Journal of CO2 Utilization 80 (2024) 102697

Contents lists available at ScienceDirect

Journal of CO2 Utilization

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

Design and synthesis of novel polycaprolactone-chitosan based scaffolds

using supercritical carbon dioxide
Milica Pantić a, Gabrijela Horvat a, Miha Berk Bevc a, Željko Knez a, b, Zoran Novak a, *
a
University of Maribor, Faculty of Chemistry and Chemical Engineering, Smetanova ulica 17, 2000 Maribor, Slovenia
b
University of Maribor, Faculty of Medicine, Taborska ulica 8, 2000 Maribor, Slovenia

A R T I C L E I N F O A B S T R A C T

Keywords: Supercritical fluid technology and supercritical carbon dioxide were used for the preparation of hybrid materials
Supercritical foams – scaffolds from PCL foams and chitosan aerogel beads. In a simple straightforward processing method, the
Aerogels advanced biomaterials for potential tissue engineering applications were fabricated, and loaded with indo­
Scaffolds
methacin as a model drug. The obtained aerogels, foams, and scaffolds were evaluated through various char­
PCL
Chitosan
acterization techniques: N2 adsorption-desorption analysis, TGA/DSC, microscope/SEM, and FTIR. The results
Indomethacin revealed that the addition of mesoporous chitosan aerogels to the macroporous structure of PCL influenced the
textural, morphological, and thermal properties of the initial foams. Unlike foams, the scaffolds showed a
mesoporous structure, and, consequently, as the aerogels share in the formulation increased, larger specific
surface areas. Scaffolds obtained under different conditions (40 ◦ C/150 bar and 60 ◦ C/200 bar) showed different
swelling behavior and degradation. Lastly, the indomethacin showed distinct release profiles, depending on the
formation and deposition conditions.

1. Introduction Processing methods with supercritical fluids have many advantages

over standard methods for the preparation of scaffolds for tissue engi­
In the last decade there has been a tremendous research effort in neering. To name just a few, the absence of toxic organic solvents, the
seeking proper tissue engineering strategies for repairing or replacing ability to control the morphology of an internal porous structure, the
damaged tissues. In most cases, these involve the creation of scaffolds, possibility to incorporate active ingredients at mild conditions while
which could be the solution to fulfilling the requirements. The most preserving their properties, low processing temperatures etc. [8]. These
important include the control of porosity and pore size, maintenance of methods overcome the limitations regarding the incorporation of ther­
mechanical properties, and material biocompatibility. molabile compounds (drugs, proteins, or other bioactive components)
A variety of scaffolds have been used to date, including natural and during processing. Besides others, supercritical fluids found application
synthetic polymers. Natural polymer scaffolds, such as hyaluronic acid as blowing agents for the preparation of foams, thus replacing hazardous
[1], collagen [2,3], and alginate [4], have been applied successfully for compounds [9]. Supercritical foaming is a simple one-step procedure for
this purpose. Moreover, synthetic polymers (e.g., polyethylene glycol, fabricating highly porous bone scaffolds from thermoplastic polyesters
polylactic acid, polyglycolide, polycaprolactone) have also been used as [10]. This method allows the formation of foams with low energy con­
scaffolds in tissue engineering [5,6]. Additionally, variety of the physi­ sumption. Moreover, the gas is removed rapidly, eliminating the need
cochemical requirements must be fulfilled, including biocompatibility for after treatments (drying or cleaning).
and biodegradability (with a matching rate of the new matrix produc­ Polysaccharide aerogels can be a great mesoporous support for
tion) upon implementation and porous structure with interconnected synthetic materials. The incorporation of aerogels in synthetic scaffolds,
pores of adequate size to allow cell adhesion, cell proliferation, and besides others, implies the replacement of standard ingredients with
subsequent tissue development. The scaffolds have to be absorbable by cleaner, biodegradable, and cheaper supports [11]. By combining
the human organism and possess the ability to bind to surrounding tis­ polysaccharide aerogels with biodegradable polyesters, hybrid scaffolds
sues [7]. can be prepared with high porosity and a mesoporous structure with

* Corresponding author.
E-mail address: zoran.novak@um.si (Z. Novak).

https://doi.org/10.1016/j.jcou.2024.102697
Received 26 July 2023; Received in revised form 18 January 2024; Accepted 29 January 2024
Available online 2 February 2024
2212-9820/© 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
M. Pantić et al. Journal of CO2 Utilization 80 (2024) 102697

good mechanical properties. Moreover, using supercritical foaming as a Na2SO4 (CAS 7757–82-6, Sigma Aldrich, p.a. purity ≥ 99.0%), Tris
processing method gives the possibility to tune the size and density of (hydroxymethyl)aminomethane, NH2C(CH2OH)3 (CAS 77–86-1, Sigma,
the prepared scaffolds [12]. purity ≥ 99%) and hydrochloric acid, HCl (Merck, Darmstadt, Germany)
Polycaprolactone (PCL) scaffolds have been investigated extensively were employed for the preparation of simulated body fluid (SBF) for the
for tissue engineering applications, due to their biocompatibility, swelling and in vitro dissolution tests. SBF with pH= 7.4 was prepared
biodegradability, structural stability, and mechanical properties [13]. by mixing 7.996 g of NaCl, 0.350 g of NaHCO3, 0.224 g of KCl, 0.174 g of
Chitosan aerogels have also been an emerging area of interest in the last K2HPO4, 0.129 g of MgCl2, 0.278 g of CaCl2 * 2 H2O, 0.071 g of Na2SO4,
decade for various applications, e.g., drug delivery systems, wound 6.057 g of NH2C(CH2OH)3 and 40 mL of 1 M HCl.
healing, and bone engineering [14]. As they form porous 3D networks
and possess excellent textural properties, they are great candidates for 2.2. Preparation methods
evaluating their incorporation in composite polymeric-based scaffolds
[15]. Chitosan aerogels themselves resulted in promising osteoblast vi­ 2.2.1. Preparation of chitosan aerogel beads
abilities and good mechanical properties [16]. A 2% (w/w) chitosan solution was prepared by dissolving a weighed
Hybrid PCL-starch scaffolds have been prepared using both xerogels amount in 0.1 M CH3COOH at room temperature. In the next step, the
[10] and aerogels [12]. While starch xerogels improved interactions solution was added dropwise to 2 M NaOH in an ethanol solution. The
between the PCL and growth factors, starch aerogels improved the po­ NaOH acted as a non-solvent for the chitosan, so the chitosan beads were
tential uses of PCL for bone regeneration significantly. Silk fibroin-PCL formed [15]. The obtained chitosan alcogel beads were cured in NaOH
scaffolds have also been fabricated and impregnated with dexametha­ in an ethanol solution for 1 h. They were afterwards transferred to ab­
sone for potential bone regeneration. Hybrid PCL-aerogels scaffolds for solute ethanol to remove the excess NaOH. After 1 h the ethanol was
all the mentioned cases were prepared from powdered mixtures. While changed with a fresh quantity, and the alcogel beads were kept in
the PCL was in a powder form, both the starch and silk fibroin aerogels ethanol until drying. In the final step a supercritical drying technique
were prepared in the form of microparticles (from water-in-oil emul­ was employed to obtain aerogel beads. All the samples were subjected to
sions) [17]. already optimized conditions for polysaccharide gel drying at 120 bar,
Chitosan aerogel incorporation can have an immense influence on 40 ◦ C for 6 h [20].
the textural, mechanical, and structural properties of supercritical foams
prepared from PCL. The aim of the following work was to develop hybrid 2.2.2. Preparation of supercritical foams and scaffolds
materials - scaffolds made from PCL foams and chitosan aerogels. The PCL foams were obtained using the gas foaming technique [21]. The
novelty of this technique is reflected in using a simple, straightforward polymer was fixed in cylindrical glass molds and placed together in a
processing method. This made the preparation of scaffolds easily high-pressure autoclave. The working parameters were set to 40 ◦ C/150
manageable, uncomplicated, and permissive. The method consisted of bar and 60 ◦ C/200 bar, to obtain foams under different conditions. The
mixing PCL pellets and previously prepared chitosan aerogel beads system was kept under constant temperature and pressure for 4 h before
further subjected to supercritical carbon dioxide. The prepared scaffolds depressurization. The depressurization rate was chosen to avoid damage
were deposited with indomethacin simultaneously with their formation. to the porous network of the prepared aerogel beads and form appro­
Indomethacin is a non-steroidal anti-inflammatory drug (NSAID, BSC II priate pores in the foams. It was set to 3 bar/min. The obtained foams
model drug) with very low aqueous solubility (5 μg/mL) and limited were removed from the glass molds and used for further characteriza­
solubility in supercritical carbon dioxide [18]. It is effective in the tion. The PCL-chitosan scaffolds were obtained in the same manner and
management of rheumatoid arthritis, ankylosing spondylitis, osteoar­ under the same conditions. The chitosan aerogel beads were synthesized
thritis, and acute gout [19]. The incorporation of indomethacin may in the first step, independently from the preparation of the resulting
lead to different release profiles, resulting in different tissue regenera­ scaffolds. Once the chitosan aerogel beads were obtained, they were
tion outcomes. The resulting scaffolds were, therefore, evaluated placed together into a glass mold with the polymer at different ratios:
regarding their textural, morphological, and thermal properties, as well 1:10, 1:20, and 1:40 (w/w). The following steps were identical to the
as their swelling behavior and indomethacin release profiles. steps applied for the preparation of foams.

