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RESEARCH ARTICLE
An injectable chitosan/laponite hydrogel synthesized via
hybrid cross-linking system: A smart platform for cartilage
regeneration
Fatemeh Ranjbardamghani
1
| Niloofar Eslahi
2
| Reza Jahanmardi
1
1
Department of Polymer Engineering, Science
and Research Branch, Islamic Azad University,
Tehran, Iran
2
Department of Textile Engineering, Science
and Research Branch, Islamic Azad University,
Tehran, Iran
Correspondence
Niloofar Eslahi, Department of Textile
Engineering, Science and Research Branch,
Islamic Azad University, Tehran, Iran.
Email: niloofar.eslahi@srbiau.ac.ir
Abstract
Thermo-responsive polymeric hydrogels have received great attention in recent
years. The current study aimed to fabricate and characterize injectable chitosan/
laponite (CS-L) hydrogels using a hybrid cross-linking method by genipin and β-gly-
cerophosphate (BGP). Fourier transform infrared analysis confirmed the ionic and
covalent interactions between the employed materials in the hydrogels. Scanning
electron microscope images showed a decrease in the mean pore size from 129 to
83 μm after the incorporation of 1% laponite into the hydrogels. Energy dispersive
X-ray analysis proved the uniform distribution of laponite in the hydrogels. Besides,
the gelation time of the chitosan/laponite hydrogels declined in comparison to the
chitosan hydrogel owing to the presence of abundant hydroxyl groups in the laponite
structure. Rheological investigations revealed 600% improvement in the storage
modulus after the incorporation of 1% laponite. The compression test results similarly
showed that the elastic modulus and compressive strength of CS-L1% were signifi-
cantly enhanced in comparison with pristine polymeric hydrogel. Non-toxicity and
antibacterial properties of the hydrogels demonstrated 95% cell viability and 99%
antibacterial activity, respectively. In conclusion, the obtained results confirmed that
the introduced hybrid hydrogels are appropriate candidates for cartilage tissue engi-
neering applications.
KEYWORDS
cartilage tissue engineering, chitosan hydrogel, laponite, smart biomaterials
1|INTRODUCTION
Polymer hydrogels, with a cross-linked macromolecular structure,
have been effectively utilized in tissue regeneration and drug delivery
due to their hydrophilicity, biodegradation properties, releasing bio-
chemical factors and simulating cellular milieus.
1–3
Injectable hydro-
gels are considered appropriate scaffolds for bone and cartilage
tissue regeneration because of their ability to provide homogenous
cell distribution before injection and in situ forming after injection,
which results in the complete filling of irregular defects. These scaf-
folds provide a minimally invasive implantation procedure.
4
Smart
nanohybrid hydrogels could endure sol–gel transition when exposed
to external stimuli, such as temperature, pH, electrical or ionic
strength alteration, leading to in-situ gelation of the injected sol
inside the body.
5
Several chemical and physical cross-linking proce-
dures such as the Michael reaction, “Click”reaction, Schiff base reac-
tion, enzymatic and UV reaction have been employed in hydrogels.
6–8
Chemical reactions between a cross-linker and a polymer lead to a
stable network, while physical interactions such as Van der Waals
and hydrogen bonds result in a weaker network. In thermo-
responsive hydrogels, the sol–gel transition occurs through tempera-
ture alteration.
9
Received: 16 January 2023 Revised: 19 March 2023 Accepted: 22 March 2023
DOI: 10.1002/pat.6051
Polym Adv Technol. 2023;1–14. wileyonlinelibrary.com/journal/pat © 2023 John Wiley & Sons Ltd. 1
Chitosan is a biocompatible and biodegradable polymer with
important biological features such as hemostatic and antibacterial
properties.
10,11
Chitosan having structural similarity with glycosamino-
glycans (GAGs) has been endorsed for cartilage tissue engineering
applications. The modulatory effect of chitosan on different inflamma-
tory cells has been reported in several studies.
12
This natural polysac-
charide accompanied by BGP (a polyol salt), is intensely assessed as a
thermosensitive hydrogel in biomedical applications.
13,14
However,
lack of mechanical strength usually restricts the practical application
of chitosan hydrogels as cartilage tissue regenerating scaffolds. The
introduction of inorganic nanoparticles including clay nanoparticles
has been shown to increase the stiffness of the hydrogels.
15
Laponite is a synthetic smectite clay with diverse technological
and biomedical applications in pharmaceutics and cosmetics. Laponite
is a disk-shaped nano-crystal with a high aspect ratio. These disks can
intensely interact with different types of materials such as natural or
synthetic polymers, small molecules, ions, and various inorganic mate-
rials. Laponite can be functionalized with chemical groups and is also
biodegradable in physiological conditions. The incorporation of this
nanomaterial has been shown to improve the strength and stiffness of
the polymeric background.
