Received: 9 February 2021 Revised: 30 March 2021
DOI: 10.1002/app.50848
ARTICLE
Genipin-crosslinked chitosan hydrogels: Preliminary
evaluation of the in vitro biocompatibility and
biodegradation
Nga T. N. Vo1 | Lei Huang2
1
Katarina Novakovic
1
School of Engineering, Newcastle
University, Newcastle Upon Tyne, UK
2
Translational and Clinical Research
Institute, Newcastle University, Newcastle
Upon Tyne, UK
Correspondence
Nga T. N. Vo, School of Engineering,
Newcastle University, Newcastle Upon
Tyne NE1 7RU, UK.
Email: n.vo2@ncl.ac.uk
Funding information
Engineering and Physical Sciences
Research Council, Grant/Award Number:
EP/N033655/1; Research Excellence
Academy, Newcastle University
1 | INTRODUCTION
An increased understanding of chronopharmacokinetic
and circadian rhythm of diseases has upraised the impor-
tance of advanced drug delivery systems, which can
mimic the symptomatic requirement of diseases and
deliver on demand in a site-specific manner. Smart
hydrogels have emerged as a promising option in this
regard. 'Smart' refers to their ability to respond to
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
© 2021 The Authors. Journal of Applied Polymer Science published by Wiley Periodicals LLC.
1 of 11 wileyonlinelibrary.com/journal/app
Accepted: 31 March 2021
| Henrique Lemos2 | Andrew L. Mellor2 |
Abstract
Genipin-crosslinked chitosan hydrogels have gained attention as promising drug
delivery vehicles. To bring them one step closer to clinical applications, this study
evaluates the hydrogels' in vitro biocompatibility and biodegradation. Cytotoxicity
test on 3T3 fibroblasts shows that the cells retain normal adhesive properties and
high viability on gels with 3.1 and 4.4 mM genipin but not on gels with 1.7 mM
genipin. The hydrogels with highest genipin content (4.4 mM) are studied further
in the presence of RAW 264.7 macrophages and DC 2.4 dendritic cells. In both
cases, cell viability is preserved and interferon-β gene transcription is enhanced
while over-production of five inflammatory cytokines is not detected, suggesting
the hydrogels' immune stimulating property and potential as vaccine carriers. The
biodegradation is monitored efficiently using the hydrogels' intrinsic fluorescence
upon crosslinking with genipin. Addition of poly (ethylene glycol) to form a semi-
interpenetrating hydrogel is found to enhance the cytocompatibility to 3T3 cells
and delay the degradation. Collectively, our findings suggest that chitosan-genipin
hydrogels can be considered as biocompatible materials, with great potential for
vaccine delivery applications.
KEYWORDS
biocompatibility, biomaterials, crosslinking, degradation
targeted stimuli by adjusting their physical or chemical
behaviors, leading to the controlled release of therapeutic
agents.1 Various stimuli have been explored for modulat-
ing drug delivery, both internal and external to the body,
such as pH,2 temperature,3 light,4 electric field,5 redox,6
reduction,7 enzymes,8 and ultrasound.9 Among polymeric
constituents used for the preparation of smart hydrogels,
chitosan, a cationic polysaccharide produced from chitin,
is attracting particular attention. Its biocompatibility,
J Appl Polym Sci. 2021;138:e50848.
https://doi.org/10.1002/app.50848

VO ET AL.
biodegradability, and low toxicity, makes it widely stud-
ied as a polymeric backbone in hydrogels' development.10
Its solubility in aqueous solution depends on pH due to
the presence of primary amine groups, and acidic solu-
tion (approximately pH 4.5–5.5) is used in preparation of
chitosan solutions.11 Due to its hydrophilic nature and
low mechanical resistance, chitosan molecules need to be
crosslinked, either physically or chemically, to form a sta-
ble hydrogel.12 Physically crosslinked hydrogels are
formed by secondary interactions, including electrostatic
attraction,13,14 hydrogen bonding, and hydrophobic inter-
action.15 Even though there are some well-known advan-
tages of physical hydrogels including the absence of
harsh chemical crosslinker, no chemical modification
required, ease of fabrication process, minimal toxicity,
and good biocompatibility, their use in biomedical appli-
cations is limited due to the nature of reversible and
unstable networks, low mechanical strength, and uncon-
trolled dissolution/degradation/matrix porosity.16–18
In chemically crosslinked hydrogels, the dominant
interactions forming the network are covalent crosslinking
between multiple active amino and hydroxyl groups of
chitosan and functional groups of crosslinkers (such as
glutaraldehyde,19 formaldehyde,20 and epoxy compounds21).
