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Zhou, A. Chen, X. Xuan, Y. Li, X. Guo, J. Zheng, J. Xiao and J. Wu, J. Mater. Chem. B, 2019, DOI:
10.1039/C8TB02590H.
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Page 1 of 24 Journal of Materials Chemistry B
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Zwitterionic Poly(Sulfobetaine Methacrylate) Hydrogels with Optimal

DOI: 10.1039/C8TB02590H
Mechanical Properties for Improving Wound Healing In Vivo
Journal of Materials Chemistry B Accepted Manuscript

Huacheng He1#, Zecong Xiao2#, Yajiao Zhou2, Anqi Chen2, Xuan Xuan2, Yanyan Li2, Xin Guo2,
Jie Zheng3*, Jian Xiao2*, Jiang Wu1*
1College
of Chemistry and Materials Engineering
Wenzhou University, Wenzhou, Zhejiang 325027, P.R. China
Published on 21 January 2019. Downloaded by Iowa State University on 1/22/2019 8:53:46 AM.
2School
of Pharmaceutical Sciences
Wenzhou Medical University, Wenzhou, Zhejiang 325035, P.R. China
3Department of Chemical and Biomolecular Engineering
The University of Akron, Akron, OH 44325, USA
*Corresponding Author: J.Z: [email protected]; J.X: [email protected]; J.W.

([email protected])
# The authors contribute equally to this work
1
Journal of Materials Chemistry B Page 2 of 24
Abstract View Article Online

DOI: 10.1039/C8TB02590H
Zwitterionic hydrogels, as highly hydrated and soft materials, have been considered as
promising materials for wound dressing, due to their unique antifouling and mechanical

properties. While the viscoelasticity and softness of zwitterionic hydrogels are hypothetically
essential for creating adaptive cellular niches, the underlying mechanical-regulated wound
healing mechanism still remains elusive. To test this hypothesis, we fabricated zwitterionic
poly(sulfobetaine methacrylate) (polySBMA) hydrogels with different elastic modulus
prepared at different crosslinker contents, and then applied the hydrogels to full-thickness
cutaneous wounds in mice. In vivo wound healing studies compared the mechanical cue-
induced effects of soft and stiff polySBMA hydrogels on wound closure rate, granulation tissue
formation and collagen deposition. Collective results showed that the softer and more
viscoelastic hydrogels facilitated cell proliferation, granulation formation, collagen
aggregation, and chondrogenic ECM deposition. Such high wound healing efficiency by the
softer hydrogels is likely attributed to stress dissipation by expanding cell proliferation the up-
regulation of blood vessel formation, and the enhanced polarization of M2/M1 macrophages,
both of which would provide more oxygen and nutrients for cell proliferation and migration,
leading to enhanced wound repair. This work not only reveal a mechanical property-wound
healing relationship of zwitterionic polySBMA hydrogels, but also provide a promising
candidate and strategy for the next-generation of wound dressings.
Keywords: zwitterionic hydrogels, non-fouling, elastic modulus, protein adsorption, wound

dressing
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1. Introduction View Article Online

DOI: 10.1039/C8TB02590H
Wound dressing is a key approach to address a grand challenge of wound healing for
different traumatic, thermal, acute, and chronic wounds affecting millions of people globally1.

Generally speaking, wound healing is a very sophisticated process that require different tissues
and cells to cooperate and communicate in a way to realize cell proliferation and tissue
remodeling (e.g. fibroblast migration, endothelial cell angiogenesis, and epithelial cell re-
epithelization) after hemostasis and inflammation.2 Despite of different wound types (e.g.,
traumatic, acute, chronic, exuding, and dry wounds), the design of nontoxic/nonallergenic
wound dressings usually should satisfy several combinatory factors: protection of wounds from
bacterial infection, high adsorption ability, improved cell proliferation, enhanced anti-
inflammatory, desirable humidity environment, and/or ease of removal and replacement
without pain.3, 4
Among different wound dressing materials, hydrogels are considered as the most
promising wet-and-soft materials for wound dressing because their high water content,
adequate mechanical/elastic, stimuli-responsive, and some self-recovery/self-healing
properties5-8, allowing them to well mimic wound soft tissues. Most of soft tissues (e.g. skin,
blood vessel and nerves) not only possess high mechanical toughness and extensibility, but
also exhibit the strain-induced viscoelastic behavior9. However, conventional hydrogels suffer
from weak mechanical properties and poor mechanical responsive at different external stimuli10,
which greatly limits their uses as wound soft tissue substitutes. Extensive studies have reported
that biomechanics of hydrogels and other soft materials (e.g. topographical and intrinsic
structure, and mechanical toughness/strength/modulus/elasticity) is critical not only for serving
as supporting substrates to retain tissue integrity and cell activity11, but also for the transversion
of biophysical cues to biochemical responses that regulate cell behaviors for tissue
regeneration.12 These cross-talk between biophysical and biochemical stimuli initiates cell
proliferation, migration, differentiation, and remodeling during the wound healing process.13,
14 Specifically, chemical (e.g. polymer components, crosslinkers) and mechanical (e.g.
toughness, stiffness, modulus) properties of hydrogels (not limited to wound dressings)
generally have influence on cell behaviors and wound repair effect.15 For instance, hydrogels
with optimal mechanical properties can better stimulate keratinocytes proliferation/migration,
angiogenesis and neovascularization, and bFGF and TGF-β1 secretion, enhance blood vessel
formation, re-epithelialization, extracellular matrix synthesis and remodeling, thus promoting

