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.     

Effects of mechanical stimulation induced by compression and medium perfusion on cardiac tissue engineering

Cardiac tissue engineering presents a challenge due to the complexity of the muscle tissue and the need for multiple signals to induce tissue regeneration in vitro. We investigated the effects of compression (1 Hz, 15% strain) combined with fluid shear stress (10^?2–10^?1 dynes/cm²) provided by medium perfusion on the outcome of cardiac tissue engineering. Neonatal rat cardiac cells were seeded in Arginine‐Glycine‐Aspartate (RGD)‐attached alginate scaffolds, and the constructs were cultivated in a compression bioreactor. A daily, short‐term (30 min) compression (i.e., “intermittent compression”) for 4 days induced the formation of cardiac tissue with typical striation, while in the continuously compressed constructs (i.e., “continuous compression”), the cells remained spherical. By Western blot, on day 4 the expression of the gap junction protein connexin 43 was significantly greater in the “intermittent compression” constructs and the cardiomyocyte markers (α‐actinin and N‐cadherin) showed a trend of better preservation compared to the noncompressed constructs. This regime of compression had no effect on the proliferation of nonmyocyte cells, which maintained low expression level of proliferating cell nuclear antigen. Elevated secretion levels of basic fibroblast growth factor and transforming growth factor‐β in the daily, intermittently compressed constructs likely attributed to tissue formation. Our study thus establishes the formation of an improved cardiac tissue in vitro, when induced by combined mechanical signals of compression and fluid shear stress provided by perfusion. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

A validated model of GAG deposition,cell distribution,and growth of tissue engineered cartilage cultured in a rotating bioreactor

In this work a new phenomenological model of growth of cartilage tissue cultured in a rotating bioreactor is developed. It represents an advancement of a previously derived model of deposition of glycosaminoglycan (GAG) in engineered cartilage by (i) introduction of physiological mechanisms of proteoglycan accumulation in the extracellular matrix (ECM) as well as by correlating (ii) local cell densities and (iii) tissue growth to the ECM composition. In particular, previously established predictions and correlations of local oxygen concentrations and GAG synthesis rates are extended to distinguish cell secreted proteoglycan monomers free to diffuse in cell surroundings and outside from the engineered construct, from large aggrecan molecules, which are constrained within the ECM and practically immovable. The model includes kinetics of aggregation, that is, transformation of mobile GAG species into immobile aggregates as well as maintenance of the normal ECM composition after the physiological GAG concentration is reached by incorporation of a product inhibition term. The model also includes mechanisms of the temporal evolution of cell density distributions and tissue growth under in vitro conditions. After a short initial proliferation phase the total cell number in the construct remains constant, but the local cell distribution is leveled out by GAG accumulation and repulsion due to negative molecular charges. Furthermore, strong repulsive forces result in expansion of the local tissue elements observed macroscopically as tissue growth (i.e., construct enlargement). The model is validated by comparison with experimental data of (i) GAG distribution and leakage, (ii) spatial‐temporal distributions of cells, and (iii) tissue growth reported in previous works. Validation of the model predictive capability—against a selection of measured data that were not used to construct the model—suggests that the model successfully describes the interplay of several simultaneous processes carried out during in vitro cartilage tissue regeneration and indicates that this approach could also be attractive for application in other tissue engineering systems. Biotechnol. Bioeng. 2010. 105: 842–853. © 2009 Wiley Periodicals, Inc.     

Flow characterization of a wavy-walled bioreactor for cartilage tissue engineering

Cartilage tissue engineering requires the use of bioreactors in order to enhance nutrient transport and to provide sufficient mechanical stimuli to promote extracellular matrix (ECM) synthesis by chondrocytes. The amount and quality of ECM components is a large determinant of the biochemical and mechanical properties of engineered cartilage constructs. Mechanical forces created by the hydrodynamic environment within the bioreactors are known to influence ECM synthesis. The present study characterizes the hydrodynamic environment within a novel wavy-walled bioreactor (WWB) used for the development of tissue-engineered cartilage. The geometry of this bioreactor provides a unique hydrodynamic environment for mammalian cell and tissue culture, and investigation of hydrodynamic effects on tissue growth and function. The flow field within the WWB was characterized using two-dimensional particle-image velocimetry (PIV). The flow in the WWB differed significantly from that in the traditional spinner flask both qualitatively and quantitatively, and was influenced by the positioning of constructs within the bioreactor. Measurements of velocity fields were used to estimate the mean-shear stress, Reynolds stress, and turbulent kinetic energy components in the vicinity of the constructs within the WWB. The mean-shear stress experienced by the tissue-engineered constructs in the WWB calculated using PIV measurements was in the range of 0-0.6 dynes/cm2. Quantification of the shear stress experienced by cartilage constructs, in this case through PIV, is essential for the development of tissue-growth models relating hydrodynamic parameters to tissue properties.     

