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Biomedical subjects

Y Kuboki

Publications and source records attributed to Y Kuboki.

At least 19 recordsLinked to original sources
PubMed2003

In vitro effect of platelet-derived growth factor-BB on collagen synthesis and proliferation of human periodontal ligament cells.

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.

Becaplermin↗
PubMed2003

Laser-perforated membranous biomaterials induced pore size-dependent bone induction when used as a new BMP carrier.

Previously we found that laser perforation of a collagen membrane (35 microm thickness, Koken Co., Tokyo) produced an effective bone morphogenetic protein (BMP) carrier, if the created pore sizes were larger than 0.5 mm. In this study we applied the same technique to create pores of 0.2 and 1.0 mm in a thicker (1.2 mm thickness) porous biodegradable membrane made of polylactic acid and an epsilon-caprolactone copolymer (PLA-CL) to obtain an effective membranous BMP carrier with higher mechanical strength. Pieces of PLA-CL (0.5 x 1.0 x 0.12 cm) combined with rhBMP-2 (5 microg) were implanted subcutaneously into rats and processed for analyses at 1-3 weeks. The laser-perforated PLA-CL membranes equipped with 1.0 mm pores induced mineralization beginning from the margins of the pores judging from the X-ray patterns, but bone formation seemed to proceed irregularly inside the pores. In the perforated PLA-CL membrane with 1.0-mm pores bone formation did not significantly increase compared with the nonperforated one. This was due to the fact that the PLA-CL membrane was already a porous structure (85% porosity). In contrast with laser-perforated PLA-CL 0.2 mm pores, bone was induced on the collagen fibers and fiber bundles inside the pores. The different patterns of bone formation between the PLA-CL membranes with 1.0 and 0.2 mm pores seemed to be related to the active formation of perpendicular collagen fibers through the 0.2 mm pores.

Animals↗
PubMed2002

Histological characterization of the early stages of bone morphogenetic protein-induced osteogenesis.

On the basis of currently available knowledge, we hypothesize that the initial bone formation, as induced by bone morphogenetic protein (BMP), is influenced by the chemical composition and three-dimensional spatial configuration of the used carrier material. Therefore, in the current study, the osteoinductive properties of porous titanium (Ti) fiber mesh with a calcium phosphate (Ca-P) coating (Ti-CaP), insoluble bone matrix (IBM), fibrous glass membrane (FGM), and porous particles of hydroxy apatite (PPHAP) loaded with rhBMP-2 were compared in a rat ectopic assay model at short implantation periods. Twelve Ti-CaP, 12 IBM, 12 FGM, and 12 PPHAP implants, loaded with rhBMP-2, were subcutaneously placed in 16 Wistar King rats. The rats were sacrificed at 3, 5, 7, and 9 days post-operative, and the implants were retrieved. Histological analysis demonstrated that IBM and Ti-CaP had induced ectopic cartilage and bone formation by 5 and 7 days, respectively. However, in PPHAP, bone formation and cartilage formation were seen together at 7 days. At 9 days, in Ti-CaP, IBM, and PPHAP, cartilage was seen together with trabecular bone. At 9 days, in FGM, only cartilage was observed. Quantitative rating of the tissue response, using a scoring system, demonstrated that the observed differences were statistically significant (Wilcoxon rank sum test, p < 0.05). We conclude that IBM, CaP-coated Ti mesh, FGM, and PPHAP provided with rhBMP-2 can indeed induce ectopic bone formation with a cartilaginous phase in a rat model at short implantation periods. Considering the different chemical composition and three-dimensional spatial configuration of the carrier materials used, these findings even suggest that endochondral ossification is present in rhBMP-2-induced osteogenesis, even though the amount of cartilage may differ.

Animals↗
PubMed2002

Bone induction in subcutaneous tissue in rats by a newly developed DNA-coated atelocollagen and bone morphogenetic protein.

A unique biomaterial, a mixture of DNA and collagen (DNA/collagen), was developed and its efficacy as a carrier matrix for bone morphogenetic protein (BMP) was evaluated histologically. The material was prepared as a composite of DNA from salmon milt and pepsin-digested type I collagen (atelocollagen) from bovine dermis. Phase-contrast and fluorescence microscopy showed atelocollagen fibres with DNA coating. The dose-response and time-course of bone induction by BMP in DNA/collagen (5 x 10 x 1 mm) in the subcutaneous tissue was investigated in 20 male Wistar rats. The BMP/DNA/collagen induced new bone in a dose-dependent manner (0, 25, 50 or 100 micrograms of BMP). Histological examination in the time-course study showed that the BMP (100 micrograms)/DNA/collagen induced bone formation, while the DNA/collagen alone resulted in the accumulation of fibroblasts. These results indicate that the DNA/collagen is effective as a carrier matrix for BMP. It provides a cell anchorage for differentiation of osteoblasts and is absorbed as bone matures.

