An in situ dressing material containing a multi‐armed antibiotic for healing irregular wounds

Acute and infected wounds resulting from accidents, battlefield trauma, or surgical interventions have become a global healthcare burden due to the complex bacterial infection environment. However, conventional gauze dressings present insufficient contact with irregular wounds and lack antibacterial activity against multi‐drug‐resistant bacteria. In this study, we develop in situ nanofibrous dressings tailored to fit wounds of various shapes and sizes while providing nanoscale comfort and excellent antibacterial properties. Our approach involves the fabrication of these dressings using a handheld electrospinning device that allows for the direct deposition of nanofiber dressings onto specific irregular wound sites, resulting in perfect conformal wound closure without any mismatch in 2 min. The nanofibrous dressings are loaded with multi‐armed antibiotics that exhibit outstanding antibacterial activity against Staphylococcus aureus (S. aureus) and methicillin‐resistant S. aureus. Compared to conventional vancomycin, this in situ nanofibrous dressing shows great antibacterial performance against up to 98% of multi‐drug‐resistant bacteria. In vitro and in vivo experiments demonstrate the ability of in situ nanofibrous dressings to prevent multi‐drug‐resistant bacterial infection, greatly alleviate inflammation, and promote wound healing. Our findings highlight the potential of these personalized nanofibrous dressings for clinical applications, including emergency, accident, and surgical healthcare treatment.

by patients are different from one setting to another, ranging from burn, chronic, surgical, and traumatic wounds caused by accident or battlefield. [4,5]The inability to give customized and personalized treatment in diverse patient wounds has hampered attempts to apply wound dressing for practical healthcare. [6,7]A number of studies have mainly used 3Dprinted hydrogels or microneedles to achieve controlled drug delivery or precise treatment for wound healthcare. [8,9]lthough some of these techniques have utility for clinical translations, the equipment of 3D printing is bulky in size and needs a complex instrument. [10,11]Additionally, the patterned design cannot suit irregular wounds with varying sizes, depths, and locations, as no two wounds are the same. [12,13]atients may feel uncomfortable owing to the huge mismatch between the hard stressing and surrounding tissues during wound healing. [14,15]In light of these challenges, there is a pressing need for innovative and personalized wound dressing solutions that can address the diverse requirements of individual patients.
[21] However, such scaffolds are limited by their uniform size and must be better suited for personalized wound care in practical applications. [22,23]To advance this technique and provide support for personalized wound healthcare, there is a need for conformal, portable electrospinning nanofibers that are fully compatible with different geometries of wounds. [24,25]Moreover, with the widespread of multi-drug resistant bacteria, the antimicrobial activity of wound dressing is another tough issue for personalized wound care.Methicillin-resistant Staphylococcus aureus (MRSA), the leading cause of wound infections, is increasingly overcoming currently available drugs such as vancomycin, daptomycin, and linezolid. [26]ith the increasing difficulty of semisynthetic antibiotics discovery, there is an urgent demand for antibacterial dressing in personalized wound healthcare. [27]his study introduces a novel approach to wound dressing based on a handheld electrospinning device that creates personalized nanofibrous dressings with high conformality and flexibility.The wound dressings can be directly electrospun onto the wound sites, fully covering them.To achieve this, we use Food and Drug Administration (FDA)-approved poly(ε-caprolactone) (PCL) as the electrospinning material and incorporate multi-armed antibiotics (E-4PBA) as the antibacterial agent against wound infections, as illustrated in Scheme 1.The nanofibrous dressing closely adheres to the wound surface under electrostatic force.Our results demonstrate that the personalized nanofibrous dressings exhibit excellent biocompatibility and effective antibacterial performance, greatly accelerating wound healing while providing conformality for different wound types.Detailed histological evaluations indicate the essential benefits of our personalized, antibacterial, and deposited nanofibrous dressing technologies.

