encoding enhanced green fluorescent protein and combined the vector with CaP. Then, this combination was mixed with bovine type I atelocollagen. After incubating this mixture in phosphate-buffered saline, the amount of the plasmid DNA in the supernatant was low when the plasmid DNA was combined with CaP. Furthermore, the plasmid DNA, which was combined with CaP, was stable in DNase digestion in vitro. The plasmid vector with or without CaP, together with the atelocollagen, was transplanted subcutaneously or injected in the bone marrow of the femurs of rats. Then, the fluorescence was observed under a confocal laser scanning microscope and the fluorescence intensity in the tissue homogenates was measured. In these animal experiments, the fluorescence was extensive when the plasmid DNA was combined with CaP. These results indicate that our formula, collagen/CaP/DNA, appeared efficient for in vivo gene transfer. Keywords:: calcium phosphate, plasmid, gene transfer, atelocollagen, enhanced green fluorescent protein

Reduction of loading force to bone induces osteopenia. Although the tail suspension model is the frequently used osteopenia model, this model burdens the animals with nonphysiologic blood distribution and systemic stress. We developed a new simple animal model for osteopenia under reduced loading. Both hind legs of male ICR mice (8 weeks old) in the experimental group were inserted into plastic tubes, which then were connected with wires of three sizes. This apparatus completely immobilized both femurs while allowing the tibias to move. Animals were pair-fed and sacrificed on Days 3, 7, 10, and 14 after immobilization. Bone mineral density measurement with dual energy xray absorptiometry revealed bone loss in the immobilized femurs. Histomorphometric analysis showed increased bone resorption and decreased bone formation, which started from Day 7 and continued until Day 14, resulting in structural disorders in the cancellous bone. Osteoclast population increase before osteoblast population decrease revealed that osteoclasts initially affect the process of this type of osteopenia. Our immobilization model is simple, easy to use, well-tolerated by the animal, and has potential for evaluating the therapeutic effects of drugs to treat osteopenia caused by reduced loading.

INTRODUCTION: Although previous studies have shown that maxillary molar extraction in ovariectomized (OVX) animals causes mandibular loss of bone, it is still questionable as to whether estrogen deficiency affects mandibles with functional occlusion. MATERIALS and METHODS: To answer this question, 13-week-old female Sprague‐Dawley rats were bilaterally OVX or sham-operated. After 109 days, the bone mineral density (BMD) of the femurs and mandibles was measured using dual-energy X-ray absorptiometry (DEXA) and peripheral quantitative computed tomography (pQCT). RESULTS: In DEXA analysis, although the BMD of the total mandible of the OVX rats was similar to that of the sham-operated rats, the BMD of the condylar region in the OVX rats had decreased by 12.5%. In pQCT analysis, decrease in trabecular BMD of the mandibular bone was detectable but low in the molar region (maximal 13%), whereas no difference was seen in cortical BMD. In the femurs, the trabecular bone prominently decreased in OVX rats (30% decrease in pQCT analysis) as previously reported. CONCLUSION: This study revealed regional differences in the mandibular bone decrease in OVX rats. Although the mechanism of low susceptibility of the mandible to estrogen-deficient conditions remains unknown, it is likely that mechanical stress derived from functional occlusion is preventing bone loss in this pathological condition. Furthermore, this study demonstrated the advantage of pQCT in analyzing rat mandibular bone. Oral Diseases (2003) 9, 24–28

