Startseite Janus nanofiber membrane films loading with bioactive calcium silicate for the promotion of burn wound healing
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Janus nanofiber membrane films loading with bioactive calcium silicate for the promotion of burn wound healing

  • Jin Wang , Long Li , Shanbo Ma , Xiaodi Guo , Meiling Zheng , Shan Miao EMAIL logo und Xiaopeng Shi EMAIL logo
Veröffentlicht/Copyright: 10. Juni 2025
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Abstract

Janus nanofiber membrane films (JNMF) are known for their asymmetric properties. This includes protecting the wound from external liquid contamination and efficiently soaking up excess fluids. The morphology of these membranes was meticulously analyzed using scanning electron microscopy, while their water absorption potential, water contact angle, and crucial mechanical properties were rigorously tested. With increasing calcium silicate (CS) concentration in JNMF, the tensile strain first increased and then decreased. When CS content was 15%, its peak value was 38.28%, indicating the best performance. In vitro tests confirmed that JNMF loaded with Si4+ effectively promote cell adhesion, multiplication, and directional migration. In vivo studies have shown that JNMF possesses anti-inflammatory properties, promotes angiogenesis, and facilitates the regeneration of epidermal tissue. Immunohistochemical staining underscored the JNMF ability to accelerate wound healing by upregulating CD31 and α-SMA expression. As a result, JNMF presents itself as a promising material for the treatment of severe burn wounds.

Keywords: Janus nanofiber membrane films; silicon ions; burn wound; histopathological; immunohistochemical

Abbreviations

CS

calcium silicate

FTIR

Fourier transform infrared spectroscopy

HEF

human epidermal fibroblasts

HUVEC

human umbilical vein endothelial cells

JNMF

Janus nanofiber membrane films

PLA

polylactic acid

PVA

polyvinyl alcohol

SEM

scanning electron microscopy

WCA

water contact angle

XRD

X ray derivatization

1 Introduction

The skin is the largest organ of the human body, and its primary function is to protect the body from external stimuli and pathogens. However, due to its location on the outermost surface of the body and its delicate nature, it is prone to injuries caused by various accidents. The process of wound healing generally involves four stages: hemostasis, inflammation, proliferation, and remodeling (1,2). The normal inflammatory response during the inflammatory phase facilitates the removal of necrotic tissue and harmful substances, thereby creating favorable conditions for subsequent cell proliferation and reconstitution (3). However, excessive inflammation can trigger macrophages to secrete a significant amount of inflammatory cytokines and hydrolases, resulting in edema, suppuration, and even wound necrosis, which pose challenges to the process of wound healing (4). The majority of dressings currently available in the market fail to effectively prevent the ingress of external bacteria during the wound healing process, leading to delayed wound healing. Consequently, an ideal wound dressing should create a microenvironment that promotes optimal wound healing by encompassing multiple functionalities, including hemostasis, anti-inflammatory properties, promotion of tissue regeneration, and bacteriostatic effects (5).

In the realm of electrospinning, significant advancements have broadened the horizons for developing advanced wound dressings. Technology is progressing from basic single-fluid blending processes to more sophisticated techniques such as bi-fluid coaxial electrospinning, side-by-side electrospinning, and multi-fluid triaxial systems. Additionally, tri-layer Janus structures and their combinations are being explored to produce novel types of nanofibers (6–10). A key advantage of these intricate internal nanostructures lies in their capacity to tailor multiple components for specific synergistic effects. This progress facilitates the customization of nanofibers with various components designed to function collaboratively for enhanced wound healing. Our research builds upon these innovations by introducing a novel approach that capitalizes on the sequential collection of nanofibers containing diverse components derived from a single-fluid process. This method not only enables the tailoring of multiple components but also provides the substantial benefit of customizing the composition of nanofibers according to the specific requirements of wound healing. By integrating these recent developments in electrospinning with our innovative methodology, we aspire to expand the possibilities for designing and enhancing wound dressing functionality. This forward-thinking strategy holds considerable potential to make a significant impact in this field and improve outcomes for patients suffering from various types of wounds.

