Bioreactors For Tissue Engineering Research Articles

A novel all-in-one bioreactor for vascular tissue engineering

Aims: Multiple efforts were made to develop small-diameter tissue engineered vascular grafts by using different bioreactor systems. However, there is still an extensive need for a compact all-in-one system that allows multiple and simultaneous processing. The aim of this project was to develop a new device to fulfill the major requirements of an ideal bioreactor.

Bone Tissue Engineering Bioreactors: A Role in the Clinic?

Tissue engineered bone grafts have the potential to be used to treat large bone defects due to congenital abnormalities, cancer resections, or traumatic incidents. Recent studies have shown that perfusion bioreactors can be used to generate grafts of clinically relevant sizes and shapes. Despite these scientific and technological successes, there is uncertainty regarding the translational utility of bioreactor-based approaches due to the perceived high costs associated with these procedures. In fact, experiences over the past two decades have demonstrated that the widespread application of cell-based therapies is heavily dependent on the commercial viability. In this article, we directly address the question of whether bioreactors used to create bone grafts have the potential to be implemented in clinical approaches to bone repair and regeneration. We provide a brief review of tissue engineering approaches to bone repair, clinical trials that have employed cell-based methods, and advances in bioreactor technologies over the past two decades. These analyses are combined to provide a perspective on what is missing from the scientific literature that would enable an objective baseline for weighing the benefit of extended in vitro cultivation of cells into functional bone grafts against the cost of additional cultivation. In our estimation, the cost of bioreactor-based bone grafts may range from $10,000 to $15,000, placing it within the range of other widely used cell-based therapies. Therefore, in situations where a clear advantage can be established for engineered grafts comprising patient-specific, autologous cells, engineered bone grafts may be a clinically feasible option.

Hydrodynamics and bioprocess considerations in designing bioreactors for cardiac tissue engineering

Introduction Cardiovascular disease (CVD) is a leading cause of mortality in most developed countries accounting for 30% of all deaths worldwide in 2008 [1]. Current trends in obesity and the lack of a healthy lifestyle will likely lead to an increase in total population suffering from heart diseases, causing associated healthcare

The Importance of Bicarbonate and Nonbicarbonate Buffer Systems in Batch and Continuous Flow Bioreactors for Articular Cartilage Tissue Engineering

In cartilage tissue engineering an optimized culture system, maintaining an appropriate extracellular environment (e.g., pH of media), can increase cell proliferation and extracellular matrix (ECM) accumulation. We have previously reported on a continuous-flow bioreactor that improves tissue growth by supplying the cells with a near infinite supply of medium. Previous studies have observed that acidic environments reduce ECM synthesis and chondrocyte proliferation. Hence, in this study we investigated the combined effects of a continuous culture system (bioreactor) together with additional buffering agents (e.g., sodium bicarbonate [NaHCO₃]) on cartilaginous tissue growth in vitro. Isolated bovine chondrocytes were grown in three-dimensional cultures, either in static conditions or in a continuous-flow bioreactor, in media with or without NaHCO₃. Tissue constructs cultivated in the bioreactor with NaHCO₃-supplemented media were characterized with significantly increased (p<0.05) ECM accumulation (glycosaminoglycans a 98-fold increase; collagen a 25-fold increase) and a 13-fold increase in cell proliferation, in comparison with static cultures. Additionally, constructs grown in the bioreactor with NaHCO₃-supplemented media were significantly thicker than all other constructs (p<0.05). Further, the chondrocytes from the primary construct expanded and synthesized ECM, forming a secondary construct without a separate expansion phase, with a diameter and thickness of 4 mm and 0.72 mm respectively. Tissue outgrowth was negligible in all other culturing conditions. Thus this study demonstrates the advantage of employing a continuous flow bioreactor coupled with NaHCO₃ supplemented media for articular cartilage tissue engineering.

Design of Bioreactor Control System for Ligament Tissue Engineering

An electromechanical control system of a novel bioreactor for ligament tissue engineering, combined with culture medium perfusion control, cyclic mechanical loading and displacement coordinated control, strains and temperature measure and control, based on programmable logical controller (PLC), was designed and implemented. Well-controlled mechanical stimulations (resolution of &lt;0.01mm for translational and &lt;0.1° for rotational strains, cyclic loading frequency of up to 1 Hz) could be applied to the growing tissue, especially to the tissue engineered anterior cruciate ligaments (ACLs). The novel control system could complete detection and control functions of multi-dimensional mechanical strain in the same axis (resolution of 0.01N for tension and 0.01Nm for torsion strains). An online parameter measuring device of PO2 ，which did not consume oxygen and independent of flow rate, and were incorporated into the culture medium recirculation loop, was designed. The displacement and mechanical stimulate coordinated control strategies were achieved. The aim of this control system is to meet the functions of bioreactor for the ACLs tissue engineering in both research and clinical applications.