3
For instance, laponite was physically
added into the silated hydroxypropyl methyl cellulose (Si-HPMC)
hydrogel for subcutaneous implantation of human nasal chondrocytes
and it was found that this interpenetrating network induced extracel-
lular matrix formation.
16
The biological interactions of laponite with
human mesenchymal stem cells at whole-transcriptome level were
also investigated by high-throughput sequencing (RNA-seq). Impor-
tant biophysical and biochemical cellular pathways activated by nano-
silicates were determined, leading to stem cell differentiation and
consequent osteogenic and chondrogenic lineages.
17
The current study aimed to fabricate an injectable chitosan/
laponite hydrogel using a hybrid cross-linking method by genipin and
BGP. Glycerophosphate can induce a thermo-responsive effect in the
chitosan hydrogels, while genipin, a naturally extracted agent, supplies
co-crosslinking of both in situ physical and covalent crosslinking and
also introduces more dimensional stability to the structure.
18,19
Geni-
pin is recognized as a favorable crosslinking agent due to its facile
reaction with polymers comprising primary amino groups and its negli-
gible cytotoxicity compared to common chemical crosslinkers.
20,21
It
should be noted that the negative charge of the surface and the posi-
tive charge of the inner side of laponite results in controlled gelation
of the hydrogel.
22
Moreover, laponite incorporation leads to an
enhancement in the mechanical strength of the provided hydrogels.
23
Therefore, the simultaneous applications of these components (includ-
ing BGP, genipin, and laponite) in the chitosan-based hydrogel could
improve the physicochemical and thermo-gelation properties of the
final composite hydrogels. The chemical, morphological, physical, rhe-
ological, and mechanical properties of the hydrogels were investigated
by Fourier transform infrared (FTIR), scanning electron microscope
(SEM), X-ray diffraction analysis (XRD), rheological analysis, and com-
pression tests, respectively. Moreover, the toxicity and antibacterial
activity of the hydrogels were evaluated for practical applications.
2|MATERIALS AND METHODS
2.1 |Materials
Chitosan (Mw 200,000–350,000), BGP and genipin were purchased
from Sigma-Aldrich (USA). Laponite
®
RD was provided by BYK
(Germany). Other chemicals such as dimethyl sulfoxide (DMSO), acetic
acid, glutaraldehyde and EtOH (70% and 99%) were obtained from
Merck (Germany). Phosphate buffered saline (PBS) (1X, pH 7.4) was
obtained from Sigma-Aldrich (USA). Luria Broth (LB) culture media
containing different bacterial strains with Staphylococcus aureus
(ATCC@25923TM) and Escherichia coli (ATCC@25922TM) was used
for antibacterial evaluations. RPMI1640 culture media, fetal bovine
serum (FBS), penicillin/streptomycin, and 0.25% Trypsin–EDTA solu-
tion were purchased from Gibco (UK).
2.2 |Preparation of hydrogel scaffolds
First, 40 mL of chitosan solution (2% wt) in 0.1 M acetic acid and
10 mL of BGP in PBS buffer (50% wt) were prepared via magnetic
stirring for 4 h at room temperature (RT, 25C, relative humidity of
60%). 0.04 g genipin was added to 4 mL PBS:DMSO (1:3) solution and
mixed for 30 min at RT. To prepare hydrogels, BGP solution was
mixed with the chitosan solution (at a volume ratio of 4:1) in an ice
bath on a magnetic stirrer. Then, different amounts of laponite, that is,
0, 1, and 2 wt% were added to the chitosan/BGP solution and the
resulting mixture was exposed to ultrasound (80 kHz) for 15 min for
uniform dispersion of laponite. Afterwards, genipin solution (5 mM),
as the second crosslinking agent, was added to mixtures and mixed
for 2 h in an ice bath. Finally, the solutions were kept in an incubator
(37C) to induce gelation. Synthesized hydrogel samples with different
laponite contents were referred to as CS-L0%, CS-L1%, and CS-L2%.
2.3 |Hydrogels characterization
2.3.1 | Gelation time
To investigate the gelation time of the hydrogels, the solution was
incubated at 37C in a glass vessel and the gelation time was deter-
mined using an inverted tube test as described elsewhere.
24
2.3.2 | FTIR
Fourier transform infrared analysis was used to study the chemical
structure of the prepared hydrogels using Fourier transform infrared
spectroscopy (Bruker Tensor 27, Japan). For this purpose, the hydro-
gels were first lyophilized and after mixing with KBr powder, the sam-
ple pellet was prepared and detected in the wavenumber range of
4000–400 cm
1
.
