Freeze-Thaw Hydrogel Fabrication Method Basic Prin

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Freeze–thaw hydrogel fabrication method: basic principles, synthesis

parameters, properties, and biomedical applications
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Page 1 of 32 AUTHOR SUBMITTED MANUSCRIPT - MRX-126599.R2
IOP Publishing Materials Research Express

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Mater. Res. Express (2022) XXXXXX https://doi.org/XXXX/XXXX
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Freeze–thaw hydrogel fabrication method: basic
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10 principles, synthesis parameters, properties, and
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biomedical applications
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16 William Xaveriano Waresindo1,2, Halida Rahmi Luthfianti1,2, Aan Priyanto1,2, Dian
17 Ahmad Hapidin2, Dhewa Edikresnha2,4, Akfiny Hasdi Aimon2, Tri Suciati3 and
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Khairurrijal Khairurrijal2,4,5,*
20 1 Doctoral
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Program of Physics, Faculty of Mathematics and Natural Sciences, Institut Teknologi
21 Bandung, Jalan Ganesha 10, Bandung 40132, Indonesia
22 2 Department of Physics, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung,
23 Jalan Ganesha 10, Bandung 40132, Indonesia
24 3
Department of Pharmaceutics, School of Pharmacy, Institut Teknologi Bandung, Jalan Ganesha 10,
25 Bandung 40132, Indonesia
26 4 University Center of Excellence – Nutraceutical, Bioscience and Biotechnology Research Center,
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Institut Teknologi Bandung, Jalan Ganesha 10, Bandung 40132, Indonesia
5 Department of Physics, Faculty of Science, Sumatera Institute of Technology, Jl. Terusan Ryacudu,
Lampung 35365, Indonesia

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31 *E-mail: krijal@itb.ac.id
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Received xxxxxx
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Accepted for publication xxxxxx
34 Published xxxxxx
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36 Abstract
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38 Hydrogel is being broadly studied due to their tremendous properties, such as swelling
39 behavior and biocompatibility. Numerous review articles have discussed hydrogel polymer
40 types, hydrogel synthesis methods, hydrogel properties, and hydrogel applications. Hydrogel
41 can be synthesized by physical and chemical cross-linking methods. One type of the physical
42 cross-linking method is freeze-thaw (F–T), which works based on the crystallization process
43
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of the precursor solution to form a physical cross-link. To date, there has been no review
44 paper which discusses the F–T technique specifically and comprehensively. Most of the
45 previous review articles that exposed the hydrogel synthesis method usually mentioned the F–
46 T process as a small part of the physical cross-linking method. This review attempts to
47 discuss the F–T hydrogel specifically and comprehensively. In more detail, this review covers
48 the basic principles of hydrogel formation in an F–T way, the parameters that influence
49 hydrogel formation, the properties of the hydrogel, and its application in the biomedical field.
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Keywords: hydrogel, freeze-thaw, process parameters, solution parameters, hydrogel properties, biomedical applications
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research on hydrogels have grown steadily due to their
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1. Introduction auspicious potential. They have been applied in many
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58 medical, engineering, and electronics industries, e.g., drug
Hydrogels are cross-linked polymer with 3D-structure
59 delivery system, wound healing, tissue engineering, brain
composed of hydrophilic groups and very flexible due to
60 tissue, wastewater treatment, batteries, polymer electrolyte,
their large water content [1–3]. In recent years, studies and
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AUTHOR SUBMITTED MANUSCRIPT - MRX-126599.R2 Page 2 of 32
Mater. Res. Express (2022) XXXXXX Waresindo et al

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3 smart supercapacitor, flexible sensors and actuators [4–12]. except covalent cross-linking [31]. However, the chemical
4 Hydrogels are flexible material, so they are easy to shape, agents are usually poisonous, so they must be neutralized
5 depending on the desired application. Hydrogels can be prior to application [32]. For biomedical applications,
6 applied in the form of gel gradients, anisotropic gels, gel chemical aspects must be considered in synthesizing
7 patterns, gel wrinkles, nanoparticle gel, and tube structures hydrogels. This chemical reaction may cause damage to the
8 [13]. The promising potential of hydrogels has attracted biocompatibility of the hydrogel, so the physical cross-
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9 many parties to develop fabrication methods to obtain linking mechanism could be a promising option.
10 maximum results. Physical crosslinking has become a popular method
11 Hydrogels can be prepared from natural and synthetic because of the simplicity of the synthesis process. Also, it
12
polymers [14]. Natural polymers that can be made into can produce hydrogels with promising properties without
13
hydrogels are as follows: alginate, gelatin, chitosan, starch, chemical cross-linking agents, thus avoiding potential
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gellan gum, and cellulose [15–20]. While the types of cytotoxicity from unreacted chemical crosslinkers [33].
15
synthetic polymers that are currently widely used, for Among the physical cross-linking methods, the F–T process
16
example, polyvinyl alcohol, polyvinyl pyrrolidone, is the most broadly used recently. The advantages of the F–T
17
18 polyacrylic acid, polyaniline, and polyacrylamide, and method are that it is simple and it requires no specific
19 polyethylene glycol [21–26]. Both types of hydrogels have equipment for hydrogel synthesis [34]. This method could
20 advantages and disadvantages. For biomedical applications, produce hydrogel with highly biocompatible, high
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21 hydrogels based on natural polymers are preferred over mechanical properties, an excellent swelling ratio, and non–
22 synthetic ones because they are biocompatible, toxic [35–38].
23 biodegradable, and non-toxic to the human body [27]. Numerous review papers about hydrogels have been
24 However, synthetic polymer-based hydrogels feature better published with specific issues. Several review papers about
25 mechanical properties for industrial applications. hydrogels have been reviewed thoroughly e.g., the raw
26 Hydrogels can be classified into physical and chemical materials for making hydrogels (synthetic and natural
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methods based on the way in which the cross-links are
formed [28]. A more detailed and comprehensive comparison
of these fabrication methods is presented in table 1. The
an polymers), the properties of the hydrogels, the potential
applications of the hydrogels, and the synthesis methods of
the hydrogels [39–43] However, a review article that
30 physical cross-linking at the molecular scale involves non- comprehensively describes the hydrogel formation scheme
31 covalent interactions, e.g., freeze-thaw (F–T), stereo complex using the F–T method has yet to be found. Therefore, this
32 formation, hydrogen bonding, or chain entanglements, while review aims to explain the formation mechanism of the
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33 chemical cross-linking is often fabricated by chemical hydrogel using the F–T method, the effect of various process
34 grafting, radiation, and polymerization [29,30]. Hydrogel parameters on the properties of the developed hydrogel, and
35 fabrication techniques that use chemical cross-linking tend to its application in several biomedical fields. Moreover, the
36 produce stable hydrogels with high mechanical strength, high highlights of the potential for future development of F–T
37 superabsorbent properties, and are insoluble in all solvents hydrogel are also provided.
38
39 Table 1. Comparison of hydrogel fabrication methods
40Crosslinking Materials and Crosslinking Crosslinking Highlights and challenges Applications Ref.
41Methods Agent Temperature and Time
42Free radical -Materials: AA/gelatin. The temperature and Hydorgel had pH sensitivity and non-Fickian Water soluble drugs [44]
43polymerization -Crosslinking agent: APS, EGD. time of gel formation are drug release. However, the toxic, unreacted carrier.
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45 °C for 1 h, 50 °C for monomer residues require a cleaning time of

