Biomedical subjects

Junzo Tanaka

Publications and source records attributed to Junzo Tanaka.

At least 55 records · Page 3Linked to original sources

PubMed2003

Hydroxyapatite-coated tendon chitosan tubes with adsorbed laminin peptides facilitate nerve regeneration in vivo.

On the inner surface of tendon chitosan tubes having a triangular shape and a hydroxyapatite coating (t-chitosan/HAp tube), laminin-1 and laminin peptides (YIGSR, IKVAV) have been adsorbed in order to develop nerve growth conduits. The mechanical property, biocompatibility and efficacy of these tubes for nerve regeneration were examined. Step-1: bridge grafting (15 mm) into the sciatic nerve of Sprague-Dawley (SD) rats was carried out using either t-chitosan or t-chitosan/HAp tubes having either a circular or triangular cross section (N=12 in each group). Specimens were taken after 2-, 4-, 6- and 8-week post-implantation (N=3 in each group) for histology determinations. Step-2: t-chitosan/HAp tubes having a triangular cross section with adsorbed laminin-1, CDPGYIGSR or CSRARKQAASIKVAVSAD, as well as control tubes without pre-adsorption were used for implantation (N=18 in each group). Isografting was also carried out (N=6). Histological evaluation was carried out similarly as in Step-1. Furthermore, evoked muscle and sensory nerve action potentials were recorded, and the percentage of myelinated axon area measured at 10 mm distance of the distal anastomosed site in the experimental, control and isograft groups after 12 weeks (N=6 in each group). The results of histological findings, as well as mechanical properties, suggest that a triangular tube shape with a HAp coating benefits nerve regeneration. The effect of laminin peptides (YIGSR, followed by IKVAV) to enhance the growth of regenerating axons has been found comparable with intact laminin-1. Although histological regeneration in both the YIGSR- and laminin-1-treated t-chitosan/HAp tubes matches the isografts, the functional recovery is however delayed.

Animals↗

PubMed2003

Transcriptional regulation of human beta-galactoside alpha2,6-sialyltransferase (hST6Gal I) gene in colon adenocarcinoma cell line.

Previous studies have shown that hST6Gal I mRNA is overexpressed in colorectal cancer tissues compared with non-malignant or benign tissue. Moreover, Form 1 (hepatic form) mRNA isoform had a marked tendency to accumulate in colon cancer [Int. J. Cancer 88 (2000) 58-65]. These findings suggest that the transcriptional regulation of Form 1 is altered during malignant transformation. We report here transcriptional regulation of the hST6Gal I gene in colon adenocarcinoma cell lines. We characterized P1 promoter region, which regulates Form 1 mRNA expression, using luciferase assays. The result indicates that the nt-156 to -1 region is important for transcriptional activity of hST6Gal I gene in colon adenocarcinoma cell lines. The nt-156 to -1 region contains HNF1 recognition element. Mutation of the HNF1 site reduced luciferase activity by about 80% compared with the wild-type construct, suggesting that HNF1 site is involved in the transcription of Form 1 mRNA in colon cancer cells.

Adenocarcinoma↗

PubMed2003

DNA microarray analysis of human gingival fibroblasts from healthy and inflammatory gingival tissues.

In the inflammatory gingival tissues of patients with periodontitis, cytokines such as interleukin (IL)-1 alpha, IL-1 beta, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha have been detected. Gingival fibroblasts are the major constituents of gingival tissue. We recently demonstrated that lipopolysaccharide (LPS) from periodontopathic bacteria induces inflammatory reactions in various tissues via CD14 and/or Toll-like receptors (TLRs) in gingival tissues [Biochem. Biophys. Res. Commun. 273 (2000) 1161]. To confirm this, we examined the expression of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF-alpha, CD14, TLR2, and TLR4 in human gingival fibroblasts (HGFs) obtained from patients with healthy or inflammatory gingiva using DNA microarray analysis. We also studied the expression levels of these proteins by flow cytometric analysis (FACS). The expression levels of all eight genes in the HGFs of the Inflammatory group were significantly higher than those in the Healthy group on DNA microarray analysis. FACS revealed that the expression levels of all eight proteins on the HGFs of the Inflammatory group were higher than those on the Healthy group. Our data indicated that these eight proteins in HGFs are involved in inflammatory conditions in the gingiva, including periodontal disease. Our results suggested that these eight proteins, in turn, act directly or indirectly on the immune response by activating host cells involved in inflammatory processes.

