Article

www.acsnano.org

Sequential Release of Small Extracellular

Vesicles from Bilayered Thiolated Alginate/
Polyethylene Glycol Diacrylate Hydrogels for
Scarless Wound Healing
Yifan Shen,# Guanzhe Xu,# Huanxuan Huang,# Kaiyang Wang, Hui Wang, Meidong Lang,* Hong Gao,*
and Shichang Zhao*
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ABSTRACT: Excessive scar formation has adverse physiological and

psychological effects on patients; therefore, a therapeutic strategy for rapid
wound healing and reduced scar formation is urgently needed. Herein,
bilayered thiolated alginate/PEG diacrylate (BSSPD) hydrogels were fabricated
for sequential release of small extracellular vesicles (sEVs), which acted in
different wound healing phases, to achieve rapid and scarless wound healing.
The sEVs secreted by bone marrow derived mesenchymal stem cells (B-sEVs)
were released from the lower layer of the hydrogels to promote angiogenesis
and collagen deposition by accelerating fibroblast and endothelial cell
proliferation and migration during the early inflammation and proliferation
phases, while sEVs secreted by miR-29b-3p-enriched bone marrow derived
mesenchymal stem cells were released from the upper layer of the hydrogels
and suppressed excessive capillary proliferation and collagen deposition during
the late proliferation and maturation phases. In a full-thickness skin defect
model of rats and rabbit ears, the wound repair rate, angiogenesis, and collagen deposition were evaluated at different time
points after treatment with BSSPD loaded with B-sEVs. Interestingly, during the end of the maturation phase in the in vivo
model, tissues in the groups treated with BSSPD loaded with sEVs for sequential release (SR-sEVs@BSSPD) exhibited a more
uniform vascular structure distribution, more regular collagen arrangement, and lower volume of hyperplastic scar tissue than
tissues in the other groups. Hence, SR-sEVs@BSSPD based on skin repair phases was successfully designed and has
considerable potential as a cell-free therapy for scarless wound healing.
KEYWORDS: thiolated alginate/polyethylene glycol diacrylate hydrogels, sequential release, small extracellular vesicles, scarless,
wound healing

scar (HS) formation,9 Complications of HS, including itching,

W ound healing is a complex and orderly physiological

process.1−3 When the integrity of the skin is
damaged, the body relies on an intricate interplay
among numerous biological pathways and various cell types to
compression, functional limitations, and anatomical deform-
ities, continue to irritate patients after the initial damage.10,11
Surgical resection is the most common therapeutic approach
restore tissue integrity and function.4,5 The process of skin for scarring; however, patients must go through the healing
wound healing is often accompanied by scar formation in process again, and the clinical therapeutic effect is often
adults.6 In the initial phase of healing, highly active fibroblasts unsatisfactory.10,12 Therefore, a therapeutic strategy allowing
promote wound closure and healing,7 but in the late phase of
wound healing, excessive deposition of the collagen and Received: September 13, 2020
extracellular matrix (ECM) caused by highly active fibroblasts Accepted: March 8, 2021
leads to inappropriate scar formation,8 which affects the Published: March 16, 2021
functional recovery of the original tissue. In particular,
excessive collagen and ECM deposition during wound healing
in severely burned patients results in keloid and hypertrophic

© 2021 American Chemical Society https://dx.doi.org/10.1021/acsnano.0c07714

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rapid wound healing without scar formation is urgently
needed; such a strategy would benefit millions of patients
and has tremendous medical value.
Recently, in tissue regeneration, considerable attention has
been devoted to small extracellular vesicles (sEVs),13 which
have diameters between 30 and 200 nm, act as vehicles of
bioactive proteins, DNAs, mRNAs, and noncoding RNAs, and
are increasingly considered important mediators of intercellular
communication,14 potential therapeutic bioactive factors, and
useful as continuation of stem cell therapy.15 Importantly,
scholars found that sEVs can be engineered or modified in a
modular fashion to improve therapeutic effects and overcome
disadvantages.2,16−19 Liu et al. found that engineered human
adipose stem cell sEVs delivered miR-21-5p to promote the
healing of diabetes-associated skin wounds.20 Ding et al.
suggested that sEVs derived from deferoxamine precondi-
tioned stem cells could promote wound healing by increasing
proangiogenic ability.21 MicroRNAs (miRNAs) are a class of
small noncoding RNAs that play pivotal roles in multiple
biological events, including cell proliferation, differentiation,
and apoptosis. MiR-29b regulates mitochondrial dysfunction
and apoptosis in HEI-OC1 cells through the SIRT1/PGC-1α
signaling pathway.22 MiR-29b-3p contained in sEVs released
by human bone-marrow-derived mesenchymal stem cells
(hBMSCs) is a potential target for regulating aging-related
insulin resistance.23 In addition, previous studies demonstrated
that miR-29b suppressed collagen deposition and fibrotic gene
expression in scar tissues24,25 and might be a therapeutic target
for scarred wound healing.26−29 Therefore, miR-29b-3p-
enriched bone marrow derived mesenchymal stem cells (B-
miR-29b-sEVs) might be a potential therapeutic intervention
to inhibit HSs.
Simple biological methods cannot easily simultaneously
promote wound healing and inhibit scar formation.30,31 The
wound healing process typically comprises the coagulation,
inflammation, proliferation, and maturation phases.32 The
wound healing response is dynamic, and the expression and
production of growth factors and cytokines are strictly
spatiotemporally regulated to ensure that the correct wound
healing events occur in the correct order.33,34 In the
coagulation phase, the platelets release vasoactive substances
such as 5-hydroxytryptamine (5-HT) and prostaglandins
(PGs), which further constrict the blood vessels and slow
blood flow. Simultaneously, the release of phospholipids and
adenosine diphosphate induces additional platelet aggrega-
tion.35 Vasoconstriction leads to local tissue ischemia, resulting
in the release of histamine and other vasoactive substances,
further leading to local vasodilation on the wound surface in
the inflammatory phase.36 During the early proliferation phase,
vascular endothelial growth factor (VEGF) promotes tissue
repair by expanding endothelial cells and increasing vascular
permeability.37 Basic fibroblast growth factor (bFGF) and
platelet derived growth factor (PDGF) mainly regulate ECM
component synthesis and deposition, but they can also
enhance wound angiogenesis.38 PDGF stimulates macrophages
and the secretion of other growth factors important in the
healing process as well as some matrix molecules such as
fibronectin, collagen and proteoglycan.39 Dysregulation of the
wound repair process leads to excessive collagen and ECM
deposition; thus, normal tissue is replaced by permanent scar
tissue. During the late proliferation and maturation phases,
various cytokines, such as transforming growth factor β1
(TGFβ1), VEGF, bFGF and PDGF, cause excessive fibrosis
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and vascularization, which can instead result in HS
formation.40 Therefore, sequential release of different bioactive
factors to fulfill the requirements of different wound healing
phases may be a strategy to promote scarless wound healing.
In the present study, we combined material technology and
biological methods to achieve optimal therapeutic outcomes.
