Introduction

Human skin serves multiple vital functions, such as protecting the body from bacterial invasion, regulating body temperature, preserving moisture, and acting as the body’s largest interface with external environment [1]. When the skin suffers severe trauma, the wound healing process does not recover naturally, orderly, and perfectly. Various adverse factors can lead to irregular and not ideal wound healing. In such cases, despite the use of surgical sutures, wound dressings, and other instruments to perform surgical closure and isolate the wound from microbial contamination, remains the gold standard for treating skin injuries. However, this approach demands significant clinical costs and patient burdens [2, 3]. Decellularized extracellular matrix materials, as naturally-derived cell scaffolding carriers, exhibit excellent biocompatibility and biodegradability, which have been widely used in the treatment and care of various wounds and burns, such as Biodesign™ (COOK Corp., USA) and Gentrix® (Integra Corp., USA) [4, 5]. These extracellular matrix materials retain a suitable spatial structure for cell growth, and offer a rough, porous fibrous morphology that provides porosity and a permeable environment, contributing to the acceleration of wound healing [6,7,Wound model on the Bama miniature pig

All the animal experiments were complied with the guidelines of the Tian** Medical Experimental Animal Care, and animal protocols were approved by the Institutional Animal Care and Use Committee of Yi Shengyuan Gene Technology (Tian**) Co., ltd (No. YSY-DWLL-2023228). Two pigs after being anaesthetized by isoflurane, the skin defect was created by 20 mm biopsy punching on the back of the pig, then the animal models could be divided into four groups. (1) No further treatment as control groups. (2) The wound was covered by Tegaderm™. (3) The wound was covered by SIS. (4) The wound was covered by SIS/PAA/LAP. After implanting the materials, the implanted materials were fixed and covered by the sterile bandage to prevent the implant from drop** or separating from the wound. All the implanted materials were refreshed every two days, followed by the standard of care. At 3-, 6-, 9-, and 12-day post-surgery, the gross appearances of the wound regeneration were estimated, among them, the harvested located tissues were fixed in 10% formalin and sectioned for H&E staining, Masson’s trichrome staining, and TNF-α/CD163/DAPI (MA5-44021, ThermoFisher; EPR19518, Abcam) and VEGF/α-SMA/DAPI (ab2350, Abcam; MA1-06110, ThermoFisher) immunohistochemical staining. The images were captured on the digital slice scanning equipment. For reverse transcription-polymerase chain reaction (RT-PCR), the primers were designed and listed in Table S1, the harvested granulation tissues (1 mm×1 mm×1 mm) were immersed in Trizol (1 mL, Invitrogen, USA) to extract the total RNA. Gene expressions were calculated by 2−ΔΔCt method, and expressed in relation to the expression of a housekee** GAPDH gene.

Statistical analysis

All quantitative data was represented as mean ± standard deviation (SD). Statistical analysis was carried out using SPSS software by one-way analysis of variance (ANOVA). Differences between groups of *P < 0.05 were considered statistically significant, **P < 0.01 were considered highly significant. ***P < 0.001 were considered very highly significant.

Results and discussion

Preparation and characterization on the SIS/PAA/LAP wound dressing

The LAP nanoparticles owned particle size of approximately 10 nm. TEM images showed specific elements such as Mg, Si, Na, and O (Fig. S1). We firstly investigated the gelation properties of the LAP nanoparticles based on electrostatic interactions. As shown in Fig. S2, when the LAP concentrations in water were low (e.g., 2 wt.% and 4 wt.%), the electrostatic interactions between LAP nanoparticles were weak to make gel formation difficult. As the concentration increased, gel formation became evident, and the gelation time decreased with higher nanoparticle concentrations. When the LAP concentration reached 10 wt.%, the gelation time declined to 86.75 ± 6.70 s. Then, we tested the tissue shear adhesion of PAA hydrogels formed by cross-linking with different concentrations of AA, according to ASTM F2255. The results showed that with increasing AA concentration, the shear adhesion strength increased accordingly. At 40 wt.%, the adhesion reached 23.59 ± 5.59 kPa. Considering the higher adhesion strength, we chose a 40 wt.% concentration of AA for further experiments.

