脱细胞牛颈静脉血管支架的内皮化研究

目的 探讨在脱细胞牛颈静脉血管支架上进行内皮细胞再种植的可行性.方法 将人骨髓间充质干细胞(BMDCs)诱导分化为内皮种子细胞后种植在脱细胞牛颈静脉血管支架上,分为动态培养和静态培养2组.培养7 d后,对标本进行病理和扫描电镜观察.结果 静态培养的血管支架表面形成连续的单细胞层.动态培养后血管支架表面的细胞仍有50%残留,沿流场方向排列.结论 人骨髓间充质干细胞诱导分化的内皮种子细胞在脱细胞牛颈静脉血管支架上可以黏附生长.     

不同应力环境对组织工程血管形成的影响

目的探讨体内不同应力环境对组织工程血管形成的影响。方法清洁级犬10只,体重15~20kg;切取一侧隐动脉10cm,消化、离心收集内皮细胞和平滑肌细胞,与154g/L型胶原蛋白凝胶均匀混合,种植于猪小肠黏膜下层(small intestinal submucosa,SIS)组织膜,包绕直径3mm、管壁厚度0.2mm的聚乙烯管,制备血管组织工程支架30支。将血管组织工程支架分别植于犬皮下、股四头肌和股动脉管壁外,即皮下组、肌内组和动脉周围组(n=10)。术后4周,取出培养组织行大体和组织学观察,并行组织工程血管形成评分及抗静水压检测。结果三种不同应力环境下的培养组织,在管腔结构、细胞生长增殖和组织塑形方面有明显差异。皮下组管腔结构不完整,内皮及平滑肌种子细胞增殖弱,SIS支架材料分解慢,腔面无内皮细胞覆盖;肌内组有完整管状结构形成,种子细胞有一定程度生长增殖,腔面见稀疏内皮细胞覆盖,少许SIS支架材料未分解,管壁细胞和纤维结构排列无序;动脉周围组可见完整血管样结构形成,种子细胞生长增殖良好,管腔有完整内皮细胞覆盖,平滑肌细胞形态、分布良好。皮下组、肌内组和动脉周围组组织工程血管形成总评分分别为5.529±0.272、8.875±0.248和14.824±0.253分,组间差异均有统计学意义(P<0.05);抗静水压三组分别为67.0±5.8、104.0±7.6和247.0±3.5kPa,动脉周围组与皮下组和肌内组比较,差异均有统计学意义(P<0.01)。结论应力刺激对体内细胞增殖和组织工程血管形成有明显影响。     

凝胶包埋兔脂肪基质细胞接种三维支架动态构建组织工程化骨

目的 探讨高效的细胞种植方法,提高工程化骨构建质量.方法 成骨诱导化脂肪基质细胞,制备含rhBMP-2的纳米化壳聚糖/胶原蛋白/β磷酸三钙(Cs-Col-β-TCP)支架,构建工程化骨.静态种植静态培养(A组),振荡种植静态培养(B组),凝胶+振荡种植静态培养(c组),凝胶+静态种植静态培养(D组).第1、4、 8、 12、16、20、24、28天,扫描电镜、共聚焦显微镜观察,检测细胞存活率、细胞增殖、碱性磷酸酶、DNA、骨钙素水平.结果 C组细胞分布均匀、呈三维内生长、细胞外基质丰富,细胞存活率、增殖活力、碱性磷酸酶、DNA、骨钙素水平均高于其他组(79.53±2.67、0.59±0.20、65.16±6.85、207.62±19.36、55.22±8.51,P<0.05).结论 凝胶联合振荡种植可提高细胞接种效率及增强诱导成骨活性.     

小口径组织工程血管体外构建的研究

目的 探讨间充质干细胞体外构建组织工程血管的可行性.方法 将体外培养扩增的犬骨髓间充质干细胞(MSCs)定向分化为平滑肌样细胞和内皮样细胞,接种于ε-己内酯/L-丙交酯(PCLA)支架上,将其置于生物反应器内,在搏动性力学(100±20/55±20)mm Hg(1 mm Hg=0.133 kPa)刺激条件下培养.3 d后行血管组织学检测.结果 血管支架拉伸强度6.1 MPa;骨髓间充质干细胞成功定向分化为平滑肌样细胞和内皮样细胞;血管腔内表面完全为细胞覆盖,表面的细胞沿液体流动的方向分布;种植的部分细胞已经渗透入血管壁内.结论 骨髓间充质干细胞可作为种子细胞,与PCLA支架在生物反应器内构建组织工程血管.     