The Role of Bioreactors in Tissue Engineering for Musculoskeletal Applications

Tissue engineering involves using the principles of biology, chemistry and engineering to design a ‘neotissue’ that augments a malfunctioning in vivo tissue. The main requirements for functional engineered tissue include reparative cellular components that proliferate on a biocompatible scaffold grown within a bioreactor that provides specific biochemical and physical signals to regulate cell differentiation and tissue assembly. We discuss the role of bioreactors in tissue engineering and evaluate the principles of bioreactor design. We evaluate the methods of cell stimulation and review the bioreactors in common use today.

A positron emission tomography approach to visualize flow perfusion in hollow-fiber membrane bioreactors

Despite the success of hollow-fiber membrane bioreactors in tissue engineering, few evaluations of steady- and pulsatile-flow perfusion through these bioreactors have been made. Such evaluations are vital to the optimization of bioreactor culture conditions. In this study, positron emission tomography (PET) was proposed and used to visualize steady- and pulsatile-flow perfusion in hollow-fiber membrane bioreactors for tissue-engineering applications. PET is a noninvasive method that allows measuring the spatial distribution of a radioactive tracer by detecting its activity within porous scaffolds. A radioactive tracer, 18-fluoro-deoxy-glucose ((18)FDG), was injected into a fluid circuit having a hollow-fiber membrane bioreactor with gel-devoid or gel-filled extracapillary space. Dynamic PET scans of the inlet section were acquired and followed by volumetric PET scans of the whole bioreactor. Results were used to reconstruct dynamic and volumetric two- and three-dimensional images. Pulsatile inlet flow improved the uniformity of perfusion flow within the bioreactor in comparison to the steady inlet flow. Pulsatile flow also reduced the accumulation of radioactive tracer for both gel-devoid and gel-filled bioreactors compared to the steady flow. The stability of the radioactive tracer for both conditions was evaluated. The potential of the PET approach was demonstrated by the quantification of the imaging results for steady- and pulsatile-flow perfusions that can be used for the development of bioreactors for tissue-engineering applications.

Mesenchymal stem cell culture in convection-enhanced hollow fibre membrane bioreactors for bone tissue engineering

Preparation of tissue engineered bone constructs to repair large size defects is limited by the difficult supply of oxygen and nutrients to cells deep inside the constructs. Hollow fibre membrane bioreactors (HFMBs) with medium flowing in membrane lumen have structural and functional analogy with cortical bone. In fact, membranes resemble the Haversian canals and provide for a distributed and delocalized source of nutrients and oxygen to surrounding cells. HFMBs where oxygen and nutrients diffuse to cells have been proposed for bone tissue engineering. HFMBs may also be so operated as to promote spontaneous Starling flows to enhance nutrients transfer to cells. In this study, the effect of low and high Starling flows was investigated on sheep mesenchymal stem cells (shMSC) cultured in the extracapillary space of HFMBs with respect to cell distribution, proliferation and early differentiation. After 12 days of culture, at low Starling flows cells formed thin layers around the membranes uniformly along the bioreactor. At high Starling flows, cells proliferated well and formed thick multilayer aggregates filling the space among membranes at the bioreactor outlet. These results make it possible to postulate the use of HFMBs for obtaining tissue engineered constructs for the repair of large bone defects.

Flow dynamics within a bioreactor for tissue engineering by residence time distribution analysis combined with fluorescence and magnetic resonance imaging to investigate forced permeability and apparent diffusion coefficient in a perfusion cell culture ch

This study reveals that residence time distribution (RTD) analysis with pH monitoring after acid bolus injection can be used to globally study the flow dynamics of a perfusion bioreactor, while fluorescence microscopy and magnetic resonance imaging (MRI) were used to locally investigate mass transport within a hydrogel scaffold seeded or not with cells. The bioreactor used in this study is a close-loop tubular reactor. A dispersion model in one dimension has been used to describe the non-ideal behavior of the reactor. From open-loop experiments (single-cycle analysis), the presence of stagnant zones and back mixing were observed. The impact of the flow rate, the compliance chamber volume and mixing were investigated. Intermediate flows (30, 45, 60, and 90 mL min(-1)) had no effect over RTD function expressed in reduced time (θ). Lower flow rates (5 and 15 mL min(-1)) were associated to smaller extent of dispersion. The compliance chamber volume greatly affected the dynamics of the RTD function, while the effects of mixing and flow were small to non-significant. An empirical equation has been proposed to localize minima of the RTD function and to predict Per . Finally, cells seeded in a gelatin gel at a density of 800,000 cells mL(-1) had no effect over the permeability and the apparent diffusion coefficient, as revealed by fluorescent microscopy and MRI experiments.

