Abstract
Developing biomaterials with antimicrobial and wound‐healing activities
for the treatment of wound infections remains challenging. Macrophages
play non‐negligible roles in healing infection‐related wounds. In this
study, a new sequential immunomodulatory approach is proposed to
promote effective and rapid wound healing using a novel hybrid hydrogel
dressing based on the immune characteristics of bacteria‐associated
wounds. The hydrogel dressing substrate is derived from a porcine
dermal extracellular matrix (PADM) and loaded with a new class of
bioactive glass nanoparticles (BGns) doped with copper (Cu) and zinc
(Zn) ions (Cu–Zn BGns). This hybrid hydrogel demonstrates a controlled
release of Cu^2+ and Zn^2+ and sequentially regulates the phenotypic
transition of macrophages from M1 to M2 by alternately activating
nucleotide‐binding oligomerization domain (NOD) and inhibiting
mitogen‐activated protein kinases (MAPK) signaling pathways.
Additionally, its dual‐temporal bidirectional immunomodulatory function
facilitates enhanced antibacterial activity and wound healing. Hence,
this novel hydrogel is capable of safely and efficiently accelerating
wound healing during infections. As such, the design strategy provides
a new direction for exploring novel immunomodulatory biomaterials to
address current clinical challenges related to the treatment of wound
infections.
Keywords: bioactive glass, copper, immunomodulation, infection‐related
wounds, zinc
__________________________________________________________________
The advancement of biomaterials possessing both antimicrobial and
wound‐healing properties for the treatment of wound infections
continues to pose a formidable challenge. The present study introduces
a novel sequential immunomodulation strategy predicated on the immune
characteristics of bacterial‐associated wounds. This approach employs
an innovative hybrid hydrogel dressing designed to expedite the healing
process of infected wounds effectively and rapidly.
graphic file with name ADVS-11-2302674-g009.jpg
1. Introduction
The skin is the largest organ in the body and serves as the first line
of defense against the external environment.^[ [42]^1 ^] Meanwhile,
microorganisms that breach this barrier via infection of wounds induce
severe inflammation, negatively impacting healing and potentially
causing life‐threatening conditions.^[ [43]^2 ^] It is therefore
essential to develop novel multifunctional bioactive materials capable
of eliminating infections and promoting wound healing and tissue
regeneration. Accordingly, several wound‐dressing materials, including
nanofibrous membranes and hydrogels, have been developed.^[ [44]^3 ^]
Hydrogels are highly permeable, biocompatible materials with hydrating
properties. As such, they are promising candidates for healing wounds
caused by infections.^[ [45]^4 ^] However, given that conventional
hydrogel dressings lack antimicrobial activity, they are often loaded
with antibiotics, which can lead to bacterial resistance and
hepatic/nephrotoxicity, ultimately impairing wound healing.^[ [46]^5 ^]
Hence, an urgent need exists for the development of novel
antibiotic‐free hydrogel wound dressings.
Macrophages mediate immune responses, antibacterial activity, and wound
healing.^[ [47]^6 ^] Moreover, they exhibit phenotypic plasticity and
can be categorized as M1 (proinflammatory) or M2 (anti‐inflammatory)
based on their responses to diverse microenvironments.^[ [48]^7 ^]
Microbial infections accompanied by biofilm formation favor the
polarization of macrophages toward the M2 phenotype. However, this
creates an immunosuppressive microenvironment that fails to eliminate
bacteria and can inhibit wound healing.^[ [49]^8 ^] Therefore,
reversing the immunosuppressive microenvironment of the infected skin
is necessary to ensure bacterial clearance and wound healing. During
the early stages of infection, M1 macrophage polarization inhibits
bacterial growth and eliminates infections,^[ [50]^8 , [51]^9 ^] which
also effectively reduces biofilm formation.^[ [52]^10 ^] Therefore,
targeting proinflammatory macrophages could reverse immunosuppression
and promote antimicrobial activity. Indeed, an early inflammatory
response is essential for preventing infection, removing tissue debris,
and wound healing.^[ [53]^11 ^] Meanwhile, prolonged M1 macrophage
dominance results in excessive or chronic inflammation, which damages
tissues, accelerates scar formation, and impedes wound healing.^[
[54]^12 ^] Accordingly, during tissue repair, following an early
transitional inflammatory phase, macrophages switch to the M2‐type. In
addition to suppressing M1 macrophages, anti‐inflammatory factors such
as interleukin (IL)−4 and IL‐10 secreted by M2 macrophages promote
tissue repair and maturation, as well as the stabilization of vessels,
thereby accelerating the process of wound healing.^[ [55]^13 ^] Hence,
early onset inflammation promotes the clearance of infection and tissue
repair, while wound healing requires a timely shift from an activated
(M1) to an alternative (M2) macrophage response. Therefore, sequential
immunomodulatory strategies are required for bacterial clearance, wound
healing, and tissue regeneration. As such, during the early stages of
infection‐induced skin injury, biomaterials should induce M1
polarization to reduce bacteria proliferation and facilitate tissue
repair. However, in the later stages, M2 macrophage polarization must
be stimulated to promote wound healing. Hence, it is crucial to develop
innovative biomaterials that align with this physiological process in
order to enhance the efficacy of infection‐related wound healing.
Trace elements like copper (Cu) and zinc (Zn) are critically involved
in cell metabolism, tissue regeneration, and immunity.^[ [56]^8 ,
[57]^14 ^] In fact, Cu^2+ possesses strong bactericidal and
antimicrobial activities against diverse bacteria, including
antibiotic‐resistance bacteria.^[ [58]^15 ^] In addition, Cu^2+
secretes pro‐inflammatory factors, thereby promoting the formation of a
pro‐inflammatory microenvironment.^[ [59]^9 ^] In contrast, Zn^2+ has
good angiogenic and tissue‐repair properties. Moreover, Zn^2+ interacts
with immune cells to facilitate the formation of an anti‐inflammatory
microenvironment.^[ [60]^16 ^] Considering that Cu^2+ and Zn^2+
regulate the immune responses in different ways, their sequential
controlled release of both these ions at different stages of injury,
that is, the release of Cu^2+ during the early stage and Zn^2+ during
the later stage, could exert dual‐temporal bidirectional
immunoregulatory functions.
In this study, we designed and synthesized an innovative hydrogel
comprising a Zn‐containing bioactive glass nanoparticle (Zn‐BGn) core
covered with a porous layer of Cu‐containing bioactive glass,
designated Cu–Zn BGn. During the early stages, the outer Cu layer of
the Cu–Zn BGn releases Cu^2+ to induce a proinflammatory response,
thereby inhibiting infection. At the later stages, the Zn‐containing
core releases Zn^2+ to form an anti‐inflammatory microenvironment and
promote tissue repair. Moreover, Cu–Zn BGns are uniformly loaded into a
porcine dermal extracellular matrix‐derived hydrogel (PADM@CZ, Scheme
[61]1 ). The results revealed that the PADM@CZ hydrogel significantly
reduced the toxic effects of nanoparticles and accelerated wound
healing. Because molecules on the extracellular matrix (ECMs) mimic the
cellular microenvironment, integrating mechanical and chemical cues
could aid in the adhesion, proliferation, and differentiation of cells,
thus facilitating homologous wound healing.^[ [62]^17 ^] Hence, the
PADM@CZ hydrogel has the potential to exhibit superior antimicrobial
and immunomodulatory properties and improved bacterial clearance, thus,
promoting wound healing by reshaping immune homeostasis.
Scheme 1.
Scheme 1
[63]Open in a new tab
a) Schematic of PADM@CZ hydrogel synthesis and b) the dual‐temporal
bidirectional immunomodulatory effects of PADM@CZ.
2. Results and Discussion
2.1. Characterization of Hydrogel
TEM images revealed the apparent spherical core–shell structure of the
Cu–Zn BGns, with Cu ions dominating the outer layer as identified by
energy dispersive spectroscopy (EDS), and Zn ions primarily located in
the inner layer (Figure [64]S1a,b, Supporting Information). Dynamic
light scattering (DLS) analysis confirmed that the Cu–Zn BGns had a
narrow size distribution with an average hydrodynamic diameter of
229.8 nm (Figure [65]S1c, Supporting Information).