Optimization of culture media to enhance the growth of tissue engineered cartilage

Tissue engineering is a promising option for cartilage repair. However, several hurdles still need to be overcome to develop functional tissue constructs suitable for implantation. One of the most common challenges is the general low capacity of chondrocytes to synthesize cartilage-specific extracellular matrix (ECM). While different approaches have been explored to improve the biosynthetic response of chondrocytes, several studies have demonstrated that the nutritional environment (e.g., glucose concentration and media volume) can have a profound effect on ECM synthesis. Thus, the purpose of this study was to optimize the formulation of cell culture media to upregulate the accumulation of cartilaginous ECM constituents (i.e., proteoglycans and collagen) by chondrocytes in 3D culture. Using response surface methodology, four different media factors (basal media, media volume, glucose, and glutamine) were first screened to determine optimal media formulations. Constructs were then cultured under candidate optimal media formulations for 4 weeks and analyzed for their biochemical and structural properties. Interestingly, the maximal accumulation of proteoglycans and collagen appeared to be elicited by different media formulations. Most notably, proteoglycan accumulation was favored by high volume, low glucose-containing DMEM/F12 (1:1) media whereas collagen accumulation was favored by high volume, high glucose-containing F12 media. While high glutamine-containing media elicited increased DNA content, glutamine concentration had no apparent effect on ECM accumulation. Therefore, optimizing the nutritional environment during chondrocyte culture appears to be a promising, straight-forward approach to improve cartilaginous tissue formation. Future work will investigate the combined effects of the nutritional environment and external stimuli.     

Development of large engineered cartilage constructs from a small population of cells

Confronted with articular cartilage's limited capacity for self‐repair, joint resurfacing techniques offer an attractive treatment for damaged or diseased tissue. Although tissue engineered cartilage constructs can be created, a substantial number of cells are required to generate sufficient quantities of tissue for the repair of large defects. As routine cell expansion methods tend to elicit negative effects on chondrocyte function, we have developed an approach to generate phenotypically stable, large‐sized engineered constructs (≥3 cm²) directly from a small amount of donor tissue or cells (as little as 20,000 cells to generate a 3 cm² tissue construct). Using rabbit donor tissue, the bioreactor‐cultivated constructs were hyaline‐like in appearance and possessed a biochemical composition similar to native articular cartilage. Longer bioreactor cultivation times resulted in increased matrix deposition and improved mechanical properties determined over a 4 week period. Additionally, as the anatomy of the joint will need to be taken in account to effectively resurface large affected areas, we have also explored the possibility of generating constructs matched to the shape and surface geometry of a defect site through the use of rapid‐prototyped defect tissue culture molds. Similar hyaline‐like tissue constructs were developed that also possessed a high degree of shape correlation to the original defect mold. Future studies will be aimed at determining the effectiveness of this approach to the repair of cartilage defects in an animal model and the creation of large‐sized osteochondral constructs. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Design and characterization of a modified T‐flask bioreactor for continuous monitoring of engineered tissue stiffness

Controlling environmental conditions, such as mechanical stimuli, is critical for directing cells into functional tissue. This study reports on the development of a bioreactor capable of controlling the mechanical environment and continuously measuring force‐displacement in engineered tissue. The bioreactor was built from off the shelf components, modified off the shelf components, and easily reproducible custom built parts to facilitate ease of setup, reproducibility and experimental flexibility. A T‐flask was modified to allow for four tissue samples, mechanical actuation via a LabView controlled stepper motor and transduction of force from inside the T‐flask to an external sensor. In vitro bench top testing with instrumentation springs and tissue culture experiments were performed to validate system performance. Force sensors were highly linear (R² > 0.998) and able to maintain force readings for extended periods of time. Tissue culture experiments involved cyclic loading of polyurethane scaffolds seeded with and without (control) human foreskin fibroblasts for 8 h/day for 14 days. After supplementation with TGF‐β, tissue constructs showed an increase in stiffness between consecutive days and from the acellular controls. These experiments confirmed the ability of the bioreactor to distinguish experimental groups and monitor tissue stiffness during tissue development. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Shear stress magnitude and duration modulates matrix composition and tensile mechanical properties in engineered cartilaginous tissue

