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Kazuhisa Bessho

Publications and source records attributed to Kazuhisa Bessho.

At least 19 recordsLinked to original sources
PubMed2007

BMP-2 promotes differentiation of osteoblasts and chondroblasts in Runx2-deficient cell lines.

To investigate the molecular mechanism underlying the differentiation of osteoblasts and chondroblasts, we established a clonal cell lines, RD-C6, from Runx2-deficient mouse embryos. RD-C6 cells expressed almost undetectable levels of phenotypes related to osteoblast and chondroblast differentiation at basal culture condition, whereas treatment with recombinant human bone morphogenetic protein-2 (rhBMP-2) or transduction of BMP-2 by adenovirus effectively induced this cell line to express mRNA related to the differentiation of osteoblasts and chondroblasts including alkaline phosphatase, osteocalcin, and osterix. Transduction of Runx2 also induced the expression of these mRNA in RD-C6 cells. BMP-2 transduction increased expression levels of mRNA for Msx2 and Dlx5, but Runx2 transduction induced no significant increases in expression levels of these mRNA. Microarray analysis using RD-C6 cells with or without rhBMP-2 treatment demonstrated that BMP-2 upregulated 66 genes including 13 transcription-related molecules such as Id1, Id2, Id4, Hey1, Smad6, Smad7, and Msx2. To confirm bone and cartilage formation ability of RD-C6 cells, we transplanted RD-C6 cells into the peritoneal cavity of athymic mice using diffusion chambers with rhBMP-2. RD-C6 cells generated unmineralized cartilage but not bone. These results indicate that BMP-2 induces Runx2-deficient cells to express markers related to osteoblast and chondroblast differentiation using a Runx2-independent pathway, but it failed to induce these cells to differentiate into bone-forming osteoblasts and mature chondrocytes.

Animals↗
PubMed2006

Simultaneous gene transfer of bone morphogenetic protein (BMP) -2 and BMP-7 by in vivo electroporation induces rapid bone formation and BMP-4 expression.

BACKGROUND: Transcutaneous in vivo electroporation is expected to be an effective gene-transfer method for promoting bone regeneration using the BMP-2 plasmid vector. To promote enhanced osteoinduction using this method, we simultaneously transferred cDNAs for BMP-2 and BMP-7, as inserts in the non-viral vector pCAGGS. METHODS: First, an in vitro study was carried out to confirm the expression of BMP-2 and BMP-7 following the double-gene transfer. Next, the individual BMP-2 and BMP-7 plasmids or both together were injected into rat calf muscles, and transcutaneous electroporation was applied 8 times at 100 V, 50 msec. RESULTS: In the culture system, the simultaneous transfer of the BMP-2 and BMP-7 genes led to a much higher ALP activity in C2C12 cells than did the transfer of either gene alone. In vivo, ten days after the treatment, soft X-ray analysis showed that muscles that received both pCAGGS-BMP-2 and pCAGGS-BMP-7 had better-defined opacities than those receiving a single gene. Histological examination showed advanced ossification in calf muscles that received the double-gene transfer. BMP-4 mRNA was also expressed, and RT-PCR showed that its level increased for 3 days in a time-dependent manner in the double-gene transfer group. Immunohistochemistry confirmed that BMP-4-expressing cells resided in the matrix between muscle fibers. CONCLUSION: The simultaneous transfer of BMP-2 and BMP-7 genes using in vivo electroporation induces more rapid bone formation than the transfer of either gene alone, and the increased expression of endogenous BMP-4 suggests that the rapid ossification is related to the induction of BMP-4.

Animals↗
PubMed2006

Gastric inhibitory polypeptide as an endogenous factor promoting new bone formation after food ingestion.

Calcium plays a fundamental role as second messenger in intracellular signaling and bone serves as the body's calcium reserve to tightly maintain blood calcium levels. Calcium in ingested meal is the main supply and inadequate calcium intake causes osteoporosis and bone fracture. Here, we describe a novel mechanism of how ingested calcium is deposited on bone. Meal ingestion elicits secretion of the gut hormone gastric inhibitory polypeptide (GIP) from endocrine K cells in the duodenum. Bone histomorphometrical analyses revealed that bone formation parameters in the mice lacking GIP receptor (GIPR(-/-)) were significantly lower than those of wild-type (GIPR(+/+)) mice, and that the number of osteoclasts, especially multinuclear osteoclasts, was significantly increased in GIPR(-/-) mice, indicating that GIPR(-/-) mice have high-turnover osteoporosis. In vitro examination showed the percentage of osteoblastic cells undergoing apoptosis to be significantly decreased in the presence of GIP. Because GIPR(-/-) mice exhibited an increased plasma calcium concentration after meal ingestion, GIP directly links calcium contained in meal to calcium deposition on bone.

