Supercritical uid technologies and tissue engineering scaolds

Robin A. Quirk
a
, Richard M. France
a
, Kevin M. Shakeshe
b,
*
, Steven M. Howdle
c
a
RegenTec Ltd, BioCity Nottingham, Pennyfoot Street, Nottingham NG1 1GF, UK
b
School of Pharmaceutical Sciences, The University of Nottingham, University Park, Nottingham NG7 2RD, UK
c
School of Chemistry, The University of Nottingham, University Park, Nottingham NG7 2RD, UK
Received 23 October 2003; accepted 18 December 2003
Abstract
Supercritical uid (SCF) processing methods possess advantages over standard processing methods for the production of
scaolds for use in tissue engineering. Advantages include the absence of organic solvents, the ability to incorporate delicate bio-
logicals without loss of activity, and control over the morphology of an internal porous architecture. This review describes SCF
processing methods of relevance to tissue engineering and controlled release strategies, with focus on the incorporation of bioactives
such as protein growth factors.
2004 Elsevier Ltd. All rights reserved.
Keywords: Supercritical uid; Carbon dioxide; Tissue engineering; Biodegradable polymers; Scaolds; Growth factor release
1. Introduction
Tissue engineering seeks to repair or replace damaged
tissues using a combination of cell/molecular biology
and materials chemistry/engineering. Over the past
decade there has been an intense research eort in this
area, which has lead to the generation of a portfolio of
tissue engineering strategies. These include, (i) direct
implantation of isolated or cultured cells, (ii) implanta-
tion of tissues which have been pre-cultured in vitro, and
(iii) direct in situ tissue regeneration [1,2]. In many
applications a scaold is required to provide the means
for cell/tissue delivery to the repair site and to improve
and control the environment for cell growth and tissue
maturation.
Current challenges to this eld include the provision
of adequate and appropriate cell sources (e.g. auto-
logous vs. allogeneic vs. xenogeneic cells [3], mature vs.
adult stem vs. embryonic stem cells [4,5], etc), vascu-
larisation of engineered tissues [1], improved scaold
materials, appropriate bioreactors and economical scale
up and process automation [6].
The scaold environment should be able to present
and deliver combinations of delicate biological materi-
als such as cell adhesion motifs, growth factors,
angiogenic factors, dierentiation factors and immuno-
suppressive or anti-inammatory agents [1,2,7,8]. For
all these factors, temporal and spatial control of release
are crucial to successful tissue repair and regeneration.
The delivery of, for example, growth factors from the
scaold should be controlled such that the factors are
delivered in appropriate concentrations at the required
time in the regeneration process, as is the case in nat-
ural wound healing processes [8,9]. In addition, any
scaold processing should not alter the activity of any
active biological agent, e.g. denaturation of proteins
through exposure to heat or organic solvents.
A variety of scaolds have been used to date,
including natural or synthetic polymers that generally
form either hydrogel or monolithic solid polymer scaf-
folds [1,3,10]. Injectable hydrogel systems are particu-
larly attractive for non-invasive approaches to in situ
tissue repair but often lack the structural stability of
solid monolithic scaolds. However, solid monolithic
porous scaolds are frequently dicult to eectively
seed with cells due to their often tortuous matrices, and
will require surgically-invasive implantation.
Natural polymer scaolds, including hyaluronic acid
(HyA) [11,12], collagen [13,14], alginate [15] and brin
[16], have all been successfully used for various tissue
*
Corresponding author. Tel.: +44-0-115-951-5104; fax: +44-0-115-
951-5110.
E-mail address: kevin.shakeshe@nottingham.ac.uk (K.M. Shake-
she).
1359-0286/$ - see front matter 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.cossms.2003.12.004
Current Opinion in Solid State and Materials Science 8 (2004) 313321
targets. Natural scaolds can be processed to retain
much of the biological information they contain, or
modied by enzymatic means to include a variety of
biological agents [17,18]. Degradation of natural scaf-
folds is controlled by enzyme release from cells within
the matrix. These materials can, however, suer from
problems such as poor mechanical strength, interbatch
variation, immunogenicity and potential disease trans-
mission [19,20].
