Angiogenesis-Enhanced Biomimetic Nanofiber Dressings: VEGF-Infused Electrospun Membranes for Targeted Wound Healing and Tissue Regeneration

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Angiogenesis-Enhanced Biomimetic Nanofiber Dressings: VEGF-Infused Electrospun Membranes for Targeted Wound Healing and Tissue Regeneration

https://doi.org/10.21203/rs.3.rs-5280160/v1

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Biomimetic dressings are widely regarded as optimal medical dressings for promoting wound healing. Researchers are endeavoring to develop a new class of dressings that can fend off bacterial infections, accelerate tissue regeneration, and perform specialized tasks to facilitate wound healing and repair. This study presents an electrospinning nanofiber membrane-building approach based on natural biopolymers and the addition of vascular endothelial growth factor (VEGF) to boost angiogenesis and promote wound healing. The composite nanofiber membrane made using the electrospinning technique can retain its physicochemical properties and biological function. It has strong biocompatibility and is appropriate for wound repair because of its improved mechanical and biomedical qualities. The designed nanofiber membrane regulates the release of highly concentrated VEGF to target the wound specifically. It facilitates the interchange of nutrients and oxygen, encourages endothelial cell migration and proliferation, and forms the vascular lumens, all of which help to speed up tissue regeneration and wound healing. As a result, VEGF@PAG nanofiber dressings have the potential to overcome the drawbacks of conventional patches and emerge as the most beneficial alternatives for wound healing and associated biological applications.

Biomedical Engineering

electrospinning nanofibers

VEGF

PLGA

angiogenesis

wound repairing

The largest organ in the human body, the skin, serves as the body's first line of defense regarding protection, immunology, thermoregulatory, and sensory barrier functions. ^[1] When this barrier is compromised, foreign pathogens can readily enter the body through the vulnerable interface, increasing the risk of significant water loss in wound inflammation, hemorrhagic shock in an emergency, and even death. Wound healing is a complex and dynamic process, and to maintain the integrity of the skin and the adjacent tissue and function is very important. ^[2] Self-grafts are the gold standard for skin treatment since they are accepted, but they are scarce and can induce donor-site morbidity and scarring. Allotransplantation and xenotransplantation incur disease transmission and immunological rejection hazards. ^[3] Interactive wound repair dressings are widely used to promote healing and prevent infection and further damage. However, the current wound dressing is only suitable for superficial wounds, which hinders the regeneration of epithelial tissue and can lead to increased scarring and bacterial growth in the process of frequent replacement, which easily causes wound adhesion and secondary trauma. ^[4] In addition, long-term use of traditional antibiotics for skin damage repair may also cause an ecological imbalance of pathogenic microorganisms, resulting in drug resistance. ^[5] Therefore, developing new wound dressings that can reduce wound infection and complications and promote cell and tissue regeneration has important clinical significance for skin repair.

The nanoscale interwoven fibers produced by electrospinning technology have the ability to uniformly distribute different functional molecules (e.g., antibiotics, growth factors, and antioxidants) within a skin extracellular matrix (ECM)-like three-dimensional staggered nanofiber structure. ^[6] Notably, the electrospinning nanofiber retains flexibility, large specific surface area, and ECM-like 3D fiber structure while exhibiting the softness, good air permeability, adjustable wettability, and biocompatibility of comparable hydrogel system tissues. ^[7-8] This can help to promote long-term release and effectively address the issue of low application efficiency brought on by uneven functional molecule distribution or inadequate load. ^[9] Furthermore, the distinct benefits of continuous, direct preparation and high controllability of electrospinning nanofiber offer further opportunities for its wound healing use. ^[10] Loading drugs into the inside or surface of nanofibers can effectively enhance the drug load and improve the controlled release of drugs, achieving breakthroughs in the application of efficient wound dressings. ^[11]

