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This case report describes the treatment of a woman who lost a central incisor. The socket developed severe tissue defects. She rejected hard and soft tissue management and the use of biomaterials. The lateral incisor was moved mesially with orthodontic treatment. The tissue defects were filled with the alveolar bone of the moved tooth and adequate bone volume was generated behind it. An implant was placed in the space that was generated without any tissue augmentation. The moved tooth had sound periodontal tissue and was restored without preparation. The horizontal tooth movement enabled an esthetic outcome with minimal intervention.

Objective In this study, we investigated the effects of enamel matrix derivative (EMD) on osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) expression of osteoblasts in the presence of lipopolysaccharide (LPS). Study design OPG and RANKL gene expression and protein synthesis of MC3T3-E1 osteoblastic cells were examined by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Results LPS inhibited OPG gene expression and protein synthesis, and stimulated RANKL gene expression and soluble RANKL synthesis. EMD enhanced OPG gene expression and protein synthesis, and inhibited RANKL gene expression and soluble RANKL synthesis. Furthermore, EMD neutralized the effects of LPS on OPG and RANKL expression in osteoblasts. Conclusions EMD might regulate the function of osteoblasts by elevating the ratio of OPG/RANKL gene expression, which is downregulated by LPS, and suppress the induction of osteoclastogenesis. Thereby, EMD might contribute to periodontal tissue regeneration.

Dentin phosphoprotein, the major noncollagenous protein in dentin, has effects on differentiation of odontoblast-like cells. This study was designed to investigate the effect of the protein on apoptosis of the cells. The odontoblast-like cells were prepared from the pulp cells of rat incisors. Apoptosis was detected by measuring caspase-3 activity by using DEVD-AMC as a fluorescent substrate. The cells formed calcification nodules in the presence of 2-glycerophosphate and expressed dentin sialophosphoprotein. Apoptosis was not observed in the cells through the differentiation stages. Then, apoptosis was induced by raising inorganic phosphate concentration in the medium. Elevation of phosphate concentration to 5 mM reduced the number of viable cells and increased caspase-3 activity, indicating the induction of apoptosis. Addition of bovine dentin phosphoprotein in the medium suppressed phosphate-induced apoptosis. Phosvitin and poly(Asp) had similar antiapoptotic effects.

OBJECTIVES: Platelet-derived growth factor (PDGF)-BB is a polypeptide growth factor which has been shown to stimulate periodontal regeneration. In this study, we investigated the time- and dose-dependent effect of PDGF-BB on the proliferation and collagen synthesis of human periodontal ligament (PDL) cells. MATERIALS AND METHODS: For the proliferation assay, PDL cells were cultured in 0.01–10 ng ml−1 of PDGF-BB for 12 or 24 h, and cell numbers were counted. For the collagen synthesis assay, PDL cells were cultured in 0.1–10 ng ml−1 of PDGF-BB for 1 to 24 h. The ratio of collagen content in total protein was evaluated, and the gene expression of type I collagen was assessed quantitatively by Northern blotting analysis. RESULT AND CONCLUSIONS: PDGF-BB stimulated the proliferation of PDL cells in a time- and dose-dependent manner with the maximum effect at 10 ng ml−1. PDGF-BB induced the collagen synthesis of PDL cells with the maximum effect for 24-h treatment, and 1 ng ml−1 of PDGF-BB. PDGF-BB exhibits an inverse dose-dependent effect on proliferation and collagen synthesis by PDL cells. These findings suggest that PDGF-BB is one of the important regulators of the maintenance of the extracellular matrix in PDL, and may play an important role in the regeneration of PDL.

In this study, we investigated the effect of type I collagen on dentin matrix protein-1 (Dmp-1) and osteocalcin (OCN) gene expression of dental pulp cells. The mRNA level of Dmp-1 gene was down-regulated; however, OCN gene expression was up-regulated by the culture of dental pulp cells with type I collagen. These findings imply that type I collagen regulates mRNA level of Dmp-1 and OCN gene that are predominantly expressed in active odontoblasts. The change of gene expression by type I collagen was suppressed by the blocking of collagen-integrin interaction. We could conclude that the effect of type I collagen was mediated via binding of collagen to integrin receptors.

A method of individual vehicle detection using grayscale images acquired from a high position is proposed for guidance of incoming vehicles to vacant cells in a parking lot and other similar purposes. With the proposed method, each image region corresponding to a cell is fragmented according to density (gray level), and the distribution of segment area is analyzed to decide if a vehicle is present. Reference images taken in vacant state are not needed, hence the method can be easily applied to parking lots in continuous service. Shape features are not employed, hence detection is performed independent of car shape. The proposed method was tested on an actual outdoor parking lot during 4 days with different weather conditions from sunrise through sunset. The results confirmed the efficiency of the proposed method, with the detection rate being over 98.7%. © 2001 Scripta Technica, Electron Comm Jpn Pt 3, 84(10): 25–34, 2001

