Ⅱ型胶原微图案化对猪关节软骨细胞行为的影响  

Effects of collagen Ⅱ micropatterning on behavior of porcine articular chondrocyte

作　　者：李裕民 龚逸鸿[1] 

机构地区：[1]中山大学工学院,广州511400

出　　处：《南方农业学报》2017年第12期2273-2280,共8页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(51103182);汤臣倍健营养科学研究基金项目(39000-71010287)

摘　　要：【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,H_2O_2∶H_2SO_4)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%CO_2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300μm)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。【Objective】The effects of topological structure in regulating biomaterial surface on basic growth and function 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(H_2O_2∶H_2SO_4).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% CO_2,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 chondrocytes 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 materials.【Conclusion】Regular COL Ⅱ sub-micron topological structure established through μCP technology has promoting effects in adhesion spreading and

关 键 词：猪 关节软骨细胞 Ⅱ型胶原(COLⅡ) 微图案 微接触印刷(μCP技术) 

分 类 号：S828[农业科学—畜牧学]