Experimental Study Of Feasibility To Construct Vascularized Tissue Engineered Adipose With HADSCs Modified By Human VEGF165 Gene And A Novel 3-D Silk Fibroin Scaffold

Objective:To explore the feasibility to construct vascularized tissue engineered adipose with hADSCs modified by human VEGF165 gene and a novel 3-D silk fibroin scaffold.Methods:Isolated, cultured human adipose derived stem cells to the third generation(P3 hADSCs), and seeded them on a novel 3-D silk fibroin scaffold modified by chitosan (CS/SF).MTT test was used to detect their adhesion and proliferation. After adipogenic induction for 14 days, cell adhesion and growth were respectively observed by electron microscopy and optical microscope,to evaluate the biocompatibility of CS/SF.2. P3 hADSCs infected by lentivirus vector carrying human recombinant VEGF165 gene, were implanted on CS/SF and cultured in vitro. The experiment was divided into three parts. Firstly, the infected and uninfected P3 hADSCs were used as experimental group 1(E1)and control group 1 (C1),both groups were induced by adipogenic differentiation medium for 14 days, which were identified by Oil red O staining to evaluate if the adipogenic ability were affected by lentivirus. Secondly:the infected and uninfected P3 hADSCs, which were regarded as experimental group2 (E2)and control group2(C2),were seeded on CS/SF,MTT assay was selected to detect the proliferation after infected by lentivirus. Thirdly, after adipogenic induction for 14 days, E2 and C2 were regarded as E3 and C3, oil red O staining and PPARy-2 gene tested by RT-PCR were employed to explore the adipogenic ability on CS/SF after infected by lentivirus.3.Experimental study in vivo.24 female Wistar rats were divided into two groups,12 in each one. E3 and C3 cultured in adipogenic differentiation medium for 5 days in vitro were respectively transplanted under subcutaneous layer in the rats.8 weeks and 12 weeks later,6 complexes in each group were taken out and examined, scaffold degradation was observed at the same time. The expression of VEGF165 and PPARy-2 were detected by Western Bolt.Results 1. hADSCs could adhere and proliferate well on CS/SF. After adipogenic induction for 14 days, oil red O staining showed that there existed a large number of mature adipocytes on CS/SF. Mature adipocytes were also found through scanning electron microscopy.2.After infected by Lentivirus for 72 h, weak green fluorescence appeared in E1,96h later the fluorescence enhanced obviously. After adipogenic induction for 14 days,a lot of mature adipocytes were seen in E1 and C1, no statistical difference existed in the two groups (P>0.05), this implied that lentivirus infection had no effect on adipogenic ability. MTT test showed that there was no statistical difference between E2 and C2 after lentivirus infecting.(P>0.05).Oil red O staining and RT-PCR both showed a large number of mature fat cells in E3 and C3,and there were no any statistical difference(P>0.05).3.8 weeks and 12 weeks after implanted, Western Blot results showed that the grafts in E3 expressed VEGF165,while no expression were found in C3.Both E3 and C3 expressed PPARy-2, the amount of expression in E3 was much more than that in C3 (P< 0.05).Scanning electron microscopy, oil red O staining and HE staining all showed that E3 and C3 both generated a lot of fat cells, while the amount in E3 was more than that in C3 (P< 0.05).Conclusions 1. CS/SF possessed outstanding biocompatibility, hADSCs could adhere and proliferate well on it.2. hADSCs infected by lentivirus vector carrying human recombinant VEGF165 gene could successfully reconstruct tissue engineering adipose with CS/SF in vitro.3. hADSCs infected by lentivirus vector carrying human recombinant VEGF165 gene and CS/SF could not only express VEGF165 and PPARy-2 sustainably in rats,but also promote to reconstruct tissue engineered adipose,this result might lay a solid foundation to reconstruct vascularized tissue engineered fat in vivo in the future.