Biomedical subjects

Masanori Kikuchi

Publications and source records attributed to Masanori Kikuchi.

17 recordsLinked to original sources

PubMed2006

Guided bone regeneration with beta-tricalcium phosphate and poly L-lactide-co-glycolide-co-epsilon-caprolactone membrane in partial defects of canine humerus.

This study was performed to evaluate the effect of betatricalcium phosphate and poly L-lactide-co-glycolide-coepsilon- caprolactone (TCP/PLGC) membrane in the repair of partial bone defects in canine proximal humerus. Three adult mixed-breed dogs were used during the experimental period. The length of the defect was quarter of the full length of humerus, and width of the defect was quarter of middle diameter of the lateral aspect of humerus. The humeri of each dog were divided into treatment (TCP/ PLGC) and control groups. The defect was covered with TCP/PLGC membrane in treatment group. To evaluate regeneration of the bone, computerized tomography (CT) and histopathologic examination were performed. The radiopaque lines were appeared at the original defect sites in TCP/PLGC group but below the original site in control at 4th week. Radiopacity and thickness of the defect sites, and radiopaque lines were more increased at 8th week than those of 4th week. Histopathologic findings revealed fibrous connective tissue migration into the defect and the migration inhibited the structure of new cortex to be placed in the original level in control whereas new cortex growth was found in the level of original line in TCP/ PLGC group. However, the new cortical bone in the TCP/ PLGC group was thinner and less organized than the adjacent intact cortex, and the amount of new cancellous bones were also scanty. The result suggested that TCP/ PLGC membrane is a good guided bone regeneration material to restore the original morphology of humerus in partial defect.

Absorbable Implants↗

PubMed2005

Computer-simulated bone architecture in a simple bone-remodeling model based on a reaction-diffusion system.

Bone is a complex system with functions including those of adaptation and repair. To understand how bone cells can create a structure adapted to the mechanical environment, we propose a simple bone remodeling model based on a reaction-diffusion system influenced by mechanical stress. Two-dimensional bone models were created and subjected to mechanical loads. The conventional finite element method (FEM) was used to calculate stress distribution. A stress-reactive reaction-diffusion model was constructed and used to simulate bone remodeling under mechanical loads. When an external mechanical stress was applied, stimulated bone formation and subsequent activation of bone resorption produced an efficient adaptation of the internal shape of the model bone to a given stress, and demonstrated major structures of trabecular bone seen in the human femoral neck. The degree of adaptation could be controlled by modulating the diffusion constants of hypothetical local factors. We also tried to demonstrate the deformation of bone structure during osteoporosis by the modulation of a parameter affecting the balance between formation and resorption. This simple model gives us an insight into how bone cells can create an architecture adapted to environmental stress, and will serve as a useful tool to understand both physiological and pathological states of bone based on structural information.

Bone Remodeling↗

PubMed2005

Bone repair analysis in a novel biodegradable hydroxyapatite/collagen composite implanted in bone.

The purpose of this study was to evaluate a biodegradable hydroxyapatite/collagen composite and to examine the use of the calcium ion contained for bone formation and growth. Surgical holes were prepared in the femora and tibiae of beagle dogs, and were filled with the hydroxyapatite/collagen composite labeled with alizarin red. After 4 weeks, calcein was administered to the experimental dogs. After 1 additional week, the femora and tibiae were removed surgically and fixed in formalin. Light microscopy and confocal laser scanning microscopy were used to examine the surgical holes with their implanted materials and the surrounding bone. There were only a few inflammatory cells adjacent to the hydroxyapatite/collagen composite. The newly formed bone in the cortical bone was stained with calcein, which binds to serum calcium, and new bone near the hydroxyapatite/collagen composite in the holes was stained positive for alizarin red, which binds to the calcium in the hydroxyapatite/collagen composite. In addition, osteoblasts near the hydroxyapatite/collagen composite as well as newly formed bone adjacent to the osteoblasts showed alizarin red staining, but the new bone at a distance from the hydroxyapatite/collagen implant reacted only to calcein staining. These results, using the tissue labeling method with calcein and alizarin red, suggested that the calcium bound to the alizarin red released from the hydroxyapatite/collagen composite materials might have been translocated to sites of new bone formation. The present experiment showed that the novel hydroxyapatite/collagen composite is a useful implant material for bone augmentation and that the calcium in the newly formed bone might have been released from the implant.

Absorbable Implants↗

PubMed2005

Development of a novel biomaterial, hydroxyapatite/collagen (HAp/Col) composite for medical use.

