International Journal of Biological Macromolecules 205 (2022) 595–603

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

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

Coordination with zirconium: A facile approach to improve the mechanical

properties and thermostability of gelatin hydrogel
Fan Zheng a, 1, Xiao Yang a, 1, Jiao Li b, Zhenhua Tian c, Bo Xiao a, Shixiong Yi a, *, Lian Duan a, *
a
State Key Laboratory of Silkworm Genome Biology, College of Sericulture, Textile and Biomass Sciences, Southwest University, Chongqing 400715, PR China
b
Stomatological Hospital of Chongqing Medical University, Chongqing 401147, PR China
c
College of Bioresources Chemical and Materials Engineering, Shaanxi University of Science & Technology, Xi'an 710021, PR China

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

Keywords: The poor mechanical property and thermostability restricted applications of gelatin hydrogel. Herein, a facile
Gelatin hydrogel and inexpensive approach of immerging cooling induced gelatin hydrogels into Zr(SO4)2 dilute solution was
Zirconium coordination applied to overcome these shortages. After this treatment, the micropores in hydrogel decreased to tens of mi­
crons while the water content slightly decreased. XPS results revealed that the coordination bonds formed be­
tween amino or carboxyl groups of gelatins and Zr4+. After immerging in 0.06 M Zr4+ solution, mechanical tests
showed that the elastic modulus, compressive modulus and compressive strength of hydrogel were about 400,
1192 and 476 kPa, respectively, which were approximate 100, 11 and 5 times larger than those of pure gelatin.
The DSC data indicated that the thermoreversible temperature of triple helix structure in gelatin was improved
from about 30 ◦ C to 55 ◦ C. More importantly, the rheological temperature sweep test revealed that hydrogels
with 0.06 M Zr4+ treatment can maintain the hydrogel state without melting even at 80 ◦ C. CCK-8 tests and
Calcein-AM/PI double-stain experiments demonstrated Zr4+ coordination was non-cytotoxic. These promising
data indicated this nontoxic method was efficient and had potential to fabricate gelatin related materials for
further application.

1. Introduction dimensional network of gelatin generates due to the entanglements

Gelatin is a common biopolymer and mainly extracted from skin,
bone or tendons of vertebrate. The developed stock farming and fishing
industry provide extensive sources and thus the supply of gelatin is
abundant with relatively low price [1]. Gelatin has diverse functional
properties, such as emulsification [2], film formation [3] and desirable
biocompatibility [4]. These functions endow gelatin with widely
application potential in many fields, including food industry [5], cos­
metics [6], biomedical applications [7,8] and pharmaceutical industry
[9,10].
As the hydrolytic product of collagen, the molecular weight of
gelatin is inhomogeneous while the typical collagen triple helix struc­
ture is partially reserved in molecular conformation [11]. Gelatin has
desirable solubility in hot water due to the transformation of triple helix
structure into random coil. The gelatin solution can convert to hydrogel
with decreasing temperature. During the cooling process, the random
coil structure of gelatin reverts to triple helix structure and the three-
and hydrogen bonds. The gelatin hydrogels with desirable biocompati­
bility can load and effectively transfer bioactive materials, intelligently
respond to stimuli [12], and thus has potential to be used as the drug
delivery [13,14], scaffold [15], wearable electronic devices [16,17] and
wound healings [18,19]. However, the mechanical properties and
thermostability of cooling induced gelatin hydrogels are undesirable.
The helix-coil transition of gelatin leads to the destruction of gelatin
network and the melting of hydrogel at about 30 ◦ C [20]. Besides, the
network sustained by entanglements and hydrogen bonds is fragile
when faces the external forces. These disadvantages seriously restrict the
further application of gelatin hydrogels [21].
Numerous studies focus on weak mechanical property and undesir­
able thermostability of gelatin hydrogels. Different novel gelatin
hydrogels are prepared according to the design philosophy of double
network [22], double cross-linking [23], Hofmeister effect [24] or hy­
bridization [25]. These methods can reinforce the mechanical properties
of gelatin hydrogels, but the complex procedures, expensive cost of

