Reactive & Functional Polymers 73 (2013) 923–928

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Reactive & Functional Polymers

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

Physically crosslinked hydrogels from polysaccharides prepared

by freeze–thaw technique
Hongbin Zhang ⇑, Fei Zhang, Juan Wu
Advanced Rheology Institute, Department of Polymer Science and Engineering, School of Chemistry and Chemical Technology, Shanghai Jiao Tong University, Shanghai 200240, China

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

Article history: Natural polysaccharides are abundant, inexpensive, renewable, modifiable and have biodegradable and
Available online 5 January 2013 biocompatible characteristics. Polysaccharides offer a very promising source for materials of tomorrow.
This review addresses recent progress in polysaccharide-based cryogels, one kind of novel physical
Keywords: hydrogels prepared by freeze–thaw technique under mild conditions and in the absence of organic sol-
Cryogels vents and toxically crosslinking agents. Polysaccharides used to fabricate physical cryogels here are, hya-
Polysaccharides luronan, carboxymethylated curdlan, carboxymethylated cellulose, xanthan, b-glucan, locust bean gum,
Gelation
starch (amylose, amylopectin and their mixtures), maltodextrins and agarose. Composite cryogels of
Natural polymers
Biomaterials
based on polysaccharides and polyvinyl alcohol are also introduced. Various physically crosslinked cryo-
gels from polysaccharides with tunable structural, mechanical, biological properties as well as multiple
applications are considered and the investigations of the formation mechanism for these cryogels are also
addressed.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction matrix. The use of crosslinking agent or the existence of reacting

The term hydrogel describes three-dimensional network struc-
tures obtained from a class of synthetic and/or natural polymers
which can absorb and retain significant amount of water or biolog-
ical fluids [1–3]. The hydrogel structure is created by the hydro-
philic groups or domains present in a polymeric network upon
the hydration in an aqueous environment. There has been increas-
ing interest in biopolymer hydrogels due to their inherently desir-
able biocompatibility and degradability, environmentally friendly
characteristics and bioactivities. Hydrogels have wide potential
applications in the fields of food, biomaterials, agriculture, water
purification, etc. Recently, intense effects are devoted to develop-
ing novel hydrogels for applications as biomaterials for drug deliv-
ery [4,5], tissue engineering [6,7] sensors [8], purification [9,10],
wound dressing [11,12] and catalyst [13].
On basis of the manners of crosslinking among the macromole-
cules in hydrogels, two main categories can be classified by
whether the crosslinking is chemically or physically based
[14,15]. Chemical crosslinking is assuredly a highly versatile
method to create hydrogels with satisfying and tailored mechani-
cal performances [16]. However, the crosslinking agents used are
often toxic compounds, which have to be extracted from the gels
before used. Moreover, crosslinking agents can give unwanted
reactions with the bioactive substances present in the hydrogel
⇑ Corresponding author.
E-mail address: hbzhang@sjtu.edu.cn (H. Zhang).
byproducts in final hydrogels cannot be totally avoided in the
process of synthesizing hydrogels by chemical reaction. These will
impair the biocompatibility and endow the hydrogels with risk in
both short and long-term applications, especially in biomedical
aspects [17].
Such adverse effects are avoided with the use of physically
crosslinked gels. Physical hydrogels, especially some based on nat-
ural biopolymers are good alternatives and are thought to be
promising materials with hugely potential applications in biomed-
ical field because the gel formation can be often carried out under
mild conditions and in the absence of organic solvents and toxi-
cally crosslinking agents [18]. One of these hydrogels with consid-
erable potential of biomaterials is physical cryogel prepared by
freeze–thaw technique, especially the gel based on polysaccha-
rides, due to their well documented biocompatibility, low or
non-toxicity and degradability under physiological conditions
either enzymatically or chemically [19–21]. This paper reviewed
the recent developments on polysaccharide-based novel physical
cryogels with emphasis on their fabrication, property and gelation
mechanism.
2. General introduction to polymeric cryogels
The cryogels result from cryogelation or cryostructuring that a
specific type of gelation that occurs upon cryogenic treatment of
the initial solution or dispersions potentially capable of forming a
gel [20]. The polymeric cryogels are macroporous heterophase gels