In this study, we demonstrated that type I collagen matrix induced the expression of osteoblastic phenotypes of bone marrow cells, and that antibone sialoprotein (BSP) monoclonal antibody suppressed the expression of these phenotypes. On the other hand, BSP accelerated the expression of osteoblastic phenotypes of bone marrow cells. The adherent bone marrow cells were harvested from rat femur and cultured on type I collagen matrix gels in medium containing 15% fetal calf serum, neither beta-glycerophosphate nor glucocorticoid. Cells showed osteoblastic phenotypes (high alkaline phosphatase activity, osteocalcin synthesis, and responsiveness against parathyroid hormone) on collagen matrix gels at week 3 after the inoculation, and simultaneously, BSP was detected in the conditioned medium by Western blotting using an anti-BSP monoclonal antibody. However, cells in the conventional culture dishes did not show osteoblastic phenotypes during the experimental period. To investigate the physiological function of BSP in osteoblastic differentiation, bone marrow cells were cultured on collagen matrix with an anti-BSP monoclonal antibody for 3 weeks. This treatment suppressed the expression of the osteoblastic phenotypes, and the effect of the antibody was abolished by the addition of bovine bone BSP. Furthermore, bovine bone BSP stimulated the expression of osteoblastic phenotypes of bone marrow cells. Our results indicate that BSP plays a crucial role in the expression of osteoblastic phenotypes of bone marrow cells.

In this paper, the authors describe a vehicle parking space monitoring system which uses a CCD camera. Image processing is used to report the presence of a vehicle in the individual spaces. The system is able to operate under various weather conditions and in both full daylight and artificial night lighting.

Bone marrow cells are multipotent cells. When bone marrow cells were cultured with type I collagen matrix gels, they showed high alkaline phosphatase activity, collagen synthesis, and formed mineralized tissues. Furthermore, cells expressed osteocalcin and bone sialoprotein genes, which are osteoblast-specific genes. These findings indicate that type I collagen matrix gels induce osteoblastic differentiation of bone marrow cells. Type I collagen interacts with the α 2 β 1 integrin receptor on the cell membrane and mediates extracellular signals into cells. DGEA peptide is a cell-binding domain of type I collagen molecule. When collagen–integrin interaction was interrupted by the addition of Asp-Gly-Glu-Ala (DGEA) peptide to the culture, the expression of osteoblastic phenotypes of bone marrow cells was inhibited. Furthermore, anti-α 2 integrin antibody, which interacts with α subunit of integrin and blocks the binding of integrin with collagen, suppressed the expression of osteoblastic phenotypes. These findings imply that collagen-α 2 β 1 integrin interaction is an important signal for the osteoblastic differentiation of bone marrow cells. J. Cell. Physiol. 184:207–213, 2000. © 2000 Wiley-Liss, Inc.

We investigated activity of bone sialoprotein (BSP) to mediate attachment of cells to hydroxyapatite using a model peptide, Glu7-Pro-Arg-Gly-Asp-Thr, which contains a putative hydroxyapatite-binding site (poly-Glu) and a cell-attachment site. The peptide has affinity to hydroxyapatite with a dissociation constant of 13.5 microM. The peptide affected in vitro mineralization in a gel system, indicating interaction between this peptide and calcium phosphate. The osteoblastic cell line MC3T3-E1 was incubated with hydroxyapatite powder coated with the peptide or proteins. Attachment of the cells was observed on the powder coated with BSP, but not on the powder coated with serum albumin. The cells were attached to the powder coated with the peptide. The cells were flattened on the powder, and pseudopods developed. The attachment of the cells was inhibited by an excessive amount of Gly-Arg-Gly-Asp-Ser peptide. In conclusion, BSP mediated attachment of osteoblastic cells to hydroxyapatite, and this activity could be accomplished only by the poly-Glu sequence and the Arg-Gly-Asp sequence.

In this study, we demonstrated that bone marrow stromal cells maintained on type I collagen matrix induced bone in vivo. The formed bone contained bone marrow, and the process of bone formation occurred without cartilage formation. Bone marrow stromal cells differentiated into osteoblasts on type I collagen matrix in vitro, but types II, III, and V collagens did not possess this activity. These findings imply that type I collagen matrix offers a suitable environment for the induction of osteoblastic differentiation in vitro and osteogenesis in vivo.

Many traffic accidents are caused by drowsiness while driving. The purpose of this research is to detect a driver's blinking using facial images obtained by a TV camera and to estimate the driver's arousal level to prevent the occurrence of an accident due to drowsiness. This report presents the method used to obtain the facial images, which are not affected much by the change of the ambient light by day and by night owing to the infrared light sources. This paper also describes the method for extracting the blinks and measuring the blinking durations, which are not influenced very much by individual differences in eye shape. Furthermore, we experiment with the relation between blinking durations and arousal levels as compared with vehicle deviation from the lane and driver's answer to the question about drowsiness. We also examine a method for estimating arousal level which reflects individual characteristics.

