Research Article
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Inflammation Self-Limiting Electrospun Fibrous Tape via
Regional Immunity for Deep Soft Tissue Repair
Qimanguli Saiding, Zhengwei Cai, Lianfu Deng, and Wenguo Cui*
Regional innate and adaptive immunity lies behind most inflammatory selflimiting conditions, where chemokines
and inflammatory cytokines secreted by
resident inflammatory cells will establish
an inflammatory signal concentration
gradient to drive the migration of bloodderived immune cells to the injury site
and quickly remove necrotic cells and
tissue fragments.[3] Once their job is done,
the immune cells will withdraw from
inflamed tissues, promoting tissue repair
responses.[4] However, potential systemic
(aging, obesity, combined diseases) and
local (hypoxia, infection, foreign bodies,
and necrotic tissue fragments) stimuli
often lead to regional immunity dysregulation and inevitably failure of inflammation
self-limiting.[5] Over-activated immune
cells persistently secrete excessive inflammatory cytokines and chemokines to
amplify the positive feedback of the chemotaxis cascade, which, as a consequence,
results in unrestricted inflammation and
impaired tissue remodeling.[6]
The failure of inflammation self-limiting after soft tissue injuries such as skin, fat and muscle
caused by surgery, trauma or congenital diseases will hinder
its structural and functional reconstruction.[7] Scholars have
proposed strategies to modulate regional innate and adaptive
immunity in the past few years by promoting macrophage
polarization through cell-targeted immune regulation,[8] such
as neutrophils, monocytes and macrophages, and capturing
overexpressed inflammatory cytokines and chemokines.
For example, Lohmann and colleagues reported a hydrogel
dressing made of heparin and derivatives to efficiently scavenge chronic wound chemokines and inflammatory cytokines,
successfully limiting local inflammation and solving the poor
diabetic wound healing.[9] However, due to the deep anatomical
position and particular tissue structure, deep soft tissues such
as muscle, fat, and ligaments are easy to produce in a large
number of pro-inflammatory chemokines and inflammatory
cytokines after injury and tend to evolve into a vicious circadian
fashion of inflammatory signals due to the relatively enclosed
microenvironment and low intervenability.[10] Therefore,
applying the above-mentioned regional immunity modulating
strategies for deep soft tissues will be less effective because of
the off-target of immune cells and damage to normal tissue
cells, low efficiency of immunological regulation, and rapid
degradation of hydrogel scaffolds.[11] Although researchers try to
Overexpression of inflammatory cytokines and chemokines occurs at deep
soft tissue injury sites impeding the inflammation self-limiting and impairing
the tissue remodeling process. Inspired by the electrostatically extracellular
matrix (ECM) binding property of the inflammatory signals, an inflammation
self-limiting fibrous tape is designed by covalently modifying the thermosensitive methacrylated gelatin (GelMA) and negatively charged methacrylated
heparin (HepMA) hydrogel mixture with proper ratio onto the electrospun
fibrous membrane by mild alkali hydrolysis and carboxyl-amino condensation reaction to restore inflammation self-limiting and promote tissue repair
via regional immunity regulation. While the GelMA guarantees cell compatibility, the negatively charged HepMA successfully adsorbs the inflammatory
cytokines and chemokines by electrostatic interactions and inhibits immune
cell migration in vitro. Furthermore, in vivo inflammation self-limiting and
regional immunity regulation efficacy is evaluated in a rat abdominal hernia
model. Reduced local inflammatory cytokines and chemokines in the early
stage and increased angiogenesis and ECM remodeling in the later phase
confirm that the tape is an approach to maintain an optimal regional immune
activation level after soft tissue injury. Overall, the reported electrospun
fibrous tape will find its way into clinical transformation and solve the challenges of deep soft tissue injury.
1. Introduction
Acute inflammation is usually self-limiting and is naturally
attenuated after eliminating harmful stimulation, followed by
restoring homeostasis and initiating tissue repair to protect the
injured tissue from over-activated “inflammatory fire”.[1] The
key to self-limiting of inflammation is the chemotactic effect
of inflammatory chemokines (such as (macrophage chemoattractant protein (MCP), macrophage inflammatory protein
(MIP)) and inflammatory cytokines (such as interlukin-8 (IL-8),
IL-1, and tumor necrosis factor-α (TNF-α)) on immune cells.[2]
Q. Saiding, Z. Cai, L. Deng, W. Cui
Department of Orthopaedics
Shanghai Key Laboratory for Prevention and Treatment of Bone and Joint
Diseases
Shanghai Institute of Traumatology and Orthopaedics
Ruijin Hospital
Shanghai Jiao Tong University School of Medicine
197 Ruijin 2nd Road, Shanghai 200025, P. R. China
E-mail: wgcui@sjtu.edu.cn
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/smll.202203265.
DOI: 10.1002/smll.202203265
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achieve deep soft tissue inflammation self-limiting by intraperitoneal injection or oral administration of drugs or liposomes
that can neutralize inflammatory chemokines and cytokines,
the effectiveness is far lower than expected due to the short
half-life of oral application, fast metabolism of intraperitoneal
injection and low drug utilization.[12] Given that, rapid, effective, and stable removal of pro-inflammatory chemokines and
cytokines and breaking the vicious cycle is the key to realizing
the self-limiting of deep soft tissue inflammation.
