Experiment Study Of Nanofibrous Composite Biomaterials For Small-diameter Tissue Engineering Blood Vessel

PartⅠPreparation and characterization of poly(L-lactide-co-ε-caprolactone) nanofibrous membranes by electrospinningObjectiveTo prepare P(LLA-CL) nanofibrous membranes by electrospinning process and investigate the influence of processing parameters on the structure of the membranes.MethodsHFIP was used as the solvent, the P(LLA-CL) nanofibrous membranes with different surface morphology were fabricated via altering P(LLA-CL) solution concentration, applied voltage, polymer and capillary-collector distance. The morphology and diameter of the fibers were observed using a scanning electron microscope (SEM).Results(1) Beaded fibers were observed when P(LLA-CL) solution concentration was 4%, while mostly smooth fibers were obtained above this concentration, and the fiber diameter increased from 357±127nm to 904±98nm with the concentration of P(LLA-CL) increasing from 6% to 12%(P<0.05). When the solution concentration was 14%, the fibers cannot be formed due to the high viscosity. (2) The fiber diameter decreased from 520±64nm to 305±58nm while increasing the applied voltage from 1kV/cm to 2kV/cm (P＜0.05). (3) The distance between capillary tip and collector played a much smaller role, there was no significant difference between the distance of 15cm and 10cm (P>0.05). (4) When polymer flow rate was 2ml/h, the smooth fibers were obtained, while polymer flow rate was 4ml/h, the fiber was bonding, and there was no apparent pore.ConclusionsThe diameter of fibers increases with the increasing concentration of P(LLA-CL) solution and decreases with the increasing applied voltage. There is no significant difference in the diameter with the distance between capillary tip and collector increasing, but when the distance is shorter and flow rates are higher, significant amounts of fiber bonding are noticeable. Part II Preparation and characterization of poly(L-lactide-co-ε-caprolactone)/fibrinogen blended nanofibrous membranes by electrospinningObjectiveTo fabricate a novel composite fibrous membranes by electrospinning a synthetic biodegradable polymer [P(LLA-CL)] with a natural protein (fibrinogen), study the structure and properties of P(LLA-CL)/fibrinogen blended scaffolds.Methods(1) We prepared composite nanofibrous membranes by co-electrospinning blend of P(LLA-CL) (8%solution) and Fibrinogen (100mg/ml solution) at different volume ratios of 4:1,2:1,1:1,1:2,1:4. (2) The morphology of the nanofibers was observed by a scanning electron microscope (SEM). (3) Surface chemistry of the nanofibers was characterized using Fourier transform infrared spectra (FITR) and X-ray photoelectron spectroscopy (XPS). (4) Hydrophilicity was evaluated by water contact angle. (5) A mechanical testing was performed to measure the effect of Fibrinogen on the scaffold mechanical properties. (6) Adhesion, proliferation and morphology of Human umbilical vein endothelial cells (HUVECs) on the composite fibrous membranes were also studied by WST-8 assay and SEM.Results(1) The fiber diameter gradually decreased with increasing fibrinogen content (P<0.05). (2) The porosity and hydrophilicity significantly improved as the fibrinogen content increased (P＜0.05). (3) Deposition of fibrinogen amino groups on the surfaces was confirmed by FTIR spectra and XPS. (4)Mechanical testing demonstrated that increasing the fibrinogen content in the blended scaffolds could decrease their tensile strength and elongation at breakage but increase the Young's modulus (p<0.05). However, the tensile strength showed different behavior. The blended scaffolds with PLCL/fibrinogen volume ratios ranging from 4:1 to 1:1 have enhanced mechanical strength and maintained a relatively high elasticity profiles. (4) The scaffolds containing P(LLA-CL) provided a better cell adhesion environment than membranes containing fibrinogen(P<0.05). However, addition of fibrinogen to P(LLA-CL) membranes enhanced the cell proliferation compared with P(LLA-CL) alone(P＜0.05), with P(LLA-CL)/fibrinogen (2:1) scaffolds showing the highest cell proliferation rate assessed by cell viability assays and SEM observations.ConclusionsOur results suggest that nanofibrous membranes electrospun from the combination of synthetic polymers and natural proteins have