A transparent osmotic wound dressing ATKPAD promotes healing by retaining cytokines and inducing hypoxia-driven inflammation

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Modern wound dressings promote healing by maintaining a moist, hypoxic environment. ATKPAD is a novel wound dressing that accelerates healing developed by Okamoto Industries, Inc. Composed of reduced starch syrup and a semi-permeable membrane, it was launched in Japan as a Class III advanced medical device in 2024. ATKPAD is unique in its transparency, allowing clear wound visibility, and selectively absorbs wound exudate through the combined effects of osmotic pressure and its semi-permeable membrane. However, the detailed molecular and cellular mechanisms of ATKPAD remain unclear. In this study, we examined its effect on acute wound healing. We analyzed the in vitro permeability of cytokines and growth factors and evaluated granulation tissue formation, angiogenesis, neutrophil infiltration, and cytokine and growth factor production in vivo . We found that cytokines and growth factors were selectively retained by ATKPAD. Its application enhanced granulation tissue formation and neutrophil infiltration, accompanied by increased hypoxia marker expression. These findings suggest that ATKPAD promotes wound healing by creating a hypoxic microenvironment that enhances early-phase inflammation while preserving key bioactive factors at the wound site. Biological sciences/Cell biology Biological sciences/Molecular biology Health sciences/Medical research Physical sciences/Materials science ATKPAD HIF-1α Inflammation Wound healing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION The wound-healing process involves overlapping phases of inflammation, proliferation, and remodeling, driven by multiple factors such as cytokines and growth factors 1 . In particular, a smooth transition from the inflammatory phase to the proliferative phase is a critical step in wound healing 2 . During the inflammatory phase, neutrophils are recruited to the wound site, where they secrete pro-inflammatory cytokines such as TNF-α and IL-6 to initiate immune responses and recruit macrophages and fibroblasts 3 . These cells, in turn, promote tissue regeneration by enhancing granulation tissue formation and angiogenesis 4 . A moist wound environment, facilitated by various therapeutic approaches such as wound dressings, is essential for the successful progression of the wound-healing process, leading to expedited wound healing, reduced infection rates, and lower overall treatment costs 5 . This moist environment helps to preserve wound exudate, which contains cytokines and growth factors crucial for cell migration, proliferation, and tissue regeneration 6 . Recently, numerous types of wound dressings—such as polyurethane films, polyurethane foams, hydrocolloids, hydrogels, and alginates—have been developed to aid in collagen synthesis, promote faster re-epithelialization, create hypoxic conditions, and decrease wound bed pH, ultimately leading to reduced infection rates 7 . Effective wound care requires not only moisture control and infection prevention but also the preservation of endogenous healing factors such as cytokines and growth factors. However, due to the material properties of existing wound dressings, there is concern that they also absorb healing-promoting components, including exudates such as growth factors. Additionally, in most cases, it is difficult to observe the wound site after applying these dressings. While polyurethane films offer good visibility, their limited capacity to absorb exudate can be a disadvantage in wounds with heavy drainage. To address this challenge, we developed an osmotic absorptive wound dressing prototype—the ATKPAD—consisting of a polyvinyl alcohol (PVA) semi-permeable film and a hypertonic gel composed of reduced starch syrup and sodium alginate. In this study, we examined the wound-healing effects of a new dressing with characteristics such as selective absorption and transparency. ATKPAD, a newly developed wound dressing inspired by clinical doctors' interest in applying food-grade osmotic dehydration sheets to wound care, is composed of a mixture of reduced starch syrup and sodium alginate, encapsulated in a semi-permeable membrane, a transparent PVA film 8 – 10 . ATKPAD absorbs wound exudate through the osmotic pressure generated by reduced starch syrup, while sodium alginate contributes to gel stabilization and facilitates exudation within the pad. This structure enables the selective absorption of low-molecular-weight substances while preserving essential bioactive molecules that may support the wound-healing process. Recently, several studies have focused on using wound dressings as novel drug delivery systems 11 ; however, ATKPAD represents a new concept in dressings, designed to retain wound-healing factors such as cytokines and growth factors. While preliminary studies in swine models have suggested the healing potential of ATKPAD, its immunological and molecular mechanisms of action remain poorly understood. In this study, we investigated the wound-healing mechanisms of ATKPAD using a murine model, with a particular focus on the inflammatory phase, during which exudate production is abundant. Our findings revealed that ATKPAD promotes wound healing by enhancing early neutrophilic inflammation, suggesting that it may serve as a suitable dressing material for the early inflammatory phase following injury. RESULTS Selective retention of cytokines and growth factors by osmotic absorptive dressing films: an in vitro evaluation The permeability of bioactive wound-healing molecules was evaluated using an in vitro two-chamber assay system, as described in Fig. 1A. After 24 h, a significant increase in fluid volume in the upper chamber was observed with PVA membranes, indicating effective osmotic water migration (Fig. 1B). Polyethylene terephthalate (PET) and polycarbonate (PC) membranes exhibited less fluid movement. This differential fluid movement also influenced cytokine retention, as described below. PC membranes permitted upward diffusion of IL-8, TNF-α, and IL-6 (Fig.1C). In contrast, PET and PVA membranes effectively retained these cytokines in the lower chamber, with PVA showing the strongest retention. Similar trends were observed for angiogenesis-related growth factors, vascular endothelial growth factor (VEGF) and PECAM-1 (Fig.1D). While PC membranes allowed significant migration, PVA membranes blocked upward transfer and maintained higher concentrations in the lower chamber. Overall, the PVA film demonstrated a superior ability to retain both inflammatory and angiogenic cytokines compared with PET. This indicated a dual function of fluid absorption and cytokine preservation. ATKPAD promotes granulation tissue through regulation by moisture conditions Previous studies indicated that vascular-rich granulation tissue is essential for successful wound healing 13 . In mice applied with ATKPAD, granulation area was found to be significantly increased compared with mice applied with a film dressing on days 5 and 7 (Fig. 2A, B). Next, as an alternative indicator of wound healing, we examined the number of CD31-positive vessels. CD31-positive vessel counts tended to increase in ATKPAD-applied mice compared with film-applied mice on day 5 (Fig. 2C). Next, we examined the production of VEGF at wound sites after ATKPAD application. As shown in Fig. 2D, there was a significantly increased synthesis of VEGF in ATKPAD-applied mice at several time points. Promoted accumulation of neutrophils and production of inflammatory cytokines at the wound sites in ATKPAD-applied mice In acute wounds, early-phase neutrophil accumulation was associated with the promotion of the healing process 12,14 . As shown in Fig. 3A, in the ATKPAD-applied group, the number of neutrophils was significantly higher than in the film-applied group on day 1, whereas there was no obvious difference in the number of total leukocytes. Similar results were obtained from immunohistochemical analysis; that is, the density of Ly6G-positive neutrophils in the granulation tissue on day 5 was also higher in the ATKPAD-applied group (Fig. 3B). As neutrophils are a principal source of inflammatory cytokines, the levels of TNF-α and IL-6 were subsequently measured (Fig. 3C). The production of IL-6 was significantly higher in the ATKPAD group from days 1 and 3. Additionally, the production of TNF-α was significantly higher in the ATKPAD group compared with the film group on day 3. ATKPAD increases inflammatory cytokines by retaining ATP at the wound site To investigate the mechanisms of neutrophil recruitment by ATKPAD, we measured CXCL1 and CXCL2, chemokines well known to induce neutrophil migration 15 . CXCL1 (12 h and 24 h) and CXCL2 (24 h) levels were significantly elevated, consistent with increased neutrophil chemoattraction in the ATKPAD group compared with the film group (Fig. 4A). Additionally, the concentration of ATP in tissue, released in response to cellular damage and known to promote neutrophil migration 16 , was significantly higher in the ATKPAD group (Fig. 4B). As HIF-1α induced by hypoxia also promotes neutrophil recruitment 17 , we next examined HIF-1α levels in the wound tissue. The expression of HIF-1α mRNA was higher in the ATKPAD group at 6 and 24 h after wounding (Fig. 4C). DISCUSSION Our results