As a commonly used tissue engineering scaffold, electrospun fibrous membranes with high surface area,[13] biodegradability,[14] and adjustable mechanical strength[15] are widely used
in deep soft tissue engineering and translational medicine in
recent years.[16] However, owing to the inert property of the synthetic polymers used in electrospinning, the obtained fibrous
membranes are generally hydrophobic. They lack biological
adhesion sites, hindering their inflammation self-limiting efficacy by regulating regional immunity. On the contrary, they will
be recognized as foreign bodies and become a noxious stimulus that induces persistent inflammation.[17] Researchers have
tried to limit the inflammatory reaction caused by electrospun
fibrous membranes by surface modification of bioactive agents
such as proteins, peptides, and nucleic acids or drug loading.[18]
For instance, Zhang et al. designed an intelligent oxidative
stress-responsive electrospun polyester membrane to release
anti-inflammatory drugs to limit local inflammation to prevent
tendon adhesion.[19] On the other hand, Wen et al. reported
electrospun fibrous vascular scaffolds loaded with microRNAs
that can realize the self-limiting of local inflammation after
vascular injury by promoting the M2 phenotype polarization of
macrophages, making them a bioactive substitute for artificial
small diameter vessels.[20] However, the low modification rate of
macromolecular substances, easy-to-lose bioactivity, and drug
burst release of the reported approaches usually fail deep soft
tissue inflammation self-limiting caused by chemokines and
inflammatory factors.[21]
Based on the fact that the overexpressed inflammatory
cytokines and chemokines are the main reason for the unrestricted inflammation and impaired tissue repair,[22] the present
study innovatively proposed an electrospun fibrous membrane
tape that can quickly and effectively capture chemokines and
inflammatory cytokines from the injury site modulating the
tissue-specific regional immunity to promote tissue regeneration. First, poly(lactic acid) (PLA) fibrous membrane was prepared by electrospinning technology, and photosensitive double
bonds were induced on its surface through mild alkali hydrolysis and N-(3-dimetthyl-aminopropyl)-N-ethyl carbodiimide
(EDC)/N-hydroxy sulfosuccinimide (NHS) carboxyl-amino
condensation reaction. Then the thermosensitive GelMA and
negatively charged HepMA hydrogel mixture with proper ratio
was sprayed on the surface of the pretreated PLA fibrous membrane and stored at low temperature to form a stable ready-touse inflammatory self-limiting tape. In vitro, apart from the cellular adhesion, proliferation, and migration effects, the tape’s
inflammatory factor and chemokine adsorption ability were
detected by lipopolysaccharide (LPS)-induced inflammatory
response and protein chip technology. A rat abdominal hernia
model was established as an example of deep soft tissue injury,
and the scavenging ability of inflammatory chemokines and
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cytokines at the injury site in the early stage was investigated by
inflammatory histological analysis. Then the tissue remodeling
potential in the later phase was evaluated as an index of early
inflammation self-limiting and regional immune regulation of
the reported tape. Collectively, this engineered tape will pave
the way for custom-made remodeling biomaterials for deep
soft tissue injury and other various reconstruction applications
(Scheme 1).
2. Results and Discussion
The emerging field of biofabrication has its aim of engineering
“smart” platforms for tissue regeneration. Gelatin is an attractive base material considering its limited antigenicity, bioactive sequences, and matrix metalloproteinase (MMP) sensitive
degradation sites. Besides, introducing functional groups can
be initiated to create complex tissue analogs, such as methacryloyl side groups to render gelatin with specific properties such
as photosensitivity.[23] On the other hand, heparin is a linear
polysaccharide and participates in many biological processes
through its interaction with various proteins, exhibiting attractive properties, such as anticoagulant activity, growth factor
binding and cell apoptosis, making them excellent candidates
for biomedical applications.[24] The negative potential that heparin carries is a unique advantage for electrostatic interactions.
In addition, heparin can also be modified chemically to make it
possible for more complex material designs, such as methacryloyl side groups again to render heparin with photocrosslinkable properties. Given the biocompatibility and electrostatic
adsorption of positively charged inflammatory signals, the
GelMA and HepMA were selected for our system. The chemical
synthesis process of GelMA and HepMA, pre-tape modification,
and tape application processes are presented in Figure 1A. Poly
(lactic acid) (PLA), a biodegradable polyester, is used for various
applications due to its mechanical strength, controllable degradability and modifiability.[25] Considering these advantages, PLA
was chosen for preparing an electrospun fibrous membrane
as a backbone of the tape. Once the membrane was obtained,
the modification process was carried out and the new chemical bonds generated are displayed in Figure 1B. The surface of
the electrospun fibrous membrane was activated by alkali, and
the double bond was induced to covalently graft the hydrogel
system. The macroscopic pictures showed that the soft and sagging PLA fibrous membrane became firmer and stiffer after
the GelMA/HepMA hydrogel mixture was sprayed (Figure 1C),
and microscopic structure change was detected by scanning
electron microscopy (SEM). The smooth, straight PLA fibers
slightly curved after activation, while the PLA-GelMA/HepMA
(PGH) tape presented a honeycomb-like appearance indicating
that the porous structure of the fibrous membrane was not
damaged during the surface modification process. Figure 1D
shows the overall application scenario of this newly developed
inflammation self-limiting ready-to-use tape. In brief, the pretape (without photocrosslinking) stored at 4 °C melts slowly
at body temperature and was crosslinked by 365 nm UV light.
Then the tape’s backing can be peeled, achieving a firm adhesion between the tape and living tissues. All the procedures
were conducted with the approval of the volunteer.
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Scheme 1. Illustration of the inflammation self-limiting tape preparing process. A) The chemical components and newly formed chemical bonds during
the pre-tape preparation. B) The in vivo deep soft tissue injury application procedure of the pre-tape. C) The cellular mechanism of the inflammation
self-limiting tape via regional immunity when applied to deep soft tissue injury.
2.1. Synthesis and Characterization of the Tape
It’s of great importance to investigate various properties of a
newly fabricated biomaterial to make it more applicable in
desired settings. After observing the macroscopic and microscopic structural changes during the modification process, we
further looked into the physicochemical characteristics of the
tape. First, as the most critical consideration for this tape, the
ratio of the GelMA and HepMA had to be optimized based on
their zeta potential to meet the requirements of tape–protein
electrostatic interactions. The zeta potential of pure GelMA
was 4.2 mV, while it was −53.8 mV for pure HepMA. The zeta
potential value decreased from 0.6 to −4.3 mV by increasing
the HepMA by 1% in the hydrogel system. The zeta potential continued to decline as the HepMA percentage increased.
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However, the potential value difference between 10% and 20%
HepMA became less significant, indicating that negative potential reached a relative platform period. Under the principle
of ensuring a strong negative charge as well as cell compatibility, 10% HepMA ratio was selected for the hydrogel mixture.
Besides, side effects such as anti-coagulation should be taken
into consideration when applied in vivo,[26] therefore, the 10%
HepMA is also a safer choice than the 20% one for this tape
preparation (Figure 2A).
