| BackgroundOsteosarcoma is the most common tumor with higher malignant degree in musculosketal systems. Its survival rate in 5 years is very low(20%). With advancement of fundamental research and development of neo-adjuvant chemotherapy, modern aim to treat Osteosareoma has transferred saving of life through limb-amputation to lengthening of survival time and limb-salvage together. The effective devitalization of bone lesions is a key to the rate of recurrence and survival in comprehensive treatment of Osteosarcoma. Cryosurgery with liquid nitrogen has been used in Osteosarcoma over 30 years, succeeded in good control of local recurrence, and taken less effection on function of bone and joint. The higher status of cryosurgery has been built in the field of treatment of Osteosarcoma. Soaking of liquid nitrogen is applied to bone lesions of 54 cases of Osteosarcoma.The cryotherapy has showed good clinical outcome. In spite of summarizing the abundant clinical experience, We lack enough knowledge of biological safety. We don't know ultrosturcture change of Osteosarcoma cells with whole neclears after soaking of liquidnitrogen which only become much more smaller. As far we don't recognize the development of these cells if they are induced to apoptosis. This experiment will be concentrated on over-mentioned problem.Material and Method1. Specimen collectedAccording to dialognosis of clinic and X-rays and pathological examination, there is 21 patients of osteosarcoma IIB grade, from March 2000 to December 2001, including 15 male patients and 6 female patients. Patients age is from 15 years old to 25 years old. Tumors of 15 cases lie in the destal extremity of femur, 6 in the proximity extremity of tibial. Diamenter of tumors in from 4cm to 6cm.Our experiment material is made up of biopsy tissue before chemotherpy of ostesarcoma. in order to advoid micro-structural and ultrostructural damage of cells.Three specimen (1cm in volume) are cutted in growing zone of tumors through biopsy. One specimen is fixed for stain with HE by 10% formalin; another is rapidly cutted to several strips, and fixed by 2.5% glutaraldehyde. Third specimen is put into metal container, and soaked by liquid nitrogen for minutes (three times repeated). At last it is rapidly cutted to several strips (1 mm3 in volume), again fixed by 2.5% glutaraldehyde.2. Paraffin specimen preparedAll specimens are fixed with 10% formalin embedded in paraffin, sectioned and stained with HE, The specimens are determined by histological examination.3. Ultrostructural specimen preparedThe specimens are fixed in 2.5% glutaraldehyde, postfixed in Osmium tetroxide, dehydrated with propylene oxide, embedded in Epon 812, Sectioned with ultramicrotome, and stained by lead citrate and observed by JEM-1200EX electron microscope at magnification of 4000 to 8000 times.ResultsWe observe micro-structural and ultrostructural change of osteosarcoma cells before and after soaking of liquid nitrogen by microscopy and transmssion electron microscopy. It is suggested that some tumor cells with whole neclear only become much more smaller than normal ones. Through observing the smaller cells, we come to find the typical apoptosic phenomenon by transmission electron microscope: cell become small. Chromatin is getting more thinner and denser, and lies in neclear evelope, which seems crescent-like. Microvillus disappears. Cell organelles seem normal. Mitochondria becomes small and splity. Neclear breakes into pieces. Martix is concentrated. Endoplasmic reticulum expandes and fuses with part of oplasmic membrane, which likes hollow vacanic opening. Some endoplasmic recticulums become ballon-like. Oplasmic membrane obviouly curls and folds inside, and cover some matrix and pieces of neclear. The whole cell is damaged to form a series of dense apoptosis body in unequal size covered by oplasmic membrane.DiscussionIt is observed that steosarcoma cells after soaking of liquid nitrogen are not only necrotized. by physical and che... |
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