Cells, Cultured↗

PubMed2003

Tendon chitosan tubes covalently coupled with synthesized laminin peptides facilitate nerve regeneration in vivo.

We have developed tendon chitosan tubes having the ability to bind peptides covalently, and the effectiveness of laminin peptides coupled to these tubular wall on nerve regeneration was examined in vivo. Bridge graft implantation (15 mm) into the sciatic nerve of SD rats was carried out using chitosan tubes having a triangular cross section containing either covalently bound intact laminin or the laminin peptides CDPGYIGSR or CSRARKQAASIKVAVSAD or being nontreated (N = 20 in each group). As a control, isografting (N = 5) was carried out. Three rats in each experimental group were sacrificed for histology observations after 1, 2, 4, 6, and 8 weeks. The total area of regenerating tissue in the tube and the length of the area where regenerating tissue attached to the inner surface of the tube were measured. In five rats from each experimental and control group, the latency quotient between the implanted and the nontreated site was determined 12 weeks after implantation. Furthermore, the percentage of myelinated axon area was measured at a 10-mm distance from the distal anastomosed site. Histological findings suggest that the immobilized laminin, confirmed by immunostaining as long as 12 weeks postoperatively, as well as laminin oligopeptides may effectively assist nerve tissue extension. According to statistical analysis of the percentage neural tissue found in relation to evoked action potentials, the sequential treatments with YIGSR first followed by IKVAV matched the effectiveness of intact laminin in enhancing nerve regeneration. However, when compared with that after isografting, the enhancement of regenerated axon growth was less sufficient.

Absorbable Implants↗

PubMed2003

Physical and biological evaluations of sintered hydroxyapatite/silicone composite with covalent bonding for a percutaneous implant material.

A composite (HA/silicone) of hydroxyapatite (HA) microparticles with an average diameter of 2.0 micro m covalently linked to a silicone substrate has been developed, and its physical and biological properties as a percutaneous soft-tissue-compatible material have been evaluated. In tensile property measurement, samples of HA/silicone and the original silicone were similar in tensile strength, ca. 7.8 MPa, and elongation at break, ca. 570%. It was found that chemical surface modification with HA particles presented no mechanical disadvantage. In an adhesive-tape peeling test, scanning electron microscopic (SEM) observation showed that HA particles coupled directly to the substrate were not removed. HA particles may bond strongly with the substrate. In human periodontal ligament fibroblast attachment and proliferation experiments, the number of cells attached to HA/silicone was 14 times greater than that attached to the original silicone after 24 h of incubation. The value on HA/silicone was ca. 80% versus that on a tissue-culture plastic used as a positive control. After 72 h of incubation, the number of cells grown on HA/silicone increased to the level of the positive control. In observation of fluorescence microscopy stained by Hoechst 33342, cells appeared to tightly adhere to HA particles coupled to the silicone sheet due to intact nuclear morphology. Observation of cells by fluorescence dye with rhodamin phalloidin showed an extensive F-actin cytoskeleton on HA/silicone. In a 4-week animal implant test, force required to pull out the HA/silicone sheet was 15 times that of the original silicone. HA-particle coating on silicone with covalent linkage gave the inert surface bioactivity. The HA composite thus effectively prevents germ infection percutaneously.

Animals↗

PubMed2003

Physical properties of type I collagen extracted from fish scales of Pagrus major and Oreochromis niloticas.

Type I collagens were extracted from fish scales of Pagrus major and Oreochromis niloticas as a possible underutilized resource for medical materials. The fish scales were demineralized with EDTA and digested by pepsin. The resultant type I collagens contained more than 33.6% of glycine as the most abundant amino acid. The denaturation temperatures of the collagens from P. major and O. niloticas were 303 and 308K, respectively, both of which were relatively lower than that of porcine dermis collagen (314K). CD spectra indicated that the denaturation temperatures were dependent on the amount of hydroxyproline, rather than proline residues. Raman spectra also indicated that the relative intensities of Raman lines at 879 and 855cm(-1) assigned to Hyp and Pro rings were changed due to the contents of the imino acids. Significantly, the content of sulphur-containing methionine was higher in the fish scales than in porcine dermis. The enthalpy and entropy estimated from thermal analyses could be correlated to amino acid sequences (Gly-Pro-Hyp) of type I collagens and the number of methionine amino acid residues.