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2 h, 55 °C for 3 h, 60 °C almost 2 weeks.
45 for 4 h, and 65 °C for 24
46 h.
47Grafting and free -Materials: BSG/AA/MBAm. Heated in water bath at The resulting hydrogel was highly porous and pH Drug delivery. [45]
48radical -Crosslinking agent: PPS. 55 °C, increased to 70 ° sensitive. It also performed the Korsmeyer-
49polymerization C for 24 h and then Peppas drug release, followed by a non-fickian
cooled at RT. diffusion mechanism.
50Ionizing radiation -Materials: AgNPs/PVP/PVA. Iradiated using 60Co at Without a crosslinking agent, it is effective Wound dressing [46]
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-Crosslinking agent: - 25 kGy with dose level against gram-positive and gram-negative bacteria
52 of 1.58 kGy/h. and their resistance. However, its mechanical
53 properties still need to be investigated further.
54One-pot synthesis -Materials: rGO/gelatin. Heated in an oil bath at It is a green and facile method without using Tissue engineering [47]
-Crosslinking agent: - 95 °C for 24 h. chemical cross-linkers or organic solvents, but at and drug delivery.
55 high concentrations, graphene can be toxic.
56Stereocomplex -Materials: L-lactide (LA) and Mixing PDLA and The produced hydrogel featured ideal Bone regeneration [48]
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57formation DLA/PEG/gelatin. PLLA solutions to obtain interconnectivity, abundant porosity, and proper
58 -Crosslinking agent: genipin. a stereocomplex at room size, which are useful for nutrient supply, cell
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1
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3 temperature. The gelatin proliferation, and calcium deposition. However,
4 solution was added at monitoring degradation and controlling polymer
5 0°C and then frozen at - constitution so that the rate of degradation is
20°C. Lastly, the perfectly consistent with new bone formation is
6 hydrogel was immersed still challenging.
7 in the genipin solution
8 for 24 h.
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9 Hydrogen bonding -Materials: GelMA/ TA. GelMA hydrogels were Hydrogel exhibited superior mechanical Wound dressing [49]
10 -Crosslinking agent: APS, formed by adding APS properties, excellent biocompatibility, and the
TEMED. and TEMED to the best recovery results in skin and gastric wounds.
11 GelMA solution at 37 °C However, the strong hydrogen bonding
12 for several minutes. interaction between GelMA and TA can suppress
13 After gelation, the the hydrogel structure thereby limiting its water
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14 hydrogel was immersed loading capacity.
in the TA solution for a
15
predetermined time, then
16 the free TA was rinsed
17 with deionized water.
18Chain entanglement -Materials: AA/SNs/OP-10/AC The solution was The synthesized hydrogel had a unique swelling Tissue engineering [50]
19and free-radical /SDS. ultrasonicated in an ice behavior, excellent flexibility, and mechanical and wound dressing
20polymerization -Crosslinking agent: APS. water bath for 15 min. strength. However, its biocompatibility and
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Then the solution was biodegradability still need to be investigated
21 heated to 70 °C with an further.
22 oil bath for 2-6 h under
23 N2 atmosphere, and the
24 polymerization process
was carried out using
25
APS.
26Freeze-thaw
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-Materials: PVA/GO/aloevera
-Crosslinking agent: -
an
The freezing temperature The hydrogel was produced without a Burn
at −15 ℃ for 20 h and crosslinking agent, had excellent hydrophilicity, dressing
thawing at 5 ℃ for 4 h. viability up to 295%, was able to reduce bacteria
Repeated for 3 cycles. up to 99.94%, and had mechanical properties like
wound [51]
skin tissue.
30
Abbreviations: AA: acrilic acid, APS: ammonium persulfate, EGD: ethylene glycol dimethacrylate, BSG: Basil Seed Gum, MBAm: N, N-Methylene-bis-acrylamide, PPS:
31potassium per sulfate, AgNPs: silver nanoparticles, rGO: reduced graphene oxide, GelMA: gelatin methacrylate, TA: tanic acid, TEMED: tetramethylethylenediamine, SNs:
32silica nanoparticles, OP-10: octylphenol polyoxyethylene ether, AC: acryloyl chloride, SDS: sodium dodecyl sulfate, GO: graphene oxide.
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34 been published in the last few decades spread across several
35 2. Freeze-thaw method subject areas (figure 1). Recent developments in the F–T
36 The F–T method is basically a physical crosslinking
method are to produce artificial glenoid labrum-based
37 method without involving chemical covalent bonding agents.
hydrogels, extracellular matrix, wound healing materials,
38 The mechanism of physical hydrogel formation can be done
delivery of herbal medicines, flexible supercapacitors,
39 through electrostatic schemes, hydrogen bonds, or
flexible electrodes, heavy metal ion removers, and absorbent
40 hydrophobic forces between polymer chains. While this
materials [34,57–63].
41
scheme requires a polymer network that meets the following
42 2.1 Basic principle of the freeze-thaw method
conditions: (1) strong interchain interactions to form stable
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collocations in the molecular network; and (2) polymer As one of the physical cross-linking methods to fabricate
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networks must encourage access to and residence of water in hydrogels, F–T has two stages of hydrogel formation, i.e.,
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the hydrogel [52]. the freezing stage of the precursor solution under 0 ℃, and
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47 Peppas first introduced the F–T method in 1975, which the thawing stage at room temperature. The main idea is to
48 studied the super-molecular structure of PVA-based control the ice crystallization process (freezing) and the
49 hydrogels [53]. By that time, the F-T process was becoming formation of an ordered structure (thawing) so that the
50 increasingly popular. Willcox (1999) utilized the F–T hydrogels possess optimal properties [64]. Hydrogels are
51 method to investigate the microstructure of PVA hydrogels made of polymers which are selected based on their superior
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52 [54]. The crystallinity properties of the hydrogels produced properties and applications, e.g., polyvinyl alcohol with
53 by the F–T method have also been described in full [55]. biocompatible properties is very suitable for biomedical
54 Using the F–T method, hydrogels can be made from natural applications, polyaniline as a conductive polymer is a
55 polymers such as polysaccharides instead of synthetic ones potential candidate for supercapacitor applications,
56 [56]. The high interest in research on F–T hydrogels is polyacrylamide can be applied to water treatment because of
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3 its ability to thicken suspended solids, and cellulose-based eventually forms a three-dimensional matrix which makes
4 hydrogel for agricultural applications [25,65–67]. the solvent trapped in the matrix, resulting in a gel phase
5 [70].
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7 2.3 Hydrogel formation parameters
8
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9 The produced hydrogel properties are influenced by the
10 synthesis method and the original properties of the
11 precursors. According to Hassan & Peppas [71], the F–T
12 hydrogel properties depend on the molecular weight of the
13 polymer, the solution concentration, F–T temperature, time,
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14 and the number of cycles. A summary of various parameters
15 for the synthesis of F–T hydrogels is given in table 2.
16 Although most of the polymers for the F–T method rely on
17 PVA, there are several other polymeric materials such as
18 chitosan, alginate, gelatin, several types of gums, к-
19 carrageenan, and glucomannan that can be made into
20 hydrogels using the same method. Some of these polymers
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21 share the same characteristics, which are hydrophilic
22 polymers with many hydrophilic functional groups (–OH, –
23 COOH, –NH2) in the chain structure. The large amounts of
24 hydrophilic groups and the freezing process in the F–T
25 method allows the polymer chains to be bonded non-
26
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Figure 1. Publication trends (a) and subject areas (b)
an covalently through the formation of hydrogen bonds
(intramolecular, intermolecular I, and intermolecular II) [72].
Furthermore, with this physical bond, the polymer chains can
analyzed by Scopus database with the keywords "Freeze form a 3-dimensional matrix capable of trapping solvents
30 Thaw Hydrogel", last updated on 13 October 2022. between cross-linking points to produce a gel phase.
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32 2.2 Polymers cross-linking mechanism on hydrogel
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2.3.1 The molecular weight of the polymer. The sum of
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34 Polymers are materials consisting of long chains of each atomic weight of the atoms making up the molecule is
35 molecules with repeating units [68]. The intrinsic structure of known as the molecular weight (MW) of the polymer, which
36 polymers, such as bonds, molecular chain arrangement, and indicates the average length of the bulk resin polymer chain
37 molecular weight, influences their properties [69]. Bonds [73]. The MW values are expressed in terms of distribution
38 determine the stability of atoms and molecules’ bond . ranges due to their different molecular weights, even the
39 Regular and irregular structures affect the strength and polymers in a particular class [74].
40 toughness of the polymer. Molecular weight influences the The MW of a polymer is closely related to its viscosity and
41 viscoelasticity and other physical properties of the polymer. permeability. Compared to low molecular weight polymers,
42 Atoms in individual polymer molecules are connected by high molecular weight polymers have a higher permeability
43 reduction factor and greater viscosity [75]. The MW of the
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strong covalent bonds, whereas intermolecular forces link

44 polymer molecules. During the hydrogel formation process polymer has been shown to play an essential role in forming
45 using the F–T method, physical cross-linking occurs due to the gel structure [76]. Ma et al. have reported the effect of
46 the crystallization of the polymeric solution. MW variations on the PVA hydrogel properties produced by
47 The cross-linking mechanism is illustrated in figure 2a. controlling the entanglement density [77]. The increase in
48 The interaction of the hydroxyl groups in the polymer chain molecular weight led to an increase in the stable hydrogel
49 with water molecules causes hydrogen bonds to form. The cross-linked network, gel concentration, and enhanced tensile
50 freezing process of the polymer solution induces crystal strength. However, increasing the molecular weight of PVA
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growth which acts as a cross-linking point between polymer could decrease the swelling ability of the hydrogel.
52 chains. The thawing treatment relaxes the polymer chains so
53 that they can move freely again. The F–T cycle is repeated
54 continuously causing more and more cross-link points to be
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formed. The point of crosslinking of the polymer chains
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55 Figure 2. (a). Cross-linking mechanism of a hydrogel during the F–T process. (b). Hydrogel microstructure changes with
56 variations in molecular weight. (c). Hydrogel morphology of different F–T cycles periods.
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5

1
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3 As shown in figure. 2b, the variation in molecular weight cross-links in the hydrogel. This crystallization occurs during
4 affects the microstructure of PVA hydrogel [77]. Low the freezing process of the polymer solution. The frozen
5 molecular weight PVA has a shorter chain length, allowing temperature parameters, freezing time, and the repetition of
6 them to move freely in the solvent. The obtained hydrogel the F–T cycle are the factors affecting the properties of the
7 has fewer microcrystalline, cross-linking points, and produced hydrogel.
8 hydrogen bonds because it is only affected by the interchain. Kim and colleagues found that various temperatures of
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9 The chain length between the cross-links of low molecular PVA solutions during the freezing process could induce the
10 weight PVA is also greater than that of high molecular hydrogel to form a stiffness gradient [70]. Low temperatures
11 weight PVA. In high molecular weight PVA, the formation cause more cross-linking points of the polymer chains to
12
of hydrogen bonds that occur are intrachain and interchain. form, so the hydrogel becomes stiffer. Furthermore,
13
Thus, the number of cross-linking points, microcrystalline, Figueroa−Pizano et al. reported the swelling ability of
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and hydrogen bonding is more significant [77]. Besides, Xia chitosan/PVA-based hydrogels at different freezing
15
et al. asserted that the PVA molecular weight must be well- temperatures (-4, -20, and -80℃) [84]. Based on their study,
16
determined [78]. PVA with a molecular weight of too low lower freezing temperatures reduced the pore size of the
17
18 cannot form a cross-linking, while a molecular weight of too hydrogel. Thus, the swelling ability decreased due to the
19 high will produce bubbles in the solution that are difficult to increased cross-linking point between PVA chains. Freezing
20 remove. time during the F–T steps can affect the formation of
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21 freezable water coagulation.
22 2.3.2 Solution concentration. The F–T method begins with Nakano and Nakaoki investigated the coagulation size of
23 the preparation of a polymer solution. The concentration of a freezable water in PVA hydrogels during the F–T cycle
24 polymer solution is expressed as the weight ratio of the variations [85]. When the F–T cycle period is short (figure
25 polymer to the solvent. Decreasing the polymer 2c), the coagulation of freezable water, the crystallites, and
26 concentration in the solution will decrease the viscosity of the PVA domain of the amorphous material swells that
27
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29
the solution and reduce the cross-linking density of the
hydrogel, which results in a softer gel [79,80]. Previous
studies have shown the effect of variations in solution
an contain non-freezable water. The cause is the interaction
between polymer chains that yields very few cross-linking
points. The higher number of the F–T cycle period triggers
30 concentration of the polymer on hydrogel properties. Syifa et more cross-linking points in the polymer. Thus, the water
31 al. found that with decreasing PVA concentration, the molecules in the swollen PVA region and non-freezable
32 resulting hydrogel became softer but the swelling value water turn into freezable water, enlarging the water domain
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33 increased [80]. Research by Waresindo and Edikresnha that can be frozen.
34 showed the same conclusion when loading PVA hydrogel The number of F–T cycles promotes the gelation of PVA
35 with guava leaf extract and red betel extract [79,81]. Hou and solution and increases the mechanical properties of PVA
36 colleagues reported the effect of different concentrations of hydrogel. Holloway’s group reported the phase separation
37 dimethyl sulfoxide (DMSO) solution on the transparency, and crystallization of PVA hydrogels during the F–T process
38 mechanical properties, and crystallinity of PVA hydrogels [86]. Samples were frozen for 21 hours at 20 ℃ and thawed
39 prepared by the F–T method [82]. They found that the for three hours at room temperature for up to ten cycles. The
40 increase in DMSO concentration or decrease in PVA resulted compression modulus value increases as the number of F–T
41 in better transparency of the PVA hydrogel. The hydrogen cycles increases from cycle one to cycle six. After six cycles,
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bond between the DMSO molecule and the −OH group of the compression modulus value tends to be stable. Li et al.
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the PVA chain inhibits crystal growth in the two-dimensional have come to a similar conclusion when observing the effect
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direction, resulting in a smaller crystal volume. The small of the number of F–T cycles on the mechanical and
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crystal volume eases the light penetration and enhances the conductive properties of PVA/PANI hydrogel for
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transparency value. Moreover, Joshi’s group examined the supercapacitor applications [87].
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48 effect of PVA solution concentration on its rheological They found that the F–T process can produce a
49 properties during the F–T gelation process [83]. Hence, the microstructure such as spongy bone in hydrogel samples that
50 PVA hydrogel has better gel strength because increasing the has increased mechanical and electrochemical properties.
51 concentration of PVA is aligned with the density of the Their variation of F–T cycles resulted in significant
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52 cross-linked network. differences in tensile strength values. Five F–T cycles have a
53 tensile strength value of 16.3 MPa with an elongation at
54 2.3.3 Temperature, duration, and number of F–T cycles. break of 407%, while zero cycles only have a tensile strength
55 The F–T method relies on a crystallization process to form value of 5.3 MPa with an elongation at break of 250%.
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3 Table 2. The effect of hydrogel formation parameters using the F–T technique on hydrogel properties
4 Polymer Molecular Concentration Temperature, time, and Hydrogels Properties Ref.
5 Weight number of FT cycles
6 PVA 145,000 19 wt.% Freeze at −20 ℃ for 30 min, During the first cycle, some parts of the [54]
thaw at 5 ℃ for 30 min. chain crystallize, forming major crystal
7 junctions of 3 to 8 nm in size. An
Repeated for 12 cycles.
8 irregular mesh separates these crystals by
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9 an average distance of 30 nm.
10 PVA/CNC - PVA 10 wt.% Freeze at −20 ℃ for 18 h, thaw The percentage of crystallinity of the [88]
11 CNC 0, 5, 10, 20 wt. at RT for 4 h. Repeated for 5 hydrogel samples was seen to decrease at
% cycles. all loadings. There is an increase in water
12 absorption when the CNC concentration
13 is increased. The high CNC content
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14 means the hydrogel has better structural
15 integrity.
16 PVA/Hemicellulose - The 10 mL mixed Freeze at −20 ℃ for 10 h, thaw The increasing number of F–T cycles [89]
s/Chitin solution (3.75 wt.%) at RT for 1 h. Repeated for 0, 1, resulted in a stiffer structure, more stable
17
with the proportion 3, 5, 7, and 9 cycles. thermal properties, and a higher degree
18 of 1:1:1. of crystallinity. In contrast, after 3 F–T
19 cycles, a lamellar structure was formed
20 so that the swelling ratio of the hydrogel
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21 equilibrium decreased.
Starch/PVA/LRD - Variated of LDR Freeze at −20 ℃ for 10 h, thaw Starch/PVA/LRD has good [38]
22
content from 0% to at 25 ℃ for 2 h. Repeated for 3 compatibility. The melting temperature
23 12.5% of a polymer cycles. decreases, but thermal stability is
24 basis improved with an increase in the LRD
25 content. The hydrogel reswelling ratio
26 has the highest value at the LRD level of
27
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PVA/CAF PVA:195,000
g/mol
13 w/v %, (10 g
PVA, and 3 g CAF).
an
Fast freezing using liquid
nitrogen for 20 min, thaw at 4
10%.
The mechanical properties, crystallinity,
and degree of swelling were increased in
[90]
CAF:194.19 g/mol ℃ for 4 h. Repeated for 2 hydrogels by the orientation method.

