ENDOTHELIAL CELL CULTURE AND SEEDING OF PROSTHETIC VASCULAR GRAFTS: AN EXPERIMENTAL STUDY

Current synthetic vascular prostheses do not acquire lining of vascular endothelium in humans or dogs. Endothelial seeding of vascular grafts has been proposed as a means of reducing the thrombogenicity of these grafts. We examined feasibility of cultivating endothelial cells (EC) by tissue culture technique and their subsequent seeding onto small diameter polytetra fluoroethylene (PTFE) grafts. Twenty adult dogs underwent common carotid artery interposition with 4 mm PTFE grafts. Ten dogs received seeded and the remaining ten received unseeded grafts. Grafts were removed at 4 and 12 weeks and their gross/morphological features compared. Cumulative patency rates for seeded grafts were 70% as compared to unseeded ones 30%. Seeded grafts were completely surfaced with a mono-layer of endothelium by 4 weeks. Small graft patency appears to be related to the establishment of an endothelial surface, the development of which is clearly facilitated by seeding with autogenous endothelium.KEY WORDS: Endothelial cell seeding, Vascular grafts     

CD34+干细胞的分化及其在人工血管内皮化中的应用

目的:探讨外周血、骨髓和脐血中CD34 细胞作为人工血管内皮化的种子细胞来源问题.方法:采集犬外周血和骨髓及人脐血,经免疫磁珠分离出内皮细胞祖细胞,内皮细胞生长因子(VEGF)诱导分化为内皮细胞并扩增,光镜、扫描电镜和免疫细胞化学鉴定;将培养细胞种植于人工血管,扫描电镜观察.结果:经流式细胞仪测定,分离后的细胞中CD34 细胞:外周血(26.30±2.42)% 、骨髓(41.84±3.65)%、脐血(74.62±4.46)%,骨髓和脐血中CD34 细胞数量明显多于外周血,且增殖、分化能力强;CD34 细胞培养2周细胞长满瓶底并达到增殖高峰,细胞呈"铺路石"状排列.Ⅷ因子,CD31免疫细胞化学染色均为阳性,透射电镜细胞胞浆内可见W-P小体;扫描电镜下,内皮细胞平铺于人工血管表面,成单层排列,细胞排列无轴向性.结论:外周血、骨髓和脐血经免疫磁珠分离系统可分离出CD34 细胞,CD34 细胞经VEGF诱导可定向分化为内皮细胞.骨髓和脐血CD34 细胞可作为人工血管内皮化的种子细胞来源.     

血管平滑肌细胞和内皮细胞联合种植生物可降解材料的组织工程血管研究

目的探讨应用组织工程学方法构建人工血管片。方法应用聚(乙交酯/丙交酯)二元共聚物(PLGA)无纺网织片作为细胞支架,体外逐层顺序种植血管平滑肌细胞、内皮细胞制作组织血管片。免疫组织化学方法鉴定平滑肌细胞、内皮细胞;扫描电镜观察组织血管片生长情况;放射免疫方法检测内皮细胞分泌内皮素、前列腺素E的功能。结果免疫组织化学染色鉴定平滑肌细胞肌动蛋白染色阳性,内皮细胞凝血Ⅷ因子相关抗原染色阳性。组织血管片在扫描电镜下显示出细胞融合,连接成片。放射免疫方法可以检测出平滑肌细胞和内皮细胞联合培养的组织片分泌内皮素(229pg/ml±34pg/ml),分泌前列环素的代谢产物6-酮-前列腺素F1α(402pg/ml±108pg/ml)。结论应用PLGA无纺网织片作为细胞支架,体外逐层种植血管平滑肌细胞、内皮细胞制作的组织血管片,基本具备正常血管组织基本形态结构和功能。     

Comparative study on seeding methods of human bone marrow stromal cells in bone tissue engineering

Background In general the traditional static seeding method has its limitation while the dynamic seeding method reveals its advantages over traditional static method. We compared static and dynamic seeding method for human bone marrow stromal cells (hBMSCs) in bone tissue engineering.Methods DNA assay was used for detecting the maximal initial seeding concentration for static seeding. Dynamic and static seeding methods were compared, when scaffolds were loaded with hBMSCs at this maximal initial cell seeding concentration. Histology and scanning electron microscope(SEM) were examined to evaluate the distribution of cells inside the constructs. Markers encoding osteogenic genes were measured by fluorescent RT-PCR. The protocol for dynamic seeding of hBMSCs was also investigated.Results DNA assay showed that the static maximal initial seeding concentration was lower than that in dynamic seeding. Histology and SEM showed even distribution and spread of cells in the dynamically seeded constructs, while their statically seeded counterparts showed cell aggregation.Fluorescent RT-PCR again showed stronger osteogenic potential of dynamically seeded constructs.Conclusion dynamic seeding of hBMSCs is a promising technique in bone tissue engineering.     

