Experimental Study Of The New Kind Of Tissue Engineering Cartilage Charged IGF-1&BMP-2

PurposeArticular cartilage is vulnerable to injury and has a limited capacity for self-repair. Experimental approaches toward treatment of damaged articular cartilage have increasingly focused on cell-based therapies. In this regard, Adipose Tissue-derived Stem Cells (ADSCs) provide an attractive alternative to mature chondrocytes that must be isolated from a very limited supply of healthy articular cartilage. ADSCs can be obtained relatively easily from fat tissue and have the capacity for differentiation into the cell types characteristic of various mesenchymal tissues, including cartilage and bone. Delivery of ADSCs to cartilaginous lesions has not yielded satisfactory regeneration of articular cartilage. One possible problem is that there is insufficient local stimulation of the implanted cells by the protein factors necessary to drive differentiation in vivo. Gene transfer might be adapted as a means to provide sustained synthesis of bioactive transgene products within cartilaginous lesions; the delivery of the appropriate stimulatory factors in this manner may enable synthesis of an improved cartilaginous repair tissue. The development of in vitro systems of chondrogenesis has been important to the identification of protein factors that can promote chondrocyte differentiation of ADSCs and improved cartilage repair in vivo. Related studies have been useful to the elucidation of the chondrogenic potential of other growth factors, including TGF-β₁, TGF-β₂, TGF-β₃, Fibroblast growth factor (FGF), bone morphogenetic protein-2(BMP-2), BMP-6, and insulin-like growth factor-1 (IGF-1). Here, we report that delivery of IGF-1 &BMP-2 in order can induce chondrogenesis of ADSCs in aggregate culture.Materials and methods1. Study of the isolation culture and biological characteristics of rabbit Adipose Tissue-derived Stem Cells.a. Fat tissue aspirate of New Zealand white rabbits was digested by typeâ… collagenase and cultured in plastic culture bottles.b. neuron-differentiation of rabbit adipose mesenchymal stem cells in vitro by GM-1 and mercaptoethanolc. The cells were examined by invert microscope, the growth curve was drawn and flow cytometry.2. The effect of Insulinlike Growth Factor-1 and Bone Morphogenetic Protein-2 gene in order transfer on the proliferation of rabbit Adipose Tissue-derived Stem Cells and its chondrocyte differentiation potential.a. Amplification and purification of plasmid DNA of pcDNA3.1+IGF-1.b. Amplification and purification of Ad-vector Ad-BMP-2.c. ADSCs was transfected by IGF-1 gene and its identification.d. ADSCs with IGF-1 gene was transfected by BMP-2 gene as follow.e. The cells were examined by invert microscope and the Identification of chondro-nodus.f. Drawing the ADSCs growth curve which charged IGF-1 and IGF-I&BMP-2.g. Collagenâ…¡were analysied by Western blot and immunofluorescence.h. MMP-3 was analysied by Western blot.i. Ultramicrostructure of ADSCs was observed by transmission electron microscope.3. Construction the 3D tissue engineering cartilage with biological complex bracket and ADSCs a. prepare the biological complex bracket with chitosan and gelatinum.b. Co-culture the bracket with ADSCs charged IGF-1&BMP-2.c. the recombination of bracket and ADSCs charged IGF-1&BMP-2 was observed by scanning electron microscopeResults1. Success of the isolation culture of rabbit Adipose Tissue-derived Stem Cells.a. After the digestion of collagenaseâ… , the obtained cell mostly is the circular mononuclear cell. ADSCs had a similar long-spindle morphology and tightly arrayed to grow, showed no morphological changes in passage cultivation.b. The induced rabbit adipose mesenchymal stem cells expressed the neuron-specific marker NSE.c. Growth curve showed the proliferation ability of ADSCs is higher than MSCs.2. The effect of Insulinlike Growth Factor-1 and Bone Morphogenetic Protein-2 gene in order transfer on the proliferation of rabbit Adipose Tissue-derived Stem Cells and its chondrocyte differentiation potential.a. Amplification and purification of plasmid DNA of pcDNA3.1-IGF-1 were success.b. Amplification and purification of Ad-BMP-2were success.c. stable transfection IGF-1 to ADSCs was success.d. chondro-nodus is show up after gene transfection.e. the ADSCs still have a high reproductive activity after gene transfection.g. ADSCs transfected with IGF-1 gene & BMP-2 showed stable expression of collagenâ…¡.h. ADSCs transfected with IGF-1 gene & BMP-2 showed the expression of MMP-3 decreased.i. Ultramicrostructure of ADSCs showed constructive metabolism active by transmission electron microscope. 3.Construction the 3D tissue engineering cartilage with biological complex bracket and ADSCsa. The biological complex bracket with chitosan and gelatinum was susscefully reconstruction.b. the cell has a fine condition on the bracketConclusion1. The combination of digestion of collagenaseâ… and adherent culture is an effective method to isolate ADSCs from fat tissue aspirate. ADSCs has higher proliferation ability and can be used in tissue engineering.2. ADSCs transfected with IGF-1&BMP-2 gene showed stable expression of collagenâ…¡.3. The biological complex bracket with chitosan and gelatinum is compatible well with ADSCs.