J.

of Supercritical Fluids 97 (2015) 238–246

Contents lists available at ScienceDirect

The Journal of Supercritical Fluids

journal homepage: www.elsevier.com/locate/supflu

Bio-safe fabrication of PLA scaffolds for bone tissue engineering by

combining phase separation, porogen leaching and scCO2 drying
Aurelio Salerno a,∗ , Mar Fernández-Gutiérrez b,c ,
Julio San Román del Barrio b,c , Concepción Domingo a
a
Institute of Materials Science of Barcelona (ICMAB-CSIC), Campus de la UAB s/n, Bellaterra 08193, Spain
b
Institute of Polymer Science and Technology, CSIC, Madrid 28006, Spain
c
CIBER-BBN, Health Institute Carlos III, Spain

a r t i c l e i n f o a b s t r a c t

Article history: The present study reports a three-step bio-safe process for fabricating porous polylactic acid (PLA) scaf-
Received 24 July 2014 folds with a biomimetic design of the porosity and the architecture and applications in bone tissue
Received in revised form 28 October 2014 engineering (bTE). The process starts with a PLA–ethyl lactate solution and involves the combination
Accepted 28 October 2014
of thermal induced phase separation (TIPS) and supercritical CO2 (scCO2 ) drying techniques for the
Available online 18 December 2014
development of a nano-fibrous architecture. In a third step, it was further combined with a gelatin
leaching technique to create an additional interconnected network of large pores of appropriate size
Keywords:
and distribution.
Biomimetic scaffold
Bone tissue engineering
The effect of the size of the gelatin particles, selected in three different ranges: 100–200, 200–400 and
Ethyl lactate 400–600 ␮m, on the morphology, porosity and textural features of PLA scaffolds is assessed by means of
Phase separation scanning electron microscopy analysis, gravimetric measurements, image analysis and low-temperature
scCO2 drying N2 adsorption. Finally, in vitro cell culture tests are carried out by using human osteosarcoma cell line
MG63 in order to evaluate the biological response of the different scaffolds prepared and to assess their
potential for bTE applications.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction regeneration in 3D. In particular, it has been demonstrated that

Bone tissue engineering (bTE) is an increasingly growing
research field, which has as the ultimate goal to develop novel ther-
apeutic treatments for bone tissue loss or failure, two important
human health problems. One of the most promising bTE approaches
involves the combination of cells, such as stem cells and osteoblasts,
and three-dimensional (3D) porous scaffolds [1,2]. For successful
bone tissue regeneration, the scaffold must be designed to act as
a synthetic extracellular matrix (ECM) analogue, providing a 3D
biomimetic structure for cell growth and new tissue development
in vitro and/or in vivo.
The rational design of ECM mimicking scaffolds is based in the
evidence that, in the native bone, the cross-talk between cells
and ECM determines the correct new bone tissue development
and functionality [3]. From morphological and structural points of
view, biomimetic scaffolds must be designed with a hierarchical
porous architecture to facilitate cell colonization and guide tissue
∗ Corresponding author. Tel.: +34 935801853.
E-mail address: asalerno@icmab.es (A. Salerno).
porous scaffold having a high porosity and an interconnected net-
work of large pores, in the order of several hundreds of microns,
promote the in vitro bone cell invasion and differentiation, as well
as the in vivo bone tissue infiltration [1–3]. Concomitantly, the
presence of a nano-scale fibrous pore wall texture, mimicking the
fibrous architecture of native collagen, is a determinant key to pro-
mote bone cell adhesion, migration and osteogenic differentiation
[4–8].
The possibility to design and fabricate biocompatible and
biodegradable scaffolds with a biomimetic design of porosity
and architecture by means of bio-safe and reliable approaches
is also a critical step towards the manufacture of bTE prod-
ucts and their translation into the market [9]. The thermally
induced phase separation method (TIPS) represents one of the
most important approaches used for fabricating nano-scale fibrous
porous polymeric scaffolds for bTE [5,8]. The basic principle of
this technique is that, by changing appropriately the tempera-
ture of a homogeneous polymer–solvent solution, it is possible
to decrease polymer–solvent affinity and to induce solution
gelation by the formation of interpenetrated networks of polymer-
rich and polymer-lean phases. Most importantly, in the case of

