组织工程皮肤生物反应器的研究与设计

目的设计一套生物反应器,能针对不同支架材料--细胞复合物进行构建组织工程皮肤.方法根据皮肤的自身生长特点和不同支架材料-细胞复合物的特性,模拟皮肤的生长环境和力学环境,通过生物反应器解决组织工程皮肤构建中支架的装夹和气液界面问题.结果生物反应器由控制系统和生物反应器主体两部分构成,能提供对多种皮肤细胞复合物的动态培养.结论皮肤生物反应器能够满足不同组织工程皮肤产品的需要.能够形成气液界面和模拟生物力学的刺激.     

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。     

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。     

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。     

成骨细胞在旋转壁式生物反应器内的大规模扩增

目的 进行SD大鼠成骨细胞在旋转壁式生物反应器内大规模扩增培养的研究。方法利用微载体悬浮培养法在旋转壁式生物反应器内大规模扩增成骨细胞，检测其组织形态和生物功能后。作为接种到支架材料上并于反应器内三维环境中培养组织工程骨的种子细胞。结果成骨细胞在生物反应器中每代可以扩增十倍以上，同时经过倒置相差显微镜、SEM(扫描电镜)以及ALP(碱性磷酸酶)和MTT等生物学性能检测后，发现在旋转壁式生物反应器中三维培养的成骨细胞各种生物指标性能良好。结论新型旋转壁式生物反应器可以提供低剪切力的培养环境，而且细胞之间有三维联系的机会，成骨细胞表现出良好的体外扩增能力，适于建立一种理想的成骨细胞体外扩增的三维培养体系。     

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.     

肝细胞体外培养平台的仿生设计与构建

构建一种能够更好地模拟肝细胞生长环境的体外培养平台。利用先进制造方法中的快速成型技术和微压印技术制作生物反应器和三维支架。将三维支架置于生物反应器中,与蠕动泵、氧和器、储液罐构建肝细胞的体外的动态培养系统。研究培养液流速、氧流量与氧含量的关系并进行参数优化,利用细胞支架复合体进行细胞培养。细胞培养结果显示该系统能够维持肝细胞的正常存活和生长。所设计并实现的动态灌注培养系统,通过调整流速和氧流量,可具有良好的氧合效果和较高的效率,能够更好的模拟肝细胞的体内生长环境。     

中空纤维生物反应器的构建及其体外灌流实验

设计原代培养的乳猪肝细胞构建生物反应器,并观察肝细胞生长特点,进行体外灌流实验。采用改良的原位胶原酶灌流法分离乳猪肝细胞及间质细胞,在中空纤维舱的纤维外间隙中共培养,并间断旋转抑制细胞贴壁。用该生物反应器建立人工肝系统,对肝硬化病人的腹水进行体外灌流实验。结果表明,乳猪原代肝细胞平均产量为(6.29±0.37)×108细胞,肝细胞的活性为84%;在纤维外间隙内间断转动培养3 h后肝细胞聚合成球。电镜可见,多个肝细胞聚合成团生长,培养的肝细胞有功能性结合,并向组织型转化。测定中空纤维舱内连续48 h培养液尿素浓度,证实肝细胞聚合体有很好的尿素合成功能。体外灌流后,生物反应器组腹水总胆红素下降,A ST升高,与对照组相比有显著性差异。生物反应器组葡萄糖浓度下降,对照组无显著变化,两组间有显著性差异。通过本组实验,证实了我们设计的生物反应器所构成的生物型人工肝(BAL)是有效的。     

自制双腔生物反应器构建组织工程骨软骨复合体的体外性能

背景:关节软骨损伤往往并发软骨下骨损伤形成骨软骨复合缺损,其治疗仍为骨科急待解决的问题,利用组织工程学构建骨软骨复合体为治疗该类疾患提供了新思路。目的:探讨利用自行设计制造的双腔搅拌式生物反应器构建一体化组织工程骨软骨复合体的可行性。方法:在双腔搅拌式生物反应器内对复合于β-磷酸三钙支架材料的羊骨髓间充质干细胞同时进行成骨和成软骨诱导,并根据施加剪切应力分为动态培养组和静态培养组。利用MTT试验、RT-PCR和扫描电镜检测骨髓间充质干细胞体外增殖和诱导分化情况。结果与结论:MTT试验和扫描电镜结果显示,骨髓间充质干细胞增殖良好。成骨和成软骨相关基因RT-PCR检测结果表明,骨髓间充质干细胞诱导分化良好,动态培养组要优于静态培养组。提示利用自行设计制作的双腔搅拌式生物反应器进行骨软骨复合体的体外构建是可行的,力学刺激环境下的构建效果要优于静态环境。     

