Hisatomo Kondo

Temporal Changes of mRNA Expression of Matrix Proteins and Parathyroid Hormone and Parathyroid Hormone–Related Protein (PTH/PTHrP) Receptor in Bone Development

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.

Clinical application of removable partial dentures using thermoplastic resin. Part II: Material properties and clinical features of non-metal clasp dentures

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.

Molecular and cell biological properties of mouse osteogenic mesenchymal progenitor cells, Kusa

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 condition inducing mineralization, with ascorbic acid and β-glycerophosphate. Kusa-O, a low osteogenic subline of Kusa, had high alkaline phosphatase activity but slow accumulation of calcium deposits even in the inducing condition. These two Kusa sublines differed in the expression of the osteogenic marker genes, osteocalcin and osteopontin, during 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.

PDGF-induced PI3K-mediated signaling enhances the TGF‑β‑induced osteogenic differentiation of human mesenchymal stem cells in a TGF-β-activated MEK-dependent manner

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.

A simple murine model for immobilization osteopenia.

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.

VEGF-C and TGF-β reciprocally regulate mesenchymal stem cell commitment to differentiation into lymphatic endothelial or osteoblastic phenotypes

The direction of mesenchymal stem cell (MSC) differentiation is regulated by stimulation with various growth factors and cytokines. We recently established MSC lines, [transforming growth factor-β (TGF-β)-responsive SG-2 cells, bone morphogenetic protein (BMP)-responsive SG-3 cells, and TGF-β/BMP-non-responsive SG-5 cells], derived from the bone marrow of green fluorescent protein-transgenic mice. In this study, to compare gene expression profiles in these MSC lines, we used DNA microarray analysis to characterize the specific gene expression profiles observed in the TGF-β-responsive SG-2 cells. Among the genes that were highly expressed in the SG-2 cells, we focused on vascular endothelial growth factor (VEGF) receptor 3 (VEGFR3), the gene product of FMS-like tyrosine kinase 4 (Flt4). We found that VEGF-C, a specific ligand of VEGFR3, significantly induced the cell proliferative activity, migratory ability (as shown by Transwell migration assay), as well as the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 in the SG-2 cells. Additionally, VEGF-C significantly increased the expression of prospero homeobox 1 (Prox1) and lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1), which are lymphatic endothelial cell markers, and decreased the expression of osteogenic differentiation marker genes in these cells. By contrast, TGF-β significantly increased the expression of early-phase osteogenic differentiation marker genes in the SG-2 cells and markedly decreased the expression of lymphatic endothelial cell markers. The findings of our study strongly suggest the following: i) that VEGF-C promotes the proliferative activity and migratory ability of MSCs; and ii) VEGF-C and TGF-β reciprocally regulate MSC commitment to differentiation into lymphatic endothelial or osteoblastic phenotypes, respectively. Our findings provide new insight into the molecular mechanisms underlying the regenerative ability of MSCs.

Effects of apatite particle size in two apatite/collagen composites on the osteogenic differentiation profile of osteoblastic cells.

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.

Examination of the Position Accuracy of Implant Abutments Reproduced by Intra-Oral Optical Impression

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.

Impact of the Static and Radiofrequency Magnetic Fields Produced by a 7T MR Imager on Metallic Dental Materials

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.

Impacts of palatal coverage on bolus formation during mastication and swallowing and subsequent adaptive changes

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.