Preparation and characterization of personalized nanofibrous dressings
The nanofibrous dressings were prepared by a handheld electrospinning device consisting of a minimized spinneret and a miniaturized electrospinning system.The minimized electrospinning spinneret integrates the feeding pump of the electrospinning solution with a high-voltage power supply for the electrospinning process (Figure S1).The PCL nanofibers exhibit good directional properties and can be directly electrospun onto the hand (Figure 1A).The electrospinning position, deposited area, and amount of nanofibers can be easily controlled by regulating the distance and spraying angle of electrospinning.Figure 1B shows the formation process of nanofibers by a handheld electrospinning device.In a typical electrospinning process, the electrospinning solution is pumped and deformed into the Taylor cone at the needle tip under the high electrostatic forces (Movie S1).After the evaporation of solvent and elongation of fibers, solid nanofibers are deposited onto a grounded collector from the minimized spinneret.Using this handheld electrospinning device, we directly electrospun nanofibers onto the tip of the thumb, and the nanofibers can fully cover the thumb without any mismatch.The nanofibrous dressing exhibits excellent conformability and comfortability and can even replicate biometric fingerprints, as shown in Figure 1C.This result provides strong evidence that nanofiber dressings provide excellent wound encapsulation on both large skin wounds and microscopic skin wounds, a property that offers a unique technological advantage in the future of internal wound care.Furthermore, randomly deposited nanofibers can be patterned by screen-printed liquid metal electrodes.Figure 1D shows the electrospinning process of patterned nanofibers collected onto conductive and grounded liquid metal electrodes.These results confirm the precise deposition of nanofibers by the handheld electrospinning device, which demonstrates significant implications for personalized healthcare with different wounds and patients.
The present study chose FDA-approved PCL as the electrospinning material for nanofibrous dressing. [28]We previously identified a new class of multi-armed antibiotics of E-4PBA that presented effective antibacterial properties against Gram-positive bacteria by inhibiting the assembly of functional cell walls. [29]Here, antibiotic molecule E-4PBA was incorporated into PCL nanofibers to serve as antibiotics for preventing bacterial infections (Figure S2).To obtain the ideal antibacterial performance of nanofibrous dressing, PCL nanofibers loaded with different contents of E-4PBA were fabricated by a handheld electrospinning device (Figure S3).The morphology of electrospun nanofibers was characterized by scanning electron microscopy (SEM) and presented a smooth surface and large porosity (Figure 1E).These porous uniform nanofibers can provide a similar topography with an extracellular matrix, which could significantly accelerate the wound healing process.As presented in Figure 1F, the diameter of in situ electrospun nanofibers was investigated to evaluate the electrospinning capacity.With the increasing E-4PBA contents, the homogeneous PCL nanofibers showed no obvious changes in diameters within the 300-500 nm range.We also tested the fluorescence spectrum of E-4PBA, which shows an excitation peak at 490 nm (Figure S4).Based on the fluorescent property of E-4PBA, we investigated the distribution of E-4PBA molecules in PCL nanofibers by confocal laser scanning microscopy.Confocal fluorescent images confirmed the homogenous distribution of E-4PBA molecules in the PCL nanofibers (Figure 1G).Thus, based on handheld electrospinning devices and E-4PBA molecules, directly deposited fluorescent nanofibers were developed for personalized healthcare and wound healing management.

S C H E M E 1
Schematic illustration of personalized nanofibrous dressing for wound healing.Antimicrobial molecule multi-armed antibiotics (E-4PBA) are incorporated into poly(ε-caprolactone) (PCL) nanofibers to protect skin wounds from bacterial infection.Based on a handheld electrospinning device, antibacterial PCL&E-4PBA nanofibrous dressings are directly electrospun onto the wound sites with excellent conformability.These advanced forms can provide personalized wound care and clinical tools for accident/battlefield rescue and surgical emergencies.

