pubs.acs.

org/acsapm Article

Tough and Biocompatible Hydrogel Tissue Adhesives Entirely Based

on Naturally Derived Ingredients
Ziyi Xu, Haihui Zhang, Yuan Huang, Hao Zhong, Peiwu Qin, Sibo Cheng, Yuenan Wang,
and Canhui Yang*
Cite This: ACS Appl. Polym. Mater. 2024, 6, 1141−1151 Read Online

ACCESS Metrics & More Article Recommendations *

sı Supporting Information
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

ABSTRACT: Hydrogel tissue adhesives have tremendous poten-

tial applications in biological engineering. Existing hydrogel tissue
Downloaded via NEWCASTLE UNIV on February 14, 2024 at 18:40:55 (UTC).

adhesives generally do not have adequate mechanical robustness

and acceptable biocompatibility at the same time. Herein, we
report a one-step method to synthesize tough and biocompatible
hydrogel tissue adhesives entirely made of naturally derived
ingredients. We select two natural polymers, chitosan and gelatin,
to construct the backbone and a bioderived compound, genipin, as
the cross-linker. We show that, upon gelation, genipins cross-link
chitosan and gelatin to form two interpenetrated networks and
interlink them to tissue surfaces. Meanwhile, hydrogen bonds form
in the matrix to strengthen the networks and at the interface to
strengthen the adhesion between the hydrogel and tissue. Furthermore, we elaborately use high initial polymer contents to induce
topological entanglements in the polymer networks to toughen the hydrogel. The resulting chitosan−gelatin hydrogel provides a
tough matrix, and the robust covalent interlinks and hydrogen bonds provide a strong interface, achieving a tensile strength of ∼190
kPa, a fracture toughness of 205.7 J/m2, a mode I adhesion energy of 197.6 J/m2, and a mode II adhesion energy of 51.2 J/m2. We
demonstrate that the hydrogel tissue adhesive is injectable, degradable, and noncytotoxic and can be used for the controlled release
of the anticancer drug cisplatin. All-natural ingredient-based tough and biocompatible hydrogels are promising as tissue adhesives for
biomedical and related applications.
KEYWORDS: hydrogel, tissue adhesive, natural polymer, toughness, biocompatibility

■ INTRODUCTION
Adhesives that can adhere strongly to tissues have tremendous
typically on the order of 10 J/m2,16,17 which greatly limits the
applications. Highly desired is to develop new polymer-based
potential applications in biological engineering as diverse as tissue adhesive materials that are soft but can adhere strongly
to tissues.18
wound dressing,1 tissue repair,2 drug delivery,3 biomedical
A hydrogel is a three-dimensional polymer network
devices,4 and bioelectronic interfaces.5 Among the many types
infiltrated with a large amount of water. Hydrogels can be
of adhesives, polymer-based tissue adhesives are appealing
categorized as natural, synthetic, and hybrid hydrogels
alternatives to sutures in various types of wound closure, such
depending on the sources of the polymers.19 Tough hydrogel
as those in the skin,1 bone,6 vessels,7 heart,8 and organ
adhesion has two prerequisites: a strong interface and tough
transplantation,9 owing to their better hemostatic effect, less
matrix. A strong interface prevents interfacial crack prop-
equipment required, faster operation speed, and noninvasive
agation and enables efficient load transfer to elicit dissipation
nature.10,11
in the bulk, and a tough matrix dissipates energy to toughen
Over the past few decades, polymeric tissue adhesives have
the adhesion. Following this basic mechanical principle,
been extensively studied. In general, an ideal tissue adhesive is
significant progress has been made recently in forming tough
expected to possess the attributes of excellent biocompatibility,
adhesion between hydrogels and tissues using various tough
ease of use, biodegradability, and robust adhesion.12,13
Cyanoacrylate has long been the strongest commercially
available tissue adhesive, but it is cytotoxic and rigid and Received: August 19, 2023
generates enormous heat during its immediate polymerization Revised: December 8, 2023
upon exposure to water. Other commercial tissue adhesives Accepted: December 8, 2023
such as the fibrin glue14 and the polyethylene glycol−based Published: December 26, 2023
adhesives15 are soft and biocompatible, but the interfacial
adhesion is weak and the toughness of the matrix is low,

© 2023 American Chemical Society https://doi.org/10.1021/acsapm.3c01926

1141 ACS Appl. Polym. Mater. 2024, 6, 1141−1151
ACS Applied Polymer Materials pubs.acs.org/acsapm Article

Figure 1. Tough and biocompatible hydrogel tissue adhesive with injectability and degradability. The precursor contains naturally derived
ingredients of chitosan, gelatin, and genipin and is shear-thinning to be injectable. After injection, genipin cross-links chitosan and gelatin to form
two interpenetrated networks and interlinks them to the amino group-rich tissue surface. Meanwhile, hydrogen bonds form to strengthen the
networks and the interface between the hydrogel and tissue. In addition, topological entanglements form to further toughen the networks. Upon
separation, the robust interfacial adhesion elicits enormous dissipation in the hydrogel matrix, resulting in a tough adhesion. After degradation, the
hydrogel disintegrates and the tough adhesion vanishes. The chemical structures of the ingredients, the cross-link, the hydrogen bonds, and the
reactions between genipins and amino groups are illustrated.

hydrogels.20−27 However, most of the tough hydrogels consist
of synthetic polymers, e.g., polyacrylamide, that are prepared
from cytotoxic raw materials, e.g., acrylamide,28,29 making
biocompatibility an indispensable concern.
In terms of biocompatibility, natural polymers are more ideal
materials of choice for tissue adhesives and have been widely
used to synthesize hydrogels.30 Nevertheless, existing natural
polymer-based hydrogel tissue adhesives are often mechan-
ically weak and cannot form a tough adhesion with tissues.
Besides, the synthesis usually involves other cytotoxic
components as initiators and cross-linkers, e.g., glutaraldehyde
and carbodiimide,31 and the synthetic procedures such as
prolonged ultraviolate illumination and heating are not friendly
1142
to tissues (Table S1).32 Moreover, the preparation process can
be cumbersome, so the raw natural polymers need to be
modified with functional groups such as dopamine, vinyl
groups, adipic acid dihydrazide, or aldehyde moieties. There-
fore, developing tough hydrogel tissue adhesives entirely based
on naturally derived ingredients via facile and biocompatible
processes is significant but remains a central challenge.
Herein, we report a simple one-step method to synthesize
tough hydrogel tissue adhesives entirely based on naturally
derived ingredients. Note that we aim to propose a general
synthetic strategy instead of new chemistries. We use the well-
known natural polymers chitosan and gelatin to construct the
polymer backbone and the naturally derived compound
https://doi.org/10.1021/acsapm.3c01926
ACS Appl. Polym. Mater. 2024, 6, 1141−1151
ACS Applied Polymer Materials pubs.acs.org/acsapm Article

Figure 2. Mechanical characterizations. (a) Photographs showing a dumbbell-shaped hydrogel (φ70) being stretched to a strain of 2. (b) Nominal
uniaxial tensile stress−strain curves of hydrogels with different water contents. (c) Tensile moduli and works of fracture of various hydrogels. (d)
Photographs showing a cylindrical hydrogel (φ60, diameter: 1 cm, height: 5 mm) being compressed and springing back. (e) Nominal compressive
stress−strain curves of hydrogels with different water contents. (f) Compressive moduli and works of fracture of various hydrogels. (g) Photographs
showing the unnotched/notched hydrogels (φ70) at different deformation states. (h) Pure shear stress−strain curves of unnotched (solid curves)
and notched (dashed curves) hydrogels. (i) Fracture energies of various hydrogels.

