胶原/壳聚糖复合纳米纤维膜引导骨再生的细胞学研究  

Effect of Electrospun Collagen/Chitosan Composite Nanofibrous Membrane on Osteogenesis for Bone Regeneration

作　　者：李晓静[1,2] 高波 董研[1] 苟中入[3] 苗滪汶 

机构地区：[1]浙江大学医学院附属第二医院口腔矫形科,杭州310009 [2]浙江大学医学院附属儿童医院口腔科,杭州310006 [3]浙江大学浙江加州国际纳米技术研究院,杭州310029

出　　处：《中国生物医学工程学报》2018年第1期79-85,共7页Chinese Journal of Biomedical Engineering

基　　金：杭州市科技局科技发展计划项目(20090833B02);卫生部省部共建项目(WKJ2009-2-033)

摘　　要：采用静电纺丝技术制备胶原/壳聚糖复合纳米纤维膜,研究其作为引导骨再生生物膜的细胞生物相容性及诱导成骨性。以乙酸为溶剂,聚环氧乙烯(PEO)为增塑剂,采用静电纺丝技术制备胶原纳米纤维膜及不同比例的胶原/壳聚糖复合纳米纤维膜(胶原、壳聚糖、PEO质量比5∶1∶4,5∶2∶3,5∶4∶1),电子显微镜观察4种纳米纤维膜的表面形态;将骨髓间充质干细胞种植于胶原纳米纤维膜及表面形态较好的胶原/壳聚糖纳米纤维膜上,通过MTT法、碱性磷酸酶检测、细胞内胶原检测、免疫荧光染色及茜素红染色法观察,研究其细胞生物相容性及诱导成骨性。扫描电子显微镜观察胶原纳米纤维膜及质量比为5∶1∶4的胶原/壳聚糖复合纳米纤维膜的纤维光滑,直径均一。MTT法检测显示,胶原纳米纤维膜和胶原/壳聚糖复合纳米纤维膜均可促进骨髓间充质干细胞的粘附和增殖。细胞培养14 d后,胶原/壳聚糖复合纳米纤维膜上细胞内胶原含量检测为2.02 mg/gport,高于胶原纳米纤维膜组的1.63 mg/gport胶原含量(P<0.05),且胶原/壳聚糖复合纳米纤维膜上细胞内碱性磷酸酶、骨钙素及钙化结节的形成均高于胶原纳米纤维膜组。胶原/壳聚糖复合纳米纤维膜可促进骨髓间充质干细胞的增殖和分化,有望应用于骨再生的研究。The aims of this study are to prepare collagen/chitosan composite nanofibrous membranes,and to examine their biocompatibility and effect of osteogenic differentiation for bone marrow mesenchymal stem cells（ BMSCs）. Take acetic acid as solvent,poly（ ethylene oxide）（ PEO） as plasticizer,the collagen nanofibrous membranes（ the mass rate of collagen/PEO,4∶ 1） and various collagen/chitosan nanofibrous membranes（ the mass rate of collagen/chitosan/PEO,5∶ 1∶ 4,5∶ 2∶ 3,5∶ 4∶ 1） were fabricated through electrospinning technique.The surface morphology of membranes were observed by scanning electron microscopy（ SEM）. BMSCs were cultured on the collagen nanofibrous membranes and the optimized collagen/chitosan nanofibrous membranes,the cell proliferation and osteogenic makers including alkaline phosphate（ ALP）,collagen content,osteocalcin（ OCN） and mineralized matrix deposits were detected respectively. SEM showed collagen nanofibers and collagen/chitosan nanofibers of mass ratio of 5 ∶ 1 ∶ 4 had a mean fiber diameter. Both collagen nanofibrousmembranes and collagen/chitosan nanofibrous membranes supported adhesion,proliferation and osteogenic differentiation of the BMSCs. The collagen/chitosan nanofibrous membranes induced higher proliferation of BMSCs than collagen nanofibrous membranes. The collagen contents were higher in cells cultured on the collagen/chitosan nanofibrous membranes for 14 days as compared to collagen nanofibrous membranes（ p 0. 05）. Similarly,cells cultured on collagen/chitosan nanofibrous membranes had the higher expression of ALP,OCN and mineralized matrix deposits than that on collagen nanofibrous membranes. Our results demonstrated that the collagen/chitosan composite nanofibrous membranes promoted adhesion,proliferation and osteogenic differentiation of BMSCs,implying their promising applications for bone regeneration.

关 键 词：静电纺丝 胶原 壳聚糖 纳米纤维 骨再生 

分 类 号：R318[医药卫生—生物医学工程]