Numerical simulation of fluid field and in vitro three-dimensional fabrication of tissue-engineered bones in a rotating bioreactor and in vivo implantation for repairing segmental bone defects

In this paper, two-dimensional flow field simulation was conducted to determine shear stresses and velocity profiles for bone tissue engineering in a rotating wall vessel bioreactor (RWVB). In addition, in vitro three-dimensional fabrication of tissue-engineered bones was carried out in optimized bioreactor conditions, and in vivo implantation using fabricated bones was performed for segmental bone defects of Zelanian rabbits. The distribution of dynamic pressure, total pressure, shear stress, and velocity within the culture chamber was calculated for different scaffold locations. According to the simulation results, the dynamic pressure, velocity, and shear stress around the surface of cell-scaffold construction periodically changed at different locations of the RWVB, which could result in periodical stress stimulation for fabricated tissue constructs. However, overall shear stresses were relatively low, and the fluid velocities were uniform in the bioreactor. Our in vitro experiments showed that the number of cells cultured in the RWVB was five times higher than those cultured in a T-flask. The tissue-engineered bones grew very well in the RWVB. This study demonstrates that stress stimulation in an RWVB can be beneficial for cell/bio-derived bone constructs fabricated in an RWVB, with an application for repairing segmental bone defects.     

Modeling evaluation of the fluid-dynamic microenvironment in tissue-engineered constructs: a micro-CT based model

Natural cartilage remodels both in vivo and in vitro in response to mechanical stresses, hence mechanical stimulation is believed to be a potential tool to modulate extra-cellular matrix synthesis in tissue-engineered cartilage. Fluid-induced shear is known to enhance chondrogenesis in engineered cartilage constructs. The quantification of the hydrodynamic environment is a condition required to study the biochemical response to shear of 3D engineered cell systems. We developed a computational model of culture medium flow through the microstructure of a porous scaffold, during direct- perfused culture. The 3D solid model of the scaffold micro-geometry was reconstructed from 250 micro-computed tomography (micro-CT) images. The results of the fluid dynamic simulations were analyzed at the central portions of the fluid domain, to avoid boundary effects. The average, median and mode shear stress values calculated at the scaffold walls were 3.48, 2.90, and 2.45 mPa respectively, at a flow rate of 0.5 cm(3)/min, perfused through a 15 mm diameter scaffold, at an inlet fluid velocity of 53 microm/s. These results were compared to results estimated using a simplified micro-scale model and to results estimated using an analytical macro-scale porous model. The predictions given by the CT-based model are being used in conjunction with an experimental bioreactor model, in order to quantify the effects of fluid-dynamic shear on the growth modulation of tissue-engineered cartilage constructs, to potentially enhance tissue growth in vitro.     

Coupling curvature-dependent and shear stress-stimulated neotissue growth in dynamic bioreactor cultures: a 3D computational model of a complete scaffold

The main challenge in tissue engineering consists in understanding and controlling the growth process of in vitro cultured neotissues toward obtaining functional tissues. Computational models can provide crucial information on appropriate bioreactor and scaffold design but also on the bioprocess environment and culture conditions. In this study, the development of a 3D model using the level set method to capture the growth of a microporous neotissue domain in a dynamic culture environment (perfusion bioreactor) was pursued. In our model, neotissue growth velocity was influenced by scaffold geometry as well as by flow- induced shear stresses. The neotissue was modeled as a homogenous porous medium with a given permeability, and the Brinkman equation was used to calculate the flow profile in both neotissue and void space. Neotissue growth was modeled until the scaffold void volume was filled, thus capturing already established experimental observations, in particular the differences between scaffold filling under different flow regimes. This tool is envisaged as a scaffold shape and bioprocess optimization tool with predictive capacities. It will allow controlling fluid flow during long-term culture, whereby neotissue growth alters flow patterns, in order to provide shear stress profiles and magnitudes across the whole scaffold volume influencing, in turn, the neotissue growth.     