genipin as the cross-linker. Chitosan, with marked biocompat-
ibility, biodegradability, nontoxicity, and cell binding ability,33
has been widely used in biomedical engineering.34−36 Gelatin
produces nontoxic and biocompatible products after degrada-
tion.37 Genipin is extracted from plants and exhibits excellent
biocompatibility38 and has been adopted as the cross-linking
agent for pharmaceutical applications.39 Although chitosan,
gelatin, and genipin have been used as constituent materials for
hydrogels, previously obtained hydrogels are mechanically
weak and cannot afford tough tissue adhesives (Table S2).
It has been noted recently that the mechanical properties of
hydrogels are profoundly affected by the synthesis con-
ditions.40−42 In this work, we rationalize the synthetic
procedures such that, upon gelation, genipin covalently
cross-links chitosan and gelatin to form two interpenetrated
networks and interlinks the two networks to tissues. The
resulting chitosan−gelatin hydrogel provides a tough matrix,
and the robust interlinks provide a strong interface. We use
high initial polymer contents such that topological entangle-
ments are formed during the formation of the network to
further toughen the hydrogel. The chitosan−gelatin hydrogel
achieves a tensile strength of ∼190 kPa, a fracture toughness of
1143
205.7 J/m2, a mode I adhesion energy of 197.6 J/m2, and a
mode II adhesion energy of 51.2 J/m2. Moreover, we show that
the hydrogel tissue adhesive is injectable, biodegradable, and
noncytotoxic and can be used for the controlled release of the
anticancer drug cisplatin.
■ RESULTS AND DISCUSSION
Principle of the Tough and Biocompatible Hydrogel
Tissue Adhesive. The principles of the tough and
biocompatible hydrogel tissue adhesive are illustrated in Figure
1. The precursor contains long-chain natural polymers chitosan
and gelatin and naturally derived cross-linker genipin. Chitosan
is a natural cationic polysaccharide consisting of D-glucos-
amine and N-acetyl-D-glucosamine units.43 Gelatin is a
mixture of peptide hydrolyzed from collagen.44 Genipin is an
aglycone derivative obtained from the enzymatic hydrolysis of
geniposide with β-glucosidase.45,46 In general, the hydrogel
precursor is viscous and non-Newtonian, featuring shear-
thinning, i.e. the viscosity reduces under shear strain. The
injectability is particularly advantageous in the scenarios of
tissue adhesives that the precursor can well conform to the
https://doi.org/10.1021/acsapm.3c01926
ACS Appl. Polym. Mater. 2024, 6, 1141−1151
ACS Applied Polymer Materials pubs.acs.org/acsapm Article

Figure 3. Tissue adhesion characterizations. (a) Schematics of the 90° peel test. (b) Force/width versus displacement curves for different
hydrogels. (c) Mode I adhesion energies of different hydrogels. (d) Lap-shear test of a layer of hydrogel sandwiched between two layers of porcine
skin. (e) Nominal stress−strain curves of different hydrogels under lap-shear. (f) Shear strengths and mode II adhesion energies of various
hydrogels under lap-shear. Photographs showing the robust adhesion of the hydrogel with (g) pig heart, (h) liver, and (i) bone.