Sodium alginate (SA), a natural marine polysaccharide, has
been widely applied in tissue engineering due to its excellent
biocompatibility.41−44 Our previous study indicated that
thiolated alginate in situ cross-linked (SA-SS-SA) hydrogels
are potential hemostatic biomaterials.45 However, as a
hemostatic agent, SA-SS-SA hydrogels degrade in liquid within
48 h. By adjusting the grafting rate and optimizing the PEG
ratio, hydrogels were fabricated to meet wound dressing
requirements. Different thiolated alginate/PEG diacrylate (SA-
SH/PEG-DA) ratios endowed the lower and upper layers of
the hydrogel with different mechanical strengths and
degradation rates, leading to spatial and temporal differences
in bioactive factor release behavior. Therefore, bilayered SA-
SH/PEG-DA hydrogels with different degradation rates for the
sequential release of sEVs were fabricated as wound dressings.
Based on the above rationale, an advanced strategy to
fabricate bilayered thiolated alginate/PEG diacrylate hydrogels
loaded with sEVs for sequential release was developed in this
study, aiming to overcome the problem of scar formation in
wound healing. Bilayered thiolated alginate/PEG diacrylate
(BSSPD) loaded with sEVs for sequential release (SRsEVs@
BSSPD) hydrogels were designed based on different phases of
wound healing; these hydrogels not only promoted wound
healing but also inhibited HS, as validated by in vitro and in
vivo studies.
RESULTS AND DISCUSSION
The skin is not only a vital organ for protecting internal
environmental stability, defending against invasion from
outside the body, evacuating moisture, and regulating body
temperature but also an immune system component.46,47
When damage to the skin barrier occurs and invasion leads to a
wound, a complex healing cascade is activated, which can
quickly restore the integrity and barrier function of skin
tissue.47 As noted above, the dynamic wound healing process
encompasses multiple overlapping events and involves a
complex array of cellular and biochemical reactions,48
including inflammatory cell recruitment, matrix deposition,
epithelialization, and scar formation. Scars consist of fibrous
hyperplastic tissue with immature collagen. In this study, we
combined material technology with sEVs therapy to rapidly
promote wound healing and inhibit HS formation. sEVs
therapy was used as the basis for modulating biological
functions, while the BSSPD hydrogel was applied to allow
sequential release of sEVs to meet the requirements during
different repair phases.
Identification of sEVs. Recently, therapeutic sEVs have
been considered a continuation of stem cell therapy, which
might be a valuable direction for scarless wound healing.49 EVs
comprise four main types: exosomes, microvesicles, large
oncosomes, and apoptotic bodies.50,51 Exosomes secreted by
stem cells can promote angiogenesis and bone regenera-
tion.52,53 Compared with traditional stem cell therapy, sEVs
have the advantages of high stability, a lack of tumorigenicity,
easy storage, and quantitative use.14 A recent study confirmed
that MSC-derived exosomes can inhibit the acute inflamma-
tory response in the wound microenvironment, accelerate
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Figure 1. (A) Representative TEM image of B-sEVs. (B) Presence of sEVs surface marker proteins (CD9, Alix, and Tsg101). (C) Size
distribution of sEVs. (D) Image of hBMSCs. (E) Uptake of sEVs by EA.hy926 cells and HFF-1 cells.
angiogenesis, promote epithelial cell proliferation and migra-
tion, and regulate collagen regeneration and deposition.54
After isolation of sEVs from the cell culture supernatant, the
sEVs secreted by BMSCs (B-sEVs) were characterized in terms
of morphology, size, and surface marker expression. Trans-
mission electron microscopy (TEM) observations revealed
that the B-sEVs had a round spherical or cup-shaped
morphology. Western blot analysis showed that B-sEVs were
positive for expression of the exosomal marker proteins Alix,
TSG101, and CD9 (Figure 1B). Nanoparticle tracking analysis
(NTA) showed that the sEVs were 30−100 nm in diameter
(Figure 1A, C). These results indicated that the collected B-
sEVs possessed typical morphological and molecular features.
Successful delivery into key cells involved in wound healing,
such as endothelial cells and fibroblasts,55 is a prerequisite to a
mechanistic understanding of sEV therapy.56 A membrane-
labeling technique was used in which the PKH-26 red
fluorescent exosome dye binds a green fluorescent dye
(PKH-67) with a longer lipid tail on the lipid bilayer of the
sEV membrane to produce a yellow-orange fluorescence.
Confocal micrographs showed obvious evidence of the cellular
attachment and internalization of B-sEVs by EA.hy926 cells
and HFF-1 cells, and PKH-26-sEVs (red) were observed to
surround the nucleus (blue). The cytoskeleton is stained green
in Figure 1E.
Mechanism of miR-29b-3p and B-miR-29b-sEVs in
Scarless Wound Healing In Vitro. miRNAs play a critical
role by binding to the 3′UTR of target genes or suppressing
their transcription to reduce their expression level.57−59 In
previous studies, miR-29b was considered a key molecule in
inhibiting tumor angiogenesis.60 MiR-29b expression is
significantly reduced in several tumors and is closely related
to prognosis.61 Coincidentally, the level of miR-29b is also
closely related to the occurrence of scarring.62 MiR-29b-3p was
predicted by three major miRNA target databases that were
used to identify possible targets. Based on the intersection of
the miRNAs predicted as targets by the three databases, we
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narrowed our investigation and selected 52 highly possible
targets (Figure 2A). Based on the intersection of 52 target
genes and the antifibrogenic gene sets (Figure 2B), 6 putative
genes were selected from the various candidates. Among these
6 putative genes, Col1A1 was identified as a target gene of
miR-29b-3p based on bioinformatic algorithms. Bioinformatic
algorithms showed that miR-29b-3p can bind to the 3′UTR of
Col1A1 (Figure 2C). The luciferase reporter assay indicated
that miR-29b-3p mimics had an inhibitory effect on Col1A1-
3′UTR expression levels but not on Col1A1-3′UTR mutant
(Mut) expression levels. These results confirmed that Col1A1
is a direct target gene of miR-29b-3p and can be inhibited by
miR-29b-3p.
Effective miRNA delivery to target cells is a major problem
that restricts miRNA-based clinical applications.63 As nano-
carriers, sEVs protect key molecules, particularly RNA-like
molecules, from being degraded by enzymes in plasma and
triggering an immune response; they also help key molecules
to penetrate various biofilms.64 Previous studies demonstrated
that the use of sEVs as therapeutic carriers had high application
potential and great value for the treatment of various
diseases.65,66 Researchers have developed a variety of physical
and chemical methods to package exogenous interfering RNAs
and chemical drugs into sEVs.67,68 In our study, miR-29b-3p
was overexpressed by lentivirus-mediated transfection to
generate engineered sEVs (Figure 2D). The miR-29b-3p
lentiviral vector was obtained from Oligobio (Beijing, China).
Lentiviral transfection was performed according to the
manufacturer’s protocol. In brief, hBMSCs were incubated in
lentivirus-containing supernatant with 5 mg/mL of polybrene
for 24 h. After infection for 48 h, hBMSCs were selected with
puromycin dihydrochloride (Thermo Fisher). In general, when
the miRNA target level in producer cells changed, the
corresponding miRNA level in sEVs showed the same change
trend. MiRNA sorting to exosomes is modulated by cell
activation-dependent changes in miRNA expression levels in
the producer cells.69−71 Therefore, miRNA-29b-3p levels in
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Figure 2. (A) miR-29b-3p target prediction via the miRWalk, miRDB, and TargetScan databases. (B) Searching for miR-29b-3p target genes
via the intersection of possible miR-29b-3p targets and a collagen deposition gene set. (C) Binding sequences of Col1A1 and miR-29b-3p.