30 cycles. This increase is due to the alignment of
31 the polymer chains that occurs during the
F–T process. The increasing number of
32
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cycles causes more polymer alignment to
33 occur.
34 Sericin/PVA - 2% w/v Sericin Freeze at −80 ℃, thaw at RT. The SS/PVA hydrogel showed excellent [91]
35 2% w/v PVA Repeatede for 4 cycles. swelling ability and hydrophilicity for its
36 The volume ratios of porous structure. PVA blending
sericin and PVA effectively improved sericin’s thermal
37 were 4:0, 4:1, 4:2, stability and enhanced sericin’s
38 4:4, 0:4. mechanical properties but did not affect
39 sericin and PVA’s crystallinity.
40 PU/PVA PU: 9.06 KDa 3 % w/v PU and 3 % Freeze at −20 ℃ for 16 h, thaw The PU/PVA hydrogel has a porous [92]
41 PVA:130,000 w/v PVA. PU at RT for 8 h. Repeated for 3 structure with different surface
g/mol concentration in cycles. morphology where the surface pore size
42 PU/PVA solutions and swelling ratio increase with
43
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was varied from 0% increasing in PU concentration.

44 to 50%.
45 PVA/CNC PVA: 85,000- 9 wt.% PVA and 1 Freeze at −20 ℃ for 12 h, thaw Increasing the CNC concentration [93]
46 124,000 g/mol wt.% CNC. PVA at RT for 6 h. Repeated for 3 enhances the capacity of water
weight varied from cycles. absorption and decreases the modulus of
47 20 g to 3 g. CNC compression and optical transparency.
48 weight varied from 0
49 to 7 g.
50 PAA/PVA PVA: 85,000- 10 wt.% PVA Freeze at −20 ℃ for 12 h, thaw The variation in acrylic acid [94]
51
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124,000 g/mol with variations in at RT for 6 h. Repeated for 3 concentration affects the swelling and
PAA concentration. cycles. thermal properties of the PAA/PVA
52 hydrogels. The increase in acrylic acid
53 concentration resulted in increased
54 stability, swelling, and thermal
55 transparency but decreased optical
56 transparency.
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PVA 146,000-186,000 9 % w/v Freeze at −23 ℃ for 16 h, thaw The variation in the number of F–T [95]
57
g/mol at 25 ℃ for 8 h. Repeated for 3 cycles affects the physico-chemical and
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1
2
3 cycles. viscoelastic properties of the hydrogels.
4 Increasing the number of F–T cycles
5 causes an increase in the degree of
crystallinity. Hydrogel crystallinity is
6 closely related to its ability to absorb
7 water.
8 PVA/GLE -PVA: 89,000- 10 wt.% PVA Freeze at −25 ℃ for 20 h, thaw An increase in GLE concentration causes [79]
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9 98,000 g/mol with variations in at RT for 4 h. Repeated for 6 an enhance in pore size, degree of
10 GLE composition. cycles. swelling, and weight loss.
CS/PVA -CS: 637,000 Da. - CS: 2 wt.%. Freeze at −4 ℃, −20 ℃, and The CS content, the number of F-T [84]
11 -PVA: 89,000 – - PVA: 10 wt.%. −80 ℃ for 4, 8, 12, and 20 cycles, and the freezing temperature
12 98,000 Da. - CS/PVA ratio: hours. Degas at atmospheric significantly affected the degree of
13 75/25, 50/50, and pressure for 2 h. Repeated for 4, swelling. Pore size increases with CS
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14 25/75. 5, and 6 cycles. content, but lower temperatures or longer
15 freezing times result in higher porosity
and smaller pores.
16 Alginate 80,000 – 120,000 0.5 and 1 wt.% Freeze at −25 ℃ for 24 h and The F–T treatment increased the [96]
17 Da thaw at 4 ℃ for 24 h. Repeated hydrogel storage modulus by almost 100
18 for 1, 2, 3, 4, 5 and 6 cycles. times. The properties of the resulting
19 alginate gel, such as dynamic modulus
20 and gel syneresis, were influenced by the
us
pH value, number of F–T cycles, alginate
21 concentration, and ionic strength.
22 Because of its soft structure and melting
23 behavior during storage, it has the
24 potential for new applications in
25 biomedical fields.
Gelatin Type A, 225 Gelatin 10 wt.% Freeze at −20 ℃, −78.5 ℃, and The combination of fast freezing and [97]
26
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bloom food-grade
an
−198 ℃ for 18 h and thaw at 4 slow thawing treatment conditions
℃ and 20 ℃ for 8 h. Repeated resulted in a uniform polymer network
for 1, 2, 3, 4, and 5 cycles. structure after several F–T cycles. The
gelatin hydrogel showed an optimized
30 fine inner structure (pore size = 23.72 ±
20.61 µm2), stable latent heat of fusion
31 (248.8 J/g), constant total moisture
32 content (about 90%), high cooling
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33 efficiency, and relatively stable
34 mechanical strength (46 kPa).
35 Curdlan gum/ к- - 1 wt.% Curdlan gum Freeze at −16 ℃ for 18 h and Compared to curdlan gels formed with [98]
carrageenan/ is combined with thaw at 25 ℃ for 6 h. Repeated guar, к-carrageenan, or locust bean gum,
36 Xanthan/ Guar/ other biopolymers for 1, 2, 3, 4, and 5 cycles. the curdlan/xanthan composite hydrogel
37 Locust bean gum (1:1 ratio) each proved to be the most resilient,
38 having a exhibiting zero syneresis with superior
39 concentration of 1 consistency in viscosity, heat stability,
wt.%. storage and loss modulus, gel strength,
40
and adhesion.
41 Curdlan gum 1.9 × 106 Da 1 – 5 wt.%. Freeze at −20 °C for 4, 12, 24 The reversible macromolecular [99]
42 and 48 h, and thawed at 25 °C conformational change and phase
43
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for 4 h. Repeated for 1, 2, 3, separation of curdlan provide a gel with

44 and 4 cycles. reversible transparency, adjustable
volume change, and mechanical strength.
45
KGM/Zein KGM: 1.050 × 5 % (w/v) total KGM Freeze at −18 ℃ for 24 h and The presence of zein strengthens the [100]
46 106 Da and zein content. thaw at 25 ℃ for 3 h. effect of F–T treatment on increasing G′
47 With different ratio gel. The F–T treatment resulted in a
48 of KGM/Zein. coarse network structure, increased the
49 G′ of the composite gel, partially
50 destroyed the original crystalline domain
of the composite gel, and increased the
51
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initial degradation temperature.

