Physical Hydrogels of Oxidized Polysaccharides and [611771]

materials
Article
Physical Hydrogels of Oxidized Polysaccharides and
Poly(Vinyl Alcohol) for Wound Dressing Applications
Raluca Ioana Baron1,*, Madalina Elena Culica1, Gabriela Biliuta1, Maria Bercea1,
Simona Gherman2, Daniela Zavastin2, Lacramioara Ochiuz2
, Mihaela Avadanei1
and Sergiu Coseri1,*
1“Petru Poni” Institute of Macromolecular Chemistry of Romanian Academy, 41 A, Grigore Ghica Voda Alley,
700487 Iasi, Romania; [anonimizat] (M.E.C.); [anonimizat] (G.B.);
[anonimizat] (M.B.); [anonimizat] (M.A.)
2Faculty of Pharmacy, “Grigore T. Popa” University of Medicine and Pharmacy Iasi, 16th University Str.,
700115 Iasi, Romania; [anonimizat] (S.G.); daniela.zavastin@umfiasi.ro (D.Z.);
[anonimizat] (L.O.)
*Correspondence: [anonimizat] (R.I.B.); [anonimizat] (S.C.); Tel.: +40 232 217454 (S.C.);
Fax:+40 232 211299 (R.I.B.)
Received: 22 April 2019; Accepted: 10 May 2019; Published: 13 May 2019
/gid00030/gid00035/gid00032/gid00030/gid00038/gid00001/gid00033/gid00042/gid00045 /gid00001
/gid00048/gid00043/gid00031/gid00028/gid00047/gid00032/gid00046
Abstract: Two natural polymers, i.e., cellulose and water soluble pullulan, have been selectively
oxidized employing the TEMPO-mediated protocol, to allow the introduction of C 6-OOH groups.
Thereafter, the composite hydrogels of poly(vinyl alcohol) (PVA) and di erent content of the oxidized
polysaccharides were prepared by the freezing /thawing method. The Fourier transform infrared
spectroscopy (FTIR) has been used to discuss the degree of interaction between the hydrogels
constituents into the physical network. The homogeneity of the prepared hydrogels as revealed
by the SEM show an excellent distribution of the oxidized polysaccharides inside the PVA matrix.
The samples exhibit self-healing features, since they quickly recover the initial structure after being
subjected to a large deformation. The cell viability was performed for the selected hydrogels, all of
them showing promising results. The samples are able to load L-arginine both by physical phenomena,
such as di usion, and also by chemical phenomena, when imine-type bonds are likely to be formed.
The synergism between the two constituents, PVA and oxidized polysaccharides, into the physical
network, propose these hydrogels for many other biomedical applications.
Keywords: oxidized cellulose; oxidized pullulan; poly(vinyl alcohol); hydrogel; self-
healing; cytotoxicity
1. Introduction
Hydrogels are remarkable polymeric networks classified as: “two- or multicomponent systems
consisting of a three-dimensional network of polymer chains and water that fills the space between
macromolecules” [ 1]. Due to their intrinsic properties, and their sensitive behavior to the fluctuations
of the environment stimuli, hydrogels are extensively used as biomaterials exhibiting excellent
biocompatibility. They can be used as sca olds, providing structural integrity to tissues, controlled
release of drugs and proteins to tissues. Moreover, they could serve as adhesives between tissue
and material surfaces. Therefore, hydrogels’ properties are crucial for tissue engineering as well as
other applications in the biomedical field. Properties such as swelling behavior, mechanical strengths
and biocompatibility are drastically dictated by the nature of the constituents. Synthetic hydrogels
include poly(vinyl alcohol) (PVA), poly(ethylene glycol), poly(vinyl pyrrolidone), or poly(acrylic acid).
They have the advantages of easiness of processing and a wide versatility of mechanical and chemical
Materials 2019 ,12, 1569; doi:10.3390 /ma12091569 www.mdpi.com /journal /materials

Materials 2019 ,12, 1569 2 of 15
properties. Natural hydrogels show indisputably preferences when biodegradability, biocompatibility
and superior cell adhesion properties are required. Proteins and polysaccharides are the two most
important classes of natural polymers used in hydrogels fabrication. In the last years, a combination
between synthetic and natural polymers has been increasingly used for the hydrogel preparation in
order to combine the advantages of the two original polymer classes. Polysaccharides especially have
gained a lot of consideration due to their abundance and renewability, being used in their native form
or as a specific derivative. For example, the oxidation of polysaccharides represent one of the most
suitable approaches to introduce new functionalities, i.e., aldehyde, ketone or carboxylic [ 2–5], that are
able to serve for further derivatisation or as anchoring sites of di erent molecules, broadening the
applications area of these products [ 6–9]. In our very recent studies, we have reported the preparation
and characterization of oxidized cellulose (or oxidized pullulan)—PVA hydrogels with di erent
content of oxidized polysaccharides [ 10,11]. Aiming to further explore the usefulness of these versatile
materials, in this paper we present new findings on the synthesis, characterization and application
of these hydrogels, for wound dressing applications. The main advantages when using the oxidized
cellulose or pullulan for the hydrogels preparation, are: i) Water is used as a sole solvent for both
components: Oxidized polysaccharide and PVA, avoiding the use of complicated solvent mixtures
required by the unoxidized cellulose, and ii) due to the extremely high number of COOH groups
incorporated after oxidation, the oxidized polysaccharide component takes over the role of crosslinking
agent, not requiring the presence of an additional, often toxic reagent. To test the e ciency of the
synthesized hydrogel for drug loading and release, we propose the use of L-arginine, because it is an
amino acid involved in the synthesis of collagen, re-epithelialization and tissue reorganization [ 12–14].
2. Materials and Methods
2.1. Materials
Poly(vinyl alcohol), molecular weight M wof 8.9 104–9.8104g/mol, 99% hydrolized, Avicel®
PH 101 purified microcrystalline cellulose, were purchased from Sigma-Aldrich (Vienna, Austria).
Pullulan, M w=1.5105g/mol (TCI Europe, Bruxelles, Belgium) was dried overnight under vacuum
at 100C prior to use.
2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO), sodium bromide, 9% (wt.) sodium hypochlorite
and other chemicals and solvents were of pure grade (Sigma Aldrich), and used for pullulan oxidation
without further purification.
Cytotoxicity tests were done using the following materials and reagents: Dulbecco’s Modified
Eagle Medium (DMEM), which comprise of: 4500 mg /mL glucose, 110 mg /L sodium pyruvate and
0.584 mg /L L-glutamine); Bovine Fetal Serum (BFS), heat inactivated, non-USA origin, sterile-filtered;
Penicillin /Streptomycin /Neomycin (P /S/N) solution, (5000 units penicillin, 5 mg streptomycin and
10 mg neomycin /mL), sterile-filtered; Phosphate Bu ered Saline (PBS) solution, sterilised, suitable for
cell culture); 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, (MTT).
N!-nitro-L-arginine methyl ester hydrochloride (NO 2-Arg-OMe, 98%, M W=269.69). All other
chemicals and reagents were from the analytical grade. The in vitro drug release studies were realized
on a spectrophotometer Specord 250, Analytic Jena (Jena, Germany). All materials and reagents were
purchased from Sigma-Aldrich unless otherwise mentioned.
2.2. Preparation of Oxidized Polysaccharides
Cellulose (Avicel®PH 101) and pullulan were used to prepare 6-carboxy corresponding counter
partners. The protocol involves the presence of TEMPO (0.5 mmol /g polysaccharide), NaBr (8 mmol /g
polysaccharide) and NaClO (8 mmol /g polysaccharide), at room temperature and pH =10. The detailed
information could be found in a previous work [8].

