gels

Article
Influence of Gel Stage from Cellulose Dissolution in
NaOH-Water System on the Performances of Cellulose
Allomorphs-Based Hydrogels
Diana Elena Ciolacu 1, * , Daniela Rusu 2 , Raluca Nicoleta Darie-Niţă 3 , Daniel Tîmpu 3 and Florin Ciolacu 4, *

1 Department of Natural Polymers, Bioactive and Biocompatible Materials,

“Petru Poni” Institute of Macromolecular Chemistry, 700487 Iasi, Romania
2 Department of Physics of Polymers and Polymeric Materials,
“Petru Poni” Institute of Macromolecular Chemistry, 700487 Iasi, Romania; [email protected]
3 Department of Physical Chemistry of Polymers, “Petru Poni” Institute of Macromolecular Chemistry,
700487 Iasi, Romania; [email protected] (R.N.D.-N.); [email protected] (D.T.)
4 Department of Natural and Synthetic Polymers, “Gheorghe Asachi” Technical University of Iasi,
700050 Iasi, Romania
* Correspondence: [email protected] (D.E.C.); [email protected] (F.C.);
Tel.: +40-332-880-220 (D.E.C.); +40-232-278-683 (ext. 2200) (F.C.)

Abstract: Novel hydrogels were prepared starting from different cellulose allomorphs (cellulose I,
II, and III), through a swelling stage in 8.5% NaOH aqueous solution, followed by freezing at low
temperature (−30 ◦ C), for 24 h. After thawing at room temperature, the obtained gels were chemical
cross-linked with epichlorohydrin (ECH), at 85 ◦ C. The swelling degrees of the hydrogels were
investigated, and a complex dependence on the type of the cellulose allomorph was found. Moreover,
the gel stage has been shown to play a key role in the design of hydrogels with different performances,
following the series: H-CII > H-CI > H-CIII. The correlations between the allomorph type and the
morphological characteristics of hydrogels were established by scanning electron microscopy (SEM).
Citation: Ciolacu, D.E.; Rusu, D.;
Darie-Niţă, R.N.; Tîmpu, D.; Ciolacu,
The hydrogel H-CII showed the biggest homogeneous pores, while H-CIII had the most compacted
F. Influence of Gel Stage from pores network, with small interconnected pores. The rheological studies were performed in similar
Cellulose Dissolution in NaOH-Water shear regimes, and a close correlation between the strength of the gel structure and the size of the
System on the Performances of gel fragments was observed. In the case of hydrogels, it has been shown that H-CII is softer, with
Cellulose Allomorphs-Based a lower resistance of the hydrogel (G′ ) above the oscillation frequencies tested, but it maintains its
Hydrogels. Gels 2022, 8, 410. https:// stable structure, while H-CIII has the highest modulus of storage and loss compared to H-CI and
doi.org/10.3390/gels8070410 H-CII, having a stronger and more rigid structure. The X-ray diffraction (XRD) method showed
Academic Editor: Yi Cao that the crystalline organization of each type of allomorph possesses a distinctive diffraction pattern,
and, in addition, the chemically cross-linking reaction has been proved by a strong decrease of
Received: 20 May 2022
the crystallinity. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy
Accepted: 24 June 2022
provided clear evidence of the chemical cross-linking of cellulose allomorphs with ECH, by the
Published: 29 June 2022
alteration of the crystal structure of cellulose allomorphs and by the formation of new ether bands.
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in Keywords: cellulose allomorphs; gel stage; gel fracturing; cross-linking; hydrogels; rheology
published maps and institutional affil-
iations.

1. Introduction
Copyright: © 2022 by the authors.
Since their development in the late 1960s, hydrogels have distinguished themselves
Licensee MDPI, Basel, Switzerland. by a network-like three-dimensional (3D) structure, as well as by their unique properties,
This article is an open access article which gives them competitive advantages compared to the other materials, making them a
distributed under the terms and promising material-platform for various applications [1–3]. The undeniable versatility of
conditions of the Creative Commons hydrogels in terms of their synthesis, composition, and physical and chemical properties
Attribution (CC BY) license (https:// has led to a considerable attention from both research and industry and has created wide
creativecommons.org/licenses/by/ applicative potential, from the agricultural and industrial to the biomedical field. A key
4.0/). feature of hydrogels is their ability to absorb large quantities of water, which makes

Gels 2022, 8, 410. https://doi.org/10.3390/gels8070410 https://www.mdpi.com/journal/gels

Gels 2022, 8, 410 2 of 21

them soft, pliable, and compatible with most of the living tissues and thus of certain
interest for the biomedical field [4,5]. In addition, their viscoelastic nature reduces any
possible damage to the surrounding tissue, after potential implantation [6]. Moreover, the
mechanical properties are comparable to those of soft tissue, a feature which renders them
particularly attractive to tissue engineers [7].
Using the natural polymers in the preparation of the hydrogels represents an attractive
option over the most of the synthetic counterparts, due to the wide design possibilities of
some specific compositions and properties, providing, besides the support function, key
features such as biodegradability, biocompatibility, and low latent toxicity [8,9]. Moreover,
hydrogels based on natural polymers can be employed either for the sustained and con-
trolled (stimuli-responsive systems) delivery of active pharmaceutical principles (mainly
drugs and proteins) to different cultures and tissues, or as scaffolds for tissue engineer-
ing [10–14].
In this regard, cellulose, the main constituent of plants fibers and the most abundant
natural polymer, with benefits such as low cost, renewability, availability, biodegradability,
and non-toxicity, represents one of the most frequently used polymers in the preparation of
hydrogels [15]. Cellulose and its derivatives have proven to be versatile polymers, which
provide a good platform for the building of 3D networks and various possibilities to design
the physico-chemical features of hydrogels, in terms of mechanical behavior, swelling
ability, and sensibility to external stimuli [7,9,16].
Cellulose displays a highly ordered crystalline structure, based on extensive intra- and
intermolecular hydrogen bonds, and is able to crystallize into various allomorphic forms
that differ in unit cell dimensions and chain polarity (cellulose I, II, III, and IV) [17,18]. The
structural differences of cellulose allomorphs, in terms of crystalline and amorphous contents
and the sizes and shapes of the crystallites corresponding to the allomorphic forms of cellulose,
are considered to be important parameters in the realization of functional materials.
Cellulose I (CI), or native cellulose, represents the prevalent form in plants. This con-
tains highly ordered (crystalline) structures, made of tightly packed linear chains, disposed
of in a parallel arrangement and held together by inter- and intramolecular hydrogen
bonds and also, disordered regions (amorphous) [18]. The natural form of cellulose was
found to be a composite of two coexisting crystalline forms, such as cellulose Iα (a tri-
clinic lattice with one chain per unit cell) and cellulose Iβ (a monoclinic lattice with two
chains per unit cell) [19]. Cellulose II (CII) is obtained by regeneration from various media
or by mercerization in an aqueous NaOH solution [20,21]. CII displays an anti-parallel
arrangement of the chains and possesses a superior chemical reactivity, as compared to
cellulose I. Moreover, the transition from cellulose I to cellulose II is not reversible, and
this implies that cellulose II is a stable form as compared with the metastable cellulose I.
Another crystalline form, known as cellulose III (CIII), can be derived from both cellulose
I and II, using different treatments [22–24]. Thus, CIII can be prepared either by soaking
cellulose samples in anhydrous liquid ammonia at −80 ◦ C or in organic amine.
Each of these three allomorphic forms of cellulose, with the advantages and limitations
of their structural architectures, which are reflected in their accessibility and reactivity,
present a wide and interesting array of potential applications in different domains.
The variations found between the crystalline and amorphous regions, within the
cellulose structure, are the result of both the origin and history of the applied treatments.
Many reagents are able to penetrate only the amorphous part of the material, and most of
the reactions will take place in this area or at the interface with the crystalline ones. Thus,
to increase the chemical reactivity of cellulose it is important to make more accessible the
crystalline regions of cellulose to reagents, by swelling and de-crystallization methods.
Due to the fact that important applications of cellulose involve its regeneration in
several morphologies such as fibers, films, membranes, nanoparticles, or bulky objects
(sponges and aerogels), the dissolution of cellulose is a crucial and challenging step [25,26].
Cellulose is difficult to dissolve in water or most organic solvents due to its characteristic
structure with close packing of macromolecular chains and a strong intra- and interchain
Gels 2022, 8, 410 3 of 21

