生物血管基质材料的抗原表达及力学特性

了解胰蛋白酶处理前后版纳微型猪近交系血管的抗原表达及力学特性，为异种移植及猪血管用于血管组织工程基质材料提供资料，取猪颈动脉，胰蛋白酶预处理猪血管前后不同时相点取标本石蜡包埋，采用组织形态学方法结合计算机图像分析，对猪血几何形态及纤维结构进行形态定量学研究，分别测定胰蛋白酶作用前后猪动脉压力直径关系，冰冻切片，用亲和免疫组化法检测α-gal抗原表达。结果表明α-gal抗原仅表达于血管内皮细胞，胰蛋白酶预处理血管后，未见α-agl抗原表达，血管内细胞包括内皮细胞，平滑肌细胞已去除，HE染色未见纤维断裂，血管无明显形变，处理前后血管顺应性相差不显著，提示经胰蛋白酶预处理的猪血管抗原性大大降低，其力学特性相差不显著，可用于血管基质材料。     

生物血管主要组织相容性复合体抗原表达及内皮化的实验研究

为了解胰蛋白酶处理前后版纳微型猪近交系血管的主要组织相容性复合体 (Major histocom patibilitycomplex,MHC)表达及去细胞后重新内皮化情况 ,为异种移植及猪血管用于血管组织工程提供资料 ,取猪颈动脉 ,胰蛋白酶预处理猪血管前后 Western Blot法检测 MHC抗原表达 ,在自行设计制作的新型动力性生物反应器中 ,用原代培养的内皮细胞种植在去细胞血管基质材料表面 ,扫描电镜检测内皮化效果。结果表明 ,胰蛋白酶预处理血管后 ,未见 MHC抗原表达 ,扫描电镜可见血管腔内皮细胞形态正常 ,沿血管长轴分布 ,提示经胰蛋白酶预处理的猪血管 MHC抗原大大降低 ,人内皮细胞能成功内皮化 ,可望构建实用的组织工程血管     

全生物化组织工程血管构建的初步研究

为探索在体外初步构建全生物化组织工程血管,采用以酶消化为主的方法制备猪颈总动脉脱细胞支架,再种植犬胸主动脉的平滑肌细胞,培养四周。经组织学染色和电镜观察显示:在猪颈总动脉脱细胞支架上,犬血管平滑肌细胞大量生长,组织学和电镜结构与正常血管壁结构类似。结果表明,我们初步成功地构建了全生物化组织工程血管,为临床血管替代物的基础研究提供了有意义的实验资料。     

组织工程化血管移植实验研究

目的 通过体外构建、体内培养的方法 构建组织工程化血管,并将其植入动物的股动脉,探讨利用小口径组织工程化血管进行血管移植的可能性.方法 首先用Brdu标记所有种子细胞,将血管平滑肌样细胞和血管内皮样细胞分层种植于胶原包埋PGA的复合支架表面;然后将细胞和支架的复合体种植于动物皮下构建组织工程化血管,并移植到股动脉;最后行HE染色及免疫荧光等相关检查,了解移植后血管的特点及细胞来源.同时以移植物为单纯支架在皮下培养所形成的管状结构作为对照组.结果 移植后组织工程化血管通畅无血栓形成.HE染色见血管壁和正常生理性血管壁的结构相似.可分内膜、中膜和外膜三层.种植2周后Brdu标记细胞的免疫荧光观察证实血管壁细胞来源于所种植的种子细胞.结论 利用骨髓间充质干细胞分化而来的种子细胞和胶原包埋的PGA支架可以构建组织工程化小口径血管,而且可以移植入动物体内.     

犬小口径组织工程化血管移植

目的 研究利用小口径组织工程化血管进行血管移植的可能性。方法 来源于骨髓间充质干细胞分化的血管平滑肌样细胞和血管内皮样细胞分层种植于胶原包埋的PGA复合支架上;将复合体种植于动物皮下构建初级组织工程化血管,然后移植组织工程化血管与股动脉,行相关检查,了解移植后血管的特点及细胞来源。结果 组织工程化血管移植物通畅,管壁无瘤样扩张,血管搏动佳,内膜光滑,无血栓形成。HE染色见血管壁和正常生理性血管壁的结构相似,分内膜、中膜和外膜三层,;免疫荧光观察证实血管壁细胞来源于所种植的种子细胞。结论 组织工程血管可以移植于动物体内,该复合体具有与天然血管相似的结构和功能。     

