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ABSTRACT: In previous studies, we have reported that the BMP-2-derived peptide KIPKASSVPTELSAISTLYL, corresponding to BMP-2 residues 73-92, binds to a BMP-2-specific receptor, and elevates both alkaline phosphatase activity and osteocalcin mRNA in the murine mesenchymal cell line, C3H10T1/2. This 73-92 peptide conjugated to a covalently crosslinked alginate gel induced ectopic bone formation in rat calf muscle, and activated osteoblasts to promote the repair of rat tibial bone defects. Here, we report repair of 20-mm long rabbit radial bone defects using the 73-92 peptide combined with a porous alpha-tricalcium phosphate (TCP) scaffold. In vitro, the 73-92 peptide was released from the porous alpha-TCP scaffold over more than one week. In vivo, radiomorphometric analysis showed that the 73-92 peptide combined with the porous alpha-TCP scaffold promoted calcification in the implanted area in a dose-dependent manner, and that 5 mg of the 73-92 peptide induced connection of 20-mm long defects, defects of critical size, 12 weeks after implantation. Histological examination revealed newly formed bone and a marrow cavity in the implanted area. The area of bone denser than 690 mg/cm(3) induced by the 73-92 peptide was nearly equal to that of the contralateral radius.
Journal of Biomedical Materials Research Part A 07/2006; 77(4):700-6. · 2.63 Impact Factor
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ABSTRACT: A novel synthetic peptide corresponding to BMP-2 residues 73-92 that can induce bone formation and can form a conjugate with a carrier to localize its effect has been reported previously. The synthetic peptide was bound to a BMP-2-specific receptor, and it elevated both the alkaline phosphatase activity and the osteocalcin mRNA in the murine multipotent mesenchymal cell line, C3H10T1/2. The 73-92 peptide also induced ectopic bone formation when conjugated to a covalently crosslinked alginate gel and implanted into a rat's calf muscle. Here, it is reported that the 73-92 peptide-conjugated alginate gel particles significantly promoted the repair of rat tibial bone defects, whereas the alginate gel sponge that the peptide was conjugated with was less effective. Further acceleration and denser bone regeneration was achieved when the 73-92 peptide-conjugated alginate gel particles were coimplanted with syngeneic rat bone-marrow stromal cells. Therefore, the 73-92 peptide can induce differentiation of osteoblast precursor cells into osteoblasts, and can activate osteoblasts to promote the repair of bone defects.
Journal of Biomedical Materials Research Part A 02/2005; 72(1):77-82. · 2.63 Impact Factor
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ABSTRACT: Bone morphogenetic protein-2 (BMP-2) promotes the formation and regeneration of bone and cartilage, and therefore constitutes the most promising candidate for a bone repair material. However, it also has a wide range of functions, such as in organogenesis and apoptosis. Therefore, we investigated a novel synthetic peptide corresponding to residues 73-92 of BMP-2. This peptide bound to a BMP-2-specific receptor and elevated both alkaline phosphatase activity and osteocalcin mRNA in the murine cell line, C3H10T1/2. The 73-92 peptide also induced ectopic calcification when conjugated to a covalently crosslinked alginate gel. Here we report that the 73-92 peptide-conjugated alginate gel showed prolonged ectopic calcification for up to 7 weeks in rat calf muscle. In contrast, rhBMP-2-impregnated collagen gel showed maximum ectopic calcification at 3 weeks, and the calcified products that had formed disappeared after 5 weeks. Histological examination showed that the 73-92 peptide-conjugated alginate gel induced many osteoblast-like cells and few osteoclasts. In contrast, rhBMP-2-impregnated collagen gel induced many osteoclasts. These results suggest that the 73-92 peptide on alginate gel remains active at the implanted site, continuously induces differentiation of osteoblast precursor cells into osteoblasts, and activates osteoblasts to promote ectopic calcification.
