Identification of type A and type C virus particles in BF murine osteosarcoma.

Two types of virus particles, intracisternal type A and extracellular type C with budding, were detected in the same cells of BF osteosarcoma, its cultured cell lines, and their BFO tumors in CBA mice. The type C particles were approximately 100 microns in diameter. The buoyant density of the virions was 1.16g/ml in sucrose and 1.07 g/ml in Ficoll. A 72S RNA was demonstrated by gel electrophoresis, but no DNA was detected. Reverse transcriptase activity was also demonstrated in detergent-treated virions. Thus, the particles seem to be RNA virus. Cellular transformation and focus formation were observed after rat and mouse embryo cell monolayers were infected with the virus. The same kind of osteosarcoma was produced by inoculation of cloned transformed cells (BFOSV) of CBA embryo cells into CBA mice. Thus, the virus seems to be an oncornavirus.

Fluorescence polarization immunoassay for cortisol.

A fluorescence polarization immunoassay for serum cortisol was established using cortisol 21-amine which was readily coupled with fluorescein isothiocyanate. The proposed method is sufficiently sensitive, reliable, specific and simple for routine determination of serum cortisol. The assay is rapid, without separation of antibody-bound and free ligands. The minimal amount of cortisol detected was 1.5 ng/tube and the measurable range was from 1.5 to 100 micrograms/dl. There was a good correlation between values obtained from radioimmunoassay and the proposed method.

Enzyme immunoassay for cortisol in serum using cortisol 21-amine.

Cortisol 21-amine (21-amino-11beta,17-dihydroxy-4-pregnene-3,20-dione) was prepared and an enzyme immunoassay for cortisol in serum was established using cortisol 21-amine conjugated with alkaline phosphatase. The minimal amount of cortisol detected was 1ng/tube and the measurable range was from 1 to 80 microgram/d1, using 10 mu 1 of serum sample. This enzyme immunoassay satisfied the standard criteria of dilution, accuracy and precision. The values correlated well with those obtained by radioimmunoassay. This enzyme immunoassay is applicable to the routine determination of serum cortisol in any clinical laboratory. Cortisol 21-amine was found to be a useful derivative for preparing cortisol-enzyme conjugate in enzyme immunoassay.

Characteristics of osteosarcoma cells in culture.

Osteosarcoma cells (BFO cell line) were successfully maintained in tissue culture for 3 years. BFO cells showed 100 per cent tumorigenicity by the isologous implantation, and almost the same histological features as the original BF osteosarcoma. BFO cells synthesize and secrete large quantities of alkaline phosphatase both in vitro (cell culture) and in vivo (tumor bearing mice). BFO cells showed a suppression in synthesizing the osteoinductive factor in vitro, but regained the capacity to synthesize it when implanted back into an isologous host. The cells showed rapid growth, a serum requirement, and no contact inhibition. Doubling time was 8.6 hours in a logarithmic growth phase. Cell cycle analyses by pulse labeling of 3H-thymidine was performed after synchronization of the cells by double treatment with an excess thymidine.

Establishment and alkaline phosphatase activity of clonal cell lines of murine osteosarcomas. A preliminary study.

Clonal cell lines of murine osteosarcomas were established and have been maintained in vitro for over a year. By implanting these cultured cells into mice, osteosarcomas, whose histological picutres were exactly the same as those of the original tumors, were easily reproduced. The cultured cells of murine osteosarcomas contain an extremely high level of alkaline phosphatase activity. The cells also secrete a great amount of extracellular alkaline phosphatase in culture media.

Studies on a factor responsible for new bone formation from osteosarcoma in mice.

The bone inducing factor derived from BF osteosarcoma was purified in the following manner. Step 1. The sarcoma, grown in CBA mice, was excised and lyophilized. Step 2. The powder was washed with chilled acetone. Step 3. The acetone-treated powder was then homogenized with chilled distilled water. Step 4. Washing with 0.15M KCl. Step 5. The precipitate was incubated in in 0.2 N NH2OH, pH7.0, for 48 H at 25 degrees. After Step 5, the bone-forming activity showed a slight increase; however, the factor remained insoluble. The properties of the factor were as follows. The factor is relatively relatively heat stable; the osteogenic activity survived the treatment at 75 degrees for 15 min or at 55 degrees for 19 h. The activity was easily lost by mechanical shaking. Incubation with DNase, RNase, neuraminidase, chondroitinase ABC and beta-galactosidase left the osteogenic activity intact, but treatment with either pronase or collagnease destroyed this activity. The results suggest that the factor may be a protein. The activity was seen with the lyophilized BF osteosarcoma cells (without matrix), and it is probable that the factor was exclusively synthesized in the cells. The bone formation, observed across a millipore filter when living BF osteosarcoma enclosed in a millipore chamber was implanted in mice, suggests the synthesis and secretion of the factor from the cells.

