| Autologous and allografts are widely used to reconstruct small diameter blood vessel currently.However,they can't meet the requirements of small-diameter vascular graft due to inadequate sources and potential immunogenicity respectively.Recently surgeons have to turn to synthetic vascular grafts made from expanded polyethylene terephthalate (Dacron) or polytetrafluoroethylene (ePTFE).Although these synthetic vascular grafts are successfully used for large blood vessel replacements, they have never been proved successful while they are applied to small-diameter blood vessel (inner diameter<6 mm) for their risk of thrombosis and poor biomechanics.Fortunately,It is vascular tissue engineering that provides a feasible solution to small-diameter vascular graft.Therefore,it is particularly important to fabricate a biodegradable and appropriately biomechanical scaffold to overcome above problems.In our previous studies,an RGD spider silk protein(pNSR32)/polycaprolactone(PCL) nanofiber membrane was prepared by electrospinning.However,pNSR32 is hydrophobic as well as PCL.It is reported that hydrophilic scaffolds was more suitable for tissue engineering application.Gelatin(Gt) with low immunogenicity and hydrophilcity was used to improve biocompatibility of the scaffolds.This study was aimed to fabricate a small diameter(inner diameter<6mm) vascular engineering scaffold that combined pNSR32,PCL and various content of Gt though electrospinning apparatus which was equipped with a rotating mandrel.Physicochemical characteristics and biocompatibility of pNSR32/PCL/Gt scaffold in vitro and in vivo were investgated.We got the results as following:1. Collagenase digestion combined with tissue attached method was established to isolate rat aorta endothelial cells(SDRAECs).Cell morphology was viewed under inverted microscope and by eosin staining.The passage 5 cells were identified by immunoreactivity with factorⅧantigen.The cells were shaped as triangle or polygonal and showed cobblestone appearance with contact inhibition.The purity of passaged endothelial cells were all reached to 100%. 2. Based on our previous research of electrospun pNSR32/PCL membrane,different content of Gt which was usually used to improve the hydrophilic were added to electrospin into five kinds of nanofibrous turbular scaffolds(A-E) with a inner diameter of 3 mm and length of 3 cm at the following parameters:voltage 80kV,polymer solution concentration 30%,feeding rate 80μL/min,curing distance 20cm,rotating speed 2 rpm.The scaffolds was listed as following order:A.pNSR32/PCL/Gt(0:100:0), B.pNSR32/PCL/Gt(5:95:0),C.pNSR32/PCL/Gt(5:85:10),D.pNSR32/PCL/Gt(5:75:20),E. pNSR32/PCL/Gt(5:65:30).3.Physicochemical characteristics of A-E scaffolds.Fiber diameter,porosity,pore diameter,water uptake were raised and contact angle was decreased with the increase of Gt concentration.Fourier transform infrared spectroscopy(FTIR) test shown that there were no new bonds formation among pNSR32, PCL and Gt.Meanwhile,scaffold conformation could not change after treatment of ethanolthe.X-ray diffraction(XRD) analysis indicated the addition of pNSR32 and Gt can increase the crystallinity of PCL scaffold. The degradation rate of scaffolds in PBS was slower than in multi-enzyme solution and degradation rate was positively correlated with the content of Gt.The ultimate tensile strength of pNSR32/PCL/Gt(5:85:10) scaffold was 2.67■0.22 MPa and the elongation at break was 117±15%at dry state.After 24h immersion in normal saline,the ultimate tensile strength decreased to 1.59±0.16MPa,the elongation increased to 177%±13%.The suture strength of pNSR32/PCL/Gt(5:85:10) scaffolds were 3.6±1.2N, whcih were up to the requirements of operation.4.Cytocompatibility of SDRAECs on electrospun scaffolds was assessed.Firstly, extraction fluid was prepared to culture SDRAECs to evaluate cytotoxicity by MTT test.The results showed that all scaffolds were no cytotoxicity.MTT test was also carried out to investigate the growth and proliferation of SDRAECs on different electrospun nanofibrous scaffolds,the rate of cell growth on B scaffolds was higher than that on A scaffold.The C scaffolds exhibited the highest cell proliferation. SDRAECs morphological characterization on the C scaffolds were studied by SEM,DAPI-stained,HE-stained of frozen sections,the results confirmed that the pNSR32/PCL/Gt(5:85:10) scaffold support SDRAECs adhesion,growth,proliferation.Immunohistochemistry results suggested that SDRAECs on C scaffolds could express the FactorⅧnormally.Cell cycle test and immunofluorescence results showed that SDRAECs on C scaffolds had faster rate of growth and better phenotype maintenance than that on TCP.In a word,the inrorporation of Gt(especially at 10wt%content) improved cytocompatibility of scaffolds.The pNSR32/PCL/Gt(5:85:10) scaffold would be very preferable for small diameter vascular tissue engineering.5. The hemocompatibility of electrospun scaffold was assessed through hemolysis experiments,recalcification clotting time,dynamic clotting time test,platelet adhesion experiments.The results indicated that hemolysis rate of electrospun A-E scaffolds were 0.26%,0.13%,0.26%,0.55%,0.91%,which meet the biomaterial requirements.The recalcification clotting time was similar with that of A scaffolds but longer than that of B scaffolds.The results of dynamic clotting time test and platelet adhesion experiments all indicated anticoagulation of C stent was superior to that of B and A scaffolds. In brief,the pNSR32/PCL/Gt (5:85:10) scaffold owned good hemocompatibility in vitro.6. Evaluation of genetic toxicity on pNSR32/PCL/Gt (5:85:10) scaffolds.Results of mouse bone marrow micronucleus test showed micronucleus rate of extraction fluid of scaffold(2.3%o±0.9%o) were significantly lower than positive control group (23.3%o±4.2%o) but similar with negative control group(2.8%o±1.3%o).Comet assay confirmed that extraction fluid of scaffolds did not cause DNA damage of SDRAECs.7. Biocompatibility of pNSR32/PCL/Gt (5:85:10) scaffolds in vivo was evaluated by SD rat muscle implants and acute toxicity reaction after intraperitoneal injection of extraction fluid to Kunming mice.The results showed that extraction fluid can't cause acute toxicity reaction with mice,the weight of mice always increased after 24h,48h,72h,96h. General observation and pathological analysis after implantation for 3d,7d,15d,30d respectively.The results shown that after implantation for 30d,SD rats not only pNSR32/PCL/Gt(5:85:10) scaffolds group but also control group all survived normally.Inflammatory response of pNSR32/PCL/Gt(5:85:10) scaffolds were almost disappeared which was consistent with blank group and medical collagen group.pNSR32/PCL/Gt(5:85:10) composite nanofiber scaffold remarkable biocompatibility for tissue engineering small diameter vascular graft. Conclusion:The Characteristics of the pNSR32/PCL scaffolds has been improved by incorporation of gelatin. Especially when the content of gelatin was 10%(wt/wt).In summary, electrospun pNSR32/PCL/Gt(5:85:10) composite nanofibrous small diameter vascular scaffolds showed better physicochemical properties as well as biocompatibility than others, which was feasible for clinical as a small-diameter vascular tissue engineering scaffold material. |
