血管内皮细胞促进组织工程骨血管化的研究进展

组织工程骨的血管化问题严重制约了大块组织工程骨的发展和运用.血管内皮细胞对组织工程骨血管化具有重要促进作用,根据血管化过程中血管内皮细胞的来源不同,将血管化过程分血管发生和血管生成两种形式.成骨细胞和血管内皮细胞共同培养时可以相互促进生长.目前对组织工程骨血管化进行了血管内皮细胞新的来源,细胞混合培养和体内试验等方面的探索.     

间充质干细胞诱导分化为血管内皮细胞的研究进展

间充质干细胞(Mesenchymal stem cells,MSCs)是一种起源于中胚层的成体干细胞,可以向所有中胚层来源的细胞及部分外胚层来源的细胞分化,且MSCs具有来源广泛、易于体外分离培养、免疫原性低,移植后无免疫排斥反应和易于基因转染等特点。血管内皮细胞属于中胚层细胞,MSCs可定向诱导分化为血管内皮细胞,在体外构建成理想的血管移植物,用于心脏组织工程瓣、血管组织工程以及再生医学领域。本文就间充质干细胞的生物学特性及诱导分化为内皮细胞的相关研究以及研究中存在的问题作一综述。     

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     

小口径生物型人工血管移植后的血管内皮再生

背景：经过生物化改造的人工血管特性更接近人体血管,移植后自体化程度也较高,但人工血管的内皮再生是解决血管长期通畅的关键。 目的：观察新型小口径生物型人造血管移植后不同时期移植材料的组织相容性及移植血管壁内膜再生的组织病理学变化。 方法：建立犬颈总动脉-人造血管端端连续缝合的动物模型。 结果与结论：①光镜：移植后12周于吻合口处见新内膜表面有不连续的内皮细胞生长;移植后6个月通畅的整段管腔内面均可见内皮细胞生长;移植后1.5年管腔通畅,部分内膜组织呈慢性炎症表现。②电镜：移植后12周新生血管内皮细胞排列规则,从吻合口向移植血管中段爬行;移植后6个月内皮细胞从吻合口向移植血管中段爬行,移植血管中段呈跳跃式片状生长的内皮细胞群落,细胞排列更致密,形态更接近成熟血管内皮细胞;移植后1.5年整段血管内壁均有致密内皮细胞覆盖,部分内膜组织呈慢性炎症表现。说明新型小口径生物型人造血管新生内皮形成早,血管内膜重构能力强,生物相容和稳定性好。     

构建组织工程化血管的内皮细胞来源

获得足够数量和功能正常的种子细胞是组织工程构建的基本前提。组织工程化血管构建的种子细胞包括血管内皮细胞、平滑肌细胞和成纤维细胞。仅就维持血管通畅方面起主要作用的血管内皮细胞来源及其近期相关研究予以综述。     

新的组织工程种子细胞--内皮祖细胞

内皮祖细胞作为一种在血管领域的干细胞在组织工程中有着广阔的应用前景.内皮祖细胞取材方便,参与新生血管的形成,具有成熟内皮细胞相似的特性.因此,内皮祖细胞可能是组织工程血管、血管植入物再内皮化以及构建组织工程器官血管网络的种子细胞的理想来源.简单介绍了内皮祖细胞的来源、特性以及体外扩增技术,并对其在组织工程中应用的研究进展做一回顾.     

组织工程化血管构建中有关VEGF和bFGF的研究进展

内皮细胞是血管组织工程的重要种子细胞,小肠粘膜下基质(SIS)的生物活性和力学特性日益引起人们的关注,细胞因子的生物活性成分及其在组织工程化血管构建中的作用可以从不同角度、不同水平进行研究.本文主要从以下两个方面进行综述:SIS中所含生长因子的含量、类型和分布;内皮细胞中碱性成纤维细胞生长因子和血管内皮细胞生长因子的分泌情况,并着重对剪切力作用下内皮细胞分泌活性的变化进行综述,从而为组织工程化血管的基础研究提供参考.     