Hematoxylin and eosin (HE) and 4′6‐diamino‐2‐phenylindole (DAPI)
staining confirmed the complete removal of porcine skin cells with an
intact extracellular structure (Figure [66]S2a,b, Supporting
Information), consistent with a previous study.^[ [67]^17 ^]
Representative SEM images of PADM and PADM@CZ (1%, 5%, and 10%) are
presented in Figure [68] 1a. Similar to the ECM and tendon hydrogels,
the PADM and PADM@CZ hydrogels had a reticular and closely packed fiber
architecture, thereby indicating that extracellular collagen digested
by pepsin could reassemble into collagen‐like fibers.^[ [69]^18 ^] In
addition, the SEM results confirmed the incorporation of Cu–Zn BGns
into the PADM@CZ hydrogel. However, at higher particle concentrations,
particularly at 10%, the original structure of the PADM became
obscured, which could potentially impact its functionality.
Consequently, we tested the mechanical properties of hydrogels at
varying particle concentrations (Figure [70]1b). Hydrogels
incorporating 1% and 5% Cu–Zn BGns, particularly the latter, exhibited
superior mechanical strength compared to pure PADM hydrogels. However,
the inclusion of 10% particles reduced the mechanical strength of the
PADM hydrogels. Consequently, PADM@CZ (5%) was selected for subsequent
analysis. Additionally, throughout the rheometer test, the storage
modulus (G′) of the hydrogels remained constant and consistently higher
than their loss modulus (G′′), indicating stable gelation and favorable
mechanical properties (Figure [71]1c).
Figure 1.
Figure 1
[72]Open in a new tab
Characterization of Cu–Zn BGns and PADM@CZ hydrogel. a) SEM images of
various PADM@CZ hydrogels (1%, 5%, and 10%). b) Typical compressive
stress–strain curves of various PADM@CZ hydrogels (1%, 5%, and 10%). c)
Storage modulus (G′) and loss modulus (G′′) of PADM, PADM@Zn, and
PADM@CZ hydrogels. d) FTIR spectra, e) XRD patterns of Zn‐BGns,
Cu‐Zn‐BGns, and various hydrogels. XPS spectra of the fully scanned
region f) and the g) Zn 2p region and Cu 2p region of PADM@CZ
hydrogels. h,i) Concentration of released Cu^2+ and Zn^2+ of the h)
PADM@Zn and i) PADM@CZ. j) Cumulative release of Cu^2+ and Zn^2+ in
PADM@CZ.
Fourier‐transform infrared analysis (FTIR) was employed to characterize
the PADM and PADM@CZ hydrogels (Figure [73]1d). FITR spectra of the
PADM hydrogels showed peaks corresponding to collagen fibers within the
PADM, including amide I at 1650 cm^−1, II at 1550 cm^−1, and III at
1240 cm^−1.^[ [74]^19 ^] The FTIR spectrum of the PADM@CZ hydrogel
showed no noticeable differences or shifts in the bands, specifically
the amide I band corresponding to the triple helical morphology of the
collagen. The characteristic peaks of BGns were at 812 cm^−1 due to the
symmetric stretching vibrations band of Si─O─Si, and at 1299–900 cm^−1
due to the asymmetric vibration bands of Si─O─Si (bridging bonds) and
the Si─O─ (non‐bridging bonds).^[ [75]^20 ^] Therefore, the FTIR
spectra validated the successful fabrication of PADM@CZ hydrogels. XRD
analysis also provides information on the axial arrangement of collagen
fibrils and the three‐stranded helical conformation of the molecules.^[
[76]^21 ^] Meanwhile, the PADM is primarily composed of type I
collagen. The XRD patterns (Figure [77]1e) of the PADM before and after
incorporating Cu–Zn BGns showed similar curves. In addition, no
significant changes were observed in the shape or positions of the
three diffraction peaks specific to PADM. The XRD results revealed that
the incorporation of Cu–Zn BGns into the PADM hydrogel did not
deteriorate the natural conformation, retaining the biological
properties of PADM. Moreover, X‐ray photoelectron spectroscopy (XPS)
suggested the presence of Cu and Zn elements in the C, N, and O
matrices of PADM, indicating the incorporation of Cu–Zn BGns
(Figure [78]1f). The relatively low XPS spectra of Cu 2p and Zn 2p
showed that the Cu–Zn BGns were embedded into the inner framework of
the PADM@CZ hydrogel (Figure [79]1g), which could not be fully
confirmed by XPS surface element analysis.
The water retention properties of the PADM and PADM@CZ hydrogels are
presented in Figure [80]S2c (Supporting Information). Both hydrogels
lost all their water content after approximately 30 h at the
restrictive temperature (37°C), with no significant differences between
them. Thus, incorporating the Cu–Zn BGns did not alter the
water‐retention properties of the PADM hydrogel. Hence the PADM
hydrogel should be capable of retaining moisture in the wound and
promoting wound healing for a longer duration.^[ [81]^22 ^]
Furthermore, the hydrogels might have the capacity to absorb some of
the leaked fluids from the wound defects, thereby promoting wound
healing.^[ [82]^23 ^]
Moreover, an increased rate of PADM and PADM@CZ hydrogel degradation
was observed with increasing temperature (Figure [83]S2d, Supporting
Information). Both hydrogels completely degraded when incubated at 42
°C for 2 d. However, at the same time points, the blank and PADM@CZ
hydrogels at 25 °C and 37 °C retained 0.46 and 0.54 g, respectively.
Next, the hydrogels were digested with trypsin to analyze the rate of
hydrogel degradation. Figure [84]S2e (Supporting Information) shows the
minimum difference in the rates of PADM and the PADM@CZ hydrogel
degradation in trypsin and phosphate‐buffered saline (PBS). The
similarity in the degradation rates can be attributed to the fact that
Cu–Zn BGns and PADM hydrogels primarily interact via adsorption and the
enzymes failed to degrade nanoparticles.^[ [85]^24 ^] However, a
significant increase in the rate of hydrogel degradation was observed
with trypsin compared with PBS alone. This can be attributed to the
decomposition of decellularized porcine skin into collagen fibers
during hydrogel preparation and the presence of proteins degraded by
trypsin in the PADM hydrogel.^[ [86]^25 ^] These results indicate that
the proteins in hydrogels can be degraded by locally secreted
proteases, thereby promoting the secretion of the active ingredients.
Indeed, Cu^2+ and Zn^2+ were sequentially released from the PADM@CZ
hydrogels (Figure [87]1h,i). The Cu^2+ concentration was higher during
the early stage, specifically on Day 1, compared to Zn^2+. However,
after Day 2, the Zn^2+ concentration was higher than that of Cu^2+.
Notably, it was not until day 5 that the cumulative concentration of
Cu^2+ slightly decreased below that of Zn^2+ (Figure [88]1j). Hence,
upon exposure to the solution, the outer porous layer of Cu degraded,
followed by slow degradation of the dense Zn layer.
2.2. Antibacterial Activity and Mechanisms of PADM@CZ Hydrogel
Methicillin‐resistant Staphylococcus aureus (MRSA) was used to test the
antibacterial properties of the PADM@CZ hydrogel in vitro. First, the
growth curves of the bacteria were plotted under different conditions
based on the absorbance of the bacterial suspension at the onset. The
results revealed a significant reduction in the amplification rates of
bacteria treated with the PADM@CZ hydrogel. However, treatment with the
PADM@Zn hydrogel yielded limited inhibitory effects on bacterial growth
(Figure [89]S3, Supporting Information). Next, cells from different
treatment groups were stained with live/dead dyes and observed under a
fluorescence microscopy (Figure [90] 2a). Significant cell death and
red staining of MRSA were observed in the PADM@CZ group. Furthermore,
relatively few dead bacterial cells were observed in the control and
PADM@Zn groups, thereby indicating that the PADM@Zn hydrogel exerted a
weak bactericidal effect. Next, we used the spread‐plate method (SPM)
to quantify the colonies. The survival rate of bacteria in the PADM@CZ
group decreased from 100.15% ± 6.45% to 23.85% ± 6.39%, whereas no
significant reduction in the survival rate was observed in the other
groups (Figure [91]2b; Figure [92]S4a, Supporting Information). These
results revealed that the PADM@CZ hydrogel exhibited a strong
bactericidal activity, which was primarily attributed to the presence
of Cu^2+ rather than Zn^2+.
Figure 2.
