关键词: 骨形态发生蛋白7, 钽, 软骨细胞, 胶原Ⅱ型, 葡糖氨基聚糖类

Abstract: Objective： To study the influence of bone morphogenetic protein-7 (BMP-7) on chondrocyte secretion and expression of type Ⅱ collagen (Col-Ⅱ), aggrecan (AGG) and SRY-related high mobility group-box gene 9 (Sox9) mRNA in porous tantalum-chondrocyte composites. Methods: The articular chondrocytes were isolated from 3-week-old New Zealand immature rabbits and identified. The 2nd generation of chondrocytes with 1×10⁶/mL inoculate concentration was seeded in porous tantalum and divided into 4 groups, and control group (tantalum/chondrocyte), 50 μg/L BMP-7 group (50 μg/L BMP-7/tantalum/chondrocyte), 100 μg/L BMP-7 group (100 μg/L BMP-7/tantalum/chondrocyte), and 200 μg/L BMP-7 group (200 μg/L BMP-7/tantalum/chondrocyte). The proliferation of chondrocytes was measured by CCK-8 assay. The chondrocyte growth and morphology were observed by scanning electron microscopy (SEM). The synthesis of glycosaminoglycan (GAG) in chondrocytes was tested by dimethyl methylene blue (DMMB) colorimetric quantification method. Col-Ⅱ, AGG and Sox9 mRNA in chondrocytes were detected by realtime PCR. Results: The chondrocytes were spindle--shaped in 24 hours of primary cell culture and most cells became polygonal shaped in 4 days. The chondrocytes were affirmed by alcian blue, safranin O and Col-Ⅱ immunocytochemistry staining. The result of CCK-8 assay showed that the level of cell proliferation in 100 μg/L BMP-7 groups were higher than those in the other groups (P<0.05). The chondrocytes implanted into porous tantalum scaffolds with BMP-7 had better functions, by which cytoplasmic processes developed and extended to the surface and inner of porous tantalum by SEM observation. DMMB quantitative determination of GAG showed that GAG amount of chondrocytes in 100 μg/L BMP-7 groups was significantly higher than those in the other groups (P<0.05). The expressions of Col-Ⅱ, AGG and Sox9 mRNA in chondrocytes were up-regulated in the experimental groups, compared with the control group and the best effect appeared when concentration of BMP-7 was 200 μg/L. (P<0.05). Conclusion: BMP-7/tantalum/chondrocytes composites enhanced in vitro chondrocyte proliferation and extracellular matrix greatly, and can promote chondrogenic gene expression.

Key words: Bone morphogenetic protein 7, Tantalum, Chondrocytes, Collagen type Ⅱ, Glycosaminoglycans