An intramedullary free vascularized fibular graft combined with pasteurized autologous bone graft in leg reconstruction for patients with osteosarcoma.

The use of pasteurized autologous bone graft has been an innovation in limb-salvage surgery; however, its principal disadvantage is fracture, infection, pseudoarthrosis, and bone resorption. We present two cases in which an intramedullary free vascularized fibular graft combined with pasteurized autologous bone graft was performed for immediate femur or tibia reconstruction following osteosarcoma resection. The rationale of this method is to combine the mechanical strength of a pasteurized bone with the biological activity of a vascularized bone. The pasteurized bone graft provides bone stock and early stability and the addition of the vascularized bone graft substantially facilitates host-pasteurized bone union. This combination procedure may be a recommended option for reconstruction of the lower leg, preserving knee joint function for patients with osteosarcoma.

Hypoxia markers in human osteosarcoma: an exploratory study.

Neoplastic cells growing under hypoxic conditions exhibit a more aggressive phenotype by activating a cascade of molecular events partly mediated by hypoxia-inducible transcription factor (HIF-1alpha) and vascular endothelial growth factor (VEGF). The roles of these markers have been studied previously in several cancer lines. We ascertained the frequency of HIF-1alpha expression, VEGF expression, the degree of neovascularization, and cell proliferation in osteosarcoma samples. Samples from osteosarcoma patients were assessed for HIF-1alpha and VEGF protein expression using immunohistochemistry, neovascularization using antibodies for Factor VIII, and cell proliferation using the Ki-67 labeling index. Associations between these parameters and clinical features were examined. HIF-1alpha staining was positive in 35% of patients and metastases were present in 61% of these HIF-1alpha-positive patients. VEGF protein expression was detected in 69% of patients, 92% of whom were female. We observed an insignificant trend for a higher frequency of VEGF expression in the high-grade as compared to low-grade osteosarcoma. We observed no association between vascular density and proliferation index and any clinical parameters. We found an association between HIF-1alpha expression and metastatic disease and between VEGF expression and female gender.

Askin tumor with metastasis to the scalp: a histochemical, immunohistochemical and ultrastructural study.

A 29-year-old woman presented with facial edema, and imaging disclosed a tumor extending from the anterior chest wall to the anterosuperior aspect of the mediastinum. Transbronchial cytology of the primary tumor and biopsy of the metastatic scalp lesion were performed. Histologically, the tumor consisted of closely packed small round cells. The neoplastic cells generally had round nuclei, finely dispersed chromatin, and small to prominent nucleoli. Histochemically, the cytoplasm of the neoplastic cells contained abundant glycogen and stained with Grimelius silver. Immunohistochemically, the neoplastic cell membranes reacted with CD99 (MIC2) and the neoplastic nuclei reacted with Fli-1, but various other markers, including lymphocyte and skeletal muscle markers, were not detected. No neoplastic cells were also reactive for chromogranin A, synaptophysin, and neurofilament. Ultrastructurally, some neoplastic cells had delicate cytoplasmic processes and contained membrane-bound dense core granules in the cytoplasm. Even if results are immunohistochemically negative for neuroendocrine markers, the combination of immunohistochemistry of CD99 (MIC2) and Fil-1 may be useful in diagnosing Askin tumor or its metastatic lesion.

VEGF expression in osteosarcoma correlates with vascular permeability by dynamic MRI.

Dynamic enhanced magnetic resonance imaging has been used to assess tumor angiogenesis in osteosarcoma. Vascular endothelial growth factor has been shown to correlate with pulmonary metastasis and a poor prognosis in osteosarcoma. The purpose of this investigation was to determine whether vascular endothelial growth factor expression in osteosarcoma correlates with vascular permeability detected by dynamic enhanced magnetic resonance imaging and to explore the role of dynamic enhanced magnetic resonance imaging as a noninvasive means of assessing tumor angiogenic activity. Fifty-five osteosarcoma patients with osteosarcoma enrolled in a treatment protocol that included dynamic enhanced magnetic resonance imaging. In 15 patients, tumor tissues were available for vascular endothelial growth factor immunohistochemical studies. A two-compartment model used the exchange rate constants (kep) between the plasma and tumor compartments to quantify vascular permeability during dynamic magnetic resonance imaging studies. Immunohistochemical staining for vascular endothelial growth factor was graded according to the intensity and number of positively stained cells. Vascular endothelial growth factor-positive tumors showed higher mean vascular permeability when compared with vascular endothelial growth factor-negative tumors. Vascular permeability also correlated with increasing vascular endothelial growth factor expression. The preliminary results in this study show an association between vascular endothelial growth factor and dynamic MR signal enhancement in osteosarcoma. Dynamic enhanced magnetic resonance imaging should be investigated as a means to prognosticate osteosarcoma patients with osteosarcoma according to their tumor angiogenic activity.

