Permeability evaluation of 45S5 Bioglass

s
-based scaffolds for
bone tissue engineering
Ignacio Ochoa
a
, Jose A. Sanz-Herrera
a
, Jose M. Garca-Aznar
a
, Manuel Doblare
a
,
Darmawati Mohamad Yunos
b
, Aldo R. Boccaccini
b,
a
Aragon Institute of Engineering Research (I3A), Group of Structural Mechanics and Materials Modelling (GEMM), Centro de Investigacion Biomedica en Red en Bioingenier a,
Biomateriales y Nanomedicina (CIBER-BBN), Universidad de Zaragoza, 50018 Zaragoza, Spain
b
Department of Materials, Imperial College London, South Kensington Campus, London SW7 2BP, UK
a r t i c l e i n f o
Article history:
Accepted 29 October 2008
Keywords:
Bioglass
s
scaffolds
Permeability
Darcys equation
Tissue engineering
a b s t r a c t
Permeability is a key parameter for microstructural design of scaffolds, since it is related to their
capability for waste removal and nutrients/oxygen supply. In this framework, Darcys experiments were
carried out in order to determine the relationship between the pressure drop gradient and the uid ow
velocity in Bioglass
s
-based scaffolds to obtain the scaffolds permeability. Using deionised water as
working uid, the measured average permeability value on scaffolds of 9095% porosity was
1.9610
9
m
2
. This value lies in the published range of permeability values for trabecular bone.
& 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Tissue engineering is an emerging eld aiming at developing
biological substitutes that restore and improve the functions of
diseased human tissue or organs (Langer and Vacanti, 1993; Chan
and Mooney, 2008). One signicant branch of tissue engineering
involves the use of high-porosity scaffolds that act as temporary
3D templates for cell adhesion, proliferation, migration and
ultimately the formation of new tissues (Hutmacher et al.,
2007). In the case of bone tissue engineering, osteoprogenitor
cells should be delivered to the sites required for bone regenera-
tion using the scaffold as an alternative to an autograft.
The viability of the construct depends on its rapid vasculari-
sation upon implantation (Chan and Mooney, 2008; Hutmacher
et al., 2007).
Scaffolds need to be in a porous form in order to support high
number of cells and allow for vascularisation upon implantation.
Moreover, the pores must be open and large enough so that cells
can easily migrate through the scaffolds (Hutmacher et al., 2007).
Porosities higher than 80% and pore sizes in the range
100500mm are considered for applications in bone tissue
engineering, but the precise pore dimension depends on the
specic application (Guarino et al., 2007). When cells have
attached to the material surface there must be enough space
and channels to allow for nutrient ingress, waste delivery, protein
transport and vascular growth to occur, functions which are
obtainable with an interconnected network of pores.
Porous scaffolds such as 45S5 Bioglass
s
-based glass-ceramic
foams (Chen et al., 2006) and similar inorganic bioactive silicate
scaffolds (Vitale-Brovarone et al., 2007; Wu et al., 2007) fabricated
by the replication method are attracting increasing attention due
to their excellent biocompatibility and bioactivity coupled with
adequate mechanical properties. These scaffolds induce a strong
bond to bone when implanted through the formation of a
hydroxyapatite layer on their surfaces. Moreover, bioactive glass
of composition 45S5 Bioglass
s
(in wt%: 45%SiO
2
, 24.5%Na
2
O,
24.4%CaO and 6%P
2
O
5
) exposes critical concentrations of Ca, Si, Na
and P ions, which have been shown to activate genes in osteoblast
cells which stimulate new bone formation in vivo (Xynos et al.,
2001). In addition, bioactive glass containing tissue engineering
constructs have been shown to stimulate angiogenesis in vitro and
in vivo (Day et al., 2004).
The permeability of scaffolds, a property directly related to the
degree of pore interconnectivity, is a key factor inuencing the
scaffolds ability to enhance tissue regeneration. Permeability
quanties the ability of a porous medium to transmit uid
through its interconnected pores or channels when subjected to
pressure. Permeability, therefore, controls the nutrient ow to
cells that migrate through the scaffolds. Studies have found that
cell growth into a scaffold depends on how well nutrients can
permeate through the porous structure during the cell culture
process (Li et al., 2003; Botchwey et al., 2003; Grimm and
Williams, 1997). Many studies have been carried out to evaluate
and characterise the macroporous structure of scaffolds and in
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0021-9290/$ - see front matter & 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jbiomech.2008.10.030

Corresponding author. Tel.: +442075946731.