Antibacterial activity of personalized nanofibrous dressing
In the wound management process, bacterial infections are the primary cause of delaying chronic wound healing. [30]We examined the antibacterial activity of nanofibrous dressings loaded with antibiotic E-4PBA molecules, which provides a new alternative to overcome antibiotic resistance.As shown in Figure 2A, the antibacterial performance of nanofibrous dressings was evaluated by exposing them to bacteria suspension of S. aureus and MRSA, the most prevalent bacteria strains in skin wounds.Due to the inherent advantages of a high surface area/volume ratio and effective porosity, nanofibrous dressings can provide an effective physical barrier against the attack of bacteria.As an antibacterial dressing, the E-4PBA can be easily released from PCL nanofibers and interact well with the cell membrane (Figure 2B).All bacteria were stained green, indicating the excellent binding interaction of E-4PBA to the cell membrane.After being treated with nanofibrous PCL dressings loaded with 6%E-4PBA for 24 h, no bacteria clones were observed on the agar plates compared with the control group.These results demonstrated that nanofibrous dressings displayed outstanding performance for bactericidal effect and inhibition of bacterial growth (Figure 2C).To further characterize the antibacterial behavior of nanofibrous dressings, the morphology of treated S. aureus and MRSA was investigated by transmission electron microscopy (TEM) and SEM.When exposed to PCL-6%E-4PBA nanofibrous dressings, the cell walls of bacteria were thickened with concave membranes, while the control group showed a clear boundary and flat septum (Figure 2D).The morphology of S. aureus and MRSA cultured on the surface of nanofibrous dressing was then characterized by SEM.Due to the highly porous nanostructure, bacteria cells could be easily attached to the nanofibers, which significantly built a protective barrier against bacteria.Moreover, the E-4PBA accumulated on the surface of S. aureus and MRSA bacteria cells, suggesting the nanofibrous dressings' damage to the bacteria (Figure 2E).Consequently, the PCL nanofibrous dressings loaded with E-4PBA demonstrated strong antibacterial ability for preventing bacterial infections of S. aureus and MRSA.

Biocompatibility of personalized nanofibrous dressing
The biocompatibility of wound dressing is key in promoting wound recovery during wound treatment. [31]PCL nanofibers  loaded with 6%E-4PBA were directly electrospun onto the cell culture dish, and human umbilical vein endothelial cells (HUVECs) and NIH-3T3 cells were directly cultured on the surface of nanofibrous after fibronectin incubation (Figure 3A).Cell viability was examined to determine the cell compatibility of nanofibrous dressing by a cell counting kit 8 (CCK-8) assay and live/dead staining.As shown in Figure 3B, HUVEC and 3T3 cells cultured on the nanofibers exhibited good cell viability and no obvious variation with increased E-4PBA content.The cytotoxicity of nanofibrous dressings was determined by live/dead imaging, with pure PCL nanofibers as a control.After culturing for 3 days, most HUVEC and 3T3 cells were alive and stained in green, indicating the outstanding cytocompatibility of nanofibrous dressings (Figure 3C).The adhesion and proliferation of cultured cells were further evaluated by confocal fluorescent imaging after phalloidin/DAPI staining, and the fluorescent images showed good biocompatibility for cell growth and migration (Figure 3D).The morphology of HUVEC and 3T3 cells was also characterized by SEM, which exhibits that cells cultured on nanofibrous membranes attach to nanofibers with high viability (Figure S5).Furthermore, we examined the in vivo biocompatibility of nanofibrous dressings by implanting nanofibers in the peritoneal cavity for 14 days.Hematoxylin and eosin (H&E) staining images of the heart, liver, spleen, lung, and kidney showed no obvious difference in both groups (Figure 3E).These data proved that the nanofibrous dressings with porous nanostructures exhibited good biocompatibility and facilitated cell growth and proliferation, suggesting promising candidates for wound dressing.