2RANJBARDAMGHANI ET AL.
2.3.3 | XRD
The X-ray diffraction analysis was carried out using an XRD device
(D8 Advance, USA) at a voltage of 40 kV and a current of 30 mA using
a Cu lamp and Cu Kαradiation (λ=1.54 Å) to determine the crystal-
linity of the prepared hydrogels. The size of crystals was also calcu-
lated using the Bragg equation as follows:
nλ¼2dsinθð1Þ
Where λis the X-ray wavelength, dis the spacing of the diffract-
ing planes, and θ(Bragg angle) is the angle between the incident rays
and the diffracting planes.
25
2.3.4 | SEM/EDX
To evaluate the morphology of the prepared hydrogels, SEM test was
employed. For this purpose, first, the hydrogels were completely
lyophilized and SEM images (VEGA, TESCAN, Czech Republic),
coupled with the energy dispersive X-ray analysis (EDX) were taken
from the cross-sections of the dehydrated samples after coating them
with gold at a voltage of 20 kV.
2.3.5 | Swelling ratio
To measure the adsorption rate of the prepared hydrogels, first, the
initial weight of the hydrogels (W
i
) was calculated. Afterwards, hydro-
gels were immersed in 5 mL of PBS at 37C. Then, the samples were
weighed after distinct time points (W
t
) and the swelling ratio was
measured using the following equation:
Swelling ratio ¼WtWi
Wi100 ð2Þ
2.3.6 | Rheological study
The oscillatory rheological properties of the hydrogels were tested
on the RS6000 Rheometer (Anton Paar Ltd., Austria) using a disk-
type parallel plate (15 mm in diameter). To evaluate thermogelling
properties, the storage modulus (G0) and loss modulus (G00 )were
recorded during a temperature sweep from 15 to 50C at a rate of
1C/min. A time sweep measurement was also carried out at a con-
stant angular frequency of 1 Hz to determine the gelation time at
physiological temperature (37C). Besides, frequency sweep mea-
surements in the linear viscoelastic region were performed to evalu-
ate the dynamic viscoelastic properties at 37C, over a wide range of
frequencies.
2.3.7 | Injectability and compression test
The injectability of the hydrogels was evaluated using a Universal test-
ing machine (Hounsfield-H10Ks, USA). Briefly, the materials were
placed in 3 mL plastic syringes and then fixed between the upper com-
pression platen and lower tensile grips. The gels were then injected
through a medical catheter (5-French Beacon) at RT. The injection rate
was controlled by changing the cross speed of the compression plate
to achieve the desired flow rates. Besides, a compression test was per-
formed on cylindrical specimens (10 mm in diameter and 5 mm in
height) at a strain rate of 20 mm.min
1
. Elastic modulus was calculated
from the slopes of stress versus strain curves at strain levels between
0–10% and compressive strength was determined at a strain of 80%.
2.3.8 | MTT assay
For in vitro studies, hydrogels were sterilized and kept for a defined
period in culture media. Cytotoxicity testing of the hydrogels was inves-
tigated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT, Sigma, USA) assay. In this research, human chondrocyte
C28/I2 cells (NCBI C620) obtained from the Pasteur Institute of Iran
cell bank were used. First, the samples of 1 1cm
2
were incubated at
37C in 1 mL of RPMI 1640 culture medium (Sigma) for 96 h. After-
wards, the culture media was extracted from the wells containing sam-
ples and transferred into a 96-well plate. Each well was seeded with
cells at a density of 1 10
4
chondrocyte cells per ml and filled with
fresh media. 3 and 7 days after incubating cells with hydrogel extracts,
the media was removed and 100 μL of a 5 mg/mL MTT solution was
added to each well and incubated at 37C for 4 h. After removing the
solution, 200 μL of DMSO (Sigma, USA) was added to each well and
incubated for 15 min at 37C to eliminate MTT. Finally, the optical den-
sity (OD) were measured at 570 and 670 nm. Pure culture medium
under similar conditions was employed as the control. MTT test was
performed in triplicate for each sample and the mean and standard
deviation was reported. The well with increased cell density exhibits
higher OD compared with the other wells. Cell viability percentage was
calculated according to the following equation:
Cell viability ¼Mean OD of samples
Mean OD of control 100 ð3Þ
2.3.9 | Cell attachment
Chondrocytes (4 10
4
cells per 100 μL of culture medium) were cul-
tured on each hydrogel sample and incubated at 37C. After 48 h, cul-
ture media was removed and un-attached cells were washed for 30 s
with PBS. Then, the hydrogels were fixed with 3.5% glutaraldehyde,
dehydrated in serial ethanol solutions of 20%, 40%, 60%, 80%, and
96% and sputter-coated with gold for SEM analysis (VEGA TS5136M,
TESCAN, USA).