Some dental implants are coated with hydroxyapatite (HA), which preferentially binds to bone. Several matrix proteins have an arginine-glycine-aspartic acid (RGD) sequence where cells attach via an integrin receptor. We hypothesized that coating an HA surface with an RGD-containing peptide might enhance the attachment and differentiation of osteoblasts. The HA disks (diameter 34 mm, thickness 1 mm) were treated with a solution (50 mM Tris/HCl and 150 mM NaCl, pH 7.4) containing the peptide EEEEEEEPRGDT, in which the E repetition exerts a high affinity to HA. After washing with phosphate-buffered saline, KUSA/A1 mouse osteoblastic cells were inoculated onto the HA surface and cultured. After 30 min, the number of cells attached to the surface was counted. The DNA content and alkaline phosphatase (ALP) activity were measured after 10 days in culture. Expression of bone matrix proteins was also examined by means of reverse transcriptase-polymerase chain reaction at 7 days; the mineralized area of the culture was also evaluated by staining with Alizarin Red S after 10 days. Treatment with the peptide stimulated cell attachment and increased DNA content and ALP activity. Furthermore, matrix protein expression and mineralized nodule formation were enhanced to a greater extent on the peptide-treated surface than on the nontreated surface. Our results indicate that coating an HA surface with RGD-containing peptide enhances osteoblast attachment and differentiation. This peptide treatment of HA-coated implants may stimulate the osseointegration of the implants.

One G protein-coupled receptor (GPCR) can activate more than one G protein, but the physiologic importance of such activation has not been demonstrated in vivo. We have generated mice expressing exclusively a mutant form of the PTH/PTHrP receptor (DSEL) that activates adenylyl cyclase normally but not phospholipase C (PLC). DSEL mutant mice exhibit abnormalities in embryonic endochondral bone development, including delayed ossification and increased chondrocyte proliferation. Analysis of the differentiation of embryonic metatarsals in vitro shows that PTH(1–34) and forskolin inhibit, whereas active phorbol ester stimulates, hypertrophic differentiation. Thus, PLC signaling via the PTH/PTHrP receptor normally slows the proliferation and hastens the differentiation of chondrocytes, actions that oppose the dominant effects of PTH/PTHrP receptors and that involve cAMP-dependent signaling pathways.

This position paper reviews physical and mechanical properties of thermoplastic resin used for non-metal clasp dentures, and describes feature of each thermoplastic resin in clinical application of non-metal clasp dentures and complications based on clinical experience of expert panels. Since products of thermoplastic resin have great variability in physical and mechanical properties, clinicians should utilize them with careful consideration of the specific properties of each product. In general, thermoplastic resin has lower color-stability and higher risk for fracture than polymethyl methacrylate. Additionally, the surface of thermoplastic resin becomes roughened more easily than polymethyl methacrylate. Studies related to material properties of thermoplastic resin, treatment efficacy and follow-up are insufficient to provide definitive conclusions at this time. Therefore, this position paper should be revised based on future studies and a clinical guideline should be provided.

Summary Prosthetic treatment with dentures is often required for the elderly who have reduced swallowing function. Therefore, it is important to understand the relationship between denture-wearing and feeding function from the perspective of swallowing. To clarify changes in bolus transport during feeding in elderly edentulous patients with or without complete dentures. Subjects were 15 elderly edentulous volunteers who were treated with maxillary and mandibular complete dentures. The test food was 10 g of minced agar jelly containing barium sulphate with a particle diameter of 4·0–5·6 mm. Lateral videofluoroscopy was performed to assess the position of the leading edge of the bolus, the bolus volume in each area at swallow onset, bolus transit time and the mandibular position during pharyngeal swallowing. There were significant changes between the bolus transport with and without dentures. Without dentures, the leading edge of the bolus at swallow onset fell from the valleculae area to the hypopharynx, and the bolus volume in the hypopharynx increased. Bolus transit time increased in the oral cavity, valleculae and hypopharynx. The mandibular position shifted anterosuperior direction. The results arose owing to anatomical changes in the oral and pharyngeal structure and the following functional changes: poor food manipulation, poor bolus formation and delayed swallowing reflex. Removing dentures in elderly edentulous individuals influences bolus transport during feeding, resulting in the exacerbation of the reduced swallowing reserve capacity that accompanies ageing, and may increase the risk of dysphagia.

