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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
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.
The loss and damage of tissues cause serious health prob- mensional (3D) biohybrid structure of a scaffold and cul-
lems (1). In the US, almost one-half of the costs for medical tured cells or tissue. Furthermore, non implantable tissue
treatments are spent on implant devices annually (2). World- structures can be applied as external support devices (e.g.,
wide, 350 billion USD are expended for substitute of organs an extracorporal liver support when a compatible donor or-
(3). The substitution of tissues (such as bone or cartilage) or gan is not readily available [4, 5]) or engineered tissues can
joints with allograft materials includes the risk of infections be used as in vitro physiological models for studying dis-
by viruses (such as HIV, hepatitis C) or a graft rejection. Ar- ease pathogenesis and developing new molecular therapeu-
tificial implants such as those used in knee or hip replace- tics (e.g., drug screening [5, 6]).
ment, have limitations due to their limited lifespan, insuffi- The generation of 3D tissue substitutes in vitro requires
cient bonding to the bone, and allergic reactions caused by not only a biological model (e.g., an adequate source for
material abrasion. New therapy concepts for practical medi- proliferable cells with appropriate biological functions, a
cal applications are required. To this end, tissue engineered protocol for proliferating cells while maintaining the tissue-
substitutes generated in vitro could open new strategies for specific phenotype), but also the further development of new
the restoration of damaged tissues. The goal of tissue engi- culture strategies including bioreactor concepts (5, 7, 8).
neering can be defined as the development of cell-based Bioreactors are well established for the cultivation of mi-
substitutes to restore, maintain or improve tissue function. crobes or mammalian cells under monitored and controlled
These substitutes should have organ-specific properties with environmental and operational conditions (e.g., pH, temper-
respect to biochemical activity, microstructure, mechanical ature, oxygen tension, and nutrient supply) up to an indus-
integrity and biostability (2). Cell-based concepts include the trial scale. However, as individual cells are mostly applied,
(i) direct transplantation of isolated cells, (ii) implantation these concepts are inapplicable to 3D tissue constructs. Fur-
of a bioactive scaffold for the stimulation of cell growth thermore, each type of tissue construct (e.g., skin, bone,
within the original tissue and (iii) implantation of a three di- blood vessel, and cartilage) will likely require an individual-
ized bioreactor design (7). Therefore, tissue-specific biore-
* Corresponding author. e-mail: poertner@tuhh.de actors should be designed on the basis of comprehensive
phone: +49-40-42878-2886 fax: +49-40-42878-2909 understanding of biological and engineering aspects. Addi-
235
236 PRTNER ET AL. J. BIOSCI. BIOENG.,
tionally, typical engineering aspects such as reliability, repro- cell densities have been associated with an enhanced tissue
ducibility, scalability and safety should be addressed (5, 8). formation including cartilage matrix production, bone min-
Several bioreactor systems have been developed and usually eralization and cardiac tissue structure formation (2325).
the expectations are very high (5, 6). The question is, how- On the other hand, an inhomogeneous distribution of cells
ever, whether bioreactors can indeed fulfil these expecta- within the scaffold can significantly affect the tissue proper-
tions. ties. Several techniques for cell seeding were discussed by
In this review, key technical challenges are identified and Martin et al. (5).
an overview of existing culture systems and bioreactors used Critical issues of all bioreactor concepts involve mass
for tissue engineering is provided. These topics have been transfer problems (e.g., oxygen and nutrient supply, and re-
addressed to some extent by several authors (4, 5, 716). moval of toxic metabolites). The size of most engineered
Therefore, they will be discussed only briefly. Particular tissues is limited as they do not have their own blood system
focus will be given to the interaction between biological and and the cells are only supplied by diffusion (6, 26). Oxygen
engineering aspects. Using cartilage tissue formation as an supply is particularly critical, as only cell layers of 100200
example, it will be shown how an increased fundamental m can be supplied by diffusion (27). However, as tissue
understanding of biological, biochemical, and engineering constructs should have larger dimensions, mass-transfer lim-
aspects can significantly improve the properties of 3D tissue itations represent one of the greatest engineering challenges.
