Abstract
Background/Aim
This study investigated the therapeutic potential of lipophosphonoxin (LPPO), an antibacterial agent, loaded into polycaprolactone nanofiber dressings (NANO-LPPO) for full-thickness wound healing. Using a porcine model, we aimed to assess the impact of areal weight of the dressing (10, 20 and 30 g/m2) on wound-healing outcomes and validate findings from previous murine studies.
Materials and Methods
Full-thickness wounds were created on porcine skin and treated with the NANO-LPPO dressings of differing thickness. Positive control (Aquacel Ag+) and standard control (Jelonet) groups were included for comparison. Wound-healing progression was evaluated macroscopically and on the histological level.
Results
Macroscopic observations indicated no signs of infection in any group, with wounds covered by scabs by day 14. Thicker dressings (areal weights of 30 and 20 g/m2) demonstrated superior performance in promoting the formation of granulation tissue and healing compared to the thinner version (areal weight of 10 g/m2). LPPO-loading enhanced scaffold wettability and biodegradability without impairing healing outcomes. Both control groups exhibited similar healing characteristics.
Conclusion
The findings underscore the importance of optimizing dressing thickness for effective wound healing. NANO-LPPO dressings exhibit translational potential as a therapeutic option for full-thickness wounds, warranting further preclinical and regulatory evaluation to support clinical application.
Keywords: Wound healing, active wound dressing, wound treatment, antibacterial agent, skin, regeneration, repair
Introduction
Full-thickness skin wounds present a significant clinical challenge, particularly when complicated by bacterial infections, such as those caused by Staphylococcus aureus (1). Effective wound dressings must not only control infection but also support tissue repair and regeneration (2). Electrospun nanofiber scaffolds, composed of biocompatible and biodegradable materials, represent a promising platform for advanced wound management. Among these, polycaprolactone (PCL)-based nanofibers have garnered attention due to their favorable mechanical properties, biodegradability, and ability to mimic the extracellular matrix structure (3). Recently, our team developed a novel lipophosphonoxin (LPPO)-loaded PCL nanofiber dressing (NANO-LPPO) that demonstrated potent antibacterial activity and healing promotion in a murine model of S. aureus-induced wound infection (4). The murine model provided critical insights into the antibacterial efficacy and biocompatibility of NANO-LPPO. The dressing exhibited sustained release of LPPO, which created a prolonged antibacterial environment, supported fibroblast function, and promoted keratinocyte health without interfering with the biological processes critical for wound healing. These results validated the efficacy of NANO-LPPO in reducing bacterial load and enhancing granulation tissue formation.
However, murine models, while invaluable for initial screenings, have inherent limitations due to anatomical, physiological, and immunological differences compared to human skin. Thus, translation of findings from murine studies to clinical settings requires further validation in models more closely resembling human skin (5). Porcine models are widely recognized as the gold standard for preclinical wound-healing studies due to their high similarity to human skin in terms of structure, thickness, collagen content, and healing mechanisms (6,7). Unlike murine skin, which heals predominantly by contraction, porcine skin exhibits re-epithelialization and granulation tissue formation similar to human wound healing (8). Even though experiments in mouse models can demonstrate reduced wound contraction (9), the porcine model remains superior due to its closer anatomical and physiological similarity to human skin. Furthermore, porcine models facilitate the study of larger wound areas, allowing the evaluation of dressing performance under clinically relevant conditions, such as prolonged dressing wear and handling. Importantly, the porcine immune response to bacterial infections mimics that of humans more closely than murine systems, making it a superior model for studying antimicrobial interventions (10).
In this study, we aimed to validate the efficacy of NANO-LPPO in a porcine model of full-thickness non-infected wounds. By utilizing the porcine model, we sought to assess the dressing's performance in terms of biocompatibility, biodegradation, and wound-healing promotion under conditions that better replicate human skin biology. This work not only bridges the translational gap between murine studies and clinical application but also provides a robust framework for further studies, including potential infection models, to fully elucidate the clinical potential of NANO-LPPO as a therapeutic dressing for complex wound management.
Materials and Methods
LPPO. The second-generation LPPO DR-6180 (Figure 1) was synthesized in multigram quantities according to the procedure reported elsewhere (11).
Figure 1.
Chemical structural formula of the tested second-generation lipophosphonoxin (LPPO) DR-6180. R=C15H31.
