DOI: 10.5301/IJAO.2011.6333
Int J Artif Organs 2011 ; 34 ( 3): 259- 270
REVIEW
Bioreactors for bone tissue engineering
Benoît Carpentier1, Pierre Layrolle2, Cécile Legallais1
1
2
UMR CNRS 6600, Biomechanics and Bioengineering, University of Technology of Compiègne, Compiègne - France
Inserm U957-LPRO Faculté de Medecine, Nantes - France
ABSTRACT:
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.
Key words: Bioreactors, Bone, Tissue engineering, Scaffolds, Finite element modeling, Mesenchymal stem cells
Accepted: December 31, 2010
INTRODUCTION
Today, autologous bone grafting is still considered to be
the “gold standard” for reconstructing large bone defects
(1). Autologous and allogenic sources of bone tissue are
the most commonly used reconstruction methods. However, both of these procedures have significant drawbacks
and adverse effects, potentially leading to major, debilitating complications (such as chronic pain, limited availability, graft rejection and disease transfer).
Different sorts of biomaterials have inspired increasing
interest in reconstructive indications. Nevertheless, synthetic bone substitutes alone have insufficient osteogenic
properties for regenerating large bone defects (1-4). The
aim of bone tissue engineering is to produce bio-hybrid
implantable bone substitutes with osteoconductive and
osteointegrative properties ex vivo in order to repair or re-
place bone tissue that is damaged or missing after transplantation (5, 6). A synoptic chart showing the requisite
stages for producing bone substitutes using isolated bone
marrow stem cells is provided in Fig. 1. The implantable
construct is thus made of a three-dimensional (3D) scaffold, autologous osteogenic cells, and the neosynthesized
extracellular matrix.
Several types of natural (2, 7-10) and synthetic (11-17)
biomaterials have been tested so far as cell-seeded implantable scaffolds (Fig. 2). Composites (18, 19) and more
recently hydrogels (17, 20-26) are also under study. The
choice of biomaterial and its formulation is a decisive step
that has an impact on cell growth and differentiation in vitro
as well as on bone tissue formation and the behavior of the
construct in vivo. The scaffold is effectively in direct contact with the cells and should therefore be suitable for cell
© 2011 Wichtig Editore - ISSN 0391-3988
259
Bioreactors for bone tissue engineering
Fig. 1 - Producing bone tissue engineered constructs.
adhesion and proliferation. For certain clinical applications,
the scaffold must have high mechanical properties. More
than anything, it must be biocompatible and, if possible,
biodegradable.
To date, different cell types have been used for bone tissue engineering. Cell lineages are usually used to check
the biocompatibility of a substrate, to better understand
certain phenomena (e.g., the interaction between the biomaterial and cells, cell adhesion, cell seeding, etc.) and to
characterize new culture devices (13, 14, 18, 27). They are
indeed readily available and easy to culture in vitro. They
are, however, a transitory step that has to give way to more
functional and applicable choices.
Primary cells are another option. The issue with this cell
source is its availability. Culturing this type of cells for a
long period of time is also a complicated task. Mesenchymal stem cells (MSCs) – isolated from animal or human
bone marrow (7, 8, 11, 12, 28-30) or adipose tissue (31,
32) – are the most promising cells and are frequently used.
MSCs proliferate very well in culture for up to 50 passages
and are able to differentiate into different lineages under
biochemical and mechanical cues. For instance, MSCs
have been differentiated into osteoprogenitors, chondrocytes or adipocytes under specific culture conditions. Moreover, the aim is ultimately to use the patient’s own stem
cells as the cell source as a means of avoiding implant
rejection in bone reconstruction clinical applications. Other
types of stem cells harvested from the patient’s body may
naturally serve as alternative cell sources (33, 34).
Cells are cultured using culture media supplemented or not
with osteogenic factors (35). The factors that are able to
promote cell differentiation and orient cells towards osteo260
Fig. 2 - Different types of biomaterial scaffolds for bone tissue engineering: a) Calcium phosphate ceramic granules, b) Rapid prototyped porous titanium, c) Biodegradable polymer microfibers and d)
hydrogel with cross-linking, cell adhesion and enzymatic cleaving
sites.
blastic phenotypes are dexamethasone, ascorbic acid and
β-glycerophosphate. They lead to an increase in early and
late bone markers and to a rise in matrix production and
mineralization. Some teams have tried to replace these
factors by shear stress while others use them in combination with mechanical loading in order to synergistically
enhance the bone tissue formation.
Biochemical and biophysical stimulations of cell proliferation and differentiation are considered to be important parameters and powerful ways to improve bone formation ex
vivo (36). Bioreactors are therefore a key technology when
it comes to promoting the production of bone substitutes
containing mature and differentiated cells capable of producing both the organic and mineralized components of
the extracellular matrix (ECM) within the 3D environment
of the scaffold.
A bioreactor is a vessel in which biochemical or biological
processes involving cells, organisms or biochemically active substances are carried out under closely monitored and
tightly controlled operating conditions (e.g., temperature,
pH, flow rate, nutrient and oxygen supply, etc.). Bioreactors are typically used in the treatment of waste water (37,
38), for mass propagation of plants (39, 40), large-scale
production of pharmaceuticals (41) and recombinant proteins (42) as well as in the food industry (43). This technology is now being developed for tissue engineering (7, 8,
44-48).
© 2011 Wichtig Editore - ISSN 0391-3988
Carpentier et al
After emphasizing the specificity of bone tissue, this paper
reviews the dynamic cell culture systems that have been
developed so far and applied to bone tissue engineering.
A brief overview is also given of the value of computational
fluid dynamics (CFD) modeling, as well as of the various
models that have been developed in this field – emphasizing their relevance in better understanding the 3D environment in which the cells are placed.
Bone tissue
Bones are highly complex organs made of various tissue
types (i.e. bone tissue, bone marrow, vascular tissue, etc.).