http://dx.doi.org/10.1016/j.supflu.2014.10.029
0896-8446/© 2014 Elsevier B.V. All rights reserved.
 A. Salerno et al. / J. of Supercritical Fluids 97 (2015) 238–246
semi-crystalline polymers, the appropriate polymer–solvent
choice and the control of the temperature and the kinetics of
the process promote the crystallization of the polymer rich phase
through the formation of a nano-scale fibrous architecture [5,8].
The final scaffold is achieved by the selective solvent extraction
and the drying of the gel. Moreover the solvent choice for the TIPS
process is an important and critical issue, as solvent residues can
affect the biocompatibility and biological response of the scaffolds.
We have recently reported a novel bio-safe process for the
design and fabrication of nano-scale fibrous polylactic acid (PLA)
aerogels with controlled morphology and structural properties
[10]. The process involved the use of ethyl lactate (EL) as a green sol-
vent to process PLA, whereas the supercritical CO2 (scCO2 ) solvent
extraction technique was applied for gel drying. EL, which is a Food
and Drug Administration approved additive in the food industry,
is a biodegradable compound and does not have potential health
risks [11]. Furthermore, the combination of the scCO2 drying pro-
cess and the TIPS method to a PLA–EL solution has made possible
the preparation of polymeric fibrous aerogels avoiding the collapse
of fibres network during solvent extraction [10].
This work represents a step beyond to previously developed
TIPS processes for PLA scaffolds preparation, since the system
here designed has and additional predesigned interconnected net-
work of large pores together with the nano-scale fibrous structure,
thus adequate for bTE. To achieve this goal, the TIPS method is
first combined with particulate leaching techniques, followed by a
scCO2 drying process. In particular, biocompatible and biodegrad-
able gelatin particles are selected as the solid porogen [12]. The
use of potentially toxic chemicals during the fabrication route is
thus so circumvented. Furthermore, the particulate leaching tech-
nique helps in achieving a fine control over the architecture of the
porous scaffold network by the appropriate selection of the con-
centration and size distribution of the particulate porogen [13,14].
Fig. 1. Scheme of the preparation of biomimetic PLA scaffolds by TIPS process combined with porogen leaching and scCO2 drying techniques.
239
In this work, gelatin particles with three different size ranges:
100–200, 200–400 and 400–600 ␮m were used. Finally, we char-
acterize the in vitro biocompatibility of the as prepared scaffolds
by using human osteosarcoma cell line MG63 (MG63) to evaluate
prepared PLA scaffolds potential for bTE applications.
2. Experimental
2.1. Materials
PLA with an 80/20 L/DL ratio, a molecular weight of 200 kDa
and an inherent viscosity at midpoint of 3.8 dL/g, was provided
by Purac Biochem (Gorinchem, The Netherlands). EL (photore-
sist grade; purity ≥99.0%) and ethanol (99.5%) were provided by
Sigma–Aldrich (Madrid, Spain) and used without further purifica-
tion. Gelatin particles (Merck, Darmstadt, Germany) were used as
the particulate porogen. CO2 (99.95 wt.%, Carburos Metálicos) was
used as the drying agent.
2.2. PLA scaffolds fabrication
The process for the fabrication of the porous nano-scale fibrous
PLA scaffolds is reported in the scheme of Fig. 1. In step 1, a homo-
geneous solution of PLA in EL (5%, w/v) was prepared at 70 ◦ C
under magnetic stirring for 8 h. Concomitantly, gelatin particles
were sieved into three different size ranges, namely 100–200 ␮m,
200–400 ␮m range and 400–600 ␮m range, and were used to con-
trol the size of the pores of the fabricated scaffolds. In step 2, the
sieved particles were manually mixed with the polymeric solution
(0.8%, w/v). Next, the as prepared mixture was placed in an alu-
minium mould, measuring 6 mm in diameter and 15 mm height,
and slight compressed to promote the compaction of the mix-
ture and the contact between adjacent gelatin particles. Solution
240 A. Salerno et al. / J. of Supercritical Fluids 97 (2015) 238–246
gelation was further induced by keeping the samples at 6 ◦ C for
3 h (step 3). Once the setting of the gel was achieved (Fig. 1), the
EL and gelatin particles were sequentially extracted by soaking the
gel first in ethanol for 1 h at room temperature and then in water
at 40 ◦ C for 2 days (step 4). Finally the scaffolds were processed
by means of a scCO2 drying process (step 5). To this purpose, the
samples were first soaked into excess of ethanol and the process
repeated three times for complete water exchange. The as obtained
PLA alcogels were placed inside of a 114 mL volume high-pressure
autoclave (TharDesign, Pittsburgh, USA) on the top of a metal-
lic support to allow for the addition of a magnetic stirrer at the
bottom of the vessel. This configuration is expected to improve flu-
ids mixing and to reduce equilibrium time [15]. The temperature
was then increased up to 39 ◦ C and liquid CO2 was pumped inside
the vessel up to a pressure of 19 MPa to ensure the achievement
of supercritical conditions without crossing the glass transition
temperature of PLA. Samples were held at these conditions for
90 min, time after which pure CO2 was flushed inside the vessel for
15 min and, finally, the vessel was depressurized to the ambient in
approximately 1 h.
2.3. Characterization of the morphological and structural
properties of PLA scaffolds
Scanning electron microscopy (SEM) analysis was used to
assess the morphology of the fabricated porous scaffolds. Sam-
ples were cross-sectioned and gold sputtered prior to be
analyzed with SEM equipment (QUANTA 200F FEG-ESEM, FEI,
The Netherlands).
The characteristics of the pore structure of the scaffolds were
determined by porosity measurements, image analysis (Image J® )
and low temperature N2 adsorption–desorption analysis. Two dif-
ferent gravimetric measurements were used to assess the overall
porosity of the scaffolds. In the first method, the overall porosity
Fig. 2. SEM images showing the morphology of PLA scaffolds as a function of the size range of gelatin particles used as solid porogen.
was determined from the dry mass and the volume of the scaffolds
using Eq. (1) [9]:
   
