Study On Construction Of Tissue-engineered Cartilage By AMSCs Combined With PHBV

Partâ… The isolation,cultivation and chondrogenic differentiation of human adipose-derived stem cells in vitroObjective To master the method of isolation and culture of human adipose-derived mesenchymal stem cells,and evaluate their capacity of proliferation and potential of chondrogenic differentiation in vitro.Methods Human subcutaneous adipose tissue obtained from liposuction,the cells were isolated by using 0.1%collagenase typeâ… , then the cells were cultured and expanded in DMEM with 10%new-born calf serum.Morphology of cells was observed with inverted microscopy. The CD29 and CD44 expression of the 6th passage cells were detected by flow cytometry.The 6th cells induced Chondrogenesis with DMEM containing of 1%new-bom calf serum,TGF-β1 10μg/L,Insulin 6.25mg/L, Transferrin 6.25mg/L,Dexamethasone 1×10^-7mol/L and ascorbic acid-2-phosphate 50mg/L.Chondrogenesis was assessed using Alcian blue staining,Toluidine blue staining,Safranin O/Fast Green staining, collagenâ…¡immunohistochemistry and Aggrecan immunofluorescence at 0,14 days.The mRNA expressions of Col2al was detected by RT-PCR and the protein expressions of Col2al and Aggrecan were detected by Western-blot at 0,14 days after initial chondrogenic induction of AMSCs.Results Primary cells began to adhere after 24 hours cultured with DMEM contains 10%new-born calf serum,and then become fusiform shape or polygon shape.Primary cells reached 90-100%confluence and presented colony whirlpool After 10-12 days cultured.Morphological change was not found within 10 passages.The induced cells presented round,triangular or multangular shape.The extracellular matrix was stained positively for Alcian blue,Toluidine blue and Safranin O at 14 days after initial chondrogenic induction.RT-PCR indicated that the induced cells could express Col2al mRNA at 14days,but no express at 0 day.Western-blot indicated that the induced cells could express the proteins of Col2al and Aggrecan at 7,14 days,only very weak at 0 day.Conclusion Adipose-derived stem cells from subcutaneous adipose tissue obtained by liposuction have great capacity of proliferation and potential of chondrogenic differentiation in vitro.Adipose-derived stem cells are an alternative source for seed cells of tissue engineering of cartilage.Part II The Biocompatibility and capacity of chondrogenic differentiation of adipose-derived mesencllymal stem cells in PHBV scaffoldsObjective To explore chondrogenic differentiation capability of human adipose-derived mesenchymal stem cells cultured and induced in PHBV scaffold.Methods PHBV scaffolds were sterilized and modified by poly-Lysine in preparation.The 6th passage Adipose-derived mesenchymal stem cells were collected and inoculated into PHBV with concentration of 2×10⁷/L per scaffold,followed by the chondrogenic differentiation medium as used in part.Noninduction groups were set as the contorl.The complex of cells and scoffolds were assessed by using acridine orange staining,scanning electron microscope,HE staining,collagenâ…¡immunohistochemistry staining and Aggrecan immunof luorescence.The mRNA expression of Col2al was detected by RT-PCR.Results Cells can attached and proliferate into PHBV.Poly-Lysine can strengthen the hydrophilia and adsorptivity of PHBV.HE staining showed some round cells in pores of scaffolds.The extracellular matrix was stained positively for collagenâ…¡immunohistochemistry staining. Acridine orange staining and scanning electron microscope showed many spherical cells distributed in pores and on the surface of the scaffolds. Much more extracellular matrices were found in the induced groups than the control groups.Aggrecan were expressed positively.RT-PCR indicated that the induced groups could express Col2al mRNA,and no express in control groups.Conclusion Human adipose-derived mesenchymal stem cells could be cultured in PHBV scaffolds and express chondrocyte phenotype under the chondrogenic differentiation environment in vitro.Partâ…¢The capacity of chondrogenesis of adipose-derived mesenchymal stem cells in PHBV scaffolds in vivoObjective To explore heterotopic chondrogenesis of differentiated adipose-derived mesenchymal stem cells in PHBV scaffolds in vivo.Methods Adipose-derived mesenchymal stem cells seed in PHBV were induced or noninduced for two weeks according to the Chapterâ…¡. The induced and noninduced complexes of cells and PHBV scaffolds were implanted into the subcutaneous in the back of nude mice.The complexes of cells and scoffolds were terminated in vivo,assessed by using HE staining,Toluidine blue staining,Safranin O staining,collagenâ…¡immunohistochemistry and Aggrecan immunofluorescence at 8,12 weeks after they were impanted in vivo.The protein expressions of Col2al and Aggrecan were detected by Western-blot complex were impanted in vivo.Results There was no postoperative inflammatory reaction were observed of the nude mice.The induced groups could keep their original size.The noninduced groups absorbed gradually.The extracellular matrix of the induced groups was stained positively for Toluidine blue,Safranin O,collagenâ…¡and Aggrecan at 8,12 weeks postoperatively.There still had PHBV residue after 8 and 12 weeks postoperatively,a few cartilage lacunae were found in the lower of the complexs.Western-blot indicated that the induced groups implanted in vivo could express Col2al and Aggrecan at 8,12 weeks postoperatively.Conclusion Adipose-derived mesenchymal stem cells combined with PHBV scaffolds induced in vitro have the capability of Heterotopic chondrogenesis in vivo.