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。     

Numerical model and experimental validation of microcarrier motion in a rotating bioreactor

The equations of motion for microcarriers in a rotating bioreactor have been formulated and trajectories obtained using numerical techniques. An imaging system was built to validate the results by direct observation of microcarrier trajectories in the rotating frame of reference. The microcarrier motion observed by this imaging system was in excellent agreement with the numerical predictions of that motion. In the rotating frame of reference, microcarriers with density greater than the surrounding fluid medium followed a circular motion relative to the culture medium combined with a persistent migration and eventual collision with the outer wall of the reactor. However, for microcarrier density less the fluid medium, their circular motion migrated toward the central region of the reactor. When multiple microcarrier beads that are lighter than water are inserted into the reactor, the centrally directed migration results in the formation of clusters that are stabilized by tissue bridges formed by osteoblasts seeded onto the microcarriers. This system offers unique opportunities to monitor tissue synthesis on microcarriers using real-time optical techniques and to optimize the bioreactor operating conditions for exploiting this technology to study early bone tissue synthesis in vitro.     

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。     

Use of a rotating bioreactor toward tissue engineering the temporomandibular joint disc

This objective of this study was to determine the effects of a rotating bioreactor in temporomandibular joint (TMJ) disc tissue engineering. Porcine TMJ disc cells were seeded at a density of 20 million cells/mL onto nonwoven poly(glycolic acid) (PGA) scaffolds in spinner flasks for 1 week and then cultured either under static conditions or in a rotating bioreactor for a period of 6 weeks. A series of analyses was performed, including mechanical testing, measurement of cellularity, quantification of matrix biosynthesis with a hydroxyproline assay and enzyme-linked immunosorbent assays, and observation of matrix distribution with immunohistochemistry. Between the bioreactor and static cultures, there were marked differences in gross appearance, histological structure, and distribution of collagen types I and II. Engineered constructs from the bioreactor contracted earlier and to a greater extent, resulting in a denser matrix and cell composition. In addition, immunostaining intensity was generally uniform in static constructs, in contrast to higher intensity around the periphery of bioreactor constructs. Moreover, bioreactor constructs had higher amounts of collagen II than did static constructs. However, differences in total matrix content and compressive stiffness were generally not significant. On the basis of the results of this study there is no clear benefit from use of the rotating bioreactor, although a sequence of static culture followed by rotating bioreactor culture may prove in the future to be more beneficial than either alone.     

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.     

组织工程生物反应器的生物力学

<正>组织工程,是指用工程科学和生命科学的原理和方法,制备组织和器官替代物,以恢复、维持或改善人体组织、器官的功能,是一个发展迅速、意义深远的生物医学工程应用领域。目前,组织工程化皮肤产品已正式进入临床应用,培育的组织工程骨骼、软骨、血管、皮肤以及神经组织正在进行体内实验,组织工程肝脏、胰脏、乳房、心脏、手指、角膜等也可以在实验室里构建生长。组织工程已形成一个发展中的产业。但是目前组织工程距离广泛应用于临床、成为社会经济新的增长点还有相当长的路要走。阻碍组织工程发展和临床应用的主要因素至少包括两点:(1)对调控组织的功能化培养的特定物理-生物化学因素知之还少;(2)高昂的生产成本和缺乏商业化的功能性组织工程产品。     

Three-dimensional cardiac tissue engineering using a thermoresponsive artificial extracellular matrix

The purpose of this study was to try to reconstitute three-dimensional cardiac tissue using a thermoresponsive artificial extracellular matrix, poly (N-isopropylacrylamide)-grafted gelatin (PNIPAM-gelatin), as the scaffold. PNIPAM-gelatin solution gels almost immediately when heated above 34 degrees C. We thought this property could become advantageous as scaffolding for reconstituting three-dimensional tissue. Because PNIPAM-gelatin solution gels so quickly, all seeded cells in PNIPAM-gelatin solution would become entrapped and uniformly distributed toward three dimensions. Thus it would be possible to reconstitute three-dimensional tissue by a very simple method of mixing cells and PNIPAM-gelatin solution. Fetal rat cardiac cells were mixed with PNIPAM-gelatin solution, incubated at 37 degrees C to allow the mixture to gel, and cultured in vitro. To define suitable culture conditions the following parameters were tested: (1) PNIPAM-gelatin concentration, 0.04 approximately 0.125 mg/ml; (2) cell seeding density, 1 approximately 50 x 10(6) cells/ml; and (3) addition or not of hyaluronic acid. With a PNIPAM-gelatin concentration of 0.05 mg/ml, a cell seeding density of 50 x 10(6) cells/ml, and the addition of hyaluronic acid, tissue was reconstituted and it contracted synchronously. After hematoxylin and eosin staining, the cells reconstituted three-dimensional tissue, and the tissue cross-section was approximately 60 microm thick.     

