Experimental Study Of Chondrogenic Differentiation Of Adipose Derived Stem Cells Of Rat In Different Concentrations Of Gentamicin In Vitro

Objective:On clinical treatment of cartilage defect is often accompanied by the use of antibiotics.gentamicin is a broad spectrum of powerful antibiotics and commonly topical used in clinical work, often get a good efficacy.this experimental study explore the capability of adipose tissue-derived stem cells of rat differentiating into Chondrocytes in vitro. To study the effect of different concentrations (0μg/ml,10μg/ml,50μg/ml,100μg/ml,200μg/ml) of gentamicin on proliferation and chondrogenic differentiation of adipose tissue-derived stem cells of rat in vitro.Methods:Adopting three-weeks old male Sprague-Dawley rat to isolating adipose tissue from rat inguinal fat pads.Then digesed with collagenase-I and placed in Dulbeco Modified Eagle Medium (DMEM) with fetal bovine serum in primary cultivation.observed the growth and appearance of the cells by a inversion microscope.The third passage of ADSCs were divided into 5 groups:one control group(containing human adipose stem cells complete medium include H-DMEM serum medium> 10%fetal bovine serum 50nmol/L vitamin C,6.25mg/L insulin,100U/ml Penicillin,100μg/ml Streptomycin,100nmol/L Dexamethasone,10μg/L transforming growth factor (TGF)-β1,without any gentamicin) and four experimental groups(containing human adipose derived stem cells complete medium+four different concentrations of gentamicin:10μg/ml,50μg/ml,100μg/ml,200μg/ml).The cell proliferation was assessed by MTT methods, toluidine blue stain and typeⅡcollagen immunohistochemistry were applied for chondrogenic identificatio.Results:1. ADSCs cultured in vitro were in flat fusiform,fibroblast-like cells shape and stably passaged, during which ADSCs kept the same shape.2.MTT experiments showed that gentamicin at 100μg/ml,200μg/ ml inhibited cell proliferation, the remaining concentrations had no side effect on cell proliferation.3.the toluidine blue staining with the cells after chondrogenic differentiation shows:200μg/ml and 100μg/ml groups lots of cell out of stain, the other groups express positive, typeⅡcollagen immunohistochemistry staining shows:200μg/ml and 100μg/ml groups weak positive Expression, the other groups express positive.Conclusions:1.ADSCs readily available, grown rapidly and stable after culture and passage;2.ADSCs have the potential to differentiate into chondrogenic lineage;3.gentamicin in lower concentrations(10μg/ml,50μg/ml)shows no side effect on ADSCs proliferation and differentiate into chondrogenic, gentamicin in higher concentrations(100μg/ml,200μg/ml)inhibited the ADSCs proliferation and differentiate into chondrogenic.