1381-5148/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.reactfunctpolym.2012.12.014
924 H. Zhang et al. / Reactive & Functional Polymers 73 (2013) 923–928
Fig. 1. The main process of formation of the polymeric cryogel: 1, macromolecules in a solution; 2, solvent; 3, low-molecular solutes; 4, polycrystals of frozen solvent; 5,
unfrozen liquid microphase; 6, polymeric framework of a cryogel; 7, macropores; 8, solvent [22].
formed as a result of the freezing, storage in the frozen state, and
subsequent thawing of the initial solutions or dispersions in which
preconditions for structure formation and transition into a gel are
already present or are specially created [22]. An obligatory condi-
tion for the formation of heterogeneous gels is then the crystalliza-
tion (freezing) of the main bulk of the solvent. After thawing out,
cryogels, or cryostructurates are formed [23]. Forced alignment
of polymeric chains as the polymer concentration is increased by
conversion of water to ice may provide a mechanism for the forma-
tion of side-by-side associations, which then remain intact on
thawing, as the junction zones of the gel. The formation of cryogels
is schematically presented in Fig. 1.
The scope of applications of the cryotropic gelation techniques
and the gel materials is fairly broad. The unique porous structure
of polymeric cryogels proves to be useful in many cases. A large
number of publications [20,23–29] have been devoted to the use
of various cryogels as materials for biomedical and biotechnologi-
cal purposes.
3. Physical cryogels based on various polysaccharides
There has been an increased interest in physical gels due to the
relative ease of production and the advantage of not using chemi-
cally crosslinking agents [17]. Physical cryogels based on polysac-
charides, which are produced by freeze–thaw of polysaccharide
solutions, inherently have additional advantages of being biocom-
patible, biodegradable and eco-friendly. Therefore, study on the
physical cryogels based on polysaccharides currently is an area
receiving considerable attention both theoretical and applied. By
varying the kinds of polysaccharides, the nature of soluble addi-
tives, and also the regime in the cryogenic treatment (temperature
and duration of freezing, rate of thawing, the number of refreezing
cycles, etc.), it is possible to regulate and modulate the properties
of the final gels and their macro- and micro-structures. Actually
quite a lot of polysaccharides such as hyaluronan (HA), carboxyme-
thylated curdlan (CMc), carboxymethylated cellulose (CMC), xan-
than, b-glucan, locust bean gum (LBG), starch (amylose,
amylopectin and their mixtures), maltodextrins (MDs) and agarose
are capable of forming cryotropic gels. Generally, the degree of
gelation or the stability and mechanical performance of the gel
formed depends on pH, temperature for freezing, time of freezing
period and cycles of freeze–thaw, and the stability and mechanical
properties of cryogels are usually increasing with increasing the
freezing time and freeze–thaw cycles.
3.1. Various polysaccharide cryogles
HA is one of the most important and ubiquitous glycosamino-
glycan in vertebrate bodies with unbranched molecular chain com-
posed of a repeating disaccharide unit of D-glucuronic acid (GlcUA)
and N-acetyl-D-glucosamine (GlcNAc) [30,31]. Although it is
known that HA is a kind of non-gelling polysaccharide, cryotropic
gels and viscoelastic putty gels of unmodified HA are found to be
able to form under certain conditions. The viscoelastic putty gel
can be obtained by adjusting the pH of the concentrated HA solu-
tion to 2.5 at physiological ionic strength [32]. But such a putty gel
is unstable, and it will convert to the solution state again after
adjusting the pH below 2.0 or above 3.0. Therefore, this severe lim-
itation of putty gel prevents its application. The cryotropic gel of