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2.3. Preparation of the Composite Hydrogels
Oxidized polysaccharides, i.e., cellulose (OxC) or pullulan (OxP) solutions of 5% were prepared
by dissolving the dry samples in Millipore water under vigorous magnetic stirring, 2 h at room
temperature. A solution of 5% PVA was prepared dissolving PVA at 80C, in Millipore water, using
magnetic stirring for 2 h. After the complete dissolution of the parental components, mixtures of
PVA/OxC and PVA /OxP with contents of oxidized polysaccharides (expressed as weight percent) were
prepared: 0.5%, 5%, 10% and 20%.
The hydrogels were obtained by freezing /thawing the aqueous solutions of pure PVA, PVA /OxC,
and PVA /OxP mixtures using three consecutive cycles (16 h, freezing at 20C, and 8 h thawing, at
room temperature), according to a method previously reported [ 10,11]. The resulted physical networks
were dried by lyophilization.
2.4. Fourier Transform Infrared Spectroscopy
A Vertex 70 spectrometer (BrukerHamburg, Germany) was used for the Fourier transform infrared
(FTIR) spectra recording in the attenuated total reflection (ATR) configuration. All spectra were
collected at 128 scans, at a 2 cm1resolution, in the mid IR range (4000–600 cm1). The OPUS 6.5
software was used for the FTIR data processing.
2.5. Nuclear Magnetic Resonance Spectroscopy
Nuclear magnetic resonance (NMR) spectra were recorded using a Bruker Avance DRX
400 MHz spectrometer. The instrument is equipped with a 5 mm QNP direct detection probe
and z-gradients. Samples of pullulan, oxidized pullulan and oxidized cellulose were prepared in D 2O
using tetramethylsilane TMS ( =0 ppm) as an internal standard, then used to record their1H-NMR
and13C-NMR spectra.
2.6. Environmental Scanning Electron Microscopy
For the morphology observation of all hydrogel samples, an environmental scanning electron
microscope (ESEM, Quanta 200, FEI Company, Hillsboro, OR, USA) instrument was used. This
apparatus works at 5 KV in high vacuum mode, with secondary electrons. Samples were coated with a
thin layer of gold, fixed by means of colloidal silver on copper supports.
2.7. Swelling Measurements
The degree of swelling ( S, %) of samples was measured in Millipore water at 25C by weighting the
dried samples, before ( Wd) and after immersing at di erent times ( Wt). After the sample immersion for
a given time, this was extracted, the water excess being removed using a filter paper, and weighted in a
high precision balance. All experiments were done three times, the calculations took into consideration
the average values. The swelling degree ( S, %) can be determined as:
S(%)=WtWd
Wd100 (1)
2.8. Rheological Measurements
The rheological behavior was investigated by using a MCR 302 Anton Paar rheometer (Graz,
Austria) equipped with the Peltier device for the temperature control and plane geometry (the upper
plate diameter of 50 mm and the gap of 500 m). An anti-evaporation device, which realizes a saturated
atmosphere near the sample, was used in order to avoid the water evaporation.
The frequency sweep tests were carried out at 25C in the linear domain of viscoelasticity.
The self-healing was revealed at !=1 rad /s and successive strains of 1% and 400%. Creep and recovery
tests were carried out for all the samples. During the creep test, di erent shear stress values were

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applied for 30 s and the evolution of deformation was followed. After the stress cessation, the recovery
of deformation was registered.
2.9. Cytotoxicity Tests
The cytotoxicity of the selected hydrogels has been evaluated in vitro using the standard MTT
test [ 15], on the primary fibroblast cell line at passage 5, obtained from Albino rabbit dermis, according
to our previous reports [10,11].
2.10. Loading of Hydrogels With L-Arginine
The hydrogel samples were separately introduced into L-arginine solutions prepared in phosphate
buer (pH 7.4); hydrogel:L-arginine ratio, 1:1 (w /w). After 72 h stirring at 100 rpm, at 37C,
the L-arginine loaded hydrogels were centrifuged and lyophilized. To determine the concentration
of the remaining drug in supernatant, absorbance was measured. Based on the calibration curve,
the percentage of L-arginine loaded in the hydrogel ( Pl, %) (Equation (2)) and the amount of L-arginine
in each hydrogel (Equation (3)) were determined:
Pl=C0Ce
C0
100 (2)
where C0represents the initial concentration of the drug in the feed /initial solution ( g/mL), and Ce
represent the drug concentration left in the solution ( g/mL).
mu=m0
Pu
100(3)
where muis the amount of L-arginine loaded into the hydrogel (mg), m0is the initial amount of
L-arginine used for hydrogel loading (mg), and Puis the percentage of L-arginine loaded into the
hydrogel (%).
In order to establish the wavelength of L-arginine, a dilute solution of the pure drug prepared
in phosphate bu er (pH 7.4) was scanned in the UV region [ 16]. In this way, a calibration
curve could be determined. Using the pure drug, a series of seven standard solutions of known
concentration ( 5–35g/mL) in phosphate bu er were prepared and the absorbance was determined at
the fixed wavelength.
2.11. In Vitro Drug Release Studies
The experimental protocol for the in vitro drug release studies is extensively detailed in
our previous study [ 14]. The quantitative L-arginine determination was performed using the
spectrophotometric method. The amount of drug released was calculated by using the calibration
curve of L-arginine, in terms of % release (Pr) as shown below:
Pr=Ce
C0
100, (4)
where C0represent the total concentration of the drug loaded ( g/mL), while Cerepresent the
concentration of the released drug ( g/mL).
2.12. Analysis of In Vitro Drug Release Kinetics
The prediction and correlation for the in vitro L-arginine release from the modified release
hydrogels was done according to the method described by Ipate et al. [ 17]. The experimental data from
thein vitro drug release experiments were investigated using four predictable models: Zero-order,
first-order kinetics, Higuchi and Korsmeyer–Peppas models [ 18]. The MATLAB 7.1. software
(developed by The MathWorks, Inc., Budapest, Hungary) has been used for the data fitting, by linear