Absorbable Implants↗
PubMed2001

Immunohistochemical localization of amelogenin in human odontogenic tumors, using a polyclonal antibody against bovine amelogenin.

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

Amelogenin↗
PubMed2001

Osteoblast-related gene expression of bone marrow cells during the osteoblastic differentiation induced by type I collagen.

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.

Amino Acid Motifs↗
PubMed2001

Geometric effect of matrix upon cell differentiation: BMP-induced osteogenesis using a new bioglass with a feasible structure.

A new biocompatible glass, which is composed of CaO, P2O5, SiO2, and Al2O3 (abbreviated CPSA) and is characterized by higher elasticity than previous bioglass products, was molded into fibers with a diameter of 9 microm. With CPSA fibers, two geometrically different structures, balls and bundles (each 20 mg in weight), were prepared, combined with 2.2 microg of rhBMP-2 (a gift from Yamanouchi Co., Japan) and implanted subcutaneously into rats. The histology showed remarkably higher bone formation in the ball-CPSA/BMP at 2 and 4 weeks than in the bundle-CPSA/BMP. The ball-CPSA/BMP showed 10 times higher alkaline phosphatase (ALP) activity at the second week and 5 times higher osteocalcin content at the fourth week than the bundle-CSPA/BMP. Vascular development in the implants was evaluated by mRNA expression of Flt-1 and KDR, two receptors for vascular endothelial growth factor (VEGF). Both receptors showed higher expression in the case of the ball, while they were not detected in the bundle. It is concluded that the BMP-induced bone formation depends highly upon the porous vasculature-inducing geometry of the matrix, which can be constructed with the new CPSA fibers.

Alkaline Phosphatase↗
PubMed2001

Ectopic bone formation in titanium mesh loaded with bone morphogenetic protein and coated with calcium phosphate.

The osteoinductive properties of porous titanium fiber mesh, with or without a calcium phosphate coating and loaded with recombinant human bone morphogenic protein-2 (rhBMP-2) or rhBMP-2 and native bovine BMP (S-300) were investigated in a rat ectopic assay model. A total of 112 calcium phosphate-coated and 112 noncoated porous titanium implants, either loaded with rhBMP-2 and S-300 or loaded with rhBMP-2 alone, were subcutaneously placed in 56 Wistar-King rats. The rats were killed 5, 10, 20, and 40 days postoperatively, and the implants were retrieved. Histologic analysis demonstrated that all growth factor and carrier combinations induced ectopic cartilage and bone formation at 5 and 10 days, respectively. At 20 days, bone formation increased and was characterized by trabecular bone and bone marrow-like tissue. At 40 days, more lamellar bone and hemopoietic bone marrow-like tissue were present. At both times, more bone had been formed in calcium phosphate-coated implants than in noncoated samples. Further, in rhBMP-2 and S-300-loaded specimens, bone formation was higher than in rhBMP-2 only-loaded specimens. In rhBMP-2 only-loaded specimens, bone formation was mainly localized inside the mesh material, whereas in specimens loaded with both rhBMP-2 and S-300, the bone was localized inside and surrounding the titanium mesh. The histological findings were confirmed by calcium content and alkaline phosphatase activity measurements. In addition, all specimens showed osteocalcin expression as early as 5 days postoperatively. Our results show that the combination of titanium mesh with BMPs can induce ectopic bone formation and that this bone formation seems to be similar to "enchondral" ossification. In addition, a thin calcium phosphate coating can have a beneficial effect on the bone-inducing properties of a scaffold material. Finally, rhBMP-2 and native BMP act synergistically in ectopic bone induction.

Alkaline Phosphatase↗
PubMed2001

Geometry of carriers controlling phenotypic expression in BMP-induced osteogenesis and chondrogenesis.