Transforming growth factor-β (TGF-β) is a critical regulator of osteogenic differentiation and the platelet-derived growth factor (PDGF) is a chemoattractant or mitogen of osteogenic mesenchymal cells. However, the combined effects of these regulators on the osteogenic differentiation of mesenchymal cells remains unknown. In this study, we investigated the effects of TGF-β and/or PDGF on the osteogenic differentiation of human mesenchymal stem cells (hMSCs). The TGF-β-induced osteogenic differentiation of UE7T-13 cells, a bone marrow-derived hMSC line, was markedly enhanced by PDGF, although PDGF alone did not induce differentiation. TGF-β induced extracellular signal-regulated kinase (ERK) phosphorylation and PDGF induced Akt phosphorylation. In addition, the mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor, U0126, suppressed the osteogenic differentiation induced by TGF-β alone. Moreover, U0126 completely suppressed the osteogenic differentiation synergistically induced by TGF-β and PDGF, whereas the phosphoinositide-3-kinase (PI3K) inhibitor, LY294002, only partially suppressed this effect. These results suggest that the enhancement of TGF-β-induced osteogenic differentiation by PDGF-induced PI3K/Akt-mediated signaling depends on TGF-β-induced MEK activity. Thus, PDGF positively modulates the TGF-β-induced osteogenic differentiation of hMSCs through synergistic crosstalk between MEK- and PI3K/Akt-mediated signaling.

Denture adhesives, characterized as medical products in 1935 by the American Dental Association, have been considered useful adjuncts for improving denture retention and stability. However, many dentists in Japan are hesitant to acknowledge denture adhesives in daily practice because of the stereotype that dentures should be inherently stable, without the aid of adhesives. The aim of this study is to verify the efficacy of denture adhesives to establish guidelines for Japanese users. The null hypothesis is that the application of denture adhesives, including the cream and powder types, or a control (isotonic sodium chloride solution) would not produce different outcomes nor would they differentially improve the set outcomes between baseline and day 4 post-application. This ten-center, randomized controlled trial with parallel groups is ongoing. Three hundred edentulous patients with complete dentures will be allocated to three groups (cream-type adhesive, powder-type adhesive, and control groups). The participants will wear their dentures with the denture adhesive for 4 days, including during eight meals (three breakfasts, two lunches, and three dinners). The baseline measurements and final measurements for the denture adhesives will be performed on the first day and after breakfast on the fourth day. The primary outcome is a general satisfaction rating for the denture. The secondary outcomes are denture satisfaction ratings for various denture functions, occlusal bite force, resistance to dislodgement, masticatory performance, perceived chewing ability, and oral health-related quality of life. Between-subjects comparisons among the three groups and within-subjects comparisons of the pre- and post-intervention measurements will be performed. Furthermore, a multiple regression analysis will be performed. The main analyses will be based on the intention-to-treat principle. A sample size of 100 subjects per group, including an assumed dropout rate of 10 %, will be required to achieve 80 % power with a 5 % alpha level. This randomized clinical trial will provide information about denture adhesives to complete denture wearers, prosthodontic educators, and dentists in Japan. We believe this new evidence on denture adhesive use from Japan will aid dentists in their daily practice even in other countries. ClinicalTrials.gov NCT01712802 . Registered on 17 October 2012.

The aim of this study was to produce epithelial attachment on a typical implant abutment surface of smooth titanium. A challenging complication that hinders the success of dental implants is peri-implantitis. A common cause of peri-implantitis may results from the lack of epithelial sealing at the peri-implant collar. Histologically, epithelial sealing is recognized as the attachment of the basement membrane (BM). BM-attachment is promoted by activated platelet aggregates at surgical wound sites. On the other hand, platelets did not aggregate on smooth titanium, the surface typical of the implant abutment. We then hypothesized that epithelial BM-attachment was produced when titanium surface was modified to allow platelet aggregation. Titanium surfaces were coated with a protease activated receptor 4-activating peptide (PAR4-AP). PAR4-AP coating yielded rapid aggregation of platelets on the titanium surface. Platelet aggregates released robust amount of epithelial chemoattractants (IGF-I, TGF-β) and growth factors (EGF, VEGF) on the titanium surface. Human gingival epithelial cells, when they were co-cultured on the platelet aggregates, successfully attached to the PAR4-AP coated titanium surface with spread laminin5 positive BM and consecutive staining of the epithelial tight junction component ZO1, indicating the formation of complete epithelial sheet. These in-vitro results indicate the establishment of epithelial BM-attachment to the titanium surface.