Bone provides the body with internal support, protection
for vital organs, as well as attachment sites for tendons
and muscles. Bone is the main storage site for calcium and
phosphate and therefore plays a crucial role in calcium and
phosphorus homeostasis. It is a dynamic tissue which undergoes continuous remodeling throughout life. Bone tissue is a specialized form of connective tissue composed of
an abundant calcified extracellular matrix (ECM) in which
cells are embedded. Mature bones are composed of two
types of bone tissue: compact (or cortical) and cancellous
(or trabecular) tissues (Fig. 3). Bone is made principally of a
calcified ECM which is composed of organic and inorganic
parts (1). Hydroxyapatite is the main crystalline mineral.
The organic component is formed of type I collagen, glycosaminoglycans (hyaluronan, chondroitin sulfate, decorin,
biglycan, etc.), osteocalcin, osteonectin, bone sialoprotein
as well as various growth and attachment factors (1).
Bones contain four main types of cells (Fig. 3). Osteoblasts
are mononucleated cells originating from bone marrow
MSCs. They are located on the surface of osteoid zones
producing collagen ECM that is progressively mineralized. Osteoblasts are involved in the mineralization phase
through the expression, synthesis and secretion of alkaline
phosphatase (ALP). Bone lining cells are inactive osteoblasts that cover bone surfaces and act as a barrier for
certain ions. Osteocytes are mature osteoblasts which
have become trapped within the extracellular bone matrix. Osteocytes are less active than osteoblasts and are
located within lacunae. They communicate with each other
and with osteoblasts using numerous processes and gap
junctions. They are responsible for bone matrix formation
and maintenance as well as calcium homeostasis. They
have also been shown to be involved in mechanotrans-
Fig. 3 - Bone tissue and its remodeling. Trabecular bone (t) is the
reservoir of bone marrow cells (m) from which osteoblasts and osteoclasts are derived.
duction processes, regulating the responses of bone to
stress and mechanical loading. Osteoclasts are large,
multinucleated cells specialized in bone resorption, originating from the hematopoietic stem cells. They are formed
in the bone marrow by fusion of monocytes. Osteoclasts
attach to the bone surface, produce H+ ions that dissolve
the hydroxyapatite mineral and synthesize active enzymes,
such as tartrate-resistant acid phosphatase (TRAP), which
degrade the organic matrix, forming Howship’s lacunae
(35). In summary, at the early stage of osteogenesis, bone
is characterized by a relatively high cellular content with
numerous osteoprogenitors producing the ECM, while a
limited number of cells are found in mature tissue. Bone is
able to heal and is constantly remodeled by cellular activity
from the bone marrow reservoir. In this process, multiple
cell types cooperate to manufacture this complex tissue
while the extracellular matrix is degraded by multinucleated osteoclastic cells. Both forming and resorbing cells
should attach to the ECM. The balance between formation
and resorption is controlled by many cues, such as signaling molecules and biomechanical forces. Bone tissue
is also a highly vascularized tissue with blood capillaries
supplying oxygen and nutrients to the cells. It is therefore
a complex 3D tissue containing different types and high
numbers of cells at the early stage and few cells but an
abundant ECM at maturity.
Geometries for bioreactor-based systems
Bioreactors are classically subdivided into four types (44):
rotating wall vessels, spinner flasks, direct perfusion sy-
© 2011 Wichtig Editore - ISSN 0391-3988
261
Bioreactors for bone tissue engineering
recently been developed (49-50). They are of particular interest for bone tissue engineering as osteogenic cells and
osteoblasts are sensitive and responsive to mechanical stimuli. Such systems are sometimes equipped with sensors
or transducers monitoring oxygen partial pressure, pH, static pressure, temperature, and so forth. They make it possible to closely control the environment in which the cells are
placed and to automate and standardize the process.
The principles and results for each type of bioreactor are
described in Table I.
Fig. 4 - Bioreactors for culturing osteoprogenitor cells on scaffolds.
Direct perfusion bioreactors
stems and hollow fiber devices (Fig. 4). Nevertheless, none
of those applied to bone tissue engineering makes use of
hollow fibers because these systems do not easily accommodate scaffolds. Mechanical loading devices have also
The closed-loop circuit is composed of a bioreactor containing the biomaterial, one or more reservoirs, and tubing.
An oxygenator is sometimes included to improve oxygen
delivery to the cells (11). The culture medium is driven by
TABLE I - PRINCIPLES AND RESULTS FOR EACH TyPE OF BIOREACTOR
Bioreactor
Cell type
Scaffold/substrate
Authors
Direct perfusion bioreactor
Goat bone marrow stromal cells
2-6 mm BCP granules
Janssen et al. 11
Direct perfusion micro-device
MC3T3-E1
PDMS
Leclerc et al.14
Direct perfusion bioreactor
Rat bone marrow stromal cells
Titanium fiber mesh
Bancroft et al.12
Direct perfusion bioreactor
Rat bone marrow stromal cells
Datta et al.28
Rotating wall vessel
MC3T3-E1
Direct perfusion bioreactor
(oscillatory flow)
Rotating wall vessel
MC3T3-E1
Pregenerated ECM containing titanium
fiber mesh
Organoapatite-coated bioactive titanium
foam
Porous ceramic β-TCP
Human bio-derived bone scaffold
Song et al.51
Aqueous-derived porous silk scaffolds
Kim et al.29
Spinner flask
Osteoblasts isolated from craniums
of neonatal Sprague-Dawley rats
Human bone marrow mesenchymal
stem cells
Adipose-derived stem cells
Collagenous microbeads (Sigma)
Rubin et al.31
Spinner flask
Human mesenchymal stem cells
Coralline hydroxyapatite scaffolds
Mygind et al.8
Spinner flask,
Perfusion cartridge
Spinner flask,
Rotating wall vessel
Rotating wall vessel
Human mesenchymal stem cells
Collagen and silk scaffolds
Meinel et al.7
Rat bone marrow stromal cells
Porous 75:25 poly(D,L-lactic-co-glycolic
acid) biodegradable scaffolds
Hollow microspheres coated with calcium hydroxyapatite
Poly(-lactic-co-glycolic acid) (PLGA)
foam discs
Explanted specimens of cartilage, 3D
collagen gels
3D type I collagen Matrices
Sikavitsas et al46
Spinner flask
Rotating wall vessel, spinner
flask, perfusion system
Mechanical loading device
Mechanical loading device
262
Rat bone marrow stromal cells and
osteosarcoma cells (ROS 17/2.8)
Osteoblastic marrow stromal cells
Activated osteoblasts and chondrocytes
Human osteoblastic precursor cells
(hFOB 1.19)
© 2011 Wichtig Editore - ISSN 0391-3988
Spoerke et al.18
Du et al.13
Qiu et al.15
Goldstein et al.52
Meyer et al.36
Ignatius et al.50
Carpentier et al
a roller pump through the bioreactor chamber in which the
scaffold is located. The medium flows either from the top
to the bottom of the chamber (12) or in the opposite direction. An oscillatory flow has sometimes been applied, only
during cell seeding or throughout the culture phase (13).