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and nonlinear regression. Data were presented as mean standard deviation being considered as
statistically significant at p <0.05. The correlation coe cient (R2) value was considered for the model
determination, which best fits the release profile of each formula, whereas the “ n” exponent gave the
insight about the mechanism [19].
3. Results and Discussions
3.1. Synthesis and Characterization of Oxidized Polysaccharide Samples
Generally, cellulose applications are drastically restricted by its high recalcitrance to common
solvents, including water. To overcome this issue, several chemical approaches could be designed. One
convenient way to generate cellulose derivatives that are able to be processed in water, comprise of the
introduction of plenty carboxyl groups which could be done by simultaneously modifying the three
–OH groups in the anhydroglucose unit, as we had previously reported [11,20]. Conversely, pullulan
exhibits an excellent solubility in water, being suitable for reactions in the homogeneous media [ 4,5,10].
The oxidation reaction performed on pullulan employing the TEMPO-mediated protocol, allow the
formation of 6-carboxy pullulan, which is even more water-soluble than the original pullulan [21].
The general reaction scheme to oxidize the two primary –OH groups in the maltotriose repeating
unit in pullulan, as well as the primary and secondary –OH groups in cellulose is presented in Figure 1.
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3.1. Synthesis and Characterization of Oxidized Polysaccharide Samples
Generally, cellulose applications are drastically restricted by its high recalcitrance to common
solvents, including water. To overcome this issue, several chemical approaches could be designed.
One convenient way to generate cellulose derivatives that are able to be processed in water, comprise
of the introduction of plenty ca rboxyl groups which could be done by simultaneously modifying the
three –OH groups in the anhydroglucose unit, as we had previously reported [11,20]. Conversely, pullulan exhibits an excellent solubility in water, being suitable for reactions in the homogeneous media [4,5,10]. The oxidation reaction performed on pullulan employing the TEMPO-mediated protocol, allow the formation of 6-carboxy pullulan, which is even more water-soluble than the original pullulan [21].
The general reaction scheme to oxidize the two primary –OH groups in the maltotriose repeating
unit in pullulan, as well as the primary and secondary –OH groups in cellulose is presented in Figure 1.

Figure 1. General scheme for the oxidation of pullulan an d cellulose, performed in the presence of
TEMPO, NaClO, NaBr and sodi um periodate, respectively.
The carboxylated polysaccharides were dialyzed against water, and freeze dried for further use
in the hydrogel fabrication.
3.2. FTIR Spectra of the Composite Hydrogels
The infrared spectra of the hydrogels with variou s formulations, presented in Figure 2, retained
all the characteristic vibrations of the PVA matrix: The broad ν(OH) band around 3300 cm−1, the
combined deformation band δ(CH 2/CH + OH) peaking around 1418 and 1340 cm−1, and the fused
ν(COH)+ ν(COC)+ δ(CCH) band in the 1200–1000 cm−1 region [22]. Especially for the OxC-PVA
hydrogels, the symmetric stretching vibration of th e carboxylate groups is clearly observed at 1610
cm-1 for 20% OxC loading, displaced to the red from the initial 1613 cm−1 value in OxC.
Figure 1. General scheme for the oxidation of pullulan and cellulose, performed in the presence of
TEMPO, NaClO, NaBr and sodium periodate, respectively.
The carboxylated polysaccharides were dialyzed against water, and freeze dried for further use in
the hydrogel fabrication.
3.2. FTIR Spectra of the Composite Hydrogels
The infrared spectra of the hydrogels with various formulations, presented in Figure 2, retained
all the characteristic vibrations of the PVA matrix: The broad (OH) band around 3300 cm1, the
combined deformation band (CH 2/CH+OH) peaking around 1418 and 1340 cm1, and the fused
(COH) +(COC) +(CCH) band in the 1200–1000 cm1region [ 22]. Especially for the OxC-PVA
hydrogels, the symmetric stretching vibration of the carboxylate groups is clearly observed at 1610
cm1for 20% OxC loading, displaced to the red from the initial 1613 cm1value in OxC.