S
% porosity = 1 − × 100 (1)
PLA
where PLA is the density of raw PLA and S is the apparent density
of the scaffold calculated from mass and volume measurements in
dry state. The mass was measured by using a high accuracy balance
(CP224S, Sartorius, Germany), while the volume was determined
by geometrical calculation using a calliper.
In the second method, the porosity of the scaffolds was deter-
mined by using Eq. (2):
mW − mD
% porosity = × 100 (2)
(mD /PLA ) + mW
where mW and mD are the wet (water) and dry masses of the
scaffold, respectively. This second equation is based on the vol-
ume of fluid (water) entrapped in the pores of the scaffolds and,
consequently, it enables the determination of the interconnected
porosity in the scaffolds. Five measurements were carried out for
each scaffold type.
Image analysis was used to assess the mean fibres diameter,
the volume fraction of large pores as well as large pores mean size
and size distribution, volume density and wall thickness. The text-
ural properties of the scaffolds were studied by low temperature
N2 adsorption–desorption analysis. The tests were carried out at
−196 ◦ C using an ASAP 2000 (Micromeritics, Norcross, USA). Prior
to measurements, samples were dried at 40 ◦ C under reduced pres-
sure for 2 days. The specific surface area was determined by the
BET method. Mesopore volume and mean pore diameter were cal-
culated using the BJH method from the adsorption curve of the
isotherm, while micropore volume was assessed by the t-curve
method.
The static compression properties of the scaffolds were deter-
mined by using a MTS QTest 1L equipment (North Carolina, USA),
 A. Salerno et al. / J. of Supercritical Fluids 97 (2015) 238–246 241

