气管软骨细胞和羧乙基壳聚糖-羟基磷灰石泡沫支架复合及裸鼠皮下植入

Complex of tracheal chondrocytes and N-carboxyethylchitosan/hydroxyapatite scaffold： A subcutaneous implantation experiment in nude mice

目的 探讨以兔气管软骨细胞为种子细胞在自制羧乙基壳聚糖-羟基磷灰石泡沫（NCECS-HA）支架合成组织工程气管软骨的可行性.方法 通过真空冷冻干燥法制得NCECS-HA泡沫支架.从6个月大的大耳白兔取气管软骨片段,Ⅱ型胶原酶消化,将所获得第3代软骨细胞种植于NCECS-HA三维支架上.细胞-支架复合物在24孔板中培养5 d以后,将其植入裸鼠皮下8周.然后取出分别进行HE染色、Ⅱ型胶原免疫组化染色和甲苯胺蓝染色,观察软骨细胞基质分泌情况.结果 8周后,构建出组织工程气管软骨示光泽良好,甲苯胺蓝染色、Ⅱ型胶原免疫组化染色显示细胞-支架复合物中的软骨细胞可以像天然软骨一样分泌糖氨多糖和Ⅱ型胶原.结论 生物材料NCECS-HA对于兔软骨细胞有良好的生物相容性,可作为生物组织工程支架.

Objective To investigate the feasibility of tissue- engineered tracheal cartilage generated from rabbit tracheal chondrocytes using N- carboxyethylchitosan/hydroxyapatite （NCECS/HA）scaffold. Methods The NCECS/HA foam scaffold was prepared by vacuum freeze-drying. Samples of rabbit tracheal cartilage were obtained from a 6-month-old rabbit, and digested with 0.2% collagenase type Ⅱ. The third-passage chondrocytes were seeded onto the three-dimensional NCECS/HA scaffold. After 5 days of culture in 24-well plates, the tissue engineered cartilages were placed subcutaneously in a nude rat, and was extracted after 8 weeks. HE staining, collagenase type Ⅱ immunohistostaining and toluidine blue staining of the extracted cartilage were respectively performed, so as to observe the matrixes secreted on the scaffold.Results Eight weeks later, the tissue engineered tracheal cartilage showed a pearly opalescence. Toluidine blue staining and collagenase type Ⅱ staining revealed that the chondrocytes from cell-stent compounds could secrete GAG and type Ⅱ collagen as does natural tracheal cartilage. Conclusion Biomaterial NCECS/HA has a good biocompatibility with rabbit chondrocytes and may be used as bio-engineered stent.