HA, produced by once or repeating freeze–thaw, was firstly re-
ported by Okamoto et al. [33]. This kind of gel overcame the draw-
backs of putty gel due to its stronger stability under different pH or
temperature conditions. The main procedures of the preparation of
HA cryogels include acidifying HA solution and then freeze–thaw
of the acid HA solution at proper subzero temperature by once or
repeating freeze–thaw. Fig. 2 showed the appearance of a kind of
HA formless cryogel [34,35]. Experimental results indicated that
this HA weak gel showing a thermoreversible property was con-
structed by entangled bundle-like structures that could be melted
at elevated temperature above 70 °C [34,35].
Curdlan is a naturally occurring linear polysaccharide entirely
composed of 1,3-b-D-glucosidic linkages [36]. This kind of glucan
shows strong bioactivities and is capable of forming physical
hydrogels by various methods [37–39]. CMc is the carboxymethyl-
ation derivative of curdlan with good water solubility bioactivity as
well [40,41]. Similar to HA, CMc aqueous solutions can form phys-
ically crosslinked gels at low pH values by freezing (Fig. 2). The pH
has a significant influence on the gelation of CMc. 1% CMc solutions
at pH below 2.40 can form gels whereas solutions at pH of 2.94
cannot form gels. The densification and subsequent stiffness of
the hydrogels become strong and the equilibrium swelling ratios
of CMc gels decrease with increasing the number of freeze–thaw
cycles or lowering the pH values [42]. The formed CMc cryogels
can persist even when the heating temperature keeps at 70 °C for
20 h. In addition, interestingly, it is also found that CMC at a low
concentration (1%) with the same feature of having carboxylic
groups can also form hydrogels at low pH by freezing [42].
Xanthan gum is an anionic polysaccharide produced commer-
cially by bacterial fermentation. It is composed of the sugars glu-
cose, mannose, and glucuronic acid. The backbone is similar to
cellulose, with added side chains of trisaccharides. Xanthan cryogel
can be formed by directly freeze–thaw treatment, the process of
which is more convenient than that of HA or CMc cryogel in which
the acidifying solution is indispensible [43]. The mechanical prop-
erties of xanthan cryogel are determined by many process param-
eters such as freezing time, freeze–thaw cycles and freezing
temperature, similar to most of cryogels. However, the concentra-
tion-dependence of gel moduli for the xanthan cryogels is unusual,
with storage modulus G0 reaching a maximum value but then
decreasing slightly at higher concentrations as shown in Fig. 3.
Addition of sugars (sucrose, glucose, fructose, maltose) at concen-
trations up to 10 wt% has no effect on the strength of the cryogels,
but higher concentrations cause a progressive reduction in moduli,
 H. Zhang et al. / Reactive & Functional Polymers 73 (2013) 923–928
Fig. 2. Typical images of cryogels of HA (left) [35] and CMc (right) [42].
Fig. 3. Variation of storage modulus G0 with polymer concentration at 5 °C for
unfrozen xanthan solutions and for xanthan cryogels formed by a single cycle of
freezing to 20 °C [43].
with no cryogelation at sugar concentrations above 20 wt%. The
presence of Ca2+ also impairs the gel strength and can abolish
the cryogelation at the low levels of Ca2+ (4 mM).
The solutions or dispersions of many neutral polysaccharides
can also be induced to formation of physical gels by freeze–thaw
treatment. The first gelatinous or fibrous precipitate of barley b-
glucans was produced by Morgan and Ofman [44] via a freeze–
thaw cycling process. In another study, Lazaridou and Biladeris
[45] tried to reveal structure and molecular size effects on b-glucan
gelation phenomena during a repeated freeze–thaw process at low
initial polysaccharide concentrations. Their findings reveal that the