体外构建组织工程化小口径血管模型

目的体外构建组织工程化人工血管模型。方法将诱导细胞制成细胞ECMgel悬液培养,镜下观察细胞形态;将诱导的平滑肌细胞和内皮细胞悬液分层种植于支架内表面,以荧光显微镜、扫描电镜和HE染色光镜观察。结果在ECMgel凝胶中细胞生长良好;旋转培养的血管模型,质地均匀,内腔面光滑;HE染色可见血管壁三层结构:内皮细胞层、平滑肌细胞层、PGA 交联胶原层;扫描电镜可见内皮细胞融合成片;荧光显微镜观察可见均匀分布的内皮细胞。结论①ECMgel是携带细胞良好的基质;②旋转培养条件可以促进细胞的贴附和分化;③利用体外诱导的种子细胞分层种植可以构建成与天然血管结构相似的血管模型。     

不同细胞浓度种植于脱细胞血管基质上的细胞生长方式*☆

背景：对于组织工程血管而言,如何在平滑肌细胞层上成功获得致密的内皮细胞层是最为关键的。 目的：探索不同细胞种植浓度对构建全生物化组织工程血管的影响。 方法：先将不同浓度(5×105,5×107 L-1)猪血管平滑肌细胞种植在猪脱细胞血管基质上,培养3 d后再将不同浓度(5×105,5×107 L-1)内皮祖细胞接种在平滑肌细胞-血管基质复合体上,构建片状全生物化组织工程材料。 结果与结论：高浓度与低浓度平滑肌细胞在脱细胞血管基质上的细胞生长曲线相似,并且种植在孔板上和在脱细胞基质上的生长曲线亦相似,但低浓度组增殖较慢,覆盖率较低。细胞覆盖率由高到低的顺序为：高浓度内皮祖细胞+含高浓度平滑肌细胞的脱细胞基质>高浓度内皮祖细胞+含低浓度平滑肌细胞的脱细胞基质>低浓度内皮祖细胞+含高浓度平滑肌细胞的脱细胞基质>低浓度内皮祖细胞+含低浓度平滑肌细胞的脱细胞基质,且高浓度内皮祖细胞在脱细胞基质上可形成较为致密的细胞层,呈现出铺路石样生长方式。说明提高细胞接种浓度有利于其在材料表面快速形成致密的细胞层。     

组织工程血管基质的制备与改性

目的：完全脱除猪胸主动脉细胞，对脱细胞血管基质进行改性，增强基质的力学强度，制备组织工程血管支架材料。方法：取家猪的新鲜去除外膜胸主动脉20根，随机分成4组，分别采用胰蛋白酶、TritonX-100及十二烷基硫酸钠（SDS）作为脱细胞试剂对猪胸主动脉进行脱细胞处理，并对脱细胞后的血管基质进行改性，采用HE染色、弹力纤维染色、力学性能测试，以评价脱细胞效果以及材料的力学性能。结果：采用1％TritonX-100单独作用84h既能完全脱除细胞，同时又可完整保留血管基质的三维结构，对胶原纤维、弹力纤维的损伤小，是一种较理想的脱细胞方法。对脱细胞后的基质进行冷冻干燥及真空热交联处理，能有效提高材料的机械强度，冷冻干燥24h后真空120℃下热交联处理12h所获得的材料的机械强度最好，断裂强度可达到1．70MPa。结论：以脱细胞血管基质经冷冻干燥和真空热交联处理后，可以作为血管组织工程的支架材料。     

小口径组织工程血管在羊体内的远期结果

背景：对小口径组织工程血管的研究至今仍主要集中于体外构建上,体内远期结果的研究少有报道。 目的：观察脱细胞猪股动脉支架和绵羊骨髓间质干细胞体外构建的小口径组织工程血管间置于骨髓间质干细胞供体绵羊体内12个月后组织学改变。 方法：将12只成年绵羊随机分为支架组和再细胞化组,支架组将猪股动脉脱细胞后间置于绵羊右侧股动脉;再细胞化组将体外诱导培养的绵羊骨髓间质干细胞种植于脱细胞猪股动脉支架中,经过体外预适应所构建的小口径组织工程血管(直径< 6 mm)间置于骨髓间质干细胞供体绵羊左侧股动脉;将12只绵羊的自体股动脉设为对照组。12个月后切取支架组和再细胞化组的植入物及邻近受体股动脉,行苏木精-伊红染色和扫描电镜检查,观察植入物和对照组股动脉内皮细胞及中层平滑肌细胞密度,采用邻甲酚酞络合酮法测定2组植入小口径组织工程血管和受体股动脉组织的钙含量。 结果与结论：支架组和再细胞化组植入后12个月内管腔均通畅,无明显管道扩张与狭窄,无腔内血栓形成,无管壁明显增厚等改变,管道内表面均已内皮化。但2组植入物管壁均有僵硬和搏动性减弱,尤以支架组植入物管壁僵硬更明显;支架组植入物管道组织钙含量最高(P < 0.01);支架组和再细胞化组管道中层平滑肌细胞密度均低于对照组(P < 0.01)。与支架组植入物相比,再细胞化组植入物组织钙含量较低(P < 0.05),中层平滑肌细胞密度较高(P < 0.01)。说明利用脱细胞猪股动脉支架体外构建小口径组织工程血管时,提高中层平滑肌细胞密度有助于改善小口径组织工程血管的远期功能。     

