骨髓单个核细胞构建细胞化生物组织工程血管支架的研究

目的研究鼠骨髓来源平滑肌祖细胞(SPCs)和内皮祖细胞(EPCs)在细胞外基质支架上的生长特性,为生物组织工程血管寻找更新的种子细胞和支架。方法将鼠骨髓来源单个核细胞(BMCs)分别用不同的培养基进行诱导分化,于培养10 d后用免疫荧光双标技术(CD34/VWF)鉴定EPCs,用-αSMA/CD14鉴定SPC。将纤维蛋白原、层粘连蛋白和纤粘连蛋白按一定比例混合后,在新鲜大鼠血浆促凝作用下,构建毯状细胞外基质(ECM)支架,在支架表面种植诱导培养骨髓来源的第2代SPCs和EPCs。用扫描电镜和透射电镜观察和分析的支架表面结构以及细胞在支架上生长情况。结果单个核细胞经EBM-2培养基诱导,培养至10 d双荧光染色结果表明,培养贴壁细胞中约90%的EPCs呈VWF/CD34双阳性;经bFGF的M-199培养基诱导阳性率逐渐增加,培养至10 d双荧光染色结果表明,培养贴壁细胞中SPCs呈-αSMA/CD14双标阳性100%。将诱导培养的第2代SPCs,种植在支架上,4 d时倒置显微镜示SPCs在支架表面细胞融合呈现典型的"峰"、"谷"样形态,当EPCs被种植10 d时扫描电镜显示在支架表面形成一个完整的较平整的细胞平面。透射电镜发现在多层平滑肌祖细胞表面有单层内皮祖细胞,呈三维立体结构,具有天然血管的内膜和中膜,更接近天然血管的结构。结论BMCs将是组织工程血管最理想的细胞来源,细胞外基质支架能显著促进SPCs和EPCs粘附、增殖和分化,可作为SPCs和EPCs生长的载体,可作为一种新颖的合成人造血管的生物组织工程支架。     

骨髓单个核细胞构建细胞化生物组织工程血管支架的研究

目的 研究鼠骨髓来源平滑肌祖细胞（SPCs）和内皮祖细胞（EPCs）在细胞外基质支架上的生长特性，为生物组织工程血管寻找更新的种子细胞和支架。方法 将鼠骨髓来源单个核细胞（BMCs）分别用不同的培养基进行诱导分化，于培养10d后用免疫荧光双标技术（CD34／、VWF）鉴定EPCs，用α-SMA／CD14鉴定SPC。将纤维蛋白原、层粘连蛋白和纤粘连蛋白按一定比例混合后，在新鲜大鼠血浆促凝作用下，构建毯状细胞外基质（ECM）支架，在支架表面种植诱导培养骨髓来源的第2代SPCs和EPCs。用扫描电镜和透射电镜观察和分析的支架表面结构以及细胞在支架上生长情况。结果 单个核细胞经EBM-2培养基诱导，培养至10d双荧光染色结果表明，培养贴壁细胞中约90％的EPCs呈VWF／Cth。双阳性；经hFGF的M-199培养基诱导阳性率逐渐增加，培养至10d双荧光染色结果表明，培养贴壁细胞中SPCs呈α-SMA／CD14双标阳性100％。将诱导培养的第2代SPCs，种植在支架上，4d时倒置显微镜示SPCs在支架表面细胞融合呈现典型的“峰”、“谷”样形态，当EPCs被种植10d时扫描电镜显示在支架表面形成一个完整的较平整的细胞平面。透射电镜发现在多层平滑肌祖细胞表面有单层内皮祖细胞，呈三维立体结构，具有天然血管的内膜和中膜，更接近天然血管的结构。结论 BMCS将是组织工程血管最理想的细胞来源，细胞外基质支架能显著促进SPCs和EPCs粘附、增殖和分化，可作为SPCs和EPCs生长的载体，可作为一种新颖的合成人造血管的生物组织工程支架。     