Human vascular smooth muscle cells and endothelial cells cocultured on polyglycolic acid (70/30) scaffold in tissue engineered vascular graft

Background Current prosthetic, small diameter vascular grafts showing poor long term patency rates have led to the pursuit of other biological materials. Biomaterials that successfully integrate into surrounding tissue should match not only the mechanical properties of tissues, but also topography. Polyglycolic acid (70/30) has been used as synthetic grafts to determine whether human vascular smooth muscle cells and endothelial cells attach, survive and secrete endothelin and 6-keto-prostaglandin F1α (6-keto-PGF1α).Methods Endothelial cells and smooth muscle cells were isolated from adult human great saphenous vein. They were seeded on polyglycolic acid scaffold in vitro separately to grow vascular patch (Groups A and B respectively) and cocultured in vitro to grow into vascular patch (Group C). Smooth muscle cells and endothelial cells were identified by immunohistochemical analysis and growth of cells on polyglycolic acid was investigated using scanning electron microscopy. The levels of endothelin and 6-keto-PGF1α in the culturing solutions were examined by radioimmunology to measure endothelial function. Results Seed smooth muscle cells adhered to polyglycolic acid scaffold and over 28 days grew in the interstices to form a uniform cell distribution throughout the scaffold. Then seed endothelial cells formed a complete endothelial layer on the smooth muscle cells. The levels of endothelin and 6-keto-prostaglandin F1 alpha in the culturing solution were (234±29) pg/ml and (428±98) pg/ml respectively in Group C and (196±30) pg/ml and (346±120) pg/ml in Group B; both significantly higher than in Groups A and D (blank control group, all P<0.05 ). Conclusions Cells could be grown successfully on polyglycolic acid and retain functions of secretion. Our next step is to use human saphenous vein smooth muscle cells and endothelial cells to grow tubular vascular grafts in vitro.     

Experimental Study on Modified Treatment andEndothelialization of Bovine Pericardial Valves

Duringthepast20years,bovinepericardialheartvalvestreatedwithglutaraldehyde(GA)havebeenwidelyusedinclinicalpracticeduetoitsexcellenthemodynamicproperties,lowthrombogenicityandfreedomfromsubstantialstructuraldegenerationduringthefirstyearafterimplantation.Buttheapplicationofthisvalveswerelimitedbecauseofitslatefailure.PreviousstudiessuggestedthatcalcificationwasamainreasonforbioprostheticalheartvalvesfaiL"re,andthetreatmentofGAisanimper.ttautcontributingfactorofcalcificationll].ThetreatmentofG…     

骨髓基质细胞与壳聚糖-聚磷酸三钙复合材料的生物相容性

目的：研究壳聚糖-聚磷酸三钙复合材料对离体培养的骨髓基质细胞黏附及增殖的影响,探讨二者的生物相容性,为骨组织工程中生物材料的选择提供实验依据。 方法：采用冻干法制备壳聚糖-聚磷酸三钙复合材料,培养兔骨髓基质细胞,传代扩增后接种到材料表面,体外继续培养,用倒置光学显微镜、扫描电镜观察细胞的黏附和生长情况,用MTT方法检测种植后2、4、6及8 d细胞的增殖情况。 结果：种植2 d后细胞呈梭形纤维细胞样,平均每100倍视野下,有生物材料的实验组与无材料的对照组胞数分别为(360±20)个和(76±18)个,二者比较差异有显著性（P＜0.01）。MTT法检测结果显示,两组细胞均保持持续增殖。且实验组增殖最快,接种后2、4 、6及8 d光吸收值与对照组比较,差异均有显著性（P＜0.01）。扫描电镜下可见材料呈多孔网状结构,兔骨髓基质细胞紧密贴附在材料表面,细胞可沿材料的孔隙活跃生长。 结论：壳聚糖-聚磷酸三钙复合材料能促进骨髓基质细胞的增殖;兔骨髓基质细胞与复合材料具有良好的生物相容性,可作为骨髓基质细胞的载体应用于组织工程。     

