用于组织工程化培养生物反应器的研究进展

生物反应器是组织工程研究与临床应用的重要工具之一 ,近年来一直受到国内外学者和企业的广泛关注。本文系统地介绍了各种用于组织工程化培养生物反应器的研究现状。由于生物反应器的机械性能、传质以及流体剪应力等因素对培养组织的形态和功能有很大的影响 ,因比 ,深入研究和开发新型生物反应器对组织工程的研究和今后临床的应用都有着十分重要的意义     

Bioreactors for Cardiovascular Cell and Tissue Growth: A Review

Heart disease is a major cause of death in the Western world. In the past three decades there has been a number of improvements in artificial devices and surgical techniques for cardiovascular disease; however, there is still a need for novel devices, especially for those individuals who cannot receive conventional therapy. The major disadvantage of current artificial devices lies in the fact that they cannot grow, remodel, or repair in vivo. Tissue engineering offers the possibility of developing a biological substitute material in vitro with the inherent mechanical, chemical, biological, and morphological properties required in vivo, on an individual patient basis. In order to develop a true biological cardiovascular device a dynamic physiological environment needs to be created. One approach that employs the use of a simulated biological environment is a bioreactor in which the in vivo biomechanical and biochemical conditions are created in vitro for functional tissue development. A review of the current state of the art bioreactors for the generation of tissue engineered cardiovascular devices is presented in this study. The effect of the simulated physiological environment of the bioreactor on tissue development is examined with respect to the materials properties of vascular grafts, heart valves, and cardiac muscles developed in these bioreactors. © 2003 Biomedical Engineering Society. PAC2003: 8768+z, 8719Hh, 8717Ee, 8719Ff, 8780Rb     

In vitro comparison of steady and pulsatile flow characteristics of jellyfish heart valve

In examining the hydrodynamic performance of artificial heart valves in vitro, experiments are carried out under either steady or pulsatile flow conditions. Steady flow experiments are simple to set up and analysis of the data is also simple; however, their validity and accuracy have been questioned. In this study, the flow characteristics of jellyfish valves are evaluated and analyzed for steady and pulsatile flow conditions. The analysis is given in terms of velocity and shear stress distributions for a cardiac flow rate of 4.5l/min, and the corresponding steady flow rate is measured at two locations, 0.5D and 1D downstream of the valve face (D being the diameter of the pipe). At the 0.5D location, the velocity profile results obtained for both flow conditions indicated that jetting flow occurred close to the wall, and flow reversal as well as stagnation zones occurred in the core of the valve chamber. These phenomena were also evident in the shear stress profiles for both pulsatile and steady flow conditions. At this location, the maximum difference between the steady and pulsatile values of peak velocity is about 18%. However, the maximum difference between the peak shear stresses was in the range of 5%–7%. At the 1D location, the flow characteristics observed under both the pulsatile and steady flow conditions were almost identical, with a maximum difference between the peak values of less than 4%. From the data presented here, it can be stated that, at least in the initial optimization of the valve hemodynamic performance, the steady hydrodynamic evaluation of the valve could be an effective tool for analyzing the flow characteristics.     

新型生物人工肝反应器悬浮培养参数的优化测定

目的对新型生物人工肝反应器进行参数的优化测定,以寻找更适合肝细胞生长的工作条件。方法从实验和仿真两方面优化运行参数。通过调节生物反应器旋转速度、培养液流速,寻找能使细胞微载体均匀悬浮于反应器的合理运行参数。利用流体动力学仿真软件Fluent对新型生物反应器进行仿真,将仿真结果与实验结果进行对比得出合理工作范围。结果生物反应器的旋转往复周期Z越大,反应器内细胞微载体达到均匀悬浮状态时所需的培养液流速就越大,即控制培养液流动的蠕动泵转速￡越大。在确保细胞微载体均匀悬浮分布下,往复周期Z=0．5min时,没有合适的,J值满足要求;往复周期Z=0．4min时,三的工作范围为120～160r／min;往复周期Z=0．3min时,￡的工作范围为80～160r／min。结论合理的运行参数优化了生物人工肝的工作方式,为动物实验及临床实验奠定了基础。     