2. Materials and methods 2.2.3. Deposition of indomethacin

The deposition of indomethacin was conducted as a one-step pro­
2.1. Materials cessing method, together with the production of scaffolds. The entrap­
ment of the drug was performed simultaneously with the scaffold
Chitosan with medium molecular weight (CAS 9012–76-4) and formation. The chitosan aerogel beads, PCL, and indomethacin powder
polycaprolactone pellets, Mn 70000–90000 (CAS 24980–41-4), were were mixed physically and then exposed to supercritical CO2 in an
purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetic acid, autoclave. This method has previously been applied successfully to
CH3COOH (Fisher Scientific, Pittsburg, PA, USA; purity ≥ 98%), was chitosan thermosets, and resulted in indomethacin being dispersed
used for the dissolution of the chitosan. Sodium hydroxide, NaOH amorphously throughout the polymer matrix [22]. The indomethacin,
(Merck, Darmstadt, Germany; purity ≥ 98%), and absolute ethanol, chitosan aerogel beads, and PCL polymer were weighed, mixed in a ratio
C2H5OH (Merck, Darmstadt, Germany) were used for the preparation of of 1:2:20 (w/w), and placed in the glass molds. The deposition of
chitosan beads. Carbon dioxide, CO2 (Messer, Ruše, Slovenia; purity by indomethacin took place upon the formation of the scaffold and PCL
volume 99.5%) was used for supercritical drying of the chitosan beads foaming. Furthermore, during the static part of the experiment, indo­
and for supercritical foaming of the PCL. Indomethacin, C19H16ClNO4 methacin entered the chitosan aerogel beads via supercritical CO2 in the
(CAS 53–86-1, Sigma-Aldrich, St. Louis, MO, USA; purity ≥ 99%), was process of supercritical deposition. The depressurization rate was set to
tested as a model drug for deposition experiments with scaffolds. So­ 3 bar/min.
dium chloride, NaCl (CAS 7647–14-5, Pregl chemicals, Ph Eur; purity ≥
99.5%), potassium chloride, KCl (CAS 7447–40-7, Honeywell, p.a.; pu­ 2.3. Characterization methods
rity ≥ 99.5%); potassium phosphate dibasic, K2HPO4 (CAS 7758–11-4,
Fluka, p.a. purity ≥ 99.0%) and sodium bicarbonate, NaHCO3 (CAS 2.3.1. N2 adsorption-desorption analysis
144–55-8, for analysis, Merck), magnesium chloride, MgCl2 (CAS Adsorption-desorption analyses were used for the determination of
7786–30-3, Sigma Aldrich, purity ≥ 98%), calcium chloride dihydrate, textural properties (specific surface areas, pore volumes, pore diameter)
CaCl2 * 2 H2O (CAS 10035–04-8, Supelco, for analysis), sodium sulfate, of the aerogels, foams, and scaffolds. The ASAP 2020MP instrument

2
M. Pantić et al. Journal of CO2 Utilization 80 (2024) 102697

(Micromeritics, Norcross, GA, USA) was used for that purpose. The automated sampling station (Agilent 850-DS) and UV-Vis Spectropho­
samples were first degassed at 50 ◦ C for 1000 min under a vacuum until tometer (Agilent Cary 60 UV-Vis). The vessel was filled with 600 mL SBF
stable pressure was obtained. Afterwards the samples were analyzed and heated to 37 ± 0.5 ◦ C. The samples were placed into the vessel, and
using N2 as the adsorptive gas. The specific surface areas were calculated the stirring rate was set to 50 rpm. The sampling (1 mL) was performed
using the Brunauer-Emmett-Teller model (BET), while the pore volumes automatically after 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 12, 16, and 24 h, and then
and pore diameters were calculated using the Barrett-Joyner-Halenda in increments of 24 h until complete indomethacin dissolution. The
model (BJH). The determined adsorption isotherms presented the samples were analyzed automatically in the UV apparatus at 265 nm for
adsorption capacities of the prepared aerogels, foams, and scaffolds. indomethacin, and the concentration was determined from the calibra­
tion curve. The cumulative release was calculated by Eq. (2).
2.3.2. Thermogravimetric analysis and differential scanning calorimetry
c•V
Thermogravimetric analysis (TGA) and differential scanning calo­ cumulative release[%] = × 100 (2)
mt
rimetry (DSC) were performed simultaneously to determine the thermal
stability of the aerogels, foams, and scaffolds. The analyses were carried where c is the concentration of indomethacin in the release medium
out on TGA/DSC1 apparatus (Mettler Toledo, Columbus, OH, USA). The after the selected time intervals in mg/mL, V is the volume of the release
samples were cut and placed into 100 µl aluminum crucibles with medium in mL, and mt is the total amount of the drug within the release
covers. The measurements were performed under an air atmosphere, medium in mg, obtained after 9 days. The drug dissolution test was
from 30 to 600 ◦ C, with a heating rate of 10 ◦ C per minute. performed in duplicate.

