Carbohydrate Polymers 289 (2022) 119467

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Paramylon hydrogel: A bioactive polysaccharides hydrogel that scavenges

ROS and promotes angiogenesis for wound repair
Huan Lei a, b, c, Jing Zhao a, b, c, Hang Li d, Daidi Fan a, b, c, *
a
Shaanxi Key Laboratory of Degradable Biomedical Materials, School of Chemical Engineering, Northwest University, Taibai North Road 229, Xi'an 710069, China
b
Shaanxi R&D Center of Biomaterials and Fermentation Engineering, School of Chemical Engineering, Northwest University, Xi'an 710069, China
c
Biotech & Biomed Research Institute, Northwest University, Xi'an 710069, China
d
Peking University First Hospital, Department of Dermatology, Xishiku Street No.8, Beijing 100034, China

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

Chemical compounds studied in this article: The excessive inflammation, oxidative stress, and impaired angiogenesis are major factors leading to difficulties
Paramylon (PubChem SID: 405237933) in chronic wound healing. To develop bioactive materials with intrinsic antioxidant and anti-inflammatory
1,4-Butanediol diepoxyglycerol ether properties, we prepared hydrogels for the first time using paramylon secreted by Euglena gracilis which is a
(PubChem CID: 9581357)
polysaccharide has been approved by FDA as food additive. Results showed that the paramylon hydrogel has
Keywords: favourable anti-inflammatory effects and the ability to scavenge reactive oxygen species (ROS) through free
Paramylon hydrogel
radical destruction, deoxygenation, and singlet oxygen quenching, and inhibit ROS production by chelating the
Anti-inflammatory
Antioxidant
metal ions required for the formation of ROS. We found that the paramylon hydrogel could effectively reduce
Pro-angiogenesis wound inflammation and promote angiogenesis to facilitate wound repair. Furthermore, for the first time, we
Wound repair found that paramylon hydrogel could promote the formation of blood vessels via the HIF-1α-VEGF pathway.
These results indicated that the highly bioactive paramylon could be the preferred material for wound healing.

1. Introduction materials with intrinsic antioxidant, anti-inflammatory and angiogenic

Chronic wounds repair remains a huge clinical challenge due to
excessive inflammation, oxidative stress, and impaired wound angio­
genesis (Shen et al., 2021; Tu et al., 2021). For chronic inflammation and
high levels of reactive oxygen species (ROS), current biomedical mate­
rial responses include wound dressings hydrogels (Chen, Chen, et al.,
2018; Huang et al., 2018; Zhao, Zhang, et al., 2020), fibrous membranes
(Guo et al., 2020; Xia, Zhang, Yu, Pei, & Luo, 2020), etc.) or nanocarriers
loaded with anti-inflammatory agents (e.g., antibiotics (Albright et al.,
2021), curcumin (Liu et al., 2018; Shen et al., 2021)) and polymer
networks grafted or loaded with antioxidant components (e.g., epi­
gallocatechin gallate (Le Thi et al., 2020; Zhao et al., 2021), polydop­
amine (Xu et al., 2020; Yuan, Shen, & Fan, 2021), nanozyme such as
copper or iron nanoparticles with ellagic acid ligands (Li, Fu, Duan, Zhu,
& Fan, 2022; Wang et al., 2021)). Majority of these research focused on
the loaded therapeutic agents. These strategies increased the complexity
of the system, and may cause abnormal tissue metabolism due to sudden
release. On the other hand, poor angiogenesis in chronic wounds can
also delay healing with a lack of oxygen and nutrients. Bioactive
properties provide a promising solution.
In order to simplify the design of wound dressing and eliminate the
need for drug loading, we focused on those polysaccharides that have
biological activity produced by marine species. For example, fucoidan
isolated from brown algae, and kelp polysaccharides showed antioxi­
dant effects via reducing superoxide anion radicals and scavenging ROS,
as well as anti-inflammatory effects by inhibiting pro-inflammatory
factors such as IL-8 and TNF-α (Yuan, Yuan, et al., 2021). Aart A. van
Apeldoorn et al. used fucoidan prepared a microencapsulated hydrogel
encapsulating beta cells using fucoidan, which significantly reduced
oxidative stress in pancreatic islets and β-cells exposed to oxidative
stress conditions (Reys et al., 2021). Another highly promising material,
paramylon, is synthesized from naked algae (also known as Euglena
gracilis) (Barsanti & Gualtieri, 2019), which is a polysaccharide
composed of linear β-1,3-glucan (Nakashima et al., 2018). Paramylon
has been approved by the Food and Drug Administration (FDA) as a food
additive (FDA, 2018), and currently shows great promise in the food
field. Sugiyama et al. (2009) found that oral administration of para­
mylon could protect against CCl4-induced acute liver injury in rats

* Corresponding author at: Shaanxi Key Laboratory of Degradable Biomedical Materials, School of Chemical Engineering, Northwest University, Taibai North Road
229, Xi'an 710069, China.
E-mail addresses: zhaojing@nwu.edu.cn (J. Zhao), fandaidi@nwu.edu.cn (D. Fan).