利用复合生物支架材料构建全生物型小口径组织工程血管的研究

目的利用复合纤维蛋白胶生物支架材料和血管细胞成分体外构建具有完整中膜及内皮层的全生物型小口径组织工程血管。方法体外培养扩增猪主动脉血管内皮细胞及平滑细胞,将血管平滑肌细胞与猪纤维蛋白胶混合,均匀复合于犬颈总动脉脱细胞基质材料外表面,经培养后将血管内皮细胞悬液种植到基质材料的内表面,构建全生物型小口径组织工程血管。通过组织学染色观察组织工程血管的组织学结构,扫描电镜观察血管的内外表面结构。结果组织学染色结果表明构建的小口径组织工程血管具有完整的内膜层及中膜层结构;扫描电镜结果显示组织工程血管内表面覆盖着完整的内皮细胞层,血管的外表面可见分层排列的血管平滑肌细胞。结论利用纤维蛋白胶血管细胞混合液复合犬颈总动脉脱细胞基质可以构建出具有完整中膜及内膜层的全生物型小口径组织工程血管。     

利用纤维蛋白胶复合血管脱细胞基质制备全生物型小口径组织工程血管支架材料的研究

目的探讨利用猪纤维蛋白胶复合犬颈总动脉脱细胞基质制备全生物型小口径组织工程血管支架材料的方法。方法将猪纤维蛋白胶均匀复合于犬颈总动脉脱细胞基质的外表面,构建全生物型小口径复合纤维蛋白胶支架材料。通过组织学染色、扫描电镜观察及生物力学测试对复合纤维蛋白胶支架材料的性质进行检测。结果组织学染色结果表明猪纤维蛋白胶均匀分布于犬颈总动脉脱细胞基质的外表面;扫描电镜结果显示复合纤维蛋白胶支架材料的外表面光滑,质地均匀。复合纤维蛋白胶支架材料、脱细胞犬颈总动脉及新鲜犬颈总动脉3组标本的极限拉伸强度和爆裂强度相似,复合纤维蛋白胶支架材料组的轴向顺应性高于脱细胞犬颈总动脉组,而与新鲜犬颈总动脉组的顺应性相似。结论猪纤维蛋白胶可以均匀复合于犬颈总动脉脱细胞基质支架材料上,能够构建出结构均匀的全生物型小口径组织工程血管的支架材料,复合纤维蛋白胶支架材料具有合适的生物力学性质,能够满足小口径组织工程血管对支架材料的要求。     

脱细胞支架复合犬骨髓源内皮祖细胞构建组织工程血管

背景：目前临床使用的小口径（〈6cm）人工血管因生物相容性差、远期通畅率低，效果并不理想。 目的：将犬骨髓源内皮祖细胞与脱细胞血管支架动态复合培养，尝试构建一种全新的组织工程血管代用品。 设计、时间及地点：随机对照实验，细胞学、组织病理学体外观察，于2005—12／2007-12在南京大学医学院附属鼓楼医院实验室完成。 材料：通过去污剂-酶消化法制备犬颈动脉脱细胞支架；采用密度梯度离心法和内皮系条件培养法，分离扩增犬骨髓源内皮祖细胞，将扩增后的内皮祖细胞种植于脱细胞支架并置于生物反应器中动态构建组织工程血管。 方法：20只犬均暴露两侧颈总动脉及一侧股动脉，每只犬在3段血管随机移植组织工程血管、脱细胞血管支架及自体静脉，分别为组织工程血管组、脱细胞血管支架组及自体静脉组，每组的移植血管数量均为20例。 主要观察指标：对培养的内皮祖细胞进行免疫组化鉴定；血管移植术后6个月行数字减影血管造影、病理切片、扫描电镜等观察移植效果。 结果：犬骨髓单个核细胞在体外培养10d后形成“铺路石样”细胞，免疫组化结果符合内皮祖细胞表型特征；将内皮祖细胞与脱细胞支架置于生物反应器培养10d后，种子细胞在血管腔内黏附生长；犬动脉移植术6个月后，组织工程血管及白体静脉通畅率分别为85％，90％，均优于脱细胞支架移植组25％。 结论：犬骨髓源内皮祖细胞复合脱细胞血管支架，可获得一种具有良好生物相容性和通畅率的生物人工血管。     

