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。     

聚ε-己内酯组织工程血管研究进展

近年来随着心血管疾病患者不断增多,人工血管在临床上的需求不断增长。人工聚合材料聚ε-己内酯[poly(ε-caprolactone),PCL]因具有生物可降解性、良好的力学性能而成为了组织工程血管材料研究的热点。但是单一PCL血管存在疏水性强、细胞黏附亲和力差等缺陷,限制了PCL材料在组织工程领域的应用。为了增强PCL材料的生物相容性,研究者们应用了各种方法对PCL材料进行表面改性或共混成复合物。本文从PCL的表面改性、复合物等方面对其研究现状进行综述。通过表面改性后的PCL及其复合物具有较好的生物相容性,提高了材料的细胞黏附和组织生长性能,这为进一步构建理想小口径人工血管提供了一定的参考。     

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.     

基于聚己内酯纤维的组织工程支架研究进展

聚己内酯(PCL)是一种生物相容性好、可吸收、易加工改性的聚酯,材料基于PCL纤维的组织工程支架,具有比表面积高、机械性能良好与孔径、孔隙率和纤维取向等结构特征易调控等特点,被广泛应用于组织工程领域。重点综述PCL纤维的组织工程支架的主要应用缺陷(包括细胞亲和力差、降解速度过慢及机械强度低)及改进手段,同时针对基于PCL纤维的组织工程支架在皮肤、血管、神经、肌腱、韧带和软骨等组织再生的最新进展进行归纳总结,发现目前多数研究集中于通过引入生物活性物质或药物以改善细胞-支架相互作用和调控支架降解行为,或通过选取不同的纺丝工艺和参数以改变支架的物理结构,调控支架机械性能与细胞诱导行为。此外,目前多数研究仍停留在实验室阶段,利用基于PCL纤维的组织工程支架低成本、易加工的优势以加快其临床转化是未来重要的发展方向。     

脉冲电刺激对血管内皮细胞与其祖细胞黏附的影响

为探索脉冲电刺激下血管内皮细胞与内皮祖细胞(EPC)之间黏附强度的改变,诱导培养外周血EPC,荧光标记后与单层血管内皮细胞共培养,固定电压和频率为5 V和5 Hz,选择1、3、6、9 ms的脉宽对其进行干预,持续刺激24 h后检测贴壁EPC的荧光强度,以荧光比率衡量。结果显示,与对照组相比,3 ms刺激组荧光比率即显著增高,随着脉宽延长,6 ms组达到最大值,但9 ms刺激组却显著下降,提示适宜脉冲电刺激有利于血管内皮细胞与EPC之间的黏附,为电刺激促进血管新生的研究提供新的理论依据。     

内皮祖细胞在血管新生及血管组织工程化过程中的生物学意义

内皮祖细胞(EPCs)是能分化为成熟血管内皮细胞的祖细胞,参与了出生后的血管再生和受损内皮的修复过程。近年来围绕以EPCs作为种子细胞来促进血管新生、维持内皮功能完整并构建组织工程化血管方面展开了许多研究。本文就这方面的进展作一综述。     

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。     

内皮祖细胞作为组织工程瓣膜内皮种子细胞的可行性研究

目的 探讨利用外周血内皮祖细胞（EPC8）制备组织工程瓣膜的可行性。方法 分离人外周血EPCs，采用酶-去垢剂法去除新鲜猪主动脉瓣细胞制备去细胞瓣膜支架，将培养的人外周血EPCs接种到去细胞瓣膜上。结果 经酶-去垢剂法去除新鲜猪主动脉瓣细胞后，细胞成分全部去除，纤维支架保存完好。去细胞处理后瓣膜无明显细胞毒性。人外周血EPCs与去细胞瓣膜共孵育2周后，细胞紧贴瓣膜表面生长形成一层连续的单细胞层，初步生物力学测定示去细胞前与再内皮化后，瓣膜力学特性无明显改变。结论 外周血分离培养扩增得到的EPCs能够再内皮化去细胞猪主动脉瓣构建组织工程瓣膜，外周血EPCs是组织工程瓣膜内皮种子细胞的新的来源。     

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。     

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。     

Electrospun scaffolds for tissue engineering of vascular grafts

There is a growing demand for off-the-shelf tissue engineered vascular grafts (TEVGs) for the replacement or bypass of damaged arteries in various cardiovascular diseases. Scaffolds from the decellularized tissue skeletons to biopolymers and biodegradable synthetic polymers have been used for fabricating TEVGs. However, several issues have not yet been resolved, which include the inability to mimic the mechanical properties of native tissues, and the ability for long-term patency and growth required for in vivo function. Electrospinning is a popular technique for the production of scaffolds that has the potential to address these issues. However, its application to human TEVGs has not yet been achieved. This review provides an overview of tubular scaffolds that have been prepared by electrospinning with potential for TEVG applications.     