Figure 2
[93]Open in a new tab
In vitro antibacterial property of PADM@CZ. a) MRSA viability following
different interventions using live/dead staining. The green
fluorescence is indicative of live bacteria while the red fluorescence
represents dead bacteria. Scale bar: 100 µm. b) Survival rates of MRSA
following incubation with different treatments. Scale bar: 100 µm. c–e)
Assessment of ROS generation was conducted using DCFH‐DA fluorescent
probes. f,g) Detection of membrane damage in MRSA resulting from
different treatments via monitoring PI influx. h) MRSA morphology was
examined through representative TEM images after employing different
treatments. The red arrow signifies the compromised bacterial cell
membrane. Scale bar: 20 µm (upper panel); 500 nm (lower panel). The
depicted schematic offers insights into the antibacterial properties of
PADM@CZ against MRSA. b,d,g) Data are presented as mean ± SD (n = 3 per
group), with “n” denoting biologically independent experiments.
A previous study has shown that Cu^2+ uses several mechanisms to kill
bacteria, including the production of reactive oxygen specifies (ROS)
to disrupt cell membranes, inhibit metabolic processes, and induce DNA
fragmentation to kill the bacteria.^[ [94]^26 ^] Therefore, we first
determined the involvement of ROS in the antibacterial activity using
dichloride‐fluorescein diacetate (DCFH‐DA) to identify the underlying
bactericidal mechanism of the PADM@CZ hydrogels. Flow cytometry results
showed that the PADM@CZ hydrogel increased ROS levels in the bacteria
(Figure [95]2e). The intensity of DCFH‐DA fluorescence in the PADM@CZ
group was 2.1‐, 3,3‐, and 3.6‐fold higher than that in the other
groups, respectively. These results were validated by fluorescence
microscopy (Figure [96]2c,d). Next, we used propidium iodide (PI, a
nucleic acid‐binding dye) to determine whether the PADM@CZ hydrogel
could disrupt cell membranes. The results revealed that PADM@CZ
hydrogel significantly increased the permeability of the bacterial cell
membranes (Figure [97]2f).
In the PADM@CZ group, the PI intensity increased by 32.64%, 31.62%, and
27.6%, respectively, compared with the bacteria in the control, PADM,
and PADM@Zn groups, respectively. Hence, the PADM@CZ hydrogel treatment
significantly disrupted the cell membranes of the bacteria. Finally, we
quantified the fluorescence intensity of PI using a fluorescence
microplate reader; the results were consistent with those of flow
cytometry (Figure [98]2g). Additionally, MRSA was treated with
different hydrogels, and the absorbances of nucleic acids and proteins
were measured at 260 nm (A260) and 280 nm (A280) to determine cell
leakage. A detectable increase in the absorbance of nucleic acids and
proteins at 260 nm and 280 nm was observed in the PADM@CZ group
compared to the control, PADM, and PADM@Zn groups (Figure [99]S4b,
Supporting Information). Furthermore, TEM images showed shrunken,
blurred borders and crumpled appearance of bacterial membranes in the
PADM@CZ group. Smooth, intact, and well‐defined cell membranes were
observed in the control, PADM, and PADM@Zn groups (Figure [100]2h).
These results demonstrated that the PADM@CZ hydrogels exerted a good
anti‐bacterial effect in vitro. That is, the metal ions are degraded
and continuously released by the PADM@CZ hydrogels in an acidic
microenvironment during bacterial infection. During the early stages of
infection, Cu^2+ is predominantly released, along with trace amounts of
Zn^2+. These metal ions, specifically Cu^2+, act as powerful
bactericidal agents and disrupt bacterial cell membranes, resulting in
leakage of cellular contents and alterations in biochemical processes.
Subsequent induction of ROS production induces DNA damage, leading to
cell death (Figure [101]2i).
2.3. Immunomodulatory Role of the PADM@CZ Hydrogel
Macrophages are important effectors of the innate immune system and are
involved in immune regulation and elimination of microbes, thereby
defending the body against infections.^[ [102]^6 , [103]^27 ^] Studies
have shown that polarized macrophages are divided into M1 and M2
types.^[ [104]^28 ^] Proinflammatory M1 macrophages aid in forming a
proinflammatory microenvironment by producing several proinflammatory
cytokines, including tumor necrosis factor‐α (TNF‐α), IL‐6, and
specific surface markers or proteins (CCR7 and inducible nitric oxide
synthase [iNOS]).^[ [105]^29 ^] In contrast, M2 macrophages create an
anti‐inflammatory microenvironment by secreting anti‐inflammatory
cytokines, such as IL‐4, CD206, and IL‐10.^[ [106]^30 ^] Meanwhile,
bacterial biofilm secretions, including extracellular polymers, etc.,
can impede bacterial clearance and prevent the M1 polarization of
macrophages, thereby suppressing (or silencing) the immune response.^[
[107]^31 ^] Additionally, excess toxins secreted by bacteria are
detrimental to the activity and function of macrophages, suppressing
local immune responses against infections.^[ [108]^32 ^] Thus,
reversing the immunosuppressive microenvironment at the infection site
by stimulating M1 macrophage polarization may be a promising approach
for eliminating infections.
We next explored the immunomodulatory effects of the various hydrogels
on macrophages. In the PADM@CZ group, a significant increase in the
secretion of IL‐6 and TNF‐α by macrophages was observed on Days 1–7,
and a decrease in the secretion of IL‐1β and IL6 was observed on Days
9–13 (Figure [109]5a,b). Furthermore, an increase in the secretion of
IL‐4 and IL‐10 was observed on Days 9–13 in the PADM@CZ group (Figure
[110]S5c,d, Supporting Information). These results demonstrated that
the PADM@CZ hydrogel could transition from a pro‐ to an
anti‐inflammatory immune response over time.
Figure 5.
Figure 5
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Assessment of the bactericidal impacts of different groups on MRSA
through macrophage‐mediated mechanisms. a) Representative fluorescence
staining of the bacterial phagocytosis formed by the MRSA (green; the
white arrow points to the bacteria inside the cell) phagocytosed by
macrophages (red) cultured with various samples (left panel: Day 5
hydrogel extracts; right panel: Day 11 hydrogel extracts). Scale bar:
40 µm. b) Flow cytometry plots depicting the phagocytic clearance of
bacteria by macrophages cultured with various samples (left panel: Day
5 hydrogel extracts; right panel: Day 11 hydrogel extracts). c)
Quantitative analysis of MRSA colonies that had survived phagocytosis
by macrophages. d) Quantification of fluorescence intensity after
phagocytosis of MRSA by macrophages cultured with various samples. e)
Schematics illustrating the ability of PADM@CZ to sustain the
proinflammatory phenotypes of macrophages. c,d) Data presented are
expressed as mean ± SD (n = 3 per group), with “n” denoting
biologically independent experiments.
RNA‐sequencing (RNA‐seq) was performed on RAW 264.7 cells treated with
Day 5 and 11 PADM@CZ hydrogel extracts for 24 h. Correlation analysis
showed a high level of consistency among samples in each treatment
group, suggesting comparable gene expression between the groups (Figure
[112]S6a,d, Supporting Information). We then screened for
differentially expressed genes (DEG) as per the following criteria:
P < 0.05 and fold change (FC) ≥ 2. A total of 1028 DEGs were identified
in the Day 5 PADM@CZ group, compared to the control group, of which 743
were upregulated, and 285 were downregulated (Figure [113]S6c,
Supporting Information). In the Day 11 PADM@CZ group, 199 DEGs were
identified, of which 95 were upregulated and 104 were downregulated
(Figure [114]S6f, Supporting Information). Finally, heat maps of the
DEGs from the Day 5 PADM@CZ and Day 11PADM@CZ groups compared to the
corresponding control groups were constructed (Figure [115] 3a,b),
revealing an increase in CCR7 and iNOS (M1 macrophage markers)
expression and a decrease in the expression of CD206 and IL‐10 (M2
macrophage‐related genes) in the Day 5 PADM@CZ group. In contrast, in
the Day 11 PADM@CZ group, an increase in CD206 and IL‐10 and a decrease
in CCR7 and iNOS expression was observed, indicating that macrophages
treated with PADM@CZ hydrogels were polarized to an anti‐inflammatory
M2 type.
Figure 3.
