Computational modeling of adherent cell growth in a hollow-fiber membrane bioreactor for large-scale 3-D bone tissue engineering

The use of hollow-fiber membrane bioreactors (HFMBs) has been proposed for three-dimensional bone tissue growth at the clinical scale. However, to achieve an efficient HFMB design, the relationship between cell growth and environmental conditions must be determined. Therefore, in this work, a dynamic double-porous media model was developed to determine nutrient-dependent cell growth for bone tissue formation in a HFMB. The whole hollow-fiber scaffold within the bioreactor was treated as a porous domain in this model. The domain consisted of two interpenetrating porous regions, including a porous lumen region available for fluid flow and a porous extracapillary space filled with a collagen gel that contained adherent cells for promoting long-term growth into tissue-like mass. The governing equations were solved numerically and the model was validated using previously published experimental results. The contributions of several bioreactor design and process parameters to the performance of the bioreactor were studied. The results demonstrated that the process and design parameters of the HFMB significantly affect nutrient transport and thus cell behavior over a long period of culture. The approach presented here can be applied to any cell type and used to develop tissue engineering hollow-fiber scaffolds.     

A biaxial rotating bioreactor for the culture of fetal mesenchymal stem cells for bone tissue engineering

The generation of effective tissue engineered bone grafts requires efficient exchange of nutrients and mechanical stimulus. Bioreactors provide a manner in which this can be achieved. We have recently developed a biaxial rotating bioreactor with efficient fluidics through in-silico modeling. Here we investigated its performance for generation of highly osteogenic bone graft using polycaprolactone–tricalcium phosphate (PCL–TCP) scaffolds seeded with human fetal mesenchymal stem cell (hfMSC). hfMSC scaffolds were cultured in either bioreactor or static cultures, with assessment of cellular viability, proliferation and osteogenic differentiation in vitro and also after transplantation into immunodeficient mice. Compared to static culture, bioreactor-cultured hfMSC scaffolds reached cellular confluence earlier (day 7 vs. day 28), with greater cellularity (2×, p < 0.01), and maintained high cellular viability in the core, which was 2000 μm from the surface. In addition, bioreactor culture was associated with greater osteogenic induction, ALP expression (1.5× p < 0.01), calcium deposition (5.5×, p < 0.001) and bony nodule formation on SEM, and in-vivo ectopic bone formation in immunodeficient mice (3.2×, p < 0.001) compared with static-cultured scaffolds. The use of biaxial bioreactor here allowed the maintenance of cellular viability beyond the limits of conventional diffusion, with increased proliferation and osteogenic differentiation both in vitro and in vivo, suggesting its utility for bone tissue engineering applications.     

3D bone tissue growth in hollow fibre membrane bioreactor: implications of various process parameters on tissue nutrition

New experimental evidence shows that hollow fibre membrane bioreactor (HFMB) may be applied to grow bulky bone tissues which may then be implanted into patients to repair skeletal defects. To design effective bone tissue engineering protocols, it is necessary to determine the quantitative relationships between the cell environment and tissue behaviour in HFMBs and their relationship with nutrient supply. It is also necessary to determine under what conditions nutritional limitations may occur and, hence, may cause cell death. These require that the appropriate bioreactor conditions for generating neotissues, and the nutrient transfer behaviour and chemical reaction during cell growth and extracellular matrix formation are studied thoroughly. In this paper, we aim to use an existing mathematical framework to analyse the influence of various relevant parameters on nutrient supply for bone tissue growth in HFMB. We adopt the well-known Krogh cylinder approximation of the HFMB. The model parameters (e.g., cell metabolic rates) and operating conditions for the mathematical model have been obtained from, or correspond to, in-house experiments with the exception of a few variables which have been taken from the literature. The framework is then used to study oxygen and glucose transport behaviour in the HFMB. Influence of a number of important process parameters, e.g., reaction kinetics, cell density, inlet concentration of nutrients, etc, on the nutrient distributions have been systematically analysed. The work presented in this paper provides insights on unfavourable system designs and specifications which may be avoided to prevent mass transfer limitations for growing bone tissues in HFMB.     