As these commonly used crosslinkers are highly toxic and
free unreacted crosslinkers and other auxiliary molecules
cannot be completely excluded, leading to potential toxic
effects even if purification step is applied,13 there is an
imperative need to utilize bio-safe crosslinkers for the prep-
aration of chitosan hydrogels, such as genipin,22 dialdehyde
starch,23 tannic acid,24 and proanthocyanidin.25 Among
these natural-derived crosslinkers, the use of genipin is of
increasing interest. As a natural product extracted from gar-
denia fruit and commonly used as a food dye and in herbal
medicine, the cytotoxicity of genipin is found to be approxi-
mately 10,000 times less than that of glutaraldehyde.26,27
Another major advantage of using genipin is its high selec-
tivity, as primary amino groups ( NH2) is the only site
targeted by genipin, while the nucleophilic OH groups
( OH and COOH) do not react with genipin.28 Recently,
we reported the development of semi-interpenetrating
(semi-IPN) hydrogels composed of chitosan, genipin and
poly (ethylene glycol) (PEG).29 The crosslinking reaction
between primary amino groups of chitosan and genipin
was found to correlate with the development of secondary
amine and heterocyclic amine ring-stretching while PEG
addition (up to a critical concentration) may interact with
chitosan via attractive intermolecular hydrogen bonding.
The pH-responsiveness was confirmed, as the resulting
hydrogels swell in acidic and shrink in basic/neutral condi-
tions, in line with chitosan pKa values. The hydrogels' phys-
ical and chemical behaviors, including porosity, swelling,
mechanical strength, and gelation kinetics, were evaluated
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as a function of genipin and PEG content. We found that
increasing genipin content (from 3.1 to 6.3 mM) significantly
increased the elastic modulus (from ~ 445 to ~ 1100 Pa)
and reduced the average pore size (from ~ 24 to ~ 18 μm)
as well as swelling ratio (from ~ 22% to ~ 15%, in pH 2 gly-
cine buffer), while addition of PEG up to 1.9 mM generated
the same effect as increasing genipin content. Thus, addi-
tion of PEG is favorable, not only to simply achieve extra
level of control in hydrogels' microstructure, but also to
reduce cost of fabrication process by cutting down the
amount of genipin used.
Following display of many promising properties as a
controlled drug delivery platform, it is now highly impor-
tant to assess the biocompatibility of these hydrogels. In
order to reach clinical applications, a biomedical system
should be non- or low-cytotoxic and enzymatically or
hydrolytically degradable.30 However, a literature search
revealed only a handful of in vitro biocompatibility studies
involving genipin-crosslinked chitosan hydrogels, even less
with the presence of PEG. A genipin-crosslinked chitosan
film with PEG (molecular weight 600 g mol−1) added as a
plasticizer was prepared using three-dimensional
(3D) printing and a nontoxic nature was confirmed with
more than 90% of human skin fibroblasts viable on the
matrix.31 Chang et al. reported a study on genipin-
crosslinked carboxylic acid-terminated mPEG-grafted
chitosan hydrogels for use as a brain tumor model.32 They
seeded glioblastoma cells on hydrogel surfaces in 96-well
plates and assessed cell spheroids grown on the gel surfaces.
It is reported that the number of gel-exposed cells remained
similar or slightly higher than the number of seeded cells
after 1 day incubation. The gels promoted stable glioblas-
toma spheroid formation within 2–3 days, showing poten-
tial as a high-throughput screening system for cancer drugs.
Even though the biocompatibility of individual com-
ponents is widely reported, the biocompatible nature of
resulting hydrogels is not fully understood, particularly
their immunoregulatory properties. Known as an immu-
nostimulatory compound, chitosan would induce
interferon-β (IFN-β), interferon gamma-induced protein
10 (IP-10) and interleukin-1ra (IL-1ra) via activating cyto-
plasmic DNA sensor (cGAS) and stimulator of interferon
genes pathway or interleukin-1β (IL-1β) and prostaglan-
din E2 via NOD-like receptor protein 3 pathway.33–35
Upon crosslinking reaction with genipin, the immunoreg-
ulatory properties of chitosan-genipin hydrogels remain
unknown. Furthermore, the in vitro biodegradation was
most often monitored by means of gravimetric measure-
ments, which are not efficient. In the present work, the
biocompatibility of these materials is explored inclusively
with a broad range of inflammatory cytokines (tumor
necrosis factor-α (TNF-α), interleukin-6 [IL-6], IP-10,
IL-1β, and IFN-β), and the biodegradation is evaluated
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3 of 11 VO ET AL.

efficiently in a minimally invasive manner by monitoring
the decay of the hydrogels' intrinsic fluorescence. As it is
crucial to assess the biocompatibility toward cells that are
similar to those exposed to the material in the clinical set-
ting, three different cell types, which serve as master regu-
lators in inflammation response (fibroblast, macrophages,
and dendritic cells), were used in direct contact with
hydrogel surfaces. The degradation of these hydrogels was
studied in lysozyme solution. The work presented aims to
bring these genipin-crosslinked chitosan hydrogels one
step closer to clinical applications.
