Biomedical subjects

Yoshinori Kuboki

Publications and source records attributed to Yoshinori Kuboki.

9 recordsLinked to original sources

PubMed2006

Differentiation of mesenchymal stem cells into osteoblasts on honeycomb collagen scaffolds.

Tissue engineering using living cells is emerging as an alternative to tissue or organ transplantation. The adult mesenchymal stem cells can be differentiated into multilineage cells, such as adipocytes, chondrocytes, or osteoblasts when cultured with specific growth factors. In the present investigation, we have studied the effect of honeycomb collagen scaffolds for the adhesion, differentiation and proliferation of bone marrow-derived mesenchymal stem cells into osteoblasts. Mesenchymal stem cells were isolated from 6-week old albino rat femur bone marrow, and cultured in alpha-MEM medium without beta-glycerophosphate and dexamethasone. Honeycomb collagen discs were prepared from bovine dermal atelocollagen, cross-linked by UV-irradiation and sterilized by heat. The honeycomb discs were placed on the culture dishes before seeding the stem cells. The cells attached quickly to the honeycomb collagen scaffold, differentiated and proliferated into osteoblasts. The differentiated osteoblasts were characterized by morphological examination and alkaline phosphatase activity. The osteoblasts also synthesized calcium-deficient hydroxyapatite (pseudo-hydroxyapatite) crystals in the culture. The mineralization was confirmed by Von Kossa staining and the crystals were analyzed by X-ray diffraction. Light microscopy and DNA measurements showed that the differentiated osteoblasts multiplied into several layers on the honeycomb collagen scaffold. The results demonstrated that the honeycomb collagen sponge is an excellent scaffold for the differentiation and proliferation of mesenchymal stem cells into osteoblasts. The data further proved that honeycomb collagen is an effective substrate for tissue engineering applications, and is very useful in the advancing field of stem cell technology and cell-based therapy.

Alkaline Phosphatase↗

PubMed2004

Enhanced cementum formation in experimentally induced cementum defects of the root surface with the application of recombinant basic fibroblast growth factor in collagen gel in vivo.

BACKGROUND: Therapies using biologically active, soluble factors such as growth factors or cytokines have been investigated for potential clinical use in regenerating lost periodontal tissue due to periodontitis. Basic fibroblast growth factor (bFGF, FGF-2) is a multifunctional growth factor that has a variety of effects including induction of proliferation and morphogenesis in a wide range of cells and tissues including periodontal ligament tissue. METHODS: In this study, we examined the effects of bFGF on the regeneration of cementum and periodontal ligament in experimentally induced partial defects in a beagle dog model. bFGF in a collagen gel was applied to the defects and root surfaces, and the teeth were replanted. RESULTS: Eight weeks post-surgery, formation of cementum on denuded dentin was enhanced by application of 0.1, 1, or 5 microg of bFGF in a collagen gel compared to collagen gel containing vehicle. Histological analyses revealed that at 4 weeks post-surgery, random periodontal ligament fibers had bound to dentin, but were attached only to denuded dentin to which 0.1, 1, or 5 microg of bFGF in collagen gel had been applied. At 8 weeks post-surgery, we observed the formation of dense fibers bound to alveolar bone and newly synthesized cementum in teeth treated with 1 microg of bFGF. CONCLUSION: These results suggest that basic fibroblast growth factor in a collagen gel is a suitable therapy for damaged periodontal ligament and could lead to readily achievable methods of treatment for periodontal disease.

Alveolar Process↗

PubMed2003

Failure to induce supracrestal bone growth between and around partially inserted titanium implants using bone morphogenetic protein (BMP): an experimental study in dogs.