In this study, we purified C-terminal propeptide of type I collagen (PICP) from the conditioned medium of osteoblastic MC3T3-E1 cells by chromatographic and Agarose gel extraction procedures. PICP was confirmed to be present in bone by Western blotting using a specific antibody, and was proved to be synthesized by osteoblasts with metabolic labeling. PICP promoted cell-attachment of osteoblastic MC3T3-E1 cells. We conclude that PICP is synthesized by osteoblasts and stored in bone, and that it plays a role in the maintenance of bone cells on bone matrix.

Bone sialoprotein (BSP) containing an Arg-Gly-Asp cell-binding sequence was purified from bovine bone 4 M guanidine-HCl extract after HCl demineralization by a series of chromatographic procedures. When this protein was coated on culture dishes in the presence of type I collagen, it increased both DNA content and alkaline phosphatase (ALP) activity in osteoblast-like MC3T3-E1 cells, and stimulated calcification in the cells, whereas fibronectin, another cell-binding protein, showed a marked increase in the DNA content but had little effect on the ALP activity. These findings suggest that BSP is mitogenic for preosteoblasts and differentiating the cells into osteoblasts, thereby stimulating bone calcification.

In-gel administration of recombinant BMP 2 (rBMP 2) to cultured bone marrow cells induces differentiation into osteoblasts much more efficiently than the conventional addition of rBMP 2 to the culture medium. We recently found that bone marrow cells cultured on collagen-gel could form bone-like calcified nodules withintwo weeks after confluence without the addition of the strong differentiationinducer dexamethasone. Using these cells on collagen-gel culture, the present studysought to evaluate the effects of rBMP 2 on cell differentiation. rBMP 2 was added to cultured rat bone marrow cells under three different conditions:(1) cellswere cultured in α-MEM with 15% fetal calf serum (standard medium) which contained various concentrations rBMP 2, (2) cells were cultured in standard medium which contained 10-8M dexamethasone (Dex), 10 mM β-glycerophosphate (β-GP), and rBMP 2 at different concentrations, and (3) cells were cultured on collagen-gel which contained rBMP 2 at different concentrations. In the standard medium (condition 1), the addition of rBMP 2 did not induce the cells to differentiate to form calcified nodules within a week, although both alkaline phosphatase activity and DNA content increased in a dose-dependent manner. In contrast, the addition of rBMP 2 as a component of collagen-gel (condition 3) markedly accelerated the phenotype expression of alkaline phosphatase and osteocalcin, as well as calcified nodule formation. This is the first report to demonstrate that collagen-gel enhances the effect of BMP in vitro. A similar effect wasobserved with rBMP 2 inthe presence of Dex and β-GP (condition 2).

The effect of the extracellular matrix on differentiation of bone marrow stromal cells into osteoblasts was studied in culture by morphological and biochemical analyses. Inoculation of bone marrow cells on a type I collagen gel produced calcified nodules in approximately 2 weeks, whereas inoculation on conventional plastic dishes failed to produce any calcified structures even after prolonged culturing. By transmission electron microscopy, the calcified nodules were shown to be similar in structure to the mineralized bone in animal tissue, in terms of hydroxyapatite deposits in matrix vesicles and collagen fibrils. Furthermore, the cells in the calcified nodules exhibited high levels of alkaline phosphatase activity and secreted a large amount of osteocalcin, indicating that the differentiation of bone marrow stromal cells into osteoblasts definitely occurred through the effect of the type I collagen gel. In contrast, when bone marrow cells were cultured on dishes coated with type I or IV collagen, they failed to produce any nodules, high alkaline phosphatase activity or osteocalcin. It was concluded that collagen in gel form playsan important role in the differentiation of bone marrow stromal cells to osteoblasts.

The bone-inducing efficiency in rat tissues of bone morphogenetic protein (BMP) was compared between the normal and ovariectomized (OVX) animal. In the calvaria of OVX rats, BMP induced a smaller amount and a slower process of bone formation than that of the control rats (sham operated). In the subcutaneous tissue of the OVX rats, BMP induced no bone formation in terms of alkaline phosphatase activity and calcium content, while in the same tissue of the control rats, BMP induced active bone formation. In an attempt to clarify the mechanism behind the reduced effect of BMP in the OVX rats, bone -marrow stromal cells of the OVX and SHAM rats were compared for their abilities to differentiate into osteoblasts. Using a culture system, it was shown that the bone-marrow stromal cells from the OVX rats exhibited much less alkaline phosphatase activity and osteocalcin synthesis than those from control rats. The results indicate that ectopic response to BMP is reduced in OVX rats, although still significantly active in isotopic response for bone formation. This change may be ascribed to the impaired recruitment of osteoblasts compared with normal animals.