Among perfusion systems, two configurations – packed
and fluidized beds – have been tested for bone tissue engineering applications. Fluidized bed bioreactors can only
be used when the biomaterial consists of microparticles
or granules. The flow – directed from the bottom to the
top of the bioreactor – has to be set at a level that is high
enough to mobilize the microparticles (53). Packed bed
configurations are more commonly used to perfuse beds
of microparticles or one-piece scaffolds. Bancroft et al investigated the effect of flow perfusion on marrow stromal
osteoblasts cultured on 3D titanium fiber mesh scaffolds
(12). Mineralized matrix production was drastically increased compared to constructs cultured in static conditions.
Leclerc et al demonstrated the importance of fluid flow and
shear stress on cell differentiation and the expression of
differentiation markers (14). Murine calvarial osteoblastic
MC3T3-E1 cells were cultured dynamically and submitted
to different flow rates within polydimethylsiloxane (PDMS)
microdevices. ALP activity – an early marker for cell differentiation – was significantly enhanced when a flow was
applied. In another publication, Datta et al showed that bone-like ECM produced in vitro together with fluid-induced
shear stress synergically enhanced the osteoblastic differentiation of rat marrow stromal cells (28). Pregenerated
ECM yielded to a significant increase in mineralized matrix
deposits in ECM containing titanium fiber meshes compared to constructs that were previously denatured. Du et al
designed a compact perfusion system using an oscillatory
flow (13). MC3T3-E1 cells were seeded and cultured for six
days in porous β-TCP cylindrical ceramics with a final culture medium volume of 1.5 mL. The oscillatory perfusion
was shown to improve cell seeding efficacy, scaffold cellularity and early osteogenesis. The biological assays 3(4,
5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
staining and Calcein-AM/propidium iodide double staining
demonstrated homogenous seeding, proliferation and viability of the cells throughout the ceramics. On the contrary,
the cells died in the center of the scaffolds under static
conditions. These studies have therefore demonstrated the
benefits of using perfusion bioreactors together with scaffolds for uniform seeding, cell culturing and differentiation
of cells into osteogenic lineage.
Rotating wall vessels
Rotating wall vessels are composed of an immobile inner
cylinder and a rotating outer hollow cylinder. The culture medium is located in between. The vessel wall is rotated at a
particular rate making it possible to create microgravity. At
that particular rotation rate, the drag force, centrifugal force, and net gravitational force on the scaffold are balanced,
leading to a free-fall state of the construct within the culture
medium and providing a dynamic culture environment with
low shear stress and high mass transfer (44). Nevertheless,
the scaffold, generally in the form of particle beads, should
have a density comparable to cell culture media unless high
rotating speeds have to be applied. Scaffolds made of biopolymers are particularly well adapted to rotating wall vessels due to their relative density, which is close to 1. On the
contrary, the use of hydroxyapatite and related calcium phosphate ceramics or titanium beads with densities of around
3 and 4 in rotating wall vessels have been accompanied by
many problems. These materials have been prepared as hollow beads in order to counterbalance the density mismatch
with the culture media.
Many authors have evaluated the feasibility of employing
rotating wall vessels for culturing osteoblasts or stem cells.
Better results have repeatedly been obtained compared to
static culture. Increased bone marker expression and nodule mineralization, for instance, have indeed been reported. Song et al seeded and cultured osteoblasts on human
bio-derived bone scaffolds within a rotating wall vessel for
three weeks (51, 53). The proliferation was higher compared to static conditions and spinner flasks. More collagen
fibers, mineralized nodules and newly-formed osteoid
tissue were observed. Fluid-induced shear stress led to
increased ALP expression and accelerated nodule mineralization. Yet Sikavitsas et al documented contradictory
results (46). Within a rotating wall bioreactor, Spoerke et
al cultured MC3T3 cells on organoapatite-coated titanium
foams which were successfully colonized (18). Cell culture
studies using a rotating wall vessel showed that rat bone
marrow stromal cells and osteosarcoma cells (ROS 17/2.8)
attached to hollow microspheres coated with calcium
hydroxyapatite and formed ECM containing 3D aggregates that experienced low shear stress (15). Mineralized nodules were observed within the aggregates. Rotating wall
vessels are therefore interesting, dynamic ex vivo systems
for simulating microgravity and studying its effects on bone
physiology.
© 2011 Wichtig Editore - ISSN 0391-3988
263
Bioreactors for bone tissue engineering
Spinner flasks
A spinner flask bioreactor or stirred bioreactor consists
of a closed tank containing a stirring system. The constructs are suspended from the cap by means of metal
stems. Such systems have been used to seed cells and
subsequently culture them in vitro. Medium stirring enhances cell transfer to and within the construct, increases nutrient and oxygen delivery to the cells via convection and creates shear stress that could be valuable
to induced cell differentiation. However, it may also
provoke detrimental turbulence and cell shear stress.