Cartilage tissue‐engineering strategies aim to produce a functional extracellular matrix similar to that of the native tissue. However, none of the myriad approaches taken have successfully generated a construct possessing the structure, composition, and mechanical properties of healthy articular cartilage. One possible approach to modulating the matrix composition and mechanical properties of engineered tissues is through the use of bioreactor‐driven mechanical stimulation. In this study, we hypothesized that exposing scaffold‐free cartilaginous tissue constructs to 7 days of continuous shear stress at 0.001 or 0.1 Pa would increase collagen deposition and tensile mechanical properties compared to that of static controls. Histologically, type II collagen staining was evident in all construct groups, while a surface layer of type I collagen increased in thickness with increasing shear stress magnitude. The areal fraction of type I collagen was higher in the 0.1‐Pa group (25.2 ± 2.2%) than either the 0.001‐Pa (13.6 ± 3.8%) or the static (7.9 ± 1.5%) group. Type II collagen content, as assessed by ELISA, was also higher in the 0.1‐Pa group (7.5 ± 2.1%) compared to the 0.001‐Pa (3.0 ± 2.25%) or static groups (3.7 ± 3.2%). Temporal gene expression analysis showed a flow‐induced increase in type I and type II collagen expression within 24 h of exposure. Interestingly, while the 0.1‐Pa group showed higher collagen content, this group retained less sulfated glycosaminoglycans in the matrix over time in bioreactor culture. Increases in both tensile Young's modulus and ultimate strength were observed with increasing shear stress, yielding constructs possessing a modulus of nearly 5 MPa and strength of 1.3 MPa. This study demonstrates that shear stress is a potent modulator of both the amount and type of synthesized extracellular matrix constituents in engineered cartilaginous tissue with corresponding effects on mechanical function. Biotechnol. Bioeng. 2009; 104: 809–820 © 2009 Wiley Periodicals, Inc.     

Effects of oxygen on engineered cardiac muscle

Concentration gradients associated with the in vitro cultivation of engineered tissues that are vascularized in vivo result in the formation of only a thin peripheral tissue-like region (e.g., approximately 100 microm for engineered cardiac muscle) around a relatively cell-free interior. We previously demonstrated that diffusional gradients within engineered cardiac constructs can be minimized by direct perfusion of culture medium through the construct. In the present study, we measured the effects of medium perfusion rate and local oxygen concentration (p(O2)) on the in vitro reconstruction of engineered cardiac muscle. Neonatal rat cardiomyocytes were seeded onto biodegradable polymer scaffolds (fibrous discs, 1.1 cm diameter x 2 mm thick, made of polyglycolic acid, 24 x 10(6) cells per scaffold). The resulting cell-polymer constructs were cultured for a total of 12 days in serially connected cartridges (n = 1-8), each containing one construct directly perfused with culture medium at a flow rate of 0.2-3.0 mL/min. In all groups, oxygen concentration decreased due to cell respiration, and depended on construct position in the series and medium flow rate. Higher perfusion rates and higher p(O2) correlated with more aerobic cell metabolism, and higher DNA and protein contents. Constructs cultured at p(O2) of 160 mm Hg had 50% higher DNA and protein contents, markedly higher expression of sarcomeric alpha-actin, better organized sarcomeres and cell junctions, and 4.5-fold higher rate of cell respiration as compared to constructs cultured at p(O2) of 60 mm Hg. Contraction rates of the corresponding cardiac cell monolayers were 40% higher at p(O2) of 160 than 60 mm Hg. The control of oxygen concentration in cell microenvironment can thus improve the structure and function of engineered cardiac muscle. Experiments of this kind can form a basis for controlled studies of the effects of oxygen on the in vitro development of engineered tissues.     

