体外构建组织工程化肌腱的初步研究

目的探讨利用可吸收生物材料聚羟基乙酸(polyglycolic acids ,PGA)和肌腱细胞在体外构建组织工程化肌腱的可能性. 方法取肌腱细胞经体外培养、扩增至第2代,与PGA混合培养形成细胞-生物材料复合物,体外培养1周后分组,用U形弹簧给细胞-PGA复合物持续张力后体外培养(n=5)为实验组;没有外加张力的作为对照1组(n=4);单纯PGA作为对照2组(n=3),每2天换液1次.分别于体外培养2、4、6周取组织做组织学、免疫组化检测,同时对第6周的标本进行透射电镜和生物力学检测. 结果第2周时,3组标本在大体、组织学等方面无明显差异,组织学上主要是未降解的PGA纤维.第4周时,实验组和对照1组均有新生肌腱组织形成,HE染色和免疫组化检测均提示胶原纤维形成,而对照2组的PGA已大部分降解.第6周时,对照1组形成的肌腱组织比实验组的粗,透射电镜检测两组标本形成的胶原纤维有周期性横纹,与对照1组相比,实验组形成的胶原纤维沿纵轴排列有序更接近正常肌腱组织,生物力学检测显示实验组的最大应力(1.107±0.327 N/mm2)明显强于对照1组(0.294±0.138 N/mm2)(P<0.05). 结论应用组织工程技术在体外可以构建肌腱组织,施加周期性的应力可能更有利于肌腱组织的形成.     

模拟微重力培养对大鼠甲状旁腺细胞形态及分泌功能的影响

目的模拟微重力培养甲状旁腺细胞,观察细胞形态及细胞分泌功能,以探讨甲状旁腺细胞更为优良的培养方法。方法雄性Wistar大鼠,37只,体质量150~200 g。1%戊巴比妥钠(50 mg/kg)腹腔注射麻醉。手术切取甲状旁腺并经病理学证实。用胶原酶Ⅱ消化获得甲状旁腺细胞后,按培养条件不同分为普通培养组(单纯甲状旁腺细胞静置培养)、聚羟基乙酸(polyglycolic acid,PGA)支架培养组(甲状旁腺细胞接种在PGA支架上静置培养)、微重力培养组(甲状旁腺细胞和PGA支架在模拟微重力环境中共培养)3组。于细胞培养第1、3、5、7天时测量各组细胞培养液中甲状旁腺激素(parathyroid hormone,PTH)的浓度,于倒置相差显微镜下观察甲状旁腺细胞的生长形态、吖啶橙/碘化丙啶染色细胞并计算细胞存活率。结果普通培养组的细胞在第7天时大部分细胞形态良好,部分细胞团中心出现坏死灶。PGA支架培养组在第7天时支架上的细胞大部分沿PGA纤维纵轴方向向两极伸展,相邻支架被细胞外基质连接。微重力培养组的细胞在培养第7天时细胞仍呈圆形,相互聚集成团,并逐渐增大;培养液中的PTH浓度也明显高于PGA支架培养组和普通培养组(P0.05),且存活率也明显高于另外两组(P0.05)。结论模拟微重力培养甲状旁腺细胞,能使细胞保持良好形态,并显著提高细胞活力及存活率,从而为细胞输注移植治疗甲状旁腺功能减退症提供优良的供体细胞,PGA支架可以作为甲状旁腺细胞培养的良好载体。     

利用聚羟基乙酸构建组织工程皮肤的实验研究

目的 以聚羟基乙酸(PGA)为培养支架，用组织工程方法在体外构建双层、有活性皮肤。方法应用酶消化法分别获取人成纤维细胞(FB)和表皮角质形成细胞(KC)，体外扩增培养。接种HF与PGA上1周后，在复合物表面接种KC，共培养1周将复合物置于气-液界面继续培养。于不同时间段取材。行组织学、超微结构及免疫组织化学检测。结果接种表皮角质形成细胞在“FB-PGA”复合物表面1周后可见2～3层连续的HK在复合物上生长良好；4周时PGA已部分降解，表皮分层分化较完善，抗involucrin染色阳性，透射电镜观察可见表皮细胞分层分化良好。细胞的相邻面有桥粒连接。结论 采用组织工程技术可以在体外构建双层、有活性的人工皮肤；组织工程皮肤具有和正常皮肤相似的组织学特征及生化成分。     