(D) Luciferase reporter activity of hsa-miR-29b-3p and Col1A1. (E) Lentiviral transduction efficiency. (F) Relative miRNA expression levels
compared with those in unmodified hBMSCs. (G, H) Akt, Smad3, Erk1/2, and TGF β1 phosphorylation levels in EA.hy926 and HFF-1 cells.

sEVs extracted from miR-29b-3p-enriched hBMSCs were also
high. Quantitative real-time polymerase chain reaction (qRT-
PCR) analysis confirmed significant upregulation of miR-29b-
3p in miR-29b-3p-enriched hBMSCs and B-miR-29b-sEVs
(Figure 2F).
One classical marker of HS is disproportionate fibrosis.55
Excessive ECM production, deposition and contraction
eventually lead to morphological changes and dysfunction at
the organ level.72,73 Excessive VEGF production and aberrant
vascularization promote scar tissue formation through various
mechanisms, such as compromised homeostasis and impaired
degradation and excessive accumulation of ECM.40 Scarless
healing and significantly reduced angiogenic responses are
common features of oral and fetal wounds.74 The protein levels
of p-type AKT and p-type Erk1/2 in EA.hy926 cells were
increased by treatment with B-sEVs. Similarly, the protein
levels of TGF-β1 and p-type smad3 in HFF-1 cells treated with
B-sEVs were increased (Figure 2G). By contrast, treatment
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with B-miR-29b-sEVs downregulated the expression of those
proteins (Figure 2H). Regarding the mechanism of B-sEVs,
vascularization and fibrosis were promoted via activation of the
PI3K/Akt, Erk1/2, and Smad3/TGFβ1 signaling pathways. In
contrast, inhibition of these signaling pathways was observed in
the B-miR-29b-sEVs group. The PI3K/Akt, Erk1/2, and
Smad3/TGFβ1 signaling pathways are the most important
signaling pathways in the progression of vascularization and
fibrosis. A recent study demonstrated the suppressive effect of
miR-29b on the essential activity of Smad3 in TGF-β1-
mediated fibrosis.62 TGF-β1/Smad3 expression varies in
distinct stages of human growth; the TGF-β1/Smad3 gene is
not expressed in the fetus, possibly explaining one mechanism
of scarless wound healing in fetal skin.75 Future treatments
may be developed by investigating the molecular basis of
wound healing, such as blocking “pro-scarring” molecules such
as TGF-β1 and promoting the expression of TGF-β3, which
aims to create an environment similar to that of the scarless
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Figure 3. Proliferation of EA.hy926 cells (A) and HFF-1 cells (B) incubated under different conditions. (C) Effects of the control, B-sEVs, B-
miR-29b-sEVs and B-NC-sEVs treatments on EA.hy926 and HFF-1 cell migration and EA.hy926 cell tube formation. Quantitative evaluation
of EA.hy926 (D) and HFF-1 (E) cell migration. (F) Quantitative evaluation of nodes. VEGF secretion from EA.hy926 cells (G) and Col I
secretion from HFF-1 cells (H). Immunofluorescence staining images of EA.hy926 (I) and HFF-1 cells (J).

healing process.75 These results imply that B-miR-29b-sEVs
might be a basis for the treatment of HS.
To further verify the hydrolytic action of B-sEVs and B-miR-
29b-sEVs, additional in vitro evidence was provided. Pro-
liferation (Figure 3A,B), migration, tube formation, and
VEGF/COL I expression and secretion were promoted in
the B-sEVs and B-NC-sEVs groups of EA.hy926 and HFF-1
cells. In contrast, the related functions were inhibited in the B-
miR-29b-sEVs group of these two cell lines. On day 7, the
number of proliferating cells in the B-miR-29b-sEVs group was
significantly lower than that in the other groups. Similarly, both
the number of cells passing through the Transwell insert
membrane and the number of blood vessels on the Matrigel
were lower in the B-miR-29b-sEVs group than in the other
groups. (Figure 3C−F). The concentrations of VEGF and Col
I in the cells were determined by enzyme-linked immuno-
sorbent assay (ELISA) (Figure 3G,H). After culture for 3 days,
the supernatant of EA.hy926 cells in the B-sEVs and B-NC-
sEVs groups had higher VEGF and COL I concentrations than
that from the corresponding groups of HFF-1 cells. Treatment
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with B-miR-29b-sEVs inhibited VEGF and COL I expression.
Immunofluorescence (IF) assays confirmed the ELISA results
(Figure 3I,J). The standard curve for ELISA is shown in Figure
S1A,B. Studies have shown that miR-29b mimics can
significantly inhibit the excessive proliferation of blood vessels
and collagen deposition in endothelial cells and fibroblasts.62
The inhibitory effect of B-miR-29b-sEVs on EA.hy926 and
HFF-1 cells further demonstrated the feasibility of treatment
with engineered sEVs.
Characterization of Hydrogels. Figure 4A shows a
general view of the BSSPD hydrogels. Raman spectroscopy
of SA-SH/PEG-DA (Figure 4B) showed no absorption peak at
approximately 524 cm−1, indicating that no disulfide bonds
existed in the SA-SH/PEG-DA hydrogels. Figure 4C and
Figure S2A show the FTIR spectra of SA-SH/PEG-DA. Peaks
at wavenumbers of 1722 and 1105 cm−1 belonged to the
characteristic peaks of carbonyls (PEG-DA) and ethers (PEG-
DA), respectively. Combined, the Raman spectra and FTIR
spectra results proved that SA-SH and PEG-DA were cross-
linked by thiol−ene click chemical bonds rather than disulfide
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Figure 4. (A) A photograph of SA-SH/PEG-DA hydrogel biolayers. (B) Raman spectra of SA-SH/PEG-DA hydrogels. (C) FTIR spectra of
SA-SH/PEG-DA hydrogels. (D) Swelling rate of SA-SH/PEG-DA hydrogels. (E) Scanning electron micrographs of lyophilized SA-SH/PEG-
DA hydrogels. (F) Fluorescent sEV tracer experiment: Blue indicates the nucleus, red indicates B-sEVs, and green indicates B-miR-29b-
sEVs. (G) Statistical analysis of the fluorescent sEV tracer experiment. (H) Curves showing the release of B-sEVs and B-miR-29b-sEVs in
PBS. (I) Statistical analysis of hydrogel degradation in PBS and cell culture medium. (J) Images of hydrogel degradation in PBS and cell
(EA.hy926) culture medium. (K) Reaction equation and diagram of the release assay.

bonds. The SA-SH-3/PEG-DA-700 hydrogels had the same
cross-linking bonds and similar chemical structures as the SA-
SH-3/PEG-DA-250 hydrogels, and the difference was the
molecular weights of the PEG-DAs. However, with the
increase of molecular weight of PEG-DA, the hydrophilicity
of its molecular chain gradually decreased which resulted in the
different hydrophilicity of the two SA-SH/PEG-DA hydrogel
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networks. The swelling rate of the SA-SH-3/PEG-DA-700
hydrogel with more hydrophobic network was lower than that
of SA-SH-3/PEG-DA-250 (Figure 4D and Figure S2B), and
the difference in swelling rate between them was more than
50%. Hydrogel group cross-linked by PEG-DA-700 had a
flatter micromorphology than PEG-DA-250 group (Figure 4E
and Figure S2C). PKH-76 is a green fluorescent dye that can
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Figure 5. (A) Changes in the wound closure areas. (B) Simulation plots. (C) Quantitative evaluation of the wound closure rate. (D, E)
Images and quantitative analysis of H&E staining.
be used to label living cells. In this study, lipid molecules with
sEV membrane structures were labeled with green fluores-
cence. The upper and lower layers of the hydrogel were loaded
separately with 1 × 1010 B-sEVs and B-miR-29b-sEVs. Two
different labeled sEVs were loaded into the hydrogels: B-miR-
29b-sEVs were stained with PKH-76 (green), and B-sEVs were
stained with PKH-76 (red). The fluorescent images (Figure
4F) and statistical analysis (Figure 4G) showed that the
number of B-sEVs increased slowly with increasing immersion
time. After 7 days, B-miR-29b-sEVs began to accumulate
gradually. Both sEVs surrounded the nucleus (blue). The
amount of sEVs released was detected by an ELISA kit.