52 Abbreviations: CNC: Cellulose Nanocrystal, PU: Polyurethane, LRD: Laponite RD, CAF: Caffeine, GLE: Guava Leaves Extract, KGM: Konjac
53 Glucomannan
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1
2
3 3. Properties of hydrogel
4 where V1 in equation 1 refers to the initial water volume,
5 The crucial properties of hydrogels, e.g., morphology and V2 recorded as the total volume of water when the hydrogel
6 porosity, swelling ability, mechanical properties, thermal is put into the beaker, and V3 refers to water volume after the
7 properties, and biocompatibility, have become the focus of
hydrogel is removed.
8 research in recent decades. These properties can be adjusted
Freeze-thawed hydrogels with a macropores structure
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9 based on the requirements of the desired application,
previously have been investigated by Waresindo et al. [79].
10 selection of appropriate materials, and synthesis methods.
Other research conducted by Li et al. demonstrated a high
11
porosity hydrogel using agarose (AG) as a pore-inducing
12 3.1 Morphology and porosity
agent [114]. As given in figure 3b, the AG transformed the
13
structure of the PVA matrix from initially dense to becoming
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The bulk hydrogel structure is a solid polymer that has
14
absorbed water and has nano-sized pores in the network porous. The addition of AG concentration enhanced the
15
16 [101]. Because pore size modulation and distribution are hydrogels’ pore diameter (20-200 µm).
17 critical for controlling hydrogel properties, they have been The percentage of porosity calculated using equation 1
18 widely used in tissue engineering, wound dressings, and drug showed that the levels of porosity varied from 56% (pure
19 delivery systems [102–105]. The hydrogels porosity is PVA), 69% (AG 2 wt.%), 78% (AG 4 wt.%), and 81% (AG
20 formed by the formation of cross-links between polymer 6 wt.%). In this case, AG not only initiated pore formation
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21 chains [106]. The pore diameter (𝑑) can be classified into by initiating nucleation and growth of ice particles, but it also
22 nanopores, micropores, and macropores, which are 𝑑 < improved mechanical properties by increasing PVA
23 0.1 𝑛𝑚, 0.1 < 𝑑 < 100 𝑛𝑚, and 𝑑 > 100 𝑛𝑚, respectively crystallinity and hydrogen bonding between molecules. The
24 [107]. pores in the hydrogel promote cell growth, vascularity, and
25 Figure 3a shows the non-porous hydrogels matrix that can nutrient diffusion. The increase in pore size aids the
26 be in the form of a tight cross-link, and there are only small efficiency of angiogenesis in the hydrogel, provides faster
27
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29
pores with a size of tens of nanometers, for example, the
alginate network [108,109]. The hydrogel matrix can also
an swelling kinetics, and improves absorbency compared to
non-porous hydrogels [115].
have macroscopic pores with sizes in the range of 10-500 µm
30 [110]. It has been mentioned in the previous section that the 3.2 Swelling ability
31 hydrogel pore size can be influenced by the parameters of the The most exciting property to study in hydrogels is their
32 hydrogel formation process.
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33 swelling ability. Swelling is a continuous transition from an
Process parameters that increase the number of cross- unbreakable or partially rubber-like state of glass to a relaxed
34 linking points will make the hydrogel stronger, but the pore
35 rubber-like state [116]. When the hydrogel meets the
size will decrease. Several methods have been developed to solution, it begins to absorb the liquid, which allows the
36 adjust the pore size of the hydrogel, for example, gas
37 hydrogel to expand. Hydrogels have the ability to absorb
foaming, reverse casting, particle leaching, and freeze-drying
38 water and expand up to 1000 times their dry weight [101]. In
[111]. Among the several methods mentioned above, freeze–
39 addition to the parameters mentioned earlier, the swelling
drying is the most widely used. The main principle of the
40 degree and the change in hydrogel dimensions can also be
freeze–dry way is a fast–cooling process, which creates
41 influenced by the hydrophilic/hydrophobic balance of the
thermodynamic instability, resulting in phase separation. The
42 hydrogel, the degree of cross-linking, the level of ionization,
43 solvent content in the hydrogel is sublimated in a vacuum,
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and its interaction with the counterion [117].

44 leaving a cavity in the previously occupied area [112]. The swelling mechanism of the hydrogel is shown in
45 Hydrogel porosity can be measured using the liquid figure 3c [43]. The swelling of the hydrogel works based on
46 displacement method assisted by a solvent that does not react the principle of osmotic pressure (the difference in the
47 with hydrogel, water, or ethanol/methanol [113]. The solvent concentration of mobile ions present in the dry hydrogel
48 used must be able to reach the entire void left to properly structure and then moves to the surrounding solution). The
49 map the pores of the hydrogel. As a result, the size of the swelling mechanism is also influenced by changes in the
50 solvent molecules has a significant impact on the accuracy of acidity of the solvent, which causes changes in the level of
51
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the hydrogel porosity. Tao et al. reported the equation to ionization of functional groups so that the hydrogel volume
52 calculate hydrogel porosity as follows [91]: becomes larger [118].
53 Porosity =
V1 −V3
× 100% (1)
V2 −V3
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Figure 3. (a). Porous and non-porous hydrogels structures. (b). SEM images of (i) pure PVA hydrogels and (ii-iv)
41
microporous hydrogels, reprinted with permission from Elsevier, license number: 5403040213213 [114]. (c). The hydrogels
42
swelling mechanism through volume phase transition. (d). The swelling ratio and weight loss of various gelatin/PVA
43
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44 hydrogels, reprinted with permission from Elsevier under a CC BY-NC-ND license [119].
45
46
47 The swelling ratio of hydrogels can be determined using where 𝑊𝑑 is the weight of the freeze-dried hydrogels, while
48 the gravimetric method. García-Astrain et al. have reported 𝑊𝑠 corresponded to the weight of the swollen hydrogels
49 the procedure for measuring the swelling ratio value of the [120].
50 alginate-based hydrogel by immersing the freeze-dried The gravimetric method can also be used to calculate the
51
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hydrogels into three different solutions [120]. The degradation of the hydrogel, which is expressed as weight
52 equilibrium swelling was assumed to be reached when the loss. After measuring the hydrogel weight to determine the
53 weight of the hydrogel was no longer increased. By using swelling value, the sample was re-dried until the weight was
54 equation 2, the swelling ratio value can be determined as stable. Then, by using equation 3, we can determine the
55 follows: percentage of weight loss [121].
56 𝑊 −𝑊
𝑆𝑤𝑒𝑙𝑙𝑖𝑛𝑔 𝑟𝑎𝑡𝑖𝑜 = 𝑠 𝑑 × 100%, (2) 𝑊𝐿 =
𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 −𝑊𝑒𝑖𝑔ℎ𝑡 𝑎𝑓𝑡𝑒𝑟 𝑑𝑒𝑔𝑟𝑎𝑑𝑎𝑡𝑖𝑜𝑛
× 100% (3)
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𝑊𝑑
57 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡
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1
2
3 Thangprasert et al. in their research, have successfully in value with an increase in the concentration of the cross-
4 fabricated gelatin/PVA hydrogels using the F–T method for linking.
5 tissue engineering applications [119]. Further
6 characterizations of the swelling ratios and weight loss of the 3.3 Mechanical properties
7 hydrogel were investigated, and the results are depicted in
8 The mechanical properties of hydrogels play a key role in
figure 3d. The swelling ability of the hydrogels was observed
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9 biological performance. Among the most critical parameters
from one to 180 minutes. In general, all samples showed a
10 significant swelling rate ranging from 1 to 30 minutes, then
in hydrogel design, the structure and conformation of
11 reached swelling equilibrium (no longer increasing in
hydrogels greatly influence their mechanical properties as
12 well as cell adhesion, proliferation, and differentiation [124].
weight). Various compositions of gelatin and PVA yielded
13 The mechanical properties of the hydrogel include
different impacts on the swelling ability of the hydrogels.
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14 compressive strength, ultimate tensile strength, and modulus
Hydrogels with a gelatin/PVA composition of 100:0 and
15 of elasticity [64,125].
70:30 had a higher swelling ratio than the other samples.
16 The best hydrogel mechanical properties are made of
Sample 0:100, which is pure PVA, showed the lowest
17 synthetic polymers (chemical bonds are stronger than natural
18 swelling value because it has a dense hydrogel matrix and a
polymers) and produced by chemical cross-linking. [33,126].
19 higher cross-linking degree. The ability of PVA to form
However, with physical treatment (e.g., F–T or anneal
20 −OH bonds between the chains will decrease due to the
swelling) and raw material from natural and synthetic
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21 addition of gelatin concentration, which increases the pore
polymers mixture, it has also been reported to yield a
22 size [92].
hydrogel with good mechanical properties [26].
23 Fan and colleagues also observed the swelling ratio of
The mechanical properties of the hydrogel can be
24 PVA/chitosan/gelatin hydrogel with chitosan/gelatin
measured using a universal testing system and rheometer or
25 concentration ratios ranging from 1: 3, 1: 2, 1: 1, 2: 1, to 3: 1
compressive testing [37,119]. Recently, Fang’s group
26 [122]. They found that the capacity of the hydrogel to absorb
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water was 20–40 times its weight. The swelling rate of the
hydrogel gradually increased after 6 to 12 hours of
immersion and began to slow down after 24 hours. The
an introduced a novel high-strength poly
liquid)/acrylamide (AM)/PVA hydrogel (LAHPs) using the
F–T method for antibacterial application [127]. The
(ionic
composition of the hydrogels (VBIMBr: AM: PVA were

30 equilibrium swelling state was achieved after 48 hours of
0:0.5:2,0.3:0.3:2, 0.5:0.5:2, 1:1:2, 2:2:2) were marked as
31 immersion. Increasing the chitosan concentration caused the
LAPH0, LAPH1, LAPH2, LAPH3 and LAPH4, respectively.
32 hydrogel swelling ratio to increase because chitosan can
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The mechanical properties of the hydrogels they measured
33 cause the hydrogel structure to become looser, and the
34 are presented in figure 4.
macromolecular chains in the system can be more easily
35 The tensile stress-strain curves in figure 4a indicate a
extended. The developed hydrogel then has large pores, and
36 significant increase in the value of the elongation at break
the swelling ratio will be greater too.
37 and maximum stress when poly (ionic liquid) concentration
Hydrogel degradation is expressed in weight loss
38 is increased. Increasing mechanical properties are associated
percentage, as given in figure 3d. The time intervals for
39 with the increasing number of hydrogen bonds formed
weight loss observation were 1, 2, 4, 8, 16, 32, and 64 days.
40 All hydrogel samples with various concentrations of
between poly (ionic liquid) and PVA [128]. The tensile force
41 gelatin/PVA showed high weight loss values and started to
can be absorbed by hydrogen bonds, resulting in better
42 mechanical properties [129]. It is proven that the LAPH3
increase from day one to day four. On the fifth day, the 100:0
43 sample with the highest concentration of poly (ionic liquid)
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(pure gelatin) and 70:30 hydrogel samples were utterly

44 had a maximum stress of 0.87 MPa and an elongation at
degraded. This degradation occurred in the absence of PVA
45 break of up to 265.92%.
content in sample 100:0, and only a little PVA in sample
46 In line with the tensile test results, the compressive
70:30.
47 strength test in figure 4b indicates that as poly (ionic liquid)
48 The cross-linking in PVA is very important in maintaining
increases, the compressive stress of LAPH also increases.
49 the physical shape of the hydrogel. This is evidenced by the
The compressive stress of the LAPH3 sample was 1.61 MPa,
50 measurement of weight loss for the other three samples
while the compressive strain was 7.83%. Furthermore,
51 (50:50, 30:70, and 0:100), which retained their shape during
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LAPH3 can also withstand the car’s pressure and maintain its
52 the degradation test [119]. Sittiwong’s group, who have
original shape, as shown in figure 4e. The LAPH3 strip is
53 observed the effect of cross-linking ratio variations on PVA
also so elastic that it can stretch up to twice its length (figure
54 hydrogels, showed a similar conclusion [123]. Increasing the
4f) and is very soft and easy to tie and stretch (figure 4g).
55 cross-linking ratio caused the hydrogel to be denser and
When attached to the hand, as shown in figure 4h, LAPH3
56 decreased the porosity. The results of the observation of the
can remain adhered to without falling due to its stretchability
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57 swelling and weight loss properties also showed a decrease

and specific viscosity values.
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11

1
2
3 The mechanical properties of the FT hydrogel can also be hydrogel that is more robust and capable of efficiently
4 tuned by designing the dual network of hydrogel for potential dissipating energy throughout the network when subjected to
5 biomedical applications [130]. This double network (DN) external forces [133]. These advantages make DN hydrogels
6 method was previously introduced by Gong et al. and very potent for tissue engineering applications and drug
7 produced a very high mechanical strength of the hydrogel delivery. However, to be of concern, DN hydrogels that are
8 [131]. DN hydrogels are usually made from a network of intended for tissue engineering must have biocompatibility
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9 stiff, short-chain polymers and then interconnected with a properties that support cell growth, migration, differentiation,
10 soft and flexible polymer network [132]. and proliferation.
11 Compared with conventional hydrogels, DN hydrogel
12
combinations synergize with each other to produce a
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54 Figure 4. Mechanical properties of LAPHs with different amounts of VBIMBr: (a). Tensile stress-strain curves, (b).
55 Compressive stress-strain curves. Images of LAPH3: (c). Unstretched and stretched, (d). Under pressure, (e). Under car
56 pressure, (f). Before and after stretching by hand, (g). Knotted and stretched, (h). Taped to the hand, reprinted with
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57 permission from Elsevier, license number: 5403031260318 [127].