showed that the ATKPAD's PVA membrane achieved this dual function: it facilitated the osmotic absorption of excess fluid while minimizing the loss of essential cytokines and growth factors. Furthermore, ATKPAD application significantly enhanced granulation and angiogenesis, primarily by promoting neutrophilic inflammation. This report is the first to reveal the mechanism of the novel wound dressing ATKPAD. These results may contribute to the effective utilization of ATKPAD in wound management. Wound-healing results from interactions among cytokines, growth factors, angiogenesis, and the extracellular matrix 18 . Wound dressings maintain a moist and hypoxic wound environment, which helps preserve healing factors and promote angiogenesis, thereby facilitating effective wound healing 6 . However, to our knowledge, no other dressing integrates both high visibility and superior exudate absorption while also supporting wound healing. Although one advantage of film dressings is that they allow clinicians to inspect the wound bed without removing the dressing, they are not suitable for highly exudative wounds 19 . Additionally, several highly absorbent wound dressings have been used all over the world, such as hydrogels, hydrofibers, alginate dressings, and polyurethane foams 20 . However, observation of the wound after these applications at the wound sites is difficult. The present study demonstrated that ATKPAD absorbed wound exudate, while maintaining wound visibility comparable with that of polyurethane film. Furthermore, in vitro experiments suggested that ATKPAD has the potential to regulate the moist wound environment, while retaining both inflammatory and angiogenic mediators at the wound site. This mechanism enables the selective absorption of low-molecular-weight substances, while preserving essential bioactive molecules that may support the wound-healing process. These findings suggested that ATKPAD is a novel wound dressing that combines enhanced visibility with the ability to maintain a favorable microenvironment for wound repair. Promotion of granulation tissue formation and angiogenesis is a key strategy in wound management 21 . Neutrophils play a critical role in initiating these processes during the early inflammatory phase of wound healing by clearing debris and releasing growth factors and cytokines 22 . Neutrophil-derived cytokines such as TNF-α and IL-6 contribute significantly to this phase; TNF-α has been reported to promote angiogenesis 23 , while IL-6 induces VEGF production 24,25 . In this study, ATKPAD appeared to enhance neutrophil recruitment on day 1 that was accompanied by a transient increase in TNF-α and IL-6 levels. This early inflammatory response was followed by enhanced granulation tissue formation and angiogenesis. Consistent with these findings, the production of VEGF, a key factor in angiogenesis 21 , was elevated in the ATKPAD-treated group, further supporting its potential to promote vascularization and tissue regeneration. As in the present study, Takeuchi et al. demonstrated that the application of a hydrogel dressing to diabetic mice led to an increase in neutrophils and VEGF, thereby promoting wound healing 26 . ATKPAD may promote wound healing through mechanisms similar to those proposed in the concept of moist wound healing, such as enhanced neutrophil recruitment and preservation of growth factors 27 . However, direct comparisons with other products were not performed. Further studies are warranted to clarify the relative efficacy of ATKPAD compared with other wound dressings. Wound dressings with low oxygen permeability can help to create a hypoxic wound environment 20 . Sano et al. reported that wound dressings with low oxygen permeability promoted granulation tissue formation and angiogenesis compared with those with high oxygen permeability 28 . HIF-1α is known as an inducible factor produced in hypoxic environments during the wound-healing process. Under normoxia conditions, HIF-1α is rapidly degraded, resulting in low protein levels and transcriptional activity whereas, under hypoxic conditions, its expression and activity are upregulated 29 . Previous studies have shown that HIF-1α accumulates gradually in response to ischemic or hypoxic stimuli, typically over a period of several hours to days 30–32 . In the context of wound healing, it has been reported that HIF-1α accumulation increases over the first 4 days post-injury and subsequently declines 33 . Based on this, we analyzed the high expression of HIF-1α mRNA in ATKPAD group at 6 and 24 h after injury, suggesting that the hypoxic environment was induced by ATKPAD, in contrast with the oxygen-permeable Tegaderm ®34 . HIF-1α activated the anaerobic glycolysis pathway to supplement ATP production under hypoxic conditions 35 . Furthermore, HIF-1α induced the gene expression of CXCL1 and CXCL2 36 . In this study, the observed increase in ATP and CXC chemokine levels in the ATKPAD group further supported the hypothesis that the hypoxic environment created by ATKPAD may enhance neutrophilic inflammation, thereby contributing to improved wound healing. Although the present study did not investigate the selective absorption of ATKPAD in vivo , it may serve as a promising contact layer for emerging therapies such as growth factor sprays (e.g., Fibrast ® spray) and cell suspension treatments (e.g., RECELL ® ) 37,38 . Further research is needed to optimize the combined use of ATKPAD with these advanced wound management strategies. CONCLUSION This study demonstrated that ATKPAD promotes acute wound healing by inducing early neutrophilic inflammation through a hypoxic environment, while also offering good visibility and absorbing exudate. As a novel dressing, it addresses the limitations of existing materials like polyurethane films and hydrocolloids. However, as ATKPAD was only introduced in Japan in 2024, its optimal indications are still under investigation. Further studies using diverse wound models are needed. MATERIALS AND METHODS Ethical statement This study was performed in strict accordance with the Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology in Japan, 2006. All experimental procedures involving animals followed the Regulations for Animal Experiments and Related Activities at Tohoku University, Sendai, Japan and were approved by the Institutional Animal Care and Use Committee at Tohoku University (No. 2023-093-01). All methods were performed in accordance with the relevant guidelines and regulations, and all efforts were made to minimize the suffering of the animals. The study was carried out in compliance with ARRIVE guidelines (https://arriveguidelines.org/). Animals Here, 8- to 9-week-old male C57BL/6J mice were purchased from CLEA Japan (Tokyo, Japan). All mice were kept in a specific pathogen-free environment, and food and water were always available. All experiments were performed under anesthesia, and every effort was made to minimize the suffering of the animals. Preparation of ATKPAD for mice ATKPAD (Okamoto Industries, Tokyo, Japan) is a sheet consisting of a gel-like absorbent material made from reduced starch syrup and sodium alginate, encapsulated in a PVA semi-permeable membrane film (Fig. 5A). ATKPAD adheres to the wound surface without the need for adhesive due to osmotic pressure, and swells as it absorbs exudate into its inner material (Fig 5B). To evaluate the wound-healing mechanism of ATKPAD in vivo , we prepared ATKPAD for mice as described below. ATKPAD for mice was composed of a semi-permeable membrane pouch made of PVA film and a liquid absorber. The liquid absorber was prepared by dissolving sodium alginate (Maikon no Kohara Co., Osaka, Japan) into reduced starch sugar (Mitsubishi Corporation Life Sciences Holdings Ltd., Tokyo, Japan). The PVA film was fabricated at a thickness of 17 μm by a casting method using a solution prepared by dissolving PVA resin (saponification degree: £98.0 mol%; Kuraray Co., Ltd., Tokyo, Japan) in purified water. Two sheets of PVA film were stacked and heat-sealed to form a pouch including 40 mg of liquid absorber by heat sealer FS-215 (FUJIIMPULSE Co., Ltd., Osaka, Japan) (Supplementary Fig. 1). ATKPAD for mice was sterilized by γ-ray irradiation (25 kGy). Preparation of the membrane-based assay system An in vitro two-chamber assay system (Neuro Probe, Inc.) was established to assess the permeability of bioactive wound-healing molecules (Figure 2a). Three types of semi-permeable membranes were tested: polycarbonate (PC; Neuro Probe, Inc) membranes with 3-µm pores (2 × 10⁶ pores/cm²), polyethylene terephthalate (PET; Lumirror™ 12P60, TORAY Industries, Inc.), and PVA (Okamoto Industries, Inc.). PC membranes served as a positive control due to their high protein permeability. Cytokine migration assay To test cytokine permeability, human growth factor and angiogenesis-related cytokine panels (BioLegend, Lots B335808 and B363859) were used. A 1:4 diluted cytokine solution (25 µL) was placed in the lower chamber, and a hypertonic solution (stock:pyrogen-free water = 5:7) was added to the upper chamber. After 24 h of incubation at 37 °C, fluids from both chambers were collected (n = 6) and analyzed using multiplex bead-based assays. Concentrations were expressed in ng/mL. Cytokine concentrations were quantified using a LEGENDplex™ human growth factor and angiogenesis-related cytokines panel (BioLegend) according