GelMA was previously reported to exhibit temperature sensitivity, as it is solid at low temperature and melts into liquid
while temperature elevates.[27] This property made it possible
to prepare a ready-to-use pre-tape that can be stored at low
temperatures before in vivo applications. The temperaturedependent gelation process was tested by rheology study
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Figure 1. Modification process of the inflammation self-limiting tape. A) The chemical synthesis process of GelMA and HepMA by gelatin and heparin
respectively. B) The graphic illustration of chemical bond introduction on the electrospun fibrous membrane. C) SEM images of the membrane activation process and the obtained pre-tape. D) The in vivo application process of the ready-to-use inflammation self-limiting tape.
and the Figure 2B illustrates that under room temperature
(5–21 °C), the hydrogel mixture exhibited solid-like behavior
as the storage modulus (G′) exceeded the loss modulus (G″),
confirming the possibility of storing pre-tape in low-temperature conditions. When the temperature continued to increase,
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the hydrogel mixture displayed a yielding behavior. At 37 °C, a
shear deformation was observed as the G″ curve crossed and
exceeded the corresponding G′ curve, indicating a transition
to liquid-like behavior. However, once exposed to 365 nm UV
light, the shear deformation appeared again, accompanied by
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Figure 2. Characterization of the inflammation self-limiting tape. A) Zeta potential of different GelMA/HepMA ratios. B) Rheology results of the
temperature-dependent gelation process of the pre-tape and photocrosslinking induced gelation process of the tape. C) Stress–strain studies of the
PLA fibrous membrane and PGH tape. D) FTIR results of the PGH tape. E) WCA results of the PLA fibrous membrane and the PGH tape. F) In vitro
degradation study of the PGH tape. G) In vitro tissue adhesive investigation under different circumstances.
G′ surpassing G″, demonstrating the light crosslinking induced
gelation after a quick liquid phase. From the results, it can be
deduced that the hydrogel mixture melts at body temperature
and fills the defect when applied to the injury site, followed
by photocrosslinking to achieve tape-tissue adhesion and presumably inflammation self-limiting and regional immunity
regulation.
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The deep soft tissues in the body usually endure mechanical
support to maintain the various physiological activities. For
instance, the abdominal muscles present different mechanical
properties to meet required physiological activities such as
jumping or coughing, during which the abdominal pressure is
claimed to reach tens to hundreds of kilopascals.[28] A critical
role of tissue engineering scaffolds is to provide the required
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mechanical support during the repair and regeneration of
damaged tissues, which requires the engineered scaffolds to
have a certain mechanical strength. To detect the mechanical
supportive potential of the tape, a stress–strain study was conducted. The results in Figure 2C reveal the increased tensile
strength of the PGH tape (4.01 MPa) compared with that of
PLA (2.27 MPa). The tensile strength of pure GelMA and the
GelMA/HepMA mixture are also given in Figure 2C and the
results were 2.61 and 8.29 kPa, respectively. The enhanced
mechanical strength of the tape is presumably the result of
both non-covalent and covalent crosslinking between hydrogel–
fiber and hydrogel–hydrogel. Therefore, it’s reasonable to think
that the tape is strong enough to protect the injured tissue from
suffering second damage during its remodeling process.
For a chemical modification process, it’s crucial to test the
formation of anticipated bonds or groups, which on the one
hand, is essential for making the synthesis process clearer,
and on the other hand, is necessary for material producibility.
The success of new bond generation after modification was
confirmed by Fourier transform infrared absorption spectroscopy (FTIR) and the peaks are displayed in Figure 2D. Apparently, post photocrosslinking step, the broad and deep peak at
3210 cm−1 confirmed the introduction of OH from GelMA. In
comparison, the new peak at 1635 cm−1 testified the carbon–
carbon double bonds (CC) of methacrylic anhydride in both
GelMA and HepMA. To analyze the surface wettability of the
tape, which is vital for cell adhesion and tissue compatibility,
the water contact angle (WCA) study was conducted, and the
results are given in Figure 2E. As the PLA is a hydrophobic
polymer, the WCA of PLA fibrous membrane was 129 ± 2°
while the WCA was 59.3 ± 7.2° for PGH. The sharp decrease
in WCA was strong evidence for successful modification of
GelMA and HepMA mixture since the hydrogel is hydrophilic
in nature.
The bio-scaffolds lose their mechanical strength as they
gradually degrade and pose a concern to successful tissue
remodeling. As the degradation process is vital for safety, the
degradability of the tape was carried out in vitro (Figure 2F).
The tape showed a slow degradation with 45% weight loss
after 55 days, while the PLA showed ≈10% mass loss, confirming the sufficient support for new tissue regeneration.
After evaluating the physicochemical properties of the tape, we
proceeded with in vitro tissue adhesion of the tape under several ordinary circumstances that could happen after implantation in vivo. As illustrated in Figure 2G, the tape adhered
to the muscle tissue firmly once photocrosslinking finished,
and the adhesion was stable even when placed in phosphate
buffer saline (PBS) or stretched and twisted. The tape’s stable
adhesion property guarantees the original implantation position and provides the required mechanical support during the
tissue repair process.
2.2. In Vitro Cellular Study
It is necessary to evaluate the cellular response to the designed
tape to avoid getting the opposite results for regional immunity
regulation.[29] Because strong negative charges will probably
inhibit cell adhesion onto the HepMA-containing platforms, the
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cellular adhesion behavior of human vaginal fibroblasts (HVFs)
on PGH and PLA-GelMA (PG) tape was tested. After 24 h of coculture, the cells presented a typical spindle-like morphology on
the PG with an aspect ratio of 7.36 ± 1.77 (Figure 3A,B). However, the cells cultured on PGH showed a more irregular morphology, mostly in polygon or ellipse shape. The aspect ratio
for this group was 2.66 ± 1.22, and the reason was under expectation, presumably because there would be a certain repulsion
between the negatively charged PGH and the cell membrane.
The cell migration distance from the surface to the deeper
position of the two groups presented no statistical difference.
The cell migration depth of PG was slightly higher than that of
PGH (Figure 3C), indirectly manifesting the above hypothesis.
Fortunately, the cells adhered to the tapes well, and long-term
survival could be predicted. Given that, the live/dead study was
further carried out to determine cytocompatibility in the long
term. As shown in Figure 3D, after 3 days of coculturing, cells
gained their normal morphologies, the cell number increased
with time, and there were almost no dead cells. However, cell
spreading and cell morphology were poor on the hydrophobic
PLA membrane compared with tissue culture plates (TCP) and
PGH groups. These results confirmed that cells could spread
and proliferate on the PGH tape, which will be necessary for
tissue repair and regeneration.
2.3. In Vitro Inflammatory Factor Adsorption Study
A critical feature of deep soft tissue injury is its exposure to
inflammatory cytokines originating from the peritoneal fluid,
destroying regional immunity.[30] The high concentration of
these cytokines causes unrestricted local inflammation and
consequently visceral adhesions after reconstruction surgery.[31]
Under this situation, tissue remodeling will be suspended or
delayed, seriously affecting body function. Thus, timely scavenging of the inflammatory cytokines is crucial for limiting
inflammatory reactions and promoting tissue regeneration.
