【作者】 方征东；

【导师】 王玉琦；

【作者基本信息】 复旦大学 ， 外科学， 2010， 博士

【摘要】 第一部分P(LLA-CL)静电纺丝工艺初步研究目的以聚左旋乳酸-己内酯共聚物[P(LLA-CL)]为原料,采用静电纺丝方法制备纤维膜,考察静电纺丝过程参数对纤维膜结构及纤维直径的影响规律。方法以六氟异丙醇为溶剂,通过调整P(LLA-CL)浓度、纺丝电压、电纺距离及溶液流速,分别制备了不同条件下的纺丝膜,通过扫描电镜(SEM)来观察纺丝膜的纤维形貌以考察这些参数对电纺膜形貌的影响。结果(1) P(LLA-CL)浓度为4%时,电纺纤维膜中有很多纺锤形的珠滴,而当浓度上升为6%时,P(LLA-CL)溶液的可纺性明显改善,电纺纤维直径由浓度6%时的357±127nm上升到浓度12%时的904±98nm(P<0.05),而聚合物浓度达到一定水平(14%)时,则难以在喷丝头形成喷丝细流,电纺不可行：(2)随着纺丝电压由1kV/cm增高到2kV/cm时,纤维平均直径由520±64nm减小至305±58nm(P<0.05)；(3)电纺距离为由15cm减至10cm时,对电纺纤维膜形貌无明显影响(P>0.05)；(4)在溶液流速为2ml/h时,可形成完好的纳米纤维形貌,但当溶液流速为4ml/h时,纤维发生明显的粘结。结论聚合物液的浓度是纺丝过程中重要影响因素,在可纺范围内,纤维直径随溶液浓度提高而增加,随着纺丝电压的增大而减小,电纺距离对纤维形貌影响不大,但电纺距离的缩小及过快的溶液流速均不利于纳米纤维膜的形成。第二部分P(LLA-CL)/纤维蛋白原纳米复合膜的制备与表征目的以合成高分子材料P(LLA-CL)与天然生物材料纤维蛋白原为原料,利用静电纺丝技术构建具有良好细胞亲和性与合适机械性能的纳米复合膜。方法(1) P(LLA-CL)溶解于六氟异丙醇中得到浓度为8%的溶液,纤维蛋白原溶解于六氟异丙醇和10×DMEM混合溶液中(体积比9：1)得到100mg/ml的溶液。把P(LLA-CL)溶液与纤维蛋白原溶液分别按4：1、2：1、1：1、1：2、1：4体积比进行混合,利用静电纺丝技术构建纳米复合膜；(2)采用SEM检测膜片的表面纤维形态,称重法计算孔隙率；(3)傅里叶全反射红外光谱(FITR)进行膜片表面基团分析；(4)X射线光电子能谱(XPS)对膜片表面元素组成进行表征；(5)水接触角测定膜片的亲水性；(6)拉伸试验检测复合膜片的力学性能；(7)以人脐静脉内皮细胞(HUVECs)种植在复合膜片上,WST-8试剂盒检测膜片对细胞对的粘附、增殖的影响,并以细胞活力实验检测细胞在膜片上生长情况,SEM观察HUVECs形态变化。结果(1) P(LLA-CL)/纤维蛋白原复合膜纤维平均直径约240纳米到450纳米,随着混纺液中纤维蛋白原浓度的增加,纤维直径逐渐减小,孔隙率增大,亲水性增加(P<0.05); (2) FITR及XPS证实复合膜片表面有纤维蛋白原氨基酸的存在；(3)拉伸试验表明在P(LLA-CL)中加入纤维蛋白原后,能降低断裂伸长率,增加杨氏模量(P<0.05),但拉伸强度表现不同,在体积比4：1复合膜片组增高(P<0.05),体积比2：1复合膜片组改变无差异(P>0.05),但当纤维蛋白原浓度增大到一定程度(1：1)则降低；(4)细胞粘附、增殖实验表明,疏水性的P(LLA-CL)支架有更好的细胞粘附能力(P<0.05),但含有纤维蛋白原的膜片有更好的细胞增殖能力(P<0.05)。其中,体积比2/1的膜片细胞增殖率最高。细胞活力实验及扫描电镜也证实在P(LLA-CL)/纤维蛋白原复合膜表面,HUVECs生长良好,近乎铺满支架表面。