2 | R E SUL T S
2.1 | Cytotoxicity toward 3T3 cells
To evaluate the cytotoxicity, 3T3 fibroblasts were cultured
on hydrogel surfaces (Figure 1). Changes in cell
morphology were assessed microscopically, using light
microscope (Figure 1(a)) and scanning electron microscope
(SEM) (Figure 1(b)) while cell viability was assessed using
an adenosine triphosphate (ATP)-based assay (Figure 1(c)).
Microscopic images show that the 3T3 cells maintain
normal morphology on hydrogels with higher genipin
content. Fibroblasts cultured on gels with 1.7 mM genipin
(F1 and F1P) remained round or oval (Figure 1(a)),
revealing a weak contact with the surface and a low per-
centage of viability (around 25%–30%, Figure 1(c)). Fibro-
blasts cultured on gels with 3.1 mM (F2 and F2P) or
4.4 mM genipin (F3 and F3P) formed elongated or spindle-
like shape, showing good spreading morphology and a
high percentage of viability (around 73%–95%). SEM
images of fibroblasts cultured on gel F3P show confluent
growth of cells on the gel surface where cells attached, flat-
tened, and spread with assembly of filopodia (Figure 1(b)).
Addition of PEG to form semi-IPN hydrogels significantly
enhanced cell viability as recorded in gels F2P and F3P,

F I G U R E 1 In vitro toxicity test using 3T3 fibroblasts cultured on chitosan-genipin hydrogels (F1, F2, and F3) and chitosan-genipin-PEG
hydrogels (F1P, F2P, and F3P) for 48 h at 37 C. (a) Light microscope images of control cells and gel-exposed cells (scale bars = 200 μm).
(b) Scanning electron microscope (SEM) images of fibroblasts cultured on sample F3P (top, scale bar = 100 μm) and its magnified section
(bottom, scale bar = 10 μm). (c) Cell viability measured by CellTiter-Glo® 2.0 assay. The luminescent intensity recorded with the control
cells was set as 100% viability. The percentages of viability of gel-exposed cells were adjusted relatively based on the control group. Data are
representative of two independent experiments (n = 6/group). Statistical significance was determined by two-tailed unpaired Student's t test;
***, p < 0.001; ****, p < 0.0001; NS, not significant [Color figure can be viewed at wileyonlinelibrary.com]
 10974628, 2021, 34, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/app.50848 by Riga Technical University, Wiley Online Library on [23/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
VO ET AL. 4 of 11

compared to gels F2 and F3, respectively, (p < 0.05)
(Figure 1(c)). In gels with the least genipin content, addition
of PEG improved cell viability (although not statistically sig-
nificant; F1 vs. F1P, p > 0.05). These findings reveal that
the hydrogels with higher genipin content or addition of
PEG present better cytocompatibility to fibroblast.
2.2 | Biocompatibility toward DC 2.4 and
RAW 264.7 cells
To test if genipin-crosslinked chitosan hydrogels induce
inflammatory cytokines, the gels with highest genipin con-
tent (4.4 mM, gel F3) were selected for further study. To
confirm the hydrogel is not toxic to DC 2.4 and RAW 264.7
cells, viability of these cells was evaluated. As shown in
Figure 2(a), crosslinked hydrogels with highest genipin
content have minimal toxicity to DC 2.4 and RAW 264.7
cells, similar to those observed in 3T3 cells. The percent-
ages of viability of gel-exposed DC 2.4 cells are 102.9%
± 26.5% (at 6 h) and 96.8% ± 20.5% (at 24 h), without sig-
nificant differences compared to the control group. The
percentages of viability of gel-exposed RAW 264.7 cells are
94.5% ± 22.2% (at 6 h) and 85.1% ± 9.7% (at 24 h), without
significant differences compared to the control group.
