静电纺丝SF/PLGA纤维支架用于血管组织工程的研究

将丝素蛋白（SF）和乳酸-羟基乙酸共聚物（PLGA）溶解在六氟异丙醇中配制成溶液,采用静电纺丝技术制备了SF/PLGA纳米纤维支架,使用扫描电子显微镜（SEM）对纤维支架进行表征,研究了聚合物溶液浓度、纺丝电压、接收距离以及体积流率对纳米纤维形态的影响,从而得到纺丝的最适宜工艺参数。考察了纤维支架表面对HUVECs细胞的相容性。结果表明：HUVECs可以在SF/PLGA纤维支架表面很好的黏附和增殖,支架具有良好的细胞相容性,在组织工程领域有良好的应用前景。     

静电纺丝制备纳米级纤维的研究

首先介绍了静电纺丝制备纳米纤维的原理及其影响因素,然后归纳、总结了当前国内外静电纺丝制备纳米纤维的研究内容,并对今后的研究提出了建设.     

再生丝素蛋白/聚乙烯醇共混纳米纤维的静电纺丝研究

以静电纺丝方法制备再生丝素蛋白/聚乙烯醇共混纳米纤维。研究了共混配比、溶液浓度、添加剂含量及电纺电压、喷丝距离等因素对纤维成形及纤维有关性能的影响。研究表明:与聚乙烯醇共混后再生丝素纤维的柔韧性有一定改善,适当增加PVA在共混物中的含量、提高纺丝液浓度以及纺丝电压有利于改善共混溶液的可纺性。另外,加入丙三醇虽可使纤维直径的均匀性有所提高,但却不利于纤维成形。     

静电纺丝PLA/丝素复合纤维膜的结构和性能

以氯仿、丙酮为混合溶剂,制得质量分数为5%的聚乳酸(PLA)纺丝液,经静电纺丝制备PLA纤维膜;以98%的甲酸为溶剂,制得丝素纺丝液,在PLA纤维膜上喷射丝素纺丝液制成PLA/丝素复合纤维膜.采用扫描电镜观察其形貌结构,并测定其微细结构、力学性能、溶失率及生物性能.结果表明:PLA/丝素复合纤维膜呈规整排列的多孔网状结构.与丝素膜相比,PLA/丝素复合纤维膜的丝素蛋白转向β折叠结构,断裂比功提高18倍,水中溶失率降低3倍,更有利于人脐静脉内皮细胞在纤维膜上的生长.     

聚ε-己内酯/聚乙二醇共混纳米纤维的静电纺丝研究

采用四氢呋喃和无水乙醇为溶剂,利用静电纺丝法制备了聚己内酯(PCL)/聚乙二醇(PEG)共混纳米纤维。研究了共混配比、溶液浓度、无水乙醇的加入以及电纺电压、接收距离等工艺参数对纤维形态和性能的影响。测试结果表明:聚乙二醇和聚己内酯以一定比例共混后改善了聚己内酯纤维毡的亲水性和细胞相容性;随着纺丝原液浓度增加,电纺产品由高分子微/纳米液滴结构渐变为珠状结构较少的平滑纤维,平均纤维直径逐渐增大;一定范围内,纤维平均直径随电压的上升而增大,但与接收距离关系不大;此外,加入无水乙醇后,共混溶液电导率增加,有利于喷射流的劈裂,减少了珠状结构的数量。     

静电纺丝制备纳米级纤维的研究

介绍了静电纺丝制备纳米纤维的原理及其影响因素,归纳、总结了当前国内外静电纺丝制备纳米纤维的研究内容,并对今后的研究提出了建议.     

SA溶液浓度对乳液静电纺丝SA/PCL纳米纤维成形的影响

乳液静电纺丝可制备同时含有亲水和亲油两相结构的复合纳米纤维。以聚己内酯(PCL)/三氯甲烷为连续相,海藻酸钠(SA)/去离子水为分散相,失水山梨醇脂肪酸酯(Span80)为乳化剂,制备油包水(W/O)型乳液,并采用乳液静电纺丝技术制得SA/PCL复合纳米纤维膜,经与Ca2+置换制得海藻酸钙(CA)/PCL复合纤维膜,研究了SA溶液浓度对纤维成形的影响。结果表明:表面活性剂Span80和分散相SA水溶液的加入可有效增加PCL的可纺性;当乳液体系中SA溶液体积一定,SA溶液浓度对SA/PCL乳液的黏度无明显影响;随SA溶液浓度的增加,SA/PCL乳液的表面张力降低,SA/PCL复合纳米纤维的直径出现极大值,但均小于仅添加Span80所得的PCL纳米纤维;SA在复合纳米纤维成形过程中会向纤维表面迁移,从而可实现SA与Ca2+交换,且离子交换后形成纤维间的粘连结构。     