动态旋转系统构建组织工程化血管模型中血管内皮细胞分泌功能监测

目的 比较静态和动态旋转系统中构建组织工程化血管模型中血管内皮细胞分泌功能。方法新型可降解材料聚羟基丁酸酯(PHB),用胶原包埋,形成多孔状PHB 胶原管形支架。分离传代、分化人脐静脉内皮细胞,接种于PHB管型支架内腔面,分别在静态、动态旋转系统中培养14 d后,测定血管内皮细胞分泌一氧化氮(NO)、前列环素(PGI2)水平。结果 在动态旋转系统构建组织工程化血管模型中血管内皮细胞分泌NO、PGI2水平显著高于静态系统,在11 d NO分别为(120.52±3.83)μmmol／L、(80.98±5.98)μmmol／L,PGI2分另9为(20.48±1.52)μg／L,(16.59±1.29)μg／L,与静态系统相比,差异有显著性(P<0.05)。结论胶原包埋PHB支架有利于细胞的黏附和生长,可作为组织工程化血管的支架材料。在动态旋转系统构建组织工程化血管模型中血管内皮细胞,具有与正常血管类似的"生理功能"。     

纳米仿生组织工程血管体外构建的实验研究

目的 利用静电纺丝 (electrospinning,ELSP) 技术构建具有纳米结构的纳米仿生组织工程血管 (nano-biomimetic-tissue engineered blood vessel,NBTEBV). 方法 6 月龄新西兰雄性家兔 30 只,体重 2.15～3.10 kg.制备兔血管内皮细胞 (vascular endothelial cell,VEC) 体外培养育种管型模具采用多排喷头ELSP混纺兔平滑肌细胞(vascularsmooth muscle cell,VSMC)悬液及仿 ECM (mimic ECM,MECM) 溶液构建 NBTEBV. NBTEBV 用生物反应器体外培养,MTT 检测静置 24 h和动态培养7 d后NBTEBV 上 VECNSMC 的成活和增殖能力,行 HE 染色、扫描电镜观察及最大抗张力检测. 结果 动态培养第7天的 NBTEBV 长 57 mm,外径 4 mm,壁厚 0.4 mm,色乳白,质地均匀,具有良好的柔韧性及弹性.静置孵育 24 h MTT 检测血管上成活细胞相对数为 3.5×105/mg,动态培养7 d 后为 8.9×106/mg.扫描电镜及 HE染色观察示NBTEBV与天然血管有相似的纳米结构及组织学特点;电镜结构呈现由100 nm 支架纤维构成的孔径约600 nm的网状结构;HE 染色示VEC 和 VSMC分层构建成血管状.动态培养第7天组织工程血管最大静水压为950 mmHg,拟生理血压下(110/70 mmHg) 管径顺应变化率 3.0%;20 mm × 5 mm 组织片最大抗张强度为 18.5 MPa. 结论 利用 ELSP技术可以将VSMC 和 MECM的支架材料同步构建成具有与天然血管相似纳米结构的组织工程血管.     

组织工程静脉体外构建条件的实验研究

目的探讨脱细胞管状基质铺植内皮祖细胞的最佳条件,在此基础上体外初步构建组织工程静脉。方法应用扫描电镜和组织学方法,比较无预衬和预衬明胶、纤维连接蛋白对铺植的影响;比较不同转速下动态铺植细胞的覆盖面积;比较静态铺植和应用自制旋转反应器动态铺植细胞的覆盖面积;比较单次铺植和多次铺植的效果应用激光共聚焦显微镜观察铺植细胞摄取乙酰低密度脂蛋白(Dil-ac-LDL)和抗血小板聚集的情况,以评价种植细胞的功能。结果纤维连接蛋白明显优于其余两种预衬方法(P<0.05),2.5 r/h时铺植细胞在形态学上优于其他各转速,动、静态铺植4 h和单、多次铺植的细胞覆盖面积差异无统计学意义(P>0.05),但后者耗时较长,易于污染。管状铺植的细胞能够摄取Dil-ac-LDL并具有抗小板黏附的功能。综合上述方法构建组织工程静脉,细胞覆盖率达84.4%±6.2%(n=12)。结论纤维连接蛋白预衬管状脱细胞基质,2.5 r/h一次动态铺植内皮祖细胞可用于体外构建组织工程静脉,其可行性有待于动物实验证实。     

全生物化组织工程血管的体外构建

目的 探讨组织工程血管的体外构建:脱细胞血管基质的制备.血管平滑肌细胞和血管内皮祖细胞的体外诱导培养和种植方法.方法 处理猪胸主动脉来获得脱细胞血管基质,采用二步种植法先后种植体外培养的平滑肌细胞和内皮细胞.结果 动脉管壁细胞全部脱除,脱细胞基质胶原蛋白含量与新鲜动脉相似,胶原纤维、弹性纤维呈网状排列,无断裂,基质保持完好.脱细胞血管基质的极限应力比新鲜动脉绀织减小20%[新鲜动脉:(1.1510±1.2870)×10-2,脱细胞血管基质:(0.9215±1.7000)×10-2,t=34.137,P<0.01].种植的平滑肌细胞和内皮细胞生长良好.结论 平滑肌细胞和内皮细胞种植于脱细胞血管基质后生长良好,可体外构建组织工程血管.     