Personalized nanofibrous dressing for in vivo wound healing
To evaluate the wound closure and healing efficiency of the personalized nanofibrous dressing, a full-thickness infected skin wound model (2 cm in diameter) was constructed on the back of the mouse.The committee of the laboratory animal center at SUSTech (NO.JY202108030) had approved all the procedures in animal experiments.The workflow for examining wound healing efficiency in a Sprague-Dawley (SD) mouse is shown in Figure 4A.Following incubation with MRSA suspension for 20 min, the infected wounds were treated with conventional gauze, PCL nanofibers, and PCL nanofibers loaded with 6%E-4PBA.Based on a handheld electrospinning device, we can completely cover irregular wound areas by adjusting the deposition of nanofibers, and the nanostructures and nanoscale sizes of deposited fibers can perfectly meet the shape of various wounds with good comfortability (Movie S2).On day 7, the infected wounds treated with PCL-6%E-4PBA exhibited significantly faster closure than gauze and PCL nanofibers groups (Figure 4B).The infected wounds treated with PCL-6%E-4PBA dressing were almost healed on day 14, while the other groups remained unhealed.Statistical results demonstrated that the wound healing process of PCL-6%E-4PBA nanofibrous dressings was greatly promoted to 96%, which is much faster than other groups (70% and 84%) on day 14 (Figure 4C).Plate photographs of bacterial clones also confirmed the good antimicrobial properties of the nanofiber dressing with significantly fewer clones than the control wound tissue.(Figure 4D).This could be attributed to the capacity that in situ electrospun nanofibrous dressings can completely cover the wound sites and provide effective antibacterial properties.Hemocompatibility is one of the most important indicators for assessing the suitability of artificial materials and medical devices for contact with blood.If the material is not blood-compatible, it may trigger adverse consequences such as thrombosis, platelet aggregation, and inflammatory reactions.The hemocompatibility of nanofibrous dressings was characterized by examining the hemolytic activity, which plays of vital role in wound treatment.After incubation with PCL nanofibrous dressings loaded with 0%, 2%, 4%, and 6% E-4PBA, the erythrocytes showed no damage and similar morphology to the control group (Figure 4E).The in situ electrospun nanofibrous dressing can provide an effective wound dressing for wound healthcare to prevent bacterial infections and greatly accelerate wound healing.
To further examine the quality of healed wound tissues, histological analysis was performed by H&E staining and Masson's trichrome staining (Figure 5A,B).In HE staining images, the group of PCL with E-4PBA nanofibrous dressing appeared to have fewer inflammatory cells and a thick epidermis than the control group.On day 14, the wound was treated with PCL with E-4PBA nanofibrous dressings, reconstructed the dermis, and recovered from damaged tissues.In Masson straining images, the group of PCL with E-4PBA nanofibrous dressing appeared to have more collagen fibers and blood vessels, which confirmed the efficiency of accelerating wound healing.Consequently, we investigated the inflammatory response of infected wounds after treatment with nanofibrous dressings, and immunohistochemistry analysis was performed on samples treated with three groups on days 4, 7, and 14.As shown in Figure 5C-E, inflammatory cytokines, including total protein, tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6), were assessed to evaluate inflammatory response.Due to the good antibacterial performance against MRSA, the PCL nanofibers loaded with 6%E-4PBA exhibited a faster-decreasing rate and less inflammatory response in the wound repair process.
Collectively, we demonstrated that personalized nanofibrous dressings, in situ electrospun by handheld electrospinning device for skin wound healthcare, enabled superior antibacterial activities and good biocompatibility for wound treatment.Compared to conventional fixed gauze and PCL nanofibers, personalized nanofibrous dressings could be directly electrospun toward the wound sites and perfectly cover different shapes of wounds with high comfortability (Figure S6).In infected mouse models, the personalized nanofibrous dressings showed a less inflammatory response and promoted wound healing efficacy.In the future, many bioactive molecules, such as proteins, drugs, and growth factors, can be easily encapsulated into nanofibrous dressings.The data suggest that personalized nanofibrous dressing provides a tunable and versatile platform for wound management and emergency healthcare.

CONCLUSION
In this study, we develop an in situ dressing material containing a multi-armed antibiotic exhibiting excellent antibacterial performance and promoting irregular wound healing.Nanofibrous dressings prepared by FDA-approved polymer combined with multi-armed molecules have effective antibacterial activities against clinical multidrug-resistant Gram-positive bacteria (S. aureus and MRSA) and good biocompatibility compared with traditional antibiotics.Moreover, the portability and flexibility of the handheld electrospinning devices allow for the direct electrospinning of dressing onto the irregular wound sites with full coverage and good comfortability.Compared with traditional gauzes and nanofiber dressing pressed onto the wound, in vitro and in vivo, studies of infected wound models demonstrate that in situ electrospun nanofibrous dressings can effectively prevent bacterial infection and promote irregular wound healing.Due to its good portability, comfortability, biocompatibility, and antibacterial properties, this nanofibrous dressing holds great promise for personalized healthcare, accident/battlefield rescue, and surgical emergency applications.

Electrospinning of personalized nanofibrous dressing
Polymer precursor solution for electrospinning was prepared by dissolving PCL powers with a mixture of DMF and THF.The E-4PBA powder was redispersed in 14 mL of a 1:1 (v/v) THF-DMF solution, to which 2 g PCL was added.Before electrospinning, the mixture of the polymer solution was bath sonicated for 10 min at room temperature (normal mode, ultrasonic cleaner; Branson 5800, Frequency: 40 kHz), and stirred overnight to obtain a homogeneous suspension of E-4PBA.The prepared electrospinning solution was transferred to a 5 mL syringe and placed into the minimized spinneret.The applied voltage and flow rate were fixed at 15 kV and 1 mL/h during in situ electrospinning.The electrospinning distance between the minimized spinneret and grounded skin was approximately 5-10 cm.The surface morphology of electrospun nanofibers was characterized by SEM (Hitachi SU8200), and the diameter distribution was analyzed by Nanomeasure software.