Animals↗

PubMed2003

The chitosan prepared from crab tendon I: the characterization and the mechanical properties.

Crystalline chitosan was prepared from crab tendon consisting mainly of chitin, including various proteins and calcium phosphates. The crab tendon has high mechanical properties due to its aligned molecular structure. Crab tendon components, i.e. proteins and calcium phosphates, were removed by deacetyl treatment using 50wt% NaOH aqueous solution at 100 degrees C, and a subsequent ethanol treatment. As judged from microscopic observations using an optical polarizer, the treated chitosan remained intact regarding its aligned molecular structure, and had a high tensile strength of 67.9+/-11.4MPa. The tensile strength was further enhanced to 235+/-30MPa by a thermal treatment at 120 degrees C, corresponding to the formation of the intermolecular hydrogen bonds.

Animals↗

PubMed2003

Transplantation of cultured bone cells using combinations of scaffolds and culture techniques.

The transplantation of cultured bone cells is expected to become a candidate for bone regeneration therapy. For the clinical application of this therapy, there remain several problems to be overcome, for example, the improvements of scaffolds and culture techniques. In this review article, two kinds of porous ceramics, a novel sintered porous hydroxyapatite and a porous beta-tricalcium phosphate (TCP), as well as a collagen-phosphosphoryn sponge are introduced as new scaffolds for bone regeneration. The former two ceramic scaffolds proved to be applicable for bone regeneration therapy. The collagen-phosphophoryn sponge proved to have bone formation ability in vivo. Moreover, for the application of this therapy to the regeneration of large bone defects, we improved the culture method by applying a low-pressure system and a perfusion system. Both culture systems accelerated the formation of bone in vivo in this transplantation model. Combinations of the scaffolds and culture techniques might be considered when designing therapeutic strategies.

Animals↗

PubMed2003

The chitosan prepared from crab tendons: II. The chitosan/apatite composites and their application to nerve regeneration.

The chitosan tubes derived from crab tendons form a hollow tube structure, which is useful for nerve regeneration. However, in order to use the chitosan tubes effectively for nerve regeneration, there remain two problems to be solved. First, the mechanical strength of the tubes is quite high along the longitudinal axis, but is somewhat low for a pressure from side. Second, the chitosan tube walls swell to reduce the inner space of the tubes in vivo. These two problems limit the clinical use of the chitosan tubes. In this study, to solve the problems, apatite was made to react with the chitosan tubes to enhance the mechanical strength of the tube walls. Transmission electron microscopy showed that apatite crystals were formed in the walls of the chitosan tubes. The c-axis of the crystals aligned well in parallel with chitosan molecules. These results indicate that the apatite crystals grow in the tubes starting from the nucleation sites of the chitosan molecules, probably by forming complexes with amino groups of chitosan and calcium ions. Further, the tubes were thermally annealed at 120 degrees C to prevent from swelling, and simultaneously formed into a triangular shape to enhance the stabilization of the tube structure. By these treatments, the hollow tubes could keep their shape even in vivo after implantation. Animal tests using SD rats further showed that the chitosan tubes effectively induced the regeneration of nerve tissue, and were gradually degraded and absorbed in vivo.

Animals↗

PubMed2003

Microstructure, mechanical, and biomimetic properties of fish scales from Pagrus major.

