Wavy-walled Bioreactor Research Articles

Type I Collagen-Based Fibrous Capsule Enhances Integration of Tissue-Engineered Cartilage with Native Articular Cartilage

Successful integration of engineered constructs with host tissues is crucial for cartilage repair, yet achieving it remains challenging. A collagen I-based fibrous capsule characterized by increased cell density and decreased glycosaminoglycan deposition usually forms at the periphery of tissue-engineered cartilage. The current study aimed to evaluate the effects of a solid fibrous capsule on construct integration with native articular cartilage. To this end, capsule-containing (CC) and capsule-free (CF) constructs were grown by culturing chondrocyte-seeded scaffolds with insulin-like growth factor-1 and transforming growth factor-β1, respectively, in a wavy-walled bioreactor that imparts hydrodynamic forces for 4weeks. The ability of harvested constructs to integrate with native cartilage was determined using a cartilage explant model. Our results revealed that adhesive stress between native cartilage and the CC constructs was 57% higher than that in the CF group, potentially due to the absence of glycosaminoglycans and increased cell density in the capsule region and deposition of denser and thicker collagen fibrils at the integration site. The present work demonstrates that the fibrous capsule can effectively enhance early integration of engineered and native cartilage tissues and thus suggests the need to include the capsule as a variable in the development of cartilage tissue engineering strategies.

Differential morphology and homogeneity of tissue-engineered cartilage in hydrodynamic cultivation with transient exposure to insulin-like growth factor-1 and transforming growth factor-β1.

Successful tissue-engineering strategies for cartilage repair must maximize the efficacy of chondrocytes within their limited life span. To that end, the combination of exogenous growth factors with mechanical stimuli holds promise for development of clinically relevant cartilage tissue substitutes. The current study aimed to determine whether incorporation of transient exposure to growth factors into a hydrodynamic bioreactor system can improve the functional maturation of tissue-engineered cartilage. Chondrocyte-seeded polyglycolic acid scaffolds were cultivated within a wavy-walled bioreactor that imparts fluid flow-induced shear stress for 4 weeks. Constructs were nourished with 100 ng/mL insulin-like growth factor-1 (IGF-1) or 10 ng/mL transforming growth factor-β1 (TGF-β1) either for the first 15 days of the culture (transient) or throughout the entire cultivation (continuous). Transiently treated constructs were found to exhibit better functional properties than continuously nourished constructs. The limited development of engineered tissues continuously stimulated by IGF-1 or TGF-β1 was related to massive growth factor leftovers in the environments that downregulated the expression of the associated receptors. Treatment with TGF-β1 eliminated the formation of a fibrous capsule at the construct periphery possibly through suppression of Smad3 phosphorylation, yielding constructs with greater homogeneity. Furthermore, TGF-β1 reversely regulated Smad2 and Smad3 pathways in articular chondrocytes under hydrodynamic stimuli partially via Smad7. Collectively, transient exposure to growth factors is likely to maintain chondrocyte homeostasis, and thus promotes their anabolic activities under hydrodynamic stimuli. The present work suggests that robust hydrodynamically engineered neocartilage with a reduced fibrotic response and enhanced tissue homogeneity can be achieved through optimization of growth factor supplementation protocols and potentially through manipulation of intracellular signals such as Smad.

Tissue Growth Modeling in a Wavy-Walled Bioreactor

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.

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

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.

Enhancing cell seeding of scaffolds in tissue engineering through manipulation of hydrodynamic parameters

The seeding of cells onto biocompatible scaffolds is a determinant step in the attainment of functional properties of engineered tissues. Efficient, fast and spatially uniform cell seeding can improve the clinical potential of engineered tissue templates. One way to approach these cell seeding requirements is through bioreactor design. In the present study, bovine chondrocytes were seeded (2.5, 5.0 or 10.0 million cells per scaffold) onto polyglycolic acid scaffolds within the hydrodynamic environments of wavy-walled and spinner flask bioreactors. Previous characterizations of the hydrodynamic environment in the vicinity of constructs cultivated in these bioreactors suggested decreased flow-induced shear stress as well as increased recirculation and magnitude of the axial fluid velocities in the wavy-walled bioreactor. Here we report more efficient and spatially uniform cell seeding in the wavy-walled bioreactor, and at intermediate initial cell densities (5 million cells per scaffold). This study constitutes an important step towards the achievement of functional tissue-engineered implants by (i) increasing our understanding of the influence of hydrodynamic parameters on the efficiency and spatial distribution of cell attachment to scaffolds and the production of extracellular matrix and (ii) introducing a comprehensive approach to the investigation of the effects of bioprocessing conditions on tissue morphology and composition.

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.

Wavy-Walled Bioreactor Supports Increased Cell Proliferation and Matrix Deposition in Engineered Cartilage Constructs

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.

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

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.

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.