2.3.3. Stereo microscope and scanning electron microscopy 3. Results

The samples were analyzed by stereo microscope (Olympus SZX10
Tokyo, Japan) and low vacuum Scanning Electron Microscopy, SEM A schematic presentation of a simple, straightforward preparation
(Sirion 400 NC, FEI, Hillsboro, Oregon, USA), to determine the procedure for scaffolds is shown in Fig. 1. The preparation of the scaf­
morphological characteristics of the aerogels, foams, and scaffolds. The folds goes by the steps presented in the scheme, which are explained in
mean pore sizes for foams were determined using Image J software. The Section 2.2.2 in detail.
preparation of samples for SEM encompassed fractionation and splatter-
coating with gold particles. The analysis was performed at an acceler­
ation voltage of 2–4 kV. 3.1. Visual appearance

2.3.4. Fourier transform infrared spectroscopy The foams and scaffolds obtained under different conditions differed
Fourier transform infrared spectroscopy (FTIR) on IRAffinity-1 s in height (the diameter of the glass mold determines the diameter of
apparatus (Shimadzu, Kyoto, Japan) was applied for the characteriza­ foams/scaffolds), color, structure, pore sizes, and pore distribution, as
tion of the aerogels, foams, scaffolds, and loaded scaffolds. The ATR-IR presented in Fig. 2a. The foams prepared at 60 ◦ C/200 bar resulted in 2-
method was applied to the aerogels, foams, and scaffolds, while the KBr fold higher forms compared to the foams prepared at 40 ◦ C/150 bar.
method was applied to the indomethacin. KBr pellet formation is a Consequently, the resulting scaffolds were also higher for the conditions
classic technique for the analysis of powder samples. The measured of higher temperatures and pressures. Furthermore, as the aerogels`
spectra of the aerogels and foams were used to identify the characteristic share in the formulation increased, the height of the scaffolds increased.
adsorption bands of the chitosan and PCL, while the spectra of the The height of the initial foams prepared at 40 ◦ C/150 bar increased
scaffolds were used for identifying the hybrid nature of the materials and by approximately 40% in scaffolds with a 10% aerogel share. The
deposition of indomethacin. maximal increase for foams prepared at 60 ◦ C/200 bar was observed for
the scaffolds with a 5% aerogel share, namely, approximately 20%. In
the latter case, the addition of 10% aerogel in the scaffolds reduced the
2.4. Swelling studies height slightly compared to the scaffolds with 5% aerogel. The higher
mass of the aerogels (due to the highest share of the aerogel compared to
A modified version of the protocol method was used for the swelling the others) led to their deeper penetration inside the scaffold. This effect
studies. The studies were performed for the foams and scaffolds to was not observed for the scaffolds prepared at 40 ◦ C/150 bar, due to
investigate how different preparation conditions and adding the chito­ seemingly smaller pores and more dense and compact structures. The
san aerogels influenced the behavior of the prepared scaffolds. Before aerogels were more concentrated in the upper half of the scaffolds due to
the measurement the samples were sterilized under a UV lamp. Samples their lower densities; however, they were also present in the lower part,
were cut to similar shapes and sizes, having similar masses, and as shown in Fig. 2b. Table 1 shows the obtained heights of prepared
immersed in a swelling medium [23]. They were maintained there for foams and scaffolds. The standard errors were no higher than 5% for all
ten weeks at a constant temperature of 37 ± 0.5 ◦ C. At selected time prepared foams/scaffolds. The diameter was 23.6 mm, determined by
intervals the samples were weighed, to monitor their mass changes. The the diameter of the glass mold.
tests were performed in duplicate. The presented results are the average
value of the measurements. The swelling ratio was calculated by Eq. (1).
3.2. N2 adsorption-desorption analysis
mt − m0
swelling ratio[%] = × 100 (1)
m0 The textural properties, e.g., specific surface areas, pore volumes,
and pore diameters, were measured using N2 adsorption-desorption
where m0 is the initial mass of the sample before the immersion in the
analysis. The determined values are shown in Table 2.
swelling medium (t = 0) in mg, and mt is the mass of the sample
Chitosan aerogel beads have already been prepared by a non-solvent
immersed in the swelling medium for the selected time t in mg.
induced phase separation method in a NaOH in water solution, followed
by a successive water-to-ethanol exchange, and, finally, supercritical
2.5. In vitro dissolution studies drying [25–27]. The reported specific surface areas ranged from 188 –
479 m2/g. The chitosan aerogel beads prepared in this work implied
For the in vitro drug dissolution studies, simulated body fluid (SBF) preparation in a NaOH in ethanol solution. The determined specific
was prepared as described in the materials section. The drug dissolution surface areas showed remarkably high values of 535 m2/g, improved
test was performed on a USP II Apparatus (Agilent 708-DS) with an compared to the reported values. Evidently, the following preparation

3
M. Pantić et al. Journal of CO2 Utilization 80 (2024) 102697

Fig. 1. Schematic presentation of the preparation procedure for hybrid materials – scaffolds from PCL foams and chitosan aerogel beads.

Table 1
Dimension of aerogels, foams, and scaffolds.
Sample Height, mm

Foam 40/150 9.1

Scaffold 40/150_2.5 10.5
Scaffold 40/150_5 11.9
Scaffold 40/150_10 15.6
Foam 60/200 18.5
Scaffold 60/200_2.5 19.6
Scaffold 60/200_5 22.5
Scaffold 60/200_10 20.7

Table 2
Specific surface areas, pore volumes, and pore diameters of aerogels, foams, and
scaffolds.
Sample Specific surface area Pore volume* Pore diameter*
(BET), m2/g (BJH), cm3/g (BJH), nm

Aerogel 528.23 ± 7.08 2.325 ± 0.09 14.35 ± 3.6

**
Foam 40/150 0.30 ± 0.06 **
Scaffold 40/ 2.88 ± 0.14 0.011 ± 0.01 12.84 ± 1.6
150_2.5
Scaffold 40/ 6.32 ± 0.23 0.027 ± 0.02 11.98 ± 3.4
150_5
Scaffold 40/ 13.24 ± 0.43 0.059 ± 0.04 12.24 ± 2.5
150_10
**
Foam 60/200 0.83 ± 0.19 **
Scaffold 60/ 4.25 ± 0.22 0.014 ± 0.02 12.72 ± 1.9
200_2.5
Scaffold 60/ 7.99 ± 0.30 0.028 ± 0.03 13.44 ± 2.5
200_5
Fig. 2. a) Foams and scaffolds prepared at 40 ◦ C/150 bar (lower), and 60 ◦ C/ Scaffold 60/ 12.42 ± 0.45 0.059 ± 0.04 14.10 ± 3.6
200 bar (upper); 2.5, 5, and 10 refer to the aerogels` share in the final formu­ 200_10
lation (%); b) cross sections of scaffolds with a 10% aerogel share (further
*exhibitive [24]
chosen for the impregnation with indomethacin) prepared at 40 ◦ C/150 bar
**undetermined
(left), and 60 ◦ C/200 bar (right).
*** 40/150 and 60/200 refer to the conditions for foams/scaffolds` preparation
(40 ◦ C/150 bar and 60 ◦ C/200 bar).
procedure led to gels with stronger networks, more resistant to ****2.5, 5, and 10 refer to the aerogels` share in the final formulation (%)
shrinkage and consequently with higher specific surface areas.
As expected, applying the BET method for measuring specific surface

4
M. Pantić et al. Journal of CO2 Utilization 80 (2024) 102697

areas did not result in valuable values, due to the macroporous nature of
the foams. On the contrary, the scaffolds showed increased specific
surface areas as the aerogels` share in the formulation increased. The
determined specific surface areas of the scaffolds were between 3 –
13 m2/g. Embedding highly mesoporous aerogels into the macroporous
foams resulted in scaffolds with measurable properties. The bigger the
aerogels` share in the scaffold, the higher the specific surface areas. As
the aerogel beads constitute only 2.5 to 10% of the overall mass, the
scaffolds’ specific surface areas were much lower than the chitosan
aerogel beads. The specific surface areas of the scaffolds are lower than
expected if the aerogel share in the scaffold’s formulation is taken into
account. The possible explanation for this irregularity could be due to
PCL’s molecule intrusion in the aerogels’ pores during the foaming. The
reported specific surface areas for PCL-starch scaffolds were much
lower, namely, 1.2–1.7 m2/g [12], while the values for PCL-silk fibroin
scaffolds were not reported [17].
As presented in Fig. 3a and b, the aerogels and scaffolds had type IV
isotherms, revealing the presence of the mesoporous structures. On the
contrary, the foams showed a flat line and the absence of a mesoporous
structure, as indicated before. Unlike the foams, the scaffolds possessed
adsorption capacities. As expected, these values were lower compared to
the aerogels, yet showing immense potential for drug entrapment.
Fig. 4a and b confirmed the highest mesoporous volumes for the chi­
tosan aerogel. As expected, the higher the aerogels’ share in the scaffolds
formulation, the higher the mesoporous volume.