Figure 3
[116]Open in a new tab
Gene expression analysis of RAW 264.7 macrophages in response to the
PADM@CZ. a,b) Microarray heat map visualizing the fold change in
expression of cell‐specific genes co‐cultured with PACM@CZ samples on
Days a) 5 and b) 11. c) Pathways upregulated in cells co‐cultured with
PACM@CZ samples on Day 5 were analyzed using the KEGG pathway method.
d) Pathways downregulated in cells co‐cultured with PACM@CZ samples on
Day 11 were analyzed using the KEGG pathway method. e) Fold change in
the expression of genes related to the NOD‐like receptor signaling
pathway in cells co‐cultured with PACM@CZ samples on Day 5. f) Fold
change in the expression of genes related to the MAPK signaling pathway
in cells cocultured with PACM@CZ samples on Day 11. g) Schematic
illustration of the dual‐temporal bidirectional immunomodulatory
effects of PADM@CZ.
Next, we performed the Kyoto Encyclopedia of Genes and Genomes (KEGG)
pathway enrichment analysis. Results showed that pathways regulating
macrophage polarization were enriched in cells treated with the PADM@CZ
hydrogel. Specifically, M1 pathways, namely, the NOD‐like and NF‐κB
signaling pathways, were upregulated significantly after treatment with
the Day 5 PADM@CZ sample (Figure [117]3c). In addition, the TNF
signaling pathway was significantly upregulated. Meanwhile, in
macrophages of the Day 11 PADM@CZ group, a decrease in the MAPK
pathway, which regulates M2 polarization, was observed, indicating
polarization toward the M2 phenotype (Figure [118]3d). Thus, the
microarray results revealed that the PADM@CZ hydrogel could induce M1
macrophage polarization at an early stage and induces M2
macrophage‐specific gene expression at later stages.
We performed real‐time polymerase chain reaction (RT‐PCR) to determine
the expression of several genes and validate the immunomodulatory
effects of the PADM@CZ hydrogel (Figure [119] 4a). RT‐PCR results
revealed a significant increase in the expression of IL‐6, CCR‐7, and
iNOS, and a decrease in that of CD206 and IL‐10, in macrophages
cultured with Day 5 PADM@CZ hydrogel. However, the opposite results
were observed for the macrophages co‐cultured with the Day11 PADM@CZ
hydrogel. That is, a significant decrease in the expression of M1
macrophage‐related genes and an increase in the expression of M2
macrophage‐related genes were observed. Immunofluorescence and flow
cytometry were performed to investigate macrophage phenotypes
(Figure [120]4b–g). A pro‐inflammatory microenvironment was established
on Day 5 using the PADM@CZ hydrogel by activating M1 macrophage
polarization and stimulating cytokine secretion. On Day 11, M2
macrophage polarization and anti‐inflammatory cytokine secretion were
induced in PADM@Zn and PADM@CZ hydrogels. Furthermore, the PADM@CZ
hydrogel regulated M1 macrophage polarization to M2 from Day 5 to 11,
demonstrating dual‐temporal bidirectional immunomodulatory effects.
These results highlighted the potential use of the PADM@CZ hydrogel for
preventing infections and promoting tissue regeneration.
Figure 4.
Figure 4
[121]Open in a new tab
The potential immunomodulatory impact of PADM@CZ hydrogels on
macrophages. a) RT‐PCR analysis results of IL‐6, CCR7, IL‐10, and
CD206. b) Immunofluorescent staining on RAW264.7 cells that were
cultured in extracts from Days 5 and 11 samples for iNOS (green), CD206
(red), and DAPI (blue). Scale bar: 20 µm. c) Scatter plots depicting
the RAW264.7 cellular surface markers CCR7 (indicative of M1
macrophages) and CD206 (demonstrative of M2 macrophages) were analyzed
via flow cytometry. Quantification of the ratio of d) iNOS and e) CD206
positive cells accomplished via immunofluorescent staining. Positive f)
CCR7 and g) CD206 cell proportion. a,d,e–g) Data are presented as mean
± SD (n = 3 per group), with “n” denoting biologically independent
experiments.
The PADM@CZ hydrogel exhibited a dual‐temporal bidirectional
immunomodulatory activity in response to varying Cu^2+ and Zn^2+
concentrations over time. However, the mechanisms underlying the
regulation of regulating macrophage phenotypes by Cu^2+ required
further investigation. Cu^2+ reportedly promotes M1 macrophage
polarization and proinflammatory cytokine secretion.^[ [122]^33 ^]
Interestingly, several studies have analyzed the response of
macrophages to Cu^2+; however, the conclusions are conflicting. These
discrepancies are likely primarily attributed to the Cu^2+
concentration. For instance, Storkánová et al. showed an increase in
the expression of IL‐10 and CD206 after treatment with low Cu^2+
concentrations (<0.64 ppm). O In contrast, a high Cu^2+ concentration
promotes the secretion of proinflammatory factors, such as TNF‐α and
CCR7, suggesting macrophage polarization toward the M1‐type.^[ [123]^34
^] In the current study, macrophages treated with Day 5 PADM@CZ
hydrogels (approximately 1.4 ppm Cu^2+), exhibited an increase in
M1‐macrophage‐related markers, which is consistent with the
relationship between the presence of Cu^2+ and its effect on M1
macrophages.
In contrast, Zn^2+ induces M2 macrophage polarization and the secretion
of anti‐inflammatory cytokines.^[ [124]^35 ^] However, Zn^2+ exerts
immunomodulatory effects in a concentration‐dependent manner. High
Zn^2+ concentrations (>65.13 ppm) promoted M1 macrophage activation,
whereas low Zn^2+ concentrations promoted the secretion of
anti‐inflammatory cytokines and inhibited proinflammatory cytokine
expression. A similar phenomenon was observed in the present study.
PADM@CZ hydrogels demonstrated slight proinflammatory effects on Day 5
(2.8 ppm Zn^2+), however, failed to mediate strong proinflammatory
effects, like Cu^2+. Furthermore, on Day 11 (0.83 ppm), as the Zn^2+
concentration decreased in PADM@CZ hydrogels, a significant increase in
the secretion of anti‐inflammatory cytokines was observed.
2.4. Macrophage‐Mediated Antibacterial Activity
Given that macrophages eliminate infections and necrotic tissues and
induce tissue repair via phagocytosis,^[ [125]^36 ^] we investigated
the effects of the PADM@CZ‐activated macrophages on bacteria. To
evaluate the effect of PADM on macrophage phagocytosis, CFDA‐SE‐labeled
MRSA was cultured with macrophages under different conditions. In
macrophages treated with the Day 5 PADM@CZ hydrogel, a significant
improvement was observed in viability and an increase in the number of
bacteria captured and phagocytosed by bacteria (green fluorescence) was
observed compared to the control, PADM, and PADM@Zn groups
(Figure [126] 5a, left panel; Figure [127]S7a, Supporting Information,
upper panel). In contrast, no differences were observed in cell
viability, or the number of bacteria captured or phagocytosed by
macrophages following treatment with Day 11 extracts (PADM@CZ, PADM,
control, and PADM@Zn) was observed (Figure [128]5a, right panel; Figure
[129]S7b, Supporting Information, upper panel). Meanwhile, flow
cytometry results revealed that in contrast to macrophages in the
control group, those in the Day 5 PADM@CZ group exhibited an increase
in the rate of MRSA phagocytosis from 4.40% to 47.8% (Figure [130]5b).
However, no difference in the rate of MRSA phagocytosis was observed in
macrophages treated with the day 11 extracts. Next, the MRSA was
cultured with macrophages for 1 h, lysed, and the number of bacteria
phagocytosed by macrophages was determined. The trend in the number of
bacteria phagocytosed by macrophages in all groups was consistent with
confocal laser scanning microscopy (CLSM) observations. That is, a
significantly higher number of bacteria was phagocytosed by macrophages
in the Day 5 PADM group than in the other groups. Macrophages engulf
and kill bacteria through various mechanisms, such as lysosomal
degradation.^[ [131]^37 ^] Therefore, SPM was used to determine the
bactericidal ability of macrophages in the cell‐bacterial co‐culture
system. A significantly reduced number of colonies were formed by
macrophages in the Day 5 PADM group, indicating the enhanced ability of
macrophages in the 5 d PADM group to eliminate bacteria (Figure
[132]S7a, Supporting Information, lower panel). Based on the
quantitative analysis, a significant decrease in the survival rates of
bacteria from 89.5% ± 2.8% (control group) to 38.7% ± 3.6% was observed
in the Day 5 PADM group (Figure [133]5c). Hence, the PADM@CZ hydrogel
modulated macrophage activity, affecting its antibacterial ability.