bonding network [18,27,28]. More recently, this common concept that cellulose–cellulose
hydrogen-bonding is the main obstacle to dissolution and the driving force of aggregation
has been questioned and, instead, it was suggested that hydrophobic interactions play a
very important role in cellulose (in)solubility [27].
There are a limited number of solvents that can directly dissolve cellulose, and be-
tween these, the most important ones are the phosphoric acid-based solvents, LiCl-based
solvents, N-methyl morpholine N-oxide/water, ionic liquids, and NaOH–water [29–32].
Nevertheless, most of these solvents are limited to a laboratory scale due to issues such as
toxicity, environmental hazard, and limited solvency [33].
One of the most common and attractive solvents of cellulose is the NaOH–water-based
system, which has gained attention due to its low cost and of the fact that is easily recyclable
and environmentally friendly.
A number of publications have reported that NaOH-water system can be a good sol-
vent for cellulose, if certain conditions are met. These conditions are: (i) the concentration
of the alkaline solution, which must be in the range of 7–10% NaOH solution, and (ii) the
temperature, which must be low enough, even below 0 ◦ C. As a result of the accomplish-
ment of these conditions, it was proved that the NaOH-water system is able to break the
intra- and intermolecular hydrogen bonds [30,34–37].
The dissolution of cellulose in NaOH-water can be achieved to a certain extent de-
pending on factors such as the molecular weight and the dimensions of the crystalline
domains [30,33,38,39]. However, most of these alkaline water-based systems allow only for
the dissolution of celluloses with a relatively low degree of polymerization, DP < 300.
Isogai and Atalla [39] investigated the possibility of dissolving various types of native
cellulose (microcrystalline cellulose, linter cellulose, softwood unbleached, and bleached
kraft pulps and softwood groundwood pulp) and of different chemically treated cellulose
(mercerized and regenerated). They observed that dissolution is possible only if cellulose is
suspended in a solution of 8.5% NaOH, at a low temperature of −20 ◦ C. After the thawing
of the frozen mass at room temperature, water is added over the obtained gel, in order to
reach a solution containing 2% cellulose in 5% aqueous NaOH.
Considering the above-mentioned dissolution method [39] and the high applicative
potential of the hydrogels, the authors intended to obtain hydrogels with controlled densi-
ties, porosities, and swelling degrees, starting from the allomorphic forms of cellulose with
different crystalline structures.
Thus, the aim of this study is to evaluate the influence of the gel structure obtained
from the cellulose-8.5% NaOH system, based on the Isogai and Atalla method, on the
performance of their corresponding hydrogels, prepared by chemical crosslinking with
epichlorohydrin (ECH).
The hydrogels were obtained starting from three allomorphic forms of cellulose (cel-
lulose I, II, and III) and were investigated by means of swelling measurements, X-ray
diffraction (XRD) analysis, scanning electron microscopy (SEM), attenuated total reflectance
Fourier transform infrared (ATR-FTIR) spectroscopy, and oscillatory rheology, in order to
establish their structure and morphology, as well as the swelling and rheological behaviors.

2. Results and Discussion

2.1. Swelling Behavior of Hydrogels
Novel hydrogels were produced by chemical cross-linking with ECH of different
cellulose allomorphs (CI, CII, and CIII), starting from a gel-like mass, obtained as an
intermediary stage within the cellulose dissolution process in NaOH-water system, at
low temperature (Figure 1a–c). One of the main interests of this study was to evaluate
the influence of the cellulose allomorphs’ structure upon the swelling capacity of their
corresponding hydrogels (Figure 1d–f) since the ability to absorb water represents one of
the most important features of these 3D materials.
Gels 2022, 8, 410 4 of 21

(a) (b) (c)

(d) (e) (f)

Figure 1. Stages in the investigation of the hydrogels based on cellulose allomorphs: (a–c) dry state of
the hydrogels H-CI, H-CII, and H-CIII, and (d–f) wet state of the hydrogels H-CI, H-CII, and H-CIII.

The three hydrogels prepared from the cellulose allomorphs H-CI, H-CII, and H-CIII
showed different swelling abilities, as reflected by the different values of the maximum
swelling degree (Qmax) and equilibrium swelling degree (Qeq) (Table 1).

Table 1. Swelling kinetic parameters of cellulose-allomorphs-based hydrogels.

Swelling Kinetic Parameters

Sample Qmax (%) Qeq (%)
ksw nsw R2
H-CI 0.291 0.386 0.999 2042 2128
H-CII 0.321 0.377 0.999 2295 2440
H-CIII 0.165 0.614 0.999 1826 1926

The graph displayed in Figure 2 shows that the hydrogel H-CII, obtained from the
cellulose II allomorph, exhibited the highest value of the swelling degree (Qmax = 2295%;
Qeq = 2440%). A medium value was recorded for the hydrogel prepared from the CI
allomorph (H-CI: Qmax = 2042%; Qeq = 2128%), while the lowest swelling values were
registered for the CIII allomorph (H-CIII: Qmax = 1826%; Qeq = 1926%). The swelling
occurred rapidly in the first 50 min and then slowly reached a constant value (Qmax)
for all the samples. The Qeq values of the never-dried hydrogels are higher than that
obtained for the Qmax of the freeze-dried hydrogels, due to the well-known hornification
Gels 2022, 8, 410 5 of 21

phenomenon [28]. However, the same descending order is maintained for both swelling
degrees, such as H-CII > H-CI > H-CIII.

Figure 2. Evolution of the swelling degree (Qmax) for the hydrogels based on cellulose allomorphs
H-CI, H-CII, and H-CIII.

The dynamic swelling behavior of the hydrogels is dependent on the relative contribution
of polymer relaxation, as well as of the penetrant diffusion [40,41]. In order to study the effect
of the hydrogel type (H-CI, H-CII, and H-CIII) on the kinetic of water uptake process, swelling
data were fitted using the power-law expression (Equation (3)) [41]. The swelling kinetic
parameters of the hydrogels based on the cellulose allomorphs are presented in Table 1.
The parameter nsw is the diffusion exponent that indicates the water transport mecha-
nism, while ksw is the swelling constant, which is related to the structural network. For
a hydrogel, (i) n = 0.5 indicates the Fickian diffusion (case I), (ii) n = 1 implies relaxation-
controlled transport mechanism (case II), and (iii) 0.5 < n < 1 indicates the non-Fickian
or anomalous transport (case III) [42]. The values of n < 0.5 show a pseudo-Fickian (less-
Fickian) diffusion [43]. The constants nsw and ksw were calculated from the slopes and
intercepts of the plots of ln (Mt/M0 ) vs. ln t.
The values of nsw obtained in the case of H-CI and H-CII hydrogels (nsw < 0.5) indicate
that the water transport mechanism follows the less Fickian diffusion, in which the rate of
diffusion is much smaller than the rate of relaxation (Rdiffusion < Rrelaxation , system controlled
by diffusion) [44]. A slight increase of nsw for H-CI indicated the fact that the swelling is
controlled more by the polymer chain relaxation rate then the water diffusion rate. For
H-CIII, it was observed that nsw > 0.5 (nsw = 0.614), which indicated a shift from the less-
Fickian diffusion (water transport mechanism) to a non-Fickian or anomalous transport
(relaxation-controlled transport mechanism). This type of diffusion indicates the fact that
the water diffusion and the polymer relaxation rate are about the same order of magnitude
(Rdiffusion ~ Rrelaxation ).
The swelling rate constant, ksw , is another important parameter that determines
the diffusional characteristics of hydrogels and a slight decrease of this parameter were
observed in the case of H-CI and H-CIII hydrogels.
For all the hydrogels, the correlation coefficients R2 are higher than 0.99, a fact that
indicates the high accuracy of the linear regression equations and good agreement between
the experimental data and the chosen model.