具有良好细胞相容性的脱细胞血管支架的制备

目的制备具有良好细胞相容性的脱细胞血管支架材料，为构建组织工程血管奠定基础。方法用含有脱氧胆酸钠、SDS、EDTA的低渗液和等渗液以及核酸酶对猪的胸主动脉进行脱细胞处理．使用HE、Movat五色法、DAPI染色及电镜观察处理结果。以MTT法分析脱细胞溶液的细胞毒性，通过种植内皮和平滑肌细胞，分析支架的细胞相容性。结果上述方法使用的脱细胞溶液细胞毒性低；经该法处理后，血管的细胞成分完全去除，胶原纤维、弹性纤维、糖蛋白等主要基质成分充分保留，组织结构完整，内皮细胞和平滑肌细胞可以在上面黏附生长，形成完整的细胞层。结论利用脱氧胆酸钠、SDS、EDTA和核酸酶联合消化的方法．可制备出基质成分充分保留、细胞相容性优良的脱细胞血管支架．适于构建组织工程血管。     

骨髓间质干细胞和脱细胞基质构建组织工程血管的动物实验

目的以骨髓细胞作为种子细胞,血管脱细胞组织基质作为支架、构建组织工程血管。方法分离小猪骨髓单个核细胞,分别置于内皮细胞培养液和平滑肌细胞培养液中培养,观察分化细胞的抗原标志;多步骤法制备犬血管脱细胞组织基质,将未经分化的骨髓单个核细胞种植于脱细胞基质构建组织工程血管,移植至骨髓供体小猪,代替部分肺动脉血管。结果小猪骨髓细胞经内皮细胞培养液或平滑肌细胞培养液培养后,分别出现成熟内皮细胞或平滑肌细胞的形态学改变,并分别表达内皮细胞或平滑肌细胞的特异性抗原;组织工程血管移植100d后管腔通畅,无血栓、钙化或动脉瘤形成,内皮细胞和平滑肌细胞分化、增殖良好;移植血管的最大负载为2．76N,最大伸长度为20．31mm。结论骨髓单个核细胞复合脱细胞基质可成功构建组织工程血管。     

可降解外套管抑制自体移植静脉内膜增生的研究

目的:研究可降解的外套管束缚自体移植静脉对减轻桥静脉内膜、中层增厚的作用,探讨其防止或延缓桥静脉再狭窄的机制。方法:建立犬自体股静脉至股动脉的端-端静脉移植模型,其中一侧桥静脉外加聚乳酸-聚乙醇酸(PLGA)做成的圆柱形外套管(外套管组),对侧单纯静脉移植作为对照组。超声多普勒观察在体移植血管的通畅性。术后2、4、6、8、24周再次手术取桥静脉。光镜观察移植静脉中段内膜、中层厚度,胶原、弹力纤维变化;透射电镜观察静脉壁基底膜、细胞排列、细胞增殖、凋亡,细胞骨架结构,细胞外基质(ECM)变化等;免疫组化观察静脉壁细胞增殖、凋亡及分布。结果:术后40条静脉均通畅,吻合口无狭窄。对照组术后2周见内膜、中层增厚;随时间延长,增厚更加明显。外套管组术后2周内膜、中层稍增厚;4~24周持续增厚,但各时间点均明显小于对照组(P<0.05,P<0.01);对照组术后2周见中层平滑肌、外膜弹力纤维断裂严重,而外套管组无明显断裂。新生静脉内膜由平滑肌细胞(SMCs)和大量ECM构成,中层主要为SMCs和ECM;术后2周,静脉壁细胞增殖和凋亡率都较高,主要分布在内膜和中层,外套管组明显低于对照组(P<0.01),4周以后两者显著下降,凋亡率高于增殖率(P<0.05)。结论:可降解外套管在术后早期可保持静脉结构的完整性,减轻管壁结构破     

