Y. Gotoh

Experimental use of fibrin glue to induce site-directed osteogenesis from cultured periosteal cells.

The purpose of this study was to determine whether a combination of fibrin glue and cultured periosteal cells will result in new bone formation at heterotopic sites in nude mice. Growing cells and developing matrices surrounding periosteal explants from the diaphyses of radii of newborn calves were minced and mixed with fibrin glue in a syringe. The cell/matrix-fibrin glue admixture was then injected into the subcutaneous space on the dorsum of athymic nude mice. After 12 weeks of implantation, gross morphology and histologic investigations showed newly formed bone structures in all cell/matrix-fibrin glue admixtures, but none in fibrin glue injected alone and used as control samples. Osteopontin, a protein important in bone development, was identified by a Western blot assay of the cell/matrix-fibrin glue composite. This study supports the feasibility of initiating site-directed formation of bone structures at heterotopic tissue sites by means of injection of cultured periosteal cells and matrix in a fibrin glue carrier. (Plast. Reconstr. Surg. 105: 953, 2000.)

Comparison of two phosphoproteins in chicken bone and their similarities to the mammalian bone proteins, osteopontin and bone sialoprotein II

Two phosphorylated proteins of approximately 66 kDa and approximately 60 kDa mass with different DEAE-Sephacel elution patterns were isolated from chicken bone and were shown to be genetically distinct by both biochemical and immunological analysis. A tryptic peptide from the 60 kDa protein was identified that was similar to a sequence of the rat bone sialoprotein II. Both proteins showed RGD inhibited cell-attachment with the MG-63 osteosarcoma cell, and the approximately 66 kDa phosphoprotein appeared to promote cell adhesion better than human vitronectin. The two phosphoproteins appear to share functional and biochemical characteristics and to be homologous to the mammalian bone phosphoproteins, osteopontin and bone sialoprotein II.

Selective extractability of noncollagenous proteins from chicken bone

Quantitative analyses of a wide variety of different solvents used for the extraction of several of the noncollagenous proteins of fully mineralized chicken bone powder were carried out to compare both the effectiveness of various procedures and the distribution of specific proteins which were solubilized. Extraction procedures included solutions of 6 M guanidine-HCl, pH 7.0, 0.5 M EDTA, pH 7.4, 0.3 N citric acid, 0.3 N HCl, 0.3 N formic acid, and 0.3 N acetic acid. Chelation of calcium ions by EDTA and dissolution of the mineral phase by acid extraction released 95% or more of the total calcium content of the bone powder by 48 hours, guanidine-HCl released less than 20% or less of the total calcium content even when extraction was carried out by 168 hours. Moreover, although guanidine-HCl solubilized a significant amount of collagen as gelatin, essentially none of the phosphoproteins, osteocalcin, or the proteoglycan decorin were solubilized, as detected by immunological techniques. In contrast, extraction of the mineralized bone powder by HCl and formic acid was very efficient in selectively solubilizing osteocalcin and osteopontin, while bone sialoprotein was selectively released by EDTA, and solubilized to a lesser extent by formic acid. Similarly, EDTA selectively removed decorin compared with HCl, formic, acetic, or citric acids. Only small amounts of osteopontin and osteocalcin were detected in the acetic acid extracts. These results provide methods for the selective solubilization of several different major, noncollagenous proteins from mineralized bone which should significantly aid in maximizing the amount of the specific protein recovered, and the ease with which the various proteins can be purified. The data also provide some insight into the intrinsic solubility characteristics of collaten, the specific noncollagenous proteins, and their potential association with each other and the mineral phase.

Ultrastructural Immunolocalization of a Major Phosphoprotein in Embryonic Chick Bone

Immunocytochemistry utilizing the protein A-gold technique was used to examine the ultrastructural cellular and extracellular distribution of a major phosphoprotein in chick bone. HCl-extracts of embryonic and neo-natal chick bones contain a major 66kD phosphoprotein (BPP) which was purified and used to raise polyclonal antibodies in rabbits. The mid-diaphyseal regions of 8-, 12- and 18-day embryonic chick tibiae were fixed with 1% glutaraldehyde and embedded in Epon or Lowicryl. Electron microscopy following incubation of tissue sections with the antibody and the protein A-gold complex revealed specific immunolabeling over the rER and Golgi apparatus of osteoblasts and over those areas of bone matrix containing Ca and P as determined by electron probe x-ray microanalysis. These included extracellular areas in the matrix undergoing early mineralization and electron dense patches occurring at the mineralization front and extending throughout the more mature bone regions. Biochemical analyses of bone tissue processed similarly to that used for immunocytochemistry confirmed the retention of phosphoprotein in the tissue. The spatial correlation of phosphoprotein in the extracellular matrix with Ca-P mineral deposits confirms an earlier report using 33Pi and radioautography and may indicate a role for phosphoproteins in calcification.