Cytokine expression was analyzed at both protein
level (measured by enzyme-linked immunosorbent assay
[ELISA]) and mRNA level (measured by reverse
transcription-polymerase chain reaction [RT-PCR]). At
protein level, Figure 2(b) shows that the measured con-
centrations of TNF-α, IL-6, and IP-10 released by gel-
exposed DC 2.4 cells at 6 h are 16.7 ± 13.6; 69.2 ± 6.9,

F I G U R E 2 Biocompatibility profiles of chitosan-genipin hydrogels (sample F3) upon incubation with DC 2.4 and RAW 264.7 cells at
37 C. assays were carried out at 6 and 24 h of incubation. (a) Cell viability measured by CellTiter-Glo® 2.0 assay. The luminescent intensity
recorded with the control cells was set as 100% viability. The percentages of viability of gel-exposed cells were adjusted relatively based on
the control group. Cytokine expression at protein level of (b) DC 2.4 and (c) RAW 264.7 cells. Cytokine expression at mRNA level of (d) DC
2.4 and (e) RAW 264.7 cells. Data are representative of two independent experiments (n = 6/group). Statistical significance was determined
by two-tailed unpaired Student's t test; **, p < 0.01; ***, p < 0.001; ND, not detected [Color figure can be viewed at wileyonlinelibrary.com]

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and 60.7 ± 3.1 pg ml−1, respectively, which are not signif-
icantly different compared to those produced by the con-
trol groups. At 24 h, the levels of IL-6 and IP-10 released
by gel-exposed DC 2.4 cells are not significantly different
while TNF-α level is significantly lower, compared to
those from control cells. In case of RAW 264.7 cells, the
measured concentrations of TNF-α, IL-6 and IP-10
released by gel-exposed cells at 6 h are 128.0 ± 21.5; 22.1
± 11.4; and 69.5 ± 16.2 pg ml−1, which are not signifi-
cantly different (in case of IL-6 and IP-10) or significantly
lower (in case of TNF-α) compared to those produced by
the control groups (Figure 2(c)). At 24 h, there is no sig-
nificant difference in levels of TNF-α, IL-6, and IP-10 pro-
duced by control cells and by gel-exposed RAW 264.7
cells. In both cell lines, the concentrations of IL-1β and
IFN-β are not detectable, indicating that the hydrogels do
not produce up-regulation of IL-1β and IFN-β in vitro.
At mRNA level, relative expression levels of TNF-α,
IL-6, IP-10, and IL-1β in gel-exposed DC 2.4 cells within
6–24 h incubation are negligible (≤3) (Figure 2(d)). Simi-
larly, mRNA expression levels of these four cytokines in
gel-exposed RAW 264.7 cells are low (≤5) (Figure 2(e)).
Interestingly, the highest level of cytokine gene expression
is found to be IFN-β. At 6 h, IFN-β gene expression levels
in gel-exposed DC 2.4 and RAW 264.7 cells are 356.2
± 65.5 and 55.7 ± 4.2, respectively. At 24 h, IFN-β gene
expression levels in gel-exposed DC 2.4 and RAW 264.7
cells are 2435.5 ± 264.5 and 181.3 ± 14.2, respectively.
Even though the up-regulation of IFN-β gene expression
within 24 h incubation is not efficient enough to produce
an elevated amount of IFN-β in culture media, these
results indicate that these crosslinked hydrogels are hypo-
inflammatory and low toxic to DC 2.4 and RAW 264.7
cells. The IFN-β gene induction found in those cells sug-
gests that the hydrogels preserve chitosan's immune stim-
ulating property to certain extent and might be used as an
immune adjuvant, but further in vivo studies are needed.
2.3 | In vitro degradation test
To assess the enzymatic degradation of hydrogels,
changes in intrinsic fluorescence of hydrogels during
incubation with lysozyme were recorded. From the 3D
scanning, a detailed plot of excitation wavelength (λEX),
emission wavelength (λEM) and relative fluorescent
units reveals that the gels have optimal excitation and
emission wavelength at 580 and 630 nm, respectively,
(Figure 3(a)). Thus, the initial fluorescence and its
changes upon exposure to lysozyme were recorded at
these wavelengths (Figure 3(b)–(f)).
As shown in Figure 3, within 1–3 days, the enzymatic
degradation of all hydrogels studied is already measurable
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VO ET AL.
as indicated by the decrease of fluorescence intensity from
100% to 70%–80%. The hydrogels incubated in PBS solution
displayed no degradation as the recorded fluorescence
intensity remained unchanged over time (data not shown).
The rate and extent of hydrogel degradation depend on the
degree of crosslinking as well as the presence of PEG. Gels
with 1.7 mM genipin (F1 and F1P) showed a significant
decrease in fluorescence during the first 5 days, while gels
with 3.1 mM genipin (F2 and F2P) or 4.4 mM genipin (F3
and F3P) had a slight decrease during the first 5 days and
remained considerably stable until Day 10 before their fluo-
rescence intensity started to decrease, reaching zero at
around Day 17 (Figure 3(b),(c)). The highest rates of fluo-
rescence loss in gels F1, F2, and F3 are 23.9%; 18.9%, and
17.5% per day, respectively; and in gels F1P, F2P, and F3P
are 28.2%; 22.3%, and 20.9% per day, respectively. Fluores-
cence intensity measurements coincide with visual observa-
tion of the degradation of hydrogels. Collectively, these
findings suggest that an increase in crosslinking density
results in a delayed degradation and a slower
degradation rate.
