修饰后几丁质作为组织工程技术中细胞载体的应用研究

Chitosan's cationic nature also allows electrosatic interaction with anionic glycosaminoglycans, gelatin and proteoglycans. In this study , Chitosan and Chitosan-gelatin complex scaffold samples were made by freezing and lyophilizing in pre-cooled glass dishes. In vitro experiment chondrocytes isolated from Changfeng crossbred porcines' ears were seeded onto chitosan and chitosan- gelatin complex scaffolds for culture in a three dimensional environment .The scaffolds of hydrophilia and adhesion to chondrocytes were observed with light microscope and scanning electron microscope. The relatively growth rate of the chondrocytes on the scaffolds were detected with MTT automated colormetric microassay .In vivo study the chondrocyte- scaffold constructs were culture in a three dimensional for seven days. Then the chondrocyte- scaffold constructs were implanted into the subcutaneous of the porcines ' abdomens. Specimens were harvested and analyzed by gross observation, histology ，type II collagen immunohistochemistry , proteoglycans levels, type II collagen mRNA and biomechanical analysis after 10 and 16 weeks in vivo respectively. The results found that chitosan-gelatin complex scaffolds have good biocompatible with chondrocytes. Chondrocytes can multiple and secrete the matrix on the porous chitosan- gelatin scaffolds. The relatively growth rate of the chondrocytes on the chitosan-gelatin complex scaffolds is above 90 percent. H.E staining showed formation of a typical cartilage, and chondrocytes are enclosed in lacuna with similar histological characteristics to natural cartilage after 16 weeks in vivo. The elasticity fibres of engineered cartilage were observed by Vehoeff's staining. Immunohistochemical staining of the neocartilage with anti- type II collagen showed the presence of type II collagen in the ECM of tissue engineered cartilage. RT-PCR detected the total RNA of engineered cartilage showing the present of II collagen mRNA. In addition, the proteoglycans levels in tissue engineered cartilage reached 90% of that founded in native auricular cartilage. Biomechanical analysis of the tissue engineered cartilage harvested at 16 weeks, the loading forces reached 85% of native auricular cartilage. The experiments demonstrated that using isolated autologous chondrocytes from porcine 's auricular cartilage and chitosan-gelatin complex scaffold, we have successfully in vivo-engineered elastic cartilage under the subcutaneous of the porcine ' abdomens. Thus ,the results of the experiment showed that chitosan-gelatin complex scaffolds may be a suitable scaffolds for tissue engineered cartilage.

经鼠尾胶原、卵磷脂及多聚赖氨酸包埋修饰的几丁质与软骨细胞体外复合培养，通过光镜及扫描电镜评价软骨细胞的吸附、生长繁殖及功能发挥情况；将经Br dU标记的软骨细胞与上鲂奘渭付≈始把趸冶┗旌虾蠡刂驳街砀共科は拢ü筇濉染色、特殊染色、免疫榛⑸锪ρЪ胺肿由镅Ф孕纬傻淖橹衅兰郏赴靥宓难芯靠僖惶跣峦揪丁