Bioreactors for Bone

Tissue Engineering
Engineering Design
SESM3001
Fras Wasim

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1 Introduction
Current medical methods for treating bone defects caused by traumatic injuries
and diseases are limited. Auto-graft transplants are most common where bone is
harvested from the iliac crest. The bone available is often limited and the
procedure often leads to medical complications such as infection and chronic
pain [1]. Tissue engineering approaches look, instead, towards in-vitro culturing
of bone marrow mesenchymal stem cells for transplant. The main aim of this
project is to design a bio-reactor to create a 1cm 3 piece of spongy bone tissue.
Currently, most tissue engineering solutions can only produce tissue thicknesses
of a few millimetres [2] . This is because of the challenges associated with
emulating in-vivo conditions. The most commonly used reactors for bone tissue
engineering include: spinner flask, rotating wall, compression, magnetic and
perfusion systems [3]. This report will investigate their advantages and
disadvantages in order to find an optimum design.

2 Design Requirements
The process of culturing bone tissue involves three phases. First, the stem cells
are seeded onto a scaffold for growth. Osteoblastic differentiation must then be
initiated by introducing stimuli and chemical agents. Once the osteoblasts have
formed, bone matrix production can occur [4]. There are several parameters that
need to be controlled to provide optimum growth conditions. These include:
oxygen and nutrient supply; pH levels; temperature; biochemical agents; and
mechanical stresses [4]. Uniformity of conditions throughout the scaffold is also
needed to form bone with consistent properties.
Firstly, when the stem cells are seeded onto the scaffold, an even distribution is
required. This is achieved by careful design of the flow conditions. The parity of
nutrient and oxygen supply is also affected by this. In static culture, only cells on
the surface of the scaffold receive high concentrations. The supply gradient
needs to be reduced in order to avoid cell necrosis [5]. Waste products also need
to be removed efficiently. However, if the rate of transfer is too high, this disrupts
cell communication deterring bone formation.
The flow also imposes viscous shear stresses on the cells. Fluidic shear stress
caused by loading in bones has been hypothesised to stimulate osteoblasts
which are responsible for matrix production [6]. When mechanical forces are
applied, the cells lay down extracellular matrix in response to the loading.
Increase in matrix production will reduce the culture time and create a more
functional tissue [7]. The required in-vivo shear stresses are estimated to be in
the range of 0.8 to 3Pa [8]. However, stimulation has been seen for values as low
as 0.01 Pa [9]. In addition to flow imposed shear stress, cyclical hydrostatic
pressure by compression as well as tensile straining has been shown to increase
cell proliferation as well [10] [11]. Flexibility in loading conditions is desirable as
optimum conditions are still unknown.

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In terms of commerciality, the overall design needs to be simple and cost

effective. It should allow for ease of use as well, requiring little expert knowledge
to operate. Sterility issues are also of utmost importance. Only materials that
can be autoclaved or chemically treated may be used. Scaffold material design is
also important and is affected by all the factors listed above; this will be
discussed further in Section 4.3.

3 Literature Review
The simplest bioreactor is the spinner flask which introduces magnetic stirring to
static culture, as shown in Error: Reference source not found. This improves mass
transfer to the periphery of the scaffold but not to the interior; cell ingrowth is
therefore limited. Spinner flask bioreactors usually rotate at 50r/min [7]. This
needs to be increased as cell necrosis still occurs in the centre of the scaffold.
However, further increases results in turbulent flow which causes high shear
stress on the periphery of the scaffold destroying cells [7] [9]. Rotating vessel
bioreactors solve this problem by creating dynamic laminar flow to reduce
diffusional limits [12]. They consist of a cylindrical outer and inner wall; the
former, the latter or both can be rotated in order to generate increased mass
transfer. The hydrodynamic forces are balanced with the gravitational forces
resulting in a microgravity environment. Despite the enhanced mass transfer,
rotating wall bioreactors have seen low success rates in comparison to spinner
flask and static cultures [9]. Some studies have shown this could be because of
wall collisions resulting in cell damage and poor cell attachment [1]. Promising
results have

Figure 2 - Schematic view of spinner flask with scaffolds attached by needles [3]
Figure 1 - Rotating Bed Bioreactor [13]
been found by attaching the scaffold rigidly as shown in Error: Reference source
not found [13]. Lighter than water scaffolds can also be used to limit the stresses
caused due to collision [1].
Perfusion systems are by far the most effective in bone tissue engineering
because they allow for the greatest penetration for nutrient and oxygen supplies.
The medium is forced through a tightly fit scaffold with highly interconnected
pores. Porosity is important for these systems; in one study, it was shown that
greater calcium deposition is seen for 75% porosity as compared to 50% [5].
Moreover, the shear stress produced by flow in perfusion can be sufficient for the
application. The flow rate can be used to alter it but this affects nutrient transfer.
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Using Equation (1), it becomes apparent that changing viscosity is a more

desirable option. In a study which incorporated dextran to alter viscosity, a 3 fold
increase in viscosity resulted in a 7 fold increase in calcium deposition [5].

du
dy

(1)

Figure 3 - Combined Perfusion and Compression System [11]

Figure 4 - Perfusion Chamber [17]
Compression systems apply loading to the scaffold material either through a
piston or by hydraulic pressure and a diaphragm. The latter is more desirable as
it allows for better sterility. These systems have also seen high success rates in
stimulating cells. Though, in order to apply the pressure, the scaffolds need to be
stronger which will affect their degradation rates. Nevertheless, compressionperfusion systems have been labelled the optimal design for bone tissue growth
[14]; an example is shown in Error: Reference source not found.