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Figure 2. The fingerprint region of attenuated to tal reflection (ATR)-FTIR spectra of the poly(vinyl
alcohol) ( PVA) hydrogels, in comparison wi th the neat PVA and OxP/OxC: ( a) OxP-PVA; ( b) OxC-
PVA.
The compatibility among the oxidized polysaccharides and PVA can be extracted from the
spectral evolution of the large ν(CO) band and here a distinction between the two types of hydrogels
is observed (Figure 2a,b). The very dynamic behavior of the ν(CO) band in the OxC-PVA samples is
due to the development of two new components, around 1048 and 1018 cm−1, even from the lowest
OxC concentration. Most probably, these two bands belong to the ν(COH) vibrations of the pending
OH groups (1048 cm−1) and to the corresponding ν(CCO) of the backbone (1018 cm−1), hydroxyls that
are engaged in strong intermolecular interactions with the three-carboxylate groups of OxC. The
redshift with 3 cm−1 of the νasym(COO−) of OxC in hydrogels and the blueshift with 11 cm−1 of ν(OH)
of PVA are additional evidences.
In OxP-PVA hydrogels, the ν(CO) band is apparently silent wi th the increasing concentration of
OxP. But the band maximum has blueshifted from 1088 cm−1 in PVA to 1093 cm−1 in OxP-20, and there
is a new component gained at 1018 cm−1 (ν(COH) of OxP shifted to the red with 6 cm−1). The
νasym(COO−) band of the OxP-PVA hydrogels is spread into a large, but weak band, centered on 1625
cm−1 in OxP-20, and that covers several species of carboxylates with various degrees of hydrogen
bonding. As OxP is mainly amorphous, as ob served from the very intense peak at 1024 cm−1 [23],
mixing with the amorphous part of the PVA is extremely efficient, and proves an excellent
compatibility among the two components.
3.3. Morphology of Hydrogels
The morphology of the prepared hydrogels was analyzed by means of ESEM. As Figure 3 shows,
the hydrogels prepared from PVA and oxidized poly saccharides, are porous networks with the pore
size ranging from about 14 to 46 µm. All hydr ogels display a homogeneous structure with no
irregular areas being observed. This can be explained by the compatibility between the two components, i.e., PVA and oxidized polysaccharide. The vast porous structure with interconnected
pores in all hydrogels samples, recommend them for biomedical applications, when biologically
active entities can easily access the inner part of the porous network.
Figure 2. The fingerprint region of attenuated total reflection (ATR)-FTIR spectra of the poly(vinyl
alcohol) (PVA) hydrogels, in comparison with the neat PVA and OxP /OxC: ( a) OxP-PVA; ( b) OxC-PVA.
The compatibility among the oxidized polysaccharides and PVA can be extracted from the spectral
evolution of the large (CO) band and here a distinction between the two types of hydrogels is observed
(Figure 2a,b). The very dynamic behavior of the (CO) band in the OxC-PVA samples is due to
the development of two new components, around 1048 and 1018 cm1, even from the lowest OxC
concentration. Most probably, these two bands belong to the (COH) vibrations of the pending OH
groups (1048 cm1) and to the corresponding (CCO) of the backbone (1018 cm1), hydroxyls that are
engaged in strong intermolecular interactions with the three-carboxylate groups of OxC. The redshift
with 3 cm1of theasym(COO) of OxC in hydrogels and the blueshift with 11 cm1of(OH) of PVA
are additional evidences.
In OxP-PVA hydrogels, the (CO) band is apparently silent with the increasing concentration
of OxP . But the band maximum has blueshifted from 1088 cm1in PVA to 1093 cm1in OxP-20,
and there is a new component gained at 1018 cm1((COH) of OxP shifted to the red with 6 cm1).
Theasym(COO) band of the OxP-PVA hydrogels is spread into a large, but weak band, centered on
1625 cm1in OxP-20, and that covers several species of carboxylates with various degrees of hydrogen
bonding. As OxP is mainly amorphous, as observed from the very intense peak at 1024 cm1[23],
mixing with the amorphous part of the PVA is extremely e cient, and proves an excellent compatibility
among the two components.
3.3. Morphology of Hydrogels
The morphology of the prepared hydrogels was analyzed by means of ESEM. As Figure 3 shows,
the hydrogels prepared from PVA and oxidized polysaccharides, are porous networks with the pore
size ranging from about 14 to 46 m. All hydrogels display a homogeneous structure with no irregular
areas being observed. This can be explained by the compatibility between the two components, i.e.,
PVA and oxidized polysaccharide. The vast porous structure with interconnected pores in all hydrogels
samples, recommend them for biomedical applications, when biologically active entities can easily
access the inner part of the porous network.

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Figure 3. SEM microphotographs (2000× magnification) of cross-sections of PVA hydrogels
containing 0.5%, 5%, 10% and 20% oxidized pullulan (OxP 0.5, OxP 5, OxP 10, OxP 20) and tricarboxy
cellulose (OxC 0.5, OxC 5, OxC 10, OxC 20), respectively.
3.4. Swelling Behavior
The high amounts of hydroxyl groups originat e from both PVA and oxidized polysaccharides,
and also carboxyl groups in oxidized polysaccharid es, have a great impact on the swelling behavior
of the prepared hydrogels. The highly hydrophilic tr ait of the hydrogels allows the water to easily
penetrate the pores of the hydrogels, Figure 4.
Figure 3. SEM microphotographs (2000 magnification) of cross-sections of PVA hydrogels containing
0.5%, 5%, 10% and 20% oxidized pullulan (OxP 0.5, OxP 5, OxP 10, OxP 20) and tricarboxy cellulose
(OxC 0.5, OxC 5, OxC 10, OxC 20), respectively.
3.4. Swelling Behavior
The high amounts of hydroxyl groups originate from both PVA and oxidized polysaccharides,
and also carboxyl groups in oxidized polysaccharides, have a great impact on the swelling behavior
of the prepared hydrogels. The highly hydrophilic trait of the hydrogels allows the water to easily
penetrate the pores of the hydrogels, Figure 4.

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0 50 100 150 200 250 3000200400600800100012001400160018002000
OxP 0.5
OxP 5
OxP 10
OxP 20
OxC 0.5
OxC 5
OxC 10
OxC 20S (%)
time (min)