* Corresponding author.
E-mail address: duan19850420@163.com (L. Duan).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.ijbiomac.2022.02.124
Received 2 December 2021; Received in revised form 9 February 2022; Accepted 19 February 2022
Available online 22 February 2022
0141-8130/© 2022 Elsevier B.V. All rights reserved.
F. Zheng et al.
reagents and potential toxicity of residues were unfavorable for the mass
manufacturing and further application.
Metal ions with vacant electron orbitals can accept lone pairs of
electrons to form coordination bonds. Wang et al. [26] reported that
metal ions coordination bonds were inexpensive and facile method to
enhance the properties of gelatin hydrogels and the effect of Fe3+ was
much better than that of Mg2+, Ca2+, Fe2+, K+ and Na+. However, some
negative factors may limit the further application of gelatin hydrogel
with Fe3+. For examples, the appearance of hydrogel changed into dark
and opacity with increasing concentration of Fe3+, restricting the
application in optical fields [27]. The tough hydrogels were not stable at
redox conditions that transformed Fe3+ to Fe2+ having poor capacity to
form robust coordination bonds [28]. Therefore, it is highly desired to
seek another colorless and stable metal ion to improve the properties of
gelatin hydrogel.
Zirconium is a colorless transitional element with approving
biocompatibility. The toxicity of zirconium has been comprehensively
evaluated in the past decades and the consensus is that zirconium-based
materials were low order of toxicity for animals [29,30]. Neither local
nor systemic adverse reaction about zirconium was observed regardless
of different adopted cell lines or animal models in experiments [29].
Therefore, it has been widely applied in vivo. For example, zirconium
based implants are designed for the repairment of teeth [31], bone [32],
knee [33] and hip [34]. Moreover, zirconium has several vacant electron
orbitals to accept lone pairs of electrons and can form stable coordina­
tion bonds [35]. The researches of Yu et al. [36,37] demonstrated that
zirconium ions can react with poly (sulfonic acid) or poly (acrylic acid)
to form stable coordination bonds and fabricate robust hydrogels. These
excellent properties suggest that zirconium might be the perfect candi­
date to overcome the disadvantages of gelatin hydrogels. However,
zirconium ions were precipitant for protein [38]. The addition of zir­
conium ions into gelatin solution usually resulted in the rapid precipi­
tation of gelatin due to the intense interactions, which hindered the
further process to obtain uniform hydrogel. To our knowledge, there was
no literature about zirconium enhanced gelatin hydrogels.
In this manuscript, we adopted a facile and inexpensive approach of
immerging cooling induced gelatin hydrogel into Zr(SO4)2 dilute solu­
tion to introduce zirconium coordination and avoid the precipitation.
The reaction mechanism between gelatin and Zr4+ was explored.
Moreover, the microstructure, thermostability, mechanical properties
and cytotoxicity of the reinforced hydrogels were evaluated to provide
fundamental data for further application.
2. Experiments and methods
2.1. Preparation of gelatin hydrogels
Type B gelatin (250 g bloom, Aladdin LTD., China) with the iso­
electric point of 4.7–5.0 was directly dissolved into deionized water at
40 ◦ C to prepare 20 wt% gelatin solution. The pH value of obtained
gelatin solution was about 6.5. Then, gelatin solution was transferred to
the Teflon mold and cooled at 4 ◦ C for 24 h to obtain cooling induced
gelatin hydrogels. The pristine gelatin hydrogels were individually
immerged into 0.03 and 0.06 mol/L Zr(SO4)2 solutions at 4 ◦ C. After