Bioreactors for Bone Tissue Engineering

Bone tissue engineering is a promising solution for patients with bone defects that require reconstruction. This regenerative therapy consists in culturing osteogenic cells on a biodegradable substrate to obtain a bio-hybrid construct that will stimulate bone healing after implantation. This multidisciplinary technology nevertheless requires further development before it can become routine clinical practice. One challenge is to achieve three-dimensional seeding and osteogenic commitment of mesenchymal stem cells on biomaterials under sterile and reproducible conditions. For this purpose, different dynamic culture systems have been developed. This paper reviews recent advances in the field of bioreactors for bone tissue engineering. The purpose of such systems is to improve nutrient delivery to the cells and generate shear stress that may promote cell differentiation into osteoblastic phenotypes. A brief overview of the value of computational fluid dynamics for understanding the cell environment is also provided. Finally, some proposals are made regarding the use of bioreactors as safe and controllable devices that will help commit cells and biomaterials for the regeneration of bone tissue.

Optimizing the medium perfusion rate in bone tissue engineering bioreactors

There is a critical need to increase the size of bone grafts that can be cultured in vitro for use in regenerative medicine. Perfusion bioreactors have been used to improve the nutrient and gas transfer capabilities and reduce the size limitations inherent to static culture, as well as to modulate cellular responses by hydrodynamic shear. Our aim was to understand the effects of medium flow velocity on cellular phenotype and the formation of bone-like tissues in three-dimensional engineered constructs. We utilized custom-designed perfusion bioreactors to culture bone constructs for 5 weeks using a wide range of superficial flow velocities (80, 400, 800, 1,200, and 1,800 µm/s), corresponding to estimated initial shear stresses ranging from 0.6 to 20 mPa. Increasing the flow velocity significantly affected cell morphology, cell-cell interactions, matrix production and composition, and the expression of osteogenic genes. Within the range studied, the flow velocities ranging from 400 to 800 µm/s yielded the best overall osteogenic responses. Using mathematical models, we determined that even at the lowest flow velocity (80 µm/s) the oxygen provided was sufficient to maintain viability of the cells within the construct. Yet it was clear that this flow velocity did not adequately support the development of bone-like tissue. The complexity of the cellular responses found at different flow velocities underscores the need to use a range of evaluation parameters to determine the quality of engineered bone.

The effects of vortex breakdown bubbles on the mixing environment inside a base driven bioreactor

The bubble-type vortex breakdown inside a cylinder with flow driven by rotation of the base, has applications in mixing. We investigate this phenomena and its effect on the environment inside an open cylinder, with potential application as a tissue-engineering bioreactor, with tissue-scaffolds of two different geometries immersed in the fluid. Addition of scaffolds induces a blockage effect, hindering the flow in the central vortex core returning to the rotating base. This promotes early onset of vortex breakdown and alters the final shape of vortex breakdown bubbles. Placement of the scaffolds centrally on the cylinder axis yields almost identical levels and distributions of shear stress between the upper and lower surfaces of scaffolds. A change from a disk shaped to an ellipsoidal scaffold, of the same size, reduces the intensity of the maximum shear stresses at the scaffold surface by up to 50%. There is a range of Reynolds numbers where increasing Reynolds number, and hence possibly increasing mixing efficiency, leads to a decrease in the maximum levels of fluid forces at the scaffold surfaces. This is an important conclusion for scaffold based tissue engineering where improved mixing is sought, but often sacrificed in favor of minimizing fluid forces.

Computational fluid dynamics modeling of momentum transport in rotating wall perfused bioreactor for cartilage tissue engineering

In this study, computational fluid dynamics (CFD) analysis of a rotating-wall perfused-vessel (RWPV) bioreactor is performed to characterize the complex hydrodynamic environment for the simulation of cartilage development in RWPV bioreactor in the presence of tissue-engineered cartilage constructs, i.e., cell-chitosan scaffolds. Shear stress exerted on chitosan scaffolds in bioreactor was calculated for different rotational velocities in the range of 33–38 rpm. According to the calculations, the lateral and lower surfaces were exposed to 0.07926–0.11069 dyne/cm 2 and 0.05974–0.08345 dyne/cm 2, respectively, while upper surfaces of constructs were exposed to 0.09196–0.12847 dyne/cm 2. Results validate adequate hydrodynamic environment for scaffolds in RWPV bioreactor for cartilage tissue development which concludes the suitability of operational conditions of RWPV bioreactor.