3
wound closure.16, 17 Hydrogels with too low or too high stiffness will compromiseDOI:
the wound
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10.1039/C8TB02590H
repair effect. These studies indicate that the presence of wound dressings allows cells to
respond micromechanical stimuli at wound sites. On the other hand, cells can also rapidly

produce extracellular matrix that will exert forces back to wound dressing materials.18, 19 Apart
from a chronic wound healing model, many previous studies have also shown that mechanical
properties of hydrogels could be used and tuned to control and stimulate cell differentiation. 20,
21
These hydrogel dressing examples have shown that biomechanics of biomaterials are
pivotal for the fate, toxicity, and function of cells and tissues during wound healing process.
However, there is a dearth of systematic research to better elucidate the poorly understood
problem of how proteins and cells respond and adapt to hydrogel-based wound dressings. An
in-depth address to this problem will provide mechanistic insights on the interactions between
wound dressings and tissues at the hydrogel-wound interface, and thus promote the
development of a new generation of wound dressings with long-term healing efficiency22.
Significant research efforts have been made to develop poly(ethylene glycol) PEG-based
polymers for wound healing, however less attention is paid to zwitterionic materials, some of
which have demonstrated their super low-fouling property in vitro and anti-inflammatory
property in vivo. Zwitterionic polymers are among the most popular antifouling materials,
because zwitterionic polymers can be more hydrophilic than PEG to strong attract a layer of
water molecules for resisting unwanted protein adsorption that will further lead to
inflammation at wounds23. Herein, we prepared zwitterionic sulfated poly(sulfobetaine
methacrylate) (polySBMA) hydrogels and applied them to full-thickness excisional acute
wound regeneration in mice, with a particular attention to wound healing efficiency in response
to mechanical softness/stiffness of polySBMA hydrogels with elastic modulus ranging from a
softness of 10 kPa to a hardness of 60 kPa. Collective results showed that the softer polySBMA
hydrogels could speed up wound healing efficiently through the intrinsic elastic impulse to
improve neovascularization. Thus, zwitterionic hydrogels exhibit great potential as promising
wound dressings.
2. Materials and Methods
2.1. Materials
[2-(Methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide(SBMA,
Mn=279.35) as monomer, Poly(ethylene glycol) dimethacrylate (PEGDMA, Mn =550) as
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crosslinker, and Photoinitiator Irgacure 2959 (I2959), as initiator, were all purchased from
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DOI: 10.1039/C8TB02590H
Sigma-Aldrich. Hematoxylin-eosin (HE) or Masson’s Trichrome Stain Kit (Sigma-Aldrich, St.

Louis, MO). Phosphate buffer saline (PBS) was purchased from Sigma-Aldrich. Horseradish

peroxidase (HRP)-conjugated goat anti-human lgG(H+L) was purchased from Beijing
Biosysthesis Biotechnology Co. Human umbilical vein endothelial cell (HUVEC) was
purchased from the Cell Storage Center of Wuhan University (Wuhan, China). Other cell
culture reagents were purchased from Invitrogen (Carlsbad, CA). Deionized water was used as
received.
2.2. Preparation of polySBMA hydrogels with different elastic modulus
Different polySBMA hydrogel were prepared using the previous method24. Here the
elastic modulus of polySBMA hydrogels was achieved by the chemical cross linker percentage.
The monomer SBMA were firstly issolved in de-ionized DI water. The corss linker (PEGDMA)
were varied from 0.1%, 0.5%, 1% to 5% (versus monomer w/w) and the initiator I2959 (final
1% versus monomer w/w) were added and complete dissolved to the above solutions at room
temperature. The final concentration of the monomer is 4 M. Then the mix solution was
transferred into a pair of glass plates separated by poly(tetrafluoroethylene) (PTFE) (with the
thickness of 3 mm or 1 mm). Next, the photo-polymerization reaction was carried out under
room temperature with 362 nm UV light SB-100P (Spectroline) for 30 mins. After the
polymerization, the hydrogels were removed from the plates by immersing in a large volume
of DI water (1 L), which was changed and kept in water every 3 h for 5 days to ensure that non-
reacted initiators or monomers were totally removed from the hydrogel.
2.3. Determination of Equilibrium water content (EWC) of the hydrogels
The hydrated hydrogels were used to quantify its EWC by weighting their mass
difference between the hydrated state and fully dried state. The weight of the hydrogels in
swollen state recorded as Ws, the lyophilized dried hydrogel recorded as Wd. The EWC is
determined by the weight change of hydrogel in swelling state and dry states using the
following Eq. (1):
(Ws ― Wd)
EWC (%) = Ws × 100% (1)
The swelling kinetics of polySBMA hydrogels were tested using gravimetric method.
Same size of prepared swollen hydrogels was firstly freed-dried Wd. Secondly immersed in
the deionized water. At each time interval, the samples were wiped with filter paper to remove
5
the appearance water. The weight of the hydrogels was measured Wt. The degreeDOI:
of 10.1039/C8TB02590H
swelling
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ratio was calculated using the following Eq. (2):