working at a cross head of 1 mm/min and equipped with a 100 N of microns, is of great importance in bTE to ensure the 3D migration
loading cell. Five cylindrical samples measuring 6 mm in diameter and colonization of bone cells and the correct development of new
and 6 mm in height were tested for each formulation. The elastic bone tissue in vitro and in vivo [1,2,12,14]. In addition, the presence
compression modulus (E) was determined as the slope of the initial of a double scale pore size distribution and a nano-scale fibrous pore
linear portion of the stress () vs. strain (ε) curve, while the com- wall texture can improve fluids transport and cell–scaffold inter-
pression yield strength ( Y ) and strain (εY ) were calculated as the actions in the entire construct [2,4,5]. To achieve this goal, in this
intercept between the elastic and plateau lines. work we proposed a three-steps process based on the combination
of TIPS, particulate leaching and scCO2 drying techniques. Although,
a similar approach has been recently reported for the preparation
2.4. In vitro cell–scaffold interaction study
of porous PLA scaffolds [13], this is the first work describing a com-
pletely bio-safe route for PLA scaffold design and fabrication.
The biocompatibility of the scaffolds was assessed in vitro by
In Fig. 2, it is reported the effect of the size of the gelatin
using MG63 cells (ECACC, Sigma–Aldrich, Madrid, Spain). Scaf-
particles on the morphology of porous PLA scaffolds prepared by
folds for cell culture experiments were sterilized by soaking
the three-step process developed in this work. As shown, scaf-
them in absolute ethanol and further washed with sterile PBS
folds have homogeneous morphologies and interconnected pores
(phosphate buffer saline, pH 7.4, Sigma) and culture medium dur-
(white arrows). Furthermore, the low magnification SEM micro-
ing 24 h. The culture medium was Dulbecco’s modified Eagle’s
graphs demonstrate that the particulate leaching technique allows
medium enriched with 4500 mg/mL of glucose (DMEM, Sigma)
modulating the size of the large-pores of the scaffolds according to
supplemented with 10% foetal bovine serum (Gibco), 200 mM l-
the size range of the gelatin particles used. The comparison of the
glutamine, 100 units/mL penicillin, 100 mg/mL streptomycin and
morphology of the scaffolds also suggests that the increase of the
1% non-essential amino acids (NEEA) modified with HEPES (all from
size range of the gelatin particles results in porous structures with
Sigma, Spain).
thicker pore walls and lower pore throats between adjacent large-
Cells were statically seeded onto the scaffolds at a density
pores. When the size of the gelatin particles increases, their contact
of 1 × 105 cell/mL. The cell–scaffold constructs were subsequently
points decreases during impingement, while the inter-particle
placed into 96-well culture plate and maintained in excess of cul-
space increases [8] producing the described effect. The interconnec-
ture medium up to 14 days and the culture medium changed at
tivity between pores obtained by the particulate leaching technique
selected time intervals. Fibrin gel was used as the control sample
can be further improved by pre-sintering the gelatin particles into
for cell culture tests.
a controlled template structure [8,16]. A close observation of the
Quantitative analysis of cell adhesion and proliferation onto the
pore walls in the scaffolds (Fig. 2) reveals that the operating condi-
scaffolds was carried out by means of the Alamar Blue test. At pre-
tions used during the TIPS–scCO2 drying steps lead to the formation
determined culture times, 100 ␮L of Alamar Blue dye (Serotec, 10%
of highly interconnected nano-scale fibrous textures.
Alamar Blue solution in phenol red free DMEM medium) was added
There are several literature studies reporting the fabrication
to the cell–scaffold constructs and the samples were incubated for
of nano-scale fibrous materials by means of a TIPS technique