肝细胞癌合并肝硬化患者肝脾联合切除术后免疫功能变化的研究

目的　探讨肝细胞癌合并肝硬化患者肝癌切除时联合脾切除术后免疫功能的变化。方法　将 16例肝癌合并肝硬化患者分成 2组 ,即肝癌切除联合脾切除组 ( 7例 )和单纯肝癌切除组 ( 9例 ) ,于术前、术后 2个月取外周血 7ml,采用流式细胞仪检测CD4、CD8、CD4 /CD8,ELISA法检测IL 2、IFN γ、IL 10。　结果　 2组患者术前CD4、CD8、CD4 /CD8、IL 2、IFN γ、IL 10水平差异无显著性 ;术后 2个月 ,切脾组CD4 ( 3 8 2 %± 3 7% )、CD4 /CD8( 1 7%± 0 3 % )高于保脾组CD4 ( 3 2 5 %± 4 0 % )、CD4 /CD8( 1 1%± 0 1% ) ,而CD8( 2 3 7%± 3 7% )低于保脾组CD8( 2 9 4 %± 4 0 % ) (P <0 0 5 ) ;切脾组IFN γ[( 10 4 4± 14 9)pg/ml]、IL 2 [( 98 6± 18 6)pg/ml]高于保脾组 [IFN γ( 70 5± 12 6)pg/ml、IL 2 ( 80 9± 13 5 )pg/ml],而IL 10 [( 5 5 5± 11 2 )pg/ml]低于保脾组 [IL 10 ( 89 4± 10 )pg/ml](P <0 0 5 )。　结论肝癌切除时联合脾切除不但没有降低机体T细胞亚群和Th细胞的平衡 ,反而促进其恢复平衡 ,并改善机体抗肿瘤免疫功能     

构建带内支撑组织工程软骨的实验研究

目的探讨以医用多孔高密度聚乙烯(Medpor)为内支撑外裹聚羟基乙酸(PGA)的支架,接种软骨细胞后,于体内构建Medpor软骨复合体的可能性。方法实验组:以直径3mm的Medpor外裹2mmPGA为支架,接种新生猪关节软骨细胞(5×107/ml),经体外培养2周及裸鼠皮下移植6周后取材,行大体、组织学、Ⅱ型胶原免疫组织化学和生物化学检测;PGA对照组:以直径8mm的单一PGA为支架,细胞接种与培养同实验组;单纯支架组:利用实验组支架,不接种细胞行体内移植。结果实验组:形成大体形态良好的Medpor-软骨复合体,内部的Medpor与外层软骨结合紧密,组织学可见成熟软骨陷窝并渗入Medpor孔隙内部、异染基质、Ⅱ型胶原表达阳性;PGA对照组:形成的软骨存在"空心"现象;单纯支架组:无软骨形成。结论利用Medpor与PGA复合的支架,接种软骨细胞后,可于体内构建出特定形状、结构与组织学良好的Medpor软骨复合体,克服了组织工程软骨的"空心"现象。     