Janus nanofiber membrane films (JNMF) are biomaterials that can imitate natural tissue structures, creating a microenvironment that guides cellular behavior and function (11). The JNMF, extensively investigated in recent years (12–17), exhibit a micro–nanofiber structure resembling the human extracellular matrix. Its remarkable ductility and tensile strength closely resemble those of human skin, enabling an optimal fit for wound coverage. Moreover, it promotes adhesion, proliferation, migration, and differentiation of skin tissue cells while facilitating gas exchange through interstitial pores (18–22). Additionally, the JNMF are lightweight and thin, thereby avoiding compression on the wound and facilitating portability (23). Currently, extensive research has been conducted both domestically and internationally in this field. Wang et al. developed a hydrophilic fiber membrane that exhibited excellent liquid absorption capabilities (24). However, this fiber membrane is susceptible to contamination from external pollutants, resulting in delayed wound healing. Deka et al. fabricated a robust hydrophobic fiber membrane which effectively resists biological adhesion, prevents water and bacterial adherence from the environment, thus promoting wound healing (25). However, the prevailing concept in wound healing currently is wet healing. This type of highly hydrophobic fibrous membrane impedes the creation of a suitable wet healing environment, thereby hindering the speed and efficacy of wound healing (26). The asymmetric bilayer structure of nanofiber membrane dressings aims to resolve the contradiction between liquid absorption, waterproofing, and antifouling simultaneously. The hydrophilic layer within the bilayer structure comes into contact with the wound, facilitating direct and rapid absorption of wound exudate while providing an appropriate moist microenvironment for wound healing. Conversely, the hydrophobic layer remains exposed to air and effectively repels foreign substances such as bacteria from infiltrating the wound site. Given these circumstances, there is an urgent need to develop nanofiber membrane dressings with an asymmetric structure that can offer a conducive microenvironment for promoting optimal wound healing.

Bioactive glass is an inorganic degradable silicate biomaterial composed of oxides such as SiO2, CaO, P2O5, and Na2O (27–29). It was discovered over 50 years ago by Professor Hench et al. at Florida State University and has since been extensively utilized for the repair of hard tissues including bone and teeth (30–33). Recent studies have demonstrated that bioactive glass possesses significant potential in soft tissue repair due to its ability to stimulate angiogenesis and expedite wound healing. Vallet-Regí et al. observed that the gradual release of Si4+ from bioactive glass promotes endothelial cell proliferation and migration, along with the expression of related cytokines like vascular endothelial growth factor (VEGF), thereby facilitating wound healing (34). Bioactive glass can protect endothelial cells under hypoxia and enhance the expression of gap junction protein CX43 in skin repair cells (endothelial cells and fibroblasts), thereby accelerating the process of vascularization (35). However, bioactive glasses are not easily applicable directly on wounds due to their granular appearance. To address this issue, bioactive glass can be integrated into the hydrophilic layer of an asymmetric fiber membrane. This nanofiber membrane can serve as a carrier for bioactive glass, gradually releasing active silicon ions to expedite wound vascularization and promote wound healing (36).

In summary, this study aims to design and prepare a bioactive nanofiber membrane dressing loaded with Si4+ with an asymmetric structure to address the key problem of burn wound repair. The bioactive nanofiber membrane dressing can efficiently manage exudate and provide waterproof and antifouling functions due to its amphiphilic characteristics. Additionally, it can promote vascularization through the release of activated Si4+ from bioactive glass. The influence of electrospinning technology on the infiltration of nanofiber membrane was studied in detail. The effect of bioactive glass loading on the release of Si4+ from the nanofiber films was investigated. To explore the regulatory effect of Si4+ release from nanofiber membrane on the biological behavior of skin tissue cells, and to explore the mechanism from the molecular and cellular levels. To verify the effect of nanofiber membrane on wound repair and reveal its mechanism by constructing a mouse skin burn model. The study protocol is shown in Figure 1.

Figure 1 (a) Preparation of CS-loaded JNMF. (b) In vitro and in vivo study of the effect of nanofibrous membrane on wound.
Figure 1

(a) Preparation of CS-loaded JNMF. (b) In vitro and in vivo study of the effect of nanofibrous membrane on wound.

2 Materials and methods

2.1 Preparation and characterization

2.1.1 Reagents

Polylactic acid (PLA) is purchased from Jinan Daigang Biomaterial Co., Ltd. Polyvinyl alcohol (PVA) is obtained from Shanghai Maclin Co., Ltd. Sodium silicate, calcium nitrate, tetrahydrofuran (THF), dimethylformamide (DMF), anhydrous ethanol, 50% aqueous solution of glutaraldehyde, and concentrated hydrochloric acid were purchased from Aladdin.

2.1.2 Preparation of spinning solution and CS nanoparticles

First, 4 mL of THF and 1 mL of DMF are mixed in a sealed bottle. Then, 1.20 g of PLA was weighed by an electronic scale and added into the bottle for stirring for 4 h. Finally, transparent PLA solution is eventually obtained. Similarly, 1.9 g of PVA was added into 12 mL distilled water at 60°C to form a transparent solution. About 3.74 g of Ca (NO3)2·4H2O was weighed and poured into a clean beaker with 40 mL distilled water. Na2SiO3·9H2O of 4.54 g was added into another clean beaker, then 40 mL distilled water was added to the beaker and stirred. The two solutions were thoroughly mixed in a magnetic stirrer and then reacted at room temperature for 1 h. After the reaction is complete, the precipitation in the mixed solution is filtered out and dried in a constant temperature oven at 45°C. After drying, white granular sediment is obtained.

