胶原-透明质酸支架的制备及其与软骨细胞复合培养的实验研究

目的制备胶原-透明质酸支架,评价其与兔髁状突软骨细胞的生物相容性,探讨其应用于关节软骨组织工程的可行性。方法冷冻干燥法制备胶原-透明质酸复合多孔海绵支架材料,将其与碳化二亚胺1-ethyl-3-（3-dimethyl aminopropyl）carbodiimide,EDC]进行化学交联。分别采用体积法和体外酶解实验测定支架材料孔隙率和降解率,扫描电镜观察交联前后支架材料的形态变化。取3周龄新西兰兔髁状突软骨细胞,体外培养5d后,甲苯胺蓝染色,倒置相差显微镜下观察行细胞鉴定。取消化后第2代软骨细胞接种至支架材料复合培养,倒置相差显微镜下观察细胞生长情况。复合培养1、3、5、7和10d后,PBS液清洗3次,置入24孔板并以0.25%胰酶和0.1%EDTA消化细胞,采集细胞进行细胞计数并绘制细胞生长曲线。另取部分样本继续培养5d,行组织学和扫描电镜观察。结果胶原-透明质酸支架材料经化学交联后,具有合适的三维多孔结构,孔隙率为83.7%,孔径100-120μm;交联后抗酶解能力显著增加。髁状突软骨细胞在其表面和内部贴附良好,形成细胞-材料复合体,细胞增殖实验显示,复合培养1d,材料中细胞数为3.7×104/支架,10d后增至8.2×104/支架。电镜观察见细胞周围有基质分泌。结论EDC交联后的胶原-透明质酸支架具有良好空间结构和生物相容性,可作为支架材料用于髁状突软骨组织工程的研究。

FABRICATION OF COLLAGEN/SODIUM HYALURONATE SCAFFOLD AND ITS BIOLOGICAL CHARACTERISTICS FOR CARTILAGE TISSUE ENGINEERING

OBJECTIVE: To develop a scaffold material containing collagen 1 and sodium hyaluronate for the cartilage tissue engineering and to evaluate its biocompatibility by using the rabbit chondrocytes derived from a mandibular condylar process. METHODS: The porous matrices containing collagen 1 and sodium hyaluronate were fabricated by the freeze-drying technique and were crosslinked by using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC). The microstructure of the scaffold was observed under the scanning electron microscope (SEM), and the enzymatic degradation test was performed to compare the ability of the scaffold resistance to collagenase before and after the crosslinking. The chondrocytes from the rabbits' condylar process were isolated and cultured before they were seeded into the scaffold, and cell attachment and proliferation were measured by the cell count 1, 3, 5, 7 and 10 days after the cell being seeded; then, the biocompatibility of the scaffold was evaluated by the light microscopic examination, histological examination, and the SEM exmination. RESULTS: The porous structure of the scaffold facilitated the penetration and attachment of the seeded cells. The porosity was 83.7% and the pore size was 100-120 microm. The cell number increased from 3.7 x 10(4) per scaffold 1 day after the cell being seeded to 8.2 x 10(4) per scaffold 10 days after the cell being seeded. The crosslinking treatment could significantly enhance the scaffold resistance to the collagenase activity. The examinations under the light microscope and SEM indicated that the chondrocyte adhered and spread well on the scaffold, and the extracellular matrices were also observed around the chondrocytes. CONCLUSION: The porous scaffold composed of collagen I and hyaluronan has an appropriate structure and a good biocompatibility for the attachment and proliferation of the chondrocytes, which can facilitate it to become a useful scaffold in the cartilage tissue engineering.