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Bioreactor design for tissue engineering

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JOURNAL OF BIOSCIENCE AND BIOENGINEERING 2005, The Society for Biotechnology, Japan
Vol. 100, No. 3, 235245. 2005
DOI: 10.1263/jbb.100.235

REVIEW
Bioreactor Design for Tissue Engineering
Ralf Prtner,1* Stephanie Nagel-Heyer,1 Christiane Goepfert,1
Peter Adamietz,2 and Norbert M. Meenen3
Technische Universitt Hamburg-Harburg, Bioprozess- und Bioverfahrenstechnik, Denickestr. 15, 21071 Hamburg,
Germany,1 Universittsklinikum Eppendorf, Institut fr Biochemie und Molekularbiologie II, Martinistr. 52,
20246 Hamburg, Germany,2 and Universittsklinikum Eppendorf, Unfall-, Hand- und
Wiederherstellungschirurgie, Martinistr. 52, 20246 Hamburg, Germany3

Received 7 March 2005/Accepted 31 May 2005

Bioreactor systems play an important role in tissue engineering, as they enable reproducible and
controlled changes in specific environmental factors. They can provide technical means to per-
form controlled studies aimed at understanding specific biological, chemical or physical effects.
Furthermore, bioreactors allow for a safe and reproducible production of tissue constructs. For
later clinical applications, the bioreactor system should be an advantageous method in terms of
low contamination risk, ease of handling and scalability. To date the goals and expectations of
bioreactor development have been fulfilled only to some extent, as bioreactor design in tissue engi-
neering is very complex and still at an early stage of development. In this review we summarize
important aspects for bioreactor design and provide an overview on existing concepts. The gener-
ation of three dimensional cartilage-carrier constructs is described to demonstrate how the prop-
erties of engineered tissues can be improved significantly by combining biological and engineering
knowledge. In the future, a very intimate collaboration between engineers and biologists will lead
to an increased fundamental understanding of complex issues that can have an impact on tissue
formation in bioreactors.

[Key words: tissue engineering, bioreactor, design considerations, cartilage]

The loss and damage of tissues cause serious health prob-
lems (1). In the US, almost one-half of the costs for medical
treatments are spent on implant devices annually (2). World-
wide, 350 billion USD are expended for substitute of organs
(3). The substitution of tissues (such as bone or cartilage) or
joints with allograft materials includes the risk of infections
by viruses (such as HIV, hepatitis C) or a graft rejection. Ar-
tificial implants such as those used in knee or hip replace-
ment, have limitations due to their limited lifespan, insuffi-
cient bonding to the bone, and allergic reactions caused by
material abrasion. New therapy concepts for practical medi-
cal applications are required. To this end, tissue engineered
substitutes generated in vitro could open new strategies for
the restoration of damaged tissues. The goal of tissue engi-
neering can be defined as the development of cell-based
substitutes to restore, maintain or improve tissue function.
These substitutes should have organ-specific properties with
respect to biochemical activity, microstructure, mechanical
integrity and biostability (2). Cell-based concepts include the
(i) direct transplantation of isolated cells, (ii) implantation
of a bioactive scaffold for the stimulation of cell growth
within the original tissue and (iii) implantation of a three di-
* Corresponding author. e-mail: poertner@tuhh.de
phone: +49-40-42878-2886 fax: +49-40-42878-2909
mensional (3D) biohybrid structure of a scaffold and cul-
tured cells or tissue. Furthermore, non implantable tissue
structures can be applied as external support devices (e.g.,
an extracorporal liver support when a compatible donor or-
gan is not readily available [4, 5]) or engineered tissues can
be used as in vitro physiological models for studying dis-
ease pathogenesis and developing new molecular therapeu-
tics (e.g., drug screening [5, 6]).
The generation of 3D tissue substitutes in vitro requires
not only a biological model (e.g., an adequate source for
proliferable cells with appropriate biological functions, a
protocol for proliferating cells while maintaining the tissue-
specific phenotype), but also the further development of new
culture strategies including bioreactor concepts (5, 7, 8).
Bioreactors are well established for the cultivation of mi-
crobes or mammalian cells under monitored and controlled
environmental and operational conditions (e.g., pH, temper-
ature, oxygen tension, and nutrient supply) up to an indus-
trial scale. However, as individual cells are mostly applied,
these concepts are inapplicable to 3D tissue constructs. Fur-
thermore, each type of tissue construct (e.g., skin, bone,
blood vessel, and cartilage) will likely require an individual-
ized bioreactor design (7). Therefore, tissue-specific biore-
actors should be designed on the basis of comprehensive
understanding of biological and engineering aspects. Addi-

235
236 PRTNER ET AL.
tionally, typical engineering aspects such as reliability, repro-
ducibility, scalability and safety should be addressed (5, 8).