Time-dependent functional maturation of scaffold-free cartilage tissue analogs

One of the most critical parameters in cartilage tissue engineering which influences the clinical success of a repair therapy is the ability to match the load-bearing capacity of the tissue as it functions in vivo. While mechanical forces are known to positively influence the development of cartilage matrix architecture, these same forces can induce long-term implant failure due to poor integration or structural deficiencies. As such, in the design of optimal repair strategies, it is critical to understand the timeline of construct maturation and how the elaboration of matrix correlates with the development of mechanical properties. We have previously characterized a scaffold-free method to engineer cartilage utilizing primary chondrocytes cultured at high density in hydrogel-coated culture vessels to promote the formation of a self-aggregating cell suspension that condenses to form a cartilage-like biomass, or cartilage tissue analog (CTA). Chondrocytes in these CTAs maintain their cellular phenotype and deposit extracellular matrix to form a construct that has characteristics similar to native cartilage; however, the mechanical integrity of CTAs had not yet been evaluated. In this study, we found that chondrocytes within CTAs produced a robust matrix of proteoglycans and collagen that correlated with increasing mechanical properties and decreasing cell-matrix ratios, leading to properties that approached that of native cartilage. These results demonstrate a unique approach to generating a cartilage-like tissue without the complicating factor of scaffold, while showing increased compressive properties and matrix characteristics consistent with other approaches, including scaffold-based constructs. To further improve the mechanics of CTAs, studies are currently underway to explore the effect of hydrodynamic loading and whether these changes would be reflective of in vivo maturation in animal models. The functional maturation of cartilage tissue analogs as described here support this engineered cartilage model for use in clinical and experimental applications for repair and regeneration in joint-related pathologies.     

Computational modeling of nutrient utilization in engineered cartilage

This study presents a mathematical model for simulating cartilaginous culture of chondrocytes seeded in scaffolds and for investigating the effects of glucose and oxygen concentration and pH value on cell metabolic rates. The model can clearly interpret the unexplained experimental observation (Sengers BG, Heywood HK, Lee DA, Oomens CWJ, Bader DL. Nutrient utilization by bovine articular chondrocytes: A combined experimental and theoretical approach. J Biomech Eng. 2005;127:758–766.), which showed that the oxygen concentration within the scaffold may increase instead of continuously decreasing in static cartilaginous culture of chondrocytes. Results from simulation demonstrate that when cells metabolize glucose and form lactate under high glucose concentration conditions, the acidity in the culture environment increases, inhibiting cell metabolic rates in the process. Consequently, the rate of oxygen consumption decreases in later stages of cell culture. As oxygen can be replenished through the free surface of the culture medium, oxygen concentration within the scaffold increases rather than decreases over time in the acidic environment. Different initial glucose concentration yields different results. In low glucose concentration conditions, oxygen concentration basically keeps decreasing with culture time. This is because the pH in the environment does not significantly change because of slower glycolysis rate in low glucose concentration cases, forming less lactic acid. From the simulation results, additional information regarding in vitro culture of chondrocytes is obtained. The correlations between nutrient consumption, lactate secretion, and pH changes during cell culture are also understood and may serve as a reference for in vitro cell culture research of tissue engineering. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 452–462, 2013     

The effect of continuous culture on the growth and structure of tissue‐engineered cartilage

The use of bioreactors for cartilage tissue engineering has become increasingly important as traditional batch‐fed culture is not optimal for in vitro tissue growth. Most tissue engineering bioreactors rely on convection as the primary means to provide mass transfer; however, convective transport can also impart potentially unwanted and/or uncontrollable mechanical stimuli to the cells resident in the construct. The reliance on diffusive transport may not necessarily be ineffectual as previous studies have observed improved cartilaginous tissue growth when the constructs were cultured in elevated volumes of media. In this study, to approximate an infinite reservoir of media, we investigated the effect of continuous culture on cartilaginous tissue growth in vitro. Isolated bovine articular chondrocytes were seeded in high density, 3D culture on Millicell? filters. After two weeks of preculture, the constructs were cultivated with or without continuous media flow (5–10 μL/min) for a period of one week. Tissue engineered cartilage constructs grown under continuous media flow significantly accumulated more collagen and proteoglycans (increased by 50–70%). These changes were similar in magnitude to the reported effect of through‐thickness perfusion without the need for large volumetric flow rates (5–10μL/min as opposed to 240–800 μL/min). Additionally, tissues grown in the reactor displayed some evidence of the stratified morphology of native cartilage as well as containing stores of intracellular glycogen. Future studies will investigate the effect of long‐term continuous culture in terms of extracellular matrix accumulation and subsequent changes in mechanical function. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Engineering superficial zone features in tissue engineered cartilage

