Bioreactors for tissue engineering

Bioreactors are essential in tissue engineering, not only because they provide an in vitro environment mimicking in vivo conditions for the growth of tissue substitutes, but also because they enable systematic studies of the responses of living tissues to various mechanical and biochemical cues. The basic principles of bioreactor design are reviewed, the bioreactors commonly used for the tissue engineering of cartilage, bone and cardiovascular systems are assessed in terms of their performance and usefulness. Several novel bioreactor types are also reviewed.     

Dynamic compression of cartilage constructs engineered from expanded human articular chondrocytes

Recent works have shown that mechanical loading can alter the metabolic activity of chondrocytes cultured in 3D scaffolds. In this study we determined whether the stage of development of engineered cartilaginous constructs (expanded adult human articular chondrocytes/Polyactive foams) regulates the effect of dynamic compression on glycosaminoglycan (GAG) metabolism. Construct maturation depended on the culture time (3-14 days) and the donor (4 individuals). When dynamic compression was subsequently applied for 3 days, changes in GAG synthesized, accumulated, and released were significantly positively correlated to the GAG content of the constructs prior to loading, and resulted in stimulation of GAG formation only in the most developed tissues. Conversely, none of these changes were correlated with the expression of collagen type II mRNA, indicating that the response of chondrocytes to dynamic compression does not depend directly upon the stage of cell differentiation, but rather on the extracellular matrix surrounding the cells.     

Bioreactor cultivation of three-dimensional cartilage-carrier-constructs

A flow-chamber bioreactor was designed for generation of three-dimensional cartilage-carrier-constructs. A specific attribute of the flow-chamber is a very thin medium layer for improved oxygen supply and a counter current flow of medium and gas. Three-dimensional cartilage-carrier-constructs were produced according to a standard protocol from chondrocytes of an adult mini-pig. The final step of this protocol was performed either in the bioreactor or in 12-well plates. The bioreactor experiments showed a significantly higher matrix thickness but a lower ratio of glycosaminoglycan to DNA. For both culture methods the constructs contained a high amount of collagen II. Appearance of the cartilage obtained in the bioreactor seemed to be closer to native cartilage with respect to distribution of the cells within the matrix, smoothness of the surface etc. All results considered the flow-chamber bioreactor is a very useful tool for generation of three dimensional cartilage-carrier constructs.     

Strain distribution in an elastic substrate vibrated in a bioreactor for vocal fold tissue engineering

A bioreactor previously described was used to quantify the shear strain along a bioengineered tissue scaffold driven at low audio frequencies (20–200 Hz). Standing wave patterns were calculated analytically by solving a classical boundary value problem for a vibrating string under tension and bending stiffness. Boundary conditions were non-traditional in that small pivot arms at the endpoints allowed neither the displacement nor the velocity to go to zero. The calculations were corroborated with stroboscopic measurement of the motion of the material in the bioreactor. Results indicate that shear strains up to 0.2 can be obtained at low frequencies (20 Hz), with a gradual decrease at higher frequencies due to the decaying amplitude response of the mechanical driver. The bioreactor may be useful for approximating the Young's modulus of the material in situ by probing for resonance frequencies in the standing wave pattern. A yet unsolved problem is a variable drag coefficient along the length of the material due to fluid turbulence in the culture medium.     

Redifferentiation of chondrocytes and cartilage formation under intermittent hydrostatic pressure

Since articular cartilage is subjected to varying loads in vivo and undergoes cyclic hydrostatic pressure during periods of loading, it is hypothesized that mimicking these in vivo conditions can enhance synthesis of important matrix components during cultivation in vitro. Thus, the influence of intermittent loading during redifferentiation of chondrocytes in alginate beads, and during cartilage formation was investigated. A statistically significant increased synthesis of glycosaminoglycan and collagen type II during redifferentiation of chondrocytes embedded in alginate beads, as well as an increase in glycosaminoglycan content of tissue-engineered cartilage, was found compared to control without load. Immunohistological staining indicated qualitatively a high expression of collagen type II for both cases.     