以线栓法构建脑梗死模型大鼠的微血管新生及电针干预效应

背景：脑梗死发生后缺血半暗带存在的状态决定了最终梗死灶的体积,在这一区域出现的选择性基因表达、蛋白合成减少、乳酸中毒和细胞毒性水肿,对神经细胞的抢救性治疗以及对神经血管单元的持续调节,成为后期神经功能恢复的结构和生理基础。 目的：以脑梗死后微血管新生过程为研究切入点,探索微血管新生随时间延续呈现的基本规律以及电针干预对局部血管增殖产生的影响。 方法：96只Wistar大鼠随机分为模型对照组和电针干预组,以线栓法复制大鼠大脑中动脉梗死模型,电针干预组在造模后即刻针刺人中穴,给予15 Hz,1 mA的电刺激,持续20 min。模型对照组同样抓取固定,但不进行任何治疗。在脑梗死后不同时间(3,6,12,24,48 h以及3,7,12 d)采用vWF、Ki67免疫荧光双标记检测血管的新生情况。 结果与结论：在大脑中动脉梗死后3,6,12 h,模型对照组梗死区周围无血管内皮细胞增殖标记,24 h出现血管内皮细胞增殖,48 h血管内皮细胞增殖增加,3 d时达到高峰,7 d时血管内皮细胞增殖水平下降,12 d时无血管内皮细胞增殖标记;在大脑中动脉梗死后3,6 h,电针干预组无血管内皮细胞增殖标记,12 h出现血管内皮细胞增殖,24,48 h血管内皮细胞增殖进一步增加,3 d时达到高峰,7 d时血管内皮细胞增殖水平下降,12 d时无血管内皮细胞增殖标记。与模型对照组比较,电针干预组血管内皮细胞增殖出现时间提前,血管内皮细胞增殖数量多于同时相模型对照组,而两组所有时相梗死区和梗死灶对侧半球均无血管内皮细胞增殖标记。结果表明电针干预可促进大脑中动脉梗死模型大鼠梗死区周围脑血管内皮细胞增殖,并将血管内皮细胞增殖出现时间提前,由此可改善脑梗死的预后。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程     

内皮祖细胞在血管组织工程的应用

内皮祖细胞是一群具有游走特性,能进一步增殖分化成为成熟内皮细胞的幼稚内皮细胞.内皮祖细胞参与了出生后缺血组织的血管发生和血管损伤后的修复.内皮祖细胞的发现为血管组织工程种子细胞增添了一个新来源.本文重点介绍成体内皮祖细胞的来源、鉴定、生物学特性、功能以及在血管组织工程中的应用.     

不同支抗控制可影响拔牙矫治中上颌第3磨牙的位置

中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Morphological and histological evaluations of 3D-layered blood vessel constructs prepared by hierarchical cell manipulation

Three-dimensional (3D)-layered blood vessel constructs consisting of human umbilical artery smooth muscle cells (SMCs) and human umbilical vascular endothelial cells (ECs) were fabricated by hierarchical cell manipulation, and their basic morphology, histology and blood compatibility were evaluated in relation to the EC layers. For the hierarchical cell manipulation, fibronectin-gelatin (FN-G) nanofilms were prepared on the surface of SMC layers to provide a cell adhesive nano-scaffold for the second layer of cells. The layer number of blood vessel constructs was easily controllable from 2 to 7 layers, and the histological evaluation, scanning electron microscope (SEM) and transmission electron microscope (TEM) observations indicated a hierarchical blood vessel analogous morphology. The immunefluorescence staining revealed homogeneous and dense tight-junction of the uppermost EC layer. Furthermore, the nano-meshwork morphology of the FN-G films like a native extracellular matrix was observed inside the blood vessel constructs by SEM. Moreover, a close association between actin microfilaments and the nano-meshworks was observed on the SMC surface by TEM. The blood compatibility of the blood vessel constructs, 4-layered SMC/1-layered EC (4L-SMC/1L-EC), was clearly confirmed by inhibition of platelet adhesion, whereas the blood vessel constructs without EC layers (4L-SMC) showed high adhesion and activation of the platelet. The 3D-blood vessel constructs prepared by hierarchical cell manipulation technique will be valuable as a blood vessel model in the tissue engineering or pharmaceutical fields.     