The increase in antimicrobial activity was primarily due to M1
macrophage polarization induced by Cu^2+ released from PADM@CZ.
Mounting evidence has shown that Cu^2+ is significantly involved in
innate immune responses. Furthermore, adequate exogenous
supplementation with Cu^2+ significantly enhances the phagocytic
activity and bactericidal ability of immune cells, such as macrophages
(Figure [134]5e).^[ [135]^8 , [136]^38 ^] Hence, low Cu^2+
concentrations alter the activity and function of the macrophages,
thereby decreasing their antibacterial activity of macrophages. This
could be the primary cause of the low phagocytic and bactericidal
abilities of the macrophages treated with Day 11 PADM hydrogels.
However, previous studies have shown that excess inflammatory responses
induced by high Cu^2+ concentrations can compromise the antimicrobial
functions of macrophages.^[ [137]^39 ^] Therefore, the PADM@CZ hydrogel
represents an “immunosuppressive self‐rescuing” biomaterial that
initially secretes high levels of Cu^2+, followed by Zn^2+ secretions,
thus activating macrophages without compromising their functions.
However, additional studies are required to determine the precise
“immunosuppressive self‐rescuing” mechanism of the PADM@CZ hydrogel.
This would aid in determining why PADM@CZ, but not PADM@Zn, can
effectively inhibit infection via macrophages.
2.5. In Vitro Biocompatibility
Biocompatibility is an important criterion for the clinical use of
hydrogels for wound dressing.^[ [138]^40 ^] Accordingly, we determined
the in vitro biocompatibility of the hydrogels using a Cell Counting
Kit (CCK‐8) assay to analyze the morphology of MC3T3‐E1 cells and
live/dead staining. CCK‐8 assay results revealed that the hydrogels
exerted no cytotoxic effects. Furthermore, the cells in all groups
showed similar proliferative behaviors after 3 and 5 d, suggesting that
the hydrogels promoted cell proliferation (Figure [139]S8a,b,
Supporting Information). Additionally, the morphology of the cells was
good (Figure [140]S8c, Supporting Information). After the cells were
cultured on different hydrogels for 24 h, live/dead staining was
performed. The living cells were stained green, and the nuclei of dead
cells were stained red. Figure [141]S8d (Supporting Information) shows
that most cells were viable after culturing with the control, PADM,
PADM@Zn, or PADM@CZ hydrogels for 24 h. Taken together, these results
indicate that the PADM@CZ hydrogels are biocompatible and facilitated
cell adhesion, spreading, and proliferation.
2.6. Promotion of Bacteria‐Associated Wound Healing by the PADM@CZ Hydrogel
Extended periods of non‐healing wounds increase the likelihood of
bacterial infections. Furthermore, such infections may compromise
immune function, which adversely affects wound healing, culminating in
pervasive infections.^[ [142]^41 ^] Hence, expeditious wound healing is
imperative for the prevention and treatment of infections. The ADM@CZ
hydrogel regulated the phenotype of immune cells in vitro via unique
ion‐releasing kinetics, thereby effectively eliminating bacterial
infections. Next, we used a mouse model of excisional skin infection to
determine the effect of the PADM@CZ hydrogel on wound healing
(Figure [143] 6a). The infected wounds gradually healed over time in
various treatment groups (Figure [144]6b). However, significant
differences in the extent of infection and the healing rate were
observed. Macroscopic infection, foci of tissue necrosis, and pus
exudation were observed in the wounds of the control, PADM, and PADM@Zn
groups, with severe infection observed on Day 4. However, when the
infection was under control, the wounds crusted and healed. In the
PADM@CZ group, the wounds were consistently treated, remained clean,
and were dry throughout the observation period. No signs of infection
or pus formation were observed. Moreover, in mice in the PADM@CZ group,
enhanced wound healing was observed, with only 7.1 ± 7.5% unhealed
wound detected on Day 14, followed by the PADM@Zn (7.50% ± 0.59%), PADM
(7.50% ± 0.59%), and control (14.10% ± 2.33%) groups (Figure [145]6c).
In addition, SPM was performed on Days 4 and 10 to evaluate the
bactericidal activity of PADM@CZ against bacteria on the wound surface.
A significant decrease in the survival rates of the bacteria was
observed upon treatment with the PADM@CZ hydrogel, and relatively
complete elimination of the bacteria was observed on Day 10
(Figure [146]6d; Figure [147]S9, Supporting Information).
Figure 6.
Figure 6
[148]Open in a new tab
PADM@CZ exhibits enhanced efficacy in promoting the healing of infected
wounds in vivo. a) Illustration of the model for mouse excisional wound
infection and associated experimental methods. b) Illustrative
photographs of the wounds encompassing diverse treatment methodologies,
alongside superimposed pictures on Days 0 (blue), 4 (orange), 7
(purple), 10 (yellow), and 14 (green). c) Area of wounds that remained
unhealed at different intervals for each group. d) Quantity of bacteria
present in the wounds across different treatment groups on Days 4 and
10. c, d) Data presented are expressed as mean ± SD (n = 3 per group),
with “n” denoting biologically independent experiments.
Wound healing is a modular process that can be classified into the
following phases: hemostasis, inflammation, proliferation, and
remodeling.^[ [149]^41b ^] Histological analysis was performed using HE
staining; the results were consistent with the wound area (Figure [150]
7a). Additionally, the length of granulation tissue in mice belonging
to different groups was measured (Figure [151]7c). Faster wound
contraction was observed in the PADM@CZ group than in the other groups
and was prominent during the early stages of wound healing. The
statistical analysis of granulation tissue width revealed that wound
contraction exhibited a higher rate in the PADM@CZ group compared to
the other groups. During the remodeling stage, it is crucial to ensure
adequate collagen deposition and remodeling to enhance the tensile
strength of the tissue and facilitate optimal healing. Hence, Masson's
trichrome staining was performed to examine the emerging collagen
fibers. On Day 14, persistent scabs consisting of dried blood, serum,
and exudates were evident within the control group, suggesting delayed
wound healing (Figure [152]7b). In contrast, the wounds that received
the PADM@CZ hydrogel treatment exhibited a distinct epidermal layer and
a notably elevated collagen volume fraction (Figure [153]7d). This
suggests that the regenerating tissue resembled healthy skin and
demonstrated enhanced healing efficacy. Angiogenesis is required for
nutrients and oxygen supply. Hence, timely and adequate angiogenesis is
required for wound healing.^[ [154]^42 ^] Angiogenesis analyzed the
abundance of neo‐angiogenesis markers, i.e., cell adhesion molecule1
(CD31) and α‐smooth muscle actin (α‐SMA), in wound sections of mouse
skin.^[ [155]^43 ^] An increase in CD31 and α‐SMA expression was
observed in the wound tissues of mice in the PADM@CZ group compared
with the other groups (Figure [156]S10, Supporting Information). These
results demonstrated the superior wound‐healing ability of the PADM@CZ
hydrogels.
Figure 7.
Figure 7
[157]Open in a new tab
PADM@CZ hydrogel‐induced acceleration of the wound repair. a) H&E
staining of wound skin tissues on Days 4 and 14. The green arrows
indicate the re‐epithelialization area, and the yellow arrows indicate
the newly formed dermal. Scale bar: 500 µm. b) Masson's trichrome
staining of wound skin tissues on Days 4 and 14. The yellow arrows
indicate the newly formed dermal. Scale bar: 500 µm. c) Corresponding
quantitative analysis of the granulation tissue width on Day 14. d)
Statistical analysis of collagen deposition during the remolding phase.
c,d) Data presented are expressed as mean ± SD (n = 3 per group), with
“n” denoting biologically independent experiments.
2.7. Effects of PADM@CZ Hydrogel on Immune Regulation In Vivo
Macrophages serve as first responders during tissue damage and are key
players in mediating the local immune response to infection. Therefore,
we investigated the immunomodulatory effects of the PADM@CZ hydrogel on
macrophages at various stages of wound healing. The wounded skin was
harvested after 4 and 14 d of treatment under different conditions, and
the proportion of macrophages was analyzed using flow cytometry
(Figure [158] 8a–c). On Day 4, a significant increase in the proportion
of M1 macrophages (CCR7^+CD206^−) and a decrease in the proportion of
M2 macrophages (CD206^+CCR7^−) were observed in the PADM@CZ group.