三种不同溶剂静电纺丝素纳米纤维的研究进展

静电纺丝素纳米纤维支架材料在组织工程领域具有广阔的应用前景。本文综述了三种不同溶剂静电纺丝素纳米纤维支架材料及其在组织工程领域的研究进展。     

静电纺再生丝素纳米纤维的结构与性能

静电纺丝获得的丝素纳米级纤维可作为细胞培养支架,用于纺丝工艺及后处理能改变丝素微细结构,影响其水溶性和力学性能。本文采用XRD、FTIR、固态13CNMR和DSC研究了不同工艺下丝素纳米纤维及经甲醇处理后的微细结构,比较了不同微细结构下的水溶性和力学性能。结果表明,电纺丝的微细结构受纺丝工艺影响,高电压、纺丝液中丝素质量分数大时纺得的电纺丝结晶度高,经甲醇处理后,β化程度提高;w(丝素)=11%、15%时制备的电纺丝断裂强度分别为8.5、11.9 cN/mm;w(丝素)=11%、19%,水溶性由51.2%下降到43.3%;w(丝素)=19%、电压32 kV制得的电纺丝甲醇处理前后水溶性从43.3%下降到6.6%,说明丝素纳米纤维结晶度提高,强度增加、水溶性下降,满足了细胞支架的要求。     

血小板裂解液大规模扩增人脐带间充质干细胞

目的用血小板裂解液(platelet lysate,PL)大规模扩增人脐带间充质干细胞(umbilical cord mesenchymal stem cell,UCMSC),并检测其生物学特性,为临床应用提供实验依据。方法分别采用PL和胎牛血清(FBS)低密度扩增人UCMSC,比较两组UCMSC的细胞形态、大小、克隆形成率、增殖能力、细胞表型和分化能力。结果 PL扩增的UCMSC形态细长;直径明显小于FBS扩增的UCMSC(P<0.05);克隆形成率与FBS扩增的UCMSC差异无统计学意义(P>0.05);有更高的细胞累积群倍数;与FBS扩增的UCMSC有相似的细胞表型;与FBS扩增的UCMSC均具有成骨、成脂诱导分化能力,但PL扩增的UCMSC成骨分化能力更强。结论 PL可取代FBS用于大规模扩增UCMSC。     

改良组织块贴壁法制备人脐带间充质干细胞

目的采用改良组织块贴壁法制备人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUCMSCs)。方法无菌条件下取足月婴儿脐带,采用改良组织块贴壁法分离间充质干细胞,台盼蓝染色法计数单个核细胞数,倒置相差显微镜下观察细胞形态,流式细胞仪检测细胞周期及免疫表型。结果原代hUCMSCs的数量为(1~5)×105个。培养至8 d,组织块边缘处有单细胞爬出;培养至2周,贴壁生长的细胞散在分布或形成小克隆;培养至20 d,细胞呈梭型、三角形或多角形。培养第20天的原代hUCMSCs的G0/G1期细胞占88.12%,S期细胞占1.23%,G2/M期细胞占10.65%,大多数细胞处于细胞增殖的潜伏期,CD105阳性率为97.65%,CD34阳性率为2.54%。结论采用改良组织块贴壁法分离了hUCMSCs,其生物学特性与骨髓间充质干细胞相似,为其临床应用奠定了基础。     

人脐血干细胞在局灶性脑缺血大鼠体内的迁移与分化

目的观察人脐血干细胞(human umbilical cord blood stem cells,HUCBSCs)在局灶性脑缺血大鼠体内的迁移与分化,探讨HUCBSCs移植对缺血性脑损伤大鼠神经功能恢复可能的作用机制。方法采用线栓法制备大鼠大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)再灌注模型,将造模成功的20只大鼠随机分为2组:移植组15只,造模成功后24 h,经尾静脉移植1 ml(2×106个)DAPI/CM-Dil荧光染料标记的HUCBSCs;模型组5只,造模成功后24 h,经尾静脉移植等量生理盐水。分别于移植前及移植后3、7、15 d进行神经功能缺损评分(neurological severity score,NSS),并取脑组织,制备冰冻切片,HE染色观察脑组织形态,荧光显微镜观察HUCBSCs在脑组织中的迁移和分布,免疫荧光法检测脑组织中巢蛋白(Nestin)和神经元特异性烯醇化酶(neuron specific enolase,NSE)的表达。结果移植组大鼠HUCBSCs移植后7 d起,其改良的NSS(m NSS)显著低于对照组(P<0.05)。移植后15 d,大鼠脑组织病理改变明显减轻。移植后3 d起,可见病灶及其周围部位有阳性细胞存在,随着时间的推移,脑缺血病灶部位的阳性细胞增多;移植后7 d,迁移至病灶部位的细胞数增加;移植后15 d,迁移至病灶部位的细胞数量达观察期内的高峰,各时间点阳性细胞数差异有统计学意义(P<0.05)。移植后15 d,大鼠脑组织中CM-Di I/NSE-FITC和CM-Di I/Nestin-FITC双阳性细胞率分别为85.2%和81.6%。结论 HUCBSCs经静脉移植后,能在大鼠体内存活,并向损伤部位迁移,具有向神经元细胞方向分化的潜能,对脑缺血大鼠的神经功能恢复具有促进作用。     