Some previously reported studies mainly focused on antagonizing or blocking specific receptors and neutralizing target
ligands. But, in vitro, inflammatory signal redundancy results
are usually hard to translate into the in vivo circumstances.
However, as a well-known fact, the inflammatory cytokines and
chemokines can bind to electrostatically extracellular matrix
(ECM) glycosaminoglycans (GAGs) through electrostatic interactions since some proinflammatory cytokines are positively
charged, such as IL-1, IL-6, IL-8 and TNF-α.[32] They can interact
with negatively charged ECM components and this will provide
a vision of specific GAG–protein interaction for therapeutic
exploitation. Therefore, it’s hopeful that the negatively charged
PGH tape absorbs the positively charged inflammatory factors
and regulates the regional innate and adaptive immunity to
promote tissue repair. To test the inflammatory factor adsorption capability of the prepared tape, we first inducted the macrophages to secrete the inflammatory cytokines and chemokines
by stimulating with LPS in vitro. Then, the prepared PGH and
the control group PG were allowed for cytokine adsorption in
the transwell plates for 24 h, followed by protein chip testing
to determine the cytokine concentration in the supernatant
(Figure 3E). As displayed in Figure 3F and Figure 4, some of
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Figure 3. In vitro cellular studies. A) The cytoskeleton staining for early cell adhesive investigation. B) Aspect ratio for cells cultured on PG and PGH
tapes for 24 h. C) The cell migration distance from the top of the tape after 24 h of co-culture. D) Live/dead study for cell compatibility investigation.
E) Graphic illustration of the in vitro inflammatory cytokine and chemokine adsorption study. F) The inflammatory cytokine and chemokine adsorption
results by protein chip study. *p < 0.05, ns: not significant, comparison between PG and PGH.
the tested inflammatory cytokines such as colony stimulating
factor (CSF), IL-1b, TNF-α and proinflammatory chemokines
including MCP-1, MIP-1a and MIP-1b between PG and PGH
groups exhibited statistical difference, demonstrating the successful adsorption by PGH tape. The other tested cytokines, for
example, some anti-inflammatory cytokines (IL-4, IL-10) and
other proinflammatory chemokines, showed no significant difference between the two tested groups, and the reason for that
may include the protein charge and the secreted amount in the
supernatant. As a proof of concept, the most common proinflammatory cytokines and chemokines displayed significant differences indicating the potential inflammatory limiting effect of
the tape.
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2.4. Cell Migration Study
Immune cells take part in inflammatory processes through
rapid, directed migration, which is called chemotaxis, toward
injury sites. The migration experiences 3D movement in the
circulatory system or body fluids and 2D migration within the
damaged tissues. Recruited immune cells will work together to
orchestrate the larger immune response. Given that, sweeping
the local inflammatory signals away from injury sites is the first
step to preventing persistent chemotaxis and direct regional
immunity to tissue repair transition. To test the cell migratory
effects of the tape in vitro, both 3D and 2D cell migration studies
were carried out with a conditioned medium obtained by treating
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Figure 4. Graphic illustration of the in vitro inflammatory cytokine and chemokine adsorption study by protein chip test. *p < 0.05, **p < 0.01,
***p < 0.001, ns: not significant, comparison between PG and PGH.
the inflammatory cell with different tapes. The 3D cell migration
results are given in Figure 5A. After 2 h of co-culture, the THP-1
(human monocytes) cells migrated from the upper chamber of
the transwell to the lower chamber, and the results were directly
detected under light microscopy since the monocytes are suspension cells and in the LPS group migrated cells are much more
than three other groups due to the high concentration of inflammatory cytokines and chemokines. In the PGH group, however,
the migrated cell number sharply decreased compared with the
LPS group (p < 0.001), and it was the reduced chemotactic effect
caused by the PGH tape’s efficient cytokine adsorption. Interestingly, the PG group also presented decreased cell migration
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efficiency than the LPS group (p < 0.01). The reason might be
the unspecific protein binding of GelMA and consequently
reduced inflammatory cytokine concentration. The same results
were observed for bone marrow-derived macrophages (BMDMs)
and human vaginal fibrblasts (HVFs) migration at different time
points. Both the adhesive cells migrated under the chemotaxis
of inflammatory cytokines and chemokines onto the lower surface of the upper chamber via deformation movement.[33] The
migrated cell number were further analyzed and were presented
in Figure 5C. The cell number was the highest in the LPS group,
while it was reduced in the PGH group but is still higher than in
the Dulbecco’s Modified Eagle Medium (DMEM) control group.
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Figure 5. The in vitro cell migration study. A) 3D cell migration results performed in transwell with monocytes, macrophages, and fibroblasts. Monocyte
migration was displayed under light microscopy, while the macrophage and fibroblast migration was visualized by crystal violence staining. B) 2D cell
migration results with macrophages and fibroblasts by wound scratch assay. C) The quantitative analysis results of the 3D and 2D cell migration studies.
*p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant, comparison between sham, and PLA, PLA and PG, PLA and PGH, PG and PGH.
The produced inflammatory cytokines and chemokines cannot
be absorbed completely, so the chemotaxis still exists. The 2D
cell migration study was conducted by wound scratch assay to
confirm further the chemotactic activity induced by the conditioned medium. As shown in Figure 5B, after 12 h, the migrated
BMDMs numbers were the highest in the LPS compared with
three other groups, but no difference was observed between PG
and PGH groups (p > 0.05). Similarly, the created wound scratch
closure rate was higher in the LPS group in the HVFs migration
study. However, the closure rate difference between LPS and PG
was less significant than migrated BMDM numbers (p > 0.05).
Both the 2D and 3D cell migratory results demonstrated the
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inhibitory efficiency of the PGH tape on the immune cells, further confirming the cellular mechanism for this tape is the regulation of regional immunity.
2.5. In Vivo Animal Study
After evaluating the capability to absorb inflammatory cytokines
and inhibit cell migration in vitro, we next assessed the inflammation self-limiting and tissue regeneration efficacy of the tape
in vivo using a deep soft tissue injury model. The animal model
establishment and time intervals are given in Figure 6A. The
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Figure 6. In vivo animal studies. A) Schematic illustration of the animal model establishment and the tape application process. B) Gross appearance
of the extracted samples, HE, Masson trichrome staining results for early inflammation self-limiting evaluation. C–E) Quantitative statistics of the in
vivo histological studies (red and yellow arrows indicate giant foreign body cells and the formed capsules). *p < 0.05, **p < 0.01, ***p < 0.001, ns: not
significant, comparison between sham, and PLA, PLA and PG, PLA and PGH, PG and PGH.