to the manufacturer's instructions. The FACSCelesta instrument (BD Biosciences) was used to acquire the data, which were analyzed using Qognit software (BioLegend). Wound creation and tissue collection As previously described ,39 , mice were anesthetized with 1.0–2.0% isoflurane (Isoflurane, Mairan Pharma, Osaka, Japan) during the wounding procedure. The dorsal hair was shaved to fully expose the skin, which was then rinsed with 70% ethanol. Four full-thickness wounds extending to the panniculus carnosus layer were created on each mouse using a 6-mm diameter biopsy punch (Biopsy Punch, Kai Industries, Gifu, Japan) under sterile conditions. The wounds were covered with either a polyurethane film (Film) (Tegaderm™ Transparent Dressing, 3M Health Care, St. Paul, MN, USA) or ATK, followed by an elastic adhesive bandage (Hilate, Iwatsuki, Tokyo, Japan) to serve as an occlusive dressing. The day on which the wounds were made was designated as day 0. Dressings were first applied on day 0 and subsequently replaced every other day, specifically on days 2, 4, and 6. To evaluate wound exudate volume, the difference in weight before and after use was calculated. As the polyurethane film did not have fluid absorption capacity, weight-based evaluation was not applicable. At various time points, mice were sacrificed with an overdose of isoflurane, and a 1-cm-square section of skin was excised using scissors. The tissue was then processed for the following analyses. Histopathology and immunohistochemistry The wounded tissues were fixed in a 4% paraformaldehyde-phosphate buffer solution and embedded in paraffin after caudocranial dissection, as previously described 40 . Sections were harvested from the central portion of the wound and stained with hematoxylin and eosin (H&E). The granulation area was determined on H&E-stained sections. For immunohistochemistry, sections were stained with anti-CD31 antibody (1:600 dilution; R&D Systems, Minneapolis, MN, USA) and anti-Ly6G Ab (clone 1A8; dilution 1:100; BioLegend) ,41 . Vascular density in five random fields (each 0.03 mm 2 ) was determined by counting the number of CD31-positive vessels, while the number of neutrophils was assessed by counting the number of Ly6G-positive polynuclear cells. Measurement of cytokines, chemokines, and growth factor concentrations in vivo The wounded tissues were homogenized in saline using a stainless mesh, and the concentrations of cytokines and chemokines in the supernatants were measured using enzyme-linked immunosorbent assay (ELISA) kits, as previously described. ELISA kits from BioLegend (San Diego, CA, USA) were used for IL-6 and TNF-α, and those from R&D Systems (Minneapolis, MN, USA) were used for CXCL1, CXCL2, and VEGF. The results were expressed as the values per tissue weight. The analysis was conducted at 12 h and on days 1, 3, and 5. Preparation of leukocytes and flow cytometric analysis Leukocytes were prepared and analyzed by flow cytometric analysis as previously described 13 and detailed in Supplementary materials. The analysis was conducted on day 1. The cells obtained from the wounded tissues were stained with 7-aminoactinomycin D (7-AAD; BioLegend), Pacific Blue-anti-CD45 monoclonal antibody (mAb; clone 30-F11, BioLegend), and FITC-anti-Ly6G mAb (clone 1A8, BioLegend). Isotype-matched irrelevant IgG was used for control staining. Neutrophils were identified as CD45 + CD11b + Ly6G + cells. The stained cells were analyzed using a BD FACS Canto II flow cytometer (BD Bioscience, Franklin Lakes, NJ, USA). The number of neutrophils was estimated by multiplying the total leukocyte count by the proportion of each fraction. Measurement of ATP levels in wound tissues Adenosine triphosphate (ATP) was measured in tissue homogenates using a Tissue ATP Assay Kit (TOYO BEINET, Tokyo, Japan) according to the manufacturer's instructions. The analysis was conducted at 6 and 24 h after wounding. Extraction of RNA and quantitative real-time RT-PCR Total RNA was extracted using Isogen (Wako Pure Chemical, Osaka, Japan), and first-strand cDNA was synthesized using a PrimeScript first-strand cDNA synthesis kit (TaKaRa Bio, Otsu, Japan) according to the manufacturer’s instructions as previously described 42 . Quantitative real-time RT-PCR was performed in a volume of 20 μL using gene-specific primers and FastStart Essential DNA Green Master (Roche Applied Science, Branford, CT, USA) in a StepOneÔ system (Thermo Fisher, Waltham, MA, USA). Primer sequences were as follows: 5ʹ-TTCTGGCCAACGGTCTAGACAAC-3ʹ (Forward) and 5ʹ-CCAGTGGTCTTGGTGTGCTGA-3ʹ (Reverse) for 18S ribosomal RNA (18S rRNA); 5ʹ -TGCGTGCATGTCTAATCTGTTCC-3ʹ (Forward) and 5ʹ-AAGATTCTGACATGCCACATAGCTC-3ʹ (Reverse) for HIF-1α. Reaction efficiency with each primer set was determined using standard amplification. Target gene expression levels and those of 18S rRNA as a reference gene were calculated for each sample using reaction efficiency. The results were analyzed using a relative quantification procedure and illustrated as relative expression compared with 18S rRNA expression. Statistical analysis Data from in vitro experiments are presented as mean ± s.e.m. Statistical comparisons among membrane types were conducted using one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test (GraphPad Prism). A P -value < 0.05 was considered statistically significant. Data from in vivo experiments were analyzed using JMP software (SAS Institute Japan, Tokyo, Japan). Data were expressed as the mean ± standard deviation (s.d.). Differences between groups were examined for statistical significance using Welch’s t -test. A P -value less than 0.05 was considered significant. Declarations ACKNOWLEDGMENTS We thank the Biomedical Research Core of Tohoku University Graduate School of Medicine for technical support. Part of this study was supported by the Support system for young researchers to use research equipment, instruments and devices at Tohoku University. AUTHOR CONTRIBUTIONS In the present study, YS, EI, and HT collected the in vivo data; TK and RA collected the in vitro data; YS, HT, YA and EK designed the research study; TK, RA, WK, IS, KS, SI, TI, HM, TA and YI analyzed the data; AK contributed to making ATKPAD for mouse for the study; and YS and YA wrote the original draft; YA and EK critically reviewed and approved the final manuscript. All authors contributed to the article and approved the submitted version. DATA AVAILABILITY STATEMENT The data that support the findings of this study are available from the corresponding author upon reasonable request. 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Andrikopoulou, E. et al. Current Insights into the role of HIF-1 in cutaneous wound healing. Curr. Mol. Med. 11 , 218–235 (2011). (DOI 10.2174/156652411795243414) (PMID: 21375491). Pichiule, P., Agani, F., Chavez, J. C., Xu, K. & LaManna, J. C. HIF-1 alpha and VEGF expression after transient global cerebral ischemia. Adv. Exp. Med. Biol. 530 , 611–617. 10.1007/978-1-4615 (2003). -0075-9_60) (PMID: 14562758). Yeh, S. H., Ou, L. C., Gean, P. W., Hung, J. J. & Chang, W. C. Selective inhibition of early—but not late—expressed HIF-1α is neuroprotective in rats after focal ischemic brain damage. Brain Pathol. 21 , 249–262. 10.1111/j.1750-3639.2010.00443.x) (2011). (PMID: 21029239). Yu, A. Y. et al. Temporal, spatial, and oxygen-regulated expression of hypoxia-inducible factor-1 in the lung. Am. J. Physiol. 275 , L818–L826. 10.1152/ajplung.1998.275.4.L818) (1998). (PMID: 9755115). Haroon, Z. A., Raleigh, J. A., Greenberg, C. S. & Dewhirst, M. W. Early wound healing exhibits cytokine surge without evidence of hypoxia. Ann. Surg. 231 , 137–147. 10.1097/00000658-200001000-00020) (2000). (PMID: 10636114). Sirvio, L. M. & Grussing, D. M. The effect of gas permeability of film dressings on wound environment and healing. J. Invest. Dermatol. 93 , 528–531. 10.1111/1523–1747.ep12284076) (1989). (PMID: 2506289). Chen, S. & Sang, N. Hypoxia-inducible factor-1: A critical player in the survival strategy of stressed cells. J. Cell. Biochem. 117 , 267–278. 10.1002/jcb.25283) (2016). (PMID: 26206147). Olbryt, M. et al. Global gene expression profiling in three tumor cell lines subjected to experimental cycling and chronic hypoxia. PLOS One . 9 , e105104. 10.1371/journal.pone.0105104) (2014 Aug 14). (PMID: 25122487) (PMCID: PMC4133353). Abdelhakim, M., Lin, X. & Ogawa, R. The Japanese experience with basic fibroblast growth factor in cutaneous wound management and scar prevention: A systematic review of clinical and biological aspects. Dermatol Ther. (Heidelb) 10 , 569–587 (2020 Aug) (DOI 10.1007/s13555-020-00407-6) (PMID: 32506250). Wood, F. M., Giles, N., Stevenson, A., Rea, S. & Fear, M. Characterisation of the cell suspension harvested from the dermal epidermal junction using a ReCell® kit. Burns 38 , 44–51. 10.1016/j.burns.2011.03.001) (2012). (PMID: 22079538). Ishi, S. et al. Cutaneous wound healing promoted by topical administration of heat-killed Lactobacillus plantarum KB131 and possible contribution of CARD9-mediated signaling. Sci. Rep. 13 , 15917. 10.1038/s41598-023-42919-z) (2023). (PMID: 37741861). Imai, T. et al. Batroxobin promotes wound healing after burn injury by enhancing blood flow. Plast Reconstr. Surg (2025). (DOI 10.1097/PRS.0000000000012137) (PMID: 40178840). Kanno, E. et al. Defect of interferon γ leads to impaired wound healing through prolonged neutrophilic inflammatory response and enhanced MMP-2 activation. Int. J. Mol. Sci. 20 , 5657. 