自体骨髓间质干细胞外基质支架在体外软骨组织工程中的应用

 目的 探讨利用自体骨髓间质干细胞外基质(autologous bone marrow mesenchymal stem cell-derived extracellular matrix,aBMSC-dECM)支架体外制备组织工程软骨的可行性。方法 取2周龄新西兰大白兔5只,分离、培养骨髓间质干细胞,原代培养4周,收集其分泌的细胞外基质,制备aBMSC-dECM支架。对支架行扫描电镜和HE染色观察。分离培养自体软骨细胞,植入支架内,48 h后对细胞-支架复合物行Live-Dead染色。分别于种植后1、2、4和6周对细胞-支架复合物(组织工程软骨)进行大体观察、体积测量、HE染色、Safranin-O染色、Ⅱ型胶原免疫组织化学染色、Real-Time PCR检测和抗压强度测试。对照为atelocollagen支架。结果 aBMSC-dECM支架呈三维多孔状海绵样结构,孔隙分布均匀,连通性较好,孔径(304.4±108.2) ?滋m,孔隙率93.3%±4.5%。与atelocollagen支架组比较,aBMSC-dECM支架组组织工程软骨呈乳白色,表面光滑有弹性,随观察时间延长体积逐渐增大,软骨细胞数量、蛋白聚糖和Ⅱ型胶原含量逐渐增多,Ⅱ型胶原及Aggrecan的mRNA持续高表达,抗压强度持续增高。结论 aBMSC-dECM支架有利于维持软骨细胞活性和生物学功能,促进组织工程软骨形成。     

兔再造阴茎模型内构建组织工程化阴茎假体的实验研究

目的 探讨于家兔再造阴茎模型内构建带内支撑组织工程化阴茎假体的可行性及其特点.方法 取成年兔耳软骨分离培养软骨细胞,制备以医用多孔高密度聚乙烯(HDPE)为内支撑,外裹聚羟基乙酸(PGA)的支架(直径3mm、长20mm柱状HDPE,外裹厚2 mm的PGA),细胞-支架复合物体外培养4周.以兔一侧腹壁浅血管为轴型血管的皮瓣形成再造阴茎模型,将经体外培养的细胞-支架复合物分别植入兔即时再造的阴茎模型内和对侧腹壁皮下,在对侧腹壁下同时植入一单纯支架.将植入体分为A组:细胞-支架复合物植入再造阴茎模型内;B组:细胞-支架复合物植入腹壁下;C组:单纯支架植入腹壁下.术后3个月将兔处死取材,标本行大体观察、组织学、组织化学、免疫组化、糖胺聚糖(GAG)含量及生物力学检测.结果 A组与B组取材标本的体积、外形与植入时相近,外层新生组织与内部HDPE结合紧密,经HE染色、Safranin O染色、Masson trichrome染色和Ⅱ型胶原免疫组化检测证实,新生组织为软骨组织,且分布相对均一完整,纤维组织长人较少,少量炎性细胞浸润;C组无软骨组织形成.A组与B组标本各项检测指标(湿重、GAG含量、抗压强度、弹性模量)差异无统计学意义(P>0.05).结论 自体软骨细胞接种于HDPE-PGA复合支架,经体外培养4周后,植于家兔即时再造阴茎模型内继续培养,可成功地构建出具有预制形态、体积、结构与组织学良好的软骨-HDPE复合体(阴茎假体).     

七氟醚增强个培养鼠背根神经节细胞γ—氨基丁酸调控的氯电流

目的　观察七氟醚 ( 0 38至 3× 10 -3 mol/L)对单个培养SD鼠背根神经节细胞 3× 10 -6mol/Lγ 氨基丁酸 (GABA)调控的氯电流影响。方法　采用膜片钳全细胞记录和“Y型管”技术。结果　0 38× 10 -3 ,0 76× 10 -3 ,1 5 2× 10 -3 ,2 2 8× 10 -3 ,3 0 4× 10 -3 mol/L七氟醚分别增强氯电流峰值高度至对照值的 149%± 2 5 % ,2 0 3%± 2 7% ,32 7%± 79% ,331%± 10 9% ,2 43%± 71%。结论　相关临床麻醉浓度的七氟醚能增强培养鼠背根神经节细胞 3× 10 -6mol/LGABA调控的氯电流     