2.1.3 Fabrication of PVA solutions containing different concentrations of CS nanoparticles

PVA of 1.9 g and 0.19 g/0.28 g/0.38 g of CS powders were poured into 12 mL water at 60°C to form PVA-10% CS solution, PVA-15% CS solution, and PVA-20% CS solution.

2.1.4 Fabrication of PLA/PVA, PLA/PVA bilayer membrane containing different concentrations of CS nanoparticles

The membrane is fabricated by a homemade electrospinning setup. The injection pump spray speed of PLA solution was adjusted to 800 μL·h−1, the voltage was 5.5–6.0 kV, the drum speed was 800 rpm, and the preset injection volume was 3,000 μL for spinning. When there is no solution in the syringe or the progress of injection pump settings is completed, take another clean glass syringe and fill it with a hydrophilic needle. Add 3.4 mL of the prepared PVA solution or PVA-10% CS solution or PVA-15% CS solution or PVA-20% CS solution to the syringe. The injection pump spray speed was adjusted to 800 μL·h−1, the voltage was 15.5–16.0 kV, the drum speed was 3,000 rpm, and the preset injection volume was 3,000 μL for spinning. When there is no solution in the syringe or the schedule set by the injection pump is completed, the spinning of the nanofibrous membrane is completed. The membrane is crosslinked by 14 mL anhydrous ethanol, 7 mL 50% aqueous solution of glutaraldehyde, and 1.5 mL concentrated hydrochloric acid.

2.1.5 Characterization

The morphology of the double-layer PVA/PLA nanofibrous membrane was characterized by scanning electron microscopy (SEM; Hitachi S4800). The double-layer membrane was sputtered with a thin layer of gold prior to SEM observation. The diameters of PVA and PLA fiber were determined from SEM micrographs using Nano measure 1.2 software. About 100 nanofibers were randomly selected for measurement. Water contact angle (WCA) measurements are made on a contact angle device (Kruss DSA 25 Contact Angle Analyzer, Germany). A drop of distilled water (2 μL) was automatically added to the surface of the sample, and the final average WCA of the two layers was obtained by measuring ten different locations on the surface of the sample. Mechanical properties of the membranes are evaluated by using an electronic tensile testing machine (Shenzhen Suns Technology Stock Co. Ltd, China).

2.2 Regulation of biological behavior of skin tissue cells by JNMF

2.2.1 Effect of JNMF on proliferation, migration, and growth morphology of human epidermal fibroblasts (HEF) and human umbilical vein endothelial cells (HUVEC)

JNMF were cut into the corresponding size by a hole punch under sterile condition, and then sterilized under ultraviolet light for 30 min. JNMF piece was implanted into the bottom of the corresponding hole plate and completely covered the bottom. HEF and HUVEC in logarithmic growth phase were digested with trypsin, collected, and centrifuged at 1,000 rpm. The supernatant was discarded, and the cells were resuspended in fresh culture medium. The cells were counted, and the cell density was adjusted.

Three experimental groups, namely the blank group, control group, and JNMF group, were established for routine culture over a period of 24 h. On the first, third, fifth, and seventh day of culture, a pre-prepared CCK8 solution (30 μL) was added to each well plate. Following a 3 h incubation period, the supernatant (100 μL) was collected into a separate 96-well plate and its absorbance at 450 nm was measured using a microplate reader. The cell proliferation rate was then calculated using the following formula: Proliferation rate% = 100% × (A intervention group − A blank group)/(A control − A blank group).

Three experimental groups were established, including a blank group, a control group, and JNMF group. After 24 h of standard culture, the liquid in each well was discarded and rinsed three times with PBS buffer before fixation with a 2% glutaraldehyde solution. The 24-well plate was then placed in a refrigerator at 4°C overnight and subsequently transferred to an electron microscope facility for scanning the following day to observe cellular growth morphology.

Cell scratch assay: When the cell density reached 80%, a sterile pre-cooled steel rod was carefully positioned above the wells, and a 200 μL pipette tip was used to create a straight scratch with moderate angle to minimize cellular damage. Subsequently, the six-well plates were placed under a microscope for documentation (timed at 0 h), followed by incubation in a cell incubator (37°C, 5% CO2). After another 24 h, photographs were taken and recorded to observe cell healing in both control and experimental groups.