constructs. Various studies showed that mechanical stimulation (e.g.,
mechanical compression, hydrodynamic pressure, and fluid
IMPORTANT ASPECTS FOR flow), which are important modulators of cell physiology,
BIOREACTOR DESIGN can have a positive impact on tissue formation (28), particu-
larly in the context of musculoskeletal tissue engineering,
With respect to tissue engineering, bioreactors are used cartilage formation, and cardiovascular tissues among others
for different purposes such as (i) cell proliferation on a (11, 2934). Despite accumulating evidence that mechani-
small scale (e.g., for individual patients) and on a large scale cal stimulation can improve the properties of engineered tis-
(e.g., for allogenic therapy concepts), (ii) generation of 3D sues, only little is known about specific mechanical forces
tissue constructs from isolated and proliferated cells in vitro or the ranges of application (i.e., magnitude, frequency, con-
and (iii) direct organ support devices (15). These bioreac- tinuous or intermittent, and duty cycle [5, 9]). Further stud-
tors should enable the control of environmental conditions ies of these factors have to be coupled with quantitative and
such as oxygen tension, pH, temperature, and shear stress computational analyses of physical forces experienced by
and allow aseptic operation (e.g., feeding and sampling). cells and changes in mass transport induced by the method
Furthermore, a bioreactor system should allow for auto- used.
mated processing steps. This is essential not only for con- Bioreactors allow for different process strategies includ-
trolled, reproducible, statistically relevant basic studies but ing the batch, fed-batch or continuous cultivation. In particu-
also for the future routine manufacturing of tissues for clini- lar, continuous perfusion enables cultivation under constant
cal application (5, 17). Besides these global requirements, and controlled environmental conditions (3538). Martin et
specific key criteria for 3D tissue constructs based on cells al. (5) summarized some of the effects of direct perfusion
and scaffolds have to be met, including the proliferation of on tissue-specific properties such as growth, differentiation
cells, seeding of cells on macroporous scaffolds, nutrient and mineralized matrix deposition by bone cells, prolifera-
(particularly oxygen) supply within the resulting tissue, and tion of human oral keratinocytes, rates of albumin synthesis
mechanical stimulation of the developing tissues (5). by hepatocytes, expression of cardiac-specific markers by
Proliferation of cells is the first step in establishing a tis- cardiomyocytes, and GAG synthesis and accumulation by
sue culture. Usually, only a small number of cells can be ob- chondrocytes. On the other hand, a bioreactor system be-
tained from a biopsy specimen and expansion up to several comes more complex as additional features such as feeding
orders of magnitude is required. The proliferation of cells is pumps, vessels for fresh and spent medium, and control
quite often accompanied by the dedifferentiation of cells. strategies are required (Fig. 1), particularly in the case of
For example, proliferating chondrocytes show a decreased mechanical stimulation. The bioreactor system has to be in-
expression level of collagen type II and an increased expres- tegrated into the entire cultivation scheme including biopsy,
sion level of collagen type I (18, 19). Small culture dishes proliferation, cell seeding, tissue formation and delivery to
(e.g., petri dishes, 12-well plates, and T-flasks) are generally the site of application (e.g., hospital). In many cases, the
used for cell expansion. As these devices allow only an in- bioreactor itself is only used for tissue formation. However,
crease in cell number by a factor of approximately 10, sev- for a wholistic approach from biopsy to the implantation of
eral subcultivations are required. These are considered as a tissue, the intire procedure should be coordinated to de-
major cause for the dedifferentiation of cells. Recent studies crease the number of steps, risk of contamination, and labor
have shown that microcarrier cultures performed in well- costs among others. This is particularly important with re-
mixed bioreactor systems can significantly improve cell ex- spect to the manufacture of engineered tissue constructs for
pansion (2022). clinical applications, in which good manufacturing practice
The cell seeding of scaffolds is an important step in estab- (GMP) requirements have to be met (7, 8).