应用骨髓基质干细胞体外构建组织工程化管状软骨的实验研究

目的以骨髓基质干细胞(bone m arrow strom a cells,BMSCs)为种子细胞,接种于管状聚羟基乙酸(PGA)支架上,在体外构建组织工程化管状软骨。方法取长枫杂交仔猪髂骨骨髓,在低糖DMEM完全培养液培养2周,传代后以浓度为5×107/m l细胞悬液均匀接种于管状PGA支架上,以高糖DMEM低血清特定培养液诱导(含胰岛素2 mg/L、转铁蛋白3 mg/L、丙酮酸100mg/L、地塞米松10-7mol/L、TGF-β10ng/L、葡萄糖4.5 mg/m l、2%胎牛血清),连续诱导培养10周,从大体、组织学和Ⅱ型胶原免疫组化对再生组织进行评价。结果6周时染色见BMSCs-PGA复合物表层为2~4层成纤维样细胞组成的软骨膜,下层为较成熟的软骨组织,软骨细胞包埋在软骨陷窝内,有很多散在PGA纤维,而在中间部分,组织量较少,结构较构散。10周时BMSCs-PGA复合物外观呈乳白色软骨样,管壁较厚,有一定的弹性,但中间部管腔塌陷较明显,苏木素-伊红染色见实验组管状BMSCs-PGA和6周时相似,但结构更致密和规则,细胞数量较正常软骨组织少,还可见少量的未降解的PGA纤维。免疫组化证实形成的组织有Ⅱ型胶原分布。结论管状BMSCs-PGA复合物在特定培养液诱导下,在体外能形成管状软骨,这为将来临床应用BMSCs作为种子细胞,修复软骨缺损或构建复合组织气管提供了实验基础和技术参数。     

猪急性创面不同部位组织匀浆液对体外培养的成纤维细胞凋亡的影响及其机制

目的　探讨猪急性创面不同部位组织匀浆液对体外培养的成纤维细胞 (fibroblast,FB)凋亡的影响。　方法　在 6只小型猪的背部造成直径 4 cm的圆形全层皮肤缺损创面 ,用取下的真皮组织进行 FB原代培养。在创面形成后第 15天 ,取创面中心及边缘新生上皮下的组织匀浆 ,离心后将上清液 - 70℃冰箱保存备用。用第 4代 FB进行实验 ,根据培养液的不同分成 7组 : 组 :DMEM+5 %胎牛血清 ; 组 :DMEM+5 %创面中心组织匀浆液 ; 组 :DMEM+5 %创面边缘组织匀浆液 ; 组 :DMEM+5 %创面中心组织匀浆液 +10μg/ ml广谱基质金属蛋白酶抑制剂 (GM6 0 0 1) ; 组 :DMEM+5 %创面边缘组织匀浆液 +10μg/ ml GM6 0 0 1; 组 :DMEM+5 %创面中心组织匀浆液 +10 ng/ ml酸性成纤维细胞生长因子 (acid fibroblast growth factor,a FGF) ; 组 :DMEM+5 %创面边缘组织匀浆液 +10 ng/ ml a FGF。在上述培养液中培养 16 h,均采用同一动物的匀浆液处理该动物来源的 FB(n=6 )。采用 PI和标记 FITC的 Annexin 作为探针 ,利用流式细胞仪检测细胞凋亡。　结果　各组细胞凋亡率分别为 : 组 4 .39%± 0 .4 1% , 组 10 .98%±1.4 2 % , 组 13.4 7%± 1.4 4 % , 组 7.2 %± 0 .4 6 % , 组 12 .1%± 0 .85 % , 组 3.9%± 0 .6 3% , 组 9.8%± 0 .5 0 % ;     