Common examples of polymers that have been electrospinning into effective tissue scaffolds include polydioxycyclohexanone, poly (ε-caprolactone), polyglycolic acid (PGA), polylactic acid (PLA), and poly (1-lactide) and its copolymer (d, L-lactide-co-ethyl ester) (PLGA), are commonly used as high surface area fiber membranes. ^[12] With its adjustable degradability, favorable biocompatibility, and high mechanical strength, in addition to its FDA approval for human treatment and other remarkable characteristics, PLGA functions admirably as a scaffold for wound healing tissues. ^[13-14] Nevertheless, PLGA's poor hydrophilicity leads to inadequate cell adhesion and infiltration outcomes. ^[15] Consequently, hyaluronic acid (HA) and gelatin, two naturally occurring biomacromolecules, were added to the scaffold to control its hydrophilic qualities and to establish an environment that is conducive to cell migration, proliferation, and growth. ^[16-17] In addition, HA and gelatin materials have natural cell recognition sites and excellent cellular affinity, which can provide a suitable microenvironment for cells, facilitate cell adhesion and migration, and thus promote wound healing. ^[18]

Reduced blood vessel growth through the process of angiogenesis is a significant factor in many non-healing wounds. ^[19] Through invasion of the wound clot and organization into a microvascular network across the granulation tissue, angiogenesis forms fresh blood vessels from existing vessels, a process that is essential to wound healing. ^[20-21] Vascular endothelial growth factor (VEGF), an essential glycoprotein with substantial biological activity, plays a pivotal role in both restorative and developmental angiogenesis within endothelial cells (EC). ^[22] By binding to particular receptors, VEGF can stimulate intracellular signaling pathways, thereby promoting the migration, proliferation, and formation of vascular lumens of EC. ^[23-24] Additionally, VEGF serves as a crucial regulator and target to maintain vascular homeostasis and enhance capillary permeability. This, in turn, stimulates the generation of fresh vascular endothelial cells and reinstates local blood flow to the skin, supplying essential nutrients and oxygen for the regeneration of epidermal and dermal cells. ^[25-26] Therefore, it facilitates the repair of skin damage and reinstates barrier function, which aligns perfectly with the intended purpose of electrospinning nanofibers for skin with this characteristic.

In this study, an ECM-like reticular nanofiber membrane (PAG) was synthesized by combining PLGA, HA, and Gelatin. Bioactive VEGF was then deposited onto the PAG to produce a nanofiber patch (VEGF@PAG), which accelerated wound healing and tissue repair and stimulated endothelial cell migration, proliferation, and tubule formation. Vascular endothelial growth factor is slowly released from the wound by VEGF@PAG, which amplifies the benefits of both materials while preserving the biomimetic qualities of electrospinning fibers and the biochemical characteristics of functional molecules. Its improved mechanical and flexible properties allow it to remain in the daily environment over time, effectively promoting wound healing and tissue repair by loading VEGF and targeting delivery to the wound. In addition, synchronizing its degradation rate with the recovery of the wound prevents secondary injury that could result from the removal of the fibrous membrane and creates space for newly growing tissue. This dressing can effectively facilitate wound repair and provide precise coverage of the lesion surface, as demonstrated by the rat-scalded skin wound model. The inexpensive and simple-to-manufacture VEGF@PAG composite system described in this study could be the optimal option for developing multifunctional bioactive fibers that promote wound healing in order to implement wound precision therapy in clinical practice.

2.1 Preparation of PAG

A solution: First, 0.06 g HA (Shanghai Macklin Biochemical Co., Ltd.) with a molecular weight of 0.8-1.5 million was weighed and dissolved in 3 mL HFIP solution and then stirred at room temperature for 24 h to add 0.3 g Gelatin (Shanghai Macklin Biochemical Co., Ltd.) to the system and continued stirring for 24 h. B solution: 0.7 g PLGA (50:50) was weighted into HFIP solution and stirred for 24 h. After mixing A and B solutions homogeneously, stirring for 24 h until a stable and homogeneous solution was obtained. Transferring the above solution into the electrospun propulsion device (specific parameters: the 9# metal needle (outer diameter 0.9 mm, inner diameter 0.57 mm), while the smoothed tip was connected to the 10 mL syringe, and the prepared PAG solution was injected into the syringe so that the metal needle was connected to the high-voltage power supply. With a grounded bowl as the receiving device, electrospinning was carried out under a receiving distance of 12 cm, a solution flow rate of 1.5 mL/h, and a voltage of 12 kV). The spinning process was observed with a tungsten halogen lamp; the collection time was about 4 h, and the thickness of the film was about 80 μm. The prepared nanofiber membrane was then dried in a vacuum oven for 12 h at room temperature to remove the residual solvent.

