| Objectives:To explore the differentiation potential of rat adipose-derived mesenchymal stem cells (ADSCs)into vascular endothelial cells and the vessel formation, and to evaluate the feasibility that porous silk fibroin scaffold and porous silk fibroin scaffold combined with cells as matrix to promote the endothelial cells generation and vasculogenesis in vivo.Methods:1. Experiments on ADSCs isolation, culture, induction and identification:The adipose tissue was obtained around epididymis region of rat and was cut into pieces and digested with 0.15 % collagenase I at 37℃for 45 minutes, and equivalent DMEM medium containing 15% fetal calf serum to terminate digestion and centrifuged 2800r for 16 minutes and cell sedimentation was inoculated in the culture plate. The growth curve of the 3rd passage ADSCs was obtained by MTT. The growth of the cells was observed by laser scanning confocal microscope. With VEGF and b-FGF as an inducing factor, the morphological change of cells and vascularization in Matrigel were observed under inverted microscope. The change of cell surface marker expression was detected by flow cytometer. The ultrastructure of the induced endothelial cell was observed by transmission electron microscopy.2. Transplantation of porous silk fibroin scaffold and Matrigel combined seeded with ADSCs and induced endothelial cells and new blood vessel formation in vivo:40 male SD rats (200g-300g, 8-12w) were assigned into 5 groups randomly: A1 control group with blank porous silk fibroin(n=8);A2 the porous silk fibroin scaffold seeded with 1×106 ADSCs(n=8);A3 the porous silk fibroin scaffold seeded with 1×106 endothelial cells induced from ADSCs(n=8);B1 control group with Matrigel(n=8);B2 Matrigel seeded with 1×106 ADSCs under induction (n=8). The cell seeded scaffolds was imbedded into the subcutaneous tissue of rats to observe new blood vessel formation in the field of transplantation. The animals in each group were sacrificed for pathological studies at 1w, 2w, 3w, 4w after operation. Tissue sections were stained with hematoxylin-eosin for histological examination. The distribution of the BrdU positive cells and FⅧ-Ag positive cells was also detected with immunohistological methods.Results:1. Experiments on ADSCs isolation, culture, induction and identification:Two hours after primary ADSCs inoculation, cells adhered to the wall, and ADSCs reached the aim of the passage on the 5th or 7th days of culture, and was digested with 0.25% trypsin-0.02% EDTA. Homogeneous long spindle cells were observed on the 3rd passage of ADSCs. MTT curve showed a slow growth phase, logarithmic growth phase and then into the plateau performance. The ADSCs was observed growing well on the porous silk fibroin scaffold in vitro. Using inverted microscope we could see that paving-stone-like cells appears after 14d induction. and ADSCs on Matrigel migrated into a group after 24 hours, and formed grid structure on the 7d. Long vasculature was observed on the 13d. Vessel branching was observed on the 22-30d. The 3rd passage ADSCs were positive for CD44 and negative for CD 31 and cell surface marker changed into CD44 negative and CD31 positive after 14d induction. W-P body was observed by transmission election microscopy.2. Transplantation of porous silk fibroin scaffold and Matrigel combined seeded with ADSCs and induced endothelial cells and new blood vessel formation in vivo:2.1 Histological examinationIn group A1, there were few blood vessels and little fibrous tissue growth, and less lymphocyte infiltration in the field of porous silk fibroin scaffold transplantation 1w after operation. The silk fibroin scaffolds remained structural integrity. New blood vessels grew in time, and more blood vessels and much fibrous tissue grew along the pore of the porous silk fibroin scaffold, and there were less lymphocytes infiltrattion and silk fibroin was not degraded at 4w.In group A2, there were few blood vessels and little fibrous tissue growth, and less lymphocyte infiltration in the field of transplantation 1w after operation. The silk fibroin scaffold was not degraded. At 4w a large amount of blood vessels and fibrous tissue regenerated along the pore of the silk fibroin scaffold, and there were much more blood vessels in large diameter, and less lymphocyte infiltration. A small amount of silk fibroin degradation was observed.In group A3, there were much more blood vessel and fibrous tissue regeneration in the field of transplantation 1w after operation. The scaffold was not degraded with little lymphocytes infiltration. At 4w, abundant blood vessels and fibrous tissue regenerated along silk fibroin scaffold. There were not only much more blood vessels of larger diameter, but rich in capillary. Lymphocytes infiltration became much less in time and a small amount of silk fibroin degradation was observed.In group A1, A2, A3, there was an increasing blood vessel regeneration at the junction region of silk fibroin scaffolds and the surrounding tissue in time.In group B1, few blood vessels grew in the field of transplantation, and there was little lymphocytic infiltration 1w after operation. At 4w a large amount of blood vessels was observed in the field of transplantation with no fibrous tissue regeneration and little lymphocytic infiltration. Matrigel could no longer be traced in the field of transplantation.In group B2, much more blood vessels regenerated and little lymphocytic infiltrated in the field of transplantation 1w after operation. Tissue boundaries surrounding Matrigel transplantation became less obvious. At 4w, a plenty of new blood vessels and more larger blood vessels could be observed in the field of transplantation. Matrigel could no longer be traced at the 4th week.2.2 Distribution of BrdU and the FⅧ-Ag positive cells1w, 2w, 3w, 4w after operation, BrdU positive cells were located with different densities in group A2, A3, and B2. the FⅧ-Ag positive cells were seen in 5 groups at 1w, 2w, 3w, 4w after operation. At 4w the quantity of FⅧ-Ag positive cells in group A1, A2, A3, B1, and B2 were 25.08±8.35/HP, 30.05±7.59/HP, 36.12±12.58/HP, 25.24±9.41/HP, 32.09±13.14/HP respectively, and the FⅧ-Ag positive cells in group A2 and A3 were significantly more than in that in group A1 (P<0.01). There were also significant difference in the FⅧ-Ag positive cells between group A2 and A3 at 4w after operation (P < 0.01). FⅧ-Ag positive cells in group B2 were significantly more than that in B1 group (P<0.01). There were no significant difference in FⅧ-Ag positive cells between group A3 and B2.Conclusion:Using the culture medium Contains VEGF and b-FGF, ADSCs may be induced to differentiate into endothelial cells in vitro. With Matrigel as an extracellular matrix, ADSCs may be induced into endothelial cells to form vessel structure and remained stable over a long period of time in vitro. Porous silk fibroin scaffold seeded with the cells or tissues have good compatibility in vivo and in vitro, and that can be used as the extracellular matrix of ADSCs induced generation of endothelial cells and blood vessels.
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