According to the curve in the figure, 80% of the sEVs of the
SA-SH-3/PEG-DA-250 hydrogel were released at day 7, and
the release became slower after that. The SA-SH-3/PEG-DA-
700 hydrogel released 80% of sEVs at day 9, and the curve
decreased after day 9. The sEVs release from the hydrogel into
the PBS was a physical diffusion process, so the sEVs release
rate of the SA-SH-3/PEG-DA-700 hydrogel with a lower
swelling rate was lower than that of the SA-SH-3/PEG-DA-250
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hydrogel (Figure 4H). To detect BSSPD degradation, BSSPD
was placed in a Transwell chamber and transferred to a well
plate for detection in PBS and medium cell culture. Figure 4I,J
shows a macroscopic illustration of the aqueous gel and the
data analysis. The upper layer is dyed blue, and the lower layer
is dyed red. In PBS, the hydrogels were degraded slowly, and
the hydrogels in the lower layer of culture medium were
degraded first and completely at day 7. The upper layer was
degraded slowly at the beginning, accelerated after day 7, and
degraded to 10% at day 14. In addition, ROS produced during
cell culture76 could cause the disruption of glycosidic bonds on
the main chain of alginate,77,78 which accelerated the
degradation of SA-SH/PEG-DA hydrogels. In general, SA-
SH-3/PEG-DA-700 hydrogels had high storage modulus
(Figure S2D), low swelling rate, and low sEVs release rate,
while SA-SH-3/PEG-DA-250 hydrogels had low storage
modulus, high swelling rate and high sEVs release rate.
Therefore, hydrogels with different sEV release rates could play
a graded release role in wound healing.
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Figure 6. (A) Reconstructed 3D images of new blood vessels and quantitative analysis of vessel number (B) and vessel area (C). (D) IHC
(CD31) and IF (CD31 and α-SMA) staining images. (E, F) Morphometric analysis of new and mature blood vessels.
In addition, the morphology of cells in these hydrogels was
not significantly altered (Figure S3A). The number of cells in
the SA-SH-3/PEG-DA-250 group and SA-SH-3/PEG-DA-700
group on days 1, 3, and 7 was not significantly different from
that in the control group (Figure S3B). SA-SH/PEG-DA
hydrogels had excellent biocompatibility and were suitable for
cell growth. Most importantly, SA-SH/PEG-DA hydrogels
were compatible with skin healing and could deliver different
sEVs to achieve sequential sEV release, indicating their
potential as dressings for scarless wound healing.
Wound Healing In Vivo. Figure 5A shows images acquired
with a digital camera, simulation diagrams (Figure 5B), and
quantitative data analysis results (Figure 5C) of the wounds in
each group at four time points. Results of the H&E staining are
shown in Figure 5D that the B-sEVs@BSSPD and SR-sEVs@
BSSPD groups exhibited significantly faster wound healing
than the others. Consistent with the quantitative analysis of the
wound healing results on days 7 and 14, the B-sEVs@BSSPD
group and SR-sEVs@BSSPD group showed significantly more
favorable results than the other groups, although the difference
between the B-sEVs@BSSPD group and SR-sEVs@BSSPD
group was not significant (Figure 5E). Regarding wound
healing, no obvious impact of the antiangiogenic, antifibrotic
effect of B-miR-29b-sEVs was seen in the SR-sEVs@BSSPD
group.
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Blood vessels in each group were systematically evaluated by
microcomputed tomography (micro-CT), immunohistochem-
istry, and immunofluorescence. The reconstructed three-
dimensional (3D) image (Figure 6A), immunohistochemical
(IHC) staining, and dual immunofluorescence staining (Figure
6D) showed that both the density and number of blood vessels
in the SR-sEVs@BSSPD group and B-sEVs@BSSPD group
were significantly higher than those in the control group,
BSSPD group, and BSSPD loaded with B-miR-29b-sEVs (B-
miR-29b-sEVs@BSSPD) group. The same results were
obtained by quantitative analysis of the area of the region
covered by new blood vessels and the number of new blood
vessels (Figure 6B,C) and the numbers of total blood vessels
and mature blood vessels (Figure 6E,F). The results of in vitro
experiments showed that B-miR-29b-sEVs had antivasculariza-
tion and antifibrotic properties. However, analysis of the in vivo
experimental results indicated that B-miR-29b-sEVs released
from the upper layer did not significantly affect wound healing
and angiogenesis in the SR-sEVs@BSSPD group.
In the main skin structure, collagen fibers are divided into
type I and type III.79 Type I collagen is thick and is the main
component of skin tissue, while type III collagen is thin and is
the main component of reticular fibers. The proportions of
collagen I and III in normal skin are affected by coordinated
expression during development, and the normal proportions of
collagen I and III maintain the normal skin tissue structure.80
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Therefore, the arrangement and distribution of collagens and
the proportions of type I and type III collagen are important
criteria for assessing wound healing.81 The Masson staining
results (Figure 7A) indicated that collagen deposition was
Figure 7. (A) Masson staining showing collagen deposition. (B)
Representative slides stained with Sirius red. Sirius red staining
can distinguish type I and type III collagen. Type I collagen is
stained yellow, and type III collagen is stained green. (C) Analysis
of the collagen I/III ratio in the five different treatment groups.
more extensive and the collagen fiber thickness was greater in
the B-sEVs@BSSPD group than in the control group, while the
collagen arrangement in the SR-sEVs@BSSPD group was more
regular and orderly. The arrangement of collagen fibers was
improved in the SR-sEVs@BSSPD group compared with the
control group and was similar to that of normal skin, indicating
the positive effect of SR-sEVs@BSSPD treatment on ECM
deposition and collagen arrangement. As shown in Figure
7B,C, the collagen I/III ratio in each group increased on day 7
due to the increase in type I collagen. The B-sEVs@BSSPD
group and SR-sEVs@BSSPD group had significantly higher
scores than the control group, BSSPD group, and B-miR-29b-
sEVs@BSSPD group. On day 14, the type I/III collagen ratios
in the three sEV treatment groups were lower than those in the
control group. Importantly, the type I/III collagen ratio in the
SR-sEVs@BSSPD group was more similar to that in normal
skin than were the ratios in the other groups.
Macrophages are indispensable for tissue repair and
remodeling and play an essential role in cutaneous wound
healing. In particular, M2 polarization exerts distinct biological
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effects during the different phases of wound healing. In the
early wound healing phase, M2 macrophages inhibit the acute
inflammatory response in the wound microenvironment,
accelerate angiogenesis, promote epithelial cell proliferation
and migration, and regulate collagen regeneration and
deposition.82 Paradoxically, the M2 phenotype is a regulator
of both healing and scar hyperplasia. Moreover, they are the
main components in the HS microenvironment and play a key
role in both initial and supporting scar formation.83 In the late
wound healing phase, the M2 phenotype promotes excessive
fibroblast proliferation, resulting in dense fibrous tissue
overgrowth and excessive collagen deposition.83 Considering
the different roles of macrophage polarization at different
phases in the physiological wound healing process, we further
explored the mechanism of action of the two types of sEVs in
the relationship between sEVs and inflammation.