Synthetic polymers, including poly(ethylene glycol)
(PEG), poly(lactic acid) (PLA), poly(glycolic acid)
(PGA), poly(lactide-co-glycolide) (PLGA), poly(capro-
lactone) (PCL) and poly(propylene fumarate), have also
been used extensively as tissue engineering scaolds
[3,10,21,22]. Resorption of poly (a-hydroxy acids) is
controlled by random hydrolysis and occurs as a bulk
degradation [23]. This degradation is exploited to provide
a controlled release mechanism for encapsulated thera-
peutics, and ensures that there is no long-term foreign
implant. Unfortunately, acidic by-products from the
breakdown of these polymers can lead to inammatory
reactions in some circumstances [24]. However, their FDA
approval and long history of use mean they remain an
enduring presence in many tissue engineering strategies.
Early research eorts in tissue engineering with syn-
thetic scaolds used degradable scaolds made from
non-woven surgical meshes. Non-woven meshes are
suited to the culture of certain tissue types, e.g. cartilage
[25,26], but lack the structural stability for many appli-
cations. A plethora of new fabrication methods have
therefore since been investigated, with the choice and
design of the scaold and processing method depending
on a number of factors.
Key requirements for scaold fabrication methods
include control of porosity/pore size, maintenance of
mechanical properties, and maintenance of material
biocompatibility. Other desirable features include avoid-
ing the use of organic solvents, or if they are required,
complete removal of solvent residues after processing.
The ability to incorporate delicate bioactives without the
denaturation that may occur upon exposure to solvents,
high temperature or shear stresses is also important.
Table 1 summarises some of the current fabrication
methods. Each method has its own particular advantages
and disadvantages, with the choice of method often
depending upon the end application. With the draw-
backs to these methods in mind, this review will focus
upon the emergence of supercritical uid (SCF) tech-
nologies as an alternative or complimentary processing
method.
2. Supercritical uid technologies
A supercritical uid (SCF) is created once a substance
is exposed to an environment where its critical temper-
ature and pressure (the intercept of which is referred to
as the critical point) are exceeded. Under these condi-
tions, the liquid and gaseous components of the material
become identical (Fig. 1), and further compression of
this uid phase will not result in liquecation. SCFs
combine the properties of the two phases from which
they are formed; they have densities and solvating
properties similar to those of liquids, but in combination
with a diusivity and viscosity comparable with that of a
gas. The solvent properties of these materials can in fact
be controlled very precisely by small manipulations in
the pressure (and therefore density) at with the SCF is
used.
CO
2
is the most common candidate for use as a
SCF due to its low toxicity, ammability and cost,
ready availability, stability, and environmental accept-
ability. In addition, the critical point conditions of
31 C and 73.8 bar are readily attainable. As such,
supercritical CO
2
(scCO
2
) has been employed in a di-
verse range of applications, including polymer synthe-
sis [34,35], drug delivery [36,37], powder production
(e.g. proteins [38,39] and ceramics [40]), powder coating
Table 1
Summary of advantages and disadvantages of current scaold fabrication methods
Fabrication method Advantages Disadvantages
Fibre bonding [27] Improved stability over non-bonded tassels and
felts
Limited application as the two polymers must
be immiscible in solvent and melt state;
no real control over porosity and pore size;
poor mechanical properties
Solvent casting/particulate leaching [28] Control over porosity and pore size ( 693%) Only produce thin membranes; use organic
solvents
Melt moulding/particulate leaching [29] Control over porosity and pore size ( 693%);
no organic solvents
High temperature required
Emulsion freeze drying [30] High volume of interconnected porosity ( 697%) User/equipment sensitive; use of organic solvents
Phase separation [31] High volume of interconnected porosity ( 697%) User/equipment sensitive; use of organic solvents
3D printing [32] High degree of spatial control of scaold
architecture
Use of organic solvent with PLA/PGA
Fused deposition modelling [33] High degree of spatial control of scaold
architecture
Polymer heated to melt state
314 R.A. Quirk et al. / Current Opinion in Solid State and Materials Science 8 (2004) 313321
[41], dyeing [42], impregnation [43], and lithography
[44].