组织工程血管支架材料

1可降解性聚乙醇酸血管外支架抑制移植静脉内膜的增生 2不同浓度细胞种植脱细胞血管基质上构建组织工程血管 3应用猪主动脉脱细胞基质制备新型组织工程血管支架：生物相容性及力学性能评价     

不同浓度细胞种植脱细胞血管基质上构建组织工程血管

背景:前期研究表明制备的脱细胞血管基质适合平滑肌细胞和内皮祖细胞生长,但是共培养模型中细胞接种密度对血管基质上细胞覆盖率的影响尚不清楚.目的:观察不同浓度度细胞种植在脱细胞血管基质上的生长情况.方法:采用两步法进行细胞种植,先将不同细胞浓度血管平滑肌细胞种植在脱细胞血管基质上,培养3 d后再将不同浓度内皮祖细胞接种在平滑肌细胞-血管基质复合体上,构建片状组织工程材料,分别在内皮祖细胞种植后3 d、1周时间段获取标本,扫描电镜观察细胞在材料上的生长情况.结果与结论:血管平滑肌细胞和内皮祖细胞在脱细胞基质表面生长良好.不同浓度细胞在基质上的排布不同;提高接种的浓度有利于在材料表面快速形成致密的细胞层.提示采用两步法以合适的浓度种植细胞于脱细胞基质上,可以构建组织工程血管.     

软骨组织工程中细胞外基质的研究

目的：分析软骨组织工程中软骨细胞、软骨基质、生长因子之间的相互作用及细胞外基质替代物的开发。 资料来源：应用计算机检索Medline1995-01／2005-12有关软骨组织工程中软骨细胞外基质的文章，检索词为“cartilage；extraeellular matrix”．并限定文章语言种类为English。 资料选择：对资料进行审阅,选取包括软骨组织工程的文章，并查阅全文。纳入标准：①软骨细胞外基质及其受体。②软骨组织重建相关生长因子。③软骨细胞外基质替代物。排除标准：Meta分析、综述文献、重复研究。 资料提炼：共收集到1 179篇关于组织工程软骨及细胞外基质的文献．纳入符合标准的文献29篇。 资料综合：软骨组织无血管，其自我修复及重建能力有限，较小的创伤就会造成严重的软骨损伤及变性。软骨基质在软骨自身动态平衡及软骨修复等方面起着非常重要的作用，软骨细胞合成、分泌软骨基质并受软骨基质的调控。重建软骨组织就必须考虑软骨细胞及其周围环境的相互作用。 结论：重建关节软骨基质、促进软骨复原，研究软骨细胞和其周围环境之间的相互作用是关键。     

脱细胞化肝脏生物衍生支架的制备及鉴定

背景:肝细胞在体外迅速失去极性及合成代谢障碍成为肝脏组织工程研究需要克服的一个难题,在体外寻找一个有利于肝细胞生长与功能维持的细胞外基质微环境成为目前研究的焦点.目的:制备肝脏去细胞化细胞外基质生物衍生支架,并对该生物衍生支架进行初步鉴定.设计、时间及地点:观察性实验,于2008-02-15/05-01在广东省脑功能修复与再生研究所完成.材料:10只雄性SD大鼠用于制备去细胞肝脏细胞外基质支架.10只雄性SD大鼠用于制备原代肝细胞.方法:将SD大鼠肝脏切成10 mm×5 mm的组织片后,经过胰酶.乙二胺四乙酸作用24 h、去垢剂曲拉通×100 72 h后得到肝脏去细胞化生物衍生支架.每只大鼠获取2×108个肝细胞,将所得原代细胞与肿脏去细胞化生物衍生支架放入含DMEM-F12,体积分数为10%胎生血清,胰岛素0.5 U/mL,地塞米松1×10-7mmol/L,表皮生长因子10 μ g/L的培养基中共培养,并与单纯原代细胞培养相比较.主要观察指标:培养14 d时组织块行苏木精-伊红染色,Masson染色进行组织学分析.并行扫描电镜观察.对培养1,3,5,7,9,14 d上清液进行白蛋白及尿素水平检测.结果:组织学榆查未见明显细胞核残存,大量胶原纤维得到保留,扫描电镜示纤维成网状排列.支架与原代肝细胞共培养上清液白蛋白和尿素水平高于单纯原代肝细胞培养(P＜0.05).结论:利用去垢剂与胰酶-乙二胺四乙酸低渗溶液处理可以有效完整去除肝脏组织块的细胞成分,较完整地保留细胞外基质;该生物衍生支架有利于肝细胞的生长及功能维持.     

