Kiyotaka Okuyama

A quantitative assay using basement membrane extracts to study tumor angiogenesis in vivo

We describe a quantitative assay for assessing tumor angiogenesis in vivo using basement membrane extracts (Matrigel). Nude mice were injected s.c. with liquid Matrigel mixed with HT1080 human fibrosarcoma cells. Since Matrigel rapidly forms a solid gel at body temperature, the gel containing tumor cells can be removed immediately and then processed for histological studies. Tumor angiogenesis was monitored quantitatively by measuring both the number and the total area of neovessels present in the gels using an image analyzer, which could be achieved approximately 72 hr later. Furthermore, HT1080 cell‐conditioned medium, which may contain various tumor‐derived factors, promoted the basement membrane degradation, migration, proliferation and tube formation of endothelial cells in vitro, as did Matrigel, although to a lesser extent. In addition, Northern blot analysis demonstrated that the amount of vascular endothelial growth factor (VEGF) mRNA in HT1080 cells was much higher than that in human fibroblasts or NIH3T3 cells. Our results suggest that angiogenesis observed in our assay may be due to the synergic effects of tumor angiogenic factors such as VEGF, and Matrigel.

Ethylene oxide sterilization of bone grafts Residual gas concentration and fibroblast toxicity

We examined the concentration of ethylene oxide in bone allografts after gas sterilization. Chips of the human femoral head were investigated. Residual gas concentration was determined by gas chromatography after the bone chips had been subjected to defatting and freeze-drying, followed by ethylene oxide gas sterilization. Bones were prepared in various ways in an attempt to reduce the concentration of residual ethylene oxide. The concentration was higher when gas sterilization was performed before freeze-drying than when it was done afterwards. An experiment performed with fibroblasts showed the high toxicity of residual ethylene oxide in bone chips, even when the concentration was very low. The growth of fibroblast was reduced more in medium which had been shaken with bones sterilized with ethylene oxide before freeze-drying than in medium which had been shaken with bones sterilized after freeze-drying. The higher residual ethylene oxide concentrations resulted in a decrease in fibroblastic culture activity. Our experiment showed the importance of reducing the residual ethylene oxide gas concentration. Defatting and freeze-drying result in lower residual ethylene oxide concentrations.