The fish scale of Pagrus major has an orthogonal plywood structure of stratified lamellae, 1-2 microm in thickness, consisting of closely packed 70- to 80-nm-diameter collagen fibers. X-ray diffraction, energy-dispersive X-ray analysis, and infrared spectroscopy indicate that the mineral phase in the scale is calcium-deficient hydroxyapatite containing a small amount of sodium and magnesium ions, as well as carbonate anions in phosphate sites of the apatite lattice. The tensile strength of the scale is high (approximately 90 MPa) because of the hierarchically ordered structure of mineralized collagen fibers. Mechanical failure occurs by sliding of the lamellae and associated pulling out and fracture of the collagen fibers. In contrast, demineralized scales have significantly lower tensile strength (36 MPa), indicating that interactions between the apatite crystals and collagen fibers are of fundamental importance in determining the mechanical properties. Thermal treatment of fish scales to remove the organic components produces remarkable inorganic replicas of the native orthogonal plywood structure of the fibrillary plate. The biomimetic replica produced by heating to 873 K consists of stratified porous lamellae of c-axis-aligned apatite crystals that are long, narrow plates, 0.5-0.6 microm in length and 0.1-0.2 microm in width. The textured inorganic material remains intact when heated to 1473 K, although the size of the constituent crystals increases as a result of thermal sintering.

Animals↗

PubMed2003

Surface modification of a porous hydroxyapatite to promote bonded polymer coatings.

Porous hydroxyapatite (Hap) blocks were sintered at several temperatures and methyl methacrylate (MMA) grafted onto the surface in a 2-step heterogeneous system as a model example for surface modification. First, sintered porous Hap was modified with 2-methacryloyloxyethylene isocyanate (MOI) monomer in anhydrous dimethyl sulfoxide using di-n-butyltin (IV) dilaurate as a catalyst and hydroquinone as an inhibitor. Amount of the introduction of MOI monomer on porous Hap was 1.62 wt % at sintered temperature 800 degrees C, 0.68 wt % at it of 1000 degrees C, and 0.59 wt % at it of 1200 degrees C. Scanning electron microscopy (SEM) showed that porous Hap pore size and shape before and after MOI treatment were unchanged. Second, graft polymerization with MMA through the vinyl bond on porous Hap was conducted using alpha,alpha'-azobis isobutyronitrile (AIBN) as an initiator. Amount of Grafted PMMA on the MOI modified porous Hap was 2.84 wt % at sintered temperature of 800 degrees C, 6.97 wt % at it of 1000 degrees C, and 6.27 wt % at it of 1200 degrees C. MOI-modified and PMMA-grafted porous Hap were characterized using Fourier transform infrared (FT-IR) spectroscopy. The compressive strength of sintered porous Hap with grafted PMMA increased about 2.7-6.7 times compared to intact porous Hap. This 2-step surface modification on porous Hap is widely applicable to graft polymerization with vinyl polymer and conjugation with a protein or an oligopeptide, such as growth factor or an adhesion molecule, to improve Hap mechanical properties and functionality.

Journal Article↗

PubMed2003

Dextran templating for the synthesis of metallic and metal oxide sponges.

Silver or gold-containing porous frameworks have been used extensively in catalysis, electrochemistry, heat dissipation and biofiltration. These materials are often prepared by thermal reduction of metal-ion-impregnated porous insoluble supports (such as alumina and pumice), and have surface areas of about 1 m(2) g(-1), which is typically higher than that obtained for pure metal powders or foils prepared electrolytically or by infiltration and thermal decomposition of insoluble cellulose supports. Starch gels have been used in association with zeolite nanoparticles to produce porous inorganic materials with structural hierarchy, but the use of soft sacrificial templates in the synthesis of metallic sponges has not been investigated. Here we demonstrate that self-supporting macroporous frameworks of silver, gold and copper oxide, as well as composites of silver/copper oxide or silver/titania can be routinely prepared by heating metal-salt-containing pastes of the polysaccharide, dextran, to temperatures between 500 and 900 degrees C. Magnetic sponges were similarly prepared by replacing the metal salt precursor with preformed iron oxide (magnetite) nanoparticles. The use of dextran as a sacrificial template for the fabrication of metallic and metal oxide sponges should have significant benefits over existing technologies because the method is facile, inexpensive, environmentally benign, and amenable to scale-up and processing.

Copper↗

PubMed2003

Application of perfusion culture system improves in vitro and in vivo osteogenesis of bone marrow-derived osteoblastic cells in porous ceramic materials.

