摘要
目的:观察钛合金表面纳米化对成骨细胞黏附的影响。方法:实验于2004-06/2005-06在上海市第六人民医院实验室完成。对SD大鼠成骨细胞进行原代培养。成骨细胞的鉴定:①倒置相差显微镜下观察细胞形态。②碱性磷酸酶染色(偶氮偶联法)。③成骨结节四环素染色:荧光显微镜观察,矿化结节呈金黄色。将成骨细胞分别与表面光滑的钛合金、表面粗糙的钛合金和表面纳米化的钛合金共同培养,一定时间后用细胞计数和扫描电镜观察等技术进行比较。结果:①材料表面特征:光滑钛合金表面除少量滑痕外无任何起伏。而粗糙钛合金表面有较大的不规则起伏,形成微米级的孔隙。纳米钛合金表面可见形成多孔纳米结构,在微米级的大孔内还有纳米级的小孔。②细胞培养和鉴定:倒置相差显微镜观察可见接种的骨组织块贴壁一两天后骨块边缘可见少量呈短梭形、三角形的细胞游离出。随培养时间延长,骨块边缘细胞增多,并向瓶壁周围移行。约2周后,细胞培养瓶底可形成一单层细胞,呈集簇状生长。未传代的细胞生长至三四周后细胞呈复层状生长,在其中心部细胞密集并可发生钙化,形成肉眼可见的散在白色点状物。③吖啶橙染色、计数及分析:随时间的延长3种材料上黏附的细胞均明显增加(P<0.01)。在1,2,4h3个同一时间段,黏附的成骨细胞数纳米>粗糙>光滑(P<0.01),且SNK检验两两比较差异显著。由此可见,成骨细胞在表面纳米化钛合金表面达到早期黏附。④电镜观察:成骨细胞与材料共培养1h后3种钛合金表面的成骨细胞数量纳米>粗糙>光滑。纳米表面的细胞大多已经伸展开来,并有较多突起,而在光滑和粗糙表面的细胞还呈球形,没有伸展开来。同时可以发现在粗糙和纳米钛合金表面的成骨细胞更倾向于黏附在材料的颗粒界面。结论:纳米化钛合金能明显促进成骨细胞的早期黏附。表面纳米化技术不仅保持材料的生物力学性能,而且使其具有纳米生物学的优点。
AIM： To observe the effect of the nano-surface on titanium alloy on the adhesion of oateoblasts. METHODS： The experiment was carried out from June 2004 to January 2005 in the laboratory of the Sixth People＇s Hospital of Shanghai city. After the osteoblasts of SD rats were primarily cultured, three methods were adopted to identify them. Inverted phase contrast microscope （IPM） was used to observe the cellular morphology. Alkaline phosphatase （ALP） staining of osteoblast and ambramycin staining of ossified nodule were also used. Golden mineralized nodule Was observed under fluorescent microscope. Then osteoblasts were cultured with three different titanium alloys including smooth （S）, rough（R） and nano-surface （N）. The adhesive osteoblasts were counted with computer and observed with scanning electron microscope （SEM）. RESULTS： （1） Characters of material surface： The three surfaces have different appearances under SEM. The S group was very smooth except some marks. The R group had bigger irregular fluctuation, which formed micrometer-grade pores. While N group had smaller regulated micrometergrade pores in which there were nanometer-grade pores. （2）Culture and identification of osteoblasts： Observation under inverted phase contrast showed that one or two days after inoculated bone tissue pieces attached to the wall, few shuttle and triangle cells dissociated. With the elongation of culture time, the cells at the edge of bone pieces increased and migrated to the wall of the bottle. About 2 weeks later, a unilaminar cells formed at the bottom of culture flask, presenting cluster-shape. Ungenerative cells presented more-layer shape 3 to 4 weeks later. At the center, cells gathered, calcified and formed scattered white-spot thing seen by naked eyes. （3）Acridine orange staining , counting and analysis： With the elongation of time , the cells adhered on the three materials increased obviously （P〈 0.01 ）. At the same time period of 1, 2 and 4 hours, the adhered osteoblasts number was nanometer 〉 rough 〉 smooth in order （P 〉 0.01）, significant difference existed in SNK test. Thus it can be seen that osteoblasts got early adhesion on the nano-surface titanium alloy. （4） Observation under electron microscope ： After osteoblasts were co-cultured with materials for one hour, the number of osteoblasts on the 3 titanium alloys was nanometer 〉 rough〉 smooth in oroler. Most of the cells on the nano-surface titanium alloy expanded with many mutations, while the osteoblasts on the smooth and rough surface still presented neurospherelike shape, without extension. It was also found that osteoblasts on the rough and nano-surface titanium alloy inclined to adhere to the interface of the materials. CONCLUSION： The earlier adhesion of osteoblast can be promoted by nanotechnology. The nano-surface technology can give the advantage of nanobiology to the material maintaining the biomechanical character.
出处
《中国临床康复》
CSCD
北大核心
2006年第25期46-48,i0003,共4页
Chinese Journal of Clinical Rehabilitation
基金
上海市纳米专项基金资助项目(T0500704)~~
