Bioreactors For Tissue Engineering Research Articles

Bioreactors for Vocal Fold Tissue Engineering.

It is estimated that almost one-third of the United States population will be affected by a vocal fold (VF) disorder during their lifespan. Promising therapies to treat VF injury and scarring are mostly centered on VF tissue engineering strategies such as the injection of engineered biomaterials and cell therapy. VF tissue engineering, however, is a challenging field as the biomechanical properties, structure, and composition of the VF tissue change upon exposure to mechanical stimulation. As a result, the development of long-term VF treatment strategies relies on the characterization of engineered tissues under a controlled mechanical environment. In this review, we highlight the importance of bioreactors as a powerful tool for VF tissue engineering with a focus on the current state of the art of bioreactors designed to mimic phonation in vitro. We discuss the influence of the phonatory environment on the development, function, injury, and healing of the VF tissue and its importance for the development of efficient therapeutic strategies. A concise and comprehensive overview of bioreactor designs, principles, operating parameters, and scalability are presented. An in-depth analysis of VF bioreactor data to date reveals that mechanical stimulation significantly influences cell viability and the expression of proinflammatory and profibrotic genes in vitro. Although the precision and accuracy of bioreactors contribute to generating reliable results, diverse gene expression profiles across the literature suggest that future efforts should focus on the standardization of bioreactor parameters to enable direct comparisons between studies. Impact statement We present a comprehensive review of bioreactors for vocal fold (VF) tissue engineering with a focus on the influence of the phonatory environment on the development, function, injury, and healing of the VFs and the importance of mimicking phonation on engineered VF tissues in vitro. Furthermore, we put forward a strong argument for the continued development of bioreactors in this area with an emphasis on the standardization of bioreactor designs, principles, operating parameters, and oscillatory regimes to enable comparisons between studies.

The Application of Bioreactors for Cartilage Tissue Engineering: Advances, Limitations, and Future Perspectives.

Tissue engineering (TE) has brought new hope for articular cartilage regeneration, as TE can provide structural and functional substitutes for native tissues. The basic elements of TE involve scaffolds, seeded cells, and biochemical and biomechanical stimuli. However, there are some limitations of TE; what most important is that static cell culture on scaffolds cannot simulate the physiological environment required for the development of natural cartilage. Recently, bioreactors have been used to simulate the physical and mechanical environment during the development of articular cartilage. This review aims to provide an overview of the concepts, categories, and applications of bioreactors for cartilage TE with emphasis on the design of various bioreactor systems.

Induction of osteogenic differentiation of bone marrow stromal cells on 3D polyester-based scaffolds solely by subphysiological fluidic stimulation in a laminar flow bioreactor

The fatal determination of bone marrow mesenchymal stem/stromal cells (BMSC) is closely associated with mechano-environmental factors in addition to biochemical clues. The aim of this study was to induce osteogenesis in the absence of chemical stimuli using a custom-designed laminar flow bioreactor. BMSC were seeded onto synthetic microporous scaffolds and subjected to the subphysiological level of fluid flow for up to 21 days. During the perfusion, cell proliferation was significantly inhibited. There were also morphological changes, with F-actin polymerisation and upregulation of ROCK1. Notably, in BMSC subjected to flow, mRNA expression of osteogenic markers was significantly upregulated and RUNX2 was localised in the nuclei. Further, under perfusion, there was greater deposition of collagen type 1 and calcium onto the scaffolds. The results confirm that an appropriate level of fluid stimuli preconditions BMSC towards the osteoblastic lineage on 3D scaffolds in the absence of chemical stimulation, which highlights the utility of flow bioreactors in bone tissue engineering.

3D printing of bioreactors in tissue engineering: A generalised approach.