Composites of bone marrow-derived osteoblasts (BMOs) and porous ceramics have been widely used as a bone graft model for bone tissue engineering. Perfusion culture has potential utility for many cell types in three-dimensional (3D) culture. Our hypothesis was that perfusion of medium would increase the cell viability and biosynthetic activity of BMOs in porous ceramic materials, which would be revealed by increased levels of alkaline phosphate (ALP) activity and osteocalcin (OCN) and enhanced bone formation in vivo. For testing in vitro, BMO/beta-tricalcium phosphate composites were cultured in a perfusion container (Minucells and Minutissue, Bad Abbach, Germany) with fresh medium delivered at a rate of 2 mL/h by a peristaltic pump. The ALP activity and OCN content of composites were measured at the end of 1, 2, 3, and 4 weeks of subculture. For testing in vivo, after subculturing for 2 weeks, the composites were subcutaneously implanted into syngeneic rats. These implants were harvested 4 or 8 weeks later. The samples then underwent a biochemical analysis of ALP activity and OCN content and were observed by light microscopy. The levels of ALP activity and OCN in the composites were significantly higher in the perfusion group than in the control group (p < 0.01), both in vitro and in vivo. Histomorphometric analysis of the hematoxylin- and eosin-stained sections revealed a higher average ratio of bone to pore in BMO/beta-TCP composites of the perfusion group after implantation: 47.64 +/- 6.16 for the perfusion group and 26.22 +/- 4.84 for control at 4 weeks (n = 6, p < 0.01); 67.97 +/- 3.58 for the perfusion group and 47.39 +/- 4.10 for control at 8 weeks (n = 6, p < 0.05). These results show that the application of a perfusion culture system during the subculture of BMOs in a porous ceramic scaffold is beneficial to their osteogenesis. After differentiation culture in vitro with the perfusion culture system, the activity of the osteoblastic cells and the consequent bone formation in vivo were significantly enhanced. These results suggest that the perfusion culture system is a valuable and convenient tool for applications in tissue engineering, especially in the generation of artificial bone tissue.

Alkaline Phosphatase↗

PubMed2003

Development of a nerve scaffold using a tendon chitosan tube.

Bridge grafting (15 mm) into the sciatic nerve of SD rats was carried out using tendon chitosan tubes having either a circular or triangular cross-section, as well as triangular tubes combined with laminin, CDPGYIGSR, or CSRARKQAASIKVAVSAD (n = 15 in each group). As a control, isografting (15 mm) was carried out in the SD rats (n = 7). Specimens were taken after 1, 2, 4, 6, and 8 weeks for histology, and nerve regeneration was evaluated electro-physiologically and histologically after 12 weeks. The mechanical strength of triangular tubes was found to be higher than circular tubes, and the inner volume of a triangular tube tends to be larger than in circular tubes. Nerve tissue regeneration along the tube wall was found in both the laminin and laminin peptide groups. According to the result of percentage neural tissue in relation to evoked action potentials, the consecutive treatments of YIGSR and IKVAV was found to match the effectiveness of intact laminin.

Animals↗

PubMed2003

Rapid and large-scale formation of chondrocyte aggregates by rotational culture.

Chondrocytes in articular cartilage synthesize collagen type II and large sulfated proteoglycans, whereas the same cells cultured in monolayer (2D) dedifferentiate into fibroblastic cells and express collagen type I and small proteoglycans. On the other hand, a pellet culture system was developed as a method for preventing the phenotypic modulation of chondrocytes and promoting the redifferentiation of dedifferentiated ones. Because the pellet culture system forms only one cell aggregate each tube by a centrifugator, the pellet could not be applied to produce a tissue-engineered cartilage. Therefore, we tried to form chondrocyte aggregates by a rotational culture, expecting to form a large number of aggregates at once. In order to increase cell-cell interactions and decrease chondrocyte-material interaction, dishes with low retention of protein adsorption and cell adhesiveness were used. In addition, rotational shaking of the dish including cells was attempted to increase the cell-cell interaction. The shaking speed was set at 80 rpm, so the cells would be distributed in the center of the dish to augment the frequency of cell-cell contact. Under these conditions, bovine articular chondrocytes started aggregating in a few hours. At 24-36 h of rotational culture, aggregates with smooth surfaces were observed. Parameters such as increase of culture time and addition of TGF-beta controlled diameters of the aggregates. There were many fusiform cells at the periphery of the aggregates, where the cells tended to form a multilayered zone in cross sections. In addition, lacune-like structure, which was almost the same as pellet culture, was observed. It was found that the internal structure of the aggregates was similar to that of pellets reported previously. Therefore, the aggregates formed by a rotational culture could become an essential component to make tissue-engineered artificial cartilage.