Bone formation in vivo occurs via two major processes, one of which depends on pre-existing cartilage, and the other does not. Bone morphogenetic proteins (BMPs) have been suggested to induce cartilage formation from non-skeletogenic mesenchymal cell population, which results in osteogenesis through the endochondral sequence. In the present study we examined if BMPs could cause direct bone formation independent of pre-existing cartilage using bovine fibrous collagen membrane (FCM) as a carrier for BMPs. Bovine metatarsal bone was extracted in 4 M guanidine HCl and BMPs were partially purified through the hydroxyapatite chromatography and the Heparin-Sepharose CL6B chromatography. The carrier was loaded with BMPs and then implanted in Wistar rats subcutaneously. The implants were fixed together with surrounding tissue every week after implantation and processed for von Kossa stain, immunohistochemistry, and electron microscopy. The phenotypes of bone and cartilage were identified histologically and immunohistochemically using antibodies against type I and type II collagen. Cartilage and bone were independently induced by 2 weeks. The bone formed directly on the collagen substrate of FCM without pre-existing cartilage. Calcification occurred in the carrier as well as the cartilage and bone matrix. The present study suggests that the BMPs induce osteogenesis in vivo independent of the endochondral sequence. © 1993 Wiley-Liss, Inc.

Histidinoalanine is a naturally occurring cross-linking amino acid found in mammalian bone, dentin, cartilage and aorta, and also in the extracellular fluid of bivalve clams. The exact components from which this amino acid derives have not been characterized in the case of bone. In this study, the origin of histidinoalanine in bone was investigated in order to clarify the possible role of the matrix component containing this amino acid in bone mineralization. An extract of bovine bone powder with 0.5 M EDTA/1M NaCl was desalted and separated by calcium-induced precipitation. The supernatant fraction was chromatographed successively on Sepharose CL-6B, hydroxyapatite, DEAE cellulose, and reverse-phase high-performance liquid chromatography columns. The fraction with the highest affinity for hydroxyapatite was found to contain the highest concentration of histidinoalanine. A phosphoprotein with a molecular weight of about 24 K that contained 1.2 mol of histidinoalanine per molecule was isolated. The amino acid composition of this fraction showed a high content of acidic amino acids and serine, at least 42% of which was phosphorylated. These results suggested a possible formation mechanism of histidinoalanine that includes the beta-elimination of phosphoserine, producing a dehydroalanine residue and an addition of histidine on it. It was concluded that this low molecular weight phosphoprotein is one of the major sources of histidinoalanine in bovine bone.

Enamel matrix derivative (EMD) plays a crucial role in periodontal tissue regeneration. However, the precise mechanism of tissue regeneration by EMD remains obscure. The purpose of this study was to clarify what factors present in EMD show bioactivity.We first examined the effect of EMD on MC3T3-E1 osteoblastic cells. To evaluate the differentiation, the expression of osteoblast-related genes was measured by reverse transcription-polymerase chain reaction, and the osteocalcin (OCN) content was measured by enzyme-linked immunosorbent assay. Alkaline phosphatase activity and the mineralization were examined histologically. EMD (intact EMD) was filtrated to separate the soluble fraction (soluble EMD), and the effects of soluble and intact EMD were examined. Neutralization of the bioactivity of EMD was performed using a polyclonal antibody against porcine transforming growth factor-beta (TGF-beta).EMD inhibited the expression of osteoblastic phenotypes, and we used the inhibitory effect of EMD on osteoblastic differentiation as a benchmark of activity of EMD. The soluble fraction separated from EMD inhibited osteoblast-related gene expression and OCN synthesis. Soluble EMD suppressed the OCN gene level within 24 hours, and the effect of soluble EMD mimicked that of TGF-beta (10 ng/ml). The antibody against TGF-beta diminished the inhibitory effect of soluble EMD on OCN gene expression.The inhibitory effect of EMD on OCN gene expression of osteoblastic cells is neutralized by the antibody against TGF-beta in it. This result might indicate that EMD contains TGF-beta and that it participates in the bioactivity of EMD.

Bovine bone marrow mesenchymal stem cells (MSCs) cultured in condensate culture, spontaneous and independent for any external biostimulants, undergo chondrogenic differentiation. In the present study, the bovine MSC chondrogenesis pathway was studied by analyzing stage-specific gene expression using quantitative "Real Time" reverse transcriptase polymerase chain reaction (qRT-PCR). Results showed that bovine MSCs underwent complete chondrogenesis; the initial stage was characterized by expression. of sox9 messenger ribonucleic acid (mRNA), followed by high transcription of chondrocyte specific genes, collagen type II and IX, biglycan and cartilage oligomeric matrix protein, and the final prehypertrophic and/or hypertrophic stage was distinguished by increased expression of collagen type X. From day 7 to day 14 of differentiation increased mRNA expression of the transforming growth factors beta1 and beta2, basic fibroblast growth factor (FGF 2), bone morphogenic protein 6 (BMP 6), insulin-like growth factors 1, parathyroid hormone related peptide and indian hedgehog (Ihh) were detected. These results suggest that these well know chondrogenic growth factors may play a role in bovine chondrogenesis in autocrine and/or paracrine manner. On day 21 of the culture, FGF 2, BMP 6 and Ihh were highly expressed, compared to cells cultured in monolayer manner, which suggests a possible function in maintaining the terminal stage of differentiation. This data extends our knowledge about the unusual species-specific bovine MSC chondrogenesis, allowing us to define the phenotype of the differentiated cells. Furthermore, this study contributes to our in understanding of known chondrogenic-growth factors in autocrine and/or paracrine manner playing a role in the spontaneous differentiation.

