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. Bioeng. 2011; 108:1159–1170. © 2010 Wiley Periodicals, Inc.     

Visualizing feasible operating ranges within tissue engineering systems using a “windows of operation” approach: A perfusion‐scaffold bioreactor case study

Tissue engineering approaches to developing functional substitutes are often highly complex, multivariate systems where many aspects of the biomaterials, bio‐regulatory factors or cell sources may be controlled in an effort to enhance tissue formation. Furthermore, success is based on multiple performance criteria reflecting both the quantity and quality of the tissue produced. Managing the trade‐offs between different performance criteria is a challenge. A “windows of operation” tool that graphically represents feasible operating spaces to achieve user‐defined levels of performance has previously been described by researchers in the bio‐processing industry. This paper demonstrates the value of “windows of operation” to the tissue engineering field using a perfusion‐scaffold bioreactor system as a case study. In our laboratory, perfusion bioreactor systems are utilized in the context of bone tissue engineering to enhance the osteogenic differentiation of cell‐seeded scaffolds. A key challenge of such perfusion bioreactor systems is to maximize the induction of osteogenesis but minimize cell detachment from the scaffold. Two key operating variables that influence these performance criteria are the mean scaffold pore size and flow‐rate. Using cyclooxygenase‐2 and osteopontin gene expression levels as surrogate indicators of osteogenesis, we employed the “windows of operation” methodology to rapidly identify feasible operating ranges for the mean scaffold pore size and flow‐rate that achieved user‐defined levels of performance for cell detachment and differentiation. Incorporation of such tools into the tissue engineer's armory will hopefully yield a greater understanding of the highly complex systems used and help aid decision making in future translation of products from the bench top to the market place. Biotechnol. Bioeng. 2012; 109: 3161–3171. © 2012 Wiley Periodicals, Inc.     

Skeletal muscle tissue engineering

The reconstruction of skeletal muscle tissue either lost by traumatic injury or tumor ablation or functional damage due to myopathies is hampered by the lack of availability of functional substitution of this native tissue. Until now, only few alternatives exist to provide functional restoration of damaged muscle tissues. Loss of muscle mass and their function can surgically managed in part using a variety of muscle transplantation or transposition techniques. These techniques represent a limited degree of success in attempts to restore the normal functioning, however they are not perfect solutions. A new alternative approach to addressing difficult tissue reconstruction is to engineer new tissues. Although those tissue engineering techniques attempting regeneration of human tissues and organs have recently entered into clinical practice, the engineering of skeletal muscle tissue ist still a scientific challenge. This article reviews some of the recent findings resulting from tissue engineering science related to the attempt of creation and regeneration of functional skeletal muscle tissue.     

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.     

A new approach to tissue engineering of vascularized skeletal muscle

Tissue Engineering of skeletal muscle tissue still remains a major challenge. Every neo-tissue construct of clinically relevant dimensions is highly dependent on an intrinsic vascularisation overcoming the limitations of diffusion conditioned survival. Approaches incorporating the arteriovenous-loop model might bring further advances to the generation of vascularised skeletal muscle tissue. In this study 12 syngeneic rats received transplantaion of carboxy-fluorescine diacetate-succinimidyl ester (CFDA)-labelled, expanded primary myoblasts into a previously vascularised fibrin matrix, containing a microsurgically created AV loop. As control cells were injected into fibrin-matrices without AV-loops. Intra-arterial ink injection followed by explantation was performed 2, 4 and 8 weeks after cell implantation. Specimens were evaluated for CFDA, MyoD and DAPI staining, as well as for mRNA expression of muscle specific genes. Results showed enhanced fibrin resorption in dependence of AV loop presence. Transplanted myoblasts could be detected in the AV loop group even after 8 weeks by CFDA-fluorescence, still showing positive MyoD staining. RT-PCR revealed gene expression of MEF-2 and desmin after 4 weeks on the AVloop side, whereas expression analysis of myogenin and MHC(embryo) was negative. So far myoblast injection in the microsurgical rat AV loop model enhances survival of the cells, keeping their myogenic phenotype, within pre-vascularised fibrin matrices. Probably due to the lack of potent myogenic stimuli and additionally the rapid resorption of the fibrin matrix, no formation of skeletal muscle-like tissue could be observed. Thus further studies focussing on long term stability of the matrix and the incorporation of neural stimuli will be necessary for generation of vascularised skeletal muscle tissue.     

3D culture of osteoblast‐like cells by unidirectional or oscillatory flow for bone tissue engineering

A medium perfusion system is expected to be beneficial for three‐dimensional (3D) culture of engineered bone, not only by chemotransport enhancement but also by mechanical stimulation. In this study, perfusion systems with either unidirectional or oscillatory medium flow were developed, and the effects of the different flow profiles on 3D culturing of engineered bone were studied. Mouse osteoblast‐like MC 3T3‐E1 cells were 3D‐cultured with porous ceramic scaffolds in vitro for 6 days under static and hydrodynamic conditions with either a unidirectional or oscillatory flow. We found that, in the static culture, the cells proliferated only on the scaffold surfaces. In perfusion culture with the unidirectional flow, the proliferation was significantly higher than in the other groups but was very inhomogeneous, which made the construct unsuitable for transplantation. Only the oscillatory flow allowed osteogenic cells to proliferate uniformly throughout the scaffolds, and also increased the activity of alkaline phosphatase (ALP). These results suggested that oscillatory flow might be better than unidirectional flow for 3D construction of cell‐seeded artificial bone. The oscillatory perfusion system could be a compact, safe, and efficient bioreactor for bone tissue engineering. Biotechnol. Bioeng. 2009;102: 1670–1678. © 2008 Wiley Periodicals, Inc.     