surface asperities of tissue for tight sealing. Besides, delivering
the hydrogel tissue adhesive through injection is much less
invasive compared to surgical operations.
During the curing process, genipin cross-links chitosan and
gelatin to form a tough interpenetrated network. The cross-
linking mechanism has three folds: two genipin molecules can
cross-link two chitosan chains, two gelatin chains, or one
chitosan chain and one gelatin chain. The underlying
chemistry is that genipins react with the primary amino groups
to form secondary amides and heterocyclic amines.47 In
addition to covalent cross-links, abundant hydrogen bonds
form between chitosan chains, gelatin chains, and chitosan and
gelatin chains to strengthen the matrix.
Since the tissue surface is generally rich in primary amino
groups, during the curing process, genipin molecules interlink
chitosan and gelatin chains to the tissue surface by forming
covalent bonds.2 Besides, hydrogen bonds also form between
chitosan and gelatin chains and the tissue surface to further
strengthen the interface. Upon separation, the robust
interfacial adhesion enables efficient stress transmission from
the interface to the bulk network, eliciting enormous
dissipation in the hydrogel matrix, such as the break of
hydrogen bonds and the distributed chain scission caused by
network imperfection,48 to register a tough adhesion.
1144
Biomedical materials have excellent degradability, and it is
necessary for them to be absorbed by the surrounding tissues
during applications to avoid surgical resection. Both chitosan
and gelatin are biodegradable. For example, the lysozyme can
degrade chitosan into low molecular weight oligomers by
cutting the β-1,4 glycosidic bonds between the D-glucosamine
and D-acetylglucosamine units under weakly acidic con-
ditions.49 As another example, the peptide chains of gelatin
will be broken into low molecular weight amino acids or
polypeptides under the action of a collagenase degradation
accelerator.50 After degradation, the hydrogel disintegrates, and
the tough adhesion vanishes spontaneously without any
cytotoxic degradation products.
Mechanical Properties of Chitosan−Gelatin Hydro-
gels. Robust mechanical properties are crucial for hydrogel
tissue adhesives to resist premature cohesive rupture. We
perform uniaxial tension, compression, and pure shear tests to
assess the mechanical properties. A dumbbell-shaped sample
can withstand a tensile strain of 200% (Figure 2a). The
synthesis conditions have prominent effects on the mechanical
properties of the hydrogels synthesized from monomers.42 We
synthesized three types of chitosan−gelatin hydrogels with
different water contents for the precursor, i.e., 60, 70, and 80%.
The hydrogel precursor becomes too viscous to manipulate
when the water content is below 60%. The uniaxial tensile
https://doi.org/10.1021/acsapm.3c01926
ACS Appl. Polym. Mater. 2024, 6, 1141−1151
ACS Applied Polymer Materials
Figure 4. Rheological characterizations. (a) Photographs showing the sol−gel transition of precursors in a bottle (top row) and a star-shaped mold
(bottom row). (b) Storage modulus (G’) and loss modulus (G”) vary with time. (c) Viscosity varies with time. (d) Gelation time (T1) and
saturation time (T2) for different hydrogels.
stress−strain curves show that the hydrogel with 60% water
content possesses the highest mechanical strength (Figure 2b),
whereas the rupture strains are mostly the same, ∼220%. The
strengths are 192.16, 53.96, and 29.93 kPa, the tensile moduli
are 74.02, 20.19, and 9.91 kPa, and the works of fracture are
166.27, 61.74, and 15.49 kJ/m3 for the water contents of 60,
70, and 80%, respectively (Figure 2c).
The strength decreases with the water content since the
density of polymer chains decreases while the modulus does
not. For a hydrogel consist of Gaussian chains, the modulus is
negatively proportional to water content φ
E (1 )NkT
where N is the number of polymer chains per unit volume, k is
the Boltzmann constant, and T is the absolute temperature.
Taking the water contents of 60 and 80% as examples, the
theoretical ratio of Eφ60/Eφ80 is 2, which is much lower than the
d d
experimental value, 7.47. The giant deviation between theory
and experiment manifests the effects of synthesis conditions
and is affected by the topological entanglements, which
function as additional cross-links to increase the effective
value of N, during the formation of the hydrogel network.40
Figure 2d shows that a cylindrical sample can sustain
compression and return to its original state. Under the
compression test, similarly, the hydrogel with 60% water
content exhibits the highest compressive strength, while the
rupture strains of all hydrogels are about 80% (Figure 2e). The
compression moduli are 217.01 79.55, and 31.84 kPa, and the
works of fracture are 22.72, 14.51, and 5.76 kJ/m3 for the water
contents of 60, 70, and 80%, respectively (Figure 2f). Figure 2g
shows the images of the pure-shear samples, with/without a
notch, at different strains, and Figure 2h plots the stress−strain
curves of various unnotched (solid curves)/notched (dashed
curves) hydrogels. Figure 2i shows the histogram of the
average fracture energies of the different hydrogels. Again,
pubs.acs.org/acsapm Article
owing to the toughening effects of topological entangle-
ments,40 the fracture energy of the hydrogel with 60% water,
205.73 J/m2, is higher than that of the hydrogel with 80%
water, 12.42 J/m2, by 1 order of magnitude.
Adhesion Performances. The adhesion between the
chitosan−gelatin hydrogel and tissue has not been quantified
before. We perform the 90° peel test to measure the mode I
adhesion energy between the hydrogel and porcine skin
(Figure 3a). The peel force versus displacement curves of the
hydrogels with different water contents are plotted in Figure
3b. The mode I adhesion energies are 197.61 67.31, and 39.11
N/m (Figure 3c) for the hydrogels with 60, 70, and 80% water
(1)
contents, respectively.
We further perform the lap-shear test to measure the mode
II adhesion energy by sandwiching a layer of chitosan−gelatin
hydrogel between two layers of porcine skin, which are in turn
glued to two PMMA plates for chasing (Figure 3d). The
hydrogels with 70% water content exhibit the highest shear
strength (Figure 3e). The samples with 60% water content
undergo adhesive failure, and only half contain hydrogel
residues. In comparison, the samples with 70 and 80% water
contents undergo cohesive failure. The hydrogels of φ60 having
the highest modulus deform the least, while the hydrogels of
φ80 having the lowest strength and toughness dissipate the least
energy. Overall, the hydrogels of φ70 have optimal perform-
ances. Specifically, the strengths are 19.51, 26.25, and 13.75
kPa, and the mode II adhesion energies are 41.66, 51.19, and
26.73 J/m2 for the hydrogels with 60, 70, and 80% water
contents, respectively (Figure 3f).
We demonstrate the appreciable adhesion between the
chitosan−gelatin hydrogel and pig heart (Figure 3g) and liver
(Figure 3h) by laminating and then peeling the hydrogel layer
from the tissues. It can be seen that both tissues have been
substantially deformed, indicating good adhesion. Using the
hydrogel as an injectable arthrosis glue (Figure 3i), we show
1145 https://doi.org/10.1021/acsapm.3c01926
ACS Appl. Polym. Mater. 2024, 6, 1141−1151
ACS Applied Polymer Materials
Figure 5. Degradation characterizations. (a) Sequential images showing the degradation processes of different hydrogels in PBS buffer solution and
enzyme solution. (b) Percentages of degradation vary with time for different hydrogels.
that the hydrogel tissue adhesive can withstand the gravity of
100 g of weight.
Injectability. Injectability is often desired for tissue
adhesives for the sake of high moldability, minimal invasion,
and easy and effective drug/cell encapsulation. The chitosan−
gelatin hydrogels exhibit a highly tunable injectability. A light-
yellow chitosan−gelatin hydrogel precursor can be cured inside
a bottle or be injected into a start-shaped mold and then cured
(Figure 4a). We conduct rheological measurements to quantify
the injectability of the chitosan−gelatin hydrogel. The viscosity
of the chitosan−gelatin solution decreases with shear rate, a
feature of shear-thinning liquid (Figure S1). Figure 4b plots
the variations of the storage modulus (G’) and loss modulus
(G″) with time for hydrogel precursors with different water
contents. When the G” is greater than the G’, the hydrogel
precursor behaves liquid-like; when the G’ is greater than the
G”, the hydrogel precursor behaves solid-like. The intersection
between G’ and G″ determines the gelling point. The time at
the gelling point is defined as the gelling time, denoted as T1.
The final storage moduli are 76, 23, and 12 kPa, respectively,
for 60, 70, and 80% water contents. Since the hydrogel
precursor contains a cross-linker, the viscosity of the precursor
will gradually increase as the cross-linking reaction proceeds.
Figure 4c plots the variations of viscosity with time for the
different hydrogels. As expected, throughout the test, viscosity
increases monotonically with time and the viscosity of the
hydrogel with 60% water content is the highest.
The injection of the hydrogel precursor should be operated
within T1 since the precursor may lose injectability after the
gelling point. To guarantee a smooth injection process requires
not only optimization of the rheological properties of the
hydrogel precursor but also the parameters of the injection
instruments such as the injection speed and the length and
radius of the needle, as well as the environment such as
temperature and pH value. On the other hand, the hydrogel
1146
pubs.acs.org/acsapm Article
needs to be cured appreciably for reliable mechanical
robustness before functioning. In our experiments, we define
the time when the viscosity reaches 80% of the plateau
viscosity as the saturation time T2. The gelling times are 117,
871, and 1174 s and the saturation times so defined are 6593,
7089, and 7338 s for different water contents (Figure 4d). In
practice, the gelling time should be tuned according to the
specific application scenarios while the saturation time
generally should be as short as possible. Note that the cross-
linking reaction of genipin with the amino groups is benign and
thus could proceed in vivo.
Degradability. As mentioned before, in situ degradation is
advantageous for hydrogel tissue adhesives to eliminate the
need of surgical removal, which often causes secondary trauma.
Degradation mechanisms include hydrolysis, erosion, and
solubilization. In particular, chitosan is the only natural
cationic polymer that can be degraded by human enzymes
due to its reactive amino groups, and gelatin can be easily
degraded by collagenase. We carry out degradation character-
izations by soaking the chitosan−gelatin hydrogels with
different water contents in a PBS buffer solution and enzyme
solution. The sequential images in Figure 5a show the
morphological changes of different samples at different times.
Figure 5b plots the variation of the degradability with time.
For the samples in PBS solution, before functioning, the
active chemicals need to diffuse into the hydrogel network,
which is associated with diffusion of water molecules. The
imbibition of water leads to the swelling of the hydrogel.
Overall, the hydrogels with an initial water content of 70%
have a balanced chemical potential of water and network
elasticity and exhibit the smallest degradation values. The
degradation values of the samples soaked in enzyme solutions
are all smaller than those of the samples soaked in PBS
solutions since the enzyme can destroy the chemical bonds of
the backbones of chitosan and gelatin.
https://doi.org/10.1021/acsapm.3c01926
ACS Appl. Polym. Mater. 2024, 6, 1141−1151
ACS Applied Polymer Materials pubs.acs.org/acsapm Article

Figure 6. Swelling and drug release characterizations and in vitro cytotoxicity test. (a) Swelling kinetics of different hydrogels in deionized water.
The solid curves are fittings. (b) Schematics of the swelling of different hydrogels. (c) Comparison of storage modulus (G’) and loss modulus (G″)
before and after swelling. (d) Release kinetics of cisplatin of different hydrogels in PBS solution. The solid curves are fittings. The inset shows the
chemical structure of cisplatin. (e) Saturated amount of released drug versus the saturated time for different hydrogels. (f) Schematic illustrating the
diffusion of a cisplatin molecule (green circle) within the hydrogel network of mesh size ξ under the confinement of hydrogen bonds. (g) Live cell
fluorescence imaging (calcein-positive) of HEK293T cells after the treatment with different concentrations of hydrogel extract solution. (h)
Cytotoxicity test of different hydrogel extracts after culturing for 24, 48, and 72 h.