A hydroxyapatite/type I collagen (HAp/Col) composite, aligning hydroxyapatite nano-crystals along collagen molecules, has been synthesized. The biocompatibility, osteoconductivity and efficacy as an rhBMP-2 carrier of this novel biomaterial implanted in the weight-bearing site have been examined. The HAp/Col implants adsorbing 0 or 400 microg/ml of rhBMP-2 were implanted into bone defects of tibiae in 3 beagle dogs and fixed according to the Ilizarov method. As a control, bone defects of 20 mm remaining in 2 beagle dogs and the dogs were allowed to walk using a Ilizarov external skeletal fixator. The radiological and histological findings suggest that the implants induce bone remodeling units and are a superior carrier of rhBMP-2 due to the stimulation of early callus and new bone formation. As a next step, anterior fusion was carried out on 6 beagle dogs with the implants adsorbing 400 microg/ml of rhBMP-2, and 9 dogs with the implants without rhBMP-2. In 3 dogs of the rhBMP-treated group, as well as 6 dogs of the non-rhBMP-treated group, the implant was fixed with a poly-L-lactide plate. Histological and radiographical analysis suggest that enhancement of callus formation and bone bridging by rhBMP-treatment is effective to prevent collapse of the implant.

Animals↗

PubMed2004

In vitro cytocompatibility assessment of beta-tricalcium phosphate/carboxymethyl-chitin composite.

A novel bioabsorbable bone substitute composed of a beta-tricalcium phosphate (beta-TCP) and a carboxymethyl-chitin (CM-chitin) sodium has been developed. Rabbit tibia defects (4 mm in diameter) were repaired after 4 weeks more effectively by the composite compared with a sham-operation group. To further investigate the biological safety of the components, genotoxicity and carcinogenicity of an extract prepared from the composite were determined using four different in vitro assays. The main extract component was identified as CM-chitin sodium [average molecular weight (Mw) approximately 230 kDa] as determined by Fourier transform infrared spectroscopy and gel permeation chromatography analysis. The concentrations of P and Ca possibly derived from beta-TCP were 17.7 and 37.1 microg/g, respectively, as determined by inductively coupled plasma mass spectroscopy. Both the metabolic activation and nonactivation (-S9) systems of the rat microsome S9 fraction were used to perform a genotoxicity evaluation using the Ames test and chromosome aberration assay on Chinese hamster lung fibroblast cells treated with the extract. In these assays, no genotoxicity was detected with doses < or =5 mg/mL (maximum concentration). The cell transformation assay using BALB/c 3T3 cells and the metabolic cooperation assay with V79 cells both showed negative results for any tumor-promoting activity caused by the extract (approximately 5 mg/mL). These results indicate that the bioabsorbable beta-TCP/CM-chitin composite is a highly biocompatible bone substitute.

3T3 Cells↗

PubMed2004

Development of guided bone regeneration membrane composed of beta-tricalcium phosphate and poly (L-lactide-co-glycolide-co-epsilon-caprolactone) composites.

To create biodegradable and thermoplastic materials for guided bone regeneration, GBR, and guided tissue regeneration, GTR, membranes, composites of beta-tricalcium phosphate, TCP, and biodegradable polyesters, poly (L-lactide-co-glycolide-co-epsilon-caprolactone), PLGC, and poly (L-lactide-co-epsilon-caprolactone), PLCL, were prepared by a heat-kneading method. The composites maintained thermoplasticity and mechanical strength by formation of a chemical interaction between Ca on TCP and C=O on the lactide segment of PLGC or PLCL. The composites also indicated composite effects in pH auto-regulation property and elongation of biodegradation period, e.g., the composites maintained their mechanical strength up to 12 weeks after soaking in both physiological and phosphate-buffered saline, and the period was sufficient time to use for GBR and GTR membranes. Animal tests for GBR indicated that the present composite membrane successfully regenerated beagles' mandible defects 10 x 10 x 10 mm3 in size. These results suggested that the TCP/PLGC bioresorbable composites could be utilized for GBR and GTR therapy.

Animals↗

PubMed2004

Glutaraldehyde cross-linked hydroxyapatite/collagen self-organized nanocomposites.