3D printing is a rapidly evolving field for biological (bioprinting) and non-biological applications. Due to a high degree of freedom for geometrical parameters in 3D printing, prototype printing of bioreactors is a promising approach in the field of Tissue Engineering. The variety of printers, materials, printing parameters and device settings is difficult to overview both for beginners as well as for most professionals. In order to address this problem, we designed a guidance including test bodies to elucidate the real printing performance for a given printer system. Therefore, performance parameters such as accuracy or mechanical stability of the test bodies are systematically analysed. Moreover, post processing steps such as sterilisation or cleaning are considered in the test procedure. The guidance presented here is also applicable to optimise the printer settings for a given printer device. As proof of concept, we compared fused filament fabrication, stereolithography and selective laser sintering as the three most used printing methods. We determined fused filament fabrication printing as the most economical solution, while stereolithography is most accurate and features the highest surface quality. Finally, we tested the applicability of our guidance by identifying a printer solution to manufacture a complex bioreactor for a perfused tissue construct. Due to its design, the manufacture via subtractive mechanical methods would be 21-fold more expensive than additive manufacturing and therefore, would result in three times the number of parts to be assembled subsequently. Using this bioreactor we showed a successful 14-day-culture of a biofabricated collagen-based tissue construct containing human dermal fibroblasts as the stromal part and a perfusable central channel with human microvascular endothelial cells. Our study indicates how the full potential of biofabrication can be exploited, as most printed tissues exhibit individual shapes and require storage under physiological conditions, after the bioprinting process.

Differentiation Potential of Early- and Late-Passage Adipose-Derived Mesenchymal Stem Cells Cultured under Hypoxia and Normoxia.

With an increasing focus on the large-scale expansion of mesenchymal stem cells (MSCs) required for clinical applications for the treatment of joint and bone diseases such as osteoarthritis, the optimisation of conditions for in vitro MSC expansion requires careful consideration to maintain native MSC characteristics. Physiological parameters such as oxygen concentration, media constituents, and passage numbers influence the properties of MSCs and may have major impact on their therapeutic potential. Cells grown under hypoxic conditions have been widely documented in clinical use. Culturing MSCs on large scale requires bioreactor culture; however, it is challenging to maintain low oxygen and other physiological parameters over several passages in large bioreactor vessels. The necessity to scale up the production of cells in vitro under normoxia may affect important attributes of MSCs. For these reasons, our study investigated the effects of normoxic and hypoxic culture condition on early- and late-passage adipose-derived MSCs. We examined effect of each condition on the expression of key stem cell marker genes POU5F1, NANOG, and KLF4, as well as differentiation genes RUNX2, COL1A1, SOX9, COL2A1, and PPARG. We found that expression levels of stem cell marker genes and osteogenic and chondrogenic genes were higher in normoxia compared to hypoxia. Furthermore, expression of these genes reduced with passage number, with the exception of PPARG, an adipose differentiation marker, possibly due to the adipose origin of the MSCs. We confirmed by flow cytometry the presence of cell surface markers CD105, CD73, and CD90 and lack of expression of CD45, CD34, CD14, and CD19 across all conditions. Furthermore, in vitro differentiation confirmed that both early- and late-passage adipose-derived MSCs grown in hypoxia or normoxia could differentiate into chondrogenic and osteogenic cell types. Our results demonstrate that the minimal standard criteria to define MSCs as suitable for laboratory-based and preclinical studies can be maintained in early- or late-passage MSCs cultured in hypoxia or normoxia. Therefore, any of these culture conditions could be used when scaling up MSCs in bioreactors for allogeneic clinical applications or tissue engineering for the treatment of joint and bone diseases such as osteoarthritis.

Computational modeling of media flow through perfusion-based bioreactors for bone tissue engineering.

Bioreactor system has been used in bone tissue engineering in order to simulate dynamic nature of bone tissue environments. Perfusion bioreactors have been reported as the most efficient types of shear-loading bioreactor. Also, combination of forces, such as rotation plus perfusion, has been reported to enhance cell growth and osteogenic differentiation. Mathematical modeling using sophisticated infrastructure processes could be helpful and streamline the development of functional grafts by estimating and defining an effective range of bioreactor settings for better augmentation of tissue engineering. This study is aimed to conduct computational modeling for newly designed bioreactors in order to alleviate the time and material consuming for evaluating bioreactor parameters and effect of fluid flow hydrodynamics (various amounts of shear stress) on osteogenesis. Also, biological assessments were performed in order to validate similar parameters under implementing the perfusion or rotating and perfusion fluid motions in bioreactors' prototype. Finite element method was used to investigate the effect of hydrodynamic of fluid flow inside the bioreactors. The equations used in the simulation to calculate the velocity values and consequently the shear stress values include Navier-Stokes and Brinkman equations. It has been shown that rotational fluid motion in rotating and perfusion bioreactor produces more velocity and shear stress compared with perfusion bioreactor. Moreover, implementing the perfusion together with rotational force in rotating and perfusion bioreactors has been shown to have more cell proliferation and higher activity of alkaline phosphatase enzyme as well as formation of extra cellular matrix sheet, as an indicator of bone-like tissue formation.