E-mail address: a.boccaccini@imperial.ac.uk (A.R. Boccaccini).
Journal of Biomechanics 42 (2009) 257260
most cases porosity, pore size and interconnectivity are used as
characterisation parameters (Hutmacher et al., 2007; Guarino
et al., 2007; Chen et al., 2006; Vitale-Brovarone et al., 2007; Wu
et al., 2007; Karageorgiou and Kaplan, 2005). It has been reported,
however, that these parameters may not be sufcient for a
complete understanding of the scaffold behaviour and there has
been some contradictions when attempting to correlate scaffolds
pore structure to their biological performance (Li et al., 2003). For
example, scaffolds with high porosity are expected to perform
better than those of lower porosity. However, biological studies
have not always shown this to be the case (Li et al., 2003). The
intrinsic permeability of porous media has been suggested to be a
more relevant parameter to characterise scaffolds (Li et al., 2003;
Sell et al., 2008). Intrinsic permeability is independent of both
the uid used for the measurement and the thickness of the
porous medium.
Several permeability measurement systems have been devel-
oped for determining the permeability of scaffolds. Haugen et al.
(2004) used a gravity-driven system to measure the permeability
of ceramic-based foams using the height of the water pipe and
uid properties. The same gravity-driven method was used by
Kohles et al. (2001) to measure the permeability of cancellous
bone from mature bovine. An elevated reservoir was used to drive
water through the bone sample and into a collection reservoir.
Swider et al. (2007) have developed a high-resolution MRI
methodology for characterising the permeability and uid
velocity within a material of interconnected porosity while
Maxwell and Wei (2007) used dry air as the uid medium
allowing rapid measurement operations. Recently, the perme-
ability of a commercial bioceramic scaffold has been evaluated
through numerical modelling by Sanz-Herrera et al. (in press).
Methods such as physical/chemical gas absorption, helium
pycnometry, mercury intrusion porosimetry and epitermal neu-
tron porosimetry can deliver information on density, pore size and
porosity of a porous medium. They do not provide, however, direct
measurements on intrinsic permeability. Moreover, some of the
measurements such as mercury intrusion porosimetry involve
high pressures that could destroy the usually fragile structure of
tissue engineering scaffolds.
In the present investigation, the permeability of Bioglass
s
-
derived scaffolds intended for bone tissue engineering and
fabricated by the foam replica technique (Chen et al., 2006) has
been measured for the rst time using deionised water and a
device using a peristaltic pump.
2. Materials and methods
Bioglass
s
-based foams were fabricated by the foam replica method described
in detail elsewhere (Chen et al., 2006). Briey, a polyurethane (PU) foam, which
serves as a sacricial template, is coated with a Bioglass
s
slurry using particles of
mean size o5mm. The coating process leads to Bioglass
s
particles adhering on the
PU foam surfaces and forming a homogeneous coating. After drying in normal air,
the PU foam is burned out slowly at 4001C to minimize damage to the Bioglass
s
coating. Once the PU template has been completely removed the scaffold is
sintered at 1100 1C for 1h to the desired density using a predetermined and
optimized heat-treatment schedule. During this process, the glass also partially
crystallizes which leads to a scaffold of suitable structural integrity for bone tissue
engineering (Chen et al., 2006).
The microstructure of the scaffold used in this investigation is shown in Fig. 1,
where a hexahedral geometry of the struts can be observed. Details about the pore
structure, mechanical properties, bioreactivity and biocompatibility in osteoblast
cell culture on these scaffolds have been presented in previous studies (Boccaccini
et al., 2007; Chen and Boccaccini, 2006; Chen et al., 2008).
Permeability tests were performed following the Darcys law:
k
mt
A
Q
Dp
(1)
where k is the intrinsic permeability (m
2
), t the specimen thickness (m), A the
cross-sectional area (m
2
), Q the ow rate (m
3
/s), Dp the pressure drop (Pa) and m
the dynamic uid viscosity (Pa s). Nevertheless, it should be pointed out that the
Darcys law is not adequate for very high velocities within the scaffold, e.g. ow
values lower than 500mL/min are recommended for the analyzed scaffold.
Specically, Darcys law is not valid when the so-called interstitial Reynolds
number is higher than 8.6 for a linearity error higher than 10% (Scheidegger, 1974).
The permeability rig used to measure the intrinsic permeability (m
2
) is shown
schematically in Fig. 2. As mentioned above, the intrinsic permeability is a
parameter independent of the uid used to measure it, and it is related to the
degree of interconnectivity of the porous scaffold. Then, a pressure-induced
permeability test was performed using deionized water (m 10
3
Pa s). The uid
was moved with the use of a peristaltic pump and was taken from an open
reservoir to the air (see Fig. 2). Because of the peristaltic pulse provided by the
pump, a uid damper KH-07596-20 Pulse Dampener (Coleparmer, Masterex) was
used to get a continuous ow through the circuit. The scaffold samples were
placed in the permeability chamber where the pressure drop was measured. The
permeability chamber was a cross-sectional area reductor available to place the
scaffold sample. This chamber exhibits two different diameters, d
1
5.30mm and
d
2
8mm, and the scaffold is located at the interface between both sections to
avoid its relative movement due to the uid stream. The pressure drop was
measured between two points at the inlet and outlet of the reductor with the use
of a pressure meter Digitron 2080P (Digitron
s
Instrumentation). Due to the setup
of the test, the measured pressure drop is attributed to the scaffold microstructure
(Dp) and the section change (Dp
Sec
). Therefore,
Dp
Total
Dp Dp
Sec
(2)
ARTICLE IN PRESS
Fig. 1. Bioglass
s
scaffold microstructure.
Fig. 2. Experimental rig used for permeability test to measure Darcys perme-
ability.
I. Ochoa et al. / Journal of Biomechanics 42 (2009) 257260 258
with Dp
Total
being the measured pressure drop by the pressure meter and (Dp
Sec
)
can be given by the following equation:
Dp
Sec