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
VOL. 100, 2005 BIOREACTOR DESIGN FOR TISSUE ENGINEERING 237
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.
ing-wall vessel (23), in which a construct remains in a state stress induced by perfusion, shear stress or gliding forces
of free-fall through the medium with a low shear stress and induced by mechanical impact, and pulsation flow) are ap-
a high mass transfer rate. This system has a wide range of plied, the exact local conditions experienced by the cells
practical applications (5, 7). A multipurpose culture system have to be determined. Therefore, experimental studies
was introduced by Minuth et al. (35) for perfusion cultures should be supported by simulation methods such as the
under organotypic conditions. Several tissue carriers can be computational fluid dynamics (CFD) or finite-element ap-
placed inside a perfusion container. Depending on the type proach. Several examples underline the potential of an inte-
of tissue-specific cell supports can be selected. A perfused grated study of mechanical and biomechanical factors that
flow-chamber bioreactor with a new concept for aeration control the functional development of tissue engineered con-
has been introduced recently (20), in which tissue-specific structs (6066). This approach will significantly improve
inserts for various types of tissue (e.g., cartilage, skin, and bioreactor design in the near future.
bone) can be applied. As an attempt to evaluate and compare different tissue
Besides these examples of multipurpose bioreactors, nu- engineering reactors, Omasa et al. (67) applied the analytic
merous of tissue-specific culture systems have been sug- hierarchy process (AHP). For evaluating a reactor for a bio-
gested. These are well reviewed with respect to specific tis- artificial liver (BAL) support system, five criteria were iden-
sues (5, 6, 811, 14, 15, 26, 50, 5759). Most of them are tified (namely, safety, scalability, cell growth environment,
custom-made; only very few have been commercialized. 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
MODELLING OF BIOREACTOR SYSTEMS
FOR TISSUE ENGINEERING tool for decision making in tissue engineering, not only for
BAL systems but for tissue engineering reactors in general.
The appropriate molecular and macroscopic architecture
of 3D tissue constructs is essential to producing a phenotyp-
INTERACTION BETWEEN BIOLOGICAL AND
ically appropriate tissue (6). However, in many cases this is
ENGINEERING ASPECTS IN GENERATION
not realized. The solution may lie in providing appropriate
OF ARTIFICIAL CARTILAGE
physiological conditions during cultivation. In many cases,
the culture systems and bioreactors were not optimized in The following considerations are intended to demonstrate
this respect. Parameters such as perfusion rate, flow condi- how the interdisciplinary application of biological and engi-
tions, shear stress, and compression magnitude were varied, neering knowledge can significantly improve the properties
quite often by a trial-and-error approach. Furthermore, dif- of tissue engineered 3D cartilage constructs. Severe health
ferent conditions have to be examined accurately with re- problems can arise from a lack or damage of hyaline carti-
gard to their effect. For example, hydrostatic pressure ap- lage in joints, particularly in knee joints (68). The methods of
plied during cartilage culture can lead to an improved mass tissue engineering enable the generation of cartilage tissue
transfer of small and large molecules into the cartilage ma- in vitro and provide new strategies to restoring damaged
trix, but can also induce a mechanical stimulation of embed- cartilage (9, 36, 58, 69). Whereas many approaches to gen-
ded cells. These two effects have to be examined separately. erating tissue-engineered cartilage are based on either algi-
Particularly, when if forces (e.g., hydrostatic pressure, shear nate encapsulation or the use of bioresorbable scaffolds (9,
VOL. 100, 2005 BIOREACTOR DESIGN FOR TISSUE ENGINEERING 239
19, 58, 59, 69, 70), recently, a new concept involving the oxygen-enriched medium. For a static culture system, the
use of a bone substitute carrier covered with a layer of oxygen supply of a cartilage pellet from the medium, which
tissue-engineered cartilage without scaffolds has been pre- is not flowing (static medium layer, Fig. 3A) was calculated
sented (17). This concept consists of four steps: (i) ex- with the following assumptions: (i) the medium is in contact
planted articular chondrocytes are expanded in a monolayer with the atmosphere via a gas-liquid interface and the oxy-
culture and then seeded on a solid carrier to form a primer gen concentration at the surface is in equilibrium with the
layer; (ii) simultaneously, chondrocytes are suspended in atmosphere. (ii) The oxygen concentration cR in the static
alginate gel and cultivated for two weeks to allow the for- medium layer can be described by one-dimensional diffu-
mation of a pericellular matrix; (iii) redifferentiated chon- sion (D= diffusion coefficient in the medium).
drocytes are then eluted out of the alginate gel and sedi-
cR 2cR
mented on the primer layer; (iv) the biphasic constructs are - = D ------------
---------- (1)
further cultivated for three weeks. Below, mainly different t x2
aspects of step iv are addressed. A similar concept was sug- (iii) The oxygen concentration cP within a pellet can be de-
gested by Waldman et al. (71). scribed by Eq. 2.
