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Silver stained nucleolar organizer regions (AgNOR) have revealed differences in the biological behavior of certain entities. This study involves a morphometric analysis of AgNOR in 6 central, classic osteosarcomas and 6 parosteal osteosarcomas. There was a statistically significant difference in the number of AgNOR per nucleus between central and parosteal osteosarcomas. Single AgNOR volume was smaller in central osteosarcomas as compared to parosteal osteosarcomas. However, this difference did not reach statistical significance. The parameter AgNOR number per nucleus revealed a cut off value such that 100% of central osteosarcoma cases lay above this value and 100% of parosteal osteosarcoma cases lay below this value. AgNOR demonstration involves a simple technique which can be performed on formalin fixed, paraffin embedded file material. Thus, it may be prudent to routinely assess AgNOR as a contributor to the determination of the pathophysiology of osteosarcomas.

Chinese hamster ovary (CHO) cells were transfected with the complementary DNA (cDNA) for bone morphogenetic protein-4 (BMP-4) that was derived from the poly A-RNA of a murine (Dunn) osteosarcoma. A clonal line of the transfected, BMP-4 gene expressing cells was expanded and inoculated into the hindlimbs of nude mice to produce BMP-4 secreting tumors. The aims of this study were to investigate the systemic effects of endogenous BMP-4 production on skeletal growth and radiologic density, the local effects of BMP-4 on bone at the tumor-bone interface, and the changes in tumor histology as a result of transfecting CHO cells with the gene for BMP-4. In control mice, mock vector-transfected CHO cells were inoculated in the same manner. Three weeks after the mice received the transfected cells, neither enhanced skeletal growth nor a change in bone density was noted. However, at the site where the bone was in contact with the tumor, new cartilage and bone were consistently observed. The murine BMP-4 transfected CHO tumor contained spicules of new bone that have been described as a histologic feature characteristic of osteosarcoma.

Microfilaments, especially actin, have been demonstrated in a variety of noncontractile cells. In earlier studies, the quantity as well as the distribution of these microfilaments has been used to differentiate normal murine fibroblasts from virally transformed cells. This study examines these differences in cells from spontaneously occurring, human osteosarcomas and normal human fibroblasts by transmission election microscopy and heavy meromyosin (HMM) decoration. Both the tumor cells and the normal fibroblasts were found to have subcortical bands of 7-nm microfilaments that labelled with HMM and were 150-300 nm in thickness. There was a central reticular pattern of microfilaments predominantly composed of 7-nm filaments in the fibroblasts but with a larger proportion of 10-nm filaments in the osteosarcoma cells. Arrowhead formation was present after mild Triton X-100 extraction and HMM decoration on only the 7-nm microfilaments, in both types of cells. Differences in the quantity of 10-nm filaments between cells in culture from spontaneously occurring human sarcomas and normal fibroblasts may account for differential surface responses, e.g., contact inhibition and saturation density. Unlike some evidence from viral transformation models, the data from this study do not support the hypothesis that tumorgenicity is linked mechanistically to decreases in polymerized cellular actin, at least not in cells from spontaneously occurring human sarcomas. The cellular behavior of human sarcomas, both in vitro and in vivo, may be a manifestation of differences both in structural proteins and cell surface proteins.

In this review are discussed a small group of bone tumors that present the greatest and sometimes unresolved problems in diagnosis, not only for the clinician but also for the pathologist, even with full knowledge of the clinical and roentgenologic presentation. The following neoplastic lesions are described in detail: cartilage-forming tumors, bone-forming tumors (with special emphasis on aggressive or malignant osteoblastoma), and round-cell tumors of bone. This last group presents a special challenge to the pathologist even after histopathologic examination. Some newer, more sophisticated methods are discussed, especially those applied in the differential diagnosis of Ewing's sarcoma and tumors of neuroectodermal origin.

An animal model for human osteosarcoma was established in newborn Syrian golden hamsters by injecting productively infected TE-85 cells (cultured human osteosarcoma cells) adjacent to the femur. Tumors were palpable 10 to 15 days following cell injection and enlarged progresively until the animal died (mean survival time was 36 days). The tumor take was 100% and all animals developed pulmonary metastases. Osteoid and bone were identified in sections of the tumor by light and electron microscopy. Invasion of the marrow spaces and adjacent skeletal muscle by the malignant osteoblasts, anaplastic sarcoma cells and multinucleated giant cells was frequently observed. The tumor was transplantable. TE-85 cell surface antigens were demonstrable by immunofluorescence on the surface of the cells of the induced tumors. The present tumor model had unique immunologic characteristics in that no antibodies were demonstrable in the sera of the tumored animals.

Although elevated alkaline phosphatase levels in osteosarcoma have been shown to be related to prognosis, the functional significance is unclear. Human osteosarcoma cells in tissue culture retain detectable amounts of alkaline phosphatase activity. In this study the specific activity of that enzyme was compared in 13 osteosarcoma tissue culture lines and 13 normal skin fibroblast lines derived from the same patients. Osteosarcoma cells had significantly higher baseline alkaline phosphatase levels and could be stimulated with hydrocortisone to produce more enzymatic activity than the fibroblast lines. Activity was localized ultracytochemically to the cell membrane and to many small intracellular vesicles in stimulated osteosarcoma cells. These observations aid in the differentiation of osteosarcoma and fibroblast lines in tissue culture and suggest an association between elevated alkaline phosphatase levels and metabolic abnormalities in patients with osteosarcoma.

Histophysiology, ultrastructure, chemical analyses of transplants and implants of Dunn and Ridgway mouse osteosarcomas demonstrate that tumorigenesis is a manifestation of deranged morphogenesis in developing mesenchymal cell populations. The end product of development is defective, incompletely calcified, disorganized bone without any inclusions of bone marrow tissue. When Dunn osteosarcoma is freeze-dried and then implanted, the tumor is resorbed and replaced by deposits of normal cartilage, bone, and bone marrow. Freeze-dried Ridgway osteosarcoma is replaced only by a fibrous connective tissue scar. Disaggregated Dunn tumor osteoblasts synthesize a trypsin-labile collagenase-resistant cell surface localized bone morphogen. Tumor matrix stroma, prepared by sequential chemical extraction of soluble non-collagenous proteins also contains significant quantities of the same bone morphogen. Tumor tissue pulverized to particle size as small as 44 micrometer3 transmitted bone morphogen more rapidly than intact tumor tissue. The total tumor cell and stroma mediated bone morphogen produces three times more normal bone than normal cortical bone matrix. Our working hypothesis is that a normal bone morphogenetic polypeptide (BMP) is synthesized by Dunn osteosarcoma cells and retained by the tumor matrix stroma. Neither the mechanism of transmission nor the mesenchymal cell receptor sites of BMP are known.

Electron microscopic study of 9 osteosarcomas corroborates the light microscopic observation that cells with osteoblastic, chondroblastic and fibroblastic features occur in osteosarcomas. Two types of multinucleated giant cells were observed, one was osteoclast-like. The other had anaplastic and degenerative changes. The intercellular matrix is composed of collagen fibers, amorphous ground substance with osteoid and tumor bone. Contrary to many previous electron microscopic reports, osteosarcomas contain more than one cell type.