【作者】 刘莹；

【导师】 战德松；

【作者基本信息】 中国医科大学 ， 口腔临床医学， 2010， 硕士

【摘要】 目的本实验用新型医用无镍不锈钢BIOSSN4浸提液培养小鼠L929成纤维细胞,通过MTT法检测细胞增殖活力并计算相对增殖率,从而对这种合金的生物相容性进初步检测,并与传统使用的医用316L不锈钢,金合金的生物相容性进行比较和评价,为该材料的临床应用提供生物学依据。方法一、实验材料将实验试样分为：A组BIOSSN4不锈钢,B组：316L不锈钢,C组：金合金,D组：铅。将A-D组材料分别制成半径5mm、厚1mm的圆形蜡片,根据不同合金的要求铸造形成圆形金属片,打磨抛光后超声清洗15min；去离子水冲洗数遍后置于玻璃小瓶中,将金属片于121℃高压灭菌后待用。无菌下,将A-D组试样分别在超净工作台中,按浸液与试样表面积之比为0.55ml/cm2的比例加入RPM11640培养液,置于37℃,95%湿度,5％CO2培养箱中浸提72小时,即得到材料浸提液。二、实验方法取96孔培养板,每孔加入细胞悬液100ul。在1-4组孔中分别加入A-D组材料浸提液100μl,在第5组孔内加入即RPMI1640培养液100μl后,放入培养中培养。分别于培养后的1-3天各取一块培养板,在倒置相差显微镜下观察活胞的形态。在每孔中加入5mg/ml的MTT液20μl,继续培养4小时后取出。吸培养液,在每孔中加入DMS0150μl,用自动酶标仪检测吸光度值。将各组的吸度值作单因素方差分析和均数间两两比较的统计学分析。计算各组的细胞相对增殖率(RGR)并作细胞毒性级评定。结果一、细胞的形态学观察培养24小时,阳性对照组中贴壁生长的细呈梭形或多角形,悬浮的细胞呈圆形或不规则形,胞质内出现空泡及颗粒状物。其余各组的细胞几乎全部贴壁生长,形态正常,胞质均匀。培养72小时,阳性对照组中的细胞大部分脱落,胞质,胞核浓缩,甚至可见许多质膜崩解,核破裂的死亡细胞。其余各组细胞大部分继续贴壁生长,细胞密度增加,可见散的悬浮的退化细胞及个别的死亡细胞。二、统计学处理结果金属材料组及阴性对照组与阳性对照组之间的光度值的差异均有统计学意义(P<0.05或P<0.01)。三种金属材料组的细胞相对增殖率由大到小的顺序依次为：金合金,BIOSSN4无镍不锈钢,316L不锈钢。BIOSSN4无镍不锈钢,金合金对细胞均有极轻微的毒性,316L不锈钢对细胞表现出1级细胞毒性,但统计学分析结果显示：三种金属材料组与阴性对照组的吸光度值之间的差均无统计学意义(P>0.05)。结论本实验证实了BIOSSN4无镍不锈钢具有极微弱的细胞毒性,但符合材料临床应用的生物相容性要求,为其应用提供了一定的生物学依据。更多还原

【Abstract】 ObjectivesIn this experiment, we investigate the cytotoxicity aim of a new medical nickel-free stainless steel (BIOSSN4). L-929 cell line of mice fibroblasts was cultivated with the infusions of BIOSSN4.The proliferation of the cells were investigated using a tetrazolium-based colorimetric assay (MTT assay) and compared with traditional 316L medical stainless steel, Au-alloy, in order to provide a biological basis for the clinical application.Methods1.The experiment materials:The experiment materials were divided into:A set:BIOSSN4 stainless steel, B set:316L stainless steel, C set:Aualloy, D set:Pb. A-D set materials were made into some wafers that diameters were 10mm, the thickness was lmm.After disinfected and sterilized, the materials were put into super clean bench. Then we used the RPMI1640 culture liquids to prepare the extraction liquids of those materials.2.The experiment methods:To take 96 bores culture plates, and add the cell suspending liquids 1 OOul into each bore. Respectively add the A-D set materials extracting liquids 100ul into the Groups 1-4bores, and add RPM11640 culture liquids 100ul into group 5bore, then put them into the culture box. Respectively take out a culture plate after educating 1-3day, and observe the changes of cell form by upside down and discrepancy microscope. To add 5mg/ml MTT liquids 20ul into each bore, and take out the plate after educating 4hours continuously. To suck out the liquids and join DMSO 150ul, into each bore. To examine the optical densisity by automatic enzyme immunoassay analyzer, and calculate the rate of cells relative growth(RGR) and assess the cytotoxic rank of each materials.Results1.The cell’s morphological observation:The first day in the positive control the cells growing on the plate showed bursiform or polygon shape, and the supending cells showed circular or irregular form, and appeared vacuoles or grains in the cytoplasm. The cells of the rest groups all almost grew with attaching on the plate, and with enmorphism and the steady cytoplasm. The third day, in the positive control the cells almost shed off, which cytoplasm and cell mucleus concentrated, and even there were many dead cells which plasmalemma collapsed and karyotheca solved. The cells of the rest groups all almost grew with attaching on the plate continuously, and the cells density creased. There were few suspending degenerate cells or dead cells.2. The results of statistical analysis:There were a significant difference in the optical densities between metal material groups or the negative control (P<0.05 or P<0.01). According to the rate of cell’s relative growth of the three metal material groups, the sequence form big to small was: the Au alloy, BIOSSN4 stainless steel,316L stainless steel. BIOSSN4 showed slight cytotoxicity,316L stainless steel showed 1-class cytotoxicity. But the statistical analysis result manifested:there were not significant difference in the optical densities between the third metal material groups and the negative control (P>0.05).ConclusionThis experiment results clarifies:BIOSSN4 only have very faint cytotoxicity, which meets the requirement for clinical application. These results provide a biological basis for clinical application更多还原