Differences Of Rat Adipose Derived Stromal Cells Osteogenesis In Different Induction Time

OBJECTIVE: To observe the effect of induction time selection on the differentiation of rat adipose derived stromal cells(ADSCs)into osteoblasts.METHODS: The adipose derived stromal cells(ADSCs)come from Sprague-Dawley rat,without sex restriction,weighing 150-200 g(Offered by the Experimental Animal Center of Shanxi Medical University).Take fat from the groin.ADSCs were isolated, purified, and they were incubated.The passage 3 adipose derived stromal cells can be used for experiment.Groups:the induced group and the non-induced group.The induced group is divided intoA,B,C three groups.The induced group was added into conditioned medium containing 1×10-8 mol/L dexamethasone,10 mmol/L beta-sodium glycerophosphate and 50 mg/L ascorbic acid at the same time of seeding(A),at 50%-60%confluence(B),at 85%-90%confluence(C),respectively.The A, B, C group of non-induced group was cultured in ordinary medium.Calcium-cobalt staining,Alizarin red staining and alkaline phosphatase activity detection were conducted to evaluate osteogenesis in 7th ,14th ,21th and 28th day after induction.Observe the growth condition and appearance changes of ADSCs by the inverted microscope.RESULT:1.Under the same initial seeding density, growth rate of induced group is lightly slower than non-induced group,s on the same time.2.After calcium-cobalt staining,a majority of cells in induced group were positively staine,there are some cell particles stained from light brown to brownish-black in cytoplasm. Under the same initial seeding density and on the same time , the quantity of positive cells change more while the confluence is higher.And the quantity of positive cells reached the peak at 85%-95% confluence.The non-induced group has no positive cells.3.Red nodules were seen after alizarin red stain. Some litter discrete red nodes appeared on the 7th day in the induced group,then the larger and more one formed on the 28th day.At 85%-95%confluence,nodes formed more early in induced group and the quantity was larger.There is no obvious node formed in the non-induced group. 4.Intracellular alkaline phosphatase activity detection showed that intracellular alkaline phosphatase activity markedly increased after inducing by osteogenic inducer than non-induced group.CONCLUSION:1. ADSCs can differentiate into osteoblasts after induction.2.Under the same initial seeding density,the higher the ADSCs contact with each other the stronger the osteogenesis is.