2.2.2 JNMF promoted angiogenesis of HUVEC

JNMF were cut into 15 mm diameter discs (the diameter of a 24-well plate) with a hole punch under sterile conditions, sterilized under UV light for 30 min, and JNMF discs were implanted into the bottom of the 24-well plate. Matrix glue was prepared on an ice bath with a 1:9 matrix glue. Then, 200 μLL was absorbed and added to a 24-well plate without JNMF and left in a cell incubator for 30 min. After matrix gel solidification, HUVEC in logarithmic growth phase were taken, digested with trypsin, collected, and centrifuged at 1,000 rpm·min−1, and the supernatant was discarded to resuspend cells. Cells were counted, the density was adjusted to 1 × 104 cells per well, and each group was set up with three multiple wells to set the blank group, control group, and nanofiber membrane group. The cells were placed in the incubator for 48 h. Cell lumen formation was observed under a microscope.

2.2.3 Immunofluorescence staining was used to detect the expression level of CD31 protein

Immunofluorescence was employed to assess the expression of CD31 protein. Cells were fixed with PBS three times for 3 min each, followed by fixation with 4% paraformaldehyde for 15 min and subsequent washing with PBS three times for 3 min each. Permeabilization was achieved by adding Triton X-100 for 10 min, followed by drying and washing with PBS three times for 3 min each. Subsequently, the cells were blocked without washing using a solution containing 5% BSA for 40 min. Primary antibody (CD31 at a dilution of 1:200) was added and incubated overnight at 4°C; it was then removed and rewarmed the next day for 1 h before being washed with PBS three times for 5 min each. The cells were incubated in the dark with a fluorescent secondary antibody (1:200) at room temperature for 1 h, followed by three washes with PBS for 5 min each. Subsequently, the cells were stained with DAPI for 8 min and washed again with PBS three times for 5 min each. Finally, the slices were sealed using an anti-fluorescence quench agent, observed under a fluorescence microscope, photographed, and the results were statistically analyzed based on optical density value/area.

2.2.4 JNMF promoted the expression of α-SMA in HEF and VEGF in HUVEC

The HEF and HUVEC samples were collected, followed by addition of TRIzol lysate and homogenization. Subsequently, chloroform was added, and the mixture was centrifuged at room temperature for 5 min. The upper aqueous phase was carefully transferred, after which isopropyl alcohol was added at room temperature for 10 min. Following centrifugation, the supernatant was discarded, and 75% ethanol was added to the pellet with subsequent shaking to ensure proper mixing. After another round of centrifugation at 4°C, the supernatant was removed and the EP tube containing RNA pellet was air-dried before being dissolved in diethyl pyrocarbonate water for complete dissolution of RNA. Finally, RNA concentration measurement took place along with primer sequence design, cDNA synthesis through reverse transcription, polymerase chain reaction (PCR); and analysis of data using appropriate software to obtain △Ct (cycle number) values. Consequently, mRNA expression levels of α-SMA in HEF cells as well as VEGF in HUVEC cells were determined.

2.3 Experimental study on the repair of burn wound with JNMF

To establish a murine burn wound model, the experimental protocol underwent review and approval by the Animal Care and Use Committee of Air Force Medical University. Animals were anesthetized using isoflurane inhalation and depilated with a razor and depilating cream. The self-made scald instrument was activated and allowed to reach the working temperature (100°C). The pre-prepared abrasive tool was then attached, ensuring contact between the mold port and the animal’s skin for 4 s, resulting in the formation of a burn wound measuring 1.5 cm × 1.5 cm. Thirty mice (n = 10) were randomly assigned to three groups: control group (untreated), gauze group (gauze), and JNMF group (nanofiber material). The wound was covered with a 2 cm × 2 cm dressing and secured with a medical breathable bandage. The wound was photographed at specific time intervals (1, 6, 8, 11, and 21 days), and the wound area was quantitatively analyzed using Image-Pro Plus software. Histopathological examination of the wound tissue was conducted at predetermined time points for histological analysis. HE staining and Masson staining were employed to assess wound healing and collagen deposition, respectively, while CD31 and α-SMA immunohistochemical staining were performed on Days 11 and 21 wounds to investigate the mechanisms underlying wound healing. Real-time quantitative polymerase chain reaction was utilized to explore the expression of inflammation-related genes during the process of wound healing.

2.4 Statistical analysis

The experiments were repeated a minimum of three times (n ≥ 3) and the results were presented as mean ± standard deviation. One-way ANOVA was employed to determine the significance levels between the control and experimental groups (*P < 0.05, **P < 0.01, ***P < 0.001).