诱导立体网架上成纤维细胞表达成骨表型及其机制的研究

目的探讨诱导条件下的成纤维细胞在三维结构的聚乙醇酸（PGA）网架上表达成骨表型的可行性,以及肿瘤坏死因子α（TNF-α）对成纤维细胞骨形态发生蛋白（BMP）受体表型表达的影响。方法分离、纯化人皮肤成纤维细胞,实验用第2代细胞：（1）种植成纤维细胞于PGA网架上,进行体外旋转培养,并用含TNF-α（50U／ml）和BMP-2（0．1μg／ml）的条件培养液进行诱导。于1d,3、6周后利用倒置相差显微镜、扫描电镜、四环素荧光标记、茜素红染色方法观察细胞生长、骨样组织形成和矿化物沉积情况。上清液生化检测分析成骨性标志物分泌情况;（2）接种成纤维细胞于预置玻片或75cm。培养瓶中,用含TNF-α（50U／ml）的条件培养液进行一次性或连续性干预,采用逆转录聚合酶链反应和免疫组织化学技术,于2．4、6、8d后分别检测BMPⅠ型受体（BMPR-ⅠA和BMPR-ⅠB）mRNA表达及蛋白形成状况。结果诱导3周后三维网架上的成纤维细胞表达成骨表型,分泌成骨细胞特征性标志物：骨钙素（OCN）和骨特异性碱性磷酸酶（B-AKP）;分泌大量细胞外基质形成骨样组织;平面培养发现,TNF-α（50U／ml）连续干预8d可增高BMPR-ⅠB的mRNA表达和蛋白合成。结论三维立体网架上的成纤维细胞在诱导条件下能够向成骨型细胞转化,形成骨样组织,有希望作为一种新的成骨型细胞的种子细胞源;TNF-α为BMP-2的靶向作用提供条件,TNF-α和BMP-2联合应用是调节成纤维细胞表型转化的诱导条件之一。     

血管平滑肌细胞在几种基质膜片上生长情况及三种接种培养方法的比较

目的 筛选适合于组织工程血管构建的可降解基质材料及最佳的接种培养方式。方法 将血管平滑肌细胞接种于几种现有的可降解基质材料上，采用MTT法观察细胞在几种基质材料上的生长情况；比较静态、水平摇床、旋转接种三种接种培养方式。结果 细胞相容性比较好的材料有：PGA、胶原加黏多糖、脱钙骨等。三种接种方法的接种率为：静态1．5％。水平摇床7．2％，旋转接种培养53．5％。结论 PGA、胶原加黏多糖、脱钙骨三种材料适合于组织工程血管再造的应用。而三种接种培养方式以旋转接种培养的效率最高。     

多孔明胶微载体旋转培养hMSC的研究

[目的]观察利用多孔微载体结合旋转细胞培养系统(rotary cell culture system,RCCS)培养人骨髓间充质干细胞(human mesenchymal stem cell,hMSC)对其增殖的影响。[方法]体外分离hMSC,扩增至第2代(P2)后分为两组。实验组采用多孔明胶微载体CultiSpher G在RCCS内进行动态培养,对照组则继续静止培养。应用倒置显微镜、扫描电镜对微载体表面的细胞粘附、生长情况进行观察,并在不同的时相点取样,行细胞计数、MTT检测,了解细胞的增殖情况。[结果]接种24 h后,大部分细胞贴附于微载体表面,随培养时间延长细胞数量增多并分泌大量基质。MTT检测及细胞计数提示细胞对数生长期较静止培养方法延长,达到生长高峰时旋转培养方法收获细胞总量为接种时10.75倍,而静止培养方法为3.19倍;细胞周期检测两种方法差别不大,大部细胞均处在S1期。[结论]Culti Spher G微载体旋转培养系统是体外扩增hMSC的有效方法,利用该系统可为工程化组织的构建提供大量特性稳定的种子细胞。     

Tissue engineered cartilage generated from human trachea using DegraPol scaffold.

