Ⅱ型胶原微图案化对猪关节软骨细胞行为的影响

目的]明确调控生物材料表面拓扑结构对猪关节软骨细胞基础生长行为和功能的影响,为关节软骨组织缺损修复提供理论依据.方法]利用食人鱼溶液(Piranha,H2O2:H2SO4)对普通玻片进行氧化,以紫外光刻法制备具有不同尺度条带微图案的Si印模,经氧气等离子体处理和II型胶原(COLII)浸润后,通过微接触印刷(μCP技术)将COLII固定在氧化玻片材料表面,将猪关节腔软骨细胞接种于微图案化的支架材料上,经37 ℃、5% CO2培养箱培养后采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)比色法和二乙酸荧光素(FDA)荧光染色法分别观察软骨细胞的增殖生长情况.结果]经食人鱼溶液处理后玻片表面羟基化程度提高,亲水性明显增强.通过μCP技术能成功将COLII印刷在羟基化玻片表面上,且条带均匀、整齐.将猪软骨细胞接种到构建的取向性COLII微图案材料上进行培养,发现COLII能为猪软骨细胞提供黏附位点,且取向性COLII微图案对软骨细胞的增殖生长有促进作用,但不同尺度(100、200和300 μm)线宽对软骨细胞的增殖生长存在明显差异,表现为线宽越宽,软骨细胞的增殖生长能力越弱.猪软骨细胞在COLII微图案材料上生长状况良好,随着培养时间的延长,软骨细胞多呈多边形;其软骨细胞培养效果优于无规则的涂覆COLII微图案材料.结论]利用μCP技术成功构建的规整COLII微米级拓扑结构对猪关节软骨细胞的黏附铺展、增殖行为有促进作用,且线宽尺度与软骨细胞增殖生长能力呈反比趋势.即通过人为调控生物材料的表面活性拓扑结构,在一定程度上能有效调控或影响关节软骨细胞的生长行为.

Effects of collagen II micropatterning on behavior of porcine articular chondrocyte

Objective]The effects of topological structure in regulating biomaterial surface on basic growth and func-tion of porcine articular chondrocyte were studied to provide reference for articular cartilage tissue repair.Method]First of all,the common glasses were oxidized by piranha solution(H2O2:H2SO4). UV lithography was used to prepare Si stamp with strip micropattern of various scales.Then the stamps were soaked in collagen II(COL II)solution after oxygen plasma treatment,COL II was fixed on the surface of oxidized glass by microcontact printing(μCP technique).Porcine articular chondrocyte was inoculated onto the microppatternized support. Finally,after being cultured in incubator with 37℃and 5% CO2,the proliferation and growth of chondrocytes were observed by both 3-(4,5-dimethylthiazole-2)-2,5-diphenyl tetrazazole bromide(MTT)and fluorescence staining of diacetate(FDA).Result]Hydroxylation degree of glass surface increased after treated by piranha solution,and hydrophilicity obviously enhanced.COL II was successfully printed on the surface of hydroxylated glass by μCP technique,and the strip was uniform and neat.The porcine chondro-cytes were inoculated into the constructed orientated COL II micropattern materials. The results indicated that COL II could provide adhesion sites for chondrocytes,and the orientated COL II micropattern was helpful for proliferation and growth of chondrocytes.However,different scales(100,200 and 300 μm)of strip width had significant differences on the proliferation and growth of chondrocytes.The wider the line width,the lower the proliferation ability of chondrocytes. Porcine chondrocytes were in good condition on COL II micropattern materials,and the chondrocytes were polygonal as the time was prolonged.The chondrocyte culture effects were superior to the non-regular coated COL II micropattern mate-rials.Conclusion]Regular COL II sub-micron topological structure established through μCP technology has promoting ef-fects in adhesion spreading and proliferation of porcine articular chondrocyte.And line width size is inversely proportional to proliferation ability of chondrocyte.The growth behavior of chondrocytes can be regulated by artificially regulating the surface active topological structure of biological materials.