strength of the cereal b-glucan cryogels increases with decreasing
polysaccharide molecular size and with increasing initial solution
concentration, number of freeze–thaw cycles, and trisaccharide
segments in the polymeric chains [45]. The effects of various poly-
ols (fructose, glucose, sorbitol, sucrose, and xylose) on the cryo-
structurization of b-D-glucan isolates were also investigated by
Lazaridou et al. [46].
LBG, tara gum (TG) and guar gum (GG) are categorized as galac-
tomannan polysaccharides which consist of a 1,4-b-D-mannose
backbone and 1,6-a-D-galactose side chains. It is reported that
LBG hydrogels can be prepared by freeze–thaw, as shown in the re-
sults of frequency sweep shown in Fig. 4a and b [47,48]. However,
the gelation is not observed for TG and GG (Fig. 4c and d), indicat-
ing the formation of cryogel is closely related to detailed molecular
925
conformation and structures [48]. The gelation of LBG is acceler-
ated when the sol is annealed at a high temperature, suggesting
that molecular conformation in the sol state also affects the forma-
tion of cryogel [47]. The effects of various sugars like sucrose, glu-
cose, fructose or sorbitol on the mechanical properties of LBG
cryogel were studied by Doyle et al. [49]. It is found that gel
strength shows an initial increase and subsequent decrease with
increasing concentration of sugar. The initial increase in gel
strength is attributed to ‘concentrated effect’ of sugar which can
absorb large amount of water and promote the association of poly-
mer chains; the subsequent decrease is tentatively ascribed to
inhibition of polymer–polymer association by binding of sugar
molecules to the polymer chains as the content of sugar is too
large.
Starch is one of the most widely used food hydrocolloids. It has
been known that aqueous pastes of various starches undergo cer-
tain physical changes as a result of freezing, frozen storage and
subsequent thawing [50]. Such a low-temperature treatment of
concentrated pastes gives rise to cryotropic gel formation, the final
products of which are in the form of sponge-like textures [51,52].
The morphology of cryogels depends on the starch properties
(amylopectin/amylose ratio), its concentration in the gelatinized
system to be frozen, conditions of cryogenic processing, the
presence of soluble admixtures, and so on [50]. For the dilute
and semi-dilute aqueous dispersion or solutions of starch, the
consequences of cryogenic treatment for these systems have been
also studied [51–53]. Some of the work focuses on the dynamics of
the formation of cryogenically induced amylopectin cryoprecipi-
tates [50] and the results show that cryoprecipitation phenomena
can be observed to be most extensive at temperatures 1–2 °C
below the melting point of the frozen system of low concentration
amylopectin aqueous solution. The effects caused by cryogenic
treatment for low concentration solutions of their artificial
mixtures with various amylopectin/amylose ratios were studied
[53]. It is concluded that promoting effects of the freeze–thaw
influence on the polymer–polymer association and the pres-
ence of some synergism in the mutual interaction of these
polysaccharides.
Maltodextrins (MDs) are the products of a partial controlled
chemical or enzymatic degradation of starches isolated from di-
verse plant sources [54,55]. Cryogenic treatment of MD aqueous
systems results in the formation of precipitates or gels, whose yield
and thermal characteristics (fusion temperature and enthalpy) de-
pend on the initial polymer concentration and conditions of freez-
ing, frozen storage, and thawing [54]. Similar to starch-based
cryogels [50,51,53], the MD cryogel is thermoreversible and the
main features of MD cryostructuration turn out to be similar to
the cryogels like amylopectin [50] and LBG [48,56].
926 H. Zhang et al. / Reactive & Functional Polymers 73 (2013) 923–928