Tissue engineering strategies for cartilage generation--micromass and three dimensional cultures using human chondrocytes and a continuous cell line

Utilizing ATDC5 murine chondrogenic cells and human articular chondrocytes, this study sought to develop facile, reproducible three-dimensional models of cartilage generation with the application of tissue engineering strategies, involving biodegradable poly(glycolic acid) scaffolds and rotating wall bioreactors, and micromass pellet cultures. Chondrogenic differentiation, assessed by histology, immunohistochemistry, and gene expression analysis, in ATDC5 and articular chondrocyte pellets was evident by the presence of distinct chondrocytes, expressing Sox-9, aggrecan, and type II collagen, in lacunae embedded in a cartilaginous matrix of type II collagen and proteoglycans. Tissue engineered explants of ATDC5 cells were reminiscent of cartilaginous structures composed of numerous chondrocytes, staining for typical chondrocytic proteins, in lacunae embedded in a matrix of type II collagen and proteoglycans. In comparison, articular chondrocyte explants exhibited areas of Sox-9, aggrecan, and type II collagen-expressing cells growing on fleece, and discrete islands of chondrocytic cells embedded in a cartilaginous matrix.     

深低温冻存组织工程化软骨在喉狭窄功能重建中的应用

目的：探讨低温保存组织工程化软骨在喉狭窄功能重建中的应用价值。方法：取3周龄新西兰兔关节软骨细胞,体外培养,取第2代对数生长期培养细胞,制成细胞悬液,调整软骨细胞悬液浓度约为5×10^7个／ml左右,接种于PGA三维支架材料上,复合物体外培养2周后冻存,冻存6个月后解冻复苏,再行体外培养观察,2周后接种于已建立的喉甲状软骨缺损模型的软骨缺损处,并设对照组。术后12周取材,行大体及组织学观察。结果：经低温冻存的组织工程化软骨生长良好,组织学观察有软骨形成,与周围软骨组织结合紧密,与非冻存组相比差异无统计学意义。结论：深低温冻存对组织工程化软骨的生物活性无明显的影响,低温冻存的组织工程化软骨可用于喉软骨缺损的修复,重建喉功能。     

An intelligent bioreactor system for the cultivation of a bioartificial vascular graft

Cardiovascular disease is the most common cause of death, accounting for 31% of deaths worldwide. As purely synthetic grafts implicate concomitant anticoagulation and autologous veins are rare, tissue‐engineered vascular grafts are urgently needed. For successful in vitro cultivation of a bioartificial vascular graft, the suitable bioreactor should provide conditions comparable to vasculogenesis in the body. Such a system has been developed and characterized under continuous and pulsatile flow, and a variety of sensors has been integrated into the bioreactor to control parameters such as temperature, pressure up to 500 mbar, glucose up to 4.5 g/L, lactate, oxygen up to 150 mbar, and flow rate. Wireless data transfer (using the ZigBee specification based on the IEEE 802.15.4 standard) and multiple corresponding sensor signal processing platforms have been implemented as well. Ultrasound is used for touchless monitoring of the growing vascular structure as a quality control before implantation (maximally achieved ultrasound resolution 65 μm at 15 MHz). To withstand the harsh conditions of steam sterilization (120°C for 20 min), all electronics were encapsulated. With such a comprehensive physiologically conditioning, sensing, and imaging bioreactor system, all the requirements for a successful cultivation of vascular grafts are available now.     

A bioreactor system for interfacial culture and physiological perfusion of vascularized tissue equivalents

A pivotal requirement for the generation of vascularized tissue equivalents is the development of culture systems that provide a physiological perfusion of the vasculature and tissue-specific culture conditions. Here, we present a bioreactor system that is suitable to culture vascularized tissue equivalents covered with culture media and at the air–medium interface, which is a vital stimulus for skin tissue. For the perfusion of the vascular system a new method was integrated into the bioreactor system that creates a physiological pulsatile medium flow between 80 and 120 mmHg to the arterial inflow of the equivalent's vascular system. Human dermal microvascular endothelial cells (hDMECs) were injected into the vascular system of a biological vascularized scaffold based on a decellularized porcine jejunal segment and cultured in the bioreactor system for 14 days. Histological analysis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining revealed that the hDMECs were able to recolonize the perfused vascular structures and expressed endothelial cell specific markers such as platelet endothelial cell adhesion molecule and von Willebrand factor. These results indicate that our bioreactor system can serve as a platform technology to generate advanced bioartificial tissues with a functional vasculature for future clinical applications.     

