| Vascular tissue engineering to address the sources of small-diameter vascular graft provides an effective way to repair vascular injuries,and is the research hotspot in recent years. Scaffold material in tissue engineering blood vessels play a key role, not only has direct support for cell characteristics, but also affected cell growth, migration, differentiation and to determine new organizational structure. Composite material is blood vessel tissue engineering scaffold material in recent years the focus of research and trends.Spider silk fibers is the protein itself, become an interest materials of tissue engineering in recent years,because of their remarkable mechanical properties, biodegradability and good biocompatibility. According to the structure and function of spider silk properties, Recombinant spider silk proteins was prepared in our lab through genetic engineering technology and spider silk protein gene sequences in the repeat function of the introduction of RGD (arginine-glycine-aspartic acid) tripeptide codon to construct and express the RGD-recombinant spider silk proteins (named pNSR32, molecular weight of approximately 102KD), bio-active molecules RGD tripeptide adhesion proteins are able to provide cell-surface receptor proteins recognize specific peptide sequence of the smallest, is currently the most widely used and most effective adhesion-promoting peptide. Polycaprolactone (PCL) is a new kind of aliphatic polyester, with its good mechanical properties, non-toxic, plastic and other excellent performance is widely used in the field of biological materials and Chitosan (CS) has excellent biocompatibility and degradation.The composite nano-fibers of small diameter pNSR32/PCL/CS stent was prepared by electrospinning,using pNSR32, PCL, and CS three kinds of materials, the evaluation of physical and chemical properties and biocompatibility were done.A new stent biological materials with comprehensive excellent performance was prepared succesfully.The optimal ratio and process parameters of pNSR32/PCL/CS composite nano-fibers vascular stent are:the mass ratio of pNSR32, PCL and CS is 10/80/10, the concentration of the electricity spinning liquid is 30%, voltage 80kV, curing distance 20cm, extrusion rate of 5ml/h, rotor diameter of Receiver is 4mm.The fiber pNSR32/PCL/CS composite nano-fibers of small diameter stents is uniform.Fourier transform infrared spectroscopy indicated that pNSR32 with PCL and CS were blended in the form of physic. The porosity and hydrophilicity of pNSR32/PCL/CS composite nano-fibers of small diameter vascular stent is 89.6% and 85.3% respectively, which is conducive to enhancing cell affinity. Mechanical performance test results show that, the suture strength of pNSR32/PCL/CS accord with the requirements of suture in operation, the stent tensile strength,Young's modulus and elongation at break of pNSR32/PCL/CS are better than porcine coronary artery. The degradation behavior of pNSR32/PCL/CS composite scaffold was conducted through the residual weight percentage of visits in vitro using phosphate solution containing porcine pancreatic lipase of the phosphate solution.The results show that pNSR32/PCL/CS composite scaffold material has a certain degree of degradation rate, but the degradation of slow hydrolysis faster than the hydrolysis rate.The blood compatibility of pNSR32/PCL/CS stent was evaluated by hemolysis experiments, dynamic clotting time test, platelet adhesion experiments and re-calcification clotting time, the experimental results show that the stent hemolysis rate was 1.5%, less than 5%, accord with bio-material requirements; pNSR32/PCL/CS decline in the previous 30minBCI faster, and is then slowly decline; compared with the PCL and PCL/CS, pNSR32/PCL/CS composite scaffold for adhesion of platelets less; the time of re-calcification of pNSR32/PCL/CS is significantly longer than that of PCL, the foregoing experimental results show that small-diameter vascular stent pNSR32/PCL/CS has good blood compatibility.The in vitro biocompatibility of pNSR32/PCL/CS composite scaffolds in this study using NIH-3T3 mouse fibroblasts.Different cellular aspects were analyzed in order to know the cell viability during cell culture on pNSR32/PCL/CS composite scaffolds:scaffolds cytotoxicity,adhesion, proliferation, morphology, and secret ion of bFGF.MTT assay results showed that comparing to that of PCL scaffolds, toxic rank of pNSR32/PCL/CS and pNSR32/PCL composite scaffolds was below 1 level, satisfying the requirements of biomaterials. SEM and HE results indicated that comparing with PCL scaffolds, pNSR32/PCL/CS and pNSR32/PCL composite scaffolds with RGD had a better cell adhesion and nanofibre scaffolds higher than porous scaffolds.. Cells on the scaffolds can form multi-layered cells on the surface. Cells had a more orientated growth on nanofibre scaffolds,but only on the surface,no into the pore. Cells on all scaffolds growed normally, excepting slower on PCL scaffolds The pNSR32/PCL/CS composite nanofiber membrane began proliferating rapidly thereafter are more slowly because of space constraints cell proliferation rate. Immunohistochemistry results indicated that cells on the scaffolds can normally proliferate and secret bFGF.All results provided evidences of good adhesion, growth, viability, morphology and normal physiologic function of cells on pNSR32/PCL/CS composite scaffolds.Therefore, it can be provisionally concluded that pNSR32/PCL/CS composite scaffold is a suitable and biocompatible material to be further used in the areas of tissue engineering.Histocompatibility and toxic reaction of scaffolds were evaluated by implanting into SD mouse in vivo and examined at different periods. Results indicated that after implanting 30d, inflammatory response of pNSR32/PCL/CS composite scaffolds and collagen both subsided, according to evaluation standards, its I-IIgrade,indicating qualification. Order of histocompatibility was:collagen(medical grade)> pNSR32/PCL/CS composite scaffolds>PCL scaffolds. The acute genneral toxicity was experimented by injected stent leaching liquor into rat.The results show that there is no acute toxicity reaction with rats, the body weight of mice increased to some extent injected extracts of the stent after 24h,48h,72h. It can be provisionally concluded that pNSR32/PCL/CS composite scaffold had favourable biocompatibility which made it possible to be tissue engineering materials.
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