However, by Day 14 of treatment, the PADM@CZ hydrogel induced M2
macrophage polarization. Immunofluorescence was performed on wounded
skin at the same time points for iNOS and CD206. The results validated
the observed macrophage polarization trend (Figure [159]8d–f). That is,
the PADM@CZ group had the highest M1/M2 ratio on Day 4 and M2/M1 ratio
on Day 14 among all groups. Moreover, the observed patterns in
inflammation‐related markers, including IL‐6 and TNF‐α (Figure
[160]S11, Supporting Information), exhibited congruence with the
polarization state of macrophages.
Figure 8.
Figure 8
[161]Open in a new tab
Impact of the PADM@CZ hydrogel on macrophage polarization in vivo. a)
Flow cytometry data of M1 and M2 cells retrieved from the wound tissue
4‐ and 14 d post‐injury. b,c) Statistical analysis concerning the
proportion of b) M1 and c) M2 macrophages. d) Immunofluorescence
staining of tissue sections at the wound site for iNOS (M1 marker) and
CD206 (M2 marker) on Days 4 and 14 subsequent to the injury. Scale bar
250 µm. e,f) Statistical analysis concerning the proportion of e) iNOS
and f) CD206 positive macrophages. b,c,e,f) Data presented are
expressed as mean ± SD (n = 3 per group), with “n” denoting
biologically independent experiments.
Furthermore, the immune cell composition in the spleen was investigated
during wound treatment under various conditions. On Day 4, the
CD8^+/CD4^+ ratio was significantly higher in the spleens of mice in
the PADM@CZ group, indicating that the PADM@CZ hydrogel improved
general immunity (Figure [162]S12a,b, Supporting Information). An
activated immunity is beneficial to antibacterial effects, which is
mainly attributed to Cu^2+ released by the PADM@CZ hydrogels.^[
[163]^44 ^] However, on Day 14, the CD8^+/CD4^+ ratio significantly
decreased in the PADM@CZ group compared to Day 4, indicating that a
sustained release of Zn^2+ inhibited excess immune activation, thereby
accelerating wound healing. In addition, during the pre‐existing period
of the PADM@CZ‐treated group (Day 4), a notable augmentation in Th1
(CD4^+CCR5^+) infiltration and a substantial reduction in Tregs
(CD4^+Foxp3^+) infiltration were observed (Figure [164]S12c–f,
Supporting Information). These findings underscore the significance of
PADM@CZ in modulating the equilibrium between immune cells that promote
activation and those that suppress immune response, thereby augmenting
the effectiveness of humoral immunity. Furthermore, HE staining
revealed no abnormalities or damage to mouse major organs, indicating
that the PADM@CZ exhibited good biocompatibility in vivo (Figure
[165]S13, Supporting Information). Taken together, these results
suggest that the PADM@CZ hydrogel possesses good antibacterial and
immunomodulatory abilities in vivo and accelerates wound healing by
promoting angiogenesis.
The challenge of effectively treating infection‐associated wounds has
been a persistent concern for researchers. Despite the development of
numerous therapeutic approaches, the presence of stubborn bacteria and
dysregulated immune responses significantly impede their sustained
effectiveness.^[ [166]^45 ^] Macrophages, which play a pivotal role in
the processing of internal antigens and the initiation of adaptive
immunity against bacterial pathogens.^[ [167]^46 ^] M1 macrophages are
crucial in the initiation of pro‐inflammatory immune responses,
bacterial phagocytosis, antimicrobial agent release, and bacterial
antigen presentation.^[ [168]^47 ^] Nevertheless, the persistence of
infection prompts the polarization of immune cells toward an
anti‐inflammatory phenotype. This transition from M1 to M2 macrophages
is marked by impaired antigen processing and presentation.^[ [169]^45a
^] The bioactive ions released by PADM@CZ primarily consist of Cu^2+
and Zn^2+, both of which hold significant significance in immune
activity. The biological impacts of Cu^2+ are intricately linked to its
concentration; at suitable levels, Cu^2+ demonstrates efficacy in
eradicating bacteria and stimulating angiogenesis, thereby expediting
the process of wound healing. Nevertheless, an excessive immune
response can lead to a disruption of redox equilibrium, exacerbating
immune disorders and consequently perpetuating the persistence of
infection. Research has demonstrated that an imbalanced redox
microenvironment at the infection site not only facilitates the
formation of biofilms but also hinders the antimicrobial immune
response.^[ [170]^48 ^] Conversely, the interaction between Zn^2+ and
immune cells promotes the development of an anti‐inflammatory
microenvironment, alongside the beneficial angiogenic and tissue repair
attributes of Zn^2+. Hence, in the current investigation, it was
observed that PADM@CZ exhibited a sequential release of Cu^2+ and
Zn^2+. By conducting RNA sequencing on RAW264.7 macrophages, it was
discovered that PADM@CZ effectively stimulated antibacterial pathways,
particularly the NOD‐like receptor signaling pathway, during the
initial phase (Day 5). The in vitro assays demonstrated that PADM@CZ
could induce a pro‐inflammatory phenotype and enhance the phagocytosis
of RAW264.7 cells. The in vivo flow cytometry analysis demonstrated
that PADM@CZ exhibited the ability to induce macrophage M1 phenotype
polarization and enhance the CD8T/CD4T ratio during the early stage
(Day 4), which plays a crucial role in bacterial presentation and
immune activation. However, it is noteworthy that during the late
stage, PADM@CZ primarily modulates the immune system employing Zn^2+,
aiming to prevent excessive immune activation and mitigate the risk of
exacerbating immune disorders. Overall, this research will inspire
continued innovation in the design of biomaterials with
immunostimulatory effects until this new approach to immunotherapy is
implemented.
3. Conclusion
Herein, we developed a PADM@CZ hydrogel to promote wound healing
following infection by releasing Cu^2+ and Zn^2+ sequentially. In
vitro, the PADM@CZ hydrogel exhibited a good dual‐temporal
bidirectional immunomodulatory effect and regulates M1/M2 macrophage
polarization from Days 5 to 11 by alternately activating the NOD‐like
pathway and inhibiting MAPK signaling pathways. Furthermore, the
superior immunomodulatory, antibacterial, and transient direct
bactericidal abilities of the PADM@CZ hydrogel exerted synergistic
anti‐infection effects in the wound infection model. However, our study
did not comprehensively examine the mobilization and functions of
immune cells, such as DC cells and NK cells, in the initial phase of
wound healing in infected wounds, in addition to macrophages. Moreover,
the molecular mechanisms underlying these processes were not explored.
In our upcoming research phase, we will specifically focus on
investigating these aspects to gain a better understanding of the
factors that contribute to the enhancement of healing in infected
wounds through the use of biomaterials. Nevertheless, this novel
dual‐temporal bidirectional immunomodulatory hydrogel exhibits good
antibacterial and wound healing‐ effects and can be applied to design
and synthesize biomaterials that regulate immune responses based on the
immune profile of a target disease.
4. Experimental Section
Synthesis and Characterization of Cu–Zn BGns
A modified Stöber method was used to synthesize the Cu–Zn BGns.^[
[171]^49 ^] First, TEOS was mixed in ethanol (solution A) with ammonia,
deionized water (DI‐H[2]O), and ethanol (solution B) with stirring.
Calcium nitrate tetrahydrate was added after 30 min. Subsequently, zinc
nitrate was added, and the reaction was allowed to proceed for 90 min
before centrifugation at 8000 rpm for 15 min. The deposits were
collected, washed with DI‐H[2]O thrice, and calcined at 700 °C for 2 h
to obtain Zn‐BGns. Next, the Zn‐BGns were dissolved in DI‐H[2]O
(solution C) with ammonia, CTAB, ethanol, and DI‐H[2]O (solution D) by
stirring for 30 min, followed by a reaction with TEOS for 15 min. Next,
we added Cu/ascorbic acid complex precursors,^[ [172]^50 ^] and the
mixture was stirred for 24 h. The colloids were centrifuged at 8000 rpm
for 15 min, collected, washed with DI‐H[2]O twice and ethanol once, and
dried at 60 °C overnight. Finally, Cu–Zn BGns were calcined at 700 °C
for 2 h. The morphology and constituents of nanoparticles were
determined using SEM (S4800, Hitachi, Japan) and TEM (JEM2100, Hitachi,
Japan) with EDS. The chemical composition and state of the
nanoparticles were analyzed using XRD (Bruker, Billerica, USA), XPS
(ESCALAB 250Xi, Thermo Fisher, USA), and FTIR (Magna‐IR 750, Thermo
Fisher, USA). DLS measurements were performed by a Wyatt Mobius DLS
instrument.