𝑊𝑡 ― 𝑊𝑑

Swelling ratio (%) = 𝑊𝑑 × 100% (2)
The losing rate of the hydrogel varied by days was achieved by weighting hydrogels
everyday recorded as We and the initial swollen hydrogel recorded as Wi. Then the water
retaining content is obtained using following Eq. (3):
𝑊𝑖 ― 𝑊𝑒
Water retaining content (%) = 𝑊𝑖 × 100% (3)
2.4. Morphology of polySBMA hydrogels
The morphology of the hydrogels was examined by scanning electron microscopy

(SEM). Swollen polySBMA hydrogel samples were firstly frozen in liquid nitrogen then
lyophilized overnight for at least 24 h. Before SEM, the cross sectional part of dried hydrogel
was coated with gold by ion sputtering. SEM analysis proceeded with an acceleration voltage
of 10 keV and a probe current of 25 mA (Hitachi S-3000 SEM).
2.5. Compressive mechanical tests of hydrogels
Swollen hydrogels immersing in water for more than 5 days with cylindrical shape (8
mm in diameter and 3 mm in thickness) were placed on the compression plate. Five disks of
each hydrogel were compressed to failure at a compressive strain rate at 1 mm/min using
Instron 3343 (Instron Co, U.S.A) with a 10 N load cell at the room temperature. The modulus
was calculated from the linear portion of the stress-strain curve.
2.6. Protein adsorption measurement
Nonspecific protein adsorption to hydrogels were determined by quantifying HRP-

conjugated anti-IgG adsorption using Tissue culture polystyrene (TCPs) as control. The
samples were firstly incubated with 1 μg/ml anti-IgG for 1.5 h followed by six times rinsing
with PBS. The TCPs and the hydrogels were then removed to 24-well plates separately.
Secondly, 1 ml o-phenylenediamine (OPD 1 μg/ml ) in 0.1M citrate phosphate buffer (pH 5.0),
containing 0.03% hydrogen peroxide was added. Next, this enzyme-substrate activity reaction
was stopped by adding an equal volume of H2SO4 (2M) after 15 min. At last, the relative protein
adsorption percentage compared to TCPs is measured by tangerine color at 492 nm.
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2.7. In vitro HUVEC cell surface attachment assay View Article Online
DOI: 10.1039/C8TB02590H
TCPs and polySBMA hydrogels were placed individually in the 24-well plates. Being
irradiated under 30mins UV light, 105 cells/mL HUVEC cells were seeded onto the samples in

RPMI 1640 medium. After the cells growing for 24 h at 37°C, 5% CO2 and 100% humidity,
the samples were photographed at 4 x Nikon Eclipse TE2000U microscope.
2.8. In vivo wound healing model and analysis

The animal models were followed by the same procedure as our previous paper25. 6 to 7
weeks male C57BL/6 mice weight 25 g were purchased from the Animal Center of Chinese
Academy of Sciences, Shanghai, China. The experiment animals were carried out with the
National Institutes of Health Guide Concerning the Care and Use of Laboratory Animals. All
animal experiments were performed in accordance with the guidelines approved by the Animal
Experimentation Ethics Committee of Wenzhou Medical University, Wenzhou, China. Mice
were maintained for at least 7d before the experiment on a standard diet and water was provided
freely available. Temperature (23 ℃ -25 ℃ ), humidity (35−60%), and photoperiod (12 h
light/dark cycle) were kept constant. Firstly, the mouse were anaesthetized by an
intraperitoneal injection of 4% chloral hydrate (0.1 ml/10g) and the dorsal area was shaved.
0.5-mm-thick silicone donut-shaped splints (the external diameter of 16 mm, the internal
diameter of 8 mm) were fixed on either side of the dorsal midline using 6-0 Prolene suture.
Two full-thickness cutaneous wounds were made using a 6 mm round skin biopsy punch
(Acuderm® inc., Ft Lauderdale, FL, USA) on each side of the dorsal midline. Secondly, the
wound site were measured using digital camera take photos to determine the original wound
area. The wound was then treated with our sample hydrogels. Each sample used 7 animals with
14 wounds per group. All hydrogel dressings were cut into 7 mm diameter dish with punch
immediately before applying to the wound. The hydrogels dressing was last covered with
TegadermTM transparent dressing (3M Health Care, Germany) to prevent from infection and
wrapped in a thin layer of self-adhesive bandages to deter chewing of the splints. At days 0, 7,
10, 14, and 17 post-treatment, the wound closure rate was determined by measuring the wound
area by Image-Pro plus to trace the wound margin. (wound closure rate % = (wound areaday0 -
wound areaday#)/wound areaday0×100%). At day 7 or 20 post-surgery C57BL/6 mice were
anesthetized with 4% chloral hydrate and sacrificed by cervical dislocation. The wound site
was excised, and the tissue was processed for histological evaluation.
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2.9. Histological analysis View Article Online