Fig. 4. Pore size distribution (a) aerogels, foams, and scaffolds and (b) foams
and scaffolds. Foams 40/150 and 60/200 had fewer than two points available
for the calculations.

3.3. Thermogravimetric analysis and differential scanning calorimetry

Thermal analyses of aerogels, foams, and scaffolds were performed

through simultaneous Thermogravimetry and Differential Scanning
Calorimetry.
Table 3 summarizes the thermogravimetric analysis, namely, the
percentage of the degraded mass of the samples for the tested temper­
ature ranges. The chitosan aerogels degraded less compared to the foams
and scaffolds, namely, 64%. The foams prepared at 40 ◦ C/150 bar and
60 ◦ C/200 bar showed almost complete degradation, 99% and 98%,
respectively. Lastly, the scaffolds revealed higher stability compared to
the foams. As the aerogels` share in the formulation increased, the
thermal degradation of the prepared scaffolds decreased. The

Table 3
Overall mass degradation of aerogel, foams, and scaffolds.
Sample Degradation, %

Aerogel 64.2
Foam 40/150 99.2
Scaffold 40/150_2.5 96.6
Scaffold 40/150_5 96.3
Scaffold 40/150_10 92.0
Foam 60/200 98.2
Scaffold 60/200_2.5 95.3
Scaffold 60/200_5 92.4
Fig. 3. Adsorption isotherms of (a) aerogels, foams, and scaffolds and (b) foams
Scaffold 60/200_10 90.2
and scaffolds.

5
M. Pantić et al. Journal of CO2 Utilization 80 (2024) 102697

degradation of foams compared to scaffolds with 10% aerogel was Fig. 5b shows the curves for foam and scaffolds prepared at 40 ◦ C/
approximately 8% higher. When comparing the scaffolds with the same 150 bar. The first endothermic peak at 67 ◦ C for all the samples pre­
composition of aerogels prepared under different conditions, slightly sented the melting peak of the PCL [29]. It can be seen that the melting
higher stability was observed for the scaffolds prepared at 60 ◦ C/ peak was not affected by the presence of the chitosan aerogel. The
200 bar. second endothermic peak, occurring between 400 and 411 ◦ C, can be
The main mass degradation of all samples occurred at higher tem­ ascribed to the complete decomposition of the PCL. PCL degrades by
peratures, corresponding to peaks obtained in the Differential Scanning specific chain end scission [30]. Namely, the peak can be associated with
Calorimetry curves. The curves presented by Fig. 5a-c agree with the hydrophilic ester group breakage and long-chain hydrocarbyl rupture,
statements regarding the thermogravimetric analysis. which is in accordance with the literature [31]. The decomposition of
Fig. 5a shows the curve for chitosan aerogels. The endothermic peaks the PCL occurred slightly earlier for the scaffolds, due to the presence of
at 110 ◦ C can be attributed to water evaporation during thermal scan­ the chitosan aerogels. Lastly, the small exothermic peaks of the scaffolds
ning (traces of moisture present in the aerogels due to storage). The occurring at 265 ◦ C can be attributed to the decomposition of the chi­
second thermal event, expressed by the exothermic peaks at 248 ◦ C, may tosan’s amine functional groups. Fig. 5c shows the curves for the foam
be related to the decomposition of the amine functional groups in the and scaffolds prepared at 60 ◦ C/200 bar. Similarly to the foams and
chitosan [28]. scaffolds prepared at 40 ◦ C/150 bar, two endothermic peaks are visible.
The first one occurred at 67 ◦ C for both the foam and scaffolds, pre­
senting the melting peak of the PCL, as mentioned before. The chitosan
aerogels did not affect the melting peak of the PCL, as described previ­
ously. The aerogels` share in the formulations affected the second
endothermic peak occurring between 399 to 415 ◦ C, while the
exothermic peak for the scaffolds, due to the decomposition of the chi­
tosan’s amine functional groups, occurred at 265 ◦ C.
Apparently, adding chitosan aerogels (different shares and prepara­
tion conditions) did not affect the melting temperature of the PCL. On
the other hand, the exothermic peaks attributed to the decomposition of
the amine functional groups for all the scaffolds occurred at higher
temperatures compared to the aerogel beads, namely, 265 ◦ C and
248 ◦ C, respectively. This means that, by introducing aerogels into the
scaffold, the amine functional groups were protected, so the decompo­
sition was postponed slightly to higher temperatures. However, the final
decomposition of the scaffolds was affected by the aerogels` share,
leading to decomposition at slightly lower temperatures, namely,
399–400 ◦ C and 411–415 ◦ C, respectively.

3.4. Microscopic and scanning electron microscopy measurements

The structure of the prepared foams and scaffolds was investigated

through optical and SEM micrographs.
Fig. 6 presents the structures of supercritical foams and supercritical
scaffolds detected by a stereo microscope. The foams prepared at 40 ◦ C/
150 bar (A) show clearly a more compact structure with smaller gaps,
while the foams prepared at 60 ◦ C/200 bar (B) have much bigger gaps.
The PCL evokes structurally different foams under different conditions.
The foam prepared at 40 ◦ C/150 bar had a mean pore size of 0.25 mm,
with a pore size distribution ranging from 0.1 mm to 0.45 mm. In the
case of the foam prepared at 60 ◦ C/200 bar, the mean pore size was
1.8 mm, with a pore size distribution from 1 mm to 2.5 mm. The
foaming performed at 60 ◦ C/200 bar led to the formation of 7-fold
bigger pores on average than those performed at 40 ◦ C/150 bar.
Furthermore, the structure of the scaffolds was changed due to the
addition of the aerogels (C, D). The PCL shaped foams all around the
aerogels, seeming like the aerogels fitted in the already existing pores.
The bigger the aerogels` share in the formulations, the more dense and
compact scaffolds were formed.
The structure of the PCL foams is macroporous (optical micro­
graphs), while the structure of the chitosan aerogel beads is mainly
mesoporous (adsorption isotherms). As expected, the structure of the
obtained scaffolds is a combination of macroporous and mesoporous,
opening the opportunity for a wide range of applications. Fig. 7 shows
the SEM micrographs of the prepared foams and scaffolds. The pores
were visible by stereo microscope, as well as by SEM on 400 x magni­
fication (A, B). However, the size of the pores varied. Since the foams
had the macroporous structures, both scaffolds had a part without the
presence of meso- or micropores coming from the foam part (C, D).
Fig. 5. DSC curves for (a) aerogel, (b) foam and scaffolds prepared at 40 ◦ C/ However, the mesoporous structure present in the scaffolds was coming
150 bar, and (c) foam and scaffolds prepared at 60 ◦ C/200 bar. from the aerogel part (E, F). The highly interconnected network of pores

6
M. Pantić et al. Journal of CO2 Utilization 80 (2024) 102697

Fig. 6. Optical micrographs; A and B: the structures of the foams prepared at 40 ◦ C/150 bar and 60 ◦ C/200 bar, respectively; C and D: the structures of the scaffolds
prepared at 40 ◦ C/150 bar and 60 ◦ C/200 bar, respectively.

