Biomedical subjects

Y Kuboki

Publications and source records attributed to Y Kuboki.

At least 37 records · Page 2Linked to original sources

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↗

PubMed1998

Properties and cytotoxicity of water soluble Na2O-CaO-P2O5 glasses.

Various compositions of Na2OCaO-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 HPO(2-)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.

Biocompatible Materials↗

PubMed1998

Carrier-dependency of cellular differentiation induced by bone morphogenetic protein in ectopic sites.

Partially purified bone morphogenetic protein (BMP) was delivered into two different types of carriers, porous particles of hydroxyapatite (PPHAP) and particles of insoluble bone matrix (IBM), and the ossification process was examined after subcutaneous implantation of the BMP/PPHAP and BMP/IBM in rats. The ossification in the BMP/PPHAP system was predominantly direct through bone formation similar to intramembranous ossification, whereas in the BMP/IBM system it was predominantly endochondral. The differences observed between the BMP/PPHAP and the BMP/IBM indicate the importance of the structure and nature of the carrier in the process of bone induction. The findings suggest that bone and cartilage differentiation is controlled not only by the regulation factor (BMP), but also by its interaction with the carrier, and that the BMP-induced cell differentiation is dependent upon the microenvironment derived from the carrier.

Animals↗

PubMed1998

A high-density rice genetic linkage map with 2275 markers using a single F2 population.

A 2275-marker genetic map of rice (Oryza sativa L.) covering 1521.6 cM in the Kosambi function has been constructed using 186 F2 plants from a single cross between the japonica variety Nipponbare and the indica variety Kasalath. The map provides the most detailed and informative genetic map of any plant. Centromere locations on 12 linkage groups were determined by dosage analysis of secondary and telotrisomics using > 130 DNA markers located on respective chromosome arms. A limited influence on meiotic recombination inhibition by the centromere in the genetic map was discussed. The main sources of the markers in this map were expressed sequence tag (EST) clones from Nipponbare callus, root, and shoot libraries. We mapped 1455 loci using ESTs; 615 of these loci showed significant similarities to known genes, including single-copy genes, family genes, and isozyme genes. The high-resolution genetic map permitted us to characterize meiotic recombinations in the whole genome. Positive interference of meiotic recombination was detected both by the distribution of recombination number per each chromosome and by the distribution of double crossover interval lengths.

Chromosome Mapping↗

PubMed1998

Regeneration of periodontal ligament and cementum by BMP-applied tissue engineering.

Previously, we demonstrated that the inductive properties of bone morphogenetic protein (BMP) highly depend on the nature of the carrier material used for implantation. In this paper, we show that administration of BMP incorporated in a fibrous collagen membrane can help to regenerate periodontal ligament and cementum both in cat canines and in monkey molars. The partially purified bovine BMP was combined with one or two layers of a fibrous collagen membrane. Although the single layer approach showed partial regeneration of periodontal defects, it also quite often led to ankylosis. The double layer technique in artificially prepared class III furcation defects in monkey molars gave favorable results. After 12 wk, not only the alveolar process but also the periodontal ligament and cementum had regenerated along the entire treated dentin surface. Collagen fibers were arranged more or less perpendicular to the surface of the new cementum. Ankylosis was not seen. It is concluded that the double-layer approach is superior to the single-layer technique in regenerating cementum.

Animals↗

PubMed1998

Conformation of dentin phosphophoryn adsorbed on hydroxyapatite crystals.

Phosphophoryn, the major noncollagenous protein of dentin, was adsorbed on synthetic hydroxyapatite crystals and analyzed by high-resolution solid-state nuclear magnetic resonance (NMR) spectroscopy. Binding of the protein was inhibited by acidic polypeptides, especially by a phosphorylated peptide. After phosphophoryn was incubated with the crystals, the crystals were collected and analyzed by 13C-cross-polarization magic-angle-spinning NMR. Several signals could be assigned to carbons of aspartic acids, taking advantage of the unique amino acid composition of this protein. Chemical shifts of signals of aspartic acids are known to reflect secondary structure of the polypeptide. The chemical shifts obtained from the phosphophoryn indicate that the secondary structure of this protein on the crystal was near to a beta-sheet structure. This result is consistent with the result for poly(Asp) adsorbed on the crystals. The beta-sheet-like structure enables phosphophoryn to extend on the crystal surface and to cover the surface with only a small number of the molecules, resulting in the high inhibitory effect of this protein on crystal growth.

Adsorption↗

PubMed1998

Effect of some physico-chemical properties of matrix on lengthwise and oriented growth of octacalcium phosphate crystal.

Relationship between some physicochemical properties of matrix, such as viscosity, density or structure of the framework, and crystal growth of octacalcium phosphate (OCP) was studied using various concentrations of polyacrylamide gels at 37 degrees C and at pH 6.5. Reaction was carried out in a model system of enamel formation, where calcium solution (30 mM) and phosphate solution (5 mM) were separated by a cation selective membrane with polyacrylamide gel on the PO4 side. OCP grew in ribbon-like morphology in 5-20% polyacrylamide gels. Crystal size of OCP decreased with an increase in gel concentration. In 30% gel, growth of OCP was disturbed, In 7.5% polyacrylamide gel, 1% albumin reduced the crystal size of OCP. In contrast, 1% enamel proteins did not much reduce the crystal size of OCP and OCP crystallized in its characteristic ribbon-like morphology.

Acrylic Resins↗

PubMed1998

pH and carbonate levels in developing enamel.