Figure 4. Swelling behavior of PVA hydrogels containi ng 0.5%, 5%, 10% and 20% oxidized pullulan
(OxP 0.5, OxP 5, OxP 10, OxP 20) and tricarbo xy cellulose (OxC 0.5, OxC 5, OxC 10, OxC 20),
respectively.
Even though from the SEM observations we coul d not find any significan t differences in pore
size in the case of the oxidized pullulan vs. tricar boxylated cellulose hydrogel s, the swelling behavior
measurements have revealed a remarkable feat ure, namely, the hydrogels made from PVA/OxC
possess much higher swelling values than those ma de from PVA/OxP. Moreover, the swelling values
increased, as the amount of OxC in the hydrogel increased. The higher swelling ability of the PVA/OxC hydrogels can be explained by the presen c e o f a m u c h h i g h e r n u m b e r o f h y d r o p h i l i c
carboxyl in these hydrogels as compared with PVA/OxP hydrogels. Another observation from SEM
microphotographs can be made, namely: The pore si ze of the hydrogels varies with the amount of
oxidized polysaccharides added during synthesis [10, 11]. Larger size pores favor the swelling degree
of the hydrogels, and as a consequence a higher load of drug, but at the same time, will contribute to
a faster drug release [24].
3.5. Rheological Behavior
In frequency sweep tests, the elastic (G’) and viscous (G”) moduli were measured and these
parameters give information about the reversibly stored deformation energy and the irreversibly
dissipated energy during one cycle, respectively. Fo r all investigated hydrogels, the G’ values were
higher than the G” ones and they are nearly independent on ω, suggesting that the network is formed.
Figure 5 shows the evolution of the viscoelastic moduli, G’ and G”, as a function of the oscillation frequency, ω, for two selected samples, OxC 10 and OxP 5, for which the highest values of viscoelastic
parameters were obtained (Figure 6).
Figure 4. Swelling behavior of PVA hydrogels containing 0.5%, 5%, 10% and 20% oxidized pullulan
(OxP 0.5, OxP 5, OxP 10, OxP 20) and tricarboxy cellulose (OxC 0.5, OxC 5, OxC 10, OxC 20), respectively.
Even though from the SEM observations we could not find any significant di erences in pore
size in the case of the oxidized pullulan vs. tricarboxylated cellulose hydrogels, the swelling behavior
measurements have revealed a remarkable feature, namely, the hydrogels made from PVA /OxC possess
much higher swelling values than those made from PVA /OxP . Moreover, the swelling values increased,
as the amount of OxC in the hydrogel increased. The higher swelling ability of the PVA /OxC hydrogels
can be explained by the presence of a much higher number of hydrophilic carboxyl in these hydrogels
as compared with PVA /OxP hydrogels. Another observation from SEM microphotographs can be
made, namely: The pore size of the hydrogels varies with the amount of oxidized polysaccharides
added during synthesis [ 10,11]. Larger size pores favor the swelling degree of the hydrogels, and as a
consequence a higher load of drug, but at the same time, will contribute to a faster drug release [24].
3.5. Rheological Behavior
In frequency sweep tests, the elastic (G’) and viscous (G”) moduli were measured and these
parameters give information about the reversibly stored deformation energy and the irreversibly
dissipated energy during one cycle, respectively. For all investigated hydrogels, the G’ values were
higher than the G” ones and they are nearly independent on !, suggesting that the network is formed.
Figure 5 shows the evolution of the viscoelastic moduli, G’ and G”, as a function of the oscillation
frequency,!, for two selected samples, OxC 10 and OxP 5, for which the highest values of viscoelastic
parameters were obtained (Figure 6).

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Figure 5. The viscoelastic moduli (G’ and G”) as a function of the oscillation frequency for hydrogels
with 10% OxC (red full symbols) and 5% OxP (blue open symbols) at 25 °C ( γ = 1%).
.
Figure 6. (a) Elastic modulus (G’) and ( b) complex viscosity ( η*) for the investigated samples with
different content of oxidized polysaccharides (% Oxy) at 25 °C ( ω = 1 rad/s, γ = 1%).
Very recently, we have shown that there is an optimum amount of oxidized polysaccharide for
which the intermolecular interactions with PV A determine the formation of a dynamic network
[10,11]. These physical hydrogels present a self -healing behavior when they are submitted to
successive low (1%) and high (400%) strains, as shown in Figure 7. The structure is perturbed by applying a high shear strain and it is recovered instantaneously after healing at a low strain value, being re-stabilized through physical interactions. The rheological feature of the composite hydrogels based on PVA and oxidized polysaccharides make them of interest for wound dressing applications
[25].

Figure 5. The viscoelastic moduli (G’ and G”) as a function of the oscillation frequency for hydrogels
with 10% OxC (red full symbols) and 5% OxP (blue open symbols) at 25C (
=1%).
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Figure 5. The viscoelastic moduli (G’ and G”) as a function of the oscillation frequency for hydrogels
with 10% OxC (red full symbols) and 5% OxP (blue open symbols) at 25 °C ( γ = 1%).
.
Figure 6. (a) Elastic modulus (G’) and ( b) complex viscosity ( η*) for the investigated samples with
different content of oxidized polysaccharides (% Oxy) at 25 °C ( ω = 1 rad/s, γ = 1%).
Very recently, we have shown that there is an optimum amount of oxidized polysaccharide for
which the intermolecular interactions with PV A determine the formation of a dynamic network
[10,11]. These physical hydrogels present a self -healing behavior when they are submitted to
successive low (1%) and high (400%) strains, as shown in Figure 7. The structure is perturbed by applying a high shear strain and it is recovered instantaneously after healing at a low strain value, being re-stabilized through physical interactions. The rheological feature of the composite hydrogels based on PVA and oxidized polysaccharides make them of interest for wound dressing applications
[25].

Figure 6. (a) Elastic modulus (G’) and ( b) complex viscosity ( *) for the investigated samples with
dierent content of oxidized polysaccharides (% Oxy) at 25C (!=1 rad /s,
=1%).
Very recently, we have shown that there is an optimum amount of oxidized polysaccharide for
which the intermolecular interactions with PVA determine the formation of a dynamic network [ 10,11].
These physical hydrogels present a self-healing behavior when they are submitted to successive low
(1%) and high (400%) strains, as shown in Figure 7. The structure is perturbed by applying a high
shear strain and it is recovered instantaneously after healing at a low strain value, being re-stabilized
through physical interactions. The rheological feature of the composite hydrogels based on PVA and
oxidized polysaccharides make them of interest for wound dressing applications [25].

Materials 2019 ,12, 1569 10 of 15
Materials 2019 , 12, x FOR PEER REVIEW 9 of 15

Figure 5. The viscoelastic moduli (G’ and G”) as a function of the oscillation frequency for hydrogels
with 10% OxC (red full symbols) and 5% OxP (blue open symbols) at 25 °C ( γ = 1%).
.
Figure 6. (a) Elastic modulus (G’) and ( b) complex viscosity ( η*) for the investigated samples with
different content of oxidized polysaccharides (% Oxy) at 25 °C ( ω = 1 rad/s, γ = 1%).
Very recently, we have shown that there is an optimum amount of oxidized polysaccharide for
which the intermolecular interactions with PV A determine the formation of a dynamic network
[10,11]. These physical hydrogels present a self -healing behavior when they are submitted to
successive low (1%) and high (400%) strains, as shown in Figure 7. The structure is perturbed by applying a high shear strain and it is recovered instantaneously after healing at a low strain value, being re-stabilized through physical interactions. The rheological feature of the composite hydrogels based on PVA and oxidized polysaccharides make them of interest for wound dressing applications
[25].