Dynamics of Swelling and Drug Release. Hydrogels are
ideal depots of therapeutic agents. Loading drugs into the
hydrogel matrix endows a hydrogel tissue adhesive with
additional functionalities such as accelerated wound healing
and antibacterial properties. Upon deployment, the hydrogel
tissue adhesives often undergo swelling, which enhances the
release of loaded drugs. Figure 6a plots the variations of the
swelling ratios of different hydrogels with time. The swelling
ratio is defined as Δm/m, where Δm is the change of mass and
m is the original mass. Assuming a free diffusion process, we
can fit the experimental data to the following formula:
(x / )
y = (1 e )
where x represents the swelling time, y represents Δm/m, α is a
dimensionless fitting parameter, and β is a fitting parameter
with a unit of time. Specifically, β represents the characteristic
swelling time. The characteristic times are 38.5, 37.1, and 18.4
h for 60, 70, and 80% water contents, respectively. For the
samples of size L ∼ 4 mm and the diffusion coefficient of water
D ∼ 10−10 m2/s, we estimate the characteristic swelling time to
be L2/D ∼ 105 s, which is in the same order of magnitude with
the experiments.
Experimental results show that the higher the initial water
content, the higher the saturated swelling ratio, which is
consistent with the literature.42 A higher initial polymer
content (i.e., a lower initial water content) will lead to more
topological entanglements between polymer chains, which act
as additional cross-links to strengthen the polymer network to
resist swelling (Figure 6b). As expected, both the storage
modulus (G’) and loss modulus (G”) decrease after swelling
(Figure 6c). Nevertheless, due to the topological entangle-
ments, the equilibrium storage modulus decreases with the
(2)
increase in the initial water content. The storage moduli after
swelling equilibrium are 829.8, 360.4, and 172.9 Pa for 60, 70,
and 80% water contents, respectively.
A piece of therapeutic agent-loaded hydrogel tissue adhesive
is a delivery platform for local administration. We loaded the
chitosan−gelatin hydrogel with cisplatin and characterized the
drug release. Cisplatin is a chemotherapy medication widely
used in clinics to treat a number of cancers. The molecular
1147 https://doi.org/10.1021/acsapm.3c01926
ACS Appl. Polym. Mater. 2024, 6, 1141−1151
ACS Applied Polymer Materials
structure of cisplatin is the square planar coordination complex
cis-[Pt(NH3) 2Cl 2 ]. Because the release of cisplatin is
accompanied by swelling, it can be seen from Figure 6d that
the release kinetics of cisplatin is similar to the swelling
kinetics. The highest water content (φ80) with the highest
swelling ratio gives the fastest release rate and the highest
saturated drug release ratio. Fitting the drug release data to the
above exponential formula, with x representing the drug release
time and y representing the drug release, gives the character-
istic times of 15.4, 12.9, and 9.0 h for 60, 70, and 80% water
contents, respectively.
The saturated drug release ratios are 47.1, 38.7, and 28.2 for
80, 70, and 60% water contents, respectively (Figure 6e). The
amino groups of cisplatin form hydrogen bonds with the
functional groups on the chitosan−gelatin network. When the
initial water content is high, the mesh size of the hydrogel
network is large and the confinement of cisplatin is small due
to the larger distance. The hydrogel with a higher initial water
content has faster swelling kinetics and a larger saturated
swelling ratio, which further facilitates the release of cisplatin
(Figure 6f). The controlled drug release and the good adhesion
of the tough and biocompatible chitosan−gelatin hydrogel to
native tissue are expected to be potentially used for the
targeted elimination of tumors. Besides, the chitosan−gelatin
hydrogel can be loaded with therapeutic agents on demand for
specific applications.
An ideal biomaterial should be nontoxic. We investigated the
cytotoxicity of the chitosan−gelatin hydrogels by evaluating
the in vitro cell viability of the extracted media leached from
the chitosan−gelatin hydrogels with different water contents.
We used fluorescence imaging and lactate dehydrogenase
(LDH) assays and observed the living cells through confocal
laser scanning microscopy (CLSM). The CLSM images
demonstrate that cell viability is not significantly affected
after culturing with different concentrations of extracted
hydrogel media compared to the control group (Figure 6g).
We further analyze the cell viability by LDH release when
treating cells with different concentrations of hydrogel extracts
for 24, 48, and 72 h. Cell viabilities of all hydrogel precursor-
treated media are higher than 80% (Figure 6h), which are
comparable to that of the untreated controls. Moreover, we
conduct a one-way variance analysis using IBM SPSS Statistics
and find no significant difference (P > 0.05) in cell viability
between the samples with different concentrations of hydrogel
extracts and the control group. These results confirm that the
chitosan−gelatin hydrogel possesses excellent biocompatibility.
The mechanical properties of the chitosan−gelatin hydrogel
should be decent to meet many biomedical applications,
although they are still relatively weak compared to those of
tough synthetic tough hydrogels. The main reason is that the
hydrogel network of our hydrogel is amorphous, entirely made
of natural polymers, and covalently cross-linked without any
further treatment, for the sake of biocompatibility and ease of
synthesis. The bulky side chains and the strong intra/
interchain interactions restrict the flexibility of the polymer
chains such that the configuration of the polymer chain is more
difficult to change to adapt to the applied force. Besides, the
natural polymer-based network is formed by preformed long-
chain polymers so that the network topology is much less
flexible. Subject to an applied force, the natural polymer-based
network more easily concentrates stress and causes the nearby
chains to break. The mechanical properties of natural polymer-
based hydrogels could be improved via, e.g., modification of
1148
pubs.acs.org/acsapm Article
functional groups, introduction of nanocrystalline domains, or
constructing networks with enormous entanglements.41 A
