{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,26]],"date-time":"2026-03-26T15:07:54Z","timestamp":1774537674067,"version":"3.50.1"},"reference-count":50,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2026,3,26]],"date-time":"2026-03-26T00:00:00Z","timestamp":1774483200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Provectus Biopharmaceuticals Inc.","award":["CON31319"],"award-info":[{"award-number":["CON31319"]}]},{"name":"Shriners Children Hospital","award":["SHC 87110-TX-23"],"award-info":[{"award-number":["SHC 87110-TX-23"]}]},{"name":"Remembering the 15 endowment"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Medicina"],"abstract":"<jats:p>Background and Objectives: Burn wounds are associated with delayed healing, infection, and pathological scarring. Effective repair requires tightly regulated immune and oxidative stress responses, including macrophage polarization. This study evaluated the association of the photosensitizer Rose Bengal, delivered in a hydrogel vehicle, with macrophage polarization and oxidative stress after burn injury. Materials and Methods: Three female red Duroc pigs underwent full-thickness contact burns followed by excision and autografting. Wounds received 20% Pluronic F-127 hydrogel containing 0.1% Rose Bengal sodium, hydrogel alone, or PBS (phosphate-buffered saline) on days 1, 7, and 14 post-burn. Biopsies from days 7 and 120 were analyzed by immunohistochemistry for pan-macrophage marker, CD206 (M2 macrophages), CD3E (T-cell infiltration), and 4-hydroxynonenal (4-HNE; oxidative stress marker). Mean fluorescence intensity was analyzed using two-way ANOVA with Tukey\u2019s post hoc test (mean \u00b1 SD, p &lt; 0.05). Results: At day 120, Rose Bengal treatment showed higher pan-macrophage expression (0.80 \u00b1 0.07) compared with PBS (0.62 \u00b1 0.10; p = 0.0034), whereas the difference versus hydrogel (0.68 \u00b1 0.07; p = 0.0628) was not significant. CD206 expression was similarly higher in Rose Bengal-treated wounds (0.77 \u00b1 0.06) compared with PBS (0.62 \u00b1 0.05; p = 0.0277); hydrogel also differed from PBS (p = 0.0287), without a difference between hydrogel and Rose Bengal. For CD3E, a significant main effect of treatment was observed (F(2,12) = 8.346, p = 0.0054), with lower values in Rose Bengal versus PBS at day 120 (p = 0.0360). No differences in 4-HNE were detected. Conclusions: Rose Bengal\u2013hydrogel treatment was associated with increased macrophage presence and enhanced M2 polarization without increased T-cell infiltration. Effects were significant versus PBS but not hydrogel, suggesting Rose Bengal may contribute to a pro-regenerative immune microenvironment without excessive adaptive activation.<\/jats:p>","DOI":"10.3390\/medicina62040629","type":"journal-article","created":{"date-parts":[[2026,3,26]],"date-time":"2026-03-26T13:50:15Z","timestamp":1774533015000},"page":"629","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["The Association of Rose Bengal with Macrophage Polarization and Oxidative Stress Response in Full-Thickness Excisional and Grafted Burn Wounds: A Porcine In Vivo Study"],"prefix":"10.3390","volume":"62","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-2487-9009","authenticated-orcid":false,"given":"Julia","family":"Kleinhapl","sequence":"first","affiliation":[{"name":"Department of Surgery, University of Texas Medical Branch, Galveston, TX 77555, USA"},{"name":"Division of Plastic, Aesthetic and Reconstructive Surgery, Department of Surgery, Medical University of Graz, 8036 Graz, Austria"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9371-9078","authenticated-orcid":false,"given":"Juquan","family":"Song","sequence":"additional","affiliation":[{"name":"Department of Surgery, University of Texas Medical Branch, Galveston, TX 77555, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ye","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Surgery, University of Texas Medical Branch, Galveston, TX 77555, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3221-513X","authenticated-orcid":false,"given":"Kan","family":"Nakamoto","sequence":"additional","affiliation":[{"name":"Department of Anesthesiology, University of Texas Medical Branch, Galveston, TX 77555, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-8342-0420","authenticated-orcid":false,"given":"Gabor","family":"Toro","sequence":"additional","affiliation":[{"name":"Department of Surgery, University of Texas Medical Branch, Galveston, TX 77555, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0353-0165","authenticated-orcid":false,"given":"Isabelle","family":"Bergman","sequence":"additional","affiliation":[{"name":"John Sealy School of Medicine, University of Texas Medical Branch, Galveston, TX 77555, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ludwik K.","family":"Branski","sequence":"additional","affiliation":[{"name":"Department of Surgery, University