DOI: 10.1039/C8TB02590H
For histological preparation the skin was fixed in 4% paraformaldehyde in 0.01M

phosphate buffered saline (PBS, pH=7.4) overnight and dehydrated in graded ethanol series

then embedded in paraffin. The tissue was sectioned into 5-μm thickness slices for
Hematoxylin and Eosin (H&E) (Beyotime Institute of Biotechnology, China) staining and for
collagen formation by Masson’s trichrome staining (Beyotime), using standard reported
procedures26. Sections were analyzed and images were captured using a Nikon ECLPSE 80i
(Nikon, Japan).
2.10. Immunofluorescent staining
The CD-31 (ab28364, Abcam), Ki67(ab15580, Abcam), CD 163 (ab182422, Abcam) and
CD 68(ab955, Abcam) were conducted by respective antibody the same with our previous
paper.23 The Skin tissue sections, prepared by a microtome to 5-μm thickness, were
deparaffinized and rehydrated, and then immersed in 3% H2O2 and 80% carbinol for 15 min at
room temperature to blocked the endogenous peroxidase activity. The tissue sections were
heated to antigen recovery in 10 mM sodium citrate buffer (pH 6.0) and after washing the
samples were blocked using 5% bovine serum albumin (BSA) (Beyotime) for 30 min at room
temperature. Skin sections (on 7 day post wound) were stained with rabbit polyclonal anti-
CD31 (1:200), anti-Ki67 (1:200), rabbit polyclonal anti-CD 163 (1:200) and mouse
monoclonal anti-CD 68 (1:100) diluted in phosphate-buffered saline (PBS) contained 1%
bovine serum albumin (BSA) overnight at 4°C. Fluorescence secondary antibodies IgG Alexa
Fluor® 488 and goat anti-mouse IgG Alexa Fluor® 647 (ab150083, 1:1500, Abcam) were
diluted with phosphate-buffered saline (PBS) respectively then incubated at 37°C for 60 min.
Followed by DAPI stained nuclear 5 min and coverslipping with Anti-fluorescent quencher.
The fluorescent images were taken by Nikon confocal laser microscope (Nikon, A1 PLUS,
Tokyo, Japan).
2.11. Statistical Analysis
All the data were expressed as the means ± standard deviations (SD). The statistical data
were compared using unpaired-student’s t-test. Significant difference was considered if the
two-tailed P-values were <0.05. For all test, *P<0.05, **P<0.01 and ***P<0.001.
8
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DOI: 10.1039/C8TB02590H
3. Results and Discussion

3.1. Physicochemical properties of polySBMA hydrogels
To fabricate the hydrogels with controllable mechanical properties, here we used different
crosslinking percentage (0.1%, 0.5%, 1%, and 5%) to tune polySBMA hydrogels from soft to
stiff. Figure 1A represents the typical compressive strain-stress curves of polySBMA
hydrogels. As crosslinkers increased from 0.1% to 5%, polySBMA hydrogels significantly and
monotonically increased their compressive stress from 0.015 Mpa to 0.35 Mpa (Figure 1A)
and elastic modulus from 6.7 kpa to 50.4 kpa (Figure 1B) at a compressive strain of 80%
without broken. For the convenience of system notation, based on compressive mechanical
properties of polySBMA hydrogels, polySBMA hydrogels with low elastic modulus of <12
kPa prepared at 0.1% and 0.5% crosslinkers are defined as soft-1 and soft-2 hydrogels, while
the remaining two hydrogels with high elastic modulus of >48 kPa prepared at 1% and 5% are
defined as stiff-1 and stiff-2 ones. Figure 1C also showed that on one hand, four polySBMA
hydrogels were highly hydrated with consistent high EWC of > 80%, demonstrating the
hydrophilic nature of polySBMA hydrogels. On the other hand, increase of crosslinker contents
also slightly reduced EWC by 10% simply because of the more compact and tightly
interpenetrating network. In parallel, Figure 1D shows the swelling kinetics of four hydrogels
in PBS solution. It can be seen that all hydrogels swell rapidly to achieve equilibrium swelling
within ~4 h. Consistently, increase of crosslinkers reduced hydrogel swelling, accordingly the
swell ratios were 1392.2% for soft-1 (0.1%), 965.0% for soft-2 (0.5%), 542.8% for stiff-1 (1%),
and 469.0% for stiff-1 (5%), respectively. This phenomenon became even more pronounced
for soft-1 hydrogels, which swollen almost 3-times higher than stiff hydrogels. Thus, the
introduction of crosslinkers enables to greatly suppress the expansion of gel networks. As
expected, the swollen transparent hydrogels become mechanically weak, due to the breaking
of hydrogen bonds and lower polymer volume fraction.
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DOI: 10.1039/C8TB02590H

Figure 1. Compressive properties of zwitterionic polySBMA hydrogels prepared by different

cross-linking density from 0.1% to 5%. (A) Compressive stress-strain curves, (B) Elastic
modulus, (C) Equilibrium water content (EWC), and (D) Swelling kinetics of as-prepared
polySBMA hydrogels.
Figure 2A showed the optical property of the four polySBMA hydrogels at the as-prepared
state. Except for the stiff-2 hydrogel (5% of crosslinkers) showing semitransparent optical
property, the other three hydrogels exhibited optical transparency. SEM images in Figure 2B
showed the internal morphology of four polySBMA hydrogels. It can be seen clearly that each
individual hydrogel possessed relatively uniform pore structure, but pore size distribution
decreased from 20 μm to 5 μm as crosslinkers. The highly porous structures in all hydrogels
resemble natural macromolecular cellar matrix systems, suitable for cells proliferation and
migration, as well as oxygen permeation especially for chronic wound as wound dressing to
improve neovascularization. Figure 2C showed that due to the strong ionic solvation of
zwitterionic polymers, all polySBMA hydrogels enable to retain high water content of ~70%
at room temperature for 7 days. Such high water retention property of Soft-1, Soft-2, Stiff-1
and Stiff-2 hydrogels makes advantages for keeping the wound site moist.
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DOI: 10.1039/C8TB02590H