Figure 7. Self-healing behavior of OxC 10 and OxP 5 hydrogels when successive strains of 1% and
400% were applied ( !=1 rad /s, 25C).
During the creep test, a constant stress is applied for 30 s and the hydrogel shows a time-dependent
increase in the strain (Figure 8a). The creep curves comprise the following components of the strain:
The instantaneous, the retardation and the viscous parts. After the stress cessation, firstly the
instantaneous part is recovered, then the retardation component, and finally it rests the viscous strain.
Materials 2019 , 12, x FOR PEER REVIEW 10 of 15
Figure 7. Self-healing behavior of OxC 10 and OxP 5 hy drogels when successive strains of 1% and
400% were applied ( ω = 1 rad/s, 25 °C).
During the creep test, a constant stress is a pplied for 30 s and the hydrogel shows a time-
dependent increase in the strain (Figure 8a). The cr eep curves comprise the following components of
the strain: The instantaneous, the retardation and the viscous parts. After the stress cessation, firstly
the instantaneous part is recovered, then the reta rdation component, and finally it rests the viscous
strain.

Figure 8. The behavior of OxC 10 and OxP 5 samples in creep and recovery tests: ( a) A shear stress of
50 Pa was applied during the creep test; ( b) the total elastic recovery ( γrec, %) as a function of the
applied shear stress during creep test for OxC 10 and OxP 5 hydrogels.
For shear stress up to 30 Pa, a hi gh elasticity of hydrogels can be observed (Figure 8b), when the
recovered strain (the sum of the instantaneous and the retardation components) represents more than
85% from the maximum value achieved during the creep test. Bellow 40 Pa, the OxC samples show
a higher elastic recovery as compared with the Ox P ones, the network is better stabilized through
intermolecular interactions. Above 50 Pa, the behavi or is opposite, OxP containing network is more
elastic, but for all types of hydrog els the elastic recovery decreases as the shear stress exceeds this
limit.
3.6. Cytotoxicity Assays
Several atributes are required for hydrogels in or der to be applied in the tissue-engineering
scaffold, such as high porosity and pore interconnectivity, which also promote the cell proliferation
and differentiation. These atributes also play a crucial role for the in-flow of nutrients or vascular in-growth, and the elution of metabolic waste and biodegradation [26].
The hydrogels prepared from PVA and carboxylated polysaccharides were in vitro cytotoxicity
tested, according to the ISO 10993-5:2009 standard recommendations [15]. The results, depicted in
Figure 9, reveal that all investigated samples are non-cytotoxic. As a general observation, the PVA/OxP hydrogels keep the cells metabolic activity in time. The best cytocompatibility has been
found for the sample with OxP 10. In this case, after 72 h of exposure with cells, the OxP 10 sample
kept cell viability over 90%. This metabolic activity of the cells culture express their ability to adapt to in the vitro conditions. The MTT results reco mmend these hydrogels for biomedical applications,
since they do not release any cytotoxic compounds.
Figure 8. The behavior of OxC 10 and OxP 5 samples in creep and recovery tests: ( a) A shear stress
of 50 Pa was applied during the creep test; ( b) the total elastic recovery (
rec, %) as a function of the
applied shear stress during creep test for OxC 10 and OxP 5 hydrogels.
For shear stress up to 30 Pa, a high elasticity of hydrogels can be observed (Figure 8b), when the
recovered strain (the sum of the instantaneous and the retardation components) represents more than
85% from the maximum value achieved during the creep test. Bellow 40 Pa, the OxC samples show
a higher elastic recovery as compared with the OxP ones, the network is better stabilized through
intermolecular interactions. Above 50 Pa, the behavior is opposite, OxP containing network is more
elastic, but for all types of hydrogels the elastic recovery decreases as the shear stress exceeds this limit.
3.6. Cytotoxicity Assays
Several atributes are required for hydrogels in order to be applied in the tissue-engineering
scaold, such as high porosity and pore interconnectivity, which also promote the cell proliferation
and di erentiation. These atributes also play a crucial role for the in-flow of nutrients or vascular
in-growth, and the elution of metabolic waste and biodegradation [26].
The hydrogels prepared from PVA and carboxylated polysaccharides were in vitro cytotoxicity
tested, according to the ISO 10993-5:2009 standard recommendations [ 15]. The results, depicted in
Figure 9, reveal that all investigated samples are non-cytotoxic. As a general observation, the PVA /OxP

Materials 2019 ,12, 1569 11 of 15
hydrogels keep the cells metabolic activity in time. The best cytocompatibility has been found for
the sample with OxP 10. In this case, after 72 h of exposure with cells, the OxP 10 sample kept cell
viability over 90%. This metabolic activity of the cells culture express their ability to adapt to in the
vitro conditions. The MTT results recommend these hydrogels for biomedical applications, since they
do not release any cytotoxic compounds.
Materials 2019 , 12, x FOR PEER REVIEW 11 of 15
24h 48h 72h020406080100120Cell viability (%)
T i m e Control
OxC 5
OxP 5
OxC 10
OxP 10
OxC 20
OxP 20M T T