Figure 3(d)–(f) shows the effect addition of PEG has
on degradation in relation to genipin content. In gels
with 1.7 mM genipin, addition of PEG showed little effect
on degradation as gels F1 and F1P had similar degrada-
tion patterns (Figure 3(d)). In gels with 3.1 mM genipin,
addition of PEG slightly retarded degradation as gels F2
and F2P started to degrade at Days 10 and 12, respec-
tively, (Figure 3(e)). This is possibly due to PEG filling up
the spaces within the gel network and binding to the
hydroxyl groups on chitosan chains via hydrogen bond-
ing, resulting in a denser structure suppressing the mobil-
ity of polymer chains. In gels with 4.4 mM genipin,
addition of PEG did not further delay degradation as gels
F3 and F3P started to degrade at Days 12 and 10, respec-
tively, (Figure 3(f)). It can be postulated that in this case,
PEG interacts with chitosan, reducing the number of
effective crosslinks between chitosan and genipin,29 and
confining the genipin's suppression effect on degradation.
Once the hydrogels start to degrade, the increased hydro-
philicity induced by PEG addition appears to accelerate
the degradation, resulting in a faster degradation rate
observed in PEG-added hydrogels compared to non-PEG-
added hydrogels, regardless of genipin content.
3 | DISCUSSION
Although chitosan membrane was reported to have no
toxic effects on fibroblast cells in vitro,36 chemical
crosslinking is known to induce cytotoxic effect and
requires investigation. As shown in the literature,
crosslinking with glutaraldehyde caused harmful irritation
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VO ET AL. 6 of 11

F I G U R E 3 Fluorescence contour map of chitosan-genipin-poly (ethylene glycol) (PEG) hydrogels (sample F3P) (a). Upon exposure to
lysozyme, the fluorescence of hydrogels was recorded at excitation/emission wavelength of 580/630 nm. Fluorescence of hydrogels grouped
by presence of PEG: (b) chitosan-genipin hydrogels and (c) chitosan-genipin-PEG hydrogels. Fluorescence of hydrogels grouped by genipin
content: Gels with (d) 1.7 mM genipin, (e) 3.1 mM genipin, and (f) 4.4 mM genipin. Data are representative of two independent experiments
(n = 4 at each data point). Statistical significance was determined by two-way ANOVA with multiple comparisons; *, p < 0.05; **, p < 0.01;
***, p < 0.001; ****, p < 0.0001; NS, not significant [Color figure can be viewed at wileyonlinelibrary.com]

to cultured cells and reduced cell viability.37,38 Our micro-
scopic images and viability assay show that hydrogels with
higher genipin content (3.1 or 4.4 mM) present better
cytocompatibility to fibroblasts, in contrast to the counter-
parts with 1.7 mM genipin (Figure 1). Our previous study
has evaluated the relation between genipin content and
elastic modulus using small deformation rheology mea-
surements.29 We found that increasing genipin content
from 3.1 to 6.3 mM significantly increased elastic modulus
(from ~ 445 to ~ 1100 Pa) and shortened the linear visco-
elastic region by 50%, suggesting a positive correlation
between genipin content and hydrogels' rigidity. In light of
these recent findings, the better compatibility of 3T3 cells
on gels with higher genipin content may be postulated to
relate to the higher stiffness of these gels. As fibroblasts'
behaviors were only assessed in terms of morphology and
viability by means of microscopy and ATP-based assay,
further investigation into cell-gel interface is needed, such
as cell migration and differentiation by means of
immunostaining,39 substrate thickness and bulk/interfa-
cial stiffness,40 surface patterning and topography.41
The present study, based on cell viability data
(Figure 1(c)), provides evidence to support the addition
of PEG. Our previous study has shown that the pres-
ence of PEG (1.2 mM) increased the elastic modulus
(from ~ 220 to ~ 445 Pa) and rigidity of the network,29
which may contribute to the enhanced
cytocompatibility of PEG-added hydrogels. Another
key factor that affects cell responses is surface wettabil-
ity. Cells show favorable adhesion to moderate hydro-
philic surfaces compared to hydrophobic or extremely
hydrophilic surfaces.42–45 The increase in hydrophilic-
ity of hydrogel surfaces by addition of PEG may pro-
mote cell adhesion and growth. Even though further
in vivo studies are needed, these results provide evi-
dence to support the hydrogel design for a specific
application. For instance, these results suggest that for
implantation, a stiffer gel with higher genipin content
and good mechanical robustness would produce favor-
able cellular responses.