Bioreactors in Tissue Engineering

AbstractTissue engineering is a promising interdisciplinary scientific field of regenerative medicine. Aiming at the structural and functional restoration of damaged tissues and organs, it possesses a role of significant socioeconomical impact. In the course towards the ultimate goal of artificially constructed natural organs, our knowledge of the elementary constitutive components of living organisms and the intrinsic mechanisms that drive their interactions is greatly enhanced. Bioreactors are valuable tools providing the technological means to investigate fundamental issues for basic research and to improve tissue‐engineering products for clinical applications. They are devices enabling the in vitro simulation of the in vivo biological, physical and mechanical environment of growing tissues. In this review paper, we discuss the general demands defining the design considerations for modern bioreactor systems. These criteria originate from physiological characteristics of the cells and biochemical and mechanical properties of the extracellular matrix (ECM). In this context, we present an overview of the various bioreactor systems dedicated to the study of specific functional tissues developed by numerous research groups.

Bioactive polymer/extracellular matrix scaffolds fabricated with a flow perfusion bioreactor for cartilage tissue engineering

In this study, electrospun poly(ɛ-caprolactone) (PCL) microfiber scaffolds, coated with cartilaginous extracellular matrix (ECM), were fabricated by first culturing chondrocytes under dynamic conditions in a flow perfusion bioreactor and then decellularizing the cellular constructs. The decellularization procedure yielded acellular PCL/ECM composite scaffolds containing glycosaminoglycan and collagen. PCL/ECM composite scaffolds were evaluated for their ability to support the chondrogenic differentiation of mesenchymal stem cells (MSCs) in vitro using serum-free medium with or without the addition of transforming growth factor-β1 (TGF-β1). PCL/ECM composite scaffolds supported chondrogenic differentiation induced by TGF-β1 exposure, as evidenced in the up-regulation of aggrecan (11.6 ± 3.8 fold) and collagen type II (668.4 ± 317.7 fold) gene expression. The presence of cartilaginous matrix alone reduced collagen type I gene expression to levels observed with TGF-β1 treatment. Cartilaginous matrix further enhanced the effects of growth factor treatment on MSC chondrogenesis as evidenced in the higher glycosaminoglycan synthetic activity for cells cultured on PCL/ECM composite scaffolds. Therefore, flow perfusion culture of chondrocytes on electrospun microfiber scaffolds is a promising method to fabricate polymer/extracellular matrix composite scaffolds that incorporate both natural and synthetic components to provide biological signals for cartilage tissue engineering applications.

Aligned poly(L‐lactic‐co‐e‐caprolactone) electrospun microfibers and knitted structure: A novel composite scaffold for ligament tissue engineering

We developed a novel technique involving knitting and electrospinning to fabricate a composite scaffold for ligament tissue engineering. Knitted structures were coated with poly(L-lactic-co-e-caprolactone) (PLCL) and then placed onto a rotating cylinder and a PLCL solution was electrospun onto the structure. Highly aligned 2-microm-diameter microfibers covered the space between the stitches and adhered to the knitted scaffolds. The stress-strain tensile curves exhibited an initial toe region similar to the tensile behavior of ligaments. Composite scaffolds had an elastic modulus (150 +/- 14 MPa) similar to the modulus of human ligaments. Biological evaluation showed that cells proliferated on the composite scaffolds and they spontaneously orientated along the direction of microfiber alignment. The microfiber architecture also induced a high level of extracellular matrix secretion, which was characterized by immunostaining. We found that cells produced collagen type I and type III, two main components found in ligaments. After 14 days of culture, collagen type III started to form a fibrous network. We fabricated a composite scaffold having the mechanical properties of the knitted structure and the morphological properties of the aligned microfibers. It is difficult to seed a highly macroporous structure with cells, however the technique we developed enabled an easy cell seeding due to presence of the microfiber layer. Therefore, these scaffolds presented attractive properties for a future use in bioreactors for ligament tissue engineering.