人脐静脉内皮细胞用于构建组织工程心脏瓣膜的实验研究

目的：以人脐静脉血管内皮细胞(HUVECs)为种子细胞，体外构建组织工程心脏瓣膜(TEHV)，并对其分泌纤溶物质——组织型纤溶酶原激活物(t-PA)及其抑制物(PAI-1)的作用进行观察。方法：获取并扩增HUVECs，种植在猪脱细胞主动脉瓣叶上，体外静态构建TEHV。观察内皮细胞的形态学变化和生长状况。收集瓣膜培养液，采用发色底物法检测瓣膜内皮细胞分泌的t-PA和PAI-1活性。结果：以猪脱细胞主动脉瓣叶作支架，HUVECs做种子细胞，体外成功构建TEHV，种植的HUVECs在瓣膜表面长成一层连续的细胞层，生长状态良好。瓣膜表面的内皮细胞能够分泌纤溶物质t-PA和PAI-1。结论：HUVECs种植在猪脱细胞瓣膜支架上可以构建TEHV，且瓣膜内皮细胞能够分泌t-PA和PAI-1。     

Morphological Characteristics of the Endothelial Ceils of Corneas of Rabbits Preserved by Desiccation

The endothelial cells of the corneas of rabbits preserved by desiccation for vari-ous duration showed no hexagonal form of normal cells but broad bean-like nuclci afterdouble staining with alizarin red and trypan blue.Scanning electron microscopic examina-tion revealed interdigitation at the intercellular junction together with intercellularvacuoles and cellular edema.Transmission electron microscopic examination showedincomplete nuclear membrane,karyopyknosis,vacuoles and remnant organellae in thecytoplasm.Partial penetrating keratoplasties were done in 37 rabbits(37 eyes)with pre-served corneas.The grafts remained transparent for a minimum of 4 days to a maximumof over 1 year,and 7 grafts remained transparent for more than 30 days.Staining ofthe transparent grafts revealed normal hexagonal endothelial cells in mosaic arrange-ment.We assumed that after transplantation of the preserved cornea the endothelial cellsregained their activities owing to the nutrient effect of the aqueous humor.     

体外扩增恒河猴自体内皮细胞人工血管移植研究

目的为自体内皮细胞移植的临床应用奠定理论基础。方法将恒河猴表浅静脉内皮细胞在体外培养１３．９±１．４天;扩增培养的自体细胞衬里于用纤维蛋白胶预衬的膨体聚四氟乙烯（ｅＰＴＦＥ）人工血管,继续培养９天。１５只恒河猴随机分为两组。实验组（１０只）用衬有自体静脉内皮细胞的人工血管置换骼总动脉;对照组（５只）用未衬里内皮细胞的人工血管置换。结果细胞数增加１４８±８９倍。所培养的细胞为二倍体细胞,纯度９９％。原代及传代细胞培养上清液中６－酮－前列腺素Ｆ１ａ（６－ｋｅｔｏ－ＰＧＦ１ａ）和ＶｏｎＷｉｌｅｂｒａｎｄ因子（ｖＷＦ）含量差异无显著意义。细胞种植后２小时和９天,人工血管腔面见一层均匀的基质,基质表面有一层连续的内皮细胞单层。内皮细胞紧密排列。呈梭状,形态饱满。实验组除１只于术后１２小时因近端吻合口破裂腹腔内出血死亡,１只人工血管于术后４周阻塞外,其余８只恒河猴人工血管术后４周均保持通畅,内膜下组织厚度为８０±１２μｍ;对照组５条人工血管全部阻塞。结论自体内皮细胞移植可有效地提高人工血管的通畅率。     

恒河猴自体静脉内皮细胞种植ePTFE人工血管后置换髂总动脉的实验研究

:15只恒河猴随机分为 2组 :实验组 10例 ,其人工血管均经自体静脉内皮细胞种植 ;对照组 5例 ,其人工血管均未经任何处理。实验组、对照组手术方法相同。将长 6cm、直径 3mmePTFE(膨体聚四氟乙烯 )人工血管近端与腹主动脉行端 -侧吻合、远端与髂总动脉行端 -端吻合。术后当日、1周及 4周 ,所有恒河猴均行静脉法数字减影造影术 ,术后 4周行彩色B型超声波检查 ,然后取出所有对照组及 9例实验组恒河猴腹腔内之人工血管 ,标本送光镜、扫描电镜及透射电镜检查。结果表明 ,术后 4周 ,对照组人工血管均不通畅 ,而实验组有 8条人工血管通畅良好。B超见全部对照组及 1例实验组人工血管腔内形成血栓 ,实验组 8条通畅之人工血管均保持较高的血流量。扫描电镜见实验组全部人工血管腔内均有一层完整融合的内皮细胞单层 ,而对照组人工血管腔内未见有内皮细胞。     