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1
2
3 3.4 Thermal properties where ∆𝐻𝑓 is the melting enthalpy, and ∆𝐻𝑓0 refers to the
4 melting enthalpy of a fully crystalline polymer (for PVA
5 The characterization of hydrogel thermal properties aims
138,60 J/g) [135]. Chee and co-workers have published their
6 to investigate the state of the polymer and evaluate the
work, focused on the effect of orientation on the properties of
7 interactions between polymer molecules in the hydrogels
F–T PVA hydrogel [90]. Their hydrogel samples were
8 [89]. The measurement of the thermal properties can be done
prepared with different F–T cycles and uniaxial orientation
pt
9 using a differential scanning calorimetry (DSC). The
cycles (100% stretching strain per cycle). The analysis of
10 resulting DSC curves allow us to determine the glass
thermal properties was carried out using the DSC2920
11 transition temperature (𝑇𝑔 ), the melting temperature (𝑇𝑚 ), the
modulated DSC from the TA instrument. Samples were
12 temperature of crystallization (𝑇𝑐 ), and calculate the degree
13 weighed around 7-10 mg with a ramp heating mode from 20
of crystallinity (𝑋𝑐 ) of the hydrogel sample [65,134]. The
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14 ℃ to 280 ℃ and a heating rate of 10 ℃/min under nitrogen
degree of crystallinity (𝑋𝑐 ) can be calculated using equation gas flow. The DSC curve of PVA hydrogels with different
15 4 below,
16 ∆𝐻𝑓
F–T cycles and orientation is shown in figure 5a.
17 𝑋𝑐 = , (4)
∆𝐻𝑓0
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Figure 5. (a). Thermal properties of PVA hydrogels, reprinted with permission from Elsevier, license number:
55
56 5403001347033 [90]. (b). Degree of crystallinity of PVA and PVA/CNC, reprinted with permission from Elsevier, license
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57 number: 5403010400871 [134].

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1
2
3 Table 3. Melting temperature (T𝑚 ), the heat of fusion (∆H𝑚 ) and temperature of crystallization (T𝑐 ) of pure PVA hydrogels
4 and PVA/CNC hydrogels determined by the DSC technique, reused with permission from Elsevier, license number:
5 5403010400871 [134].
6 Compositions Number of F–T cycles
7
8 3 F–T 5 F–T
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9
𝑇𝑚 (℃) ∆𝐻𝑚 (J/g) 𝑇𝑐 (℃) 𝑇𝑚 (℃) ∆𝐻𝑚 (J/g) 𝑇𝑐 (℃)
10
PVA5 230 75.1 204 230 77.5 204
11 PVA5/CNC1 229 74.2 208 229 75.0 208
12 PVA 7.5 233 81.4 204 233 79.5 207
13 PVA7.5/CNC1 233 75.2 207 231 75.1 207
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14 PVA10 233 77.1 201 233 78.4 202
15 PVA10/CNC1 232 71.2 206 232 75.1 207
16
17
18 The 𝑇𝑔 of PVA was seen at the peak of 40-64 ℃, which When the PVA concentration increased from 7.5 to 10%,
19 was represented by relaxation 𝛼. The solvent cast PVA the degree of crystallinity stopped increasing or began to
20 sample had a 𝑇𝑔 of 63.84 ℃, then when the sample was decrease. This is because inhibition of polymer chain folding
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21 freeze-thawed, the temperature decreased to 39.93 ℃. and crystal formation due to large polymer chain
22 According to Nugent and co-workers, the plasticization entanglement increases when the polymer concentration is
23 effect due to the presence of water in the hydrogel sample is increased [137]. The addition of CNC increased mutual
24 the main cause of this decrease in 𝑇𝑔 value [136]. The interactions compared to independent associations, resulting
25 in a decrease in PVA crystallinity. A decrease in hydrogel
relaxation in the PVA crystal domain (β-relaxation) causes
26 crystallinity due to differences in the number of F–T cycles
27
28
29
the presence of broad peaks observed at temperatures around
140 ℃ due to the evaporation of the remaining water in the
hydrogel sample [137].
an has previously been reported by Ricciardi et al. [55]. They
found that the hydrogel crystallinity increased with the
increasing number of F–T cycles. However, the crystallinity
30 The melting temperature (𝑇𝑚 ) of the PVA crystal domain
was relatively static after five or more cycles.
31 was observed at a peak of 223 ℃, which was relatively large
32 and sharp [138]. Increasing the FT cycle and orientation
3.5 Biocompatibility
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33 cycle causes an increase in the endothermic PVA curve to
34 become sharper and a shift of the peak to a lower For biomedical applications, hydrogels must have
35 temperature. In line with Chee’s group, the thermal biocompatibility properties. Naahidi et al. defined
36 properties of PVA hydrogels have also been reported by biocompatibility as “a biomaterial’s ability to perform with
37 Butylina et al. [134]. They made PVA hydrogels/cellulose an appropriate host response in the specific application”
38 nanocrystals (CNC) using the F–T technique with varying [140]. Biocompatibility consists of biosafety and bio-
39 numbers of cycles and cellulose concentrations. Thermal functionality parameters. Biosafety is the local and systemic
40 properties were analyzed by weighing the sample mass and response of the host to the presence of the applied material
41 then characterized using a Mettler Toledo DSC821e tool. (the material must be non-toxic, non-mutated, and non-
42 The characterization results are listed in table 3. carcinogenic). On the other hand, bio-functionality is the
43
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Hydrogels samples with cycle variations of three times ability of a material to function as expected in a particular
44 and five times both showed an increase in their melting application [43]. The biocompatibility of hydrogels can be
45 temperature when the PVA concentration was increased from evaluated through the hematolysis test, systemic allergy test,
46 5 to 7.5%, then when the PVA concentration was increased pyrogen test, conjunctival stimulation test, toxicity test, and
47 to 10%, the melting temperature stopped increasing and cell growth [141]. It has been mentioned before that the
48 might even decrease. The addition of CNC decreased the hydrogel is made using F–T without involving toxic cross-
49 melting point of all samples except for 7.5% PVA hydrogel linking agents, so it can be said that this hydrogel is very safe
50 samples made with three F–T cycles. These results have been to use for biomedical applications [79,81,103].
51
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confirmed in the previous studies by Roohani et al. and The cytotoxicity test of hydrogel samples can be carried
52
Abitol et al. [88,139]. The decrease in melting point is out by in vitro cell viability using the 3–(4, 5–dimethyl2–
53
caused by the strong interaction between the polymer matrix thiazolyl) –2, 5–diphenyl–2H–tetrazolium bromide (MTT)
54
and the cellulose surface, limiting the formation of large assay [142]. Cytotoxicity levels can be classified based on
55
polymer crystal domains by the polymer matrix chain. The the percentage of cells that survived during the MTT assay,
56
degree of crystallinity is calculated using equation 4, and the
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57 also known as the relative growth rate (RGR), which can be

58 results are shown in figure 5b. calculated using the following equation 5. [143].
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14

1
2
3 RGR =
𝑂𝑝𝑡𝑖𝑐𝑎𝑙 𝑑𝑒𝑛𝑠𝑖𝑡𝑦 𝑣𝑎𝑙𝑢𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
(5) network polyvinyl alcohol/gelatin/glycerol (PVA/GEL/GL)
4 𝑂𝑝𝑡𝑖𝑐𝑎𝑙 𝑑𝑒𝑛𝑠𝑖𝑡𝑦 𝑣𝑎𝑙𝑢𝑒 𝑜𝑓 𝑛𝑒𝑔𝑎𝑡𝑖𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙
organohydrogel made using the F–T technique [144]. The
5 The RGR percentage values greater than 100, 75–99, 50–
samples prepared were hydrogel PVA/GEL/GL, PVA/GL,
6 74, and 25–49 can be classified as non–poisonous, slightly
and GEL/GL by freezing at −25 ℃ for 12 hours and
7 poisonous, moderately poisonous, and severely poisonous,
thawing at room temperature for two hours.
8 respectively. Liu et al. have successfully reported a double
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9 The procedure for cytotoxicity analysis using the MTT
10 assay was started by culturing the control group, the blank
11 group, and the experimental group of the three types of
12 hydrogel samples, each consisting of 4 samples, in a cell
13 culture incubator at 37 ℃ with 5% CO2 for 24 hours. Then
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14 L929 mouse fibroblasts in the logarithmic growth phase and
15 good conditions were installed on 96-well cell culture plates
16 at a cell volume of 5 × 103 cells/well, and the medium
17 content per well was 200 μL. Subsequently, the 96-well plate
18 after 24 hours of culture was replaced with an
19 organohydrogel extract solution, which had a concentration
20 of 0.2 g/mL, and incubation was continued for 24 hours. 20
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21 μL of MTT (5 mg/mL in PBS) was added to each well of a
22 96-well plate in a dark place, then incubated for 4 hours at 37
23 ° C in a CO2 incubator. After that, the MTT and culture
24
solution were discarded.
25
The interaction between cell culture and MTT will
26
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28
29
produce purple formazan. Therefore, 150 μL dimethyl
sulfoxide (DMSO) is used, which can dissolve this formazan.
The sample solution was then cultured for 24 hours, and an
an
30 optical density (OD) measurement was carried out, but
31 previously 96-well plates were shocked for 10 minutes. After
32 the OD value is obtained, the RGR percentage/cell viability
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33 can be calculated using equation 5. Cell morphology and the
34 percentage of cell viability are shown in figure 6.
35 Figure 6a shows L929 cells incubated for 24 hours and 48
Figure 6. (a). The cell morphology and viability of L929
36 hours. All samples showed good viability, but there was an
cells observed by light microscopy. (b). The Cell viability of
37 increase in the number of L929 cells when incubated for 48
L929 cells, reprinted with permission from Elsevier, license
38 hours. In figure 6b, after 24 hours of incubation, L929 cells
number: 5403010834918 [144].
39 in all samples showed a cell viability percentage above 90%.
40 However, after 48 hours of incubation, only L929 cells in the
41 PVA/GL organohydrogel extract solution showed a decrease To give a more detailed comparison in section 3, we have
42 in the percentage of cell viability, which was below 90%. summarized several studies regarding the characterization of
43 the hydrogel properties produced by the F–T method and
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These characterization results indicated that the developed