Finite element study of scaffold architecture design and culture conditions for tissue engineering

Tissue engineering scaffolds provide temporary mechanical support for tissue regeneration and transfer global mechanical load to mechanical stimuli to cells through its architecture. In this study the interactions between scaffold pore morphology, mechanical stimuli developed at the cell microscopic level, and culture conditions applied at the macroscopic scale are studied on two regular scaffold structures. Gyroid and hexagonal scaffolds of 55% and 70% porosity were modeled in a finite element analysis and were submitted to an inlet fluid flow or compressive strain. A mechanoregulation theory based on scaffold shear strain and fluid shear stress was applied for determining the influence of each structures on the mechanical stimuli on initial conditions. Results indicate that the distribution of shear stress induced by fluid perfusion is very dependent on pore distribution within the scaffold. Gyroid architectures provide a better accessibility of the fluid than hexagonal structures. Based on the mechanoregulation theory, the differentiation process in these structures was more sensitive to inlet fluid flow than axial strain of the scaffold. This study provides a computational approach to determine the mechanical stimuli at the cellular level when cells are cultured in a bioreactor and to relate mechanical stimuli with cell differentiation.     

Integration of hollow fiber membranes improves nutrient supply in three-dimensional tissue constructs

Sufficient nutrient and oxygen transport is a potent modulator of cell proliferation in in vitro tissue-engineered constructs. The lack of oxygen and culture medium can create a potentially lethal environment and limit cellular metabolic activity and growth. Diffusion through scaffold and multi-cellular tissue typically limits transport in vitro, leading to potential hypoxic regions and reduction in the viable tissue thickness. For the in vitro generation of clinically relevant tissue-engineered grafts, current nutrient diffusion limitations should be addressed. Major approaches to overcoming these include culture with bioreactors, scaffolds with artificial microvasculature, oxygen carriers and pre-vascularization of the engineered tissues. This study focuses on the development and utilization of a new perfusion culture system to provide adequate nutrient delivery to cells within large three-dimensional (3D) scaffolds. Perfusion of oxygenated culture medium through porous hollow fiber (HF) integrated within 3D free form fabricated (FFF) scaffolds is proposed. Mouse pre-myoblast (C2C12) cells cultured on scaffolds of poly(ethylene-oxide-terephthalate)-poly(butylene-terephthalate) block copolymer (300PEOT55PBT45) integrated with porous HF membranes of modified poly(ether-sulfone) (mPES, Gambro GmbH) is used as a model system. Various parameters such as fiber transport properties, fiber spacing within a scaffold and medium flow conditions are optimized. The results show that four HF membranes integrated with the scaffold significantly improve the cell density and cell distribution. This study provides a basis for the development of a new HF perfusion culture methodology to overcome the limitations of nutrient diffusion in the culture of large 3D tissue constructs.     

成骨细胞培养在骨组织工程和运动领域的研究进展

背景：成骨细胞在不同的培养环境、细胞因子刺激下增殖、分化及功能不同。 目的：总结成骨细胞体外培养技术与成骨细胞增殖速度的研究进展。 方法：检索1990/2011PubMed数据及万方数据库有关运动、成骨细胞培养和骨组织工程等方面的文献,英文检索词为“exercise, Osteoblast raise method”,中文检索词为“运动,成骨细胞,骨组织工程”。排除与研究目的无关和内容重复者。保留29篇文献做进一步分析。 结果与结论：骨组织工程构建中种子细胞的选择方式、支架材料的不同形式、细胞因子、培养环境、力学因素都对成骨细胞的培养有重要影响。构建骨组织工程中成骨细胞在不同的培养环境、细胞因子刺激下会有不同的增殖能力和分化结果。成骨细胞的增殖速度与旋转壁式生物反应器提供的低剪力环境、适宜的细胞因子、细胞支架的选择密切相关。 关键词：成骨细胞;培养;骨组织工程;运动;细胞增殖 doi:10.3969/j.issn.1673-8225.2012.11.033     

New multi-cue bioreactor for tissue engineering of tubular cardiovascular samples under physiological conditions