2Q
2
r
p
1
d
2
1

1
d
2
2
!
(3)
Different uid ow regimes were applied controlling the ow rate with the
peristaltic pump on two specimens of the Bioglass
s
foams. The thickness of the
samples were 8.52 and 8.11mm for samples 1 and 2, respectively, being in both
cases the cross-sectional area A pd
2
2
/4. The corresponding DpQ curves were
obtained for both samples and the intrinsic permeabilities were estimated from
that data.
3. Results and discussion
The relationship between the ow rate and measured pressure
drop is depicted in Fig. 3 for the two samples investigated.
Because of the geometry of the reductor tube and the applied
ow rates, the pressure drop due to the sectional reduction
is negligible being, therefore, the measured pressure drop
mainly related to the inclusion of the scaffold and consequently
the experimental value is valid for the measurement of the
intrinsic permeability.
From the data in Fig. 3 and Eq. (1), the permeability values for
the two samples tested were found to be 1.8510
9
and
2.0710
9
m
2
, respectively. Averaging for Bioglass
s
-based foams
of 9095% of porosity, the permeability is thus k 1.9610
9
m
2
.
It should be noted that the values shown in Fig. 3 are
exclusively valid for deionised water. However, the intrinsic
permeability values obtained from the data in Fig. 3 are
independent of the uid, they only depend on the scaffold pore
structure. It is therefore instructive to compare the values of
permeability determined for the present Bioglass
s
scaffolds with
data available in the literature. For the case of bone, for example,
values between k 2.010
9
and 9.510
9
m
2
have been
presented for cancellous bovine bone (Kohles et al., 2001) in the
range of porosities 8090%. In addition, values of k 7.2210
9
and 5.1310
9
m
2
have been found for human cancellous bone of
the vertebral body and proximal femur, respectively, although
high variations from these values may be obtained dependent on
the site region and overall porosity (Nauman et al., 1999).
Moreover, Shimko et al. (2005) found values of intrinsic perme-
ability in the range k 5.210
10
6.210
10
m
2
for tantalum
scaffolds in the porosity range 9095%. Several other values have
been reported for scaffolds by Haddock et al. (1999), who
measured values in the range k 1.6310
10
8.6210
10
m
2
.
Additionally, Li et al. (2003) established k 2.1310
10
m
2
for
porous biomaterials of 70% porosity. As a result, the measured
intrinsic permeability in the present experiments is in the range of
values reported by several authors on both bone and scaffolds. In
this context, it should be highlighted that the intrinsic perme-
ability is a function of the pore morphology, interconnection and
pore size as well as overall porosity. This fact shows that scaffolds
of the same porosity may result in different values of the intrinsic
permeability due to differences of the microstructural design of
the pore structure and morphology. However, if we consider a
specic family of scaffolds containing a regular microstructure for
a wide range of porosities, for instance those fabricated using
rapid prototyping, permeability can be shown with enough
accuracy to be an increasing function correlating with the third
power of porosity (Sanz-Herrera et al., 2008). Therefore, to get a
high overall permeability, high-porosity scaffolds are recom-