immerging for 5 days, the Zr4+ treated gelatin hydrogels were rinsed and
subsequently dialyzed with deionized water (replaced 3 times a day) for
3 days to obtain hydrogels with neutral pH value.
2.2. Measurement of water content
The classic gravimetric method [39] was adopted to evaluate the
water content of gelatin hydrogels. The wet weights (Ww) of hydrogels
were obtained after wiping the clinging water while the dry weights
(Wd) were obtained after lyophilization. Water content was acquired
from Eq. (1):
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International Journal of Biological Macromolecules 205 (2022) 595–603
Water content (%) = (Ww − Wd )/Wd × 100 (1)
2.3. Scanning electron microscopy (SEM) and energy dispersive system
(EDS)
Fresh cut edge of lyophilized gelatin hydrogels was treated by
spray‑gold and then investigated by Phenom Pro (Fei Company, USA).
On the other hand, the corresponding elements mapping of Carbon (C),
Nitrogen (N), Oxygen (O) and Zirconium (Zr) were collected by EDS.
2.4. X-ray photoelectron spectroscopy (XPS) measurements
Fresh cut edge of lyophilized gelatin hydrogels was investigated by
PHI 1600 ESCA system (Perkin-Elmer, USA). The enveloped 1 s level of
C, N, O and 3d Zr levels were separated into corresponding peaks using
Avantage 5.9 software (Thermo Fisher Scientific, USA).
2.5. Fourier transform infrared spectroscopy (FTIR) measurements
The grinded lyophilized gelatin hydrogels were mixed with KBr to
prepare compound pellets under high pressure. Subsequently, the pellets
were investigated the absorption FTIR spectra from 4000 to 400 cm− 1 by
Nicolet iS10 FTIR spectrometer (Thermo Fisher Scientific, USA).
2.6. X-ray diffraction (XRD) analysis
Grinded lyophilized gelatin hydrogels were tested by XRD-7000
(Shimadzu, Japan) with Cu Kα radiation at 40 kV. The range of 2θ
was performed from 5 to 90◦ with 25 mA electricity.
2.7. Measurement of rheological properties
The rheological property of gelatin hydrogels was evaluated by
MCR302 rheometer (Anton-Paar, Austria). Hydrogels were placed into
the parallel-plate system with 2.5 cm diameter. The dynamic frequency
sweep measurement was conducted from 0.1 to 10 Hz with the fixed
strain of 0.1% at 25 ◦ C. The temperature sweep measurement was
measured from 20 to 80 ◦ C with fixed strain of 0.1% at 5 Hz.
2.8. Compressive test
Cylindrical gelatin hydrogel with radius of 0.5 cm and height of 2 cm
was applied to evaluate the compressive property. The tests were per­
formed by E44 universal test machine (MTS Systems Co. Ltd., China)
with 0.1 cm/min compressive rate to record compressive stress-strain
curves. The compressive modulus (Es) and compressive strength (Cs)
were obtained from the curves.
2.9. Differential scanning calorimetry (DSC) measurements
Thermostabilities of hydrogels were evaluated via DSC 250 (TA in­
struments, USA). Approximate 10 mg hydrogel was encapsulated in
aluminum crucible while another aluminum crucible containing 10 mg
distilled water was set as reference. The heating process conducted from
15 to 70 ◦ C with the rate of 2 ◦ C/min to obtain DSC curves.
2.10. Toxicity
MC3T3-E1 cells suspension (500 μL, 2.5 × 104 cells/mL, ATCC,
Manassas, VA) was added into 24-well plates and incubated with gelatin
hydrogels at 37 ◦ C in a humidified atmosphere of 5% CO2. At pre­
determined time points (3 and 7 days), CCK-8 (Dojindo, Japan) was
added to each well for 2 h at 37 ◦ C. Subsequently, the cell suspension
was transferred into 96-well plates with 100 μL per well. The absorbance
at 450 nm were measured by multimode plate reader (Perkin-Elmer,
F. Zheng et al. International Journal of Biological Macromolecules 205 (2022) 595–603