The effect of bone morphogenetic protein on supracrestal bone growth around partially inserted implants in a dog model is described. The lower premolar teeth (P1, P2, P3 and P4) were extracted on both sides of the mandible in six dogs. At a surgical exposure 12 weeks later, two 10-mm turned titanium implants were partially inserted, approximately 15 mm apart, in the areas of the P1 and P3 in each side of the mandible, allowing five threads to protrude from the bone crest. A titanium mesh was fastened to the coronal aspect of the two fixtures and the space beneath the mesh was filled with bone morphogenetic protein (S300 BMP) in combination with an insoluble bone matrix carrier, or with the carrier alone. The mesh was covered with an ePTFE membrane. Thus, a space for potential bone formation was created between the two implants. The surgical flaps were coronally positioned and secured with vertical mattress sutures. After 16 weeks of healing, biopsy specimens were retrieved and examined histologically. Bone was not formed around the protruding implants or in the created space between the implants in any case. The carrier was incompletely resorbed. We conclude that supracrestal bone growth beyond the crestal limit with or without BMP in such a large space as in this experimental design may not be possible.

Absorbable Implants↗

PubMed2003

[Bone morphogenetic protein (s) (BMP): structure and new functions].

Classical function of BMP was to induce cartilage and bone when implanted into animal skin or muscle. In spite of this remarkable function, which attracted the attention of clinicians, it took 37 years to obtain a FDA approval for clinical use ever since the discovery of BMP. This situation was contrast to the discovery of insulin that went directly to clinic and to Nobel Prize within only 2 years. There were two reasons why BMP took so long time before clinical application. (1) BMP is a cytokine, which works multi-functionally and is relating with many other factors. (2) BMP requires suitable carriers for reasonable bone induction. Design of the recommendable carrier required systematic understanding of the nature of artificial ECM. In addition to the classical function of bone induction, new functions as a morphogen were comparatively described in a table. Examples of the new functions includes developmental patterning of dorso-ventral axis of vertebrate embryo, formation of morphogen gradient in Drosophila embryo and the mechanism of feather - branching morphogenesis were introduced.

English Abstract↗

PubMed2002

Characterization of mineral deposits formed in cultures of a hamster tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) double-positive cell line (CCP).

We have established tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) double-positive cell lines (CCP-2, CCP-7, CCP-8) from hamster bone marrow. Accumulation of mineral deposits was observed on the dishes when the clones were cultured in McCoy's 5A medium supplemented with 20% fetal calf serum. The materials were dissolved in 0.05 N HCl, and proteins found in the acid extracts were identified by N-terminal amino acid sequencing. The major components were bovine fetuin and prothrombin precursor. In addition, several cell-derived proteins, such as high mobility group 1 protein (HMG1), secretory leukocyte protease inhibitor (SLPI) and EPV20, a 2.0-kDa milk glycoprotein, were identified. HMG1 was detected, by immunostaining, on the cell surface of all the CCP clones. Metabolically labeled cellular sphingomyelin, sialyllactosylceramide, and proteoglycans were also found in the mineral deposits. Reverse transcription/polymerase chain reaction of CCP-2 mRNA revealed that the cells synthesized alkaline phosphatase, bone sialo protein, and osteonectin, but not matrix Gla protein, osteopontin, and type I collagen. CCP-2 cells formed tumors when injected subcutaneously into nude mice. In the tumor tissue, Alizarin-red-positive nodules surrounded by TRAP- and ALP-positive cells were observed, indicating CCP-2 cells can also induce calcification in vivo.

3T3 Cells↗

PubMed2002

Biochemical analysis of collagen in adhesive tissues formed after digital flexor tendon injuries.

Restrictive adhesion is a major problem following flexor tendon surgery. We investigated crosslinks and types of collagen in two adhesive tissue models in chickens, characterized by loose adhesion and dense adhesion. The flexor digitorum profundus tendon was cut and sutured in two ways: (1) the severed tendon was sutured outside the tendon sheath (loose adhesion model), and (2) the tendon sheath was excised and the severed tendon was sutured on the damaged bony floor (dense adhesion model). Biochemical analysis of collagen was done for each group after tenorrhaphy. In the outside-sheath group, the ratio of the collagen crosslinks, dihydroxylysinonorleucine/hydroxylysinonorleucine, was lower than that in the sheath-excised group at 12 weeks, while the collagen crosslinks ratio remained considerably high in the sheath-excised group. The ratio of type III collagen to type I collagen in the outside-sheath group was higher than that in the sheath-excised group at 12 weeks and approached an almost normal value at 24 weeks. The collagen type III/I ratio remained low in the sheath-excised group. The current study shows that biochemical properties are different between loose and dense adhesions. In dense adhesive scars, pathological collagen turnover occurs even at 24 weeks after tenorrhaphy.