Fibroblast growth factor 4 (FGF-4) plays important roles in bone development during embryogenesis. Human FGF-4 consists of 206 amino acid residues. We produced amino-terminally truncated rhFGF-4, named rhFGF-4(73-206), that increases bone mineral density when systemically administered to mice. In the present study, we examined the effects of rhFGF-4(73-206) in bone after local administration. We injected 1 microg of rhFGF-4(73-206) into tibiae of 10-week-old Sprague-Dawley rats. At days 4 and 7, sets of animals were subjected for tibial bone marrow cell culture in an osteogenic medium. The bone marrow cells from FGF-4-injected tibiae produced more alkaline phosphatase-positive cells and mineralized nodules than those from control tibiae, indicating that local injection of rhFGF-4(73-206) increased the osteoblastic population in the bone marrow. The remaining sets of animals were killed on days 7 and 10. The tibiae were then analyzed with soft X-ray, dual energy X-ray absorptiometry, peripheral quantitative computed tomography, and histomorphometry. The radiographic analyses revealed increases in trabecular bone in the tibiae but no difference in the cortical bone between the rhFGF-4(73-206) group and the control group. High bone turnover and a derived increase of trabecular bone mineral density were demonstrated by histomorphometry in the rhFGF-4(73-206) group. The present results indicate that local injection with rhFGF-4(73-206) elicited an increase in the osteogenic cell population in the tibial bone marrow, which resulted in more trabecular bone.

Adhesive restorative systems have expanded the range of possibilities for direct pulp-capping technique, with evidences of clinical success in vital pulp therapy. However, quite few studies have described the direct responses of pulp cells following the application of resinous materials to pulp exposure. To address this issue, effects of exposure to an adhesive resin, 4-methacryloxyethyl trimellitate anhydride/methyl methacrylate-tri-n-butyl borane (4-META/MMA-TBB) resin on cellular activity were investigated in an established rat dental pulp cell line (RPC-C2A). RPC-C2A cells were cultured on normal plastic plates or the disks prepared from 4-META/MMA-TBB resin (Super Bond CB) in a-MEM containing 10% FBS. After 3, 7 and 14 days, DNA content and alkaline phosphatase (ALP) activity were measured. Total RNA in each group was extracted and RT-PCR analysis was performed. Moreover, the live cell ratio was also evaluated by cytotoxicity assay after treatment with various concentrations of 4-META/MMA-TBB. At day 3, 7 and 14, amount of DNA and ALP activity of the cells on normal plastic plates and the one on the 4-META/MMA-TBB were comparable. Cells of both groups expressed mRNA of type I collagen (Coll), ALP, osteopontin (OPN), osteocalcin (OC), and bone morphogenetic protein (BMP-2). Furthermore, 4-META/MMA-TBB (10(-1)% or less) did not influence dead cell ratio in the confluent state. According to the results of these in vitro studies, exposure to this resinous material would not induce cytotoxic response in the pulp cells.