A critical parameter during the cultivation of cartilage
dcP d2cP X qmax cP
tissue is oxygen supply. The effect of oxygen during the in ---------- = Deff -----------
- ------------------- ---------------- (2)
vitro cultivation of chondrocytes is poorly understood, and dt dx2 V ks + cP
therefore it is presently a controversial issue (72). In several where X is the cell density, qmax the maximal cell-specific
studies, chondrocytes were immobilized in alginate beads oxygen uptake rate, V the volume of the cartilage and kS the
and cultivated under different oxygen concentrations in the Monod constant. (iv) The mass transfer of oxygen from the
gas phase (for review see Ref. 72). ODriscoll et al. (73) medium to the pellet is only by diffusion. At the interface,
observed a limited collagen type II production at very high oxygen concentration and slope are identical on both sides.
(90% O2) and very low (15% O2) oxygen concentrations. At the pellet wall, the slope of oxygen concentration is zero.
Domm et al. (19) showed a stimulatory effect of a decreased Figure 3 shows the calculated oxygen concentration profile
oxygen tension (5% O2) on matrix production. In a recent as a function of length scale and time with the following pa-
study of Malda et al. (18), the pellets of chondrocytes were rameters: a cell density of 2 106 cells per pellet, a maximal
suspended in a stirred bioreactor under different oxygen cell specific oxygen uptake rate of qmax = 1013 mol cell1 h1,
concentrations. They observed an increased production of kS = 1075 105 mol l1 and Deff = 2.16 105 cm2 s1, which is
glycosaminoglycan at 5% and 1% O2 (v/v) in comparison 80% of the diffusion coefficient in the medium (38). As
with aeration at 21% O2 (air). The increased glycosami- shown in Fig. 3A, oxygen concentration in the vicinity of
noglycan production is accompanied by a decrease in col- the pellet decreases very rapidly to zero within a very short
lagen type I level. On the other hand, several studies of time. This effect is mainly due to the very low diffusive
chondrocytes embedded in a 3D matrix or a scaffold have transport of oxygen through the boundary layer between the
demonstrated an enhanced matrix formation, particularly cartilage pellet and the medium.
proteoglycan synthesis under more aerobic conditions (74, Alternatively, a perfused system was modelled, in which
75). an oxygen enriched medium flows over the cartilage and the
The main difference between the applied methodologies thickness of the boundary layer over the cartilage should be
(alginate and pellet culture vs. cartilage generation in 3D significantly decreased (Fig. 3B). The oxygen concentration
scaffolds) can be observed in oxygen gradients in the vicin- cm in the flowing medium as a function of time and length
ity and within the formed cartilage (38, 76). In the case of xm can be described by Eq. 3.
alginate and pellet culture, oxygen gradients at the surface
dcm d2cm 4
of constructs can be neglected, and only oxygen limitations ----------- = D ------------
- + u --------------- (c0 cm) (3)
within the constructs are likely. For the cultivation of 3D dt dxm2 dP
scaffolds, even more significant oxygen gradients within the where U is the flow velocity, dP the pellet diameter, and c0
formed matrix are expected. the oxygen concentration at xm = 0. The results of these sim-
Another attempt to improve tissue-engineered cartilage is ulations are summarised in Fig. 3B for a flow rate of 3
the application of mechanical force during the cultivation to ml min1. These calculations show that the mass transfer
produce a phenotypically appropriate tissue. Four main types resistance in the boundary layer between the cartilage and
of force are currently used in cartilage cultivation: hydro- the medium can be significantly decreased, but still oxygen
static pressure, direct compression, and high- and low-shear concentration within the pellet decreases very rapidly.
fluid environments. All these forces have been integrated For both systems, a severe oxygen limitation has to be
into culturing devices used as bioreactors for articular carti- expected within the tissue matrix. The main reason for this
lage. The individual effects have been reviewed by Darling limitation is the very low rate of transport by diffusion.