https://doi.org/10.1016/j.carbpol.2022.119467
Received 30 December 2021; Received in revised form 2 April 2022; Accepted 4 April 2022
Available online 8 April 2022
0144-8617/© 2022 Elsevier Ltd. All rights reserved.
H. Lei et al.
through an antioxidant mechanism. These authors also showed that oral
administration of paramylon inhibited the development of atopic
dermatitis in mice (Sugiyama et al., 2010). Kengo et al. (Suzuki et al.,
2018) evaluated the remission of collagen-induced rheumatoid arthritis
(RA) in mice with naked algae powder and paramylon. Clinical signs of
arthritis as well as IL-17, IL-6 and IFN-γ levels in lymphatic tissues were
analysed to determine the effect of naked algae powder and paramylon
on the relief of RA. These researches suggested that paramylon is a
promising antioxidant and anti-inflammatory material.
We hope to leverage the biological efficacy of these algal poly­
saccharides to further improve wound repair dressings, there are no
reports on the preparation of hydrogels using paramylon, and we
speculate that due to the high crystallinity of paramylon (Nakashima
et al., 2018), it is difficult to dissolve in water, thus limiting its devel­
opment in hydrogel wound repair dressings. Considering the microen­
vironment of chronic wounds with excessive inflammation and high
levels of ROS, we believe that paramylon have the potential to shorten
the inflammatory phase of wound healing and is a promising material
for wound repair.
The preparation of hydrogels for wound repair using paramylon has
not yet been reported and the mechanisms involved are still unknown.
Therefore, in this work, we prepared paramylon hydrogels for the first
time and seek to exploit the properties of the biomaterial itself to slow
down inflammation and reactive oxygen species and promote angio­
genesis, and attempted to elucidate the mechanism behind ROS clear­
ance and angiogenesis. The results of ROS scavenging experiments
showed that the paramylon hydrogels can scavenge ROS through free
radical destruction, deoxygenation, and singlet oxygen quenching, and
the paramylon hydrogels could also inhibit ROS production by chelating
the metal ions required for the formation of ROS. During the repair of
chronic burn wounds, paramylon hydrogel promoted the formation of
blood vessels via the HIF-1α-VEGF pathway, which facilitated rapid
wound healing.
2. Materials and methods
2.1. Materials
Paramylon (Mw: 190 kDa, 98%) was provided by Yunnan Baoshan
Zeyuan algal health technology Co., Ltd. (Baoshan, China), and 1,4-
butanediol diepoxyglycerol ether (BDDE) was purchased from Macklin
Inc. (Shanghai, China). Other reagents were used as received without
further processing or purification.
2.2. Preparation of the paramylon hydrogel
First, 1 g of paramylon powder and 0.3 g of sodium hydroxide were
added to 10 mL of water and stirred thoroughly until the paramylon was
fully dissolved in a translucent solution. Then, 0.6 mL of 1,4-butanediol
diglycidyl ether (BDDE) was mixed completely into the solution, which
was left in a water bath at 50 ◦ C for 6 h to form an alkaline paramylon
hydrogel. To adjust the pH of the paramylon hydrogel, and dialyzed
against weak acid (pH 6.0) twice for 1 h each and then against neutral
water (pH 7.0) 10 times for 3–5 h each until the pH was neutral
(6.8–7.5), and the paramylon hydrogel was obtained.
The dialyzed process leads to changes in the proportional compo­
nents of the paramylon hydrogel, and we have examined changes in the
swelling, pore size and mechanical properties of the hydrogel during the
dialyzed process. In order to clarify type of hydrogel used in each
experiment, we illustrate the volume and composition of the hydrogels
used in all experiments, as shown in Supporting Table 1.
2.3. Characterization
Details on the scanning electron microscopy (SEM, Carl Zeiss,
Oberkochen, Germany) analysis, pore size determination, Fourier
2
Carbohydrate Polymers 289 (2022) 119467
transform infrared (FTIR) spectroscopy (Thermo Fisher Scientific, Wal­
tham, MA, USA) analysis, thermogravimetric analysis (TGA, SDT-Q600,
TA Instruments, USA), transmittance measurement, water retention
analysis, and swelling test can be found in the supporting information.
2.4. Mechanical properties
The analysis of mechanical properties included rheological testing
and compression testing. The rheological properties of the paramylon
hydrogels were measured with a rheometer (Anton Paar, MCR302) to
determine the transformation between the storage modulus (G′ ) and loss
modulus (G′′ ) in the critical strain region and linear viscoelastic region of
the paramylon hydrogel. Mechanical testing of the paramylon hydrogel
was performed using a universal material testing machine (INSTRON
5565, Instron, Norwood, MA, USA). For the cyclic compression tests, the
hydrogel (cylindrical, 1 cm diameter, 1 cm thickness) was compressed
for one cycle, and the modulus data were automatically calculated by
the INSTRON 5565 instrument.
2.5. Biocompatibility of the hydrogels
The biocompatibility of the paramylon hydrogels was evaluated
through analyses of the cytotoxicity of hydrogel extracts, cell adhesion
on the hydrogels, cell proliferation on the hydrogels, and blood
compatibility (haemolysis assay). Experimental details can be found in
the supporting information.
2.6. In vitro anti-inflammatory activity
In vitro anti-inflammatory experiments were conducted using mouse
monocyte macrophage leukemia cells (RAW264.7). The experiments
were grouped into control group (normal culture medium), lipopoly­
saccharide (LPS) group (concentration of 10 ng/mL of LPS, positive
control), paramylon hydrogel group (concentration of 50 mg/mL of
paramylon extract), and LPS + paramylon hydrogel group (concentra­
tions the same as the LPS group and paramylon hydrogel group). The
RAW264.7 cells were inoculated on sterile coverslips and cultured for
24 h, and the supernatant was carefully removed, and the liquid from
each group was added for another 24 h. Cell crawls were washed with
PBS, fixed with 4% paraformaldehyde, subjected to immunofluores­
cence staining (IL-10 (20850-1-AP), TNF-α(ab1793)), and then observed
under a laser confocal microscope (Nikon-A1, Japan). The levels of in­
flammatory cytokines (TNF-α and IL-10) secreted by cultured
RAW264.7 cells were determined using enzyme-linked immunosorbent
assay (ELISA) kits (Shanghai Enzyme-Linked Biotechnology Co., Ltd.,
Shanghai, China).
2.7. Antioxidant activity
The antioxidant activity assay included an in vitro free radical-
scavenging assay (superoxide radical •O2− , hydroxyl radical •OH), an
in vitro H2O2-scavenging assay, a test of metal ion (Fe2+, Cu2+)
adsorption on the hydrogel, and an in vivo ROS-scavenging assay.
To determine the ability of the hydrogels to scavenge •O2− . Hydrogel
was mixed with 12 mL of Tris-HCl (50 mM, pH 8.1), and 2 mL of py­
rogallol (3 mM) was subsequently added to the mixture, which was then
allowed to stand for 5 min in the dark. The groups were control group (1
mL of deionized water), paramylon hydrogel groups (0.1 g and 1 g of
paramylon hydrogel), and ascorbic acid group (100 μmol). The reaction
was stopped by adding 4 mL of hydrochloric acid (1 M) to each group,
and the absorbance at 299 nm was measured. The •O2− scavenging effect
( )
was calculated: Scavenging effect (%) = control × 100%.
A − Ahydrogel
Acontrol
The assay of the scavenging ability of hydrogels for •OH was modi­
fied from ref. (Zhang et al., 2020). FeSO4 (1.0 mL, 9.0 mM), hydrogel
(1.0 g) and salicylic acid (1.0 mL, 9.0 mM) were placed into a centrifuge
H. Lei et al. Carbohydrate Polymers 289 (2022) 119467

Fig. 1. Schematic illustration of the preparation of paramylon hydrogel and use as a wound repair dressing.

tube, H2O2 was added to the centrifuge tube at 37 ◦ C for 30 min, and the hydrogel was weighed and placed in CuCl2 solution with stirring at
absorbance at 510 nm was measured. The groups were control group (1 37 ◦ C. CuCl2 (100 μL) solution was removed at certain intervals, and the
mL of deionized water), paramylon hydrogel groups (0.1 g and 1 g of absorbance was read at 300 nm. A standard curve of the CuCl2 con­
paramylon hydrogel), and ascorbic acid group (100 μmol). The •OH centration versus absorbance was generated at the same time. The
scavenging effect was calculated using the following equation: amount of Cu2+ adsorbed by the hydrogel at different times was
( )
Scavenging effect (%) = Ahydrogel
A
× 100%. calculated until adsorption equilibrium was reached. Similarly, the
control
adsorption of Fe2+ (FeSO4) on the hydrogel was measured.
Paramylon hydrogel (1 g) was mixed with H2O2 (1 mM, 10 mL) and
placed at 37 ◦ C. The supernatant was collected at different time points
2.7.1. In vivo ROS-scavenging assay
and the supernatant (50 μL) was mixed with 100 μL of Ti(SO4)2 solution
All animal experiments were reviewed by the Northwest University
(1.33 mL of 24% Ti(SO4)2 solution and 8.33 mL of H2SO4 mixed with 50
Laboratory Animal Management and Ethics Committee, and complied
mL of deionized water). The absorbance at 405 nm was read after 30
with legal and institutional guidelines related to animal ethics, and were
min. The concentration of H2O2 was determined from a standard curve.
approved by the Ethics Committee (NWU-AWC-20210810R). Sprague
A 200 mg/mL CuCl2 solution was prepared, and 0.5 g of paramylon
Dawley (SD) rats (250 g) were depilated on their back. Triangles were

3
H. Lei et al. Carbohydrate Polymers 289 (2022) 119467

Fig. 2. a) FTIR spectra of paramylon and paramylon hydrogel; b) TGA curves of the paramylon hydrogels; c) weight change due to water absorption and swelling (n
= 3); d) changes in paramylon hydrogel composition after water absorption and swelling (n = 3); e) pore size of paramylon hydrogels; f) cryo-scanning electron
micrographs of a paramylon hydrogel; g) water retention of paramylon hydrogels (n = 3); h) transmittance of paramylon hydrogels; i) softness of para­
mylon hydrogels.