Animals↗

PubMed2002

Inhibition of BMP-induced ectopic bone formation by an antiangiogenic agent (epigallocatechin 3-gallate).

Epigallocatechin 3-gallate (EGCG), which is one of the components of green tea, was recently shown to inhibit endothelial cell growth in vitro and angiogenesis in vivo [5]. We have previously shown that bone and cartilage formation by bone morphogenetic protein (BMP) is highly dependent on the geometry of the carrier (vasculature-inducing or -inhibiting geometry [2]. To verify the function of angiogenesis in the BMP induction system, we examine in this article whether inhibition of angiogenesis enhances chondrogenesis and suppresses osteogenesis. Fibrous glass membrane used as a BMP carrier was mixed with 1.2 micrograms rhBMP-2 and 1-10 micrograms of EGCG and was implanted into rats subcutaneously. As the dose of EGCG increased, alkaline phosphatase activity and calcium content were decreased, whereas the type II collagen content was increased. The results clearly indicated that inhibition of vascularization enhanced chondrogenesis and suppressed osteogenesis.

Alkaline Phosphatase↗

PubMed2002

Geometry of artificial ECM: sizes of pores controlling phenotype expression in BMP-induced osteogenesis and chondrogenesis.

This study aims to elucidate the feasible geometry of the scaffolds in bone and periodontal tissue engineering. Several biomaterials with different geometries are compared in terms of their patterns of ectopic BMP-induced chondrogenesis and osteogenesis. The materials include a honeycomb-shaped hydroxyapatite (HCHAP) with different tunnel sizes, a laser-perforated collagen membrane (LPM), and CPSA bioglass fibers. Implanted pellets were removed at 1-4 weeks and analyzed for bone and cartilage formation histologically and biochemically. Porous particles of hydroxyapatite (PPHAP), porous blocks of hydroxyapatite (PBHAP), and LPM did not induce detectable cartilage formation. In straight tunnel structures with various diameters in honeycomb-shaped hydroxyapatite (HCHAP), tunnels with smaller diameters (approximately 0.1 mm) induced cartilage followed by bone formation, while one with a larger diameter (0.35 mm) directly induced bone formation within the tunnels. It is concluded that the "vasculature-inducing geometry" of the carrier as an ECM is crucially important for osteogenesis.

Animals↗

PubMed2002

Pleiotrophin regulates bone morphogenetic protein (BMP)-induced ectopic osteogenesis.

We previously isolated pleiotrophin (PTN) from bovine bone as a protein and showed that it stimulated osteoblastic growth and differentiation. Further details of its function, however, have not been fully clarified. The aim of this paper was to elucidate the effects of PTN on bone morphogenetic protein (BMP)-induced ectopic osteogenesis. Recombinant human BMP (rhBMP)-2 (1.2 microg) was combined with a fibrous glass membrane, which had been established as an effective carrier. Various amounts of the purified bovine PTN (5, 10, 50, and 100 microg) or rhPTN (5 and 10 microg) were added to the rhBMP-2/carrier composites and implanted into rats subcutaneously as reported. It was found that the amount of bone induced in the system increased with the addition of 10 microg of either purified PTN or rhPTN. However, the amount of bone decreased with the addition of 50 or 100 microg of purified PTN dose-dependently, as judged by both alkaline phosphatase activity and calcium content in the retrieved implants. It was concluded that purified PTN or rhPTN, at ratios of concentration of 10-100 microg of PTN to 1.2 microg of rhBMP-2 in the carrier, regulated the ectopic bone-inducing activity of rhBMP-2.

Animals↗