组织工程化旋转生物反应器研究进展

概述了组织工程化水平旋转生物反应器的工作原理、培养环境、应用现状和发展趋势。水平旋转生物反应器为体外培养动物细胞保持其正常形态、结构、功能和遗传特性提供了一种新手段，得天独厚的微重力、高效物质传递和低剪应力环境、多孔立体网状支架材料、在线监测和控制细胞三维生长等优势，为离体细胞重建组织、实现人工构建组织和器官有望成为现实。     

组织工程化旋转生物反应器研究进展

概述了组织工程化水平旋转生物反应器的工作原理、培养环境、应用现状和发展趋势。水平旋转生物反应器为体外培养动物细胞保持其正常形态、结构、功能和遗传特性提供了一种新手段,得天独厚的微重力、高效物质传递和低剪应力环境、多孔立体网状支架材料、在线监测和控制细胞三维生长等优势,为离体细胞重建组织、实现人工构建组织和器官有望成为现实。     

小口径组织工程血管生物反应器的制造

生物反应器在构建小口径组织工程血管中起着重要的作用.本文设计并造了能够模拟人体小口径动脉脉动流的生物反应器.该反应器的波形发生器输出成年人左心室容积变化信号驱动直线电机作为动力源,通过调节后负荷,从而产生近生理的脉动流.特殊设计的旋转培养室可以对三维的管壁支架进行二次的细胞接种.并且使得旋转接种和脉动培养能够连续进行.与现有的组织工程血管生物反应器相比,在理论和实践上有较大的创新性.     

利用hBMSCs在生物反应器中构建小口径血管的实验研究

目的采用改进的生物反应器系统,应用人骨髓间充质干细胞(hBMSCs)构建小口径的组织工程血管。方法设计一套血管生物反应器系统,采用有限元方法对组织工程小血管托架材料进行分析,从而设计一套用于构建直径为2mm的小血管托架;收集人的原代骨髓基质干细胞进行体外扩增和培养,选用第3代细胞与聚羟基乙酸酯(PGA)复合后置于血管生物反应器中动态培养;培养4周后,对材料复合物取材,进行大体观察、HE染色、扫描电镜和平滑肌免疫组化等指标检测。结果血管色泽明亮,有一定的弹性,用镊子反复压下血管能够反弹恢复原样;细胞分泌的胶原基质排列较规则,免疫组化结果表明血管含有平滑肌弹性肌动蛋白的成分。结论改进的血管生物反应器能模拟血管的力学环境,并能利用hBMSCs成功构建组织工程化小血管组织。     

心肌组织工程生物反应器研究进展

组织工程的研究主要围绕种子细胞、生物材料和组织构建这三个基本要素而展开。组织构建技术是组织工程研究的核心。组织工程生物反应器是一种体外构建人体组织的系统装置。心肌组织工程在替代和维持梗塞的心肌组织功能,并进而治愈疾病以最大限度地挽救病人生命方面可能发挥巨大作用。主要介绍了国内外工程化心肌组织体外构建技术,特别是用于构建工程化心肌组织的心肌组织工程生物反应器研究方面的进展。     

心脏瓣膜组织工程支架的制备及细胞种植

为了探讨心脏瓣膜组织工程支架的制备及细胞种植 ,我们将新鲜猪心瓣膜用胰蛋白酶及 DNA酶消化去除细胞 ,光镜和电镜观察其结构 ,并将人脐静脉内皮细胞株、猪颈动脉内皮细胞和狗肌成纤维细胞滴种在脱细胞猪心瓣膜上 ,HE和免疫组化染色观察。结果显示胰蛋白酶联合 DNA酶消化去除了所有细胞 ,而瓣膜的三维结构保持完好。种植的人内皮细胞几乎完全覆盖了瓣膜的表面 ,猪内皮细胞在瓣膜上呈斑块状生长 ,狗肌成纤维细胞不但生长于瓣膜表面 ,且渗透到基质内部生长。这表明 ,胰蛋白酶联合 DNA酶消化是一种较为理想的猪瓣膜脱细胞方法 ;人和猪血管内皮细胞及狗肌成纤维细胞均能在猪瓣膜支架上较好地黏附和生长。     

Versatile fabrication of vascularizable scaffolds for large tissue engineering in bioreactor