mechanical property-biodegradability conflict exists in which
the mechanical properties will deteriorate when the hydrogel
network undergoes biodegradation. Nevertheless, the conflict
can be circumvented by tailoring the function times. For
instance, the characteristic time needed for appreciable
biodegradation can be rationally tuned so that the hydrogel
can maintain its mechanical robustness within a certain time
window. The proposed strategy, in principle, applies to other
biopolymers rich in amino groups. For example, carboxymethyl
chitosan can be used and the obtained genipin cross-linked
carboxymethyl chitosan−gelatin hydrogel exhibits decent
mechanical properties and good adhesion to the liver (Figure
S2).
■ CONCLUSIONS
In conclusion, we have reported tough and biocompatible
hydrogel tissue adhesives completely based on naturally
derived ingredients. The hydrogel contains a tough matrix
consisting of two interpenetrating networks of chitosan and
gelatin cross-linked by genipin, which also covalently interlinks
the hydrogel matrix to tissue surfaces. Topological entangle-
ments are formed to toughen the chitosan−gelatin hydrogel
matrix and thus the adhesion by increasing the initial polymer
contents. We have shown that the chitosan−gelatin hydrogel is
injectable and biodegradable with tailorable injection and
biodegradation dynamics. Moreover, we have demonstrated
the drug loading and release performances as well as excellent
biocompatibility of the chitosan−gelatin hydrogel. This work
puts a step toward tough and biocompatible hydrogel tissue
adhesives entirely based on naturally derived ingredients for
injectable drug-loaded hydrogel materials for biomedicine and
engineering.
■ EXPERIMENTAL SECTION
Materials. We procured chitosan (CS, H1928001) and acetic acid
(G2121128) from Aladdin, gelatin (Gel, CLP813) from Shanghai
Biad Pharmaceutical Technology Co., Ltd., and genipin (E100770)
from Anergy Co., Ltd. All chemicals were obtained without further
purification.
Preparation of Hydrogels. First, gelatin, chitosan, and 1 mL of
deionized water were mixed in a 4 mL centrifuge tube at 65 °C to
dissolve gelatin. Then, a genipin solution was added at a
concentration of 0.035 g/mL. Finally, 20 μL of glacial acetic acid
was added to dissolve chitosan and catalyze the chemical reaction
between genipin and the amino groups on the polymer chains. The
pH value of the precursor was ∼7. After curing, the once light-yellow
liquid-state precursor became a dark-green solid-state hydrogel. The
masses of gelatin Ma1 = 0.83 g, Ma2 = 0.42 g, and Ma3 = 0.27 g,
chitosan Mb1 = 0.067 g, Mb2 = 0.034 g, and Mb3 = 0.022 g, and genipin
solution Vc1 = 25.6 μL, Vc2= 12.8 μL, and Vc3 = 8.5 μL were used to
prepare the hydrogels with water contents of 60, 70, and 80%,
respectively. The water content was quantified by weight percentage.
Rheological Tests. Rheological measurements were conducted to
evaluate the gelling processes of the precursors and the viscoelastic
properties of the cured hydrogels by using a rheometer (TA
Instruments HR30) equipped with a Peltier plate for temperature
control. A parallel steel geometry (diameter: 20 mm) was used for all
experiments. Oscillatory measurements were performed at a constant
frequency of 1 Hz and a small strain of 0.1%. The precursor was
added to the loading platform of the rheometer, and then the gelation
process was monitored by measuring the evolutions of storage and
loss moduli (G’ and G”, respectively) as a function of time at 37 °C.
To prevent the hydrogel from dehydration during the test, a
https://doi.org/10.1021/acsapm.3c01926
ACS Appl. Polym. Mater. 2024, 6, 1141−1151
ACS Applied Polymer Materials
humidifier was used to pump humid moisture during the test. Each
test was repeated at least three times.
Mechanical Tests. For compression, a cylindrical acrylate mold
with dimensions of 10 mm in diameter and 5 mm in height was
prepared by using a laser cutter (CMA0604-B-A), and the precursor
was poured into the mold to prepare cylindrical samples. The samples
were loaded to a mechanical testing machine (Instron 5966) with a
100 N load cell and compressed at a loading velocity of 30 mm/min
at 25 °C. The force−displacement curves were recorded to calculate
the stress−strain curves. Unless otherwise specified, nominal stress
and nominal strain were used throughout the text.
For uniaxial tension, a large 2 mm-thick sheet was prepared,
incubated for 30 min at 65 °C, and aged for 20 min at 25 °C. Then,
dumbbell-shaped samples with a gauge length of 12 mm and a width
of 2 mm were cut from the sheet. The samples were loaded to a
mechanical testing machine (Instron 5966) with a 100 N load cell and
elongated at a loading velocity of 30 mm/min at 25 °C. The force−
displacement curves were recorded to calculate the stress−strain
curves. The work of fracture is defined as the energy per unit volume
needed to break the hydrogel and calculated as the area underneath
the stress−strain curve
c
Wf = ( )d
0 (3)
where εc is the rupture strain.
To measure the fracture toughness, two sets of samples (width ×
length × thickness: 1 × 7 × 0.2 cm3) of identical dimensions were
prepared, with one being precut with a single-edge notch along the
middle line of the sample and the other being intact. For the notched
samples, we made a precrack of 20 mm using a razor blade. After the
sample was loaded to the testing machine, the effective width (along
the loading direction) of the sample is 10 mm. The size of the
precrack was much larger than the width while much smaller than the
length of the sample. The unnotched samples are stretched along the
direction of length (may or may not up to rupture), and their nominal
stress−strain curves were measured. The notched samples were
stretched until the notch started to propagate catastrophically at a
critical strain εc. The fracture toughness of the hydrogel is calculated
by
c
=H ( )d
0 (4)
where H is the width of the sample along the loading direction at the
undeformed state and σ is the nominal stress.
The 90° peel test was conducted to measure the mode I adhesion
energy. A piece of chitosan−gelatin hydrogel sample (width × length
× thickness: 2 × 10 × 0.1 cm3) was in situ synthesized on pigskin,
which was fixed on an acrylic plate. A sheet of nonwoven fabric (width
× length: 2.5 × 20 cm2) was used as the soft but inextensible backing.
