IGF-1 Induces And Enhances Chondrogenesis Of AMSCs In Vitro And In Vivo

Chapter1 Isolation, culture and identification of human adipose-derived mesenchymal stem cellsObjective: To explore the strategies of isolation, culture, and identify the expression of cell surface markers of human adipose-derived mesenchymal stem cells (AMSCs) obtained from solid adipose.Methods: Solid subcutaneous adipose was obtained from patients undergoing hip surgery, and attached cells were obtained from adipose by using Collagenase I. Cells were cultured in DMEM containing 10% new-born calf serum. Cells proliferation was detected by means of MTT, and growth curve was made. Expression of cell surface marker CD29 and CD44 were detected.Results: A few of adherent cells were observed after cultured 24h later. Cells proliferated fast and exhibited spindle-shape after cultured 1 week. Cell adherence and proliferation were speeded up after passaged. Growth curve exhibited that the passaged 6 and passaged 3 AMSCs had high reproductive activity, while passaged 9 AMSCs had low reproductive activity. Flow cytometry showed that 97% passage 6 AMSCs expressed CD29, and 95% AMSCs expressed CD44.Conclusion: High purity mesenchymal stem cells were easy to isolated from solid adipose. The mesenchymal stem cells had high reproductive activity, and may be a kind of ideal seeding cells for cartilage tissue engineering.Chapter 2 Effect of IGF-1 on chondrogenic differentiation of human AMSCsObjective: To explore the possibility of inducing AMSCs chondrogenic differentiation by using IGF-1 and the interaction with TGF-β1 in induction.Methods: AMSCs was obtained, and 2×10~5cells/cm~2 AMSCs were seeded in culture flask. Insulin-free chondrogenic media containing IGF-1 or(and) TGF-β1 was used to induce AMSCs. Cell aggregation was observed. 2 weeks later, cells were harvested and stained by using toluidine blue and collagen II antibody. RT-PCR was used to detect the expression of collagen II, aggrecan, Sox9 mRNA. Western blot was applied to detect the synthesis of collagen II and aggrecan.Results: AMSCs aggregated to radiate cell masses after chondrogenic media was added. The AMSCs of IGF+TGF group aggregated to several macroscopic cell masses. After induced for 2 weeks, the cells were positive to toluidine blue stain and collagen II stain. Result of RT-PCR and Western blot revealed that the expression of collagen II, aggrecan, Sox9 mRNA and expression of collagen, collagen II of IGF group were significantly higher than that of control group, the expression of cartilage-related gene and protein of IGF+TGF group were significantly higher than that of IGF group and TGF group; while no significant difference was found between IGF group and TGF group.Conclusion: IGF-1 can induce AMSCs chondrogenic differentiation independent of TGF-β1. IGF-1 and TGF-β1 have additional effect on chondrogenic differentiation of AMSCs.Chapter 3 Chondrogenic inducing of AMSCs in P(LLA-co-CL) scaffolds in vitroObjective: To explore the opportunity of P(LLA-co-CL) used as material of scaffolds for cartilage tissue engineering, and the methods of construct tissue engineered cartilage with AMSCs in vitro.Methods: P(LLA-co-CL) scaffolds were fabricated and inoculated with AMSCs, and different chondrogenic inductor or media were used according to group. 2 weeks later, scaffolds were scan by using electron microscope to survey the adhersion and proliferation of AMSCs. Constructs were sliced and HE stain, toluidine blue stain, collagen II stain were applied. RT-PCR was applied to detect the expression of collagen II, aggrecan, Sox9 mRNA. Western blot was applied to detect the synthesis of collagen II and aggrecan.Results: Cell suspension was absorbed in P(LLA-co-CL) scaffolds quickly. The result of electron microscope showed that AMSCs adhered to P(LLA-co-CL) scaffolds and growed on the surface of the scaffolds. HE stain showed that a lot of cells existed in the three induced groups and rare cells existed in control group. The cells and extracellular matrix were positive to toluidine blue stain and collagen II stain in the three induced groups while negative in control group. RT-PCR revealed that the expression of collagen II, aggrecan and Sox9 mRNA in IGF group and TGF group were significantly higher than that in control group; and the expression of collagen II, aggrecan and Sox9 mRNA in IGF+TGF group were significantly higher than that in IGF group and TGF group. Western blot revealed that expression of collagen II and aggrecan in IGF group and TGF group were significantly higher than that in control group; and the expression of collagen II and aggrecan in IGF+TGF group were significantly higher than that in IGF group and TGF group.Conclusion: The P(LLA-co-CL) scaffolds can provide a place for AMSCs to adhere, differentiate to chondrocyte, and synthesize extracellular matrix. IGF-1 can induce chondrocytic differentiation of AMSCs in 3D condition indepentant of TGF-βsignal and had additional effect on chondrocytic differentiation when TGF-β1 was used in combination.Chapter 4 The effect of IGF-1 on chondrogenesis of human AMSCs in vivoObjective: To explore chondrogenesis of induced AMSCs in vivo, and the influence of additive effect of chondrogenesis on cartilage formation in vivo.Methods: AMSCs were seeded on P(LLA-co-CL) scffolds, Insulin-free chondrogenic media containing IGF-1 or(and) TGF-β1 was used. After 2 weeks, the compounds were transplanted in subcutaneous tissue of nude mice. The activity and incision of the mice were observed. The compounds were harvested and HE stain, collagen II stain, toluidine blue stain were applied. Western blot was applied to detect the expression of collagen II and aggrecan.Results: The foodintake and activity were good after being transolanted and incision primary healed. The diameter of compounds was measured and the diameter of IGF+TGF group was larger than that of IGF group and TGF group. HE stain revealed that cartilage lacunas were found in the compounds of IGF group, TGF group and IGF+TGF group, while no cartilage lacuna was found in that of control group. The cells and extracelluar metrix of the three induced groups were positive to toluidine blue stain and collagen II stain. Result of Western blot revealed that collagen II and aggrecan synthesized in IGF+TGF group were higher than IGF group and TGF group, while no significant difference was found between IGF group and TGF group.Conclusion: AMSCs induced with IGF-1 can construct cartilage in vivo. High quality cartilage can be constructed when IGF-1 was used combined with TGF-β1. P(LLA-co-CL) scaffolds are absorbable, and can be used as scaffolds for cartilage tissue engineering.