Bone marrow mesenchymal stem cells (MSCs) are candidate cells for cartilage tissue engineering. This is due to their ability to undergo chondrogenic differentiation after extensive expansion in vitro and stimulation with various biomaterials in three-dimensional (3-D) systems. Collagen type II is one of the major components of the hyaline cartilage and plays a key role in maintaining chondrocyte function. This study aimed at analyzing the MSC chondrogenic response during culture in different types of extracellular matrix (ECM) with a focus on the influence of collagen type II on MSC chondrogenesis. Bovine MSCs were cultured in monolayer as well as in alginate and collagen type I and II hydrogels, in both serum free medium and medium supplemented with transforming growth factor (TGF) beta1. Chondrogenic differentiation was detected after 3 days of culture in 3-D hydrogels, by examining the presence of glycosaminoglycan and newly synthesized collagen type II in the ECM. Differentiation was most prominent in cells cultured in collagen type II hydrogel, and it increased in a time-dependent manner. The expression levels of the of chondrocyte specific genes: sox9, collagen type II, aggrecan, and COMP were measured by quantitative "Real Time" RT-PCR, and genes distribution in the hydrogel beads were localized by in situ hybridization. All genes were upregulated by the presence of collagen, particularly type II, in the ECM. Additionally, the chondrogenic influence of TGF beta1 on MSCs cultured in collagen-incorporated ECM was analyzed. TGF beta1 and dexamethasone treatment in the presence of collagen type II provided more favorable conditions for expression of the chondrogenic phenotype. In this study, we demonstrated that collagen type II alone has the potential to induce and maintain MSC chondrogenesis, and prior interaction with TGF beta1 to enhance the differentiation.

OBJECTIVE: Pluripotent mesenchymal stem cells (MSC) have been isolated and well characterized from several tissue sources, including bone marrow stroma. MSC from different animals showed slight differences in morphology and in the potential to differentiate. In the present study, we isolated MSC from bovine bone marrow and induced chondrogenesis in order to establish a new experimental model of stem cell research. METHODS: Bone marrow was harvested from 8 calves. For inducing chondrogenesis, MSC were cultured in pellet culture system in a chemically defined medium supplemented with 0 and 10 ng/mL of transforming growth factor beta1 (TGF-beta1). Chondrogenic differentiation was evaluated by histological, immunohistochemical, and in situ hybridization techniques. The degrees of genes expression were measured by quantitative RT-PCR. RESULTS: Metachromatic alcian blue staining and immunoreactivity for type II collagen were detected in both pellet groups (0 and 10 ng/mL TGF-beta1) after 7 days of culturing. In situ hybridization demonstrated strong expression of type II collagen and aggrecan mRNAs in the round cells located at the center region of pellets and at densely organized areas. On the other hand, type I collagen mRNA was strongly expressed in the superficial layer of the pellets. After 20 days of pellet culture, expression of type II collagen mRNA in the cells which were not treated by TGF-beta1 was 1.7-fold higher compared with that treated by TGF-beta1. CONCLUSION: Independent, spontaneous chondrogenesis of bovine MSC in pellet culture occurred without addition of any external bioactive stimulators, namely factors from TGF-beta family, which were previously considered necessary.

Matrix extracellular phosphoglycoprotein (MEPE)/ osteoblast/osteocyte factor 45 (OF45) is a recently isolated RGD-containing matrix protein that acts as the tumor-derived phosphaturic factor in oncogenic hypophosphatemic osteomalacia. It is also highly expressed by osteoblasts and osteocytes. We examined the regulation of MEPE/OF45 mRNA expression in osteoblastic cells derived from high-density cultures of primary rat bone marrow stromal cells incubated with dexamethasone, beta-glycerophosphate, and ascorbic acid. The level of MEPE/OF45 mRNA in these cells was down-regulated by the addition of fibroblast growth factor 2 (FGF2) for 48 h. These effects were observed in a dose-dependent manner between 2 and 10 ng/mL. FGF2 also reduced the expression of osteocalcin mRNA in these cells. In contrast, bone sialoprotein mRNA expression was increased by FGF2, while alpha1(I) procollagen mRNA expression was not altered. Additionally, neither Runx2 and osterix mRNA expression nor cell proliferation were affected by the addition of FGF2 in these high-density cultures, indicating that regulation by FGF2 may not be dependent on these transcription factors or on the proliferation of cells. Experiments using actinomycin D indicated that FGF2 decreased the stability of the MEPE/OF45 mRNA. Moreover, inhibition of a specific mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase (MEK) by PD98059 blocked FGF2-regulated MEPE/OF45 expressions, indicating that this regulation requires the MAPK pathway. These results suggest that MEPE/OF45 gene is one of the targets of FGF2 and may play an important role during bone formation and calcification.