Our earlier studies showed that the surface of developing and calcifying enamel changes its pH alternatively along the tooth axis when stained with pH indicating dyes. Based on the pH conditions, the enamel at this stage was distinguished as neutral zone (N1 and N2) and acid zone (A1 and A2). The aim of the present study was to correlate changes of pH with proteolytic activity and crystal size of the calcifying bovine enamel. Specimens of developing bovine enamel were separated into four maturing stages using pH staining methods. Crystal chemistry of the developing enamel was investigated using thermogravimetry (TGA), ICP emission spectrometry, X-ray diffractometry (XRD) and infrared spectroscopy (IR). Previous biochemical analysis of proteolytic enzyme activity from enamel indicated that the optimal pH of the major protease was approximately pH 6.0, coinciding with the pH of the A1 zone. IR, TGA and XRD analyses showed that most of the organic components of the enamel decomposed at 580 degrees C. Higher levels of carbonate were observed in the secretory stages than in mature enamel. The Ca/P molar ratio of the enamel apatite was lower than the stoichiometric value of 1.67. These results suggest that growth and maturation of enamel apatite crystals is related to a decrease in the carbonate level and appear to be related to the alternative calcification and decomposition of enamel proteins.

Ameloblasts↗

PubMed1998

Two new in vitro calcification systems showing the higher calcifiability of enamel proteins than dentin and bone matrices.

One of the difficulties in simulating in vivo calcification by in vitro experiments is how to prepare and apply a suitable calcifying solution. We have previously developed an entirely new model system consisting of 40% acrylamide gel blocks that contains matrix proteins and is immersed in fetal calf serum at 37 degrees C. (40% gel system) for 18 hr (Connect. Tissue Res., 33, 185, 1995). The gels were analyzed for immobilized calcium. In this system bovine enamel proteins (0.1% in the gel) showed the highest calcifiability among the tested matrices, followed by insoluble bovine dentin, bone and skin collagens. The 40% gel system provides a barrier for high molecular weight inhibitor molecules in the body fluid. The new calcifying system developed in this study consists of the matrix protein sealed in dialysis tubing within a glass chromatography column that was eluted with a calcifying solution. In this system (dialysis tubing system), again the enamel protein showed higher calcifiability than dentin, bone and skin collagens. It was also shown that enamel proteins became not only a reversible opaque gel, but also a relatively-irreversible coagulant, if the solution contained calcium and phosphate ions at concentration below saturation (1 mM calcium and 1 mM phosphate). With both systems combined, deposition and crystal growth of minerals in enamel proteins will be better understood than with previous methods.

Acrylic Resins↗

PubMed1998

[Bone matrix proteins].

Bone matrix is composed of collagen and non-collagenous proteins. The collagen is mainly type I collagen. Characteristics of bone collagen are in posttranslational modifications and utilization of transcriptional elements in the promoter. The non-collagenous proteins are acidic Ca-binding proteins: bone Gla protein(BGP), bone sialoprotein (BSP), osteopontin, osteonectin etc. BGP and BSP are specific to bone, and other proteins are present also in non-mineralized tissues. BGP functions in suppression of excessive mineralization. BSP and osteopontin are sialoproteins containing a RGD cell-attachment sequence and poly(acidic amino acid) sequences. BSP is present in sites of bone formation. Osteopontin is involved in attachment of osteoclasts to bone surface.

Animals↗

PubMed1997

Measurement of histidinohydroxylysinonorleucine and hydroxyproline in skin collagen by reversed-phase high-performance liquid chromatography after 9-fluorenylmethyl chloroformate labeling.

A novel, highly sensitive method to quantify histidinohydroxylysinonorleucine (HHL), a trifunctional type of cross-link in skin collagen, was developed. HHL in skin hydrolysates labeled with 9-fluorenylmethyl chloroformate (FMOC-Cl) was separated by reversed-phase high-performance liquid chromatography. Mass spectrometric analysis revealed that two FMOCs were bound to two primary amino acid residues, histidine and hydroxylysine, but not to lysine residue in one HHL molecule. Hydroxyproline was simultaneously measured to express the molar ratio of HHL to collagen. The detection range of HHL was from 1 to 10 pmol and that of hydroxyproline from 1 to 50 pmol. A 6-mm punch-biopsied human skin sample contained 0.40 to 0.69 mol of HHL per one molecule of collagen. This sensitive method is useful as it is rapid and can be used to examine the aging process or the change of HHL content in skin collagens of various pathologic states.

Chromatography, High Pressure Liquid↗

PubMed1997

Bone mass loss due to estrogen deficiency is compensated in transgenic mice overexpressing human osteoblast stimulating factor-1.

Osteoblast stimulating factor-1 (OSF-1) stimulates in vitro proliferation and differentiation of osteoblastic cells, and its gene is expressed in the bone and brain tissues in mammals and amphibians. To evaluate the in vivo function of OSF-1 in bone metabolism, transgenic mice overexpressing the human osf-1 gene driven by the osteocalcin promoter were generated. Femoral bone mineral content was increased in transgenic mice relative to wild-type controls as estimated by ash assay, depending on the transgene copy number per cell. In ovariectomized mice, bone mass loss due to estrogen deficiency was observed in both transgenic and control mice but bone mass was still higher in transgenic mice than in controls. Bone mass in ovariectomized transgenic mice was comparable to that in wild-type mice without ovariectomy. These observations indicate that OSF-1 may direct in vivo appositional bone formation by increasing osteoblast activity rather than decreasing osteoclast activity, suggesting a new way to treat osteoporosis with OSF-1.

Animals↗