3 Results and discussion

3.1 Preparation and characterization of JNMF

We successfully prepared JNMF with different CS loading concentrations of PLA/PVA-0% CS, PLA/PVA-10% CS, PLA/PVA-15% CS, and PLA/PVA-20% CS. The SEM images showed that the diameter of PLA fibers was thick and unevenly arranged, with a diameter of 1.12 μm. The diameter of PVA fiber was smaller and more uniform, with a diameter of 0.85 μm. The reason for this phenomenon may be that the speed set in the spinning process of PLA is relatively small, only 800 rpm. The spinning solution speed of PVA is 3,000 rpm. So, the higher the rotational speed, the smaller the diameter of the fibers, and the more evenly the fibers are arranged. In addition, the voltage also affected the diameter of the fiber. The voltage of PLA spinning was only set at 5.50–6.00 kV, while that of PVA spinning is set at 15.5–16.0 kV. The higher voltage may also account for the smaller diameter of the fibers. At the same time, it was found that the fiber diameter also increased with the increase of CS concentration, 10%, 15%, 20% corresponding to 0.80, 0.81, 0.83 μm. This was because of the doped CS encapsulated in the fiber (37–40) (Figure 2a–f).

Figure 2 Preparation of JNMF containing different concentrations of CS: (a) PLA, (b) PVA, (c) PVA-10% CS, (d) PVA-15% CS, (e) PVA-20% CS, and (f) mean diameters of fibers containing different concentrations of CS.
Figure 2

Preparation of JNMF containing different concentrations of CS: (a) PLA, (b) PVA, (c) PVA-10% CS, (d) PVA-15% CS, (e) PVA-20% CS, and (f) mean diameters of fibers containing different concentrations of CS.

Then the average diameter of all the fibers was measured, which further verified the result that the diameter of PLA fiber was larger than that of PVA fiber. To explore whether CS was successfully reconstituted into the fiber and the distribution of elements in the fiber, we also mapped PLA/PVA composite fiber membrane containing 15% CS under SEM. The test results are shown in Figure 2. Figure 3a shows a picture of PLA/PVA-15%. Figure 3b shows the distribution of Si4+ in the fiber. Since only CS contains Si element, CS has been successfully synthesized into PLA/PVA fiber membrane, and the distribution is relatively uniform (41,42). Figure 3c shows the distribution of element C in the fibers, and the general morphology of the fibers can be vaguely observed.

Figure 3 (a) SEM photo of PLA/PVA-15% CS, (b) Si element distribution diagram. (c) C element distribution map, (d) FTIR spectrum of CS and PLA/PVA composite JNMF containing different concentrations of CS, and (e) XRD of PLA/PVA composite JNMF with different concentrations of CS.
Figure 3

(a) SEM photo of PLA/PVA-15% CS, (b) Si element distribution diagram. (c) C element distribution map, (d) FTIR spectrum of CS and PLA/PVA composite JNMF containing different concentrations of CS, and (e) XRD of PLA/PVA composite JNMF with different concentrations of CS.

Figure 4 shows that for JNMF, the initial WCA on the PLA side is 120°, and the contact angle barely changes within 10 s. The average WCA on the PLA side of several JNMF was 117.6°, showing significant hydrophobicity. The initial WCA on the PVA-CS side was different. With the increase of compound CS concentration, the initial WCA was 82.3°, 77.7°, 68.1°, and 68.0°, respectively. The WCA of several PVA fibers decreased almost to zero within 5–6 s or remained unchanged after decreasing to a certain extent, indicating that PVA fibers had obvious hydrophilicity. In addition, increasing CS concentration can reduce the size of the initial WCA within a certain range, indicating that CS can improve the hydrophilicity of PVA fibers (43).

Figure 4 Effects of different concentrations of CS on the contact angle of PLA/PVA-CS JNMF.
Figure 4

Effects of different concentrations of CS on the contact angle of PLA/PVA-CS JNMF.