In the present study we have developed a multi-cue bioreactor (MCB) that is capable of delivering a range of stimuli to assist the development of a tissue-engineered construct. The MCB provides an accurate and utilizable computer-controlled pulsatile pump and strain induction mechanism and it has the capability of applying physiological conditions to samples. The device described here emulates the pressure and straining environment found at the aortic root. This function, along with an integral perfusion and sterile containment system, allows for long-term culture and whole-tissue testing capability. Aortic and pulmonary arteries were obtained from freshly isolated porcine hearts and subjected to various loading regimens (Deltapressure/flow/force). Through analyzing data acquired by the MCB transducer array it was possible to differentiate the dynamic mechanical properties of the tissue types tested. In addition, the MCB illustrates a novel concept in cardiovascular tissue engineering: being able to support long-term tissue culture of cell-seeded substrates while they are under the influence of mechanical cues. After 7 days of pulsation in the MCB cell alignment was observed. The MCB represents a versatile model that will enable the development of tissue engineering not only for cardiovascular tissue, but for all tubular tissues such as esophageal, tracheal, and bronchial systems.     

Development of a novel pulsatile bioreactor for tissue culture

The construction of tissue-engineered parts such as heart valves and arteries requires more than just the seeding of cells onto a biocompatible/biodegradable polymeric scaffold. It is essential that the functionality and mechanical integrity of the cell-seeded scaffold be investigated in vitro prior to in vivo implantation. The correct hemodynamic conditioning would lead to the development of tissues with enhanced mechanical strength and cell viability. Therefore, a bioreactor that can simulate physiological conditions would play an important role in the preparation of tissue-engineered constructs. In this article, we present and discuss the design concepts and criteria, as well as the development, of a multifunctional bioreactor for tissue culture in vitro. The system developed is compact and easily housed in an incubator to maintain sterility of the construct. Moreover, the proposed bioreactor, in addition to mimicking in vivo conditions, is highly flexible, allowing different types of constructs to be exposed to various physiological flow conditions. Initial verification of the hemodynamic parameters using Laser doppler anemometry indicated that the bioreactor performed well and produced the correct physiological conditions.     

Human hepatocyte functions in a crossed hollow fiber membrane bioreactor

An important challenge in liver tissue engineering is the development of bioartificial systems that are able to favour the liver reconstruction and to modulate liver cell behaviour.A crossed hollow fiber membrane bioreactor was developed to support the long-term maintenance and differentiation of human hepatocytes. The bioreactor consists of two types of hollow fiber (HF) membranes with different molecular weight cut-off (MWCO) and physico-chemical properties cross-assembled in alternating manner: modified polyetheretherketone (PEEK-WC) and polyethersulfone (PES), used for the medium inflow and outflow, respectively. The combination of these two fiber set produces an extracapillary network for the adhesion of cells and a high mass exchange through the cross-flow of culture medium. The transport of liver specific products such as albumin and urea together with the transport of drug such as diazepam was modelled and compared with the experimental metabolic data. The theoretical metabolite concentration differed 7.5% for albumin and 5% for urea with respect to experimental data. The optimised perfusion conditions of the bioreactor allowed the maintenance of liver functions in terms of urea synthesis, albumin secretion and diazepam biotransformation up to 18 days of culture. In particular the good performance of the bioreactor was confirmed by the high rate of urea synthesis (28.7 μg/h 10⁶ cells) and diazepam biotransformation. In the bioreactor human hepatocytes expressed at high levels the individual cytochrome P450 isoenzymes involved in the diazepam metabolism. The results demonstrated that crossed HF membrane bioreactor is able to support the maintenance of primary human hepatocytes preserving their liver specific functions for all investigated period. This device may be a potential tool in the liver tissue engineering for drug metabolism/toxicity testing and study of disease pathogenesis alternatively to animal experimentation.     

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     

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。     

微载体培养技术在骨与软骨组织工程研究中的应用☆

背景：骨与软骨组织工程学中增殖种子细胞和保持细胞特定表型是其难点,微载体生物反应器培养系统提供了很好的条件来解决这个问题。 目的：分析近年来国内外骨、软骨细胞微载体培养的研究进展,为骨与软骨细胞微载体培养技术和组织工程研究提供理论基础。 方法：由第一作者在2010-11进行检索。检索数据库：PubMed数据库(网址http://www.ncbi.nlm.gov/PubMed);万方数据库(网址http://www.wanfangdata.com.cn),资料的检索时间范围为1967/2011。英文检索词为“microcarrier,cartilage,tissue engineering”,中文检索词为“微载体,软骨,组织工程学”。排除与本文无关及陈旧、重复的文章,共保存32篇文献做进一步分析。 结果与结论：在微载体培养系统中,可较好的调控骨与软骨细胞培养条件,能在短时间内大量的增殖,并能保持其细胞的表型,甚至出现表型增强现象,在骨、软骨组织工程学研究和临床应用中有着巨大潜力。     