Antibacterial evaluation of nanofibrous dressing
The antibacterial performance of nanofibrous dressing was investigated by culturing S. aureus and MRSA on the nanofibrous membranes in an LB broth medium at 37 • C. The suspensions of S. aureus and MRSA containing 10 6 CFU/mL were added into a 6-well plate, and the nanofibrous membranes were placed at the bottom of the plate.After incubation in a 37 • C bacteriological incubator for 24 h, the bacterial suspensions were collected and observed by confocal microscopy (Zeiss LSM710).After incubation with nanofibrous dressing, the bacterial suspensions were smeared into LB agar.The conventional plate counting method was used for evaluating the antibacterial activities of nanofibrous dressing by colony counting.The morphology of S. aureus and MRSA treated with nanofibrous dressings was characterized by SEM (Hitachi SU8100) and TEM (Talos F200X) after glutaraldehyde fixation and gradient dehydration.

4.4
In vitro biocompatibility evaluation HUVEC and NIH-3T3 cells were used to evaluate the cell viability of nanofibrous dressings by CCK-8 assay (Dojindo).Before cell culture, the nanofibers were incubated with fibronectin (Thermo Fisher) to increase cell adhesion for 1 h.HUVEC and NIH-3T3 cells were cultured on nanofibrous membranes with a 5 × 105 cells/cm 2 density and stained with a Live/dead kit (Invitrogen) after 72 h culturing.Confocal microscopy (Zeiss LSM710) was used to observe the morphology of cultured cells after phalloidin/DAPI staining.SEM images of cells cultured on the surface of nanofibrous membranes were captured at an accelerating voltage of 5 kV after the paraformaldehyde fixation and gradient dehydration.The biocompatibility of nanofibrous dressings was investigated by implanting nanofibrous dressings into the abdominal cavity of the SD mouse.After 1 month of implantation, the major organs, including the heart, liver, spleen, lung, and kidney, were fixed and stained by HE histological analysis.

Personalized nanofibrous dressing for wound healing
Twenty-four Sprague Dawley female rats (8-week-old, 200-250 g) were randomly divided into three groups after purchasing from Guangdong Medical Experiment Animal Center.The committee of the laboratory animal center at SUSTech (NO.JY202108030) had approved all the procedures in animal experiments.After anaesthetization with 1% urethane, a full-thickness wound (2 cm in diameter) was symmetrically generated on the back of the SD mouse.The infected wound model was constructed by incubating with MRSA bacteria (10 6 CFU/mL, 200 μL) for 20 min after dripping in the MRSA bacteria, and then infected wounds were covered by gauze, PCL nanofibers, and E-4PBA nanofibrous dressings.On days 0, 4, 7, and 14, the infected wounds of rats in the three groups were photographed and measured using Image J software.After treatment on days 0, 4, 7, and 14, the rats were executed, and the skin tissue at the wound site was cut and soaked in 4% paraformaldehyde overnight.After dehydration overnight and embedded in wax blocks, the skin tissues were cut into 5-10 μm thickness and stained with H&E and Masson's trichrome.Stained sections were dehydrated with pure alcohol and then made transparent with xylene.A drop of Canada gum was placed on the clear sections and sealed with a coverslip.After the gum dries slightly, labels are applied and the sectioned specimens are ready for use.Inflammatory cytokines including TNF-α, IL-6 as well as the total protein were collected and investigated by GraphPad Prism 7.0 software.All quantitative data are expressed as mean ± standard deviation.Statistical analyses were performed with a two-sided p < 0.05 as statistically significant.Graphs were plotted using GraphPad 7.0 Prism.