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ventral abdominal hernia repair model was adopted as a deep
soft tissue injury and performed on Sprague Dawley (SD) rats.
The rats showed no irritation, discomforted behavior, or altered
food and water intake behavior during the whole process. All
the materials applied to the hernia injury defect showed good
interactions with the abdominal muscle tissues. No signs of
abscess, hematoma, edema, or infection of the gross tissues
were observed at injury sites. Histological analysis was performed to detect the inflammatory reaction, tape integration,
and tissue repair. The adhesion score results in Figure 6C, it
can be concluded that there was no difference between sham
and PLA groups, while the difference was significant between
the PLA groups. Besides, the score in the PGH group was
much lower than that in the PG group (p < 0.05). These were
the macroscopic index for regional immune regulation of
the PGH tape in the early phase. The HE staining results in
Figure 6B showed foreign body giant cells (FBGs) infiltration
into the implant site on day 7 post-injury (dpi). Despite the
excellent tissue integration of all the four groups, some presented better-limited inflammation than others. For instance,
the FBCs in PG and PGH groups were much less than PLA
group (p < 0.01), though there was no significant difference
between the PG and PGH groups. On day 14, the FBGs in
the PGH group further decreased compared with three other
groups, and there was a significant difference between the
PG and PGH groups (p < 0.05). In the blank group, however,
there were few FBGs within 14 days (Figure 6D). To assess the
capsule formation caused by early inflammation, Masson’s trichrome staining study was performed on day 14 post-injury.
The results in Figure 6E illustrated that the PLA group developed a much thicker capsule around implants while the sham
and PGH groups showed reduced capsule thickness. The capsule in the PG group was thinner than that in PLA (p < 0.05)
but still thicker than in the PGH group (p < 0.05). These results
further confirmed the inflammation limiting effect of PGH in
vivo.
To test the in vivo inflammatory factor scavenging capability
of the tape in the deep soft tissue injury, in which situation the
continuously persisted cytokines and chemokines hinder the
tissue repair process, we tested the factors within wound tissues by protein chip assay. The in vivo inflammatory cytokine
and chemokine adsorption results in Figure 7 showed that the
typical proinflammatory cytokines and chemokines such as
IL-1b, TNF-α, MCP-1, MIP-1a, Rantes, and growth related oncogene (GRO) displayed significant statistical difference between
the two groups demonstrating the successful inflammatory
factor scavenging by the tape. In accordance with the in vitro
studies, some of the tested anti-inflammatory cytokines showed
no significant difference between two groups which may contribute to the later stage tissue regeneration and repair.
The initial inflammatory response was also analyzed by specific expression of CD11b and iNOS (Figure 8A). The images
of immunofluorescence staining showed that the PLA group
was heavily infiltrated with CD11b+ neutrophils (Figure 8A).
Quantitative analysis confirmed that the PGH group showed
a lower level than the PLA group (p < 0.01) after 7 days, but
the difference was not statistically significant compared with
the PG group (p > 0.05) (Figure 8C). The PLA membrane
similarly triggered more intense iNOS expression than the
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PGH group (p < 0.05) while presenting a comparable level
with the PG group (p > 0.05). Due to the PGH tape’s inflammatory cytokine adsorption ability, the accumulated inflammatory cells at the injury site were reduced, which was also
confirmed by immunohistochemical staining (Figure 8B).
Consistently, the expression of proinflammatory IL-1, IL-6,
and TNF-α was higher in the PLA and PG groups than in
the control group and PGH on day 14 (Figure 8E–G). The
expression difference between PG and PGH groups demonstrated the successful scavenging of inflammatory cytokines
and chemokines from injury sites. Regarding the IL-1 and
TNF-α, PLA and PG groups displayed a significant difference
(p < 0.01), probably because of PG’s unspecific and temporary absorption of some cytokines. The sham group showed
a relatively mild inflammatory reaction throughout the early
inflammatory process.
The inflammatory reaction at injury sites plays a crucial role
in deep soft tissue repair outcomes.[34] The tissue remodeling
was consecutively analyzed after achieving the early inflammation self-limiting via regional immune regulation. Evaluation of
the newly formed tissues 28 days post-injury revealed a faster
angiogenesis and ECM remodeling in the PGH group. α-SMA
immunofluorescence staining was visualized to quantify blood
vessel ingrowth in tapes and PLA membrane throughout integration (Figure 9A). The PGH group exhibited more intense
fluorescence staining and more newly formed blood vessel
numbers than PLA and sham groups (p < 0.01). PLA and sham
groups showed comparable blood vessel intensity but at a very
low level. The reason why the difference between PGH and PG
was not significant (p > 0.05) is that the GelMA component in
the PG tape has excellent cell adhesion and migration effects
on endothelial cells, which resulted in enhanced angiogenesis
(Figure 9C). The collagen I (COL I) expression levels were visualized to evaluate ECM remodeling (Figure 9B). The images of
COL I immunofluorescence staining demonstrated that after
28 days of implantation, the PGH tape induced much more
organized collagen fiber deposition than the three other groups.
The quantitative analysis in Figure 9D illustrated that sham,
PLA, and PG groups showed no difference regarding COL I
deposition (p > 0.05). The deep soft tissue injury environment
achieved granulation tissue maturation, vascularization, and
ECM remodeling as a secondary effect of early inflammatory
self-limiting. Although the early inflammation is not intense
in the sham group, wound healing is delayed because of the
deep anatomical structure, mechanical support deficiency and
lack of nutrition and oxygen. The PLA group, however, has sufficient mechanical strength, but the local inflammatory reaction was aggregated due to its hydrophobicity, which severely
impairs tissue repair. On the other hand, the PG group exhibited much better biocompatibility than the PLA one, thanks to
the GelMA. Still, the overexpressed inflammatory cytokines and
chemokines could not be scavenged and consequently failed
in early inflammation self-limiting. In this way, the minor
early inflammation self-limiting difference between the PG
and PGH groups further expanded in the later stage of tissue
regeneration.