Figure 2. Physical properties of zwitterionic polySBMA hydrogels prepared at different cross-

linking densities from 0.1% to 5% (namely as soft-1, soft-2, stiff-1, and stiff-2 hydrogels). (A)
Visual inspection of appearance, (B) SEM images (scale bar=50 μm), and (C) water retaining
capacity of the four polySBMA hydrogels.
3.2. In vitro antifouling properties of polySBMA hydrogels

Antifouling properties of polySBMA polymers are critical for wound healing23. Our
previous work has demonstrated that polySBMA polymer brushes could achieve ultralow
fouling level of protein adsorption (<0.3 ng/cm2) and also excellent hydrophilicity, salt
resistance, and biocompatibility27-29. Different from previous work focusing on antifouling
property of polySBMA brushes, here we examined the protein adsorption on polySBMA
hydrogels using enzyme-linked immunosorbent assay (ELISA), where tissue culture
polystyrene (TCPs) was used as control to set a complete monolayer protein adsorption (100%).
As shown in Figure 3A, as compared to TCPs, all polySBMA hydrogels significantly reduce
adsorbed protein amounts to 5%-20%. Stiffer polySBMA hydrogels exhibited the slightly
better protein resistance than softer hydrogels, probably because higher elastic modulus and
the more compact porous structures prevent protein adsorption on and adaption to hydrogel
surface24. Further data analysis results showed that under the consideration of error bar, no
significant difference (ns) between soft (0.1% and 0.5%) and stiff (1% and 5%) hydrogels. To
11
further test the ability of polySBMA hydrogel to resist cell attachment, a cell assayDOI:
was carried
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10.1039/C8TB02590H
out to probe the additional antifouling property of the hydrogels. GFP-expressed HUVEC cells
were used to co-culture with polySBMA hydrogels at 37° C for 24 h. After culturing, hydrogels

were gently washed with PBS to remove any unbound or weakly bound cells. Finally, the
images of four hydrogels were taken under a fluorescent microscope with the same excitation
light and exposure time. As shown in Figure 3B, in sharp contrast to TCPs whose surface was
covered by an almost full layer of cells, all polySBMA hydrogels exhibited neglectable cell
attachment. Even though the polySBMA hydrogels had been soaked in serum-containing
medium for two weeks, polySBMA hydrogels, regardless of their mechanical softness or
stiffness, were able to kept their non-fouling property and did not allow cell adhesion on their
surfaces, demonstrating their intrinsic nature of antifouling property.
Figure 3. Antifouling properties of zwitterionic polySBMA hydrogels prepared at different

cross-linking density from 0.1% to 5%. (A) IgG protein adsorption and (B) Cell adhesion (scale
bar=500 μm) on all polySBMA hydrogels relative to TCPs.
3.3. In vivo wound regeneration properties of polySBMA hydrogels

After polySBMA hydrogels have demonstrated their excellent antifouling properties to
resist both protein adsorption and cell adhesion in vitro, here we applied polySBMA hydrogels
to full-thickness cutaneous wounds in C57BL/6 mice, and then examined their wound
efficiency as the effect of elastic modulus. At a first glance, both visual images of wound bed
closure (Figure 4A) and the corresponding mimetic trace of wound bed closure (Figure 4B)
were used to assess the in vivo wound-healing efficacy of the polySBMA hydrogels at different
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time points during 20-days treatment. Compared to a control group treated with PBS,Viewall
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DOI: 10.1039/C8TB02590H
polySBMA hydrogels showed the enhanced wound regeneration behavior and no obvious sign
of inflammation or infection near the wound area. It appears that the growth of new epidermis

extended to the center of all hydrogel treated wounds, resulting in a reduction in wounded area.
Among four polySBMA hydrogel treated groups, the wounds treated by soft-1 and soft-2
hydrogels regenerated faster than stiff-1 and stiff-2 hydrogels. Specifically, after 20-days
treatment, soft-1 and soft-2 polySBMA hydrogels enable to completely heal the wounds with
almost scarless, while siff-1 and stiff-2 treated wounds still remained obvious residual wound
area. Quantitatively, Figure 4C analyzed the restoration area ratio of wound bed. Consistent
with visual inspection, soft-1 and soft-2 hydrogels showed significantly higher wound closure
rate than stiff-1 and stiff-2 hydrogels at every check point of days. The final wound closure
rates of soft hydrogels were 98.1% and 93.2%, as compared to those of stiff-1 (87.5%), stiff-2
(89.5%), and control (85.0%) hydrogels. The ~10% enhancement in wound regeneration of
soft polySBMA hydrogels could be attributed to soft mechanical properties, high water content,
and nanomorphological structures, all of which are more compatible with soft skin, thus
providing a “suturing” effect at the wound site to enhance wound contraction and closure
during the early stage of the wound-healing process. In Figure 4C, on day 20, there exists
significant differences between soft-2 and stiff-1 gels and between soft-2 and stiff-2 gels, in
which both p values were lower than 0.01 (“*” indicates p<0.05). However, the results on day
17 did not show obvious improvement between soft-2 and stiff-1/stiff-2 gels. In general, both
soft hydrogels showed significant enhancement of wound regeneration than both stiff
hydrogels on day 17 and day 20. This result further confirms the wound healing efficiency is
sensitive to the mechanical property of the hydrogels.
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DOI: 10.1039/C8TB02590H