Synthesis of PADM@CZ Hydrogel
After rinsing with sterile water for 3 h, the porcine skin tissue was
subjected to repeated freeze/thaw cycle procedures with liquid
nitrogen. The tissues were then agitated at 120 rpm and 25 °C to remove
the subcutaneous tissue. The samples were incubated with 0.1% Triton
X‐100/PBS (v/v) for 12 h and 0.1% sodium dodecyl sulfate for 6 h.
Subsequently, they were lyophilized, powdered, and digested with pepsin
in an acidic environment (pH 2‐3) for 10 min.^[ [173]^51 ^] Cu–Zn BGns
solution was added dropwise, and the mixture was quickly stirred. The
gel was stored at 4 °C and digested for 2 h until it became translucent
and viscous. Next, PBS was added, the osmotic pressure was adjusted,
and the pH was adjusted to 7‐8 using precooled 10 m NaOH. Finally, to
prepare PADM@CZ hydrogels, the gels were incubated at 37 °C for 20 min,
freeze‐dried, and compressed. The final products obtained, namely
PADM@CZ (1%), PADM@CZ (5%), and PADM@CZ (10%), were contingent upon the
mass differentials between the added particles and the lyophilized
hydrogel powder. The PADM and PADM@Zn hydrogels were prepared using the
same method, however, Cu–Zn BGns were not added or replaced with
Zn‐BGns.
Characterization of PADM@CZ Hydrogels: Characterization
HE and DAPI staining were performed to assess the removal of cellular
and nuclear particles. The samples were dehydrated using a
critical‐point dryer, and the structural characteristics of the
hydrogels were analyzed using SEM. Finally, the chemical compositions
of the materials were characterized using XPS, XRD, and FTIR
spectroscopy.
Mechanical Property Tests
To assess the mechanical properties of the hydrogels, their compression
capabilities were analyzed using a universal testing machine (Instron
5567, USA). Cylindrical hydrogels measuring 10 mm in height and 5 mm in
diameter were fabricated for testing. Compression tests were performed
at a controlled speed of 5 mm min^−1 until a maximum strain of 75% was
achieved. Rheological tests were performed using a rheometer (MARS 60,
USA) to measure the mechanical properties of the hydrogels. Hydrogel
circles with diameters of ≈1.5 cm were prepared for testing. The test
was performed with gap values of 1–2 mm, 1 Hz frequency, 1% strain, and
a 130 s time oscillation scan.
Water Retention
PADM, PADM@Zn, and PADM@CZ hydrogels were incubated at 37 °C. Their
masses were measured at set time points until no change in mass was
observed. Water retention in the hydrogels was calculated using
Equation ([174]1):
[MATH: Waterretentionrate=W2/<
msub>W1×100% :MATH]
(1)
where W [2] corresponds to the weight of the hydrogel at each time
point, and W [1] corresponds to its initial weight.
Biodegradability of Hydrogels
Next, the biodegradability of the PADM, PADM@Zn, and PADM@CZ hydrogels
were determined under various conditions. The samples were incubated in
microcentrifuge tubes at different temperatures (25 °C, 37 °C, or 42
°C) and stirred slowly, along with subsequent regular observation. The
liquid from the samples was aspirated, and the remaining samples were
weighed. Next, hydrogels were solidified and incubated in PBS, followed
by incubation with or without trypsin at 37 °C. The solution was
removed from the centrifuge tubes and the hydrogels were rinsed with
PBS daily. Finally, the samples were dried and weighed.
Measurement of Ion Release
To investigate the ion release behavior of the PADM@CZ hydrogel, the
same mass of each hydrogel was placed in 10 mL of simulated body fluid
(SBF, PH1820, Scientific Phygene, China). Next, the release medium was
completely removed and equal volumes of SBF were changed daily. The Cu
and Zn ion concentrations were measured using an inductively coupled
plasma atomic emission spectrometer (X Series 2, Thermo Fisher
Scientific, USA). The PADM and PADM@Zn hydrogels, which were used as
control groups, were evaluated using the same method.
Cells and Bacteria
Mouse‐derived macrophages (RAW264.7) and MC3T3‐E1 osteoblast precursor
cells were obtained from the Shanghai Institute of Cell Biology. Cells
were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented
with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. MRSA
(ATCC 43300) was purchased from the American Type Culture Collection
and was cultured in tryptic soy broth (TSB; Hopebio).
Preparation of Sample Extracts
The PADM, PADM@Zn, and PADM@CZ hydrogels were sliced into small
sections measuring 10 × 10 × 5 mm and immersed in DMEM supplemented
with 10% FBS and 1% penicillin/streptomycin. The cultures were
maintained at 37 °C for 14 d with daily medium renewal. To explore the
distinct impacts of each sample on cells, extracts were collected daily
during the incubation period and stored at 4 °C for subsequent
analysis.
In Vitro Antibacterial Tests: Direct Antibacterial Assay
MRSA frozen cultures were incubated in TSB at 37 °C overnight on a
shaker, diluted at a 1:10,000 ratio, and cultured until the logarithmic
growth phase. Subsequently, 500 µL of bacterial suspensions containing
1 × 10^6 colony forming units (CFUs) mL^−1 were cultured in hydrogel
extracts (Days 1, 2, 3, 4, and 5). The effects of the materials on
bacterial growth were evaluated using a growth curve assay and a
microplate reader. Absorbance was measured at 600 nm from 0 to 24 h.
All experiments were conducted in triplicates and repeated three times.
Next, we co‐cultivated bacteria with a diverse set of hydrogels for
24 h, followed by SPM and quantification of the CFUs on agar plates.
Additionally, we analyzed the antibacterial activity by live/dead
staining. Briefly, we added 500 µL of combined dye (Syto9 and PI) in
different samples and cultured them for 15 min. Bacteria were collected
on glass slides and visualized under a fluorescence microscope.
Apoptosis and damage to cell membranes were analyzed; detached bacteria
were stained for 15 min, centrifuged, washed with cold PBS, and
collected. The bacteria were analyzed by flow cytometry.
Estimation of ROS Levels
DCFH‐DA (a ROS probe) was used to measure ROS levels in cells. The
bacteria were cultured for 24 h and incubated with 10 × 10^−6 m DCFH‐DA
for 30 min. A fluorescence microscope was used to measure ROS levels in
the cells. The fluorescence intensity was measured with the aid of a
microplate reader at λ [ex]/λ [em] = 485 nm/525 nm. The ROS levels in
bacteria from all groups were measured in triplicates. Additionally,
the bacteria were stained and analyzed using a flow cytometer.
Membrane Damage and Leakage Assay
Bacteria were cocultured for 24 h, stained with 2.5 µg mL^−1 PI for
30 min, centrifuged, washed, and analyzed by flow cytometry. Similarly,
bacteria were incubated with PI for 30 min, and the fluorescence
intensity of these samples was measured at λ [ex]/λ [em] =
535 nm/615 nm using a microplate reader. Next, we evaluated the leakage
of cellular contents under various treatments. To this end, the
bacterial supernatant was centrifuged at a rate of 8000 × g for 10 min,
and the absorbance was measured at 260 nm (A260, nucleic acids) and
280 nm (A280, proteins) using a spectrophotometer. All experiments were
performed in triplicate.
TEM
TEM was performed to analyze changes in the ultrastructure of the
bacterial membranes. Briefly, bacteria (1 × 10^6 CFUs mL^−1) were
treated with various hydrogels for 24 h, resuspended in 1 mL of
fixative, and treated with osmium tetroxide. The bacteria were then
dehydrated using an ethanol gradient, embedded and cut using a diamond
knife. The resulting sections were placed on mesh copper mesh grids,
stained with lead citrate, and visualized by TEM.
In Vitro Immunomodulatory Assays: Enzyme‐Linked Immunosorbent Assay (ELISA)
RAW 264.7 cells were cultured with hydrogel extracts (Days 5 and 11)
for 24 h and the cell culture supernatants were harvested for measuring
IL‐1β, TNF‐α, IL‐6, and IL‐10 levels using ELISA (MultiSciences
Biotech, China).