Fluid-induced low shear stress improves cartilage like tissue fabrication by encapsulating chondrocytes

In the recent years, there has been considerable development in the regenerative medicine, which aims to repair, regenerate, and improve injured articular cartilage. The aim of the present study was to investigate the effect of flow-induced shear stress in perfusion bioreactor on alginate encapsulating chondrocytes. The shear stress imposed on the cells in the culture chamber of bioreactor was predicted with computational fluid dynamic. Bovine nasal chondrocytes were isolated and expanded to obtain a pellet. The cell pellet was resuspends in alginate solution, transferred to the culture chamber, and dynamically cultured under direct perfusion. At the end of culture, tissue constructs were examined histologically and by immunohistochemistry. The results of computational fluid dynamic modeling revealed that maximum wall shear stress was 4.820 × 10^?3 Pascal. Macroscopic views of the alginate/chondrocyte beads suggested that it possessed constant shape but were flexible. Under inverted microscope, round shape of chondrocyte observed. Cell distribution was homogeneous throughout the scaffold. Tissue construct subjected to shear showed morphological features, which are characteristic for natural cartilage. Immunohistochemistry results revealed immunopositivity for type II collagens in tissue constructs samples. Flow induced shear stress in the perfusion bioreactor and chnondrocyte encapsulation provide environment to support cell growth, and tissue regeneration and improve cartilage like tissue fabrication.     

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.     

Human mesenchymal stem cell position within scaffolds influences cell fate during dynamic culture

Cell-based tissue engineering is limited by the size of cell-containing constructs that can be successfully cultured in vitro. This limit is largely a result of the slow diffusion of molecules such as oxygen into the interior of three-dimensional scaffolds in static culture. Bioreactor culture has been shown to overcome these limits. In this study we utilize a tubular perfusion system (TPS) bioreactor for the three-dimensional dynamic culture of human mesenchymal stem cells (hMSCs) in spherical alginate bead scaffolds. The goal of this study is to examine the effect of shear stress in the system and then quantify the proliferation and differentiation of hMSCs in different radial annuli of the scaffold. Shear stress was shown to have a temporal effect on hMSC osteoblastic differentiation with a strong correlation of shear stress, osteopontin, and bone morphogenic protein-2 occurring on day 21, and weaker correlation occurring at early timepoints. Further results revealed an approximate 2.5-fold increase in cell number in the inner annulus of TPS cultured constructs as compared to statically cultured constructs after 21 days. This result demonstrated a nutrient transfer limitation in static culture which can be mitigated by dynamic culture. A significant increase (P < 0.05) in mineralization in the inner and outer annuli of bioreactor cultured 4 mm scaffolds occurred on day 21 with 79 ± 29% and 53 ± 25% mineralization area, respectively, compared to 6 ± 4% and 19 ± 6% mineralization area, respectively, in inner and outer annuli of 4 mm statically cultured scaffolds. Surprising lower mineralization area was observed in 2 mm bioreactor cultured beads which had the highest levels of proliferation. These results may demonstrate a relationship between scaffold position and stem cell fate. In addition the decreased proliferation and matrix production in statically cultured scaffolds compared to bioreactor cultured constructs demonstrate the need for bioreactor systems and the effectiveness of the TPS bioreactor in promoting hMSC proliferation and differentiation in three-dimensional scaffolds.     

Three-dimensional cell seeding and growth in radial-flow perfusion bioreactor for in vitro tissue reconstruction

Radial-flow perfusion bioreactor systems have been designed and evaluated to enable direct cell seeding into a three-dimensional (3-D) porous scaffold and subsequent cell culture for in vitro tissue reconstruction. However, one of the limitations of in vitro regeneration is the tissue necrosis that occurs at the central part of the 3-D scaffold. In the present study, tubular poly-L-lactic acid (PLLA) porous scaffolds with an optimized pore size and porosity were prepared by the lyophilization method, and the effect of different perfusion conditions on cell seeding and growth were compared with those of the conventional static culture. The medium flowed radially from the lumen toward the periphery of the tubular scaffolds. It was found that cell seeding under a radial-flow perfusion condition of 1.1 mL/cm2 x min was effective, and that the optimal flow rate for cell growth was 4.0 mL/cm2 x min. At this optimal rate, the increase in seeded cells in the perfusion culture over a period of 5 days was 7.3-fold greater than that by static culture over the same period. The perfusion cell seeding resulted in a uniform distribution of cells throughout the scaffold. Subsequently, the perfusion of medium and hence the provision of nutrients and oxygen permitted growth and maintenance of the tissue throughout the scaffold. The perfusion seeding/culture system was a much more effective strategy than the conventional system in which cells are seeded under a static condition and cultured in a bioreactor such as a spinner flask.     