OBJECTIVE: To date numerous attempts have been undertaken to conquer the challenging problem of reconstructing long segmental tracheal defects, as yet without lasting success. Recently, employing concepts of tissue engineering in animals, cartilage-like constructs were transplanted in vivo. However, both the feasibility of fabricating tracheal replacements and the use of human tracheal chondrocytes (HTC) for tissue engineering are still under investigation. In this study, we optimized isolation and cultivation techniques for human tracheal cartilage, assessing the feasibility of seeding these cells onto a novel, three-dimensional (3-D) polyester-urethane polymer (DegraPol). METHODS: Human tracheal cartilage was harvested from the trachea of lung donors, digested in 0.3% collagenase II, and the condrocytes serially passaged every 7-9 days. Cells were also cultivated over agar plate during the total 6-8 weeks expansion phase. Thereafter, chondrocytes were seeded onto DegraPol (pore sizes 150-200 microm) with a seeding density of 2.4 x 10(7)/ml, and chondrocyte-polymer constructs maintained during in vitro static culture. RESULTS: HTC displayed stable proliferation kinetics in monolayer culture with positive expression of collagen type II. Following polymer seeding, both cellular proliferation and extracellular matrix (ECM) production, as measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and glycosaminoglycan assays, continued over extended culture. Active growth of HTC on DegraPol was further demonstrated by Alcian blue staining, with the histomorphological appearance of the construct resembling that of native cartilage. Scanning electron microscopy showed chondrocyte growth and ECM synthesis both on the surface and inside the porous scaffold, with a dense cell layer on the surface of the scaffold and a lower cell distribution in the scaffold's interior. CONCLUSIONS: The harvested chondrocytes from human trachea cartilage expand well in vitro and possess the ability to form new cartilage-like tissue when seeded onto DegraPol matrix. However, improved culture conditions are needed to permit cellular growth throughout cell-polymer constructs.     

聚羟基烷酸酯与绵羊骨髓基质干细胞相容性的研究

目的评价聚羟基烷酸酯(PHBV)作为组织工程支架与绵羊骨髓基质干细胞(BMSCs)的生物相容性。方法原代培养绵羊BMSCs,传至2～3代后,接种至PHBV膜和泡沫样三维支架上,光镜和扫描电镜观察细胞形态,计数1、2、6 h时的细胞黏附率;并以接种至培养板上细胞为对照组,每日细胞计数,绘制生长曲线;按培养液量与支架体积10 mL／cm3为标准浓度制备浸提液,并制备标准浓度1／16～16倍的浸提液,以MTT法检测细胞毒性;流式细胞仪分析接种到材料上的细胞周期,计算增殖指数;BMSCs接种于PHBV三维支架上4、8、12 d,以Hoechst33258荧光法定量测定细胞内DNA含量, BCA法测定蛋白质含量。结果第3代BMSCs接种至PHBV膜上2 h后即大部黏附,黏附率75．6％,与对照组相比差异无统计学意义,绘制生长曲线见细胞生长与对照组无差异;MTT法检测见9个浓度梯度的浸提液毒性均为0级;光镜和扫描电镜观察见细胞接种于PHBV膜上2 h后大部分黏附,3 d后伸展良好,呈纺锤形或梭形,在三维支架的孔隙内立体生长,1周开始细胞间连接,3周广泛连接,分泌大量基质;流式细胞分析见接种于材料上的细胞周期无变化;接种至PHBV三维支架上的细胞内DNA、蛋白质浓度与对照组比较无差异。结论PHBV作为BMSCs的组织工程支架材料,具有良好的生物相容性。     