Fig. 4. The frequency sweeps of G0 (s), G00 (4) and g (h) of 3% LBG cryogel by five freeze–thaw cycles (a), and the frequency–storage modulus G0 curves of 3% LBG (b), 3% TG
(c), 5% GG (d) solutions after different freeze–thaw cycles (The respective symbol in figures (c) and (d) denotes the same one as in figure (b).) [48].

In addition, agarose, the predominant component of agar, made
up of the repeating monomeric unit of agarobiose, can also form
hydrogels by directional freezing [57].
3.2. Composite cryogels based on polysaccharides and polyvinyl
alcohol (PVA)
Many polysaccharides have been added to the polyvinyl alcohol
(PVA) aqueous solution to form composite cryogels which combine
the unique properties of both materials. A series of physically
crosslinked complex hydrogels of PVA and sodium carboxymethyl-
cellulose (CMC) have been prepared via physical mixing and a
freeze–thaw technique [58]. In this composite hydrogel, PVA crys-
talline regions serve as physical crosslinks and the interactions be-
tween CMC and PVA result in intramolecular entanglements. A
network structure model of the composite cryogel is proposed in
Fig. 5. It is also reported that composite cryogels containing PVA
and different amounts of dextran or chitosan could be prepared
using the freeze–thaw method [59]. It is suggested that the pres-
ence of dextran favors the crystallization process of PVA while
chitosan seems to perturb the formation of PVA crystallites leading
to a material with a less regular structure. Sodium alginate has also
been introduced into PVA solution to fabricate composite cryogels
[60,61]. In addition, some composite cryogels based on polysaccha-
rides and PVA, for example, PVA/water-soluble chitosan hydrogels
[62] and PVA/CMC [63], have been prepared by a combination of
irradiation and freeze–thaw. It is found that the physical properties
of the hydrogels can be improved when the combinational meth-
ods were used.
4. Formation mechanisms of the physical cryogels of
polysaccharides
The nature of the intermolecular bonds stabilizing the junction
knots of the three-dimensional polymer network in polymeric
physical cryogels depends on the structures of the gel-forming
polymers. Among polymeric cryogels, the physicochemical bases
of cryogelation of PVA solutions have long been investigated
[23,64,65]. Briefly, under freeze–thaw treatment, PVA chains are
able to form ordered structures known as microcrystalline zone
[66], acting as junction knots of the network. At these knots, PVA
chains form numerous hydrogen bonds. As most of the polysaccha-
rides with capability of formation of cryogels have a larger amount
of hydroxyl and carboxyl groups in the backbone of polymer
chains, it is also suggested that the nature of intermolecular links
in junction zones of most polysaccharide cryogels is hydrogen
bonding [20]. However, the exact interaction model and forming
mechanism of these cryogels are varied.
For polysaccharides of b-glucan [44,45], starch (amylose, amy-
lopectin or their mixtures) [50–52], agarose [57], MDs [54] and
LBG [47,48,56], the microcrystallinity zones may also function as
the physical network junction knots. These polysaccharides are
highly crystalline, and the solutions for many of them can form
gels at temperatures above 0 °C, which is very similar to the behav-

