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。     

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。     

组织工程心脏瓣膜的构建：现状与前景

背景：目前临床上应用的心脏生物瓣和机械瓣都存在一些缺陷和不足,而组织工程心脏瓣膜有可能避免这些问题的出现,成为瓣膜替代物的理想选择。 目的：探讨构建组织工程心脏瓣膜的实验研究进展。 方法：应用数据库检索的方法分析关于组织工程心脏瓣膜的实验研究文献,组织工程心脏瓣膜的三大要素为种子细胞、支架材料和细胞种植。 结果与结论：心脏瓣膜修复和置换是目前治疗心脏瓣膜性疾病的主要外科手段。目前,主要用于构建组织工程心脏瓣膜的种子细胞有血管内皮细胞、内皮祖细胞以及骨髓间充质干细胞等。经脱细胞处理的支架具有良好的生物力学性能和组织相容性,细胞种植后支架表面会形成一层连续的细胞层,其构建的组织工程心脏瓣膜是可行的。组织工程心脏瓣膜有着良好的应用前景,但目前还有很多问题需要解决,还处于研究的初级阶段。 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：     

修复运动性肌腱损伤组织工程化肌腱及种子细胞和支架材料

背景：获得大规模、具有再生活力的种子细胞以及具有与正常人体肌腱组织相接近的力学性能的理想支架材料是当前组织工程化肌腱研究面临的最为关键的限制性因素。 目的：总结和分析组织工程肌腱研究中的种子细胞和支架材料的研究进展。 方法：查阅近年来肌腱组织工程研究的相关文献,综合国内外最新研究成果,就肌腱组织工程中合适的种子细胞来源、研究更为理想的支架材料及组织相容性等方面的进展进行概述。 结果与结论：肌腱组织工程中常用的种子细胞有间充质干细胞、肌腱干细胞及胚胎干细胞等,可以向骨、软骨和脂肪分化,修复肌腱损伤的理想细胞。肌腱组织工程支架材料有天然材料及人工合成材料等,肌腱组织工程支架材料应有良好的生物相容性和适度的机械性能,复合材料将是肌腱组织工程支架材料研究的重点。 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      

初级组织工程小口径血管的制备

目的 探讨利用组织工程技术构建小口径组织工程化血管的可行性.方法 将犬骨髓细胞诱导为平滑肌细胞和内皮细胞后,用Brdu标记平滑肌细胞,DAPI标记内皮样细胞,观察细胞标记的成功率;血管平滑肌样细胞和血管内皮样细胞分层种植于胶原包埋PGA的复合支架表面:将细胞和支架的复合体种植于犬皮下,并设立对照组,分别于植入后的第4、6、8周取出植入皮下的组织工程化血管,进行相关检查分析.结果 免疫荧光提示Brdu、DAPI标记种子细胞效果良好;核呈蓝色荧光的内皮样细胞均匀排列在支架表面:组织工程化血管材料植入动物皮下取出后观察,实验组管壁结构比较清晰,细胞数量也已减少、胶原成分增多,管壁分层明显,可见内膜、中膜和外膜结构,对照组无此特点;免疫荧光观察,8周实验组组织工程血管壁内有Brdu标记的种子细胞存在.结论 以动物的皮下为生物反应器,采用静态培养的方式可强化组织工程血管壁,该组织的结构与天然血管相似,种子细胞同时参与初级组织工程小口径血管的构建.     

生物反应器在组织工程研究中的应用

生物反应器在组织工程研究中的应用非常广泛,从最初的种子细胞增殖、分化,到关键的组织体外构建,都可以利用生物反应器来模拟细胞和组织在体内的生长环境,提高工程化组织构建的效率。本文结合本期发表的6篇相关文献,介绍了组织工程中研究中生物反应器的作用,建议通过包括生物力学在内的多学科的研究手段,获得调控不同类型细胞生长和组织构建的关键参数,实现功能化组织工程的研究目标。     