BACKGROUND: The effect of the geometry of extracellular matrices on bone morphogenetic protein (BMP)-induced osteogenesis has not been systematically studied. Geometry is crucially important for the scaffold in bone and joint tissue engineering. The purpose of this study was to elucidate principles of geometry of matrices in designing new scaffolds and matrices for use in reconstruction of bone and joints. METHODS: More than ten biomaterials with different geometries, including a unique device of honeycomb-shaped hydroxyapatite, were combined with BMPs of recombinant (rhBMP-2) or natural bovine origin (S300 BMP cocktail) and implanted subcutaneously into 4-week-old Wistar-King rats. The implanted pellets were removed at 1-4 weeks and analyzed for bone and cartilage formation by histological and biochemical methods. RESULTS: BMP-induced bone and cartilage induction was highly dependent on the geometric properties of the carrier. Some carriers such as porous particles or blocks of hydroxyapatite induced osteogenesis directly, without detectable chondrogenesis, whereas other carriers such as fibrous glass membrane induced cartilage exclusively. Still other carriers induced mostly cartilage followed by bone formation. Solid particles of hydroxyapatite and fibrous glass membrane with too tight a meshwork did not induce bone or cartilage. The optimal pore size for bone-forming efficacy in porous blocks of hydroxyapatite was a diameter of 300-400 microm. In straight tunnel structures with various diameters in honeycomb-shaped hydroxyapatite, tunnels with smaller diameters (90-120 microm) induced cartilage followed by bone formation, whereas those with larger diameters (350 microm) induced bone formation directly within the tunnels. CONCLUSIONS: BMP carriers were classified into three types: bone-inducing, cartilage-inducing, and cartilage-bone-inducing. From the analysis of causative factors inducing osteogenesis and chondrogenesis in the BMP system, we concluded that the geometry of the carrier is crucially important and vasculature-inducing geometry should be considered in designing effective scaffolds for bone formation. We propose a classification of geometry of the artificial extracellular matrices that is useful for designing a scaffold for tissue engineering of bone and related tissues. CLINICAL RELEVANCE: Conventional requisites of the BMP carriers for clinical use have mainly concerned the affinities of carriers with cells and biomolecules and their mechanical strength. The vasculature-inducing geometry of carriers adds a new criterion in designing systems for effective bone and joint reconstruction. The geometries of porous structures-their sizes, continuity, and straightness as verified by hydroxyapatite in this study-will be applicable for other biomaterials for clinical reconstruction therapy.

Animals↗
PubMed2000

A scaffold protein in the c-Jun NH2-terminal kinase signaling pathways suppresses the extracellular signal-regulated kinase signaling pathways.

We previously reported that c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1) functions as a putative scaffold factor in the JNK mitogen-activated protein kinase (MAPK) cascades. In that study we also found MEK1 and Raf-1, which are involved in the extracellular signal-regulated kinase (ERK) MAPK cascades, bind to JSAP1. Here we have defined the regions of JSAP1 responsible for the interactions with MEK1 and Raf-1. Both of the binding regions were mapped to the COOH-terminal region (residues 1054-1305) of JSAP1. We next examined the effect of overexpressing JSAP1 on the activation of ERK by phorbol 12-myristate 13-acetate in transfected COS-7 cells and found that JSAP1 inhibits ERK's activation and that the COOH-terminal region of JSAP1 was required for the inhibition. Finally, we investigated the molecular mechanism of JSAP1's inhibitory function and showed that JSAP1 prevents MEK1 phosphorylation and activation by Raf-1, resulting in the suppression of the activation of ERK. Taken together, these results suggest that JSAP1 is involved both in the JNK cascades, as a scaffolding factor, and the ERK cascades, as a suppressor.

Animals↗
PubMed2000

Effects of geometry of hydroxyapatite as a cell substratum in BMP-induced ectopic bone formation.

Three different types of porous hydroxyapatite with pore sizes of 100-200 micrometer in diameter-porous particles of hydroxyapatite (PPHAP), porous blocks of hydroxyapatite (PBHAP), and honeycomb-shaped hydroxyapatite (HCHAP)-were compared in terms of their abilities to induce osteogenesis when implanted subcutaneously with recombinant human BMP-2 into rats and extracted at 1, 2, 3, and 4 weeks. Histologically, direct bone formation occurred in PPHAP and PBHAP while only endochondral ossification took place in HCHAP. Interestingly, cartilage in the central zones and bone in the orifice zones of the tunnels of the HCHAP were observed at 2 weeks. After 3 weeks, the cartilage disappeared and bone formation occurred throughout the inner surface of the tunnels of the HCHAP, always leaving space for capillaries within the tunnels. Alkaline phosphatase activity and osteocalcin content were the highest in HCHAP among the three hydroxyapatite implants. These results clearly indicate that BMP-induced bone formation is highly dependent on the geometry of the carrier, which provides feasible structural factors for vascularization.