Several bioreactor systems have been developed and usually
the expectations are very high (5, 6). The question is, how-
ever, whether bioreactors can indeed fulfil these expecta-
tions.
In this review, key technical challenges are identified and
an overview of existing culture systems and bioreactors used
for tissue engineering is provided. These topics have been
addressed to some extent by several authors (4, 5, 716).
Therefore, they will be discussed only briefly. Particular
focus will be given to the interaction between biological and
engineering aspects. Using cartilage tissue formation as an
example, it will be shown how an increased fundamental
understanding of biological, biochemical, and engineering
aspects can significantly improve the properties of 3D tissue
constructs.
IMPORTANT ASPECTS FOR
BIOREACTOR DESIGN
With respect to tissue engineering, bioreactors are used
for different purposes such as (i) cell proliferation on a
small scale (e.g., for individual patients) and on a large scale
(e.g., for allogenic therapy concepts), (ii) generation of 3D
tissue constructs from isolated and proliferated cells in vitro
and (iii) direct organ support devices (15). These bioreac-
tors should enable the control of environmental conditions
such as oxygen tension, pH, temperature, and shear stress
and allow aseptic operation (e.g., feeding and sampling).
Furthermore, a bioreactor system should allow for auto-
mated processing steps. This is essential not only for con-
trolled, reproducible, statistically relevant basic studies but
also for the future routine manufacturing of tissues for clini-
cal application (5, 17). Besides these global requirements,
specific key criteria for 3D tissue constructs based on cells
and scaffolds have to be met, including the proliferation of
cells, seeding of cells on macroporous scaffolds, nutrient
(particularly oxygen) supply within the resulting tissue, and
mechanical stimulation of the developing tissues (5).
Proliferation of cells is the first step in establishing a tis-
sue culture. Usually, only a small number of cells can be ob-
tained from a biopsy specimen and expansion up to several
orders of magnitude is required. The proliferation of cells is
quite often accompanied by the dedifferentiation of cells.
For example, proliferating chondrocytes show a decreased
expression level of collagen type II and an increased expres-
sion level of collagen type I (18, 19). Small culture dishes
(e.g., petri dishes, 12-well plates, and T-flasks) are generally
used for cell expansion. As these devices allow only an in-
crease in cell number by a factor of approximately 10, sev-
eral subcultivations are required. These are considered as a
major cause for the dedifferentiation of cells. Recent studies
have shown that microcarrier cultures performed in well-
mixed bioreactor systems can significantly improve cell ex-
pansion (2022).
The cell seeding of scaffolds is an important step in estab-
lishing a 3D culture in a macroporous scaffold. Not only
seeding at high cell densities, but also a homogenious distri-
bution of cells within the scaffold is essential. High initial
J. BIOSCI. BIOENG.,
cell densities have been associated with an enhanced tissue
formation including cartilage matrix production, bone min-
eralization and cardiac tissue structure formation (2325).
On the other hand, an inhomogeneous distribution of cells
within the scaffold can significantly affect the tissue proper-
ties. Several techniques for cell seeding were discussed by
Martin et al. (5).
Critical issues of all bioreactor concepts involve mass
transfer problems (e.g., oxygen and nutrient supply, and re-
moval of toxic metabolites). The size of most engineered
tissues is limited as they do not have their own blood system
and the cells are only supplied by diffusion (6, 26). Oxygen
supply is particularly critical, as only cell layers of 100200
m can be supplied by diffusion (27). However, as tissue
constructs should have larger dimensions, mass-transfer lim-
itations represent one of the greatest engineering challenges.
Various studies showed that mechanical stimulation (e.g.,
mechanical compression, hydrodynamic pressure, and fluid
flow), which are important modulators of cell physiology,
can have a positive impact on tissue formation (28), particu-
larly in the context of musculoskeletal tissue engineering,
cartilage formation, and cardiovascular tissues among others
(11, 2934). Despite accumulating evidence that mechani-
cal stimulation can improve the properties of engineered tis-
sues, only little is known about specific mechanical forces
or the ranges of application (i.e., magnitude, frequency, con-
tinuous or intermittent, and duty cycle [5, 9]). Further stud-
ies of these factors have to be coupled with quantitative and
computational analyses of physical forces experienced by
cells and changes in mass transport induced by the method
used.