Kim et al cultured human bone marrow mesenchymal
stem cells on aqueous-derived porous silk scaffolds
(15 mm in diameter, 5 mm thick, 900-1000 µm pore
size) using a spinner flask bioreactor (29). Enhanced
cell proliferation and differentiation (increased ALP activity and matrix mineralization) were obtained compared to static conditions. This was accounted for by the
increase in fluid-induced shear stress. As stated above,
Song et al have obtained better results with rotating
wall vessels than with spinner flasks while culturing
osteoblasts on human bio-derived bone scaffolds (51).
Sikavitsas et al investigated the effect of cell culture
conditions on the proliferation and differentiation of
rat bone marrow stromal cells seeded on 3D porous
75:25 poly(D, L-lactic-co-glycolic acid) biodegradable
scaffolds (46). Cell proliferation, ALP activity, osteocalcin secretion and calcium deposition were all enhanced when constructs were cultured in spinner flasks
compared to static conditions and cultures in rotating
wall vessels. Histological sections showed inhomogeneous cell distribution with a concentration of cells
and mineralization at the exterior of the foams at three weeks. Rubin et al studied the feasibility of using
injectable collagenous microbeads as a scaffold for
adipose-derived stem cells (31). Adipose-derived stem
cells isolated from human adipose tissue were cultured in a spinner flask. The cells attached, proliferated
and maintained a high level of viability. Once exposed
to osteogenic culture medium, they differentiated into
osteoblasts. Using coralline hydroxyapatite scaffolds,
Mygind et al cultured human mesenchymal stem cells
for three weeks in spinner flasks (8). Dynamic culture
resulted in increased proliferation, differentiation and
distribution of cells within the scaffolds. In their study, Meinel et al compared the effectiveness of culturing
264
human mesenchymal stem cells on three different protein scaffolds (unmodified and cross-linked collagen
and silk) in static conditions, spinner flasks or perfused
cartridges (7). Total calcium content and ALP activity
per unit of DNA were higher for constructs cultured in
spinner flasks than those in static conditions. Total calcium content was also higher than in the constructs
cultured in perfused cartridges. Certain histological
differences between both dynamic systems were also
observed: bone rods appeared at the periphery of the
constructs when spinner flasks were used, whereas a
randomly-distributed mineralized matrix materialized
throughout the construct when flow perfusion was applied.
Mechanical loading devices
Mechanical loading devices have recently been developed. The aim of using this type of system is to reproduce the mechanical stimuli found in vivo. As with
fluid shear stress, the application of mechanical stimuli greatly influences bone metabolism in vivo and applying such loads to constructs cultured in vitro may
have beneficial effects on in vitro tissue formation. The
consequences of compression, stretching and cyclic
strains on bone cells have been reported in a couple of
publications (49, 50).
Meyer et al proposed and designed a bioreactor-based
system making it possible to apply cyclic strains to 3D
constructs (49). Its effects on tissue specimens containing osteoblasts and chondrocytes were investigated.
Physiological loads (2000 µstrain) led to an increase in
cellularity and ECM proteins. The aim of the study by
Ignatius et al was to assess the effect of cyclic loading
on human osteoblastic precursor cells (hFOB 1.19) seeded on 3D type I collagen matrices (50). The constructs
were stretched daily by the application of cyclic uniaxial
strains. The expression of genes implicated in cell proliferation, differentiation, and matrix production was compared to that of unstretched controls. Cyclic stretching
increased cell proliferation and enhanced the expression
of all the investigated genes to some extent. Mechanical
loading devices will certainly attract more interest in future
studies as mechanical cues are particularly important in
bone physiology. However, these dynamic systems require the use of “soft scaffolds” such as hydrogels, polymers
or collagen that can support compressive loading.
© 2011 Wichtig Editore - ISSN 0391-3988
Carpentier et al
Advantages of dynamic culture within
bioreactors
In addition to providing sterile housing, culturing cells in
dynamic conditions has several advantages. The aforementioned studies have shown that convection forces are
crucial for obtaining homogenous cell seeding throughout
scaffolds (47, 48), increasing mass transport and promoting cell proliferation and differentiation (7, 12, 28, 46).
Homogenous cell seeding on and in 3D
scaffolds
Cell seeding corresponds to the distribution and dissemination of harvested and isolated cells within a scaffold. As
it is the first step in creating a hybrid biomaterial ex vivo,
careful attention must be paid to this critical and decisive
stage. Achieving homogenous cell distribution at a high
density enhances the likelihood of obtaining well-organized tissue formation throughout the scaffold. Harvested
cells are usually sub-cultured in vitro in order to obtain a
large number of cells and high density after cell seeding.
While static loading of cells is the most commonly used
seeding technique, numerous studies have documented
low seeding efficiency and inhomogeneous cell distribution within scaffolds (13, 47) under these circumstances.
Moreover, non-uniform cell colonization of the scaffolds
may lead to localized matrix deposits and substitutes that
are arranged spatially in an unsatisfactory manner.
Many authors have shown that cell proliferation and matrix
deposition in static culture is curtailed and limited to the
periphery of the scaffolds (54) while dynamic conditions
were able to improve cell ingrowth and favor homogenous
cell distribution within the construct (47, 48, 52). Wendt developed an original technique to enhance the homogeneity
of cell seeding based on an oscillating perfusion of cell suspensions through the scaffolds (46, 47).
Increase in mass transport
thors have shown enhanced cell viability, survival and cellularity at the center of scaffolds when convection forces
were applied compared to static culture (13, 47, 55). Direct
perfusion systems are often more efficient than spinner flasks and rotating wall vessels as the medium is compelled
to penetrate inside the scaffold to its core.