A C K N O W L E D G M E N T S
We thank the National Key R&D Program of China (2022YFB3804700), the National Natural Science Foundation of China (52203243, 22234004, 21761142006, 21535001, and 81730051), Guangdong Innovative and Entrepreneurial Research Team Program (2019ZT08Y191), the Shenzhen Science and Technology Program (KQTD20190929172743294 and JCYJ20200109141231365), the Chinese Academy of Sciences (QYZDJ-SSW-SLH039), Shenzhen Key Laboratory of Smart Healthcare Engineering (ZDSYS20200 811144003009), Guangdong Major Talent Introduction Project (2019CX01Y196), Tencent Foundation through the XPLORER PRIZE for financial support.Ruihua Dong, Mian Chen, and Yuexiao Jia contributed equally to this work.We also thank for SUSTech Core Research Facilities.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.

F
I G U R E 1 Fabrication and characterization of personalized nanofibrous dressings.(A) Picture of in situ electrospinning of poly(ε-caprolactone) (PCL) nanofibers onto the hand by a handheld electrospinning device.(B) Captured picture of in situ electrospinning Taylor cone and jets showing nanofiber formation.(C) Picture of PCL nanofibers electrospinning on the tip of the thumb.The partially enlarged image shows the excellent conformability of electrospun nanofibers with a clear biometric fingerprint.(D) Picture of nanofibrous patterned dressings prepared by surface topography of SUSTech based on the liquid metal electrode collectors.(E) Scanning electron microscopy (SEM) images showing nanofibrous morphology of PCL nanofibers with different contents of multiarmed antibiotics (E-4PBA).(F) Diameter distribution of PCL nanofibers prepared from various contents of E-4PBA: 0%, 2%, 4%, and 6%.(G) Confocal images of in situ electrospun nanofibers show the smooth and evenly distributing fluorescent E-4PBA in the PCL nanofibers.

F
I G U R E 2 Concept and characterization of antibacterial activities by in situ electrospun nanofibrous dressings.(A) Schematic overview of antibacterial activities by nanofibrous dressings against Gram-positive bacteria.(B) Confocal images of Staphylococcus aureus (S. aureus) and Methicillin-resistant S. aureus (MRSA) treated with poly(ε-caprolactone) (PCL) nanofibers containing 6% multi-armed antibiotics (6%E-4PBA).(C) Pictures of bacterial colonies by S. aureus and MRSA bacteria after treatment with nanofibrous dressings.(D) Transmission electron microscopy (TEM) images of normal and nanofiber-treated S. aureus and MRSA bacteria showing flat septum in dividing cells.(E) Scanning electron microscopy (SEM) images of S. aureus and MRSA bacteria showing the morphologies of the bacterial cell membrane.

F I G U R E 3
In vitro biocompatibility evaluation of personalized nanofibrous dressings.(A) Schematic illustration of the experimental procedure to culture cells on the in situ electrospun nanofibrous dressings.(B) Cell viability of nanofibrous dressings for human umbilical vein endothelial cells (HUVEC) and NIH 3T3 cells after incubation with 24 h by cell counting kit 8 (CCK-8) assay.(C and D) Live/dead staining images and confocal microscope images of HUVEC and NIH 3T3 cells showing the cytotoxicity and cell morphologies of in situ electrospun nanofibrous dressings.(E) HE staining of rats' heart, liver, spleen, lung, and kidney after 1-month implantation of nanofibrous dressings.

F I G U R E 4
In vivo wound healing performance of nanofibrous dressings in an infected wound model.(A) Schematic illustration of the infected wound healing process in rats' skin wound model.(B) Representative pictures of methicillin-resistant Staphylococcus aureus (MRSA)-infected wounds treated with commercial gauze, fixed poly(ε-caprolactone) (PCL) nanofibers, and in situ electrospun nanofibrous dressing on days 0, 4, 7, and 14. (C) Wound healing percentage of skin area on days 0, 4, 7, and 14 for each group (n = 6).*p < 0.05 and * *p < 0.01.(D) Representative plate photographs of bacteria clones after gauze and nanofibrous dressing treatments.(E) Hemolytic activity evaluation of different nanofibrous dressings showing good hemocompatibility with rat red blood cells.ND: not detected; NC: negative control group; PC: positive control group.

F I G U R E 5
Histological assessment of infected wound site during the wound healing process.(A) Hematoxylin and eosin (H&E) staining images of skin tissues from diverse groups on days 7 and 14. (B) Corresponding Masson's staining images of skin wounds from different groups on days 7 and 14. (C-E) Quantitative analysis of inflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), as well as total protein caused by MRSA infections in three groups.*p < 0.05 and * *p < 0.01.