Bioreactors allow for different process strategies includ-
ing the batch, fed-batch or continuous cultivation. In particu-
lar, continuous perfusion enables cultivation under constant
and controlled environmental conditions (3538). Martin et
al. (5) summarized some of the effects of direct perfusion
on tissue-specific properties such as growth, differentiation
and mineralized matrix deposition by bone cells, prolifera-
tion of human oral keratinocytes, rates of albumin synthesis
by hepatocytes, expression of cardiac-specific markers by
cardiomyocytes, and GAG synthesis and accumulation by
chondrocytes. On the other hand, a bioreactor system be-
comes more complex as additional features such as feeding
pumps, vessels for fresh and spent medium, and control
strategies are required (Fig. 1), particularly in the case of
mechanical stimulation. The bioreactor system has to be in-
tegrated into the entire cultivation scheme including biopsy,
proliferation, cell seeding, tissue formation and delivery to
the site of application (e.g., hospital). In many cases, the
bioreactor itself is only used for tissue formation. However,
for a wholistic approach from biopsy to the implantation of
tissue, the intire procedure should be coordinated to de-
crease the number of steps, risk of contamination, and labor
costs among others. This is particularly important with re-
spect to the manufacture of engineered tissue constructs for
clinical applications, in which good manufacturing practice
(GMP) requirements have to be met (7, 8).
VOL. 100, 2005
FIG. 1. Set-up of flow-chamber-bioreactor system consisting of flow chamber with inserts for tissue constructs, conditioning vessel, peristaltic
pump, humidifier, exhaust flask, and medium exchange bottle.
CULTURE SYSTEMS AND BIOREACTORS
USED IN TISSUE ENGINEERING
The engineering of 3D tissue constructs include cell main-
tenance, proliferation and tissue formation. An overview of
culture systems and bioreactors used for these purposes is
shown in Fig. 2. Cell maintenance and proliferation is usu-
ally performed in culture systems developed for the mono-
layer culture of adherent cells (T-flasks, petri dishes, multi
well plates). These systems enable sterile handling proce-
dures and are easy to use, disposable and low-cost (39).
Moreover, they require individual handling as for example
in medium exchange and cell seeding; their usefulness is
limited when large quantities are required (7). This can be
overcome to some extent using sophisticated robotics (5).
Furthermore, controlling environmental parameters includ-
ing pH, pO2, and temperature is generally impossible. A fur-
ther drawback is the limited increase in cell number (ap-
proximately 1020 times during cultivation). The generation
of a large number of cells requires several enzymatic sub-
cultivation steps accompanied by an increase in passage
number and cell dedifferentiation.
In recent studies, small well-mixed bioreactors (e.g., shake
flasks, stirred vessels, and super spinner) have been sug-
gested for cell proliferation, in which the cells are grown on
microcarriers (2022). These systems were used in the culti-
vation of encapsulated cells (18, 19) or neural stem cells in
single-cell suspension culture (40).
In fixed bed and fluidized bed bioreactors, cells are immo-
bilized in macroporous carriers. The carriers are arranged in
a column either packed (fixed bed) or floating (fluidized
bed). The column is permanently perfused with a condi-
tioned medium from a medium reservoir, mostly in a circu-
lation loop. These types of reactor are very efficient for the
long-term cultivation of mammalian cells for the production
of biopharmaceuticals (e.g., monoclonal antibodies, recom-
BIOREACTOR DESIGN FOR TISSUE ENGINEERING 237
binant drugs including tPA, and EPO) or recombinant retro-
viruses for gene therapy (4143). With respect to tissue en-
gineering they have been investigated for several applica-
tions including the cultivation of liver cells as an extra-
corporal liver device (4, 10, 37), proliferation of stem cells
(4446), cultivation of cardiovascular cells (11) and carti-
lage cells (9). As most of the scaffolds have large, inter-
connected pores, during seeding, cells are distributed quite
uniformly. During cultivation, medium flow through a con-
struct enhances the mass transfer of substrates, particularly
oxygen to immobilized cells when interconnected cell free
pores are available.
In membrane bioreactors including hollow-fiber reactors
(47), the miniPerm system (48) or the tecnomouse (49),
cells are cultivated at tissuelike densities in a compartment,
which contains one or several types of membrane for nu-
trient and oxygen supply and removal of toxic metabolites.
Hollow-fiber systems are widely used in the production of
biopharmaceuticals including monoclonal antibodies. Several
examples of modified membrane bioreactors exist for the
3D culture of tissue cells including hepatocytes (4, 5054),
skin cells (55) or other human cells (49, 56).
Most of the culture systems and bioreactors discussed so
far were first developed for the cultivation of mammalian
cells and adapted to the engineering of 3D tissue constructs.