Fig. 1. (a) Appearance of gelatin hydrogels with various concentration of Zr4+. (b-d) SEM images of lyophilized gelatin hydrogels with (b) 0, (c) 0.03 and (d) 0.06 M
Zr4+. (scale bar = 300 um). (e-g) Corresponding elements mapping of Carbon (e), Zirconium (f) and image merging of SEM and element mapping (g) on cut edge of
gelatin hydrogels with 0.06 M Zr4+ (scale bar = 200 um).

osmotic pressure was one of the reasons for the variated water content
Table 1
[40]. Water in pure gelatin hydrogels was electrolyte free. After
Water contents of different gelatin hydrogels.
immerging into Zr(SO4)2 solution, the interior of hydrogel was deion­
Concentration of Zr(SO4)2 solution (M) 0 0.03 0.06 ized water while the exterior of hydrogel was Zr(SO4)2 solution.
Water content (%) 82.6 ± 1.9 72.9 ± 1.0 68.5 ± 2.6 Therefore, driven by the osmotic pressure, fresh water flowed from the
interior to the exterior of hydrogel, which caused the shrinkage of
hydrogel. On the other hand, Zr(SO4)2 solution can reduce the re­
USA). Besides, the MC3T3-E1 cells suspension was incubated with
pulsions of hydrogel network due to the charge screening effect [41],
gelatin hydrogels in 24-well plates for 3 or 7 days, respectively and then
which also caused the deswelling of gelatin hydrogel. To sum up, the
double stained by Calcein-AM and Propidium Iodide solution. The
decreased water content can be comprehended as the synergetic results
growth of cells was observed by fluorescent microscope.
of osmotic pressure and charge-screening effect.
The SEM images of pure gelatin hydrogel exhibited large lamella
3. Results and discussion
structure and pores with diameter of hundreds of microns (Fig. 1b).
After Zr4+ treatment, the diameter of pores obviously decreased to tens
3.1. Appearance, water content and microstructure of gelatin hydrogels
of microns (Fig. 1c and d), which was similar to the variation of cross-
linked gelatin hydrogel [42]. Therefore, the immerging treatment may
The digital photographs of different gelatin hydrogels were shown in
generate cross-linked bridges in gelatin hydrogels. The corresponding
Fig. 1a. It was clearly that all gelatin hydrogels were light yellow and
elements mapping of nitrogen, oxygen (not given), carbon (Fig. 1e),
transparent, suggesting the uniform microstructure without precipita­
zirconium (Fig. 1f) and merging of SEM and elements mapping (Fig. 1g)
tion. On the other hand, the size of hydrogel obviously decreased after
in the cut edge of cross-linked gelatin hydrogel demonstrated that Zr4+
coordinating with Zr4+. To evaluate the size variation, water content of
could uniformly infiltrate into the interior of hydrogel. Hence, the
hydrogel was studied and listed in Table 1. The water contents decreased
variated micromorphology of hydrogels might be due to the interactions
from 82.6% to 68.5% when the dosage of Zr4+ increased to 0.06 M. The

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Fig. 2. XPS spectra of gelatin hydrogels treated by 0 and 0.06 M Zr4+ and the schematic of coordination. (a) Wide scan XPS spectrum. (b) High resolution C 1 s
spectra with deconvolution. (c) High resolution N 1 s spectra with deconvolution. (d) High resolution O 1 s spectra with deconvolution. (e) High resolution spectra of
Zr 3d. (f) The schematic of coordination between gelatin and Zr4+ at neutral pH.
between Zr4+ and gelatin.
3.2. XPS spectra analysis
The interactions between gelatin and Zr4+ were evaluated by XPS
spectra to give an insight into the mechanism. The typical carbon
contamination (C–C) was corrected to 284.8 eV and all spectra were
adjusted via this correction. In wide scan spectrum (Fig. 2a), it was
clearly that the pure gelatin exhibited peaks at 285, 400 and 531 eV
attributing to C 1 s, N 1 s and O 1 s, respectively. After the immerging
treatment, peak corresponding to Zr 3d at 184 eV generated. Moreover,
the intensities of peaks attributed to carbon, nitrogen and oxygen all
decreased dramatically. Cao et al. [43] reported that the amino groups
and carboxyl groups in protein were the active sites for Zr4+ coordina­
tion. Wang et al. [44] reported that coordination caused the decreased
peak intensities of carbon, nitrogen and oxygen in XPS spectra. There­
fore, the variated spectrum of Zr4+ treated hydrogel might be explained
by the coordination between gelatin and zirconium.
Deconvolution was applied to further analyze high resolution XPS
spectra. C 1s spectrum of pure gelatin hydrogel contained 3 peaks at
284.8, 286.1 and 288.0 eV, representing C-C/C-H, C-O/C-N and O=C-
NH, respectively (Fig. 2b). These peaks had no banding energy shift after
immerging in Zr4+ solution. However, the intensities of C-O/C-N and
O=C-NH decreased dramatically. Area ratio of peaks at 286.1 and 288
eV to peak at 284.8 eV obviously decreased from 1.07 and 0.88 to 0.64
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International Journal of Biological Macromolecules 205 (2022) 595–603
and 0.55, respectively. This variation suggested that the amino groups
and carboxyl groups may coordinate with Zr4+. The N 1 s spectrum of
pure gelatin contained 2 peaks at 399.8 and 400.2 eV, attributing to
N–H and C–N, respectively (Fig. 2c). The treatment by Zr4+ resulted in
0.1 and 0.3 eV upshift of binding energies for N–H and C–N peaks,
respectively. This variation proved that the amino groups of gelatin took
part in coordination [45]. The O 1 s spectrum in Fig. 2d showed gelatin
contained 2 peaks (531.4 and 532.4 eV) representing O–H and C–O,
both of which had 0.3 eV upshift of binding energies after Zr4+ treat­
ment. Moreover, a new peak generated at 530.8 eV. Wang et al. [46]
reported that the new peak attributed to the bond of metal‑oxygen when
the carboxyl groups of protein coordinated with metal ions. Therefore,
coordination between Zr4+ and carboxyl groups in gelatin was
confirmed, too. The spectra of Zr 3d contained 2 peaks at 185.5 and
183.1 eV, representing the Zr 3d5/2 and 3d3/2, respectively (Fig. 2e).
Area ratio of these two peaks was around 2: 3 and the spin-orbit sepa­
ration was 2.4 eV, indicating that zirconium was still the Zr4+ oxidation
state in hydrogels and the coordination number was 4 [47]. To sum up,
the mechanism of coordination in gelatin hydrogel with neutral pH was
revealed and shown in Fig. 2f. The coordination bonds formed compli­
cated cross-linking among gelatin networks, which may benefit the
improvement of hydrogel properties. The previous research [26] sug­
gested that metal ions can coordinate with carboxyl groups in gelatin.
The XPS results revealed that not only the carboxyl groups, but also the
amino groups in gelatin coordinated with metal ions, which
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Fig. 3. Characterization of gelatin hydrogels with 0, 0.03 and 0.06 M Zr4+. (a) FTIR spectra. (b) XRD spectra.