Exploitation of the unique properties of SCFs has led
to the development of a number of polymer processing
strategies. There are many excellent reviews outlining
these processes in more detail [36,45], so, as this review is
concerned primarily with the processing of tissue engi-
neering scaolds, the brief discussion of these methods
will be limited to manipulation of such materials. As
such, there are three main types of processing strategy in
use:
1. rapid expansion of supercritical solutions (RESS),
2. particles from gas saturated solutions (PGSS),
3. antisolvent techniques.
2.1. RESS technique
In polymer processing, this method is often used to
generate microparticles for drug delivery applications
[46,47]. Here, the polymer and drug must dissolve in the
SCF. This mixture is then rapidly expanded into a low
temperature and pressure environment. This leads to a
rapid decline in the solubility of the polymer in the SCF,
and crystallisation of the solute as micro- or nanopar-
ticles of a narrow size distribution. RESS can be used
for any polymer with sucient solubility in the SCF.
Unfortunately, most polymers and pharmaceuticals
have a very low solubility in scCO
2
, except for their low
molecular weight fractions. Despite these drawbacks,
this approach has already been used to generate P
DL
LA
microparticles containing a cholesterol-lowering statin
agent [48].
2.2. PGSS technique
The eect of scCO
2
on many polymers is to lower
their glass transition temperatures (T
g
) and, if the T
g
is
reduced below the operating temperature, the material
will plasticize, or liquefy. This is because of the very high
solubility of the dense gas in the polymer. Some poly-
mers are in fact able to absorb large concentrations
(1040 wt%) of CO
2
[45], which enables the polymer to
liquefy at a temperature well below its T
g
. The SCF is
therefore applied under pressure into the polymer until a
gas saturated solution is formed. When depressurized
through a nozzle, the gas comes out of liqueed poly-
mer, and the polymer foams, or forms into well dened
particulates. This process has the advantages that the
starting material does not have to be soluble in the SCF,
and that no organic solvents are required during pro-
cessing. This technique has been previously employed to
fabricate microparticles of nifedipine. In this example,
the drug molecule itself was shown to plasticize in the
presence of the SCF, thus leading to production of ne
particles with enhanced release properties [49]. Addi-
tionally, a mixture of PEG and nifedipine was found to
micronise to particles. A variant of the process devel-
oped by Shine and Gelb has also been developed to
microencapsulate viral materials within polymers. The
technique was termed polymer liquecation using SC
solvation (PLUSS) [45].
2.3. Antisolvent techniques
The Gas Antisolvent (GAS) technique [36,50] em-
ploys a SCF to act as an antisolvent and precipitate a
solute from an organic solvent. As the liquid phase is
soluble with the SCF, it expands upon its addition,
which results in a decreased capacity of the solvent to
support polymer dissolution. This results in the precip-
itation of microparticles, the size of which can be con-
trolled by adjusting the temperature, pressure or rate of
gas addition. This strategy has been used to prepare
P
L
LA [51] and PEG/PLA blend nanoparticles [52]
for incorporation and delivery of insulin, with activity
of the protein being shown to be maintained dur-
ing processing. Other related antisolvent techniques in-
clude precipitation with compressed antisolvents (PCA)
[53,54], supercritical antisolvent (SAS) processing [55],
aerosol spray extraction system (ASES) [56], and solu-
tion enhanced dispersion by SCFs (SEDS) [57].
3. Uses of SCFs in tissue engineering
Supercritical uids have been applied in recent years
to developing tissue engineering strategies. The impetus
for embracing these processing methodologies has ran-
ged from their potential ability to create scaolds with
controlled porosities [58], the creation of encapsulated
growth factors [59,60], pharmaceuticals [36] and plas-
mids [61] for controlled delivery to developing tissues,
the removal of residual solvents following other fabri-
cation methods [62], and even the treatment of tissue
samples to improve their biointegration (e.g. bone del-
ipidation to reduce the immunogenicity of allogeneic
Fig. 1. Chronological images of a view cell showing the creation of the
single supercritical uid phase (image c) by taking liquid CO
2
above its
critical temperature and pressure (images a and b); with subsequent
lowering temperature (and hence pressure) the process is reversed
(images d and e).
