The Study Of The ADSCs Differentiate To Chondrocyte Ablity Under Micromass Culture Condition Induced By BMP-7

ObjectiveCompare the ability of the adipose derive stem cells differentiate into cartilage cells under two ways culture plate or micromass in the presence of BMP-7 and explore the effect of different ways of cultivating for the stem cells differentiate into cartilage capacity.To find a better way of cultivating to promote the differentiation of adipose derive stem cells.MethodCell isolation and amplification Wistar rat adipose tissue was resected from the renal region of seven adult male rats,The obtained tissue was chopped into small pieces of about 1 mm~3,and the extracellular matrix was digested for 20 min at 37_C with 0.1%collagenase.terminate the digestion with Dulbecco's modified Eagle's medium(D-MEM,Gibco,Paisley,UK) supplemented with 10%fetal bovine serum The cell suspension was centrifuged at 1500r for 10 min,and the pellet was resuspended in culture medium,which was composed of Dulbecco's modified Eagle's medium(D-MEM,Gibco,Paisley,UK) supplemented with 10%fetal bovine serum The stromal cells were then plated in tissue culture flasks。The primary cells(P0) were cultured until fusion,after which they were harvested by treatment with trypsin (0.25%)/EDTA.the passages 4 cells were resuspended at concentration of 1×10~7/ml, and culture in the presence of BMP-7 by different ways plane or micromass.Relevant target detectionl inverted microscope was used to observe the primary cells and induced cells proliferation.The CD29,CD34 and CD44 CD45 expressions of the ADSC cells were detected by flow cytometry.2 to detection theproliferation ability of stem cells by MTT method.3 to identified theⅡcollagen protein expression by the immunohistochemical staining.after induced 14,21 days,to detect the cartilage-specific aggrecan and typeⅡcollagen mRNA expression.at the same time to detect the type X collagen mRNA expression which is a sign of hypertrophic chondrocytes.Result.The morphological characteristics of ADSC,It can be seen 24 hours after inoculation there is a small number of adherent cells,form spindle and polygonal short, after 48 hours the cells markedly increased about nine days and they are fusion more than 90%.。Induced by 7 days the plane culture group the cells were spindle.the micromass culture group was short spindle.21 days after induction the two groups are round cell morphology.Flow cytometry characterization of ADSCs.ADSCs were stained with surface markers CD29 express 80.2%,C34 express 43.2%,CD34,CD45 express less than 2%.MTT test results show that the trend of the proliferation of the plane culture group cell was flat.after 10 days enter the platform period.the MTT value of the Micromass culture group was higher than the group of plane.there were statistically significant between them.it is suggessed that Micro-group cells prolifration was significantly faster than the training group plane.and the Group of micromass culture has no obvious plateau.The finding of typeⅡimmunohistochemistry stain showed that cells express strong positive for typeⅡcollagen in the micromass culture system whereas it was weakly positive in plate culture system;The specific mRNA for cartilage typeⅡcollagen and aggrecan mRNA were expressed in two systems after culture 14,21 days by RT-PCR,but the micromass group is strong,typeⅩcollagen mRNA expressed weaker in the plate culture system than in the micromass culture system.ConclusionIn the presence of BMP-7 the adipose derived stem cell can differenciate to chondrocyte,The micromass group can promote the ADSCs proliferation and chondrogenic differentiation better than the plate culture systems and can maintain the phenotype of chondrocytes,it is the optimal choice for cartilage tissue enginerring.