Fig. 5. Network models of CMC/PVA complex gels (a) in HCl (0.1 M, pH 1.2) and (b) in phosphate buffered saline (PBS) (0.1 M, pH 7.4) [58].
 H. Zhang et al. / Reactive & Functional Polymers 73 (2013) 923–928 927

OH CH2OH
O
O OH OH

O O
O
C NHCOCH3
O O

H
H

O O
C CH2OH
O O
O HO

HO O O
OH NHCOCH3

Fig. 6. Hydrogen bonding between –COOH groups in HA cryogels [35].

iors of PVA. These cryogels are mostly thermally reversible and
their melting temperature and melting enthalpy can be detected
by DSC, polarized optical microscopy and other methods. For
example, the crystalline microstructures of agarose cryogels can
be detected by polarized optical microscopy. The color change
from blue to yellow in polarizing light micrograph of LBG cryogels
indicates that molecular chains of LBG are fabricated in a cross-
linked mesh structure and that each thread aligns along the fiber
axis direction [47]. These phenomena are reflection of crystalline
characteristics which have been shown in PVA cryogels. Therefore
the mechanism of these polysaccharides is thought to be similar to
that of PVA, and the microcrystalline zones stabilized by hydrogen
bonds are responsible for the formation of these gels.
Although the xanthan cryogel shared some features of LBG
cryogel, Giannouli and Morris [43] pointed out that there are some
significant differences between the two systems. Firstly, the solu-
tions of LBG at certain concentration can form a network structure
gradually at temperature above 0 °C, suggesting that freezing sim-
ply accelerates the process. Solutions of xanthan, by contrast re-
main stable on prolonged storage at positive temperature.
Another difference is that LBG exist in solution as disordered coils,
whereas xanthan, except at high temperature and low ionic
strength, adopts an ordered stiff, rod-like conformation [67,68].
These ordered conformations make xanthan solutions form a cho-
lesteric mesophase at certain concentration only under which the
solution is isotropic [69]. Thereby it is proposed that forced align-
ment of xanthan chains as the polymer concentration is increased
by freezing water to ice may provide a mechanism for the forma-
tion of side-by-side associations, which then remain intact on
thawing, as the junction zones of the cryogel.
The exact forming mechanism of HA cryogel has not been fully
understood [34,35]. The complexity of forming mechanism of HA
cryogel might be mainly derived from its chemical structure, which
includes not only massive –OH groups as in PVA and galactoman-
nan, but also –COO and –NHCH3 groups along with possible
hydrophobic regions. Okamoto et al. tentatively suggested that
the formation of HA cryogel was a cooperative phenomenon result-
ing from the interaction of hydrophobic and hydrogen bonds be-
tween HA molecules. Collins and Birkinshaw presumed that
either a crystallization process or at least a process analogous to
crystallization occurred during the formation of HA cryogel [34].
However, the results of X-ray RD, DSC and polarized microscopy
showed that the cryogels had no typical characters of crystalline
structures [35], indicating that the crystallization may contribute
little to the formation of cryogel. The fact that protonation of the
polyanion of HA is indispensable to the gelation of HA. The inter-
molecular and intramolecular hydrogen bonding induced from –
COOH in HA chains may play an important role in respect to the
network formation and stabilization of HA gel, and the possible
pattern is shown in Fig. 6. The gelation of CMc and CMC [42] that
have carboxylic groups forming hydrogels at low pH by freeze–
thaw may be the same with that of HA cryogel. In fact, lowering
the pH of CMC aqueous concentrated solution (>10%) can directly
induce the gel formation. This mechanism involves replacing the
sodium in CMC with hydrogen in the acid solution to promote
hydrogen bonding, thus decreasing the solubility of CMC thereby
resulting in the formation of an elastic hydrogel [70].
5. Conclusion and outlook
As a class of fascinating materials, hydrogels hold potential
application in many fields, such as scaffolds for tissue engineering,
vehicles for drug delivery, sensors, and catalysis. This review sum-
marized the representative works on physical hydrogels from var-
ious polysaccharides in recent years. Physical hydrogels from
polysaccharides prepared by freeze–thaw have shown some im-
proved properties over traditional polysaccharide hydrogels ob-
tained by chemical crosslinking or cryogels from synthetic
polymers with being more biocompatible and biodegradable.
Polysaccharide cryogels by freeze–thaw technique undoubtedly
belong to the category of physically crosslinked gels whose three
dimensional structure is stabilized mainly by multiple interchain
hydrogen bonds in the junction zones of the polymeric network.
While xanthan cryogel can be formed by directly freeze–thaw
treatment, protonation of the anionic polysaccharides such as
HA, CMc and CMC, all of which have carboxylic groups on the back-
bone, is indispensable to the association of chains via hydrogen
bonding. Although cryostructurates in these cryogels (zones of
microcrystallinity) resembling the much more well-studied gels
of PVA were not found, the feature of hydrogen bonding in polysac-
charide cryogels were experimentally evident. For neutral polysac-
charides like b-glucan, starch (amylose, amylopectin or their
mixtures), agarose, maltodextrin and LBG, the microcrystallinity
zones may also function as the physical network junction knots.
The gelation of these polysaccharides is similar to that of PVA in
which hydrogen bonding between hydroxy groups is predominant.
928 H. Zhang et al. / Reactive & Functional Polymers 73 (2013) 923–928
Although there are many novel hydrogels being reported every
year, the lack of molecular level understanding of the gelation
mechanism is still a problem. Subtle physicochemical mechanisms
of gelation processes and the exact interaction models in the cryo-
gel have not yet been adequately studied, though the need for
these studies is obvious. Nevertheless, efforts are being made and
progresses are on the way.
In some cases, e.g., vehicles for drug delivery in comparison to
scaffolds for tissue engineering, highly mechanical performance
of hydrogels may not be the most important or necessary. How-
ever, it is still a big challenge to rationally design and fabricate a
physical hydrogel that is of satisfying mechanical behavior without
any chemical modification, simultaneously retaining its inherent
biocompatibilities, biodegradability, environmentally friendly
characteristics and bioactivities. As a class of promising biomateri-
als, the application of physical hydrogel from polysaccharides
would be extended especially in the fields where highly mechani-
cal performance is not requested. Due to the important role of
hydrogel in supramolecular chemistry, condensed matter physics
and material science, more and more researchers will pursue in
this field. Therefore, the field of physical hydrogels, especially poly-
saccharide cryogels, will experience intense investigation and ex-
pand rapidly.
Acknowledgements
The authors are thankful for the financial support for this work
from the National Natural Science Foundation of China (Grant No.
21074071) and the Shanghai Leading Academic Discipline Project
(No. B202).
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