细胞在小口径组织工程血管中的抗钙化作用

背景：小口径组织工程血管的远期结果研究极少,尚未见到研究组织工程血管分子水平、离子水平远期结果和平滑肌细胞与钙化关系的报道。 目的：利用脱细胞猪股动脉基质作为支架和犬血管壁细胞作为种子细胞体外构建小口径组织工程血管,植入种子细胞供体犬股动脉部位6个月,观察植入物中层平滑肌细胞与钙化的关系。 方法：12只实验犬被随机分为支架组(n=6)和再细胞化组(n=6),自体股动脉被作为对照组;支架组犬接受猪股动脉经脱细胞后的基质支架植入双侧股动脉,再细胞化组犬接受受体血管壁细胞共同培养、联合种植于脱细胞的猪股动脉基质并体外预适应后植入血管壁细胞供体双侧股动脉位置;6个月后测定植入物和对照组股动脉组织钙含量、平滑肌密度和病理学变化。 结果与结论：小口径组织工程血管植入后6个月检查见2组植入物无明显狭窄和扩张,扫描电镜示内表面均已完全内皮化,有管壁僵硬和局部钙化斑块形成,以上改变以支架组植入物更明显。支架组管道组织钙含量显著高于再细胞化组和自体股动脉(P < 0.01),再细胞化组植入物组织钙含量亦显著高于自体股动脉(P < 0.01);病理学检查示再细胞化组植入物平滑肌密度高于支架组(P < 0.01),再细胞化组和支架组植入物平滑肌密度均低于对照组(P < 0.01);超声检查见2组管道植入术后即刻与6个月后舒缩幅度较邻近自体股动脉舒缩幅度小,有部分管道无舒缩功能。结果提示,猪股动脉常规脱细胞方去获得的基质作为支架体外构建组织工程血管时,平滑肌细胞难于迁移至支架中层,中层平滑肌密度低,植入体内6个月后中层平滑肌细胞密度仍低,平滑肌细胞有抗血管钙化作用。     

脱细胞血管组织基质的制备和平滑肌细胞的种植

目的组织工程血管的研制成功将为先天性心脏病外科提供一种新的理想的修补材料,本文研究血管脱细胞组织基质的制备,平滑肌细胞的体外培养和种植方法.方法取成年羊胸主动脉,用胰酶消化去除细胞成分,保存弹性蛋白和胶原蛋白,获得血管脱细胞组织基质,经点状注射法,种植体外培养的小牛平滑肌细胞.结果羊主动脉经脱细胞处理,得到的组织基质最大负载下降20%,最大伸长无显著改变;脱细胞组织基质的胶原蛋白含量与新鲜主动脉相似;基质的纤维结构完整,为网状或多孔状;种植的小牛平滑肌细胞生长良好.结论采用多步骤方法获得的血管脱细胞组织基质可用作为制备组织工程血管的支架,适宜于血管平滑肌细胞的生长.     

豚鼠肠系膜淋巴管平滑肌细胞的培养

目的 :培养豚鼠肠系膜淋巴管平滑肌细胞。方法 :肠鼠肠系膜淋巴管用胰蛋白酶消化二次 ,第一次消化 15min ,除去肠系膜、淋巴管外膜等组织 ,第二次消化中膜的平滑肌 ,3 0～ 45min后 ,吸出未消化的组织 ,获得平滑肌细胞。常规培养、传代。结果 :培养细胞经光镜、电镜和荧光显微镜可观察到平滑肌的典型的形态和结构。结论 :所培养细胞为平滑肌细胞 ,此法可应用于管径较小的淋巴管和血管平滑肌细胞的培养。     