Fig. 4. Mechanical properties of gelatin hydrogels. (a) Elastic modulus. (b) Viscous modulus. (c) Image of pure gelatin hydrogel with pressure. (d) Image of 0.06 M
Zr4+ treated gelatin hydrogel with pressure. (e) Compressive stress-strain curves. (Es: compressive modulus; Cs: compressive strength).

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Fig. 5. Thermal stability of gelatin hydrogels with different dose of Zr4+. (a) DSC curves of gelatin hydrogels. Rheological temperature sweep curves of (b) pure
gelatin hydrogel, (c) gelatin hydrogel with 0.03 M Zr4+ and (d) gelatin hydrogel with 0.06 M Zr4+.
supplemented the reaction mechanism between gelatin and metal ions.
3.3. FTIR and XRD spectra analysis
Fig. 3a listed the FTIR spectra of gelatin hydrogels. The absorption
peaks of gelatin were located at 1634, 1558 and 1246 cm− 1, associating
with the amide I, II and III [48]. After Zr4+ treatment, these absorption
peaks attributed to gelatin had no obvious shift, indicating the un­
changed molecular structure of gelatin. On the other hand, some new
peaks emerged. The peak corresponding to Zr–O (around 616 cm− 1)
[49] also demonstrated the coordination between carboxyl groups and
Zr4+. The peaks located at 974 and 1052 cm− 1 was the v1-SO42− and v3-
SO42− band [50], respectively. Olivera et al. [51,52] reported that SO42−
were able to form the coordination bridges. In this case, SO42− may also
participate in the coordination. The XRD spectra were shown in Fig. 3b.
Pure gelatin hydrogels showed a sharp peak at around 7◦ and a broad
peak at around 20◦ . The sharp peak was associated with the triple helix
structure with the diameter of 1.4 nm [53,54]. The broad peak mainly
correlated with the distance (0.44 nm) between adjacent amino acids in
helix region of gelatin [53,55]. For comparison, the broad peak of
hydrogels treated by Zr4+ had no variation, suggesting the unchanged
helix structure. On the other hand, the new sharp peak related to
amorphous zirconium emerged at around 5◦ [49]. This new peak
covered the previous sharp peak of gelatin at 7◦ and demonstrated the
amorphous state of zirconium in hydrogels.
3.4. Mechanical properties
Rheology is a common method to assess mechanical property of
gelatin hydrogel. Both elastic modulus (G') and viscous modulus (G") of
gelatin hydrogel were constant from 0.1 to 10 Hz (Fig. 4a and b).
Moreover, the G' values were much larger than G" values for all hydro­
gels. These data indicated that gelatin hydrogels with or without Zr4+
treatment were all solid [56]. On the other hand, it was clearly that the
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moduli of hydrogel were Zr4+ dosage dependent. G' value of pure gelatin
hydrogels was about 4 kPa. For hydrogel treated by 0.03 M Zr4+, G'
value sharply increased to more than 100 kPa. Moreover, G' value of
hydrogel with 0.06 M Zr4+ further increased to about 400 kPa and was
approximate 100 times larger than that of pristine gelatin hydrogel. The
reinforcement could be explained by complex coordination of Zr4+. The
XPS data demonstrated that the amino groups and carboxyl groups
formed complex coordination bonds with Zr4+. Several adjacent gelatin
molecules can be cross-linked together via coordination bonds. The
entangled network of pure gelatin hydrogels was reinforced by the
complex coordination and thus the moduli of hydrogels were improved.
As a contrast, the common cross-linking agent, such as N-(3-Dimethy­
laminopropyl)-N9-ethylcarbodiimide hydrochloride (EDC), can form
cross-linking bridge between 2 molecules, resulting in only 10 times
increased G' value for gelatin hydrogels [57]. Literatures also reported
some other technologies to improve mechanical property of gelatin
hydrogel, but they usually restricted by the limited efficiency or rigorous
processing steps. For example, G' value of gelatin hydrogels mixed with
nanofiber cellulose were no more than 10 kPa [58]. The G' value of
reported strong gelatin hydrogels, such as hydrogels enhanced by Hof­
meister effect [24], carrageenan and potassium sulfate [56], multi-
interpenetrating network [59], usually were about 10 kPa. These com­
parisons demonstrated that immerging gelatin hydrogel into Zr(SO4)2
solution was efficient to improve the mechanical property.
The compressive experiments were also performed to assess me­
chanical property of hydrogel. The photographs of hydrogels with finger
pressing were shown in Fig. 4c and d. The pure gelatin hydrogel was
fragile but Zr4+ treated hydrogel was still intact, revealing the more
solid network in hydrogel. The stress-strain curves of different gelatin
hydrogels were listed in Fig. 4e. The compressive resistant ability of
hydrogels obviously ascended with increasing Zr4+ dosage. The
compressive modulus of 1192.6 kPa and compressive strength of 476.1
kPa for hydrogel treated by 0.06 M Zr4+ exhibited 11.4 and 5.0 times
increase compared to 95.5 and 79.2 kPa for the pure gelatin. The
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Fig. 6. Cytotoxicity of gelatin hydrogels. (a) Proliferation of MC3T3-E1. Cell viabilities were examined by CCK-8 and values represented mean ± SD for 6 duplicates.
(b) Merged fluorescent microscopy images of MC3T3-E1 with double-stained by Calcein-AM/PI (scale bar = 400 um).