Osteochondral tissue coculture: An in vitro and in silico approach

Osteochondral tissue engineering aims to regenerate functional tissue-mimicking physiological properties of injured cartilage and its subchondral bone. Given the distinct structural and biochemical difference between bone and cartilage, bilayered scaffolds, and bioreactors are commonly employed. We present an osteochondral culture system which cocultured ATDC5 and MC3T3-E1 cells on an additive manufactured bilayered scaffold in a dual-chamber perfusion bioreactor. Also, finite element models (FEM) based on the microcomputed tomography image of the manufactured scaffold as well as on the computer-aided design (CAD) were constructed; the microenvironment inside the two FEM was studied and compared. In vitro results showed that the coculture system supported osteochondral tissue growth in terms of cell viability, proliferation, distribution, and attachment. In silico results showed that the CAD and the actual manufactured scaffold had significant differences in the flow velocity, differentiation media mixing in the bioreactor and fluid-induced shear stress experienced by the cells. This system was shown to have the desired microenvironment for osteochondral tissue engineering and it can potentially be used as an inexpensive tool for testing newly developed pharmaceutical products for osteochondral defects.     

Computational fluid dynamics modeling of steady-state momentum and mass transport in a bioreactor for cartilage tissue engineering

Computational fluid dynamics (CFD) models to quantify momentum and mass transport under conditions of tissue growth will aid bioreactor design for development of tissue-engineered cartilage constructs. Fluent CFD models are used to calculate flow fields, shear stresses, and oxygen profiles around nonporous constructs simulating cartilage development in our concentric cylinder bioreactor. The shear stress distribution ranges from 1.5 to 12 dyn/cm(2) across the construct surfaces exposed to fluid flow and varies little with the relative number or placement of constructs in the bioreactor. Approximately 80% of the construct surface exposed to flow experiences shear stresses between 1.5 and 4 dyn/cm(2), validating the assumption that the concentric cylinder bioreactor provides a relatively homogeneous hydrodynamic environment for construct growth. Species mass transport modeling for oxygen demonstrates that fluid-phase oxygen transport to constructs is uniform. Some O(2) depletion near the down stream edge of constructs is noted with minimum pO(2) values near the constructs of 35 mmHg (23% O(2) saturation). These values are above oxygen concentrations in cartilage in vivo, suggesting that bioreactor oxygen concentrations likely do not affect chondrocyte growth. Scale-up studies demonstrate the utility and flexibility of CFD models to design and characterize bioreactors for growth of tissue-engineered cartilage.     

A dynamical study of the mechanical stimuli and tissue differentiation within a CaP scaffold based on micro-CT finite element models

The control of the mechanical stimuli transmitted to the cells is critical for the design of functional scaffolds for tissue engineering. The objective of this study was to investigate the dynamics of the mechanical stimuli transmitted to the cells during tissue differentiation in an irregular morphology scaffold under compressive load and perfusion flow. A calcium phosphate-based glass porous scaffold was used. The solid phase and the fluid flow within the pores were modeled as linear elastic solid material and Newtonian fluid, respectively. In the fluid model, different levels of viscosity were used to simulate tissue differentiation. Compressive strain of 0.5% and fluid flow with constant inlet velocity of 10 μm/s or constant inlet pressure of 3 Pa were applied. Octahedral shear strain and fluid shear stress were used as mechano-regulatory stimuli. For constant inlet velocity, stimuli equivalent to bone were predicted in 80% of pore volume for the case of low tissue viscosity. For the cases of high viscosity, fluctuations between stimuli equivalent to tissue formation and cell death were predicted due to the increase in the fluid shear stress when tissue started to fill pores. When constant pressure was applied, stimuli equivalent to bone were predicted in 62% of pore volume when low tissue viscosity was used and 42% when high tissue viscosity was used. This study predicted critical variations of fluid shear stress when cells differentiated. If these variations are not controlled in vitro, they can impede the formation of new matured tissue.     