Bone tissue engineering in osteoporosis

Osteoporosis is a polygenetic, environmentally modifiable disease, which precipitates into fragility fractures of vertebrae, hip and radius and also confers a high risk of fractures in accidents and trauma. Aging and the genetic molecular background of osteoporosis cause delayed healing and impair regeneration. The worldwide burden of disease is huge and steadily increasing while the average life expectancy is also on the rise. The clinical need for bone regeneration applications, systemic or in situ guided bone regeneration and bone tissue engineering, will increase and become a challenge for health care systems. Apart from in situ guided tissue regeneration classical ex vivo tissue engineering of bone has not yet reached the level of routine clinical application although a wealth of scaffolds and growth factors has been developed. Engineering of complex bone constructs in vitro requires scaffolds, growth and differentiation factors, precursor cells for angiogenesis and osteogenesis and suitable bioreactors in various combinations. The development of applications for ex vivo tissue engineering of bone faces technical challenges concerning rapid vascularization for the survival of constructs in vivo. Recent new ideas and developments in the fields of bone biology, materials science and bioreactor technology will enable us to develop standard operating procedures for ex vivo tissue engineering of bone in the near future. Once prototyped such applications will rapidly be tailored for compromised conditions like vitamin D and sex hormone deficiencies, cellular deficits and high production of regeneration inhibitors, as they are prevalent in osteoporosis and in higher age.     

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。     

Bone marrow tissue engineering

The creation of mixed hematopoietic chimerism has become an important clinical strategy for tolerance induction for cellular and organ transplantation, and for the treatment of numerous hematopoietic diseases. Clinical success has been limited however, by host immune response and by competition from host hematopoiesis. Recent data suggests that limited donor stem cell engraftment after minimally myeloablative hematopoietic stem cell (HSC) transplantation may in part be due to MHC associated microenvironmental mismatch resulting in a competitive disadvantage for donor HSC. A strategy to overcome this barrier to stable mixed hematopoietic chimerism would involve concurrent transplantation of a donor bone marrow microenvironment. To test this possibility, we set out to develop a method to tissue engineer a bone marrow microenvironment. One to two murine femurs were mechanically crushed to a fine suspension and were combined in vitro with various delivery vehicles. These constructs were transplanted into syngeneic animals in locations that are known to support transplantation of other tissues. Although bone formation was observed with several conditions, bone marrow formation was noted only within the small bowel mesentery when type I collagen was used as the delivery vehicle. No bone marrow formed when the vehicle was changed to polyglycolic acid or type IV collagen. We have demonstrated that the small bowel mesentery can support bone marrow formation under specific in vivo conditions. Future work will focus on strategies for transplantation of an engineered donor bone marrow environment to facilitate creation of allogeneic mixed hematopoietic chimerism.     

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.     

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.     

Three-dimensional-construct bioreactor conditioning in human tendon tissue engineering

Human tendon tissue engineering attempts to address the shortage of autologous tendon material arising from mutilating injuries and diseases of the hand and forearm. It is important to maximize the tissue-engineered construct's (TEC's) biomechanical properties to ensure that the construct is in its strongest possible state before reimplantation. In this study, we sought to determine the bioreactor treatment parameters that affect these properties. Using small- and large-chamber three-dimensional-construct bioreactors (SCB and LCB, respectively), we applied cyclic axial load to TECs comprising reseeded human flexor and extensor tendons of the hand. First, small-sample pilot studies using the LCB were performed on matched-paired full-length flexor tendons to establish proof of concept. Next, large-sample studies using the SCB were performed on matched-paired extensor tendon segments to determine how reseeding, load duty cycle, load magnitude, conditioning duration, and testing delay affected ultimate tensile stress (UTS) and elastic modulus (EM). We found that compared with reseeded matched-paired controls under dynamic-loading at 1.25?N per TEC for 5 days, (1) acellular TECs had lower UTS (p=0.04) and EM (p<0.01), (2) unloaded TECs had lower UTS (p=0.01) and EM (p=0.02), (3) static-loaded TECs had lower UTS (p=0.01) and EM (p<0.01), (4) TECs conditioned for 3 days had lower UTS (p=0.03) and EM (p=0.04), and (5) TECs conditioned for 8 days had higher UTS (p=0.04) and EM (p=0.01). However, TECs conditioned at higher loads (2.5?N per TEC) and lower loads (0.625?N per TEC) possessed similar UTS (p=0.83 and p=0.89, respectively) and EM (p=0.48 and p=0.89, respectively) as controls stimulated with 1.25?N per TEC. After cycle completion, there is attrition of UTS (p=0.03) and EM (p=0.04) over a 2-day period. Our study showed that the material properties of human allograft TECs can be enhanced by reseeding and dynamic-conditioning. While conditioning duration has a significant effect on material properties, the load magnitude does not. The issue of attrition in biomechanical properties with time following cycle completion must be addressed before bioreactor preconditioning can be successfully introduced as a step in the processing of these constructs for clinical application.     