纳米纤维支架的制备及其在组织工程皮肤中的应用

本文通过综述现阶段纳米纤维支架的制备方法、可用材料及制备后的生物功能修饰等方面的研究进展,为设计真正意义的组织工程皮肤纳米纤维支架提供理论帮助。多聚物纳米纤维支架能够提供三维空间结构,并且能够调节细胞行为,具有传递生物分子的潜能,因此它们在组织工程应用中具有广泛的前景。现在能应用多种方法及材料制备纳米纤维结构,但是通过现有的方法和材料还不能将纳米纤维的所有优点完全体现出来,并构建成功一个真正意义上的纳米纤维三维支架结构。所以对纳米纤维支架制备技术的不断改进和对应用的材料体系的深入了解,将对未来临床成功地应用聚合纳米纤维组织工程支架奠定坚实基础。     

Electrospun sulfated silk fibroin nanofibrous scaffolds for vascular tissue engineering

One of the major downfalls of tissue-engineered small-diameter vascular grafts is the inability to obtain a confluent endothelium on the lumenal surface. Loosely attached endothelial cells (ECs) are easily separated from the vessel wall when exposed to the in vivo vascular system. Thus any denuded areas on the lumenal surface of vascular grafts may lead to thrombus formation via platelet deposition and activation. If the denuded areas could express anticoagulant activity until the endothelial cell lining is fully achieved, it may greatly improve the chances of successful vascular reconstruction. In this study, we fabricate sulfated silk fibroin nanofibrous scaffolds (S-silk scaffolds) and assess the anticoagulant activity and cytocompatibility of S-silk scaffolds in vitro in order to improve the antithrombogenicity and get some insights into its potential use for vascular tissue engineering. Sulfated silk fibroin was prepared by reaction with chlorosulphonic acid in pyridine, and then was developed to form an S-silk scaffold by electrospinning technique. FTIR analyses identified the successful incorporation of sulfate groups in silk fibroin molecules. It was found that the anticoagulant activity of S-silk scaffolds was significantly enhanced compared with silk fibroin nanofibrous scaffolds (Silk scaffolds). Vascular cells, including ECs and smooth muscle cells (SMCs), demonstrated strong attachment to S-silk scaffolds and proliferated well with higher expression of some phenotype-related marker genes and proteins. Overall, the data in this study suggest the suitability of S-silk scaffolds used along with vascular cells for the development of tissue-engineered vascular grafts.     

应用脱细胞血管基质构建组织工程血管的初步研究

目的： 采用脱细胞处理的猪胸主动脉血管基质作为支架材料，接种人脐带血管内皮细胞，构建组织工程血管。方法: 以新鲜猪胸主动脉为原材料， 1% Triton X-100为脱细胞试剂，制备脱细胞血管基质；采用冷冻干燥和热交联法对脱细胞血管基质进行改性，并进行组织学观察及力学性能测定。用人脐带内皮细胞经培养和扩增后，再与所制备的脱细胞血管基质进行复合培养，光学显微镜及扫描电镜观察内皮细胞生长状况。结果: 1% Triton X-100处理84h的猪胸主动脉，既能完全脱除血管中细胞，同时又完整保留血管基质的三维结构；对脱细胞血管基质冷冻干燥24 h，120 ℃下热交联12 h，能有效提高材料的机械强度，断裂强度可达到1.70 MPa。扫描电镜下可见，脱细胞血管基质与内皮细胞复合培养7 d，已形成典型血管内膜样结构。结论: 经改性后的脱细胞血管基质与内皮细胞具有良好的相容性，用其作为支架材料与内皮细胞复合培养，有望应用于构建组织工程化血管。     

Biodegradable porous scaffolds for tissue engineering

 Three-dimensional scaffolds play an important role in tissue engineering as an adhesive substrate for implanted cells and a physical support to guide the formation of new organs. The scaffolds should facilitate cell adhesion, promote cell growth, allow the retention of differentiated cell functions, and be biocompatible, biodegradable, highly porous with a large surface-to-volume ratio, mechanically strong, and malleable. A number of biodegradable three-dimensional scaffolds have been developed for tissue engineering. This paper reviews some of the recent events in the development of these scaffolds. Received: March 6, 2002     

聚乙烯醇-壳聚糖-胶原组织工程支架的研究

目的 以聚乙烯醇（PVA）、壳聚糖（Cs）和胶原（Col）为主要原料制备PVA-Cs—Col复合支架,并研究其作为组织工程支架材料的可行性。方法 把聚乙烯醇、壳聚糖和胶原按一定配比复合,测定复合材料的含水率、膨胀率和力学性能,扫描电镜观察材料横截面的组织形态。结果 不同分子量PVA与不同质量的cs和Col复合,得到的复合支架材料湿态抗张强度为5．70MPa,含水率在60．15％-72．50％,膨胀率在185．33％～317．57％。不同配比的复合支架具有不同的内部组织形态结构。结论 PVA—Cs—Col复合支架材料具有较高的含水率和适宜的膨胀率,内部孔洞丰富,Cs：PVA：Col的质量比为30：15：0．20时,复合支架综合性能较佳,适合用于组织工程支架材料。     

