医用多孔钽材料对大鼠软骨细胞增殖、细胞周期及凋亡的影响

Effect of porous tantalum material on chondrocytes proliferation,cell cycle and apoptosis on rats

[目的]观察国产多孔钽材料对大鼠软骨细胞增殖、细胞周期及凋亡的影响，为其作为软骨组织工程支架材料临床应用提供实验依据。[方法]选用3周龄SD大鼠双侧股骨头、膝关节分离软骨组织，经Ⅱ型胶原酶消化，饱和湿度培养，传代。倒置显微镜下逐日观察细胞生长状态。待第2代细胞生长至80％左右时，通过甲苯胺蓝染色、番红O-固绿染色以及Ⅱ型胶原免疫细胞化学染色进行软骨细胞鉴定。以F12-DMEM为浸提介质分别制备100％、50％、25％多孔钽浸提液，软骨细胞加不同浓度多孔钽浸提液作为实验组，未加浸提液的软骨细胞作为对照组；MTr法检测多孔钽浸提液对软骨细胞增殖的影响及细胞毒性检测。将第2代软骨细胞接种到多孔钽支架上进行体外三维培养，而对照组是将软骨细胞接种到无支架的培养板上，倒置相差显微镜观察两组软骨细胞生长状态；胰酶消化复合于材料上的软骨细胞，流式细胞仪检测软骨细胞的细胞周期变化及细胞凋亡。[结果]软骨细胞接种初期大小不等，呈圆形悬浮于培养液中，24h后贴壁细胞伸展呈三角形、多角形等。第7—-9d待细胞生长呈现融合时即可传代。甲苯胺蓝染色显示软骨细胞胞浆内可见蓝紫色颗粒，番红O-固绿染色显示胞浆呈绿色、细胞核呈紫红色，Ⅱ型胶原免疫细胞化学染色显示阳性信号位于胞浆及部分胞膜，以上方法证实了分离培养的细胞为软骨细胞；bwr法检测显示随着培养时间的延长，各浓度浸提液组细胞生长与对照组无明显差异，软骨细胞形态正常，贴壁增殖良好，相同时间点不同浓度的浸提液与对照组间细胞增殖差异无统计学意义（P〉0．05），提示软骨细胞在多孔钽浸提液中生长、增殖状态良好且多孔钽材料无毒性。多孔钽支架材料外观灰色光亮，表面及断面可见分布均匀的蜂窝状孔隙，孔径针尖大，支架材料不透光，将分离培养的第2代软骨细胞接种到多孔钽支架上后，复合培养24h后，倒置相差显微镜可见材料边缘有少量软骨细胞黏附，细胞呈多角形；随着培养时间增加，材料边缘及培养板底部细胞黏附的数量逐渐增多，边缘软骨细胞生长良好；流式细胞仪检测显示实验组与对照组细胞均为正常二倍体细胞，两组细胞周期分布相似，实验组及对照组的细胞凋亡率及坏死细胞率差异均无统计学意义（P〉0．05）。[结论]国产多孔钽材料无毒性，对软骨细胞增殖、细胞周期及凋亡无明显影响，适合作为软骨组织工程支架材料。

[ Objective ] To study the effect of domestic porous tantalum material on the chondrocyte proliferation, cell cycle and apoptosis, and to provide the experimental data for the clinical application. [ Method ] The 3 weeks born SD rat were select- ed and cartilage of bilateral femoral head and knee were resected. Chondrocytes were digested by type Ⅱ collagen enzyme diges- tion method, and cultured at saturated humidity and passaged. The growth of cells were observed under inverted phase contrast microscope, then the 2nd generation of chondrocytes covering the 80% were identified with toluidine blue staining, safranine O - solid green staining and type Ⅱ collagen immunocytochemistry staining. The different concentrations of porous tantalum leaching liquor（ 100% ,50% and 25% ） were prepared with medium F12 - DMEM. The chondrocytes were cultured in leaching liquor of different concentrations were used as experimental group and control group was without leaching liquor. MTT assay was applied to detected the cell proliferation ability and cytotoxicity in the leaching liquor. The 2nd generation of chondrocytes were seeded on porous tantalum scaffolds,with three -dimensional cultivation, while the cells of control group were seeded on cell panels. Theseeded chondrocytes were digested by trypsin and the cell cycle and apoptosis were detected by flow cytometry. [ Result ] In early days the chondrocytes differed in shape and suspended in a culture solu- tion,and triangle and polygon in 24 hours later. The chondrocytes were passaged till cell fusion in 7 - 9 days. Toluidine blue staining showed there were bluish violet granules within the chondrocyte cy-toplasm. There were greenish within the chondrocyte cytoplasm and purphsh red wihtin the nucleus by safranine O - solid green staining. Type Ⅱ collagen immunocytochemistrical staining showed positive signals in the cytoplasm and part of the cytomem- brane. The MTT method showed that the number of cells was increasing with time, the chondrocytes growing normally and having good adherent proliferation abilities. At the same time point, there were no significant difference in the cell proliferation ability between the different concentrations of leaching liquor groups and control group （ P 〉 0.05 ）. The method showed cell prolifera- tion and growing normally in the leaching liquor, and the porous tantalum having no toxicity. Porous tantalum scaffolds were gray and light- proof, the surface and cross section were evenly distributed needlepoint cellular porosity. The 2nd generation of chon- drocytes were planted in porous tantalum scaffolds, and 24 hours later, a few polygon chondrocytes were found adhering to the edge of porous tantalum scaffold materials under inverted microscope. With the increasing of time, many of chondrocytes could be found adhering to the edge of materials and the bottom of cell panels under inverted microscope, and the cells growing normally. Flow cytometry showed that there was normal diploid cells and similar cell cycle distribution in both groups. There were no sig- nificant difference in the rates of apoptosis and necrosis cells between the experimental group and control group （ P 〉 0.05 ）. [ Conclusion ] The domestic porous tantalum has non - toxicity, not a marked effect on chondrocytes proliferation, cell cycle and apoptosis, which make it suitable candidate as a cartier for cartilage tissue engineering.