血管组织工程的研究与进展

背景：血管组织工程是指利用血管壁的正常细胞和生物可降解材料来制备、重建和再生血管替代材料的科学。近年来,组织工程学技术的发展推动了组织工程化血管的研究,已成为今后血管替代物的重要方向。 目的：综述血管组织工程的相关临床及基础研究进展。 方法：检索SCI数据库2001/2010有关血管组织工程的文献,检索词为“组织工程血管(tissue-engineered vascular);组织工程(tissue engineering);血管(vascular);支架材料(scaffold materials);支架(scaffolds);种子细胞(seed cell);细胞外基质(extracellular matrix, ECM);血管支架(vascular scaffold);高分子材料(polymer materials);复合材料(composite materials);纳米(nanometer);生物材料(biological materials)”,对血管组织工程的临床及基础文献进行分析。 结果与结论：血管组织工程研究的内容主要有种子细胞、细胞外基质替代物以及组织工程血管三维培养。血管组织工程所应用的种子细胞包括自体血管壁细胞、胚胎干细胞和骨髓间充质干细胞,还包括内皮细胞,平滑肌细胞及成纤维细胞等众多组织细胞。在组织工程血管构建中血管组织微环境是活细胞在体外生长所需的支持物,是种子细胞生长增殖的三维空间,便于细胞黏着、生长、进行新陈代谢。因此,组织工程血管需要具有良好的生物相容性,可塑性强,来源广泛,有一定的抗张强度和无免疫原性的支架材料。根据来源和性能,目前研究应用的材料分为天然生物生材料和合成材料两种。     

体外构建工程化心肌组织的研究与应用

背景：“工程化心肌组织”是应用组织工程的方法,构建出具有天然心肌组织特征的心肌,将它移植到体内,最终修复或完全替代病损组织。但以往研究获得的工程化心肌组织存在较多的缺陷,仍不能满足实际需要。 目的：总结体外构建工程化心肌组织方法的研究进展。 方法：由第一作者用计算机检索中国期刊全文数据库(CNKI：2000/2010)和Medline(2000/2010)数据库,检索词分别为“心肌,组织工程,心肌构建物”和“Myocardial Construction materials, Tissue Engineering, Myocardial”。从种子细胞、支架材料、体外培养环境和生物反应体系、工程化心肌组织的再血管化、移植实验5方面进行总结,对不同的种子细胞及生物支架材料和体外培养的环境、移植工程化组织的再血管化等方面进行了介绍。共检索到150篇文章,按纳入和排除标准对文献进行筛选,共纳入30篇文章。 结果与结论：适合心肌细胞生存和心肌细胞形成的新型生物活性支架,理想的种子细胞、体外培养环境与生物反应体系、移植工程化组织的再血管化,动物移植实验等方面都是组织工程化心肌组织再造的关键所在。构建的组织工程化心肌组织应同时具备良好的收缩功能、稳定的电生理特性、力学强度和柔韧性、无免疫原性及自身能血管化或在移植后能迅速血管化的条件,但未来还需进一步研究。     

组织工程皮肤的研究新进展

组织工程皮肤是指由细胞或细胞外基质或由两者共同结合组成的皮肤产品,足应用生命科学和工程学的原理与技术将种子细胞与适当的支架材料相结合构建出的用于修复、维护和改善损伤皮肤组织功能和形态的生物替代物。目前的组织工程皮肤包括自体或异体培养的表皮片、真皮替代物及含表真皮双层结构的皮肤替代物二种,已成为修复大面积、深度皮肤缺损创面最有应用前景的方法之一。皮肤组织工程的研究内容主要包括三个方面,即皮肤种子细胞培养、真皮支架材料和体外构建活性复合皮。本文即从这三方面对组织工程化皮肤的研究进展作一简要综述。     

基于计算流体力学的小血管生物反应器设计

目的：设计一套能构建内壁直径为2mm的组织工程小血管的生物反应器。方法：根据计算流体力学原理和方法对组织工程小血管托架材料进行分析，设计一套用于培养2mm小血管的生物反应器。采用压注成型技术制作了小血管的托架。结果：确定了硅胶管的尺寸结构并获得了成型产品，设计完成了培养室内相应的辅助结构，使整个反应器系统能够对PGA-细胞材料复合物进行动态培养。结论：小血管托架的流体力学仿真分析是合理的，在此基础上构建的血管生物反应器性能稳定、可靠。     

Flow Perfusion Culture of Marrow Stromal Cells Seeded on Porous Biphasic Calcium Phosphate Ceramics