Fig. 7. SEM micrographs; A and B: the structures of the scaffolds prepared at 40 ◦ C/150 bar and 60 ◦ C/200 bar, respectively (400 x magn.); C and D: nonporous
structure of the scaffolds prepared at 40 ◦ C/150 bar and 60 ◦ C/200 bar (5000 x magn.), respectively; E and F: the mesoporous structure of the scaffolds prepared at
40 ◦ C/150 bar and 60 ◦ C/200 bar (20,000 x magn.), respectively.

7
M. Pantić et al. Journal of CO2 Utilization 80 (2024) 102697

was clearly visible, which is a characteristic of aerogels. higher swelling ratio.

As shown, the foams and scaffolds prepared under different condi­
tions showed completely different behavior in the degradation medium.
3.5. Swelling studies While the samples prepared at 40 ◦ C/150 bar reached the maximal
swelling ratio later and were stable over the tested period, the samples
The swelling behavior of the foams and scaffolds was monitored over prepared at 60 ◦ C/200 bar reached maximal swelling behavior faster,
ten weeks in the prepared degradation medium (as explained in Section after which they started decomposing. Furthermore, the swelling ratios
2.1.). Fig. 8a shows the behavior of the foam and scaffolds prepared at were higher for the samples prepared at 60 ◦ C/200 bar. The exception
40 ◦ C/150 bar. As the foam and the scaffolds were introduced into the was the scaffolds with 10% aerogel, where the higher swelling ratio was
degradation medium, they swelled immediately (absorbed the liquid). observed for the scaffold prepared at 40 ◦ C/150 bar.
The swelling of the samples continued over time, and the swelling ratio
reached its maximal value after five to seven weeks, depending on the 3.6. Fourier transform infrared spectroscopy
sample. Afterward, the samples stayed stable in the degradation medium
without showing decomposition or erosion of the samples. Furthermore, Fourier transform infrared spectroscopy was employed to identify
the studies showed that the foam reached a lower swelling ratio specific bonds in the prepared scaffolds and confirm the presence of
compared to the scaffolds. The bigger the aerogels` share in the indomethacin in the loaded scaffolds.
formulation, the greater the swelling of the scaffolds. Fig. 8b shows the Fig. 9a shows the FTIR spectra of chitosan aerogel, PCL foams, and
behavior of the foam and scaffolds prepared at 60 ◦ C/200 bar. Similarly PCL-chitosan scaffolds. The spectrum depicting the chitosan aerogel da
to the samples prepared at 40 ◦ C/150 bar, they swelled after contact strong band at 3358 cm–1, presenting the stretching of N–H and O–H and
with the degradation medium. However, the foam and scaffolds pre­ the intramolecular hydrogen bonds. The band at 2866 cm–1 can be
pared under these conditions reached the maximal swelling ratio after attributed to C–H symmetric stretching. The peaks at 1647 and
one to two weeks, depending on the sample. Afterward, they started to 1589 cm–1 are characteristic of N–H and C–N amide functional groups,
decompose slowly. After ten weeks, the samples’ overall decomposition while the peaks at 1472 and 1315 cm–1 represent CH2 bending and CH3
was between 8% (foam 40/150) and 34% (scaffold 40/150_10). As in symmetrical deformations. Finally, the peaks at 1065 and 1028 cm–1 are
the previous case, the scaffolds with a bigger aerogels` share had a characteristic of C–O stretching. The spectrum of the PCL foam shows
peaks at 2941 and 2864 cm− 1, representing CH2 asymmetric stretching
and CH2 symmetric stretching, respectively. The sharp peak at
1721 cm− 1 can be attributed to C– –O carbonyl stretching. A peak at
1296 cm− 1 shows the stretching of C–O and C–C, while the peaks at
1238 and 1165 cm− 1 represent asymmetric and symmetric C–O–C
stretching. Both the chitosan aerogel and PCL foam spectra correlate
with the spectra from the literature [32,33].
Finally, when comparing the spectrum of scaffolds with the spectra
of aerogels and foams, the mentioned characteristic peaks of both chi­
tosan and PCL are present (some of which are overlapping). The spec­
trum of the scaffolds proves their PCL-chitosan composite nature.
Fig. 9b shows the FTIR spectra of indomethacin and indomethacin
loaded PCL-chitosan scaffolds. The spectrum for indomethacin shows
characteristic bands at 3848 and 2924 cm− 1 due to O–H stretching. The
peaks at 1715 and 1692 cm− 1 represent the C– –O stretching of carboxyl
acid dimer and the C– –O stretching of the benzoyl group, respectively.
The peak at 13014 cm− 1 can be attributed to C–O stretching. The data
presented are in accordance with the literature [34]. When comparing
the spectrum of the indomethacin loaded scaffolds, most of the char­
acteristic peaks of indomethacin overlap with the characteristic peaks of
chitosan and PCL. However, the peaks corresponding to the C– –O
stretching of the carboxyl acid dimer and the C– –O stretching of the
benzoyl group of indomethacin are also visible in the spectrum of the
scaffold, shifted slightly to the right (1676 and 1665 cm− 1). This is the
inevitable evidence of the presence of indomethacin in the PCL-chitosan
formulations.

3.7. In vitro dissolution studies

A model drug, indomethacin, was deposited into the scaffolds at

40 ◦ C/150 bar and 60 ◦ C/200 bar. The indomethacin, chitosan aerogel
beads, and PCL polymer were mixed in a ratio of 1:2:20 (w/w). By this
method, 4.5% of indomethacin loading was established into the scaf­
folds with 10% of aerogel beads. Considering the diffusion process, it is
expected that a higher quantity of indomethacin was concentrated in the
lower half of the scaffold.
The loaded scaffolds were examined further to determine the release
profiles of indomethacin, as shown in Fig. 10.
The release profiles from the scaffolds prepared under different
Fig. 8. Swelling profiles for foam and scaffolds prepared at (a) 40 ◦ C/150 bar, conditions are completely different. While observing Fig. 10a, in can be
(b) 60 ◦ C/200 bar. seen that the indomethacin achieved complete dissolution after 72 h (3

8
M. Pantić et al. Journal of CO2 Utilization 80 (2024) 102697

Fig. 9. (a) FTIR spectra of chitosan aerogels, PCL foams and PCL-chitosan scaffolds; (b) FTIR spectra of indomethacin and PCL-chitosan scaffolds loaded with
indomethacin.