R.A. Quirk et al. / Current Opinion in Solid State and Materials Science 8 (2004) 313321 315
grafts [63,64]). Due to the ambient processing tempera-
tures, and the potential to process polymers without
employing organic solvents, the supercritical approach
is also attractive for the potential to preserve the activity
of delicate protein molecules that may form an integral
component of the tissue regeneration process.
Tissue engineering scaolds are required to possess
both a high degree of porosity and an interconnected
pore structure. This ensures adequate cell attachment
throughout the 3D matrix, promotes both rapid angio-
genesis throughout the construct and integration of the
implant with host tissue, and facilitates cell migration
and nutrient transfer during tissue morphogenesis.
Porosity is created during the SCF process by the ther-
modynamic instability that results following depressu-
risation around the liqueed polymer [65,66]. This
results in the nucleation of the gas molecules in order to
minimize their free energy, which creates pores as the
polymer resolidies. The porosity and pore structure of
the resultant scaold can therefore be controlled by
varying the amount of gas incorporated and, as shown
in Fig. 2, its release rate from the polymer (which is in
turn related to the pressure and temperature to which
the polymer is exposed and saturated with CO
2
), or the
gas diusion rate (controlled by temperature and de-
pressurisation rate). The nucleation of pores will occur
both homo- and heterogeneously [58,67], that is, will
form either throughout the continuous polymer phase,
or at an interface within the material. Heterogeneous
nucleation occurs more readily than the homogeneous
route due to the work and free energy required in cre-
ating a volume change and forming a stable new surface.
Factors such as the inherent porosity in the pre-
processed material and residual solvents that may
plasticize and alter the surface free energy of the poly-
mer can therefore impact upon the extent of each
nucleation type, the diering rates of which inversely
aect the average pore size formed [58].
In 1990, De Ponti et al. described a gas foaming
process for fabricating scaolds from the biodegradable
poly(a-hydroxyacid)s (e.g. P
L
LA, P
DL
LA, PGA and
PLGA) commonly employed in tissue engineering re-
search [68]. Subsequently, Mooney et al. [58] exposed
either compressed polymer pellets or solvent cast discs
to CO
2
at a pressure of 55 bar at 2023 C for 72 h (see
Table 2). Although not supercritical, over prolonged
periods, CO
2
under these conditions is still able to sat-
urate the polymer and create a gas saturated polymer,
from which depressurisation/gas nucleation can result in
a vitried, porous scaold being formed. It was con-
rmed that a high amorphous fraction is a prerequisite
for this processing technique as, of the materials tested,
crystalline polymers (PGA, P
L
LA) did not form porous
structures due to their relative inability to dissolve
gases. The intrinsic viscosity of the polymer must also
be considered, as longer polymer chains, being more
entangled, display an increased resistance to expansion
during nucleation, and thus result in a smaller pore size
[72].
Two potential drawbacks that were noted with this
strategy were the formation of a non-porous skin on
most samples (which results from rapid diusion of gas
away from the surface [58,85]), and a lack of intercon-
nectivity between pores (limited to between 10% and
30% of the total pore number [21]). These problems were
successfully addressed by the introduction of NaCl
particles as a porogen prior to CO
2
exposure, which is
leached out afterwards [69]. As this process relied on the
close proximity of randomly distributed porogen, fur-
ther control over the degree of interconnection was later
achieved by fusion of adjacent salt particles following
exposure to a high humidity environment [71].
When using this strategy, it has been shown that
consideration should be given to the ratio of salt to
polymer [75] and the gas exposure time used [72], in
order to ensure that the scaolds created in this manner
possess suitable mechanical strength. This method of
scaold fabrication has been employed both for the
controlled delivery of proteins and as a support for
the 3D culture of numerous cell types (Table 2). Of
particular importance has been the demonstrated dual
delivery of two angiogenic growth factors (vascular
endothelial growth factor; VEGF, and platelet-derived
growth factor; PDGF), each with their own tailored
release proles. This was achieved by simultaneously
processing protein-encapsulated polymer microparticles
and a powder/protein mix using this gas foaming ap-
proach [74]. The microparticle-encased factor is dis-
persed evenly throughout the resultant scaold matrix
and is released as the polymer degrades, whereas the
factor that is mixed freely is associated largely with the
surface of the polymer and is therefore released more
rapidly. VEGF is an initiator of angiogenesis while
PDGF has been shown to promote maturation of blood
Fig. 2. SEM micrographs of P
D; L
LA polymer after processing in
scCO
2
at 240 bar, 35 C with (a,b) 12 min venting and (c,d) 60 min
venting.