2.2 Water contact angle test

The spinning fiber membrane was cut into 1 cm×1 cm and fixed on the operating table. Then 5 μL of PBS solution (pH = 7.4) was dropwise added to the spinning fiber. The contact angle between the droplet and the spinning fiber membrane at 0 s, 1 s, and 2.5 s was measured using the German Lauda Scientific LSA100.

2.3 Swelling rate test

The spinning film was cut to 4 cm × 4 cm and weighed (Wd), then the sample was soaked in PBS solution at 37 °C and pH = 7.4 for 8 h, and the sample was removed to weigh (Ws). The swelling ratio is calculated as follows: swelling rate (%) = [(Ws-Wd)/Ws] ×100%.

2.4 Mechanical property testing

The mechanical properties of the fiber membrane were tested by the INSTRON 5982 mechanical testing machine. Dry mechanical properties: The sample was cut into 10 mm × 70 mm dumbbell-shaped strips. The sample was held at a distance of 50 mm and stretched at a speed of 0.01N initial tension and 5 mm/min at room temperature to obtain a stress-strain curve. Wet mechanical properties: The sample was soaked in PBS (pH = 7.4) solution for 1 h, then taken out, and the moisture on the surface and edge of the membrane was absorbed with filter paper, and the test was carried out under the same test conditions as in the dry state. Three measurements were taken per sample.

2.5 Degradation in vitro

The spinning film was cut to 4 cm × 4 cm and weighed its maximum weight (Wd) after swelling, and then the sample was immersed in PBS (37°C, pH = 7.2) solution with constant temperature shaking. The sample was removed periodically, wiped dry with filter paper, and weighed (Ws) again. Its 14-day degradation behavior was measured. The degradation rate calculation formula is degradation (%) = (Ws/Wd) ×100%.

2.6 Sustained release of the drug

The fiber membrane loaded with VEGF was placed in 10 mL PBS solution (pH = 7.4, 37 °C) for sustained drug release. UV-Vis absorption spectra test for drug content in PBS solution within 72 h.

2.7 Cell viability assay

The CCK-8 method was used to detect cell proliferation. Hacat (human immortalized keratinocytes) and HUVEC (human umbilical vein endothelial cells) with a density of 4×10³ per well were seeded into 96-well plates. Cells were cultured with high-glucose medium (HG, 33mM glucose). After overnight incubation, HG-cultured cells are treated with VEGF with different concentrations for 1, 2, 4 days, respectively, followed by the addition of 10 μL of CCK8 and incubated for an additional 1h. Calculate cell viability by determining absorbance at 450 nm with a microplate reader.

2.8 Cell migration capacity assay

2×10⁴ HUVECs were inoculated in the middle of a 24-well plate without the addition of fetal bovine serum, and the cell culture was the same as the grouping. Then, different concentrations of VEGF were added to the lower chamber 24 h later. Washed the cells in the upper chamber 3 times with PBS and fixed them with 4% paraformaldehyde for 15 min. Then, the chamber was treated with crystal violet (0.1%, w/v) for 10 min, and the migrating cells were observed using light microscopy.

2.9 Capillary-like structures formation experiments

Tubular formation tests were performed in HUVEC to observe the ability to form capillary-like structures under different conditions. Briefly, the different concentrations of VEGF were added to a pre-cooled 24-well plate with 200 μL per well. Then, HUVECs at 1.5 × 10⁵ cell densities per well were seeded onto 24-well plates and cultured at 37 °C. After 6 h, the formation of capillary-like structures was observed with an inverted light microscope.