outlined on their back, and the injection sites for LPS, paramylon, and
LPS + paramylon hydrogel were at the top of the triangles. Paramylon
hydrogels are pre-granulated through a screen for injection by syringe
and then autoclaved. Fifty microlitres of LPS (10 mg/mL) was injected at
the injection site, and paramylon hydrogel was injected 2 h later (Zhao,
Huang, et al., 2020). The rats were sacrificed after 24 h and 72 h, and the
skin at the injection site was collected and stained (DHE fluorescence
stain) for ROS determination. The ROS content was also determine with
the ROS kit.
2.8. In vivo degradation
All animal experiments were reviewed by the Northwest University
Laboratory Animal Management and Ethics Committee, and complied
with legal and institutional guidelines related to animal ethics, and were
approved by the Ethics Committee (NWU-AWC-20210810R). SD rats
were subjected to subcutaneous implantation. The back of the rats was
shaved and disinfected, then the skin on the back of the rats was incised,
and the hydrogel was implanted subcutaneously on the back of the rats.
After 1, 3, 5, 7 and 9 weeks, the rats were executed and the skin on the
back at the site of the implanted hydrogel was separated. Then, the skin
tissue samples were placed in projection electron microscopy fixative for
transmission electron microscopy, hydrogels were separated and vac­
uum freeze-dried, and degradation was detected by SEM. Two rats were
assessed at each time point.
2.9. Acute wound repair: a model of full-skin defects in the rat back
A full-thickness skin defect model in rats was used to simulate acute
wounds. The experiment included a control group and a paramylon
hydrogel group. SD rats were anesthetized for dorsal debridement, and
full-thickness skin wounds (8 mm in diameter) were cut in the back of
each rat using a punch biopsy after alcohol disinfection. Parametron
hydrogel group treatment: the hydrogel was applied to the skin wound,
then covered and fixed with gauze and polyurethane film. Control group
treatment: gauze and polyurethane film only. The hydrogel was changed
daily to ensure moisture retention in the hydrogel and a moist envi­
ronment for the wound.
2.10. Chronic wound repair: a third-degree scald model on the rat back
A third-degree burn rat model was established by a tabletop
temperature-controlled scalding instrument (YSL-5Q, Jinan Yiyan
Technology Development Co., China), and the SD rats were anesthetized