Calcium phosphate ceramics have been widely used for filling bone defects to aid in the regeneration of new bone tissue. Addition of osteogenic cells to porous ceramic scaffolds may accelerate the bone repair process. This study demonstrates the feasibility of culturing marrow stromal cells (MSCs) on porous biphasic calcium phosphate ceramic scaffolds in a flow perfusion bioreactor. The flow of medium through the scaffold porosity benefits cell differentiation by enhancing nutrient transport to the scaffold interior and by providing mechanical stimulation to cells in the form of fluid shear. Primary rat MSCs were seeded onto porous ceramic (60% hydroxyapatite, 40% β-tricalcium phosphate) scaffolds, cultured for up to 16 days in static or flow perfusion conditions, and assessed for osteoblastic differentiation. Cells were distributed throughout the entire scaffold by 16 days of flow perfusion culture whereas they were located only along the scaffold perimeter in static culture. At all culture times, flow perfused constructs demonstrated greater osteoblastic differentiation than statically cultured constructs as evidenced by alkaline phosphatase activity, osteopontin secretion into the culture medium, and histological evaluation. These results demonstrate the feasibility and benefit of culturing cell/ceramic constructs in a flow perfusion bioreactor for bone tissue engineering applications.     

The importance of bicarbonate and nonbicarbonate buffer systems in batch and continuous flow bioreactors for articular cartilage tissue engineering

In cartilage tissue engineering an optimized culture system, maintaining an appropriate extracellular environment (e.g., pH of media), can increase cell proliferation and extracellular matrix (ECM) accumulation. We have previously reported on a continuous-flow bioreactor that improves tissue growth by supplying the cells with a near infinite supply of medium. Previous studies have observed that acidic environments reduce ECM synthesis and chondrocyte proliferation. Hence, in this study we investigated the combined effects of a continuous culture system (bioreactor) together with additional buffering agents (e.g., sodium bicarbonate [NaHCO?]) on cartilaginous tissue growth in vitro. Isolated bovine chondrocytes were grown in three-dimensional cultures, either in static conditions or in a continuous-flow bioreactor, in media with or without NaHCO?. Tissue constructs cultivated in the bioreactor with NaHCO?-supplemented media were characterized with significantly increased (p<0.05) ECM accumulation (glycosaminoglycans a 98-fold increase; collagen a 25-fold increase) and a 13-fold increase in cell proliferation, in comparison with static cultures. Additionally, constructs grown in the bioreactor with NaHCO?-supplemented media were significantly thicker than all other constructs (p<0.05). Further, the chondrocytes from the primary construct expanded and synthesized ECM, forming a secondary construct without a separate expansion phase, with a diameter and thickness of 4?mm and 0.72?mm respectively. Tissue outgrowth was negligible in all other culturing conditions. Thus this study demonstrates the advantage of employing a continuous flow bioreactor coupled with NaHCO? supplemented media for articular cartilage tissue engineering.     

In vitro localization of bone growth factors in constructs of biodegradable scaffolds seeded with marrow stromal cells and cultured in a flow perfusion bioreactor

Tissue engineering strategies aim at controlling the behavior of individual cells to stimulate tissue formation. This control is achieved by mimicking signals that manage natural tissue development or repair. Flow perfusion bioreactors that create culture environments with minimal diffusion constraints and provide cells with mechanical stimulation may closely resemble in vivo conditions for bone formation. Therefore, these culturing systems, in conjunction with an appropriate scaffold and cell type, may provide significant insight towards the development of in vitro tissue engineering models leading to improved strategies for the construction of bone tissue substitutes. The objective of this study was to investigate the in vitro localization of several bone growth factors that are usually associated with bone formation in vivo by culturing rat bone marrow stromal cells seeded onto starch-based biodegradable fiber meshes in a flow perfusion bioreactor. The localization of several bone-related growth factors-namely, transforming growth factor-beta1, platelet-derived growth factor- A, fibroblast growth factor-2, vascular endothelial growth factor, and bone morphogenetic protein- 2-was determined at two different time points in scaffolds cultured under perfusion conditions at two different flow rates using an immunohistochemistry technique. The results show the presence of regions positively stained for all the growth factors considered, except platelet-derived growth factor-A. Furthermore, the images obtained from the positively stained sections suggest an increase in the immunohistochemically stained area at the higher flow rate and culture time. These observations demonstrate that flow perfusion augments the functionality of scaffold/cell constructs grown in vitro as it combines both biological and mechanical factors to enhance cell differentiation and cell organization within the construct. This study also shows that flow perfusion bioreactor culture of marrow stromal cells, combined with the use of appropriate biodegradable fiber meshes, may constitute a useful model to study bone formation and assess bone tissue engineering strategies in vitro.     