Figure 5 - Comparative study between

and production
hydrostaticlevels
compression
on bone
marrow
stromalstudy
cells
Figure 6 perfusion
Osteocalcin
in a perfusion
and
compression

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The results for a perfusion and compression study are seen in Error: Reference
source not found and Error: Reference source not found. The combination of
perfusion and hydrostatic compression does not increase cell proliferation.
However, it does result in significantly quicker differentiation rates as indicated
by the osteocalcin levels in the latter diagram. Error in measurement is high so it
is difficult to tell whether these results are conclusive. A dual system is much
more complex than a single one and the costs associated must be justified
against the resulting improvements.
Finally, magnetic stimulation is also possible by attaching magnetic nanoparticles
directly to the cell membrane or to ion channel proteins. Time-varying magnetic
fields can be used to apply complex and precise loading directly to the cells. The
scaffolding does not need to be strengthened as it would in other mechanical
stimulation methods. Degradation rates can be decided more easily as a result. It
is non-invasive so it is also easier to keep sterility. In addition, spatially varying
load profiles can be given by controlling the particles magnetic properties [7].
However, studies have shown that magnetically oscillating forces do not increase
cell proliferation but only bone gene expression [15]. This is similar to
compression loading. Prediction of forces analytically can be more challenging as
well but the overall mechanical design is simple.
Cell seeding can also be
controlled to create a more even distribution.

4 Bio-Reactor Design
4.1 Main Design
Perfusion is a necessary requirement for the design of the system as it is the only
way to ensure efficient mass transfer. In terms of additional loading conditions,
compression and magnetic systems have both had positive results. Magnetic
forces, even without nanoparticle addition, can improve cell culture significantly.
In a particular study, the application of oscillating electromagnetic fields
increased matrix calcification by 5 fold [16]. Thus, all three parameters were
incorporated in the final design. Error: Reference source not found to Error:
Reference source not found show rough models of the cultivating chamber.
Further dimensions and drawings are given in the Appendix.
In order to achieve perfusion, the scaffold was made as a 11x10x10 cuboid for
clamping. It was placed in a cassette by assuming a press fit. Neoprene rings are
used to seal the cassette in the chamber; the cap at the top screws into the
outer case to create a compressive fit [17]. The rest of the materials will mainly
be made of Perspex as it is easily sterilised by ethylene oxide gas [7]. Its
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transparency might also allow for microscopy. In terms of compression, a

pneumatic system is used with two diaphragms at either side of the cassette as
shown in Error: Reference source not found. The diaphragm also screws into the
outer case to create tight seals. Finally, the magnetic forces were achieved by
two solenoids: Helmholtz coil [3]. By ensuring that the coils are wound in the
same direction and the distance,

a , between them is equivalent to the radius

of the loops, a uniform field can be generated at the mid-plane [18]. The number
of turns, N, and the current, I, in the coil can be set for a desired flux density. A
PEMF generator can be used to cause sinusoidal variations in the magnetic field
which improves culture times. For cell seeding, a constant magnetic field could
also be used to adjust the distribution of cells.

5 5 B a 7
10
32

Rubber Tube
for main flow

(2)

Coi
l

Pneumatic
flow tube to
incorporate
compression
Figure 7 - Final Chamber Design

Neoprene
Rings

Diaphragm
screwed into
Coil
the side Mounting
for
pneumatic
loading

Scaffold
Cassette
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Figure 9 - Cross-sectional view with pneumatic compression system
Figure 8 - Cross-sectional view of chamber design

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Neoprene Rings for

sealing
Scaffold
holder/Casse
tte
Top
which
has
fine
threads
to
screw
and
clamp
the
scaffold

Outer casing

Diaphragms
on
screw in caps for
seal
Figure 10 - Exploded view for Chamber Design
A schematic for the overall system is also shown in Error: Reference source not
found. The details of sensors have been omitted for simplicity. For the pneumatic
system, a 4/2 directional control valve is prescribed. When the valve is in the left
setting, the compressor would increase the pressure on left hand diaphragm.
When it is in its right setting, the right side will be pressurised. Solenoid valves
allow direct control of the loading. Check valves will also be required at all
entrances to the culture chamber to allow for load holding.