Production of shear stress and promotion of cell
differentiation
Bone cells have been shown to be highly sensitive and responsive to both mechanical forces and fluid shear stress,
in vivo as well as in vitro. Shear stress has been identified
as a parameter influencing cell metabolism. On bone cells,
it has for instance been shown to increase production of
nitric oxide (56, 57) and prostaglandin E2 (57).
Physiologically, interstitial fluid flows through canalicular
channels present in bone during cyclic loading. The data
from Durr et al suggest that the intensity of shear stress
generated on bone cells is proportional to the rate of loading (58) and that recovery periods between loading phases increase the effectiveness of osteogenesis and bone
formation. Mimicking in vivo stimuli such as shear stress
while culturing stem or bone cells ex vivo might thus help
generate bone-like tissue.
Along similar lines, a number of studies have shown the effect of fluid-induced shear stress on cell differentiation (13,
14, 28, 59). It has effectively been reported that dynamic
culture increases bone marker synthesis (52, 55, 60) (ALP,
osteocalcin, etc.) and enhances mineralized matrix deposition (12, 61). Fluid flow is responsible for the creation of
shear stress that has a direct impact on signal transduction
and cell metabolism.
Current limitations and potential use of modeling
approaches
Need for optimization
Adding convection forces to diffusion improves both nutrient and oxygen supply to the cells, and waste removal,
which may significantly enhance cell proliferation and metabolism (52, 55). Convection forces reduce nutrient and
oxygen gradients within culture systems leading to more
evenly distributed supplies. It helps medium components
penetrate into and to the core of the biomaterial. Many au-
Dynamic culture appears essential for obtaining homogenous cell seeding throughout 3D scaffolds, improving oxygen and nutrient delivery to the cells and producing shear
stress as well as promoting cell differentiation. Such culture systems should, however, be operated in a way that
makes it possible to have sufficient oxygen and nutrient
© 2011 Wichtig Editore - ISSN 0391-3988
265
Bioreactors for bone tissue engineering
Fig. 5 - The different steps in computational fluid dynamics for modeling
perfusion bioreactors: a) Computer
Assisted Design to create the geometry; b) 3D representation of all the
elements; c) Mesh implementation
for finite element or finite volume method; d) visualization of flow patterns,
pressure or shear forces (not shown)
in the bioreactor. Here, the entrance
only is shown, highlighting the presence of high recirculation.
transport without damaging the cultured bio-hybrid material. As a compromise has to be reached, it is essential that
the appropriate culture conditions be determined for each
system and each application.
Certain studies have compared the effectiveness of direct
perfusion systems, rotating wall vessels, and spinner flasks
in terms of cell seeding, cell proliferation, differentiation or
matrix production, and mineralization (7, 46, 51, 53). The
best results are typically obtained while using perfusion systems. Regarding spinner flasks and rotating wall vessels,
as the results are somewhat contradictory, a consensus
has not been reached. In any case, they lead to less evenly
distributed cells and both the expression of bone markers
and matrix mineralization are lower than what is obtained
using perfusion-based systems. From the available data,
mechanical loading devices appear to be likewise a precious means of promoting bone tissue formation but the
optimization of these dynamic systems requires further engineering and testing.
Numerical modeling of culture systems and
constructs
Modeling biological, chemical or physical events would
be a valuable tool for biologists who use dynamic culture
devices. These models could describe flow, mass transfer
266
and possible biological activities in the system under study.
Although modeling cannot replace experimentation, conducting such comprehensive work before starting a series
of experiments would be a way to gain time and reduce the
final number of trials.
Modeling the system that serves to engineer bone tissue
makes it possible to fully characterize the experimental
conditions. Many parameters are interdependent and have
a direct impact on cell culture. For instance, modifying fluid
flow leads to changes in pressure, shear stress and concentrations that need to be characterized since bone cells are very sensitive and responsive to mechanical stimuli
such as pressure and shear stress.
Modeling fluid motion within the chamber is also a means of
ensuring that there are no recirculation and dead volumes.
Such areas may have a very negative impact on nutrient
and oxygen delivery to the cells that are located there. Modeling is therefore essential while designing the geometry
of a culture device. As an example, Figure 5 shows the
different steps from the computer assisted design (CAD) of
a bioreactor to the simulation of culture medium flow at its
entrance. Computational fluid dynamics (CFD) evidenced
major recirculation zones that led to the initial design to be
changed before manufacturing the bioreactor.
Pierre et al have concentrated their interest on the transport phenomena occurring within perfusion bioreactors
© 2011 Wichtig Editore - ISSN 0391-3988
Carpentier et al
(62). Limiting factors may indeed impact cell proliferation,
differentiation and viability. A two-dimensional mathematical model, based on stationary mass and momentum
transport has been proposed and numerically solved. The
results show that fluid velocity is a critical parameter influencing oxygen concentration at the cell surface and
therefore cell metabolism. A compromise between oxygen
delivery – which has to be high enough – and shear stress
has to be reached in order to achieve suitable culture conditions.
Kwon et al conducted a numerical (CFD) analysis of a rotating wall bioreactor to evaluate oxygen transport (63).
Their study shows that the rotating speed for appropriate
suspension needs to be increased as aggregate size increases, leading to higher mean oxygen concentrations.
In one study, the authors used the Lattice-Boltzmann
method to simulate the flow conditions in perfused cellseeded cylindrical scaffolds (64). The scaffold architecture
was defined using microcomputed tomography imaging
and shear stress was estimated at various media flow rates by multiplying the symmetric part of the gradient of the
velocity field by the dynamic viscosity of the cell culture
media. Based on these few examples, the need for comprehensive numerical and mathematical models is obvious in the
field of bone tissue engineering. Such models either attempt to better describe and understand cell consumption,
nutrient and oxygen delivery, and protein synthesis, or instead focus on mechanical aspects such as static pressure
or fluid-induced shear stress generated at the cell surface.