Preparation of nanofibrous material and loading with LPPO. Nanofibrous wound dressings were prepared by blending electrospinning technology (12,13). Three nanofibrous PCL materials with different thicknesses (areal weights of 10, 20 and 30 g/m2) were prepared without LPPO whereas three PCL materials (areal weights of 10, 20 and 30 g/m2) were loaded with LPPO at 7 wt%. The concentration was calculated based on dry matter, excluding the solvent. Firstly, LPPO was prepared by dissolving 1.2 g of LPPO in 82.8 g chloroform/ethanol (8:2) solvent mixture (Penta s.r.o., Prague, Czech Republic). Ultrasound was then applied for 30 min to enhance the solubilization of LPPO and ensure homogeneity of the solutions using an ultrasonic bath (Shesto, Watford, UK). Then 16 g of PCL (Merck, Darmstadt, Germany) with an average molecular weight Mn of 45,000 was dissolved in the LPPO solution. The polymer concentration used for obtaining continuous fibrous meshes was 16 wt%. All materials were electrospun using a Nanospider NS 1WS500U (Elmarco a.s., Liberec, Czech Republic) on polypropylene spunbond microfibrous nonwoven material (Pegatex S; 20 g/m2; fiber diameter 20 µm; PFNonwovens Czech, Prague, Czech Republic). For electrospinning, a positive voltage was applied to the instrument’s wire (i.e. the spinning electrode) and a negative voltage was applied to the collector; the potential difference was 50 kV. The distance between the spinning electrode and the collector was 180 mm. The temperature during the experiments was 22±5˚C with a relative humidity of 30±5% for pure PCL nanofibers and 20% for PCL nanofibers with LPPO. Areal weight was controlled by changing the supporting nonwoven material withdrawal speed: 33 mm/min for 30 g/m2 NANO; 48 mm/min for 20 g/m2 NANO; 75 mm/min for 10 g/m2NANO; 9 mm/min for 30 g/m2 NANO-LPPO; 14 mm/min for 20 g/m2 NANO-LPPO; and 32 mm/min for 10 g/m2 NANO-LPPO. Produced samples were finally sterilized by ethylenoxide according to ČSN EN ISO 11135 for 12 h at room temperature. All the sterilized samples were then vented at room temperature for 2 weeks.
Porcine model. All experiments were carried out according to the guidelines for the care and use of experimental animals and approved by the Resort Professional Commission of the CAS for Approval of Projects of Experiments on Animals (Reference number: AVCR 5853/2023 SOV II). Two 3-year-old miniature pigs (castrated males, mean weight±standard deviation= 98.1±2.97 kg) of the Libechov breed (IAPG CAS, v.v.i.; PIGMOD; http://www.iapg.cas.cz/en/) were used for the experimental study. Minipigs were chosen as a suitable model given their considerable similarity to human anatomy, physiology and pathophysiology of the skin. The animals were pre-medicated with intramuscular application of a mixture (TKX) containing 4 mg/kg tiletamine and 4 mg/kg zolazepam (Zoletil 100; Virbac, Carros, France), 10 mg/kg ketamine (Narketan 10; Vetoquinol UK Ltd, Towcester, UK) and 2 mg/kg xylazine (Rometar 2%; Spofa, Prague, Czech Republic). For analgesia, intramuscular application of 150 mg tramadol hydrochloride (Tramal; STADA Arzneimittel AG, Bad Vilbel, Germany) was used. After intravenous cannulation and intubation, isoflurane (1.5%) was used for inhalation anesthesia. Seven days after skin wound creation, the wound dressing cover was replaced under analgosedation induced by intramuscular administration of the TKX mixture combined with Tramal. At day 14, the animals were deeply anesthetized by intramuscular application of TKX mixture combined with intravenous propofol application. Immediately after, the animals were sacrificed by exsanguination, followed by the collection of wound samples.
Wounds. On day 0, the day of wounding, pigs were anesthetized, and hairs were clipped from the cervical to lumbar dorsum, and the skin was scrubbed with iodine solution followed by an ethanol rinse. Wounds were made on the backs of the pigs as shown in Figure 2. The full-thickness wounds were created using a scalpel. We tested three NANO dressings with LPPO and three NANO dressings without LPPO. Aquacell Ag+ (Convatec, Deeside, UK) was used as the positive control, and Jelonet (Smith & Nephew, Hull, UK) was used as the standard dressing. Figure 2 also illustrates the placement of the different dressings on the respective wounds.
Figure 2.