The Fourier transform infrared spectroscopy (FTIR) spectra of the PLA/PVA JNMF and CS are shown in Figure 3d. The figure shows the top to bottom IR spectra of the PLA/PVA composite JNMF containing different concentrations of CS. In the infrared spectrum of CS, there is a peak at 474 cm−1, which belongs to the characteristic peak of Si–O–Si bond. There is a broad peak at 3,463 cm−1, which belongs to the characteristic peak of the hydroxyl group. This peak of the hydroxyl group indicates that the CS powder contains crystalline water. The infrared spectra of PLA/PVA showed a broad peak at 3,385 and 2,943 cm−1, which are characteristic peaks of hydroxyl and methylene in PVA, respectively. There is a peak at 1,095, 1,382 and 1,735 cm−1, which belonged to the characteristic peaks of ether bond, methyl group, and carbonyl group in PLA, respectively. This shows that PLA and PVA have been successfully combined. There is no peak at 474 cm−1, indicating that there is no CS in PLA/PVA composite membrane. The infrared spectra of PLA/PVA-10% CS, PLA/PVA-15% CS, and PLA/PVA-20% CS show a wide peak at 3,385 and 2,943 cm−1, which was characteristic peaks of hydroxyl and methylene in PVA, respectively. There is a peak at 1,095, 1,382 and 1,756 cm−1, which belonged to the characteristic peaks of ether bond, methyl group, and carbonyl group in PLA, respectively. This indicates that PLA and PVA exist in these three composite membranes. There is a small spike at 474 cm−1 in all three spectra, indicating that CS has been successfully recombined into the PLA/PVA nanofibrous membrane (44,45).

JNMF were tested by X-ray derivatization (XRD) and the effect of CS concentration on the crystallinity of nanofiber was studied. The test results are shown in Figure 3e. The PLA/PVA JNMF without CS show amorphous state. The peaks of the four curves did not change much, indicating that the concentration of CS particles had no effect on the size of the peaks. However, the formation of hydrogen bond between CS, PLA, and PVA resulted in the decrease of crystallinity, increase of peak width, and decrease of height of PLA/PVA composite JNMF (46).

Then the effects of CS on mechanical properties of PLA/PVA JNMF were studied. The results showed that when the concentration of CS is 0%, the mechanical properties of PLA/PVA composite membrane are better, the fracture stress is 3.38 MPa, and the fracture strain is 67.20%. However, with the increase of the concentration of composite CS, the fracture stress of the three kinds of composite membranes was 1.63, 1.20, and 0.69 MPa, respectively, showing a gradual downward trend, indicating that the doping of CS reduced the elasticity of the membranes to a certain extent. The fracture strain was 62.13%, 64.16%, and 38.28%, respectively, showing a trend of increasing first and then decreasing. The fracture stress–strain data of the four JNMF are shown in Table 1. The stress–strain curves of the four JNMF are shown in Figure 5a. Through the analysis of the data, JNMF with composite concentration of 15% CS can reach the maximum strain and small stress in the three kinds of membranes with CS added, indicating that its mechanical properties in the three kinds of membranes are the best (47–49).

Table 1

Fracture stress and strain of electrospun PLA/PVA fiber films with varying concentrations of CS

CS (%) Rupture stress (MPa) Fracture strain (%)
0 3.380 67.264
10 1.632 62.138
15 1.203 64.169
20 0.687 38.281
Figure 5 Mechanical properties and water absorption test results of JNMF. (a) Stress–strain curves of PLA/PVA nanofiber membranes with different concentrations of CS. (b) Liquid adsorption performance test of PLA/PVA JNMF with different concentrations of CS.
Figure 5

Mechanical properties and water absorption test results of JNMF. (a) Stress–strain curves of PLA/PVA nanofiber membranes with different concentrations of CS. (b) Liquid adsorption performance test of PLA/PVA JNMF with different concentrations of CS.

As can be seen from Figure 5b, the liquid absorption ratio of PLA/PVA JNMF was about ten times of its own weight, up to 11.53 g·g−1, showing good liquid absorption performance. However, with the increase of CS concentration in JNMF, the liquid absorption ratio decreased, and the two showed a negative correlation trend (50).

3.2 Regulation of biological behavior of skin tissue cells by JNMF

Electron microscopy revealed that after 48 h of culture in JNMF, cells displayed well-extended morphology with significantly increased migration distance (P ≤ 0.01) and wound healing ability (Figure 6a and b) (51), indicating that JNMF effectively promoted HEF and HUVEC proliferation, growth, stretchability conducive to tissue formation, and vascular endothelial cell development.

Figure 6 Investigation of cellular responses to JNMF: (a) JNMF facilitated the migration of HEF and HUVEC and (b) the impact of JNMF on the morphology of HEF and HUVEC (*P ≤ 0.05, **P ≤ 0.01).
Figure 6

Investigation of cellular responses to JNMF: (a) JNMF facilitated the migration of HEF and HUVEC and (b) the impact of JNMF on the morphology of HEF and HUVEC (*P ≤ 0.05, **P ≤ 0.01).