Figure 4. In vitro wound healing efficacy of zwitterionic polySBMA hydrogels prepared at

different cross-linking densities. (A) photographs of excised wounds, (B) schematic of wound
closure trace, and (c) quantitative evaluation of wound closure rate for all hydrogel- and saline
(control)-treated wounds on day 0, 7, 10, 14, 17 and 20 in each group. Error bars indicate SD.
Significant differences between sample means are indicated: ** P <0.01, * P <0.05 where n>6.
Considering that a typical wound healing process comprises granulation tissue

formation, re-epithelialization, and collagen deposition, here we used the H&E staining of
wound treated with polySBMA hydrogels on day 7 and day 20 to evaluate the granulation and
epidermal formation. As shown in Figure 5A, H&E stained sections of the wounded skins
treated with polySBMA hydrogels exhibited a markedly higher epidermis and granulation
construction than that of control group. Specifically, on day 7, hydrogel-treated groups
developed a full length of epidermal layer, while the control group induced the less
epithelialization. On day 20, hydrogel-treated groups were able to connect tightly regenerated
dermis and fill with appendants under a fully healed epithelialization layer, while the control
group still had larger unhealed wound areas with a thin granulation construction. Similar to our
earlier in vivo wound closure results, while the H&E staining for all groups showed no
pathological changes, the softer hydrogels had the smaller wound closure area and the thicker
granulation formation than the stiffer hydrogels. Furthermore, H&E staining was performed on
the wound cross sections to identify and quantify at the center of the wound. (Figure 5B). The
results show that the mean length of granulation gap at day 7/day 20 for wounds treated with
wounds treated with soft-1, soft-2, stiff-1, stiff-2, and PBS was 3.0/1.2, 3.4/1.8, 3.8/2.5, 4.0/2.0
14
and 4.5/3.2 μm, respectively (Figure 5C). The mean granulation tissue thickness
DOI:was with
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10.1039/C8TB02590H
average 1.6/1.6, 1.5/1.3, 1.4/1.2, 1.3/1.2, and 1.0/0.7 μm for wounds treated with soft-1, soft-
2, stiff-1, stiff-2, and PBS was at day 7/day 20, respectively (Figure 5D). Altogether, these

results suggest that the softer polySBMA hydrogels significantly accelerate skin wound healing
via improved granulation tissue formation and epithelial tissue regeneration, relative to the
stiffer hydrogels.
Figure 5. H&E staining of wound sections obtaining from different zwitterionic polySBMA
hydrogels and control groups day 7 and day 20 post-operation. (A) H&E stained images for
skin wounds treated with polySBMA hydrogels and PBS on day 7 and day 20. (B) schematic
measurement for granulation tissue thickness and granulation gap30, 31. (C) Quantification of
granulation tissue gap and thickness on day 7 and 20. **P < 0.01, *P < 0.05, n> 5.
In the final remodeling stage of wound healing, a mass deposition of collagen (a predominant
structural protein in skin) is necessary to reconstruct dermal tissue at wound sites effectively.
Masson’s trichrome staining (MTS) on day 7 and day 20 were used to determine the
histological collagen deposition (staining in blue). As shown in Figure 6A, on day 7, all
polySBMA-treated wounds were covered by a more intense blue color as compared to the
control group, demonstrating that collagen deposition was most enhanced in hydrogel-treated
wounds, leading to the thicker wound granulation formation and the smaller wound gap. On
day 20, the accelerated granulation tissue formation and collagen deposition became even more
pronounced. Among four hydrogel-treated wounds, it can be seen that the softer hydrogel
groups not only had the extensive and thick collagen deposition both in wound center and
15
wound edge reflected by the deep blue staining, but also the most orderly collagen arrangementView Article Online
DOI: 10.1039/C8TB02590H
compared with other groups at both 7 and 20 days. We also observed that the soft hydrogel-
treated wounds had more densely packed collagen fibers with parallel arraignment as compared

to the stiff hydrogel-treated wounds with loosely packed collagen fibers running in irregular
arrangements, or control with nothing. Quantitatively, Figure 6B shows the statistical analysis
of collagen deposition on day 20. Clearly, the mean collagen deposition density in PBS-, soft-
1-, soft-2-, stiff-1-, and stiff-2-treated wounds was 4539.4±1386.6, 42882.3±15839.2,
15887.5±6386.6, 11528.3±1380.3, and 6091.7±1526.0 /mm2, respectively. So, the soft
hydrogel-treated wounds represented 3-7 fold increase in collagen deposition, relative to the
wounds treated with stiff hydrogels. These results confirm further the effectiveness of wound
healing by wound remodeling via more collagen deposition. It is important to mention that,
collective data from Figure 4 (wound closure rate and image), Figure 5 (granulation
formation), Figure 6 (collagen deposition) indicate that the wound in the control group on day
20 did not completely close, and the partial wound closure is likely due to the contraction effect
the wound. We should also note that severed muscle and deeply stain with trypan does not
necessarily indicate the existence of normal tissue, instead they could be an indicator of newly
regenerated skins as well in our case.
16
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DOI: 10.1039/C8TB02590H