of Texas Medical Branch, Galveston, TX 77555, USA"},{"name":"Shriners Children\u2019s Texas, Galveston, TX 77550, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2972-3440","authenticated-orcid":false,"given":"Steven E.","family":"Wolf","sequence":"additional","affiliation":[{"name":"Department of Surgery, University of Texas Medical Branch, Galveston, TX 77555, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3657-0633","authenticated-orcid":false,"given":"Amina El","family":"Ayadi","sequence":"additional","affiliation":[{"name":"Department of Surgery, University of Texas Medical Branch, Galveston, TX 77555, USA"},{"name":"Shriners Children\u2019s Texas, Galveston, TX 77550, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,3,26]]},"reference":[{"key":"ref_1","unstructured":"American Burn Association (2026). Burn Incidence & Treatment in the U.S. Fact Sheet, American Burn Association. Available online: https:\/\/www.ameriburn.org\/prevention\/burn-incidence-and-treatment-in-the-united-states."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Markiewicz-Gospodarek, A., Kozio\u0142, M., Tobiasz, M., Baj, J., Radzikowska-B\u00fcchner, E., and Przekora, A. (2022). Burn Wound Healing: Clinical Complications, Medical Care, Treatment, and Dressing Types: The Current State of Knowledge for Clinical Practice. Int. J. Environ. Res. Public Health, 19.","DOI":"10.3390\/ijerph19031338"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Burgess, M., Valdera, F., Varon, D., Kankuri, E., and Nuutila, K. (2022). The Immune and Regenerative Response to Burn Injury. Cells, 11.","DOI":"10.3390\/cells11193073"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Radzikowska-B\u00fcchner, E., \u0141opuszy\u0144ska, I., Flieger, W., Tobiasz, M., Maciejewski, R., and Flieger, J. (2023). An Overview of Recent Developments in the Management of Burn Injuries. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms242216357"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1016\/j.addr.2017.09.018","article-title":"Burn injury: Challenges and advances in burn wound healing, infection, pain and scarring","volume":"123","author":"Wang","year":"2018","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"960","DOI":"10.1111\/wrr.13226","article-title":"Outcomes of dermal substitutes in burns and burn scar reconstruction: A systematic review and meta-analysis","volume":"32","author":"Verwilligen","year":"2024","journal-title":"Wound Repair Regen."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"599","DOI":"10.1038\/s41580-024-00715-1","article-title":"Cellular and molecular mechanisms of skin wound healing","volume":"25","author":"Martin","year":"2024","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Zieli\u0144ska, M., Paw\u0142owska, A., Orze\u0142, A., Sulej, L., Muzyka-Placzy\u0144ska, K., Baran, A., Filipecka-Tyczka, D., Paw\u0142owska, P., Nowi\u0144ska, A., and Bogus\u0142awska, J. (2023). Wound Microbiota and Its Impact on Wound Healing. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms242417318"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1427","DOI":"10.1016\/S0140-6736(16)31406-4","article-title":"Hypertrophic scarring: The greatest unmet challenge after burn injury","volume":"388","author":"Finnerty","year":"2016","journal-title":"Lancet"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Downer, M., Berry, C.E., Parker, J.B., Kameni, L., and Griffin, M. (2023). Current Biomaterials for Wound Healing. Bioengineering, 10.","DOI":"10.3390\/bioengineering10121378"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"137633","DOI":"10.1016\/j.ijbiomac.2024.137633","article-title":"Extracellular matrix-based biomaterials in burn wound repair: A promising therapeutic strategy","volume":"283","author":"Song","year":"2024","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"492","DOI":"10.1089\/wound.2012.0435","article-title":"Innate and Adaptive Immune Responses in Wound Epithelialization","volume":"3","author":"Strbo","year":"2014","journal-title":"Adv. Wound Care"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"314","DOI":"10.1038\/nature07039","article-title":"Wound repair and regeneration","volume":"453","author":"Gurtner","year":"2008","journal-title":"Nature"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2891","DOI":"10.1016\/j.ymthe.2022.07.016","article-title":"Macrophages as a therapeutic target to promote diabetic wound healing","volume":"30","author":"Sharifiaghdam","year":"2022","journal-title":"Mol. Ther."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Li, M., Hou, Q., Zhong, L., Zhao, Y., and Fu, X. (2021). Macrophage Related Chronic Inflammation in Non-Healing Wounds. