Osteogenic Differentiation Of Rat Adipose Derived Mesenchymal Stem Cells In Nanohydroxyapatite/collagen/L-poly Lactic Acid

Bone defects were serious problems for bone surgery. The main treatment methods, such as autograft,allograft,bone-grafting materials et al, couldn't completely meet the clinical needs. The bone tissue engineering provided novel methods for healing bong defects. It consisted of three phrases, namely the choosing and culture of seeding cells,placing the seeding cells in the scaffolds and implanting the compositesinto the body. According to the principle, we would study the ability ofthe rat adipose derived mesenchymal stem cells (ADMSCs) innanohydroxyapatite/collagen/L-poly lactic acid to construct bone tissue.Chapterâ… Experimental study of the isolation,cultureand biologicalcharacteristics of rat adipose derived mesenchymal stem cellsObjectives: To investigate methods for the isolation, culture andbiological characteristics of ADMSCs in order to provide seeding cellsfor bone tissue engineering.Methods: The rat inguinal adipose tissue was excised and digestedwith 0.075% collagenase, and then the cells were cultured in DMEM with10% new-born calf serum. The morphology of rat ADMSCs wasobserved under inserted microscope,light microscope with Gimmsastaining and transmission electron microscope. The seeding efficiency ofthe 3^rd passage was detected by cell counting. The growth curve of the3^rd,5^th and 10^th passage cells were drawn using MTT. The 5^th passagecells were examined using flow cytometric methods about CD antigens:29,44,34, the markers of the mesenchymal stem cells.Results: Primary cells began to adhere to the plate and appeared tri-angular or multiangular after 2 days and fibroblast-like in 4 to 5 days.They confluenced more than 90% after 7 to 9 days like a whirlpool andwholly covered the plate in 10 to 12 days. Observed under the lightmicroscope with Gimmsa staining, the cells appeared fibroblast-like withthe purple nucleolus and pink cytoplasm. Observed under thetransmission electron microscope, the nucleolus appeared large with relatively undeveloped organelles. The seeding efficiency of the 3^rd passage cells was 33%±2%,75%±1% and 92%±2% after being seeded 2,6 and 12 hours. The cells almost adhere to the plate in 24 hours. The growth curve showed that cells of the 3^rd,5^th and 10^th passage cells proliferated rapidly, there were no differences between them. Flow cytometry showed that 95% and 96% of the 5^th cells expressed CD29 and CD44 respectively, only 5% of the cells expressed CD34.Conclusions : The characteristics of cells obtained from rat adipose tissue were similar to the mesenchymal stem cells and could be a candidate for tissue engineering seeding cells.Chapterâ…¡Osteogenic differentiation of rat adipose derived mesenchymal stem cells in vitroObjectives: To study the osteogenic differentiation potential of rat ADMSCs in vitro and make preparations for further research about bone tissue construction in vivo.Methods: The 3^rd passage rat ADMSCs were cultured in DMEM supplemented with 10% new born calf serum,dexamethasone,ascorbic acid,β-glycerophosphat and vitamin D₃ to be induced into osteoblast. The morphology of cells after osteoinduction were observed under inserted microscope,light microscope with Gimmsa staining and transmission electron microscope. The Alkaline Phophatase staining was verified by Gomori staining and the levels of Alkaline Phophatase were measured. The formation of calcium nodules were confirmed by Alizarin red S and tetracycline fluorescence staining .The expression of bone gla protein mRNA was examined by RT-PCR. The secretion of collagen typeâ… was verified by immunocytochemistry staining and Western blotting.Results: After osteogenic induction, the cells became larger and cuboidal. Osteoinducted for 7 days, observed under light microscope with Gimmsa staining, the cells became larger with purple nucleolus and pink cytoplasm, while the nucleolus appeared large with relatively developed organelle observed under the transmission electron microscope. The Gomori staining appeared positive after osteogenic induction for 7 days and the positive ratio was 80%±2% after osteoinduction for 14 days. The level of Alkaline Phophatase gradually became higher in 14 days. The Alizarin red S and tetracycline fluorescence staining both appeared positive, which verified the formation of calcium nodules, when osteoinducted 21 days. The expression of bone gla protein mRNA showed positive at 280bp after osteogenic induction for 14 and 21 days. There was more expression for 21 days. The secretion of collagen typeâ… was positive with immunocytochemistry staining after osteoinduction for 7 and 14 days. The Western blotting verified the secretion was more for 14 days.Conclusions: The rat ADMSCs could be osteoinduced to osteoblast in vitro and be used to construct bone tissue.Chapterâ…¢Bone tissue construction in vivo from rat adipose derived mesenchymal stem cells in nanohydroxyapatite/ collagen/L-poly lactic acid scaffoldObjectives: To explore the bone tissue construction in vivo from rat ADMSCs in nanohydroxyapatite/collagen/L-poly lactic acid and make preparations for an animal model.Methods: The 3^rd passage rat ADMSCs were seeded and osteoinduced in nanohydroxyapatite/collagen/L-poly lactic acid in vitro to construct cells-scaffold composites, then the composites were implanted into rat back muscles for 4 weeks,8 weeks and 12 weeks. The composites and products were observed under light microscope with HE staining and scanning electron microscope. The cells were examined with BrdU immunocytochemistry staining.Results: The rat ADMSCs could adhere on the surfaces and grow into the holes of the scaffold. The bone tissue were successfully constructed for 4 weeks,8 weeks and 12 weeks. The BrdU immunocytochemistry staining was positive.Conclusions: The nanohydroxyapatite/collagen/ L-poly lactic acid was an ideal scaffold. The rat ADMSCs could be used to construct bone tissue in vivo.