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Y Kuboki

Publications and source records attributed to Y Kuboki.

At least 19 recordsLinked to original sources
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↗
PubMed2000

Preliminary communication. MRNA expression of MT1-MMP, MMP-9, cathepsin K, and TRAP in highly enriched osteoclasts cultured on several matrix proteins and ivory surfaces.

We demonstrated that the transcriptional expression of MT1-MMP, MMP-9, cathepsin K, and TRAP in highly enriched osteoclasts were regulated by different matrix proteins that bind to integrin on osteoclast, such as collagen type I (CoI), fibronectin (FN), vitronectin (VN), osteopontin (OPN), and ivory. Results suggested that the OPN-integrin alphavbeta3 binding plays a more important role than CoI-alpha2beta1 binding in the regulation of osteoclast activity.

Animals↗
PubMed1999

Prostaglandin E1 and recombinant bone morphogenetic protein effect on strength of hydroxyapatite implants.

Although combinations of hydroxyapatite (HAP) and bone morphogenetic protein (BMP) are expected to provide potent alternatives to autogenous bone grafts, it is still anticipated that substances that act synergistically with BMP will be found because the inducing potential of purified BMP in bone is not strong enough. We already have shown that prostaglandin (PG) E1 has a strong and dose-dependent synergistic effect on the osteoinductive activity induced by recombinant human (rh) BMP and that it enhances osteoconduction even when used alone. In this study, porous HAP rods were treated as follows: (1) without PGE1 or rhBMP (control group); (2) with varying concentrations of PGE1; and (3) with varying concentrations of PGE1 combined with 1 microg of rhBMP-2. The rods were subperiosteally implanted on the cranial bone of rabbits to evaluate the effect of these treatments on the mechanical strength of the implanted HAP rods. The HAP rods were removed 3, 6, or 9 weeks after implantation and subjected to mechanical strength determinations. The control group (no addition of BMP to the rods) showed no significant increase in three-point bending strength or in compression strength compared to pre-implantation. On the other hand, PGE1 combined with rhBMP had a strong and dose-dependent effect on the mechanical strength of HAP, increasing it significantly, especially compression strength. PGE1 also increased mechanical strength even when used alone. Histological examination revealed that PGE1, whether or not it was combined with rhBMP, increased bone formation into the pores of HAP and consequently increased the mechanical strength of porous HAP.

Alprostadil↗
PubMed1998

BMP-induced osteogenesis on the surface of hydroxyapatite with geometrically feasible and nonfeasible structures: topology of osteogenesis.

Bone morphogenetic protein (BMP) is known to require a suitable carrier to induce ectopic bone formation in vivo. Hydroxyapatite ceramics have been reported to be effective in some forms but ineffective in others as a carrier of BMP-induced bone formation. In this study we compare three geometrically different forms of hydroxyapatite to examine their functions as carriers of BMP-induced bone formation. A fraction containing all the active BMPs (BMP cocktail) was partially purified from a 4M guanidine extract from bovine bone by a three-step chromatographic procedure. The BMP cocktail was combined with each of three forms of hydroxyapatite--solid particles (SPHAP), porous particles (PPHAP), and coral-replicated porous tablets (coral-HAP)--and implanted subcutaneously into rats. Both the PPHAP and coral-HAP systems induced osteogenesis 2 weeks after implantation, as evidenced by morphological and biochemical observations. Details of the osteogenetic process were followed by double-fluorescence labeling in the coral-HAP system to confirm bone formation on the surface of hydroxyapatite. However, there was no evidence of osteogenesis or chondrogenesis in the SPHAP system. The results indicate that the geometry of the interconnected porous structure in PPHAP and coral-HAP create spaces for vasculature that lead to osteogenesis while the smooth structure and close contact of particles in SPHAP inhibit vascular formation and proliferation of mesenchymal cells, preventing bone and cartilage formation. It was concluded that the geometrical structure in hydroxyapatite ceramics that induces vasculature is crucial as a carrier for BMP-induced bone formation.

Alkaline Phosphatase↗
PubMed1998

Type X collagen is not localized in hypertrophic or calcified cartilage in the developing rat trachea.

Our previous studies have shown that rat tracheal chondrocytes become larger and hypertrophic, and that the cartilage matrix calcifies during development. Type X collagen is a short collagen molecule identified in hypertrophic and calcified cartilage in the growth plate of long bones during endochondral ossification. The present study was designed to investigate the distribution of type X collagen in rat tracheal cartilage during development before and after hypertrophization and calcification. Tracheas from postnatal Wistar rats, newborn, and at 4, 8 and 10 weeks were fixed along with hind limbs from newborn rats. Serial sections were made and adjacent sections were processed for von Kossa staining or immunohistochemistry for type X collagen. In addition, the immunoreactivity to type II collagen was examined as a control. The anti-type X collagen antibody stained hypertrophic and/or calcified cartilage in the newborn rat tibia. The immunoreaction for type X collagen was localized in the uncalcified peripheral region of tracheal cartilage in 4, 8 and 10-week-old rats. In contrast, the anti-type X collagen antibody did not show immunoreactivity to hypertrophic or calcified cartilage in the central region of the 10-week-old rat tracheal cartilage. The present study has suggested that type X collagen is not involved in hypertrophization of chondrocytes or calcification of the matrix in developing rat tracheal cartilage.

Animals↗
PubMed1998

Involvement of CD14 on human gingival fibroblasts in Porphyromonas gingivalis lipopolysaccharide-mediated interleukin-6 secretion.

The lipopolysaccharides (LPS) of Porphyromonas gingivalis are implicated in the initiation and development of periodontal diseases. However, the mechanisms underlying P. gingivalis LPS-mediated periodontal destruction are still unknown. Here, it was found that P. gingivalis LPS activates human gingival fibroblasts (HGF) to release interleukin 6 (IL-6) via CD14. Flow-cytometric analysis showed that HGFs bind to fluorescein-isothiocyanate (FITC)-labelled LPS, and express CD14 on their surfaces. The binding of FITC LPS was competitively suppressed by unlabelled synthetic lipid A as well as by LPS. LPS-induced IL-6 production was inhibited by anti-CD14 monoclonal antibody in a dose-dependent manner. The binding of FITC LPS to HGF was abrogated by anti-CD14 monoclonal antibody. Engagement of LPS initiated the protein tyrosine phosphorylation of several intracellular proteins including extracellular signal-regulated kinase (ERK) 1 and 2, and these events were suppressed by the anti-CD14 monoclonal. These results suggest that CD14 is a cell surface binding site for LPS and is involved in the LPS-mediated activation of HGF.

Antibodies, Monoclonal↗