2.10 Expression of VEGFR1 protein in cells

HUVEC cells were seeded into 24-well plates at a density of 5 × 10⁴ cells/well for 24 h, then added with VEGF at different concentrations and incubated for 24 h. Cells were washed twice with PBS and fixed with 4% paraformaldehyde. Cells were washed thrice with PBS, and the samples were treated using Triton to block with a blocking solution for 1 h. First incubated with VEGFR1 antibody (primary antibody) overnight at 4 °C, then incubated with fluorescently labeled secondary antibody (antibody of primary antibody) in the dark for 2 h. Cells were washed three times with PBS, and an antiquencher agent was added. VEGFR1 protein expression fluorescence was visualized by a fluorescence microscope.

2.11 Construction of traumatic rat models

In this study, 18 SD male rats, equally divided into 3 groups, were used to construct the model. Isoflurane was used to anesthetize the rat. The back of the rat was exposed with the help of an animal shaving machine, and the surgical range was marked with a marker. Using a sterile scalpel and scissors, a square, full-thickness skin with a side length of about 2 cm was removed. The control group did not undergo treatment and recovered naturally, and the PGA group and the VEGF@PAG group were treated with spinning film PAG and spinning film VEGF@PAG, respectively. All animal handling procedures were performed in accordance with the guidelines approved by the Ethics Committee of the People's Hospital of Guangxi Zhuang Autonomous Region (KY-ZC-2022-149). This study adhered to the relevant animal welfare guidelines to ensure ethical treatment of the animal subjects involved.

2.12 Wound closure ratio detection

IPad is used to take pictures of the wound on days 0, 6, 9, 14, and 18 postoperatively. ImageJ is used to measure wound size. Wound healing rate (%) =[(S0-St)/S0] ×100%， S0 is the initial wound size, and St is the wound size at each time point.

2.13 Histological staining

For histological evaluation, after sacrificing rats on days 9 and 18 postoperatively, wound tissue was collected and fixed in 4% paraformaldehyde. The harvested sample was dehydrated and embedded in paraffin and cut into 5 μm sections. H&E was used to visualize the tissue, while collagen deposition was evaluated using Masson trichromatic staining. H&E-stained sections were observed with a scanner, and Masson staining was observed using an upright microscope.

2.14 Statistical analysis

Statistical analyses were conducted using GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA). A t-test and two-way ANOVA were used to determine statistical differences between the groups (n.s. means no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

3.1 Fabrication and Characterization of VEGF@PAG nanofiber membrane

The bioactive and biocompatible nanofiber membranes were fabricated by incorporating biodegradable PLGA, HA, and gelatin derived from natural polymer materials, followed by the addition of VEGF. This dressing has the potential to facilitate wound tissue repair and mitigate the risk of secondary injury that may occur during treatment. (Scheme 1)

Spinning fiber membrane scaffolds (PAGs) containing HA, Gelatin, and PLGA were prepared by electrospinning technology, as shown in Figure S1 (supporting information), and the morphology of the materials was characterized by SEM (Figure 1a). PAG fibers were typically non-directional and exhibited a distinct three-dimensional porous structure with a diameter of 200-300 nm and a uniform distribution, as indicated by the results. In addition to promoting drug loading and release, this structure aids in the reduction of wound exudate and promotes the process of wound healing. Proper surface hydrophilicity of epidermal wound repair materials is essential for biological function. It can affect the adsorption of nutrients on the surface of the material and promote the adhesion and proliferation of cells, which is one of the indispensable characteristics of wound repair materials. In order to evaluate the hydrophilic properties of the surface of the PAG fiber membrane, a static water contact angle (WCA) experiment was performed. As shown in Figure 1b, when the liquid was added, the WCA on the PAG fiber membrane was measured as 127.3°. With increasing time, the WCA decreased, and the liquid was absorbed entirely within 2.5 seconds, showing excellent super-hydrophilic properties of PAG membranes. This result could be attributed to the great hydrophilicity of HA and gelatin materials. Subsequently, we conducted an evaluation of the swelling ratio, which is a vital indicator of the dressing's wettability as it guarantees a moist environment that promotes the recovery of skin wounds. Following soaking, certain fibers started to break down, the fiber diameter thickened, and the PAG spinning fiber membrane showed signs of swelling. In the early stages, the PAG swelling ratio in the PBS solution was relatively rapid, and it eventually reached nearly 70% in one hour (Figure 1c). As the amount of time increased, the swelling ratio subsequently started to slow down and reached 95% after 8 hours. The limited expansion of the spun fiber membrane network may be attributed to the hydrophobic nature of the PLGA material. By retaining the hydrophilicity of PAG without compromising its mechanical properties, the nanofiber membrane ensures that the HA and gelatin exhibit strong hydrophilicity while maintaining excellent structural stability. This is a critical characteristic in the process of skin repair. The aforementioned demonstrates that the swelling ratio of the PAG is suitable, which may facilitate wound healing by establishing a hydrophilic environment, preventing excessive swelling from deforming the fiber membrane and diminishing the wound's protective effect.

