Mycoplasma-mediated bone resorption in bone organ cultures.

Mycoplasmas have been identified as one of many aetiological factors associated with experimental or human joint disease. Mycoplasma hyorhinis and M. arthritidis, but not M. pulmonis were found to cause significant release of calcium from murine long bone explants. The resorption process is inhibited by calcitonin, acetazolamide and by indomethacin. Mycoplasma-derived bone resorbing activity (M-BRA) is not an endotoxin as its effect is equally potent in cultures of bones obtained from endotoxin-responsive and -unresponsive mice. M-BRA is a high molecular weight compound resistant to proteases and heat but sensitive to hyaluronidase, lipase, detergents and in part to alkali and acid conditions. The active component is associated with the particulate fraction of the mycoplasma and its yield is enhanced by sonication. The damage to the subchondral bone in arthritis associated with a mycoplasma infection may be caused by a potent bone resorption inducing agent of mycoplasma origin.

Acquisition of resistance to 6-azauridine through DNA amplification in neoplastic but not normal osteoblasts.

In this communication, we have characterized the resistance to AZUrd in tumorigenic mouse C3H-OS osteosarcoma cells and non-tumorigenic MC3T3-E1 osteoblast cells. DNA and RNA blot analysis showed a 30-fold increase in UMP synthase specific DNA and a 10-fold increase in mRNA, respectively, in resistant versus non-resistant C3H-OS cells. No corresponding increases in either UMP synthetase DNA or mRNA were evident in resistant MC3T3-E1 osteoblasts. Karyotype analysis of MC3T3-E1 and C3H-OS cells revealed translocations in the resistant cells. Regardless of drug-sensitive or resistant phenotype, the normal and neoplastic cells exhibited aneuploidy which was significantly more pronounced in the non-resistant tumor cells. Additionally, the number of chromosomes decreased in all resistant cells whether normal or neoplastic. We conclude that genomic instability in neoplastic cells is a prerequisite for the generation of drug resistant variants via the process of gene amplification.

Involvement of the plasmin system in dissociation of the insulin-like growth factor-binding protein complex.

A variety of treatments, including acid, heparin, and proteases, are known to free insulin-like growth factors (IGFs) from their binding proteins (IGFBPs). However, the physiologically relevant mechanism regulating the interaction of IGFs and IGFBPs is unknown. We report here the ability of plasmin to dissociate IGFs from IGFBPs. In chromatographic experiments, plasmin completely dissociated complexes of [125I] IGF-I-BP and [125I]IGF-II-BP formed with purified decidual IGFBP (hIGFBP-1) or IGFBPs present in medium conditioned by human osteosarcoma MG-63 cells. Plasmin dissociation of IGF-BP complexes was dose dependent. Neither plasminogen nor plasminogen activators (PAs) alone affected dissociation; however, activation of plasminogen to plasmin by either urokinase PA or tissue-type PA resulted in the dissociation of IGF-BP complexes. Plasmin dissociated immunoreactive and bioactive IGF from IGFBP equivalent to approximately 70% and approximately 60% of the acid control value, respectively. In medium conditioned by MG-63 cells, dissociation of IGF-BP complexes was catalyzed by PAs secreted by MG-63 cells, principally urokinase PA. Limited plasmin degradation of IGF was suggested by chromatographic experiments involving [125I] IGF. Treatment of uncomplexed IGF-I with plasmin concentrations equivalent to those in chromatographic experiments did not result in a significant loss of bioactivity, although a 2-fold increase in the plasmin concentration resulted in a approximately 20% loss of activity. Similar plasmin treatment of equimolar concentrations of hIGFBP-1 resulted in a marked degradation of IGFBP, with loss of IGF-binding ability. In vitro experiments confirmed plasmin dissociation of bioactive IGF-I from hIGFBP-1. In MG-63 cells, IGFBPs can form an IGF reservoir in the pericellular space surrounding the cells by combining IGFs with IGF-BP to form complexes that are incapable of binding to the IGF receptors. The secretion of PAs by osteosarcoma cells and the availability of plasminogen in the extravascular tissues indicate the possibility of a regulatory system in osteosarcoma cells in which pericellular plasmin affects the availability of IGFs to their membrane receptors.

Bone resorption in osteogenic sarcoma--II. Resorption of the bone collagenous matrix by tumor cells, normal fibroblasts and macrophages.

