钛表面粗糙度对牙周韧带细胞骨钙素表达的影响

Influence of the surface roughness of titanium on the expression of osteocalcin in human periodontal ligament cells

目的:以骨钙素为细胞分化指标,观察牙周韧带细胞在不同粗糙度纯钛表面的分化特征。方法:实验于2005-01/12在赣南医学院科研中心完成。①将纯钛棒制备成直径10mm、厚2mm的钛片,共24个,分为A、B、C、D4组,每组6个样品。4组样品分别采用100目、280目、400目、800目水砂纸进行打磨,然后采用如下方法对钛片进行处理:A组:单纯的机械处理;B组:单纯的机械处理+65%的硝酸(100℃,1h)处理;C组:单纯的机械处理+100μm的Al2O3喷砂处理;D组:单纯的机械处理+100μm的Al2O3喷砂处理后+65%的硝酸(100℃,1h)处理。所有样品均经过灭菌处理。②取无龋坏、无牙周病的正畸牙,磷酸盐缓冲液冲洗3遍,采用组织块培养法进行体外培养。刮取牙根中1/3处的牙周膜,剪成大小为1mm3的组织块,均匀地铺于培养瓶底面。待组织块贴牢瓶底后轻轻翻转培养瓶,继续培养。待细胞从组织块边缘游离出后,胰蛋白酶分散,消化传代。观察牙周韧带细胞的体外培养特征。③将传至第3代的牙周韧带细胞,以2×108L-1的浓度分别接种于4组样品表面,免疫荧光技术观察在不同粗糙度的商用纯钛表面的牙周韧带细胞骨钙素的表达。结果:①4组样品的粗糙度分别是(0.5995±0.0083),(0.4065±0.0046),(0.3588±0.0118),(0.0087±0.0022)μm,经方差分析,P<0.01。组与组之间比较经q检验,P<0.01。②牙周韧带细胞在形态上为长梭形的成纤维细胞样细胞。5～6d后,细胞从组织块边缘游离出来,游离细胞呈长梭形。传代后的牙周韧带细胞呈长梭形,少量呈多角形。③免疫学显示细胞抗角蛋白抗体阴性,抗波形蛋白阳性,证实细胞为中胚层来源的结缔组织成纤维细胞。牙周韧带细胞在纯钛表面培养第7天,细胞在各组样品的纯钛表面增殖良好。在每组纯钛表面上的细胞均形成与钛片的条形生长,生长方向与钛片最终打磨方向一致。A组纯钛表面上的细胞形态清晰,部分荧光染色呈颗粒状,细胞表达骨钙素;B组纯钛表面上的细胞以树突状突起融合在一起,细胞表达骨钙素;C组纯钛表面上的细胞密度增加。D组纯钛表面上的细胞间隙难以区分,呈片状生长,细胞染色增强。结论:纯钛表面粗糙度越小,牙周韧带细胞可能表达骨钙素越高,牙周韧带细胞矿化能力可能越强。

AIM： To observe the expression of osteocalcin （OC） in periodontal ligament cells （PDLCs） attaching to the surface of pure titanium with different roughness commercially. METHODS： The experiment was conducted in the Center of Scientific Research, Gannan Medical College between January and December 2005. （1） Pure titanium was made into 24 titanium pieces with the diameter of 10 mm and thickness of 2 mm, which were then divided into four group A, B, C and D with 6 samples in each group. Samples in four groups were polished by waterproof abrasive paper of 100 meshes, 280 meshes, 400 meshes and 800 meshes respectively, and then treated respectively as the following. Group A： treated simply by machinery. Group B： treated by machinery and 65% of nitric acid at 100 ℃ for one hour. Group C： treated by machinery and Al2O3 sand blasting of 100 μm. Group D： treated by 65% nitric acid at 100 ℃ for one hour following treatment by machinery and Al2O3 sand blasting of 100 μm. All samples were treated by germicidal method. （2）The orthodontic tooth without dental caries or periodontopathy were selected and washed with phosphate buffer （PB） for three times and then were cultured in vitro with serial cultivation. 1/3 of alveolodental ligament in the dental root was obtained by scraping and then were sheared into pieces of 1 mm^3 to evenly pave at the bottom of culture flask. The culture flask was slightly turned over after the sample pieces firmly adhered to the bottom to continue the culture. PDLCs were dissociated from the tissue mass and the trypsinase scattered, and the passage were digested. The in vitro cultural characteristics of PDLCs were studied. （3） PDLCs of tertiary generation were inoculated on the surface of samples in four groups at the concentration of 2×10^8 L^-1. The expression of OC on the surface of commercially used pure titanium with different roughness was detected by immunofluorescent technique. RESULTS： （1）The rugosity of samples in four groups were （0.599 5 ±0.008 3）,（0.406 5±0.004 6）,（0.358 8±0.011 8）,（0.008 7±0.002 2） μm respectively, which were analyzed by analysis of variance, P 〈 0.01. Differences among groups were compared with q test, P 〈 0.01. （2） PDLCs were long-shuttle shaped fibroblast-like ceils. After 5-6 days, PDLCs were dissociated from the tissue mass, The dissociated cells were in long-shuttle shape, and the configuration of the ceils was legible. PDLCs were in long-shuttle shape after generation, on the same time, few PDLCs were in polygon shape. （3）Anti-keratin antibody was negative, and the anti-wave protein antibody was positive. It was proved that the ceils were fibroblasts of connective tissue derived from mesoblast. After cultured on the surface of pure titanium with different roughness for 7 days, PDLCs in each group proliferated well to match with the direction of pure titanium polishing. PDLCs of group A were clear in shape, and partial fluorescent dyeing showed granule shape, and PDLCs expressed OC. PDLCs of group B were fused together with the dendritic protuberance, and PDLCs expressed OC. The density of PDLCs in group C increased, which it was difficult to distinguish the cell clearance at the PDLCs of group D, and cells fully grew by the slice shape, and the dyeing boosted up. CONCLUSION： The smaller the roughness on the surface of pure titanium was, the higher the expression of OC Of PDLCs may be, and the stronger the mineralizing ability of PDLCs may be.