They then need to be compared with experimental data for
further validation.
Summary and considerations for the future
Recent advances in bone tissue engineering have led orthopedic and maxillofacial surgeons to consider introducing these techniques into their clinical practice. However,
the reconstruction of extended bone defects still remains
challenging. Some clinical studies with low numbers of patients have been reported using this approach but the outcomes were inconsistent and showed low efficacy in bone
regeneration (65, 66). The reasons for the limited clinical
success may include several factors in the multidisciplinary field of bone tissue engineering.
Specially designed bioreactors may reproduce the forces
encountered by cells during bone tissue development as
well as improving the diffusion of nutrients and oxygen
through the 3D cell-laden constructs. Another challenge
for bone tissue engineering is the 3D organization of different cell types in the scaffold as well as vascularization
and degradation at the expense of ECM production. Controlling these factors may deliver tissue constructs most
resembling natural bone tissue.
Culturing 3D constructs of cells and hydrogels using bioreactors should recreate the biomechanics of bone tissue. A
system that applies force to mesenchymal stem cells, osteoclasts, and endothelial cells within cross-linked hydrogels
would certainly be helpful for understanding the formation
and remodeling of bone tissue.
The concept of growing a bone tissue equivalent ex vivo
using multiple cell populations within 3D biomaterials under dynamic conditions is ambitious and requires a multidisciplinary approach in order to be successful. Growing
bone tissue equivalents may also serve as alternatives for
in vivo testing and may thus reduce animal experimentations.
Bone tissue engineering still lacks proof of clinical efficacy,
possibly due to the inadequate design of the tissue engineered constructs compared to natural tissue. Further advances in this field may help in the understanding of stem
cell differentiation in relation to the micro-environment and
lead to the development of tissue engineered bone grafts
with more predictable clinical performances. This would be
of great benefit for patients.
ACKNOWLEDGEMENTS
The authors would like to thank Dr J. Sohier for the SEM image
of biodegradable polymer microfibers used in Figure 4.
Financial support: This work was supported financially by both the
Inserm National Program for Research in Osteoarticular diseases
(PRO-A) and the National Research Agency (ANR TecSan) through
the ATOS project.
Conflict of interest statement: None of the authors has any competing interest to declare.
© 2011 Wichtig Editore - ISSN 0391-3988
267
Bioreactors for bone tissue engineering
LIST OF ABBREVIATIONS
ALP = Alkaline phosphatase
BCP = biphasic calcium phosphate
BMSC = Bone marrow stromal cell
CAD = Computer assisted design
CFD = Computational fluid dynamics
DNA = Deoxyribonucleic acid
ECM = Extracellular matrix
HA = Hydroxyapatite
hMSC = Human mesenchymal stem cell
MSC = Mesenchymal stem cell
PDMS = Polydimethylsiloxane
PLGA = Poly(-lactic-co-glycolic acid)
TCP = Tricalcium phosphate
TRAP = Tartrate resistant acid phosphatase
3D = Three-dimensional
Address for correspondence:
Cécile Legallais
University of Technology of Compiègne
UMR CNRS 6600, Biomechanics and Bioengineering
Centre de recherche de Royallieu
BP 20529
60205 Compiègne Cedex, France
e-mail: [email protected]
REFERENCES
1. Kneser U, Schaefer DJ, Polykandriotis E, Horch RE. Tissue
engineering of bone: the reconstructive surgeon’s point of
view. J Cell Mol Med 2006; 10: 7-19.
2. Giannoudis PV, Dinopoulos H, Tsiridis E. Bone substitutes:
An update. Injury 2005; 36: S20-7.
3. Laurencin C, Khan Y, El-Amin SF. Bone graft substitutes. Expert Rev Med Devices 2006; 3: 49-57.
4. Fellah BH, Gauthier O, Weiss P, Chappard D, Layrolle P.
Osteogenicity of biphasic calcium phosphate ceramics
and bone autograft in a goat model. Biomaterials 2008; 29:
1177-88.
5. Petite H, Viateau V, Bensaï W, et al. Tissue-engineered bone
regeneration. Nat Biotechnol 2000; 18: 959-963.
6. Khan Y, Yaszemski MJ, Mikos AG, Laurencin CT. Tissue
engineering of bone: material and matrix considerations. J
Bone Joint Surg (Am) 2008; 90 (Suppl 1): 36-42.
7. Meinel L, Karageorgiou V, Fajardo R, et al. Bone tissue engineering using human mesenchymal stem cells: effects of
scaffold material and medium flow. Ann Biomed Eng 2004;
32: 112-22.
8. Mygind T,Stiehler M, Baatrup A, et al. Mesenchymal stem
cell ingrowth and differentiation on coralline hydroxyapatite
268
scaffolds. Biomaterials 2007; 28: 1036-47.
9. Ben-Nissan B. Natural bioceramics: from coral to bone and
beyond. Curr Opin Solid State Mater Sci 2003; 7: 283-8.
10. Turhani D, Watzinger E, Weissenböck M, et al. Analysis of
cell-seeded 3-dimensional bone constructs manufactured in
vitro with hydroxyapatite granules obtained from red algae.
J Oral Maxillofac Surg 2005; 63: 673-81.
11. Janssen FW, Oostra J. Oorschot Av, van Blitterswijk CA. A
perfusion bioreactor system capable of producing clinically
relevant volumes of tissue-engineered bone: In vivo bone
formation showing proof of concept. Biomaterials 2006; 27:
315-23.
12. Bancroft GN, Sikavitsas VI, van den Dolder J, et al. Fluid
flow increases mineralized matrix deposition in 3D perfusion
culture of marrow stromal osteoblasts in a dose-dependent
manner. Proc Natl Acad Sci USA 2002; 99: 12600-5.
