纤维连接蛋白表面修饰后聚酯纤维韧带细胞相容性的变化

Biocompatibility of ligament material by fibronectin surface modification

目的:观察纤维连接蛋白表面预处理增加新型聚酯纤维韧带(ligament advanced reinforcement system,LARS)的细胞相容性的作用。方法:实验于2005-09/11在上海复旦大学放射医学研究所骨细胞实验室完成。①实验分组:采用纤维连接蛋白表面修饰LARS人工韧带材料,为蛋白处理组。LARS人工韧带材料不做任何处理,为未处理组。蛋白处理组和未处理组在培养后2,4,6,8,10,12d测定成骨细胞数量、MTT法测定吸光度(A值)、测定碱性磷酸酶值(A值)。②实验评估:成骨细胞和与LARS人工韧带材料体外共培养。培养后3,7d扫描电镜观察成骨细胞在两组材料上的形态。培养后7d行碱性磷酸酶染色,观察细胞的形态及细胞和材料的细胞相容性情况。结果:①成骨细胞的数量:细胞在接种到材料后即开始增殖,在1～3d内增殖较缓慢,自第4天起细胞增殖迅速,蛋白处理组至第8天达到增殖高峰,未处理组至第10天才达到增殖高峰。蛋白处理组材料和未处理组材料上细胞增殖基本符合正常细胞增殖。蛋白处理组材料细胞对数增长期较未处理组细胞增殖期长,且最高峰值也高(P<0.05)。②成骨细胞的增殖能力:培养后2,4,6,8,10,12d细胞增殖能力迅速增加,两组细胞增殖能力到第6天达最高峰,以后逐渐降低,在每个时间段蛋白处理组材料上的细胞的增殖能力均高于未处理组,差异有显著性意义(P<0.05)。③成骨细胞的功能:培养后2,4,6,8,10,12d细胞碱性磷酸酶值随时间推移基本稳定,略有下降,两组间差异无显著性意义(P=0.46)。④扫描电镜下成骨细胞形态:培养后3d,未处理组材料上细胞沿材料表面和纤维孔径之间生长,细胞形态尚不典型。蛋白处理组材料上细胞形态比较典型,具有伪足和触角结构,细胞表面具有大量毛刺样结构。培养后7d,蛋白处理组材料和未处理组材料上细胞生长情况均较好,可见细胞大量汇合,沿纤维表面和纤维间隙之间生长,可见细胞在蛋白处理组材料上数量更多,汇合面积更大。⑤成骨细胞的碱性磷酸酶染色:成骨细胞与LARS人工韧带支架材料共培养后,培养后2d起在材料的周边可见梭形和三角形的细胞游离出,进而贴壁汇合,数量逐渐增多,细胞生长情况良好,尤其是在蛋白处理组可发现更明显的细胞黏附生长情况。结论:LARS人工韧带在体外实验中表现出良好的生物相容性,采用纤维连接蛋白表面预处理的方法简便、有效,能够有效增加材料的细胞相容性。

AIM： To verify the biocompatibility of ligament advanced reinforcement system （LARS） artificial ligament material by surface modification using fibronectin. METHODS： The experiment was carried out in the Laboratory of Osteocytes, Institute of Radiology at Fudan University from September to November in 2005. ①The study was divided into experiment group and control group according to whether LARS artificial ligament material was modified with fibronectin on surface. At 2, 4, 6, 8, 10, 12 days of culture, the osteoblasts were counted, alkaline phosphatase （ALP） value was measured, and MTT colorimetric method was used to determine the absorbance （A value）. ②Osteoblasts were cultured in vitro onto the surface of LARS artificial ligament material. On the 3^rd and 7^th days of culture, scanning electron microscope was used to identify the morphology of cells on the materials with or without fibronectin surface modification. On the 7^th day of culture, ALP stain was used to observe the morphology of cells and the biocompatibility of materials. RESULTS： ①Count of osteoblasts： Cells began to proliferate as soon as cultured on the materials, the growth was slow at 1-3 days of culture, rapid from the 4^th day, and reached a peak on the 8^th day of experiment group and 10^th day of control group. Both groups met the normal cell proliferation, and the increased logarithmic phase of cell in experiment group was longer than that of control group, and the peak was also higher （P 〈 0.05）.②Preliferation of osteoblasts： The proliferation increased and peaked on the 6^th day, then began to decrease gradually. At each point of culture, the cells of experiment group proliferated more rapidly than those of control group, and there were significant differences （P 〈 0.05）. ③Function of osteoblasts： With the prolong of culture time, ALP value remained stable and then a little decreased, but there was no difference between the two groups （P =0.46）. ④Morphology of osteoblasts： on the 3^rd day, the cells of control group grew along the material surface and fiber pore, while those of experiment group were typical to appear pseudopodia and tentacle, together with a great quantity of sentus-like structure on surface. On the 7^th day, the cells of both groups grew well along the fiber surface and interspace with confluence occurred, cells proliferated better on the ligament material using fibronectin modification than that without.⑤ALP stain of osteoblasts： On the 2^nd day, fusiform and triangle cells educed from the surrounding of materials, then adhered and coalesced. The adhesion and proliferation of cells were more obvious in the experiment group. CONCLUSION： LARS artificial ligament shows fine biocompatibility through in vitro culture experiment, and the surface fibronectin modification is a convenient and efficient way to enhance the biocompatibility of artificial ligament material.