Journal of Biomedical Materials Research Part A 08/2004; 70(1):115-21. · 2.63 Impact Factor
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ABSTRACT: Apoptosis as well as cell growth and cell differentiation play an important role in the maintenance of homeostasis in multicellular organisms. Disruption of apoptosis causes serious diseases, such as cancer, chronic inflammatory diseases, and autoimmune diseases; therefore, the control of apoptosis is one of the most promising therapeutic approaches to these apoptosis-disrupted diseases. Apoptosis is mediated by soluble factors, which belong to the TNF superfamily, such as TNF-alpha, FasL, and TRAIL. Here, we report that we deduced ligand-binding domains based on the structure of apoptosis ligand-receptor complex, and the synthetic peptide corresponding to residues 91-102 of DR5 indeed showed specific binding to TRAIL molecule and inhibited TRAIL-induced cell death both in L929 cells and in HeLa cells. The other death receptor-derived peptides, which are the corresponding regions of TNFR1 and Fas, also showed specific binding to TNF-alpha and FasL and inhibited the ligand-induced cell death, respectively. These results suggest that the position of the ligand-binding region is conserved among these death-receptor family members, whereas the primary amino acid sequence determines ligand specificity.
Biochimica et Biophysica Acta 07/2004; 1699(1-2):131-7. · 4.66 Impact Factor
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ABSTRACT: Bone morphogenetic protein-2 (BMP-2) promotes the formation and regeneration of bone and cartilage, and also participates in organogenesis, cell differentiation, cell proliferation, and apoptosis. BMP-2 has two epitopes referred to as the "wrist epitope" and the "knuckle epitope". The wrist epitope is thought to bind to BMP receptor IA and the knuckle epitope to BMP receptor type II. However, the precise receptor-binding region in BMP-2 has not yet been clarified. Here, we report that a synthetic peptide, KIPKASSVPTELSAISTLYL, corresponding to residues 73-92 of the knuckle epitope of BMP-2, elevated alkaline phosphatase (ALP) activity in the murine multipotent mesenchymal cell line, C3H10T1/2. The 73-92 peptide significantly inhibited the binding of rhBMP-2 to both BMP receptors type IA and type II. The 73-92 peptide also promoted the expression of osteocalcin mRNA and induced ectopic calcification when it was immobilized on a covalently cross-linked alginate gel and implanted into rat calf muscle. The X-ray diffraction (XRD) pattern of the calcified product was identical to that of the rat tibia, and the major peaks were attributed to hydroxyapatite. These results indicate that the 73-92 peptide may be one of the receptor-binding sites on BMP-2 and may stimulate bone precursor cells to induce calcification.
Biochimica et Biophysica Acta 10/2003; 1651(1-2):60-7. · 4.66 Impact Factor
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Shiho Hirohara,
Makoto Obata, Atsuhiro Saito,
Shin-ichi Ogata,
Chikara Ohtsuki,
Suguru Higashida,
Shun-ichiro Ogura,
Ichiro Okura,
Yuko Sugai,
Yuji Mikata,
Masao Tanihara,
Shigenobu Yano
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ABSTRACT: Thirty-two glycoconjugated porphyrins were synthesized by a modification of Lindsey method in the presence of Zn(OAc)(2).2H(2)O as a template. The Zn(2+) ion template strategy improved the yield about three-fold in the case of meta-substituted tetraphenylporphyrins. In addition, free-base porphyrins were obtained almost quantitatively by demetalation with 4 M HCl. Sixteen deacetylated glycoconjugated porphyrins were tested as candidate photodynamic therapy (PDT) drugs using HeLa cells. Most of the deacetylated glycoconjugated porphyrins showed higher cellular uptake than tetraphenylporphyrin tetrasulfonic acid (TPPS), and 5,10,15,20-tetrakis[4-(beta-D-arabinopyranosyloxy)phenyl]porphyrin (p-5d) in particular showed 18.5-fold higher uptake than TPPS. The photocytotoxicity of 5,10,15,20-tetrakis[4-(beta-D-glucopyranosyloxy)phenyl]porphyrin (p-5a), p-5d and TPPS was examined with HeLa cells, using a light dose of 16 J/cm(2). These photosensitizers had no cytotoxicity in the dark, but their photocytotoxicity increased in the order of TPPS < p-5a < p-5d. These results suggest p-5d is a good candidate for a PDT drug.
Photochemistry and Photobiology 80(2):301-8. · 2.41 Impact Factor