Gene Expression
Microarray analysis was performed to determine the gene expression
patterns of macrophages. Briefly, RAW 264.7 cells were seeded at a
density of 5 × 10^5 cells per well and cultured with PADM@CZ hydrogel
extracts prepared on Days 5 and 11 for 24 h. Next, total cellular RNA
was isolated using TRIzol reagent based on the manufacturer's
guidelines. RNA‐seq and bioinformatics analysis were performed by
Shanghai Novelbio Ltd. The KEGG pathway and Gene Ontology enrichment
analyses were performed on the DEGs.
RT‐PCR
RAW 264.7 cells were cultured and collected after 24 h. Total RNA was
isolated using TRIzol. Gene expressions were normalized to housekeeping
genes (ACTB). The primer sequences are shown in Table [175]S1
(Supporting Information).
Immunofluorescence
Briefly, cells were incubated with Day 5 or 11 PADM, PADM@Zn, or
PADM@CZ hydrogel extracts for one day. Subsequently, the cells were
collected, fixed, and blocked before staining with antibodies against
iNOS (ab210823) and CD206 (ab64693). Next, cells were incubated with
corresponding secondary antibodies and counterstained with DAPI for
1 h. The cells were observed and photographed using CLSM.
Flow Cytometry Assay
Flow cytometry was performed using antibodies against CCR7 and CD206 to
determine the proportion of M1 and M2 macrophages. Briefly, macrophages
were cultured with Days 5 or 11 hydrogel extracts for 24 h, blocked for
30 min, and stained with allophycocyanin (APC)‐conjugated CD206 and
phycoerythrin (PE)/Cy7‐conjugated CCR7. Finally, the cells were washed,
resuspended in 500 µL of PBS, and analyzed by flow cytometry.
Macrophage‐Mediated Bactericidal Assay
Fluorescence‐labeled MRSA was used to quantitatively analyze the
phagocytosis of bacteria by macrophages treated with various hydrogels.
Briefly, 1 mL of 2 × 10^9 CFU mL^−1 MRSA was stained using 400 µL of
1 µg mL^−1 CFDA‐SE for 30 min in the dark. Excess dye was removed, and
the fluorescence‐labeled MRSA was resuspended in PBS for subsequent
experiments. Next, 1 × 10^5 RAW264.7 cells per well were treated with
Days 5 or 11 hydrogel extracts from the four groups for 24 h,
harvested, and cultured in new well plates. Next, RAW264.7 cells were
cultured with ≈1 × 10^7 CFU mL^−1 fluorescence‐labeled MRSA for 2 h and
stained with Dil (Beyotime) for 15 min, following the manufacturer's
instructions. Finally, the cells were washed and imaged using CLSM.
Following the same procedure, the efficiency of macrophage bacterial
phagocytosis was evaluated by flow cytometry without staining the cell
membrane. Additionally, to determine the bactericidal effect of
macrophages on MRSA, RAW264.7 cells were lysed using 1% Triton X‐100 to
release the bacteria, which were counted via the SPM. Thereafter, to
determine the phagocytosis of bacteria by RAW264.7 cells,
fluorescent‐bacteria and RAW264.7 cells were cocultured for 2 h, and
the extracellular fluorescent‐MRSA was quenched using trypan blue.
Phagocytosis of MRSA by macrophages was measured at excitation/emission
wavelengths of 485/520 nm with a microplate reader.
In Vitro Cytocompatibility
: Following the manufacturer's instructions, cytotoxicity was analyzed
by staining the cells with a live/dead staining kit. Briefly, MC3T3‐E1
cells were seeded on the hydrogels and cultured for 24 h. Additionally,
a CCK‐8 assay was performed to evaluate the biocompatibility of the
hydrogels. Briefly, cells were seeded in 96‐well plates at a density of
5 × 10^3 cells per well and cultured for 24 h, and 100 µL of the
different hydrogel extracts were added to the wells. At pre‐set time
points, the medium was replaced with a CCK‐8 mixture solution (10%
CCK‐8 solution and 90% DMEM medium) and incubated for 2 h. Finally, the
absorbance was measured at 450 nm using a microplate reader (BioTek).
The measurements were repeated thrice for each group. For live‐cell
staining, the cells were washed and incubated with calcein and PI (for
dead cells) for 30 min.
Cell Morphology
MC3T3‐E1cells were cultured for 1 d, washed three times with PBS, fixed
with paraformaldehyde, and permeabilized using Triton X‐100. Next,
actin was stained with fluorescein isothiocyanate‐phalloidin. Finally,
the nuclei were stained with DAPI and observed via CLSM.
In Vivo Evaluation: Mouse Wound Model and Treatment
The Animal Ethics and Welfare Committee of Changzheng Hospital approved
all animal experiments (approval number 2023‐564). All surgical
procedures were conducted following the established guidelines. Male
ICR mice (4–5 weeks old, 18–20 g) were used to create a wound infection
mouse model. First, the dorsal surfaces of the mice were depilated.
Next, 8 mm diameter wounds were created, and 10 µL of 2 × 10^9 CFU
mL^−1 bacterial solution was inoculated. The next day, the mice were
randomly categorized into the control (Ctrl), PADM, PADM@Zn, or PADM@CZ
(n = 10 per group) groups. Briefly, MRSA‐infected wounds were treated
with control (PBS), PADM, PADM@Zn, or PADM@CZ (this treatment was
performed only once in the entire experimental cycle). The mice were
provided with food and water ad libitum. Moisture‐, temperature‐, and
light‐controlled (12 h light‐dark cycle) microinsulators were used to
house the mice. Digital photographs were captured daily, and the area
of the wounds was measured using the “Image J” software. The percentage
of the wound area was calculated using Equation ([176]2):
[MATH: Scratchopenarea%=(remainingwoundarea/
originalwoundareavehicle)×100 :MATH]
(2)
Microbiological and Histological Analyses
Mice from each group were euthanized on days 5 and 11 post‐surgery, and
infected tissues were collected and homogenized with 5 mL of PBS in
order to evaluate the bacterial load. Next, the wounds with surrounding
tissues were harvested for histological evaluation, including HE
staining and Masson's trichrome staining. Immunofluorescence was
performed to determine the expression of endogenous iNOS, CD206, CD31,
and α‐SMA. Immunohistochemistry was used performed to measure IL‐6 and
TNF‐α levels.
To evaluate immune infiltrate phenotyping, spleen cells were treated
with CD45 (AF488; Biolegend, cat. no. 160305, clone S18009D), CD3 (PE;
Biolegend, cat. no. 100205, clone 17A2), CD4 (BV605; Biolegend, cat.
no. 100547, clone RM4‐5), CD8a (PercP/Cy5.5; Biolegend, cat. no.
100733, clone 53‐6.7), CCR5 (PercP/Cy5.5; Biolegend, cat. no. 107016,
clone HM‐CCR5), CD8a (BV421; Biolegend, cat. no. 126419, clone MF‐14)
antibodies on ice. After 30 min, the cells were washed twice with PBS
and subjected to flow cytometry. Finally, major organs such as the
heart, spleen, lung, liver, and kidney were isolated for further
examinations.
Statistical Analysis
Numeric data were reported as mean ± standard deviation (SD), unless
specified otherwise. A two‐tailed Student's t‐test was used to compare
two groups, while multiple comparisons were conducted using one‐way
analysis of variance (ANOVA) with Tukey's post hoc test. Excel 2016 and
GraphPad Prism 9 were used for all calculations and statistical
analyses. Statistical significance was set at P < 0.05.
Conflict of Interest
The authors declare no conflict of interest.
Author Contributions
S.H., S.L., and Q.L. contributed equally to this work. S.H.:
conceptualization, methodology, investigation, writing‐original draft,
data curation. S.L.: conceptualization, methodology, validation,
investigation. Q.L.: conceptualization, methodology, investigation,
data curation. E.X.: investigation, data curation, methodology. L.M.:
methodology, investigation. X.M.: methodology, investigation. Z.X.:
conceptualization, resources. C.Z.: conceptualization, methodology,
investigation, funding acquisition. X.L.: conceptualization, resources,
supervision, funding acquisition, writing‐review & editing. G.X.:
conceptualization, resources, supervision, project administration,
funding acquisition, writing‐review & editing.
Supporting information
Supporting Information
[177]Click here for additional data file.^ (5.6MB, pdf)
Acknowledgements