Status of methylthioadenosine phosphorylase and its impact on cellular response to L-alanosine and methylmercaptopurine riboside in human soft tissue sarcoma cells.

The aim of this study was to examine the expression of methylthioadenosine phosphorylase (MTAP) in 21 fresh tumor samples from patients with soft tissue sarcomas (STS) and 11 human soft tissue sarcoma cell lines, and to determine if loss of expression of this enzyme was correlated with increased sensitivity to L-alanosine and/or 6-methylmercaptopurine. We used a polyclonal antibody to measure the expression of MTAP in soft tissue sarcoma cell lines and in fresh tumor samples. Transfection of the HT-1080 cell line with a plasmid containing the cDNA for the MTAP gene was also performed to generate cell lines for in vitro and in vivo comparative sensitivity studies. MTAP was not expressed in 8 of 21 fresh STS tumors. The expression of MTAP was also not detectable in 3 of the 11 soft tissue sarcoma cell lines (HT-1080, HS42, and M-9 110). These three cell lines were more sensitive to L-alanosine, a potent inhibitor of de novo AMP synthesis, and to an inhibitor of de novo purine nucleotide synthesis, 6-methylmercaptopurine riboside (MMPR). The IC50 values for L-alanosine and MMPR were >20-fold lower in MTAP-deficient cells than in MTAP-positive cells. Restoration of MTAP into HT-1080 MTAP-deficient cells also led to decreased sensitivity to L-alanosine and MMPR. An in vivo study using HT-1080 cell tumors with and without MTAP expression confirmed that tumors lacking MTAP were more sensitive to L-alanosine than tumors expressing MTAP. These results provide the basis for selective therapy using inhibitors of de novo purine nucleotide synthesis such as L-alanosine or MMPR to treat patients with STS lacking this enzyme.

Mechanism of apoptotic resistance of human osteosarcoma cell line, HS-Os-1, against irradiation.

In our previous study, we examined reactive oxygen species (ROS) formation in T lymphocytes following 5 Gy of irradiation. Using a CCD camera system, we monitored fluorescence in T lymphocytes loaded with the succinimidyl ester of Dichlorodihydrofluorescein diacetate (H2DCFDA), which is non-fluorescent until oxidized by ROS. We found that ROS formation occurred immediately after irradiation, continued for several hours, and resulted in oxidative DNA damage. Therefore, the origin of the hyper-radiosensitivity of T lymphocytes seemed to be the high production of ROS in the mitochondrial DNA following irradiation. In this study, we examined radiation-induced ROS formation, oxidative DNA damage, early apoptotic changes, and mitochondrial membrane dysfunction in the human osteosarcoma cell line HS-Os-1, which was established from an osteoblastic tumor that arose in the left humerus of an 11-year-old girl and was already morphologically characterized in vitro and in vivo. We found that ROS formation and oxidative DNA damage were actually scarcely seen after irradiation of up to 30 Gy in these cells; that mitochondrial membrane potential was preserved; and that apoptotic changes were not demonstrated despite the relatively high-dose irradiation of 30 Gy. Therefore, the origin of the close similarity of radiosensitivity between adult articular chondrocytes and the human osteosarcoma cell line HS-Os-1, is considered to involve the low degree of ROS formation following irradiation; the similarity possibly results from the strong scavenging ability of these two kinds of cells for free radicals including hydroxyl radicals.

Mechanism of hydrogen peroxide-induced apoptosis of the human osteosarcoma cell line HS-Os-1.