A major challenge in cartilage tissue engineering is the need to recreate the native tissue's anisotropic extracellular matrix structure. This anisotropy has important mechanical and biological consequences and could be crucial for integrative repair. Here, we report that hydrodynamic conditions that mimic the motion‐induced flow fields in between the articular surfaces in the synovial joint induce the formation of a distinct superficial layer in tissue engineered cartilage hydrogels, with enhanced production of cartilage matrix proteoglycan and Type II collagen. Moreover, the flow stimulation at the surface induces the production of the surface zone protein Proteoglycan 4 (aka PRG4 or lubricin). Analysis of second harmonic generation signature of collagen in this superficial layer reveals a highly aligned fibrillar matrix that resembles the alignment pattern in native tissue's surface zone, suggesting that mimicking synovial fluid flow at the cartilage surface in hydrodynamic bioreactors could be key to creating engineered cartilage with superficial zone features. Biotechnol. Bioeng. 2013; 110: 1476–1486. © 2012 Wiley Periodicals, Inc.     

Increased rate of chondrocyte aggregation in a wavy-walled bioreactor

A novel wavy-walled bioreactor designed to enhance mixing at controlled shear stress levels was used to culture chondrocytes in suspension. Chondrocyte aggregation in suspensions mixed at 30, 50, and 80 rpm was characterized in the wavy-walled bioreactor and compared with that in conventional smooth-walled and baffled-walled spinner flask bioreactors. Aggregation was characterized in terms of the percentage of cells that aggregated over time, and aggregate size changes over time. The kinetics of chondrocyte aggregation observed in the bioreactors was composed of two phases: early aggregation between 0 and 2 h of culture, and late aggregation between 3 and 24 h of culture. At 50 rpm, the kinetics of early aggregation in the wavy-walled bioreactor was approximately 25% and 65% faster, respectively, than those in the smooth-walled and baffled-walled spinner flask bioreactors. During the late aggregation phase, the kinetics of aggregation in the wavy-walled bioreactor were approximately 45% and 65% faster, respectively, than in the smooth-walled and baffled-walled spinner flasks. The observed improved kinetics of chondrocyte aggregation was obtained at no cost to the cell survival rate. Results of computerized image analysis suggest that chondrocyte aggregation occurred initially by the formation of new aggregates via cell-cell interactions and later by the joining of small aggregates into larger cell clumps. Aggregates appeared to grow for only a couple of hours in culture before reaching a steady size, possibly determined by limitations imposed by the hydrodynamic environment. These results suggest that the novel geometry of the wavy-walled bioreactor generates a hydrodynamic environment distinct from those traditionally used to culture engineered cartilage. Such differences may be useful in studies aimed at distinguishing the effects of the hydrodynamic environment on tissue-engineered cartilage. Characterizing the wavy-walled bioreactor's hydrodynamic environment and its effects on cartilage cell/tissue culture can help establish direct relationships between hydrodynamic forces and engineered tissue properties.     

In silico multi‐scale model of transport and dynamic seeding in a bone tissue engineering perfusion bioreactor

Computer simulations can potentially be used to design, predict, and inform properties for tissue engineering perfusion bioreactors. In this work, we investigate the flow properties that result from a particular poly‐L ‐lactide porous scaffold and a particular choice of perfusion bioreactor vessel design used in bone tissue engineering. We also propose a model to investigate the dynamic seeding properties such as the homogeneity (or lack of) of the cellular distribution within the scaffold of the perfusion bioreactor: a pre‐requisite for the subsequent successful uniform growth of a viable bone tissue engineered construct. Flows inside geometrically complex scaffolds have been investigated previously and results shown at these pore scales. Here, it is our aim to show accurately that through the use of modern high performance computers that the bioreactor device scale that encloses a scaffold can affect the flows and stresses within the pores throughout the scaffold which has implications for bioreactor design, control, and use. Central to this work is that the boundary conditions are derived from micro computed tomography scans of both a device chamber and scaffold in order to avoid generalizations and uncertainties. Dynamic seeding methods have also been shown to provide certain advantages over static seeding methods. We propose here a novel coupled model for dynamic seeding accounting for flow, species mass transport and cell advection‐diffusion‐attachment tuned for bone tissue engineering. The model highlights the timescale differences between different species suggesting that traditional homogeneous porous flow models of transport must be applied with caution to perfusion bioreactors. Our in silico data illustrate the extent to which these experiments have the potential to contribute to future design and development of large‐scale bioreactors. Biotechnol. Bioeng. 2013; 110: 1221–1230. © 2012 Wiley Periodicals, Inc.