In mammalian cells, several observations indicate not only that phosphate transport probably regulates local inorganic phosphate (Pi) concentration, but also that Pi affects normal cellular metabolism, which in turn regulates apoptosis and the process of mineralization. To elucidate how extracellular Pi regulates cellular functions of pre-osteoblastic cells, we investigated the expression of type III sodium (Na)-dependent Pi transporters in rat bone marrow stromal cells and ROB-C26 pre-osteoblastic cells. The mRNA expression level of gibbon ape leukemia virus receptor (Glvr)-2 was increased by the addition of Pi in rat bone marrow stromal cells, but not in ROB-C26 or normal rat kidney (NRK) cells. In contrast, the level of Glvr-1 mRNA was not altered by the addition of extracellular Pi in these cells. The induction of Glvr-2 mRNA by Pi was inhibited in the presence of cycloheximide (CHX). Moreover, mitogen-activated protein kinase (MEK) /extracellular-signal-regulated kinase (ERK) pathway inhibitors; U0126 (1.4-diamino-2, 3-dicyano-1, 4-bis [2-amino-phenylthio] butadiene) and PD98059 (2′-Amino-3′-methoxyflavone) inhibited inducible Glvr-2 mRNA expression, but p38 MEK inhibitor SB203580 [4-(4′-fluorophenyl)-2-(4′-methyl-sulfinylphenyl)-5-(4′pyridyl) imidazole] did not inhibit the induction of Glvr-2 mRNA expression, suggesting that extracellular Pi regulates de novo protein synthesis and MEK/ERK activity in rat bone marrow stromal cells, and through these, induction of Glvr-2 mRNA. Although Pi also induced osteopontin mRNA expression in rat bone marrow stromal cells but not in ROB-C26 and NRK cells, changes in cell viability with the addition of Pi were similar in both cell types. These data indicate that extracellular Pi regulates Glvr-2 mRNA expression, provide insights into possible mechanisms whereby Pi may regulate protein phosphorylation, and suggest a potential role for the Pi transporter in rat bone marrow stromal cells. J. Cell. Physiol. 198: 40–47, 2004. © 2003 Wiley-Liss, Inc.

Objectives: To evaluate the antibacterial activity of resin composite containing Maillard reactive product (MRP) and hydrogen peroxide (H 2 O 2 ) on Streptococcus mutans ( S. mutans ), and to investigate the antibacterial mechanism involved. Methods: The growth of S. mutans was investigated after dental resin containing H 2 O 2 in the presence and absence of MRP, was immersed into bacterial solution. The effect of MRP on H 2 O 2 degradation was examined by the measurement of H 2 O 2 content. Results: The resin composite containing MRP and H 2 O 2 showed stable antibacterial activity compared with resin containing H 2 O 2 only, and the effect of MRP was speculated to be the suppression of H 2 O 2 degradation, and the presence of H 2 O 2 correlated with the antibacterial activity of resin composite. These results indicated that the antibacterial activity of resin composite containing MRP and H 2 O 2 on S. mutans was dependent on the presence of H 2 O 2 , and MRP suppressed the degradation of H 2 O 2 after combination with H 2 O 2 . EDTA also suppressed the degradation of H 2 O 2 . Conclusions: An antibacterial effect of resin composite containing MRP and H 2 O 2 on S. mutans was observed. The effect of MRP on H 2 O 2 might be a metal chelating action. Application of resin composite containing MRP and H 2 O 2 to a caries dentine could be an alternative therapy to or serve as an additional minimally invasive antibacterial treatment.

This paper presents the improvement of robustness of the vision system for lane mark detection using the wide dynamic range vision sensor. The performance of the lane mark detection was evaluated by experiments for the road scenes such as a tunnel exit or bridge crossing road, in comparison with the vision system using a conventional TV camera. A conventional edge-based image recognition technique was used for lane mark detection. The experimental results have confirmed the robustness of the vision system with wide dynamic range vision sensor.

Bone marrow contains multipotent cells that differentiate into fibroblasts, adipocytes, and osteoblasts. Recently we found that type I collagen matrix induced the osteoblastic differentiation of bone marrow cells. Three weeks after cells were cultured with type I collagen, they formed mineralized tissues. In this study, we investigated the expression of osteoblast-related genes (alkaline phosphatase, osteocalcin, bone sialoprotein, osteopontin, and cbfa-1) during the osteoblastic differentiation. The expression of alkaline phosphatase and osteopontin genes increased time-dependently during the osteoblastic differentiation. Osteocalcin and bone sialoprotein genes were expressed in cells that formed mineralized tissues, and both were expressed only after cells reached the mineralized tissue-formation stage. On the other hand, the cbfa-1 gene was expressed from the early differentiation stage. The Asp-Gly-Glu-Ala (DGEA) amino acid domain of type I collagen interacts with the alpha2beta1 integrin receptor on the cell membrane and mediates extracellular signals into cells. When the collagen-integrin interaction was interrupted by the addition of DGEA peptide to the culture, the expression of osteoblastic phenotypes of bone marrow cells was inhibited. These findings imply that the collagen-alpha2beta1 integrin interaction is an important signal for the osteoblastic differentiation of bone marrow cells.