As immunological responses are responsible for
adverse outcomes upon injection or implantation of bio-
materials, including inflammation and fibrosis, initial
evaluation of biocompatibility of chitosan-genipin hydro-
gels toward macrophages (RAW 264.7) and dendritic cells
(DC 2.4) were carried out. The results show that the

7 of 11
hydrogels with highest genipin content (4.4 mM) preserve
cell viability and do not cause over-production of five
inflammatory cytokines (Figure 2), suggesting their hypo-
inflammatory behavior. Interestingly, gel-exposed cells
expressed an elevated amount of IFN-β gene, especially
in DC 2.4 cells with IFN-β mRNA expression level of
~ 2400 at 24 h incubation (Figure 2(d)). These results
indicate that crosslinking reaction between chitosan and
genipin yields a biocompatible product without
compromising the biocompatibility of each component
even when high amount of genipin is used, providing
extra evidence to support the use of genipin for medical
purposes.46–49 The IFN-β gene induction found in gel-
exposed cells, suggests the immune stimulating property
of the hydrogels and the potential to act as an immune
adjuvant in vaccine delivery, even though further in vivo
studies are required.
Previous studies have commonly used the weight loss
to monitor the degradation.50–52 However, gravimetric
measurement is a highly invasive procedure with several
intermediate steps (such as transferring gels between
enzyme solution and balance, removing excess solvent,
and a gel fragmentation during the handling) which
could affect the results. In this study, we show for the
first time, to our knowledge, the use of the hydrogels'
intrinsic fluorescence to track the enzymatic degradation
efficiently in a minimally invasive manner. The hydro-
gels' fluorescence is a result of highly π-π* conjugated
derivatives formed by the crosslinking reaction between
chitosan and genipin.53 Consistent with previous
reports,53,54 our results confirm the presence of a fluores-
cence peak at the excitation/emission wavelengths of
370/470 nm. However, we find that the hydrogels exhibit
a stronger red fluorescence at 580/630 nm, which were
then used as optimal excitation/emission wavelengths for
fluorescence characterization (Figure 3(a)).
Enzymatic degradation is a significant source of
chitosan depolymerization, in which chitosan is degraded
by lysozyme and bacterial enzymes present in the
colon.55–57 Thus, lysozyme was the choice of enzyme in
this study. The active site of lysozyme is found to consist
of six subsites, which bind to the D-glucosamine units of
chitosan and form enzyme-material complex.58 The
cleavage of glycosidic linkage occurs when the alternate
sites of lysozyme interact with acetamide side chains of
N-acetyl glucosamine units of chitosan. Simultaneously
with chitosan chain scission, cleavage and/or destruction
of its functional groups (amino, carbonyl, and hydroxyl)
occur.59 In this study, the degradation process shows a
biphasic pattern, which involves a gradual decrease in
fluorescence within the first few days, followed by a sud-
den loss in fluorescence until the matrix is completely
broken down. The first phase of the degradation process
10974628, 2021, 34, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/app.50848 by Riga Technical University, Wiley Online Library on [23/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
VO ET AL.
exhibits moderate fluorescent loss, possibly due to the
initial diffusion of lysozyme from surrounding solution to
the gel matrix and the gradual recognition of acetamide
side chain of N-acetyl glucosamine units to activate its
hydrolysis activity. Once cleavage of glycosidic linkage
occurs, the degradation products, including low MW
chitosan, chitogligomer, and N-acetyl glucosamine D-res-
idues, may be subsequently released and dissolved into
the surrounding media.60 As lysozyme solution is
refreshed after each fluorescence reading and the hydro-
gel networks become loosened over time, the hydrolysis
activity is accelerated, leading to an abrupt decrease in
fluorescence intensity, which coincides with the sudden
mass loss observed visually.
As expected, increasing genipin content prolonged
the degradation with a slower degradation rate, due to
the higher crosslinked network suppressing the mobility
of polymer chains (Figure 3(b),(c)). Moreover, chitosan
chains bridged by genipin form a cyclic crosslinking
structure with increased stereohindrance for the penetra-
tion of lysozyme due to the bulky heterocyclic-structure
of genipin.61 These observations are consistent with pre-
vious reports,61–64 indicating that genipin concentration
is a suitable variable to modulate the hydrogels' degrada-
tion rate. We also uncover the effect of addition of PEG
on degradation. Addition of PEG delays the hydrogel deg-
radation to some extent. Addition of PEG showed the
most distinct impact on degradation of hydrogels
employing moderate genipin concentration (F2 and F2P,
Figure 3(e)), compared to the counterparts with lowest
(F1 and F1P, Figure 3(d)) or highest (F3 and F3P,
Figure 3(f)) genipin content. This suggests existence of
an optimal range of genipin concentration beyond which
PEG addition may lead to a reduced number of effective
crosslinking between chitosan and genipin. These find-
ings support the results from our previous study, which
revealed the link between PEG content and the sudden
change in hydrogels' swelling capacity, gelation kinetics
and mechanical strengths.29 Methodology used to follow
the degradation of theses gels in vitro is an innovative
and yet simple approach to deploy the intrinsic fluores-
cence of the chitosan-genipin links for tracking the bio-
degradation in vitro, and possibly in vivo too.