Morphological and Histological Evaluations of 3D-Layered Blood Vessel Constructs Prepared by Hierarchical Cell Manipulation

Three-dimensional (3D)-layered blood vessel constructs consisting of human umbilical artery smooth muscle cells (SMCs) and human umbilical vascular endothelial cells (ECs) were fabricated by hierarchical cell manipulation, and their basic morphology, histology and blood compatibility were evaluated in relation to the EC layers. For the hierarchical cell manipulation, fibronectin-gelatin (FN-G) nanofilms were prepared on the surface of SMC layers to provide a cell adhesive nano-scaffold for the second layer of cells. The layer number of blood vessel constructs was easily controllable from 2 to 7 layers, and the histological evaluation, scanning electron microscope (SEM) and transmission electron microscope (TEM) observations indicated a hierarchical blood vessel analogous morphology. The immunefluorescence staining revealed homogeneous and dense tight-junction of the uppermost EC layer. Furthermore, the nano-meshwork morphology of the FN-G films like a native extracellular matrix was observed inside the blood vessel constructs by SEM. Moreover, a close association between actin microfilaments and the nano-meshworks was observed on the SMC surface by TEM. The blood compatibility of the blood vessel constructs, 4-layered SMC/1-layered EC (4L-SMC/1L-EC), was clearly confirmed by inhibition of platelet adhesion, whereas the blood vessel constructs without EC layers (4L-SMC) showed high adhesion and activation of the platelet. The 3D-blood vessel constructs prepared by hierarchical cell manipulation technique will be valuable as a blood vessel model in the tissue engineering or pharmaceutical fields.     

Regression of blood vessels in the ventral velum of Xenopus laevis Daudin during metamorphosis: light microscopic and transmission electron microscopic study

Structural changes of the ventral velum of Xenopus laevis tadpoles from late prometamorphosis (stage 58) to the height of metamorphic climax (stage 62) were examined by light and transmission electron microscopy. Special emphasis was given to the blood vessel regression. Early changes of velar capillaries were formation of luminal and abluminal endothelial cell processes, vacuolation, and cytoplasmic and nuclear chromatin condensation. At the height of metamorphic climax, transmission electron microscopy revealed apoptotic endothelial cells with nuclear condensation and fragmentation, intraluminal bulging of rounded endothelial cells which narrowed or even plugged the capillary, and different stages of endothelial cell detachment ('shedding') into the vessel lumen. These changes explain the 'miniaturisation' of the velar microvascular bed as well as the typical features found in resin-casts of regressing velar vessels which have been observed in a previous scanning electron microscopy study of the ventral velum.     

Tissue-engineered vascular grafts as in vitro blood vessel mimics for the evaluation of endothelialization of intravascular devices

The accelerating use of minimally invasive procedures for the treatment of cardiovascular disease, and the commensurate development of intravascular devices such as stents, has lead to a high demand for preclinical assessment techniques. A 3-dimensional in vitro blood vessel mimic (BVM) would be ideal for device testing before animal or clinical studies. This is possible based on current capabilities for the creation of tissue-engineered vascular grafts (TEVGs). Using an established method of pressure-sodding human endothelial cells onto a polymer scaffold, a BVM was created in an in vitro bioreactor system under flow. Scanning electron microscopy and immunohistochemistry verified a cellular lining and revealed a luminal monolayer of endothelial cells. After BVM development, bare metal stents were deployed. Stented and unstented BVMs were evaluated using fluorescent nuclear staining and optical coherence tomography (OCT). En face and cross-sectional evaluation of bisbenzimide-stained nuclei revealed cellular coverage of the stent surfaces. Cross-sectional evaluation using OCT also illustrated a cellular layer developing over the stent struts. These data support the use of TEVGs as in vitro BVMs for pre-clinical evaluation of the endothelial cell response to stents and endovascular devices.     

