| Objective By compare the difference of secretory function and related genes expressionof porous tantalum-chondrocytes co-cultured in different concentrations of BMP-7,toexplore the feasibility of porous tantalum-chondrocytes co-cultured to construct tissueengineered cartilage,provide experimental evidence to cartilage defects of cartilage repairtissue engineering.Methods1Cultured chondrocytes and groups:3-week-old infant rabbits,take the kneecartilage primary culture,good condition with third-generation cellular concentration of1×106/ml cells were seeded in porous tantalum metal,divided into four groups:①controlgroup (cartilage cells co-cultured with porous tantalum);②50ng/ml group (cartilage cellsco-cultured with porous tantalum metal, adding rhBMP-7in the medium concentration of50ng/ml);③100ng/ml group (cartilage cells co-cultured with porous tantalum metal,adding rhBMP-7in the medium concentration of100ng/ml);④200ng/ml group (cartilagecells co-cultured with porous tantalum,adding rhBMP-7concentration of200ng/ml).2Cartilage cells identified:Alcian blue staining, safranin-O staining, Line type II collagenimmunocytochemistry.3CCK-8assay with different concentrations of BMP-7poroustantalum-chondrocyte growth and proliferation.4Scanning electron microscopy ofchondrocytes in each group porous tantalum growth.5Dimethyl methylene blue (DMMB)colorimetric quantification method of porous tantalum-chondrocyte glycosaminoglycanfor quantitative analysis.6Type II collagen and Sox9immunocytochemistry staining andstatistical analysis of its results.7Real Time-PCR detection of COL-II, AGG and Sox9mRNAexpression.Results1Chondrocytes morphological characteristics and identification:3rd chondrocytesseeded6~8h after the completion of the majority of adherent cells,24h most of the cellswere short spindle,triangular growth;3d,the number of cells increased significantly.Alcian Blue staining, Safranin-O staining, type II collagen immunohistochemical stainingshow have positive results.2CCK-8method to detect BMP-7porous tantalum-cartilagecell growth proliferation.BMP-7can promote the proliferation of porous tantalum-cartilage cells,and100ng/ml group have the best effection,but the proliferation of 200ng/ml was slightly inhibited.3Scanning electron microscopy of cartilage cell growthin each group,chondrocytes stretch on the surface of the porous tantalum,a large numberof cells connected and extracellular matrix secreted by mutual into a film,laminatedgrowth,covering the porous tantalum surface.4Dimethyl methylene blue (DMMB)colorimetric quantification of glycosaminoglycan(GAG) quantitative detection:5d and14d,50ng/ml and100ng/ml the GAG secretion of chondrocytes group higher than thecontrol group, and100ng/ml group GAG content group and the control group comparedwith a statistically significant difference, and following the extension of incubation time,the secretion of GAG increased obviously.5Col-II and Sox9immunocytochemistryresults: IOD value of Col-II,Sox9expression of100ng/ml are the largest, which indicatesthat100ng/ml group secrete Col-II the most quantity.6Real Time-PCR detection COL-II,AGG and Sox9mRNA expression of chondrocytes:In the group of50ng/ml,100ng/mlgroup and200ng/ml group,the relative expression of Col-II, AGG and Sox9mRNAamount compared with the control group were statistically significant (P<0.05), and withthe increase of the concentration of BMP-7mRNA expression is also a correspondingrise. In the14d, the COL-II, AGG and Sox9mRNAexpression decreased relative amountwhen compared to the first5d.Conclusion1BMP-7could promote the proliferation of porous tantalum compositecultured cartilage cells and concentration of100ng/ml BMP-7proliferation best.2BMP-7promote porous tantalum-chondrocytes secrete COL-II,AGG,in which the concentrationof100ng/ml of BMP-7is the optimal concentration.3BMP-7could promote theexpression of porous tantalum-chondrocyte gene function COL-II, AGG and Sox9, andwith increasing concentrations of upregulation enhanced. |
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