In vitro study of the effect of cyclic strains on the dermal fibroblast (GM3384) morphology--mapping of cell responses to strain field

Cells can respond to mechanical forces and actively interact with mechanical stimulations in vitro. Understanding the effect of mechanical loading on cell morphology signifies a critical biomechanics issue in tissue engineering. In this study, human dermal fibroblasts (GM3384) underwent cyclic strain. This was done by culturing a monolayer of the cells onto a transparent membrane and applying a cyclic stress using a computer controlled bioreactor. The cells were mechanically stimulated at around 7% strain with 1 cycle per minute for 2 days. Finite element analysis (FEA) was then employed to characterize the strain field across the substrate membrane in the bioreactor. The results showed that strain distribution were non-uniform in the substrate membrane. The mapping of cell morphology with the strain field revealed that the cells exposed to the equibiaxial strain exhibited the classical spindle morphology while the cells subjected to uniaxial strain changed to a polygonal morphology. It is concluded that the nature of the strain has significant impact on the final cell morphology.     

三维立体空间动态培养及骨髓间充质干细胞软骨向分化的研究

目的三维立体空间动态诱导骨髓间充质干细胞软骨向分化，分析培养状态对软骨向分化的影响。方法分离培养骨髓间充质干细胞（MSC），以海藻酸钠凝胶载体诱导软骨向分化，于转壁反应器制造周期应力性三维立体环境，比较凝胶立体结构、细胞聚集状态、细胞构型等因素对软骨向分化的影响，分别以凝胶微球悬浮高密度细胞、凝胶包埋离心细胞团、凝胶覆盖细胞及普通细胞培养等方式．进行空间动态诱导培养：同时各培养方式分别设立相应静止培养方式作为对照。10d后观察大体和组织学形态．测量生物化学指标并筛选。结果三维诱导方式比平面诱导有效，动态诱导环境优于静止诱导环境．结合三维立体空间培养和动态环境可进一步提高软骨诱导效率。其中．凝胶微球悬浮高密度细胞的培养方式分化效果最好．优于凝胶包埋细胞团的培养方式。结论海藻酸钠凝胶微球立体动态培养可有效诱导MSC的软骨向分化．为改进软骨组织工程种子细胞的立体空间动态培养提供新思路。     

Engineering of rat articular cartilage on porous sponges: effects of tgf-beta 1 and microgravity bioreactor culture

The objective of this study was to develop an engineered rat hyaline cartilage by culturing articular chondrocytes on three-dimensional (3D) macroporous poly(DL-lactic-co-glycolic acid) (PLGA) sponges under chondrogenic induction and microgravity bioreactor conditions. Experimental groups consisted of 3D static and dynamic cultures, while a single cell monolayer (2D) served as the control. The effect of seeding conditions (static vs. dynamic) on cellularization of the scaffolds was investigated. MTT assay was used to evaluate the number of viable cells in each group at different time points. Formation of a hyaline-like cartilage was evaluated for up to 4 weeks in vitro. While 2D culture resulted in cell sheets with very poor matrix production, 3D culture was in the favor of tissue formation. A higher yield of cell attachment and spatially uniform cell distribution was achieved when dynamic seeding technique was used. Dynamic culture promoted cell growth and infiltration throughout the sponge structure and showed the formation of cartilage tissue, while chondrogenesis appeared attenuated more towards the outer region of the constructs in the static culture group. Medium supplemented with TGF-beta 1 (5 ng/ml) had a positive impact on proteoglycan production as confirmed by histochemical analyses with Alcian blue and Safranin-O stainings. Formation of hyaline-like tissue was demonstrated by immunohistochemistry performed with antibodies against type II collagen and aggrecan. SEM confirmed higher level of cellularization and cartilage tissue formation in bioreactor cultures induced by TGF-beta 1. The data suggest that PLGA sponge inside rotating bioreactor with chondrogenic medium provides an environment that mediates isolated rat chondrocytes to redifferentiate and form hyaline-like rat cartilage, in vitro.     