TISSUE ENGINEERING BIOREACTORS: POTENTIAL APPLICATIONS AND SCALE UP STRATEGY

Tissue engineering bioreactors have been used in order to achieve production of artificial tissue, increasing cell proliferation capacity and yield and/or in vitro tissue/disease modelling. Although it is still discussing how to obtain functional and vascular tissue with these bioreactors, preclinical and clinical studies are ongoing. Tissue engineering bioreactors have been used as lab-scale bioreactors until now. Crucial potential application areas can be created by increasing the production capacity and bioprocess efficiency of these bioreactors. In this review, recent bioreactor technologies such as spinner flask, rotating wall/bed, hollow fiber membrane, perfusion and mechanical stimuli bioreactors are briefly presented in terms of their potential applications in medical field especially in the scope of scale-up approaches such as bubble column, stirred tank, membrane, air lift, fluidized packed and bed bioreactors.
 Peer Review History: 
 Received 1 May 2020; Revised 11 June; Accepted 6 July, Available online 15 July 2020
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 Name: Dr. Sally A. El-Zahaby
 Affiliation: Pharos University in Alexandria, Egypt
 E-mail: sally.elzahaby@yahoo.com
 
 Name: DR Viney Chawla
 Affiliation: University Institute of Pharmaceutical Sciences
 Baba Farid University of Health Sciences
 Sadiq Road, Faridkot-Punjab 151203
 E-mail: drvineychawla@gmail.com
 
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Channeling Effect and Tissue Morphology in a Perfusion Bioreactor Imaged by X-Ray Microtomography.

Perfusion bioreactors for tissue engineering hold great promises. Indeed, the perfusion of culture medium enhances species transport and mechanically stimulates the cells, thereby increasing cell proliferation and tissue formation. Nonetheless, their development is still hampered by a lack of understanding of the relationship between mechanical cues and tissue growth. Combining tissue engineering, three-dimensional visualization and numerical simulations, we analyze the morphological evolution of neo-tissue in a model bioreactor with respect to the local flow pattern. NIH-3T3 cells were grown under perfusion for one, two and three weeks on a stack of 2mm polyacetal beads. The model bioreactor was then imaged by X-ray micro-tomography and local tissue morphology was analyzed. To relate experimental observations and mechanical stimulii, a computational fluid dynamics model of flow around spheres in a canal was developed and solved using the finite element method. We observe a preferential tissue formation at the bioreactor periphery, and relate it to a channeling effect leading to regions of higher flow intensity. Additionally, we find that circular crater-like tissue patterns form in narrow channel regions at early culture times. Using computational fluid dynamic simulations, we show that the location and morphology of these patterns match those of shear stress maxima. Finally, the morphology of the tissue is qualitatively described as the tissue grows and reorganizes itself. Altogether, our study points out the key role of local flow conditions on the tissue morphology developed on a stack of beads in perfusion bioreactors and provides new insights for effective design of hydrodynamic bioreactors for tissue engineering using bead packings.

A Multimodal Stimulation Cell Culture Bioreactor for Tissue Engineering: A Numerical Modelling Approach

The use of digital twins in tissue engineering (TE) applications is of paramount importance to reduce the number of in vitro and in vivo tests. To pursue this aim, a novel multimodal bioreactor is developed, combining 3D design with numerical stimulation. This approach will facilitate the reproducibility between studies and the platforms optimisation (physical and digital) to enhance TE. The new bioreactor was specifically designed to be additive manufactured, which could not be reproduced with conventional techniques. Specifically, the design suggested allows the application of dual stimulation (electrical and mechanical) of a scaffold cell culture. For the selection of the most appropriate material for bioreactor manufacturing several materials were assessed for their cytotoxicity. Numerical modelling methods were then applied to the new bioreactor using one of the most appropriate material (Polyethylene Terephthalate Glycol-modified (PETG)) to find the optimal stimulation input parameters for bone TE based on two reported in vitro studies.