Fiber membrane dressings with suitable mechanical properties can provide great support for the wound environment and adhesion of cells, thereby offering a good healing environment for wounds. Subsequently, the dry and wet mechanical properties of the dressing were tested. As shown in the stress-strain curves of Figures 1d and 1e, Young's modulus of PAG was 238.67 MPa in the dry state and 38.50 MPa in the wet state, which could basically meet the requirements of the mechanical properties of materials in various tissues and various motion states of the human body, and would not limit daily activities.

It is critical that the rate of regenerative repair of wound tissue matches the degradation time of the fibrous membrane. As shown in Figure 1f, PAG degraded faster on the first day and relatively slowed thereafter. The prepared fiber membrane would degrade to less than 50% within six days and would be broken in about 13 days, which meant that the degradation degree was higher than 70%. Generally, the wound healing cycle is about 7-13 days. Therefore, the PAG fiber membrane can provide the necessary support for tissues in the early stage of treatment and offer new space for tissues and cells, which are basically degraded in the later stage. This rate of degradation is well adapted to the growth rate of the tissue. Moreover, as PAG is continuously degraded, its pore size gradually increases, which is conducive to cell migration, internal growth, and proliferation, further accelerating the healing process of wounds.

3.2 Effectively released VEGF-enhanced keratinocyte and EC proliferation

After loading VEGF, its presence in the fiber network could be observed by SEM images. The standard curve of VEGF was prepared by an enzyme-linked immunosorbent assay. The absorbance of the supernatant at 450 nm was detected by UV-Vis absorption spectra tests, and the concentration was calculated according to the standard curve so that the encapsulation efficiency of VEGF in VEGF@PAG was calculated to be 95.64% (Figure 2a), indicating that the fiber membrane has an ultra-high loading efficiency for VEGF. In order to study the controlled drug release of synthesized VEGF@PAG dressing, the VEGF release ability of the dressing was evaluated by UV-vis absorption spectra in a PBS environment (pH = 7.4, 37°C). From Figure 2b, it could be observed that VEGF was released quickly in the beginning, and the drug release rate gradually decreased after eight hours. The drug could be effectively released 52.039 ± 3.919% within 72 h, which might be due to the fact that VEGF on the surface of PAG in the early stage was released first, and VEGF inside the fiber was gradually released under the action of osmotic pressure. These findings demonstrated that VEGF could sustain its activity on the PAG fiber membrane for an extended period and be progressively released to promote wound healing. Next, the toxicity and biocompatibility of the VEGF@PAG by CCK-8 assay (Cell Counting Kit-8 assay) were used to detect the proliferation of Hacat and HUVEC cell lines. After treating the cells with VEGF separately, there was no significant change in the number of cells on the first day. The cells began to proliferate significantly over time (Figure 2c&d). With the increased concentration of added VEGF, the cell proliferation increased significantly, and the cell cultured with 1000 ng/mL VEGF had the highest proliferation capacity. After four days of incubation, the viability of the Hacat and HUVEC cell lines increased to 170% and 174%, respectively. These results suggest that nanofiber membranes can mimic the ECM environment and release VEGF to induce the proliferation of Hacat and HUVEC cells for skin repair.