mended. With increasing porosity, however, the apparent
scaffold stiffness will decrease according to the square of porosity
(Sanz-Herrera et al., 2008). Thus, scaffold design should consider
an optimal porosity enabling sufciently high permeability for
waste removal and nutrients supply and adequate stiffness to
sustain the loads transmitted to the scaffold from the surrounding
healthy bone. Another point of interest related to the intrinsic
scaffold permeability is the attachment and migration of cells to
the scaffold surface. This mechanism seems to be dependent on
both the bulk biomaterial stiffness (among many other effects)
(Discher et al., 2005) and the available specic surface. The
specic surface is not directly related to the permeability although
it is inuenced by permeability since the specic surface is a
function of the microstructural design of the scaffold and porosity,
which determine the overall permeability, as mentioned above.
Usually, higher porosities (for the same size specimen) lead to
lower available specic surface, and consequently less space for
cell attachment and proliferation. Thus, scaffold design is a
complex task which depends on the specic bone tissue
engineering application being the permeability an important
parameter which inuences others and consequently the nal
success of the scaffold in a given certain application.
Bioglass
s
foams fabricated by the replica method have been
shown to be remarkably similar in their interconnected pore
microarchitecture to trabecular bone, e.g. by micro-CT imaging
(Boccaccini et al., 2007). The foam replica technique is in fact a
highly versatile method to produce scaffolds of designed pore
geometry which depends only on the original structure of the
sacricial PU foam (Chen et al., 2006; Vitale-Brovarone et al.,
2007; Wu et al., 2007; Muthutantri et al., 2008). The present study
has conrmed for the rst time quantitatively the resemblance of
the Bioglass
s
-based scaffold pore interconnectivity with that of
trabecular bone via the measurement of permeability.
4. Conclusions
Highly porous Bioglass
s
scaffolds (9095% porosity) fabri-
cated by the foam replica technique were investigated in terms of
their permeability. The experiments were carried out in a
dedicated rig designed for permeability assays to measure Darcys
permeability values. The measured average permeability,
k 1.9610
9
m
2
, is closely within the range of the reported
experimental data for human trabecular bone, conrming that
these scaffolds have transport properties as well as pore structure
close to trabecular bone and, therefore, represent interesting
articial extracellular matrix structures for the bone tissue
engineering applications.
ARTICLE IN PRESS
Q

p

[
P
a
]
0
100
200
300
400
500
600
700
50 ml/min
Sample 1
Sample 2
100 ml/min 200 ml/min 300 ml/min 400 ml/min
Fig. 3. Relationship DpQ for two Bioglass
s
scaffold samples tested, being the
uid deionised water.
I. Ochoa et al. / Journal of Biomechanics 42 (2009) 257260 259
Conict of interest statement
None.
Acknowledgement
DMY acknowledges nancial support from the Ministry of
Science, Technology and Environment of Malaysia (MOSTI). Dr. Ian
Thompson (Kings College London, UK) is acknowledged for
providing the glass powder used in this investigation.
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