10.3390/ijms20225657) (2019). (PMID: 31726690). Sato, Y. et al. Limited role of Mincle in the host defense against infection with Cryptococcus deneoformans . Infect. Immun. 88 , e00400–e00420 (2020). (DOI 10.1128/IAI.00400 – 20) (PMID: 32868343). Additional Declarations Competing interest reported. The authors declare that this study received research funding and experimental materials from Okamoto industries, inc. Supplementary Files Supplementaryfigure1.tif Supplementary Fig. 1. Preparation of ATKPAD for mice. Two sheets of PVA film were stacked and heat-sealed to create a pouch (outer dimensions: 20 × 20 mm; inner dimensions: 10 × 10 mm), enclosing 40 mg of a liquid-absorbing material composed of sodium alginate and reduced starch syrup. Graphicalabstract.tif Cite Share Download PDF Status: Published Journal Publication published 06 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 26 May, 2025 Editor assigned by journal 26 May, 2025 Submission checks completed at journal 25 May, 2025 First submitted to journal 22 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6729108","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":461419267,"identity":"bda7eecf-e2fb-456d-9c04-7578be41a5cf","order_by":0,"name":"Yuki Sato","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIie2RMUsDMRTHXwikS2zWO07Qj5CSQYWznyXhIC5FCo6WGih0irjab6HfIHBwXSquji2Ck8NNcsMJ3uENXXI6CuY3vRfej/9LAhAI/EkIuK5C2xJAAv3u8E9K1A6N7uUvFegUktA9xQt/0cpBPZ4zwLk4r9JLRjccqhkMTvyKc2iZRbEh6nUi9VV8azmyBeAz41MujEMGR9xRISYyVw/PlMOBAcxdjwL1TaOwj+S0U9Bnr9IsBiRvU0gCrfJkOe5LiTdv0qnlOl4tiBhZrdXKFtP8sIi8dxmutSjL+pqxwWK3rdJU3dHscfc+SzPfix236bKt9r+uOYwy7lGOPOkAY58SCAQC/44vdJ9aAmXa5dkAAAAASUVORK5CYII=","orcid":"","institution":"Department of Translational Science for Nursing, Tohoku University Graduate School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Yuki","middleName":"","lastName":"Sato","suffix":""},{"id":461419268,"identity":"fe2872c1-b63f-425d-abe2-e3bd25faafa5","order_by":1,"name":"Eiki Ito","email":"","orcid":"","institution":"Department of Plastic and Reconstructive Surgery, Tohoku University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Eiki","middleName":"","lastName":"Ito","suffix":""},{"id":461419269,"identity":"ed6cc0e8-c030-491d-8f0d-f0974c88ca7f","order_by":2,"name":"Hiromasa Tanno","email":"","orcid":"","institution":"Department of Translational Science for Nursing, Tohoku University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hiromasa","middleName":"","lastName":"Tanno","suffix":""},{"id":461419270,"identity":"bcef7fdb-389a-4c91-846d-779c3d31f5a5","order_by":3,"name":"Takashi Kanno","email":"","orcid":"","institution":"Laboratory for Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Takashi","middleName":"","lastName":"Kanno","suffix":""},{"id":461419271,"identity":"449286ee-0e53-4ece-84ce-7b615d926eda","order_by":4,"name":"Ryuto Arai","email":"","orcid":"","institution":"Laboratory for Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ryuto","middleName":"","lastName":"Arai","suffix":""},{"id":461419272,"identity":"88213faa-f7ea-492a-9049-cd484b17faec","order_by":5,"name":"Ayato Kaneta","email":"","orcid":"","institution":"Okamoto Industries, Inc.","correspondingAuthor":false,"prefix":"","firstName":"Ayato","middleName":"","lastName":"Kaneta","suffix":""},{"id":461419273,"identity":"79d83ae4-d1c9-418e-b3cb-24921bc7402b","order_by":6,"name":"Wakana Kamada","email":"","orcid":"","institution":"Department of Translational Science for Nursing, Tohoku University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Wakana","middleName":"","lastName":"Kamada","suffix":""},{"id":461419274,"identity":"9322fb34-24d4-4a42-90fa-504de4167796","order_by":7,"name":"Ikue Sone","email":"","orcid":"","institution":"Department of Plastic and Reconstructive Surgery, Tohoku University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ikue","middleName":"","lastName":"Sone","suffix":""},{"id":461419275,"identity":"2d75f455-2214-4619-afbf-4a8785569bec","order_by":8,"name":"Ko Sato","email":"","orcid":"","institution":"Department of Clinical Microbiology and Infection, Tohoku University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ko","middleName":"","lastName":"Sato","suffix":""},{"id":461419276,"identity":"ad8ba20a-5a3b-4b29-a635-6bdeee98aa07","order_by":9,"name":"Shinyo Ishi","email":"","orcid":"","institution":"Department of Plastic and Reconstructive Surgery, Tohoku University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shinyo","middleName":"","lastName":"Ishi","suffix":""},{"id":461419277,"identity":"a66b966b-6e56-4847-8784-3a893ba26c72","order_by":10,"name":"Toshiro Imai","email":"","orcid":"","institution":"Department of Plastic and Reconstructive Surgery, Tohoku University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Toshiro","middleName":"","lastName":"Imai","suffix":""},{"id":461419278,"identity":"09033ae5-8a1b-4a1e-9a37-c019a7be7e73","order_by":11,"name":"Hiromu Matsunaga","email":"","orcid":"","institution":"Department of Plastic and Reconstructive Surgery, Tohoku University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hiromu","middleName":"","lastName":"Matsunaga","suffix":""},{"id":461419279,"identity":"c55f259b-17ea-492f-b2e5-725248762c47","order_by":12,"name":"Tetsuji Aoyagi","email":"","orcid":"","institution":"Department of Clinical Microbiology and Infection, Tohoku University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Tetsuji","middleName":"","lastName":"Aoyagi","suffix":""},{"id":461419280,"identity":"9f594350-280d-4cc2-8983-f6cb2dd6ed9c","order_by":13,"name":"Yoshimichi Imai","email":"","orcid":"","institution":"Department of Plastic and Reconstructive Surgery, Tohoku University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yoshimichi","middleName":"","lastName":"Imai","suffix":""},{"id":461419281,"identity":"b76729ff-1a80-4b28-a2f5-2655adcc1e56","order_by":14,"name":"Yoshiyuki Adachi","email":"","orcid":"","institution":"Laboratory for Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yoshiyuki","middleName":"","lastName":"Adachi","suffix":""},{"id":461419282,"identity":"bd1d1a52-c06c-4dd5-ac20-7d0e2707cfa3","order_by":15,"name":"Emi Kanno","email":"","orcid":"","institution":"Department of Translational Science for Nursing, Tohoku University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Emi","middleName":"","lastName":"Kanno","suffix":""}],"badges":[],"createdAt":"2025-05-23 04:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6729108/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6729108/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-026-41264-1","type":"published","date":"2026-03-06T15:59:12+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83441017,"identity":"2e8a626a-f2ac-4cc1-b837-28e625fc1261","added_by":"auto","created_at":"2025-05-26 09:35:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1144830,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e permeability assay for angiogenesis cytokines and growth factors. (\u003cstrong\u003ea\u003c/strong\u003e) Schematic and image of the cytokine permeability assay system. The device consists of upper and lower chambers separated by a test membrane. A hypertonic solution in the upper chamber induces osmotic water flow, while the lower chamber contains a cytokine solution. The schematic shows the configuration and fluid flow direction under the osmotic gradient. (\u003cstrong\u003eb\u003c/strong\u003e) Fluid accumulation in chambers after 24-h incubation. The fluid volume was measured in both chambers. A significant increase in upper chamber volume was observed with the PVA membrane, indicating osmotic water migration. (\u003cstrong\u003ec\u003c/strong\u003e) Inflammatory cytokine distribution across membranes. Cytokine concentrations were measured in the upper and lower chambers after incubation. PC membranes allowed upward migration of several cytokines, whereas the PET and PVA membranes retained cytokines in the lower chamber. (\u003cstrong\u003ed\u003c/strong\u003e) Distribution of angiogenesis-related cytokines. Concentrations of VEGF and PECAM-1 were assessed in both chambers. PC membranes permitted upward migration, while PET and PVA membranes inhibited cytokine movement. PVA membranes showed enhanced retention in the lower chamber. Data are presented as mean ± s.e.m. (n = 6). Statistical analysis was performed using one-way ANOVA with Bonferroni correction. ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ns, not significant.\u003c/p\u003e","description":"","filename":"Figure117.png","url":"https://assets-eu.researchsquare.com/files/rs-6729108/v1/34be2023cbe8cabe4a769d21.png"},{"id":83440743,"identity":"839c9d17-14cd-4e63-864b-a4c6b46fe6be","added_by":"auto","created_at":"2025-05-26 09:27:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2853507,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of ATKPAD application on wound healing and angiogenesis. Wounds were created on the backs of mice and, immediately after wounding, ATK or film was applied to the base of the wounds. Dressings were first applied on day 0 and subsequently replaced every other day, specifically on days 2, 4, and 6. (\u003cstrong\u003ea\u003c/strong\u003e) Hematoxylin and eosin staining of wound tissue on day 5 post-wounding. Arrowheads indicate the epithelial edges, and dotted lines outline the granulation tissue. (\u003cstrong\u003eb\u003c/strong\u003e) The graph shows the granulation area of wound tissues on days 5 and 7 after wounding (n = 8 per group). (\u003cstrong\u003ec\u003c/strong\u003e) The number of vessels stained with anti-CD31 antibody on day 5. Arrowheads indicate CD31-positive vessels. The vascular density per mm\u003csup\u003e2 \u003c/sup\u003ewas determined by counting the positive vessels (n = 8 wounds per group). (\u003cstrong\u003ed\u003c/strong\u003e) VEGF levels in the wounded tissue homogenates were measured on days 1, 3, and 5. Four wounds were created in one mouse, two of which were combined into one sample, and four mice were analyzed in each group (n = 4 mice per group). Each column represents the mean ± s.d. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure217.png","url":"https://assets-eu.researchsquare.com/files/rs-6729108/v1/cee4e71f4ac02e899760bdcd.png"},{"id":83440740,"identity":"7ee144c7-5c66-46f5-ac18-37329505453d","added_by":"auto","created_at":"2025-05-26 09:27:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4491531,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of ATKPAD on the accumulation of neutrophils and synthesis of inflammatory cytokines. (\u003cstrong\u003ea\u003c/strong\u003e) The numbers of leukocytes and neutrophils in the wound tissue were analyzed using flow cytometry. The graph shows the number of leukocytes and neutrophils on day 1 (n = 4 or 5 mice per group). (\u003cstrong\u003eb\u003c/strong\u003e) Immunohistochemical staining of Ly6G-positive neutrophils. Arrowheads indicate Ly6G-positive neutrophils. The accompanying graph shows neutrophil density (cells/mm²), quantified by counting Ly6G-positive cells (n = 4 per group). (\u003cstrong\u003ec\u003c/strong\u003e) IL-6 and TNF-α levels in the wounded tissue homogenates were measured on days 1, 3, and 5. Four wounds were created in one mouse, two of which were combined into one sample, and four mice were analyzed in each group (n = 4 mice per group). Each column represents the mean ± s.d. *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure313.png","url":"https://assets-eu.researchsquare.com/files/rs-6729108/v1/f7d6c677199070b3f24863be.png"},{"id":83440741,"identity":"4451035d-c929-4411-96fe-4dec292506f2","added_by":"auto","created_at":"2025-05-26 09:27:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":210802,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of ATKPAD on neutrophil chemoattractants and wound tissue hypoxia. (\u003cstrong\u003ea\u003c/strong\u003e) Production of CXCL1 and CXCL2 was measured at 12 and 24 h after wounding. Four wounds were created in one mouse, two of which were combined into one sample, and four mice were analyzed in each group (n = 4 mice per group). (\u003cstrong\u003eb\u003c/strong\u003e) ATP levels in the wounded tissue homogenates were measured at 6 and 24 h after wounding (n = 4 mice per group). (\u003cstrong\u003ec\u003c/strong\u003e) HIF-1α mRNA expression in the wounded tissues at 6 and 24 h. Four wounds were created in one mouse, two of which were combined as one sample and were analyzed in each group (n = 4 per group). Each column represents the mean ± s.d. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure410.png","url":"https://assets-eu.researchsquare.com/files/rs-6729108/v1/c6e144a657a4654a0405ced5.png"},{"id":83440744,"identity":"8256edda-c6be-4baf-8c25-9d7b3c20cd6a","added_by":"auto","created_at":"2025-05-26 09:27:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3118588,"visible":true,"origin":"","legend":"\u003cp\u003eStructure and features of ATKPAD. (\u003cstrong\u003ea\u003c/strong\u003e) ATKPAD (A: Absorbent, T: Transparent, K: Keeper) comprises absorbent materials, specifically reduced starch syrup and sodium alginate, enclosed within a semi-permeable PVA membrane. The semi-permeable membrane selectively absorbs water and volatile odorants such as ammonia and amines, from the wound exudate, effectively trapping them within the pad. Despite its visual capacity, ATKPAD enhances absorption by facilitating moisture evaporation from the opposite side. (\u003cstrong\u003eb\u003c/strong\u003e) Macroscopic photographs of ATKPAD. ATKPAD is transparent, enabling continuous wound observation without removing the dressing. It swells upon absorbing exudate.\u003c/p\u003e","description":"","filename":"Figure59.png","url":"https://assets-eu.researchsquare.com/files/rs-6729108/v1/a84b58d08a088652a04e6e9b.png"},{"id":104250773,"identity":"2050a44c-6fba-4312-a1be-42c627f4ad82","added_by":"auto","created_at":"2026-03-09 16:08:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12426747,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6729108/v1/79890cd3-52da-4f72-ae97-521a28b6ed22.pdf"},{"id":83440746,"identity":"d57cb81d-0dc1-4506-8a89-be84b2de620f","added_by":"auto","created_at":"2025-05-26 09:27:01","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5746922,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig. 1. \u003c/strong\u003ePreparation of ATKPAD for mice. Two sheets of PVA film were stacked and heat-sealed to create a pouch (outer dimensions: 20 × 20 mm; inner dimensions: 10 × 10 mm), enclosing 40 mg of a liquid-absorbing material composed of sodium alginate and reduced starch syrup.\u003c/p\u003e","description":"","filename":"Supplementaryfigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-6729108/v1/aeeea1f5611158777bc856f0.tif"},{"id":83441019,"identity":"45495b02-355e-4161-9356-20c9690caef1","added_by":"auto","created_at":"2025-05-26 09:35:01","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":475211,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-6729108/v1/21b601b4c4159bc9394f543f.tif"}],"financialInterests":"Competing interest reported. The authors declare that this study received research funding and experimental materials from Okamoto industries, inc.","formattedTitle":"A transparent osmotic wound dressing ATKPAD promotes healing by retaining cytokines and inducing hypoxia-driven inflammation","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe wound-healing process involves overlapping phases of inflammation, proliferation, and remodeling, driven by multiple factors such as cytokines and growth factors\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In particular, a smooth transition from the inflammatory phase to the proliferative phase is a critical step in wound healing\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. During the inflammatory phase, neutrophils are recruited to the wound site, where they secrete pro-inflammatory cytokines such as TNF-α and IL-6 to initiate immune responses and recruit macrophages and fibroblasts\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. These cells, in turn, promote tissue regeneration by enhancing granulation tissue formation and angiogenesis\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA moist wound environment, facilitated by various therapeutic approaches such as wound dressings, is essential for the successful progression of the wound-healing process, leading to expedited wound healing, reduced infection rates, and lower overall treatment costs\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. This moist environment helps to preserve wound exudate, which contains cytokines and growth factors crucial for cell migration, proliferation, and tissue regeneration\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Recently, numerous types of wound dressings\u0026mdash;such as polyurethane films, polyurethane foams, hydrocolloids, hydrogels, and alginates\u0026mdash;have been developed to aid in collagen synthesis, promote faster re-epithelialization, create hypoxic conditions, and decrease wound bed pH, ultimately leading to reduced infection rates\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Effective wound care requires not only moisture control and infection prevention but also the preservation of endogenous healing factors such as cytokines and growth factors. However, due to the material properties of existing wound dressings, there is concern that they also absorb healing-promoting components, including exudates such as growth factors. Additionally, in most cases, it is difficult to observe the wound site after applying these dressings. While polyurethane films offer good visibility, their limited capacity to absorb exudate can be a disadvantage in wounds with heavy drainage. To address this challenge, we developed an osmotic absorptive wound dressing prototype\u0026mdash;the ATKPAD\u0026mdash;consisting of a polyvinyl alcohol (PVA) semi-permeable film and a hypertonic gel composed of reduced starch syrup and sodium alginate. In this study, we examined the wound-healing effects of a new dressing with characteristics such as selective absorption and transparency.\u003c/p\u003e \u003cp\u003eATKPAD, a newly developed wound dressing inspired by clinical doctors' interest in applying food-grade osmotic dehydration sheets to wound care, is composed of a mixture of reduced starch syrup and sodium alginate, encapsulated in a semi-permeable membrane, a transparent PVA film\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. ATKPAD absorbs wound exudate through the osmotic pressure generated by reduced starch syrup, while sodium alginate contributes to gel stabilization and facilitates exudation within the pad. This structure enables the selective absorption of low-molecular-weight substances while preserving essential bioactive molecules that may support the wound-healing process. Recently, several studies have focused on using wound dressings as novel drug delivery systems\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e; however, ATKPAD represents a new concept in dressings, designed to retain wound-healing factors such as cytokines and growth factors.