Recently we found that the carboxyl-terminal propeptide of type I collagen (c-propeptide) is a major secretory protein of osteoblasts. Mature osteoblasts secreted 64 nM c-propeptide, and it was reported that 40 nM c-propeptide inhibited collagen synthesis at 80% of the control level. In this study, we investigated the effect of c-propeptide on collagen synthesis of preosteoblasts and osteoblasts, and found that preosteoblasts downregulated collagen synthesis by 40 nM c-propeptide, but osteoblasts were not affected by the same condition. When the binding activities of c-propeptide for preosteoblasts and osteoblasts were compared, osteoblasts showed weak affinity to c-propeptide compared with preosteoblasts, and the number of receptors for c-propeptide decreased in osteoblasts. These results imply that a decrease of receptors in osteoblasts might reduce the sensitivity of osteoblasts to c-propeptide.

Various compositions of Na2O–CaO–P2O5 glasses are prepared to estimate glass formation, dissolution properties and cytotoxicity. In the wide composition range of 40 mol% of P2O5 or more, clear glass samples were obtained. The estimated glass forming region was consistent with other ternary phosphate glass systems. The glass transition temperatures and crystallization temperatures decreased with increasing P2O5 content and increased with CaO content. Dissolution properties in distilled water and simulated body fluid (SBF) were measured. In distilled water, CaO free glasses showed extremely fast dissolution. The dissolution rate decreased with increasing CaO content and decreasing P2O5 content. This composition effect results from cross-link formation between the non-bridging oxygens of two different chains by Ca2+ ions which improves the phosphate network strength. In SBF, the dissolution rate followed a similar trend, but glass dissolution was suppressed. This suppression occurred due to the existence of soluble species of glass such as Na+, Ca2+ and HPO2-4. The cytotoxicity decreased with increasing CaO content and with decreasing PO2.5 content. This was the result of a change in pH and ion concentration in the medium.

Phase-contrast and fluorescence microscopic observation showed that DNA added in the cell-culture medium for fibroblasts localized just on the surface of fibroblasts. The DNA bound to fibroblasts was found to be eluted by treating with collagenase. The suppression for the proliferation of fibroblasts by external DNA was confirmed with microscopic observation for the cells cultured in the presence and absence of DNA. Proliferation of the cells decreased from 412 to 155% by the addition of DNA. These results indicate that DNA has an affinity for collagen, the most major extracellular-matrix produced by fibroblasts, and suppresses the growth of fibroblasts.

Chondroadherin, which is reported to be synthesized by chondrocytes and to promote their attachment, was purified from bovine bone. It was a minor component of bone organic matrix, and was present in the 4 M guanidine extract of demineralized bone. Chondroadherin promoted attachment of osteoblastic cells to solid-state substrates, and bound to collagen. Binding of chondroadherin to collagen was significantly higher than that of osteonectin or decorin. These findings imply that chondroadherin may play a role in maintaining bone cells on the collagen matrices of bone.

Bone sialoprotein is unique to bone and dentin, but its precise role in these tissues is still unknown, although several hypotheses have been presented. We chose ectopic chondro- and osteogenesis induced by bone morphogenetic protein (BMP) as a model system to examine the role of this protein. Partially purified bovine BMP obtained by a three-step chromatographic procedure contained all the active BMPs (natural BMP cocktail). It was combined with insoluble bone matrix and subcutaneously implanted into rats. Expression of bone sialoprotein (BSP) in the implants was followed by using a monoclonal antibody as previously reported. Immunostaining studies showed BSP in the osteoblasts lining the new bone surface at 5 weeks. Western blotting showed 53 and 30 kDa bands, instead of the 57 kDa band normally found in rat femur. These two fragments were metabolically labeled with [3H]proline. The total amount of the fragments rapidly increased after 3 weeks, and at 5 weeks was 3 times as high as that at 2 weeks and still increasing. This time-dependent change was almost parallel to that of osteocalcin. The amount of bone estimated in terms of calcium content increased until 3 weeks and was remained at a plateau thereafter. Alkaline phosphatase activity was prominent only in the first 3 weeks. It was concluded that the 53 and 30 kDa BSP fragments might contribute to maintenance or remodeling in BMP-induced ectopic bone formation.

Bone marrow cells have multipotency for differentiation. Recently we found that type I collagen matrix induced the osteogenic differentiation of bone marrow cells. In this study we showed that TGF-beta enhanced the effect of collagen matrix on bone marrow cells and accelerated the osteogenic differentiation. These results imply that TGF-beta might play a crucial role in the osteogenic differentiation of bone marrow cells during embryogenesis and the repair of bone.