The sample was loaded onto an Instron 5966 machine with a 100 N
load cell and pulled at a loading speed of 30 mm/min. The peel
force−displacement curves were recorded to calculate the mode I
adhesion energy. As the peel displacement increases, the peel force
increases and then plateaus at a steady state value. At the steady state,
the mode I adhesion energy is calculated as
Fss
I =
W (5)
where Fss is the steady-state peel force and W is the width of the
sample.
The lap-shear test was conducted to measure the mode II adhesion
energy. A layer of 0.5 mm-thick chitosan−gelatin hydrogel was
prepared in situ between two pieces of porcine skin. Two acrylic plates
were glued to the porcine skin pieces and then clamped to the Instron
5966 machine with a 100 N load cell and pulled at a loading speed of
30 mm/min. The lap-shear force−displacement curves were recorded
to calculate the mode II adhesion energy. The mode II adhesion
energy is calculated as
1149
pubs.acs.org/acsapm Article
c
II =t ( )d
0 (6)
where t is the thickness of the sandwiched hydrogel layer at the
undeformed state, γc is the nominal rupture shear strain, and τ is the
nominal shear stress.
Swelling Test. The initial masses of the hydrogel samples with
different water contents were measured. Then, the samples were
soaked in phosphate buffered saline (PBS) buffer solutions. After
some time, the weights of the hydrogels were measured again. The
selected time intervals were 0.5, 1, 2, 3, 6, 9, 24, 30, 48, 56, 68, 72, 84,
96, 120, 150, 180, 200, and 250 h. Each measurement was repeated
three times.
Degradation Test. Cubic samples (1 × 1 × 1 cm3) were
immersed in ethanol for 24 h to remove the unreacted contents.
Then, the samples were freeze-dried for 48 h and weighed.
Subsequently, the samples were immersed in pure PBS solution
(control group) and PBS solution containing lysozyme (1 mg/mL)
and collagenase (0.16 mg/mL) (test group).51,52 The samples were
cultured at 37 °C, and the solutions were renewed every 2 days.
Finally, the samples were taken out at different times, washed with
ethanol for 3 h, and freeze-dried for 24 h. The percentage of weight
loss was calculated using the following formula:
Mf
remaining weight = × 100%
Mi (7)
where “Mi” is the initial weight of the freeze-dried hydrogel before
degradation and “Mf” is the weight of the freeze-dried hydrogel after
degradation. Each test was repeated at least five times.
In Vitro Drug Release. Cisplatin was selected as the model drug
in this study. To prepare the drug-loaded hydrogels, 4 μL of cisplatin
solution (2.5 mg/mL) was added to the hydrogel precursors. The
concentration of cisplatin in the resulting hydrogels is 10 μg/mL.
Hydrogel samples (0.15 g) were immersed in 50 mL of PBS solution
(pH 6.8) at 37 °C. Then, 5 mL of the solution was taken out at
different times, namely, 0.5, 1, 2, 3, 6,12, 24, 30, 36, 48, 60, 72, 84, and
96 h; meanwhile, 5 mL of fresh PBS was added. The abstracted
solution was added with 500 μL of concentrated nitric acid, sealed,
and placed in an oven at 65 °C overnight. Finally, the platinum (Pt)
concentration in the sample was detected by inductively coupled
plasma-mass spectrometry (ICP-MS) to obtain the drug release curve.
In Vitro Cytotoxicity Test. The cytotoxicity detection kit
(Roche) was used to measure the in vitro cytotoxicity of hydrogels
according to the supplier’s protocol. The principle of measurement is
based on lactate dehydrogenase (LDH) release from damaged cells to
media. The cell viability was calculated by the percentage of treated
cells with respect to untreated cells. The hydrogel (5 g) was first
washed with PBS (pH 7.4) three times. Next, the hydrogel was
incubated with Dulbecco’s Modified Eagle Medium (Gibco) with
10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL
streptomycin (Thermo Fisher Scientific) for 30 min at 37 °C. To
obtain the hydrogel extract stock solution, different concentrations of
hydrogel were immersed with 20 mL of complete growth media for 24
h at 37 °C and then passed through a 0.22 μm filter. NIH-3T3 cells
(ATCC) were plated in a 96-well plate (Corning) at a density of 5000
cells per well and incubated for 24 h at 37 °C and 5% CO2 in a
humidified incubator. Cells were treated with 100 μL of different
concentrations of hydrogel extract solutions and cultured for 24, 48,
and 72 h. Each hydrogel extract (50 μL) was transferred to a new 96-
well plate and mixed with 50 μL of reaction mixture for 30 min.
Absorbance was measured at 490 nm by using a plate reader (Tecan
Microplate Reader Spark). For fluorescence imaging, the cells were
transfected with TagBFP fluorescent proteins and cultured with
different concentrations of hydrogel extract solutions (0, 60, and 80%)
for 48 h and then imaged by a confocal microscope (Nikon A1R SI
Confocal).
https://doi.org/10.1021/acsapm.3c01926
ACS Appl. Polym. Mater. 2024, 6, 1141−1151
ACS Applied Polymer Materials
■ ASSOCIATED CONTENT
* Supporting Information
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsapm.3c01926.
Variations of viscosity with shear rate, performances of
the carboxymethyl chitosan−gelatin hydrogel, prepara-
tion and properties of natural polymer-based hydrogels
and hydrogels based on chitosan, gelatin, and genipin,
fitting parameters for swelling and drug release (PDF)
■ AUTHOR INFORMATION
Corresponding Author
Canhui Yang − Shenzhen Key Laboratory of Soft Mechanics
and Smart Manufacturing, Department of Mechanics and
Aerospace Engineering, Southern University of Science and
Technology, Shenzhen 518055, China; orcid.org/0000-
0001-5674-834X; Email: yangch@sustech.edu.cn
Authors
Ziyi Xu − Shenzhen Key Laboratory of Soft Mechanics and
Smart Manufacturing, Department of Mechanics and
Aerospace Engineering, Southern University of Science and
Technology, Shenzhen 518055, China
Haihui Zhang − Institute of Biopharmaceutical and Health
Engineering, Tsinghua Shenzhen International Graduate
School, Tsinghua University, Shenzhen 518055, China
Yuan Huang − Shenzhen Key Laboratory of Soft Mechanics
and Smart Manufacturing, Department of Mechanics and
Aerospace Engineering, Southern University of Science and
Technology, Shenzhen 518055, China
Hao Zhong − Shenzhen Key Laboratory of Soft Mechanics
and Smart Manufacturing, Department of Mechanics and