RANJBARDAMGHANI ET AL.3
2.3.10 | Antibacterial evaluations
The shaking flask method was used to evaluate the antibacterial prop-
erties of the hydrogels, which is a quantitative antibacterial examina-
tion for defining the antibacterial activity of hydrogels under dynamic
contact conditions.
26
First, circular samples with 1 cm diameter were
incubated in a flask including 10 mL LB culture media containing dif-
ferent bacterial species with Staphylococcus aureus (S. aureus)
(ATCC
@
25923™) and Escherichia coli (E. coli) (ATCC
@
25922™), at the
concentration of 1.5 10
6
CFU as gram-positive and gram-negative
colonies, respectively. Then, the flask was kept in a rotary stirrer at
200 rpm, 37C for 24 h. In order to count bacterial colonies, 0.1 mL of
the solution was removed from the flask and diluted to a suitable vol-
ume using distilled water and then added to the agar culture medium.
Samples without laponite were considered as controls. In the end, the
number of colonies grown on the agar plate was counted for each
dilution and the quantitative amount of bacterial reduction was mea-
sured based on the following equation:
Reduction%¼C0C
C0100 ð4Þ
In the mentioned equation, C
0
and C denote the colonies, which
are grown on the control sample and antibacterial samples,
respectively.
2.3.11 | Statistical analysis
All experiments were done in triplicate and the data were shown as
mean and standard deviation (SD). Comparison between different
groups was performed by multifactorial one-way analysis of variance
(ANOVA). The p-value of 0.05 was considered to be statistically
significant.
3|RESULTS AND DISCUSSION
3.1 |Gelation properties of the hydrogels
Gelation time is an essential characterization of injectable hydrogels.
BGP was applied to not only induce a thermo-responsive effect in
chitosan hydrogels but also to keep the chitosan solution at the bio-
logical pH (7). It has been proved that a higher BGP concentration
offers a shorter gelation time.
27
As a similar case, chitosan-placental
ECM biocomposite was developed benefiting from BGP crosslinks
which provided a thermos-responsive behavior for this scaffold and
directly affected the gelation time at biological temperature.
28
Never-
theless, an increased concentration of BGP might have a toxic effect
on the cells.
29
Thus, it is important to control and detect its optimal
concentration. In this study, genipin was also used as another cross-
linking agent for inducing chemical interactions between the polymer
FIGURE 1 Schematic of crosslinking mechanism in the hydrogels.
4RANJBARDAMGHANI ET AL.
chains and improving mechanical stability. Figure 1depicts the possi-
ble crosslinking mechanism in the introduced hydrogel, which is com-
posed of two reactions: (1) the electrostatic interactions between
amine groups of chitosan and phosphate groups of BGP acting as an
ionic crosslinking agent, (2) a nucleophilic substitution reaction involv-
ing the replacement of the ester group on the genipin by a secondary
amide bond with chitosan. The second reaction is complicated by the
oxygen radical-induced polymerization of genipin resulting in a blue
color.
30
After the incorporation of laponite, the hydroxyl groups on its
surface can form hydrogen bonds with functional groups of chitosan
hydrogel. It was found that exfoliated laponite particles may act as
multifunctional crosslinkers in forming composite hydrogels, and the
polymer chains were anchored to the particles to assemble a network
structure. The faces of laponite are negatively charged while their
slices are positively charged. These properties allow them to form a
hydrogel with controlled gelling properties resembling a “house of
cards”structure.
31
Table 1shows the calculated gelation time for each formulation
using an inverted test tube. According to the obtained results, the
incorporation of laponite into the chitosan decreased the gelling time
of the hydrogels. Accordingly, the gelation time of hydrogel without
laponite was 15 min, in which the addition of 1 wt% laponite to the
hydrogel formulation, reduced the gelation time to 5 min. This might
be caused owing to the presence of abundant hydroxyl groups in the
laponite structure which led to an increase in physical interactions
between laponite and chitosan molecules.
32
3.2 |FTIR
Fourier transform infrared spectra of chitosan/laponite hydrogels are
exhibited in Figure 2A. The characteristic peaks of crosslinked chito-
san can be seen around 3500–3300 cm
1
, which is related to N H
and OH groups. Also, two peaks at 1664 and 1595 cm
1
are associ-
ated with C O and NH
2
groups of chitosan, respectively.
33
Further-
more, 1030 and 958 cm
1
peaks correspond to PO
42
and HPO
4
groups of BGP in the hydrogels, respectively.