\u003c/p\u003e \u003cp\u003eWhile preliminary studies in swine models have suggested the healing potential of ATKPAD, its immunological and molecular mechanisms of action remain poorly understood. In this study, we investigated the wound-healing mechanisms of ATKPAD using a murine model, with a particular focus on the inflammatory phase, during which exudate production is abundant. Our findings revealed that ATKPAD promotes wound healing by enhancing early neutrophilic inflammation, suggesting that it may serve as a suitable dressing material for the early inflammatory phase following injury.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eSelective retention of cytokines and growth factors by osmotic absorptive dressing films: an \u003cem\u003ein vitro\u003c/em\u003e evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe permeability of bioactive wound-healing molecules was evaluated using an \u003cem\u003ein vitro\u003c/em\u003e two-chamber assay system, as described in Fig. 1A. After 24 h, a significant increase in fluid volume in the upper chamber was observed with PVA membranes, indicating effective osmotic water migration (Fig. 1B). Polyethylene terephthalate (PET) and polycarbonate (PC) membranes exhibited less fluid movement. This differential fluid movement also influenced cytokine retention, as described below. PC membranes permitted upward diffusion of IL-8, TNF-\u0026alpha;, and IL-6 (Fig.1C). In contrast, PET and PVA membranes effectively retained these cytokines in the lower chamber, with PVA showing the strongest retention. Similar trends were observed for angiogenesis-related growth factors, vascular endothelial growth factor (VEGF) and PECAM-1 (Fig.1D). While PC membranes allowed significant migration, PVA membranes blocked upward transfer and maintained higher concentrations in the lower chamber. Overall, the PVA film demonstrated a superior ability to retain both inflammatory and angiogenic cytokines compared with PET. This indicated a dual function of fluid absorption and cytokine preservation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eATKPAD promotes granulation tissue through regulation by moisture conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious studies indicated that vascular-rich granulation tissue is essential for successful wound healing\u003csup\u003e13\u003c/sup\u003e. In mice applied with ATKPAD, granulation area was found to be significantly increased compared with mice applied with a film dressing on days 5 and 7 (Fig. 2A, B). Next, as an alternative indicator of wound healing, we examined the number of CD31-positive vessels. CD31-positive vessel counts tended to increase in ATKPAD-applied mice compared with film-applied mice on day 5 (Fig. 2C). Next, we examined the production of VEGF at wound sites after ATKPAD application. As shown in Fig. 2D, there was a significantly increased synthesis of VEGF in ATKPAD-applied mice at several time points.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePromoted accumulation of neutrophils and production of inflammatory cytokines at the wound sites in ATKPAD-applied mice\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn acute wounds, early-phase neutrophil accumulation was associated with the promotion of the healing process\u003csup\u003e12,14\u003c/sup\u003e. As shown in Fig. 3A, in the ATKPAD-applied group, the number of neutrophils was significantly higher than in the film-applied group on day 1, whereas there was no obvious difference in the number of total leukocytes. Similar results were obtained from immunohistochemical analysis; that is, the density of Ly6G-positive neutrophils in the granulation tissue on day 5 was also higher in the ATKPAD-applied group (Fig. 3B). As neutrophils are a principal source of inflammatory cytokines, the levels of TNF-\u0026alpha; and IL-6 were subsequently measured (Fig. 3C). The production of IL-6 was significantly higher in the ATKPAD group from days 1 and 3. Additionally, the production of TNF-\u0026alpha; was significantly higher in the ATKPAD group compared with the film group on day 3.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eATKPAD increases inflammatory cytokines by retaining ATP at the wound site\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the mechanisms of neutrophil recruitment by ATKPAD, we measured CXCL1 and CXCL2, chemokines well known to induce neutrophil migration\u003csup\u003e15\u003c/sup\u003e. CXCL1 (12 h and 24 h) and CXCL2 (24 h) levels were significantly elevated, consistent with increased neutrophil chemoattraction in the ATKPAD group compared with the film group (Fig. 4A). Additionally, the concentration of ATP in tissue, released in response to cellular damage and known to promote neutrophil migration\u003csup\u003e16\u003c/sup\u003e, was significantly higher in the ATKPAD group (Fig. 4B). As HIF-1\u0026alpha; induced by hypoxia also promotes neutrophil recruitment\u003csup\u003e17\u003c/sup\u003e, we next examined HIF-1\u0026alpha; levels in the wound tissue. The expression of HIF-1\u0026alpha; mRNA was higher in the ATKPAD group at 6 and 24 h after wounding (Fig. 4C).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur results showed that the ATKPAD\u0026apos;s PVA membrane achieved this dual function: it facilitated the osmotic absorption of excess fluid while minimizing the loss of essential cytokines and growth factors. Furthermore, ATKPAD application significantly enhanced granulation and angiogenesis, primarily by promoting neutrophilic inflammation. This report is the first to reveal the mechanism of the novel wound dressing ATKPAD. These results may contribute to the effective utilization of ATKPAD in wound management.\u003c/p\u003e\n\u003cp\u003eWound-healing results from interactions among cytokines, growth factors, angiogenesis, and the extracellular matrix\u003csup\u003e18\u003c/sup\u003e. Wound dressings maintain a moist and hypoxic wound environment, which helps preserve healing factors and promote angiogenesis, thereby facilitating effective wound healing\u003csup\u003e6\u003c/sup\u003e. However, to our knowledge, no other dressing integrates both high visibility and superior exudate absorption while also supporting wound healing.\u0026nbsp;Although one advantage of film dressings is that they allow clinicians to inspect the wound bed without removing the dressing, they are not suitable for highly exudative wounds\u003csup\u003e19\u003c/sup\u003e. Additionally, several highly absorbent wound dressings have been used all over the world, such as hydrogels, hydrofibers, alginate dressings, and polyurethane foams\u003csup\u003e20\u003c/sup\u003e. However, observation of the wound after these applications at the wound sites is difficult. The present study demonstrated that ATKPAD absorbed wound exudate, while maintaining wound visibility comparable with that of polyurethane film. Furthermore, \u003cem\u003ein vitro\u003c/em\u003e experiments suggested that ATKPAD has the potential to regulate the moist wound environment, while retaining both inflammatory and angiogenic mediators at the wound site. This mechanism enables the selective absorption of low-molecular-weight substances, while preserving essential bioactive molecules that may support the wound-healing process. These findings suggested that ATKPAD is a novel wound dressing that combines enhanced visibility with the ability to maintain a favorable microenvironment for wound repair.\u003c/p\u003e\n\u003cp\u003ePromotion of granulation tissue formation and angiogenesis is a key strategy in wound management\u003csup\u003e21\u003c/sup\u003e. Neutrophils play a critical role in initiating these processes during the early inflammatory phase of wound healing by clearing debris and releasing growth factors and cytokines\u003csup\u003e22\u003c/sup\u003e. Neutrophil-derived cytokines such as TNF-\u0026alpha; and IL-6 contribute significantly to this phase; TNF-\u0026alpha; has been reported to promote angiogenesis\u003csup\u003e23\u003c/sup\u003e, while IL-6 induces VEGF production\u003csup\u003e24,25\u003c/sup\u003e. In this study, ATKPAD appeared to enhance neutrophil recruitment on day 1 that was accompanied by a transient increase in TNF-\u0026alpha; and IL-6 levels. This early inflammatory response was followed by enhanced granulation tissue formation and angiogenesis. Consistent with these findings, the production of VEGF, a key factor in angiogenesis\u003csup\u003e21\u003c/sup\u003e, was elevated in the ATKPAD-treated group, further supporting its potential to promote vascularization and tissue regeneration. As in the present study, Takeuchi \u003cem\u003eet al.\u003c/em\u003e demonstrated that the application of a hydrogel dressing to diabetic mice led to an increase in neutrophils and VEGF, thereby promoting wound healing\u003csup\u003e26\u003c/sup\u003e. ATKPAD may promote wound healing through mechanisms similar to those proposed in the concept of moist wound healing, such as enhanced neutrophil recruitment and preservation of growth factors\u003csup\u003e27\u003c/sup\u003e. However, direct comparisons with other products were not performed. Further studies are warranted to clarify the relative efficacy of ATKPAD compared with other wound dressings.