DegraPol支架上培养人气管软骨细胞体外合成组织工程软骨  

Tissue engineered cartilage generated from human tracheal chondrocyte using DegraPol scaffold

作　　者：杨林[1,3] 纪玲[2,3] 王正[1] Walter Weder 

机构地区：[1]暨南大学第二临床医学院深圳市人民医院胸外科,518020 [2]暨南大学第二临床医学院深圳市人民医院检验医学部,518020 [3]Division of Thoracic Surgery,Laboratory of Tissue Engineering,University Hospital of Zurich,Switzerland

出　　处：《中国医药生物技术》2007年第1期18-22,共5页Chinese Medicinal Biotechnology

基　　金：国家自然科学基金(30500499)

摘　　要：目的探讨以人气管软骨细胞(HTC)为种子细胞在新型生物材料DegraPol泡沫支架上体外合成组织工程气管软骨的可行性。方法从肺移植供体(9例)取人气管软骨片段,胶原酶消化,将所获软骨细胞传代培养,将传代至6～8代的HTC种植到DegraPol支架上体外静态培养。种植前行II型胶原免疫组织化学染色。于体外静态培养第2小时末和第3、7、21、42天取HTC-DegraPol复合物用噻唑蓝(MTT)法测定细胞增殖活性;培养3周后取HTC-DegraPol复合物制备组织学检测标本行阿辛蓝染色观察硫酸软骨素分泌情况,并制备扫描电镜标本观察HTC在DegraPol支架中培养后的超微结构。结果HTC在传代培养中显示稳定的增殖活性并分泌II型胶原。种植到DegraPol支架中后培养2h和3、7、21、42d时MTT法测定的A值分别为0.112±0.004、0.151±0.021、0.170±0.035、0.176±0.023和0.213±0.023(每个时点n=4)。培养21、42d与培养2h比较,活性HTC数量差异均有统计学意义(P<0.05)。阿辛蓝染色显示HTC-DegraPol复合物的基质分泌硫酸软骨素,证实HTC-DegraPol复合物具有软骨样结构。扫描电镜观察显示新生软骨在DegraPol材料表面和中央均有形成,但以表面为主。结论HTC可以在体外良好扩增并以多孔生物材料DegraPol为支架体外合成组织工程气管软骨,但培养条件应进一步优化。Objective To assess the feasibility of tissue engineering of cartilage by seeding human tracheal chondrocytes (HTC) onto a 3-D polyesterurethane polymer (DegraPol) scaffold. Methods Human tracheal chondrocytes were harvested from the trachea of 9 lung donors, digested in 0.3% collagenase II, and serially passaged every 7-9 days. Then the sixth to eighth passage cells were seeded onto DegraPol scaffolds and cultivated in vitro. The proliferation of the HCT was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylter-trazolium bromide (MTT) assay at 2 hours, and 3, 7, 21, and 42 days. Three weeks after the cultivation, the secretion of chondroitin sulfate in the chondrocyte-DegraPol constructs was detected using alcian blue staining, and the ultrastructure of the constructs was observed under an electron microscope. Results The HTC proliferated stably in monolayer culture with positive expression of collagen type II. The A values at 2 hours, and 3, 7, 21, and 42 days were 0.112±0.004、0.151±0.021、0.170±0.035、0.176±0.023 and 0.213±0.023, respectively (n=4 for each time point). The number of active HTC at 21 and 42 days was significantly higher than that at 2 hours (P<0.05). The alcian blue staining showed that the matrix of the chondrocyte-DegraPol constructs secreted chondroitin sulfate, and the constructs had a cartilage-like structure. Electron microscopy showed that cartilages generated both outside and inside the scaffold, and most of them distributed on the surface of the DegraPol. Conclusions The chondrocytes harvested from the human tracheal cartilage can be amplified well in vitro and form cartilage-like tissues on a DegraPol matrix. However, the culture conditions should be improved for cellular growth throughout a cell-polymer construct.

关 键 词：组织工程 器官培养技术 软骨细胞 气管 

分 类 号：R318.1[医药卫生—生物医学工程]