Aerospace Engineering, Southern University of Science and
Technology, Shenzhen 518055, China
Peiwu Qin − Institute of Biopharmaceutical and Health
Engineering, Tsinghua Shenzhen International Graduate
School, Tsinghua University, Shenzhen 518055, China;
orcid.org/0000-0002-7829-8973
Sibo Cheng − Suzhou Soft Intelligent Materials Co. Ltd.,
Suzhou 215123, China
Yuenan Wang − Department of Therapeutic Radiology, Yale
University, New Haven, Connecticut 06511, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsapm.3c01926
Author Contributions
C.Y. and Z.X. conceived the research ideas and designed the
research. Z.X. designed, synthesized, and characterized the
mechanical properties and drug release performances. H.Z.
conducted the cytotoxicity tests. Y.H. synthesized and
characterized the carboxymethyl chitosan-based hydrogels.
Z.X. analyzed the experimental results with inputs from other
authors. Z.X. drafted the manuscript. C.Y. revised the
manuscript with inputs from other authors. C.Y. supervised
the study.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This work is supported by the National Key Research and
Development Program of China (grant 2023YFB3812500), the
Science, Technology, and Innovation Commission of Shenz-
1150
pubs.acs.org/acsapm Article
hen Municipality (ZDSYS20210623092005017), the National
Natural Science Foundation of China (no. 12302212), the
Natural Science Foundation of Guangdong Province
(2022A1515010601), and the Stable Support Plan Program
of Shenzhen Natural Science Fund Grant
(20200925174603001). The authors acknowledge the assis-
tance of SUSTech Core Research Facilities.
■ REFERENCES
(1) Li, J.; Celiz, A. D.; Yang, J.; Yang, Q.; Wamala, I.; Whyte, W.;
Seo, B. R.; Vasilyev, N. V.; Vlassak, J. J.; Suo, Z.; Mooney, D. J. Tough
adhesives for diverse wet surfaces. Science 2017, 357 (6349), 378−
381.
(2) Yuk, H.; Varela, C. E.; Nabzdyk, C. S.; Mao, X.; Padera, R. F.;
Roche, E. T.; Zhao, X. Dry double-sided tape for adhesion of wet
tissues and devices. Nature 2019, 575 (7781), 169−174.
(3) Li, J.; Mooney, D. J. Designing hydrogels for controlled drug
delivery. Nat. Rev. Mater. 2016, 1 (12), 16071.
(4) Feiner, R.; Engel, L.; Fleischer, S.; Malki, M.; Gal, I.; Shapira, A.;
Shacham-Diamand, Y.; Dvir, T. Engineered hybrid cardiac patches
with multifunctional electronics for online monitoring and regulation
of tissue function. Nat. Mater. 2016, 15 (6), 679−685.
(5) Yuk, H.; Wu, J. J.; Zhao, X. H. Hydrogel interfaces for merging
humans and machines. Nat. Rev. Mater. 2022, 7 (12), 935−952.
(6) Hoffmann, B.; Volkmer, E.; Kokott, A.; Augat, P.; Ohnmacht,
M.; Sedlmayr, N.; Schieker, M.; Claes, L.; Mutschler, W.; Ziegler, G.
Characterisation of a new bioadhesive system based on poly-
saccharides with the potential to be used as bone glue. Journal of
Materials Science-Materials in Medicine 2009, 20 (10), 2001−2009.
(7) Giraudo, M. V.; Di Francesco, D.; Catoira, M. C.; Cotella, D.;
Fusaro, L.; Boccafoschi, F. Angiogenic Potential in Biological
Hydrogels. Biomedicines 2020, 8 (10), 436.
(8) Hong, Y.; Zhou, F.; Hua, Y.; Zhang, X.; Ni, C.; Pan, D.; Zhang,
Y.; Jiang, D.; Yang, L.; Lin, Q.; Zou, Y.; Yu, D.; Arnot, D. E.; Zou, X.;
Zhu, L.; Zhang, S.; Ouyang, H. A strongly adhesive hemostatic
hydrogel for the repair of arterial and heart bleeds. Nat. Commun.
2019, 10, 2060.
(9) Liu, K.; Yang, H.; Huang, G. B.; Shi, A. H.; Lu, Q.; Wang, S. P.;
Qiao, W.; Wang, H. H.; Ke, M. Y.; Ding, H. F.; Li, T.; Zhang, Y. C.;
Yu, J. W.; Ren, B. Y.; Wang, R. F.; Wang, K. L.; Feng, H.; Suo, Z. G.;
Tang, J. D.; Lv, Y. Adhesive anastomosis for organ transplantation.
Bioact. Mater. 2022, 13, 260−268.
(10) Ghobril, C.; Grinstaff, M. W. The chemistry and engineering of
polymeric hydrogel adhesives for wound closure: a tutorial. Chem. Soc.
Rev. 2015, 44 (7), 1820−1835.
(11) Ma, Y.; Yao, J.; Liu, Q.; Han, T.; Zhao, J.; Ma, X.; Tong, Y.; Jin,
G.; Qu, K.; Li, B.; Xu, F. Liquid Bandage Harvests Robust Adhesive,
Hemostatic, and Antibacterial Performances as a First-Aid Tissue
Adhesive. Adv. Funct. Mater. 2020, 30 (39), 2001820.
(12) Ahmed, E. M. Hydrogel: Preparation, characterization, and
applications: A review. J. Adv. Res. 2015, 6 (2), 105−121.
(13) Zhou, L.; Dai, C.; Fan, L.; Jiang, Y.; Liu, C.; Zhou, Z.; Guan, P.;
Tian, Y.; Xing, J.; Li, X.; Luo, Y.; Yu, P.; Ning, C.; Tan, G. Injectable
Self-Healing Natural Biopolymer-Based Hydrogel Adhesive with
Thermoresponsive Reversible Adhesion for Minimally Invasive
Surgery. Adv. Funct. Mater. 2021, 31 (14), 2007457.
(14) Sierra, D. H. Fibrin Sealant Adhesive Systems: A Review of
Their Chemistry, Material Properties and Clinical Applications. J.
Biomater. Appl. 1993, 7 (4), 309.
(15) Wallace, D. G.; Cruise, G. M.; Rhee, W. M.; Schroeder, J. A.;
Prior, J. J.; Ju, J.; Maroney, M.; Duronio, J.; Ngo, M. H.; Estridge, T.;
Coker, G. C. A tissue sealant based on reactive multifunctional
polyethylene glycol. J. Biomed. Mater. Res. 2001, 58 (5), 545−555.
(16) Thistlethwaite, P. A.; Luketich, J. D.; Ferson, P. F.; Keenan, R.
J.; Jamieson, S. W. Ablation of persistent air leaks after thoracic
procedures with fibrin sealant. Annals of Thoracic Surgery 1999, 67
(2), 575−577.
https://doi.org/10.1021/acsapm.3c01926
ACS Appl. Polym. Mater. 2024, 6, 1141−1151
ACS Applied Polymer Materials
(17) Dastjerdi, A. K.; Pagano, M.; Kaartinen, M. T.; McKee, M. D.;
Barthelat, F. Cohesive behavior of soft biological ″glues″. Experiments
and modeling. 2012, 8 (9), 3349−3359.
(18) Giri, T. K.; Thakur, D.; Alexander, A.; Ajazuddin; Badwaik, H.;
Tripathi, D. K. Alginate based Hydrogel as a Potential Biopolymeric
Carrier for Drug Delivery and Cell Delivery Systems: Present Status
and Applications. Curr. Drug Delivery 2012, 9 (6), 539−555.
(19) Buwalda, S. J.; Boere, K. W. M.; Dijkstra, P. J.; Feijen, J.;
Vermonden, T.; Hennink, W. E. Hydrogels in a historical perspective:
From simple networks to smart materials. J. Controlled Release 2014,
190, 254−273.
(20) Yang, J.; Bai, R.; Chen, B.; Suo, Z. Hydrogel Adhesion: A
Supramolecular Synergy of Chemistry, Topology, and Mechanics.
Adv. Funct. Mater. 2020, 30 (2), 1901693.
(21) Yang, C. H.; Suo, Z. G. Hydrogel ionotronics. Nat. Rev. Mater.
2018, 3 (6), 125−142.
(22) Yuk, H.; Zhang, T.; Lin, S. T.; Parada, G. A.; Zhao, X. H.
Tough bonding of hydrogels to diverse non-porous surfaces. Nat.