Figure 9. The most significant results of the cyto toxicity evaluation, performed on the
PVA/carboxylated polysaccharide hydrogels by using the MTT assay.
3.7. Adsorption and Release of L-Arginine
Based on the calibration curve, presented in the Supplementary Materials (Figure S1), L-arginine
hydrogel loading and drug release calculations were made, Table 1.
Table 1. L-arginine hydrogel loading.
Sample m hydrogel , mg m L-arg, mg % P u ± SD m L-arg Loaded, mg
OxC0.5 142 142 12.92 ± 0.51 18.34 ± 0.72
OxC5 193 193 15.32 ± 0.09 29.57 ± 0.18
OxC10 166 166 13.40 ± 0.21 22.25 ± 0.34 OxC20 201 201 16.36 ± 0.42 32.89 ± 0.84
OxP0.5 170 170 18.44 ± 0.57 31.35 ± 0.96
OxP5 178 178 15.36 ± 0.32 28.72 ± 0.59
OxP10 200 200 5.84 ± 0.40 11.69 ± 0.79
OxP20 198 198 12.48 ± 0.47 24.70 ± 0.94
PVA 205 205 11.64 ± 0.32 23.86 ± 0.65
The results of the L-arginine loading in oxidized cellulose and PVA hydrogels are consistent
with the observed results in the FTIR and SEM dete rminations. Good dispersion of oxidized cellulose
in PVA correlated with the hydrogel surface uniformi ty (SEM) led to a uniform loading of L-arginine
i n h y d r o g e l s . F r o m t h e d a t a p r e s e n t e d , i t i s n o ted that the percentage of L-arginine increases
gradually as the percentage of oxid ized cellulose increases. From the SEM analysis it can be observed
that there is a proportional increase between the amount of oxidized cellulose and the pore size,
which allowed the diffusion of L-arginine into the porous structures of the hydrogels as the sizes
increase.
The loading of hydrogels based on the oxidized pullulan and PVA is correlated with a number
of physical and chemical factors. The small percen tage of oxidized pullulan (0.5%) allowed a good
dispersion of it in the amorphou s part of the PVA matrix. The multitude of small pores (according to
SEM morphology) determined an increase in the amou nt of L-arginine loaded on this hydrogel. As
shown in the previous sections (FTIR, SEM), increasing the amount of the oxidized pullulan in the
Figure 9. The most significant results of the cytotoxicity evaluation, performed on the PVA /carboxylated
polysaccharide hydrogels by using the MTT assay.
3.7. Adsorption and Release of L-Arginine
Based on the calibration curve, presented in the Supplementary Materials (Figure S1), L-arginine
hydrogel loading and drug release calculations were made, Table 1.
Table 1. L-arginine hydrogel loading.
Sample mhydrogel , mg mL-arg , mg % P uSD mL-arg Loaded, mg
OxC0.5 142 142 12.92 0.51 18.34 0.72
OxC5 193 193 15.32 0.09 29.57 0.18
OxC10 166 166 13.40 0.21 22.25 0.34
OxC20 201 201 16.36 0.42 32.89 0.84
OxP0.5 170 170 18.44 0.57 31.35 0.96
OxP5 178 178 15.36 0.32 28.72 0.59
OxP10 200 200 5.84 0.40 11.69 0.79
OxP20 198 198 12.48 0.47 24.70 0.94
PVA 205 205 11.64 0.32 23.86 0.65
The results of the L-arginine loading in oxidized cellulose and PVA hydrogels are consistent with
the observed results in the FTIR and SEM determinations. Good dispersion of oxidized cellulose in
PVA correlated with the hydrogel surface uniformity (SEM) led to a uniform loading of L-arginine in
hydrogels. From the data presented, it is noted that the percentage of L-arginine increases gradually as
the percentage of oxidized cellulose increases. From the SEM analysis it can be observed that there is a
proportional increase between the amount of oxidized cellulose and the pore size, which allowed the
diusion of L-arginine into the porous structures of the hydrogels as the sizes increase.

Materials 2019 ,12, 1569 12 of 15
The loading of hydrogels based on the oxidized pullulan and PVA is correlated with a number
of physical and chemical factors. The small percentage of oxidized pullulan (0.5%) allowed a good
dispersion of it in the amorphous part of the PVA matrix. The multitude of small pores (according
to SEM morphology) determined an increase in the amount of L-arginine loaded on this hydrogel.
As shown in the previous sections (FTIR, SEM), increasing the amount of the oxidized pullulan in
the PVA matrix, will cause an increase on the optical density, resulting in a gradual decrease in drug
loading, as shown in Table 1.
3.7.1. In Vitro L-Arginine Release Studies
Figure 10 shows the release profile of L-arginine from hydrogels based on natural polymers with
the modified structure and PVA at the physiological pH for normal tissue (7.4). It can be seen that the
L-arginine release rate is higher for hydrogels with large pore sizes (OxC20_L-arg), as compared with
those with the reduced pore size (OxP0.5_L-arg).
Materials 2019 , 12, x FOR PEER REVIEW 12 of 15
PVA matrix, will cause an increase on the optical density, resulting in a gradual decrease in drug
loading, as shown in Table 1.
3.7.1. In Vitro L-Arginine Release Studies
Figure 10 shows the release profile of L-arginine from hydrogels based on natural polymers with
the modified structure and PVA at the physiological pH for normal tissue (7.4). It can be seen that the
L-arginine release rate is higher for hydrogels with large pore sizes (OxC20_L-arg), as compared with those with the reduced pore size (OxP0.5_L-arg).