316 R.A. Quirk et al. / Current Opinion in Solid State and Materials Science 8 (2004) 313321
Table 2
Summary of some SCF processes reported for the fabrication of tissue engineering scaolds
Scaold material Pre-process
presentation
CO
2
process conditions Scaold characterisation Demonstrated
application (if any)
Comments Ref.
Pressure
(bar)
Temp
(C)
Exposure
time (h)
Pr. release
rate (min)
Porosity (%) Ave pore
size (lm)
P
DL
LA (50:50) Solvent cast discs 55 2023 72 0.25 PLGA97 10100 Skin layer on most sample
surfaces
[58]
P
DL
LA P
L
LA72
P
L
LA P
DL
LA67
PLGA (50:50) Heat compression
moulded discs
55 2023 72 0.25 PLGA94 100500 PGA can be mixed with PLGA to
regulate porosity
[58]
PGA PGA0 (PLGA)
Skin layers on sample surfaces
PLGA (85:15) Polymer pellets mixed
with NaCl
55 Room 48 8596.5 193439 Smooth muscle
cell culture
No skin present [6971]
Interconnected pores
Salt fusion further enhances
interconnectivity
PLGA
(85:15) +alginate
Compression
moulded with NaCl
59 Room 20 2 93 Mineralized for
bone engineering;
VEGF delivery
Scaolds mineralized by incuba-
tion in simulated body uid
[59]
VEGF over 70% active for up to
12 days.
44% incorp eciencylosses
during leaching/mineralization
P
DL
LA Compression
moulded with and w/o
NaCl and alginate
59 Room Up to 48 110 PLGAs 95 360 VEGF delivery Very rapid pressure release
reduces porosity by 2%
[72,73]
PLGA (50:50;
75:25 and 85:15)
P
DL
LA0
Short exposure times (<6 h)
creates fragile scaolds
PGA
PGA0
VEGF over 100% active com-
pared with controls, with incorp
eciency 90% and 72% for PLGA
85:15 and 75:25 respectively
PLGA (75:25;
85:15)
Double emulsion
microspheres and
particles with alginate
55 Room 72 0.25 Dual delivery of
PDGF and VEGF;
angiogenesis
Release proles controlled by the
respective growth factor distribu-
tions throughout polymer
[74]
PLGA (75:25) Double emulsion
microspheres and
NaCl
55 Room 1216 Rapid >94 DNA delivery Salt:polymer ratio between 25
29:1 required to give interconnec-
tivity and mechanical strength
[61,75]
PLGA (80:20;
65:35)
Emulsion with aque-
ous protein phase
80 35 24 0.170.2 bFGF delivery Residual solvent above acceptable
USP levelsfurther solvent
removal required
[76]
P
DL
LA Polymer powders 172 37 <1 2120 100500
and
Hydroxyapatite
composites; pro-
tein delivery (inc.
BMP-2 and pleio-
trophin); bone
engineering; liver
One-step process [60,7782]
PLGA (75:25)
0.055
Interconnected pores created
without need for porogens PCL
Enzyme activity 100% retained
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vessel networks by recruitment of smooth muscle cells.
Dual delivery of these two factors each, with distinct
release kinetics, promoted the formation of a vascular
network in vivo to a greater extent than either factor
delivered alone. Such complex release kinetics will prove
essential in order to mimic the intricate cascades of
growth factors that are stimulated at precise stages
during tissue formation and repair [8,86]. It should be
pointed out that the drawback of the salt leaching route
to controlled porosity is that the leaching stage does
remove some of the incorporated bioactive material.