2.2. Morphological Investigation of the Hydrogels

SEM is a relatively simple morphological analysis technique that requires minimum
sample preparation and provides solid and realistic information regarding the spatial
arrangement of any given material [28]. Therefore, SEM was used to study the surface
features of the hydrogels based on cellulose allomorphs and to collect data with respect
Gels 2022, 8, 410 6 of 21

to the appearance, integrity, porosity, and degree of uniformity of the hydrogels. This
technique offers essential details regarding the morphological aspects of the hydrogels,
which ranged from millimeters down to micrometers.
It is expected that the different types of the allomorph used in the hydrogels will
be reflected in different morphologies, as was also observed in the investigations on the
swelling behavior (Figure 3).

(a) (b)

(c)
Figure 3. SEM micrographs of the hydrogels based on cellulose allomorphs: (a) H-CI, (b) H-CII, and
(c) H-CIII.

The analysis of SEM micrographs evidenced the fact that the 3D polymeric matrices
with dissimilar homogeneities and had varying porous structures, and they differed from
one another in terms of shape, dimension, and distribution of pores. The average pore size
was determined from the SEM micrographs, by measuring 50 randomly chosen pores.
The H-CI hydrogel shows interconnected pores of a roughly circular shape, dis-
persed in a quasi-homogeneous, compact matrix. The average diameter of the pores is
86.2 ± 12.7 µm, confirming a tightly packed morphology, a fact that is explains the relatively
small amount of water absorbed by this 3D matrix, as was confirmed by the swelling data.
In comparison, the H-CII hydrogel has bigger, interconnected, and ovoid-pores dis-
tributed along a more homogeneous surface. The average diameter of these pores is
108.2 ± 15.3 µm, thus explaining the more relaxed morphology of these hydrogels, which
is able to incorporate a larger quantity of water.
The H-CIII hydrogel has the most compacted pores network, with small intercon-
nected pores (an average diameter of 67.5 ± 12.6 µm), information that correlates well
Gels 2022, 8, 410 7 of 21

with the swelling data, with H-CIII having the lowest swelling degree compared to the
other two hydrogels.
All the observations from the SEM micrographs are consistent with the information data
on the swelling behavior, confirming a direct dependence between the pore size and the
swelling degree of the hydrogels; thus, the bigger the pore size, the higher the swelling degree.

2.3. Crystallinity in Cellulose Allomorphs and Cellulose-Based Hydrogels

The differences in the swelling behaviors of the hydrogels were initially attributed to
the differences in the structural organization of the starting materials (cellulose allomorphs),
which is why a complex investigation of the supramolecular structure by XRD of both the
starting materials and the hydrogels was performed.
The XRD diffractograms characteristic of cellulose allomorphs (CI, CII, and CIII), as
well as those of the corresponding hydrogels (H-CI, H-CII, and H-CIII), are presented
in Figure 4. Several structural changes that occur during the mercerization process are
highlighted in Figure 4 and consist of a decrease in the degree of crystallinity of CII
compared to CI and a complete transformation of CI into the CII allomorph (Figure 4b).
It has been observed that the transition process from CI to CII is accompanied by
modifications of the intensities corresponding to different crystallographic planes. The
diffraction peaks characteristic of CI are presented in Figure 4a and appear at Bragg angles
(2θ) of 14.6◦ , 16.2◦ , and 22.5◦ , which are typical for the crystallographic planes, (110),
(1–10), and (200), respectively. In the case of the CII, three characteristic cellulose lattice
planes were identified in the XRD diffraction pattern (Figure 4b), and these appear at
12.1◦ assigned to the (110) plane, at 20.1◦ for the (1–10) plane, and at 21.8◦ for (200) plane,
respectively. Generally, CII is characterized by a slightly larger peak (1–10) on the left and a
slightly smaller (200) on the right, a well-known difference that demonstrates the complete
transformation of a crystalline structure of CI to a crystalline structure of CII [17]. This fact
was also observed in our study, when it was found that CII contains a (1–10) peak with a
much higher intensity than the (200) peak, proving a complete transformation of CI to the
lower free energy crystalline pattern, CII. The diffractogram of CIII (Figure 4c) presents one
peaks at 11.8◦ (110 plane) and an overlapping of the planes (1–10) and (200), at 2θ of 21.1◦ .
It is obvious that the crystalline organization of each type of allomorph has a distinctive
diffraction pattern, with specific variations of both the intensities of the diffraction peaks
and the positions of the Bragg angles, at which their characteristic maxima are placed.
The diffractograms displayed in Figure 4d–f show a significant decrease of the intensi-
ties of all the peaks characteristic of the main crystallographic planes, an overlapping of the
planes (1–10) with (200), and a displacement of the plane (110) towards lower values of the
angle 2θ. These modifications indicate that the chemical cross-linking has been achieved in
all hydrogels, accompanied by the damage of the initial crystalline structure of cellulose.
The synthesis of hydrogels from different types of cellulose allomorphs proceeds with
the major impairment of the crystalline organization. All the prepared hydrogels, regardless
of the initial allomorph, lead essentially to the same structure specific to a predominantly
amorphous cellulose, with peaks that have become wider and weaker. However, the
presence of a certain crystallinity was observed, demonstrated by the presence of the peaks
corresponding to the characteristic crystallographic planes of CII allomorph.
Thus, the diffraction peaks characteristic of H-CI (Figure 4d) appear at 9.39◦ for
the crystallographic plane (110), while for the crystallographic planes (1–10) and (200) a
Bragg angle of 20.1◦ was recorded. The same transformation was observed also for H-CII
(Figure 4e), where the two characteristic peaks were identified at 2θ of 9.21◦ and 20.1◦ ,
while in the case of H-CIII (Figure 4f), the peaks appeared at 2θ of 9.47◦ and 20.1◦ .
Gels 2022, 8, 410 8 of 21

250 100

22.5
200 80
Intensity, a.u.

Intensity, a.u.
150 60

20.1
100 40
14.6
18.1
16.2
50 20
19.3 20.6 9.39
0 0
10 15 20 25 30 5 10 15 20 25 30
2θ, ° 2θ, °

(a) (d)
250 100

200 80
Intensity, a.u.
Intensity, a.u.

150 20.1 60
20.1
21.8
100 40
18.3

50 12.1 20
19.1 9.21

0 0
10 15 20 25 30 5 10 15 20 25 30
2θ, ° 2θ, °

(b) (e)

250 100

200 80
Intensity, a.u.

Intensity, a.u.

21.1
150 60

100 40 20.1

50 20 18.3
11.8 19.2
9.47
0 0
10 15 20 25 30 5 10 15 20 25 30
2θ, ° 2θ, °

(c) (f)

Figure 4. The deconvoluted XRD diffractograms of (a–c) cellulose allomorphs (CI, CII, and CIII) and
(d–f) hydrogels obtained from the corresponding cellulose allomorphs (H-CI, H-CII, and H-CIII).

XRD studies of cellulose-allomorphs-based hydrogels confirm that chemically cross-

linked hydrogels undergo distinct transformations. The sharp reduction of the crystallinity
recorded for the case of the chemically cross-linked hydrogels is a fact confirmed also
byθ other authors [28]. In the initial cellulose, the chains are packed in a highly orderly
manner with a compact structure, while in the case of hydrogels, the crystalline struc-
ture was destroyed by the cross-linking reaction, which disrupts the self-association and
the packing of the cellulose chains, consequently leading to an increase of the sample’s
hydrophilicity [20,28].
Gels 2022, 8, 410 9 of 21

The changes recorded by the cellulose-allomorphs-based hydrogels (H-CI, H-CII, and

H-CIII), regarding their crystallinity index (CrI) and the crystallite dimension (Dhkl) are
presented in Table 2. The crystallinity index was determined by using Equation (4), while
the crystallite size (Dhkl) in the direction perpendicular to the analyzed lattice planes with
the Miller indices (hkl) was evaluated by using Scherrer’s equation (Equation (5)) [41,42].