Objective: Enamel matrix derivative (EMD) has been clinically used to promote periodontal tissue regeneration. The purpose of the present study is to clarify EMD affects on osteoblastic cells and bone regeneration. Materials and Methods: Mouse osteoblastic cells (ST2 cells and KUSA/A1 cells) are used in culture experiments. After cells were treated with EMD, cell growth was evaluated with DNA measurement, 5-bromo-2′-deoxyurydine (BrdU) incorporation assay. Measurement of alkaline phosphatase (ALP) activity and mineralized-nodule (MN) formation, Northern blotting analysis and zymography are also performed. In addition, EMD was applied to a rat skull defect and the defect was radiographically and histologically evaluated 2 weeks after the application. Results: EMD did not stimulate ST2 cell growth; however, it enhanced KUSA/A1 cell proliferation. Although EMD stimulated ALP activity in both the cells, ALP activity in KUSA/A1 cells was affected to a much greater degree. Corresponding to the increase in ALP activity, MN formation in KUSA/A1 cells was enhanced by EMD. EMD stimulated osteoblastic phenotype expression of KUSA/A1 cells such as type I collagen, osteopontin, transforming growth factor beta 1 and osteocalcin. EMD treatment also stimulated matrix metalloproteinase production in KUSA/A1 cells. Although the effects of EMD on osteoblastic cells depend on cell type, the overall effect of EMD on osteoblastic cells is stimulatory rather than inhibitory. Finally, EMD application to a rat skull defect accelerated new bone formation. Conclusion: These results indicate that EMD affects osteoblastic cells and has potential as a therapeutic material for bone healing.

Expression of parathyroid hormone and parathyroid hormone-related protein (PTH/PTHrP) receptor is one of the osteoblastic phenotypes; however, it has not been clear whether this phenotype expression is a marker of immature or mature osteoblasts. We examined the temporal expression pattern of PTH/PTHrP receptor in bone development in vivo and in vitro compared with the expression of other osteoblastic phenotypes: osteopontin (OPN), bone sialoprotein (BSP) and osteocalcin (OC), alkaline phosphatase (ALP), and mineralization. Total RNA was extracted from rat calvariae, and cell culture of rat bone marrow at different developmental stages and then Northern blot hybridization were performed. Mineralization was detected with contact microradiography (CMR) in calvaria or with Alizarin Red S staining in bone marrow cell culture. Both in calvaria and in marrow cell culture, extensive expression of OPN, BSP, type I collagen (COL I), and ALP coincided with the onset of mineralization, and OC expression was observed after mineralized tissue formation. Notably, PTH/PTHrP receptor was expressed at an early developmental stage (prenatal day 14 in calvaria, day 5 in culture) when mineralized tissue was not formed and other osteoblastic phenotypes were scarcely detected. Further study in cell culture revealed that the fold increase in cyclic adenosine monophosphate (cAMP) in response to PTH was elevated with the advance in the culture stage. These results indicate that mRNA expression of PTH/PTHrP receptor could be the early differentiation marker in osteoblastic lineage and that the levels of cAMP production in response to PTH represent the stage of osteoblastic differentiation.

Background: Resonance frequency analysis (RFA) is applied to evaluate implant stability, and the clinical relevance of this application is accepted. However, a discrepancy between resonance frequency (RF) and other parameters of implant stability such as implant insertion torque and bone mineral density at the insertion site has been reported. In addition, the relation between RF and histological implant–bone contact has not been well documented. Purpose: To explain this discrepancy and to clarify the relation between RF and histological implant–bone contact, we conducted the present study. Materials and methods: A hydroxyapatite-coated implant, 4 mm diameter and 10 mm length, was used. We placed the implant in a small plastic box vertically and fixed the implant in the box with small screws at different height positions. An ‘Osstell’ transducer was mounted on the implant and RF was measured with or without loosening the screws. Twenty-four implants were placed in the tibiae of four mini-pigs. The animals were sacrificed 1, 2 and 4 weeks after the placement, and the RF of each implant was measured. Ground sections were prepared and implant–bone contact was histomophometrically measured. Results: Loosening the screw at the neck region of the implant remarkably decreased RF compared with the screws of the other regions. Correlation between RF and implant–bone contact, which was measured all around the implant, was not significant (r=0.221, P=0.299). However, the correlation coefficient increased to ‘0.361’ when implant–bone contact was measured at the neck of the implant (P=0.0835), although these two parameters were not statistically correlated. Conclusions: Although RF did not correlate with histological implant–bone contact, the present results demonstrated that a connection between the implant and bone at the neck region of the implant affects RF the most effectively, further suggesting the superiority of RFA in the process of implant treatment and the follow-up. The present results could explain the discrepancy between RFA and other parameters of implant stability.