reinforced compressive resistance of hydrogels also can be attributed to
the coordination bonds between Zr4+ and gelatin. By contrast, the lit­
eratures reported the double cross-link by graphene oxide and glutar­
aldehyde increased the compressive modulus and compressive strength
of gelatin hydrogels by 2.9 and 2.0 times, respectively [23]. The two
parameters of gelatin hydrogels respectively increased by 4.1 and 0.2
times after hybridizing with borosilicate bioactive glass [60]. To sum up,
the promising data demonstrated that Zr4+ treatment was efficient
method to improve mechanical property of gelatin hydrogel.
3.5. Thermostability
The cooling induced gelatin hydrogels maintained their morphology
by entanglements of triple helix structure and hydrogen bonds, which
were unstable and thermoreversible. When the temperature increased to
about 30 ◦ C, gelatin hydrogels melted and transformed into solution
again [61]. The invertible thermostability of gelatin hydrogel went
against the further application. DSC tests can reflect the structure vari­
ation during heating process. As shown in Fig. 5a, there were two peaks
respectively located at 29 and 31 ◦ C on DSC curve of pure gelatin
hydrogels. Djabourov et al. [62] reported that the hydrogen bonds in
gelatin were destructed at around 30 ◦ C and the further heating caused
the conformation change of partly reserved collagen triple-helix struc­
tures, resulting in the two adjacent peaks in differential melting curve of
gelatin hydrogel. Therefore, melting of pure gelatin hydrogels mainly
attributed to the destructed hydrogen bonds and variated triple helix
structures. For hydrogel treated by 0.03 M Zr4+, the two peaks located at
44 and 50 ◦ C, respectively. When the dose of Zr4+ increased to 0.06 M,
the two peaks distributed at 55 and 58 ◦ C, respectively. This trend
suggested that not only the mechanical properties of gelatin hydrogels,
but also the thermostability was improved by the Zr4+ treatment. The
improved thermostability can be comprehended as the result of complex
coordination bond strengthened triple-helix structure and network in
hydrogel.
Rheological temperature sweep tests were adopted to evaluate the
stability of mechanical properties with heating process. As shown in
Fig. 5b, G' values of pure gelatin hydrogel were constant and order of
magnitude larger than G" values before the temperature reaching about
30 ◦ C, suggesting the stable hydrogel state. With the further ascending
temperature, the G' values sharply decreased and became much lower
than G" value. This trend was coincided with the corresponding DSC
curve and can be explained by the melting of hydrogel. For gelatin
hydrogel with 0.03 M Zr4+, the change of both moduli was similar to
that of pristine hydrogel except for increased melting temperature at
about 60 ◦ C (Fig. 5c). The improved melting temperature also coincided
with the corresponding DSC curve and could be ascribed to the coor­
dination bond strengthened triple-helix structure and network. More­
over, the melted hydrogel with 0.03 M Zr4+ still had several orders of
magnitude larger G' values than melted pristine gelatin hydrogel, sug­
gesting the coordination still existed in the viscous melting solution. In
Fig. 5d, the modulus variation of hydrogel with 0.06 M Zr4+ was much
different from the other two groups. There was no obvious decrease of
modulus during the heating process, implying the sustained hydrogel
state without melting. Previous researches of Jiang et al. [63,64]
demonstrated the thermostability of zirconium coordination was higher
than 80 ◦ C. Therefore, the sustained hydrogel state at high temperature