乳鼠成骨细胞与珊瑚-羟基磷灰石支架材料在旋转式生物反应器中培养的实验研究

目的探讨联合运用成骨细胞、珊瑚-羟基磷灰石(CHA)支架材料和自行研制的旋转式生物反应器在体外构建组织工程化骨的可行性.方法运用骨片组织培养法,分离培养Wistar乳鼠的成骨细胞,传至第3代后,形成成骨细胞-CHA复合物,然后在自行研制的生物反应器中培养.通过茜素红(ARS)染色,MTT法及其他组织化学的方法来研究在生物反应器中培养14 d时的成骨潜能.结果成骨细胞和CHA支架材料有良好的生物相容性.在生物反应器中,成骨细胞-CHA复合物培养至第14天时,CHA明显促进了细胞的增殖,ARS染色呈弱阳性反应,同时,碱性磷酸酶(ALP)的分泌减少.结论CHA材料和生物反应器可以为成骨细胞提供良好的生物学及力学环境,三者联合运用有希望成为构建组织工程骨的理想方法.     

旋转灌注式生物反应器系统构建及在骨组织工程中的应用

构建一种旋转灌注式生物反应器系统，并设计制造具有相互连通微管道结构的大段支架。生物反应器在旋转的同时实现对大段支架的灌注，系统内外气体的实时交换通过安装在反应器两端的半透膜和可透气的蠕动泵软管实现。容器内的氧气和营养物质在得到充分混合之后，连续不断地输送到支架微管道内。使黏附在微管道内的细胞获得充足营养的同时受到一定流体剪应力的刺激，调节细胞功能的发挥。该系统克服了静态培养中存在的各种缺点，改善了培养环境，增加了培养过程的可控性，有助于促进黏附在微管道内部的细胞增殖、分化和产生大量基质。将成骨细胞／支架结构体在该生物反应器系统中培养14d后，用扫描电镜观察细胞在大段支架微管道内的生长情况，结果表明，支架微管道内有大量细胞长入。     

Tubular perfusion system for the long-term dynamic culture of human mesenchymal stem cells

In vitro culture techniques must be improved to increase the feasibility of cell-based tissue engineering strategies. To enhance nutrient transport we have developed a novel bioreactor, the tubular perfusion system (TPS), to culture human mesenchymal stem cells (hMSCs) in three-dimensional scaffolds. This system utilizes an elegant design to create a more effective environment for cell culture. In our design, hMSCs in the TPS bioreactor are encapsulated in alginate beads that are tightly packed in a tubular growth chamber. The medium is perfused by a peristaltic pump through the growth chamber and around the tightly packed scaffolds enhancing nutrient transfer while exposing the cells to shear stress. Results demonstrate that bioreactor culture supports early osteoblastic differentiation of hMSCs as shown by alkaline phosphatase gene expression. After 14 and 28 days of culture significant increases in the gene expression levels of osteocalcin, osteopontin, and bone morphogenetic protein-2 were observed with bioreactor culture, and expression of these markers was shown to increase with media flow rate. These results demonstrate the TPS bioreactor as an effective means to culture hMSCs and provide insight to the effect of long-term shear stresses on differentiating hMSCs.     

Enhancement of viability of muscle precursor cells on 3D scaffold in a perfusion bioreactor

The aim of this study was to develop a methodology for the in vitro expansion of skeletal-muscle precursor cells (SMPC) in a three-dimensional (3D) environment in order to fabricate a cellularized artificial graft characterized by high density of viable cells and uniform cell distribution over the entire 3D domain. Cell seeding and culture within 3D porous scaffolds by conventional static techniques can lead to a uniform cell distribution only on the scaffold surface, whereas dynamic culture systems have the potential of allowing a uniform growth of SMPCs within the entire scaffold structure. In this work, we designed and developed a perfusion bioreactor able to ensure long-term culture conditions and uniform flow of medium through 3D collagen sponges. A mathematical model to assist the design of the experimental setup and of the operative conditions was developed. The effects of dynamic vs static culture in terms of cell viability and spatial distribution within 3D collagen scaffolds were evaluated at 1, 4 and 7 days and for different flow rates of 1, 2, 3.5 and 4.5 ml/min using C2C12 muscle cell line and SMPCs derived from satellite cells. C2C12 cells, after 7 days of culture in our bioreactor, perfused applying a 3.5 ml/min flow rate, showed a higher viability resulting in a three-fold increase when compared with the same parameter evaluated for cultures kept under static conditions. In addition, dynamic culture resulted in a more uniform 3D cell distribution. The 3.5 ml/min flow rate in the bioreactor was also applied to satellite cell-derived SMPCs cultured on 3D collagen scaffolds. The dynamic culture conditions improved cell viability leading to higher cell density and uniform distribution throughout the entire 3D collagen sponge for both C2C12 and satellite cells.     

