| OBJECTIVETo investigate a suitable vector of tissue engineering through binding calcium alginate hydrogel with transforming growth factor beta 1(TGF-β1),which can promote the chondrocytes' proliferation,and to understand the role of TGF-β1 played in the differentiation of in vitro cultured adipose-derived stem cells in calcium alginate hydrogel into chondrocytes,thus providing experimental references for the repair of articular cartilage defects.EXPERIMENTAL METHODSUnder sterile condition,10mL adipose tissues harvested from bilateral inguinal groove were cut into several pieces(about 1 mm3) and then digested using 0.1% collagenase and 0.25%pancreatin,shaking 15 minutes at 37℃.Basic medium was added to terminate digestion process,followed by the centrifugation and supernatant removal.Then cells were resuspended with basic medium and inoculated in the culture flask with 5%CO2 saturated humidity at 37℃.The culture media were renewed 20 hours later,and then changed every 3 days.Cells were subcultured when they reached an 80%-90%confluence at 7-10 days.Adipose-derived stem cells at passage 4 were selected,digested and resuspended with basic medium.Calcium alginate hydrogel (Matrixkit) were located on the 6-well plate and rinsed with complete culture medium three times;Using sterile syringe,cell suspension was added dropwise onto the surface concave of calcium alginate hydrogel,5×106/ml,and were cultured with 10% FBS-DMEM complete medium containing 5%CO2 at 37℃.Half of basic medium was exchanged to induced medium(50mg/L TGF-β1),and completely changed to induced medium 24 hours later.Afterwards the culture medium was renewed every 3 days.EXPERIMENTAL RESULTS 1.Cellular proliferation detectionThe cells began to proliferate following the inoculation,MTT value was shown to be increasing with the time going2.microscopic observationsFollowing the inoculation of cellular suspension,the cells were round and adhered on the vector.Along with the time prolonging,the cells gradually covered the whole gap and were embedded in the matrix cells secreted.There were tight binds between cells and between cells and vector.3.Hematoxylin-eosin(HE) stainHE stain results have showed that a large amount of extracellular matrices were produced at 3 weeks of the culture,they were closely connected.4.Electron microscopic observationCalcium alginate hydrogel has many reticular pores,which is beneficial for the substance-energy exchange and accordingly promote the cellular growth,as well as for the metabolite excretion.Cells have shown to adhere on calcium alginate hydrogel with extension and good growth.In addition,cells were embedded in the matrices cell secreted and were closely connected with each other.5.TypeⅡcollagen immunohistochemistry detectionImmunohistochemical results have demonstrated that the cell-vector compound was positive for typeⅡcollagen.Conclusion1.Calcium alginate hydrogel can produce a three-dimensional culture for cells,its great surface area and many inner pores both fit the cellular adhesion and nutrition exchange,also benefit the cells adhesion,growth and proliferation.2.TGF-β1 can promote the in vitro culture of chondrocytes and the differentiation of adipose-derived stem cells into chondrocytes on calcium alginate hydrogel vector. |