days) for the scaffold prepared at 40 ◦ C/150 bar, while, in the case of the where approximately 90% of the model drug was released. It can be
scaffold prepared at 60 ◦ C/200 bar, it reached complete dissolution concluded that the pore structure and their arrangement influenced the
after 168 h (7 days). When comparing these two release profiles, it can indomethacin deposition and, later, its dissolution highly.
be said that, in the case of the scaffolds prepared at 40 ◦ C/150 bar, a
more controlled release was achieved, while the release profile of the 4. Discussion
scaffold prepared at 60 ◦ C/200 bar seemed burst. However, while
observing Fig. 10b and the release profile for the scaffold prepared at The thermal stability, blend compatibility, and low melting point
60 ◦ C/200 bar, the controlled release of indomethacin was achieved in (59–64 ◦ C) of polycaprolactone have inspired huge interest for its
the first 8 h. various applications, such as packaging, loose-fillers, biomedical de­
As the results from the swelling studies showed and in vitro disso­ vices, and scaffolds [35]. As a semi-crystalline polyester with
lution studies confirmed, the scaffolds prepared under different condi­ outstanding mechanical properties, good biocompatibility and degrad­
tions led to a completely different material. The scaffolds prepared ability, PCL is applied widely for tissue engineering [36]. It is important
under lower conditions of pressure and temperature (40 ◦ C/150 bar) to emphasize that the main requirements for ideal scaffolds include the
resulted in materials with a postponed release of the model drug, while presence of macropores (100–1000 µm), as well as high inter­
the scaffolds prepared at higher conditions of pressure and temperature connectivity [37]. The main objective of the following work was to
(60 ◦ C/200 bar) gave materials with controlled release over the first 8 h, prepare PCL-chitosan scaffolds from PCL foams and chitosan aerogel

9
M. Pantić et al. Journal of CO2 Utilization 80 (2024) 102697

Fig. 10. (a) Release profiles of indomethacin from scaffolds prepared at 40 ◦ C/150 bar and 60 ◦ C/200 bar until complete dissolution; (b) Release profiles of
indomethacin from scaffolds prepared at 40 ◦ C/150 bar in the first 8 h.

beads, and to investigate their potential as biomaterials for tissue en­ surface areas and adsorption capacities of the prepared scaffolds
gineering applications. The preparation process included the synthesis increased with the addition of chitosan aerogels. The TGA/DSC analyses
of chitosan aerogel beads and the supercritical foaming of PCL under showed that the addition of chitosan aerogels did not affect the melting
two different conditions: 40 ◦ C/150 bar and 60 ◦ C/200 bar. The prep­ point of the PCL, set at 67 ◦ C for all formulations. However, the addition
aration of composite materials, so-called PCL-chitosan scaffolds, took of chitosan aerogels to the formulations improved the thermal stability
place under the same conditions used for obtaining PCL foams. The of scaffolds slightly, and postponed the decomposition of the chitosan
conditions for scaffold preparation were chosen based on extensive amine functional groups. By comparing foams and scaffolds prepared
literature research. Firstly, the conditions 40 ◦ C/150 bar were chosen, under different conditions with the same composition, it can be
to respond to the conditions used to prepare the PCL-aerogel formula­ observed that the materials prepared at higher temperatures and pres­
tions available in the literature [12,17]. On the other hand, the higher sures (60 ◦ C/200 bar) showed slightly higher thermal stability. The
pressure and temperature conditions (60 ◦ C/200 bar) were chosen Optical and SEM micrographs revealed the macroporous and meso­
potentially to instigate the higher solubility of the model drug indo­ porous structures of the prepared scaffolds. However, the structures of
methacin, and operate at temperatures close to the melting point of the scaffolds prepared under different conditions were different. The scaf­
PCL. The conditions of slow depressurization rate and high saturation folds prepared at higher temperatures and pressures (60 ◦ C/200 bar)
times were chosen based on the data that pore sizes increase with the had larger pores and a wider pore size distribution compared to those
increase of depressurization time and longer saturation times [38,39]. prepared at lower temperatures and pressures (40 ◦ /150 bar). These
Larger pores in the formulations were desirable for fitting in the chitosan results agree with the literature, where it was shown that, with the in­
aerogels beads. crease of the foaming temperature, the increase of the pore size occurred
The N2 adsorption-desorption analysis revealed that the specific as a consequence of the decrease of the polymer stiffness [40].