\u003c/p\u003e\n\u003cp\u003eWound dressings with low oxygen permeability can help to create a hypoxic wound environment\u003csup\u003e20\u003c/sup\u003e. Sano et al. reported that wound dressings with low oxygen permeability promoted granulation tissue formation and angiogenesis compared with those with high oxygen permeability\u003csup\u003e28\u003c/sup\u003e. HIF-1\u0026alpha; is known as an inducible factor produced in hypoxic environments during the wound-healing process. Under normoxia conditions, HIF-1\u0026alpha; is rapidly degraded, resulting in low protein levels and transcriptional activity whereas, under hypoxic conditions, its expression and activity are upregulated\u003csup\u003e29\u003c/sup\u003e. Previous studies have shown that HIF-1\u0026alpha; accumulates gradually in response to ischemic or hypoxic stimuli, typically over a period of several hours to days\u003csup\u003e30\u0026ndash;32\u003c/sup\u003e. In the context of wound healing, it has been reported that HIF-1\u0026alpha; accumulation increases over the first 4 days post-injury and subsequently declines\u003csup\u003e33\u003c/sup\u003e. Based on this, we analyzed the high expression of HIF-1\u0026alpha; mRNA in ATKPAD group at 6 and 24 h after injury, suggesting that the hypoxic environment was induced by ATKPAD, in contrast with the oxygen-permeable Tegaderm\u003csup\u003e\u0026reg;34\u003c/sup\u003e. HIF-1\u0026alpha; activated the anaerobic glycolysis pathway to supplement ATP production under hypoxic conditions\u003csup\u003e35\u003c/sup\u003e. Furthermore, HIF-1\u0026alpha; induced the gene expression of CXCL1 and CXCL2\u003csup\u003e36\u003c/sup\u003e. In this study, the observed increase in ATP and CXC chemokine levels in the ATKPAD group further supported the hypothesis that the hypoxic environment created by ATKPAD may enhance neutrophilic inflammation, thereby contributing to improved wound healing.\u003c/p\u003e\n\u003cp\u003eAlthough the present study did not investigate the selective absorption of ATKPAD \u003cem\u003ein vivo\u003c/em\u003e, it may serve as a promising contact layer for emerging therapies such as growth factor sprays (e.g., Fibrast\u003csup\u003e\u0026reg;\u003c/sup\u003e spray) and cell suspension treatments (e.g., RECELL\u003csup\u003e\u0026reg;\u003c/sup\u003e)\u003csup\u003e37,38\u003c/sup\u003e. Further research is needed to optimize the combined use of ATKPAD with these advanced wound management strategies.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThis study demonstrated that ATKPAD promotes acute wound healing by inducing early neutrophilic inflammation through a hypoxic environment, while also offering good visibility and absorbing exudate. As a novel dressing, it addresses the limitations of existing materials like polyurethane films and hydrocolloids. However, as ATKPAD was only introduced in Japan in 2024, its optimal indications are still under investigation. Further studies using diverse wound models are needed.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eEthical statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in strict accordance with the Fundamental Guidelines for\u003c/p\u003e\n\u003cp\u003eProper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology in Japan, 2006. All experimental procedures involving animals followed the Regulations for Animal Experiments and Related Activities at\u003c/p\u003e\n\u003cp\u003eTohoku University, Sendai, Japan and were approved by the Institutional\u003c/p\u003e\n\u003cp\u003eAnimal Care and Use Committee at Tohoku University (No. 2023-093-01). All methods were performed in accordance with the relevant guidelines and regulations, and all efforts were made to minimize the suffering of the animals. The study was carried out in compliance with ARRIVE guidelines (https://arriveguidelines.org/).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHere, 8- to 9-week-old male C57BL/6J mice were purchased from CLEA Japan (Tokyo, Japan). All mice were kept in a specific pathogen-free environment, and food and water were always available. All experiments were performed under anesthesia, and every effort was made to minimize the suffering of the animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of ATKPAD for mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eATKPAD (Okamoto Industries, Tokyo, Japan) is a sheet consisting of a gel-like absorbent material made from reduced starch syrup and sodium alginate, encapsulated in a PVA semi-permeable membrane film (Fig. 5A). ATKPAD adheres to the wound surface without the need for adhesive due to osmotic pressure, and swells as it absorbs exudate into its inner material (Fig 5B). To evaluate the wound-healing mechanism of ATKPAD \u003cem\u003ein vivo\u003c/em\u003e, we prepared ATKPAD for mice as described below. ATKPAD for mice was composed of a semi-permeable membrane pouch made of PVA film and a liquid absorber. The liquid absorber was prepared by dissolving sodium alginate (Maikon no Kohara Co., Osaka, Japan) into reduced starch sugar (Mitsubishi Corporation Life Sciences Holdings Ltd., Tokyo, Japan). The PVA film was fabricated at a thickness of 17 \u0026mu;m by a casting method using a solution prepared by dissolving PVA resin (saponification degree: \u0026pound;98.0 mol%; Kuraray Co., Ltd., Tokyo, Japan) in purified water. Two sheets of PVA film were stacked and heat-sealed to form a pouch including 40 mg of liquid absorber by heat sealer FS-215 (FUJIIMPULSE Co., Ltd., Osaka, Japan) (Supplementary Fig. 1). ATKPAD for mice was sterilized by \u0026gamma;-ray irradiation (25 kGy).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of the membrane-based assay system\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn \u003cem\u003ein vitro\u003c/em\u003e two-chamber assay system (Neuro Probe, Inc.) was established to assess the permeability of bioactive wound-healing molecules (Figure 2a). Three types of semi-permeable membranes were tested: polycarbonate (PC; Neuro Probe, Inc) membranes with 3-\u0026micro;m pores (2 \u0026times; 10⁶ pores/cm\u0026sup2;), polyethylene terephthalate (PET; Lumirror\u0026trade; 12P60, TORAY Industries, Inc.), and PVA (Okamoto Industries, Inc.). PC membranes served as a positive control due to their high protein permeability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytokine migration assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo test cytokine permeability, human growth factor and angiogenesis-related cytokine panels (BioLegend, Lots B335808 and B363859) were used. A 1:4 diluted cytokine solution (25 \u0026micro;L) was placed in the lower chamber, and a hypertonic solution (stock:pyrogen-free water = 5:7) was added to the upper chamber. After 24 h of incubation at 37 \u0026deg;C, fluids from both chambers were collected (n = 6) and analyzed using multiplex bead-based assays. Concentrations were expressed in ng/mL. Cytokine concentrations were quantified using a LEGENDplex\u0026trade; human growth factor and angiogenesis-related cytokines panel (BioLegend) according to the manufacturer\u0026apos;s instructions. The FACSCelesta instrument (BD Biosciences) was used to acquire the data, which were analyzed using Qognit software (BioLegend).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWound creation and tissue collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs previously described\u003csup\u003e,39\u003c/sup\u003e, mice were anesthetized with 1.0\u0026ndash;2.0% isoflurane (Isoflurane, Mairan Pharma, Osaka, Japan) during the wounding procedure. The dorsal hair was shaved to fully expose the skin, which was then rinsed with 70% ethanol. Four full-thickness wounds extending to the panniculus carnosus layer were created on each mouse using a 6-mm diameter biopsy punch (Biopsy Punch, Kai Industries, Gifu, Japan) under sterile conditions. The wounds were covered with either a polyurethane film (Film) (Tegaderm\u0026trade; Transparent Dressing, 3M Health Care, St. Paul, MN, USA) or ATK, followed by an elastic adhesive bandage (Hilate, Iwatsuki, Tokyo, Japan) to serve as an occlusive dressing. The day on which the wounds were made was designated as day 0. Dressings were first applied on day 0 and subsequently replaced every other day, specifically on days 2, 4, and 6. To evaluate wound exudate volume, the difference in weight before and after use was calculated. As the polyurethane film did not have fluid absorption capacity, weight-based evaluation was not applicable. At various time points, mice were sacrificed with an overdose of isoflurane, and a 1-cm-square section of skin was excised using scissors. The tissue was then processed for the following analyses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathology and immunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe wounded tissues were fixed in a 4% paraformaldehyde-phosphate buffer solution and embedded in paraffin after caudocranial dissection, as previously described\u003csup\u003e40\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eSections were harvested from the central portion of the wound and stained with