favorable mechanical and biological properties, which might be useful for tissue engineering.Part III Development and evaluation of a small diameter tubular scaffolds of electrospun poly(L-lactide-co-ε-caprolactone)/fibrinogen blended fibersObjectiveTo fabricated a small diameter tubular scaffolds by electrospinning P(LLA-CL) with fibrinogen. The physico-mechanical property and biocompatibility of the P(LLA-CL)/ fibrinogen scaffolds were examined.Methods(1) A small diameter tubular scaffolds were fabricated by co-electrospinning blend of P(LLA-CL) (8%solution) and Fibrinogen (100 mg/ml solution) at volume ratios of 2:1. The 3-dimensional structure of small-small diameter tubular scaffolds was observed by a scanning electron microscope (SEM). (2) Biocompatibilities of the tubular scaffolds were evaluated in vivo and in vitro by the means of acute hemolysis test and cytotoxicity test, short-term test of subcutanous implantation. (3) The compliance, burst pressure, and suture retention strength were measured in vitro by insufflation and pull-through techniques in order to evaluate the biomechanical properties of the tubular scaffolds.Results(1)The tubular scaffolds(5～8 cm in length and approximately 5 mm in inner diameter) have randomly oriented nanofibrous structure with a well interconnected network of pores, the diameters of the fibers at the outer surfaces were 318±56 nm and the average pore diameters were 4.56±1.23μm; (2) Hemolysis rate was 2.87±0.49%; (3) There was no difference of cytotoxicity test between the P(LLA-CL)/fibrinogen tubular scaffolds and negative control group(P>0.05). (4) Upon placement in rat subcutaneous pouches, the scaffolds were gradually biodegraded with little inflammatory reaction. (5) The burst pressure and suture retention strength of P(LLA-CL)/fibrinogen tubular scaffolds were 2970±363mmHg,217±17g, respectively, which are comparable with those of native arteries. The compliance was 136%/100mmHg, stiffness (β) was 43.43, which was better than the value of ePTFE.ConclusionsThe P(LLA-CL)/fibrinogen tubular scaffolds are biocompatible and possess biomechanical properties which are comparable with those of native arteries.Part IV Initial experimental study on the replacement of canine carotid artery with tissue engineering blood vesselObjective:To evaluate the possibility of vascular endothelial cells seeding on the surface of the P(LLA-CL)/fibrinogen scaffolds, and investigate the patency and remodeling of the endothelialized scaffolds in vivo.Methods(1) Endothelial cells were harvested by the in situ application of type I collagenase to canine saphenous vein under anesthesia. The vascular endothelial cells were cultured and mass cultured. (2) In vitro endothelialization of the small-diameter tubular scaffolds were achieved by seeding ECs on the P(LLA-CL)/fibrinogen or P(LLA-CL) scaffolds. (3) Tissue engineered blood vessels were used to repair the man-made default of carotid artery in canine. The study was divided into two groups:one is the scaffold with endothelialization, the other is the scaffold without endothelial cells seeding. (4) The patency of TEBV was evaluated by Doppler and DSA postoperatively. (5) The remodeling in vivo was evaluated by histologically examination and immunohistochemistry.Results(1) The vascular endothelial cells were isolated and cultured successfully. (2) Confluent lining of vascular ECs on the inner surface of the P(LLA-CL)/fibrinogen was observed, ECs coverage was 99.3±0.2%,while ECs coverage on the P(LLA-CL) was only 38.2±6.5%(P<0.05). (3) The process of operation was satisfied, one canine died caused by anastomosis bleeding, one case of wound infection. (4) Thrombus formation was observed in all the control group. Among the 9 survival cases in experiment group, thrombus formation was observed in 2 cases, the residual 7 cases keep patency. (5) The TEBVs showed unform layered tissue with endothelial cells, the vascular smooth muscle cells and extracellular matrix were comparable to native artery in 8 weeks postoperation.ConclusionsP(LLA-CL)/fibrinogen scaffolds provides structural basis for vascular ECs seeding. The endothelialized scaffolds can withstand the shear stress of blood flow, and can be transformed to similar structures of native arterial walls under the hemodynamic condition in 8 weeks postoperation.