Effects of perfusion and dynamic loading on human neocartilage formation in alginate hydrogels

Dynamic loading and perfusion culture environments alone are known to enhance cartilage extracellular matrix (ECM) production in dedifferentiated articular chondrocytes. In this study, we explored whether a combination of these factors would enhance these processes over a free-swelling (FS) condition using adult human articular chondrocytes embedded in 2% alginate. The alginate constructs were placed into a bioreactor for perfusion (P) only (100?μL/per minute) or perfusion and dynamic compressive loading (PL) culture (20% for 1?h, at 0.5?Hz), each day. Control FS alginate gels were maintained in six-well static culture. Gene expression analysis was conducted on days 7 and 14, while cell viability, immunostaining, and mechanical property testing were performed on day 14 only. Total glycosaminoglycan (GAG) content and GAG synthesis were assessed after 14 days. Col2a1 mRNA expression levels were significantly higher (at least threefold; p<0.05) in both bioreactor conditions compared with FS by days 7 and 14. For all gene studies, no significant differences were seen between P and PL treatments. Aggrecan mRNA levels were not significantly altered in any condition although both GAG/DNA and (35)S GAG incorporation studies indicated higher GAG retention and synthesis in the FS treatment. Collagen type II protein deposition was low in all samples, link protein distribution was more diffuse in FS condition, and aggrecan deposition was located in the outer regions of the alginate constructs in both bioreactor conditions, yet more uniformly in the FS condition. Catabolic gene expression (matrix metalloproteinase 3 [MMP3] and inducible nitric oxide synthase [iNOS]) was higher in bioreactor conditions compared with FS, although iNOS expression levels decreased to approximately fourfold less than the FS condition by day 14. Our data indicate that conditions created in the bioreactor enhanced both anabolic and catabolic responses, similar to other loading studies. Perfusion was sufficient alone to promote this dual response. PL increased the deposition of aggrecan surrounding cells compared with the other conditions; however, overall low GAG retention in the bioreactor system was likely due to both perfusion and catabolic conditions created. Optimal conditions, which permit appropriate anabolic and catabolic processes for accumulation of ECM and tissue remodeling for neocartilage development, specifically for humans, are needed.     

Design of a high-throughput flow perfusion bioreactor system for tissue engineering

Flow perfusion culture is used in many areas of tissue engineering and offers several key advantages. However, one challenge to these cultures is the relatively low-throughput nature of perfusion bioreactors. Here, a flow perfusion bioreactor with increased throughput was designed and built for tissue engineering. This design uses an integrated medium reservoir and flow chamber in order to increase the throughput, limit the volume of medium required to operate the system, and simplify the assembly and operation.     

Design and validation of a bi-axial loading bioreactor for mechanical stimulation of engineered cartilage

A mechanical-conditioning bioreactor has been developed to provide bi-axial loading to three-dimensional (3D) tissue constructs within a highly controlled environment. The computer-controlled bioreactor is capable of applying axial compressive and shear deformations, individually or simultaneously at various regimes of strain and frequency. The reliability and reproducibility of the system were verified through validation of the spatial and temporal accuracy of platen movement, which was maintained over the operating length of the system. In the presence of actual specimens, the system was verified to be able to deliver precise bi-axial load to the specimens, in which the deformation of every specimen was observed to be relatively homogeneous. The primary use of the bioreactor is in the culture of chondrocytes seeded within an agarose hydrogel while subjected to physiological compressive and shear deformation. The system has been designed specifically to permit the repeatable quantification and characterisation of the biosynthetic activity of cells in response to a wide range of short and long term multi-dimensional loading regimes.     

Hepatic differentiation of human embryonic stem cells is promoted by three-dimensional dynamic perfusion culture conditions

The developmental potential of human embryonic stem cells (hESCs) holds great promise to provide a source of human hepatocytes for use in drug discovery, toxicology, hepatitis research, and extracorporeal bioartificial liver support. There are, however, limitations to induce fully functional hepatocytes on conventional two-dimensional (2D) static culture. It had been shown that dynamic three-dimensional (3D) perfusion culture is superior to induce maturation in fetal hepatocytes and prolong hepatic functions of primary adult hepatocytes. We investigated the potential of using a four-compartment 3D perfusion culture to induce hepatic differentiation in hESC. Undifferentiated hESC were inoculated into hollow fiber-based 3D perfusion bioreactors with integral oxygenation. Hepatic differentiation was induced with a multistep growth factor cocktail protocol. Parallel controls were operated under equal perfusion conditions without the growth factor supplementations to allow for spontaneous differentiation, as well as in conventional 2D static conditions using growth factors. Metabolism, hepatocyte-specific gene expression, protein expression, and hepatic function were evaluated after 20 days. Significantly upregulated hepatic gene expression was observed in the hepatic differentiation 3D culture group. Ammonia metabolism activity and albumin production was observed in the 3D directed differentiation culture. Drug-induced cytochrome P450 gene expression was increased with rifampicin induction. Using flow cytometry analysis the mature hepatocyte marker asialoglycoprotein receptor was found on up to 30% of the cells in the 3D system with directed hepatic differentiation. Histological and immunohistochemical analysis revealed structural formation of hepatic and biliary marker-positive cells. In contrast to 2D culture, the 3D perfusion culture induced more functional maturation in hESC-derived hepatic cells. 3D perfusion bioreactor technologies may be useful for further studies on generating hESC-derived hepatic cells.     