4 | CONCLUSION
The hydrogel bioactivity is a critical design parameter
which must be fully investigated for a successful clinical
application. This study provides initial evaluation of bio-
logical functions of chitosan-genipin hydrogels, including
biocompatibility toward three different cell lines and bio-
degradation under enzymatic activity. Our results show

VO ET AL.
that hydrogels with higher genipin content (3.1 or
4.4 mM) support better 3T3 fibroblasts' adhesion and via-
bility, compared to the hydrogels with 1.7 mM genipin.
The hydrogels with highest genipin content (4.4 mM) do
not cause the over-production of five inflammatory cyto-
kines (TNF-α, IL-6, IP-10, IL-1β, and IFN-β) in RAW
264.7 macrophages and DC 2.4 dendritic cells while pre-
serving good cell viability. These results highlight the
good biocompatibility of the crosslinked hydrogels, even
when high amount of crosslinking agent genipin is used.
The increase of IFN-β gene expression found in RAW
264.7 and DC 2.4 cells suggests the potential of these
materials as an immune adjuvant, however further
in vivo studies are required to understand the signaling
pathway of these materials. The hydrogel's ability to fluo-
resce is beneficial, not only for tracking biodegradation
in vitro, but also for prospective monitoring of conforma-
tion changes, distribution, and degradation in living bod-
ies, without the need for a fluorescent probe. Addition of
PEG to form a semi-IPN network is found to enhance the
cytocompatibility to 3T3 fibroblasts and delay the enzy-
matic degradation to some extent. These findings encour-
age further developments and in vivo validations of these
materials, including use in the field of vaccine delivery.
5 | EXPERIMENTAL SECTION
5.1 | Materials
Chitosan (medium molecular weight [MW], 82%
deacetylation, viscosity 420 centipoise, product number
448877, lot number STBG5137V), PEG (MW 6000 g mol−1,
product number 81255, lot number BCBT1548), genipin
(MW 226 g mol−1, product number G4796, lot number
116M4706V), glacial acetic acid, ethanol, and hexa-
methyldisilazane (HMDS) were supplied from Sigma–
Aldrich. 3T3 mouse fibroblast cells, RAW 264.7 macrophage
TABLE 1 Compositions of feed solutions of chitosan, PEG, and genipin
Initial feed solutiona
Sample name VChitosan (ml)
F1
F2
F3
1.0
F1P
F2P
F3P
Abbreviation: PEG, poly (ethylene glycol).
a
Chitosan 1.5% (wt/vol); PEG 5% (wt/vol), and genipin 0.5% (wt/vol).
b
0.2 ml of deionized water was added instead of PEG solution to preserve desired concentrations of genipin and enable direct comparison of samples.
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8 of 11
cells and DC 2.4 dendritic cells were from Huang and
Mellor's lab (Newcastle University, UK). The complete cul-
ture medium used was Dulbecco's modified Eagle medium
(DMEM) or Roswell Park Memorial Institute (RPMI)–1640
medium, supplemented with fetal calf serum (10%), L-
glutamine (2 mM), penicillin (100 U ml−1) and streptomy-
cin (100 U ml−1). Phosphate buffered saline (PBS), trypsin
0.05%–EDTA 1X solution and trypan blue solution (0.4%)
were supplied from Fisher Scientific. CellTiter-Glo® 2.0
assay was supplied from Promega. ELISA kits for TNF-α,
IL-6, and IL-1β were supplied from Biolegend, while ELISA
kits for IP-10 and IFN-β were supplied from R&D System.
Complementary DNA kits for RT-PCR were supplied from
Clontech.
5.2 | Synthesis of hydrogel samples
Chitosan (1.5% wt/vol) was dissolved in acetic acid
solution (1% vol/vol) and sterilized by autoclaving
(136 C, 90 min). PEG (5% wt/vol in deionized water)
and genipin (0.5% wt/vol in deionized water) were ster-
ilized using 0.22 μm syringe filters. In multi-well
plates, a mixture of chitosan, PEG and genipin solu-
tions was added to each well (Table 1). The plates were
incubated at 37 C for 24 h to facilitate the gelation.
Following, the gel-coated wells were washed with PBS
for 2 h and incubated with culture mediums for 24 h at
37 C (for in vitro toxicity and cytokine analysis). For
testing in vitro degradation, once removed from the
oven, the plates were directly exposed to lysozyme
solution (as detailed in Section 2.6).