However, besides some exceptions, they can hardly be used
in the generation of implantable tissue constructs. Each type
of tissue intended for implantation (e.g., skin, heart valve,
blood vessel, and cartilage) requires a different geometric
structure and a specific bioreactor design, particularly when
mechanical stimulation has to be provided. Finally, engi-
neered constructs have to be transferred from the culture
system to the patient after cultivation. Therefore, several tis-
sue-specific culture systems and bioreactors have been sug-
gested.
One of the most prominent culture systems is the rotat-
238 PRTNER ET AL.
FIG. 2. Cell culture systems used in tissue engineering.
ing-wall vessel (23), in which a construct remains in a state
of free-fall through the medium with a low shear stress and
a high mass transfer rate. This system has a wide range of
practical applications (5, 7). A multipurpose culture system
was introduced by Minuth et al. (35) for perfusion cultures
under organotypic conditions. Several tissue carriers can be
placed inside a perfusion container. Depending on the type
of tissue-specific cell supports can be selected. A perfused
flow-chamber bioreactor with a new concept for aeration
has been introduced recently (20), in which tissue-specific
inserts for various types of tissue (e.g., cartilage, skin, and
bone) can be applied.
Besides these examples of multipurpose bioreactors, nu-
merous of tissue-specific culture systems have been sug-
gested. These are well reviewed with respect to specific tis-
sues (5, 6, 811, 14, 15, 26, 50, 5759). Most of them are
custom-made; only very few have been commercialized.
MODELLING OF BIOREACTOR SYSTEMS
FOR TISSUE ENGINEERING
The appropriate molecular and macroscopic architecture
of 3D tissue constructs is essential to producing a phenotyp-
ically appropriate tissue (6). However, in many cases this is
not realized. The solution may lie in providing appropriate
physiological conditions during cultivation. In many cases,
the culture systems and bioreactors were not optimized in
this respect. Parameters such as perfusion rate, flow condi-
tions, shear stress, and compression magnitude were varied,
quite often by a trial-and-error approach. Furthermore, dif-
ferent conditions have to be examined accurately with re-
gard to their effect. For example, hydrostatic pressure ap-
plied during cartilage culture can lead to an improved mass
transfer of small and large molecules into the cartilage ma-
trix, but can also induce a mechanical stimulation of embed-
ded cells. These two effects have to be examined separately.
Particularly, when if forces (e.g., hydrostatic pressure, shear
J. BIOSCI. BIOENG.,
stress induced by perfusion, shear stress or gliding forces
induced by mechanical impact, and pulsation flow) are ap-
plied, the exact local conditions experienced by the cells
have to be determined. Therefore, experimental studies
should be supported by simulation methods such as the
computational fluid dynamics (CFD) or finite-element ap-
proach. Several examples underline the potential of an inte-
grated study of mechanical and biomechanical factors that
control the functional development of tissue engineered con-
structs (6066). This approach will significantly improve
bioreactor design in the near future.
As an attempt to evaluate and compare different tissue
engineering reactors, Omasa et al. (67) applied the analytic
hierarchy process (AHP). For evaluating a reactor for a bio-
artificial liver (BAL) support system, five criteria were iden-
tified (namely, safety, scalability, cell growth environment,
mimicking native liver functions and handling). On the ba-
sis of these criteria, six different types of BAL systems were
successfully ranked using AHP. The process is a valuable
tool for decision making in tissue engineering, not only for
BAL systems but for tissue engineering reactors in general.
INTERACTION BETWEEN BIOLOGICAL AND
ENGINEERING ASPECTS IN GENERATION
OF ARTIFICIAL CARTILAGE
The following considerations are intended to demonstrate
how the interdisciplinary application of biological and engi-
neering knowledge can significantly improve the properties
of tissue engineered 3D cartilage constructs. Severe health
problems can arise from a lack or damage of hyaline carti-
lage in joints, particularly in knee joints (68). The methods of
tissue engineering enable the generation of cartilage tissue
in vitro and provide new strategies to restoring damaged
cartilage (9, 36, 58, 69). Whereas many approaches to gen-
erating tissue-engineered cartilage are based on either algi-
nate encapsulation or the use of bioresorbable scaffolds (9,
VOL. 100, 2005
19, 58, 59, 69, 70), recently, a new concept involving the
use of a bone substitute carrier covered with a layer of
tissue-engineered cartilage without scaffolds has been pre-
sented (17). This concept consists of four steps: (i) ex-
planted articular chondrocytes are expanded in a monolayer
culture and then seeded on a solid carrier to form a primer
layer; (ii) simultaneously, chondrocytes are suspended in
alginate gel and cultivated for two weeks to allow the for-
mation of a pericellular matrix; (iii) redifferentiated chon-
drocytes are then eluted out of the alginate gel and sedi-
mented on the primer layer; (iv) the biphasic constructs are
further cultivated for three weeks. Below, mainly different
aspects of step iv are addressed. A similar concept was sug-
gested by Waldman et al. (71).
