Repair Of Soft Tissue Defect In Rats With Tissue-engineered Adipose Constructed By Co-culture Of VEGF165 Transfected HADSCs And HUVEC

Objective:1. To explore the best conditions of lentiviral vector infecting human adipose mesenchymal stem cells(hADSCs) and adipogenic differentiation ability before and after infected; 2. To study the feasibility of adipose tissue engineering constructed by seeding hADSCs and human umbilical vein endothelial cells(HUVEC) on silk fibroin scaffold; 3. To investigate the repair of soft tissue defect in rat by tissue-engineered adipose which were constructed by VEGF165 transfected hADSCs and HUVEC.Methods:1. hADSCs was extracted from human normal adipose tissue, culture and amplification. Identification of multi-directional differentiation ability by adipogenic and osteogenic induction, Flow cytometry was used to detect expression of surface molecule CD34, CD44, CD45, CD90, which is associated with the surface of the stem cells. Adipose tissue-derived stromal cells were infected for 8 hours by lentiviral with enhanced green fluorescent protein (EGFP) at different MOI. Observing effect of infection with inverted fluorescent microscope; the infection rate was analyzed by FCM(flow cytometry); comparing proliferation ability of hADSCs between infected before and after by MTT; after inducted for 14days, stained by Oil red O, the adipogenic ability was analyzed between infection and no infection by The Image-Pro Plus Image analysis software; 2. Determination of amplification of HUVEC and surface molecules CD31, CD34, the proliferation curve of hADSCs and HUVEC were measured. After seeded on silk fibroin scaffold, co-cultured cells were observed under scanning electron microscope. The cell-scaffold complexes were cultured in adipogenic introduce medium for 14 days, then Oil red O was adopted to detect adipogenic cells. The expression of specific gene PPARy-2 was tested through RT-PCR.3. In vivo:18 Wistar rats were needed in the study, made of soft tissue defect model on hindlimbs. Experimental design is divided into three groups, group Ⅰ:co-culture system composed of HUVEC and VEGF165-hADSCs; group Ⅱ:only VEGF165-hADSCs. Both of groups were seeded on the silk fibroin scaffold, and transplanted on the site of soft tissue defect of rats. Another rats of soft tissue defect as control group Ⅲ. After 4 weeks and 8weeks, observated the growth situation of complexes, and compared the wet weight. The growth cells on silk fibroin scaffold materials were observed under scanning electron microscope. Oil red O and HE staining, compared two groups situation of adipogenic and vascularization.Results:1.hADSCs were isolated and cultured, flow cytometry tested CD44 and CD90 positive, CD34 and CD45 negative, with ability of multi-directional differentiation. The infection efficiency was 0.24%,13.48%,40.33%,89.27%,40.33%,98.49% and 99.11% when lentiviral carried EGFP infected hADSCs on the condition of MOI 1,10,25,50,75,100,150. After infected with MOI 50, comparing proliferation and adipogenesis of hADSCs before and after infection, there was no statistically significant difference (P> 0.05).2. Surface moleculars CD31 (+) and CD34 weakly positive were tested by Flow cytometry. MTT showed the proliferation rate of HUVEC was faster than hADSCs, with the ratio of 1:4, HUVEC and hADSCs were co-cultured for three days, the number of two kind cells was close. Seeded co-culture system on silk fibroin scaffolds, and adipogenic induction for 14 days, PPARD-2 gene expression was determined by RT-PCR. Oil red O staining showed that both groups can induce a large number of fat cells.3. after 4w and 8w, the gross shows both of groups can be repair soft, tissue defect, Compare the wet weight of two groups construction, VEGF165-hADSCs-HUVEC group> VEGF165-hADSCs group. Red lipid drops were found on Frozen sections stained by oil red O. The grow station of cells in co-culture group was obviously better than relatively simple hADSCs group Under the electron microscope. HE staining show that two groups have different degrees of vascularization, but the number of blood vessels of VEGF165-hADSCs-HUVEC group was obviously more than simple VEGF165-hADSCs group.Conclusions:1. MOI 50 was optimal conditions for lentiviral vector infecting hADSCs, the proliferation and adipogenesis of hADSCs had no obvious effect before and after infection.2. hADSCs could be cultured together with HUVEC at ratio of 1:4, and combined with silk fibroin, successfully construct engineered adipose.3. Soft tissue defect could be repaired effectively by the tissue engineered adipose, which was constructed by VEGF165 transfected hADSCs and HUVEC seeding on porous silk fibroin sponge.