Scheme of positions of open full-thickness excisions and the applied wound dressings. Full-thickness skin excisions were made on the back of two pigs and dressed using either the tested NANO/NANO-LPPO dressings or a commercial dressing as a positive/negative control (Aquacel/Jelonet).
Histology of open wounds. Skin-wound specimens were removed from sacrificed pigs and routinely processed for histological staining (fixation in 4% buffered formaldehyde; dehydration using increasing concentrations of ethanol; paraffin embedding; sectioning; and staining). Depa-raffinized sections (3 µm-thick) were stained with hematoxylin-eosin (basic staining), and with Sirius Red (collagen staining).
Re-epithelialization of the epidermis, presence of inflammatory cells (polymorphonuclear leukocytes), fibroblasts, formation of lumenized vessels, and deposition of new collagen were qualitatively evaluated. Collagen maturation was assessed under polarized light. All evaluations were conducted in a blinded fashion to ensure unbiased interpretation of the results. Photographs were collected under identical conditions (light /time exposure) with an Olympus BX51 microscope equipped with Olympus DP23 CCD camera (Olympus, Tokyo, Japan) and QuickPHOTO Micro (Promicra, Prague, Czech Republic) software.
Results
Wounds. Representative photographs of wounds are shown in Figure 3. The excisions in all treatment groups were similar in size and appearance at the start of the study. No presence of infection was recorded throughout the observation period. By day 14, wounds in all groups were covered with scabs, indicating progression in the healing process. The positive control group treated with Aquacel Ag+ and the standard control group treated with Jelonet exhibited similar macroscopic healing characteristics to each other, further supporting the comparability of the tested conditions.
Figure 3.
Open full-thickness wounds/excisions at the day of surgery (0 d) and after 14 days (14 d) of healing with the tested wound dressings. Full-thickness skin excisions were created on the backs of two pigs and treated with either NANO/NANO-LPPO dressings or positive/negative controls (Aquacel/Jelonet).
Histology. Representative microphotographs are shown in Figure 4. On day 14, histological analysis revealed well-formed granulation tissue, rich in high-caliber vessels. The acute inflammatory phase had concluded, with a well-defined demarcation line separating vital tissue from the scab. Epidermal regeneration was incomplete in both the control and positive control wounds. The center of the wound remained unbridged by an epithelial sheet, and the demarcation line, primarily composed of neutrophil granulocytes, persisted over the granulation tissue. In contrast, wounds treated with the thinner NANO dressing (areal weight of 10 g/m2) showed reduced formation of granulation tissue, with persisting acute inflammation in central areas, whereas both thicker NANO dressings (areal weights of 20 and 30 g/m2) stimulated granulation tissue development with resolved acute inflammatory response also in the wound centers.
Figure 4.
Representative microphotographs of tissue from wounds treated with either NANO/NANO-LPPO dressings or positive/negative controls (Aquacel/Jelonet) collected 14 days after full-thickness skin excision. Tissues were stained with hematoxylin-eosin (HE) and examined under light microscopy. GT: Granulation tissue; WC: wound center; WE: wound edge. Scale bar=100 μm.
Polarized light analysis (Figure 5) confirmed that the NANO dressings did not impair collagen remodeling, although the interface with intact dermis (characterized by thick collagen fibers) remained clearly visible, as scars never fully replicate normal dermis. Notably, both thicker NANO dressings (areal weights of 20 and 30 g/m2) exhibited the most mature granulation tissue among the test wounds, particularly when NANO was loaded with LPPO. This finding indicates a more advanced stage of granulation tissue maturation.
Figure 5.
Representative microphotographs of tissue from wounds treated with either NANO/NANO-LPPO dressings or positive/negative controls (Aquacel/Jelonet) collected 14 days after full-thickness skin excision. Tissues were stained with Sirius Red (SR) and analyzed under polarized light. GT: Granulation tissue; WE: wound edge. Scale bar=100 μm.