一种体外贴壁细胞应变加载装置的研制

目的开发一套新型的应变加载装置,用于贴壁细胞力学生物学研究。方法该装置基于基底形变加载技术,采用可控制编程器驱动步进器,引起硅橡胶小室变形,实现多单元大应变的细胞加载;研制该装置,检测机械性能;建立硅橡胶小室的三维模型,利用有限元技术对硅橡胶小室进行仿真,分析该小室的应变场均匀性问题;采用该装置对骨髓间充质干细胞(bone marrow stromal cells,BMSCs)加载5%机械应变,频率0.5 Hz,2 h/d,持续5 d,并在倒置显微镜下观察细胞形态的变化。结果所研制的适用于体外细胞加载装置可对3组细胞加载基底实现最大至50%机械单向应变;在10%应变范围内,硅橡胶小室底部的均匀应变场面积占比保持在50%以上,保证了细胞受力均匀; BMSCs形态发生明显变化,排列方向趋于垂直主应变加载方向。结论该装置运行可靠,应变范围宽,频率可调,操作方便,可同时对多组细胞培养基底进行应变加载,为细胞力学生物学研究提供了便利条件。     

Tissue-engineering bioreactors: a new combined cell-seeding and perfusion system for vascular tissue engineering

One approach to the tissue engineering of vascular structures is to develop in vitro conditions in order ultimately to fabricate functional vascular tissues before final implantation. In our experiment, we aimed to develop a new combined cell seeding and perfusion system that provides sterile conditions during cell seeding and biomechanical stimuli in order to fabricate autologous human vascular tissue in vitro. The cell seeding and perfusion system is made of Plexiglas and is completely transparent (Berlin Heart, Berlin, Germany; University Hospital Benjamin Franklin, Berlin, Germany). The whole system consists of a cell seeding chamber that can be incorporated into the perfusion system and an air-driven respirator pump connected to the bioreactor. The cell culture medium continuously circulates through a closed-loop system. We thus developed a cell seeding device for static and dynamic seeding of vascular cells onto a polymeric vascular scaffold and a closed-loop perfused bioreactor for long-term vascular conditioning. The cell seeding chamber can be easily connected to the bioreactor, which combines continuous, pulsatile perfusion and mechanical stimulation to the tissue-engineered conduit. Adjusting the stroke volume, the stroke rate, and the inspiration/expiration time of the ventilator allows various pulsatile flows and different levels of pressure. The whole system is a highly isolated cell culture setting, which provides a high level of sterility and a gas supply and fits into a standard humidified incubator. The device can be sterilized by ethylene oxide and assembled with a standard screwdriver. Our newly developed combination of a cell seeding and conditioning device provides sterile conditions and biodynamic stimuli for controlled tissue development and in vitro conditioning of an autologous tissue-engineered vessel.     

Tubular perfusion system culture of human mesenchymal stem cells on poly-L-lactic acid scaffolds produced using a supercritical carbon dioxide-assisted process

In vitro human mesenchymal stem cell (hMSC) proliferation and differentiation is dependent on scaffold design parameters and specific culture conditions. In this study, we investigate how scaffold microstructure influences hMSC behavior in a perfusion bioreactor system. Poly-L-lactic acid (PLLA) scaffolds are fabricated using supercritical carbon dioxide (SC-CO(2) ) gel drying. This production method results in scaffolds fabricated with nanostructure. To introduce a microporous structure, porogen leaching was used in addition to this technique to produce scaffolds of average pore size of 100, 250, and 500 μm. These scaffolds were then cultured in static culture in well plates or dynamic culture in the tubular perfusion system (TPS) bioreactor. Results indicated that hMSCs were able to attach and maintain viability on all scaffolds with higher proliferation in the 250 μm and 500 μm pore sizes of bioreactor cultured scaffolds and 100 μm pore size of statically cultured scaffolds. Osteoblastic differentiation was enhanced in TPS culture as compared to static culture with the highest alkaline phosphatase expression observed in the 250 μm pore size group. Bone morphogenetic protein-2 was also analyzed and expression levels were highest in the 250 μm and 500 μm pore size bioreactor cultured samples. These results demonstrate cellular response to pore size as well as the ability of dynamic culture to enhance these effects. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A:2563-2572, 2012.