Except for the cytokine scavenging capacity of the tape, the
ready-to-use nature of the tape is also an advantage. Storing in
the low temperature as a pre-tape and on-demand use in time
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Figure 7. Graphic illustration of the in vivo inflammatory cytokine and chemokine adsorption study by protein chip test. *p < 0.05, **p < 0.01, ***p <
0.001, ns: not significant, comparison between PG and PGH.
and space is a significant superiority of this tape for clinical
translation. Considering some critical settings, such as first
aid in a car accident or severe trauma, wound treatment on the
battlefield or defect filling after tumor resection, this cytokine
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scavenging tape is of particular value to rescue a limb, an
organ or even a life.[35] The reported design includes no drug
or bioactive ingredients that the efficiency should be considered when applied to actual wound circumstances. Despite the
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Figure 8. A) Immunofluorescence staining results for CD 11b+ neutrophils and iNOS+ macrophages. B) Immunohistochemical staining for IL-1, IL-6,
and TNF-α. C,D) Quantification for immunofluorescence intensity of CD 11b and iNOS. E–G) IHC score for IL-1, IL-6, and TNF-α. *p < 0.05, **p < 0.01,
***p < 0.001, ns: not significant, comparison between sham, and PLA, PLA and PG, PLA and PGH, PG and PGH.
safe components, convenience and potential clinical value, the
tape’s inflammation self-limiting and tissue repair efficiency
need to be evaluated in bigger animals that simulate human
body physiology with more specific injury models.
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3. Conclusion
In the present study, we successfully prepared an inflammation
self-limiting electrospun fibrous tape to reduce overexpressed
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Figure 9. In vivo tissue remodeling analysis. A) Immunofluorescence staining for α-SMA to evaluate angiogenesis at injury sites. B) Immunofluorescence staining for COL I to evaluate ECM remodeling at injury sites. C,D) Quantification of immunofluorescence intensity for α-SMA and COL I.
*p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant, comparison between sham, and PLA, PLA and PG, PLA and PGH, PG and PGH.
inflammatory cytokines and chemokines to achieve deep soft
tissue repair via regional immunity. The negatively charged
GelMA/HepMA hydrogel mixture is covalently modified onto
the electrospun fibrous membrane to form a pre-tape. After
photocrosslinking, the tape showed a strong mechanical
strength and increased surface wettability compared with PLA
fibrous membrane. The reported tape presented well the cell
adhesion and proliferation properties and successfully adsorbed
several positively charged cytokines in vitro. After applying to a
rat abdominal hernia model, the tape was more dominant in
local inflammation self-limiting during the early stages than
the PLA or PG groups, and it provided strengthened mechanical support for the remodeling of hernia defects compared
with the sham group. In the later stage, consistently, the tape
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promoted better angiogenesis and ECM remodeling within the
defect area thanks to earlier regional immunity regulation than
the three other groups. To sum up, we anticipate the electrospun fibrous tape will shed light on the valuable strategies for
deep soft tissue repair via regulating regional immunity.
4. Experimental Section
Chemicals and Reagents: PLA was purchased from Jinan Daigang
Biomaterial Co., Ltd; gelatin, heparin (MW, 14000), methacrylic
anhydride (MA), NaOH, anhydrous ethanol, hexafluoroisopropanol
(HFIP), 2-(N-morpholino)ethanesulfonic acid buffer (MES,), N-(3dimethylaminopropyl)-N-ethyl
carbodiimide
(EDC),
N-hydroxy
sulfosuccinimide (NHS), and Dulbecco’s PBS (DPBS) were purchased
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from Sigma-Aldrich Chemical Co. DMEM, fetal bovine serum (FBS),
streptomycin, penicillin, and other cell culture reagents were received
from Gibco, USA. Live/Dead reagent and phalloidin were obtained from
Invitrogen.
Synthesis and Tape Preparation: GelMA and HepMA were prepared
as in other studies reported by the authors’ research group.[36] Briefly,
for GelMA synthesis, 10 g gelatin was dissolved in 100 mL DPBS under
mechanical stirring at 50 °C. On being completely dissolved, 4 mL MA
was added drop by drop for the next 1 h. Then extra 100 mL DPBS was
added and stirred for half an hour, followed by dialysis for three days
under mechanical stirring at 37 °C. For HepMA synthesis, 1 g heparin
was completely dissolved in 50 mL deionized water (DI water) under
mechanical stirring at 4 °C, and 8 mL MA was added drop by drop. Next,
6 mL 5 m NaOH was added into the reaction system for termination and
stirred overnight at 4 °C. Then the system was washed with precooled
anhydrous ethanol right before being centrifuged for 15 min, followed by
dialysis for three days under mechanical stirring at room temperature.
After dialysis, the GelMA and HepMA were freeze-dried for four days
and stored at −20 °C for the subsequent studies.
To prepare electrospun fibrous tape, PLA electrospun fibrous
membrane was fabricated as previously described in another study.[37]
Briefly, 1 g PLA was dissolved in HFIP under mechanical stirring
overnight at room temperature, and the electrospinning process was
carried out under the following parameters: 15 kV high voltage, 0.8 mL h−1
pumping speed, 20 cm collection distance, and 400 rpm collector
rolling speed. The obtained fibrous membrane was stored at −20 °C
for the subsequent studies. For fibrous tape preparation, circular (for
in vitro cellular studies) or rectangular (for tape characterization or in
vivo animal studies), shaped membranes were treated with 0.05 m
NaOH for 30 s to conduct the esterification reaction and washed with
DI water three times. 120 mg EDC and 180 mg NHS were dissolved in
20 mL 0.05 m MES buffer was added onto the membrane for activation.
Washed with DI water three times, 0.5 mg mL−1 GelMA precursor was
added and allowed for a 1 h reaction to induce a double bond. 10%
GelMA (with a 0.2% lithium phenyl-2,4,6-trimethylbenzoylphosphinate,
LAP, a kind of photoinitiator) or the mixed GelMA/HepMA (with a 0.2%
LAP) precursor was sprayed onto the fibrous membrane and stored as
a pre-tape for in vivo studies and crosslinked by 365 nm blue light to
form the fibrous tape for physicochemical characterization as well as
for in vitro studies. PLA fibrous membrane modified with pure GelMA
was named PG, while a membrane with mixed GelMA and HepMA as
PGH. For the human in vivo applications studies, the Medical Ethics
Committee of International Peace Maternity and Child Health Hospital,
Shanghai Jiao Tong University School of Medicine approved the studies
according to the principles of (1) Guidance and Regulations on Ethical
Review of Drug and Clinical Trials promulgated and implemented by
the State Drug and Food Administration; (2) Declaration of Helsinki;
and (3) International Ethical Guide to Biomedical Research on Human
Body (project number: GKLW(2018-30)). The first author of this work
voluntarily provided the back of her hand to conduct the application
process with full willingness and a dedicative spirit.