34
Cross-linking proce-
dure of chitosan with genipin resulted in the formation of amine III
and amide groups, which are observed at 1581 and 1666 cm
1
corre-
sponding to the stretching vibration of N H and C O vibration. As
for CS-L1%, the band at 1100 cm
1
is related to the Si O Si groups
indicating the incorporation of laponite into the chitosan structure.
35
As for CS-L2%, increasing the laponite concentration compared to
CS-L1% resulted in the shifting of amide I and II bands towards the
higher wavenumbers. Also, the stretching vibration peaks of N H and
OH shifted to higher wavenumber from 3425 cm
1
(CS-L0%) to
3434 cm
1
(CS-L1%) in accordance with engagement of amine groups
of chitosan and hydroxyl groups of laponite. This change is followed
by shifting back to a lower wavenumber (3425 cm
1
) as the laponite
content reaches 2% owing to the excess unreacted hydroxyl groups in
the hydrogel.
3.3 |XRD
X-ray diffraction analysis patterns of the hydrogels are illustrated in
Figure 2B. CS-L0%. It can be seen that the CS-L0% (pure chitosan)
hydrogel exhibits two peaks in the region of 2θ=20and 6,
which are related to the crystalline regions of the biopolyme.
36
According to the literature, pure laponite has two peaks in the
regions of 6.3and 60, corresponding to (001) basal planes and
(060) lateral planes of the clay, respectively.
37
The obtained results
reveal that by the incorporation of laponite into the hydrogels,
these characteristic peaks of laponite nano-sheets are observed in
accordance with former studies. Also, increasing the percentage of
laponite caused a growth in the intensity of these peaks. The
FIGURE 2 (A) Fourier transform infrared spectra and (B) X-ray
diffraction analysis patterns of the hydrogels.
TABLE 1 Gelation time of the final hydrogels.
Sample Gelation time (min)
CS-L0% 15
CS-L1% 5
CS-L2% 7
RANJBARDAMGHANI ET AL.5
calculated crystallite size (according to the Equation 1) shows an
increase after laponite addition from 38.24 nm for CS-L0% to
60.43 nm and 57.25 nm for CS-L1% and CS-L2% samples, respec-
tively. Moreover, the crystallinity of the hydrogels enhances from
23.5% (without laponite) to 28% for CS-L1% and 32% for CS-L2%.
This enhancement is probably attained through re-orientation of
polymeric chains due to the electrostatic forces between laponite
nanoparticles and chitosan.
38
3.4 |SEM/EDX
Scanning electron microscope images of chitosan-based hydrogels are
illustrated in Figure 3. The cross-sections represent homogeneous and
porous network structures with interconnected porosity. As can be
seen, the addition of laponite to the hydrogel enhanced the pore dis-
tribution and caused a decrease in average pore size from 129 to
108 and 83 μm for CS-L0%, CS-L1% and CS-L2% samples, respec-
tively. Other studies have also confirmed that the amount of laponite
had a significant effect on the morphology and uniformity of the poly-
mers.
39
The decrease in the pore size can be the consequence of
increased physical interactions between the components by laponite
incorporation.
40
It should be pointed that the uniform morphology
and interconnected structure of composites are initially affected by
primary physical interactions and chemical crosslinking generated via
BGP and genipin governing the crosslinking mechanism, respectively.
9
To examine the distribution of laponite in the hydrogels, the
lyophilized hydrogels were investigated with EDX analysis. The map-
ping results in Figure 4prove the presence of Si in the nanocomposite
hydrogels. It can be seen that laponite nanoplates are uniformly dis-
tributed in CS-LP1%, while the distribution of laponite in CS-LP2% is
relatively heterogeneous. The aggregation of laponite can be attrib-
uted to the excess amount of laponite in the chitosan matrix. The
homogeneous distribution of laponite in the CS-LP1% hydrogel is
beneficial in maintaining the integrity of the materials.
23
3.5 |Swelling
The swelling capacity of hydrogels exerts a profound effect on their
biological and mechanical properties. The swelling ratio of the
hydrogelsisshowninFigure5. According to the obtained results,
an increase in laponite concentration leads to lower swelling from
52% for CS-L0% to 42% for CS-L1% and 30% for CS-L2% after
24 h. These results confirm that the physical properties of the
hydrogels depend on the laponite content. A higher amount of clay
could increase the physical crosslinking, together with preventing
the dissolution of hydrogel in water because of the noncovalent
interactions between the polymer and clay.
41
The effect of inor-
ganic particles on the properties of chemically crosslinked hydrogels
has been considered previously. Swelling results of PVA/laponite
membranes displayed a reduction in swelling with increasing
FIGURE 3 Scanning electron microscope images and pore size distribution histograms of the hydrogels: (A) CS-L0%, (B) CS-L1%, and
(C) CS-L2%.