The in vitro flow cytometry results shown in Figure 8A,B
indicate that B-miR-29b-sEVs induced M1 macrophage
polarization and B-sEVs induced M2 macrophage polarization.
Moreover, B-miR-29b-sEVs reversed B-sEV-induced M2
macrophage polarization, as M2 macrophages were depleted
and M1 macrophages were enriched (Figure 8C−E). The
relative expression levels of genes, as determined by RT-PCR,
also proved the immunomodulatory function of B-miR-29b-
sEVs and B-sEVs. Anti-inflammatory genes (Arg1 and IL-4)
were upregulated by B-sEVs, and proinflammatory genes (IFN-
γ and IL-1β) were upregulated by B-miR-29b-sEVs (Figure
8F,G). Consistent with the flow cytometry results, proin-
flammatory genes were upregulated and anti-inflammatory
genes were downregulated in the B-sEVs/B-miR-29b-sEV
group. These results indicated different immunomodulatory
functions of B-miR-29b-sEVs and B-sEVs and suggested that
B-miR-29b-sEVs attenuated B-sEV-induced M2 polarization.
The immunomodulatory function was further characterized
in vivo by immunofluorescence staining (Figure 8H). At 7 days,
M2 polarization was significantly enhanced in the B-sEVs@
BSSPD and SR-sEVs@BSSPD groups compared to that in the
other groups, while the high CD206 expression was decreased
in the B-miR-29b-sEVs@BSSPD group. M2 macrophage
polarization was suppressed by B-miR-29b-sEVs, thus exacer-
bating the proinflammatory response in the early phase and
inhibiting rapid epithelialization and wound healing. As the
time of wound healing is prolonged and the inflammatory
response continues, the number of macrophages remains
high.84,85 Here, as the healing process was delayed and a large
number of macrophages were recruited to the wound site,
CD206 expression in the B-miR-29b-sEVs@BSSPD group was
found to be high on day 14. In contrast, M2 polarization of
macrophages was weakened in the B-sEVs@BSSPD and SR-
sEVs@BSSPD groups on day 14, as the wounds in these two
groups were almost completely closed. Interestingly, M2
polarization of macrophages was much weaker in the SR-
sEVs@BSSPD group than in the B-sEVs@BSSPD group
(Figure 8I) due to the introduction of B-miR-29b-sEVs in
the later phase. Based on these in vitro and in vivo results,
sequential release of B-sEVs and B-miR-29b-sEVs by SR-
sEVs@BSSPD led to M2 polarization in the early phase and
attenuated M2 polarization in the later phase. Considering the
biological phase of wound healing, rapid initiation of wound
healing and subsequent inhibition of excessive collagen
production and fibrosis could be achieved by the immunor-
egulatory function of SR-sEVs@BSSPD. Therefore, sequential
release of sEVs from BSSPD could account for both the speed
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Figure 8. (A) Histograms of CCR7 and CD206 expression after treatment with B-sEVs or B-miR-29b-sEVs for 48 h, as determined by flow
cytometry. (B) Quantitative analysis of the data in the histograms. (C) Histograms of CCR7 and CD206 expression after treatment with B-
sEVs or B-miR-29b-sEVs and sequential release of B-sEVs (48 h) and B-miR-29b-sEVs (48 h) for 96 h, as determined by flow cytometry. (D)
Quantitative analysis of the data in the histograms. (E) Analysis of the M2/M1 ratio. (F, G) Relative gene expression levels of Arg1, IL-4,
IFN-γ, and IL-1β. (H) Immunofluorescence staining of macrophages in vivo on days 7 and 14 (DAPI: blue, CD206: green). (I)
Immunofluorescence analysis of M2 polarization.

and the quality of wound healing to promote wound healing
and reduce HS.
Rodent models of full-thickness skin wounds have
fundamental biological and structural limitations in their
ability to faithfully represent scarring in humans.86 For
instance, rats have a panniculus carnosus, a muscle that
promotes wound contraction, in their dorsal skin.87 However,
in human wounds, the healing process is driven mainly by
granulation and reepithelialization. Additionally, after wound
healing, scars on the dorsal skin of rats rapidly shrink and are
not obvious, hindering long-term studies on scar reconstruc-
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tion.88 Therefore, the classical rat model of full-thickness skin
defects has natural limitations in the evaluation of hypertrophic
scarring. A previous study showed that in rabbit ears,
epidermal hyperplasia occurs after wound healing, which is
considered to be similar to hyperplastic scarring in humans.89
Therefore, we used rabbit ear scar models to evaluate the effect
of different treatments on scarless wound healing. The wound
healing rates in the B-sEVs@BSSPD and SR-sEVs@BSSPD
groups were consistent with the results in Sprague−Dawley
(SD) rats (Figure 9A,B). The collagen in the SR-sEVs@
BSSPD-treated rabbit ears was more orderly and more similar
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Figure 9. (A) Changes in the wound closure areas in the rabbit ear were observed 0, 1, 2, and 4 weeks after surgery. (B) Traces of wound
closures. (C) Masson staining of wounds. (D) Ultrasonography of wounds: (a) Control, (b) BSSPD, (c) B-sEVs@BSSPD, (d) B-miR-29b-
sEVs@BSSPD, and (e) SR-sEVs@BSSPD. (E) Quantitative analysis of the SEI.
to that in normal skin sections than was the collagen in the
other treatment groups (Figure 9C). In addition, the skin in
the SR-sEVs@BSSPD group had a lower protrusion height
than that in the B-sEVs@BSSPD and B-miR-29b-sEVs@
BSSPD groups (Figure 9D). Doppler ultrasound was further
utilized to measure scar thickness. Quantitative Doppler
ultrasound analysis showed that the scar elevation index
(SEI) in the SR-sEVs@BSSPD group was smaller than that in
the other four groups, indicating minimal scar formation in this
group (Figure 9E). In brief, SR-sEVs@BSSPD reduced HS
formation without affecting the wound closure speed.
CONCLUSION
Herein, we designed a strategy for the development of scarless
wound healing dressings and the etiological treatment of scars
based on sequential sEVs releasing from SH/PEG-DA
hydrogels. Bilayered SA-SH/PEG-DA hydrogels were fabri-
cated by thiol−ene click chemistry, exhibiting promising
wound protection ability and sequential sEVs release. In the
coagulation, inflammation, and early proliferation phases of
wound healing, B-sEVs released from the lower layer of the
hydrogels promoted angiogenesis and collagen deposition,
which accelerated wound healing. MiR-29b-3p was secreted by
B-miR-29b-sEVs to suppress endothelial cell and fibroblast
proliferation and migration and Col1A1 expression in
fibroblasts by inhibiting the PI3K/Akt, Erk1/2, and Smad3/
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TGF-β1 signaling pathways. In contrast, in the late
proliferation and maturation phases of wound healing, B-
miR-29b-sEVs released from the upper layer of the hydrogels
inhibited excessive angiogenesis and collagen deposition, thus
reducing the formation of HSs. Collectively, these biomedical
functions of SR-sEVs@BSSPD constitute an effective sympto-
matic treatment strategy that aims to improve skin
regeneration and inhibit HS.