Characterization of the Electrospun Fibrous Tape: Fibrous membranes
or tape samples were freeze-dried and sprayed with an AuPd layer
(≈1 nm thick) to improve the surface conductance. The surface images
were obtained by a scanning electron microscope (SEM, Hitachi,
SU5000).
Zeta Potential Measurement: The Zeta potential of different hydrogel
precursors was measured using a Malvern Zetasizer Nano ZS (Malvern
Instruments). Pure GelMA and a series of mixed hydrogel precursors
containing different concentrations of HepMA were prepared to
determine the most appropriate HepMA content for fibrous tape
preparation.
Rheology Analysis: Rheology analysis was conducted to test the
gelation process as the GelMA presents temperature sensitivity during
the temperature change. The gelation process was also analyzed under
365 nm UV light irritation except for the temperature. The testing
process was carried out with a Rheostress RS100 rheometer equipped
with parallel plates at a 10% strain, 1 Hz frequency, and a 0.5 mm gap
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for 350 s. The gel point was determined when the storage modulus (G′)
surpassed the loss modulus (G″).
Mechanical Stress–Strain Study: The PLA fibrous membrane and PGH
fibrous tape were cut into dumbbell-shaped specimens (4 cm × 1 cm).
Mechanical stress–strain tests were performed using a universal testing
machine (Heng Yi) at a 5 mm min−1 (n = 3). The compression modulus
was measured using the same universal testing machine for pure
hydrogel mechanical strength testing.
Fourier Transform Infrared Absorption Spectroscopy: The chemical bond
formation was tested in the different samples using Fourier transform
infrared absorption spectroscopy (FTIR, Nicolet 6700). The PLA fibrous
membrane and PGH fibrous tape were freeze-dried before being cut into
1 × 1 cm pieces before the test.
Water Contact Angle: The samples were cut into small strips (2 cm ×
1 cm) and placed on glass slides. The WCA was detected by a DSA100
(Germany) at room temperature (n = 3).
Degradation Test: Both PLA fibrous membrane and PGH fibrous tape
samples were cut into 1 cm × 1 cm pieces and weighed (W1). Then the
samples were immersed in a 4 mL PBS solution containing 0.01 mg mL−1
type I collagenase in a 37 °C shaking incubator for 60 days (n = 6). At
given time points, the samples were taken out and weighed again (W2).
Then the degradability was calculated by the following equation:
Weight loss ( % ) = ( W2 − W1) /W1× 100%
(1)
In Vitro Tissue Adhesion Assay: The tissue adhesion property of the
tape was determined in vitro using rat abdominal muscle. The pre-tape
was placed on the defect area of the muscle, and after photocrosslinking,
the tissue was soaked in PBS to test its stability in the body fluid. Then,
the tape-tissue system underwent stretching and twisting to detect its
adhesion stability under extreme mechanical pressure.
Cell Adhesion: HVFs were used to study their growth and adhesion
behavior on the electrospun fibrous tape. The cell extraction procedure
was described in the earlier study.[38] The circular tapes with a diameter
of 1 cm were placed in a 24-well plate and sterilized under the ultraviolet
light front and back for 12 h. HVFs of second to third passages were
cultured on the disinfected tapes with a 2 × 105 cells/mL concentration
and placed at 37 °C in a humidified incubator with 5% CO2. After 24 h,
samples were washed with PBS and fixed with 4% paraformaldehyde
(PFA, Thermo Fisher) for 30 min followed by treatment with 0.1% Triton
X-100 (Sigma-Aldrich) for 15 min. Then, phalloidin and DAPI were
sequentially used to label skeleton and nuclei. Confocal laser scanning
microscope analysis (CLSM, LSM800, ZEISS, Germany) was conducted,
and the obtained 2D and 3D cell images were further analyzed by
Image J software. Cell aspect ratio (CAR) was calculated as the ratio of
the major axis to the minor axis from 10 single cells for each sample.
The cell migration distance was obtained by measuring the distance
of the nearest tape surface and the lowest cell nuclei. Nuclei less than
10 µm from the surface were considered on the tape surface and not
migrated. Image J software was used for the calculation.
Live/Dead Assay: To test long-term cell activity, live/dead assay was
conducted after three days of co-culturing of HVFs on the disinfected
tapes. Cells were washed with sterile DPBS three times right before
incubation in a 37 °C, 5% CO2 humidified incubator with a live/
dead kit treatment. Then the samples were observed under confocal
laser scanning microscope analysis (CLSM), and images were further
analyzed by Image J software.
In Vitro Inflammatory Adsorption Study: The inflammatory
factor adsorption study was carried out by activating BMDM with
lipopolysaccharide (LPS, Sigma-Aldrich). First of all, mouse BMDM was
obtained from the bone marrow cells under the previously described
procedures.[39]
The femurs and tibias of naive mice were isolated, and the
surrounding tissues were cleaned. After washing with sterile PBS three
times, both ends of the bones were cut off, and the cavity was flushed
with sterile PBS until it became whitish. The red blood cells in the bone
marrow cells were removed with red blood cell lysis buffer and incubated
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in RPMI-1640 medium (Gibco, USA) supplemented with 10 ng mL−1
macrophage colony-stimulating factor (M-CSF, Sigma Aldrich) for three
days. Unattached cells were discarded on day 4 by medium change,
and adherent BMDMs were harvested for the cellular experiments.
1 × 105 BMDMs were cultured in 6-well plates, and the next day 2 mL
DMEM supplemented with 100 ng mL−1 LPS were changed to activate
BMDMs for 4 h. The PG or PGH tapes were placed in the 6-well
Transwell (Corning, USA) upper chamber and placed onto the activated
BMDMs. After 24 h, the supernatant of each group was collected, and
the inflammatory cytokines and chemokines were detected by a Luminex
protein biochip testing system (Bio-Plex MAGPIX System, Bio-Rad)
with a test kit (Bio-Plex Pro Mouse Cytokine Grpl Panel 23-plex, Wayen
Biotechnologies, Shanghai) according to the manufacturer’s instructions.
Briefly, the supernatants were incubated in 96-well plates embedded with
microbeads for 1 h followed by incubation with detection antibody for
30 min. Subsequently, streptavidin-PE was added to each well for 10 min
and values were read using the Bio-Plex MAGPIX System (Bio-Rad).