组织工程心脏瓣膜构建中内皮细胞增殖的影响因素

目的:研究影响种植在脱细胞猪主动脉瓣叶上的新生牛主动脉内皮细胞(BAECs)增殖的因素。方法:以脱细胞猪主动脉瓣叶为支架,在其上种植BAECs。实验分3组:A组单次种植和多次重复(间隔24 h)种植内皮细胞,细胞总数相同;B组细胞培养液含和不含内皮细胞生长物质(ECGS);C组种植前分别以PBS和 FCS预处理12 h;通过细胞计数,观察种植细胞增殖的变化。结果:多次重复连续种植较单次种植的细胞数明显增加(P<0.05);培养液中加ECGS组细胞计数明显比未加ECGS组高(P<0.05);细胞种植前以FCS浸泡者比以PBS浸泡者细胞数增加(P<0.05)。结论:单独种植内皮细胞于脱细胞猪主动脉瓣叶上时,将瓣叶支架以FCS浸泡,培养液中加ECGS,多次重复、间隔24 h种植细胞能明显促进内皮细胞的增殖。     

3D打印明胶海藻酸钠凝胶支架对人牙髓细胞的黏附增殖作用

目的 探讨3D打印的明胶海藻酸钠凝胶支架对人牙髓细胞(HDPCs)的细胞毒性,及不同接种方法对细胞黏附生长的差异.方法 组织块酶消化法分离培养HDPCs,利用Bioplotter三维生物打印机进行明胶海藻酸钠凝胶支架的3D打印,选取第3代HDPCs,Cell Counting Kit-8试剂检测不同浓度材料浸提液对细胞增殖的影响,扫描电镜观察、台盼蓝染色计数比较直接滴加低浓度细胞悬液与高浓度细胞浓缩液接种法,对细胞在材料表面黏附与增殖作用的差异.结果 不同浓度的材料浸提液均对HDPCs细胞毒性为0,具有促进细胞增殖的作用.HDPCs在3D打印的明胶海藻酸钠凝胶支架上生长良好,先滴加高浓度的细胞浓缩液可以更有效的促进细胞对材料的黏附,5d后材料表面的细胞计数结果较滴加低浓度细胞悬液显著增加,差异具有统计学意义(P＜0.05).结论 3D打印的明胶海藻酸钠凝胶支架具有良好的生物相容性,具有促进HDPCs增殖的作用,可作为牙再生的支架材料,选择高浓度细胞浓缩液接种细胞更有利于细胞黏附.     

人脐静脉内皮细胞的培养及鉴定

目的　探讨新生儿脐静脉内皮细胞的培养及鉴定方法 ,建立血管内皮细胞培养模型 ,为体外研究血管内皮细胞提供实验手段。方法　采用胶原酶Ⅱ灌流消化法培养人脐静脉内皮细胞 ,当原代培养细胞 80 %以上汇合后 ,用胰蛋白酶消化传代 ;根据细胞生长特点、形态特征、细胞表型和分泌蛋白流式细胞术 (FM术 )免疫荧光检查对细胞进行鉴定。结果　种植在培养瓶中的内皮细胞 12小时贴壁生长 ,48～ 72小时生长最快 ,7～ 10天汇合。内皮细胞呈接触抑制生长、呈鹅卵石样外观 ,FM术检测CD3 1和VⅢ -R -Ag均为阳性表达。 结论　消化酶灌注脐静脉消化内皮细胞是获取血管内皮细胞的一种好方法 ,可靠性大 ,成功率高 ,可以构建体外研究血管内皮细胞的模型     

直接染色法观察组织工程心脏瓣膜构建中种植细胞的生长

为了快速观察组织工程心脏瓣膜构建中种植细胞的生长状况，采用去细胞猪主动脉瓣叶为支架，将新生牛主动脉内皮细胞种植于其上，第3、7天取瓣叶行H－E直接染色，并以常规切片H－E染色和扫描电镜作为对照。结果发现，该3种方法观察所见细胞第3、7天的生长疏密状况基本同步。提示，瓣叶H－E直接染色可方便快捷地观察种植细胞的生长状况。