Mater. 2016, 15 (2), 190−196.
(23) Gao, Y.; Han, X.; Chen, J.; Pan, Y.; Yang, M.; Lu, L.; Yang, J.;
Suo, Z.; Lu, T. Hydrogel-mesh composite for wound closure. Proc.
Natl. Acad. Sci. U.S.A. 2021, 118 (28), No. e2103457118.
(24) Freedman, B. R.; Kuttler, A.; Beckmann, N.; Nam, S.; Kent, D.;
Schuleit, M.; Ramazani, F.; Accart, N.; Rock, A.; Li, J. Y.; Kurz, M.;
Fisch, A.; Ullrich, T.; Hast, M. W.; Tinguely, Y.; Weber, E.; Mooney,
D. J. Enhanced tendon healing by a tough hydrogel with an adhesive
side and high drug-loading capacity. Nat. Biomed. Eng. 2022, 6 (10),
1167−1179.
(25) Liu, J.; Qu, S.; Suo, Z.; Yang, W. Functional hydrogel coatings.
Natl. Sci. Rev. 2021, 8 (2), nwaa254.
(26) Zhang, D.; Yang, F.; He, J.; Xu, L.; Wang, T.; Feng, Z.-Q.;
Chang, Y.; Gong, X.; Zhang, G.; Zheng, J. Multiple physical bonds to
realize highly tough and self-adhesive double-network hydrogels. ACS
Applied Polymer Materials 2020, 2 (3), 1031−1042.
(27) Zhang, Y.; Ren, B.; Xie, S.; Cai, Y.; Wang, T.; Feng, Z.; Tang, J.;
Chen, Q.; Xu, J.; Xu, L. Multiple physical cross-linker strategy to
achieve mechanically tough and reversible properties of double-
network hydrogels in bulk and on surfaces. ACS Appl. Polym. Mater.
2019, 1 (4), 701−713.
(28) Chen, Q.; Zhu, L.; Zhao, C.; Wang, Q.; Zheng, J. A robust, one-
pot synthesis of highly mechanical and recoverable double network
hydrogels using thermoreversible sol-gel polysaccharide. Advanced
materials 2013, 25 (30), 4171−4176.
(29) Chen, Q.; Chen, H.; Zhu, L.; Zheng, J. Fundamentals of double
network hydrogels. J. Mater. Chem. B 2015, 3 (18), 3654−3676.
(30) Yuan, L.; Wu, Y.; Fang, J.; Wei, X. J.; Gu, Q. S.; El-Hamshary,
H.; Al-Deyab, S. S.; Morsi, Y.; Mo, X. M. Modified alginate and
gelatin cross-linked hydrogels for soft tissue adhesive. Artificial Cells
Nanomedicine and Biotechnology 2017, 45 (1), 76−83.
(31) Zhu, W.; Li, Y. L.; Liu, L. X.; Chen, Y. M.; Wang, C.; Xi, F.
Supramolecular Hydrogels from Cisplatin-Loaded Block Copolymer
Nanoparticles and alpha-Cyclodextrins with a Stepwise Delivery
Property. Biomacromolecules 2010, 11 (11), 3086−3092.
(32) Josa-Cullere, L.; Llebaria, A. In the Search for Photocages
Cleavable with Visible Light: An Overview of Recent Advances and
Chemical Strategies. Chemphotochem 2021, 5 (4), 298−316.
(33) Tang, W.; Wang, J.; Hou, H. W.; Li, Y.; Wang, J.; Fu, J. A.; Lu,
L.; Gao, D. D.; Liu, Z. M.; Zhao, F. Y.; Gao, X. Q.; Ling, P. X.; Wang,
F. S.; Sun, F.; Tan, H. N. Review: Application of chitosan and its
derivatives in medical materials. Int. J. Biol. Macromol. 2023, 240,
No. 124398.
(34) Huang, W.; Cheng, S.; Wang, X.; Zhang, Y.; Chen, L.; Zhang,
L. Noncompressible Hemostasis and Bone Regeneration Induced by
an Absorbable Bioadhesive Self-Healing Hydrogel. Adv. Funct. Mater.
2021, 31 (22), 2009189.
(35) Feng, Y.; Gao, H. L.; Wu, D.; Weng, Y. T.; Wang, Z. Y.; Yu, S.
H.; Wang, Z. Biomimetic Lamellar Chitosan Scaffold for Soft Gingival
Tissue Regeneration. Adv. Funct. Mater. 2021, 31 (43), 2105348.
1151
pubs.acs.org/acsapm Article
(36) Pok, S.; Vitale, F.; Eichmann, S. L.; Benavides, O. M.; Pasquali,
M.; Jacot, J. G. Biocompatible Carbon Nanotube-Chitosan Scaffold
Matching the Electrical Conductivity of the Heart. ACS Nano 2014, 8
(10), 9822−9832.
(37) Dong, Y.; Sigen, A.; Rodrigues, M.; Li, X.; Kwon, S. H.; Kosaric,
N.; Khong, S.; Gao, Y.; Wang, W.; Gurtner, G. C. Injectable and
Tunable Gelatin Hydrogels Enhance Stem Cell Retention and
Improve Cutaneous Wound Healing. Adv. Funct. Mater. 2017, 27
(24), 1606619.
(38) Yu, Y. B.; Xu, S.; Li, S. M.; Pan, H. Genipin-cross-linked
hydrogels based on biomaterials for drug delivery: a review. Biomater.
Sci. 2021, 9 (5), 1583−1597.
(39) Chang, W. H.; Chang, Y.; Chen, Y. C.; Sung, H. W.
Hemoglobin polymerized with a naturally occurring crosslinking
agent as a blood substitute: In vitro and in vivo studies. Artificial Cells
Blood Substitutes and Biotechnology 2004, 32 (2), 243−262.
(40) Kim, J.; Zhang, G. G.; Shi, M. X.; Suo, Z. G. Fracture, fatigue,
and friction of polymers in which entanglements greatly outnumber
cross-links. Science 2021, 374 (6564), 212−216.
(41) Nian, G.; Kim, J.; Bao, X.; Suo, Z. Making Highly Elastic and
Tough Hydrogels from Doughs. Adv. Mater. 2022, 34 (50), 2206577.
(42) Sun, X. J.; Rao, P.; He, X. T.; Yang, C. H.; Hong, W.
Chemically identical gels I-under-crosslinked networks. J. Mech. Phys.
Solids 2023, 175, No. 105278.
(43) Suh, J. K. F.; Matthew, H. W. T. Application of chitosan-based
polysaccharide biomaterials in cartilage tissue engineering: a review.
Biomaterials 2000, 21 (24), 2589−2598.
(44) Wang, S.; Li, K.; Zhou, Q. High strength and low swelling
composite hydrogels from gelatin and delignified wood. Sci. Rep.
2020, 10 (1), 17842.
(45) Paik, Y. S.; Lee, C. M.; Cho, M. H.; Hahn, T. R. Physical
stability of the blue pigments formed from geniposide of gardenia
fruits: Effects of pH, temperature, and light. J. Agric. Food. Chem.
2001, 49 (1), 430−432.
(46) Butler, M. F.; Ng, Y. F.; Pudney, P. D. A. Mechanism and
kinetics of the crosslinking reaction between biopolymers containing
primary amine groups and genipin. J. Polym. Sci., Part A: Polym. Chem.
2003, 41 (24), 3941−3953.
(47) Cui, L.; Jia, J. F.; Guo, Y.; Liu, Y.; Zhu, P. Preparation and
characterization of IPN hydrogels composed of chitosan and gelatin
cross-linked by genipin. Carbohydr. Polym. 2014, 99, 31−38.
(48) Yang, C. H.; Yin, T. H.; Suo, Z. G. Polyacrylamide hydrogels. I.
Network imperfection. J. Mech. Phys. Solids 2019, 131, 43−55.
(49) Nordtveit, R. J.; Varum, K. M.; Smidsrod, O. Degradation of
partially N-acetylated chitosans with hen egg white and human
lysozyme. Carbohydr. Polym. 1996, 29 (2), 163−167.
(50) Pang, J. H.; Wischke, C.; Lendlein, A. In vitro Degradation
Analysis of 3D-architectured Gelatin-based Hydrogels. MRS Adv.
2020, 5 (12−13), 633−642.
(51) Tanuma, H.; Kiuchi, H.; Kai, W. H.; Yazawa, K.; Inoue, Y.
Characterization and Enzymatic Degradation of PEG-Cross-Linked
Chitosan Hydrogel Films. J. Appl. Polym. Sci. 2009, 114 (3), 1902−
1907.
(52) Gorgieva, S.; Kokol, V. Preparation, characterization, and in
vitro enzymatic degradation of chitosan-gelatine hydrogel scaffolds as
potential biomaterials. J. Biomed. Mater. Res. Part A 2012, 100A (7),
1655−1667.
https://doi.org/10.1021/acsapm.3c01926
ACS Appl. Polym. Mater. 2024, 6, 1141−1151