Osteogenic induction by cell-free material from murine osteosarcoma and its cultured cell line.

A bone-inducing factor was detected in murine osteosarcoma and its cultured cells. The factor was partially purified from the osteosarcoma and its biological properties were examined. The ectopic formation of the bone with hematopoietic marrow was observed in situ histologically 4 weeks after inoculation of the cell-free material intramuscularly or subcutaneously. Non-osteogenic tumors do not produce this factor. This factor seems to be a protein which is relatively resistant to heating yet labile to mechanical shaking.

Solubilization and concentration of a bone-inducing substance from a murine osteosarcoma.

The osteogenic factor from murine osteosarcoma was soluble in 4M guanidine-HCl. The precipitate that appeared after dialysis of the guanidine solution against deionized water showed a higher osteogenetic activity than did the initial dry powder of tumor tissue. Electron-microscopically, this bone-inducing fraction seems to be homogenous. Neither a fibrous nor collagenous cross-banded structure was observed in the fraction. Since the chemical analysis of the fraction also showed a relatively low content of hydroxyproline, the osteogenic factor is probably not collagen.

Human bone matrix gelatin as a clinical alloimplant. A retrospective review of 160 cases.

Bone matrix gelatin, prepared by sequential chemical treatment including decalcification with 0.6 N hydrochloric acid [9], was used as an alloimplant for the treatment of benign bone tumours, tumorous conditions of bone, acetabular dysplasia, delayed union, traumatic bone defects and other disorders. The bone matrix gelatin implanted into bone defects was incorporated successfully in 98% of implantations, excluding cases of infection, tumour recurrence and recurrence of tumorous conditions. The material was also implanted into ten bone sites as an onlay but in five it was resorbed without new bone formation. The incorporation of the bone matrix gelatin into the recipient bed was completed from 6 to 33 months (average 14.9 months) after implantation. Wound infection complicated 5 of 165 implantations (3%) in previously uninfected sites. Low grade fever persisting after the tenth post-operative day (a probable sign of immunological reaction) occurred in 4 of 160 implantations (2.5%), excluding cases of infection. Alloimplants of bone matrix gelatin are thus effective in the treatment of bone defects. The risk of complication such as rejection or infection is low.

Biochemical stability of a bone-inducing substance from murine osteosarcoma.

Devitalized cultured cells of a murine osteosarcoma preserved strong osteogenic activity even after incubation in neutral and acidic buffer at 37 degrees for six days. This observation suggested that an osteosarcoma-derived osteogenic factor was considerably stable to endogenous proteases, or that osteosarcomas do not synthesize enzymes for degradation of the osteogenic factor. EDTA decalcification or extraction of the osteosarcoma at 37 degrees for six days had no apparent effect on the osteogenic factor. Disulfide-bond reducing agents (2-mercaptoethanol and dithiothreitol) completely inactivated the osteogenic activity and in the presence of guanidine HCl, the loss of activity was irreversible by reoxidation. There findings support the view that disulfide-bonds in the molecular structure of the osteogenic factor are essential for the biological activity.

Partial purification of bone-inducing substances from a murine osteosarcoma.

Bone-inducing substance from a murine osteosarcoma (BFO osteosarcoma) was partially purified by biochemical procedures, i.e., extraction by 4M Gu-HCl, fractionation by ethanol, precipitation in neutral buffer solution with low ionic strength and two steps of gel filtration. The yield of the final preparation was 2 mg from 10 g (wet weight) of BFO osteosarcoma. The preparation constantly induced ectopic bone in situ when implanted into allogenic mice. SDS polyacrylamide electrophoresis revealed that the biologically active fraction contained two distinct substances, the molecular weights of which were 19,500 and 22,500, respectively. Further purification of the bone-inducing substance is now in progress.