Extended-term culture of bone cells in a compartmentalized bioreactor

A specialized bioreactor is used to grow mineralizing, collagenous tissue up to 150 microm thick from an inoculum of isolated murine (mouse calvaria MC3T3-E1, American Type Culture Collection (ATCC) CRL-2593) or human (hFOB 1.19 ATCC CRL-11372) fetal osteoblasts over uninterrupted culture periods longer than 120 days (4 months). Proliferation and phenotypic progression of an osteogenic-cell monolayer into a tissue consisting of 6 or more cell layers of mature osteoblasts in the bioreactor was compared with cell performance in conventional tissue-culture polystyrene (TCPS) controls. Cells in the bioreactor basically matched results obtained in TCPS over a 15-day culture interval, but loss of insoluble extracellular matrix and an approximate doubling of apoptosis rates in TCPS after 30 days indicated that progressive instability of cultures maintained in TCPS with periodic refeeding but without subculture. In contrast, stable cultures were maintained in the bioreactor for more than 120 days, suggesting that extended-term tissue maintenance is feasible with little or no special technique. Transmission electron microscopy ultramorphology of tissue derived from hFOB 1.19 recovered from the bioreactor after only 15 days of culture showed evidence of osteocytic-like processes and gap junctions between cells like those observed in vivo, in addition to elaboration of the usual osteoblastic markers such as alkaline phosphatase activity and mineralization (alizarin red). Thus, the bioreactor design based on the principle of simultaneous growth and dialysis was shown to create an extraordinarily stable peri-cellular environment that better simulates the in vivo condition than conventional tissue culture. The bioreactor shows promise as a tool for the in vitro study of osteogenesis and osteopathology.     

Comparison of scaffolds and culture conditions for tissue engineering of the knee meniscus

The menisci of the knee are semilunar fibrocartilaginous structures critical in load bearing, shock absorption, stability, and lubrication. In this study, two commonly used biomaterials, a hydrogel (agarose) and a nonwoven mesh polymer [poly(glycolic acid); PGA], were compared for suitability as scaffold materials for tissue engineering the knee meniscus. In addition, a rotating wall bioreactor culture of both scaffold materials was compared with static cultures. Constructs were cultured for up to 7 weeks in static and rotating wall bioreactor culture. Cell numbers were 22 times higher in PGA than agarose after 7 weeks in culture. Static PGA scaffolds had more than twice the amount of sulfated glycosaminoglycans and three times the amount of collagen compared to static agarose constructs at week 7. The rotating wall bioreactor was not found with increase matrix production or cell proliferation significantly over static cultures.     

Coculture strategies in bone tissue engineering: the impact of culture conditions on pluripotent stem cell populations

The use of pluripotent stem cell populations for bone tissue regeneration provides many opportunities and challenges within the bone tissue engineering field. For example, coculture strategies have been utilized to mimic embryological development of bone tissue, and particularly the critical intercellular signaling pathways. While research in bone biology over the last 20 years has expanded our understanding of these intercellular signaling pathways, we still do not fully understand the impact of the system's physical characteristics (orientation, geometry, and morphology). This review of coculture literature delineates the various forms of coculture systems and their respective outcomes when applied to bone tissue engineering. To understand fully the key differences between the different coculture methods, we must appreciate the underlying paradigms of physiological interactions. Recent advances have enabled us to extrapolate these techniques to larger dimensions and higher geometric resolutions. Finally, the contributions of bioreactors, micropatterned biomaterials, and biomaterial interaction platforms are evaluated to give a sense of the sophistication established by a combination of these concepts with coculture systems.     