Approaches for poly(a-hydroxyacid) scaold pro-
duction using scCO
2
have been reported by Hile et al.
[76]. Here, a water-in-oil emulsion, consisting of an
aqueous protein (basic broblast growth factor; bFGF)
phase and an organic polymer solution phase, is exposed
to a SCF in order to (i) extract the organic solvent
and precipitate the polymer, (ii) saturate the polymer
phase and then, upon pressure release, (iii) create a
porous structure. Reported residual solvent levels were,
however, above US Pharmacopoeia levels, and further
solvent removal is therefore required before these con-
structs would be suitable for in vivo use.
The method of Howdle et al. [61,87] is based upon the
PGSS approach and completely avoids the use of sol-
vents. Scaolds such as those shown in Fig. 3 are instead
produced in a one-step process, a protein being rst
homogeneously distributed throughout the plasticized
polymer by use of an impeller device [83,88]. The rate of
depressurisation has been shown to aect the pore size
distribution of the resultant matrices, with a faster de-
pressurisation (2 min) creating smaller pores. The
process also leads to the generation of micropores
throughout the scaold i.e. interconnectivity is created
without the use of porogens. This approach has been
used to create P
DL
LA biocomposites containing high
loadings of calcium hydroxyapatite [87]. The enzyme
ribonuclease A was also incorporated and released over
an 80 day period, with activity being completely retained
following exposure to the processing conditions [61].
Fig. 3. Fabrication of cylindrical porous P
D; L
LA scaolds by SCF
processing of the polymer within a shaped mould. T
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318 R.A. Quirk et al. / Current Opinion in Solid State and Materials Science 8 (2004) 313321
Successful bone formation has been demonstrated fol-
lowing the culture of osteoprogenitor cells on these
scaolds in both in vitro and in vivo models using a
range of both adhesion peptide [77] and growth factor
modications [7880]. An alternative to the creation of
solid scaolds is to force the polymer through an orice
during depressurisation and to thus fabricate micro-
particles [89]. Microparticle-based scaolds are nding
application as injectable systems for cell and growth
factor delivery within the body direct to a site of injury
or repair [90].
Despite poly(a-hydroxyacid)s remaining the most
popular choice of scaold material (and therefore fea-
turing heavily in SCF fabrication strategies), other
polymers are also beginning to be processed into 3D
matrices using this route. These include the crystallisa-
tion of swollen, crosslinked low density poly(ethylene)
using SC propane to create porous structures for the
culture of hepatocyte HepG2 cells [91], and the produc-
tion of solid structures using hyaluronic acid-derivatives
[84]. HyA is a naturally-occurring glycosaminoglycan,
and is increasing in popularity as a bioresorbable scaold
material. Its use to date has been limited by its high
solubility in aqueous solutions at body temperature; an
eect that can be remedied by the creation of polymers of
HyA esters [92]. These materials have been used in the
areas of bone [93] and liver [94] regeneration, and in
urethral reconstruction [95]. Employing an antisolvent
(SAS) SCF process, these polymers can be fabricated
into sponges, threads, and microparticles, with negligible
residual solvent being evident after processing.
4. Summary
In recent years, SCFs have been employed to address
the drawbacks of existing scaold fabrication methods.
Key advantages are to avoid the use of conventional
solvents or, if required, to eliminate organic solvent
residues. Additionally, they can be used to create
biocomposites and/or preserve protein activity, and to
exert a high level of control over scaold porosity
and architecture. Numerous processing strategies are
emerging which rely on SCFs for either their plasticizing
or antisolvent properties. However, despite the obvious
advantages, there are some limitations. The control over
internal scaold architecture cannot approach that of
the 3D printing technique, and the range of polymer
types for which SCFs are applicable might be limiting in
some applications, particularly where high mechanical
strength is required. Further process optimisation is
therefore still required, but the future of SCFs looks
bright, and the versatility of SCF processing will ulti-
mately result in innovative combinations with other
scaold fabrication methodologies.
Acknowledgements
We thank JJA Barry and MMCG Silva for their help
and advice. SMH is a Royal Society-Wolfson Research
Merit Award Holder.
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