Experimental Study On Chondrogenesis And HIF-1α Expression Of Rabbit Adipose-derived Mesenchymal Stem Cells By Dynamic Compression Combined With IGF-1 Gene Transfection

ObjectiveTo investigate the effects on HIF-1αexpression of rabbit adipose-derived mesenchymal stem cells by cyclically dynamic compression combined with IGF-1 gene transfection, and to explore the mechanism on promoting chondrogenesis by HIF-la.Methods1.The posterior subcutaneous adipose tissue of adult New Zealand white rabbits was digested with type I collagenase, and ADSCs were harvested。2.After being transfected with pcDNA3.1-IGF-1, RT-PCR was used to analyze the expression level of IGF-1 in pcDNA3.1-IGF-1-ADSCs.3.All cells were seeded onto chitosan/gelatin scaffolds with a density of 5×107 cells/ml, and divided into groups:Group A (control), non-transfected ADSCs; Group B (IGF-1), hIGF-1 gene transfected ADSCs; Group C (loading), untransfected ADSCs with stimulation of cyclically compressive loading; Group D (loading+IGF-1), hIGF-1 gene transfected ADSCs and loading stimulation. The dynamic compression was carried out with a bio-reactor (BIODYNAMIC TM5110, Bose Corporation) at a frequency of 0.1 Hz, and sinusoidal strain amplitudes of 2%(2% at 0.1 Hz). The dynamic load was performed every 20 minutes,4 hours per day for 7 days.4.Macroscopical observation, and to assess the compressive mechanical properties of cell/scaffold constructs.5.Scanning electron microscopy (SEM) was performed to evaluate the morphological appearance of ADSCs and the deposited matrix within the chitosan/gelatin scaffold. 6.The MTT assay was used to express the cell proliferation, meanwhile the Vybrant(?) CM-Dil cell-labeling solution was used to observe the proliferation and distribution of the cell.7.The total amount of GAG was evaluated using a ((1,9-dimethyl) methylene blue) DMMB assay after papain digestion of the constructs.8.The expression of related genes, IGF-1, HIF-1α, typeⅡcollagen (COLⅡ) and Sox-9, was quantified by sybr-green real time PCR.Results1.The ADSCs digested with typeⅠcollagenase was isolated successfully. The cells had a similarly long-spindle morphology, and showed no morphological changes in passage cultivation.2.The stable expression of pcDNA3.1-IGF-1 cell lines were established successfully, and showed stable expression of IGF-1 in mRNA level.3.The surfaces of the cell/scaffold constructs were smooth and shiny, and had a good elasticity in group D. Comparison of elastic modulus:Group A