design-biaxial-mechanical-loading-bioreactor-for-tissue

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

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.

Increased rate of chondrocyte aggregation in a wavy-walled bioreactor.

The wavy-walled bioreactor (WWB) possesses a novel geometry comprised of walls with sinusoidal waves that mimic baffles in an effort to promote mixing. This geometry provides a unique hydrodynamic environment suitable for the cultivation of mammalian cells and tissues and the investigation of fluid mechanical effects on cell and tissue growth and development. In the present study, mixing in WWB was characterized and compared to that in a conventional spinner flask (SF). The key parameters included in this characterization were mixing time, residence time distribution (RTD), and dissolved oxygen concentration during engineered cartilage tissue cultivation. Factors that influenced mixing in WWB included wave amplitude, agitation rate, and the ratio of the impeller diameter to the tank diameter (D/T). Data obtained from RTD and acid base neutralization studies confirmed the presence of different mixing zones in WWB. A theoretical comparison of WWB to a baffled spinner flask (BSF) using computational fluid dynamics (CFD) modeling predicted that while enhanced mixing was achieved in wavy-walled and BSF bioreactors, the shear stresses applied on tissue constructs were 15% lower in WWB. Improved mixing was achieved in WWB compared to the SF at similar D/T ratios, verified by improved oxygen transport and increased dispersion. However, for lower D/T ratios mixing in WWB was not necessarily improved. This study demonstrated the importance of characterization of mixing by showing the impact of even minor changes in bioreactor geometry and operating conditions.

Characterization of mixing in a novel wavy-walled bioreactor for tissue engineering.

Hydrodynamic forces in bioreactors can decisively influence extracellular matrix deposition in engineered cartilage constructs. In the present study, the reduced fluid shear, high-axial mixing environment provided by a wavy-walled bioreactor was exploited in the cultivation of cartilage constructs using polyglycolic acid scaffolds seeded with bovine articular chondrocytes. Increased growth as defined by weight, cell proliferation and extracellular matrix deposition was observed in cartilage constructs from wavy-walled bioreactors in comparison with those from spinner flasks cultured under the same conditions. The wet weight composition of 4-week constructs from the wavy-walled bioreactor was similar to that of spinner flask constructs, but the former were 60% heavier due to equally higher incorporation of extracellular matrix and 30% higher cell population. It is most likely that increased construct matrix incorporation was a result of increased mitotic activity of chondrocytes cultured in the environment of the wavy-walled bioreactor. A layer of elongated cells embedded in type I collagen formed at the periphery of wavy-walled bioreactor and spinner flask constructs, possibly as a response to local shear forces. On the basis of the robustness and reproducibility of the extracellular matrix composition of cartilage constructs, the wavy-walled bioreactor demonstrated promise as an experimental cartilage tissue-engineering vessel. Increased construct growth in the wavy-walled bioreactor may lead to enhanced mechanical properties and expedited in vitro cultivation.

Wavy-walled bioreactor supports increased cell proliferation and matrix deposition in engineered cartilage constructs.

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.

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

Physical forces experienced by engineered-tissues during in vitro cultivation influence tissue growth and function. The hydrodynamic environment within bioreactors plays a decisive role in providing the necessary physical stimuli and nutrient transport to support tissue development. Our overall goal is to investigate interrelationships between the local hydrodynamic environment in the bioreactor and the structural and functional tissue properties in order to optimize the production of clinically relevant engineered-tissues. To this end, we used computational fluid dynamics (CFD) modeling to characterize the complex hydrodynamic environment in a wavy-walled bioreactor used for cultivation of tissue-engineered cartilage constructs and examined the changes in the flow field due to the presence of constructs. The flow-induced shear stress range experienced by engineered constructs cultivated in the wavy-walled bioreactor (0-0.67 dyn/cm(2)) was found to be significantly lower than that in the spinner flask (0-1.2 dyn/cm(2)), and to be modulated by the radial or axial position of the constructs. These CFD results are validated by experimental particle-image velocimetry (PIV) measurements previously reported by our group. Results from the present study indicate that the location of constructs in the bioreactor not only affected the magnitude and distribution of the shear stresses on the constructs, but also other hydrodynamic parameters, such as the directional distribution of the fluid velocity and the degree of fluid recirculation, all of which may differentially influence the development of tissue-engineered constructs.

Location of scaffolds in bioreactors modulates the hydrodynamic environment experienced by engineered tissues.