Three osteogenic sarcoma cell lines of human and canine origin were compared to normal fibroblastic cells and peptone-induced murine peritoneal macrophages in terms of bone collagenous matrix (BCM) resorption capacity. The dissolution of the BCM was measured in an in vitro system consisting of the tested cells and live or killed [3H]-proline-labeled fetal mouse long bones. Experiments with osteogenic sarcoma cells revealed a paradoxical phenomenon indicating an inverse relationship between the number of tumor cells and the rate of collagen resorption from live bones. On the other hand, collagen matrix of devitalized bones, particularly those denatured by exposure to heat, is strongly resorbed by osteogenic sarcoma cells even in the presence of serum. Contrary to osteogenic sarcoma cells, normal fibroblasts do not resorb collagen from either live or killed bones, regardless of the devitalization method and culture conditions utilized. Macrophages resorb collagenous matrix from live bones, but their collagen resorption activity from devitalized bones depends greatly on choice of the incubation condition. Results have shown that osteogenic sarcoma tumor cells, when acting alone, may not have the capacity to destroy healthy bone. We suggest, therefore, that bone destruction seen in osteogenic sarcoma patients depends on the metabolic condition of the affected bone, and the interaction between the tumor and normal host cells and tissues.

Bone resorption in osteogenic sarcoma. I. Release of calcium by tumor cells, normal fibroblasts, and macrophages.

The bone calcium resorption activities of three different types of cells that are present either within or in the vicinity of an osteogenic sarcoma tumor mass were examined: osteogenic sarcoma cells, normal fibroblasts, and macrophages. Release of calcium was measured in bone organ cultures in live and killed 45Ca-labeled fetal or newborn mouse long bones. The bone explants, tibiae and humeri, were co-cultured with various numbers of effector cells in microwell plates. Cultured osteogenic sarcoma cells resorb calcium from both live and heat-devitalized bones in a cell-number-dependent manner. A reduced effect of the tumor cells on killed bone suggests that the release of calcium from living bone by tumor cells is mediated partially through stimulation of the endogenous bone-resorbing systems. Fibroblasts also resorb calcium both from live and killed bones but at a lower rate than osteogenic sarcoma cells. Peptone-elicited peritoneal macrophages are capable of sustaining calcium resorption from live bones as well as from bones devitalized by a mild nondenaturating method. Macrophages, however, failed to resorb calcium from heat-killed bones. The release of osseous calcium and bone damage associated with osteogenic sarcoma are manifestations of the resorbing properties and interactions among the tumor, bone, and host tumor infiltrating cells.

Delayed growth inhibition of human leukaemic cells in culture following treatment with aminonucleoside of puromycin.

The effects of the aminonucleoside analogue of puromycin (AMS) on the growth of the CCRF-CEM human leukaemic lymphoblast cell line have been studied. CEM lymphoblasts are capable of growth in the presence of AMS for a limited period of time, in a manner similar to other transformed cell lines in culture. However, a delayed effect of AMS was found to take place at the time of the third cell division after application of the drug. This phenomenon of delayed growth inhibition has not yet been described for any other type of cell. The delayed inhibition of CEM cells caused by AMS manifests regardless of the subsequent presence or absence of the drug. The magnitude of the delayed inhibition is linearly dependent on the concentration of AMS during the initial incubation period. Cell proliferation during drug application is not necessary for establishing the inhibition. Following the inhibition period, the cells resume a normal rate of growth.

On the nature of stress fractures.

It is felt that stress fractures are caused by excessive, repetitive muscle forces acting across the affected bone. These fractures should be suspected in participants of athletic endeavors who present with a history of persistent, focal, activity-related pain regardless of their stage of physical conditioning. Associated physical findings are localized tenderness and swelling without evidence of a generalized systemic response. Bone scans and serial roentgenograms including specialized views may be required for documentation. Limitation of the athletic activity is the hallmark of the treatment program.

Delayed surgical repair of ruptured ligaments: a comparative biomechanical and histological study.

The importance of prompt surgical repair of complete ligament ruptures was quantitated using 38 rabbits with standardized transection injuries ofthe medial collateral ligament of one knee. Tensile testing of the mechanical properties, coupled with histological evaluation allowed assessment of the integrity of suture repairs performed immediately after ligament injury, versus those with 3 and 10 day delay times as well as those with non-operative management. Surgical repair performed within the first 3 days is beneficial in terms of final repair strength. Irreversible ligament strength and stiffness deficits progressively accumulate during the 10 days immediately following injury. Surgical repair after more than 10 days postinjury is contraindicated since the postoperative mechanical properties of the healed ligament do not differ from those for non-operative injury management.

Metabolic and endocrine alterations in osteosarcoma patients.

Eighteen patients with primary osteosarcoma were studied for abnormal endocrinologic functions. Oral glucose tolerance tests revealed abnormal glucose, insulin, or growth hormone response curves in 78% of the study population. Elevated somatomedin values were noted in 72% of the patients tested. No significant deviations were observed in serum cortisol, estradiol, testosterone, follicle stimulating hormone, and luteinizing hormone levels. Likewise, lipid screening and 24 hour 17-hydroxy and 17-keto steroid excretion levels were normal. Statistically these derangements were unrelated, leading to the hypothesis that some additional factor or factors are present and responsible for the abnormalities noted. These deviations, found in association with primary osteosarcoma, constitute a new "paraneoplastic syndrome."

Repair of acromioclavicular separation using a dacron prosthesis graft.