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。     

Differentiation of mammalian skeletal muscle cells cultured on microcarrier beads in a rotating cell culture system

The growth and repair of adult skeletal muscle are due in part to activation of muscle precursor cells, commonly known as satellite cells or myoblasts. These cells are responsive to a variety of environmental cues, including mechanical stimuli. The overall goal of the research is to examine the role of mechanical signalling mechanisms in muscle growth and plasticity through utilisation of cell culture systems where other potential signalling pathways (i.e. chemical and electrical stimuli) are controlled. To explore the effects of decreased mechanical loading on muscle differentiation, mammalian myoblasts are cultured in a bioreactor (rotating cell culture system), a model that has been utilised to simulate microgravity. C₂C₁₂ murine myoblasts are cultured on microcarrier beads in a bioreactor and followed throughout differentiation as they form a network of multinucleated myotubes. In comparison with three-dimensional control cultures that consist of bioreactor exhibit an attenuation in differentiation. This is demonstrated by reduced immunohistochemical staining for myogenin and α-actinin. Western analysis shows a decrease, in bioreactor cultures compared with control cultures, in levels of the contractile proteins myosin (47% decrease, p<0.01) and tropomyosin (63% decrease, p<0.01). Hydrodynamic measurements indicate that the decrease in differentiation may be due, at least in part, to fluid stresses acting on the myotubes. In addition, constraints on aggregate size imposed by the action of fluid forces in the bioreactor affect differentation. These results may have implications for muscle growth and repair during spaceflight.     

Bone tissue engineering using polycaprolactone scaffolds fabricated via selective laser sintering

Polycaprolactone (PCL) is a bioresorbable polymer with potential applications for bone and cartilage repair. In this work, porous PCL scaffolds were computationally designed and then fabricated via selective laser sintering (SLS), a rapid prototyping technique. The microstructure and mechanical properties of the fabricated scaffolds were assessed and compared to the designed porous architectures and computationally predicted properties. Scaffolds were then seeded with bone morphogenetic protein-7 (BMP-7) transduced fibroblasts and implanted subcutaneously to evaluate biological properties and to demonstrate tissue in-growth. The work done illustrates the ability to design and fabricate PCL scaffolds with porous architecture that have sufficient mechanical properties for bone tissue engineering applications using SLS. Compressive modulus and yield strength values ranged from 52 to 67 MPa and 2.0 to 3.2 Mpa, respectively, lying within the lower range of properties reported for human trabecular bone. Finite element analysis (FEA) results showed that mechanical properties of scaffold designs and of fabricated scaffolds can be computationally predicted. Histological evaluation and micro-computed tomography (microCT) analysis of implanted scaffolds showed that bone can be generated in vivo. Finally, to demonstrate the clinical application of this technology, we designed and fabricated a prototype mandibular condyle scaffold based on an actual pig condyle. The integration of scaffold computational design and free-form fabrication techniques presented here could prove highly useful for the construction of scaffolds that have anatomy specific exterior architecture derived from patient CT or MRI data and an interior porous architecture derived from computational design optimization.     

Collagen expression in tissue engineered cartilage of aged human articular chondrocytes in a rotating bioreactor

This study describes the culture and three-dimensional assembly of aged human articular chondrocytes under controlled oxygenation and low shear stress in a rotating-wall vessel. Chondrocytes cultured in monolayer were released and placed without any scaffold as a single cell suspension in a rotating bioreactor for 12 weeks. Samples were analyzed with immunohistochemistry, molecular biology and electron microscopy. During serial monolayer cultures chondrocytes dedifferentiated to a "fibroblast-like" structure and produced predominantly collagen type I. When these dedifferentiated cells were transferred to the rotating bioreactor, the cells showed a spontaneous aggregation and formation of solid tissue during the culture time. Expression of collagen type II and other components critical for the extracellular cartilage matrix could be detected. Transmission electron microscopy revealed a fine network of randomly distributed collagen fibrils. This rotating bioreactor proves to be a useful tool for providing an environment that enables dedifferentiated chondrocytes to redifferentiate and produce a cartilage-specific extracellular matrix.     

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。     

Bone tissue engineering using human adipose-derived stem cells and honeycomb collagen scaffold

Human adipose-derived stem cells (ASCs) have the capacity to regenerate and the potential to differentiate into multiple lineages of mesenchymal cells. The aim of this study was to investigate the possibility of using honeycomb collagen scaffold to culture ASCs in bone tissue engineering. The osteogenic capacity of ASCs in vitro, was confirmed by histology and measuring the expression of cbfa-1. After that, ASCs were cultured for up to 14 days in the honeycomb scaffold to allow a high density, three-dimensional culture. Scanning electron microscopy data showed that the scaffold was filled with the grown ASCs, and calcification, stained black with von Kossa, was confirmed. Furthermore, The ASC-loaded honeycomb collagen scaffolds cultured for 14 days were subcutaneously transplanted into nude mice, and excised after 8 weeks. Bone formation in vivo was examined using HE stain, von Kossa stain, and osteocalcin immunostain. Those histological views showed significant positive stains in the samples of osteogenic medium in the three types of stain. These results suggest that this carrier is a suitable scaffold for ASCs and will be useful as a three-dimensional bone tissue engineering scaffold in vitro and in vivo.