To control the mechanical properties and biodegradability of self-organized hydroxyapatite/collagen (HAp/Col) nanocomposites, cross-linkage was introduced into the composites with glutaraldehyde (GA). The HAp/Col composite suspensions, prepared by a simultaneous titration method and aged for 3h, were cross-linked with the reagents for 10min under vigorous stirring. The precipitates obtained were filtrated and compacted by dehydration under a uniaxial pressure. The particle size distribution, 3-point bending strength, contained water amount and swelling ratio of the composites were examined as a function of cross-linkage amount; the biodegradability was estimated by animal tests using rabbits. As regards the cross-linked composites, no long-rage alignment of HAp crystals along collagen molecules was found with a transmission electron microscope, suggesting that the cross-linking reagents suppressed their long-range self-organization mechanism. The 3-point bending strength increased with the GA content and took a maximal value at 1.35mmol/g(col). The animal tests indicated no toxicity and osteoclastic resorption with good osteoconductivity. The resorption rate was decreased with increasing GA concentration. These results suggest that GA cross-linkage controls mechanical properties and resorption rate without reducing high biocompatibility of the composite.

Animals↗

PubMed2004

Development of a hydroxyapatite/collagen nanocomposite as a medical device.

The effect of cross-linking of a hydroxyapatite/collagen (HA/Col) nanocomposite, in which HA nanocrystals and collagen fibers are aligned like natural bone by a self-organization mechanism between HA and collagen in vitro, on mechanical properties was examined. The influence of degree of cross-linking, as well as rhBMP-2 preadsorption to the composite on the substitution pattern and rate with bone, was examined. In Experiment 1, anterior fusion was carried out at the C3-C4 vertebrae on 10 dogs and they were implanted as follows: without cross-linking and without adsorbed rhBMP-2 (three dogs), with cross-linking and without adsorbed rhBMP-2 (three dogs), without cross-linking and with adsorbed rhBMP-2 (two dogs), and with cross-linking and adsorbed rhBMP-2 (two dogs). Implants were removed from each dog for histology determinations after 12, 16, and 24 weeks in the non-rhBMP-treated groups, and after 16 and 24 weeks in the rhBMP-treated groups. In Experiment 2, the HA/Col composites with cross-linking and both with and without rhBMP-2 pretreatment were implanted into a bone defect of 20 mm made in the central part of tibiae in dogs (N = 3 in each group). As a control, bone defects of 20 mm remained without implantation (N = 3). The dogs were allowed to walk using an Ilizarov extra skeletal fixator. The implants were removed after 12, 16, and 24 weeks from one dog in each group. The cross-linking of the HA/Col composite was effective in controlling both the mechanical strength and bioresorbability. A "self-organization process" on the HA/Col implant surface resulted in the formation of bone remodeling units in and around the implant. Radiographic and histological findings suggest that a combined treatment of cross-linking of the HA/Col composite with preadsorption of rhBMP-2 molecules may be a very suitable replacement of existing ceramic systems in the anterior fusion of the cervical spine, as well as inlay grafting of bone defects in weight-bearing sites.

Animals↗

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

Synthesis and structural analysis of five novel oligosaccharides prepared by glucosyltransfer from beta-D-glucose 1-phosphate to isokestose and nystose using Thermoanaerobacter brockii kojibiose phosphorylase.

Five novel oligosaccharides (tetra-, penta- and hexa-saccharides) were synthesized by glucosyltransfer from beta-D-glucose 1-phosphate to isokestose (O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-alpha-D-glucopyranoside) or nystose (O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-alpha-D-glucopyranoside) using Thermoanaerobacter brockii kojibiose phosphorylase. The oligosaccharides were identified as 2(2-alpha-D-glucopyranosyl)(m)isokestose; [O-alpha-D-glucopyranosyl-(1-->2)](m)-O-[beta-D-fructofuranosyl-(2-->1)](2)-alpha-D-glucopyranoside: m=1, 2, and 3, and 2(2-alpha-D-glucopyranosyl)(n)nystose; [O-alpha-D-glucopyranosyl-(1-->2)](n)-O-[beta-D-fructofuranosyl-(2-->1)](3)-alpha-D-glucopyranoside: n=1 and 2 using gas liquid chromatography analysis of the methyl derivatives, and MALDI-TOF-MS and NMR measurements of the newly formed oligosaccharides. 1H, 13C NMR signals of each saccharide were assigned using 2D-NMR techniques, including COSY, HSQC, HSQC-TOCSY, HMBC, CH(2)-selected E-HSQC, and CH(2)-selected E-HSQC-TOCSY.

Bacteria, Anaerobic↗

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↗

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↗

PubMed2002

Development of an artificial vertebral body using a novel biomaterial, hydroxyapatite/collagen composite.

