Enhancement by bFGF of osteogenesis induced by rhBMP-2 in rats.

Subcutaneous implantation of bone morphogenetic protein (BMP) combined with a fibrous glass membrane (FGM) induces cartilage formation in the entire inner area of the membrane within 2 wk. It has been hypothesized that a tight FGM network (1 microm exclusion size) provides immature cells with spaces for penetrating into the membrane, but not for vascular formation, at least until 2 wk. To test this hypothesis, basic fibroblast growth factor (bFGF), known to be a potent stimulant of capillary formation, was applied to the implant. BMP was combined with FGM in the presence or absence of bFGF, and then implanted subcutaneously into the backs of rats. The bFGF-supplemented implant caused 1.3 times higher alkaline phosphatase activity and 3 times higher calcium contents at 2 wk, whereas type II collagen contents decreased, thus indicating that bFGF enhances bone formation in BMP/FGM implants. These results suggest that bFGF induces faster and stronger invasion of capillaries into the FGM and destroys its tight network, resulting in acceleration of the ossification process.

Starch debranching enzyme (R-enzyme or pullulanase) from developing rice endosperm: purification, cDNA and chromosomal localization of the gene.

Starch debranching enzyme (R-enzyme or pullulanase) was purified to homogeneity from developing endosperm of rice (Oryza sativa L. cv. Fujihikari) using a variety of high-performance liquid chromatography columns, and characterized. A cDNA clone encoding the full length of the rice endosperm debranching enzyme was isolated and its nucleotide sequence was determined. The cDNA contains an open reading frame of 2958 bp. The mature debranching enzyme of rice appears to be composed of 912 amino acids with a predicted relative molecular mass (Mr) of 102,069 Da, similar in size to its Mr of about 100,000 Da estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The amino acid sequence of rice debranching enzyme is substantially similar to that of bacterial pullulanase, while it bears little similarity to that of bacterial isoamylase or to glycogen debranching enzymes from human muscle and rabbit muscle. Southern blot analyses strongly suggest that the debranching enzyme gene is present as a single copy in the rice genome. Analysis by restriction fragment length polymorphism with a probe including the 3'-untranslated region of cDNA for rice debranching enzyme confirmed that the debranching enzyme gene is located on chromosome 4.

Developmental and differential regulations in gene expression of Xenopus pleiotrophic factors-alpha and -beta.

Using conserved nucleotide sequences in mammalian osteoblast specific factor-1 (OSF-1) coding regions, we isolated two kinds of cDNA clones from the Xenopus brain library. The encoded proteins, named Xenopus pleiotrophic factors (X-PTFs)-alpha and -beta, were 65 and 87% homologous to human midkine and OSF-1, respectively. In the adult frog, X-ptf-alpha was expressed in the ovary, brain, eye, bone, heart and lung, whereas X-ptf-beta was expressed in the brain, eye and bone. By in situ hybridization of the tailbud embryo, X-ptf-alpha mRNA was detected rather broadly in the head/tail regions including the central nervous system (CNS), whereas X-ptf-beta mRNA was restricted to the CNS, particularly in the hind-brain. During embryogenesis, X-ptf-alpha mRNA was detected in the one-cell stage embryo, whereas only zygotic expression was observed in X-ptf-beta. X-ptf-beta mRNAs contained approximately 79 bp tandem repeats in the 3'-untranslated region, complementary to those found in retinoic acid cellular receptor mRNA and in the sense strand of short interspersed repeat transcripts in X. laevis.

Bone induction of hydroxyapatite combined with bone morphogenetic protein and covered with periosteum.

Using a rabbit model, we evaluated the role of the periosteum in bone induction using hydroxyapatite ceramic pellets, some of which had been coated with bone morphogenetic protein. Eighteen rabbits were divided into two groups, a control group that received pellets soaked in phosphate-buffered saline alone and another group for which the pellets had been supplemented with bone morphogenetic protein. After making a skin incision on the head of each rabbit, two caudally pedicled periosteal flaps measuring 1 cm in width and 2.5 cm in length were elevated. These flaps were wrapped around hydroxyapatite pellets manufactured from limestone and fixed with sutures. After implantation, both groups of rabbits were returned to their cages, maintained for 3, 6, or 9 weeks [every group consisted of 3 rabbits (6 pellets)], and then sacrificed. In this study, the extent of bone induction, which was measured with a dual x-ray densitometer, that resulted from covering the hydroxyapatite pellet with periosteum alone (control group) was minimal. On the contrary, since osteogenesis from the periosteum toward the pores of the pellet was observed in the bone morphogenetic protein group much more than in the control group, the usefulness of our technique was confirmed. However, active osteogenesis was observed with subperiosteal implantation of the hydroxyapatite-bone morphogenetic protein complex in the bone morphogenetic protein group, but the osteogenesis observed was not distributed over the entire pellet.

A new method for in vitro calcification using acrylamide gel and bovine serum.

To investigate the mechanism of biological calcification in vitro, a model system consisting of an acrylamide gel block (1 x 3 x 3 mm) and fetal bovine serum was developed. Mineral deposition was induced in gel blocks which were immersed in 300 microliters of fetal bovine serum at 37 degrees C for 7 days in a CO2 incubator. X-ray diffraction indicated that the mineral was hydroxyapatite with low crystallinity. Effects of the concentration of acrylamide gel, the partial pressure of CO2 and matrix proteins within the gel on the mineral formation were investigated. In the gel concentration range of 10-60%, the largest amount of crystal grew in 40% acrylamide gel, where the serum protein did not penetrate. With an increase in the partial pressure of CO2 the Ca content in the gel block increased, reached the highest level at about 3.5% CO2 and then began to decrease. In 40% gel and at 5% CO2, the mineral formation was enhanced by phosvitin, phosphophoryn, demineralized dentin powder and alkaline phosphatase. Mineral deposition occurred around the collagen fibers immobilized in 40% acrylamide gel. These results indicate that 1) a putatively serum-derived inhibitor of calcification with high-molecular weight was prevented from penetrating into the 40% acrylamide gels, 2) immobilized polyanionic proteins and alkaline phosphatase were able to increase mineral deposition and 3) the partial pressure of CO2 greatly influenced the mineral deposition. It was concluded that this gel system is useful to investigate the mechanism of biological calcification in vitro.

X-ray photoelectron spectroscopic studies of the adsorption of salivary constituents on enamel.

Time-dependent change of adsorption of salivary components on the outermost surface layer of enamel was studied by x-ray photoelectron spectroscopy. Adsorption of proteinaceous components, as monitored in terms of the relative mass of nitrogen, was detected within 30 min, increased with time, and reached a plateau at 90 min. Thus, the ratio of nitrogen to calcium in the two-hour sample increased to about 240 times that in the control sample. The ratio of carbon to nitrogen on the surface decreased to about one-half of that in the control sample. The data established the time required for equilibrium between the proteinaceous component in saliva and the amount of material adsorbed onto the tooth surface.