脱细胞支架复合兔骨髓间充质干细胞构建组织工程血管

背景:目前临床使用的小口径(<6 mm)人工血管因生物相容性差、远期通畅率低,效果并不理想.因此,学术界一直致力于寻找具有正常血管生物学功能的血管代用品,组织工程血管的构建与功能研究已成为目前热门研究课题.目的:将兔骨髓间充质干细胞与脱细-胞血管支架动态复合培养体外构建组织工程血管,通过体内移植实验,探讨该组织工程血管的组织相容性及通畅率.设计、时间及地点:随机对照实验,细胞学、组织病理学观察,于2006-01/2008-06在南京大学医学院附属鼓楼医院实验室完成.材料:通过去污剂-酶消化法制备兔腹主动脉脱细胞支架;采用密度梯度离心法结合贴壁分离培养法,分离扩增兔骨髓间充质干细胞,将扩增后的干细胞静态种植于脱细胞支架后置于生物反应器中动态培养构建组织工程血管.方法:60只兔随机均分为3组,剪取一段腹主动脉长约1.0 cm,再将移植血管以8/0聚丙烯线间断外翻吻合到腹主动脉上.组织工程血管组:受体为对应抽取骨髓干细胞的实验兔,以组织工程血管为移植血管;脱细胞血管支架组:以脱细胞处理的同种异体腹主动脉为移植血管;同种异体血管组:以同种异体新鲜腹主动脉作为移植血管.主要观察指标:对培养的骨髓间充质干细胞进行免疫组化鉴定;血管移植后3个月行数字减影血管造影、病理切片、扫描电镜等观察移植效果.结果:兔骨髓间充质干细胞在体外培养8 d后形成漩涡状排列,免疫组化结果符合间充质干细胞表型特征:将间充质干细胞与脱细胞支架置于生物反应器培养12 d后,种子细胞在血管腔内黏附生长;血管移植3个月后,组织工程血管组、脱细胞血管支架组通畅率分别为90%,80%,均优于同种异体血管组(25%).移植3个月后苏木精一伊红染色及扫描电镜结果显示,组织工程血管组形成清晰的内、中、外膜3层结构,形态接近正常动脉,内皮细胞覆盖完整;脱细胞血管支架组血管内表面内皮细胞覆盖不完整,伴有附擘血栓形成,内膜轻度增生,伴炎性细胞浸润:同种异体血管组内膜极度增厚、坏死,管腔明显狭窄,伴不同程度的血栓机化.结论:将兔骨髓间充质干细胞复合脱细胞血管支架上,可获得一种具有良好生物相容性和通畅率的生物人工血管.     

血管组织工程实验研究

目的获得血管组织工程的实验依据。方法获取大鼠血管内皮（VEC）和平滑肌细胞（VSMCs）,进行培养扩增,然后接种到血管组织工程支架上;将VSMCs-血管支架移植到大鼠体内,观察移植后大鼠活体和组织病理反应。结果培养的细胞符合血管种子细胞的形态特征;VSMCs接种到血管组织工程支架上获得成功;VSMCs-血管支架移植到大鼠体内10d后,支架被纤维组织包裹,支架内见VSMCs生长,支架中央管腔存在,但与正常血管结构有很大差别,而且不具备血管功能。结论利用血管组织工程支架构建的大鼠VSMCs-血管支架移植到大鼠皮下是可行的;人工构建血管具有类似血管雏形,但尚不具备血管的正常结构。     

Vascular engineering for bypass surgery

Since the introduction of synthetic vascular grafts in the 1960s, only two-stage endothelial cell seeding has demonstrated any significant improvement over conventional vascular grafts, and its benefits have yet to be demonstrated on a large scale. Tissue engineering is a rapidly expanding field with great potential, but efforts to construct tissue-engineered arterial grafts have, to date, yielded little clinical success. This review explores the latest approaches to the construction of a superior vascular graft, along with its potential for use in the clinic in the future.     