4 h. Subsequently, 100 ␮L of the reaction media for each test sample
[5,8,12,17,18]. In some of these works, the studied material is PLA,
was extracted and transferred to a 96-well plate. The fluorescence
since this polymer is biodegradable and can be processed to prepare
emission was measured at 590 nm ex and 630 nm em on a Biotek
nano-fibres starting from a wide range of solvents and composi-
Synergy HT. The remaining samples were washed with PBS twice to
tions [5,17,18]. As reported by Shao and co-workers, during the
remove any residual reagent, and 1 mL of the culture medium was
early stage of polymer crystallization from a solution, the PLA
added to continue cell culture experiments. This step was done at
condenses first in the form of amorphous nano-particles of about
1, 3, 7, and 14 days. Analysis of variance (ANOVA) of the results was
20 nm in diameter and, further, it crystallizes by creating a fibrous
performed with respect to control with a p < 0.05 significance level.
structure that is initiated from the central nuclei and grew radi-
Cell adhesion, morphology and colonization into the differ-
als outward [17]. The final morphological, structural and thermal
ent scaffolds were assessed by optical, fluorescence and SEM
properties of the samples are therefore dependent on two main
microscopy analyses at 7 days of culture. In particular, the medium
aspects, the gelation conditions and the drying method. Regarding
was removed, the samples were washed with PBS, the cells were
the first aspect, Ma and Zhang investigated in detail the gelation
fixed with a solution of 3.7 vol% formaldehyde in PBS for 10 min,
behaviour of PLA in different solvents, such as tetrahydrofuran
permeabilized with 0.5% Triton in PBS for 30 min, and finally
and N,N-dimethylformamide, and by using polymer concentrations
washed twice with PBS. The nuclei were stained with a solution
in the range of 1–7.5% [18]. The authors reported that solution
of the blue fluorescent dye Hoechst 33342 (1 mL/1000 mL in PBS,
gelation occurred for several solvents and solvent mixtures, while
Sigma) and actin filaments were stained with a solution of the red
gelation time and temperature, as well as polymer concentration,
staining agent phalloidin (1 mL/100 mL in PBS, Molecular Probes)
were key factors affecting the formation of the nano-scale fibrous
and incubated for 30 min at room temperature. Then, samples were
structure. For instance, they demonstrated that at a high gelation
washed with 0.1% Tween 20 in PBS and dried at room tempera-
temperature polymer crystallization occurs before liquid–liquid
ture. All samples were examined by epifluorescence microscopy
phase separation, thus leading to a platelet-like morphology. Con-
using a NIKON ECLIPSE TE2000-S fluorescence microscope. Poly-
versely, at a low temperature, for example below 19 ◦ C in the
meric samples were examined by optical microscopy using the
case of PLA–tetrahydrofuran solution, gelation was induced by
same microscope after fixation with formaldehyde. For SEM, the
liquid–liquid phase separation followed by crystallization of the
samples were freeze-dried and subsequently, the seeding surface
polymer rich phase, finally, leading to a nano-scale fibrous mor-
gold-sputtered prior to be analyzed by the microscope.
phology [18]. The morphological characteristics observed in the
samples prepared in this work evidence that the gelation tempera-
3. Results and discussion ture used, which was more than 20 ◦ C lower than the gelation point
of the starting solution [10], was sufficient to ensure that crystal-
3.1. Morphology and structural properties of PLA scaffolds lization occurs after phase separation, hence achieving the fibrous
structure.
The possibility to design and fabricate porous scaffolds with Another critical aspect regarding the fabrication of nano-scale
interconnected porosity and large pores, of the order of hundreds fibrous polymeric scaffolds is related to the process of gel drying.
242 A. Salerno et al. / J. of Supercritical Fluids 97 (2015) 238–246