3.3 VEGF promotes vascular EC migration and angiogenesis

Endothelial cells are essential building blocks in blood vessels and play a vital role in vascularization. Subsequently, the endothelial cell migration of Hacat and HUVEC was assessed by a transwell migration assay. The experimental results showed that VEGF promoted the migration of hypoxic cells, and the mobility of cells showed a significant increase with the increase in VEGF concentration (Figure 3a). Compared with the number of Hacat cells migrated in the control group, the number of G + 10 ng/mL VEGF, G + 100 ng/mL VEGF and G + 1000 ng/mL VEGF groups increased by 2.9, 5.6, and 6.5 times, respectively (220.7 ± 36.5 vs. 641.3 ± 66.5 (***p = 0.000656), 1240.7 ± 94.9 (****p = 0.000064), 1433.3 ± 85.7 (****p = 0.000023), respectively. Figure 3c). The same trend was observed in HUVEC cells (Figure 3c). The number of migrated HUVEC cells in the G + 10 ng/mL VEGF, G + 100 ng/mL VEGF, and G + 1000 ng/mL VEGF groups increased by 1.2, 4.2, and 5.2 times, respectively, when compared to the number migrated in the control group (313.0 ± 78.9). The respective migrated cell numbers were 386.7 ± 24.7 (p = 0.197761), 1301.3 ± 124.8 (***p = 0.000317), and 1616.7 ± 146.1 (***p = 0.000169).

HUVEC possesses the potential of stem cells, as evidenced by its capacity to divide and migrate in response to angiogenic signals swiftly. Consequently, we utilized HUVEC to examine the capacity for capillary-like structure formation under various conditions. The number of tube-like structure formations generated on HUVEC tends to increase with the increase of VEGF concentration (Figure 3b). Compared with the number of tubes per field in the control group (62.0 ± 3.6), the number of tubes per field in the G + 1000 ng/mL VEGF group increased to 140.3 ± 1.5 (****p = 0.000004, Figure 3d). The experimental outcomes described above demonstrated that VEGF can stimulate angiogenesis during the injury repair process and laid the groundwork for additional functional verification.

VEGF is a necessary regulator for normal angiogenesis, and VEGFR1 is the most potent mitotic receptor for recruiting hematopoietic precursors and monocytes at the site of pathological pro-inflammatory responses, thereby promoting angiogenesis. HUVEC cells were co-incubated with VEGF and VEGFR1 antibodies, and the localization of VEGFR1 protein and its expression in HUVEC cells were visualized by fluorescence immunoassay. The experimental results showed that the green fluorescence in the field became brighter with the concentration of VEGF increasing, indicating increased expression of VEGFR1 protein (Figure 4a). To further examine the findings, flow cytometry was used to perform a comprehensive quantitative analysis of the fluorescence data, which verified that the expression of VEGFR1 protein rose as VEGF levels increased (Figure 4b). The results demonstrate that VEGF can promote the proliferation of HUVEC and Hacatl cells, implying that the VEGF@PAG nanofiber membrane might encourage angiogenesis and cell migration, which in turn could assist in the healing of wounds.

3.4 Effects of VEGF@PAG dressing on wound healing in rats

To demonstrate the clinical utility of VEGF@PAG, we developed the models of cutaneous wound injury on the back of rats and assessed the in vivo wound healing efficacy (Figure 5a). As shown in Figure 5b, the wound area of all groups of rats gradually decreased within 18 days in the in vivo wound healing experiment. Following an 18-day treatment period, the VEGF@PAG group of rats exhibited complete closure of their wounds. However, the control group (which received natural restoration) and the PAG group continued to have visible wounds and scabs at the site of the lesions. We conducted quantitative analysis according to the measured wound area and further statistically analyzed the recovery effect of rats in different treatment groups. The treatment effect of the VEGF@PAG group and the PAG group was significantly better than that of the control group, with the healing rate of the VEGF@PAG group reaching 77.8 ± 6.7% (Figure 5c) at day 9. The wounds in the PAG group (*p = 0.042878) and the VEGF@PAG group (*p = 0.037795) were significantly more minor than those in the control group at day 18. The healing ratio of the VEGF@PAG group approached 100% on day 18, confirming the ability of VEGF@PAG to accelerate wound healing. Additionally, the reactive oxygen species (ROS) at the site of injury on day 9 and day 18 of treatment were determined in various groups. ROS expression in wound tissue cells was significantly reduced upon VEGF@PAG contact with the tissue (Figure S2, Supporting Information), suggesting that VEGF@PAG may enhance the healing process of inflammatory lesions by absorbing excessive intracellular ROS in response to oxidative stress in the local wound area.