Intima/medulla reconstruction and vascular contraction–relaxation recovery for acellular small diameter vessels prepared by hyperosmotic electrolyte solution treatment

This study aims at the evaluation of blood vessel reconstruction process of decellularized small diameter vessels prepared by a hyperosmotic electrolyte solution treatment not only histologically but also physiologically in rat transplantation model. Complete cell removal by a hyperosmotic electrolyte solution treatment was confirmed by hematoxylin/eosin staining and scanning electron microscopic observation. All acellular vessels transplanted into the rat abdominal aorta were patent up to 14 months. One week post-transplantation, the vWF-positive cells were observed on the luminal surface but the layer formation did not complete. Five weeks following transplantation, the vWF-positive endothelial cells were located on the intima consistent with intact endothelial cells. Beneath the endothelial cells, α-SMA-positive smooth muscle cells were distributed. The harvested vessels displayed formation of tunica intima (endothelial cells) and tunica medulla (smooth muscle cell) layers. We also examined the physiological properties of the vessels 12 months post-transplantation using a wire myograph system. The transplanted vessels contracted upon addition of norepinephrine and relaxed upon addition of sodium nitroprusside as well as the native vessels. In conclusion, the acellular vessels prepared with hyperosmotic electrolytic solution showed excellent and long-term patency, which may be related to the successful preservation of vascular ECM. In addition, the acellular vessels revealed the intima/medulla regeneration with the physiological contraction–relaxation functions in response to the each substance.     

The endoendothelial layer of normal and injured vascular endothelium--an electron microscope study

The ultrastructural appearance of the endoendothelial lining of normal and injured small blood vessels has been examined after staining with alcian blue, ruthenium red or colloidal iron hydroxide. An endoendothelial layer is constantly present in all types of small blood vessel but its appearance and staining reactions are variable and unpredictable. Although normally visible only on the luminal aspect of endothelial cells, study of inflamed vessels suggests that it also covers the deep aspect of endothelium. The endoendothelial layer persists apparently unchanged after mild or severe injury to endothelium. It appears to be to some degree movable and can be displaced by emigrating leucocytes, but rapidly regains its original position and appearance after the escaping cell has passed. No change in the appearance of the endoendothelial layer could be detected which might account for either the adhesion of leucocytes to vascular endothelium in areas of acute inflammation or of lymphocytes to the surface of high endothelial venules in lymphoid tissues.     

Immunohistochemical evaluation of factor VIII related antigen,filament proteins and lectin binding in haemangiomas

Summary Immunohistochemical identification of factor VIII related antigen (F VIII RAG) filament proteins (actin, myosin, filamin, vimentin and desmin) and lectin binding patterns of Con A, PNA, SBA, WGA, RCA-1, UEA-1 and DBA in the endothelial cells and the musclar layers of haemangiomas and normal blood vessels are reported, using paraffin sections with the HRP method.The endothelial cells of haemangiomas were usually strongly positive to F VIII RAG as were those from capillary vessels and other small vessels. Some of the endothelium from haemangiomas and angiokeratomas was negative for factor VIII. The vessel walls of hemangiomas showed staining slightly positive for microfilaments (actin, myosin, filamin). The smooth muscle layer in small vessels showed a more marked staining with actin. Vimentin and desmin reactions in the vessel walls of, haemangioma and in normal vessels were slight or moderate. UEA-1 lectin binding was constantly positive in endothelial cells from hemangiomas and in blood vessels. SBA and WGA binding appeared in the border layer of endothelium in haemangiomas and normal vessels.     

兔骨髓内皮祖细胞在组织工程血管构建中的实验研究

建立体外分离、培养及鉴定兔骨髓血内皮祖细胞（endothelial progenitor cell,EPCs）的方法,并探讨其在血管组织工程构建过程中的功能。采用密度梯度离心法分离单个核细胞,经培养鉴定为EPCs后作为种子细胞接种于人纤维连接蛋白包被（FN）的组织工程血管支架上,加入血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）进行体外诱导培养,同时设置未包被纤维连接蛋白及未添加血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）的培养方法作为对照组,体外培养10 d后,对构建的组织工程血管进行鉴定分析。分离培养的骨髓单个核细胞呈典型的＂铺路石样＂外观。经免疫荧光检测、细胞吞噬功能鉴定为内皮祖细胞;种植细胞10 d后结果显示：加入纤维连接蛋白和血管内皮生长因子的血管支架可见细胞种植密度明显高于对照组,扫描电子显微镜观察到,血管内腔面较为完整的覆盖内皮细胞。HE染色显示：内皮细胞在血管支架上成活并较为均匀;免疫组化结果显示分化为成熟血管内皮细胞并表达VEGFR-2、vWF、CD34。兔骨髓单个核细胞体外培养可以诱导分化为内皮祖细胞,血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）和纤维连接蛋白（FN）的组合更有利于内皮祖细胞在血管支架上增殖和分化,为人工血管制备创造了条件。     