The results of the cell proliferation test, SEM analysis, and scratch test demonstrated that cells cultured in JNMF exhibited a rapid growth trend on Day 3 and significantly higher cell proliferation on Days 5 and 7 compared to the control group (P ≤ 0.01) (Figure 7a and b) (52,53). Additionally, utilizing the 2−∆∆CT method for determining relative gene expressions levels, we found a significant reduction in α-SMA expression among HEFs cultured on our JNMF (P ≤ 0.05), whereas VEGF expression was notably increased among HUVECs under similar conditions (P ≤ 0.01) (Figure 7c and d) (54).

Figure 7 JNMF facilitated cellular proliferation and enhanced protein expression: (a) JNMF promote the proliferation of HEF, (b) JNMF promote the proliferation of HUVEC, (c) JNMF promote α-SMA expression in HEF, and (d) JNMF promote VEGF expression in HUVEC (*P ≤ 0.05, **P ≤ 0.01).
Figure 7

JNMF facilitated cellular proliferation and enhanced protein expression: (a) JNMF promote the proliferation of HEF, (b) JNMF promote the proliferation of HUVEC, (c) JNMF promote α-SMA expression in HEF, and (d) JNMF promote VEGF expression in HUVEC (*P ≤ 0.05, **P ≤ 0.01).

The results from this study demonstrate that cells cultured on JNMF exhibit an elongated morphology with increased formation of cell tubes when compared to those in the normal control group. Furthermore, by enhancing CD31 expression which regulates endothelial cell connectivity and communication, we observed significant promotion of endothelial cell survival and proliferation (P ≤ 0.01). These findings highlight the effective stimulation of tube formation and angiogenesis by our JNMF in both HEF and HUVEC cultures as depicted in Figure 8a–d (55,56).

Figure 8 JNMF facilitate angiogenesis: (a) immunofluorescence was used to detect CD31 protein expression, (b) tube formation assay in HUVEC, (c) statistical results of CD31 protein expression, and (d) statistical results of tube formation in HUVEC (*P ≤ 0.05, **P ≤ 0.01).
Figure 8

JNMF facilitate angiogenesis: (a) immunofluorescence was used to detect CD31 protein expression, (b) tube formation assay in HUVEC, (c) statistical results of CD31 protein expression, and (d) statistical results of tube formation in HUVEC (*P ≤ 0.05, **P ≤ 0.01).

3.3 In vivo evaluation of JNMF for promoting burn wound healing

Severe burn wounds are often accompanied by copious exudate and a high susceptibility to bacterial infection. Therefore, we established a mouse model with second-degree burns to validate the wound healing and antibacterial efficacy of asymmetric dressings (Figure 9a). In vivo animal experiments showed that during the wound healing process, the exudate produced by nanofiber membrane groups was absorbed by JNMF, while the gauze group had the opposite effect, and even more serious inflammatory reaction occurred to produce pus (Figure 9c). After 11 days of treatment with different dressings, the area of burn wound in nanomembrane group was significantly reduced, which was more than that in gauze group and blank group. On the 21st day of treatment, the nanomembrane-treated wounds were almost completely healed, and the local hair regeneration was strong. This suggests that nanofilms have the potential to promote burn wound healing (Figure 9b and d).

Figure 9 The in vivo assessment of burn wound healing in mice. (a) Results of HE staining of normal skin tissue and burn wound skin tissue (1. Normal skin tissue was stained with HE; 2. HE staining of burn wound tissue). (b) Wound healing process with different methods of treating wounds. (c) Exudates from wounds treated with different methods. (d) Statistical results of wound healing area of each group at different time points (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).
Figure 9

The in vivo assessment of burn wound healing in mice. (a) Results of HE staining of normal skin tissue and burn wound skin tissue (1. Normal skin tissue was stained with HE; 2. HE staining of burn wound tissue). (b) Wound healing process with different methods of treating wounds. (c) Exudates from wounds treated with different methods. (d) Statistical results of wound healing area of each group at different time points (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).

In JNMF treatment group, there was reduced granulation in the tissue, with predominant collagen formation and absence of inflammatory cells. Additionally, a lower distribution of blood vessels indicated complete healing of the skin tissue (Figure 10a) (57,58). Masson staining is commonly used to observe the development of cutaneous muscle fibers and collagen fibers. The results show that the expression of collagen fibers in the new skin tissue from JNMF group exceeded that of both the model group and control group (Figure 10b) (59). PCR results demonstrated that nanofilm treatment exhibited superior effects compared to both the model and control groups (Figure 10c) (60,61). JNMF loaded with calcium silicate (CS) not only alleviates the release of inflammatory factors but also effectively regulates the arrangement of collagen. JNMF can reduce inflammatory responses through the sustained release of silicon ions, promoting cell migration and collagen synthesis, thereby playing a significant role in wound healing. Furthermore, the bioactivity of calcium silicate further accelerates the healing process.

