软骨细胞与静电纺丝聚已内酯支架灌流培养构建组织工程软骨的实验研究

目的 采用静电纺丝聚已内酯(polycaprolactone,PCL)支架与软骨细胞复合培养,比较静态和灌流生物反应器培养条件下对细胞增殖及基质分泌的影响.方法 构建PCL支架,自制灌流生物反应器,分离兔软骨细胞,培养后接种于PCL支架,分为灌流培养组和静态培养组.在培养第3、7、14天对支架-细胞复合体行扫描电镜观察,DNA、糖胺聚糖和总胶原定量检测；在培养第14天分析软骨特异性基因表达并观察软骨基质分泌情况.结果 电镜观察PCL支架纤维直径(1.67±0.76) μm,孔径(17.65土7.11)μm,可见支架中软骨细胞黏附生长良好,灌流培养条件下细胞增殖快,且较好地保持了软骨细胞特征形态.在培养第7天,灌流培养组DNA定量高于静态培养组；在培养第3、7和14天,灌流培养组糖胺聚糖定量均高于静态培养组,灌流培养组糖胺聚糖/DNA比值均高于静态培养组.在培养第14天,灌流培养组Ⅱ型胶原、蛋白聚糖基因表达增加；软骨分化指数高于静态培养组.在培养第14天,组织学染色可见灌流培养促进细胞的增殖和渗透生长,提高了软骨基质的分泌,并见软骨陷窝样结构.结论 在灌流生物反应器培养条件下,静电纺丝PCL支架与软骨细胞复合培养可促进软骨细胞的增殖和基质的分泌,提高了组织工程软骨的质量.

Cartilage tissue engineering by electrospun PCL scaffolds seeded with rabbit chondrocytes under flow perfusion culture in vitro

Objective To investigate the chondrocyte proliferation and extracellular matrix biosynthesis of electrospun PCL scaffolds seeded with rabbit chondrocytes under flow perfusion culture in vitro. Methods Nonwoven PCL microfiber mats were fabricated, and contra-aperture cylindrical glass equipment as a perfusion bioreactor was designed and manufactured on our own. The experiment included perfusion culture group and static culture group. Primary chondrocytes were isolated from the knee joints of two-month-old New Zealand white rabbits and seeded into scaffolds. The scaffold-cell complexes were harvested at 3, 7, and 14 days of culture for scanning electron micrograph (SEM) analysis, biochemical assay, real-time PCR and histology analysis. Results Electrospun PCL scaffolds were composed of microfibers with a diameter of 1.67±0.76 μm and pores with a diameter of 17.65±7.11 μm. SEM showed a better cell proliferation with typical morphology of chondrocytes under perfusion culture. At 7 days of culture, DNA content in perfusion culture group was higher than in static culture group. At 3, 7 and 14 days of culture, compared with the static culture group, glycosaminoglycan (GAG) content and GAG/DNA ratio in perfusion culture group were higher, and the differences were statistically significant. At 14 days of culture, real-time PCR showed aggrecan and collagen type II gene expression and collagen type II to collagen type I ratio were higher in perfusion culture group than in static culture group; HE and safranin O staining showed a significant cell proliferation, infiltration, as well as extracellular matrix biosynthesis in perfusion culture group. Conclusion Under flow perfusion culture, the electrospun PCL scaffolds seeded with rabbit chondrocytes can enhance chondrocyte proliferation and extracellular matrix biosynthesis, which is a promising method for cartilage tissue engineering.