Growth factors have become an important component for tissue engineering and regenerative medicine. Insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-beta 1) in particular have great significance in cartilage tissue engineering. Here, we describe sequential release of IGF-I and TGF-beta 1 from modular designed poly(l,d-lactic-co-glycolic acid) (PLGA) scaffolds. Growth factors were encapsulated in PLGA microspheres using spontaneous emulsion, and in vitro release kinetics was characterized by ELISA. Incorporating BSA in the IGF-I formulations decreased the initial burst from 80% to 20%, while using uncapped PLGA rather than capped decreased the initial burst of TGF-beta 1 from 60% to 0% upon hydration. The bioactivity of released IGF-I and TGF-beta 1 was determined using MCF-7 proliferation assay and HT-2 inhibition assay, respectively. Both growth factors were released for up to 70 days in bioactive form. Scaffolds were fabricated by fusing bioactive IGF-I and TGF-beta 1 microspheres with dichloromethane vapor. Three scaffolds with tailored release kinetics were fabricated: IGF-I and TGF-beta 1 released continuously, TGF-beta 1 with IGF-I released sequentially after 10 days, and IGF-I with TGF-beta 1 released sequentially after 7 days. Scaffold swelling and degradation were characterized, indicating a peak swelling ratio of 4 after 7 days of incubation and showing 50% mass loss after 28 days, both consistent with scaffold release kinetics. The ability of these scaffolds to release IGF-I and TGF-beta 1 sequentially makes them very useful for cartilage tissue engineering applications.

Sequential release of bioactive IGF-I and TGF-beta 1 from PLGA microsphere-based scaffolds.

In vitro cartilage tissue engineering culture systems benefit from a fine balance of biochemical and mechanical components to maintain the chondrocyte phenotype. This balance, however, can be disrupted by using typical methods for cultivating chondrogenic cells in medium supplemented with fetal bovine serum (FBS) and growth factors. Our goal was to determine the effects of fluid-dynamic stimuli, fetal bovine serum and dexamethasone on the chondrogenesis of 14-day synoviocyte pellet cultures in the presence of TGF-beta1. We employed a pellet culture system that provides a highly cellular three-dimensional structure that permits differentiation and extracellular matrix synthesis. Our results indicated that FBS inhibited glycosaminoglycan (GAG) and type II collagen production. Interestingly, the effect of dynamic stimuli was modulated by the presence of FBS; mixed serum-free cultures had increased GAG production, whereas mixed cultures with 10% FBS exhibited less GAG production compared with their static counterparts, possibly due to pronounced suppressive effects of FBS via increased transport. Dexamethasone addition during the first week of culture resulted in enhanced extracellular matrix production and increased cellularity. Moreover, the presence of 10% FBS in addition to ITS(+) and TGF-beta1 did not significantly increase cell proliferation compared with serum-free medium. These results indicate the importance of a comprehensive analysis of growth conditions for each cell culture system.

FBS suppresses TGF-beta1-induced chondrogenesis in synoviocyte pellet cultures while dexamethasone and dynamic stimuli are beneficial.

Functional engineered cartilage constructs represent a promising therapeutic approach for the replacement of damaged articular cartilage. The in vitro generation of cartilage tissue suitable for repair requires an understanding of the complex interrelationships between environmental cues, such as hydrodynamic forces, and tissue growth and development. In the present study, engineered cartilage constructs were cultivated in four well-defined hydrodynamic environments within a bioreactor, and correlations were established between construct ultrastructural and mechanical properties and key hydrodynamic parameters. Results suggest that even for similar composition, constructs may exhibit different mechanical properties due to differences in their ultrastructure that can be modulated by hydrodynamic parameters. For example, improved mechanical properties were observed in constructs that exhibited a thick fibrous outer capsule as a result of cultivation under increased hydrodynamic shear. In particular, uniformity in the contribution of the fluid velocity vectors (axial, radial, and tangential) to the total fluid velocity and shear stress were the hydrodynamic parameters that most affected the construct properties under investigation. The correlations identified here may be useful in the development of engineered tissue growth models that inform the design of bioreactor cultivation systems toward the production of clinically relevant engineered cartilage.

Hydrodynamic parameters modulate biochemical, histological, and mechanical properties of engineered cartilage.