4
H. Lei et al.
and then dorsally debrided. To create a third-degree burn wound, a flat-
tipped aluminium metal rod (1.5 cm diameter) was electrically heated to
90 ◦ C and brought into contact with the skin of a rat's back after
debridement for 30 s. The temperature of the metal rod was monitored
throughout the injury creation by a fabricated digital computerized
multimeter. The contact pressure was 1000 g. After 3 days of burn
injury, the crusted wound was debrided to remove necrotic tissue, and a
third-degree scald model was established. Paramylon hydrogel group
treatment: the hydrogel was applied to the skin wound, then covered
and fixed with gauze and polyurethane film. Control group treatment:
gauze and polyurethane film only. The hydrogel was changed daily to
ensure moisture retention in the hydrogel and a moist environment for
the wound.
2.11. Histological evaluation
For histopathological and collagen deposition evaluation, tissues
were stained with H&E and Masson's trichrome. Collagen density was
analysed by ImageJ.
For immunofluorescence (TNF-α, HIF-α), tissues were removed from
the fixed solution for sectioning, and sections were incubated with TNF-
α (TNF-α, Abcam, ab1793), HIF-α (Bioss, bs-1407R) and VEGF (Abcam,
ab1316) respectively for 2 h at room temperature. Cell nuclei were
stained with DAPI. Photographs were taken by Nikon A1 Laser Confocal
Microscope.
2.12. Statistical analysis
At least three independent replications were performed for each data
point. SPSS software was used for statistical analyses, and the values are
represented as the means and standard deviations. The significance was
analysed including one- or two-sided t-testing with significance as fol­
lows: *, p < 0.05; **, p < 0.01; and ***, p < 0.001.
3. Results and discussion
3.1. Design strategy and preparation mechanism for paramylon hydrogels
Highly crystalline paramylon is insoluble in water, and can only be
dissolved in water by changing to an amorphous form after treatment
with a strong alkali. Therefore, to prepare paramylon hydrogel, we
chose the epoxy compound BDDE as a cross-linking agent. The epoxide
group of BDDE opens the ring and reacts with the hydroxyl group of the
paramylon molecular chain under alkaline conditions, forming inter­
molecular connections similar to “hand-holding” (Fig. 1). The pH of the
hydrogel formed under these conditions is greater than 14, and a
strongly alkaline gel is obviously not suitable as a wound repair dressing;
thus, dialysis is needed to adjust the pH of the hydrogel and to remove
most of the residual BDDE cross-linker, which is a low-toxicity com­
pound that can have harmful health effects. The washed hydrogel ab­
sorbs water and swells to a transparent state. The gel also has high
temperature stability and can maintain a gel state at 121 ◦ C, so it can be
sterilized by autoclaving. The sterile treated paramylon hydrogel can be
used to promote wound repair.
3.2. Characterization of paramylon hydrogels
3.2.1. Chemical structure
A new characteristic peak corresponding to C–O–C appeared at
1076.08 cm− 1 (Fig. 2a), while the peak at 1154 cm− 1 was attributed to
–CHOH in the paramylon hydrogel, indicating that BDDE cross-linked
paramylon successfully (Li et al., 2015; Tomihata & Ikada, 1997;
Zhang et al., 2014). Fig. 2b shows the effect of hydrogel network cross-
linking on the thermal stability of the hydrogel. Compared with that of
paramylon, the thermal decomposition temperature of paramylon
hydrogel was increased (326 ◦ C for paramylon compared to 345 ◦ C for
5
Carbohydrate Polymers 289 (2022) 119467
paramylon hydrogel), and the weight loss was reduced (11.1 ± 1.87%
residual weight for paramylon hydrogel compared to 6.9 ± 1.63% for
paramylon, *p < 0.05); thus, the cross-linking improved the thermal
stability of the hydrogel.
3.2.2. Swelling properties
A hydrogel absorbs water and swells during washing. The alkaline
paramylon hydrogel was yellow and transparent (Fig. 2c inset), and
after washing and pH adjustment, the paramylon hydrogel absorbed
water and swelled to three times its original volume and was colourless
and transparent. The colour change may be due to the oxidation of the
polysaccharide during alkaline and heating processes, while the process
of washing and swelling may reduce some of the polysaccharide mole­
cules to a transparent colour. After 150 min, the hydrogel reached
swelling equilibrium (Fig. 2c), and its volume and mass no longer
changed. The concentration of paramylon in the hydrogel was 10% at
the time of preparation and only 3.2 ± 0.48% after absorbing a large
amount of water (Fig. 2d).
3.2.3. Surface morphology and pore size
An extensive and appropriately sized pore structure ensures the
breathability of a hydrogel dressing, which is essential for wound repair
(Zhu et al., 2018). The hydrogels with 10% paramylon absorbed water
and swelled after dialysis for adjusted pH, the volume and mass
increased, and the content of paramylon in the hydrogels was main­
tained at only 3%. The pore size was measured after treated with liquid
nitrogen and then photographed by cryo-scanning electron microscopy
of cleaned samples at different times (3% paramylon when cleaned to
swelling equilibrium; 6% paramylon when cleaned for 1 h; 10% para­
mylon in unwashed hydrogel). The average pore sizes of the hydrogels
(Fig. 2e) with different concentrations of paramylon were 218.2 μm (3%
paramylon), 161.6 μm (6% paramylon) and 76.5 μm (10% paramylon).
The pore structure of the hydrogels in the wet state could be seen by
cryo-scanning electron microscopy immediately after the hydrogels
were treated with liquid nitrogen (Fig. 2f, 3% paramylon). The pore size
of the wet-state paramylon hydrogel ranges from 2 to 10 μm, and its
interior had a uniform porous microstructure and suitable bore size
which ensure breathability and nutrient transport.
3.2.4. Water retention
Rapid wound recovery requires a moist and sterile environment (Lei,
Zhu, & Fan, 2020). Therefore, a hydrogel must retain water. The water
retention capacity of the paramylon hydrogel at 4 ◦ C and 37 ◦ C is shown
in Fig. 2g. At 4 ◦ C, the hydrogel retained the most water. The water in
the hydrogel evaporated faster at 37 ◦ C. The water content of paramylon
hydrogel was maintained at 87 ± 1.9% after 7 days at 4 ◦ C, and the
water content of the hydrogel was 53.28 ± 1.5% after 3 days at 37 ◦ C.
Besides the external temperature, the rate of water evaporation is also
associated with the size of the pore size and the content of hydrophilic
groups in the hydrogel (Lei et al., 2020). Generally the smaller the pore
size and more hydrophilic groups of a hydrogel, the better the water
retention properties. The pore size range of the 3% paramylon hydrogel
was approximately 80–230 μm, which was a medium pore size range in
hydrogels, and the paramylon has a lot of hydrophilic hydroxyl groups,
which can preserve water. The results of the water retention tests show
that the paramylon hydrogel have good water retention properties. The
appropriate storage temperature for the hydrogel dressing was 4 ◦ C,
ensuring the required moisture content of the wound at human skin
temperature, and that a wound dressing made from this hydrogel should
be changed every 2–3 days.
3.2.5. Light transmission
Non-transparent wound dressings require frequent removal during
use to observe wound healing. Frequent disturbance of the wound not
only increases the likelihood of wound deterioration but also greatly
hinders temporary monitoring of the wound. Therefore, transparent
H. Lei et al.
Fig. 3. a) The G′ and G′′ of the paramylon hydrogel measured by sweeping the strain amplitudes; b) G′ and G′′ of hydrogels with different paramylon concentrations
(***p < 0.001, n = 3); c) cyclic compressive stress-strain curves of paramylon hydrogels (***p < 0.001, n = 3); d) photograph of hydrogel being compressed, bent
and stretched.
dressing materials allow for real-time wound monitoring and care
(Chen, Zeng, et al., 2018). The light transmission of the hydrogels is
shown in Fig. 2h (visible light area: 400–780 nm (Pu et al., 2017)). The
results showed that the paramylon hydrogel exhibited high trans­
mittance over 80% between 400 and 780 nm. The high transparency of
the hydrogel dressing provides real-time monitoring of wound healing,
and any type of wound deterioration including bleeding, infection or
necrosis can be visualized in real time without removing the wound
dressing, which ensures well-timed treatment.
3.2.6. Flexibility
The hydrogel is soft and conforms to the skin, as shown in Fig. 2i. It is
very flexible, adapts well to different angles at the joints and ensures a
certain fit; even if the fingers were reversed and inverted, the hydrogel
would not fall off. The explanation for the hydrogel not falling off the
finger is due to its good flexibility and the physical attraction of the
hydrogel to the skin (e.g. hydrogen bonding) (Yang, Bai, Chen, & Suo,
2020), as well as the surface tension of the water molecules in the
hydrogel on the skin.
3.3. Mechanical properties
The mechanical durability of hydrogel wound dressings is influenced
by their inherent mechanical properties. The mechanical properties of
paramylon hydrogels were evaluated by rheological analysis and
compression experiments.
The results of a strain sweep test (Fig. 3a) showed that the paramylon
hydrogel was in the gel state (G′ > G′′ ) in the linear viscoelastic region,
indicating that the hydrogel had good elasticity in the low strain state.
With the gradual increase in amplitude, G′ and G′′ intersect (G′ = G′′ ),
this point is the transformation point of the sol-gel (the intersection
point is also known as the flow point), and in the region of stress or strain
above this point, the sample produces flow. The hydrogel exhibited flow
state when G′ < G′′ . The G′ and G′′ value of the hydrogel in the linear
viscoelastic region is affected by the cross-linking density of the
hydrogel. The concentration of paramylon was 6% after acid washing
6
Carbohydrate Polymers 289 (2022) 119467
for 2 h. After water washing to neutral, the paramylon hydrogel reached
dissolution equilibrium at a paramylon concentration of 3%. The con­
centration of paramylon affects the cross-linking density of the gel. The
concentration of the prepared alkaline paramylon hydrogel was 10%,
while the washed hydrogel increased in mass and volume due to water
absorption and swelling. Therefore, the content of paramylon decreased
to 3% while the cross-linking density decreased, thereby significantly
reducing both G′ and G′′ (Fig. 3b).
This phenomenon was also demonstrated by cyclic compression ex­
periments. Washing the hydrogel decreased the content of paramylon
due to water absorption and swelling, and therefore, the compressive
stress was significantly reduced (Fig. 3c). Even though the mechanical
properties of the paramylon hydrogels were reduced (119 kPa at 80%
compressive strain) compared to those of hydrogels made of other nat­
ural materials (only 12 kPa at 80% compressive strain for collagen
composite hyaluronic acid hydrogels (Shen et al., 2021)), paramylon
hydrogels retained their original shape after extrusion, twisting, and
stretching (Fig. 3d), reflecting sufficient mechanical properties for use as
wound dressings.
3.4. Biocompatibility of the hydrogels
Fig. 4(a) shows the MTT assay results. L929 cells were incubated
with extracts of paramylon hydrogel (extraction times of 24 h and 48 h)
for 24 h. The results showed that the paramylon hydrogel was not toxic
to L929 cells and promoted cell proliferation, and the cell viability of
paramylon hydrogel extracts extracted for 48 h (128.4 ± 10.4%, 100%
cell viability in the control group using normal medium) was signifi­
cantly greater than that of the extracts extracted for 24 h (105.3 ± 7.3%)
(**p < 0.01, n = 3), indicating that the paramylon hydrogel extract was
beneficial to cell proliferation.
Fig. 4(b) shows that the cell adhesion to the paramylon hydrogel was
37.5 ± 3.7%, 46.8 ± 3.5%, 52.8 ± 4.2%, 67.8 ± 4.1%, and 80.3 ± 4.6%
at 1, 2, 3, 4, and 5 days, respectively, indicating that the number of L929
cells adhering to the paramylon hydrogel increased during the culture
period. The microscope and SEM results (Fig. 4d) show that the cells
H. Lei et al. Carbohydrate Polymers 289 (2022) 119467

Fig. 4. a) Cell viability (L929) with hydrogel extracts (**p < 0.01, n = 3), specify 100% cell viability for the control group using normal media; b) cell adhesion and
proliferation (L929) on the hydrogels (***p < 0.001, n = 3); c) Hemocompatibility of the hydrogel; d) microscope and SEM pictures of cells (L929) adhered to
paramylon hydrogels after incubation (scale bars: 100 μm and 50 μm); e) the anti-inflammatory effect of paramylon hydrogel was investigated in RAW264.7 which
were induced by LPS. Confocal images showing immunofluorescence staining with TNF-α (red), IL-10 (green) and DAPI (blue). f) Quantitative data on the area
covered by TNF-α and IL-10. We set the TNF-α and IL-10 data from the control as 100% (***p < 0.001, n = 3); g) the cytokine levels of TNF-α and IL-10 were detected
by kits (**p < 0.01, n = 3).

attached to the paramylon hydrogel at 1, 2, and 3 days and proliferated into contact with blood. The safety of hydrogels used around blood can
over time. This indicates that the paramylon hydrogels have good be assessed by hemolysis tests. Clinically applied biomedical materials,
cytocompatibility. such as wound dressings, should exhibit a hemolysis rate of less than
During contact with a wound, the hydrogel is most likely to come 5.0% (Archana, Singh, Dutta, & Dutta, 2015). Paramylon hydrogels