13. Du D, Furukawa K, Ushida T. Oscillatory perfusion seeding
and culturing of osteoblast-like cells on porous beta-tricalcium phosphate scaffolds. J Biomed Mater Res A 2008; 86:
796-803.
14. Leclerc E, David B, Griscom L, et al. Study of osteoblastic
cells in a microfluidic environment. Biomaterials 2006; 27:
586-95.
15. Qiu QQ, Ducheyne P, Ayyaswamy PS. Fabrication, characterization and evaluation of bioceramic hollow microspheres
used as microcarriers for 3-D bone tissue formation in rotating bioreactors. Biomaterials 1999; 20: 989-1001.
16. Bashur CA, Shaffer RD, Dahlgren LA, Guelcher SA, Goldstein AS. Effect of Fiber Diameter and Alignment of Electrospun Polyurethane Meshes on Mesenchymal Progenitor
Cells. Tissue Eng Part A 2009; 15: 2435-45.
17. Terraciano V, Hwang N, Moroni L, et al. Differential response
of adult and embryonic mesenchymal progenitor cells to
mechanical compression in hydrogels. Stem Cells 2007; 25:
2730-8.
18. Spoerke ED, Murray NG, Li H, Brinson LC, Dunand DC,
Stupp SI. A bioactive titanium foam scaffold for bone repair.
Acta Biomater 2005; 1: 523-33.
19. Ignjatovic N, Ninkov P, Ajdukovic Z, Vasiljevic-Radovic D,
Uskokovic D. Biphasic calcium phosphate coated with polyd,l-lactide-co-glycolide biomaterial as a bone substitute. J
Eur Ceram Soc 2007; 27: 1589-94.
20. Lutolf MP, Hubbell JA. Synthetic biomaterials as instructive
extracellular microenvironments for morphogenesis in tissue
engineering. Nat Biotechnol 2005; 23: 47-55.
21. Hartgerink JD, Beniash E, Stupp SI. Self-assembly and mineralization of peptide-amphiphile nanofibers. Science 2001;
294: 1684-8.
22. Lee KY, Mooney DJ. Hydrogels for tissue engineering. Chem
Rev 2001; 101: 1869-79.
23. Ladet S, David L, Domard A. Multi-membrane hydrogels.
Nature 2008; 452: 76-9.
24. Fellah BH, Weiss P, Gauthier O, et al. Bone repair using a
new injectable self-crosslinkable bone substitute. J Orthop
© 2011 Wichtig Editore - ISSN 0391-3988
Carpentier et al
Res 2006; 24: 628-35.
25. Vinatier C, Magne D, Moreau A, et al. Engineering cartilage
with human nasal chondrocytes and a silanized hydroxypropyl methylcellulose hydrogel. J Biomed Mater Res A 2007;
80: 66-74.
26. Trojani C, Weiss P, Michiels JF, et al. Three-dimensional culture and differentiation of human osteogenic cells in an injectable hydroxypropylmethylcellulose hydrogel. Biomaterials 2005; 26: 5509-17.
27. Dhurjati R, Liu X, Gay CV, Mastro AM, Vogler EA. Extendedterm culture of bone cells in a compartmentalized bioreactor.
Tissue Eng 2006; 12: 3045-54.
28. Datta N, Pham QP, Sharma U, Sikavitsas VI, Jansen JA,
Mikos AG. In vitro generated extracellular matrix and fluid
shear stress synergistically enhance 3D osteoblastic differentiation. Proc Natl Acad Sci USA 2006; 103: 2488-93.
29. Kim HJ, Kim UJ, Leisk GG, Bayan C, Georgakoudi I, Kaplan
DL. Bone regeneration on macroporous aqueous-derived
silk 3-D scaffolds. Macromol Biosci 2007; 7: 643-55.
30. Bianco P, Riminucci M, Gronthos S, Robey PG. Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cell 2001; 19: 180-92.
31. Rubin JP, Bennett JM, Doctor JS, Tebbets BM, Marra KG.
Collagenous microbeads as a scaffold for tissue engineering
with adipose-derived stem cells. Plast Reconstr Surg 2007;
120: 414-24.
32. Cui L, Liu B, Liu G, et al. Repair of cranial bone defects with
adipose derived stem cells and coral scaffold in a canine
model. Biomaterials 2007; 28: 5477-86.
33. Derubeis AR, Cancedda R. Bone marrow stromal cells (BMSCs) in bone engineering: limitations and recent advances.
Ann Biomed Eng 2004; 32: 160-5.
34. Derubeis AR, Mastrogiacomo M, Cancedda R, Quarto R.
Osteogenic potential of rat spleen stromal cells. Eur J Cell
Biol 2003; 82: 175-81.
35. Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation
and activation. Nature 2003; 423: 337-42.
36. Meyer U, Joos U, Wiesmann HP. Biological and biophysical
principles in extracorporeal bone tissue engineering: Part I.
Int J Oral Maxillofac Surg 2004; 33: 325-32.
37. Buttiglieri G, Peschka M, Fromel T, et al. Environmental occurrence and degradation of the herbicide n-chloridazon.
Water Res 2009; 43: 2865-73.
38. Yang Z, Zhou S, Sun Y. Start-up of simultaneous removal of
ammonium and sulfate from an anaerobic ammonium oxidation (anammox) process in an anaerobic up-flow bioreactor.
J Hazard Mater 2009; 169: 113-8.
39. Zhou LG, Wu JY. Development and application of medicinal
plant tissue cultures for production of drugs and herbal medicinals in China. Nat Prod Rep 2006; 23: 789-810.
40. Liu CZ, Murch SJ, El-Demerdash M, Saxena PK. Artemisia
judaica L.: micropropagation and antioxidant activity. J Biotechnol 2004; 110: 63-71.
41. Panagiotou G, Andersen MR, Grotkjaer T, Regueira TB,
42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. Nielsen J, Olsson L. Studies of the production of fungal
polyketides in Aspergillus nidulans by using systems biology
tools. Appl Environ Microbiol 2009; 75: 2212-20.
