皮质骨I型胶原的提取及胶原-明胶支架材料的制备

Extraction of type I collagen and preparation of a collagen-gelatin scaffold

[目的]探索一种较理想的皮质骨I型胶原的提取方法并制备胶原-明胶支架材料。[方法]以牛皮质骨为原料,经低温液氮粉碎、梯度脱水、脱脂、脱钙处理成为脱钙骨基质粉。通过酶溶解法处理脱钙骨基质粉,经溶解、离心、透析、冻干等步骤获得皮质骨胶原,SDS-PAGE电泳、氨基酸分析等方法鉴定其特征;再以胶原、明胶通过冷冻干燥技术制备神经支架材料,扫描电镜观察其内部结构。[结果]所得胶原经SDS-PAGE电泳、氨基酸分析等方法鉴定符合I型胶原特征;制备的支架材料外观呈圆柱状,内部为孔径均匀平行排列的微管结构,具有仿生效果。[结论]皮质骨中能够获得纯度较高的I型胶原,可用于胶原材料的制作。

[ Objective] To develop an ideal method for extracting type Ⅰ collagen from cortical bone and to prepare a collagen-gelatin scaffold. [ Method] The cortical bone was disintegrated into bone matrix powder in a high speed mill and was subsequently dehydrated in alcohol, decalcificated in hydrochloric acid and defatted in chloroform： methanol （ 1 ： 1, v/v）. The osscins were extracted using improved pepsin digestion method after the bone matrix powder was dissolved, centrifuged, dialyzed and lyophilized. Type Ⅰ collagen was then characterized by SDS-PAGE and amino-acid composition analysis. The biomaterial was made of type Ⅰcollagen and gelatin using freeze-drying method, and the alignment regularities of microscopic channels and their course directions were observed under the scanning electronic microscope. The size of the micropores and the factor of porosity were also measured. [ Result] The collagen extracted was confirmed to be type Ⅰ collagen by SDS-PAGE and amino-acid composition analysis. All the scaffolds looked like circular cylinder, the microscopic channels were arranged in parallel manners, and the pore sizes of the channels were uniform. [ Conclusion] Ossein extracted from cortical bone is a real type Ⅰ collagen that can be applied in the construction of collagen products.