Growth factor effects on costal chondrocytes for tissue engineering fibrocartilage

Tissue-engineered fibrocartilage could become a feasible option for replacing tissues such as the knee meniscus or temporomandibular joint disc. This study employed five growth factors (insulin-like growth factor-I, transforming growth factor-beta1, epidermal growth factor, platelet-derived growth factor-BB, and basic fibroblast growth factor) in a scaffoldless approach with costal chondrocytes, attempting to improve biochemical and mechanical properties of engineered constructs. Samples were quantitatively assessed for total collagen, glycosaminoglycans, collagen type I, collagen type II, cells, compressive properties, and tensile properties at two time points. Most treated constructs had lower biomechanical and biochemical properties than the controls with no growth factors, suggesting a detrimental effect, but the treatment with insulin-like growth factor-I tended to improve the constructs. Additionally, the 6-week time point was consistently better than that at 3 weeks, with total collagen, glycosaminoglycans, and aggregate modulus doubling during this time. Further optimization of the time in culture and exogenous stimuli will be important in making a more functional replacement tissue.     

Mesenchymal progenitor cells derived from chorionic villi of human placenta for cartilage tissue engineering

Human mesenchymal stem cells are currently being studied extensively because of their capability for self-renewal and differentiation to various connective tissues, which makes them attractive as cell sources for regenerative medicine. Herein we report the isolation of human placenta-derived mesenchymal cells (hPDMCs) that have the potential to differentiate into various lineages to explore the possibility of using these cells for regeneration of cartilage. We first evaluated the chondrogenesis of hPDMCs in vitro and then embedded the hPDMCs into an atelocollagen gel to make a cartilage-like tissue with chondrogenic induction media. For in vivo assay, preinduced hPDMCs embedded in collagen sponges were subcutaneously implanted into nude mice and also into nude rats with osteochondral defect. The results of these in vivo and in vitro studies suggested that hPDMCs can be one of the possible allogeneic cell sources for tissue engineering of cartilage.     

Development and validation of a novel bioreactor system for load- and perfusion-controlled tissue engineering of chondrocyte-constructs

Osteoarthritis is a severe socio-economical disease, for which a suitable treatment modality does not exist. Tissue engineering of cartilage transplants is the most promising method to treat focal cartilage defects. However, current culturing procedures do not yet meet the requirements for clinical implementation. This article presents a novel bioreactor device for the functional tissue engineering of articular cartilage which enables cyclic mechanical loading combined with medium perfusion over long periods of time, under controlled cultivation and stimulation conditions whilst ensuring system sterility. The closed bioreactor consists of a small, perfused, autoclavable, twin chamber culture device with a contactless actuator for mechanical loading. Uni-axial loading is guided by externally applied magnetic fields with real-time feedback-control from a platform load cell and an inductive proximity sensor. This precise measurement allows the development of the mechanical properties of the cultured tissue to be monitored in real-time. This is an essential step towards clinical implementation, as it allows accounting for differences in the culture procedure induced by patient-variability. This article describes, based on standard agarose hydrogels of 3 mm height and 10 mm diameter, the technical concept, implementation, scalability, reproducibility, precision, and the calibration procedures of the whole bioreactor instrument. Particular attention is given to the contactless loading system by which chondrocyte scaffolds can be compressed at defined loading frequencies and magnitudes, whilst maintaining an aseptic cultivation procedure. In a "proof of principle" experiment, chondrocyte seeded agarose gels were cultured for 21 days in the bioreactor system. Intermittent medium perfusion at a steady flow rate (0.5 mL/min) was applied. Sterility and cell viability (ds-DNA quantification and fluorometric live/dead staining) were preserved in the system. Flow induced shear stress stimulated sGAG (sulfated glycosaminoglycan) content (DMMB assay) after 21 days, which was confirmed by histological staining of Alcian blue and by immunostaining of Aggrecan. Experimental data on mechanotransduction and long-term studies on the beneficial effects of combined perfusion and different mechanical loading patterns on chondrocyte seeded scaffolds will be published separately.     