A strategy to determine operating parameters in tissue engineering hollow fiber bioreactors

The development of tissue engineering hollow fiber bioreactors (HFB) requires the optimal design of the geometry and operation parameters of the system. This article provides a strategy for specifying operating conditions for the system based on mathematical models of oxygen delivery to the cell population. Analytical and numerical solutions of these models are developed based on Michaelis–Menten kinetics. Depending on the minimum oxygen concentration required to culture a functional cell population, together with the oxygen uptake kinetics, the strategy dictates the model needed to describe mass transport so that the operating conditions can be defined. If c_min ≫ K_m we capture oxygen uptake using zero‐order kinetics and proceed analytically. This enables operating equations to be developed that allow the user to choose the medium flow rate, lumen length, and ECS depth to provide a prescribed value of c_min. When , we use numerical techniques to solve full Michaelis–Menten kinetics and present operating data for the bioreactor. The strategy presented utilizes both analytical and numerical approaches and can be applied to any cell type with known oxygen transport properties and uptake kinetics. Biotechnol. Bioeng. 2011; 108:1450–1461. © 2011 Wiley Periodicals, Inc.     

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.     

On‐demand three‐dimensional freeform fabrication of multi‐layered hydrogel scaffold with fluidic channels

One of the challenges in tissue engineering is to provide adequate supplies of oxygen and nutrients to cells within the engineered tissue construct. Soft‐lithographic techniques have allowed the generation of hydrogel scaffolds containing a network of fluidic channels, but at the cost of complicated and often time‐consuming manufacturing steps. We report a three‐dimensional (3D) direct printing technique to construct hydrogel scaffolds containing fluidic channels. Cells can also be printed on to and embedded in the scaffold with this technique. Collagen hydrogel precursor was printed and subsequently crosslinked via nebulized sodium bicarbonate solution. A heated gelatin solution, which served as a sacrificial element for the fluidic channels, was printed between the collagen layers. The process was repeated layer‐by‐layer to form a 3D hydrogel block. The printed hydrogel block was heated to 37°C, which allowed the gelatin to be selectively liquefied and drained, generating a hollow channel within the collagen scaffold. The dermal fibroblasts grown in a scaffold containing fluidic channels showed significantly elevated cell viability compared to the ones without any channels. The on‐demand capability to print fluidic channel structures and cells in a 3D hydrogel scaffold offers flexibility in generating perfusable 3D artificial tissue composites. Biotechnol. Bioeng. 2010;105: 1178–1186. © 2009 Wiley Periodicals, Inc.     

Flow dynamics in bioreactors containing tissue engineering scaffolds

Bioreactors are widely used in tissue engineering as a way to distribute nutrients within porous materials and provide physical stimulus required by many tissues. However, the fluid dynamics within the large porous structure are not well understood. In this study, we explored the effect of reactor geometry by using rectangular and circular reactors with three different inlet and outlet patterns. Geometries were simulated with and without the porous structure using the computational fluid dynamics software Comsol Multiphysics 3.4 and/or ANSYS CFX 11 respectively. Residence time distribution analysis using a step change of a tracer within the reactor revealed non-ideal fluid distribution characteristics within the reactors. The Brinkman equation was used to model the permeability characteristics with in the chitosan porous structure. Pore size was varied from 10 to 200 microm and the number of pores per unit area was varied from 15 to 1,500 pores/mm(2). Effect of cellular growth and tissue remodeling on flow distribution was also assessed by changing the pore size (85-10 microm) while keeping the number of pores per unit area constant. These results showed significant increase in pressure with reduction in pore size, which could limit the fluid flow and nutrient transport. However, measured pressure drop was marginally higher than the simulation results. Maximum shear stress was similar in both reactors and ranged approximately 0.2-0.3 dynes/cm(2). The simulations were validated experimentally using both a rectangular and circular bioreactor, constructed in-house. Porous structures for the experiments were formed using 0.5% chitosan solution freeze-dried at -80 degrees C, and the pressure drop across the reactor was monitored.     