10
M. Pantić et al. Journal of CO2 Utilization 80 (2024) 102697

Furthermore, less uniform pores were obtained at 60 ◦ C/200 bar. This 5. Conclusion
phenomenon can be ascribed to the PCL phase coalescing before the
crystallization of the polymer matrix, due to the use of temperatures To summarize, promising mesoporous-macroporous materials were
above the melting point of the PCL [41]. Furthermore, higher temper­ obtained by combining PCL foams and chitosan aerogel beads. The
atures led to a pale yellowish color and completely different structures, addition of aerogels to the composition of PCL foams improved the
pore sizes, and pore uniformity of the prepared scaffold. Nevertheless, textural properties (increased specific surface areas and adsorption ca­
the foaming temperature of 60 ◦ C is well below the degradation tem­ pacities) and increased the thermal stability of the foams slightly. The
perature of PCL, so the foaming should allow for the preservation of the successful deposition of indomethacin, confirmed by FTIR, was achieved
chemical structure of the polymer. in a simple process of simultaneous foaming and deposition. It was
The performed swelling studies and in vitro dissolution tests proved shown that materials with completely different properties can be pre­
that forming the scaffolds under different conditions led to materials pared simply by adjusting the temperature and pressure parameters. The
with different properties and behavior in simulated body fluids. optical and SEM micrographs gave an insight into the morphology of the
Apparently, preparation at 40 ◦ C/150 bar led to the formation of more obtained scaffolds. While the scaffolds prepared at 60 ◦ C/200 bar can be
stable scaffolds than preparation at 60 ◦ C/200 bar. The swelling studies used for the shorter-term replacement with a crucial release of the model
performed over ten weeks revealed that the scaffolds prepared at lower drug in the first 24 h, the scaffolds prepared at 40 ◦ C/150 bar can be
temperatures and pressures reached the maximal swelling ratio later, used for a longer-term replacement with a release of the drug for one
afterward showing good stability for the tested period. Contrarily, the week.
scaffolds prepared at higher temperatures and pressures reached a
higher maximal swelling ratio in a shorter time, after which they started CRediT authorship contribution statement
decomposing. The degradation of the polymeric graft is desirable, since
it should be replaced gradually by newly formed tissue, being Pantić Milica: Conceptualization, Formal analysis, Investigation,
completely resorbed finally. The most important requirement is a Methodology, Visualization, Writing – original draft. Horvat Gabrijela:
controlled degradation rate. The polymer should be resorbed fast Conceptualization, Formal analysis, Investigation, Writing – review &
enough to allow growth of the bone tissue, but slow enough to provide editing. Berk Bevc Miha: Formal analysis, Investigation. Knez Željko:
the necessary mechanical support [21]. Even though scaffolds prepared Supervision, Writing – review & editing. Novak Zoran: Conceptuali­
at 60 ◦ C/200 bar started degrading earlier, the obtained degradation zation, Resources, Supervision, Writing – review & editing.
was controlled. Lastly, simultaneous scaffold preparation and deposition
led to promising results. The main advantage of this process is that an Declaration of Competing Interest
influence on the loading can be made simply by manipulating the ratio
between the PCL, chitosan aerogel, and indomethacin. The scaffolds The authors declare that they have no known competing financial
prepared at 40 ◦ C/150 bar showed a controlled release of indomethacin interests or personal relationships that could have appeared to influence
over seven days. This is in agreement with the swelling studies, showing the work reported in this paper.
slower swelling and higher stability in the simulated body fluid. In
contrast, the scaffolds prepared at 60 ◦ C/200 bar showed burst release Data Availability
compared to the scaffolds prepared at 40 ◦ C/150 bar. Nevertheless,
these scaffolds still showed controlled release of the indomethacin over Data will be made available on request.
the first 8 h. The scaffolds prepared at 60 ◦ C/200 bar swelled more and
faster, leading to a faster release of the indomethacin. The behavior of Acknowledgments
the scaffolds in the simulated body fluids can be related to their struc­
tures. The more compact structure with a narrower pore size of scaffolds Special thanks to the Slovenian Research Agency (ARIS) for their
prepared at 40 ◦ C/150 bar led to materials with prolonged stability and financial support of Research Programme P2–0046 and Research Project
release of the model drug in the simulated body fluid. The deposition of L2–3175. The authors acknowledge the use of apparatus for supercriti­
indomethacin was influenced by the structure of the prepared scaffolds, cal drying, procured within the project "Upgrading national research
which led to different release profiles. Studies on hybrid PCL-starch and infrastructures - RIUM", which was co-financed by the Republic of
PCL-silk fibroin scaffolds reported release profiles of the tested drugs as Slovenia, the Ministry of Higher Education, Science and Innovation and
well. PCL-starch scaffolds showed comparable results to the scaffolds the European Union from the European Regional Development Fund.
prepared at 40 ◦ C/150 bar in this study [12]. The tested drug showed an
initial burst release during the first hours, and then the sustained release References
was for several days. However, the release profiles of the tested drug
from PCL-silk fibroin scaffolds were strongly influenced by the chemical [1] C. Chircov, A.M. Grumezescu, L.E. Bejenaru, Hyaluronic acid-based scaffolds for
state of the tested drug. Scaffolds showed a two-stage release consisting tissue engineering, Rom. J. Morphol. Embryol. Rev. Roum. Morphol. Embryol. 59
(2018) 71–76.
of a burst release during the first hour followed by a sustained release in [2] Y. Xie, N. Kawazoe, Y. Yang, G. Chen, Preparation of mesh-like collagen scaffolds
the following three weeks [17]. for tissue engineering, Mater. Adv. 3 (2022) 1556–1564, https://doi.org/10.1039/
Finally, the combination of aerogels and supercritical foams led to D1MA01166A.
[3] C. Dong, Y. Lv, Application of collagen scaffold in tissue engineering: recent
materials – scaffolds with outstanding properties of aerogels (high sur­
advances and new perspectives, Polymers 8 (2016) 42, https://doi.org/10.3390/
face areas, high adsorption capacities, high mesoporous volumes) and polym8020042.
good mechanical support of foams. The hybrid materials have a higher [4] A.K. Nayak, B.C. Mohanta, M.S. Hasnain, M.N. Hoda, G. Tripathi, Chapter 14 -
Alginate-based scaffolds for drug delivery in tissue engineering, in: A.K. Nayak, M.
potential for tissue engineering applications, e.g., bone regeneration
S. Hasnain (Eds.), Alginates Drug Deliv, Academic Press, 2020, pp. 359–386,
applications, regarding their textural properties (pore sizes). Scaffolds https://doi.org/10.1016/B978-0-12-817640-5.00014-5.
used for bone regeneration need porous structures with different pore [5] Z. Terzopoulou, A. Zamboulis, I. Koumentakou, G. Michailidou, M.J. Noordam, D.
ranges to allow the transport of the fluids (nutrients, metabolites, N. Bikiaris, Biocompatible synthetic polymers for tissue engineering purposes,
Biomacromolecules 23 (2022) 1841–1863, https://doi.org/10.1021/acs.
wastes) and accessibility of cells for bone ingrowth [42]. Furthermore, biomac.2c00047.
mild operation conditions (40 ◦ C/150 bar, 60 ◦ C/200 bar) create op­ [6] E.S. Place, J.H. George, C.K. Williams, M.M. Stevens, Synthetic polymer scaffolds
portunities for various bioactive components incorporation, such as for tissue engineering, Chem. Soc. Rev. 38 (2009) 1139–1151, https://doi.org/
10.1039/b811392k.
growth factors. In addition, these materials can be used for either bone [7] R. Langer, J.P. Vacanti, Tissue engineering, Science 260 (1993) 920–926, https://
healing, as implants, or as medical devices for drug release. doi.org/10.1126/science.8493529.