Decellularized matrices for tissue engineering

Importance of the field: Biomimetic scaffolds and substrates of extracellular matrices (ECMs) play an important role in the regulation of cell function and in the guidance of new tissue regeneration, as an ECM has the intrinsic cues necessary to communicate with and dictate to cells.

Areas covered in this review: This paper reviews the latest developments in ECM scaffolds and substrates obtained from decellularized tissues, organs or cultured cells and their application in tissue engineering. The ECM composition, structure, interaction with surrounding cells, preparation method and usage in the regeneration of various tissues and organs are summarised.

What the reader will gain: The advantages and challenges of decellularized matrices are highlighted.

Take home message: Similarity in the composition, microstructure and biomechanical properties of the decellularized scaffolds and substrates to those of the native tissues and organs maximizes the promotion effect in the regeneration of both structural and functional tissues and organs. Simple tissues as well as complicated organs have been decellularized and decellularization methods have been optimized to completely remove the cellular components while keeping the ECM intact.     

A dynamic distention protocol for whole‐organ bladder decellularization: histological and biomechanical characterization of the acellular matrix

A combined physical–chemical protocol for whole full‐thickness bladder decellularization is proposed, based on organ cyclic distention through repeated infusion/withdrawal of the decellularization agents through the urethra. The dynamic decellularization was intended to enhance cell removal efficiency, facilitating the delivery of detergents within the inner layers of the tissue and the removal of cell debris. The use of mild chemical detergents (hypotonic solution and non‐ionic detergent) was employed to limit adverse effects upon matrix 3D ultrastructure. Inspection of the presence of residual DNA and RNA was carried out on decellularized matrices to verify effective cell removal. Histological investigation was focused on assessing the retention of adequate structural and functional components that regulate the biomechanical behaviour of the acellular tissue. Biomechanical properties were evaluated through uniaxial tensile loading tests of tissue strips and through ex vivo filling cystometry to evaluate the whole‐organ mechanical response to a physiological‐like loading state. According to our results, a dynamic decellularization protocol of 17 h duration with a 5 ml/min detergent infusion flow rate revealed higher DNA removal efficiency than standard static decellularization, resulting in residual DNA content < 50 ng/mg dry tissue weight. Furthermore, the collagen network and elastic fibres distribution were preserved in the acellular ECM, which exhibited suitable biomechanical properties in the perspective of its future use as an implant for bladder augmentation. Copyright © 2013 John Wiley & Sons, Ltd.     

神经组织工程生物支架材料生物相容性研究进展

生物支架是对细胞外基质结构和功能的仿生,良好的生物相容性是神经组织工程支架材料的核心要素。目前神经组织工程生物支架材料主要分为两大类：天然生物材料和人工合成的可降解材料。本文就几种比较典型的神经组织工程生物材料的生物功能性进行综述。     

Contact‐free monitoring of vessel graft stiffness – proof of concept as a tool for vascular tissue engineering