Table 2. Crystallite size (D) and crystallinity index (CrI) of the cellulose allomorphs and cellulose-
based hydrogels.

Samples D(110) (nm) D(1–10) (nm) D(200) (nm) CrI (%)

CI 5.995 5.232 8.330 86.02
Cellulose allomorphs CII 5.492 7.786 8.191 82.20
CIII 4.821 8.039 8.039 70.72
H-CI 3.615 2.665 44.99
Cellulose-based hydrogels H-CII 4.349 3.147 46.62
H-CIII 3.009 2.636 40.71

Regarding the crystallinity index (CrI) of the allomorphic celluloses, there was a
decrease in it when passing from the untreated sample of CI (CrI = 86.02%) to the mercerized
CII (CrI = 82.20%) and to CIII (CrI = 70.72%). In the case of the hydrogels, the crystallinity
indexes have not varied too much between the samples, with the recorded values being
44.99% for H-CI, 46.62% for H-CII, and 40.71% for H-CIII.
A relationship between the crystallinity index and the crystallite dimensions was
observed for the studied samples. Thus, the smaller the dimensions of the crystallites, the
lower the CrI of the sample, a fact associated with an increase of the amorphous domains.
This aspect was also confirmed by other authors [45,46].
Moreover, the dramatic decrease in the crystalline indexes of cellulose-based hydrogels
is a result of chemical crosslinking between the hydroxyl groups of the cellulose chains.
However, XRD investigations have shown the same type of supramolecular organization of
hydrogels, regardless of the type of allomorph used as starting material, which does not explain
the differences in the swelling behavior of hydrogels resulting from different allomorphs.

2.4. Rheological Evaluation of the Gels and Hydrogels Based on Cellulose Allomorphs
Since the changes in the supramolecular structure do not explain the swelling behav-
ior, the explanation for the differences in the swelling behavior was searched for in the
differences recorded during the hydrogel synthesis processes.
Hydrogels were prepared starting from a swelling stage of cellulose in NaOH solutions,
at low temperatures, based on a method developed by Isogai and Atalla [39], followed by
chemical cross-linking with epichlorohydrin (ECH).
As already known, the first stage in polymer dissolution is swelling (Figure 5—stage I),
a phenomenon based on the incorporation into the polymer of a large amount of solvent.
The swelling may be limited or unlimited, similar to dissolution. The limited swelling is
the process of interaction of the polymer with the solvent, which is stopped when the
latter is absorbed by the polymer, without a spontaneous dissolution. The method of
dissolving cellulose at low temperatures involves a complete freezing stage by cooling
to very low temperatures (Figure 5—stage II), and the thawing of the obtained block
allows one to obtain the gel phase. Two phases are specific to the limited swelling process:
one represented by the polymeric matrix in which the solvent molecules diffused—the
gel—and other represented by the pure solvent or diluted solution of polymer in solvent—
the supernatant. The two phases are delimited by a separation surface and they coexist in an
equilibrium, as shown in Figure 5—stage III.
Gels 2022, 8, 410 10 of 21

Figure 5. Schematic method for the preparation of hydrogels based on cellulose allomorphs.

The first differences appeared in the aspect of the gel phase resulting from the swelling
in NaOH solution of cellulose allomorphs (Figure 6).

Figure 6. Gel phases corresponding to each type of cellulose allomorphs: CI, CII, and CIII.

The gels have different appearances, volumes, consistencies, and densities. The density
and polymerization degree corresponding to the cellulose allomorphs are presented in
Table 3.

Table 3. The density and polymerization degree corresponding to the cellulose allomorphs.

Sample CI Alkali-Treated CI CII Alkali-Treated CII CIII Alkali-Treated CIII

DP 183 171 154 149 164 158
Density (g/mL) 1.050 - 1.282 - 0.960 -

The gel resulting from the swelling of the CII allomorph is obviously the most compact,
while that of the CIII allomorph is the least dense. These aspects were reflected in their
behavior in the next stage, the mixture with the cross-linker under controlled shear regimes.
By mixing with the cross-linking agent in identical shear regimes, the cellulose-NaOH
gels are fractured into fragments of sizes correlated with the strength of the gels. The
result is a hydrogel with a high cross-linking density if the gel fragments are small in size
and with a lower cross-linking density in the case of resistant gels that fracture into large
fragments (Figure 7).
Gels 2022, 8, 410 11 of 21

Figure 7. A model for the hydrogels synthesis, as a result of the cross-linking of gels fragmented in
large or small fractions.

In order to validate the proposed model, rheological investigations were performed, which
were able to explain both the structure and the swelling behavior of the obtained hydrogels.
To highlight the strength of cellulose-NaOH gels, oscillatory amplitude sweeps tests
were carried out. The linear viscoelastic region (LVR) was determined, and the brittle
fracturing behavior of the gels was evidenced.
The (elastic) storage modulus G′ represents the elastic portion of the viscoelastic
behavior, while the (viscous) loss modulus G′′ characterizes the viscous portion of the
viscoelastic behavior.
The approximate values of G′ within the LVR, which refer to the gel strength, vary
between 6350 Pa for CI, 11,500 Pa for CII, and 3500 Pa for CIII, showing enhanced stiffness
“at rest” of the network (G′ LVR) for cellulose allomorphs CII followed by CI.
When the LVR is exceeded, G′ and G′′ are dependent on the strain amplitude, usually
decreasing with its enhancement for all gels at large deformations. The amplitude sweep
results reveal the network breakdown as the strain increases. All the studied gels of
cellulose allomorphs exhibit a “flow point” (crossover G′ = G′′ ) over 14.5%. The stress and
strain at the flow points (δf ) and (γf ), respectively, were determined at the crossover point
(G′ = G′′ ), with the resulting values being presented in Table 4 [47].

Table 4. The values of dynamic moduli at the crossover point (G′ = G′′ ), and the stress and strain at
the flow point (δf and γf ).

Sample G′ = G′′ (Pa) δf (Pa) γf (%)

CI 1072 434.9 28.57
CII 1102 491.1 31.52
CIII 1027 211.4 14.54

The experimental results show a lower strength of CIII at the flow point, with the
lowest values of the dynamic moduli, δf and γf , and the maximum structure breakdown of
this cellulose allomorph being registered at 14.54% strain deformation.
Figure 8 shows that G′ displays the sharpest downturn starting at γ = 1% for CII, thus
indicating a brittle fracturing behavior that led to the inhomogeneous breaking of gel CII
under shear into larger pieces [48].
Moreover, Figure 8 displays that the loss modulus G′′ follows an almost constant value
in the LVE region for all gels, but, unlike G′ , the G′′ curve rises sharply because initially
a few individual bonds in the network are broken, developing micro cracks, keeping the
entire surrounding material firmly together. After reaching the maximum peak before
breakdown, the gel starts to flow so the G′′ curve drops continuously and not as steep as
G′ . The sharp increase of the viscous modulus G′′ is due to the internal viscous friction of
Gels 2022, 8, 410 12 of 21

the resulting freely movable fragments around the micro cracks (Figure 8b). The individual
micro cracks continue to grow and might lead to the formation of the continuous macro
crack through the entire material that starts to flow, with the viscous behavior of the sample
dominating in this case (G′′ > G′ ) [49].

(a)

(b)

Figure 8. (a) Amplitude sweep test results (a) G′′ and G″′′ dependence of the oscillatory strain for CI,
CII and CIII; and (b) G′′ and G″′′ dependence of the shear stress for gels CI, CII, and CIII.

The shape of G′′ can be used to distinguish the behavior of the gels. The process of
structural breakdown is faster for CII and delayed for CI (and especially CIII).

′ ″
Gels 2022, 8, 410 13 of 21

The rheological measurement results offer information on the structure–property

relationship of the hydrogels. The dynamic viscoelastic properties of the formed networks
of cellulose-based hydrogels were evaluated by frequency sweep analysis (Figure 9). The
elastic features of the matrix were dominant throughout the whole measured angular
frequencies, characterized by G′ values higher than G′′ values, which demonstrates typical
gel-like behavior.