6RANJBARDAMGHANI ET AL.
laponite concentration in the membranes.
42
Ninan et al.
43
also
found that swelling capacity decreased with an increase in clay con-
centration. The swelling factor is intensely reliant on the amount of
nanoclay in the matrix and the interconnection of the pores, which
is an essential characterization for the diffusion of nutrients. The
calculated swelling ratio of hydrogels is in line with the SEM images,
FIGURE 4 Energy dispersive X-ray analysis and Si mapping of: (A) CS-L0%, (B) CS-L1%, and (C) CS-L2% hydrogels.
RANJBARDAMGHANI ET AL.7
showing a decrease in the pore size with increasing laponite
content.
3.6 |Rheological studies
To investigate the gelation temperature of the hydrogels, temperature
sweep mode was performed using a rheometer at an angular fre-
quency of 1 Hz. As can be seen from Figure 6A,B, the loss modulus
(G00) was higher than the storage modulus (G0) at lower temperatures,
indicating a fluid-like behavior. As the heating proceeded, G0increased
remarkably quicker than G00. At higher temperatures, G0dominated
G00, implying gel-like behavior. The cross-over point of G0and G00 in
the rheometer curve is known as the gelation point. Investigations of
G0and G00 changes versus temperature revealed that physicochemical
interactions of chitosan chains with genipin, BGP and laponite led to
the gelation of the hydrogels. Following the gelation, G0and the vis-
cosity of the solution increase intensively.
44
According to the results,
the gelation temperature of CS-L0% hydrogel was 36.4C. Incorpora-
tion of laponite (1%) into the chitosan structure decreased the gela-
tion temperature of the hydrogel to 31.8C owing to the additional
interactions between hydroxyl groups of laponite and amide and
hydroxyl groups of chitosan which decrease the mobility of the poly-
mer chains and consequently decrease the gelation temperature.
45
The gelation time is also an essential parameter required for the
formation of an injectable hydrogel.
4
Therefore, in order to explore
the gelation time of the hydrogel, a time sweep rheology test was
conducted at 37C in Figure 6C. For CS-L1% hydrogel, the loss modu-
lus G00 is higher than the elastic modulus G0in the first several min,
indicating the dominance of viscous properties at the beginning of the
reaction. Since the increasing rate of G0was faster than that of G00,an
intersection point emerged at approximately 5 min for the nanocom-
posite hydrogel, suggesting that the liquid-like structure was trans-
formed into a gel-like system with dominant elastic properties. The
result demonstrated that the optimum gelation time was obtained by
adjusting the laponite content in the chitosan matrix. Figure 6D shows
that complex viscosity increases gently as the temperature reaches
30C. At higher temperatures, there is a significant growth in viscosity
due to the gelation process.
To study the viscoelastic properties of chitosan-based hydrogels,
frequency sweep experiments were also performed in parallel plate
geometry at 37C from 0 to 100 Hz. Figure 6E validates the shear-
thinning behavior of the hydrogels, since there is a decline in the com-
plex viscosity by increasing frequency. Figure 6F shows that in all
samples, the values of the storage modulus G0were higher than the
loss modulus G00 indicating an elastic characteristic. The elastic proper-
ties of the prepared hydrogels prevail over the viscous properties, as
expected.
46
In addition, G0is highly dependent on the percentages of
the employed components and the gelation mechanism of the hydro-
gel. The addition of 1 wt% laponite to the formulation caused the for-
mation of a strong network of cross-linked hydrogel, which increased
the storage modulus. Nevertheless, increasing the amount of laponite
from 1 to 2 wt% has reduced the elastic modulus G0, owing to the
aggregation of the nanoplates.
3.7 |Injectability and compression properties
It is necessary to determine the injection force value which is the
required force for the injection of the hydrogel formulation at a given
injection rate via a needle with a predetermined gauge.
47
According
to a relevant study, the addition of a clay to the polyelectrolyte com-
plex and carboxymethyl cellulose hydrogel resulted in an increased
amount of injection forces.
48
Moreover, another study showed
changes in viscoelastic properties of laponite nanoparticle-silated
hydroxypropyl methyl cellulose reinforced hydrogel after laponite
addition affecting injectability.
16
Additionally, it was reported that the
genipin crosslinking agent caused sol–gel transition in the chitosan-
based hybrids after injection.