Animals↗
PubMed2006

Runx3 expression during mouse tongue and palate development.

Studies of molecular mechanisms underlying the development of the mammalian oral mucosa have revealed a major involvement of transforming growth factor beta (TGF-beta) and bone morphologic protein (BMP) signaling pathways. Here, we examined the expression of a downstream target of TGF-beta and BMPs, Runx3, in oral mucosa. Runx3 is a runt-related transcription factor that acts as a gastric tumor suppressor and regulator of growth and differentiation in mammalian gastric epithelial cells. Another member of the Runx family in C. elegans, run, is involved in the development of a functional hypodermis and gut. In this report, we examined Runx3 expression using reverse transcription-polymerase chain reaction, immnunohistochemistry, and in situ hybridization and found that Runx3 is expressed in the tongue and palate epithelium of mouse embryos from embryonic day 12.5 to 16.5. The functional relationship between Runx3 and TGF-beta/BMPs signaling in tongue and palate development is discussed.

Animals↗
PubMed2005

Human BMP-2 gene transfer using transcutaneous in vivo electroporation induced both intramembranous and endochondral ossification.

It has been generally accepted that bone morphogenetic protein-2 (BMP-2) can induce osteogenesis in skeletal muscles via endochondral ossification. However, it is not clear how the ossification process occurs after the BMP-2 gene transfer to skeletal muscles in rats using in vivo electroporation. In this study, we evaluated the ossification process by BMP-2 gene transfer using in vivo electroporation. The gastrocnemius muscles of Wistar rats were injected with human BMP-2 gene expression vector (pCAGGS-BMP-2), followed by electroporation under the condition of 100 V, 50 msec per 1 sec, x8. Light and electron microscopic and radiographic analyses were performed at 1, 3, 5, 7, and 10 days after treatment. At 7 days, no sign of cartilage and/or bone formation was detected. However, at 10 days after in vivo electroporation, soft X-ray analysis revealed small lucent areas around the plasmid-injected region. Clusters of both cartilage tissues, leading to endochondral ossification and intramembranous bones of various sizes, were observed between muscle fibers. RT-PCR detected osteocalcin mRNA, showing bone formation at 10 days. Our findings strongly suggest that BMP-2 gene transfer using in vivo electroporation induces not only endochondral ossification but also intramembranous ossification.

Animals↗
PubMed2004

Effect of phosphophoryn on rhBMP-2-induced bone formation.

In the present study, dentin phosphophoryn (DPP) derived from fresh bovine dentin was evaluated as a co-factor for recombinant human bone morphogenetic protein-2 (rhBMP-2) in rhBMP-2-induced bone formation in rats. A 5 microg amount of Escherichia coli-derived rhBMP-2 variant was combined with DPP cross-linked to type I collagen (2.4 microg DPP/360 microg collagen), acting as carrier. Next, rhBMP-2/DPP/collagen composites were implanted by onlay-grafting beneath the cranial periosteum in 4-week-old Wistar rats. Rats were sacrificed at 2 and 3 weeks after implantation. Throughout the experimental period, rhBMP-2/DPP/collagen composite induced more bone formation than the rhBMP-2/collagen composite. Moreover, the degradation rate of rhBMP-2/DPP/collagen composite in rat was faster than that of rhBMP-2/collagen composite. Neither DPP/collagen composite nor collagen alone conducted bone formation even at 3 weeks postimplantation. These results indicate that the bone-inducing activity of rhBMP-2 is enhanced by DPP as a co-factor of rhBMP-2 in vivo.

Animals↗
PubMed2004

Evaluation of titanium plasma-sprayed and plasma-sprayed hydroxyapatite implants in vivo.

In this study, bone interfacial strength and bone contact length at the plasma-sprayed hydroxyapatite (HA) and titanium plasma-sprayed (TPS) implants were evaluated in vivo. Non-coated titanium (Ti) implants were used as controls. Cylindrical coated or non-coated implants (4.0mm diameter by 8mm long) were implanted in the dogs' mandibles. Loading of the implants was performed at 12 weeks after implantation. At 12 weeks after implantation (prior to loading) and 1 year after loading, implants were evaluated for interfacial bone-implant strength and bone-implant contact length. No significant differences in interfacial bone-implant strength for all groups at 12 weeks after implantation and after 1 year loading in normal bone were found. However, bone contact length for HA implants was significantly higher than the TPS and Ti implants for both periods tested (12 weeks after implantation and 1 year after loading). It was concluded that TPS implants exhibited similar pull-out strength compared to the HA implants. In addition, the lower bone contact length on the TPS surface compared to HA surfaces did not affect the interfacial bone-implant strength for both implants.