Strategies towards Orthopaedic Tissue Engineered Graft Generation: Current Scenario and Application

Even though degradation and damage to bone and cartilage tissue can be resolved naturally, but there is a great challenge to regenerate functional tissue due to multiple pathological conditions. To treat the diseased/ damaged bone and cartilage tissue as well as to improve or maintain its natural functions and structure, there are different strategies being developed for repair and regeneration of these tissues. Various innovative researches lead to remarkable improvement in clinical outcome of defective bone and cartilage treatments. Biomaterial based scaffolds which are capable of supporting cell growth and applied for replacement of tissue in vivo for both bone and cartilage. The review also delineates about the tissue engineering bioreactors for the recreation of frameworks to recellularise the graft in vitro by presenting them to physiologically significant mechanical or potentially hydrodynamic stimulation condition. This review summarizes and discusses the strategies for regeneration and repair of bone and cartilage tissue, current scenario and application.

Fluid flow in an enclosed cavity of rotational bioreactor for bone tissue engineering

The developed mathematical model was applied for study of fluid dynamics in a rotational bioreactor for bone tissue engineering by in vitro technology. The research goal is finding an optimal mode for rotation ensuring proper cyclic loading from fluid upon the cell-seeded biomaterial. The basis for developing a mathematical model of a bioreactor was a design of rotational type biological reactor used in medical research; the liquid flow is generated through viscosity mechanism due to surface rotation. Mathematical description of flow in a reactor cavity was performed with Navier—Stokes equations. It was assumed that flow regime in the boundary layer is laminar. Numerical algorithm was accomplished using a fluid flow solver “Fluent” in the code package ANSYS-12. Four variants of generating the rotational motion in the reactor cavity were considered. A series of parametric computations was performed for the rotation frequency f in the range 0.05 ≤ f ≤ 0.25 Hz. The paper offers visualization of velocity fields in the vertical plane. The distributions for shear stress and pressure in the working zone of reactor were calculated and analyzed. Simulations demonstrated that a method of fluid rotation by driving the outer cylinder with an offset axis is the best for arranging a cyclic pressure and cyclic shear stress on the biological material.

Validation of a novel 3D flow model for the optimization of construct perfusion in radial-flow packed-bed bioreactors (rPBBs) for long-bone tissue engineering

Osteogenic cell culture in three-dimensional (3D) hollow cylindrical porous scaffolds in radial-flow packed-bed bioreactors (rPBBs) may overcome the transport limitations of static and axial perfusion bioreactors in the engineering of long-bone substitutes. Flow models of rPBBs help optimize radial flux distribution of medium and tissue maturation in vitro. Only a 2D model is available for steady flow transport in rPBBs with axisymmetric inlet and outlet accounting for the fluid dynamics of void spaces, assessed against literature information. Here, a novel 3D model is proposed for steady flow transport in the three compartments of rPBBs with a more practical lateral outlet. A 3D model of transient tracer transport was developed based on the flow model to predict bioreactor residence time distribution (RTD). Model-predicted flow patterns were validated in terms of RTD against tracer experiments performed with bioreactor prototypes equipped with commercial scaffolds for bone tissue engineering. Bioreactors were challenged with a step change in entering tracer concentration in an optimized set-up under conditions promoting uniform radial flux distribution and typical shunt flows. Model-predicted RTDs agreed well with those experimentally determined. In conclusion, tracer experiments validate the use of the 3D flow model for optimizing construct perfusion in rPBBs to engineer long-bone substitutes.