Fig. 3. Overall porosity and fraction of large-pores of PLA scaffolds as a function of

the size range of gelatin particles used as solid porogen.

Indeed, owing to the high specific surface and tiny fibrous morphol-
ogy, drying the gel in air often provides a white and dense collapsed
xerogel. This effect depends on the capillary forces exerted on
the pore walls by the evaporating liquid [10]. scCO2 drying may
overcome this problem, as it allows drying the gels through the for-
mation of a supercritical solution with the liquid solvent inside the
pores, typically ethanol [10,13]. Indeed, the scCO2 –solvent mixture
shows no surface tension and can be eliminated from the sample
by venting the pressure vessel to the ambient pressure. Neverthe-
less, it is critical the selection of the appropriate temperature and
pressure conditions to avoid both crossing the glass transition tem-
perature of the polymer and passing through the liquid state of the
supercritical mixture.
Porosity and pore structure features are important parameters
for the design of porous scaffolds for bTE. Figs. 3–5 report some
important structural parameters of the scaffolds prepared as a func-
tion of the size range of the porogen used. As shown in Fig. 3, all
the scaffolds have overall porosity values higher than 95%, while
the large-pore fraction that is ascribable to the leaching of gelatin
particles decreases from 94% for particles in the 100–200 ␮m range
down to 79% for particles in the 400–600 ␮m range. The presence
of an interconnected porosity is an essential requirement for bTE
scaffolds to allow for the 3D cell colonization and the transport
of fluids necessary for cell survival and growth. In this work, we
determined the amount of interconnected porosity by assessing
the volume percent of retained water inside the scaffolds before
scCO2 drying by following Eq. (2). In agreement to the morpholog-
ical results of Fig. 2, the results of these tests (not shown) support
the consideration that, independently of the gelatin particles used,
the scaffolds were provided of 98% interconnected open porosity.
As expected, the selection of different gelatin particle size ranges
Fig. 4. Effect of the size range of gelatin particles used as solid porogen on the (A)
allows the formation of large pores in the scaffold, in a range going
large-pore diameter and mean fibre size, (B) large-pore size distribution and (C)
from 171 ± 62 ␮m up to 440 ± 60 ␮m. Conversely, minor differ- large-pore wall thickness and density of PLA scaffolds.
ences are observed in the mean fibre size, which varies from 100 to
200 nm (Fig. 4A). This last result suggests that the addition of gelatin
as the particulate porogen to the PLA–EL solution does not affect Low temperature N2 adsorption–desorption analysis is used to
significantly the TIPS of the polymeric solution. This is because assess the nano-scale pore structure and textural characteristics of
gelatin is almost insoluble in EL as well as TIPS process can easily the scaffolds. The results of this test are reported in Fig. 5. Fig. 5A–C
take place also in the inter-particles spaces. The increase of the size reports the N2 adsorption–desorption isotherms of PLA scaffolds
range of gelatin particles induces the shift of the size distribution as a function of the gelatin particle size used during the fabrica-
of large pores to higher values (Fig. 4B) and also affects the den- tion route. For all the scaffolds, the observed isotherms have the
sity and wall thickness of large pores (Fig. 4C). In particular, as the type II trend, which is characterized by a first sharp adsorption
size range of the gelatin particles increases from 100–200 ␮m up increase at low pressure due to micropores, followed by a large
to 400–600 ␮m, the pore density decreases more than one order of mesoporous region with a modest adsorption increase with pres-
magnitude, from 1.5 × 107 down to 9.0 × 105 pores/cm3 and, con- sure and, ultimately, a sharp increase of the adsorption volume.
comitantly, the pore wall thickness increases from 6.1 ± 4.2 up to Furthermore, the curves shift to higher relative pressure values
80.9 ± 29.4 ␮m, respectively. with the increase of the size range of gelatin particles from 100–200
 A. Salerno et al. / J. of Supercritical Fluids 97 (2015) 238–246 243

Fig. 5. Results of BET analysis showing the textural features of the scaffolds as a function of the size range of gelatin particles used as solid porogen. Adsorption–desorption
curves of PLA scaffolds prepared by using gelatin size range of (A) 100–200 ␮m, (B) 200–400 ␮m and (C) 400–600 ␮m. Pore size distribution curves obtained from the (D)
adsorption and (E) desorption steps. (F) Mean nano-pore size and specific surface of the scaffolds.

to 400–600 ␮m. For all of the samples, we observed a small closed
adsorption–desorption hysteresis loop at relative pressures above
0.4, which is due to the mesopore [19]. The pore volume distribu-
tion trends obtained by the adsorption and desorption experiments
are shown in Fig. 5D and E, respectively, evidencing that the scaf-
folds have both mesopores and macropores and the pore volume
fraction corresponding to micropores and mesopores increase with
the increase of the size range of gelatin particles. The pore size and
specific surface results are reported in Fig. 5F. All of the scaffolds
have mean pore size values close to 10 nm while the specific surface
increases from 28.9 ± 0.3 up to 38.5 ± 0.2 m2 /g with the increase
of the size range of gelatin from 100–200 to 400–600 ␮m. These
results are in agreement to the morphological observations of the
cross-section of the scaffolds displayed in the SEM images of Fig. 2
as well as the porosity data of Figs. 3 and 4. Indeed, the decrease
of the large pore fraction and the increase of the thickness of the
large pore walls increase the volume fraction of nano-fibres and,
concomitantly, the specific surface of the scaffolds.
Providing an adequate mechanical support is a critical scaffold
requirement. Indeed, the mechanical properties of the scaffold may
directly affect cell biosynthesis and, concomitantly, define suitable
in vitro and/or in vivo applications [12,20]. Fig. 6A reports the  vs. ε
curves of the scaffolds as a function of the size range of gelatin par-
ticles used. In this figure, we observed the typical trend of porous
materials, which is characterized by an initial linear elastic region,
followed by a plateau and by a sudden increase of the  value as a
consequence of pores collapse and scaffold densification. The cor-
respondent mechanical properties are depicted in Fig. 6B. The value
of E decreased slightly from 1.7 ± 0.4 MPa in scaffolds prepared by
using 100–200 ␮m gelatin particles down to 1.3 ± 0.3 MPa in scaf-
folds prepared by using 400–600 ␮m gelatin particles. The increase
of the size range of gelatin particles also resulted in the increase of
εY and the correspondent decrease of  Y (Fig. 6B). These results are
in agreement with literature data, thus evidencing the decrease of
the mechanical properties of porous materials by increasing the
mean pore size and the widening of the pore size distribution [21].
3.2. In vitro cell–scaffold interaction study
The biocompatibility of PLA scaffolds was investigated in vitro
by using MG63 cells, as these cells represent a good model for the
examination of the biological response of bTE scaffolds [14]. The
results of the in vitro study are reported in Figs. 6 and 7. As shown
in Fig. 6, the intensity of the Alamar Blue fluorescence, which is