A critical parameter during the cultivation of cartilage
tissue is oxygen supply. The effect of oxygen during the in
vitro cultivation of chondrocytes is poorly understood, and
therefore it is presently a controversial issue (72). In several
studies, chondrocytes were immobilized in alginate beads
and cultivated under different oxygen concentrations in the
gas phase (for review see Ref. 72). ODriscoll et al. (73)
observed a limited collagen type II production at very high
(90% O2) and very low (15% O2) oxygen concentrations.
Domm et al. (19) showed a stimulatory effect of a decreased
oxygen tension (5% O2) on matrix production. In a recent
study of Malda et al. (18), the pellets of chondrocytes were
suspended in a stirred bioreactor under different oxygen
concentrations. They observed an increased production of
glycosaminoglycan at 5% and 1% O2 (v/v) in comparison
with aeration at 21% O2 (air). The increased glycosami-
noglycan production is accompanied by a decrease in col-
lagen type I level. On the other hand, several studies of
chondrocytes embedded in a 3D matrix or a scaffold have
demonstrated an enhanced matrix formation, particularly
proteoglycan synthesis under more aerobic conditions (74,
75).
The main difference between the applied methodologies
(alginate and pellet culture vs. cartilage generation in 3D
scaffolds) can be observed in oxygen gradients in the vicin-
ity and within the formed cartilage (38, 76). In the case of
alginate and pellet culture, oxygen gradients at the surface
of constructs can be neglected, and only oxygen limitations
within the constructs are likely. For the cultivation of 3D
scaffolds, even more significant oxygen gradients within the
formed matrix are expected.
Another attempt to improve tissue-engineered cartilage is
the application of mechanical force during the cultivation to
produce a phenotypically appropriate tissue. Four main types
of force are currently used in cartilage cultivation: hydro-
static pressure, direct compression, and high- and low-shear
fluid environments. All these forces have been integrated
into culturing devices used as bioreactors for articular carti-
lage. The individual effects have been reviewed by Darling
and Athansiou (9).
Our work started from an engineering view point, that is,
we invastigated the oxygen transport in culture systems for
the generation of cartilage pellets and within the pellet itself
theoretically. Two culture strategies were modelled, a static
culture system with the supply of oxygen through the me-
dium by diffusion and a perfused culture system with an
BIOREACTOR DESIGN FOR TISSUE ENGINEERING 239
oxygen-enriched medium. For a static culture system, the
oxygen supply of a cartilage pellet from the medium, which
is not flowing (static medium layer, Fig. 3A) was calculated
with the following assumptions: (i) the medium is in contact
with the atmosphere via a gas-liquid interface and the oxy-
gen concentration at the surface is in equilibrium with the
atmosphere. (ii) The oxygen concentration cR in the static
medium layer can be described by one-dimensional diffu-
sion (D= diffusion coefficient in the medium).
cR 2cR
- = D ------------
---------- (1)
t x2
(iii) The oxygen concentration cP within a pellet can be de-
scribed by Eq. 2.
dcP d2cP X qmax cP
---------- = Deff -----------
- ------------------- ---------------- (2)
dt dx2 V ks + cP
where X is the cell density, qmax the maximal cell-specific
oxygen uptake rate, V the volume of the cartilage and kS the
Monod constant. (iv) The mass transfer of oxygen from the
medium to the pellet is only by diffusion. At the interface,
oxygen concentration and slope are identical on both sides.
At the pellet wall, the slope of oxygen concentration is zero.
Figure 3 shows the calculated oxygen concentration profile
as a function of length scale and time with the following pa-
rameters: a cell density of 2 106 cells per pellet, a maximal
cell specific oxygen uptake rate of qmax = 1013 mol cell1 h1,
kS = 1075 105 mol l1 and Deff = 2.16 105 cm2 s1, which is
80% of the diffusion coefficient in the medium (38). As
shown in Fig. 3A, oxygen concentration in the vicinity of
the pellet decreases very rapidly to zero within a very short
time. This effect is mainly due to the very low diffusive
transport of oxygen through the boundary layer between the
cartilage pellet and the medium.