11
M. Pantić et al. Journal of CO2 Utilization 80 (2024) 102697

[8] R.A. Quirk, R.M. France, K.M. Shakesheff, S.M. Howdle, Supercritical fluid [25] F. Quignard, R. Valentin, F.D. Renzo, Aerogel materials from marine
technologies and tissue engineering scaffolds, Curr. Opin. Solid State Mater. Sci. 8 polysaccharides, N. J. Chem. 32 (2008) 1300–1310, https://doi.org/10.1039/
(2004) 313–321, https://doi.org/10.1016/j.cossms.2003.12.004. B808218A.
[9] E. Di Maio, E. Kiran, Foaming of polymers with supercritical fluids and perspectives [26] C. López-Iglesias, J. Barros, I. Ardao, F.J. Monteiro, C. Alvarez-Lorenzo, J.
on the current knowledge gaps and challenges, J. Supercrit. Fluids 134 (2018) L. Gómez-Amoza, C.A. García-González, Vancomycin-loaded chitosan aerogel
157–166, https://doi.org/10.1016/j.supflu.2017.11.013. particles for chronic wound applications, Carbohydr. Polym. 204 (2019) 223–231,
[10] L. Diaz-Gomez, A. Concheiro, C. Alvarez-Lorenzo, C.A. García-González, Growth https://doi.org/10.1016/j.carbpol.2018.10.012.
factors delivery from hybrid PCL-starch scaffolds processed using supercritical fluid [27] C. López-Iglesias, J. Barros, I. Ardao, P. Gurikov, F.J. Monteiro, I. Smirnova,
technology, Carbohydr. Polym. 142 (2016) 282–292, https://doi.org/10.1016/j. C. Alvarez-Lorenzo, C.A. García-González, Jet cutting technique for the production
carbpol.2016.01.051. of chitosan aerogel microparticles loaded with vancomycin, Polymers 12 (2020)
[11] Y. Wang, Y. Su, W. Wang, Y. Fang, S.B. Riffat, F. Jiang, The advances of 273, https://doi.org/10.3390/polym12020273.
polysaccharide-based aerogels: Preparation and potential application, Carbohydr. [28] F.S. Kittur, K.V. Harish Prashanth, K. Udaya Sankar, R.N. Tharanathan,
Polym. 226 (2019) 115242, https://doi.org/10.1016/j.carbpol.2019.115242. Characterization of chitin, chitosan and their carboxymethyl derivatives by
[12] L. Goimil, M.E.M. Braga, A.M.A. Dias, J.L. Gómez-Amoza, A. Concheiro, C. Alvarez- differential scanning calorimetry, Carbohydr. Polym. 49 (2002) 185–193, https://
Lorenzo, H.C. de Sousa, C.A. García-González, Supercritical processing of starch doi.org/10.1016/S0144-8617(01)00320-4.
aerogels and aerogel-loaded poly(ε-caprolactone) scaffolds for sustained release of [29] R. Longo, M. Catauro, A. Sorrentino, L. Guadagno, Thermal and mechanical
ketoprofen for bone regeneration, J. CO2 Util. 18 (2017) 237–249, https://doi.org/ characterization of complex electrospun systems based on polycaprolactone and
10.1016/j.jcou.2017.01.028. gelatin, J. Therm. Anal. Calorim. 147 (2022) 5391–5399, https://doi.org/
[13] R. Dwivedi, S. Kumar, R. Pandey, A. Mahajan, D. Nandana, D.S. Katti, D. Mehrotra, 10.1007/s10973-022-11225-7.
Polycaprolactone as biomaterial for bone scaffolds: review of literature, J. Oral. [30] G. Sivalingam, G. Madras, Thermal degradation of poly (ε-caprolactone), Polym.
Biol. Craniofac. Res. 10 (2020) 381–388, https://doi.org/10.1016/j. Degrad. Stab. 80 (2003) 11–16, https://doi.org/10.1016/S0141-3910(02)00376-2.
jobcr.2019.10.003. [31] Y. Zhang, Y. Wu, M. Yang, G. Zhang, H. Ju, Thermal stability and dynamic
[14] S. Takeshita, S. Zhao, W.J. Malfait, M.M. Koebel, Chemistry of chitosan aerogels: mechanical properties of poly(ε-caprolactone)/chitosan composite membranes,
three-dimensional pore control for tailored applications, Angew. Chem. Int. Ed. 60 Materials 14 (2021) 5538, https://doi.org/10.3390/ma14195538.
(2021) 9828–9851, https://doi.org/10.1002/anie.202003053. [32] G. Lawrie, I. Keen, B. Drew, A. Chandler-Temple, L. Rintoul, P. Fredericks,
[15] C. Chartier, S. Buwalda, H. Van Den Berghe, B. Nottelet, T. Budtova, Tuning the L. Grøndahl, Interactions between alginate and chitosan biopolymers characterized
properties of porous chitosan: aerogels and cryogels, Int. J. Biol. Macromol. 202 using FTIR and XPS, Biomacromolecules 8 (2007) 2533–2541, https://doi.org/
(2022) 215–223, https://doi.org/10.1016/j.ijbiomac.2022.01.042. 10.1021/bm070014y.
[16] M. Pantić, U. Maver, J. Rožanc, B. Vihar, D.C. Andrejč, Ž. Knez, Z. Novak, [33] T. Elzein, M. Nasser-Eddine, C. Delaite, S. Bistac, P. Dumas, FTIR study of
Evaluation of ethanol-induced chitosan aerogels with human osteoblast cells, Int. J. polycaprolactone chain organization at interfaces, J. Colloid Interface Sci. 273
Biol. Macromol. 253 (2023) 126694, https://doi.org/10.1016/j. (2004) 381–387, https://doi.org/10.1016/j.jcis.2004.02.001.
ijbiomac.2023.126694. [34] P. Garbacz, M. Wesolowski, DSC, FTIR and Raman spectroscopy coupled with
[17] L. Goimil, V. Santos-Rosales, A. Delgado, C. Évora, R. Reyes, A.A. Lozano-Pérez, S. multivariate analysis in a study of Co-Crystals of pharmaceutical interest,
D. Aznar-Cervantes, J.L. Cenis, J.L. Gómez-Amoza, A. Concheiro, C. Alvarez- Molecules 23 (2018) 2136, https://doi.org/10.3390/molecules23092136.
Lorenzo, C.A. García-González, scCO2-foamed silk fibroin aerogel/poly [35] M.A. Woodruff, D.W. Hutmacher, The return of a forgotten
(ε-caprolactone) scaffolds containing dexamethasone for bone regeneration, J. CO2 polymer—Polycaprolactone in the 21st century, Prog. Polym. Sci. 35 (2010)
Util. 31 (2019) 51–64, https://doi.org/10.1016/j.jcou.2019.02.016. 1217–1256, https://doi.org/10.1016/j.progpolymsci.2010.04.002.
[18] Y. Tozuka, Y. Miyazaki, H. Takeuchi, A combinational supercritical CO2 system for [36] Y. Zhu, D. Li, K. Zhang, L. Jiang, C. Shi, J. Fangteng, C. Zheng, B. Yang, H. Sun,
nanoparticle preparation of indomethacin, Int. J. Pharm. 386 (2010) 243–248, Novel synthesized nanofibrous scaffold efficiently delivered hBMP-2 encoded in
https://doi.org/10.1016/j.ijpharm.2009.10.044. adenoviral vector to promote bone regeneration, J. Biomed. Nanotechnol. 13
[19] L.I. Cabezas, V. Fernández, R. Mazarro, I. Gracia, A. de Lucas, J.F. Rodríguez, (2017) 437–446, https://doi.org/10.1166/jbn.2017.2361.
Production of biodegradable porous scaffolds impregnated with indomethacin in [37] A.A. Zadpoor, Bone tissue regeneration: the role of scaffold geometry, Biomater.
supercritical CO2, J. Supercrit. Fluids 63 (2012) 155–160, https://doi.org/ Sci. 3 (2015) 231–245, https://doi.org/10.1039/C4BM00291A.
10.1016/j.supflu.2011.12.002. [38] A. Salerno, E. Di Maio, S. Iannace, P.A. Netti, Solid-state supercritical CO2 foaming
[20] M. Pantić, Ž. Knez, Z. Novak, Supercritical impregnation as a feasible technique for of PCL and PCL-HA nano-composite: Effect of composition, thermal history and
entrapment of fat-soluble vitamins into alginate aerogels, J. Non-Cryst. Solids 432 foaming process on foam pore structure, J. Supercrit. Fluids 58 (2011) 158–167,
(Part B) (2016) 519–526, https://doi.org/10.1016/j.jnoncrysol.2015.11.011. https://doi.org/10.1016/j.supflu.2011.05.009.
[21] E. Markočič, T. Botić, S. Kavčič, T. Bončina, Ž. Knez, In vitro degradation of Poly(d, [39] K. Kosowska, J. Krzysztoforski, M. Henczka, Foaming of PCL-based composites
l-lactide-co-glycolide) Foams Processed with Supercritical Fluids, Ind. Eng. Chem. Using scCO2: structure and physical properties, Materials 15 (2022) 1169, https://
Res. 54 (2015) 2114–2119, https://doi.org/10.1021/ie504579y. doi.org/10.3390/ma15031169.
[22] K. Gong, J.A. Darr, I.U. Rehman, Supercritical fluid assisted impregnation of [40] A. Salerno, S. Zeppetelli, E.D. Maio, S. Iannace, P.A. Netti, Novel 3D porous multi-
indomethacin into chitosan thermosets for controlled release applications, Int. J. phase composite scaffolds based on PCL, thermoplastic zein and ha prepared via
Pharm. 315 (2006) 93–98, https://doi.org/10.1016/j.ijpharm.2006.02.030. supercritical CO2 foaming for bone regeneration, Compos. Sci. Technol. 70 (2010)
[23] T. Kokubo, H. Kushitani, S. Sakka, T. Kitsugi, T. Yamamuro, Solutions able to 1838–1846, https://doi.org/10.1016/j.compscitech.2010.06.014.
reproduce in vivo surface-structure changes in bioactive glass-ceramic A-W3, [41] A. Satpayeva, A. Rojas, M. Tyrka, E. Ksepko, M.J. Galotto, I. Zizovic, Supercritical
J. Biomed. Mater. Res. 24 (1990) 721–734, https://doi.org/10.1002/ foaming and impregnation of polycaprolactone and polycaprolactone-
jbm.820240607. hydroxyapatite composites with carvacrol, Processes 10 (2022) 482, https://doi.
[24] G. Horvat, M. Pantić, Ž. Knez, Z. Novak, A brief evaluation of pore structure org/10.3390/pr10030482.
determination for bioaerogels, Gels 8 (2022) 438, https://doi.org/10.3390/ [42] Processing of Materials for Regenerative Medicine Using Supercritical Fluid
gels8070438. Technology, Bioconjug. Chem. 26 (2015) 1159–1171. https://doi.org/10.1021/
bc5005922.

12