Bioreactors for bone tissue engineering

Engineering bone tissue for use in orthopaedics poses multiple challenges. Providing the appropriate growth environment that will allow complex tissues such as bone to grow is one of these challenges. There are multiple design factors that must be considered in order to generate a functional tissue in vitro for replacement surgery in the clinic. Complex bioreactors have been designed that allow different stress regimes such as compressive, shear, and rotational forces to be applied to three-dimensional (3D) engineered constructs. This review addresses these considerations and outlines the types of bioreactor that have been developed and are currently in use.

Simple Modular Bioreactors for Tissue Engineering: A System for Characterization of Oxygen Gradients, Human Mesenchymal Stem Cell Differentiation, and Prevascularization

Large-scale tissue engineering is limited by nutrient perfusion and mass transport limitations, especially oxygen diffusion, which restrict construct development to smaller than clinically relevant dimensions and limit the ability for in vivo integration. The goal of this work was to develop a modular approach to tissue engineering, where scaffold and tissue size, transport issues, and surgical implantation in vivo are considered from the outset. Human mesenchymal stem cells (hMSCs) were used as the model cell type, as their differentiation has been studied for several different cell lineages and often with conflicting results. Changes in the expression profiles of hMSCs differentiated under varied oxygen tensions are presented, demonstrating tissue-specific oxygen requirements for both adipogenic (20% O₂) and chondrogenic (5% O₂) differentiation. Oxygen and nutrient transport were enhanced by developing a bioreactor system for perfusing hMSC-seeded collagen gels using porous silk tubes, resulting in enhanced oxygen transport and cell viability within the gels. These systems are simple to use and scaled for versatility, to allow for the systematic study of relationships between cell content, oxygen, and cell function. The data may be combined with oxygen transport modeling to derive minimally sized modular units for construction of clinically relevant tissue-engineered constructs, a generic strategy that may be employed for vascularized target tissues.

Electric Field Stimulation Integrated into Perfusion Bioreactor for Cardiac Tissue Engineering

We describe herein the features of a novel cultivation system, combining electrical stimulation with medium perfusion for producing thick, functional cardiac patches. A custom-made electrical stimulator was integrated via inserting two carbon rod electrodes into a perfusion bioreactor, housing multiple neonatal Sprague-Dawley rat cardiac cell constructs between two 96% open-pore-area fixing nets. The stimulator produced adjustable stimulation waveform (i.e., duty cycle, number of stimulating channels, maximum stimulation amplitude, etc.), specially designed for cardiac cell stimulation. The cell constructs were subjected to a homogenous fluid flow regime and electrical stimulation under conditions optimal for cell excitation. The stimulation threshold in the bioreactor was set by first determining its value in a Petri dish under a microscope, and then matching the current density in the two cultivation systems by constructing electric field models. The models were built by Comsol Multiphysics software using the exact three-dimensional geometry of the two cultivation systems. These models illustrate, for the first time, the local electric conditions required for cardiomyocyte field excitation and they confirmed the uniformity of the electrical field around the cell constructs. Bioreactor cultivation for only 4 days under perfusion and continuous electrical stimulus (74.4 mA/cm², 2 ms, bipolar, 1 Hz) promoted cell elongation and striation in the cell constructs and enhanced the expression level of Connexin-43, the gap junction protein responsible for cell-cell coupling. These results thus confirm the validity of the electrical field model in predicting the optimal electrical stimulation in a rather complex cultivation system, a perfusion bioreactor.

How to Optimize Maturation in a Bioreactor for Vascular Tissue Engineering: Focus on a Decision Algorithm for Experimental Planning

Bioreactors may become essential tools for developing tissue-engineered organs from cells, with or without scaffolds. Cells seeded in these devices are expected to grow and form a tissue. Up to now, one of the main challenges to successfully develop functional organs is the structural organization of the cells into the scaffold during maturation in the bioreactor. The maturation step is affected by a set of highly interlinked dynamical variables (flow, stress, pH, temperature, and growth factors) in such a way that fixing the optimal environmental conditions becomes very complex. This work focuses on how the experimental parameters in the bioreactor can be optimized through numerical modeling to maximize tissue growth. Genetic programming (GP) and Markov decision processes (MDPs) were used in synergy to generate and take full advantage of a model of the vascular construct growth. The approach consists in formulating a model through GP to explain the growth of the construct and using MDPs to come up with a strategy to yield the best results in the experimental runs. Construct growth was improved, and the regeneration process was better understood in numerical simulations that relied on this control system. Therefore, an advanced numerical controller of this type could become an effective and inexpensive tool for planning experimental work in tissue engineering.