Despite significant progresses were achieved in tissue engineering over the last 20 years, a number of unsolved problems still remain. One of the most relevant issues is the lack of a proper vascularization that is limiting the size of the engineered tissues to smaller than clinically relevant dimensions. Sacrificial molding holds great promise to engineered construct with perfusable vascular architectures, but there is still the need to develop more versatile approaches able to be independent of the nature and dimensions of the construct. In this work we developed a versatile sacrificial molding technique for fabricating bulk, cell-laden and porous scaffolds with embedded vascular fluidic networks. These branched fluidic architectures are created by highly resistant thermoplastic sacrificial templates, made of poly(vinyl alcohol), representing a remarkable progress in manufacturability and scalability. The obtained architecture, when perfused in bioreactor, has shown to prevent the formation of a necrotic core in thick cell-laden constructs and enabled the rapid fabrication of hierarchically branched endothelium. In conclusion we demonstrate a novel strategy towards the engineering of vascularized thick tissues through the integration of the PVA-based microfabrication sacrificial approach and perfusion bioreactors. This approach may be able to scale current engineered tissues to clinically relevant dimensions, opening the way to their widespread clinical applications.     

Numerical analysis and linear theory of pulsatile flow in cylindrical deformable tubes: The testing of a numerical model for blood flow calculation

The purpose of this paper is to present standard results on the effect of nonlinearities on the computed pulsatile flow in a cylindrical distensible tube as a first stage in the calculation of flow in a tapered tube and blood flow in arteries. The calculations are made using the pressure-radius relationship of a rubber tube with no longitudinal motion and for a linearised relationship. The one-dimensional equations of motion are solved by the method of finite differences. The values of skin friction that are incorporated are determined from the vorticity and continuity equations for a rigid tube and a correction made to the current diameter at each time step. The accuracy of the results is assessed and the effect of varying parameters investigated. The method is applied to a segment of an infinite tube for which the linear analytical solution is available. The characteristics of the velocity wave calculated from an input pressure wave are presented as departures from the linear theory values of these characteristics, the wave speed, flux and transmission factor per wavelength. Computations are made at values of non-dimensional frequency (Stokes number α) of about 3 and 10. It is concluded that as far as physiological application is concerned (i.e. small amplitude and long wavelength) the results of linear theory are a very good first approximation for the cylindrical tube. At α=10, the relative departure of wave speed is about 0·5 times the relative diameter amplitude ( amplitude/mean diameter) when the pressure-radius relation is linear and the pressure and velocity waves have the same characteristics. At α=3 the corresponding wave speed departure is about 0·1 . The relative departure of the flux is less than 0·05 at α=3 and about 0·5 at α=10. The transmission coefficient has a relative departure of less than 0·05 at α=10 and its relative increase at α=3 is about 0·3 .     

生物反应器体外构建组织工程化尿路肌性管腔

背景：理想的组织工程尿道替代物应具有良好的力学特性,足以承受长时间的尿液排泄冲击,而静态培养的尿路肌性管腔强度不佳。已有研究表明,力学刺激能够促进细胞生长和细胞外基质的分泌。 目的：探讨生物反应器内构建组织工程化尿路肌性管腔的可行性。 方法：酶消化法获取脂肪干细胞,经体外培养和扩增后,流式细胞技术检测细胞表面抗原,将脂肪干细胞接种于聚羟基乙酸上,形成细胞-材料复合物,体外培养1周后,将其置于生物反应器内培养,实验组予以动态力学刺激培养;对照组为静态培养,先采用基础培养基培养3周,而后用成肌诱导培养液诱导4周,行大体观察及组织学检测。 结果与结论：流式细胞仪检测细胞表面CD90,CD44,CD105表达率分别为99.42%,98.12%,93.27%;CD34,CD45表达率分别为4.92%和0.38%,实验组培养的肌性管腔色泽明亮,管腔圆润,免疫组化染色显示,细胞材料复合物在诱导4周后,细胞表达结蛋白和α-平滑肌肌动蛋白阳性,细胞材料复合物胶原成分多。对照组构建的肌性管腔色泽暗淡,管腔轻度塌陷,细胞材料复合物胶原成分较少。提示脂肪干细胞复合聚羟基乙酸材料在生物反应器内动态培养可构建具有良好结构的尿路肌性管腔。 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;组织工程全文链接：     

组织工程生物反应器的力学环境特点

组织工程学是一门以细胞生物学和工程学为基础,应用工程学和生命科学的原理,开发器官缺损患者所需代替物,并构建和保持或增强其组织功能性的一门交叉学科。生物反应器作为组织工程中非常重要的工具,目前主要是从生物力学问题、三维空间培养问题、传质问题、培养的环境条件问题(pH、氧张力等)和物理因素(电场、磁场、应力场)等方面开展其研究。本文作者主要从生物力学角度介绍组织工程生物反应器研究的最新进展,重点探讨组织工程生物反应器的力学环境。     