Alternatively, a perfused system was modelled, in which
an oxygen enriched medium flows over the cartilage and the
thickness of the boundary layer over the cartilage should be
significantly decreased (Fig. 3B). The oxygen concentration
cm in the flowing medium as a function of time and length
xm can be described by Eq. 3.
dcm d2cm 4
----------- = D ------------
- + u --------------- (c0 cm) (3)
dt dxm2 dP
where U is the flow velocity, dP the pellet diameter, and c0
the oxygen concentration at xm = 0. The results of these sim-
ulations are summarised in Fig. 3B for a flow rate of 3
ml min1. These calculations show that the mass transfer
resistance in the boundary layer between the cartilage and
the medium can be significantly decreased, but still oxygen
concentration within the pellet decreases very rapidly.
For both systems, a severe oxygen limitation has to be
expected within the tissue matrix. The main reason for this
limitation is the very low rate of transport by diffusion.
Similar conclusions have been drawn elsewhere (38, 76). A
flow with an enriched medium, as in the perfused culture
system, can overcome this problem only to some extent by
decreasing the thckness of the boundary layer between the
tissue matrix and the medium. These findings lead to the
question of whether it is possible to predict the properties of
cartilage generated in these bioreactor systems on the basis
240 PRTNER ET AL.
FIG. 3. Modelling of oxygen profiles within tissue-engineered cartilage. (A) Calculated oxygen profile in static culture system for cultivation
of artificial cartilage pellets. (B) Calculated oxygen profile in perfusion culture system for cultivation of artificial cartilage pellets.
of the above conclusions.
This will be addressed below by comparing experimental
results from different cultivation systems. The methods will
be introduced only briefly. For details, see Ref. 17. Three-
dimensional cartilage-carrier constructs were produced ac-
cording to the protocol described above from chondrocytes
of an adult mini pig. The final step of this protocol was per-
formed either in bioreactors (in a flow-chamber or under in-
termittent hydrostatic pressure) or in 12-well plates as the
control (Fig. 4). The flow-chamber bioreactor was specially
designed for the generation of three-dimensional cartilage-
carrier constructs. A specific feature of the flow chamber is
a very thin medium layer for improved oxygen supply and a
counter current flow of the medium and gas (20). Another
bioreactor system was designed in which an intermittent hy-
drostatic pressure is applied during the cultivation. It en-
ables the cultivation of seven cartilage-carrier constructs in
parallel. The main results of the experiments are summa-
rized in Table 1.
Experiments at different oxygen concentrations (21% and
10% v/v O2) were performed in 12-well plates as the control
and the constructs were compared qualitatively and quanti-
tatively. The appearance of the cartilage obtained under de-
creased oxygen tension seemed to be closer to the native
cartilage with respect to the shape of cells, distribution of
cells within the matrix, and smoothness of the surface
among others. The thickness of the cartilage formed by free
swelling was always in the same range as that of the native
J. BIOSCI. BIOENG.,
cartilage (approximately 1 mm). For both oxygen concen-
trations the content of glycosaminoglycan (GAG) was simi-
lar, but still significantly lower than that in the native carti-
lage (data not shown). Qualitatively, the most stable attach-
ment of the cartilage on top of the carrier was found for 10%
O2 (v/v). Furthermore, the cultivated cartilage contained a
large content of collagen type II (data not shown). From
these observations, a lower oxygen concentration in the gas
phase is recommended.
Experiments in the flow-chamber bioreactor showed a
significantly higher matrix thickness but a lower content of
GAG then cultures in 12-well plates as the control. The ap-
pearance of the cartilage obtained in the bioreactor seemed
to be closer to the native cartilage with respect to the shape
of cells, distribution of cells within the matrix, and smooth-
ness of the surface among others. The cartilage obtained
from 12-well plates showed an inhomogeneous distribution
of cells, a more uneven surface and holes within the matrix.
Another important requirement for a successful implanta-
tion is the consistency of the cartilage. In the case of bio-
reactor cultures, the compaction of the cartilage and the at-
tachment between the cartilage and carrier were very good,
indicating that the construct is appropriate for implanta-
tion. In contrast, the cartilage-carrier constructs cultivated
in 12-well plates were soft and the attachment between the
cartilage and carrier was not sufficient. In this case, the car-
tilage tended to slip off the carrier with only a slight me-
chanical impact. Furthermore, the cultivated cartilage should
VOL. 100, 2005 BIOREACTOR DESIGN FOR TISSUE ENGINEERING 241

FIG. 4. Culture systems used for generation of three-dimensional cartilage carrier constructs.

contain a significant content of collagen type II. This was
confirmed qualitatively by immunohistological analysis.