四肢小口径组织工程血管的实验研究

目的研究以小肠黏膜下层组织（small—calibu tissue，SIS）为血管支架，在体内血流条件下培养小口径组织工程血管的可行性。方法自犬隐动脉分离出血管种子细胞，与Ⅰ型胶原蛋白凝胶均匀混合，种植于SIS膜表面，包绕3mm聚乙烯棒制成3层管状支架，为实验组；制成单层无细胞管状支架，为对照组。两种支架作为血管移植物，分别植入15只犬两侧股动脉缺损处进行桥接吻合。术后进行彩超、组织学评价血管的形成过程。结果术后12周，14个3层血管支架保持通畅，通畅率为93．3％，有明显的血管生物结构形成。单层血管支架5个出现部分狭窄，只有3个完全通畅，通畅率为52．6％。结论SIS膜制成的3层血管支架能直接修复血管缺损，并能在体内环境中培形成生物化的小口径组织工程血管。     

组织工程化血管构建的初步实验研究

目的探讨组织工程化血管构建的初步实验方法。方法预构具有三维结构的胶原蛋白-几丁聚糖骨架,以人血管内皮细胞、平滑肌细胞及成纤维细胞作为种子细胞,培育后二步法种植并行工程化血管成熟培养。经生物学检测,补片修补实验鼠腹主动脉人为缺损。结果人血管内皮细胞、平滑肌细胞及成纤维细胞可作为种子细胞应用;预制的三维骨架具有良好的组织和细胞兼容性,种植种子细胞后可形成良好的细胞黏附、生长;体外静态及生物反应器培养,工程组织中细胞数及细胞外基质含量明显增加（P〈0．05）;血小板凝集试验表明该工程组织具有一定的抗凝特性;修补大鼠腹主动脉缺损,术后10d可维持通畅。结论胶原蛋白-几丁聚糖预构的三维骨架可应用于血管组织工程研究,细胞种植后经成熟培养,可初步构建具有一定强度和抗凝特性的组织工程化血管。     

Biomechanics and tissue engineering

Development of artificial scaffold for musculo-skeletal applications, especially in load-bearing situations, requires the consideration of biomechanical aspects for its integrity and its function. However, the biomechanical loading could also be used to favour tissue formation through mechano-transduction phenomena. Design of scaffold could take advantages of this intrinsic mechanical loading.     

Hepatic tissue engineering

Fulminant hepatic failure (FHF) attributes to rising medical cost and accounts for many deaths each year in the United States. Currently, the only solution is organ transplantation. Due to increasing donor organ shortage, many in need of transplantation continue to remain on the waiting list. Liver Assist Devices (LADs) are being used to temporarily sustain liver function and bridge the period between FHF and transplantation. Hepatic Tissue Engineering is a step toward alleviating the need for donor organs; yet many challenges must be overcome including scaffold choice, cell source and immunological barriers. Bioreactors have aided in hepatocyte survival and have proven to sustain viable cells for several weeks. Achieving the necessary functions required for hepatic replacement is aided by the incorporation of growth factors and mitogens many that now can be bound to the polymer scaffold and released in a timely manner. Utilizing concepts such as MicroElectroMechanical systems (MEMs) technology, our laboratory is able to mimic the natural vasculature of the liver and sustain functional and viable hepatocytes. Expanding and improving upon this platform technology, advancements made will continue toward the development of a fully functioning and implantable liver.     

Bladder tissue engineering

The bladder can lose the ability to store and empty effectively as a result of numerous conditions. When conservative methods to maximize patient safety and quality of life fail, surgical reconstruction of the bladder is usually considered. Augmentation cystoplasty can be performed with the use of the small bowel, large bowel, or less often, stomach. An alternative approach, tissue engineering, identifies the body's own potential for regeneration and supports this propensity with appropriate raw materials and growth factors so that the body's original structure and function may be restored. Tissue engineering can involve the use of a scaffold or matrix alone or of cell-seeded matrices. Harvesting cells and culturing them has become an important tool in tissue engineering. Multiple possibilities for sources of cells have been investigated, including stem cells and differentiated cells from organs other than the bladder; however, to date, autologous bladder cells remain the gold standard for culture and seeding.     