To further explore wound recovery, H&E staining, and Masson staining analysis on rats' wound injury sites were performed. As shown in Figure 6a, it was observed that more inflammatory cells appeared in the wounds of rats with skin wound injury in the control group after 9 days of treatment, while inflammatory cells in the wounds of the PAG group and VEGF@PAG group had begun to decrease, fibroblasts and neo capillaries increased within evident hyperemia. After 18 days of treatment, each group's capillaries and inflammatory cells decreased, and there was no obvious hyperemia. However, the control group had fewer skin appendages, disordered structures, and unclear conditions. Compared with the control group, the VEGF@PAG group was close to normal skin, with more skin appendages and a significant decrease in capillaries. As shown in Figure 6b, it could be observed that 9 days after surgery, more collagen fibers (blue) in the VEGF@PAG group than in the PAG group and the control group, and their collagen deposition was denser and more organized, which confirmed the better wound recovery effect of rats. The analysis of the EVG staining results similarly confirmed the above results (Figure S3, Supporting Information). After 9 and 18 days of treatment, the PAG and PAG@VEGF groups showed significantly more elastic fibers (dark brown) and collagen fibers (pinks) than the control group, indicating that PAG@VEGF and PAG could accelerate the formation of collagen fibers and thus promote wound healing. The results of the immunohistochemical analysis demonstrated a substantial increase in fibroblast growth factor (FGF2) secretion in the VEGF@PAG group subsequent to treatment (Figure S4, supporting information). This increase surpassed that of both the control group and the PAG group, suggesting that the VEGF@PAG group has the capability to stimulate FGF2 production and enhance the efficacy of tissue repair.

This study used electrospinning to create VEGF@PAG, an artificial bioactive fiber membrane that is highly drug-loaded. The PLGA platform was primed with VEGF to facilitate endothelial cell migration and proliferation, the creation of a vascular lumen, and expedite tissue repair and wound healing. With its suitable rate of expansion and degradation, the nanofiber membrane VEGF@PAG permits continuous drug release while leaving the required space for the growth of new tissue. In addition to alleviating the temporal and spatial deficiencies inherent in conventional dressings, this novel bioactive scaffold prevents secondary skin injury upon removal, thereby exhibiting promising medical implications within the field of skin tissue engineering.

Ethical approval declarations

All animal handling procedures were performed in accordance with the guidelines approved by the Ethics Committee of the People's Hospital of Guangxi Zhuang Autonomous Region, with ethical approval (No: KY-ZC-2022-149) obtained from the Institutional Review Board. This study adhered to the relevant animal welfare guidelines to ensure ethical treatment of the animal subjects involved.

Acknowledgments

This study was supported by the Guangdong Basic and Applied Basic Research Foundation [2020A1515110625], the Youth Science Innovation and Entrepreneurship Talent Training Project of Nanning (20220108), and the Self-funded Scientific Research project of Guangxi Zhuang Autonomous Region Health Commission (No. Z-A20220149).

Availability of data and materials

The authors declare that all data supporting the results of this study are available within the paper and its Supplementary Information.

Declarations

The authors declare no competing interests. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Scheme 1 is available in the Supplementary Files section.

The authors declare no competing interests.

Scheme1.png
Scheme 1. The nanofiber membrane, composed of bioactive material, exhibits excellent air and moisture permeability in stretchable areas of the body. It possesses adequate mechanical strength and an extracellular matrix (ECM)-like structure that promotes cell adhesion and proliferation while minimizing the risk of rejection reactions.
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Angiogenesis-Enhanced Biomimetic Nanofiber Dressings: VEGF-Infused Electrospun Membranes for Targeted Wound Healing and Tissue Regeneration

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Abstract

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1. Introduction

2. Materials and Methods

3. Results and Discussion

4. Conclusion

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Acknowledgments

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References

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Supplementary Files

Status:

Version 1