In the third set of experiments cartilage-carrier constructs
were cultivated in a bioreactor, in which intermittent hydro-
static pressure was applied during the last week of cultiva-
tion (total time, 5 weeks). A total pressure of 3 105 Pa was
applied eight times per hour for 2.5 min during this week.
Constructs cultivated under an intermittent hydrostatic pres-
sure load and a decreased oxygen tension (10% O2 v/v)
showed a significantly higher matrix thickness and a higher
GAG content than those cultivated in 12-well plates as the
control (Table 1). The compaction of matrix as well as at-
tachment between the cartilage and carrier was evaluated
qualitatively and found to be much better in the bioreactor
cultures with intermittent hydrostatic pressure loading than
in constructs cultivated without loading in 12-well plates.
In a small animal trial, constructs were implanted into
mini pigs. One pig recieved constructs cultivated with pres-
sure loading and at 21% O2 v/v and anotther pig received
constructs cutlivated with pressure loading and at 10% O2
v/v. After surgery, none of the animals showed restrictions
in movement. In both pigs, the cultivated cartilage inte-
grated into the surrounding native cartilage without the for-
mation of gaps. The immunohistological analysis revealed
that explants cultivated at 10% O2 v/v showed a higher col-
lagen type II content than those cultivated at 21% v/v O2
(Fig. 5). The explants cultivated at 10% O2 v/v indicated a
hyaline like cartilage, whereas those cultivated at 21% O2

TABLE 1. Biochemical data of cartilage-carrier constructs cultivated in 12-well plates, in flow-chamber bioreactor,
under intermittent hydrostatic pressure, and for native cartilage (glycosaminoglycane, GAG)
O2-tension in Matrix
Passage GAG
Culture system the gas phase thickness
no. (g)
(%) (mm)
12-well plate 4 21 1.2 0.7 162 103.5
12-well plate 4 10 1.3 0.6 155.9 74.4
Flow-chamber bioreactor 6 21 1.6 0.2 142.7 9.7
12-well plate (control) 6 21 1.1 0.1 238.9 22.1
Static culture under intermittent hydrostatic pressure 3 10 1.1 0.7 466.53 7.1
12-well plate (control) 3 10 0.7 0.0 268.8 74
Native cartilage 1.0 0.0 1680.1 211.8
The cultivation principle consists of the following steps (17): (i) chondrocytes are isolated from the articular cartilage of an adult mini-pig,
expanded in a monolayer culture and then seeded on a solid ceramic carrier (diameter 4.5 mm) to form a primer layer; (ii) simultaneously, chondro-
cytes are suspended in alginate gel and cultivated for two weeks to allow the formation of a pericellular matrix; (iii) redifferentiated chondrocytes
are then recovered from the alginate gel and sedimented on the primer layer; (iv) the biphasic constructs are further cultivated for three weeks to
allow free-swelling of the cartilage matrix. This step was performed either in a 12-well plate, a flow-chamber bioreactor (flow rate, 0.5 ml/min) or
under intermittent hydrostatic pressure (total pressure of 3 105 Pa, 82.5 min per hour, applied hourly during the last week of cultivation). Three
sets of experiments were performed: (i) cultivation of cells in passage 4 in 12-well plates at 21% and 10% oxygen tensions in the headspace;
(ii) cultivation of cells in passage 6 in a flow-chamber bioreactor and in 12-well plates (control) at 21% oxygen tension; (iii) cultivation of cells in
passage 3 in a bioreactor under intermittent hydrostatic pressure and in 12-well plates (control) at 10 % oxygen tension. Native cartilage from an
adult mini-pig; diameter, 4.5 mm; average of five samples.
242 PRTNER ET AL.
FIG. 5. Images of immunohistological staining for collagen type
II of implanted cartilage-carrier construct (mini-pig) after explanation
and decalcification of carrier. Cartilage-carrier constructs were culti-
vated prior to implantation under loading with intermittent hydrostatic
pressure in a bioreactor aerated with 21% O2 (a, b) and 10% O2 (c, d).
Time of observation for the animals: 8 weeks. Bars indicate 500 m. 1,
Native cartilage; 2, newly formed cartilage; 3, bone; 4, extracted car-
rier after decalcification. The protocol for the generation of cartilage-
carrier constructs is described in Table 1.
v/v showed unsatisfactory results.
The above considerations underline the idea that engi-
neering knowledge can help improve cultivation systems
and applied strategies for tissue engineering. On the other
hand, it becomes obvious, that both engineering and funda-
mental studies on cell biology are required to further clarify
the observed effects. The theoretical simulations indicate
that even under ideal conditions (no mass transfer limitation
in the fluid phase) a severe oxygen limitation within the en-
gineered tissue should be expected. If oxygen supply would
be the limiting factor during cartilage formation, a bioreac-
tor system (flow chamber) with an improved oxygen supply
should lead to a better quality of the engineered cartilage.