(a)

(b)

Figure 9. Frequency sweep tests results of hydrogels: (a) the dependence of the storage modulus G′
″
(solid symbols) and loss modulus G′′ (open symbols) on the angular frequency; (b) the dependence
of the complex viscosity on the angular frequency.
′
All values, especially for G′ , are almost independent of the tested angular frequency,
proving that the hydrogels display an excellent structured three-dimensional (3D) network,
Gels 2022, 8, 410 14 of 21

and the applied deformation does not affect the stability of the studied hydrogels’ network
structure. The absence of a crossover frequency indicates permanent chemical cross-linking.
The graph shown in Figure 9a reveals that the structure of H-CII is softer, with a lower
resistance of the hydrogel (G′ ) over the tested oscillation frequencies compared with H-CIII,
but its stable structure is maintained. At higher angular frequencies (over 100 s−1 ), the
loss (viscous) moduli increase, although not overlapping the storage moduli, maintains the
gel-like behavior up to 500 s−1 , the highest tested angular frequency.
The hydrogel corresponding to cellulose allomorph III (H-CIII) exhibits a higher
storage and loss modulus than hydrogels H-CI and H-CII, showing a stronger and stiffer
structure. This mechanical feature is translated also into a higher curve of complex viscosity
for H-CIII (Figure 9b). The complex dynamic viscosity decreases with the increasing
oscillation frequency for all the evaluated cellulose-based hydrogels.
All the rheological results are in good correlation with the SEM and XRD data and
with those obtained for the swelling ability of the hydrogels.

2.5. ATR-FTIR Analysis of the Cellulose Allomorphs and Cellulose-Based Hydrogels

ATR-FTIR spectroscopy was used in order to prove the achievement of the cross-
linking reaction between cellulose and EPC, this being a highly effective analytical tool for
the evaluation of the surface chemical composition of materials.
The ATR-FTIR spectra of the hydrogels (H-CI, H-CII, and H-CIII) compared with their
corresponding cellulose allomorphs (CI, CII, and CIII) are shown in Figure 10.
The spectral modification in the region 1500–800 cm−1 is assigned to an alteration of
the crystalline structure of cellulose, following the alkaline treatment and, implicitly, the
chemical cross-linking of cellulose with ECH [50]. In addition, it can be observed that the
band at 1636 cm−1 , characteristic of the adsorbed water [51], is more intense in the hydrogel
spectra. This observation reflects the higher hydrophilic character of the hydrogels in
comparison with their corresponding cellulose allomorphs.
The difference spectrum [H-Ci – Ci ] has been calculated in order to highlight the
spectral changes resulting from chemical cross-linking (top spectra in Figure 10).
All the three allomorphic forms of cellulose present the decrease of the bands at:
− 994–980 cm−1 (ν(CO) at C6) with the counterpart at 3437 cm−1 (O2-H· · · O6 in-
tramolecular H-bonds);
− 1054 cm−1 (ν(CO) at C3), 3339 cm−1 (O3-H· · · O5 intramolecular H-bonds), and
3301 cm−1 (O6-H· · · O3′ intermolecular H-bonds);
− 1106 cm−1 (ν(CO) at C2) and 3296 cm−1 (coupled vibrations of O2-H· · · O6 and
O3-H· · · O5 intramolecular H-bonds) [51–54].
These changes show that in all three hydrogels, the chemical crosslinking of cellulose
with ECH takes place mainly at the secondary alcohol C2 and the primary alcohol C6 and
less at the secondary alcohol C3. The same observations were also confirmed by other
authors [38,55].
This finding can be explained by the fact that Na+ breaks the intermolecular hydrogen
bonds O2-H· · · O6′ and that the most probable location of the Na+ ion is at C2, thus
facilitating the realization of the cross-linking reaction on this site [56–59]. In addition,
many studies show that C3 carbon is the most resistant to complexation with NaOH [30],
which is also observed in our case.
In the case of CI, the large band at 1102 cm−1 indicates that the secondary alcohols C2
contributed to the formation of ether-based cross-links. Besides the very clear ν(O2-H· · · O6)
peak at 3437 cm−1 , the initial CII cellulose had an additional peak at 3480 cm−1 , which
belongs to ν(O6-H) weakly H-bonded and to some –OH groups located in the interface
and surface regions of crystallites [60]. The disappearance of both peaks upon crosslinking
certifies the formation of ether-based linkage in the less organized regions first and also
attests to the disruption of intermolecular hydrogen bonds (O6-H· · · O3′ ). In the case of
H-CIII, the intense negative band at 1160 cm−1 , characteristic of ν(COC) of glycosidic
Gels 2022, 8, 410 15 of 21

linkage, shows that the network formed through cross-linking reaction has decreased its
degrees of freedom.

Figure 10. ATR-FTIR spectra comparison of cellulose allomorphs (dark blue line) with those of their
corresponding hydrogels (red line): (A) CI and H-CI, (B) CII and H-CII, and (C) CIII and H-CIII. The
difference spectrum [H-Ci – Ci] is added to the top of each figure (blue line). The vertical dotted lines
mark the position of the characteristic bands of the chemical cross-linking of cellulose with ECH.

In the spectra of the three hydrogels, H-CI, H-CII, and H-CIII, the chemical crosslinking
of cellulose with ECH is highlighted by the formation of new bands at 2930 and 2873 cm−1,
assigned to νasym (CH2 ) and νsym (CH2 ); at 1458 cm−1, given by the δsciss (CH2 ), and at
1133 cm−1, assigned to ν(COC) of the ether linkages [61].
Because the FTIR spectra of the three hydrogels are very similar and the ν(OH)
vibration has the same position and profile in the all three spectra (~3380 cm−1 ), the
network ultimately has the same structure irrespective of the type and number of alcohol
groups involved.
Gels 2022, 8, 410 16 of 21

3. Conclusions
Cellulose-based hydrogels were prepared from three allomorphic forms of cellulose (CI,
CII and CIII) starting from a gel stage, within their dissolution process in NaOH-water system,
followed by the chemical cross-linking with ECH. Several correlations have been established
by means of the swelling behavior, XRD, SEM, ATR-FTIR, and rheological measurements.
SEM investigations evidenced 3D networks with dissimilar morphologies and homo-
geneities, depending on each cellulose allomorph, as well as a close correlation between
the type of cellulosic allomorph, swelling degree, and morphology of the porous structure.
The method of hydrogel synthesis proceeds with a major impairment of the crystalline
organization, even if the dissolution process is partial, being limited only to the swelling
phase. All hydrogels prepared regardless of the starting allomorph led to essentially the
same supramolecular organization, specific to mostly amorphous cellulose.
The gel stage in the synthesis of hydrogels from cellulose allomorphs plays a key role
in obtaining hydrogels with different performances. By swelling in NaOH solutions, at a
low temperature, cellulose allomorphs led to gels with different strengths and rheological
characteristics. By mixing with the cross-linking agent in identical shear regimes, the
cellulose-NaOH gels were fractured into fragments of sizes correlated with the strength of
the gels. The result was a hydrogel with a high cross-linking density, if the gel fragments
were small in size and with a lower cross-linking density, in the case of resistant gels that
fractured into large fragments.
It has been shown that in order to prepare hydrogels with higher characteristics than
those obtained from CI, by the method used in this study, a previous conversion of CI to
the CII allomorph is necessary. By swelling in NaOH-water, at low temperatures, CII leads
to denser gels, which are more difficult to fragment and, implicitly, to less cross-linked
hydrogels (H-CII), which have superior swelling capacity. In the case of the CIII allomorph,
the obtained hydrogels (H-CIII) demonstrated inferior swelling characteristics to those of
native cellulose (H-CI) but superior rheological and resistance properties.
In conclusion, some of the most important features of these hydrogels (controlled
porosity and swelling degree) can be tailored by using a certain type of cellulose allomorph
(C I, CII or CIII), in order to fulfill a certain set of characteristics requested by a given
application (wound dressing or tissue engineering).

