正交试验设计建立多因素的海藻酸钠复合载体立体培养软骨细胞体系  

Multifactoral sodium alginate complex carriers for 3-dimensional culture of chondrocytes： an orthogonal experiment design

作　　者：梁伟国[1] 沈雁[1] 关广雄[2] 杨小红[1] 戴丽冰[1] 梁佩红[1] 谭见容[1] 李超霞[1] 

机构地区：[1]广州市红十字会医院骨科,510220 [2]广州医学院第二附属医院

出　　处：《中华创伤骨科杂志》2008年第2期164-168,共5页Chinese Journal of Orthopaedic Trauma

基　　金：广东省卫生厅课题（2004553）;2004年广州市卫生局重点课题（2004Z005）

摘　　要：目的通过正交试验设计研究四种主要因素对软骨细胞增殖的影响，探讨海藻酸钠胶体复合载体体外培养软骨细胞增殖的最佳配比培养体系，为组织工程种子细胞的研究提供实验依据。方法建立单层贴壁和海藻酸钠复合载体立体培养细胞模型，采用正交试验4因素2-wav和4因素3-way设计，用XTT方法检测软骨细胞在一定浓度的复合因子包括碱性成纤维细胞生长因子、透明质酸钠、纤维连接蛋白、壳聚糖的完全培养基中的增殖情况；采用倒置、相差、激光共聚焦显微镜的形态学观察；采用LDH检测复合材料对细胞的毒性反应，确定体外培养体系方案。结果正交试验设计能确定各因素的有效浓度及配比方案，其中碱性成纤维细胞生长因子与透明质酸有共协同作用，在DMEM／Ham＇s F12复合培养基的海藻酸钠载体支架中培养软骨细胞能快速增殖，细胞数48h就增殖3倍多，Safranine O GAG染色显示传至9代仍然产生大量蛋白聚糖基质；LDH检测未见毒副作用；该复合载体具有良好的成形性和组织相容性。结论在DMEM／Ham＇s F12复合培养基的海藻酸钠载体中培养能快速增殖并有利于维持软骨细胞表型及功能的表达。Objective To explore the best composition of sodium alginate complex carrier containing 4 elements and their influence on chondrocyte proliferation in vitro in an orthogonal experiment design. Methods Three-dimensional models of monolayer adherent culture and sodium alginate complex carrier were established. The orthogonal design and statistics of 4 factors two-way and 4 factors three-way were conducted. The chondrocyte proliferation at certain concentrations in complete medium containing basic fibroblast growth factor （bFGF）, hyaluronic acid （HA）, fibronectin and chitosan was detected by XTT. The chondrocyte morphology was observed by inverted microscope, contrast phase microscope and laser confocal microscopy. The toxic reaction of composite materials to chondrocytes was detected by LDH. The cultural protocol in vitro was determined. Results The orthogonal experiment design could determine the effective concentration and optimal proportion protocol for every factor. The bFGF and HA showed a synergistic effect. The chondrocytes proliferated quickly in the complex medium DMEM/Ham＇s F12 with sodium alginate complex carrier scaffold. Cell proliferation increased 3 times in 48 h. Safranine O GAG staining revealed that the passage to 9th generation still produced a great quantity of proteoglycans matrix. The toxic reaction was not detected by LDH. The complex carrier had satisfactory shaping and histocompatibility. Conclusion Chondrocytes can proliferate quickly in DMEM/Ham＇s F12 nutrient medium with sodium alginate complex carrier and retain the expression of cartilage phenotype and function.

关 键 词：软骨细胞 细胞 培养的 载体 正交试验 

分 类 号：R318.0[医药卫生—生物医学工程]