壳聚糖-明胶-卡拉胶支架复合人牙龈成纤维细胞的体外研究

Properties of human gingival fibroblasts cultured on chitosan-gelatin-carrageenan scaffold in vitro

目的:研究人牙龈成纤维细胞(human gingival fibroblast cells,HGFs)在壳聚糖-明胶、壳聚糖-明胶-卡拉胶两种支架材料上生长增殖及某些细胞外基质分泌的情况,为研究组织工程化牙龈支架材料提供实验依据。方法:组织块法培养HGFs,将第4代的HGFs分别接种于两种支架材料上,以壳聚糖-明胶-卡拉胶为实验组,壳聚糖-明胶作为对照组。扫描电镜(SEM)观察两种支架材料的结构;四唑盐比色实验(MTT)法检测HGFs在两种支架材料上的增殖情况;通过酶联免疫吸附法(ELISA)检测细胞-支架复合培养液中I型胶原蛋白(Collagen I,Col I)和糖胺多糖(Glycosamlnoglycans,GAG)的含量。结果:扫描电镜显示两种支架材料均呈多孔结构,孔径50～200μm,孔隙率达90%。MTT法检测显示:培养1 d实验组与对照组吸光值(A值)差异无统计学意义(P>0.05);第3、5、7天时实验组A值明显高于对照组,差异有统计学意义(P<0.05)。ELISA结果显示,培养1 d实验组Col I和GAG的含量与对照组相比差异无统计学意义(P>0.05);第3、5、7天各时间点,实验组Col I和GAG的含量均明显高于对照组,差异有统计学意义(P<0.05)。结论:HGFs与壳聚糖-明胶-卡拉胶支架复合后,其生物学行为更为活跃,壳聚糖-明胶-卡拉胶支架比壳聚糖-明胶支架更适合作为组织工程化牙龈的生物支架材料。

AIM： To investigate the proliferation and extracellular matrixs secretion properties of human gin- gival fibroblasts （HGFs） seeded on chitosan-gelatin and chitosan-gelatin-carrageenan as scaffolds. METHODS： The fourth generation of HGFs was seeded on chitosan-gelatin-carrageenan scaffold （experimental group） and chitosan-gelat- in scaffold （control group）. The structure of the scaffolds was observed by scanning electron microscopy. HGF prolifera- tion was assessed with MTT assay and Col Ⅰ and GAG secretions in the culture supernatant were measured by ELISA. RESULTS： Both scaffolds had porous structure with an average pore diameter between 50μm- 200μm, the porosity of both scaffolds was around 90%. MTT assay showed that the number of HGFs of the experimental group was not signifi- cantly different from that of the control group after 1 day （ P 〉 0.05 ）. But there was significant difference between the two groups after 3, 5 and 7 days （P 〈 0.05）. No significant difference on Col Ⅰ and GAG secretion was recorded between the two scaffolds after a 1-day culture （P 〉 0. 05）. However, Col Ⅰ and GAG secretions of chitosan-gelatin-carrageenan group were significantly higher than those of the control scaffold when cultured for 3, 5 and 7 days （P 〈 0.05）. CONCLUSION： HGFs seeded on chitosan-gelatin-carrageenan scaffold is biologically more active. Chitosan-gelatin-carrag- eenan scaffold is more proper than chitosan-gelatin scaffold as a biological scaffold for tissue engineered gingiva.