Skin tissue engineering

The coverage of extensive wounds with viable autologous keratinocytes remains the only option of treatment if autologous donor skin is not obtainable. There is evidence that proliferating keratinocytes, as suspended cells or as a single layer, are adequate for wound closure. Understanding keratinocyte-matrix interactions not only allows us to influence keratinocyte outgrowth, adhesion, and migration, but may also guide us to modify matrix molecules for enhancing keratinocyte take. Further approaches may include the generation of genetically manipulated keratinocytes, which allow the use of an off-the-shelf epidermal replacement. As surgeons, our goal is to help burn patients with the best quality of skin in the shortest time possible. As tissue engineers, we have not achieved the goal of a universal skin product. By continually reviewing the options and using them, we can at least use the proper material in the adequate situation. Because of the limited resources, the need for comparisons of clinical effectiveness and cost are ever more important. As anatomy and physiology of engineered skin substitutes improve, they will become more similar to native skin autografts. Improvement of skin substitutes will result from inclusion of additional cell types (eg, melanocytes) and from modifications of culture media and scaffolds. Skin-substitute materials may be able to stimulate regeneration rather than repair, and tissue-engineered skin may match the quality of split-skin autografts, our present gold standard.     

Vascular tissue engineering

Reconstructive surgery using autologous vessels is the conventional approach for substitution of diseased vessels or for generation of bypass to improve blood supply downstream of stenosed vessels. In some circumstances the use of autologous material is not possible due to concomitant diseases or previous use, and artificial grafts must be used. Unfortunately, these grafts cannot substitute small-caliber arterial vessels because of thrombotic complications. The objective of tissue engineering at the vascular level is then to generate biological substitutes of arterial conduits with functional characteristics of native vessels, combining cellular components with biodegradable scaffolds. These research projects started in several laboratories, in the late 1990s, and have expanded in different directions using a number of experimental approaches. The objective of this review is to give an overview of the results so far obtained in this area of research, and to discuss the problems related to these investigations, at the experimental and clinical level. The article provides an overview of different biodegradable scaffolds used, experimental techniques for vessels maturation in vitro under mechanical stimulation, and of differentiated as well as precursors of vascular cells, which opens new opportunities for further development of this form of cell transplantation. Finally, the current available results in clinical research will be discussed.     

血管组织工程的进展

血管病变尤其是冠状动脉的闭塞性病变为人类的主要病死原因之一。其主要的治疗方法是血管搭桥手术,需要各种直径的血管移植物作为修补材料。临床已经应用的血管移植物包括自体血管、异体血管和合成材料管道。然而目前无论那种血管移植物都无法满足临床需要,临床需要一种新的血管替代物,这种管道应具有高度的组织相容性、可生     

Cardiac tissue engineering

Recent progress in implantations of differentiated cardiac and non-cardiac cells as well as adult stem cells into the heart suggests that the irreversible loss of viable cardiac myocytes that occurs during myocardial infarction can be at least partly substituted. We evaluated an alternative approach by reconstituting cardiac tissue grafts in vitro and implanting them as spontaneously and coherently contracting tissues. For this purpose we have optimized a method to generate ring-shaped three-dimensional engineered heart tissue (EHT) in vitro from neonatal rat cardiac myocytes. When subjected to isometric force measurements in organ baths, electrically stimulated EHTs exhibit a Frank-Starling behavior, a positive inotropic response to increases in extracellular calcium, a positive inotropic and lusitropic response to isoprenaline, and a negative inotropic response to the muscarinic agonist carbachol ('accentuated antagonism'). Twitch tension under maximal calcium amounts to 1-2 mN/ mm2. Importantly, passive (resting) tension is low, yielding a ratio of active/passive tension of approximately 1.5 under basal and 14 under maximal calcium. Morphologically, EHTs represent a highly interconnected three-dimensional network of cardiac myocytes resembling loose cardiac tissue with a high fraction of binucleated cardiac myocytes, strong eosin staining and elongated centrally located nuclei. Electron microscopy demonstrated well developed sarcomeric structures, T-tubules, SR vesicles, T-tubule-SR-junctions, all types of intercellular connective structures, and a basement membrane. Thus, EHTs comprise functional and morphological properties of intact, ventricular myocardium. First implantation experiments of EHTs in the peritoneum of Fischer 344 rats showed that EHTs survived for at least 14 days, maintained a network of differentiated cardiac myocytes, and were strongly vascularized. Thus, EHTs may serve as material for a novel tissue replacement approach.