Nutrient channels and stirring enhanced the composition and stiffness of large cartilage constructs

A significant challenge in cartilage tissue engineering is to successfully culture functional tissues that are sufficiently large to treat osteoarthritic joints. Transport limitations due to nutrient consumption by peripheral cells produce heterogeneous constructs with matrix-deficient centers. Incorporation of nutrient channels into large constructs is a promising technique for alleviating transport limitations, in conjunction with simple yet effective methods for enhancing media flow through channels. Cultivation of cylindrical channeled constructs flat in culture dishes, with or without orbital shaking, produced asymmetric constructs with poor tissue properties. We therefore explored a method for exposing the entire construct surface to the culture media, while promoting flow through the channels. To this end, chondrocyte-seeded agarose constructs (∅10 mm, 2.34 mm thick), with zero or three nutrient channels (∅1 mm), were suspended on their sides in custom culture racks and subjected to three media stirring modes for 56 days: uniaxial rocking, orbital shaking, or static control. Orbital shaking led to the highest construct E_Y, sulfated glycosaminoglycan (sGAG), and collagen contents, whereas rocking had detrimental effects on sGAG and collagen versus static control. Nutrient channels increased E_Y as well as sGAG homogeneity, and the beneficial effects of channels were most marked in orbitally shaken samples. Under these conditions, the constructs developed symmetrically and reached or exceeded native levels of E_Y (~400 kPa) and sGAG (~9%/ww). These results suggest that the cultivation of channeled constructs in culture racks with orbital shaking is a promising method for engineering mechanically competent large cartilage constructs.     

Growth factors for sequential cellular de- and re-differentiation in tissue engineering

A model system for the in vitro generation of cartilaginous constructs was used to study a tissue engineering paradigm whereby sequentially applied growth factors promoted chondrocytes to first de-differentiate into a proliferative state and then re-differentiate and undergo chondrogenesis. Early cultivation in medium with supplemental TGF-β1/FGF-2 doubled cell fractions in 2-week constructs compared to unsupplemented controls. Subsequent culture with supplemental IGF-I yielded large 4-week constructs with high fractions of cartilaginous extracellular matrix (ECM) and high compressive moduli, whereas prolonged culture with supplemental FGF-2 yielded small 4-week constructs with low ECM fractions and moduli. Sequential supplementation with TGF-β1/FGF-2 and then IGF-I yielded 4-week constructs with type-specific mRNA expression and protein levels that were high for type II and negligible for type I collagen, in contrast to other growth factor regimens studied. The data demonstrate that structural, functional, and molecular properties of engineered cartilage can be modulated by sequential application of growth factors.     

骨碎补结合组织工程软骨促进软骨再生及其机理研究

目的:评估骨碎补在促进软骨再生治疗中的价值及作用机制。方法:选取健康新西兰大耳白兔100只,采用随机数值表法随机分为5组,每组包含20只实验动物。A、B组:改建后进行肾上腺髓质素(adrenomedullin,ADM)移植;C组:自体骨髓充质干细胞(MSCs)复合,并进行改建ADM移植;D、E组:进行h IGF-1基因转染的MSCs复合,并改建ADM移植。B、C、E组实验动物结合喂养40%骨碎补汤,每只每日150ml,用药4周。A、D组不结合使用口服骨碎补汤。每组均在第4、8、12w时处死5只实验动物,切取关节软骨缺损部位并进行组织学观察,采用Masson三色染色法评估软骨细胞的形态变化,采用Wakitani组织学评分进行各组间形态学比较。结果:E组实验动物关节软骨的表面有比较完整的潮线,大部分比较光滑,变薄区可见零散的浅淡着色的纤维组织。在第4、8、12周,各组Wakitani组织学评分逐渐下降,但结合骨碎补的B、C、E三组下降更为显著(P0.05),说明骨碎补可显著改善软骨组织缺损。结论:采用中药骨碎补结合工程软骨可以显著提高家兔膝关节软骨缺损修复的质量,为临床使用骨碎补治疗软骨病变提供了重要的依据。     

Mathematical modelling of fibre-enhanced perfusion inside a tissue-engineering bioreactor