Figure 1a illustrated the schematic process of preparing the asymmetric SIS-based wound dressing, which involved coating the SIS surface with AA pre-polymerized solution containing LAP nanoparticles, and then subjected it to UV light exposure, resulting in the formation of SIS/PAA/LAP patch. Figure 1b showed the physical appearance of the SIS/PAA/LAP patch, by estimating its thickness, the PAA/LAP layer owned 4.93 ± 0.67 μm, demonstrating that the PAA adhered to the SIS surface without affecting the patch’s morphology, and the SIS/PAA/LAP patch exhibited good flexibility (Fig. 1c). SEM examination of the SIS/PAA/LAP revealed that the PAA/LAP coating adhered uniformly to the SIS surface (Fig. S4, Fig. 1d). Additionally, specific element map**s of LAP, such as Mg and Si, confirmed the LAP even distribution within the SIS/PAA/LAP patch. We also conducted mechanical tests on the SIS, SIS/PAA, and SIS/PAA/LAP patches. The results confirmed that the introduction of PAA and PAA/LAP coatings improved the mechanical strength. Young’s modulus and tensile strength of SIS patches increased from 215.15 ± 13.90 MPa and 17.56 ± 3.82 MPa to 360.63 ± 24.37 MPa and 48.90 ± 6.80 MPa, respectively, in the case of SIS/PAA/LAP. This asymmetric SIS/PAA/LAP structure exhibited tissue adhesion strength, as shown in Fig. S6. These results demonstrated that through selection of LAP and AA concentrations, we were able to prepare asymmetrically adhesive SIS-based wound dressings.

Fig. 1
figure 1

Preparation on the SIS/PAA/LAP wound dressing. a Schematic illustration on fabricating the SIS/PAA/LAP patch, starting from the AA pre-polymerized solution containing LAP nanoparticles coated on the SIS patch, to photopolymerization and crosslinking to form PAA/LAP adhesive layer on the SIS substrate. b Gross appearance on the SIS/PAA/LAP patch. c Flexibility of the SIS/PAA/LAP patch. d Cross-sectioned SEM image on the SIS/PAA/LAP patch. e Elemental map**s on the SIS/PAA/LAP.

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Evaluation on the tissue adhesion

We conducted systematic testing to evaluate the tissue adhesion of the wound dressings. In tissue shear adhesion tests, when the LAP concentration reached 10 wt.%, there was a significant increase in shear adhesion strength, rising from an initial value of 21.38 ± 4.94 kPa (SIS/PAA40%) to 32.98 ± 2.31 kPa (SIS/PAA40%/LAP10%) (Fig. 2(a, b)), which was attributed to the formation of a second crosslinking network and cohesion beside the PAA network. Furthermore, we compared the adhesion strength of the prepared SIS/PAA/LAP with commercial tissue adhesive products, demonstrating that its adhesion strength surpassed products such as Coseal (22.05 ± 1.65 kPa), TissuePatch (20.70 ± 2.12 kPa), and Tegaderm (7.25 ± 2.08 kPa). Additionally, we tested the burst strength according to the ASTM F2392 standard. The results revealed that the SIS/PAA/LAP had burst strength of 22.25 ± 2.50 kPa, significantly higher than Coseal (7.53 ± 1.22 kPa), TissuePatch (6.10 ± 0.62 kPa), and Tegaderm (2.50 ± 0.95 kPa) (Fig. 2(d, e)). We assumed that the combination of double network structures including LAP electrostatic interaction as the second cohesion, and PAA chemical crosslinking hydrogel as the tissue adhesion and first cohesion layers significantly enhanced the material’s mechanical strength and adhesion strength (Fig. 2f) [27]. Histological staining at 6 and 9 days revealed that the control and Tegaderm groups had obvious wound defects and lower collagen expression. In contrast, the wound gaps in the SIS, and particularly the SIS/PAA/LAP groups became narrower, with higher collagen mature (Fig. 4d, Fig. S8). This asymmetric adhesive SIS/PAA/LAP wound dressing with continuous ion release showed advantages in wound healing compared with other recent reports regarding on wound healing materials (Fig. 4e). Furthermore, in the VEGF/DAPI fluorescence staining, the SIS/PAA/LAP groups exhibited higher VEGF fluorescence intensity, being 1.84 times and 2.89 times than that of the control group at 6 and 9 days, respectively (Fig. S9). These findings suggested that the prepared SIS/PAA/LAP could firmly adhere to wound surfaces and, as LAP released active ions, to achieve excellent tissue repair results.

In pathological H&E staining of major metabolic organs such as the heart, liver, spleen, lung, and kidney at 9 days post-surgery, the tissue morphology and hematological analysis of the SIS/PAA/LAP groups showed no significant difference compared to the control groups, indicating good tissue compatibility (Fig. S10).