Figure 6. Masson’s trichrome staining (MTS) of wound sections obtaining from different
zwitterionic polySBMA hydrogels and control groups day 7 and day 20 post-operation,
showing collagen deposition and maturity. (A) MTS images for skin wounds treated with
polySBMA hydrogels and PBS on day 7 and day 20 (scale bar=500 μm), as well as a close-up
MTS images at wound center and wound edge on day 20 (scale bar=100 μm). (B)
Quantification of collagen deposit density in the wound sites on day 20 by optical blue density
(IOD), **P < 0.01, *P < 0.05, n > 5.
3.4. Soft polySBMA hydrogels accelerat more neovascularization than stiff polySBMA
hydrogels
Among the wound healing processes that could be triggered by GFs, neovascularization and
angiogenesis are crucial to tissue engineering32. The formation of new vasculature is
fundamental to powering the wound regeneration. To confirm the newly formed vessels found
in wound area, we also performed immunofluorescence staining of CD31 endothelial cell
marker to identify the new blood vessels formation on day 7. As shown in Figure 7A, wounds
17
treated with both softer hydrogels exhibited a higher amount of CD31 cells than wounds treated
View Article Online
DOI: 10.1039/C8TB02590H
with stiffer hydrogels. To further assess this outcome, the overall area covered by the CD31
cells in the hydrogel groups was quantified to determine their density in the wound area. Figure

7B showed that the density of CD31 cells was 17.21%, 13.4%, 4.3%, and 1.7% for soft-1-,
soft-2-, stiff-1-, and stiff-2-treated wounds, respectively, consistent with immunofluorescence
staining images. Thus, the softer hydrogel wound dressings will promote angiogenesis and
neovascularization, which in turn provide more oxygen and nutrients to the wounds and guide
the cells for wound remolding such as granulation and collagen formation (Figure 7C). Thus,
our results suggest that soft hydrogels accelerate wound healing via improved granulation
tissue formation and epithelial tissue regeneration, possibly by increasing collagen deposition
and the formation of new blood vessels.
Figure 7. CD31 staining for characterizing neovascularization in wound sites of different

zwitterionic polySBMA hydrogel and control group. (A) CD 31 (red) and DAPI (Blue)
staining for new blood vessels on day 7 post-wound operation. (B) Quantitative analysis of
newly formed blood vessels as measured by vascular area density on day 7. **P < 0.01, *P <
0.05, n>5. (C) Schematic of neovascularization at wound sites.
18
3.5. Soft polySBMA hydrogels enhance cell proliferation and M2/M1 macrophages than
View Article Online
DOI: 10.1039/C8TB02590H
stiff polySBMA hydrogels

In order to further understand the mechanical regulation of wound healing mechanism.

Ki-67 protein (also known as MKI67) was used as cellular maker for evaluating cell
proliferation during the wound repair process. As shown in Figure 8A, soft-1 and soft-2
dressings up-regulated cell proliferation as compared to stiff-1 and stiff-2 dressings at early
stage of wound healing on day 7, as evidenced by significant differences of *** p< 0.001
between soft-1and two stiff hydrogels and of ** p<0.01 between soft-2 and two stiff hydrogels,
respectively (Figure 8C). In addition, these cells could further secret granulation matrix and
different types of growth factors to promote wound regeneration. This enhanced cell
proliferation once again confirms a beneficial role of soft polySBMA hydrogels in wound
repair.
Macrophages activation of M2 (anti-inflammatory) versus M1 (pro-inflammatory) is

considered as an important factor to promote angiogenesis and tissue remodeling. In our
previous work, we have demonstrated that zwitterionic polySBMA hydrogel enable to pursue
the polarization of macrophages from M1 to M2 through enhanced anti-inflammatory proteins
to achieve wound pro-healing functions33. Here, we also quantified and compared M1 (stained
by CD-68, red color) and M2 macrophage (stained by CD-163, green color) of the four
hydrogels, as compared to the control group. It can be seen in Figure 8 B&D that the soft-1
and soft-2 dressings were much more active to up-regulate M2 macrophages at day 7 than the
stiff-1 and stiff-2 dressings, as evidenced by the presence of more green color in Figure 8B
and a significant difference of *** p<0.001 between soft hydrogels and stiff hydrogels in
Figure 8D. In line with wound closure rate, granulations formation, and collagen deposition,
the results of the expression of M2/M1 macrophages again confirm the positive role of soft
hydrogels in wound regeneration.
We also conducted cell toxicity experiments, when co-cultured with polySBMA