We develop a simple mathematical model for forced flow of culture medium through a porous scaffold in a tissue-engineering bioreactor. Porous-walled hollow fibres penetrate the scaffold and act as additional sources of culture medium. The model, based on Darcy's law, is used to examine the nutrient and shear-stress distributions throughout the scaffold. We consider several configurations of fibres and inlet and outlet pipes. Compared with a numerical solution of the full Navier-Stokes equations within the complex scaffold geometry, the modelling approach is cheap, and does not require knowledge of the detailed microstructure of the particular scaffold being used. The potential of this approach is demonstrated through quantification of the effect the additional flow from the fibres has on the nutrient and shear-stress distribution.     

Nanomaterial scaffolds for stem cell proliferation and differentiation in tissue engineering

Tissue engineering is a clinically driven field and has emerged as a potential alternative to organ transplantation. The cornerstone of successful tissue engineering rests upon two essential elements: cells and scaffolds. Recently, it was found that stem cells have unique capabilities of self-renewal and multilineage differentiation to serve as a versatile cell source, while nanomaterials have lately emerged as promising candidates in producing scaffolds able to better mimic the nanostructure in natural extracellular matrix and to efficiently replace defective tissues. This article, therefore, reviews the key developments in tissue engineering, where the combination of stem cells and nanomaterial scaffolds has been utilized over the past several years. We consider the high potential, as well as the main issues related to the application of stem cells and nanomaterial scaffolds for a range of tissues including bone, cartilage, nerve, liver, eye etc. Promising in vitro results such as efficient attachment, proliferation and differentiation of stem cells have been compiled in a series of examples involving different nanomaterials. Furthermore, the merits of the marriage of stem cells and nanomaterial scaffolds are also demonstrated in vivo, providing early successes to support subsequent clinical investigations. This progress simultaneously drives mechanistic research into the mechanotransduction process responsible for the observations in order to optimize the process further. Current understanding is chiefly reported to involve the interaction of stem cells and the anchoring nanomaterial scaffolds by activating various signaling pathways. Substrate surface characteristics and scaffold bulk properties are also reported to influence not only short term stem cell adhesion, spreading and proliferation, but also longer term lineage differentiation, functionalization and viability. It is expected that the combination of stem cells and nanomaterials will develop into an important tool in tissue engineering for the innovative treatment of many diseases.     

Growth factor contents of autologous human sera prepared by different production methods and their biological effects on chondrocytes

To discuss the autologous serum production for cartilage tissue engineering, we compared three kinds of sera: whole blood-derived serum (WBS), platelet-containing plasma-derived serum (PCS), and plasma-derived serum (PDS), on the growth factor contents and their biological effects on human auricular chondrocytes. EGF, VEGF and PDGF levels were highest in WBS, while PCS and PDS followed WBS. The proliferation effects of WBS were the most pronounced, followed by that of PCS, both of which realized a 1000-fold-increase in chondrocyte numbers at the third passage, whereas PDS reached it after passage 4. No significant differences were observed in histology or cartilaginous matrix measurements of tissue-engineered cartilage produced from chondrocytes cultured under different serum conditions. WBS would be clinically useful because of its potent proliferation effects, while PCS, which possibly saves the red cell concentrate, may be an option in cases where there are elevated risks of blood loss.     

Hydrostatic fluid pressure promotes cellularity and proliferation of human dermal fibroblasts in a three-dimensional collagen gel/sponge

Cell constructs and culture systems are essential components of tissue engineering. Cell constructs are usually composed of a dense population of cells, for which long-term culture is required in vitro. However, the denser construct suffers from the absence of passive nutrient supply, gas exchange, and removal of degraded debris. We have developed a novel hydrostatic pressure/perfusion (HP/P) culture system that improves the quality of neo-tissues, providing an automated affordable system for clinical applications. We evaluated the effects of HP/P on cellularity, viability, and proliferation of human dermal fibroblasts seeded in a gel/sponge construct. HP/P and perfusion promoted cell migration and significantly increased proliferation and DNA content after 4 days culture compared to the static culture. HP/P culture is beneficial for building a denser three-dimensional fibroblast construct.