Hydroxyapatite/collagen (HAp/Col) composites having a bone-like nanostructure were synthesized and shaped into implants. This study was designed to develop an artificial vertebra system using this novel implant for anterior fusion of the cervical spine. Anterior fusion was carried out on 6 beagle dogs with the implants adsorbing rhBMP-2 (400 microg/ml). and 9 dogs with the implants without rhBMP-2. In 3 dogs of the rhBMP-treated group, as well as 6 dogs of the non-rhBMP-treated group, the implant was fixed with a poly-L-lactide plate and 2 titanium screws. Implants were taken out after 13 weeks from each 3 dogs in the rhBMP(-):plate(-). rhBMP(-):plate(+) and rhBMP(+):plate(+) groups. Also, the implants were removed from each 3 dogs in the rhBMP(-):plate(+) and rhBMP(+):plate(+) groups after 24 weeks. Histological and radiographical analysis suggested that since the larger part of the composite material was absorbed within 13 weeks, reduction of the intervertebral distance was caused, and that enhancement of callus formation and bone bridging by rhBMP-treatment was effective to prevent collapse of the implant, even though an effect of anterior plate-fixation was not obvious. The HAp/Col implant adsorbing rhBMP-2 may be a suitable replacement for the existing ceramics in anterior interbody fusion of the cervical spine.

Animals↗

PubMed2002

Effect of anti-CD14 antibody on experimental periodontitis induced by Porphyromonas gingivalis lipopolysaccharide.

The lipopolysaccharide (LPS) released by Porphyromonas gingivalis, a Gram-negative bacterium found in the periodontal pockets of patients with periodontitis, induces bone resorbing activity in vivo. We previously showed that a receptor for LPS on human gingival fibroblasts and gingival epithelial cells is CD14. In this study, we established a mouse model of experimental periodontitis by applying a P. gingivalis LPS solution to the buccal region of mice. P. gingivalis LPS-induced bone resorption and interleukin-6 production in the gingival tissues were significantly inhibited by pretreatment with anti-CD14 antibody for 5 weeks prior to LPS treatment. This result suggests that anti-CD14 antibody may be usable as a prototype for the development of drugs for the treatment of periodontal disease.

Animals↗

PubMed2002

Two novel oligosaccharides formed by 1F-fructosyltransferase purified from roots of asparagus (Asparagus officinalis L.).

Two novel oligosaccharides, tetra-and penta-saccharides were synthesized by fructosyl transfer from 1-kestose to 4G-beta-D-galactopyranosylsucrose with a purified 1F-fructosyltransferase of asparagus roots and identified as 1F-beta-D-fructofuranosyl-4G-beta-D-galactopyranosylsucrose, O-beta-D-fructofuranosyl-(2-->1)-beta-D-fructofuranosyl-O-[beta-D-galactopyranosyl-(1-->4)]-alpha-D-glucopyranoside and 1F(1-beta-D-fructofuranosyl)2-4G-beta-D-galactopyranosylsucrose, [O-beta-D-fructofuranosyl-(2-->1)]2-beta-D-fructofuranosyl-O-[beta-D-galactopyranosyl-(1-->4)]-alpha-D-glucopyranoside, respectively. Both oligosaccharides were scarcely hydrolyzed by carbohydrase from rat small intestine. Human intestinal bacterial growth by 1F-beta-D-fructofuranosyl-4G-beta-D-galactopyranosylsucrose was compared with that by the tetrasaccharides, stachyose and nystose. Bifidobacteria utilized 1F-beta-D-fructofuranosyl-4G-beta-D-galactopyranosylsucrose to the same extent as stachyose or nystose. On the other hand, the unfavorable bacteria, Clostridium perfringens, Escherichia coli and Enterococcusfaecalis, that produce mutagenic substances did not use the synthetic oligosaccharide.

Animals↗

PubMed2002

Long-term durability of porous hydroxyapatite with low-pressure system to support osteogenesis of mesenchymal stem cells.

In this study, we hypothesize that loading more marrow-derived mesenchymal stem cells (MSCs) into porous material by using a low-pressure system during subculture, creating a composite which combines MSCs and a novel mechanical reinforced porous hydroxyapatite, can result in more bone tissue formation in vivo. Within 26 weeks postimplantation, we examined in vivo bone formation of the experimental group with 100 mmHg pressure applied to porous HA blocks loaded with MSCs. For in vivo testing, the 2-week subcultured HA/MSC composites were implanted into subcutaneous sites of syngeneic rats. These implants were harvested at 13 and 26 weeks after implantation. SEM showed that the pore surface is covered by osteoblasts as well as collagenous extracellular matrix at 13 weeks. Light microscopy revealed the quantity of bone at 26 weeks was greater than at 13 weeks. These results showed that the novel mechanical reinforced porous HA combined with MSC has more potential for bone formation at 100 mmHg, making this method very efficient for bone reconstruction.

Animals↗