Discussion
In the present study, we successfully compared three different areal weights (10, 20, and 30 g/m2) of NANO dressing, loaded or not with LPPO, in a standardized porcine model of full-thickness wound. Our findings revealed that thicker nanofiber dressings (areal weights of 20 and 30 g/m2) were more effective in promoting wound healing compared to the thinner version (areal weight of 10 g/m2). This observation underscores the importance of optimizing dressing thickness to achieve better clinical outcomes. Notably, when NANO was loaded with LPPO, we observed no impairment in wound healing compared to the control, further validating the biocompatibility of the antibacterial molecule. This biocompatible and biodegradable scaffold consists of a skin extracellular matrix-like (3), macroporous network of cross-linked smooth fibers [diameter around 1 µm, with pores of several micrometers (4)], with the ability to extract wound exudate and deliver oxygen (14). This unique material composition not only enhances adhesion and occlusion but also integrates seamlessly into the wound bed, reducing the frequency of dressing changes (15). Importantly, the addition of LPPO improved the wettability and biodegradability of the scaffold while preserving its structural integrity.
Interestingly, the healing-promoting effect of NANO can be further enhanced through material modifications, such as combining PCL with fish collagen and covalently bonded chito-oligosaccharides (16). These modifications may provide additional benefits, such as accelerated healing and reduced fibrosis, as demonstrated in previous studies (16,17). Another scaffold comprised two supportive PCL-chitosan layers (accelerating healing) on the sides and a polyvinyl alcohol-metformin hydrochloride (down-regulating expression of fibrosis-related genes) in the middle (18). From this point of view, fabricated scaffolds (19) are promising candidates for treatment of full-thickness wounds (reducing fibrosis/infection and facilitating repair/ regeneration). While our study focused on the use of LPPO-loaded PCL scaffolds, these advancements highlight the potential for future modifications of the dressing to achieve even better therapeutic outcomes.
Through comprehensive evaluation, we confirmed the translational potential of NANO-LPPO. The thicker scaffolds demonstrated superior performance in promoting granulation tissue formation and wound healing. This aligns with findings in a murine model (4) and underscores the relevance of porcine models as a bridge to clinical applications. Importantly, the anti-bacterial properties of LPPO-loaded dressings did not hinder the natural healing process, further supporting their safety and efficacy.
Despite the promising results, our study has several limitations. Firstly, the experiment was conducted using only two pigs, which limits the generalizability of the findings. Secondly, no statistical analysis was performed to validate the observed trends, and quantitative examination of histology was not conducted. Thirdly, we investigated only one type of wound − a full-thickness excision − without exploring other wound types or conditions. Additionally, the study was performed exclusively on sterile wounds, and we did not utilize a model of infected wounds to assess the dressing's antimicrobial efficacy under such conditions.
Moving forward, it is imperative to translate these preclinical findings into clinical practice. Regulatory approval from agencies such as the U.S. Food and Drug Administration or European Medicines Agency is a crucial next step to certifying NANO-LPPO as a medical device or drug for human use. Our results contribute to advancing wound care strategies and emphasize the importance of optimizing dressing design to enhance therapeutic efficacy. By bridging the gap between preclinical research and clinical application, this study lays the groundwork for improving outcomes in the management of complex wounds.
Patents
The NANO-LPPO dressing has been protected by submitting a national patent application in the Czech Republic under the reg. no. PV 2020-316.
Conflicts of Interest
The Authors have no conflicts of interest to declare.
Authors’ Contributions
Conceptualization: Dominik Rejman and Peter Gál. Formal analysis: Robert Zajíček. Funding acquisition: Dominik Rejman, David Lukáš and Peter Gál. Investigation and methodology: Hubert Šuca, Štefan Juhás, Jana Juhásová, Věra Jenčová, Eva Kuželová Košťáková, David Lukáš, Peter Bohuš, Robert Zajíček, Dominik Rejman, and Peter Gál. Writing – original draft: Peter Gál. All Authors read and approved the final version of the manuscript.
Acknowledgements
The Grant Agency of the Ministry of the Education, Science, Research and Sport of the Slovak Republic (under contract no. 1/0455/22, 1/0226/22 and 1/0262/24), Charles University (COOPERATIO33), and the Slovak Research and Development Agency (under contract no. APVV-20-0017 and APVV-22-0006) are appreciated for their support. The Czech Science Foundation (22-08857S), Czech Health Research Council (NW24-08-00073) and The National Institute Virology and Bacteriology Project (Programme EXCELES, Project No. LX22NPO5103) funded by the European Union NextGenerationEU program, as well as European Union NextGenerationEU through the Recovery and Resilience Plan for Slovakia under the project no. 09I03-03-V04-00075 are also gratefully acknowledged. The study was also partially supported by the European Regional Development Fund by Programme Johannes Amos Comenius (No. CZ.02.01.01/00/22_008/0004562 Project "Excellence in Regenerative Medicine") and RVO: 67985904.
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