Alkaline Phosphatase↗
PubMed2000

Carboxyl-terminal propeptide of type I collagen (c-propeptide) modulates the action of TGF-beta on MC3T3-E1 osteoblastic cells.

Previously we found that the carboxyl-terminal propeptide of type I collagen (c-propeptide) is a major secretory protein of MC3T3-E1 osteoblastic cells. In this study, we found that c-propeptide suppresses collagen synthesis and alkaline phosphatase activity of MC3T3-E1 osteoblastic cells at the early-differentiated stage in a dose dependent manner. Mature osteoblasts did not respond to c-propeptide. These findings imply that c-propeptide modulates the function of osteoblasts at an early differentiation stage. Transforming growth factor-beta (TGF-beta) is stored in bone and released from bone matrix after the resorption by osteoclasts. We investigated the effect of c-propeptide on the action of TGF-beta, and found that it enhanced the effect of TGF-beta. We conclude that c-propeptide is a physiological modulator of TGF-beta in bone metabolism.

Alkaline Phosphatase↗
PubMed2000

Type I collagen-induced osteoblastic differentiation of bone-marrow cells mediated by collagen-alpha2beta1 integrin interaction.

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 alpha 2 beta 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-alpha 2 integrin antibody, which interacts with alpha subunit of integrin and blocks the binding of integrin with collagen, suppressed the expression of osteoblastic phenotypes. These findings imply that collagen-alpha 2 beta 1 integrin interaction is an important signal for the osteoblastic differentiation of bone marrow cells.

Alkaline Phosphatase↗
PubMed2000

The effect of carboxyl-terminal propeptide of type I collagen (c-propeptide) on collagen synthesis of preosteoblasts and osteoblasts.

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.

3T3 Cells↗
PubMed2000

Bone sialoprotein (BSP) is a crucial factor for the expression of osteoblastic phenotypes of bone marrow cells cultured on type I collagen matrix.

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.

Animals↗
PubMed2000

DegraPol-foam: a degradable and highly porous polyesterurethane foam as a new substrate for bone formation.

Bone morphogenetic protein (BMP) is known to require a suitable carrier to induce ectopic bone formation in vivo. To evaluate the suitability of DegraPol-foam, a degradable, elastic, and highly porous polyesterurethane foam as carrier for BMP-induced bone formation, a fraction containing all the active BMPs (BMP cocktail) was combined with DegraPol-foam and implanted subcutaneously into rats. DegraPol-BMP scaffolds were found to induce osteogenesis 2 weeks after implantation as evidenced by morphological and biochemical observations. In addition, the osteoblast-compatibility of DegraPol-foam was examined here. In vitro, primary rat osteoblasts and osteoblasts from the human cell line (HFO1) attached and proliferated preferentially on the surface of the DegraPol-foam. Both cell types exhibited relatively high attachment and low doubling time that resulted in a confluent cell multilayer with spindle-shaped morphology on the surface of the foam. Osteoblasts produced high concentrations of collagen type I and osteocalcin, and expressed increasing levels of alkaline phosphatase (ALP) activity. Taken collectively, both osteoblasts from rat tibia and from the human cell line HFO1 showed high cell attachment and growth, and preserved their phenotype. The geometrical structure of DegraPol is a suitable carrier for BMP for the induction of bone formation.

Absorbable Implants↗
PubMed2000

Study on the secretion of cytokines from osteoblasts stimulated with prostaglandin E1.

In previous studies, prostaglandin (PG) E1 was found not only to enhance the effect of rhBMP, but also to increase orthotopic osteoconduction and enhance bone formation, even when used alone, as well as increasing the physical strength of implanted porous hydroxyapatite ceramics (HAP). In the present study, the secretion of cytokines from MC3T3-E1 cells was investigated, which is a cell line of osteoblasts derived from the mouse skull, stimulated in vitro by 10(-5) M and 10(-6) M PGE1, in order to elucidate the mechanism of action of PGE1. The cytokines examined included transforming growth factor (TGF) beta 1, interleukin (IL)-6, and IL-1 beta. The results of this study clarify the mechanism of enhancement of osteogenesis by PGE1. Elevated TGF beta 1 plays a very important role, and the findings are significant because they may provide the basis for the establishment of a rational clinical combined application of these materials.

3T3 Cells↗