49
In the present work, the gelation investigation was done at 25C
standing for injectability of hydrogels at room temperature. The injec-
tion force values of samples in Figure 7A are 17.3 ± 2.07, 18.2 ± 2.47
and 39.4 ± 4.85 for CS-L0%, CS-L1%, CS-L2%, respectively. Although
the injection force increased slightly by adding 1% laponite, CS-L1%
hydrogel could still flow easily from the catheter at a relatively low
force and be injected by hand. However, at higher percentages of
laponite (2%), injection force increased significantly compared to CS-
L0% and CS-L1% hydrogels. The mechanical aspects of clay-reinforced
hydrogels have been reported to be affected by laponite content. As a
case, dopamine-modified four-armed poly (ethylene glycol) (PEG-D4)
demonstrated an enhancement in the mechanical properties after
addition of 1% laponite owing to the increased interfacial interactions
of dopamine and laponite.
50
In another study, chemically crosslinked
polyacrylamide (PAAm) was reinforced with different contents of lapo-
nite and indicated around one order of magnitude enhancement in the
elastic modulus and compressive strength with 2 to 3 wt% laponite
incorporation in comparison with the control hydrogel.
51
FIGURE 5 The swelling ratio of the hydrogels.
8RANJBARDAMGHANI ET AL.
Figure 7B summarizes the compression properties of the hydro-
gels. The results indicate that by incorporation of laponite, the elastic
modulus and compressive strength of the samples enhance signifi-
cantly from 0.1 MPa (for CS-L0%) to 0.2 MPa (for CS-L1%) and from
0.04 (for CS-L0%) to 0.27 MPa (for CS-L1%), respectively. In accor-
dance with the rheological results, by increasing the laponite content
to higher value of 2%, the deterioration in mechanical properties
occurred. This can be rooted in the loss of structural integrity as a
FIGURE 6 Temperature sweep results for (A) CS-L0% and (B) CS-L1% hydrogels, (C) Time sweep test for CS-L1% at 37C, Complex viscosity
versus (D) temperature and (E) frequency, (F) rheological characterization of hydrogels via frequency sweep.
RANJBARDAMGHANI ET AL.9
result of laponite content being beyond the tolerance limit of the
hydrogel.
52
The compressive strengths of our samples are also higher
than some previously reported studies. For instance, chitosan hydro-
gel including alginate microspheres demonstrated compression
strength of 57.3 kPa.
53
3.8 |In vitro studies
The viability of chondrocytes on the extracted media of hydrogels
was evaluated using an MTT assay after 3 and 7 days. MTT results in
Figure 8A indicate high cell viability in all samples as confirmed in sev-
eral other studies, where chitosan and laponite have been used for an
extensive range of biological applications. Chitosan is a natural poly-
saccharide consisting of N-acetyl-D-glucosamine and D-glucosamine
units with biodegradability and nontoxicity.
2,14
According to an inves-
tigation on cartilage regeneration, chitosan-genipin microgels showed
non-toxic and complete in vivo lysozyme degradation after 28 days
leading to enhancement in cartilage repair through rat growth plate
injury.
54
Besides, laponite is a non-toxic and biocompatible silicate
clay. It has been stated that incorporating silicate nanoparticles into
polymers increases cell attachment, proliferation, and differentia-
tion.
55
Because of their layered structure, and thereby their great spe-
cific surface area, smectite minerals such as laponite exhibit
adsorptive ability, surface reactivity, and cation exchange capacity in a
polymer matrix which can explain their improved biological proper-
ties.
56
For instance, the addition of laponite in chitosan/polyaniline
hydrogel led to induction of osteogenic differentiation of stem cells,
as well as suitable viability of mouse fibroblasts (L929).
57
In another
study, it has been shown that the introduction of laponite nanoparti-
cles remarkably promoted MG63 cell viability, proliferation and
attachment.
6
As can be seen in Figure 8A, there is also no significant
difference between the studied specimens. These results validate that
the fabricated hydrogels have the potential to provide a suitable
biocompatible and nontoxic condition for the growth and proliferation
of chondrocytes.
Cell-biomaterial interaction is a sophisticated procedure that
affects cell viability, proliferation, and differentiation. Attachment to a
biomaterial surface is of vital importance to a cell as it provides signs
for migration, and cues for growth and differentiation signaling.
58
To
assess the cell adhesion properties of the hydrogels, chondrocyte
cells were cultured on the hydrogels for 48 h. The SEM results in
Figure 8B indicate appropriate cell attachment on the surface of CS-
L0%, CS-L1% and CS-L2% samples, respectively. The presence of
laponite in hydrogel structure improves cell attachment markedly,
due to the proper biological properties of this biocompatible
nanostructure.
3.9 |Antibacterial properties
As a tissue engineering scaffold, a hydrogel must possess antibacterial
activity to ban the migration and colonization of microorganisms in
the defect by creating a hurdle against external infecting microorgan-
isms.