METHODS
This study was approved by the ethics committee of Shanghai Jiao
Tong University Affiliated Sixth People’s Hospital, School of
Medicine, Shanghai Jiao Tong University. All animal experiments
were approved by the animal research ethics committee of Shanghai
Jiao Tong University Affiliated Sixth People’s Hospital and were
conducted in accordance with the National Institutes of Health Guide
for the Care and Use of Experimental Animals (Animal Welfare Ethics
acceptance number (approval number) DWLL2020-0522).
Lentivirus Transfection. The miR-29b-3p lentiviral vector was
obtained from Oligobio (Beijing, China). The transfection protocol
was performed according to the manufacturer’s instruction. In brief:
hBMSCs were incubated in retro-viral supernatant with 5 mg/mL of
polybrene for 24 h. After infection for 48 h, hBMSCs were treated
with puromycin dihydrochloride (Thermo Fisher).
Characterization of sEVs. Morphological characterization of
vesicles was performed by TEM (JEM-1400, JEOL, Japan). Western
blot analysis was conducted to validate the sEV markers Alix (Protein
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Tech, USA), TSG101 (Protein Tech, USA), and CD9 (Abcam,
USA). The size distribution of sEVs was determined by NTA.
Internalization of sEVs. Purified sEVs were labeled with the
lipophilic fluorescent PKH dye PKH-26 (Sigma-Aldrich, Germany).
The PKH26-labeled sEVs were then resuspended in the culture
medium of EA.hy926 and HFF-1 cells for 24 h. After fixation, the
sEVs were washed 3 times with PBS prior to immunostaining with
QuickBlock blocking buffer (Beyotime, China). Then, the cytoske-
leton was stained with phalloidin (1:200, Yeasen, China) for 45 min at
room temperature prior to 3 washes with PBS for 5 min each. After
the final wash, 4′,6-diamidino-2-phenylindole (DAPI; 1:2000, Solar-
bio, China) in PBS was added, and cells were stained for 10 min
before imaging via confocal microscopy (Leica, Solms, Germany).
qRT-PCR. MiR-29-3p- and RNU6B (BioTNT, Shanghai, China)
miRNA-specific qRT-PCR detection kits were used for cDNA
synthesis and qRT-PCR. MiR-29-3p expression levels were
normalized to those of RNU6B. cDNA synthesis was performed
using TransScript All-in-One First-Strand cDNA short SuperMix for
qPCR (Transgen Biotech, Beijing, China). TransStart Top Green
qPCR SuperMix (Transgen Biotech) was used for qRT-PCR, and the
results were processed by normalization.
Cell Proliferation and Migration Assays. CCK-8 assays were
used to analyze the proliferation of EA.hy926 cells and HFF-1 cells.
EA.hy926 cells were cultured at different densities and under different
conditions. EA.hy926 cells were plated in flat-bottom 96-well plates
(Corning, USA) at a density of 2 × 103 cells/well and were cultured
for 1, 3, and 7 days. HFF-1 cells (1.5 × 103) were cultured in a 96-well
plate under similar conditions. Then, 100 μL of medium containing
10% CCK-8 reagent was added to each plate. The plates were then
incubated for 2 h, and the absorbance of the sample was immediately
measured at 450 nm in a microtitration plate reader (Winooski
BioTek, USA). The migration of EA.hy926 cells and HFF-1 cells was
evaluated by a Transwell assay (3422, Corning, USA). EA.hy926 cells
(2 × 104) were seeded in the upper chambers in 200 μL of serum-free
medium. Five hundred μL of complete medium was added to the
lower chambers. Then, the cells were fixed, stained, imaged by
microscopy (Olympus IX 70, Tokyo, Japan), and counted with
ImageJ software. The migration of HFF-1 cells was evaluated with the
same method.
Tube Formation Assay. Two hundred μL of Matrigel (BD
Bioscience) was added to each well of precooled 24-well plates, and
gel electrophoresis was conducted at 37 °C for 1 h. Cells were
precultured for 48 h in complete growth medium under different
conditions. A total of 1 × 105 pretreated EA.hy926 cells were then
seeded on Matrigel-coated plates and cultured. EA.hy926 cell tube
formation was evaluated by microscopy (Olympus IX 70, Tokyo,
Japan) after 8 h of incubation. The number of tubes was analyzed with
ImageJ software.
VEGF/Col I Secretion Assay. Cells were maintained in medium
at different concentrations for 3 days, and the cells were then fixed
with paraformaldehyde (PFA), and washed with PBS, permeabilized
with 0.25% Triton X-100 in PBS, and blocked with 3% bovine serum
albumin (BSA). Anti-VEGF/Col I (1:200, ABclonal, China) anti-
bodies were added, and the cells were then incubated at 4 °C
overnight. The next day, the secondary antibodies were added, and
the cells were incubated in the dark for 1 h. Then, the cells were
washed 3 times (5 min each time) with PBS. EA.hy926 cells and
HFF-1 cells were then incubated with 5 g/mL of phalloidin (1:200,
Yeasen, China) for 45 min in the dark. After three PBS washes, the
cells were stained with DAPI (1:200, Solarbio). A confocal
microscope (Leica, Solms, Germany) was used to visualize the cells.
VEGF/Col I secretion from EA.hy926 cells/HFF-1 cells was assessed
by ELISA (NeoBioscience, China). EA.hy926/HFF-1 cells (1 × 105)
were seeded in medium at different concentrations in 6-well plates.
The supernatant of the samples was collected after 3 days of
incubation, and the VEGF/Col I concentrations were determined in
accordance with the manufacturer’s instructions.
Western Blot Analysis of EA.hy926 Cells and HFF-1 Cells.
Cultured sEVs or cells were lysed in RIPA lysis buffer containing a
supplement (Invitrogen, USA). Protein loading buffer (5×) was used
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to dilute the lysates at a ratio of 1:5. Then, the lysates were heated to
95 °C for 5 min. Protein extracts were separated by 10% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis at 120 V for 1 h
and transferred onto polyvinylidene difluoride membranes (Merck-
Millipore) at 200 mA for 1.5 h. Membranes were blocked with 5%
nonfat milk or BSA for 1 h and were then incubated with
combinations of primary antibodies overnight at 4 °C. Next,
membranes were incubated with combinations of horseradish
peroxidase-conjugated secondary antibodies for 1 h at room
temperature. The immunoreactive bands were visualized with an
enhanced chemiluminescence reagent (Thermo Fisher Scientific).
Characterization of Hydrogels. The surface morphology of the
six freeze-dried SA-SH/PEG-DA hydrogels was observed by scanning
electron microscopy (S-3400N, Hitachi, Japan) at an acceleration
voltage of 15 kV. The samples were sputtered with gold for 45 s
before visualization.
Raman spectroscopy was carried out with an inVia Reflex Raman
spectrometer (Renishaw Company, UK). The wavelength was fixed at
514.5 nm, and the scanning range was set at 4000 cm−1 to 400 cm−1.
The sample was placed on the slide for direct laser irradiation.
A Nicolet 5700 infrared spectrometer (Thermo Scientific, USA)
was used for FTIR spectroscopy. The scanning wavenumber range
was set at 4000 cm−1 to 400 cm−1.