44 organohydrogel has good biocompatibility for application to their applications, which are presented in table 4.
45 the human body [144].
46
47
48 Table 4. Hydrogel properties and their application produced by the F–T technique
49Materials Approaches Morphology and swelling Mechanical and thermal Biocompatibility Applications Ref.
50 ability
51PVA/CH/
ce
Mixing various -Uniformity and an -Incorporation of Ag in the -Absorption of serum Wound dressing [145]
52AgNPs amounts of PVA, CH, interconnected porous hydrogel network resulted in protein was increased with
and AgNPs. Freezing structure. higher tensile strength and higher the increase of CH.
53 at −20 ℃ for 18 h and -Higher swelling ratio elongation at break (Young -Good compatibility with
54 thawing at RT for 30 when CH content was modulus of 14.28 MPa). blood cells
55 min (3 cycles). increased (up to 1000%). -Inhibition of DPPH up to
56 69.2%
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57CH/PVA/ Different -Adding CeO2NPs - - Cells can be viable and Human dermal [146]
CeO2NPs concentrations of increased the porosity of multiply on both hydrogels fibroblasts
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1
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3 CeO2NPs (0, 0.5, 1%). hydrogels (from 81 to 90 (0.5% and 1%) and do not
4 Freezing at −20 ℃ for %). show significant toxicity.
5 20 h and thawing at - There was a significant - Hydrogel with 0.5%
RT for 8 h (6 cycles). increase in the swelling CeO2NPs can effectively
6 value after incubating it for inhibit MRSA growth
7 up to 5 hours, then the compared to hydrogels
8 value tended to be with 0% and 1%.
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9 constant. The added CeO2-
NPs to the hydrogel
10
structure caused an
11 increase in swelling value,
12 although it was not
13 significant.
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14PVA/n-HA/ A double network -The DN hydrogel -DN hydrogel with HA presence -Good hydrogel ability to Cartilage repair [147]
15HACC hydrogel was prepared microstructure became showed excellent mechanical promote osteoblast cell
with a variation in n- denser, and the pore size properties (ultimate tensile growth.
16 HA content (0.4, 0.6, decreased sharply. strength up to 2.70 MPa and -Cell viability was
17 or 0.8 g). Freeze at Meanwhile, the presence of compressive strength almost 75 increased with the
18 −20 ℃ for 12 h and HA did not have a vital MPa). Without HA, the increasing HA
19 thawing at 25 ℃ for effect on the DN hydrogel mechanical properties slightly concentration.
12 h (3 cycles). microstructure. decreased.
20
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-The sample showed a low -The presence of HA causeed the
21 swelling value as expected hydrogel sample to have a higher
22 for a cartilage repair residual mass during the
23 application. TG/DTG test.
24PVA/HA/ Variation of COL The addition of COL Hydrogel samples with 1% COL - Bionic cartilage [72]
25COL concentration (0, 1, 2, concentrations in the content had good crystallinity
and 3 wt.%).). Freeze hydrogel sample caused an levels so that their mechanical
26
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at −20 ℃ for 8–10h
and thawing at RT for
2 h (10 cycles).
increase in hydrophilic
groups such as amino
(−NH2) and carboxyl
groups (−COOH). Thus,
an
properties were also better
(tensile strength reached 1.62
MPa and elongation at break
reached 532%).
the hydrogel composite
30
with 3% COL content had
31 good swelling properties
32 and the highest water
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33 content.
34Kappa- Various CD - Hardness was increased by low The addition of the number Food industry [148]
35carrageenan/ concentrations (1, 2, 3, CD concentrations and then of F–T cycles increased the
CD 4, 5, and 6% w/w). decreased by increasing the CD syneresis value of K-
36 The sample was first concentrations. CD affected the carrageenan gel. The
37 stored at 4 ℃ for 16 hardness of K-carrageenan gel. network or structure of K-
38 hours and then frozen carrageenan was easily
39 for 24 hours at affected by the formation
−16 ℃. After that, the of ice crystals so that there
40 was a high-water
sample was removed
41 for 6 hours at 25 ℃ separation during the
42 and frozen again at thawing process.
43
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−16 ℃. (5 F–T
44 cycles).
45PVA/GLE The freezing - The morphology of the - An increase in the modulus of - Wound dressing [79]
temperature at −25 ℃ porous hydrogel was elasticity reaches 1000 times the
46 for 20 h, thawing at observed, which increased initial value at a strain ratio of 0-
47 24 ℃ for 4 h for 6 in size with the addition of 95% and 15 times at a strain ratio
48 cycles, and different the GLE fraction. of 50-95%.
49 GLE fractions. - The ability to swell - Shows a decrease in the degree
reaches 207%, which has of crystallinity of the hydrogel
50
potential as a wound when the concentration of GLE is
51
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dressing material. increased.

52ALG/CS-G ALG solution was The compact structure and - Hydrogel demonstrated good It was observed that all the Drug delivery [149]
53 made at 2% and then homogeneous pores were mechanical properties that are prepared formulations had
54 added CS-G with observed. suitable for skin applications with a similarity factor f2 < 50
concentration higher values of firmness, and a difference factor f1 >
55 variations ranging 15, indicating that the F–T
compressibility, compactness,
56 from 0.5% to 2%. and tackiness. technique significantly
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57 Posaconazole (POS) at
- In the thermograms, no POS
affected the POS release
2% was added to the profile. In addition,
58 temperature shift was observed,
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1
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3 matrix to observe the indicating no interaction between alginate/chitosan glutamate
4 drug release drug and polymer for all systems. showed a fairly high
characteristics. Freeze inhibition zone value in C.
5 at -20 ℃ for 18 h and parapsilosis culture.
6 thawing at RT for 6 h,
7 repeated for 3 cycles.
8 PVA/CAF 10% PVA/CAF was Increasing the F-T cycle The highest crystallinity and The drug delivery system Drug delivery [150]
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rapidly frozen using and stretching cycle Young's modulus were obtained best fits the Hixson-
9 liquid nitrogen for 20 resulted in a stiffer PVA at 36% and 1462 MPa, Crowell drug release
10 min and thawed at 4 ℃ hydrogel. SEM images respectively, for samples that model. The rate of drug
11 for 4 h, repeated with show the difference underwent 2 F-T cycles and 2 release increases with the
different F-T cycles between pure PVA stretching cycles. increase in the surface area
12 and stretching cycles. hydrogel and PVA/CAF of the stretched hydrogel.
13 hydrogel. During the F-T
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14 cycle, CAF tends to form
long, needle-shaped
15 crystals. The degree of
16 swelling increased with the
17 increasing alignment of the
PVA polymer chains.
18
PVA/PCE The freezing - The internal morphology - The compression modulus of Composite hydrogel has Wound dressing [81]
19 temperature at −25 ℃ observed using SEM did composite hydrogels tends to antibacterial activity
20 for 20 h and thawing show some phase decrease with increasing PCE against S. aureus and P.
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21 at 27 ℃ for 4 h for 6 separation between the fraction; this is due to the aeruginosa. The release of
cycles. Different PCE liquid and solid phases in presence of an ethanol fraction PCE from the hydrogel
22 fractions. the hydrogel composite. that does not freeze during the F– followed a pseudo-Fickian
23 - The degree of swelling of T process.
diffusion model.
24 the composite hydrogel -PCE was stable in the range of
25 showed an average value room temperature, to body
26 above 200%, which temperature as no significant
27
28CS/metal ions 0.45% CS was loaded The
29(Ag2++, 2+Ca2+, with
indicated a good ability to mass
absorb wound exudate.
SEM
different confirmed the intertwined
results
an
observed.
decomposition
-
was
In vitro studies show that Chronic and infected [151]

gauze can temporarily wound healing
30Zn , Cu ) combinations of ions hydrogel network that was release some metal ions on
and their respective lyophilized and filled in the demand, and the released
31
concentrations, then mesh holes of the gauze, metal ions show
32 placed in the middle of indicating that the CS/M
n+
effectiveness in killing
dM
33 medical gauzes.
hydrogel was successfully bacteria and accelerating
coated on the gauze after cell migration. In vivo
34 Freezing at −20 ℃
the F–T process. studies reveal that metal
35 overnight and thawing ion-filled gauze can
at RT.
36 efficiently promote the
healing of infected wounds
37 by promoting granulation
38 formation, collagen
39 deposition and maturation,
re-epithelialization,
40 angiogenesis, and
41 inhibiting inflammation
42 through the regulation of
43 inflammatory factor
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expression.
44Abbreviations: CS: Chitosan, AgNPs: Silver Nanoparticles, CeO2NPs: Cerium Oxide Nanoparticles, n-HA: Nano Hydroxyapatite, HACC: 2-hydroxypropyltrimethyl
45ammonium chloride chitosan, HA: Hyaluronic Acid, COL: Collagen, CD: Cyclodextrins, GLE: Guava Leaf Extract, ALG: Sodium Alginate, CS-G: Chitosan Glutamate, CAF:
46Caffeine, PCE: Piper crocatum extract.
47
48
49 to be avoided in order to be suitable for application in the
50 4. Biomedical application of F–T hydrogel biomedical field. In addition, the need for biomedical
51
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applications that require hydrogels with high mechanical

Hydrogel has the advantages of increased
52 properties can also be provided by the F–T method by
biocompatibility, tunable biodegradability, precise
53 optimizing the parameters of the synthesis process. The F–T
mechanical strength and a porous structure compared to other
54 method has also been proven to produce hydrogels that can
55 types of biomaterials [152]. The F–T method is basically a
load drugs, antioxidants, or antibacterial agents, making
56 physical crosslinking mechanism without involving toxic
them very useful in the biomedical field. In this section, we
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57 crosslinking agents like dialdehyde, formaldehyde, or