4. Materials and Methods

4.1. Materials
Cellulose I (CI), the microcrystalline cellulose, was purchased by Sigma-Aldrich (Saint
Louis, MO, US) under the trade name of Avicel PH-101. The cross-linking agent, epichloro-
hydrin (ECH), was purchased from Merck (Hohenbrunn, Germany) and was used without
further purification. Sodium hydroxide (NaOH) in pellets, with a purity ≥ 97%, was
supplied by Merck (Hohenbrunn, Germany).

4.2. Preparation of Cellulose Allomorphs

Cellulose II (CII), mercerized cellulose, was prepared from cellulose I by soaking in
a 17.5% NaOH solution for 24 h at 20 ◦ C, followed by a rigorous rinsing with distilled
water and drying in vacuum. Cellulose III (CIII) was prepared by soaking cellulose I in
organic amine (100% ethylenediamine) for 24 h, at room temperature. The cellulose amine
complex was washed with anhydrous methanol, and finally cellulose III samples were
dried in vacuum.

4.3. Preparation of Cellulose Hydrogels

Hydrogels based on cellulose allomorphs were prepared by a patented procedure [62].
One g of cellulose was suspended in 26.9 mL of water, 2.5 g of NaOH was then added, and the
mixture was shaken to dissolve the NaOH at room temperature, resulting in a suspension of
the cellulose in an 8.5% NaOH solution. The suspension was cooled to −30 ◦ C, up to a frozen
solid, and held at that temperature 24 h. The frozen solid was then allowed to thaw at room
Gels 2022, 8, 410 17 of 21

temperature and was transformed into a gel-like mass, and the epichlorohydrin was added
under controlled stirring, for 10 min. The obtained composition was cross-linked for 6 h
at 85 ◦ C. The obtained hydrogels were washed several times with warm water in order to
remove the excess of salts and any traces of the cross-linking agent from their structures.
The obtained hydrogels were dried in a freeze-dryer and were coded like H-CI for the
one obtained from cellulose I and, respectively, H-CII and H-CIII for those obtained from
cellulose II and cellulose III.

4.4. Degrees of Polymerization

Degrees of polymerization of cellulose (DP) were measured by the viscosity method
in 0.5 mol Cuen [63].

4.5. Swelling Behavior

The swelling studies of the hydrogels were performed by direct immersion in distilled
water, at 37 ◦ C. The hydrogels were periodically removed from the solution, gently wiped
with a soft tissue in order to remove the excess surface solution, weighed, and then placed
back into the vessel. The swelling degree (Qmax , %) of the hydrogels in distilled water was
determined gravimetrically, using the following relation:

mt − m0
Qmax (%) = × 100 (1)
m0

where mt —the weight of the swelled hydrogel at time t; and m0 —the weight of dry hydrogel.
The equilibrium swelling degree (Qeq , %) was determined for the never-dried hydro-
gels and was calculated by using the following equation:

m∞ − m0
Qeq (%) = × 100 (2)
m0

where: m∞ —the weight of the swelled hydrogel at equilibrium; and m0 —the weight of
dry hydrogel.
To determine the kinetics of solvent diffusion into the matrices (swelling), the following
equation was used [41]:
Wt
= ksw × tnsw (3)
Weq
where Wt —the amount of water absorbed by the hydrogel at time t; Weq —the amount of
water absorbed by the hydrogel at equilibrium; ksw —the swelling constant incorporating
characteristics of the macromolecular network system (min−n ); and nsw —the swelling
diffusional exponent, which is indicative of the transport mechanism.
The constants n and k were calculated from the slopes and intercepts of the plots of ln
(Wt /Weq ) vs. lnt. Equation (3) was applied in the early swelling stages (swelling degree
less than 60%), and the linearity was observed.

4.6. X-ray Diffraction Analysis (XRD)

XRD investigations were performed by using a Bruker-AXS D8 ADVANCE (Bruker
AXS GmbH, Karlsruhe, Germany) apparatus, having a transmission type goniometer
based on Ni-filtered, Cu-Kα radiation at 40 kV. The goniometer was scanned stepwise
every 10◦ from 10◦ to 40◦ within the 2θ range. The resulting diffraction patterns exhibited
peaks that were deconvoluted from a background scattering by using Lorenzian functions,
while the diffraction pattern of an artificially amorphized sample was approximated by
a Gaussian functions curve fitting analysis [17,23]. The data were obtained using XRD
Commander software (Bruker AXS GmbH, Karlsruhe, Germany) and were processed with
Gels 2022, 8, 410 18 of 21

DIFFRAC.EVA V1.1 (Bruker AXS GmbH, Karlsruhe, Germany). The crystallinity index
(CrI) was calculated by using the following equation [23]:

SC
CrI (%) = × 100 (4)
SC + SA

where SC and SA are the surfaces of the crystalline and amorphous areas, respectively.
The crystallite sizes were measured from their respective XRD patterns by using the
Scherrer equation [23]:
k×λ
Dhkl = (5)
β× cos θ
where Dhkl is the size of crystallite (nm), k is the Scherrer constant (0.94), λ is the X-ray
wavelength (0.15418 nm), β is the full-width at half-maximum of the reflection hkl, and θ is
the half of the 2θ value for the corresponding Bragg peak.

4.7. Scanning Electron Microscopy (SEM)

A thorough analysis of the internal morphology of the hydrogels was performed by
using a scanning electron microscope Quanta 200 (FEI Company, Hillsboro, OR, USA)
operating at 20 kV with secondary electrons, in low vacuum mode. SEM investigations
were focused on the morphological properties of the hydrogels, including homogeneity,
sample shape, surface changes, and porosity. The hydrogels samples were studied at
various magnifications, with each magnification being mentioned additionally to the
corresponding SEM micrograph.

4.8. Rheological Characterization

A stress-controlled Rheometer Physica MCR-301 (Anton Paar, Graz, Austria) has
been used to investigate the rheological properties of cellulose allomorphs gels and their
corresponding hydrogels. Specific oscillatory measurements were performed in dynamic
mode using parallel plate geometry with 25 mm diameter and a 3 mm gap between the
plates. Each swollen sample was placed on the lower plate, the upper plate was lowered
to the desired gap height and the sample diameter was cut to fit the used plate geometry,
removing the excess sample. In order to prevent the water evaporation, the rheological
measurements were conducted at a constant temperature (25 ± 0.1 ◦ C), and a special
system for controlling temperature and evaporation was used. Oscillatory amplitude
sweeps tests (strain between 0.001% and 100% at 10 rad/s) were carried out to determine
the linear viscoelastic region (LVR) and to characterize the gels’ microstructure and the
fracture process of the gels. The viscoelastic properties of the cellulose-based hydrogels
were investigated by frequency sweep tests in the frequency range from 0.05 to 500 s−1 ,
with a constant strain of 1%.

4.9. Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) Spectroscopy

A Vertex 70 spectrometer (Bruker, Hamburg, Germany) was used for the Fourier trans-
form infrared (FTIR) spectra recording in the attenuated total reflection (ATR) configura-
tion. All spectra were collected at 128 scans, at a 2 cm−1 resolution, in the mid IR range
(4000–600 cm−1 ). The OPUS 6.5 software was used for the FTIR data processing.

Author Contributions: Conceptualization, D.E.C. and F.C.; investigation—SEM, D.R.; investigation

—rheology, R.N.D.-N.; investigation—XRD, D.T. and D.E.C.; writing—original draft preparation,
D.E.C. and F.C.; writing—review and editing, D.E.C. and F.C.; and supervision, D.E.C. and F.C. All
authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Gels 2022, 8, 410 19 of 21

Data Availability Statement: Not applicable.