一种分离培养人脐带Wharton's jelly间充质干细胞的新方法

目的探讨一种分离培养人脐带Wharton′s jelly间充质干细胞(mesenchymal stem cell,MSC)的新方法。方法取人脐带组织,除去动静脉后剪碎成2～5 mm3,将组织碎片浸入4 g/LⅠ型胶原酶和1 g/L透明质酸酶的混合液中,于37℃处理1 h,再用0.25%胰蛋白酶在相同条件下消化30 min,得到的消化液经70μm细胞滤网过滤后,制备单个细胞悬液,培养并传代。取P1、P3、P7代脐带Wharton′s jelly MSC,绘制细胞生长曲线;取P3代细胞,采用流式细胞术检测细胞表面标记分子,并分别采用成骨和成脂诱导培养基进行成骨及成脂诱导分化,茜素红染色和油红O染色观察结果。结果 P1、P3代脐带Wharton′s jelly MSC的增殖能力强,且P1代细胞的增殖能力强于P3代,P7代细胞的增殖能力较P3代细胞有所减弱。P3代脐带Wharton′s jelly MSC高表达CD90(99.8%)、CD105(100%)和CD166(100%),低表达CD45(0.3%)、CD14(0.1%)、CD34(0.2%)和CD79a(0.3%),不表达HLA-DR。P3代Wharton′s jellyMSC经成骨诱导后,茜素红染色可见红色结节;经成脂诱导后,油红O染色可见脂质沉积。结论本方法获得的Wharton′s jelly MSC活性好,增殖能力强,为后续实验研究及临床应用提供了理想的种子细胞。     

Enhanced chondrogenic differentiation of human bone marrow mesenchymal stem cells on PCL/PLGA electrospun with different alignments and compositions

The simultaneous effect of electrospun scaffold alignment and polymer composition on chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBMMSC) is investigated. Aligned and randomly oriented polycaprolactone/poly(lactic-co-glycolic acid) (PLGA) hybrid electrospun scaffolds with two different ratios are fabricated by electrospinning. It is found that aligned nanofibrous scaffolds support higher chondrogenic differentiation of hBMMSCs compared to random ones. The aligned scaffolds show a higher expression level of chondrogenic markers such as type II collagen and aggrecan. It is concluded that the aligned nanofibrous scaffold with higher PLGA ratio could significantly enhance hBMMSC proliferation and differentiation to chondrocytes.     

脐带间充质干细胞长期体外培养的生物学特性及遗传稳定性

目的观察脐带间充质干细胞(umbilical cord mesenchymal stem cells,UC-MSCs)体外长期培养的生物学特性及遗传稳定性。方法从剖宫产足月健康新生儿脐带中分离培养UC-MSCs,并连续传代,显微镜下观察细胞形态。分别检测P3和P12代UC-MSCs的增殖能力、免疫表型、向成脂肪细胞和成骨细胞的分化诱导率、衰老情况、成纤维细胞集落形成单位(CFU-F)及染色体核型。结果 P3代UC-MSCs为形态相对均一的梭形贴壁细胞,呈平行排列生长或旋涡状生长;P12代UC-MSCs宽大扁平,贴壁性减退,漂浮细胞增多,胞浆内出现黑色颗粒和空泡;P3和P12代UCMSCs均表达CD73、CD90、CD105,不表达CD34、CD45、HLA-DR;P3较P12代UC-MSCs的细胞增殖能力及向成脂肪细胞分化诱导率均明显升高(P均0.05);向成骨细胞分化诱导率及细胞衰老率均明显降低(P均0.05);成纤维细胞集落数及染色体中期分裂相明显增多(P0.05)。结论长期体外培养的UC-MSCs生物学特性发生改变,遗传学特性稳定,但染色体中期分裂相减少。     

Computational modeling of megakaryocytic differentiation of umbilical cord blood-derived stem/progenitor cells

Quantifying the effect of exogenous parameters regulating megakaryopoiesis would enhance the design of robust and efficient protocols to produce platelets. We developed a computational model based on time-dependent ordinary differential equations (ODEs) which decoupled expansion and differentiation kinetics of cells using a subpopulation dynamic model. The model described umbilical cord blood (UCB)-derived cell's behavior in response to the external stimuli during expansion and megakaryocytic differentiation ex vivo. We observed that the rate of expansion of Mk progenitors and production of mature Mks were higher when TPO was included in the expansion stage and cytokines were added during differentiation stage. Our computational approach suggests that the Mk progenitors were an important intermediate population that their dynamic should be optimized in order to establish an efficient protocol. This model provides important insights into dynamics of cell subpopulations during megakaryopoiesis process and could potentially contribute toward the rational design of cell-based therapy bioprocesses.     