Polymer‐based microparticles in tissue engineering and regenerative medicine

Different types of biomaterials, processed into different shapes, have been proposed as temporary support for cells in tissue engineering (TE) strategies. The manufacturing methods used in the production of particles in drug delivery strategies have been adapted for the development of microparticles in the fields of TE and regenerative medicine (RM). Microparticles have been applied as building blocks and matrices for the delivery of soluble factors, aiming for the construction of TE scaffolds, either by fusion giving rise to porous scaffolds or as injectable systems for in situ scaffold formation, avoiding complicated surgery procedures. More recently, organ printing strategies have been developed by the fusion of hydrogel particles with encapsulated cells, aiming the production of organs in in vitro conditions. Mesoscale self‐assembly of hydrogel microblocks and the use of leachable particles in three‐dimensional (3D) layer‐by‐layer (LbL) techniques have been suggested as well in recent works. Along with innovative applications, new perspectives are open for the use of these versatile structures, and different directions can still be followed to use all the potential that such systems can bring. This review focuses on polymeric microparticle processing techniques and overviews several examples and general concepts related to the use of these systems in TE and RE applications. The use of materials in the development of microparticles from research to clinical applications is also discussed. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Osteogenic differentiation and osteochondral tissue engineering using human adipose‐derived stem cells

Osteogenesis and the production of composite osteochondral tissues were investigated using human adult adipose‐derived stem cells and polyglycolic acid (PGA) mesh scaffolds under dynamic culture conditions. For osteogenesis, cells were expanded with or without osteoinduction factors and cultured in control or osteogenic medium for 2 weeks. Osteogenic medium enhanced osteopontin and osteocalcin gene expression when applied after but not during cell expansion. Osteogenesis was induced and mineralized deposits were present in tissues produced using PGA culture in osteogenic medium. For development of osteochondral constructs, scaffolds seeded with stem cells were precultured in either chondrogenic or osteogenic medium, sutured together, and cultured in dual‐chamber stirred bioreactors containing chondrogenic and osteogenic media in separate compartments. After 2 weeks, total collagen synthesis was 2.1‐fold greater in the chondroinduced sections of the composite tissues compared with the osteoinduced sections; differentiation markers for cartilage and bone were produced in both sections of the constructs. The results from the dual‐chamber bioreactor highlight the challenges associated with achieving simultaneous chondrogenic and osteogenic differentiation in tissue engineering applications using a single stem‐cell source. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Perfusion seeding of channeled elastomeric scaffolds with myocytes and endothelial cells for cardiac tissue engineering

The requirements for engineering clinically sized cardiac constructs include medium perfusion (to maintain cell viability throughout the construct volume) and the protection of cardiac myocytes from hydrodynamic shear. To reconcile these conflicting requirements, we proposed the use of porous elastomeric scaffolds with an array of channels providing conduits for medium perfusion, and sized to provide efficient transport of oxygen to the cells, by a combination of convective flow and molecular diffusion over short distances between the channels. In this study, we investigate the conditions for perfusion seeding of channeled constructs with myocytes and endothelial cells without the gel carrier we previously used to lock the cells within the scaffold pores. We first established the flow parameters for perfusion seeding of porous elastomer scaffolds using the C2C12 myoblast line, and determined that a linear perfusion velocity of 1.0 mm/s resulted in seeding efficiency of 87% ± 26% within 2 hours. When applied to seeding of channeled scaffolds with neonatal rat cardiac myocytes, these conditions also resulted in high efficiency (77.2% ± 23.7%) of cell seeding. Uniform spatial cell distributions were obtained when scaffolds were stacked on top of one another in perfusion cartridges, effectively closing off the channels during perfusion seeding. Perfusion seeding of single scaffolds resulted in preferential cell attachment at the channel surfaces, and was employed for seeding scaffolds with rat aortic endothelial cells. We thus propose that these techniques can be utilized to engineer thick and compact cardiac constructs with parallel channels lined with endothelial cells. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

The role of skeletal‐muscle‐based thermogenic mechanisms in vertebrate endothermy

Thermogenesis is one of the most important homeostatic mechanisms that evolved during vertebrate evolution. Despite its importance for the survival of the organism, the mechanistic details behind various thermogenic processes remain incompletely understood. Although heat production from muscle has long been recognized as a thermogenic mechanism, whether muscle can produce heat independently of contraction remains controversial. Studies in birds and mammals suggest that skeletal muscle can be an important site of non‐shivering thermogenesis (NST) and can be recruited during cold adaptation, although unequivocal evidence is lacking. Much research on thermogenesis during the last two decades has been focused on brown adipose tissue (BAT). These studies clearly implicate BAT as an important site of NST in mammals, in particular in newborns and rodents. However, BAT is either absent, as in birds and pigs, or is only a minor component, as in adult large mammals including humans, bringing into question the BAT‐centric view of thermogenesis. This review focuses on the evolution and emergence of various thermogenic mechanisms in vertebrates from fish to man. A careful analysis of the existing data reveals that muscle was the earliest facultative thermogenic organ to emerge in vertebrates, long before the appearance of BAT in eutherian mammals. Additionally, these studies suggest that muscle‐based thermogenesis is the dominant mechanism of heat production in many species including birds, marsupials, and certain mammals where BAT‐mediated thermogenesis is absent or limited. We discuss the relevance of our recent findings showing that uncoupling of sarco(endo)plasmic reticulum Ca²⁺‐ATPase (SERCA) by sarcolipin (SLN), resulting in futile cycling and increased heat production, could be the basis for NST in skeletal muscle. The overall goal of this review is to highlight the role of skeletal muscle as a thermogenic organ and provide a balanced view of thermogenesis in vertebrates.