Acknowledgments: Mihaela Iuliana Avadanei, affiliated to “Petru Poni” Institute of Macromolecular
Chemistry, Iasi, Romania is acknowledged for ATR-FTIR spectroscopy investigations.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Ho, T.-C.; Chang, C.-C.; Chan, H.-P.; Chung, T.-W.; Shu, C.-W.; Chuang, K.-P.; Duh, T.-H.; Yang, M.-H.; Tyan, Y.-C. Hydrogels:
Properties and Applications in Biomedicine. Molecules 2022, 27, 2902. [CrossRef]
2. Zainal, S.H.; Mohd, N.H.; Suhaili, N.; Anuar, F.H.; Lazim, A.M.; Othaman, R. Preparation of cellulose-based hydrogel: A review.
J. Mater. Res. Technol. 2021, 10, 935–952. [CrossRef]
3. Gebeyehu, E.K.; Sui, X.; Adamu, B.F.; Beyene, K.A.; Tadesse, M.G. Cellulosic-based conductive hydrogels for electro-active tissues:
A review summary. Gels 2022, 8, 140. [CrossRef]
4. Rizwan, M.; Yahya, R.; Hassan, A.; Yar, M.; Azzahari, A.D.; Selvanathan, V.; Sonsudin, F.; Abouloula, C.N. pH sensitive hydrogels in
drug delivery: Brief history, properties, swelling, and release mechanism, material selection and applications. Polymers 2017, 9, 137.
[CrossRef] [PubMed]
5. Zhu, T.; Mao, J.; Cheng, Y.; Liu, H.; Lv, L.; Ge, M.; Li, S.; Huang, J.; Chen, Z.; Li, H.; et al. Recent progress of polysaccharide-based
hydrogel interfaces for wound healing and tissue engineering. Adv. Mater. Interfaces 2019, 6, 1900761. [CrossRef]
6. Mantha, S.; Pillai, S.; Khayambashi, P.; Upadhyay, A.; Zhang, Y.; Tao, O.; Pham, H.M.; Tran, S.D. Smart hydrogels in tissue
engineering and regenerative medicine. Materials 2019, 12, 3323. [CrossRef]
7. Kabir, S.M.F.; Sikdar, P.P.; Haque, B.; Bhuiyan, M.A.R.; Ali, A.; Islam, M.N. Cellulose-based hydrogel materials: Chemistry,
properties and their prospective applications. Prog. Biomater. 2018, 7, 153–174. [CrossRef]
8. Loo, S.L.; Vásquez, L.; Athanassiou, A.; Fragouli, D. Polymeric Hydrogels—A promising platform in enhancing water security
for a sustainable future. Adv. Mater. Interfaces 2021, 8, 2100580. [CrossRef]
9. Ciolacu, D.E.; Nicu, R.; Ciolacu, F. Cellulose-based hydrogels as sustained drug-delivery systems. Materials 2020, 13, 5270.
[CrossRef]
10. Hoare, T.R.; Kohane, D.S. Hydrogels in drug delivery: Progress and challenges. Polymer 2008, 49, 1993–2007. [CrossRef]
11. Kwiecień, I.; Kwiecień, M. Application of polysaccharide-based hydrogels as probiotic delivery systems. Gels 2018, 4, 47.
[CrossRef]
12. Rusu, D.; Ciolacu, D.; Simionescu, B.C. Cellulose-based hydrogels in tissue engineering applications. Cellul. Chem. Technol. 2019,
53, 907–923. [CrossRef]
13. Gomez-Florit, M.; Pardo, A.; Domingues, R.M.A.; Graça, A.L.; Babo, P.S.; Reis, R.L.; Gomes, M.E. Natural-based hydrogels for
tissue engineering applications. Molecules 2020, 25, 5858. [CrossRef] [PubMed]
14. Ciolacu, D.E.; Nicu, R.; Ciolacu, F. Natural polymers in heart valve tissue engineering: Strategies, advances and challenges.
Biomedicines 2022, 10, 1095. [CrossRef] [PubMed]
15. Kundu, R.; Mahada, P.; Chhirang, B.; Das, B. Cellulose hydrogels: Green and sustainable soft biomaterials. Curr. Res. Green Sustain.
Chem. 2022, 5, 100252. [CrossRef]
16. Onofrei, M.D.; Filimon, A. Cellulose-based hydrogels: Designing concepts, properties, and perspectives for biomedical and
environmental applications. In Polymer Science: Research Advances, Practical Applications and Educational Aspects; Méndez-Vilas, A.,
Martín, A.S., Eds.; Formatex Research Center: Badajoz, Spain, 2016; pp. 108–120.
17. Ciolacu, D.; Pitol-Filho, L.; Ciolacu, F. Studies concerning the accessibility of different allomorphic forms of cellulose. Cellulose
2012, 19, 55–68. [CrossRef]
18. Wohlert, M.; Benselfelt, T.; Wågberg, L.; Furó, I.; Berglund, L.A.; Wohlert, J. Cellulose and the role of hydrogen bonds: Not in
charge of everything. Cellulose 2022, 29, 1–23. [CrossRef]
19. Gautam, S.P.; Bundela, P.S.; Pandey, A.K.; Jamaluddin; Awasthi, M.K.; Sarsaiya, S. A review on systematic study of cellulose.
J. Appl. Nat. Sci. 2010, 2, 330–343. [CrossRef]
20. Chiriac, A.I.; Pastor, F.I.J.; Popa, V.I.; Aflori, M.; Ciolacu, D. The influence of supramolecular structure of cellulose allomorphs on
the interactions with cellulose-binding domain, CBD3b from Paenibacillus barcinonensis. Cellulose 2014, 21, 203–219. [CrossRef]
21. Mukarakate, C.; Mittal, A.; Ciesielski, P.N.; Budhi, S.; Thompson, L.; Iisa, K.; Nimlos, M.R.; Donohoe, B.S. Influence of crystal
allomorph and crystallinity on the products and behavior of cellulose during fast pyrolysis. ACS Sustain. Chem. Eng. 2016, 4,
4662–4674. [CrossRef]
22. Ciolacu, D.; Ciolacu, F.; Dumitriu, R.; Vasile, C.; Popa, V.I. Kinetics aspects in the enzymatic hydrolysis of cellulose allomorphs.
Cellulose Chem. Technol. 2007, 41, 37–42.
23. Ciolacu, D.; Gorgieva, S.; Tampu, D.; Kokol, V. Enzymatic hydrolysis of different allomorphic forms of microcrystalline cellulose.
Cellulose 2011, 18, 1527–1541. [CrossRef]
24. Wu, Q.; Xu, J.; Zhu, S.; Kuang, Y.; Wang, B.; Gao, W. Crystalline stability of cellulose III nanocrystals in the hydrothermal treatment
and NaOH solution. Carbohydr. Polym. 2020, 249, 116827. [CrossRef] [PubMed]
25. Klemm, D.; Heublein, B.; Fink, H.P.; Bohn, A. Cellulose: Fascinating biopolymer and sustainable raw material. Angew. Chem. Int.
Ed. Engl. 2005, 44, 3358–3393. [CrossRef] [PubMed]
Gels 2022, 8, 410 20 of 21