Twenty patients were treated for acute acromioclavicular separation using a Dacron vascular prosthesis and followed for an average period of 13.1 months. None demonstrated loss of motion, or muscle weakness. All resumed their prior level of athletic activity. Satisfactory results were obtained in 18 patients. Residual deformity was the primary reason for an unsatisfactory rating. Recurrence of deformity can be minimized by technical skill and perfection of the surgical reduction.

Primary osteosarcoma of the skull.

In 33 cases of primary osteosarcoma of the skull in a relatively old age group, the sex ratio and poor prognosis is the same as in osteosarcoma of long bones. However, patients in the second decade of life free of neurologic disease have a better prognosis.

Hormone suppression of DNA synthesis in cultured chondrocyte and osteosarcoma cell line.

Recent evidence suggests that endocrine factors play an important role in the natural history of osteosarcoma. The occurrence of this tumor in the metaphysis of rapidly growing adolescents, coupled with increased female survival led to the investigation of the effects of various hormones on cultured osteosarcoma cells. The in vitro effects of physiologic concentrations of human growth hormone, 17beta estradiol, and progesterone on cultured osteosarcoma cells and chondrocytes are presented. Growth hormone significantly enhances 3H-thymidine incorporation in osteosarcoma cells and chondrocytes, in the presence of human serum. The use of other sera, culture media, or heat inactivation of the human serum abolishes this effect. Estradiol and progesterone, alone, or in combination produce significant suppression of DNA synthesis in cultured tumor cells. Several sera contain a heat-labile factor which has the capacity to block the suppressive effect of estradiol. This factor could be overcome by increasing the concentration of hormone, or by heat-inactivation of the serum. The use of hormone therapy in the treatment of osteosarcoma has never been reported, despite its demonstrated value in certain other malignancies. In light of these observations and considering the poor prognosis in this disease it seems reasonable to initiate a study of adjunctive hormone therapy in osteosarcama.

The host immune response in human osteosarcoma.

A patient's immunologic response to a malignant tumor may be a major factor in determining his ultimate prognosis. An in vitro microcytotoxicity test using cultured tritiated thymidine (3HT) labeled osteosarcoma cells and autologous fibroblasts has been developed to determine the nature of this response. The role of cell mediated and serum factors has been quantitatively evaluated and the following results obtained. Osteosarcoma patients have been demonstrated to possess a normal cellular immune response which exhibits non-specific cytotoxicity in vitro. These patients can not differentiate their tumor cells from autologous fibroblasts, even though they may significantly suppress the growth of homologous tumors or fibroblasts. Serum blocking factors capable of inhibiting lymphocyte mediated cytotoxicity are occasionally noted. A reliable quantitative microcytotoxicity technique is presented which demonstrates that: (1) osteosarcoma is not due to host immuno-incompetence, (2) a common sarcoma antigen does not exist and (3) serum blocking factors may occasionally be present.

Tibiofibular synostosis: a cause of ankle disability.

A syndrome of ankle pain on weight-bearing while running due to post-traumatic ossification of the tibiofibular ligament is described. Pain is caused by failure of normal downward and lateral motion of the fibula. Treatment consists of complete excision of the synostosis, followed by cast immobilization for 3 weeks.

Assay of cell-mediated cytotoxicity by tritiated thymidine labeled tumor cells.

A microtechnique for quantitating cell-mediated cytotoxicity using tritiated thymidine labeled target cells is presented. Target cell incorporation of tritiated thymidine is augmented by using methotrexate to arrest endogenous thymidine synthesis. Complete solubilization of labeled cells is accomplished by oxidizing samples to tritiated water and carbon dioxide prior to quantitation in a scintillation counter. This versatile technique, using various combinations of lymphocytes, sera and target cells, permits the simultaneous comparison of 500 or more individual samples.

Investigation of carbohydrate metabolism and somatomedin in osteosarcoma patients.

Altered carbohydrate metabolism associated with fibrosarcomas and chondrosarcomas has been well-documented in past literature. This report describes abnormal carbohydrate metabolism in 2 osteosarcoma patients, and abnormalities in growth hormone and somatomedin serum levels. Experimental evidence is presented showing in vitro suppression of osteosarcoma tumor cell proliferation by 17 beta Estradiol. Estrogen inhibition of linear bone growth, cartilage proliferation, and somatomedin is discussed with reference to possible estrogen therapy in osteosarcoma.

Growth and hormone control mechanisms in osteosarcoma. Evidence for a new therapeutic approach.

The association between osteosarcoma and growth, including altered carbohydrate metabolism has been investigated in 5 osteosarcoma patients. Serum from an adolescent osteosarcoma patient has been noted to consistently enhance 3-H-thymidine uptake in cultured chondrocytes and tumor cells. Females survive osteosarcoma more frequently than males. In vitro serum-enhanced proliferation of osteosarcoma cells is suppressed by estradiol.