Sequential biochemical and mechanical stimulation in the development of tissue-engineered ligaments

Application of stimuli in sequence to developing cultures in vitro offers the potential to intricately direct cell development and differentiation by following the template of native tissue behavior. We hypothesize that administration of mechanical stimulation at the peak of growth factor-induced cell activity will differentiate bone marrow stromal cells (BMSCs) along a fibroblast lineage and enhance in vitro ligament development through enhanced matrix ingrowth, matrix metalloproteinase-2 (MMP-2) production, collagen type I production, and extracellular matrix (ECM) alignment. BMSC-seeded silk matrices were cultured in a static growth-factor-free environment for 5 days prior to loading into bioreactor vessels to first establish an appropriate dynamic rotational regime, as determined through assessment of cell activity, histology, and surface topography. Once the regime was determined, seeded matrices initially cultured in basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), or growth-factor-free control medium for 5 days were loaded into the bioreactor for 9 days of mechanical stimulation. Our findings indicated that the sequential application of mechanical stimulation following growth factor supplemented static culture-induced cell differentiation toward a fibroblast lineage, enhancing matrix ingrowth, cell and ECM alignment, and total collagen type I produced compared to respective static cultures. The current results suggest a dynamic culturing regime in the development of engineered tissues.     

Functional activity of hepatocytes in tissue fragments in a new bioreactor biological artificial liver

Functional activity of hepatocytes in a new bioreactor designed for culturing of liver tissue fragments under perfusion conditions was tested. Specific hepatic functions such as ammonium detoxification, urea and protein synthesis, and P-450-dependent metabolism of p-nitroanisole were maintained for 1–1.5 days. The bioreactor can be used as a bioartificial liver support apparatus. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 129, No. 6, pp. 698–700, June, 2000     

Cell-nanofiber-based cartilage tissue engineering using improved cell seeding, growth factor, and bioreactor technologies

Biodegradable nanofibrous scaffolds serving as an extracellular matrix substitute have been shown to be applicable for cartilage tissue engineering. However, a key challenge in using nanofibrous scaffolds for tissue engineering is that the small pore size limits the infiltration of cells, which may result in uneven cell distribution throughout the scaffold. This study describes an effective method of chondrocyte loading into nanofibrous scaffolds, which combines cell seeding, mixing, and centrifugation to form homogeneous, packed cell-nanofiber composites (CNCs). When the effects of different growth factors are compared, CNCs cultured in medium containing a combination of insulin-like growth factor-1 and transforming growth factor-beta1 express the highest mRNA levels of collagen type II and aggrecan. Radiolabeling analyses confirm the effect on collagen and sulfated-glycosaminoglycans (sGAG) production. Histology reveals chondrocytes with typical morphology embedded in lacuna-like space throughout the entire structure of the CNC. Upon culturing using a rotary wall vessel bioreactor, CNCs develop into a smooth, glossy cartilage-like tissue, compared to a rough-surface tissue when maintained in a static environment. Bioreactor-grown cartilage constructs produce more total collagen and sGAG, resulting in greater gain in net tissue weight, as well as express cartilage-associated genes, including collagen types II and IX, cartilage oligomeric matrix protein, and aggrecan. In addition, dynamic culture enhances the mechanical property of the engineered cartilage. Taken together, these results indicate the applicability of nanofibrous scaffolds, combined with efficient cell loading and bioreactor technology, for cell-based cartilage tissue engineering.     

Treatment of acute liver failure in pigs reduces hepatocyte function in a bioartificial liver support system

Several different types of bioartificial liver (BAL) support systems have been developed to bridge patients suffering from acute liver failure (ALF) to transplantation or liver regeneration. In this study we assessed the effects of ALF plasma on hepatocyte function in the BAL system that has been developed in our center. Pigs (40-60 kg) were anaesthetised and a total hepatectomy was performed. Cells were isolated from the resected livers and were transferred to the bioreactor of the BAL system. Twenty hours after cell isolation, hepatocytes in the BAL were tested for cell viability and functional activity by using a recirculating test medium in which assessment of LDH leakage, ammonia clearance, urea synthesis, 7-ethoxycoumarin O-deethylase (ECOD) activity and pseudocholine esterase production was performed. Subsequently, two groups were studied. In one group (I, n=5), the cell-loaded bioreactor was used to treat the donor pig, rendered anhepatic, for 24 hours. In the second group (II, n=5) the bioreactor was cultured for 24 h and served as a control. After 24 hours treatment or culturing, the cell viability count and functional activity tests were repeated. The results show that hepatocytes in the BAL remained viable after 24 h treatment of anhepatic pigs, as shown by the LDH release and pseudocholine esterase production. However, metabolic functions such as ammonia clearance, ECOD and urea synthesis were reduced after 24 h exposure of hepatocytes to autologous ALF plasma, whereas these functions were unaltered after 24 h culturing of the cells in the bioreactor.