The effect of hydrodynamic shear on 3D engineered chondrocyte systems subject to direct perfusion

Bioreactors allowing direct-perfusion of culture medium through tissue-engineered constructs may overcome diffusion limitations associated with static culturing, and may provide flow-mediated mechanical stimuli. The hydrodynamic stress imposed on cells within scaffolds is directly dependent on scaffold microstructure and on bioreactor configuration. Aim of this study is to investigate optimal shear stress ranges and to quantitatively predict the levels of hydrodynamic shear imposed to cells during the experiments. Bovine articular chondrocytes were seeded on polyestherurethane foams and cultured for 2 weeks in a direct perfusion bioreactor designed to impose 4 different values of shear level at a single flow rate (0.5 ml/min). Computational fluid dynamics (CFD) simulations were carried out on reconstructions of the scaffold obtained from micro-computed tomography images. Biochemistry analyses for DNA and sGAG were performed, along with electron microscopy. The hydrodynamic shear induced on cells within constructs, as estimated by CFD simulations, ranged from 4.6 to 56 mPa. This 12-fold increase in the level of applied shear stress determined a 1.7-fold increase in the mean content in DNA and a 2.9-fold increase in the mean content in sGAG. In contrast, the mean sGAG/DNA ratio showed a tendency to decrease for increasing shear levels. Our results suggest that the optimal condition to favour sGAG synthesis in engineered constructs, at least at the beginning of culture, is direct perfusion at the lowest level of hydrodynamic shear. In conclusion, the presented results represent a first attempt to quantitatively correlate the imposed hydrodynamic shear level and the invoked biosynthetic response in 3D engineered chondrocyte systems.     

Computational evaluation of oxygen and shear stress distributions in 3D perfusion culture systems: macro-scale and micro-structured models

We present a combined macro-scale/micro-scale computational approach to quantify oxygen transport and flow-mediated shear stress to human chondrocytes cultured in three-dimensional scaffolds in a perfusion bioreactor system. A macro-scale model was developed to assess the influence of the bioreactor design and to identify the proper boundary conditions for the micro-scale model. The micro-scale model based on a micro-computed tomography (microCT) reconstruction of a poly(ethylene glycol terephthalate)/poly(butylene terephthalate) (PEGT/PBT) foam scaffold, was developed to assess the influence of the scaffold micro-architecture on local shear stress and oxygen levels within the scaffold pores. Experiments were performed to derive specific oxygen consumption rates for constructs perfused under flow rates of 0.3 and 0.03 ml min(-1). While macro-scale and micro-scale models predicted similar average oxygen levels at different depths within the scaffold, microCT models revealed small local oxygen variations within the scaffold micro-architecture. The combined macro-scale/micro-scale approach indicated that 0.3 ml min(-1), which subjected 95% of the cells to less than 6.3 mPa shear, would maintain the oxygen supply throughout the scaffold above anoxic levels (>1%), with 99.5% of the scaffold supplied with 8-2% O(2). Alternatively, at 0.03 ml min(-1), the macro-scale model predicted 6% of the cells would be supplied with 0.5-1% O(2), although this region of cells was confined to the periphery of the scaffold. Together with local variations predicted by the micro-scale model, the simulations underline that in the current model system, reducing the flow below 0.03 ml min(-1) would likely have dire consequences on cell viability to pronounced regions within the engineered construct. The presented approach provides a sensitive tool to aid efficient bioreactor optimization and scaffold design.     

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.     

Enhancement of cell growth in tissue-engineering constructs under direct perfusion: Modeling and simulation

Perfusion bioreactors improve mass transfer in cell-scaffold constructs. We developed a mathematical model to simulate nutrient flow through cellular constructs. Interactions among cell proliferation, nutrient consumption, and culture medium circulation were investigated. The model incorporated modified Contois cell-growth kinetics that includes effects of nutrient saturation and limited cell growth. Nutrient uptake was depicted through the Michaelis-Menton kinetics. To describe the culture medium convection, the fluid flow outside the cell-scaffold construct was described by the Navier-Stokes equations, while the fluid dynamics within the construct was modeled by Brinkman's equation for porous media flow. Effects of the media perfusion were examined by including time-dependant porosity and permeability changes due to cell growth. The overall cell volume was considered to consist of cells and extracellular matrices (ECM) as a whole without treating ECM separately. Numerical simulations show when cells were cultured subjected to direct perfusion, they penetrated to a greater extent into the scaffold and resulted in a more uniform spatial distribution. The cell amount was increased by perfusion and ultimately approached an asymptotic value as the perfusion rates increased in terms of the dimensionless Peclet number that accounts for the ratio of nutrient perfusion to diffusion. In addition to enhancing the nutrient delivery, perfusion simultaneously imposes flow-mediated shear stress to the engineered cells. Shear stresses were found to increase with cell growth as the scaffold void space was occupied by the cell and ECM volumes. The macro average stresses increased from 0.2 mPa to 1 mPa at a perfusion rate of 20 microm/s with the overall cell volume fraction growing from 0.4 to 0.7, which made the overall permeability value decrease from 1.35 x 10(-2)cm(2) to 5.51 x 10(-4)cm(2). Relating the simulation results with perfusion experiments in literature, the average shear stresses were below the critical value that would induce the chondrocyte necrosis.     