Biomimetic characterization reveals enhancement of hydroxyapatite formation by fluid flow in gellan gum and bioactive glass composite scaffolds

This study presents production and biomimetic characterization of macroporous composite scaffolds based on gellan gum and nanoparticulate bioactive glass (GG-BAG) under conditions relevant for bone tissue engineering. Formation of hydroxyapatite (HAp) within the scaffolds was investigated in the simulated body fluid (SBF) during 14 days in two biomimetic bioreactors: perfusion bioreactor (1.1 ml/min SBF flowrate) and a bioreactor with coupled dynamic compression and SBF perfusion (5% strain, 0.68 Hz, 1 h on/1 h off, 1.1 ml/min SBF flowrate). HAp formation was evaluated by scanning electron microscopy (FE-SEM), energy dispersive X-ray (EDS) analysis, Fourier transform infrared spectroscopy (FTIR), and X-ray powder diffraction (XRPD). The superficial SBF velocity of 100 μm/s in perfusion bioreactors induced the formation of abundant cauliflower-like HAp crystals throughout the scaffold interior and flake-like crystals on external surfaces resulting in improved mechanical properties as compared to the initial scaffolds. The obtained results indicate potentials of macroporous GG-BAG scaffolds in conjunction with perfusion bioreactors for bone tissue engineering demonstrating high bioactivity suited for cultures of osteogenic cells.

Electrical Stimulation Optimization in Bioreactors for Tissue Engineering Applications

We review here the current research status on bioreactors for tissue engineering with cell electrical stimulation. Depending on the cell types, electrical stimulation has distinct objectives, in particular being employed both to mimic and enhance the endogenous electricity measured in the natural regeneration of living organisms as well as to mimic strain working conditions for contractible tissues (for instance muscle and cardiac tissues). Understanding the distinct parameters involved in electrical stimulation is crucial to optimize its application. The results presented in the literature and reviewed here reveal that the application of electrical stimulation can be essential for tissue engineering applications.

Histological Method to Study the Effect of Shear Stress on Cell Proliferation and Tissue Morphology in a Bioreactor.

Tissue engineering represents a promising approach for the production of bone substitutes. The use of perfusion bioreactors for the culture of bone-forming cells on a three-dimensional porous scaffold resolves mass transport limitations and provides mechanical stimuli. Despite the recent and important development of bioreactors for tissue engineering, the underlying mechanisms leading to the production of bone substitutes remain poorly understood. In order to study cell proliferation in a perfusion bioreactor, we propose a simplified experimental set-up using an impermeable scaffold model made of 2mm diameter glass beads on which mechanosensitive cells, NIH-3T3 fibroblasts are cultured for up to 3weeks under 10mL/min culture medium flow. A methodology combining histological procedure, image analysis and analytical calculations allows the description and quantification of cell proliferation and tissue production in relation to the mean wall shear stress within the bioreactor. Results show a massive expansion of the cell phase after 3weeks in bioreactor compared to static control. A scenario of cell proliferation within the three-dimensional bioreactor porosity over the 3weeks of culture is proposed pointing out the essential role of the contact points between adjacent beads. Calculations indicate that the mean wall shear stress experienced by the cells changes with culture time, from about 50mPa at the beginning of the experiment to about 100mPa after 3weeks. We anticipate that our results will help the development and calibration of predictive models, which rely on estimates and morphological description of cell proliferation under shear stress.

Mechanistic role of perfusion culture on bone regeneration

Bone tissue engineering (BTE) aims to develop engineered bone tissue to substitute conventional bone grafts. To achieve this, culturing the cells on the biocompatible three-dimensional (3D) scaffold is one alternative approach. The new functional bone tissue regeneration could be feasible by the synergetic combinations of cells, biomaterials and bioreactors. Although the field of biomaterial design/development for BTE applications attained reasonable success, development of suitable bioreactor remains still a major challenge. Tissue engineering bioreactors provide the microenvironment required for neo-tissue regeneration, and also can be used to study the physio-chemical cues effect on cell proliferation and differentiation in order to produce functional tissue. In this direction, various bioreactors have been developed and evaluated for the successful development of engineered bone tissue. Continues assessment of tissue development and limitations of the bioreactors lead to the progression of perfusion flow bioreactor system. Improvements in perfusion reactor system were able to yield multiple tissue engineered constructs with uniform cell distribution, easy to operate protocols and also effectively handled for the functional tissue development to meet the adequate supply of engineered graft for clinical application.

Bone tissue engineering in the greater omentum is enhanced by a periosteal transplant in a miniature pig model.