On the other hand, lower oxygen concentrations in the gas
phase seem to improve some matrix properties. From the
results discussed above, these discrepancies can be solved
only to some extent. With respect to important biochemical
properties, particularly the content of GAG, the constructs
from the flow chamber bioreactor showed significantly lower
values than those from the 12-well plates, probably due to a
higher, detrimental oxygen concentration in the matrix. On
the other hand, other matrix properties, particularly the at-
tachment between the cartilage and carrier was better for
constructs from the flow chamber than for those from 12-
well plates. This can be due to a better oxygen supply within
the matrix close to the surface of the carrier.
The best results were obtained from constructs cultivated
under intermittent hydrostatic pressure and a decreased oxy-
gen concentration in the gas phase. Initially, this phenome-
non is difficult to understand. As the partial pressure of oxy-
gen in the gas phase depends on total pressure, a higher
pressure should even increase oxygen concentration signifi-
cantly, leading to even worse matrix properties. However,
J. BIOSCI. BIOENG.,
further investigations showed that due to the very low mass
transfer between the static culture medium and the gas
phase, oxygen concentration in the culture medium did not
increase during intervals with a higher pressure. Therefore,
other factors that explain the significantly improved matrix
properties of constructs cultivated under intermittend hy-
drostatic pressure compared with static cultures in 12-well
plates should be identified. Probably, the mechanical stimu-
lation of chondrocytes is the main effect. However, to clar-
ify this, further biochemical and cell biological studies are
required to determine, which cell biological effects are re-
sponsible for the observed phenomena.
CONCLUSIONS
Bioreactors for the generation of 3D tissue constructs can
provide a better process control by taking into account dif-
ferent demands of cells during cultivation. Furthermore,
they can provide the technical means to perform controlled
studies aimed at understanding specific biological, chemical
or physical effects. Moreover, bioreactors enable a safe and
reproducible production of tissue constructs. An overall com-
parison of different culture methods shows clearly the ad-
vantages of bioreactor culture. Not only can the properties
of cultivated 3D tissue constructs be improved, aspects such
as safety of operation argue for the use of bioreactor sys-
tems. Furthermore, the bioreactor can be used to study
effects such as shear flow and/or hydrostatic pressure on the
generation of tissues. For future clinical applications, the
bioreactor system should be an advantageous method in
terms of low contamination risk, ease of handling and scal-
ability.
To date the goals and expectations of bioreactor develop-
ment have been fulfilled only to some extent, as bioreactor
design in tissue engineering is very complex and still at an
early stage of development. In the future, a very intimate
collaboration between engineers and biologists will lead to
an increased fundamental understanding of complex issues
that can have an impact on tissue formation in bioreactors.
On one hand, devices are required with a well-described
microenvironment of cells for fundamental studies. On the
other hand, a transition from a laboratory scale to an in-
dustrial scale will require a high adaptability of specialized
bioreactors in a standardized production process. These ad-
vances will aid in ensuring that tissue engineering fulfil the
expectations for revolutionizing medical care.
ACKNOWLEDGMENTS
We would like to thank Drs. Klaus Baumbach, Frank Feyerabend,
Jan-Philipp Petersen and Jens Schrder for scientific input and
help in animal trials, Sven Cammerer and Katja Schmid for model-
ling the cartilage reactor as well as Katharina Braun, Ditte
Siemesgelss and Richard Getto for technical support. The finan-
cial support of Biomet Deutschland GmbH, Berlin under the
BMBF grant no. 03N4012 and the city of Hamburg within the
Qualtittsoffensive Tissue Engineering is gratefully acknowl-
edged.
VOL. 100, 2005
NOMENCLATURE
c0 : oxygen concentration in the flowing medium layer
at xm = 0 m, mol l1
cm : oxygen concentration in flowing medium layer,
mol l1
cR : oxygen concentration in static medium layer,
mol l1
cP : oxygen concentration in cartilage matrix, mol l1
D : diffusion coefficient in medium, m2 s1
Deff : diffusion coefficient in cartilage matrix, m2 s1
dP : pellet diameter, m
kS : Monod constant, mol l1
qmax : maximal cell-specific oxygen uptake rate,
mol cell1 h1
u : flow velocity, m2 s1
V : volume of cartilage, l
X : cell density, cells l1
x : length, m
xm : length in medium, m
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