Aerosols↗
PubMed2004

Osteoinduction by bone morphogenetic protein 2-expressing adenoviral vector: application of biomaterial to mask the host immune response.

We constructed a human bone morphogenetic protein 2 (BMP-2)-expressing adenoviral vector, AxCABMP-2, which showed osteoinduction in immunosuppressed rats. In immunocompetent rats, new bone was not induced, because of the rapid elimination of transduced cells. Biomaterials such as collagen can be used as carriers for the delivery of DNA vectors, allowing prolonged expression of plasmid DNA in normal animals. We evaluated osteoinduction with AxCABMP-2 and atelopeptide type I collagen in immunocompetent rats. Collagen plus AxCABMP-2 (BMP group), collagen plus AxCALacZ (LacZ group), or collagen alone (CL group) was implanted into calf muscle pouches in immunocompetent rats, or AxCABMP-2 alone (injection group) was injected into the calf muscle. On days 3, 7, 14, and 21 after treatment, osteoinduction was evaluated. In the BMP group, bone formation was not observed on days 3 and 7. On day 14, radiographic formation was seen, but little bone formation was detected histologically. On day 21, new bone formation was observed both radiologically and histologically. In the other groups, osteoinduction was not found at any time. Immunohistochemical analysis on days 3 and 7 revealed decreased immunogenicity in the BMP group compared with the injection group. These findings suggested that collagen was an effective masking material for our vector.

Adenoviridae↗
PubMed2004

Acceleration effect of human recombinant bone morphogenetic protein-2 on differentiation of human pulp cells into odontoblasts.

Predictable pulp capping procedures remain problematic, possibly because of the lack of appropriate stimulating factors for dentin formation. The present study examines the ability of one such stimulating factor, bone morphogenetic protein-2, to accelerate the differentiation of human dental pulp cells into odontoblasts. The number and morphology of cells between groups treated with 0 and 100 ng/ml of human recombinant bone morphogenetic protein-2 (rhBMP-2) did not significantly differ. However, ALPase activity (a marker for biomineralization) in the group stimulated with rhBMP-2 was more than double that of the control group. We then measured the expression of mRNA encoding dentin sialophosphoprotein (DSPP) as a marker of odontoblasts in rhBMP-2-stimulated human pulp cells using a quantitative polymerase chain reaction. The expression of DSPP mRNA in cells stimulated for 24 h by 1000 ng/ml of rhBMP-2 was approximately 20-fold and 5-fold higher than that by stimulated by 10 and 100 ng/ml, respectively. These findings show that rhBMP-2 promoted the differentiation of human dental pulp cells into odontoblasts but did not affect cell proliferation, suggesting that rhBMP-2 may have therapeutic utility in vital pulp therapy.

Adult↗
PubMed2003

Ectopic bone formation by human bone morphogenetic protein-2 gene transfer to skeletal muscle using transcutaneous electroporation.

Therapy using recombinant human bone morphogenetic protein-2 (rhBMP-2) is expected to promote bone healing and regeneration. Previous studies using protein or virus vectors for direct clinical application had problems, including a lack of efficiency, safety, and simplicity of the delivery system, and required an expensive protein, carrier matrix, or antigenic viral vector. In vivo gene transfer by electroporation is a simple and inexpensive method that only requires a plasmid and an electroporation device. Here, we created a plasmid-based human BMP-2 construct (pCAGGS-BMP-2) and examined the induction of bone in the skeletal muscle of rats after transferring different doses of this plasmid (25 microg, 100 microg, and 400 microg) by transcutaneous electroporation (8 electrical pulses of 100 V and 50 msec, in 1 to 5 sessions). First, we verified the gene transfer by transcutaneous electroporation using pCAGGS-lacZ. Next, the BMP-2 gene transfer and the production and localization of BMP-2 were identified by reverse transcription-polymerase chain reaction (RT-PCR), Western blots, and immunohistochemistry. Ectopic bone formation was verified by radiography, histologic and immunohistochemical analyses, and quantitative examination. Ectopic bone formation, consisting of active osteoblasts and osteoclasts, was observed in all rats treated with electroporation. Thus, transcutaneous electroporation with pCAGGS-BMP-2 induced ectopic bone formation in the skeletal muscle of rats. This supports the possibility of applying human BMP-2 gene transfer using transcutaneous electroporation clinically.

