摘要
目的探讨丝素蛋白支架复合脂肪干细胞体外构建组织工程软骨的可行性。方法以丝素蛋白为原料制作具有仿生取向微孔结构的支架，接种第3代兔脂肪干细胞，加入成软骨诱导液进行培养。CCK-8检测诱导液对细胞增殖的影响，组织学及Ⅱ型胶原免疫组化染色观察脂肪干细胞的基质分泌，Elisa定量检测蛋白多糖和Ⅱ型胶原分泌量，实时定量PCR检测软骨特定基因Ⅱ型胶原、蛋白多糖和Sox-9的表达水平，并检测支架的力学性能。结果扫描电镜下支架纵切面为平行排列的微管样结构，横切面为椭圆或圆形孔隙结构。脂肪干细胞在支架上黏附良好，随诱导时间延长分泌大量取向分布的细胞外基质。脂肪干细胞在支架上呈对数生长趋势，成软骨诱导组吸光度值在诱导第7天、第21天高于普通培养基组。组织学及免疫组化染色结果在诱导第21天均呈阳性。诱导第21天蛋白多糖含量[（15．89±1．64）μg／mg]和Ⅱ型胶原含量[（1．89±0．28）μg／ms]高于诱导第7天[（5．02±0．91）μg／mg和（0．28±0．08）μg／mg]，第7天高于第0天[（0．77±0．25）μg／mg和（O．12±0．05）μg／ms）]。Ⅱ型胶原、蛋白聚糖、Sox-9的基因表达水平在第21天均高于第0天和第7天。第21天支架纵向压缩弹性模量为（84．41±7．12）kPa，高于第0天的（52．48±5，78）kPa和第7天的（59．30±6．43）kPa。结论取向性丝素蛋白支架接种成软骨诱导的脂肪干细胞能够在体外构建组织工程软骨，并能提高支架的力学强度。
To investigate the feasibility of fabricating an oriented scaffold combined with chondrogenic-in- duced adipose-derived stem ceils （ADSCs） for cartilage tissue engineering in vitro. Methods The silk fibroin scaffold with biomi- metic oriented microstructure was made by the directional crystallization technology. The structure of scaffold was observed by the SEM. Rabbit ADSCs of passage 3 were seeded into the scaffold, and induced by chondrogenic medium. The cell-scaffolds were detected by SEM and CCK-8, respectively. HE, toluidine blue, safranin O and type Ⅱ collagen immunohistochemical staining were also observed. The proteoglycan and type II collagen secretion was detected by Elisa method. The gene expression of type Ⅱ collagen, proteoglycans and Sox-9 was detected by the RT-PCR. Results SEM indicated that the parallel microtubule-like structure can be seen arranged in longitudinal section and the elliptical or circular pore structure in cross-section. The ADSCs uniformly ad- hered to the surface and inner pore, and secreted extracellular matrix distributed in the oriented microstructure. CCK-8 indicated the cell proliferation in induction group was higher than the general media group at the culture of 7 d and 21 d （P 〈0.05）. Safranin O, toluidine blue staining and type Ⅱ collagen immunohistochemical staining was positive at the induction of 21 d. Biochemical analysis indicated that proteoglycan and type Ⅱ collagen content at the induction of 21 d （15.89± 1.64 μg/mg, 1.89±0.28 μg/mg） was significantly higher than the content at 7 d （5.02±0.91 μg/mg, 0.28±0.08 μg/mg） and 0 d （0.77±0.25μg/mg, 0.12±0.05μg/mg）, the difference was statistically significant （P 〈0.05）. The levels of type collagen, proteoglycan, Sox-9 gene expression at the induction of 21 d were significantly higher than that of 7 d and 0 d, the difference was statistically significant （P 〈0.05）. The mechan- ical strength of the constructs of 21 d （84.41±7.12 kPa） was higher than that of 0 d （52.48±5.78 kPa） and 7 d （59.30±6.43 kPa）, the difference was statistically significant （P 〈0.05）. Conclusion The cartilage-like tissue can be constructed in vitro by oriented silk fibroin scaffold and chondrogenic-induced ADSCs, its mechanical property can also be improved.
出处
《中华骨科杂志》
CAS
CSCD
北大核心
2015年第12期1235-1242,共8页
Chinese Journal of Orthopaedics
基金
国家自然科学基金（31470937、31300798、81272046、81572154）
关键词
软骨
组织工程
丝素蛋白
干细胞
Cartilage
Tissue engineering
Silk
Stem cells