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can be attributed to the abundant coordination bonds among gelatin
network, which maintained the network without melting. To sum up,
the DSC and rheological temperature sweep tests confirmed that Zr4+
coordination definitely improved thermostability of gelatin hydrogel.
3.6. Toxicity
The toxicity of gelatin hydrogels determined the application prospect
of gelatin hydrogel. Piconi et al. [29] reported that zirconium was
nontoxic according to experiments with different cell lines and animal
models. In this manuscript, we investigated the toxicity of gelatin
hydrogels by CCK-8 test and Calcein-AM/PI double-stain. Fig. 6a listed
the proliferation of MC3T3-E1. It was clearly that there was no obvious
difference for OD values among control group, pure gelatin hydrogel and
hydrogels with 0.06 M Zr4+ after incubating for 3 and 7 days. Moreover,
the merged fluorescent microscopy images (Fig. 6b) showed MC3T3-E1
exhibited strong green fluorescence after incubating with gelatin
hydrogels for 3 days and 7 days, suggesting that cells were predominant
healthy. In the meanwhile, the density of cells obviously increased with
prolonged incubating time. The results of CCK-8 and fluorescent mi­
croscopy demonstrated the gelatin hydrogels were nontoxicity after Zr4+
treatment. These promising data indicated that this facile method had
potential for further application.
4. Conclusions
In this manuscript, cooling induced gelatin hydrogel was immerged
into Zr(SO4)2 dilute solution to improve their thermostability and me­
chanical properties. This facile and inexpensive method based on the
efficient coordination between Zr4+ and the active amino or carboxyl
groups in gelatins. The coordination bonds improved the rheological
modulus and compressive resistance of hydrogel. On the other hand,
gelatin hydrogels with 0.06 M Zr4+ can maintain the mechanical prop­
erties at relatively high temperature of 80 ◦ C without melt. Furthermore,
the thermostability and mechanical property of hydrogels can be regu­
lated by Zr4+ dosage while all the hydrogels were non-cytotoxic, sug­
gesting that this facile method can be adopted for many gelatin related
fields including food industry, cosmetic and tissue engineering.
CRediT authorship contribution statement
Fan Zheng: Data curation, Formal analysis, Investigation, Method­
ology, Writing - original draft, preparation.
Xiao Yang: Data curation, Formal analysis, Methodology, Visualiza­
tion, Writing - original draft, preparation.
Jiao Li: Investigation, Methodology.
Zhenhua Tian: Visualization, Methodology, Funding acquisition.
Bo Xiao: Supervision, Methodology, Funding acquisition.
Shixiong Yi: Conceptualization, Supervision, Visualization, Writing -
review & editing.
Lian Duan: Conceptualization, Methodology, Supervision, Visuali­
zation, Writing - original draft, Writing - review & editing, Funding
acquisition.
Declaration of competing interest
The authors declare they have no known competing financial in­
terests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This research was supported by the National Natural Science Foun­
dation of China (22008201, 82072060 and 21706151) and the Natural
Science Foundation Project of Chongqing, China (Grant No.
cstc2021jcyj-msxmX0240).
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