二次贴壁法高效制备人脐带间充质干细胞

目的采用二次贴壁法高效制备人脐带间充质干细胞(human umbilical cord derived mesenchymal stem cells,hUC-MSCs)。方法将传统组织块贴壁法弃掉的脐带组织块转移至新的培养瓶中进行二次贴壁,分离hUC-MSCs,并用无血清培养体系连续传代,镜下观察细胞形态;分别检测不同代次细胞的增殖能力、细胞周期、免疫表型、多向分化能力。结果二次贴壁法分离的hUC-MSCs在第4天即出现细胞克隆,第8天汇合度可达70%,经过二次贴壁法,1根20 cm的脐带组织共获得P3间充质干细胞(MSCs)总数为1×10~9个。二次贴壁分离的细胞增殖能力旺盛,与一次贴壁获得的干细胞同代次平均倍增时间差异无统计学意义(P0.05)。传统贴壁法与二次贴壁分离的细胞均表达超过98%的CD73、CD90、CD105和低于2%的CD34、CD45、CD14、CD79a、HLA-DR,且该细胞具有分化为成骨、成软骨和成脂能力。结论经无动物源培养体系体外扩增的二次贴壁分离方法可以获得大量具有MSCs生物学特性的hUC-MSCs,为其规模化临床应用奠定了基础。     

Characterization of CS/PVA/GO electrospun nanofiber membrane with astaxanthin

Tissue engineering has been widely used in regenerative medicine and tissue engineering scaffolds have become a new research direction for periodontal regenerative repair. We aim to develop a biological scaffold material that can support host immunity and promote periodontal regeneration. In this paper, chitosan (CS)/polyvinyl alcohol (PVA)/graphene oxide (GO)/astaxanthin (ASTA) nanofibers membranes were prepared by electrospinning. The nanofibers were characterized by scanning electron microscopy, infrared spectroscopy, mechanical testing, antibacterial testing and cytotoxicity testing. The CS/PVA/GO/ASTA nanofiber membrane had favorable micro-morphology, good mechanical properties and no cytotoxicity. This preliminary study demonstrates that the CS/PVA/GO/ASTA nanofiber membrane can be used for in vivo and in vitro experiments related to periodontal regeneration. The related mechanism of periodontal regeneration will be evaluated in future studies.     

PLGA/SF blend scaffolds modified with plasmid complexes for enhancing proliferation of endothelial cells

Biomimetic scaffolds have been investigated for vascular tissue engineering for many years. However, the design of an ideal biodegradable vascular scaffold is still in progress. The optimization of poly(lactide-co-glycolide)/silk fibroin (PLGA/SF) blend composition was performed to provide the designed scaffolds with adequate mechanical properties and favorable biocompatibility for the intended application. By systematically varying the weight ratio of PLGA and SF, we could control fiber diameter and hydrophilicity as well as mechanical properties of the fibrous scaffolds. These scaffolds with a weight ratio of PLGA/SF at 70/30 exhibited excellent performance, such as tensile strength of 1.5 ± 0.1 MPa, and elongation at break of 77.4 ± 6.4%. Therefore, PLGA/SF scaffold with a weight ratio of 70/30 was chose as the matrix because it matches at best the mechanical demands for application in vascular tissue engineering. In order to promote the endothelialization of electrospun scaffolds, we used pEGFP-ZNF580 plasmid (pZNF580) complexes to modify the electrospun scaffolds by electrospraying technique. pZNF580 complexes were prepared from pZNF580 and microparticles (MPs) of amphiphilic copolymer methoxy-poly(ethylene glycol)-block-poly(3(S)-methyl-2,5-morpholinedione-co-glycolide)-graft-polyethyleneimine. Negatively charged PLGA/SF fibers adsorbed the positively charged MPs via physical deposition and electrostatic force. Scanning electron microscope image indicated the forming of composite scaffold and MPs did not change fiber’s shape and 3-D structure. Cell culture experiments demonstrated that the scaffolds modified with MPs/pZNF580 complexes could promote human umbilical vein endothelial cell growth and inhibit human umbilical artery smooth muscle cell proliferation. Our results indicated that the composite scaffolds with MPs/pZNF580 complexes could be used as a potential scaffold for vascular tissue engineering.