Computational modeling of flow-induced shear stresses within 3D salt-leached porous scaffolds imaged via micro-CT

Flow-induced shear stresses have been found to be a stimulatory factor in pre-osteoblastic cells seeded in 3D porous scaffolds and cultured under continuous flow perfusion. However, due to the complex internal structure of porous scaffolds, analytical estimation of the local shear forces is impractical. The primary goal of this work is to investigate the shear stress distributions within Poly(l-lactic acid) scaffolds via computation. Scaffolds used in this study are prepared via salt leeching with various geometric characteristics (80–95% porosity and 215–402.5 μm average pore size). High resolution micro-computed tomography is used to obtain their 3D structure. Flow of osteogenic media through the scaffolds is modeled via lattice Boltzmann method. It is found that the surface stress distributions within the scaffolds are characterized by long tails to the right (a positive skewness). Their shape is not strongly dependent on the scaffold manufacturing parameters, but the magnitudes of the stresses are. Correlations are prepared for the estimation of the average surface shear stress experienced by the cells within the scaffolds and of the probability density function of the surface stresses. Though the manufacturing technique does not appear to affect the shape of the shear stress distributions, presence of manufacturing defects is found to be significant: defects create areas of high flow and high stress along their periphery. The results of this study are applicable to other polymer systems provided that they are manufactured by a similar salt leeching technique, while the imaging/modeling approach is applicable to all scaffolds relevant to tissue engineering.     

Prediction of the micro-fluid dynamic environment imposed to three-dimensional engineered cell systems in bioreactors

Bioreactors allowing culture medium perfusion overcome diffusion limitations associated with static culturing and provide flow-mediated mechanical stimuli. The hydrodynamic stress imposed to cells will depend not only on the culture medium flow rate, but also on the scaffold three-dimensional (3D) micro-architecture. We developed a CFD model of the flow of culture medium through a 3D scaffold of homogeneous geometry, with the aim of predicting the shear stress acting on cells as a function of parameters that can be controlled during the scaffold fabrication process, such as the scaffold porosity and the pore size, and during the cell culture, such as the medium flow rate and the diameter of the perfused scaffold section. We built three groups of models corresponding to three pore sizes: 50, 100 and 150 microm. Each group was made of four models corresponding to 59%, 65%, 77%, and 89% porosity. A commercial finite-element code was used to set up and solve the problem and to analyze the results. The mode value of shear stress varied between 2 and 5 mPa, and was obtained for a circular scaffold of 15.5 mm diameter, perfused by a flow rate of 0.5 ml/min. The simulations showed that the pore size is a variable strongly influencing the predicted shear stress level, whereas the porosity is a variable strongly affecting the statistical distribution of the shear stresses, but not their magnitude. Our results provide a basis for the completion of more exhaustive quantitative studies to further assess the relationship between perfusion, at known micro-fluid dynamic conditions, and tissue growth in vitro.     

Modeling and design of optimal flow perfusion bioreactors for tissue engineering applications

Perfusion bioreactors have been used in different tissue engineering applications because of their consistent distribution of nutrients and flow-induced shear stress within the tissue-engineering scaffold. A widely used configuration uses a scaffold with a circular cross-section enclosed within a cylindrical chamber and inlet and outlet pipes which are connected to the chamber on either side through which media is continuously circulated. However, fluid-flow experiments and simulations have shown that the majority of the flow perfuses through the center. This pattern creates stagnant zones in the peripheral regions as well as in those of high flow rate near the inlet and outlet. This non-uniformity of flow and shear stress, owing to a circular design, results in limited cell proliferation and differentiation in these areas. The focus of this communication is to design an optimized perfusion system using computational fluid dynamics as a mathematical tool to overcome the time-consuming trial and error experimental method. We compared the flow within a circular and a rectangular bioreactor system. Flow simulations within the rectangular bioreactor are shown to overcome the limitations in the circular design. This communication challenges the circular cross-section bioreactor configuration paradigm and provides proof of the advantages of the new design over the existing one.     