5.3 | Preparing cell suspension
Cells were cultured in the complete culture medium
(DMEM for 3T3 and RPMI-1640 for DC 2.4 and RAW
VPEG (ml) VGenipin (ml) [C]genipin/sample (mM)
— b
0.1 1.7
0.2 3.1
0.3 4.4
0.2 0.1 1.7
0.2 3.1
0.3 4.4

9 of 11
264.7) at 37 C in a humidified atmosphere containing 5%
CO2. The cells growing in a logarithmic phase were col-
lected for passage or seeding. The cell suspension was
centrifuged (1200 rpm, 5 min) and the cells were re-
suspended in culture medium. The total number of live
cells was counted using a trypan blue solution (0.4%) and
a hemocytometer. For all in vitro tests, the control cells
(cells seeded in wells containing only culture medium)
and treated cells (cells seeded in gel-coated wells) were
incubated at 37 C in a humidified atmosphere containing
5% CO2.
5.4 | Cytotoxicity toward 3T3 cells
To investigate the cytotoxic effect, a mouse fibroblast
cell line (3T3) was used. The morphology of 3T3 cells
attached on hydrogel surfaces (prepared in 24-well
plates) was evaluated using light microscope and SEM.
The 3T3 cells were seeded on the gel-coated plates by
dispensing cell suspension (500 μl) into each well and
gently rotating horizontally to distribute the cells evenly
over the gel surfaces. The cells were examined using an
inverted microscope every 12 h to assess the changes in
cell morphology for 48 h. At the endpoint, the culture
medium was aspirated, and the samples were fixed in
formalin for 24 h followed by dehydration using etha-
nol, HMDS gradient and air-drying, and then examined
by SEM.
To quantify cell viability, 3T3 cell suspension (100 μl)
was added on hydrogel surfaces (prepared in 96-well
plates) and the amount of ATP produced by viable cells
was measured using CellTiter-Glo® 2.0 reagent. After
48 h incubation, in each well, the reagent (100 μl) was
added. The plates were placed on an orbital shaker for
2 min and equilibrated at room temperature for 10 min
to stabilize the luminescent signals, which were then
recorded using a plate reader.
5.5 | Biocompatibility toward DC 2.4 and
RAW 264.7 cells
To evaluate the induction of inflammatory cytokines, a
mouse dendritic cell line (DC 2.4) and a mouse macro-
phage cell line (RAW 264.7) were used. The DC 2.4 or
RAW 264.7 cells were seeded on hydrogel surfaces (pre-
pared in 24-well plates) and at scheduled times, super-
natant was collected to measure cytokine production
(TNF-α, IL-6, IP-10, IL-1β, and IFN-β) by ELISA
according to manufacturer protocols while the cells
were lysed to measure cytokine gene expression by
RT-PCR. The total RNA was isolated from the lysed cells
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VO ET AL.
by trizol and the extracted RNA was reverse-transcribed
using a random hexamer cDNA RT kit (Clontech).
Quantitative RT-PCR was performed on AriaMx
RT-PCR system (Agilent) with iQ™ SYBR™ Green Sup-
ermix (Bio-rad). A house keeping gene, glyceraldehyde-
3-phosphate dehydrogenase was used as the endogenous
RNA control. Each sample was run in duplicate and
normalized to the internal control. To quantify cell via-
bility, DC 2.4 or RAW 264.7 cells were seeded on hydro-
gel surfaces (prepared in 96-well plates) and the
CellTiter-Glo® 2.0 reagent was used following the same
procedure as applied to 3T3 cells.
5.6 | In vitro degradation test
To test if the hydrogels are enzymatically degradable, we
tracked the decrease in their intrinsic fluorescence upon
exposure to lysozyme.53 Degradation was also denoted
visually. A 3D fluorescence scan with excitation range of
350–600 nm and emission range of 450–700 nm was per-
formed to determine the optimal excitation and emission
peaks of the gels prepared in 24-well black plates. Lyso-
zyme with an activity of ~ 100,000 U mg−1 was dissolved
in PBS (0.5 mg ml−1) and then added to the gel samples
(1 ml per well). The samples were then incubated at
37 C. At scheduled times, the solutions were aspirated
and the plates were read for fluorescent signals. After
each reading, the same amount of treatment solution was
added to each well. For reference purpose, the control
samples were tested under the same condition as
described above using PBS solution without adding
lysozyme.
5.7 | Statistics
Data are expressed as mean ± standard deviation. For
degradation test, two-way ANOVA with multiple com-
parisons was performed. Unpaired two-tailed Student's
t tests were performed for two group comparisons. Statis-
tical significance is defined as * p < 0.05, ** p < 0.01, ***
p < 0.001, and **** p < 0.0001. OriginPro was used to
perform all data analyses.
ACKNOWLEDGMENTS
This work was supported by the Research Excellence
Academy scheme, Newcastle University, and UK Engi-
neering and Physical Sciences Research Council (grant
number EP/N033655/1).
CONFLICT OF INTEREST
The authors declare no conflict of interest.

VO ET AL.
ORCID
Nga T. N. Vo https://orcid.org/0000-0001-8946-9312
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