Chen J, Chen H, Zhu X, et al. Long-term production of soluble human Fas ligand through immobilization of Dictyostelium discoideum in a fibrous bed bioreactor. Appl Microbiol
Biotechnol 2009; 82: 241-8.
Mazutti MA, Zabot G, Boni G, et al. Kinetics of inulinase production by solid-state fermentation in a packed-bed bioreactor. Food Chem 2010; 120: 163-73.
Martin I, Wendt D, Heberer M. The role of bioreactors in tissue engineering. Trends Biotechnol 2004; 22: 80-6.
Portner R, Nagel-Heyer S, Goepfert C, Adamietz P, Meenen
NM. Bioreactor design for tissue engineering. J Biosci Bioeng 2005; 100: 235-45.
Sikavitsas VI, Bancroft GN, Mikos AG. Formation of three-dimensional cell/polymer constructs for bone tissue engineering in a spinner flask and a rotating wall vessel bioreactor. J
Biomed Mater Res 2002; 62: 136-48.
Wendt D, Marsano A, Jakob M, Heberer M, Martin I. Oscillating perfusion of cell suspensions through three-dimensional
scaffolds enhances cell seeding efficiency and uniformity.
Biotechnol Bioeng 2003; 84: 205-14.
Wendt D, Jakob M, Martin I. Bioreactor-based engineering
of osteochondral grafts: from model systems to tissue manufacturing. J Biosci Bioeng 2005; 100: 489-94.
Meyer U, Buchter A, Nazer N, Wiesmann HP. Design and
performance of a bioreactor system for mechanically promoted three-dimensional tissue engineering. Br J Oral Maxillofac Surg 2006; 44: 134-40.
Ignatius A, Blessing H, Liedert A, et al. Tissue engineering of
bone: effects of mechanical strain on osteoblastic cells in
type I collagen matrices. Biomaterials 2005; 26: 311-8.
Song K, Liu T, Cui Z, Li X, Ma X. Three-dimensional fabrication of engineered bone with human bio-derived bone scaffolds in a rotating wall vessel bioreactor. J Biomed Mater
Res A 2008; 86: 323-32.
Goldstein AS, Juarez TM, Helmke CD, Gustin MC, Mikos
AG. Effect of convection on osteoblastic cell growth and
function in biodegradable polymer foam scaffolds. Biomaterials 2001; 22: 1279-88.
Song KD, Liu TQ, Li XQ, Cui ZF, Sun XY, Ma XH. Three-dimensional expansion: in suspension culture of SD rat’s osteoblasts in a rotating wall vessel bioreactor. Biomed Environ
Sci 2007; 20: 91-8.
Xie Y, Hardouin P, Zhu Z, Tang T, Dai K, Lu J. Three-dimensional flow perfusion culture system for stem cell proliferation inside the critical-size beta-tricalcium phosphate scaffold. Tissue Eng 2006; 12: 3535-43.
Cartmell SH, Porter BD, Garcia AJ, Guldberg RE. Effects of
medium perfusion rate on cell-seeded three-dimensional
bone constructs in vitro. Tissue Eng 2003; 9: 1197-203.
Bacabac RG, Smit TH, Mullender MG, Dijcks SJ, Van Loon
JJ, Klein-Nulend J. Nitric oxide production by bone cells is
© 2011 Wichtig Editore - ISSN 0391-3988
269
Bioreactors for bone tissue engineering
57. 58. 59. 60. 61. 270
fluid shear stress rate dependent. Biochem Biophys Res
Commun 2004; 315: 823-9.
Bakker AD. Soejima., Klein-Nulend J. Burger EH. The production of nitric oxide and prostaglandin E2 by primary
bone cells is shear stress dependent. J Biomech 2001; 34:
671-7.
Burr DB, Robling AG, Turner CH. Effects of biomechanical
stress on bones in animals. Bone 2002; 30: 781-6.
Botchwey EA, Pollack SR, Levine EM, Laurencin CT. Bone
tissue engineering in a rotating bioreactor using a microcarrier matrix system. J Biomed Mater Res 2001; 55: 242-53.
Wang Y, Uemura T, Dong J, Kojima H, Tanaka J, Tateishi T.
Application of perfusion culture system improves in vitro
and in vivo osteogenesis of bone marrow-derived osteoblastic cells in porous ceramic materials. Tissue Eng 2003;
9: 1205-14.
Sikavitsas VI, Bancroft GN, Holtorf HL, Jansen JA, Mikos
AG. Mineralized matrix deposition by marrow stromal osteo-
62. 63. 64. 65. 66. blasts in 3D perfusion culture increases with increasing fluid
shear forces. Proc Natl Acad Sci USA 2003: 100: 14683-8.
Pierre J, Oddou C. Engineered bone culture in a perfusion
bioreactor: a 2D computational study of stationary mass and
momentum transport. Comput Methods Biomech Biomed
Engin 2007; 10: 429-38.
Kwon O, Devarakonda SB, Sankovic JM, Banerjee RK. Oxygen transport and consumption by suspended cells in microgravity: a multiphase analysis. Biotechnol Bioeng 2008;
99: 99-107.
Porter B, Zauel R, Stockman H, Guldberg R, Fyhrie D. 3-D
computational modeling of media flow through scaffolds in a
perfusion bioreactor. J Biomec 2005; 38: 543-9.
Quarto R, Mastrogiacomo M, Cancedda R, et al. Repair of
large bone defects with the use of autologous bone marrow
stromal cells. N Engl J Med 2001;344:385-6.
Meijer GJ, de Bruijn JD, Koole R, van Blitterswijk CA. Cellbased bone tissue engineering. PLoS Med 2008; 29: 3053-61.
© 2011 Wichtig Editore - ISSN 0391-3988