SA-SH/PEG-DA hydrogels with different ratios were placed in PBS
buffer (pH = 7.4) at 37 °C for measurement of the swelling rate. After
swelling for 15 min, excessive liquid was absorbed, and the hydrogels
were reweighed. The swelling rate of the SA-SH/PEG-DA hydrogels
(Seq%) was calculated with the following equation:
Weq − Wd
Seq % = × 100%
Wd
where Weq is the mass of the swollen gel, and Wd is the mass of the
dry gel.
The rheological properties of the SA-SH/PEG-DA hydrogels were
evaluated with a MARS3 rotary rheometer (Thermo Scientific, USA).
Frequency conversion scanning was used to modulate the frequency
from 0.01 Hz to 10 Hz. The strain was set at 1%, and the temperature
was set at 37 °C.
Fabrication of the SR-sEVs@BSSPD Hydrogel. The photo-
initiator I2959 (1 mg) was dissolved in 400 μL of deionized water,
and 4 μL of PEG-DA (Mn = 250) was added to the solution. Then,
100 μL of an aqueous solution of B-sEVs (1 × 1011 sEV particles/mL)
was added, and the solution was stirred thoroughly again. The above
solution was then added to a sample bottle containing 5 mg of SA-SH
powder (SA:SH 3:31.35%), and the sample was immediately
irradiated under a UV lamp at 365 nm after rapid stirring for 10
min. After UV irradiation, the SA-SH/PEG-DA hydrogel was
removed and placed in a dialysis bag (MWCO = 14 kDa) for dialysis
to remove excess I2959 and PEG-DA that were not involved in the
reaction. The above method was used to add the same volume of an
aqueous solution of B-miR-29b-sEVs to 11 μL of PEG-DA (Mn =
700) to fabricate the corresponding SA-SH-3/PEG-DA-250 hydrogel.
Fabrication of the B-sEVs@BSSPD Hydrogel. The photo-
initiator I2959 (1 mg) was dissolved in 400 μL of deionized water,
and 4 μL of PEG-DA (Mn = 250) was added to the solution. Then,
100 μL of an aqueous solution of B-sEVs (1 × 1011 sEV particles/mL)
was added, and the solution was stirred thoroughly again. The above
solution was then added to a sample bottle containing 5 mg of SA-SH
powder (SA:SH 3:31.35%), and the sample was immediately
irradiated under a UV lamp at 365 nm after rapid stirring for 10
min. After UV irradiation, the SA-SH/PEG-DA hydrogel was
removed and placed in a dialysis bag (MWCO = 14 kDa) for dialysis
to remove excess I2959 and PEG-DA that were not involved in the
reaction. The above method was used to add the same volume of an
aqueous solution of B-sEVs to 11 μL of PEG-DA (Mn = 700) to
fabricate the corresponding SA-SH-3/PEG-DA-250 hydrogel.
Fabrication of the B-miR-29b-sEVs@BSSPD hydrogel. The
photoinitiator I2959 (1 mg) was dissolved in 400 μL of deionized
water, and 4 μL of PEG-DA (Mn = 250) was added to the solution.
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Then, 100 μL of an aqueous solution of B-miR-29b-sEVs (1 × 1011
sEV particles/mL) was added, and the solution was stirred thoroughly
again. The above solution was then added to a sample bottle
containing 5 mg of SA-SH powder (SA:SH 3:31.35%), and the sample
was immediately irradiated under a UV lamp at 365 nm after rapid
stirring for 10 min. After UV irradiation, the SA-SH/PEG-DA
hydrogel was removed and placed in a dialysis bag (MWCO = 14
kDa) for dialysis to remove excess I2959 and PEG-DA that were not
involved in the reaction. The above method was used to add the same
volume of an aqueous solution of B-miR-29b-sEVs to 11 μL of PEG-
DA (Mn = 700) to fabricate the corresponding SA-SH-3/PEG-DA-
250 hydrogel.
ELISA of SEVs. SR-sEVs@BSSPD hydrogels were immersed in a
six-well plate in PBS, and the plate was placed in a cell incubator for 3,
6, and 12 h and 1, 2, 3, 4, 5, 6, 7, 8, and 9 days. The materials
immersed for 9 days were dissolved, the residual content of sEVs was
measured after 9 days, and the total number of sEVs was calculated.
The numbers of sEV particles were determined using a sEV ELISA kit
for CD63 according to the instructions provided. In brief, standard
curves were generated by continuous dilution of the standard with
sEVs combined with buffer solution.
Release of Fluorescent sEVs. B-miR-29b-sEVs were stained with
PKH-67 and B-sEVs with PKH-26, and the two types of sEVs that
were successfully stained were placed on the upper and lower layers of
BSSPD in the original order. SR-sEVs@BSSPD hydrogels stained with
the sEV dye were placed in the upper compartment of the Transwell
chamber in a 6-well plate, and EA.hy926 cells (2 × 103) were added to
the lower compartment in 500 μL of complete medium. The level of
the liquid was flush with the upper and lower boundaries of the
hydrogel. The 6-well plates were incubated in cell incubators for 1, 2,
3, 4, 7, 9, 11, and 14 days. The cells were removed, and the plates
were fixed with PFA, washed three times with PBS, and
immunostained with QuickBlock blocking buffer for 10 min. The
plates were then washed with PBS 3 times for 5 min each. Finally,
after washing, DAPI was added. After staining for 10 min, the cells
were washed three times with PBS in the dark. Then, the cells were
visualized by confocal microscopy, and images were acquired. ImageJ
was used for measurement and statistical analysis based on the
proportions of the three colors in the images.
Degradation of BSSPD. The upper layer of BSSPD was stained
blue with 1,9-dimethylmethylene blue, while the lower layer of
BSSPD hydrogels was stained red with rhodamine B. The BSSPD
hydrogels were placed in the upper compartments of a 6-well
Transwell plate. EA.hy926 cells in PBS or culture medium were
seeded in the lower compartments. The level of the liquid was just
above the top of the lower compartment. The plates were then
incubated in a cell culture incubator. Images were acquired on days 0,
1, 2, 3, 5, 7, 9, 11, and 14. Statistical analysis was conducted based on
the hydrogel height.
SD Rats and Surgical Procedure. Thirty male SD rats (2
months old, 250 ± 15 g) were selected to establish a full-thickness
skin defect model in this study. BSSPD, B-sEVs@BSSPD, B-miR-29b-
sEVs, and SR-sEVs@BSSPD hydrogels were applied to the wound
surfaces on the SD rats. After the operation, the wounds were covered
with sterile gauze. All wounds were observed daily to ensure that the
materials and pressure dressing remained intact.
Microfil Perfusion and Micro-CT. SD rats were perfused with
Microfil (MV-122; Flow Tech, USA), and neovascularization was
evaluated 14 days after surgery. The experimental animals were
euthanized and were then perfused intracardially with 100 mL of
heparinized saline, and 20 mL of Microfil was continuously injected at
2 mL/min. The dye entered the neovascularized area of the wound
through the cardiac vessels. Finally, the experimental samples were
incubated in a polymerizing contrast agent at 4 °C overnight. The
next day, the wound samples were trimmed using micro-CT
instrument (SkyScan). Red indicates the new blood vessels in the
wound 14 days after surgery. SkyScan software (SkyScan Company)
was used to reconstruct 3D images of wound neovascularization. The
same section was selected, and neovascularization is marked in red in
the figure. Then, the number of new blood vessels per unit area and
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the area occupied by new blood vessels were calculated, and the data
for the five groups were analyzed with ImageJ software.