and Athansiou (9). Similar conclusions have been drawn elsewhere (38, 76). A
Our work started from an engineering view point, that is, flow with an enriched medium, as in the perfused culture
we invastigated the oxygen transport in culture systems for system, can overcome this problem only to some extent by
the generation of cartilage pellets and within the pellet itself decreasing the thckness of the boundary layer between the
theoretically. Two culture strategies were modelled, a static tissue matrix and the medium. These findings lead to the
culture system with the supply of oxygen through the me- question of whether it is possible to predict the properties of
dium by diffusion and a perfused culture system with an cartilage generated in these bioreactor systems on the basis
240 PRTNER ET AL. J. BIOSCI. BIOENG.,
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. cartilage (approximately 1 mm). For both oxygen concen-
This will be addressed below by comparing experimental trations the content of glycosaminoglycan (GAG) was simi-
results from different cultivation systems. The methods will lar, but still significantly lower than that in the native carti-
be introduced only briefly. For details, see Ref. 17. Three- lage (data not shown). Qualitatively, the most stable attach-
dimensional cartilage-carrier constructs were produced ac- ment of the cartilage on top of the carrier was found for 10%
cording to the protocol described above from chondrocytes O2 (v/v). Furthermore, the cultivated cartilage contained a
of an adult mini pig. The final step of this protocol was per- large content of collagen type II (data not shown). From
formed either in bioreactors (in a flow-chamber or under in- these observations, a lower oxygen concentration in the gas
termittent hydrostatic pressure) or in 12-well plates as the phase is recommended.
control (Fig. 4). The flow-chamber bioreactor was specially Experiments in the flow-chamber bioreactor showed a
designed for the generation of three-dimensional cartilage- significantly higher matrix thickness but a lower content of
carrier constructs. A specific feature of the flow chamber is GAG then cultures in 12-well plates as the control. The ap-
a very thin medium layer for improved oxygen supply and a pearance of the cartilage obtained in the bioreactor seemed
counter current flow of the medium and gas (20). Another to be closer to the native cartilage with respect to the shape
bioreactor system was designed in which an intermittent hy- of cells, distribution of cells within the matrix, and smooth-
drostatic pressure is applied during the cultivation. It en- ness of the surface among others. The cartilage obtained
ables the cultivation of seven cartilage-carrier constructs in from 12-well plates showed an inhomogeneous distribution
parallel. The main results of the experiments are summa- of cells, a more uneven surface and holes within the matrix.
rized in Table 1. Another important requirement for a successful implanta-
Experiments at different oxygen concentrations (21% and tion is the consistency of the cartilage. In the case of bio-
10% v/v O2) were performed in 12-well plates as the control reactor cultures, the compaction of the cartilage and the at-
and the constructs were compared qualitatively and quanti- tachment between the cartilage and carrier were very good,
tatively. The appearance of the cartilage obtained under de- indicating that the construct is appropriate for implanta-
creased oxygen tension seemed to be closer to the native tion. In contrast, the cartilage-carrier constructs cultivated
cartilage with respect to the shape of cells, distribution of in 12-well plates were soft and the attachment between the
cells within the matrix, and smoothness of the surface cartilage and carrier was not sufficient. In this case, the car-
among others. The thickness of the cartilage formed by free tilage tended to slip off the carrier with only a slight me-
swelling was always in the same range as that of the native 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 cultures with intermittent hydrostatic pressure loading than
confirmed qualitatively by immunohistological analysis. in constructs cultivated without loading in 12-well plates.