Increased smooth muscle actin expression from bone marrow stromal cells under retinoic acid treatment: an attempt for autologous blood vessel tissue engineering

Vascular replacement in vital organs is sometimes necessary for human life for example because of atherosclerosis. Blood vessel tissue engineering is applied for autologous transplantations to avoid graft rejections. Stem cells are used for blood vessel tissue engineering because they are the origin of smooth muscle cells, endothelial cells and fibroblasts. This paper shows that bone marrow stromal cells (BMSCs) can be induced to differentiate into the early stage of smooth muscle cells by using 0.01 microM retinoic acid. The differentiation of BMSCs to smooth muscle cells was detected by the expression of smooth muscle alpha actin (SM alpha-actin), the earliest smooth muscle cell marker. The SM alpha-actin marker expression was demonstrated using indirect immunofluorescence technique and Western blot analysis. The induction of BMSC to form early stages of smooth muscle cells in this study is appropriate for blood vessel tissue engineering because the early stage smooth muscle cells may be stimulated to develop vascular walls with endothelial cells using a co-culture system.     

Engineering of an elastic large muscular vessel wall with pulsatile stimulation in bioreactor

Tissue engineering offers a new approach for the construction of vascular substitutes in vitro with proper mechanical properties. Although success has been made in the engineering of small blood vessels (<6mm in diameter), it remains a challenge to engineer large vessels (>6mm in diameter) due to their insufficient biomechanical property. In the current study, an elastic large vessel wall (6mm in diameter) was engineered by loading a polyglycolic acid (PGA) unwoven fiber scaffold seeded with smooth muscle cells (SMCs) on a vessel reactor designed with dynamic culture conditions. SMCs were isolated from canine carotid artery and expanded before seeding on a PGA fiber mesh. The cell-seeded PGA mesh was then loaded on a vessel reactor and subjected to pulsatile stimuli. Grossly, an elastic vessel wall was formed after 8 weeks of dynamic engineering. Histological examination showed well-orientated smooth muscle cells and collagenous fibers in the group with dynamic culture. In addition, the phenotype of SMCs was confirmed by positive staining of smooth muscle alpha-actin and calponin. On the contrary, disorganized smooth muscle cells and collagenous fibers were observed in the group under static culture without stimuli. Furthermore, the engineered vessels under dynamic culture exhibited significant improvements on biomechanical property over the one from static culture. Our results indicate that the approach developed in the current work is efficient for large vessel engineering. This approach may also be suitable for the engineering of other tissues with muscular tubular structure.     

Smooth muscle differentiation in cultured human breast gland stromal cells

We analyzed in cultures from the human breast the potential of stromal cells resembling fibroblasts to undergo smooth muscle differentiation. The cellular components of the breast tissue from 10 biopsies were disaggregated by collagenase digestion and further purified by differential centrifugation into suspensions of single cells and intact blood vessels. These two fractions of stromal cells were plated in culture and their phenotypic traits analyzed within 24 hours. During this time, the blood vessel fraction gave rise to stromal cells with smooth muscle differentiation as judged immunocytochemically using monoclonal antibodies to alpha-/gamma-muscle actins, to alpha-smooth muscle actin, to type IV collagen, and to laminin. Furthermore, the cells of this fraction resembled smooth muscle cells based on 2D gel electrophoresis and immunoblotting determination of isoactin content. After 24 hours in culture, the single-cell fraction consisted of an almost pure population of cells not exhibiting smooth muscle differentiation but rather resembling fibroblasts. Maintenance of fibroblast-like cells without smooth muscle differentiation was possible for more than 14 days on chemically defined medium. These cells remained quiescent, as measured by cell quantification and immunoreactivity to the proliferation-associated antigen, Ki-67. Growth of these cells could be stimulated by adding serum at any time during the experimental period. Single-cell fractions from seven biopsies were allowed to grow exponentially in the presence of serum for up to 10 days, and the kinetics of smooth muscle differentiation were monitored immunocytochemically and biochemically. These experiments showed that alpha-smooth muscle actin synthesis was induced in 10 to 80% of the fibroblast-like cells after 4 to 11 days in culture. Both the final number of alpha-smooth muscle actin-positive cells and the onset of synthesis varied with the initial seeding density. Dose-response experiments (at constant cell density) revealed that serum exerted maximal effect at concentrations above 10%. It was therefore concluded that elements of smooth muscle differentiation may arise in non-smooth muscle stromal cells taken directly from human breast tissue.     