hematoxylin and eosin (H\u0026amp;E). The granulation area was determined on H\u0026amp;E-stained sections. For immunohistochemistry, sections were stained with anti-CD31 antibody (1:600 dilution; R\u0026amp;D Systems, Minneapolis, MN, USA) and anti-Ly6G Ab (clone 1A8; dilution 1:100; BioLegend)\u003csup\u003e,41\u003c/sup\u003e. Vascular density in five random fields (each 0.03 mm\u003csup\u003e2\u003c/sup\u003e) was determined by counting the number of CD31-positive vessels, while the number of neutrophils was assessed by counting the number of Ly6G-positive polynuclear cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of cytokines, chemokines, and growth factor concentrations \u003cem\u003ein vivo\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe wounded tissues were homogenized in saline using a stainless mesh, and the concentrations of cytokines and chemokines in the supernatants were measured using enzyme-linked immunosorbent assay (ELISA) kits, as previously described. ELISA kits from BioLegend (San Diego, CA, USA) were used for IL-6 and TNF-\u0026alpha;, and those from R\u0026amp;D Systems (Minneapolis, MN, USA) were used for CXCL1, CXCL2, and VEGF. The results were expressed as the values per tissue weight. The analysis was conducted at 12 h and on days 1, 3, and 5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of leukocytes and flow cytometric analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLeukocytes were prepared and analyzed by flow cytometric analysis as previously described\u003csup\u003e13\u003c/sup\u003e and detailed in Supplementary materials. The analysis was conducted on day 1. The cells obtained from the wounded tissues were stained with 7-aminoactinomycin D (7-AAD; BioLegend), Pacific Blue-anti-CD45 monoclonal antibody (mAb; clone 30-F11, BioLegend), and FITC-anti-Ly6G mAb (clone 1A8, BioLegend). Isotype-matched irrelevant IgG was used for control staining. Neutrophils were identified as CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003e cells. The stained cells were analyzed using a BD FACS Canto II flow cytometer (BD Bioscience, Franklin Lakes, NJ, USA). The number of neutrophils was estimated by multiplying the total leukocyte count by the proportion of each fraction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of ATP levels in wound tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdenosine triphosphate (ATP) was measured in tissue homogenates using a Tissue ATP Assay Kit (TOYO BEINET, Tokyo, Japan) according to the manufacturer\u0026apos;s instructions. The analysis was conducted at 6 and 24 h after wounding.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtraction of RNA and quantitative real-time RT-PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted using Isogen (Wako Pure Chemical, Osaka, Japan), and first-strand cDNA was synthesized using a PrimeScript first-strand cDNA synthesis kit (TaKaRa Bio, Otsu, Japan) according to the manufacturer\u0026rsquo;s instructions as previously described\u003csup\u003e42\u003c/sup\u003e. Quantitative real-time RT-PCR was performed in a volume of 20\u0026thinsp;\u0026mu;L using gene-specific primers and FastStart Essential DNA Green Master (Roche Applied Science, Branford, CT, USA) in a StepOne\u0026Ocirc;\u0026nbsp;system (Thermo Fisher, Waltham, MA, USA). Primer sequences were as follows: 5ʹ-TTCTGGCCAACGGTCTAGACAAC-3ʹ (Forward) and\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e5ʹ-CCAGTGGTCTTGGTGTGCTGA-3ʹ (Reverse) for 18S ribosomal RNA (18S rRNA); 5ʹ -TGCGTGCATGTCTAATCTGTTCC-3ʹ (Forward) and\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e5ʹ-AAGATTCTGACATGCCACATAGCTC-3ʹ (Reverse) for HIF-1\u0026alpha;. Reaction efficiency with each primer set was determined using standard amplification. Target gene expression levels and those of 18S rRNA as a reference gene were calculated for each sample using reaction efficiency. The results were analyzed using a relative quantification procedure and illustrated as relative expression compared with 18S rRNA expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData from \u003cem\u003ein vitro\u003c/em\u003e experiments are presented as mean \u0026plusmn; s.e.m. Statistical comparisons among membrane types were conducted using one-way analysis of variance (ANOVA) followed by Bonferroni\u0026rsquo;s multiple comparison test (GraphPad Prism). A \u003cem\u003eP\u003c/em\u003e-value \u0026lt; 0.05 was considered statistically significant. Data from \u003cem\u003ein vivo\u003c/em\u003e experiments were analyzed using JMP software (SAS Institute Japan, Tokyo, Japan). Data were expressed as the mean \u0026plusmn; standard deviation (s.d.). Differences between groups were examined for statistical significance using Welch\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test. A \u003cem\u003eP\u003c/em\u003e-value less than 0.05 was considered significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Biomedical Research Core of Tohoku University Graduate School of Medicine for technical support. Part of this study was supported by the Support system for young researchers to use research equipment, instruments and devices at Tohoku University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the present study, YS, EI, and HT collected the \u003cem\u003ein vivo\u003c/em\u003e data; TK and RA collected the \u003cem\u003ein vitro\u003c/em\u003e data; YS, HT, YA and EK designed the research study; TK, RA, WK, IS, KS, SI, TI, HM, TA and YI analyzed the data; AK contributed to making ATKPAD for mouse for the study; and YS and YA wrote the original draft; YA and EK critically reviewed and approved the final manuscript. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that this study received research funding and experimental materials from Okamoto Industries, Inc.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eEming, S. A., Martin, P. \u0026amp; Tomic-Canic, M. Wound repair and regeneration: mechanisms, signaling, and translation. \u003cem\u003eSci. 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(DOI 10.1128/IAI.00400\u0026thinsp;\u0026ndash;\u0026thinsp;20) (PMID: 32868343).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ATKPAD, HIF-1α, Inflammation, Wound healing","lastPublishedDoi":"10.21203/rs.3.rs-6729108/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6729108/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe inflammatory response after skin injury is crucial for activating the wound-healing process involving cytokine and growth factor secretion essential for tissue repair. Modern wound dressings promote healing by maintaining a moist, hypoxic environment. ATKPAD is a novel wound dressing that accelerates healing developed by Okamoto Industries, Inc. Composed of reduced starch syrup and a semi-permeable membrane, it was launched in Japan as a Class III advanced medical device in 2024. ATKPAD is unique in its transparency, allowing clear wound visibility, and selectively absorbs wound exudate through the combined effects of osmotic pressure and its semi-permeable membrane. However, the detailed molecular and cellular mechanisms of ATKPAD remain unclear. In this study, we examined its effect on acute wound healing. We analyzed the \u003cem\u003ein vitro\u003c/em\u003e permeability of cytokines and growth factors and evaluated granulation tissue formation, angiogenesis, neutrophil infiltration, and cytokine and growth factor production \u003cem\u003ein vivo\u003c/em\u003e. We found that cytokines and growth factors were selectively retained by ATKPAD. Its application enhanced granulation tissue formation and neutrophil infiltration, accompanied by increased hypoxia marker expression. These findings suggest that ATKPAD promotes wound healing by creating a hypoxic microenvironment that enhances early-phase inflammation while preserving key bioactive factors at the wound site.\u003c/p\u003e","manuscriptTitle":"A transparent osmotic wound dressing ATKPAD promotes healing by retaining cytokines and inducing hypoxia-driven inflammation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-26 09:26:56","doi":"10.21203/rs.3.rs-6729108/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-26T08:19:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-26T07:39:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-26T01:43:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-05-23T03:58:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e6b7e2ed-5f89-43bc-966a-df7cb8bfb34d","owner":[],"postedDate":"May 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":49006507,"name":"Biological sciences/Cell biology"},{"id":49006508,"name":"Biological sciences/Molecular biology"},{"id":49006509,"name":"Health sciences/Medical research"},{"id":49006510,"name":"Physical sciences/Materials science"}],"tags":[],"updatedAt":"2026-03-09T16:04:44+00:00","versionOfRecord":{"articleIdentity":"rs-6729108","link":"https://doi.org/10.1038/s41598-026-41264-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-03-06 15:59:12","publishedOnDateReadable":"March 6th, 2026"},"versionCreatedAt":"2025-05-26 09:26:56","video":"","vorDoi":"10.1038/s41598-026-41264-1","vorDoiUrl":"https://doi.org/10.1038/s41598-026-41264-1","workflowStages":[]},"version":"v1","identity":"rs-6729108","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6729108","identity":"rs-6729108","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}