Fig. 6. (A)  vs. ε curves and (B) E,  Y and εY values of the scaffolds as a function of the size range of gelatin particles used.
244 A. Salerno et al. / J. of Supercritical Fluids 97 (2015) 238–246
Fig. 7. MG63 cells proliferation over 14 days of in vitro culture as assessed by means
of Alamar Blue assay.
directly correlated to the number of viable cells in the scaffolds,
increases over the culture time for all of the different scaffolds
prepared. Nevertheless, different proliferation trends are observed,
especially in comparison with the fibrin control. In particular, at
day 1 of culture the fluorescence intensity of both control and PLA
scaffolds raises a similar value, close to 2 × 103 . This result provides
important information as it indicates that MG63 cells adhesion into
the PLA scaffolds is close to the adhesion observed in the fibrin gel,
which represents an excellent cell culture platform. Conversely, by
increasing the culture time up to 3 days, the fluorescence intensity
of the control is about sevenfold higher than that of PLA scaffolds.
Minor differences are observed for the PLA scaffolds as a function
of the size range of the porogen used. The further increase of the
culture time up to 14 days results in the increase of the number of
viable cells for all of the samples. However, the observed increase is
more pronounced in the case of PLA scaffolds rather than the con-
trol. Indeed, if compared to the value at day 3, a twofold increase of
the fluorescence intensity is obtained at day 14 for fibrin control,
while the fluorescence intensity value increases up to 8 times for
all of the PLA scaffolds prepared. It is however important to point
out that the final overall value of fluorescence for the scaffolds,
achieved after 14 days of culture, is close to the value achieved
by the cells cultured onto the control after 3 days. Furthermore, it
should be considered that cell proliferation can be strongly affected
by the structure of the supporting scaffolds. In particular, the fibrin
gel used as the control represents a 2D scaffold where cells can
Fig. 8. Optical and fluorescence images of cell–scaffold constructs showing cells distribution onto the seeding surface.
adhere and proliferate easily, even under static culture conditions,
because of their correct exposure to the culture medium. However,
after a certain culture time, cell proliferation would result in the
complete covering of the gel surface and no more space would be
available for cells to further proliferate. This consideration seems to
be supported by the trend observed in the fluorescence measure-
ments of cells cultured onto the control (Fig. 8), which evidenced
a fast increase of cell proliferation in the first week of culture fol-
lowed by a plateau in the second week. Conversely, cells seeded
onto the PLA scaffolds could have more space to grow and pro-
liferate even if their optimum exposure to the nutrients of the
culture medium could not be guaranteed by the static culture con-
ditions used in this work. In Fig. 8, the optical and fluorescence
microscope images of the cell–scaffold constructs at 7 days of cul-
ture are reported. As shown, the cells colonize and proliferate onto
the surface and also into the interior of the pores of the PLA scaf-
folds exposing to the seeding surface. In particular, nucleus staining
by DAPI, which allows clearly distinguish the cells from the sur-
rounding scaffold bulk material, demonstrates that cell adhesion
and distribution occurred quite uniformly onto the entire seeding
surface of the scaffolds. The ability of the scaffolds to support cell
adhesion was also assessed by SEM analysis and the results of this
test are reported in Fig. 9. As shown in Fig. 9A, at day one of seeding,
cells seeded onto the fibrin gel were well adhered and character-
ized by a flattened morphology. Similarly, the cells seeded onto the
PLA scaffold were adhered onto the seeding surface and colonized
extensively the pores of the scaffolds, finally indicating substantial
interaction between cells and the supporting substrate.
The design of porous scaffolds characterized by well controlled
porosity, pore size distribution and interconnectivity as well as pore
wall texture has been reported to be an essential issue in bTE [1–5].