The intermediate filament proteins of rabbit vascular smooth muscle: immunofluorescent studies of desmin and vimentin

Summary The localization of desmin and vimentin in rabbit vascular smooth muscle was studied using the fluorescent antibody technique. Desmin and vimentin were present in the smooth muscle cells of the portal anterior mesenteric vein, renal vein and renal artery. All endothelial cells and the smooth muscle cells of the abdominal aorta and main pulmonary artery contained only vimentin. The results suggest that desmin and vimentin are differentially distributed in rabbit vascular smooth muscle and that both may occur in a single blood vessel.     

Perfusion bioreactor for small diameter tissue-engineered arteries

A scaleable perfusion bioreactor has been developed for tissue engineering of small diameter arterial constructs. This modular bioreactor allows for dynamic sequential seeding of smooth muscle and endothelial cells, biomechanical stimulation of cells during culture, and monitoring of tissue growth and maturation. Bovine aortic smooth muscle and endothelial cells were seeded onto porous tubular poly(glycolic acid) nonwoven scaffolds and cultured in the bioreactor under pulsatile flow conditions for up to 25 days. Cell proliferation was more than 3-fold after 4 days, smooth muscle cells expressed differentiated phenotype after 16 days, and collagen and elastin were distributed throughout the construct after 25 days of culture. In bioreactor experiments in which the construct lumen was seeded with endothelial cells by perfusion after 13 days of smooth muscle cell culture, endothelial cell seeding efficiency was 100%, and a confluent monolayer was observed in the lumen within 48 h. These data demonstrate that this perfusion bioreactor supports sequential seeding of constructs with smooth muscle and endothelial cells. Dynamic culture under pulsatile flow leads to cellular expression of differentiated function and extracellular matrix deposition toward the development of tissue-engineered arterial constructs.     

Combining cell sheet technology and electrospun scaffolding for engineered tubular,aligned, and contractile blood vessels

Herein we combine cell sheet technology and electrospun scaffolding to rapidly generate circumferentially aligned tubular constructs of human aortic smooth muscles cells with contractile gene expression for use as tissue engineered blood vessel media. Smooth muscle cells cultured on micropatterned and N-isopropylacrylamide-grafted (pNIPAm) polydimethylsiloxane (PDMS), a small portion of which was covered by aligned electrospun scaffolding, resulted in a single sheet of unidirectionally aligned cells. Upon cooling to room temperature, the scaffold, its adherent cells, and the remaining cell sheet detached and were collected on a mandrel to generating tubular constructs with circumferentially aligned smooth muscle cells which possess contractile gene expression and a single layer of electrospun scaffold as an analogue to a small diameter blood vessel's internal elastic lamina (IEL). This method improves cell sheet handling, results in rapid circumferential alignment of smooth muscle cells which immediately express contractile genes, and introduction of an analogue to small diameter blood vessel IEL.     

骨髓间充质干细胞构建组织工程化小口径血管

目的采用在动物体外构建初级组织工程化血管、体内强化的方法,探讨构建小口径组织工程化血管的可能性.方法体外培养骨髓间充质干细胞(BMSC),用含全反式维甲酸(AT-RA)、双丁酰环磷酸腺苷(db-cAMP)的DMEM-LG培养液和含血管内皮细胞生长因子(VEGF)的培养液诱导BMSC分别向血管平滑肌样细胞和血管内皮样细胞分化.免疫荧光观察平滑肌样细胞β肌动蛋白的表达和内皮样细胞vWF的表达.电镜观察超微结构的改变.诱导的血管平滑肌样细胞和血管内皮样细胞,分层种植于胶原包埋聚乙醇酸(PGA)的复合支架表面,将细胞和支架复合体种植于动物皮下,于植入后第4、8周再次麻醉动物,取出植入皮下的组织工程化血管,行组织学检查、压力实验及免疫荧光检查.结果诱导14 d后,BMSC能够分化为血管平滑肌样细胞和血管内皮样细胞:β肌动蛋白和vWF呈阳性表达,电镜证实细胞出现了相应的形态学改变.人工血管组织学观察见管壁结构清晰.单纯支架组可承受100～150 mm Hg(1mm Hg=0.133 kPa)的血管腔内压力,实验组则均可承受200mm Hg的血管腔内压力不破裂.实验组皮下培养8周Brdu标记细胞的免疫荧光结果显示部分细胞核呈现明亮的黄绿色荧光.结论以动物皮下为生物反应器可构建出组织工程化血管,其大体结构和天然血管相似.