In the third set of experiments cartilage-carrier constructs In a small animal trial, constructs were implanted into
were cultivated in a bioreactor, in which intermittent hydro- mini pigs. One pig recieved constructs cultivated with pres-
static pressure was applied during the last week of cultiva- sure loading and at 21% O2 v/v and anotther pig received
tion (total time, 5 weeks). A total pressure of 3 105 Pa was constructs cutlivated with pressure loading and at 10% O2
applied eight times per hour for 2.5 min during this week. v/v. After surgery, none of the animals showed restrictions
Constructs cultivated under an intermittent hydrostatic pres- in movement. In both pigs, the cultivated cartilage inte-
sure load and a decreased oxygen tension (10% O2 v/v) grated into the surrounding native cartilage without the for-
showed a significantly higher matrix thickness and a higher mation of gaps. The immunohistological analysis revealed
GAG content than those cultivated in 12-well plates as the that explants cultivated at 10% O2 v/v showed a higher col-
control (Table 1). The compaction of matrix as well as at- lagen type II content than those cultivated at 21% v/v O2
tachment between the cartilage and carrier was evaluated (Fig. 5). The explants cultivated at 10% O2 v/v indicated a
qualitatively and found to be much better in the bioreactor 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. J. BIOSCI. BIOENG.,
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,
FIG. 5. Images of immunohistological staining for collagen type they can provide the technical means to perform controlled
II of implanted cartilage-carrier construct (mini-pig) after explanation
and decalcification of carrier. Cartilage-carrier constructs were culti- studies aimed at understanding specific biological, chemical
vated prior to implantation under loading with intermittent hydrostatic or physical effects. Moreover, bioreactors enable a safe and
pressure in a bioreactor aerated with 21% O2 (a, b) and 10% O2 (c, d). reproducible production of tissue constructs. An overall com-
Time of observation for the animals: 8 weeks. Bars indicate 500 m. 1, parison of different culture methods shows clearly the ad-
Native cartilage; 2, newly formed cartilage; 3, bone; 4, extracted car-
rier after decalcification. The protocol for the generation of cartilage-
vantages of bioreactor culture. Not only can the properties
carrier constructs is described in Table 1. 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
v/v showed unsatisfactory results. effects such as shear flow and/or hydrostatic pressure on the
The above considerations underline the idea that engi- generation of tissues. For future clinical applications, the
neering knowledge can help improve cultivation systems bioreactor system should be an advantageous method in
and applied strategies for tissue engineering. On the other terms of low contamination risk, ease of handling and scal-
hand, it becomes obvious, that both engineering and funda- ability.
mental studies on cell biology are required to further clarify To date the goals and expectations of bioreactor develop-
the observed effects. The theoretical simulations indicate ment have been fulfilled only to some extent, as bioreactor
that even under ideal conditions (no mass transfer limitation design in tissue engineering is very complex and still at an
in the fluid phase) a severe oxygen limitation within the en- early stage of development. In the future, a very intimate
gineered tissue should be expected. If oxygen supply would collaboration between engineers and biologists will lead to
be the limiting factor during cartilage formation, a bioreac- an increased fundamental understanding of complex issues
tor system (flow chamber) with an improved oxygen supply that can have an impact on tissue formation in bioreactors.
should lead to a better quality of the engineered cartilage. On one hand, devices are required with a well-described
On the other hand, lower oxygen concentrations in the gas microenvironment of cells for fundamental studies. On the
phase seem to improve some matrix properties. From the other hand, a transition from a laboratory scale to an in-
results discussed above, these discrepancies can be solved dustrial scale will require a high adaptability of specialized
only to some extent. With respect to important biochemical bioreactors in a standardized production process. These ad-
properties, particularly the content of GAG, the constructs vances will aid in ensuring that tissue engineering fulfil the
from the flow chamber bioreactor showed significantly lower expectations for revolutionizing medical care.
values than those from the 12-well plates, probably due to a
higher, detrimental oxygen concentration in the matrix. On ACKNOWLEDGMENTS
the other hand, other matrix properties, particularly the at-
tachment between the cartilage and carrier was better for We would like to thank Drs. Klaus Baumbach, Frank Feyerabend,
constructs from the flow chamber than for those from 12- Jan-Philipp Petersen and Jens Schrder for scientific input and
well plates. This can be due to a better oxygen supply within help in animal trials, Sven Cammerer and Katja Schmid for model-
ling the cartilage reactor as well as Katharina Braun, Ditte
the matrix close to the surface of the carrier. Siemesgelss and Richard Getto for technical support. The finan-
The best results were obtained from constructs cultivated cial support of Biomet Deutschland GmbH, Berlin under the
under intermittent hydrostatic pressure and a decreased oxy- BMBF grant no. 03N4012 and the city of Hamburg within the
gen concentration in the gas phase. Initially, this phenome- Qualtittsoffensive Tissue Engineering is gratefully acknowl-
non is difficult to understand. As the partial pressure of oxy- edged.
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,
VOL. 100, 2005 BIOREACTOR DESIGN FOR TISSUE ENGINEERING 243
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