For instance, a porosity degree of ca. 90% was found to be very
important to ensure an adequate space for cell adhesion and tissue
infiltration in three-dimensions [2]. Concomitantly, the presence
of pores in the size ranging from 100 to 500 ␮m is indicated as an
essential feature of porous scaffolds for bTE to support cell adhesion
and osteogenic expression [2]. The results of this study demon-
strate that the scaffolds prepared by the developed process meet
these criteria. Furthermore, the analysis of the results of the in vitro
biological tests provides important information about the interac-
tion between MG63 cells and the nano-scale fibrous PLA scaffolds.
Indeed, as shown in Fig. 6, the seeding efficiency and cell adhe-
sion into the porous structure of the fabricated PLA scaffolds at
day 1, which are directly correlated to the fluorescence intensity of
 A. Salerno et al. / J. of Supercritical Fluids 97 (2015) 238–246
Fig. 9. SEM images of the seeding surface of cell–scaffold constructs after 1 day
of in vitro culture: (A) fibrin gel and (B) PLA scaffold prepared by means of gelatin
particles in the 200–400 ␮m size range.
Alamar Blue test, are comparable to those achieved in the case of
the fibrin gel (Fig. 6). This important result is certainly dependent
on the proper large-pore size range of PLA scaffolds, but can be also
explained taking into account the peculiar morphology and texture
of the pore walls of the scaffolds. Indeed, it was recently demon-
strated that porous scaffolds with high specific surface enhanced
protein adsorption from the culture medium and, consequently,
improved cell adhesion, spreading and differentiation [5,22]. The
results of the Alamar Blue tests carried out in this study are in agree-
ment with the data supplied by Woo and co-workers [5,8,22] as
we observed that the PLA scaffolds prepared supported cell adhe-
sion, colonization and proliferation over 14 days of culture in vitro
(Figs. 6 and 7). As expected, the highest MG63 cells number was
achieved in the case of the fibrin gel used as control as the physical
and chemical properties of this material enhance the interaction
with transplanted cells. It is well known that, in the case of PLA
scaffolds, the extent of cell adhesion and proliferation are mainly
dictated by both the adsorption of proteins that are present in the
culture media and by the architecture of the porous network [23].
In the 3–14 days time culture range, the nano-scale fibrous PLA
scaffolds designed and fabricated in this work are able to sup-
port the continuous and progressive MG63 cells proliferation over
the entire culture time (Fig. 6). These results demonstrate that the
designed scaffolds are very promising for bTE applications, even if
studies focusing on cell differentiation and de novo ECM synthesis
are necessaries to further assess their osteogenic properties.
245
4. Conclusions
In this work a novel bio-safe process for the design and fabri-
cation of biomimetic porous PLA scaffolds for bTE is reported. The
process is based on the combination of TIPS, porogen leaching and
scCO2 drying for the fabrication of PLA scaffolds with an intercon-
nected network of large pores and nano-scale fibrous pore-wall
architecture. The operating conditions selected to achieve this goal,
namely gelation temperature during TIPS step as well as gelatin
particle size range and scCO2 drying conditions, are reported. In
particular, PLA scaffolds with overall porosity higher than 95%, large
pores with mean size ranging from 171 ± 62 ␮m to 440 ± 60 ␮m
and nano-scale fibrous architecture providing specific surfaces in
the 30–40 m2 /g range are fabricated. Finally, in vitro cell culture
tests are carried out by using MG63 cells and the results demon-
strate the biocompatibility of the as prepared scaffolds and their
potential for bTE.
Acknowledgements
The authors thank Julio Fraile for his support during the exper-
imental activity. Aurelio Salerno acknowledges the CSIC for the
financial support through a JAE-DOC contract cofinanced by the
FSE. The authors also acknowledge the financial support of the Min-
isterio de Economía y Competitividad through the research project
BIOREG (MAT2012-35161) and POLREMED (MAT2010-18155).
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