Endothelialization of embolized tumor cells during metastasis formation

The reaction of the endothelial barrier to tumor cell extravasation has been studied using electron microscopy. The model system was pulmonary metastases produced by intravenous injection of B16-F10 melanoma cells. A striking difference was observed in the behavior of the endothelial lining of arterioles versus that of capillaries.In capillaries, partial retraction of endothelial cells took place following the attachment of tumor cells. The tumor cells then immediately attached to the basement membrane and the basolateral surface of the retracted endothelial cells. The endothelial cells extended to cover the tumor cells prior to complete extravasation.In the arterioles, on the other hand, endothelial retraction did not occur following tumor cell attachment. Instead the attached tumor cell emboli became encompassed by endothelial cells, outgrowing from the intact endothelial lining of the arteriole. Owing to the proliferation of the tumor cells, tumor colonies encompassed by endothelial cells expanded within the lumen. When these intravascular growths completely filled the lumen, the tumor cells extravasated from the vessel only after the original endothelial layer became mechanically disrupted and the tumor cells thereby came into contact with the basement membrane.Part of this work was carried out during a year spent at the N.C.I, as Fogarty Scholar-in-Residence.     

Contribution of synovial lining cells to synovial vascularization of the rat temporomandibular joint

The lining layer of the synovial membrane in the temporomandibular joint (TMJ) contains two types of lining cells: macrophage‐like type A and fibroblast‐like type B cells. The type B cells are particularly heterogeneous in their morphology and immunoreactivity, so that details of their functions remain unclear. Some of the type B cells exhibit certain resemblances in their ultrastructure to those of an activated capillary pericyte at the initial stage of the angiogenesis. The articular surface, composed of cartilage and the disc in the TMJ, has few vasculatures, whereas the synovial lining layer is richly equipped with blood capillaries to produce the constituent of synovial fluid. The present study investigated at both the light and electron microscopic levels the immunocytochemical characteristics of the synovial lining cells in the adult rat TMJ, focusing on their contribution to the synovial vascularization. It also employed an intravascular perfusion with Lycopersicon esculentum (tomato) lectin to identify functional vessels in vivo. Results showed that several type B cells expressed desmin, a muscle‐specific intermediate filament which is known as the earliest protein to appear during myogenesis as well as being a marker for the immature capillary pericyte. These desmin‐positive type B cells showed immunoreactions for vimentin and pericyte markers (neuron‐glial 2; NG2 and PDGFRβ) but not for the other markers of myogenic cells (MyoD and myogenin) or a contractile apparatus (αSMA and caldesmon). Immunoreactivity for RECA‐1, an endothelial marker, was observed in the macrophage‐like type A cells. The arterioles and venules inside the synovial folds extended numerous capillaries with RECA‐1‐positive endothelial cells and desmin‐positive pericytes to distribute densely in the lining layer. The distal portion of these capillaries showing RECA‐1‐immunoreactivity lacked lectin‐staining, indicating a loss of blood‐circulation due to sprouting or termination in the lining layer. The desmin‐positive type B and RECA‐1‐positive type A cells attached to this portion of the capillaries. Some capillaries in the lining layer also expressed ninein, a marker for sprouting endothelial cells, called tip cells. Since an activated pericyte, macrophage and tip cell are known to act together at the forefront of the vessel sprout during angiogenesis, the desmin‐positive type B cell and RECA‐1‐positive type A cell might serve as these angiogenic cells in the synovial lining layer. Tomato lectin perfusion following decalcification would be a highly useful tool for research on the vasculature of the mineralized tissue. Use of this technique combined with immunohistochemistry should permit future extensive investigations on the presence of the physiological angiogenesis and on the function of the lining cells in the synovial membrane.