Figure 10 Evaluation of wound epithelialization, collagen deposition, and protein expression in distinct experimental groups. (a) Histological staining results of skin tissue in each experimental group were assessed at various time points. (b) Deposition of collagen was assessed in each group at 11 and 21 days. (c) Enhanced protein expression in the regenerated skin tissue (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).
Figure 10

Evaluation of wound epithelialization, collagen deposition, and protein expression in distinct experimental groups. (a) Histological staining results of skin tissue in each experimental group were assessed at various time points. (b) Deposition of collagen was assessed in each group at 11 and 21 days. (c) Enhanced protein expression in the regenerated skin tissue (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).

Generally, CD31 expression in the new skin tissue was low. Immunohistochemical results indicate that the 21-day nanofilm treatment group had lower CD31 expression compared to the control group. Myofibroblasts, highly active secretory cells derived from fibroblasts with the ability to synthesize collagen, exert traction on the surrounding connective tissue and promote wound contraction (Figure 11a and b). Upon completion of epithelization and cessation of myofibroblast contraction, α-SMA expression within the myofibroblasts ceases. The study results indicate a significant decrease in α-SMA expression in JNMF group compared to both the control and model groups at 21 days (Figure 11c and d) (62–64). The promotion of α-SMA and CD31 expression indicates that under the influence of the material, the ability of cells to migrate and regenerate blood vessels has been significantly enhanced. By modulating these biomarkers, JNMF provide a new strategy for improving wound healing.

Figure 11 Quantification of wound blood vessels (brown) on Days 11 and 21 was performed using immunohistochemical staining. (a) Results of immunohistochemical staining for CD31. (b) Statistical results for immunohistochemical staining of CD31. (c) Results of immunohistochemical staining for α-SMA. (d) Statistical results for immunohistochemical staining of α-SMA (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).
Figure 11

Quantification of wound blood vessels (brown) on Days 11 and 21 was performed using immunohistochemical staining. (a) Results of immunohistochemical staining for CD31. (b) Statistical results for immunohistochemical staining of CD31. (c) Results of immunohistochemical staining for α-SMA. (d) Statistical results for immunohistochemical staining of α-SMA (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).

4 Conclusion

In this study, we hypothesized that by modulating the concentration of CS doping, we could fabricate JNMF exhibiting exceptional mechanical properties, hydrophilicity, and water absorption capabilities. Furthermore, we aimed to validate their bioactivity in promoting wound healing through both in vitro and in vivo experiments. Compared to existing wound dressings, these JNMF not only demonstrate superior hydrophilicity and water absorption but also exhibit significant bioactivity that effectively facilitates wound healing. This advancement provides a novel approach for the development of advanced wound dressings. Looking ahead, our research team plans to further optimize the composition and structure of JNMF to enhance their mechanical strength and biocompatibility. We will conduct more comprehensive in vivo studies to evaluate their efficacy across various types of wounds. Additionally, we aim to explore the synergistic potential of combining JNMF with other wound dressings to improve overall healing outcomes.

# These authors have contributed equally.

  1. Funding information: This work was supported by the Innovation Program for Military Medicine Promotion Program of the Fourth Military Medical University (Project number: 2020JSTS09).

  2. Author contributions: Jin Wang: investigation, data curation, writing – original draft; Long Li: methodology, investigation; Shanbo Ma: project administration; Xiaodi Guo: methodology; Meiling Zheng: writing – original draft; Feiyan Wang: project administration; Shan Miao: conceptualization, supervision; Xiaopeng Shi: writing – review & editing, supervision.

  3. Conflict of interest: Authors state no conflict of interest.

  4. Data availability statement: All data generated or analyzed during this study are included in this published article.

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Received: 2024-07-23
Revised: 2024-10-26
Accepted: 2024-11-02
Published Online: 2025-06-10

© 2025 the author(s), published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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https://doi.org/10.1515/epoly-2024-0074
Schlagwörter für diesen Artikel
Janus nanofiber membrane films; silicon ions; burn wound; histopathological; immunohistochemical
Creative Commons
BY 4.0

Artikel in diesem Heft

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  26. Bending crack propagation and delamination damage behavior of orthogonal ply laminates under positive and negative loads
  27. Rapid Communication
  28. RAFT-mediated polymerization-induced self-assembly of poly(ionic liquid) block copolymers in a green solvent
  29. Corrigendum
  30. Corrigendum to “High-strength polyvinyl alcohol-based hydrogel by vermiculite and lignocellulosic nanofibrils for electronic sensing”
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Heruntergeladen am 3.10.2025 von https://www.degruyterbrill.com/document/doi/10.1515/epoly-2024-0074/html
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