Development of an artificial vessel lined with human vascular cells

OBJECTIVES: Thrombogenity of small-diameter vascular prostheses might be reduced by complete coverage of the luminal surface with vascular cells. We investigated cell seeding on polyurethane vascular prostheses. METHODS: Thirty polyurethane vascular prostheses were divided into 3 groups of 10 each: group A, diameter of 20 mm and gamma-sterilized; group B, diameter of 4 mm and gamma-sterilized; and group C, diameter of 4 mm and ethylene oxide sterilized. Human smooth muscle cells, fibroblasts, and endothelial cells were isolated from saphenous vein segments and expanded in culture. Five polyurethane vascular prostheses of each group were seeded with endothelial cells alone (mean, 4.8 +/- 1.2 x 10(6) cells), and the remaining 5 polyurethane vascular prostheses were preseeded with a mixed culture of fibroblasts and smooth muscle cells (mean, 7.7 +/- 2.3 x 10(6) cells), followed by endothelial cell seeding (mean, 4.4 +/- 0.9 x 10(6) cells). Seven days after cell seeding, the polyurethane vascular prostheses were perfused under a pulsatile flow (80 pulses/min, 140/80 mm Hg, and 120 mL/min) for 2 hours. Specimens were taken after each seeding procedure both before and after perfusion and then examined both with a scanning electron microscope and immunohistochemically. RESULTS: Isolated endothelial cell seeding revealed better initial adhesion in groups A and B than in group C (63% vs 33%). After 7 days, the cells had covered approximately 80% of the luminal surface in groups A and B, whereas group C cells rounded up and lost adhesion. After perfusion testing of group A and B prostheses, only 10% of the surface was still covered with endothelial cells. Preseeding with the mixed culture again revealed a better initial adhesion in groups A and B compared with that in group C (76% vs 41%). In groups A and B endothelial cell seeding (adhesion, 72%) resulted in a confluent endothelial cell layer. The results of immunohistochemical staining were positive for collagen IV, laminin, CD31, and Factor VIII. In group C only isolated cells were found after each seeding procedure, which rounded up and vanished during the next days. Perfusion testing of group A and B prostheses revealed that the confluent cell layer remained stable, with only small defects (<10% of the surface). The cells stained positivively for endothelial nitric oxide synthase. CONCLUSION: Seeding of a mixed culture out of fibroblasts and smooth muscle cells resulted in improved endothelial cell adhesion and resistance to shear stress. This outcome was caused by an increased synthesis of extracellular matrix proteins. Cell attachment was better on gamma-sterilized polyurethane vascular prostheses compared with on those undergoing ethylene oxide sterilization.     

Improving endothelial cell retention for single stage seeding of prosthetic grafts: use of polymer sequences of arginine-glycine-aspartate.

OBJECTIVE: single stage seeding within the timeframe of a typical vascular operation has not been successful. One reason for this is poor cell adherence to the graft lumen once exposed to pulsatile blood flow. In this study we have carried out investigations with the use of two different fibronectin-based peptides, fibronectin-like engineered protein polymer (FEPP) which contains multiple copies of arginine-glycine-aspartate (RGD) and fibronectin adhesion promoting peptide (FAPP) to improve cell adherence. MATERIALS AND METHODS: FAPP and FEPP were coated onto native polyurethane and heparinised polyurethane grafts. The grafts were then seeded for either 1 or 2h with human umbilical vein endothelial cells (HUVEC). After the incubation period the cells were washed off and cell retention was calculated. Cell metabolism was measured using Alamar Blue, and confirmed with scanning electron microscopy (SEM). RESULTS: heparinised grafts coated with FEPP showed the best cell retention after both 1 and 2h seeding (80+/-4% vs 81+/-3%). This graft had no significant difference in cell retention after both times whilst all the other grafts had better cell retention after a 2h seeding. The Alamar blue and SEM results confirmed cell viability and function for all graft types. CONCLUSION: heparinised graft coated with FEPP allows significant cell retention after only 1h of seeding and shows promise for single stage seeding.     

