A computational and experimental study inside microfluidic systems: the role of shear stress and flow recirculation in cell docking.

In this paper, microfluidic devices containing microwells that enabled cell docking were investigated. We theoretically assessed the effect of geometry on recirculation areas and wall shear stress patterns within microwells and studied the relationship between the computational predictions and experimental cell docking. We used microchannels with 150 microm diameter microwells that had either 20 or 80 microm thickness. Flow within 80 microm deep microwells was subject to extensive recirculation areas and low shear stresses (<0.5 mPa) near the well base; whilst these were only presented within a 10 microm peripheral ring in 20 microm thick microwells. We also experimentally demonstrated that cell docking was significantly higher (p < 0.01) in 80 microm thick microwells as compared to 20 microm thick microwells. Finally, a computational tool which correlated physical and geometrical parameters of microwells with their fluid dynamic environment was developed and was also experimentally confirmed.

Drug release from coronary eluting stents: A multidomain approach.

In this paper, starting from a consistent mathematical model, a novel computational approach is proposed for assessing some biomechanical effects on drug release from coronary drug-eluting stents (DESs), related to tissue properties, local hemodynamics and stent design. A multiscale and multidomain advection-diffusion model is formulated for describing drug dynamics in the polymeric substrate covering the stent, into the arterial wall, and in the vessel lumen. The model accounts for tissue microstructure (anisotropic drug diffusion, porosity, drug retention induced by resident proteins), macrostructure (plaque between stent and tissue), and local hemodynamics. In the case of hydrophobic taxus-based compounds, several numerical analyses have been carried out on simplified geometries by using finite element simulations, performing significant comparisons with other recent studies and highlighting general conclusions for assessing effectiveness of some modelling features as well as useful hints for optimizing drug delivery design and technology.

Potential and bottlenecks of bioreactors in 3D cell culture and tissue manufacturing.

Over the last decade, we have witnessed an increased recognition of the importance of 3D culture models to study various aspects of cell physiology and pathology, as well as to engineer implantable tissues. As compared to well-established 2D cell-culture systems, cell/tissue culture within 3D porous biomaterials has introduced new scientific and technical challenges associated with complex transport phenomena, physical forces, and cell-microenvironment interactions. While bioreactor-based 3D model systems have begun to play a crucial role in addressing fundamental scientific questions, numerous hurdles currently impede the most efficient utilization of these systems. We describe how computational modeling and innovative sensor technologies, in conjunction with well-defined and controlled bioreactor-based 3D culture systems, will be key to gain further insight into cell behavior and the complexity of tissue development. These model systems will lay a solid foundation to further develop, optimize, and effectively streamline the essential bioprocesses to safely and reproducibly produce appropriately scaled tissue grafts for clinical studies.

Engineered cartilage constructs subject to very low regimens of interstitial perfusion.

We have studied an in vitro engineered cartilage model, consisting of bovine articular chondrocytes seeded on micro-porous scaffolds and perfused with very low regimens of interstitial flow. Our previous findings suggested that synthesis of sulphated glycosaminoglycans (sGAG) was promoted in this model, if the level of shear generated on cells was maintained below 10 mPa (0.1 dyn/cm2). Constructs were stimulated with a median shear stress of 1.2 and 6.7 mPa using two independent culture chambers. Quantification of the applied stresses and of oxygen consumption rates was obtained from computational modelling. Experimentally, we set a time zero reference at 24 hours after cell seeding and total culture time at two weeks. The cell metabolic activity, measured by MTT, was significantly lower in all constructs at two weeks (-73% in static controls, -66% in the 1.2 mPa group and -60% in the 6.7 mPa group) vs. the time zero group, and significantly higher (+33%) in the 7 mPa group vs. static controls. The ratio between synthesis of collagen type II/type I, measured by Western Blot, was significantly higher in the 1.2 mPa constructs (+109% vs. the 6.7 mPa group, +120% vs. the time zero group and +286% vs. static controls). A trend of decreased alpha-actin expression was observed with increased ratio of type II to type I collagen, in all groups. These results reinforce the notion that, at early time points in culture, hydrodynamic shear below 10 mPa may promote formation of extra-cellular matrix specific to hyaline cartilage in chondrocyte-seeded constructs.

Microcirculation within grooved substrates regulates cell positioning and cell docking inside microfluidic channels.

Immobilization of cells inside microfluidic devices is a promising approach for enabling studies related to drug screening and cell biology. Despite extensive studies in using grooved substrates for immobilizing cells inside channels, a systematic study of the effects of various parameters that influence cell docking and retention within grooved substrates has not been performed. We demonstrate using computational simulations that the fluid dynamic environment within microgrooves significantly varies with groove width, generating microcirculation areas in smaller microgrooves. Wall shear stress simulation predicted that shear stresses were in the opposite direction in smaller grooves (25 and 50 microm wide) in comparison to those in wider grooves (75 and 100 microm wide). To validate the simulations, cells were seeded within microfluidic devices, where microgrooves of different widths were aligned perpendicularly to the direction of the flow. Experimental results showed that, as predicted, the inversion of the local direction of shear stress within the smaller grooves resulted in alignment of cells on two opposite sides of the grooves under the same flow conditions. Also, the amplitude of shear stress within microgrooved channels significantly influenced cell retainment in the channels. Therefore, our studies suggest that microscale shear stresses greatly influence cellular docking, immobilization, and retention in fluidic systems and should be considered for the design of cell-based microdevices.

An in silico bioreactor for simulating laboratory experiments in tissue engineering.

This paper presents a software framework for the computational modeling of tissue engineering experiments, aimed to supplement and extend the empirical techniques currently employed in tissue engineering. The code included a model of cell population dynamics coupled to a finite element model of oxygen diffusion and consumption at the macroscale level, including the scaffold and the culture medium, and at the level of the scaffold microarchitecture. Cells were modeled as discrete entities moving in a continuum space, under the action of adhesion and repulsion forces. Oxygen distribution was calculated with the transient diffusion equation; oxygen consumption by cells was modeled by using the Michaelis-Menten equation. Other phenomena that can be formulated as a differential problem could be added in a straightforward manner to the code, due to the use of a general purpose finite element library. Two scaffold geometries were considered: a fiber scaffold and a scaffold with interconnected spherical pores. Cells were predicted to form clusters and adhere to the scaffold walls. Although the code demonstrated the ability to provide a robust performance, a calibration of the parameters employed in the model, based on specific laboratory experiments, is now required to verify the reliability of the results.

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.

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.