discuss the application of F–T hydrogel in several important
58 tetramethylethylenediamine, etc. These toxic properties need
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3 biomedical fields, e.g., wound dressing, drug delivery the collagen fiber reinforcing synthesis process also begins in
4 systems, and tissue engineering. this phase.
5 Furthermore, the proliferation phase also initiates the
6 4.1 Wound dressing formation of epithelial cells, keratinocytes, and fibroblasts.
7 [156]. Wound healing ends with a regeneration stage (figure
8 Wound dressings are usually made of polymers in the
7d) where the dermal tissue recovers and there is an increase
pt
9 form of gauze, gel, hydrogel, or hydrocolloid. The hydrogel
is the most promising form and is ideal for application as a in the tensile strength of the scar tissue. Inflammatory cells
10 have also been cleared from the regenerated area while
11 wound dressing because it keeps moisture in the wound area,
helps remove wound exudate, prevents infection, and collagen is renovated to increase its tensile strength.
12
Complete wound healing may take weeks or months [160].
13 facilitates tissue regeneration [153]. A wound is damage that
Ideally, a wound management product should be able to
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14 occurs on the inside or outside of the body's tissues, which
absorb excess wound exudate and toxins, maintain good
15 can result from a collision, cut, or other damage. Injuries can
moisture between the wound surface and the wound
16 disrupt typical tissue structure and function. There are two
covering, prevent infection, prevent overheating, have good
17 types of wounds: acute wounds and chronic wounds. Acute
18 wounds heal entirely in less time, while chronic wounds take permeability, and be easily removed without aggravating the
19 longer to heal [154]. wound [161]. The scheme of using hydrogels in the wound
20 Several factors, such as wound infection, wound depth, healing process is shown in Figure 8. Open wounds (Figure
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21 foreign body interaction, stress, pressure, age, and congenital 8a) have 20 times greater water and fluid loss than normal
22 disease, can affect wound healing [155]. The wound healing skin and are very susceptible to bacterial invasion [118].
23 process begins with the exudative stage, inflammation, Applying hydrogel to an open wound (figure 8b and 8c) can
24 proliferation, and finally regenerative, as shown in figure 7 help accelerate wound healing by exchanging moisture,
25 [156]. The exudative stage (figure 7a), also known as the thereby creating a more optimal microclimate between the
26 wound and the wound dressing [162].
27
28
29
an
hemostasis and coagulation stage, is the process of stopping
blood flow within minutes by the aggregation of platelets and
the formation of fibrin clots. The interaction of blood with
The insoluble hydrophilic structure of the hydrogel has a
tremendous capacity to absorb wound exudate and allow
oxygen diffusion to promote healing [163]. Hydrogels can be
exposed collagen and other components of the extracellular
30 matrix causes the release of clotting factors that act as plugs composited with bioactive materials so that they have
31 by activated platelets [157]. antibacterial activity [79,81,164]. Hong et al. successfully
32 reported hydrogel preparations made using the F–T process
dM
The inflammatory stage (figure 7b) releases inflammatory
33 for wound dressing applications [165]. They used tannic acid
cells such as neutrophils and macrophages to the wound site.
34 (TA) as an antibacterial agent and then composited it with
Foreign bodies, bacteria and damaged endogenous tissue can
35 PVA. With variations in the composition of tannic acid,
be cleaned by the inflammatory response mechanism [158].
36 PVA/TA hydrogel composite samples were frozen for 18
Neutrophils only work in the early stages of wound healing,
37 hours at −20 ℃ then thawed at 25 ℃ (6 hours), resulting in a
38 whereas macrophages are able to survive through all phases,
from exudative to regenerative [159]. New blood vessels are hydrogel with good mechanical strength and high
39 antibacterial activity (99.9% against S. aureus).
40 formed during the proliferation phase (figure 7c). In addition,
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33 Figure 7. Illustration of the wound healing stage, reprinted with permission from Elsevier, license number: 5403000535998
34 [156].
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52 Figure 8. Hydrogel membrane application as a wound dressing.

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1
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3 4.2 Drug delivery system the extract released, and even after 3 hours, the extract
4 release was still below 50%. The reason may be that the
5 Hydrogels can be used in drug delivery systems because they extract is not well encapsulated in the PVA matrix.
6 have a three–dimensional structural network with a high– Moreover, aspects of controlled release need to be
7 water absorption capacity. This hydrogel property is useful in considered in the design of drug delivery systems. The main
8 the process of loading drugs and releasing drugs in a idea of controlled drug release is to treat the symptoms in the
pt
9 controlled manner. Drug delivery is the process of desired and targeted area [177]. Considering that the human
10 transporting pharmaceutical compounds into the body to digestive tract has a unique microenvironment, hydrogels
11 achieve the expected therapeutic effects [166]. The high- that can respond to changes in stimulus (particularly, changes
12 water content present in hydrogels helps hydrophilic in acidity levels) are needed as a controlled drug delivery
13 transport drugs. One of the physical properties of the medium [178]. Currently, PVA-based hydrogels have been
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14 hydrogel, which is related to its porosity, can affect the shown to exhibit pH-responsive properties [179,180].
15 continuous drug dissolution process [154]. However, pure PVA has the disadvantage that it is not
16 Two methods can carry out the process of loading the sensitive to changes in pH [181]. Therefore, some
17 drug onto the hydrogel. First, the polymer is mixed with the
18 researchers combine it with PAA to produce hydrogels with
drug, followed by a chemical and physical cross-linking pH-responsive properties [182,183]. Nevertheless, PAA
19 process. The second method is the hydrogel formed by
20 tends to cause irritation, thus limiting its use in the
soaking in a medicinal solution [167]. However, the drug
us
21 biomedical field.
delivery system using hydrogel media has difficulty loading Interestingly, Xie and co-workers in their research
22
hydrophobic drugs and tends to have low tensile strength
23 succeeded in developing a pH-sensitive PVA/sodium
[168]. The way to increase the efficiency of loading
24 alginate hydrogel with optimized F–T process parameters
hydrophobic drugs into the hydrogel matrix is by combining (freeze at – 18 ℃ for 22 h, thaw at RT for 2 h) [184]. As a
25
molecules with the ability to form inclusion complexes and
26 model of drug release, they used chloramphenicol, which
27
28
29
incorporate hydrophobic groups [169]. Printing of hydrogel
affecting the drug release kinetics [170].

an
supermolecules can increase drug loading efficiency without
was tested under conditions similar to the microenvironment
of the human digestive tract. In figure 9b, the cumulative
release pattern of chloramphenicol in simulated intestinal
30 As shown in figure 9a, the polymer can be composited fluid (pH 7.4) and gastric fluid (pH 1.2), confirms that the
31 with nanoparticles to form a hydrogel superstructure for hydrogel synthesized with 4 F–T cycles and 50% sodium
32 controlled drug delivery applications [171,172]. alginate exhibits sensitive behavior to changes in pH.
dM
33 Nanoparticles have been developed very massively and have Furthermore, the effect of the sodium alginate
34 the advantage of being a type of drug delivery encapsulation composition in the hydrogel matrix on the release
35 that can load small molecules, peptides, nucleic acids, characteristics is shown in figure 9c. The addition of sodium
36 proteins, and so on [173,174]. The drug release alginate is known to reduce the cumulative release of
37 characteristics of the nanoparticles-hydrogel can be modified chloramphenicol from the hydrogel matrix, making it a
38 by varying the components of the nanoparticles [175]. potential candidate for controlled drug delivery systems. The
39 Besides, the variations in hydrogel synthesis parameters main reason may be that the incorporation of sodium alginate
40 by the F–T method can affect drug release characteristics. into the PVA hydrogel matrix causes a decrease in the pore
41 The greater number of F–T cycles causes the density of the size, which in turn affects the drug release behavior [185].
42 cross-linking to increase so that the permeability of the Increasing the F–T cycle from 2 to 6 led to a reduction in the
43
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matrix decreases. Thus, the release of the drug is slower cumulative release of chloramphenicol from 83.5% to 76.6%
44 [176]. Previously, Edikresnha et al. had observed the release (figure 9d). The reason is that the density of the hydrogel
45 behavior of PVA hydrogels loaded by Piper crocatum crosslinks increases with increasing F–T cycles, as
46 extract (freeze at – 20 ℃ for 20 h, thaw at 27 ℃ for 4 h, 6 previously described [176,184].
47 cycles) [81]. They found that within the initial 20 minutes,
48 there was a short-term burst release with nearly 13-20% of
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35 Figure 9. (a). Hydrogel contains nanoparticles for drug delivery applications. (b). Cumulative release of chloramphenicol
36
from PVA/sodium alginate hydrogel in simulated intestinal and gastric fluid. (c). Cumulative release of chloramphenicol at
37
various sodium alginate mass ratios. (d). Cumulative release of chloramphenicol at different F–T cycles. Figure 9b-d were
38
reprinted with permission from Jhon Wiley and Sons, license number: 5424760125448 [184].
39
interaction and characteristics with cells when applied to
40
4.3 Tissue engineering tissue regeneration sites [191]. Previous research by Engler’s
41
42 group has successfully investigated the behavior of cells
Tissue engineering is interdisciplinary research that
43 under two-dimensional (2D) hydrogel conditions [192].
pte
combines several fields, such as materials science, cell

44 Several determinants of cell behavior include the level of
biology, and engineering, to repair damaged tissue [186].
45 stiffness, ligand density, and matrix porosity of the hydrogel.
Tissue engineering aims to mimic the extracellular matrix
46 Huebsch et al. initiated the investigation of cell behavior in
(ECM) for tissue regeneration. The hydrogel can be used for
47 tissue engineering, including skin regeneration, cartilage
3-dimensional (3D) hydrogel conditions with variations in
48 repair, bone, and vascular scaffold [7,147,187,188]. Several
the level of stiffness (2.5-110 kPa) [193]. The application of
49 methods used in the manufacture of hydrogel scaffolds
the 3D hydrogel to the subchondral bone interface is
50 include lyophilization, emulsification, solvent washing,
illustrated in figure 10a.
51
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Thangprasert et al. in their research, have reported on

foaming gas release, photolithography, microfluidics, micro
52 printing, and 3D printing [189].
gelatin/PVA hydrogel fabrication using the F–T method for
53 tissue engineering applications [119]. The F–T fabrication
Requirements that must be possessed by scaffolding
54 parameters, such as polymer concentration, freezing
materials include being biocompatible, biodegradable, sterile,
55 temperature, and the number of F–T cycles, greatly affect the
and having a structure that supports cell attachment, cell
56 resulting hydrogel properties. Based on their results,
proliferation, and differentiation [190]. Hydrogel
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57 differences in gelatin and PVA composition led to

58 compatibility and degradability are needed to see their
59
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1
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3 differences in swelling properties, degradation rates, pore components such as collagen. When bonded to PVA,
4 sizes, and biological properties (the best properties belong to collagen with a triple helical structure and a high
5 the gelatin/PVA 30:70 composition). Nevertheless, the concentration of hydrophilic groups can provide better
6 mechanical properties of the hydrogel are not sufficient to mechanical properties [194]. In addition, collagen is able to
7 support the subchondral bone interface. However, the ALP show remarkable biocompatibility, which supports cell
8 activity test, calcium content, and alizarin red confirmed the adhesion, proliferation, and differentiation in articular
pt
9 osteogenic potential and biocompatibility of the fabricated cartilage [195]. Articular cartilage plays a role in reducing
10 hydrogel samples. friction under high loads in activities of daily movement
11 Fortunately, these poor mechanical properties can be [196].
12
improved by combining PVA with extracellular matrix
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51 Figure 10. (a). Hydrogel application as tissue engineering in osteoarthritis, reprinted with permission from Elsevier under a
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52 CC BY-NC-ND license [119]. (b) Physical appearance, morphology, and crosslinking mechanism of PVA/HA/COL
53 hydrogels. (c). Friction lubrication schematic diagram of PVA/HA/COL hydrogels. (d). Average friction coefficient of
54 PVA/HA/COL hydrogels. (e). Uniaxial tensile stress-strain curves of PVA/HA/COL hydrogels, reprinted with permission
55 from Elsevier, license number: 5425211214549 [72]. (f). Uniaxial tensile stress-strain curves of PVA-HA/HACC-Cit double
56 network hydrogels. (g). Average friction coefficient of PVA-HA/HACC-Cit double network hydrogels at different loading
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57 rates, reprinted with permission from Elsevier, license number: 5425440387179 [197].
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22