Design of scaffolds for blood vessel tissue engineering using a multi-layering electrospinning technique

Aiming to develop a scaffold architecture mimicking morphological and mechanically that of a blood vessel, a sequential multi-layering electrospinning (ME) was performed on a rotating mandrel-type collector. A bi-layered tubular scaffold composed of a stiff and oriented PLA outside fibrous layer and a pliable and randomly oriented PCL fibrous inner layer (PLA/PCL) was fabricated. Control over the level of fibre orientation of the different layers was achieved through the rotation speed of the collector. The structural and mechanical properties of the scaffolds were examined using scanning electron microscopy (SEM) and tensile testing. To assess their capability to support cell attachment, proliferation and migration, 3T3 mouse fibroblasts and later human venous myofibroblasts (HVS) were cultured, expanded and seeded on the scaffolds. In both cases, the cell-polymer constructs were cultured under static conditions for up to 4 weeks. Environmental-scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), histological examination and biochemical assays for cell proliferation (DNA) and extracellular matrix production (collagen and glycosaminoglycans) were performed. The findings suggest the feasibility of ME to design scaffolds with a hierarchical organization through a layer-by-layer process and control over fibre orientation. The resulting scaffolds achieved the desirable levels of pliability (elastic up to 10% strain) and proved to be capable to promote cell growth and proliferation. The electrospun PLA/PCL bi-layered tube presents appropriate characteristics to be considered a candidate scaffold for blood vessel tissue engineering.     

血管组织工程的发展现状和趋势

心脑血管疾病是全球发病率和死亡率最高的疾病,其主要病因是动脉粥样硬化。临床上主要采用血管移植物重建病损血管,人造合成血管在大口径血管修复中取得了满意的效果,但在小口径血管修复中效果并不理想。近30年来,血管组织工程发展极其迅速,从再生的角度为血管修复提供了新的途径。本文综述血管组织工程的最新进展(体外、体内、原位血管组织工程),并对未来发展趋势进行了前瞻性展望。     

Design and fabrication of porous biodegradable scaffolds: a strategy for tissue engineering

Current strategies of tissue engineering are focused on the reconstruction and regeneration of damaged or deformed tissues by grafting of cells with scaffolds and biomolecules. Recently, much interest is given to scaffolds which are based on mimic the extracellular matrix that have induced the formation of new tissues. To return functionality of the organ, the presence of a scaffold is essential as a matrix for cell colonization, migration, growth, differentiation and extracellular matrix deposition, until the tissues are totally restored or regenerated. A wide variety of approaches has been developed either in scaffold materials and production procedures or cell sources and cultivation techniques to regenerate the tissues/organs in tissue engineering applications. This study has been conducted to present an overview of the different scaffold fabrication techniques such as solvent casting and particulate leaching, electrospinning, emulsion freeze-drying, thermally induced phase separation, melt molding and rapid prototyping with their properties, limitations, theoretical principles and their prospective in tailoring appropriate micro-nanostructures for tissue regeneration applications. This review also includes discussion on recent works done in the field of tissue engineering.     

Textile-templated electrospun anisotropic scaffolds for regenerative cardiac tissue engineering

For patients with end-stage heart disease, the access to heart transplantation is limited due to the shortage of donor organs and to the potential for rejection of the donated organ. Therefore, current studies focus on bioengineering approaches for creating biomimetic cardiac patches that will assist in restoring cardiac function, by repairing and/or regenerating the intrinsically anisotropic myocardium. In this paper we present a simplified, straightforward approach for creating bioactive anisotropic cardiac patches, based on a combination of bioengineering and textile-manufacturing techniques in concert with nano-biotechnology based tissue-engineering stratagems. Using knitted conventional textiles, made of cotton or polyester yarns as template targets, we successfully electrospun anisotropic three-dimensional scaffolds from poly(lactic-co-glycolic) acid (PLGA), and thermoplastic polycarbonate-urethane (PCU, Bionate^®). The surface topography and mechanical properties of textile-templated anisotropic scaffolds significantly differed from those of scaffolds electrospun from the same materials onto conventional 2-D flat-target electrospun scaffolds. Anisotropic textile-templated scaffolds electrospun from both PLGA and PCU, supported the adhesion and proliferation of H9C2 cardiac myoblasts cell line, and guided the cardiac tissue-like anisotropic organization of these cells in vitro. All cell-seeded PCU scaffolds exhibited mechanical properties comparable to those of a human heart, but only the cells on the polyester-templated scaffolds exhibited prolonged spontaneous synchronous contractility on the entire engineered construct for 10 days in vitro at a near physiologic frequency of ∼120 bpm. Taken together, the methods described here take advantage of straightforward established textile manufacturing strategies as an efficient and cost-effective approach to engineering 3D anisotropic, elastomeric PCU scaffolds that can serve as a cardiac patch.