Figure 11 - Schematic diagram of overall system

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4.2 Flow Characteristics

The main aim would be to achieve a laminar flow as this will create uniform
conditions and limit cell damage. For flow in circular pipes, the Reynolds number
must be below 2300 for this to occur; the density, viscosity, flow velocity and
diameter being key parameters. In this case, the flow chamber is quite small and
has abruptly changing boundaries; the flow will not be fully developed so analytic
results become difficult to retrieve. A CFD analysis is therefore required to fully
understand the flow characteristics.
In terms of design methodology, it would be necessary to see what flow rates
optimise mass transfer of nutrients first. Most studies use flow rates within the
region of 0.6ml/min to 4ml/min [8]. High rates can interfere with cell-cell
signalling mechanisms hindering growth; 9mL/min is thought to be the start of
growth inhibition [9]. After the flow characteristics have been analysed, the
geometry and diameter can be optimised. Viscosity and density of the flow
medium could also be altered if required. Shear stresses are required to be
within 3Pa; this will then set the maximum flow rate achievable.
Different flow regimes can also be used: continuous flow, oscillatory or pulsatile
flow. Pulsatile flow has shown to increase oestrogenic differentiation rates,
increasing levels of alkaline phosphatase and extracellular protein in comparison
with continuous flow [19]. Though, most studies support the use of continuous
flow regimes [9]. A combination of both should be tested and compared.

4.3 Scaffold Materials

The main design requirements for the scaffold are bio-compatibility,
biodegradability, appropriate surface chemistry, pore size, shape and mechanical
strength. Bio-compatibility is fulfilled by most metals, polymers and ceramics.
The scaffold and degradation products should not be toxic as this can provoke
inflammatory response [20]. Cell adhesion and cell activating properties are
equally as important to allow for enhanced growth and transmission of forces invitro. Bio-derived bone scaffolds have shown to be effective in enhancing
proliferation but are dismissed due to the problem of host rejection.
In addition, only polymers and some ceramics are biodegradable. The
degradation rate needs to be controlled precisely to support the tissue until it
has load bearing capacity. Though, stress shielding might occur if it bears too
much of the load. Bone remodels itself according to loading conditions; if the
scaffold takes too much of the stress, surrounding tissue may become weaker.
Thus, the mechanical strength should be similar to that of the bone tissue.
Slightly stronger material may be required due to the compression applied in the
bioreactor. The degradation rate and the mechanical strength are conflicting
parameters; increasing one negatively affects the other. Ideally, a material is
required where both these parameters can be changed independently. The time
of degradation varies for different implant locations but will be between 3-18
months [20].

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Interconnected porosity is also a requirement where pore size needs to be at

least 100 m; 200-350m has been found the optimum for bone in-growth [21].
Spongy bone requires highly porous and resistant scaffolds [20]. Its mechanical
properties vary but its Youngs Modulus is between 2 and 20MPa [21]. Increased
porosity will decrease the mechanical strength but improve the nutrient and
oxygen circulation.
Synthetic polyester based polymers such as PLA, PGA and PLGA are the obvious
choice as their degradation properties can be tailored to needs. Though, their
mechanical strength can be on the low side. For this reason, IP-CHA is chosen as
the scaffold material. It is a hydroxyapatite ceramic that has been built with
inter-porous connections to improve cell ingrowth. It has a compressive strength
of 10-12MPa, an average pore size of 150 m and a high porosity of 70%. These
are suitable parameters for cancellous bone and perfusion. It has been used in
various clinical situations and shown good biocompatibility and positive results
[22]. Hydroxyapatite is a major constituent of bone and so has been known to
improve cell proliferation.

4.4 Monitoring Methods

The common feedback variables are O2 and glucose consumption, CO2
production, pH levels and temperature. Firstly, the current design is small
enough for incubation to allow for temperature control. The others need to be
controlled by feedback. The sensors used to measure these variables can be
either invasive or non-invasive. Direct contact with the fluid or cells will improve
the quality of data; however, the sensors used must be able to withstand
sterilisation processes and once placed, re-calibration will become difficult [7].
Micro-electrodes and fibre optic sensors can be used to measure oxygen levels
respectively. Non-invasive methods include optical methods such as
spectrophotometry or fluorimetry but there is a difficulty to achieve high
sensitivity. Dyes can be used which are sensitive to different parameters.
Sterilisation issues can be avoided and calibration becomes easier [7].

5 Conclusion
To conclude, it was found that perfusion systems are necessary to supply an
even distribution of nutrients and minerals. Compression systems and magnetic
fields are also promising as they increase cell differentiation and matrix
production respectively. A system was devised which incorporated all these
designs. This allows for greater versatility and a large range of culture conditions
possible.

6 Bibliography
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in bone tissue engineering, Landes Bioscience, vol. 2, no. 4, 2012.

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[2] I. Martin, D. Wendt and M. Heberer, The role of bioreactors in tissue

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7 Appendix
Overall Chamber

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Outer Casing

Cassette/Scaffold Holder

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Neoprene Rings

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Coil Mounting (No mounting bracket for this design yet)

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