IHC and Immunofluorescence Analysis. Quantitative analysis
of IHC staining was based on previous studies.90 To assess vascular
system formation, CD31 (1:200, Abcam, Cambridge, UK) was
stained by IHC methods. CD31 (1:200, Abcam, Cambridge, UK) and
α-SAM (1:50, Abcam, Cambridge, UK) were stained with
immunofluorescence methods. Sections were observed via fluores-
cence microscopy (Olympus IX 70, Tokyo, Japan) and confocal laser
scanning microscopy (Leica, Solms, Germany). ImageJ Plus software
was used to calculate the number of blood vessels.
Histological Analysis. Samples were dehydrated in an ethanol
gradient, embedded in paraffin, and cut into 6 mm thick sections
perpendicular to the wound surface. The sections were stained with
H&E for histological evaluation. The length of the new epithelium
was determined by the changes in the wound surface as observed by
H&E staining to assess the effect of each treatment on
epithelialization of the wound surface.91 Masson’s trichrome staining
was applied to detect collagen fibers in tissues. The collagen fibers
appear blue, and the muscle fibers appear red. Sirius red staining was
used mainly to analyze the degree and characteristics of fibrosis. The
slices were dyed in Biebrich scarlet dye for 8−10 min. Two or three
containers of anhydrous ethanol were used for dehydration. The
samples were sliced, transferred to clean xylene for 5 min for clearing,
and sealed using neutral gum. Sirius red staining can visualize specific
collagen tissue; under a polarized light source, type I collagen appears
yellow or red, type III collagen appears green, and type II collagen
appears blue, green, or pale blue. ImageJ software was used to
calculate and analyze the collagen I/III ratio in tissues.
Flow Cytometric, qRT-PCR, and Immunofluorescence
Analysis of Macrophagocytes. Flow cytometric analysis: After
treatment of RAW264.7 cells with B-sEVs, B-miR-29b-sEVs, B-sEVs,
or B-miR-29b-sEVs for 48 and 96 h, cells were detached with PBS
solution (4 °C) and resuspended in cell staining buffer. After blocking
with a rat antimouse CD16/32 antibody (Biolegend, USA),
RAW264.7 cells were stained with rat antimouse CD206 Alexa
Fluor 647 antibody (BD Pharmingen, USA) and rat antimouse CCR7
APC (Biolegend, USA) for 30 min at 4 °C in the dark. The samples
were evaluated and analyzed with the CytoFLEX Platform (Beckman,
USA). For quantification of the relative gene expression levels of
Arg1, IL-4, IFN-γ, and IL-1β in RAW264.7 cells cultured with B-sEVs,
B-miR-29b-sEVs, and B-sEVs/B-miR-29b-sEVs, qRT-PCR was
applied as described above. In immunofluorescence staining images
acquired 7 and 14 days after surgery, nuclei in the rat skin specimens
appear blue, and CD206 appears green. Statistical analysis was
performed with ImageJ software.
Rabbit Model of HS and Surgical Procedure. Fifteen healthy
New Zealand white rabbits (2.5−3 kg) were obtained from HFK
(HFK Bioscience Co., Ltd., Beijing). The operation was performed in
a sterile environment after the animals were anesthetized with sodium
pentobarbital (Sigma, 30 mg/kg). The epidermis, dermis, and
pericardium of each wound were thoroughly excised. On the ventral
surface of each ear, four circular wounds 10 mm in diameter were
established away from the middle auricular artery and the
periauricular vein. We randomly divided all treated rabbits into five
groups (n = 3 per group): the BSSPD, B-sEVs@BSSPD, B-miR-29b-
sEVs, and SR-sEVs@BSSPD groups. The wounds in the control
group were untreated, and the wounds in the different treatment
groups were covered with the corresponding hydrogel. The wound
healing process was monitored and recorded every day until death 4
weeks after the operation. The scar thickness was calculated using an
ultrasound instrument (Mindray M9, Mindray, China). Scar tissue
was removed from the surrounding uninjured (2 mm) margin. The
scar tissue samples from each group were divided into three parts. The
specimens were fixed with 4% PFA for 24 h, embedded in paraffin,
sectioned transversely, and subjected to histological analysis. The SEI
was determined by Masson’s trichrome staining. The dermis in
Masson’s trichrome-stained tissues was imaged in 5 randomly selected
fields.
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Statistical Analysis. All data are shown as the mean ± standard
deviation (SD) values. Independent samples t tests were used to
compare means between two groups. One-way analysis of variance
(ANOVA) was used to determine the level of significance with
GraphPad Prism software, and P < 0.05 was considered to indicate
statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001 compared
to the control group; #P < 0.05 compared to the B-sEVs@BSSPD
group; &P < 0.05 compared to the B-miR-29b-sEVs@BSSPD group.
ASSOCIATED CONTENT
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsnano.0c07714.
Additional experimental methods and supplementary
figures. Figure S1: Elisa standard curve. Figure S2:
Further information and data plots about SA-SH/PEG-
DA hydrogels. Figure S3: Biocompatibility of SA-SH/
PEG-DA hydrogels (PDF)
AUTHOR INFORMATION
Corresponding Authors
Hong Gao − Department of Orthopaedic Surgery, Shanghai
Jiao Tong University Affiliated Sixth People’s Hospital,
Shanghai 200233, China; orcid.org/0000-0001-9347-
3293; Email: honggao630@163.com
Shichang Zhao − Department of Orthopaedic Surgery,
Shanghai Jiao Tong University Affiliated Sixth People’s
Hospital, Shanghai 200233, China;
Email: zhaoshichang0404@163.com
Meidong Lang − Shanghai Key Laboratory of Advanced
Polymeric Materials, Key Laboratory for Ultrafine Materials
of Ministry of Education, School of Materials Science and
Engineering, East China University of Science and
Technology, Shanghai 200237, China; orcid.org/0000-
0003-2053-9284; Email: mdlang@ecust.edu.cn
Authors
Yifan Shen − Department of Orthopaedic Surgery, Shanghai
Jiao Tong University Affiliated Sixth People’s Hospital,
Shanghai 200233, China
Guanzhe Xu − Shanghai Key Laboratory of Advanced
Polymeric Materials, Key Laboratory for Ultrafine Materials
of Ministry of Education, School of Materials Science and
Engineering, East China University of Science and
Technology, Shanghai 200237, China; Internet of Things
Research Center, Advanced Institute of Information
Technology, Peking University, Hangzhou 311200, China
Huanxuan Huang − Shanghai Key Laboratory of Advanced
Polymeric Materials, Key Laboratory for Ultrafine Materials
of Ministry of Education, School of Materials Science and
Engineering, East China University of Science and
Technology, Shanghai 200237, China
Kaiyang Wang − Department of Orthopaedic Surgery,
Shanghai Jiao Tong University Affiliated Sixth People’s
Hospital, Shanghai 200233, China
Hui Wang − Green Chemical Engineering Technology
Research Center, Shanghai Advanced Research Institute,
Chinese Academy of Sciences, Shanghai 201210, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsnano.0c07714
Author Contributions
#
These authors contributed equally to the work.
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Article
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors acknowledge the support of the National Natural
Science Foundation of China (nos. 51972212, 51802326, and
81572178) and Shanghai Municipal Commission of Health
and Family Planning (no. 20124356).
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6368 https://dx.doi.org/10.1021/acsnano.0c07714
ACS Nano 2021, 15, 6352−6368