Figure 10. The most relevant curves showing the in vitro profile of L-arginine from the investigated
hydrogels.
By associating natural oxidized polysaccharides with PVA, prolonged drug delivery systems are
obtained. The hydrogel based only on PVA loaded with L-arginine, released the drug almost
completely in the first 30 min (97.33% ± 0.1%), which confirms that the physical properties of PVA are not destroyed by lyophilization [27]. The morpho logy of the hydrogel network based on oxidized
cellulose and PVA, results in a more rapid release of L-arginine due to its diffusion phenomenon
from the hydrogels pores. With the increase in th e amount of oxidized cellulose from the hydrogel,
the amount of drug released increases. From Figu re 10, it can be observed that the amount of L-
arginine released by the OxC5_L-arg hydrogel, after the first 30 min, was 66.92 ± 0.51% and for the OxC20_L-arg hydrogel was 85.8 ± 0.39% and after three h the percentage increased to 83.68 ± 0.17% and 98.52 ± 0.35%, respectively. This release behavior indicates a possible physical adsorption of drug molecules to the surface of the hydrogels pores.
The release of L-arginine from hydrogels based on oxidized pullulan and PVA is directly related
to the pores morphology [10]. The results of the rele ase test show that the release rate of the drug
increases with the size of the pores. Samples OxP0 .5_L-arg and OxP5_L-arg, after 30 min, released
51,64 ± 0.46% respectively 58.28 ± 0.34%, and after 3 h 61,4 ± 0.61% and 73,68 ± 0.54%, respectively.
The OxP0.5_L-arg sample generated a prolonged L-ar ginine release profile of 90.21 ± 0.15% over an
eight-h period, which recommends it for use in pr olonged release systems. Under these L-arginine
release conditions, the OxC20_L-arg hydrogel is recommended for highly exudative wounds because this hydrogel has a high fluid uptake capacity an d an increased drug release rate. Conversely, the
OxP0.5_L-arg hydrogel prolonged release profile, can be related with its low swelling degree and
small pore sizes [28,29].
3.7.2. Analysis of In Vitro Drug Release Kinetics 406080100
0 60 120 180 240 300 360 420 480Cumulative amount of L-arg ( %)
Time (min)OxC 5_L arg
Ox C20_L arg
Ox P0.5_L arg
Ox P5_L arg
PVA_L arg
Figure 10. The most relevant curves showing the in vitro profile of L-arginine from the
investigated hydrogels.
By associating natural oxidized polysaccharides with PVA, prolonged drug delivery systems are
obtained. The hydrogel based only on PVA loaded with L-arginine, released the drug almost completely
in the first 30 min (97.33% 0.1%), which confirms that the physical properties of PVA are not destroyed
by lyophilization [ 27]. The morphology of the hydrogel network based on oxidized cellulose and
PVA, results in a more rapid release of L-arginine due to its di usion phenomenon from the hydrogels
pores. With the increase in the amount of oxidized cellulose from the hydrogel, the amount of drug
released increases. From Figure 10, it can be observed that the amount of L-arginine released by the
OxC5_L-arg hydrogel, after the first 30 min, was 66.92 0.51% and for the OxC20_L-arg hydrogel was
85.80.39% and after three h the percentage increased to 83.68 0.17% and 98.52 0.35% , respectively.
This release behavior indicates a possible physical adsorption of drug molecules to the surface of the
hydrogels pores.
The release of L-arginine from hydrogels based on oxidized pullulan and PVA is directly related
to the pores morphology [ 10]. The results of the release test show that the release rate of the drug
increases with the size of the pores. Samples OxP0.5_L-arg and OxP5_L-arg, after 30 min, released
51,64 0.46% respectively 58.28 0.34%, and after 3 h 61,4 0.61% and 73,68 0.54%, respectively.
The OxP0.5_L-arg sample generated a prolonged L-arginine release profile of 90.21 0.15% over an

Materials 2019 ,12, 1569 13 of 15
eight-h period, which recommends it for use in prolonged release systems. Under these L-arginine
release conditions, the OxC20_L-arg hydrogel is recommended for highly exudative wounds because
this hydrogel has a high fluid uptake capacity and an increased drug release rate. Conversely, the
OxP0.5_L-arg hydrogel prolonged release profile, can be related with its low swelling degree and small
pore sizes [28,29].
3.7.2. Analysis of In Vitro Drug Release Kinetics
Four drug release models, namely: Zero order, first order, Higuchi model, Korsmeyer–Peppas
model, were applied to investigate the mechanism and kinetics of the in vitro release behavior of
L-arginine from the hydrogels (Table 2). The correlation coe cient (R2) value was used to determine
the model that best fits the release, and the release exponent ” n” gave the insight about the mechanism.
The release of L-arginine from hydrogels is best described by the Korsmeyer–Peppas equation. The value
of the nparameter, between 0.5 and 1, corresponds to a non-Fickian di usion mechanism [ 30,31].
Additionally, for all samples the value for R2, between 0.9600 and 0.9998, suggests a release of L-arginine
by di usion. All of these data indicate that the release of L-arginine occurs by the di usion combined
with erosion /degradation of the polymer matrix of the drug [19,32].
Table 2. Results of curve fitting of the in vitro L-arginine release profile from hydrogels based on
oxidized natural polysaccharides and PVA.
Kinetic ModelModel
CoecientsModified Release Sample
OxC5_L-arg OxC_20_L-arg OxP0.5_L-arg OxP5_L-arg PV A
Zero-orderK0 6.1162 6.7557 5.2270 5.8099 50.12
R20.2554 0.0842 0.4616 0.3465 0.8344
First-orderK0 1.5336 3.3439 0.5627 0.8223 8.8848
R20.6919 0.9576 0.1221 0.3992 0.9789
HiguchiK0 43.682 53.335 33.340 39.559 29.76
R20.5259 0.2799 0.7224 0.6266 0.9664
Korsmeyer-Peppasn 0.52 0.49 0.57 0.65 0.7
K0 63.40 84.61 47.31 55.47 31.5
R20.987 0.9600 0.9865 0.9832 0.9998
4. Conclusions
In this paper, cellulose and pullulan have been selectively oxidized employing the
TEMPO /NaClO /NaBr protocol, in order to introduce carboxylic groups. The resulted oxidized
products were used for preparing hydrogels with PVA, employing di erent ratios of oxidized
polysaccharides /PVA. The resulted hydrogels were investigated by using spectral and microscopic
techniques and their rheological and swelling features were analyzed and further tested for the
incorporation and release of L-arginine. The loading of L-arginine takes place by physical phenomena,
such as di usion, but also by chemical phenomena, when it is possible to form imine-type bonds,
especially to materials with oxidized pullulan, which have a prolonged release of the drug to
physiological pH. These non-invasive, L-arginine loaded materials can be used as sponges to treat
heavily scarred wounds, burns or other skin conditions. In the medical field, the e cacy of these
materials could be improved by introducing into the polymeric matrix some antiseptic, antimycotic
and/or anti-inflammatory drugs.
Supplementary Materials: The following are available online at http: //www.mdpi.com /1996-1944 /12/9/1569/s1,
Figure S1: Absorption spectrum.
Author Contributions: Conceptualization, S.C. and G.B.; methodology, S.C.; software, M.A. and M.B.; validation,
M.B., S.C. and L.O.; formal analysis, R.I.B.; investigation, D.Z.; resources, S.G.; data curation, M.E.C.;

Materials 2019 ,12, 1569 14 of 15
writing—original draft preparation, S.C. and M.B.; writing—review and editing, S.C., M.B. and L.O.; visualization,
D.Z.; supervision, M.B.; project administration, S.C.; funding acquisition, S.C.
Funding: This work was supported by a grant from the Executive Agency for Higher Education, Research,
Development and Innovation Funding, UEFISCDI, project number PN-III-P4-ID-PCE-2016-0349, acronym ERAW.
Conflicts of Interest: The authors declare no conflict of interest.
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