59
Chitosan has been reported as an antibacterial agent in bio
scaffolds. For instance, the antibacterial activity was proved in
chitosan-hydrogel-coated cellulosic fabric against both E. coli and
S. aureus colonies.
60
Also, another study demonstrated the antibacter-
ial properties of genipin-crosslinked carboxymethyl chitosan compos-
ite containing tea tree oil (TTO) against mentioned colonies leading to
higher than 90% of their inhibition due to the polyphenols presence in
TTO as well as chitosan molecules protonated groups with antibacter-
ial functionality.
61
Two mechanisms have been reported for chitosan
antibacterial activity. The most important mechanism is its attachment
(due to its cationic nature) to the cell wall of a negatively charged bac-
terium that disrupts the cell by reducing subsequent permeability to
the membrane. By binding to DNA, it inhibits DNA replication and
subsequent bacterial cell death.
10,62
Another mechanism proposed for
FIGURE 7 (A) Injection force variation of the hydrogels with different laponite contents, (B) Compressive modulus and compressive strength
of hydrogels at a strain =80%. * and ** indicate statistically significant differences between groups (p value <0.05).
10 RANJBARDAMGHANI ET AL.
the antibacterial properties of chitosan is its activity as a killing agent
that produces toxins and inhibits microbial growth.
38,63
The antibacterial activity of the hydrogels was evaluated against
S. aureus and E. coli, the most important microorganisms in the wound
and body environment. The results are shown in Tables 2and 3. It can
be observed that the sample containing 1% laponite has the highest
percentage of antibacterial activity. Laponite is a synthetic nano-clay
with a chemical formula Na
0.7
+[(Si
8
Mg
5.5
Li0.3)O
20
(OH)
4
]
0.7
and its
slow degradation into non-toxic ions such as Na
+
,Li
+
,Mg
2+
, and
Si(OH)
4
can encourage the growth and osteoblast differentiation of
stem cells without any additional osteo induction.
64
Laponite nano-
plates exhibit a positive charge on the edges and a negative charge on
their surfaces, respectively. These charges provide an appropriate
condition for polar molecules such as bacteria to be loaded by
FIGURE 8 (A) MTT results of hydrogels after 3 and 7 days, (B) SEM images of chondrocytes attachment on hydrogels: (A) CS-L0%, (B) CS-
L1%, and (C) CS-L2%.
RANJBARDAMGHANI ET AL.11
laponite nanoplates.
65
On the other hand, the antibacterial properties
of chitosan/nanoparticle hybrids are affected by numerous parame-
ters, including bacterial type, concentration, growth stage, pH, zeta
potential, molecular weight, and degree of acetylation.
66
Any factor
that can upset the balance of any of these involved factors can change
the antibacterial properties of the hydrogels.
4|CONCLUSION
In this study, injectable hydrogels based on chitosan and laponite
were introduced using a hybrid crosslinking method (BGP as a physi-
cal cross-linker and genipin as a chemical covalent crosslinker). Based
on the results from the FTIR analysis, the validation of the physical
and chemical interactions between the crosslinking agents and chito-
san was confirmed. SEM images exhibited that the incorporation of
laponite into the formulation of hydrogels led to a decrease in the
pore size of hydrogels from 129 μm (for CS-L0%) to 83 μm(forCS-
L1%). EDX analysis affirmed the uniform distribution of laponite in
CS-L1% matrix. XRD investigation showed the presence of laponite
particles in the hydrogels. Rheological studies indicated that the
addition of laponite to the chitosan hydrogels resulted in a decrease
in gelation time and temperature of the hydrogels by creating hydro-
gen bonds with chitosan chains. The addition of 1% laponite
improved the elastic modulus and compressive strength of the
hydrogels up to 2 and 6 folds, respectively. Investigations of cell
adhesion onto the hydrogels showed that the presence of laponite
led to better cell adhesion. The non-toxicity (based on the MTT
assay) and antibacterial properties of the prepared hydrogels against
two types of gram-positive and gram-negative bacteria indicated the
possibility of using these injectable hydrogels as smart platforms for
cartilage tissue engineering. Discussing limitation, toxicity issues
with crosslinking agents can be solved through selection of opti-
mized concentration. Besides, further in vivo assays should be per-
formed on the designed hydrogels to evaluate their practical
potential in clinic.
CONFLICT OF INTEREST STATEMENT
The authors declare no conflicts of interest.
DATA AVAILABILITY STATEMENT
Research data are not shared.
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How to cite this article: Ranjbardamghani F, Eslahi N,
Jahanmardi R. An injectable chitosan/laponite hydrogel
synthesized via hybrid cross-linking system: A smart platform
for cartilage regeneration. Polym Adv Technol. 2023;1‐14.
doi:10.1002/pat.6051
14 RANJBARDAMGHANI ET AL.