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H. Lei et al. Carbohydrate Polymers 289 (2022) 119467

Fig. 5. a) ROS fluorescent probe (DHE) to monitor the reduction in oxidative stress after treatment with paramylon hydrogel. Fuchsia colouring indicates the
intracellular ROS content, blue colouring corresponds to DAPI. Scale bar: 50 μm. b) ROS kit to detect ROS levels in tissues after different treatments. The green dashed
line indicates the amount of ROS in normal skin tissue (***p < 0.001, n = 3). c) Hydroxyl radical-scavenging ability of paramylon hydrogel. d) •O2− -scavenging
ability of paramylon hydrogel. e) Potential mechanisms of antioxidant activity in paramylon hydrogels. f) Quantification of the residual H2O2 after reaction with
paramylon hydrogel (n = 3). g) Hydrogel adsorption to Fe2+and Cu2+ (n = 3).

induced hemolysis of 0.0023 ± 0.000056% in contact with red blood
cells in whole blood at 37 ◦ C for 60 min (Fig. 4c). Thus, paramylon
hydrogel is a nonhemolytic and safe material for contact with wounds
and blood.
These experimental results suggest that paramylon hydrogels have
optimal cytocompatibility and hemocompatibility for use as wound
dressings.
3.5. In vitro anti-inflammatory activity
Paramylon has been shown to possess anti-inflammatory effects as a
food product; however, in the field of wound repair, no study has been
conducted on whether paramylon possesses anti-inflammatory effects in
direct contact with skin. Anti-inflammatory material makes wound
rapidly pass the immune inflammatory phase, which has been a hot
topic in the field of biomedical materials. Therefore, we investigated the
in vitro anti-inflammatory effect of paramylon hydrogel.
LPS stimulates macrophages and induces transcription of inflam­
matory factors, such as tumor necrosis factor alpha (TNFα), through the
translocation of nuclear factor pathway (Schilling et al., 2018). The
proinflammatory factor TNF-α was abundantly expressed in the LPS
group compared to the normal cell control group (Fig. 4e–f). The par­
amylon hydrogel extract induced macrophages to produce the anti-
inflammatory factor IL-10 (Fig. 4e–f), and LPS-induced macrophages
produced significantly less TNF-α than the LPS-positive group after
treatment with paramylon hydrogel extract, indicating that paramylon