Fig. 4
figure 4

Wound regeneration in the rat full-thickness skin injury. a Gross appearance on the wound repair. b Real-time wound trajectory on the wound regeneration. c Wound contraction along with time. d H&E and Masson’s trichrome staining on the located wound tissues after 9-day. e Our current work compared with recent wound healing materials. N = 4, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001

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Furthermore, in a larger-scale wound defect model on the skin of miniature pig, we evaluated the effectiveness of the wound dressings in promoting tissue healing (Fig. 5a). In the overall wound repair outcome, all groups exhibited slow rate wound repair in the early stage, however, after 9 days, the wound repair results became different. At 9 days post-surgery, the SIS/PAA/LAP groups had a wound contraction rate of 40.02 ± 8.76%, significantly higher than the other groups. At day-12, the wound contraction rate of the SIS/PAA/LAP groups reached 80.75 ± 9.53%, while the control, Tegaderm, and SIS had rates of only 37.25 ± 3.77%, 46.50 ± 5.26%, and 55.50 ± 5.80%, respectively (Fig. 5(b, c)). In histological staining, we observed that the SIS/PAA/LAP wound dressing used on the wound surface did not induce adverse reactions. Additionally, the use of SIS and SIS/PAA/LAP led to a significant increase in epidermis thickness (SIS: 135.63 ± 40.86 μm, SIS/PAA/LAP: 271.75 ± 12.84 μm) compared to the control and Tegaderm groups. In Masson’s trichrome staining, the SIS/PAA/LAP groups exhibited higher levels of collagen expression, with more orderly collagen arrangement (Fig. 5(d-f)). These results confirmed that the SIS/PAA/LAP had good biocompatibility and was not only effective in promoting full-thickness skin wound repair in rats but also achieved excellent tissue repair results in a larger-scale miniature pig skin injury model.

Fig. 5
figure 5

Wound regeneration in the Bama miniature porcine skin injury. a Illustration on the experiment routine and pathological analysis. b Gross appearance on the wound repair. c Wound contraction along with time. d Epidermis thickness growth after 12-day. e H&E staining, and f Masson’s trichrome staining on the located wound tissues after 12-day. N = 4, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001

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In addition, we conducted immunohistochemical staining on the 12-day wound tissue to further analyze how SIS/PAA/LAP achieved improved tissue repair. We chose TNF-α as a typical pro-inflammatory marker and CD163 as a typical anti-inflammatory marker. Based on the expression of TNF-α/CD163 immunofluorescence, there were no significant differences in TNF-α expression among the groups. However, the SIS/PAA/LAP groups had 1.31 times the intensity of CD163 expression compared to the control, 1.28 times compared to Tegaderm, and 1.14 times compared to SIS (Fig. 6(a, c)). In terms of VEGF/α-SMA expression, the SIS/PAA/LAP groups exhibited higher expression levels of VEGF and α-SMA compared to the other groups. The VEGF was 2.75 times than that of the control, 2.79 times than that of Tegaderm, while α-SMA was 2.99 times than that of the control, 3.36 times than that of Tegaderm, and 1.41 times than that of SIS (Fig. 6(b, d)). And from the RT-PCR analysis, the specific gene expressions (e.g., TNF-α, CD163, VEGF) were also accorded with the previous findings (Fig. 6e). These results indicated that a significant inflammatory response in the early stages of wound healing [28, 29], when SIS/PAA/LAP was used as a wound dressing, the release of bioactive ions such as Mg2+ and Si4+ enabled to increase the expression levels of intercellular anti-inflammatory factors (e.g., CD163), to create a microenvironment conducive to cell migration and proliferation [27, 32, 33]. Therefore, SIS/PAA/LAP was capable of achieving excellent tissue wound repair outcomes.

Fig. 6
figure 6

Histological analysis on the wound regeneration in pig. a Immunofluorescent staining of TNF-α (red) / CD163 (green) / DAPI (blue), and b VEGF (red) / α-SMA (green) / DAPI (blue) on the located wound tissue after 12-day. c Semi-quantitative analysis of TNF-α / CD163, and d VEGF / α-SMA fluorescent intensity expression based on panel a and b, N = 4. e RT-PCR analysis of the retrieved granulation tissues for specific gene expressions. N = 3, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001

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Conclusion

In this study, the asymmetric adhesive wound dressing SIS/PAA/LAP was prepared from photopolymerizing and crosslinking AA pre-cursor solution containing LAP nanoparticles on the SIS patch. By optimizing the composition (e.g., 40 wt.% AA and 10 wt.% LAP), the resulting wound dressing exhibited excellent tissue adhesion strength (~ 33 kPa) and burst strength (~ 22 kPa), which outperformed commercially available tissue adhesives used as wound dressings. In the cell assays, rat and porcine skin injury models, the SIS/PAA/LAP demonstrated good cytocompatibility and pro-angiogenic capacity, leading to remarkable tissue repair outcomes. This SIS/PAA/LAP patch not only showed advantages as wound dressings, but also held promise for applications in surgical fields, such as general and cardiac surgery.