Bioreactors have played a crucial role in recent approaches to cartilage tissue engineering, providing an environment that promotes efficient cell seeding, nutrient and waste transport, and essential physical stimuli. This study employed a wavy-walled bioreactor to investigate the effects of the hydrodynamic environment on the properties of engineered cartilage. Its unique design provides multiple hydrodynamic environments within one setting. A tissue growth model was developed to characterize the tissue growth and extracellular matrix synthesis by chondrocytes seeded and cultivated on polyglycolic acid scaffolds in the wavy-walled bioreactor for a period of 4 weeks. This model consists of four components: 1) a computational fluid dynamics model, 2) a kinetic growth model, 3) an artificial neural network that empirically correlates hydrodynamic parameters with kinetic constants, and 4) a second artificial neural network that correlates the biochemical composition of constructs with their material properties. In tandem, these components enable the prediction of the dynamics of tissue growth, as well as the final compositional and mechanical properties of engineered cartilage. The growth model methodology developed in this study serves as a tool to predict the optimal bioprocessing conditions required to achieve desired tissue properties.

Tissue growth modeling in a wavy-walled bioreactor.

Bioreaktoren spielten eine entscheidende Rolle in den letzten Zug&auml;nge zum Knorpel Tissue-Engineering, Schaffung einer Umgebung, die effiziente Zelle Aussaat, nahrhafte und Abfall-Transport und wesentliche physikalische Reize f&ouml;rdert. Diese Studie besch&auml;ftigt wellig ummauerten Bioreaktor, die Auswirkungen der hydrodynamischen Umwelt auf die Eigenschaften von ver&auml;nderter Knorpel zu untersuchen. Das einzigartige Design bietet mehrere hydrodynamische Umgebungen innerhalb einer Einstellung. Ein Gewebe-Wachstum-Modell wurde entwickelt, um das Gewebewachstum und die Synthese der extrazellul&auml;ren Matrix zu charakterisieren, durch Chondrozyten ges&auml;t und kultiviert auf Polyglycolic S&auml;ure Ger&uuml;ste in wellenf&ouml;rmigen ummauerten Bioreaktor f&uuml;r einen Zeitraum von 4 Wochen. Dieses Modell besteht aus vier Komponenten: 1) eine numerische Str&ouml;mungsmechanik-Modell, 2) eine kinetische Wachstumsmodell, 3) ein k&uuml;nstliches neurales Netz, die empirisch hydrodynamischer Parameter mit kinetischen Konstanten korreliert und 4) eine zweite k&uuml;nstliches neuronales Netz, die die biochemische Zusammensetzung der Konstrukte mit ihren Materialeigenschaften korreliert. Im Tandem erm&ouml;glichen diese Komponenten die Vorhersage von der Dynamik der Gewebewachstum sowie die endg&uuml;ltigen kompositorischen und mechanischen Eigenschaften von ver&auml;nderter Knorpel. Die in dieser Studie entwickelte Wachstum-Modell-Methodik dient als Werkzeug vorherzusagen, die optimalen bioprocessing Bedingungen ben&ouml;tigt, um die gew&uuml;nschte Gewebe-Eigenschaften zu erreichen.

Gewebewachstum Modellierung in einem wellenf&ouml;rmigen ummauerten Bioreaktor.

Synovial membrane has been shown to contain mesenchymal stem cells. We hypothesized that an enriched population of synovial fibroblasts would undergo chondrogenic differentiation and secrete cartilage extracellular matrix to a greater extent than would a mixed synovial cell population (MSCP). The optimum doses of transforming growth factor beta 1 (TGF-beta1) and insulin-like growth factor 1 (IGF-1) for chondrogenesis were investigated. CD14-negative isolation was used to obtain a porcine cell population enriched in type-B synovial fibroblasts (SFB) from an MSCP. The positive cell surface markers in SFB were CD90, CD44, and cadherin-11. SFB and MSCP were cultured in the presence of 20 ng/mL TGF-beta1 for 7 days, and SFB were demonstrated to have higher chondrogenic potential. Further dose-response studies were carried out using the SFB cells and several doses of TGF-beta1 (2, 10, 20, and 40 ng/mL) and/or IGF-1 (1, 10, 100, and 500 ng/mL) for 14 days. TGF-beta1 supplementation was essential for chondrogenesis and prevention of cell death, whereas IGF-1 did not have a significant effect on the SFB cell number or glycosaminoglycan production. This study demonstrates that the CD14-negative isolation yields an enhanced cell population SFB that is more potent than MSCP as a cell source for cartilage tissue engineering.

CD14-negative isolation enhances chondrogenesis in synovial fibroblasts.