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H. Lei et al.
had an anti-inflammatory effect. The TNF-α and IL-10 secreted by
RAW264.7 cells were detected using the kit. The results were confirmed
by the immunofluorescence results which indicated that the paramylon
hydrogel promoted the expression of the anti-inflammatory factor IL-10
and reduced LPS-induced inflammation, and the 100 mg/mL paramylon
hydrogel extract was comparable to 1 mg/mL dexamethasone in
reducing inflammation.
We found that paramylon hydrogels have anti-inflammatory effects
and may have great potential to promote healing in wound repair. The
mechanism by which paramylon promotes the anti-inflammatory factor
IL-10 is not clear, but it has been shown that the monomeric β-1,3-glucan
of paramylon can act on the dectin receptor of macrophages and activate
IL-10 production via the splenic tyrosine kinase pathway (De Marco
Castro, Calder, & Roche, 2021). We investigated the anti-inflammatory
activity of other natural polysaccharides. The inhibitory effect of
fucoidan on lipopolysaccharide (LPS)-induced pro-inflammatory medi­
ators in BV2 microglia has been demonstrated, with fucoidan treatment
significantly inhibiting nitric oxide and prostaglandin E2 in LPS-
stimulated BV2 microglia. It also attenuated the expression of pro-
inflammatory cytokines, including interleukin-1β and TNF-α. In addi­
tion, fucoidan inhibited NF-κB activation to exhibit potent anti-
inflammatory activity (Park et al., 2011). Pleurotus citrinopileatus
mushroom polysaccharide, which has a β-1,6-glucan monomer struc­
ture, mainly triggers Dectin1 and Toll-like receptor2 signaling in mac­
rophages, leading to a reduced ability to differentiate into pro-
inflammatory (M1) macrophage subtypes under inflammatory condi­
tions, inhibits the secretion of pro-inflammatory cytokines TNF-α and
chemokines and promotes the expression of anti-inflammatory cytokine
IL-10 in LPS/IFNγ-activated macrophages (Minato, Laan, van Die, &
Mizuno, 2019).
The anti-inflammatory properties of polysaccharides are closely
related to their chemical structures, and polysaccharides with β-glucan
structures as well as β-(1,3) and (1,6) glycosidic bonds play an important
role in enhancing immunomodulatory effects and often exhibit effective
anti-inflammatory activity (Hou, Chen, Yang, & Ji, 2020). According to
our results, it appears that paramylon with β-1,3-glucan structure also
has some immunomodulatory effects, and the immunomodulatory
mechanisms of paramylon should be paid to further unveil the deeper
connection of them.
3.6. Antioxidant activity
Uncontrolled inflammation is a major factor inhibiting rapid wound
repair, and decreasing the uncontrolled inflammatory reaction can
significantly accelerate wound healing. High levels of ROS are a major
contributor to wound inflammation, as ROS inhibit wound healing
through lipid peroxidation. Therefore, the development of antioxidant
materials has been a hot topic in the field of wound repair. To date,
polysaccharides extracted from plants (Yin, Zhang, & Li, 2019), fungal
polysaccharides (Liao & Huang, 2019), and algal polysaccharides (Lu,
Lu, Chen, & Mi, 2019) have been shown to be effective ROS scavengers.
Oral paramylon can protect rats from CCl4-induced acute liver injury
through an antioxidant mechanism. In the field of wound repair, it has
not been investigated whether paramylon has antioxidant effects when
in direct contact with the skin. Therefore, we investigated the antioxi­
dant effects of paramylon hydrogels in vivo and in vitro, such as scav­
enging free radicals and scavenging ROS.
Polysaccharides can play a direct role in free radical destruction,
deoxygenation, and singlet oxygen quenching. The structural composi­
tion of polysaccharides consists of glyoxylates and monosaccharides
connected by glycosidic bonds. After the reaction between poly­
saccharides and free radicals, the free radicals burst and the poly­
saccharides are oxidized causing the glycosidic bonds to break, or they
are oxidized to become unstable ester bonds (Vreeburg, Airianah, & Fry,
2014; Zegota & Von, 1977). We characterized the polysaccharide
involvement in deoxygenation and singlet oxygen quenching via the in
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Carbohydrate Polymers 289 (2022) 119467
vitro scavenging of •OH, •O2− , and H2O2.
The ROS-scavenging effect of paramylon hydrogels was studied in
vivo. LPS is commonly used to induce inflammatory responses and ROS
production in vivo (Zhao et al., 2018). There were almost no ROS in
normal skin tissues, a large amount of ROS was produced in the tissues
after LPS induction, and the skin tissues injected with only paramylon
hydrogel showed a trace amount of ROS, probably due to a foreign body
immune response. The production of ROS in the skin tissues of the LPS-
induced and paramylon hydrogel-injected groups was significantly
lower than that of the LPS-induced group, indicating that the paramylon
hydrogel is a good ROS scavenger (Fig. 5a). The kit assay showed similar
results (Fig. 5b), and the paramylon hydrogel group was effective in
reducing ROS caused by LPS. As a potential mechanism, paramylon may
directly destroy free radicals and directly trap the ROS generated,
thereby inhibiting or slowing lipid peroxidation.
We compared the ability of paramylon hydrogels to scavenge •OH
and •O2− with ascorbic acid. Ascorbic acid is an acidic polyhydroxy
compound and is the most versatile antioxidant known, which is highly
reductive and the enediol in its molecular structure can be oxidized to a
ketone group which scavenges free radicals (Du, Cullen, & Buettner,
2012; Kim et al., 2016). As Fig. 5c shows, The paramylon content was
3% per gram of paramylon hydrogel, with 174 μmol sugar monomers.
The •O2− scavenging rate was 43.39 ± 3.43% for 174 μmol sugar
monomer and 28.25 ± 5.67% for 100 μmol ascorbic acid. Thus the •O2− -
scavenging performance of paramylon was 80.51% of that of ascorbic
acid. The •OH scavenging rate was 48.41 ± 5.53% for 174 μmol sugar
monomer and 34.55 ± 5.50% for 100 μmol ascorbic acid. Thus the •OH
scavenging performance of paramylon was 80.51% of that of ascorbic
acid. Fig. 5f shows the effect of the paramylon hydrogel on H2O2 scav­
enging, and relative equilibrium was reached after 30 min, with a
scavenging rate of 55.8 ± 2.54%. For •O2− , polysaccharides can be
oxidized to achieve ROS scavenging (Niu, Xu, Jiang, Li, & Yuan, 2017).
The hydrogen on the hydrocarbon chain of paramylon combines with
•OH to form water, which consumes •OH, while the carbon on the hy­
drocarbon chain are further oxidized to form peroxyl radicals, which are
then decompose into non-harmful products such H2O or O2 (Vreeburg
et al., 2014). The scavenging mechanism of H2O2 is similar to the
scavenging mechanism of •OH (Fig. 5e).
Polysaccharides could also inhibit ROS production by chelating the
metal ions (Fig. 5e) required for the formation of ROS. For example, the
alcoholic hydroxyl groups in the polysaccharide structure can chelate
the metal ions (such as Fe2+, Cu2+, etc.) essential for the generation of
•
OH, which finally inhibit the generation of ROS. Therefore, we exam­
ined the ability of paramylon hydrogels to chelate Fe2+ and Cu2+. As
shown in Fig. 5g, the hydrogel could adsorb Cu2+ and Fe2+, and its
colour changed from colourless transparent to blue (Cu2+) and green
(Fe2+). The hydrogel reached adsorption equilibrium after 100 min, and
the rates of Cu2+ and Fe2+ adsorption were 2.74 ± 0.135 mg ion/mg gel
and 3.52 ± 0.157 mg ion/mg gel, respectively.
The antioxidant mechanism of polysaccharides is mainly achieved
through (i) hydrogen atom transfer and (ii) single electron transfer,
where the hydrogen donating ability of the active hydroxyl and carboxyl
groups present in the polysaccharide molecule allows the poly­
saccharide to destroy free radicals or to prevent peroxide formation by
reacting with specific peroxide precursors (Mirzadeh, Arianejad, &
Khedmat, 2020). Therefore, the monosaccharide composition, structure
and conformation, polarity and intramolecular hydrogen bonding
properties of natural polysaccharides are related to their antioxidant
activity. Researchers found that mushroom polysaccharides with β(1,3)-
glucan structure have scavenging effect on hydroxyl radicals and su­
peroxide radicals and chelating ability on Fe2+ (Chen, Ju, Li, & Yu,
2012; Thetsrimuang, Khammuang, Chiablaem, Srisomsap, & Sarnthima,
2011). Ganoderma lucidum polysaccharides with β-1,3, β-1,6 or β-1,4,
β-1,6-glucan composition also showed good scavenging of free radicals
and chelation of Fe2+ (Li, Nie, Yan, Zhu, & Xie, 2009; Yan et al., 2009).
Paramylon is a polysaccharide with β-1,3-glucan structure. The hydroxyl
H. Lei et al.
Fig. 6. a) Observations during various periods of the degradation assay in vivo. b) Degradation of subcutaneously implanted hydrogels at different times. c) The
hydrogel reacted with the tissue after implantation under the skin, and the degree of the inflammatory reaction around the tissue was observed by transmission
electron microscopy. Red arrows indicate blood vessels, blue arrows indicate inflammatory cells, green arrows indicate collagen, and yellow arrows indi­
cate fibroblasts.
group of the sugar chain and the glycosidic bond can provide more
hydrogen to the •OH to generate stable radicals to stop the chain reac­
tion, and the hydroxyl group also has metal chelating ability, which
makes it inactive in the Fenton reaction with the ability to inhibit free
radical production.
Similar to the antioxidant mechanism of most polysaccharides, the
mechanism of free radical scavenging by paramylon is not specific.
3.7. Histocompatibility and in vivo degradation of hydrogels
After implantation of paramylon hydrogel under the skin, human
body fluids, some nutrients and water will enter the hydrogel void, so
the degradation of the hydrogel is divided into degradation between
voids, degradation in layers and degradation from the edge of the par­
amylon hydrogel. The hydrogel degraded over time, and the gel with a
diameter of 8 mm and a thickness of 2.5 mm was completely degraded
within 9 weeks after implantation under the skin (Fig. 6a). No redness or
swelling was observed around the gel during the degradation period, so
it was tentatively determined to have no obvious rejection reaction.
Changes in the internal morphology of the porous paramylon hydrogel
were detected by SEM (Fig. 6b). The network structure of the paramylon
hydrogel was uniformly porous, as shown in Fig. 2c. After implantation
under the skin, irregularities in the internal structure, disruption of the
coherence between the pores and the thickness between the pores were
observed over time (Fig. 6b). In the third week of degradation, the
surface structure of the hydrogel became rougher and disorder appeared
between the pores, and at 5–7 weeks of degradation, there were no pores
between the pore walls and even fragment degradation, resulting in a
large number of fragments covering the surface of the material.
To demonstrate the biological compatibility of paramylon hydrogels,
degradation site tissues were subjected to transmission electron micro­
scopy to observe changes in cell morphology and surrounding collagen.
After 1 week of subcutaneous implantation, only a very small number of
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Carbohydrate Polymers 289 (2022) 119467
inflammatory cells (neutrophils) were seen around the vessels (Fig. 6c).
The surrounding collagen fibres were crisscrossed, and spindle-shaped
fibroblasts were seen between them. Between weeks 3 and 7, no in­
flammatory cells were observed, and both collagen fibres and fibroblasts
appeared normal, indicating that the paramylon hydrogel does not cause
a strong inflammatory response and has excellent histocompatibility.
3.8. Acute wound repair: a model of full-skin defects in the rat back
A full-skin defect on the backs of rats was used to simulate an acute
wound, as shown in Fig. 7a, and on day 3, the paramylon hydrogel
dressing group had better wound contraction than the control group. At
day 7, this difference was even more pronounced (***p < 0.001). At day
12, 28% of the wounds in the control group were still unhealed, while
those in the paramylon hydrogel dressing group were completely closed
(Fig. 7b, ***p < 0.001). The wounds in the blank group did not close
until day 15, indicating that the paramylon hydrogel dressing was
effective.
The results of H&E staining are shown in Fig. 7c. H&E staining
enabled evaluation of the thickness of the granulation tissue, which
composed of capillary-rich fibrous connective tissue, inflammatory cells,
fibroblasts, collagen fibres, etc. The thickness and the abundance of
granulation tissue are the vital indicators for evaluating wound healing
(Lei & Fan, 2021; Yang, Liang, Chen, Duan, & Guo, 2022). In this study,
the thickness of granulation was higher in both dressing groups than in
the control group (*p < 0.05; Fig. 7e). We observed that the control
group was still in the inflammatory phase at day 7, with a large number
of inflammatory cells in the tissue (Fig. 7c), while there was a significant
difference between the dressing group and control group. Due to the
good anti-inflammatory effect of the paramylon hydrogel, no significant
inflammatory percolation was seen in the wounds of the dressing group.
The dressing also provided an artificially moistened microenvironment
that was more conducive to wound healing. On day 12, the epidermal
H. Lei et al. Carbohydrate Polymers 289 (2022) 119467