Hypoxia in static and dynamic 3D culture systems for tissue engineering of bone

Tissue engineering of sizeable cell-scaffold constructs is limited by gradients in tissue quality from the periphery toward the center. Because homogenous delivery of oxygen to three-dimensional (3D) cell cultures remains an unsolved challenge, we hypothesized that uneven oxygen supply may impede uniform cellular growth on scaffolds. In this study we challenged static and dynamic 3D culture systems designed for bone tissue engineering applications with a well-growing subclone of MC3T3-E1 preosteoblasts and continuously measured the oxygen concentrations in the center of cell-seeded scaffolds and in the surrounding medium. After as little as 5 days in static culture, central oxygen concentrations dropped to 0%. Subsequently, cells died in central regions of the scaffold but not in its periphery, where oxygen levels were approximately 4%. The use of perfusion bioreactors successfully prevented cell death, yet central oxygen concentrations did not rise above 4%. We conclude that 3D culture in vitro is associated with relevant oxygen gradients, which can be the cause of inhomogeneous tissue quality. Perfusion bioreactors prevent cell death but they do not entirely eliminate 3D culture-associated oxygen gradients. Therefore, we advise continuous oxygen monitoring of 3D culture systems to ensure tissue quality throughout engineered constructs.     

Formation of three-dimensional cell/polymer constructs for bone tissue engineering in a spinner flask and a rotating wall vessel bioreactor

The aim of this study is to investigate the effect of the cell culture conditions of three-dimensional polymer scaffolds seeded with rat marrow stromal cells (MSCs) cultured in different bioreactors concerning the ability of these cells to proliferate, differentiate towards the osteoblastic lineage, and generate mineralized extracellular matrix. MSCs harvested from male Sprague-Dawley rats were culture expanded, seeded on three-dimensional porous 75:25 poly(D,L-lactic-co-glycolic acid) biodegradable scaffolds, and cultured for 21 days under static conditions or in two model bioreactors (a spinner flask and a rotating wall vessel) that enhance mixing of the media and provide better nutrient transport to the seeded cells. The spinner flask culture demonstrated a 60% enhanced proliferation at the end of the first week when compared to static culture. On day 14, all cell/polymer constructs exhibited their maximum alkaline phosphatase activity (AP). Cell/polymer constructs cultured in the spinner flask had 2.4 times higher AP activity than constructs cultured under static conditions on day 14. The total osteocalcin (OC) secretion in the spinner flask culture was 3.5 times higher than the static culture, with a peak OC secretion occurring on day 18. No considerable AP activity and OC secretion were detected in the rotating wall vessel culture throughout the 21-day culture period. The spinner flask culture had the highest calcium content at day 14. On day 21, the calcium deposition in the spinner flask culture was 6.6 times higher than the static cultured constructs and over 30 times higher than the rotating wall vessel culture. Histological sections showed concentration of cells and mineralization at the exterior of the foams at day 21. This phenomenon may arise from the potential existence of nutrient concentration gradients at the interior of the scaffolds. The better mixing provided in the spinner flask, external to the outer surface of the scaffolds, may explain the accelerated proliferation and differentiation of marrow stromal osteoblasts, and the localization of the enhanced mineralization on the external surface of the scaffolds.     

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.     

Computational simulation of a magnetic microactuator for tissue engineering applications

The next generation of tissue engineered constructs (TECs) requires the incorporation of a controllable and optimized microstructure if they are to chemically, mechanically, and biologically mimic tissue function. In order to obtain TECs with optimized microstructures, a combination of spatiotemporally regulated mechanical and biochemical stimuli is necessary during the formation of the construct. While numerous efforts have been made to create functional tissue constructs, there are few techniques available to stimulate TECs in a localized manner. We herein describe the design of a microdevice which can stimulate TECs in a localized, inhomogeneous, and predefined anisotropic fashion using ferromagnetically doped polydimethylsiloxane microflaps (MFs). Specifically, a sequential magneto-structural finite element model of the proposed microdevice is constructed and utilized to understand how changes in magnetic and geometrical properties of the device affect MF deflection. Our study indicates that a relatively small density of ferromagnetic material is required to result in adequate force and MF defection (175 μm ~7% TEC strain). We also demonstrate that MF to magnet distance is more important than inherent MF magnetic permeability in determining resulting MF deflection. An experimental validation test setup was used to validate the computational solutions. The comparison shows reasonable agreement indicating a 5.9% difference between experimentally measured and computationally predicted MF displacement. Correspondingly, an apparatus with two MFs and two magnets has been made and is currently undergoing construct testing. The current study presents the design of a novel magnetic microactuator for tissue engineering applications. The computational results reported here will form the foundation in the design and optimization of a functional microdevice with multiple MFs and magnets capable of stimulating TECs in nonhomogenous and preferred directions with relevant spatial resolution.