A partially purified BMP preparation was combined with a fibrous glass membrane (FGM) or porous particles of hydroxyapatite (PPHAP), and then implanted subcutaneously into the backs of rats. As a control of these new carriers, a conventional carrier of insoluble bone matrix (IBM) was also used. These new geometrically different solid-state carriers induced tissues in quite different manners. FGM/BMP implants induced cartilage formation within the entire inner area of the membrane accompanied by a small amount of bone formation on the surface of the membrane. In contrast, PPHAP/ BMP implants induced only bone within the pores of PPHAP without any detectable cartilage formation. Enzyme-linked immunosorbent assay revealed that the type II collagen content in FGM/BMP was six times higher than that in IBM/BMP, while there was no detectable type II collagen in PPHAP/BMP. The results were explained by the geometric properties of the two distinctive carriers.

Time-dependent changes of the [3H]-proline-labeled noncollagenous proteins synthesized by the osteogenic cell-line MC3T3-E1 were analyzed over a range starting from cell confluency to 13 days after confluency during which time cells formed a bone-like structure. It was found that the 40 kDa protein on SDS-polyacrylamide gel (SDS-PAGE) remarkably increased in the cell-matrix layer at about 9 days after cell confluence, just before calcification. This protein was highly purified and was found to contain high amounts of glutamic acid, glycine, and serine. An internal amino acid sequence of this protein was revealed to be K-X-M-A-P-E-E-X-P, which showed homology with the sequence of the EF-hand domain in osteonectin/SPARC (secreted protein, acidic, and rich in cysteine). This protein co-migrated with collagen in gel filtration, and ion-exchange chromatography. Furthermore, it showed high affinity to type I collagen.

The early stage of cross-link formation in bone collagen was studied in a cell culture system. An osteogenic cell line that produces and accumulates a remarkably high amount of collagen, and that eventually forms bone-like structures, was used in this study for its time-dependent development of reducible cross-links. It was found that precursors of the cross-link, dehydro-dihydroxynorleucine and dehydro-hydroxynorleucine became detectable as soon as the cells attained a confluent state. They showed maximal amounts at days 3-5 after confluence, but substantially disappeared at day 10 after confluence. In contrast, two characteristic cross-links of bone collagen, dehydro-dihydroxylysinorleucine (dehydro-DHLNL) and dehydro-hydroxylysinorleucine (dehydro-HLNL), which were present in trace amounts at the stage of cell confluence, gradually increased in amount and reached a plateau at day 10, just when their precursors disappeared. Thus, it was found that there was a time lag of about a week between the maximal formations of precursors and cross-links of bone collagen in this system. The significance of this time lag was interpreted in terms of the minimum essential accumulation of collagen for the precursor-product transition. The ratio of dehydro-DHLNL to dehydro-HLNL was as low as 0.7 at day 3 after confluency, increased to 4.2 at day 20, the period just before mineralization began, and decreased thereafter, suggesting a qualitative change in bone collagen associated with mineralization.

著者はラット顎下腺中にPlasminogen Activator (Plasminを介しFibrinに作用する) とFibrinolyticenzyme (Fibrin直接に作用する) を認め, 下記の性質を明らかにするとともに, 既に報告されているラット顎下腺内Proteaseと比較した。(1) Plasminogen Activator (以下Activatorと略) ならびにFibrinolytic enzymeの分子量はそれぞれ28, 000, 30, 000であった。(2) 上記両酵素はAmidase活性を示したが, ActivatorはFibrin, CaseinならびにAlbumin分解活性を示さず, Fibrinolytic enzymeはTrypsin, Plasminに比し高い基質特異性を示した。(3) 両酵素はDFP, Soy Bean Trypsin Inhibitorにより活性を抑制されたが, TLCKの影響を受けなかった。(4) 上記両酵素は血管透過性を充進させなかった。(5) Activatorは既に報告されているThe kallikrein-like peptidaseと, Fibrinolytic enzymeはSalivainとそれぞれ類似性を示した。

It was found that significant precipitation occurred immediately after calcium, at a concentration as low as 2 mM, was added to a desalted solution of EDTA extract of adult bovine femur. The maximal yield of the precipitates was observed at a calcium concentration of 30 mM. These precipitates were dissolved in 0.5 M EDTA, desalted, and characterized by Sepharose CL-6B gel filtration chromatography and high performance gel-exclusion chromatography. Results revealed that the precipitates were enriched in a 40 K protein and a higher molecular weight fraction as compared with the original extract of bone proteins. The 40 K fraction was isolated and identified as osteonectin, as judged from amino acid analysis, electrophoresis, and immunodetection. The supernatant after calcium-induced precipitation predominantly contained osteocalcin and a 50 K protein that was tentatively identified as alpha 2HS protein. Osteonectin was purified from the calcium-induced precipitates from the EDTA extract of bovine bone. By calcium titration using fluorescence spectrometry, the isolated osteonectin showed high affinity to calcium ions with an apparent dissociation constant (K0.5) of 8 x 10(-7) M. Thus, the use of calcium to separate bone proteins, especially osteonectin, was proved to be a useful technique. In addition, calcium-induced precipitation of osteonectin suggested a possible in vivo mechanism via which osteonectin might interact with calcium ions and participate in the initial immobilization of calcium to induce the nucleation of calcification in bone tissue.