结论以P(LLA-CL)与纤维蛋白原共混后行静电纺丝,能构建具有良好力学性能及生物活性的纳米复合膜片。高分子合成材料P(LLA-CL)能够作为膜片的骨架提供良好的力学性能,天然材料纤维蛋白原增加了膜片的生物活性,作为组织工程支架有潜在的应用前途。第三部分小口径P(LLA-CL)／纤维蛋白原管形支架构建及生物学性能评价目的制备小口径P(LLA-CL)/纤维蛋白原管形支架,并评价支架的生物相容性和生物力学性能。方法(1)以P(LLA-CL).纤维蛋白原为原料,按体积比2：1混合后构建小口径管形支架,观察制备的小口径血管支架的大体形态并用扫描电镜观测其三维结构；(2)利用溶血试验、细胞毒性试验、皮下植入试验评价支架材料的生物相容性；(3)检测支架的爆破强度、最大缝持张力、顺应性以评价小口支架的生物力学性能。结果(1)管形支架长度在5～8cm,内径5mm左右,管壁厚度为0.4-0.5mm,支架表面呈网格状三维结构,并有大小不等、互相交通的孔隙,测算孔径的平均直径为4.56±1.23μm,表面纤维纤维平均直径318±56nm；(2)P(LLA-CL)／纤维蛋白原浸提液溶血率为2.87±0.49%。(3)细胞毒性实验示P(LLA-CL)／纤维蛋白原浸提液较阴性对照组无明显差异(P>0.05)；(4)皮下植入试验P(LLA-CL)／纤维蛋白原支架炎症反应轻微,12周时种植的材料大部分已降解,材料降解区被成纤维细胞和宿主新生胶原纤维代替；(5)P(LLA-CL)/纤维蛋白原管形支架爆破强度为2970±363 mmHg,最大缝持张力为217+17g,管腔内径变化为1.36%/100mmHg,β值为43.43。结论静电纺丝法构建的P(LLA-CL)／纤维蛋白原管形支架材料具有良好的生物相容性,并具有有类似于自体动脉的爆破强度和最大缝持张力,顺应性优于ePTFE人工血管第四部分组织工程血管置换犬颈动脉初步试验研究目的制备小口径P(LLA-CL)／纤维蛋白原管形支架,内皮化后构建组织工程血管,并以其替代犬颈动脉缺损,观察组织工程血管的在体内的通畅及重塑情况。方法(1)采用原位插管、酶消化法分离、培养和体外扩增实验犬浅静脉内皮细胞(ECs)；(2)血管内皮细胞高密度种植于P(LLA-CL)／纤维蛋白原及P(LLA-CL)管形支架内面使其内皮化；(3)以内皮化的组织工程血管修复犬颈动脉缺损,并以未种植内皮细胞的支架作为对照；(4)术后以彩色多普勒及DSA检查移植物的通畅情况；(5)组织学及免疫组织化学检测组织工程血管体内重塑情况。结果(1)成功分离、培养并在体外大规模扩增犬静脉ECs；(2)P(LLA-CL)／纤维蛋白原管形支架内面形成较完整的ECs单层,ECs覆盖率达到99.3±0.2%,而P(LLA-CL)支架ECs覆盖率只有38.2±6.5%(P<0.05)；(3)犬颈动脉修复缺损手术均顺利,术后吻合口破裂死亡1例,切口感染1例；(4)试验组术后9例存活,随访7例移植物保持通畅,2例发生血栓形成；3例对照组均发生血栓形成；(5)实验组组织工程血管术后组织学检测示内膜层完整,术后8周有类似于正常动脉壁的血管平滑肌细胞及细胞外基质。结论P(LLA-CL)／纤维蛋白原管形支架有益于内皮细胞种植,体内试验证实内皮化后构建的组织工程血管能够承受体内血流冲击,并在8周左右逐渐重塑形成有类似正常动脉壁的组织结构。更多还原

【Abstract】 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.更多还原