Animals↗
PubMed2003

Over expression of bone morphogenetic protein-3b (BMP-3b) using an adenoviral vector promote the osteoblastic differentiation in C2C12 cells and augment the bone formation induced by bone morphogenetic protein-2 (BMP-2) in rats.

BMP-3b is a novel BMP-3-related protein and its biological functions are unknown. In order to investigate the biological actions of BMP-3b, we constructed a BMP-3b-expressing recombinant adenoviral vector (AxCAKBMP-3b). We show that over expression of BMP-3b stimulated the induction of differentiation and the osteoinduction activity of a human BMP-2-expressing recombinant adenoviral vector (AxCAOBMP-2). C2C12 cells were infected in vitro with AxCAKBMP-3b, AxCAOBMP-2 and a control vector containing no foreign genes (AxCAwt). Cells infected with AxCAOBMP-2 and AxCAKBMP-3b produced more alkaline phosphatase and secreted more osteocalcin into the culture medium than cells infected with AxCAOBMP-2 and AxCAwt. When AxCAOBMP-2, AxCAKBMP-3b, and AxCAwt were injected into the calf muscles of nude rats (F 344/N Jcl-rnu), the osteoinduction seen with AxCAOBMP-2 and AxCAKBMP-3b was greater than that seen with AxCAOBMP-2 and AxCAwt.

Adenoviridae↗
PubMed2003

A bioactive bone cement containing Bis-GMA resin and A-W glass-ceramic as an augmentation graft material on mandibular bone.

The potential of a bioactive bone cement (BABC) as an onlay graft material for the mandible with and without the periosteum was investigated in rabbits. Its matrix consists of bisphenol-alpha-glycidyl methacrylate (Bis-GMA) and triethylene-glycol dimetacrylate (TEGDMA) and its filler is silane-treated CaO-SiO2-P2O5-MgO-CaF2 glass (A-W glass-ceramic) powder. The BABC was pasted onto the mandible under the periosteum in Group 1, and onto the mandible with the periosteum removed in Group 2 and allowed to set in situ. In both groups, the cement-bone interface was filled by new bone at 4, 12 and 48 weeks, and bone grew from adjacent bone tissue into the cement-soft tissue interface at 12 and 48 weeks. There were no differences in the rate of bone formation between the groups. The shearing strength increased progressively from 0.25+/-0.10 MPa (mean+/-SD) at week 1 to 7.98+/-0.62 MPa at week 48. The results suggest that the BABC has good handling properties, a high bonding strength and good biocompatibility, and that it has potential for clinical application as a substitute material for autogenous bone transplantation.

Alveolar Ridge Augmentation↗
PubMed2002

Effect of FK506 on osteoinduction by recombinant human bone morphogenetic protein-2.

FK506 is an immunosuppressant that is used widely in organ transplantation, and it has recently been recognized as effective for promoting the growth of bone grafts [J. Bone Miner. Res. 15 (2000) 1147]. In this study, we evaluated the influence of FK506 on osteoinduction by recombinant human bone morphogenetic protein-2 (rhBMP-2) using atelopeptide type I collagen as a carrier. We administered FK506 (1 mg/kg/day intramuscularly) on days -2 to 0, -2 to 7, and -2 to sacrifice. rhBMP-2 was implanted into the calf muscle of Wistar rats (thirty per group) and the implant was sampled on days 7, 14, and 21. Radiographic evaluation, histological examination, and biochemical analysis were performed. It was found that FK506 promoted the early stage of osteoinduction after short-term administration. However, long-term administration of this agent accelerated both bone formation and bone resorption. In order to use FK506 effectively for promoting bone growth, we must further examine the appropriate dose, method, and period of administration.

Alkaline Phosphatase↗
PubMed2002

Immunoselection and adenoviral genetic modulation of human osteoprogenitors: in vivo bone formation on PLA scaffold.