Animals↗

PubMed2002

An improved method to prepare hyaluronic acid and type II collagen composite matrices.

An improved method to prepare hyaluronic acid (HyA) and type II collagen composite matrices was developed. HyA and type II collagen formed polyion complex (PIC) in aqueous solution due to their opposite electrostatic charges. Therefore, optimal conditions at which PIC was not formed between HyA and type II collagen were determined in order to directly crosslink using a water-soluble carbodiimide [WSC; 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride]. HyA-collagen composite matrices were successfully obtained using WSC in the presence of 0.4M NaCl, which was found the optimal concentration to suppress PIC formation. Fourier transformed infrared spectra of the obtained matrices showed ester bond formation, indicating that the crosslinking reaction between HyA molecules, HyA and collagen molecules, and collagen molecules proceeded. The swelling ratio of a HyA-collagen composite matrix depended on the WSC concentration and HyA content. From scanning electron microscopy observations, the lyophilized matrices possess multi pore structures. The HyA-collagen composite matrices using this improved method will be useful in the preparation of cartilage regenerative scaffolds, and this methodology may be applied to other polyanion-polycation combinations as well.

Biocompatible Materials↗

PubMed2002

In vitro change in mechanical strength of beta-tricalcium phosphate/copolymerized poly-L-lactide composites and their application for guided bone regeneration.

Novel composites of bioactive beta-tricalcium phosphate [Ca(3)(PO(4))(2)] and biodegradable copolymerized poly-L-lactide (CPLA) were prepared by a heat-kneading method. The mechanical and chemical changes of the composites were evaluated in vitro by soaking in physiological saline and Dulbecco's phosphate buffered saline. When soaked in physiological saline, the 3-point mechanical strength decreased rapidly from 60 to 30 MPa in the initial 4 weeks and then gradually reached a plateau; the initial decrease in the mechanical strength was ascribed to the dissolution of beta-tricalcium phosphate from the surface. The mechanical properties evident at 8-12 weeks were sufficient for the composites to be used as a biodegradable material for regeneration of bone because the hydrolysis of CPLA was inhibited in both physiological saline and phosphate-buffered saline as a result of a pH-buffering effect. Composite membranes 250-microm thick were used to regenerate large bone defects in beagle dogs: 10 x 10 x 10 mm(3) in volume in the mandible and 20 mm in length in the tibia. The afflicted areas covered with the composite membranes were almost perfectly filled with new bone 12 weeks after the operation, whereas those covered with a CPLA membrane or without any membranes were invaded by soft tissue.

Algorithms↗

PubMed2002

Implantation study of a novel hydroxyapatite/collagen (HAp/col) composite into weight-bearing sites of dogs.

A hydroxyapatite/type I collagen (HAp/Col) composite, aligning hydroxyapatite nanocrystals along collagen molecules, has been prepared. The biocompatibility, osteoconductive activity, and efficacy as a carrier of rhBMP-2 of this novel biomaterial implanted in the weight-bearing site have been examined. The HAp/Col implants (15 mm in diameter and 20 mm in length) with a surface cross-linked layer containing rhBMP-2 (0 or 400 microg/ml) were implanted into bone defects of tibiae in three beagle dogs and fixed according to the Ilizarov method. As a control, bone defects of 20 mm in two beagle dogs did not receive implants, and the dogs were allowed to walk using an Ilizarov extraskeletal fixator. The specimens were removed from one dog in each group after 12 weeks. Also, the Ilizarov fixators in the rhBMP-treated dogs were removed after 12 weeks, after which full weight bearing started. The specimens were further taken out after 18 and 24 weeks in the rhBMP-treated and non-rhBMP-treated dogs, and after 24 weeks in the control group. The change of bone mineral density, as well as radiological and histological findings, suggest that the implants are able to induce bone remodeling units and are a superior carrier of rhBMP-2 due to the stimulation of early callus and new bone formation.

Animals↗