In our previous study, we examined radiation-induced ROS formation, oxidative DNA damage, early apoptotic changes, and mitochondrial membrane dysfunction in the human osteosarcoma cell line HS-Os-1, which was established from an osteoblastic tumor that arose in the left humerus of an 11-year-old girl and was already morphologically characterized in vitro and in vivo. We found that ROS formation and oxidative DNA damage were scarcely seen after irradiation of up to 30 Gy in these cells; that mitochondrial membrane potential was preserved; and that apoptotic changes were not demonstrated despite the relatively high-dose irradiation of 30 Gy. Based on these results, the radioresistance of the human osteosarcoma cell line HS-Os-1, was considered to arise, at least in part, from the low level of ROS formation following irradiation, which in turn may have resulted from the strong scavenging ability of the cells for free radicals, including hydroxyl radicals. Therefore, in this study, we examined the effect of exogenous hydrogen peroxide, which causes a potent oxidative stress and has been demonstrated to be a potent apoptosis-inducer in many kinds of cells. We found that addition of 1 or 10 mM hydrogen peroxide induced ROS formation, oxidative DNA damage, dysfunction of the mitochondrial membrane potential, and early apoptotic changes in the human osteosarcoma cell line HS-Os-1. We therefore concluded that intracellular ROS formation is involved in the hydrogen peroxide-induced apoptosis of HS-Os-1 cells.

Enhancement of pedicle screw stability using calcium phosphate cement in osteoporotic vertebrae: in vivo biomechanical study.

We conducted an experimental study using female beagles with and without ovariectomy-induced osteoporosis to determine the effect of calcium phosphate cement (CPC) on the mechanical stability of inserted pedicle screws. A drill hole was created from the base of the transverse process to the vertebral body; CPC was injected into the hole, and then a screw was inserted into the same hole. In the presence of osteoporosis evidenced by dual X-ray absorptiometry, the stability of the inserted screw augmented by CPC against pull-out and cephalocaudal forces were significantly greater by 28% and 54% at 1 week after operation, 48% and 71% at 2 weeks, and 56% and 68% at 4 weeks compared with those without CPC. The pull-out strength increased progressively with time after surgery, probably reflecting new-bone growth from the surrounding cancellous bone, which was in direct contact with the CPC, as shown in the histologic study. At each time point the cephalocaudal rigidity was similar and the pull-out strength greater than that for the screws inserted without CPC in nonporotic dogs. These findings suggest that CPC augments the stability of the inserted pedicle screws and increases the stiffness of fixed osteoporotic motion segments using instrumentation.

Metabolic effects of X-ray irradiation on adult human articular chondrocytes.

There have been few reports regarding the metabolic effects of X-ray irradiation on adult human articular chondrocytes. The purpose of this study was to evaluate whether exposure to X-ray irradiation during tumor surgery can cause impaired metabolism in adult articular cartilage. To achieve this we exposed cultured chondrocytes isolated from normal or degenerated cartilage to varying doses of X-ray irradiation, then measured apoptosis, and the production of chondroitin sulfates (CS), prostaglandin E2 (PGE2) and p38 mitogen-activated protein kinase (MAPK) in these cells. The number of apoptotic cells was not affected by irradiation in chondrocytes from normal or degenerated cartilage. Likewise, the production of C6S and C4S was not altered by irradiation in either group. The concentration of PGE2 in non-degenerated chondrocyte cultures did not change with radiation in a dose-dependent manner. However, the concentration of PGE2 in degenerated chondrocytes increased in a radiation dose-dependent manner. Irradiation at 10 Gy, in degenerated chondrocytes, induced remarkable activation of p38. This suggests that it is important to consider whether there is an osteoarthritic joint in the area that is to receive radiation therapy during tumor surgery.

Mechanical properties of the femur filled with calcium phosphate cement under torsional loading: a model in rabbits.

We conducted an experimental study using rabbits to determine the effect of injection of calcium phosphate cement (CPC) through a small cortical window on the torsional strength of the long bone. A drill hole created in the femoral shaft and medullary curettage before CPC injection simulates the clinical procedure for managing benign bone tumors. Torsional loading always produced a spiral fracture through the drill hole. The CPC-treated femurs, but not the polymethylmethacrylate-treated femurs, tolerated greater torsional loads and had greater energy absorption to failure at 24 h and 2 weeks postoperatively compared with the contralateral sham-operated femurs. These two parameters, however, were only 54% and 26%, respectively, of those in normal femurs at 24 h, but they were 71% and 51% of those in normal femurs at 12 weeks. Histologic sections demonstrated progressive covering of the drill hole by new bone in the CPC-treated specimens and invagination by newly formed cortical bone into the medullary cavity following sham operation. CPC increases the torsional strength of the long bones immediately after injection, although not sufficiently to preclude the need for external fixation in clinical applications. Better management of the cortical defect is needed to further improve torsional strength.