Flow rates in perfusion bioreactors to maximise mineralisation in bone tissue engineering in vitro

In bone tissue engineering experiments, fluid-induced shear stress is able to stimulate cells to produce mineralised extracellular matrix (ECM). The application of shear stress on seeded cells can for example be achieved through bioreactors that perfuse medium through porous scaffolds. The generated mechanical environment (i.e. wall shear stress: WSS) within the scaffolds is complex due to the complexity of scaffold geometry. This complexity has so far prevented setting an optimal loading (i.e. flow rate) of the bioreactor to achieve an optimal distribution of WSS for stimulating cells to produce mineralised ECM. In this study, we demonstrate an approach combining computational fluid dynamics (CFD) and mechano-regulation theory to optimise flow rates of a perfusion bioreactor and various scaffold geometries (i.e. pore shape, porosity and pore diameter) in order to maximise shear stress induced mineralisation. The optimal flow rates, under which the highest fraction of scaffold surface area is subjected to a wall shear stress that induces mineralisation, are mainly dependent on the scaffold geometries. Nevertheless, the variation range of such optimal flow rates are within 0.5–5 mL/min (or in terms of fluid velocity: 0.166–1.66 mm/s), among different scaffolds. This approach can facilitate the determination of scaffold-dependent flow rates for bone tissue engineering experiments in vitro, avoiding performing a series of trial and error experiments.     

Influence of flow rate and scaffold pore size on cell behavior during mechanical stimulation in a flow perfusion bioreactor

Mechanically stimulating cell-seeded scaffolds by flow-perfusion is one approach utilized for developing clinically applicable bone graft substitutes. A key challenge is determining the magnitude of stimuli to apply that enhances cell differentiation but minimizes cell detachment from the scaffold. In this study, we employed a combined computational modeling and experimental approach to examine how the scaffold mean pore size influences cell attachment morphology and subsequently impacts upon cell deformation and detachment when subjected to fluid-flow. Cell detachment from osteoblast-seeded collagen-GAG scaffolds was evaluated experimentally across a range of scaffold pore sizes subjected to different flow rates and exposure times in a perfusion bioreactor. Cell detachment was found to be proportional to flow rate and inversely proportional to pore size. Using this data, a theoretical model was derived that accurately predicted cell detachment as a function of mean shear stress, mean pore size, and time. Computational modeling of cell deformation in response to fluid flow showed the percentage of cells exceeding a critical threshold of deformation correlated with cell detachment experimentally and the majority of these cells were of a bridging morphology (cells stretched across pores). These findings will help researchers optimize the mean pore size of scaffolds and perfusion bioreactor operating conditions to manage cell detachment when mechanically simulating cells via flow perfusion.     

Regulation of autocrine fibroblast growth factor‐2 signaling by perfusion flow in 3D human mesenchymal stem cell constructs

Perfusion bioreactor systems play a crucial role in mitigating nutrient limitation as well as providing biomechanical stimuli and redistributing regulatory macromolecules that influence human mesenchymal stem cells (hMSC) fate in three‐dimensional (3D) scaffolds. As fibroblast growth factor‐2 (FGF‐2) is known to regulate hMSC phenotype, understanding the role of autocrine FGF‐2 signaling in the 3D construct under the different perfusion flow provides important insight into an optimal bioreactor design. To investigate FGF‐2 signaling inhibition in hMSC cultured in the porous poly(ethylene terephthalate) (PET) scaffolds perfused under two flow configurations, PD173074, an FGFR1 inhibitor, was added in growth media after 7 day of pre‐culture and its impact on hMSC proliferation and clonogenicity during the subsequent 7 days of cultivation was analyzed. Compared with control constructs in growth media, the addition of PD173074 resulted in significant reduction in hMSC proliferation and colony formation in both constructs with a more dramatic reduction in the parallel flow constructs. The results demonstrate that autocrine FGF‐2 plays a significant role in 3D scaffold and suggest modulation of the perfusion flow in the bioreactor as a strategy to influence autocrine actions and cell fate in the 3D scaffold. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012