A hybrid compliant vascular graft seeded with microvascular endothelial cells extracted from human omentum

We report the development of a hybrid vascular graft using an innovative compliant poly(carbonate-urea)urethane unlike any previous polyurethane MyoLink as a permanent scaffold. The engineered graft has a hierarchical arterial structure: a monolayer of oriented microvascular endothelial cells (MVECs) and 3-D matrix (human collagen Type 4/dermatan sulfate) bonded onto MyoLink. The grafts' clinical feasibility was evaluated by determining optimal MVEC seeding density, incubation time, viability, and adhesion of these cells when exposed to arterial shear stress. MVECs from human omentum were isolated by a new centrifugation protocol, radiolabeled and seeded onto hybrid graft 2 to 18 x 105 cells/cm(2) for 24 h at 37 degrees C and for 1 to 5 h at 6 x 105 cells/cm(2), washed 3 times, and gamma counted. Qualitative assessment of seeding density/incubation was also undertaken with scanning electron microscopy (SEM) and viability tested with a modified Alamar Blue assay. A pulsatile flow phantom was used to subject the hybrid graft (200 mm length, 5 mm internal diameter) seeded with radiolabeled MVECs (6 x 105 cells/cm(2) at 2 h) to arterial shear and dynamic scintigraphy images acquired in real time using a nuclear medicine gamma camera system during 14 h of perfusion (n = 6). The optimal seeding density was 6 x 105 cells/cm(2), and qualitative SEM confirmed this. Incubating cells for 2 h produced significantly greater cell attachment than was seen for 1 h incubation (p < 0.05), and there was no significant difference in adhesion between cells incubated over the 2 h. Exposure of grafts to acute shear stress resulted in significantly higher proportions of initial cells attached to hybrid grafts compared to native MyoLink grafts (67 +/- 3% versus 55 +/- 2%, p < 0.001). As shown here, tissue engineering of native Myolink grafts significantly reduces the seeding density and incubation time to produce a monolayer onto which cells adhere to better.     

Basic fibroblast growth factor coating and endothelial cell seeding of a decellularized heparin-coated vascular graft

The objective of this study was to determine the effect of basic fibroblast growth factor (bFGF) coating on endothelial cell seeding and proliferation on a decellularized heparin coated vascular graft and to determine the retention of seeded cells on the graft under flow conditions. Disks of heparin coated decellularized grafts were incubated for 24 h as controls or with bFGF. Human microvascular endothelial cells (HMECs) or canine peripheral blood endothelial progenitor cells (CEPC) were seeded onto the disks and incubated for 96 h or 48 h, respectively. HMECs were also seeded onto the luminal surfaces of two heparin-coated decellularized grafts for 3 h. One graft was placed in a perfusion culture system and cultured for an additional 6 h with flow and pressure. After culturing, there were 4.7 +/- 1.4 cells/mm(2) HMECs on control grafts and 11.4 +/- 1.4 cells/mm(2) in bFGF treated grafts (P < 0.05). Likewise, with CEPCs, there were 14.8 +/- 4.8 cells/mm(2) in control grafts and 33.3 +/- 7.3 cells/mm(2) in bFGF treated grafts. After only 3 h of cell attachment, 60% of HMECs were retained in the intact graft exposed flow relative to the static control graft, which is an acceptable level. These data demonstrate that bFGF coating on the heparin bound decellularized grafts significantly increases both HMEC and dog EPC proliferation and that seeded cells are stable under perfusion conditions.     

采用组织工程方法体外构建血管模型的初步实验研究

目的　探索体外构建组织工程化人工血管的可行性。方法　通过酶消化法分离牛主动脉血管内皮细胞,培养、传代,纯化。采用交联胶原包埋处理聚羟基乙酸纤维无纺网(PGA),将第3～7代血管内皮细胞接种于上述材料上,通过特制的旋转装置,缓慢动态旋转培养10天,使内皮细胞在管腔内表面附着生长,扫描电镜观察,采用6-酮-前列腺素-1α放射免疫测试药盒测定管形材料中内皮细胞释放前列环素量。结果　培养血管内皮细胞VIII因子相关抗原抗体染色呈阳性,内皮细胞在管腔内表面贴附良好,细胞之间融合成片,经10天培养,形成较完整内膜层,覆盖率为(91.2±1.5)%,前列环素生成率为（4.6±0.5）μg/cm