Recently, hydrogels (alginate, agarose, polyethylene glycol, etc.) have been investigated as promising cartilage-healing materials. To further improve cell-material interactions or mechanical properties of such hydrogel scaffolds, many materials (such as ceramics or carbon nanotubes) have been added to produce composites with tailored properties. In this study, rosette nanotubes (RNTs, self-assembled nanotubes built from DNA base pairs), hydrogels, and cells (specifically, fibroblast-like type-B synoviocytes [SFB cells] and chondrocytes) were combined via a novel electrospinning technique to generate three-dimensional implantable scaffolds for cartilage repair. Importantly, results of this study showed that electrospun RNT/hydrogel composites improved both SFB cell and chondrocyte functions. RNT/hydrogel composites promoted SFB cell chondrogenic differentiation in 2 week culture experiments. Further, studies demonstrated that RNTs enhanced hydrogel adhesive strength to severed collagen. Results of this study thus provided a nanostructured scaffold that enhanced SFB cell adhesion, viability, and chondrogenic differentiation compared to nanosmooth hydrogels without RNTs. This study provided an alternative cartilage regenerative material derived from RNTs that could be directly electrospun into cartilage defects (with SFB cells and/or chondrocytes) to bond to severed collagen and promote cell adhesion, viability, and subsequent functions.

Self-assembled rosette nanotube/hydrogel composites for cartilage tissue engineering.

The design of optimal bioreactor systems for tissue engineering applications requires a sophisticated understanding of the complexities of the bioreactor environment and the role that it plays in the formation of engineered tissues. To this end, a tissue growth model is developed to characterize the tissue growth and extracellular matrix synthesis by chondrocytes seeded and cultivated on polyglycolic acid scaffolds in a wavy-walled bioreactor for a period of 4 weeks. This model consists of four components: (1) a computational fluid dynamics (CFD) model to characterize the complex hydrodynamic environment in the bioreactor, (2) a kinetic growth model to characterize the cell growth and extracellular matrix production dynamics, (3) an artificial neural network (ANN) that empirically correlates hydrodynamic parameters with kinetic constants, and (4) a second ANN that correlates the biochemical composition of constructs with their material properties. In tandem, these components enable the prediction of the dynamics of tissue growth, as well as the final compositional and mechanical properties of engineered cartilage. The growth model methodology developed in this study serves as a tool to predict optimal bioprocessing conditions required to achieve desired tissue properties.

Article	Year
Increased rate of chondrocyte aggregation in a wavy-walled bioreactor. Biotechnology and bioengineering\| PubMed ID: 15515164	2004
Characterization of mixing in a novel wavy-walled bioreactor for tissue engineering. Biotechnology and bioengineering\| PubMed ID: 16175564	2005
Wavy-walled bioreactor supports increased cell proliferation and matrix deposition in engineered cartilage constructs. Tissue engineering\| PubMed ID: 16411815	2005
Flow characterization of a wavy-walled bioreactor for cartilage tissue engineering. Biotechnology and bioengineering\| PubMed ID: 17031866	2006
Location of scaffolds in bioreactors modulates the hydrodynamic environment experienced by engineered tissues. Biotechnology and bioengineering\| PubMed ID: 17318906	2007
Sequential release of bioactive IGF-I and TGF-beta 1 from PLGA microsphere-based scaffolds. Biomaterials\| PubMed ID: 18166223	2008
FBS suppresses TGF-beta1-induced chondrogenesis in synoviocyte pellet cultures while dexamethasone and dynamic stimuli are beneficial. Journal of tissue engineering and regenerative medicine\| PubMed ID: 18265417	2007
Hydrodynamic parameters modulate biochemical, histological, and mechanical properties of engineered cartilage. Tissue engineering. Part A\| PubMed ID: 18803479	2009
Tissue growth modeling in a wavy-walled bioreactor. Tissue engineering. Part A\| PubMed ID: 18847355	2009
CD14-negative isolation enhances chondrogenesis in synovial fibroblasts. Tissue engineering. Part A\| PubMed ID: 19382853	2009
Self-assembled rosette nanotube/hydrogel composites for cartilage tissue engineering. Tissue engineering. Part C, Methods\| PubMed ID: 20184414	2010
Modeling of bioreactor hydrodynamic environment and its effects on tissue growth. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 22692614	2012

Bahar Bilgen

Biography

JoVE Journal Publications

Other Publications