The aim of this study was to examine the potential of immunoselected genetically modified human osteoprogenitors to form bone in vivo on porous PLA scaffolds. Human osteoprogenitors from bone marrow were selected using the antibody STRO-1 utilising a magnetically activated cell separation system. The STRO-1(+) fraction isolated 7% of nucleated marrow cells and increased fibroblastic colony formation by 300% and alkaline phosphatase activity by 190% over unselected marrow cell cultures. To engineer bone tissue, STRO-1(+) culture-expanded cells were transduced with AxCAOBMP-2, an adenovirus carrying the human BMP-2 gene, injected into diffusion chambers containing porous PLA scaffolds, and implanted in vivo. After 11 weeks the presence of bone mineral was observed by X-ray analysis and confirmed for mineral by von Kossa, as well as bone matrix composition by Sirius red staining, birefringence, and type I collagen immunohistochemistry. Bone formation in vivo indicates the potential of using immunoselected progenitor cells and ex vivo gene transfer with biodegradable scaffolds, for the development of protocols for the treatment of a wide variety of musculo-skeletal disorders.

Absorbable Implants↗
PubMed2002

Adenoviral BMP-2 gene transfer in mesenchymal stem cells: in vitro and in vivo bone formation on biodegradable polymer scaffolds.

The aim of this study was to determine the feasibility of adenoviral gene transfer into primary human bone marrow osteoprogenitor cells in combination with biodegradeable scaffolds to tissue-engineer bone. Osteoprogenitors were infected with AxCAOBMP-2, a vector carrying the human BMP-2 gene. Alkaline phosphatase activity was induced in C2C12 cells following culture with conditioned media from BMP-2 expressing cells, confirming successful secretion of active BMP-2. Expression of alkaline phosphatase activity, type I collagen and mineralisation confirmed bone cell differentiation and maintenance of the osteoblast phenotype in extended culture for up to 6 weeks on PLGA porous scaffolds. In vivo implantation of adenoviral osteoprogenitor constructs on PLGA biodegradeable scaffolds, using diffusion chambers, also demonstrated bone cell differentiation and production of bone tissue. The maintenance of the osteoblast phenotype in extended culture and generation of mineralised 3-D scaffolds containing such constructs indicate the potential of such bone tissue engineering approaches in bone repair.

Absorbable Implants↗
PubMed2002

The effect of fibroblast growth factor-2 on the osteoinductive activity of recombinant human bone morphogenetic protein-2 in rat muscle.

To clarify the effect of recombinant human basic fibroblast growth factor (FGF-2) on the osteoinductive activity of recombinant human bone morphogenetic protein-2 (BMP-2) in vivo, different amounts of FGF-2 (0, 16, 80 and 400 ng, and 2, 10 and 50 micro g: n=10 in each group), BMP-2 (2 micro g) and type I collagen as a carrier were mixed and implanted into rat calf muscles. Three weeks after implantation, compared with the controls, the radiopaque shadows of the implants were increased in the 16, 80 and 400 ng FGF-2-treated groups, but decreased in the 2, 10 and 50 micro g FGF-2-treated groups. In addition, alkaline phosphatase activity was increased in the 16, 80 and 400 ng FGF-2-treated groups but decreased in the 50 micro g FGF-2-treated group. Histological examination revealed increased bone formation in the 16, 80 and 400 ng FGF-2-treated groups. These results show that combined treatment with FGF-2 and BMP-2 has a biphasic effect on osteoinductive activity, i.e. it increases with low doses of FGF-2 and decreases with high doses of FGF-2.

Animals↗
PubMed2002

Expression of bone morphogenetic protein in the course of osteoinduction by recombinant human bone morphogenetic protein-2.

To clarify the mechanism of osteoinduction by recombinant human bone morphogenetic protein-2 (rhBMP-2), we examined the time-course localization of bone morphogenetic proteins (BMPs) immunostained by an anti-BMP-2 monoclonal antibody after implantation of pellets consisting of rhBMP-2 and collagen in rat calf muscle pouch. On day 3 after implantation, BMP was detected in the entire lump, and the intensity of staining for BMP around the implant on day 7 was weaker than that on day 3. The staining for BMP decreased with time and the region of staining for BMP remained more centralized in the implant. On day 10 after implantation, BMP was observed in part of the newly induced cartilage, especially around chondrocytes. On day 14 after implantation, BMP was localized in the newly induced woven bone. On day 21, BMP staining was found in osteoblasts at the surface of the newly induced bone. Especially, the staining for BMP decreased from day 10 to day 21. These results indicate that the woven bone was replaced with mature lamellar bone from day 14 to day 21. The present findings suggest that rhBMP-2 plays an important role in osteoinduction, especially at the early stage.

Animals↗