| Objective To investigate the feasibility of constructing tissue engineered adipose with human adipose-derived stem cells (hADSCs)and Genipin cross-linked type I collagen sponge scaffoldMethods1. Isolated and cultured hADSCs to the third generation, and the cells were seeded on Genipin cross-linked type I collagen sponge scaffold. Determined cell adhesion rate, and MTT assay was used to evaluate the adhesion and proliferation of cells on the scaffold,and the toxic effects of Genipin cross-linked type I sponge collagen on hADSCs. Optical microscopy and scanning electron microscopy were utilized to observe the adhesion and growth process of hADSCs on the scaffold, and the morphological changes of cells.2Endothelial cells and adipogenic cells both induced from ADSCs were co-cultured on the scaffolds in vitro, the experiment was divided into three groups. In the experiment Group A, adipogenic cells and endothelial cells were seeded at a ratio of4:1. We seeded respectively endothelial cells and adipogenic cells on the scaffolds as Group B and C. After seeded the cells on the scaffolds,the cell-scaffold complexes were cultured in adipogenic introduce medium for5to7days, then Oil red O was adopted to detect adipogenic cells. The expression of specific gene PPARy-2was tested through RT-PCR. Then scanning electron microscope was performed to observe the cells’ growth on scaffolds.3Above three groups of complexes and another blank scaffold set as group D were transplanted under subcutaneous layer in female Wistar rats.12weeks after transplanted,the complexes were taken out from the rats and examined through the above methods. Simultaneously,the degradation was observed.Apart from that, the feasibility of contrUsting tissue engineered adipose in vivo and vascularization of tissue engineered adipose were evaluated. The expression of specific gene PPARy-2was tested through RT-PCRResults1hADSCs could adhere to the scaffold immediately after seeded and increase gradually on the scaffold,and the average adhesion rate of hADSCs on the scaffold was86.5%. Optical microscopy and scanning electron microscopy showed that hADSCs adhered on the material well. The cells increased gradually over time, and could migrate into the interior of scaffold, and distributed evenly with the passage of time when observed with optical microscopy.2In vitro experiment, cells could be seen in Group A, B and C, and they all grew well. The expression of PPARy-2was detected in Group A and B, while no expression was found in group C. Oil Red O staining result indicated that after adipogenic induction, Group A and B were successfully induced to generate a large number of mature adipocytes, meanwhile no adipocytes were found in Group C. The number of adipocytes in Group A and B showed a significant statistical difference when compared to Group C(P<0.01).3In vivo experiment,Oil red O staining was positive in Group A and B, the amount of adipocytes and new formed vessels in Group A were obviously more than that in Group B, there was a significant statistical difference between Group A and B(P<0.01). Scaffolds degraded significantly in four groups,and compared to other groups, scaffolds of Group A degraded more apparently.Conclusions1. hADSCs could adhere and proliferate well on Genipin cross-linked type I collagen sponge scaffold. The biomaterial possessed very low cytotoxicity to the cells, and the material showed outstanding biocompatibility in vitro.2. Endothelial cells induced from hADSCs and hADSCs co-cultured on Genipin cross-linked type I collagen sponges could generate mature adipocytes after adipogenic induction.3. Co-transplanting of endothelial cells and adipogenic cells both induced from hADSCs could significantly promote constructing tissue engineered adipose and angiogenesis. |
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