26. Seddiqi, H.; Oliaei, E.; Honarkar, H.; Jin, J.; Geonzon, L.C.; Bacabac, R.G.; Klein-Nulend, J. Cellulose and its derivatives: Towards
biomedical applications. Cellulose 2021, 28, 1893–1931. [CrossRef]
27. Korhonen, O.; Sawada, D.; Budtova, T. All-cellulose composites via short-fiber dispersion approach using NaOH–water solvent.
Cellulose 2019, 26, 4881–4893. [CrossRef]
28. Lindman, B.; Medronho, B.; Alves, L.; Costa, C.; Edlund, H.; Norgren, M. The relevance of structural features of cellulose and its
interactions to dissolution, regeneration, gelation and plasticization phenomena. Phys. Chem. Chem. Phys. 2017, 19, 23704–23718.
[CrossRef]
29. Liebert, T. Cellulose solvents—Remarkable history, bright future. In Cellulose Solvents: For Analysis, Shaping and Chemical
Modification, 1st ed.; Liebert, T.F., Heinze, T.J., Edgar, K.J., Eds.; ACS Symposium Series; American Chemical Society: Washington,
DC, USA, 2010; chapter 1; pp. 3–54.
30. Budtova, T.; Navard, P. Cellulose in NaOH–water based solvents: A review. Cellulose 2016, 23, 5–55. [CrossRef]
31. Shuai, J.; Wang, X. Novel solvent systems for cellulose dissolution. BioResources 2020, 16, 2192–2195. [CrossRef]
32. Acharya, S.; Liyanage, S.; Parajuli, P.; Rumi, S.S.; Shamshina, J.L.; Abidi, N. Utilization of cellulose to its full potential: A review
on cellulose dissolution, regeneration, and applications. Polymers 2021, 13, 4344. [CrossRef]
33. Kihlman, M.; Medronho, B.F.; Romano, A.L.; Germgård, U.; Lindman, B. Cellulose dissolution in an alkali based solvent: Influence
of additives and pretreatments. J. Braz. Chem. Soc. 2013, 24, 295–303. [CrossRef]
34. Wang, Y.; Deng, Y. The kinetics of cellulose dissolution in sodium hydroxide solution at low temperature. Biotechnol. Bioeng. 2009,
102, 1398–1405. [CrossRef] [PubMed]
35. Cai, J.; Zhang, L.; Liu, S.; Liu, Y.; Xu, X.; Chen, X.; Chu, B.; Guo, X.; Xu, J.; Cheng, H.; et al. Dynamic self-assembly induced rapid
dissolution of cellulose at low temperatures. Macromolecules 2008, 41, 9345–9351. [CrossRef]
36. Roy, C.; Budtova, T.; Navard, P.; Bedue, O. Structure of cellulose–soda solutions at low temperatures. Biomacromolecules 2001, 2,
687–693. [CrossRef]
37. Egal, M.; Budtova, T.; Navard, P. Structure of aqueous solutions of microcrystalline cellulose/sodium hydroxide below 0 ◦ C and
the limit of cellulose dissolution. Biomacromolecules 2007, 8, 2282–2287. [CrossRef]
38. Ciolacu, D.; Rudaz, C.; Vasilescu, M.; Budtova, T. Physically and chemically cross-linked cellulose cryogels: Structure, properties
and application for controlled release. Carbohydr. Polym. 2016, 151, 392–400. [CrossRef]
39. Isogai, A.; Atalla, R.H. Dissolution of Cellulose in Aqueous NaOH Solutions. Cellulose 1998, 5, 309–319. [CrossRef]
40. Peppas, N.A.; Franson, N.M. The swelling interface number as a criterion for prediction of diffusional solute release mechanisms
in swellable polymers. J. Polym. Sci. Pol. Phys. 1983, 21, 983–997. [CrossRef]
41. Ritger, P.L.; Peppas, N.A. A simple equation for description of solute release. II Fickian and anomalous release from swellable
devices. J. Control Release 1987, 5, 37–42. [CrossRef]
42. Bartil, T.; Bounekhel, M.; Cedric, C.; Jerome, R. Swelling behavior and release properties of pH-sensitive hydrogels based on
methacrylic derivatives. Acta Pharm. 2007, 57, 301–314. [CrossRef]
43. Ostrowska-Czubenko, J.; Gierszewska, M.; Pieróg, M. pH-responsive hydrogel membranes based on modified chitosan: Water
transport and kinetics of swelling. J. Polym. Res. 2015, 22, 1–12. [CrossRef]
44. Gierszewska-Drużyńska, M.; Ostrowska-Czubenko, J. Mechanism of water diffusion into noncrosslinked and ionically crosslinked
chitosan membranes. Prog. Chem. Appl. Chitin Deriv. 2012, 17, 59–66.
45. Poletto, M.; Ornaghi Júnior, H.L.; Zattera, A.J. Native Cellulose: Structure, characterization and thermal properties, materials.
Materials 2014, 7, 6105–6119. [CrossRef] [PubMed]
46. Kim, U.J.; Eom, S.H.; Wada, M. Thermal decomposition of native cel-lulose: Influence on crystallite size. Polym. Degrad. Stab.
2010, 95, 778–781. [CrossRef]
47. Mezger, T.G. The Rheology Handbook, 3rd ed.; Vincentz Network: Hanover, Germany, 2011; pp. 171–173.
48. Available online: https://wiki.anton-paar.com/en/amplitude-sweeps/ (accessed on 16 May 2022).
49. Mendoza, L.; Batchelor, W.; Tabor, R.F.; Garnier, G. Gelation mechanism of cellulose nanofibre gels: A colloids and interfacial
perspective. J. Colloid Interface Sci. 2018, 509, 39–46. [CrossRef]
50. Navarra, M.A.; Dal Bosco, C.; Moreno, J.S.; Vitucci, F.M.; Paolone, A.; Panero, S. Synthesis and characterization of cellulose-based
hydrogels to be used as gel electrolytes. Membranes 2015, 5, 810–823. [CrossRef]
51. Oh, S.Y.; Yoo, D.I.; Shin, Y.; Seo, G. FTIR analysis of cellulose treated with sodium hydroxide and carbon dioxide. Carbohydr. Res.
2005, 340, 417–428. [CrossRef]
52. Oh, S.Y.; Yoo, D.I.; Shin, Y.; Kim, H.C.; Kim, H.Y.; Chung, Y.S.; Park, W.H.; Youk, J.H. Crystalline structure analysis of cellulose
treated with sodium hydroxide and carbon dioxide by means of X-ray diffraction and FTIR spectroscopy. Carbohydr. Res. 2005,
340, 2376–2391. [CrossRef]
53. Marechal, Y.; Chanzy, H. The hydrogen bond network in Iβ cellulose as observed by infrared spectrometry. J. Molec. Struct. 2000,
523, 183–196. [CrossRef]
54. Watanabe, A.; Morita, S.; Ozaki, Y. Study on temperature-dependent changes in hydrogen bonds in cellulose Iβ by infrared
spectroscopy with perturbation-correlation moving-window two-dimensional correlation spectroscopy. Biomacromolecules 2006, 7,
3164–3170. [CrossRef]
55. Udoetok, I.A.; Dimmick, R.M.; Wilson, L.D.; Headley, J.V. Adsorption properties of cross-linked cellulose-epichlorohydrin
polymers in aqueous solution. Carbohydr. Polym. 2016, 136, 329–340. [CrossRef] [PubMed]
Gels 2022, 8, 410 21 of 21

56. Nishimura, H.; Sarko, A. Mercerization of cellulose. 6. Crystal and molecular structure of Na–cellulose IV. Macromolecules 1991,
24, 771–778. [CrossRef]
57. Takahashi, M.; Ookubo, M.; Takena, H. Solid state 13 C NMR spectra analysis of alkalicellulose. Polym. J. 1991, 23, 1009–1014.
[CrossRef]
58. Fink, H.P.; Walenta, E.; Kunze, J.; Mann, G. Wide angle X-ray and solid state C-NMR studies of cellulose alkalisation. In Cellulose
and cellulose derivatives: Physico-chemical aspects and industrial applications; Kennedy, J.F., Phillips, G.O., Williams, P.A., Eds.;
Woodhead Publishing: Cambridge, UK, 1995; Chapter 66; pp. 523–528.
59. Isogai, A. NMR analysis of cellulose dissolved in aqueous NaOH solutions. Cellulose 1997, 4, 99–107. [CrossRef]
60. Lee, C.M.; Kubicki, J.D.; Fan, B.; Zhong, L.; Jarvis, M.C.; Kim, S.H. Hydrogen-bonding network and OH stretch vibration of
cellulose: Comparison of computational modeling with polarized IR and SFG spectra. J. Phys. Chem. B 2015, 119, 15138–15149.
[CrossRef]
61. Socrates, G. Infrared and Raman Characteristic Group Frequencies: Tables and Charts, 3rd ed.; Wiley: Chichester, UK, 2004.
62. Ciolacu, D.E. Process for producing an absorbent material based on cellulose. Patent RO122780; State Office for Inventions and
Trademarks (OSIM), Romania, 29 January 2010.
63. Tappi T230 om-19, Viscosity of Pulp the Partent Owner Is State Office for Inventions and Trademarks (OSIM), Romania. (Capillary
Viscometer Method); TAPPI Press: Atlanta, GA, USA, 2019.