1
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3 As a natural tissue at the ends of bones in synovial joints, freeze at – 20 ℃ for 12 h, thaw at 25 ℃ for 12 h, 3 cycles,
4 articular cartilage is often damaged by arthritis, sports then soak in Na3Cit solution to obtain a second network.
5 trauma, or other conditions. When such damage occurs, the From figure 10f, it is known that the double hydrogel
6 ability to repair itself is limited given the lack of blood network structure can increase the tensile strength and
7 vessels, lymphatic vessels, and nerves in the area [198]. For elongation at break from 0.29 MPa and 345% to 2.95 MPa
8 this reason, the incorporation of collagen and PVA in the and 710%. Likewise, toughness and compressive modulus
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9 form of hydrogels was offered as candidate articular cartilage show excellent mechanical properties as well. While the
10 materials [199]. average coefficient of friction for all DN hydrogels (figure
11 Recently, a 15% PVA combined with 0.05% hyaluronic 10g) is below 0.10, which is lower than the PVA/HA/COL
12
acid (HA) and collagen (COL) whose concentration varied composite hydrogels. Moreover, excellent fatigue resistance
13
from 0 to 3%, was synthesized into a hydrogel with the and recovery properties have been demonstrated due to its
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following parameters, freeze at – 20 ℃ for 8-10 h, thaw at unique dual structure. The presence of HA in the hydrogel
15
RT for 2 h, 10 cycles [72]. As shown in figure 10b, the F–T matrix provides high tribological properties and
16
17 treatment induces polymer chains to form hydrogen bonds biocompatibility, as evidenced by cell viability and
18 with each other (intramolecular, intermolecular I and proliferation assays [197]. Thus, this dual network hydrogel
19 intermolecular II). The F–T treatment and the increase in composite is very promising for articular cartilage
20 COL content in the hydrogel matrix resulted in a porous applications.
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21 structure due to the increased number of hydrogen bonds.
22 This characteristic allows for the availability of free space for 5. Conclusion and future direction
23 water storage, which in turn can be useful for reducing
Hydrogel is a three-dimensional material that can swell up
24 friction.
to 1000 times its dry weight. The basic materials for
25 Moreover, to assess the feasibility of the F–T hydrogel
hydrogel are natural polymers (for example, alginate,
26 composite as articular cartilage, a friction lubrication test
27
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29
scheme was carried out (figure 10c). When sliding loads are
applied (loading rate 10 mm/min), the soft polymer chain
structure (PVA/HA/COL) can reduce the friction coefficient
an chitosan, gum, starch, gelatin, and cellulose) and artificial
polymers (PVA, PVP, PEG, PANI, and PAA). Hydrogel
synthesis can be carried out by the chemical cross-linking
method (covalent bonding) and the physical cross-linking
30 by up to 50% compared to pure PVA (figure 10d). The
method (non-covalent).
31 decrease in the degree of cross-linking in the longer HA
F–T is a type of physical cross-linking method that utilizes
32 chains and their lubricating effect contributed to the increase
dM
the crystallization process of precursor solutions and
33 in hydrogel softness and the decrease in the coefficient of
34 hydrogen bond interactions between polymer chains to
friction, respectively.
35 produce hydrogels. When selecting the polymer material
It should be noted that the hydrogel is a permeable three-
36 used in the F–T method, it is necessary to consider the
dimensional matrix, which allows the liquid within to be
37 structure and use of the appropriate solvent. Being a
extruded when subjected to sliding loads. The liquid then
38 hydrophilic polymer with a large amount of –OH, –COOH,
acts as a lubricant for the friction pairs, and as evidenced by
39 and –NH2 groups in its chain structure provides the
the increase in water retention (due to a more porous
40 structure), the friction coefficient also decreases
advantage that, during the freezing process, the polymer can
41 significantly. However, COL itself is known to have low
form chain associations through hydrogen bonding.
42 Meanwhile, the use of solvents has a significant effect on the
mechanical properties, and an increase in COL content does
43
pte
formation of hydrogels, considering that each of the above

not necessarily improve these mechanical properties [200].
44 polymers has a different solubility. The F–T method can
From figure 10e, it is known that the optimum tensile
45 only be carried out if the polymer has completely dissolved
strength and elongation at break are shown in hydrogel
46 so that the polymer chains with the hydrophilic functional
composites containing 1% COL, which are 1.62 MPa and
47 group can crosslink each other. Thus, the use and selection of
48 532%, respectively [72]. Therefore, modifying the dual
solvents according to the type of polymer must be carried out
49 network structure (DN) as discussed in Section 3.3, is offered
properly.
50 to improve the mechanical properties of the hydrogel.
The advantage of the F–T method is that it is easy to
51 Gan et al. in their research succeeded in developing a DN
ce
fabricate and does not involve cross-linking chemicals,

52 hydrogel based on poly(vinyl alcohol)-(nano
which sometimes have toxic properties. The hydrogel
53 hydroxyapatite)/(2-hydroxypropyltrimethyl ammonium
properties can be influenced by the parameters of the
54 chloride chitosan) abbreviated as PVA-HA/HACC-Cit, using
solution and the parameters of the synthesis process, such as
55 the F–T method [197]. They prepared the first network of
polymer molecular weight, the solution concentration,
56 PVA-HA/HACC hydrogel under the following conditions,
freezing and thawing temperatures, F–T duration, and the
Ac
57 number of F–T cycles.

58
59
60
23

1
2
3 The properties of the hydrogel can be adjusted according hydrogels with optimum properties for certain applications
4 to the actual requirements of the application. Some of the still need to be carried out.
5 most studied hydrogel properties are morphology (porosity),
6 swelling degree, degradability, mechanical properties, Acknowledgements
7 thermal properties, and biocompatibility. The hydrogel
8 This research was financially supported by the Institute of
produced by the F–T method has good biocompatibility. It is
pt
9 Research and Community Engagement, ITB Research Grant
very supportive when applied to the biomedical field, e.g.,
10 in the fiscal years of 2021 – 2022. W.X.W. and A.P.
wound dressing, drug delivery systems, and tissue
11 engineering.
gratefully acknowledges the Indonesian Endowment Fund
12 for Education (LPDP) for doctoral scholarships. H.R.L.
However, several challenges still need to be addressed for
13 acknowledges the Ganesha Talent Assistantship Research
further development of the F-T method to yield superior
cri
14 Group (GTA-RG) for the provision of doctoral scholarship.
hydrogel. Further study on the F–T method to produce
15
hydrogels with the desired properties for specific
16 Data availability statement
applications is still required. Deep research and optimization
17
on variations in solution and process parameters can be All data that support the findings of this study are
18
carried out to support this goal. For example, for wound included within the article (and any supplementary files).
19
20 dressing applications, hydrogels with good mechanical
Competing interests
us
21 properties are needed. Process parameters and solution
22 parameters are varied, which allows the formation of cross- The authors declare no conflict of interest.
23 links at the molecular level to produce an elastic, stretchable,
24 and not easily broken hydrogel.
25 As we mentioned in the previous section, one of the
26 leading users of the hydrogel product is the biomedical ORCID iDs
27
28
29
hydrogel-based materials that are used in this field must be
free from toxic substances. Both the polymer and the solvent
an
sector. In the biomedical field, toxicity is a crucial matter, so
1. William Xaveriano Waresindo: https://orcid.org/0000-
0003-0028-603X.
30 must satisfy this requirement. Fully natural polymers are 2. Halida Rahmi Luthfianti: https://orcid.org/0000-0002-
31 preferred to produce hydrogels for medical applications. 3458-0619.
32 Further research is still needed to explore the various 3. Aan Priyanto: https://orcid.org/0000-0002-7553-6446.
dM
33 sources of biopolymers and to examine their qualifications to 4. Dian Ahmad Hapidin: https://orcid.org/0000-0002-2644-
34 obtain the desired hydrogels. However, hydrogel 0296.
35 developments based on polymer blends composed of natural 5. Dhewa Edikresnha: https://orcid.org/0000-0001-6203-
36 and synthetic ones are still worthwhile to be carried out as 4343.
37 long as the quantity of the synthetic polymers is in the 6. Akfiny Hasdi Aimon: https://orcid.org/0000-0002-7168-
38 tolerance range that makes them safe to use for biomedical 0512.
39 applications. 7. Tri Suciati: https://orcid.org/0000-0002-5145-5668.
40 Many researchers have also tried to compose polymers 8. Khairurrijal Khairurrijal: https://orcid.org/0000-0002-
41 with endemic natural materials originating from various parts 9452-4192.
42
of the world with antibacterial and antioxidant activity.
43
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Freeze–thaw hydrogel fabrication method: basic principles, synthesis

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xxxx-xxxx/xx/xxxxxx 1 © 2022 IOP Publishing Ltd

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45 °C for 1 h, 50 °C for monomer residues require a cleaning time of

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57 evidenced by the increasing number of documents that have

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strong covalent bonds, whereas intermolecular forces link

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Mater. Res. Express (2022) XXXXXX Waresindo et al

Mater. Res. Express (2022) XXXXXX Waresindo et al

CAF:194.19 g/mol ℃ for 4 h. Repeated for 2 hydrogels by the orientation method.

was varied from 0% increasing in PU concentration.

Mater. Res. Express (2022) XXXXXX Waresindo et al

for 4 h. Repeated for 1, 2, 3, separation of curdlan provide a gel with

initial degradation temperature.

Mater. Res. Express (2022) XXXXXX Waresindo et al

and its interaction with the counterion [117].

Mater. Res. Express (2022) XXXXXX Waresindo et al

composition of the hydrogels (VBIMBr: AM: PVA were

(pure gelatin) and 70:30 hydrogel samples were utterly

57 swelling and weight loss properties also showed a decrease

Mater. Res. Express (2022) XXXXXX Waresindo et al

57 permission from Elsevier, license number: 5403031260318 [127].

Mater. Res. Express (2022) XXXXXX Waresindo et al

57 number: 5403010400871 [134].

Mater. Res. Express (2022) XXXXXX Waresindo et al

57 also known as the relative growth rate (RGR), which can be

Mater. Res. Express (2022) XXXXXX Waresindo et al

These characterization results indicated that the developed

Mater. Res. Express (2022) XXXXXX Waresindo et al

dressing material. increased.

Mater. Res. Express (2022) XXXXXX Waresindo et al

In vitro studies show that Chronic and infected [151]

applications that require hydrogels with high mechanical

57 crosslinking agents like dialdehyde, formaldehyde, or

Mater. Res. Express (2022) XXXXXX Waresindo et al

52 Figure 8. Hydrogel membrane application as a wound dressing.

Mater. Res. Express (2022) XXXXXX Waresindo et al

affecting the drug release kinetics [170].

combines several fields, such as materials science, cell

Thangprasert et al. in their research, have reported on

57 differences in gelatin and PVA composition led to

Mater. Res. Express (2022) XXXXXX Waresindo et al

Mater. Res. Express (2022) XXXXXX Waresindo et al

formation of hydrogels, considering that each of the above

fabricate and does not involve cross-linking chemicals,

57 number of F–T cycles.

Mater. Res. Express (2022) XXXXXX Waresindo et al

Mater. Res. Express (2022) XXXXXX Waresindo et al

373–92 [28] Ahmad S, Ahmad M, Manzoor K, Purwar R and

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biomacromolecular hydrogel classification and its Phys. 37 3438–54

chemically cross-linked acrylic acid/gelatin 923–8

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