Study On The Construction Of The Silk Fibroin Scaffolds Encoding Gene For Angiogenesis And Its Effect On Dermal Regeneration

Promoting regeneration of microvascular and enhancing the rate of vascularization are important to improve the survival rate of the skin dermal regeneration scaffold. A novel and biodegradable Antheraea pernyi silk fibroin(AP), with functions of cell targeting, endosomal escaping and nuclear localization, which was prepared by conjugating protamine sulfate(PS) encoding nuclear localization signal(NLS) to Antheraea pernyi silk fibroin(ASF) encoding RGD tripeptide sequence and His was built, and gene therapy as well as tissue-engineered scaffolds were combined to fabricate silk fibroin scaffolds encoding gene for angiogenesis in this study. The(AP+PEI) co-combine gene delivery carrier and AP/ASF/PEI layer-to-layer gene delivery carrier deliver VEGF165-Ang-1 double genes co-expression plasmid to transfect cells in situ, promoting transfected surrounding cells to secret corresponding growth factors such as VEGF and Ang-1 VEGF continuously in local, and improving the survival of angiogenesis in scaffolds effectively.First, the protamine was covalently attached to ASF by 1-ethyl-3(3-dimethylaminopropyl) carbodiimide(EDC). The cationic modified of ASF was successfully by the coupling reaction between the carboxyl of ASF and the amino of PS. The Zeta potential of AP was up to +15mv and its p I up to 8.7 when the weight rations of ASF and PS was 100/10. The AP could condense p DNA effectively when the weight rations of ASF and PS above 4/2, nd the surface complexes were positively charged.And, the(AP+PEI) co-combine gene delivery carrier and AP/ASF/PEI layer-to-layer gene delivery carrier can package p DNA and protect it from nuclease degradation efficiently. Both those complexes are uniformity and have high positive surface charge. Beside, both of those two gene carrier could reduce the cytotoxicity of PEI and show high transfection efficiency. And the transfection efficiency of AP/ASF/PEI was higher than that of(AP+PEI).Then, the(AP+PEI)/p DNA((30+10)/2) and AP/ASF/PEI/p DNA(45/45/10/2) complexes were incorporated into SF scaffolds which were prepared by freeze-drying to built a gene-activated scaffolds. The complexes could be well-distributed, and the cells could be seen completely spreading state in the interior of bracket. The released(AP+PEI)/p DNA((30+10)/2) and AP/ASF/PEI/p DNA(45/45/10/2) complexes are still capable of transfecting cells. The transfected cells secreted VEGF sustainingly with time. Meanwhile, the gene-activated scaffolds promoted the formation of blood vessels on chicken embryo villus allantois membrane(CAM) by secreting corresponding growth factor while the p DNA encoding VEGF165 and Ang-1.Further, the vessel network reconstruction behavior in vivo induced by the gene-activated scaffolds was investigated by implanted(AP+PEI)/p DNA((30+10)/2) and AP/ASF/PEI/p DNA(45/45/10/2) gene-activated SF scaffolds in rat dorsal dermal defects respectively. The results indicated that the immune response and inflammation reactions caused by gene-activated scaffolds were mild, and the repairing cells could migrate to the center of the scaffolds. The gene-activated scaffolds induced the newly-formed tissue and blood capillary to grown. The formation of capillary network and collagen fibers in the gene-activated scaffolds were significantly higher than that in SF scaffolds in the early recover time. The VEGF、Ang-1、CD31、α-SMA、PDGF、b FGF and v WF in the gene-activated scaffolds also had a higher expression than that in the SF scaffolds. This not only illustrated that gene-activated scaffolds induce cells secreting corresponding growth factor while the p DNA encoding VEGF165 and Ang-1, but also create a good micro-environment for the repairing cells migration, adhesion and growth to promote the angiogenesis.