hydrogels, to verify their biocompatibility. As shown in Fig. S1-A, as compared to the control
group, four hydrogel groups showed no obvious differences in terms of the morphology and
proliferation of Hacat cells. Furthermore, CCK-8 assay in Fig. S1-B showed that upon 24 h
incubation, cell viability for all hydrogels groups and the control group displayed no obvious
difference. Both cell toxicity and viability results indicate the good biocompatibility of
19
polySBMA hydrogels.
20
Journal of Materials Chemistry B
DOI: 10.1039/C8TB02590H
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Page 20 of 24
Figure 8. A) Immunofluorescent staining of wound sites on day 7 post-wounding forView

cell
Article Online
DOI: 10.1039/C8TB02590H
proliferation (Ki67, red) and stained nuclear with DAPI (blue). B) Immunofluorescent staining
of wound sites on day 7 post-wounding for M1 (CD-68, red), M2 macrophage (CD-163, green)
and stained nuclear with DAPI (blue). C) Quantitative evaluation of Ki67 expression of four

hydrogels and the control on day 7. D) Quantitative evaluation of M2/M1 of four hydrogels
and the control. Significant differences between samples are defined by *** P <0.001, ** P
<0.01, where n>6.
4. Conclusion
In this study, zwitterionic polySBMA hydrogels with different mechanical properties were
prepared and used as wound dressings to treat the full-thickness dermal wound in mice. First,
all polySBMA hydrogels demonstrated their high water content and retention ability, and super
antifouling properties to prevent unwanted protein adsorption and cell adhesion in vitro. Then,
the softer polySBMA hydrogels exhibited the better wound healing efficiency in vivo than the
stiffer ones, through their enhanced wound closure, accelerated granulation tissue formation,
increased collagen deposition, and improved new blood vessel formation. Finally, collective
data revealed a correlation between mechanical property and wound healing efficiency,
demonstrating a simple and possible generalizable approach to achieve the better wound
healing efficiency by simply controlling mechanical property of hydrogels as wound dressings.
Acknowledgements: J.W. and H.H. thank for the financial support from National Natural
Science Funding of China (81601615 and 81701809), the Zhejiang Qianjiang Talent project
under the Grant (QJD1803015) and Zhejiang Provincial Natural Science Foundation of China
under Grant No. LGF19H180008. J.Z. thanks a fully financial support from NSF (DMR-
1806138 and CMMI-1825122).
21
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acceptance, before technical editing, formatting and proof reading.

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Zwitterionic Poly(Sulfobetaine Methacrylate) Hydrogels with Optimal

Mechanical Properties for Improving Wound Healing In Vivo

Journal of Materials Chemistry B Accepted Manuscript

*Corresponding Author: J.Z: [email protected]; J.X: [email protected]; J.W.

# The authors contribute equally to this work

Abstract View Article Online

Journal of Materials Chemistry B Accepted Manuscript

Keywords: zwitterionic hydrogels, non-fouling, elastic modulus, protein adsorption, wound

1. Introduction View Article Online

Journal of Materials Chemistry B Accepted Manuscript

formation, re-epithelialization, extracellular matrix synthesis and remodeling, thus promoting

Journal of Materials Chemistry B Accepted Manuscript

2. Materials and Methods

Sigma-Aldrich. Hematoxylin-eosin (HE) or Masson’s Trichrome Stain Kit (Sigma-Aldrich, St.

Journal of Materials Chemistry B Accepted Manuscript

2.2. Preparation of polySBMA hydrogels with different elastic modulus

2.3. Determination of Equilibrium water content (EWC) of the hydrogels

ratio was calculated using the following Eq. (2):

Journal of Materials Chemistry B Accepted Manuscript

2.4. Morphology of polySBMA hydrogels

The morphology of the hydrogels was examined by scanning electron microscopy

2.5. Compressive mechanical tests of hydrogels

2.6. Protein adsorption measurement

Nonspecific protein adsorption to hydrogels were determined by quantifying HRP-

Journal of Materials Chemistry B Accepted Manuscript

the samples were photographed at 4 x Nikon Eclipse TE2000U microscope.

2.8. In vivo wound healing model and analysis

2.9. Histological analysis View Article Online

For histological preparation the skin was fixed in 4% paraformaldehyde in 0.01M

Journal of Materials Chemistry B Accepted Manuscript

2.10. Immunofluorescent staining

2.11. Statistical Analysis

View Article Online

3. Results and Discussion

Journal of Materials Chemistry B Accepted Manuscript

View Article Online

Journal of Materials Chemistry B Accepted Manuscript

Figure 1. Compressive properties of zwitterionic polySBMA hydrogels prepared by different

decreased from 20 μm to 5 μm as crosslinkers. The highly porous structures in all hydrogels

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Journal of Materials Chemistry B Accepted Manuscript

Figure 2. Physical properties of zwitterionic polySBMA hydrogels prepared at different cross-

3.2. In vitro antifouling properties of polySBMA hydrogels

Journal of Materials Chemistry B Accepted Manuscript

Figure 3. Antifouling properties of zwitterionic polySBMA hydrogels prepared at different

3.3. In vivo wound regeneration properties of polySBMA hydrogels

Journal of Materials Chemistry B Accepted Manuscript

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Journal of Materials Chemistry B Accepted Manuscript

Figure 4. In vitro wound healing efficacy of zwitterionic polySBMA hydrogels prepared at

Considering that a typical wound healing process comprises granulation tissue

Journal of Materials Chemistry B Accepted Manuscript

Journal of Materials Chemistry B Accepted Manuscript

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