Reconstruction of bone defects with autologous grafts has certain disadvantages. The aim of this study is to introduce a new type of living bioreactor for engineering of bone flaps and to evaluate the effect of different barrier membranes. Scaffolds loaded with bone morphogenetic proteins and bone marrow aspirate wrapped with either a collagen membrane or a periosteal flap were implanted in the greater omentum of miniature pigs. Both histological and radiographic evaluation showed proven bone formation and increased density after 8 and 16 weeks, with an enhanced effect of the periosteal transplant. The greater omentum is a suitable bioreactor for bone tissue engineering. Endocultivation is both an innovative and promising approach in regenerative medicine.

Factors Affecting Mass Transport Properties of Poly(ε-caprolactone) Membranes for Tissue Engineering Bioreactors.

High porosity and mass transport properties of microfiltration polymeric membranes benefit nutrients supply to cells when used as scaffolds in interstitial perfusion bioreactors for tissue engineering. High nutrients transport is assumed when pore size and porosity of the membrane are in the micrometric range. The present work demonstrates that the study of membrane fouling by proteins present in the culture medium, though not done usually, should be included in the routine testing of new polymer membranes for this intended application. Two poly(ε-caprolactone) microfiltration membranes presenting similar average pore size (approximately 0.7 µm) and porosity (>80%) but different external surface porosity and pore size have been selected as case studies. The present work demonstrates that a membrane with lower surface pore abundance and smaller external pore size (approximately 0.67 µm), combined with adequate hydrodynamics and tangential flow filtration mode is usually more convenient to guarantee high flux of nutrients. On the contrary, having large external pore size (approximately 1.70 µm) and surface porosity would incur important internal protein fouling that could not be prevented with the operation mode and hydrodynamics of the perfusion system. Additionally, the use of glycerol in the drying protocols of the membranes might cause plasticization and a consequent reduction of mass transport properties due to membrane compaction by the pressure exerted to force perfusion. Therefore, preferentially, drying protocols that omit the use of plasticizing agents are recommended.

Development and biological validation of a cyclic stretch culture system for the ex vivo engineering of tendons.

An innovative approach to the treatment of tendon injury or degeneration is given by engineered grafts, made available through the development of bioreactors that generate tendon tissue in vitro, by replicating in vivo conditions. This work aims at the design of a bioreactor capable of applying a stimulation of cyclic strain on cell constructs to promote the production of bioartificial tissue with mechanical and biochemical properties resembling those of the native tissue. The system was actuated by an electromagnet and design specifications were imposed as follows. The stimulation protocol provides to scaffolds a 3% preload, a 10% deformation, and a stimulation frequency rate set at 0.5, 1, and 2 Hz, which alternates stimulation/resting phases. Porcine tenocytes were seeded on collagen scaffolds and cultured in static or dynamic conditions for 7 and 14 days. The culture medium temperature did not exceed 37°C during prolonged culture experiments. The applied force oscillates between 1.5 and 4.5 N. The cyclic stimulation of the engineered constructs let both the cells and the scaffold fibers align along the strain direction in response to the mechanical stimulus. We designed a pulsatile strain bioreactor for tendon tissue engineering. The in vitro characterization shows a preferential cell alignment at short time points. Prolonged culture time, however, seems to influence negatively on the survival of the cells indicating the need of further optimization concerning the culture conditions and the mechanical stimulation.

Bioreactors for Cardiac Tissue Engineering.

The advances in biotechnology, biomechanics, and biomaterials can be used to develop organ models that aim to accurately emulate their natural counterparts. Heart disease, one of the leading causes of death in modern society, has attracted particular attention in the field of tissue engineering. To avoid incorrect prognosis of patients suffering from heart disease, or from adverse consequences of classical therapeutic approaches, as well as to address the shortage of heart donors, new solutions are urgently needed. Biotechnological advances in cardiac tissue engineering from a bioreactor perspective, in which recapitulation of functional, biochemical, and physiological characteristics of the cardiac tissue can be used to recreate its natural microenvironment, are reviewed. Detailed examples of functional and preclinical applications of engineered cardiac constructs and the state-of-the-art systems from a bioreactor perspective are provided. Finally, the current trends and future directions of the field for its translation to clinical settings are discussed.