Fig. 7. a) Photos of wounds and trajectories of wound closure for each group (n = 3). b) Wound closure rates for each group at different time points. c) H&E staining
of wounds after 7 and 12 days at low magnification (scale bar: 1 mm) and high magnification (scale bar: 100 μm) (n = 3). d) Masson's trichrome staining of a wound
site after 7 and 12 days (scale bar: 100 μm) (n = 3). e) Thickness of granulation tissue for different treatments on days 7 and 12. (*p < 0.05, n = 3). f) Analysis of
collagen staining intensity (*p < 0.05, n = 3).

tissue of the paramylon hydrogel wound dressing group was intact, and
blood vessels and other attachments were observed in the dermis,
indicating good promotion of wound healing by the paramylon
hydrogel.
The amount of collagen production at a wound site is another cri­
terion for evaluating wound repair. Masson staining was performed to
detect deposition of nascent collagen in the wound-healing area (Fig. 7d,
f). The paramylon hydrogel group was found to exhibit significantly
higher collagen deposition than the control group after 7 days and 12
days. The paramylon hydrogel promotes the proliferation of fibroblasts
and thus collagen deposition. The results showed that collagen was
mainly distributed in and around these fibroblasts, suggesting that fi­
broblasts have the ability to produce collagen.
3.9. Chronic wound repair: a third-degree scald model on the backs of
rats
Chronic wounds with a persistent inflammatory response signifi­
cantly alter cytokines, causing increased levels of inflammation,
persistent wound infection, hypoxia and poor nutrient transfer as well as

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H. Lei et al.
Fig. 8. a) Timeline of burn treatment. b) Characteristics of the outer epidermis of third-degree scald wounds. c) Characteristics of the subcutaneous tissue of third-
degree scald wounds. d) H&E staining of skin tissue with third-degree burns (scale bar: 100 μm).
difficulty in vascularization and re-epithelialization. A third-degree
scald model was developed to simulate chronic wounds (Fig. 8a), and
a scaldometer was used to create a circular scald wound (1.5 cm initial
scald diameter) (Fig. 8b), which increased in size to 2 cm in diameter by
day 5. The skin was dissected, and significant haemorrhage and oedema
were found (Fig. 8c). A large number of inflammatory cells had infil­
trated, the collagen fibres were swollen and latticed, and there was se­
vere subcutaneous bleeding from vascular damage (Fig. 8d). The
destruction of epidermal and dermal tissues leads to the accumulation of
inflammation, generating a large amount of ROS and forming a chronic
wound (Peng et al., 2021). The degree of burn is judged to be third
degree according to the “ rule of three degrees and four levels “ criteria
(supporting information, Table S1). We performed simple debridement
of the rat wounds to remove the necrotic epidermis and inner layer of
tissue and built a third-degree scald wound.
After debridement, we evaluated the ability of the paramylon
hydrogel to promote healing of third-degree scald wounds. The wound-
healing process of each rat was recorded, and the closure was traced and
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Carbohydrate Polymers 289 (2022) 119467
analysed in detail (Fig. 9a–b). The paramylon hydrogel showed a sig­
nificant promotion effect from day 10, with significant differences
compared to the control group at days 10, 15 and 20 (***p < 0.001, n =
3). The wounds in the paramylon hydrogel group were completely
closed by 20 days, while the wounds in the control group were closed at
28 days. The healing rate of the hydrogel group was enhanced by
28.57% compared to that of the control group, demonstrating the effi­
cacy of paramylon hydrogel in promoting the healing of third-degree
burns.
We verified the in vivo degradability of the hydrogel in a subcu­
taneous degradation experiment. In wound repair experiments, we
needed to change the hydrogel dressing daily, and to test whether the gel
would degrade within a day, we tested the dry weight of the used
dressing and found that the weight of the gel after application in acute
wounds lost 1.63 ± 0.07% compared to its initial dry weight, and the gel
loss was up to 3.87 ± 0.15% in chronic wounds. The reason is the area of
chronic wounds is 3.24 times larger than that of acute wounds, and
chronic wounds exude more fluid and carry enzymes, such as amylase or
H. Lei et al. Carbohydrate Polymers 289 (2022) 119467

Fig. 9. a) Photographs of third-degree burn wounds at days 0, 5, 10, 15 and 12 and traces of wound-bed closure in each treatment group (n = 3). b) In vivo third-
degree burn wound closure rates at different time points. c) H&E staining of third-degree burn wounds after 7 and 12 days at low magnification (scale bar: 1 mm) and
high magnification (scale bar: 100 μm) (n = 3). d) Thickness of granulation tissue for different treatments (***p < 0.001, n = 3). e) Masson's trichrome staining of a
third-degree burn wound site (scale bar: 100 μm) (n = 3). f) Analysis of collagen staining intensity (***p < 0.001, n = 3).

lysozyme, which can decompose more paramylon hydrogel, while the
degradation products of hydrogel are also beneficial to promote anti-
inflammation or antioxidation of wounds, thus promoting wound repair.
The thickness of granulation and the histological characteristics of
each group at different recovery periods were assessed by H&E staining
(Fig. 9c–d). The granulation thickness in the paramylon hydrogel dres­
sing group was significantly higher than that in the control group at days
5, 10, 15, and 20, and both groups exhibited significant inflammation at
day 5. In the control group, vascular bruising with focal haemorrhage
was observed, while in the paramylon hydrogel group, fibrous tissue
proliferation with new capillary formation, vasodilation, bruising and
extravasation of red blood cells was observed in the new granulation
tissue. At day 10, the epidermal structure was still missing in the control
group and the paramylon group. In the control group, multifocal
lymphocyte infiltration and multinucleated giant cell formation were
observed, while in the hydrogel group, collagen fibres proliferated,
macrophage phagocytosis was gradually weakened, macrophages were
absorbed by the body tissue, and microvessels were formed in the tissue.
At day 15, the collagen fibres in the control group were hyperplastic
with evidence of macrophage reaction, and chronic inflammatory cell
infiltration was observed. In contrast, the interstitial fibres of the gran­
ulation tissue in the paramylon hydrogel group gradually collagenized,
and the number of capillaries gradually decreased with the formation of
microvessels. At day 20, a wound epidermis began to form gradually in
the control group, and chronic inflammatory cell infiltration could still
be seen in the dermis. In the paramylon hydrogel group, the epidermis
had an intact structure without obvious inflammatory cells, and some
microvessels were formed. Masson staining (Fig. 9e–f) showed that the
collagen density in the paramylon hydrogel group was significantly
higher than that in the control group, indicating that paramylon
hydrogel accelerated the recovery of chronic wounds from third-degree
burns by promoting the deposition of collagen.

13
H. Lei et al. Carbohydrate Polymers 289 (2022) 119467

Fig. 10. a) Images showing the immunofluorescence labelling of skin wound tissues with TNF-α. b) Immunofluorescence-labelled images of skin wound for HIF-1α.
c) Immunofluorescence-labelled images of skin wound for VEGF. d) Quantitative data on the area of TNF-α coverage in the full skin defect model. We set the area of
TNF-α coverage in control group on day 3 to 100% (n = 3). e) Quantitative data on the area of HIF-1α coverage in the full skin defect model. We set the area of HIF-1α
coverage in control group on day 3 to 100% (n = 3). f) Quantitative data on the area of VEGF coverage in the full skin defect model. We set the area of VEGF coverage
in control group on day 3 to 100% (n = 3). g) Quantitative data on the area of TNF-α coverage in third-degree burns model. We set the area of TNF-α coverage in
control group on day 5 to 100% (n = 3). h) Quantitative data on the area of HIF-1α coverage in third-degree burns model. We set the area of HIF-1α coverage in
control group on day 5 to 100% (n = 3). i) Quantitative data on the area of VEGF coverage in third-degree burns model. We set the area of VEGF coverage in control
group on day 5 to 100% (n = 3). Scale bar indicates 100 μm, ***p < 0.001, **p < 0.005, *p < 0.01.