Transwell Cell Migration Assay: Before conducting the cell migration
study, various conditioned medium was first prepared as previously
described.[10] Briefly, BMDMs were incubated overnight in DMEM
containing 100 ng mL−1 LPS, and the obtained conditioned medium was
just named LPS. On the other hand, the PG or PGH were accordingly the
PG, or PGH tape treated medium for the same period, while pure DMEM
was used as blank control. THP-1 monocytes were purchased from
Procell, Wuhan, China. Transwell cell migration assay was performed
with THP-1, BMDMs and HVFs. 2 × 105 THP-1 were suspended in 200 µL
DMEM in the upper chamber of the 24-well transwell plate, while the
lower chamber was filled with a 700 µL different conditioned medium
and DMEM. After 2 h of transmigration, the upper chamber was
removed, the lower chamber was observed under a light microscope
(NIKON, Japan), and the migrated cells were counted by Image J
software. For BMDMs and HVFs migration assay, 1 × 104 cells were
suspended in 200 µL DMEM in the upper chamber while 700 µL different
conditioned medium and DMEM in the lower chamber. After incubation
of 12 and 24 h, respectively, the unmigrated cells on the upper surface of
each chamber membrane were cleaned with a cotton swab, followed by
fixation with PFA. Next, cells that migrated onto the bottom surface were
stained with Crystal Violet Stain (Servicebio, Wuhan, China) and washed
with DI water before being observed under a light microscope.[40] The
migrated cells were counted by Image J software.
Wound Scratch Assay: Both BMDMs and HVFs were cultured in
the 6-well plates, and the wound scratch was processed as previously
described.[41] Specifically, after 90% cell confluence was reached,
a 200 µL pipette was used to make a straight line, and the detached
cells were removed by washing with PBS three times. Then, cells were
refreshed with different conditioned medium and DMEM followed by
incubation for given periods.
In Vivo Animal Studies: Forty-eight male Sprague Dawley rats (n = 6)
with an average weight of 250–300 g were purchased from Beijing Vital
River Laboratory Animal Technologies Co. Ltd. The surgical procedures
were carried out in the Shanghai Branch of Beijing Vital River Laboratory
Animal Technologies Co. Ltd. Animals were hosted in specific pathogenfree facilities and housed under appropriate room temperature, normal
diet, and light/dark cycle.
Abdominal Wall Defect Model Establishment: All the animal study
protocols were conducted under strict instructions of the National
Institutes of Health’s Guide for the Care and Use of Laboratory Animals,
and the surgical procedures were approved by the Animal Ethics
Committee of the Beijing Vital River Laboratory Animal Technologies
Co. Ltd. (P2021095). The preoperative preparations were carried out
as described in another study.[42] Shortly, anesthesia was conducted
by intraperitoneal injection of 1% pentobarbital solution with a dose
of 0.1 mg g−1, and the abdominal furs of the rats were shaved and
disinfected with iodine. A vertical incision along the white line of the
abdomen incision was created, and the subcutaneous tissues were
carefully dissected. After exposing sufficient operative field, 4 cm × 1 cm
(head-to-tail) partial-thickness rectangular defects were established on
one side of the white line by removing the abdominal muscles (partial
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internal oblique muscles, external oblique muscles, and transversus
abdominis), just leaving the transversalis fascia and peritoneum. Then,
PLA fibrous membrane, PG, and PGH tapes were implanted while a
sham group was created without any treatment. After the surgery, all
animals were put back in specific pathogen-free facilities and given close
postoperative observation.
Sample Harvesting and Histological Analysis: The rats were sacrificed at
given time points, and samples were harvested for specific experimental
purposes. Signs of inflammation, hematoma, edema, or adhesion of
the gross tissues were recorded. The early inflammatory response was
analyzed by HE and Masson’s trichrome staining. The infiltrated FBGs
were counted on day 7 and day 14, while the capsule thickness on day 14
was calculated. Besides, immunofluorescence staining was conducted to
detect infiltrated neutrophils and macrophages on day 7 with anti-CD11b
(Abcam, ab1211, 1–5 µg mL−1) and anti-iNOS (Abcam, ab178945, 1:500)
antibodies, respectively. In vivo inflammatory cytokine and chemokine
adsorption assay was conducted by above mentioned Luminex protein
chip testing system (Bio-Plex MAGPIX System, Bio-Rad) with a test kit
(Bio-Plex Pro Rat Cytokine Grpl Panel 23-plex, Wayen Biotechnologies,
Shanghai) according to the manufacturer’s instructions after 7 days
post injury. Briefly, tissue samples obtained from wound sites around
the implants were lysed and centrifugated at 10 000 rpm for 15 min.
Protein concentrations were measured by a microplate reader (Bio-Rad),
and protein sample was diluted to an equal volume of 50 µL. Afterward,
protein samples were incubated in a 96-well plate embedded with
microbeads for 1 h and incubated with detection antibodies for 30 min.
Finally, streptavidin-PE was added into each well to be incubated for
10 min, and values were measured by the Bio-Plex MAGPIX System
(Bio-Rad).
Then, samples were incubated in 96-well plates embedded with
100 mg tissue sample Immunohistochemical staining was further
conducted to test the inflammatory cytokines at injury sites with IL-1
(Servicebio, Wuhan, P01584, 1:1000), IL-6 (Servicebio, Wuhan, P20607,
1:500), and TNF-α (Servicebio, Wuhan, P01375, 1:500). The samples
extracted on day 28 underwent immunofluorescence staining for
regenerative analysis to detect angiogenesis and ECM remodeling by
being incubated with anti-α-SMA (Abcam, ab124964, 1:500), anti-col
I (Abcam, ab260043, 1:30). The immunofluorescence density was
calculated by Image software.
Statistical Analysis: All the experiments were repeated three times, and
the results were displayed as the mean ± standard deviation (SD). p <
0.05 was considered to indicate statistically significant differences. The
data were analyzed by unpaired t-tests or one-way ANOVA followed by
Tukey’s post hoc test. GraphPad Prism 8.3.0 was used to process data.
Acknowledgements
Q.S. and Z.C. contributed equally to this work. This work was supported
by National Key Research and Development Program of China
(2020YFA0908200), National Natural Science Foundation of China
(51873107 and 32101104), Shanghai Municipal Education Commission—
Gaofeng Clinical Medicine Grant Support (20171906), Science and
Technology Commission of Shanghai Municipality (19440760400),
Shanghai Municipal Health Planning Commission (202140127), and
GuangCi Professorship Program of Ruijin Hospital Shanghai Jiao Tong
University School of Medicine.
Conflict of Interest
The authors declare no conflict of interest.
Data Availability Statement
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
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Keywords
electrospinning, inflammation self-limiting, regional immunity, soft
tissue, tape
Received: June 26, 2022
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