振荡流动对组织工程用灌注式生物 反应器中剪切力分布影响

目的 考察振荡流动以及三维支架孔径和孔隙率对生物反应器内流速和剪切力分布的影响,并根据理论计算结果为脱细胞骨三维支架和灌注式生物反应器制备提出优化方法。方法 针对实验室前期制备的骨组织工程用脱细胞骨三维支架和灌注式生物反应器,将脱细胞骨三维支架简化为各向同性的多孔介质,对生物反应器内的流速和剪切力分布进行理论建模。结果 振荡流作用时,多孔支架材料内速度和达西剪切力呈现一致的变化规律,不同半径处流速和达西剪切力差异减小,有利于在骨组织工程中对种子细胞进行均匀三维培养。提高入口灌流速度可提高平均达西剪切力;增加多孔支架孔径或孔隙率对支架内流速峰值影响不大,但会显著降低平均达西剪切力;提高入口振荡流动振荡频率可降低支架内流速最大峰值,显著减小不同半径处流速的差异。结论 适宜的振荡流易产生利于骨组织工程干细胞所需剪切力,研究结果有望为优化骨组织工程中种子细胞的三维培养方法提供理论指导。     

一种新型的生物反应器

本文介绍了一种应用于生物透析系统 L BDS的专用生物反应器 SBR。叙述了它的工作原理、结构组成和特点。给出了动物实验的结果。实验验证了这种专用生物反应器 ,可以大量、长期地培养肝细胞 ,并使其保持持久的活性和功能。验证了血液和肝细胞之间的免疫反应 ,并观察到了它的新陈代谢。     

Scaffolds for tissue engineering of cardiac valves

Tissue engineered heart valves offer a promising alternative for the replacement of diseased heart valves avoiding the limitations faced with currently available bioprosthetic and mechanical heart valves. In the paradigm of tissue engineering, a three-dimensional platform – the so-called scaffold – is essential for cell proliferation, growth and differentiation, as well as the ultimate generation of a functional tissue. A foundation for success in heart valve tissue engineering is a recapitulation of the complex design and diverse mechanical properties of a native valve. This article reviews technological details of the scaffolds that have been applied to date in heart valve tissue engineering research.     

Design and Hydrodynamic Evaluation of a Novel Pulsatile Bioreactor for Biologically Active Heart Valves

Biologically active heart valves (tissue engineered and recellularized tissue-derived heart valves) have the potential to offer enhanced function when compared to current replacement value therapies since they can possibly remodel, and grow to meet the needs of the patient, and not require chronic medication. However, this technology is still in its infancy and many fundamental questions remain as to how these valves will function in vivo. It has been shown that exposing biologically active tissue constructs to pulsatile pressures and flows during in vitro culture produces enhanced extracellular matrix protein expression and cellularity, although the ideal hydrodynamic conditioning regime is as yet unknown. Moreover, in vitro organ-level studies of living heart valves aimed at studying the remodeling processes require environments that can accurately reproduce in vivo hemodynamics under sterile conditions. To this end, we have developed a system to study the effects of subjecting biologically active heart valves to highly controlled pulsatile pressure and flow waveforms under sterile conditions. The device fits inside a standard incubator and utilizes a computer-controlled closed loop feedback system to provide a high degree of control. The mean pressure, mean flow rate, driving frequency, and shape of the pulsatile pressure waveform can be changed automatically in order to simulate both physiologic and nonphysiologic hemodynamic conditions. Extensive testing and evaluation demonstrated the device's ability to subject a biologically active heart valve to highly controlled pulsatile waveforms that can be modulated during the course of sterile incubation.     

一种组合式组织工程动态培养仪

随着组织工程技术应用的多样化,为不同的目标组织提供不同的培养条件,改进工程组织的化学和生物力学性能,或通过不同的培养条件诱导干细胞向不同方向分化,这些得到了学者们越来越多的关注。结合中国现有的实验室条件,中国医学科学院,北京协和医学院整形外科医院自主研发了一种组合式组织工程动态培养仪,以为不同的目标工程组织提供不同的动态培养环境。