14
H. Lei et al.
3.10. Healing factor analysis
To further investigate the biological mechanisms that promote
wound healing, we assessed the expression of characteristic healing
factors TNF-α (Fig. 10a), HIF-1α (Fig. 10b) and VEGF (Fig. 10c), during
acute and chronic wound repair.
TNF-α synthesis occurred immediately after wound formation and
was enhanced in the first few hours. TNF-α expression was high in the
early stages of wound repair and then decreased to basal levels as the
wound gradually healed. On the third day after acute wounding, the
TNF-α level in the control group was significantly higher than that in the
paramylon hydrogel group (Fig. 10d), which was attributed to the
bacterial barrier effect of the hydrogel at a certain thickness preventing
wound infection and the anti-inflammatory effect of paramylon. At day
5 after chronic wound formation, all groups showed more significant
TNF-α expression than the acute wounds due to the ulceration of tissues
producing large amounts of ROS or to proteases removing damaged
tissues and macrophages consistently being in a proinflammatory state.
The control group presented a typical process of higher long-term TNF-α
expression in chronic wounds (Fig. 10g). The expression of TNF-α in the
paramylon hydrogel group was significantly lower than that in the
control group at day 10 and decreased rapidly over time.
The increasing expression of HIF-1α in the body under hypoxia is
involved in some biological effects such as erythropoiesis and angio­
genesis (Chen et al., 2021; Zhang, Li, Ma, He, & Li, 2021). Additional
studies have demonstrated that non-hypoxic stimuli also increase HIF-
1α expression in a cell-specific manner, and that some of these increases
are equal to or greater than the hypoxia-induced increases (Zhang et al.,
2019).
We first discovered that the paramylon hydrogel could promote the
expression of HIF-1α. Fig. 10e shows that the expression of HIF-1α was
significantly higher in the paramylon hydrogel group than in the control
group at day 3 after acute wounding. The expression of HIF-1α in
chronic wounds was significantly higher than that in acute wounds
(Fig. 10h) because chronic wounds promote the production of HIF-1α in
response to the hypoxic environment due to the high production of ROS.
The expression of HIF-1α in the control group was maintained at high
levels for 20 days, indicating that ROS production was still high in the
wounds. The high expression in the paramylon hydrogel group in
chronic wounds at day 5 may be due to a combination of the hypoxic
environment and the promotion of HIF-1α by paramylon, with expres­
sion reaching even 3 times that of the control group. To the best of our
knowledge, our results demonstrate for the first time that the direct
stimulation of HIF-1α expression by paramylon at the wound site. The
potential mechanism is that some of the polymeric polysaccharides with
β-1,3 glucan monomers can promote HIF-1α expression by binding to
HIF-1α specific DNA sequences and strongly activating HIF-1α tran­
scription (Zhang et al., 2019). The paramylon hydrogel also possessed
ROS scavenging properties (Section 3.6), thus HIF-1α expression in the
paramylon group decreased significantly on day 10, probably because
paramylon hydrogel improved the hypoxic environment, and HIF-1α
expression at this time was influenced by the effect of paramylon on HIF
transcription. The expression of HIF-1α was almost absent in the para­
mylon hydrogel group at day 12 after acute wounding. On day 15 of
chronic wound repair, HIF-1α expression in the paramylon group was
significantly less than in the control group, which we attribute to the fact
that the wound was already in a normoxic state, with a dramatic
reduction in wound size to 30% of the initial size and a reduced contact
area with the paramylon hydrogel, and therefore a weakened effect on
HIF-1α transcription.
The HIF-1α pro-angiogenesis does not act directly, but rather pro­
motes erythropoiesis and angiogenesis via the HIF-1α-VEGF pathway, so
we examined the expression of VEGF in wound tissue. The results
showed that VEGF and HIF-1α exhibited similar trends (Fig. 10f, i), and
the VEGF was expressed in large amounts in the early stages of wound
repair, which promoted neovascularization. We suggest that the pre-
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Carbohydrate Polymers 289 (2022) 119467
expression of VEGF (5–10 days) was regulated by HIF-1α, as the
expression of both HIF-1α and VEGF in the paramylon hydrogel group
was significantly higher than in the control group, and VEGF is a clear
downstream factor of HIF-1α, therefore the paramylon does promote the
HIF-1α-VEGF pathway. And at the later stage (15 days), there may be
other regulatory pathways or cumulative effects from the earlier stage,
so that there may be some degree of expression of VEGF even if HIF-1α
expression was not high. The results also corresponded to the histolog­
ical H&E staining results showing a large amount of angiogenesis in the
paramylon hydrogel group at day 15.
We have innovatively elucidated that the mechanism for promoting
wound repair by paramylon hydrogels is to reduce inflammation at the
wound site by decreasing TNF-α expression and to promote angiogenesis
by promoting HIF-1α expression and thus activating the HIF-1α-VEGF
pathway. Due to the physiological characteristics of chronic wounds
exhibiting a large inflammatory response, oxidative stress, and impaired
wound angiogenesis, paramylon hydrogels showed a greater potential in
the repair of chronic wounds than of acute wounds.
4. Conclusion
To the best of our knowledge, this is the first study to prepared a
paramylon hydrogel for wound repair. The hydrogel exhibits a favour­
able pore structure to ensure permeability, and its good mechanical
properties, cytocompatibility and hemocompatibility satisfy the basic
requirements for a wound dressing. Notably, paramylon hydrogels could
inhibit ROS production by directly neutralizing free radicals and
chelating metal ions required for ROS formation, leading to ROS scav­
enging and effective anti-inflammation. In addition, the paramylon
hydrogel can promote angiogenesis through the HIF-1α-VEGF pathway.
It is expected that paramylon which has good anti-inflammatory, anti­
oxidant properties and promote angiogenesis will become a popular
bioactive material in the field of promoting wound repair. In the future,
paramylon can be further developed as a matrix for encapsulating cells
or bioactive molecules, which would have good potential in the field of
tissue engineering scaffolds or oral treatment of gastrointestinal
inflammation, and the mechanism of anti-inflammatory and antioxidant
properties of paramylon is expected to be further clarified in future
studies.
Data availability
All relevant data supporting the findings of this study are either
included within the article and its Supplementary Information files or
available upon request from the corresponding author.
CRediT authorship contribution statement
Huan Lei: Methodology, Data curation, Writing – original draft. Jing
Zhao: Conceptualization, Writing – review & editing. Hang Li: Re­
sources, Visualization. Daidi Fan: Supervision, Project administration.
Declaration of competing interest
The authors declare no known competing financial interests in this
work.
Acknowledgments
We sincerely thank Prof. Yuanguang Li of East China University of
Science and Technology for providing paramylon. This work was sup­
ported by National Natural Science Foundation of China (No.
21838009), the National Key R&D Program of China (No.
2019YFA0905200), the Xi'an Science and Technology Project
(20GXSF0004, 20191422315KYPT014JC016) and Natural Science
Foundation of Shaanxi Province (No. 2020JQ-570).
H. Lei et al.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.carbpol.2022.119467.
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