Tissue‐engineered vessel grafts have to mimic the biomechanical properties of native blood vessels. Manufacturing processes often condition grafts to adapt them to the target flow conditions. Graft stiffness is influenced by material properties and dimensions and determines graft compliance. This proof‐of‐concept study evaluated a contact‐free method to monitor biomechanical properties without compromising sterility. Forced vibration response analysis was performed on human umbilical vein (HUV) segments mounted in a buffer‐filled tubing system. A linear motor and a dynamic signal analyser were used to excite the fluid by white noise (0–200 Hz). Vein responses were read out by laser triangulation and analysed by fast Fourier transformation. Modal analysis was performed by monitoring multiple positions of the vessel surface. As an inverse model of graft stiffening during conditioning, HUV were digested proteolytically, and the course of natural frequencies (NFs) was monitored over 120 min. Human umbilical vein showed up to five modes with NFs in the range of 5–100 Hz. The first natural frequencies of HUV did not alter over time while incubated in buffer (p = 0.555), whereas both collagenase (?35%, p = 0.0061) and elastase (?45%, p < 0.001) treatments caused significant decreases of NF within 120 min. Decellularized HUV showed similar results, indicating that changes of the extracellular matrix were responsible for the observed shift in NF. Performing vibration response analysis on vessel grafts is feasible without compromising sterility or integrity of the samples. This technique allows direct measurement of stiffness as an important biomechanical property, obviating the need to monitor surrogate parameters. Copyright © 2016 John Wiley & Sons, Ltd.     

Alteration of the extracellular matrix and alpha‐gal antigens in the rat lung scaffold reseeded using human vascular and adipogenic stromal cells

Regenerated organs are expected to solve the problem of donor organ shortage in transplantation medicine. One approach to lung regeneration is to decellularize the organ and reseed it with selected cells. An advantage of the procedure is reduced immunogenicity, because all cells can be theoretically replaced by autologous cells. However, little is known regarding the extracellular matrix (ECM) damage during decellularization and ECM reconstruction process in the organ regeneration. We aimed to evaluate ECM damage and reconstruction of the decellularized–recellularized rat lung, including the removal of alpha‐gal xenoantigens. Rat lungs were perfused with sodium dodecyl sulfate and Triton X‐100 via the pulmonary artery, after which the decellularized scaffold was reseeded with rat or human endothelial cells and adipose‐derived stem cell (ASCs). The ECM and alpha‐gal antigen were evaluated using immunohistochemistry, western blotting, and a glycosaminoglycan assay. Alcian blue staining revealed increased production of proteoglycan following the addition of ASCs to the rat lung recellularized with rat lung microvascular endothelial cells. Glycosaminoglycan levels decreased in the decellularized lung and increased in the recellularized lung, especially in the ASC‐treated group. Immunohistochemical expression of the alpha‐gal protein was decreased to an undetectable level in the decellularized lung tissue and disappeared after recellularization with human cells. In western blot analysis, the bands of alpha‐gal protein almost disappeared after recellularization with human cells. In conclusion, characteristics of the regenerated ECM might depend on the species and type of cells used for recellularization. Therefore, alpha‐gal antigen might be eliminated after a prolonged culture, when using human cells.     

Improvement of the in vivo cellular repopulation of decellularized cardiovascular tissues by a detergent‐free,non‐proteolytic,actin‐disassembling regimen

Low immunogenicity and high repopulation capacity are crucial determinants for the functional and structural performance of acellular cardiovascular implants. The present study evaluates a detergent‐free, non‐proteolytic, actin‐disassembling regimen (BIO) for decellularization of heart valve and vessel grafts, particularly focusing on their bio‐functionality. Rat aortic conduits (rAoC; n = 89) and porcine aortic valve samples (n = 106) are decellularized using detergents (group DET) or the BIO regimen. BIO decellularization results in effective elimination of cellular proteins and significantly improves removal of DNA as compared with group DET, while the extracellular matrix (ECM) structure as well as mechanical properties are preserved. The architecture of rAoC in group BIO allows for improved bio‐functionalization with fibronectin (FN) in a standardized rat implantation model: BIO treatment significantly increases speed and amount of autologous medial cellular repopulation in vivo (p < 0.001) and decreases the formation of hyperplastic intima (p < 0.001) as compared with FN‐coated DET‐decellularized grafts. Moreover, there are no signs of infiltration with inflammatory cells. The present biological, detergent‐free, non‐proteolytic regimen balances effective decellularization and ECM preservation in cardiovascular grafts, and provides optimized bio‐functionality. Additionally, this study implies that the actin‐disassembling regimen may be a promising approach for bioengineering of acellular scaffolds from other muscular tissues, as for example myocardium or intestine. Copyright © 2017 John Wiley & Sons, Ltd.