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.681710"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Hesketh, M., Sahin, K.B., West, Z.E., and Murray, R.Z. (2017). Macrophage Phenotypes Regulate Scar Formation and Chronic Wound Healing. Int. J. Mol. Sci., 18.","DOI":"10.3390\/ijms18071545"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"249","DOI":"10.22203\/eCM.v024a18","article-title":"Reactive oxygen species (ROS)\u2014A family of fate deciding molecules pivotal in constructive inflammation and wound healing","volume":"24","author":"Bryan","year":"2012","journal-title":"Eur. Cell Mater."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"114004","DOI":"10.1016\/j.biopha.2022.114004","article-title":"The initiation of oxidative stress and therapeutic strategies in wound healing","volume":"157","author":"Wang","year":"2023","journal-title":"Biomed. Pharmacother."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"e70330","DOI":"10.1111\/iwj.70330","article-title":"Reactive Oxygen Species and Antioxidants in Wound Healing: Mechanisms and Therapeutic Potential","volume":"22","author":"Ukaegbu","year":"2025","journal-title":"Int. Wound J."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/1010-6030(89)85002-6","article-title":"Rose Bengal","volume":"47","author":"Neckers","year":"1989","journal-title":"J. Photochem. Photobiol. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Osta\u0144ska, E., Barna\u015b, E., Bartusik-Aebisher, D., Dynarowicz, K., Szpunar, M., Skr\u0119t-Magier\u0142o, J., and Aebisher, D. (2022). Histopathological Analysis of the Effect of Photodynamic Action on Post-Chemotherapy Excised Breast Cancer Tissue. Medicina, 58.","DOI":"10.3390\/medicina58060700"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1016\/j.biomaterials.2014.10.058","article-title":"Photo-induced inhibition of Alzheimer\u2019s \u03b2-amyloid aggregation in vitro by rose bengal","volume":"38","author":"Lee","year":"2015","journal-title":"Biomaterials"},{"key":"ref_23","first-page":"3","article-title":"Detection of Human Brucellosis by Brucellacapt and Rose Bengal Test in the Endemic Area","volume":"4","author":"Dicle","year":"2023","journal-title":"New Trends Med. Sci."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"387","DOI":"10.1016\/j.ajo.2019.08.027","article-title":"Rose Bengal Photodynamic Antimicrobial Therapy (RB-PDAT) for Patients with Progressive Infectious Keratitis: A Pilot Clinical Study","volume":"208","author":"Naranjo","year":"2019","journal-title":"Am. J. Ophthalmol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1845","DOI":"10.1167\/iovs.19-26604","article-title":"Enhancing Rose Bengal-Photosensitized Protein Crosslinking in the Cornea","volume":"60","author":"Wertheimer","year":"2019","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"645","DOI":"10.1002\/lsm.22066","article-title":"A photoactivated nanofiber graft material for augmented Achilles tendon repair","volume":"44","author":"Ni","year":"2012","journal-title":"Lasers Surg. Med."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1177\/1553350618824448","article-title":"Photochemical Tissue Bonding Technique for Improving Healing of Hand Tendon Injury","volume":"26","author":"Ding","year":"2019","journal-title":"Surg. Innov."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Percie du Sert, N., Hurst, V., Ahluwalia, A., Alam, S., Avey, M.T., Baker, M., Browne, W.J., Clark, A., Cuthill, I.C., and Dirnagl, U. (2020). The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. PLoS Biol., 18.","DOI":"10.1371\/journal.pbio.3000410"},{"key":"ref_29","unstructured":"Wachtel, T.L., McCahan, G.R., Watson, W.I., and Gorman, M. (2026, March 18). Determining the Surface Areas of Miniature Swine and Domestic Swine by Geometric Design\u2014A Comparative Study, Available online: https:\/\/apps.dtic.mil\/sti\/tr\/pdf\/AD0757600.pdf."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"637","DOI":"10.2131\/jts.41.637","article-title":"Body surface area measurement in laboratory miniature pigs using a computed tomography scanner","volume":"41","author":"Itoh","year":"2016","journal-title":"J. Toxicol. Sci."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1119","DOI":"10.1016\/j.burns.2008.03.013","article-title":"A porcine model of full-thickness burn, excision and skin autografting","volume":"34","author":"Branski","year":"2008","journal-title":"Burns"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"683","DOI":"10.1080\/08998280.2021.1953867","article-title":"Management of full-thickness skin grafts","volume":"34","author":"Davis","year":"2021","journal-title":"Proc. Bayl. Univ. Med. Cent."},{"key":"ref_33","unstructured":"American Veterinary Medical Association (2020). AVMA Guidelines for the Euthanasia of Animals: 2020 Edition, American Veterinary Medical Association. Available online: https:\/\/www.avma.org\/sites\/default\/files\/2020-02\/Guidelines-on-Euthanasia-2020.pdf?utm_source=chatgpt.com."},{"key":"ref_34","unstructured":"Bergman, I. (2026). Mean-Intensity-Fluorescence Macro for Immunofluorescence Images in Fiji, GitHub. Available online: https:\/\/github.com\/BellaBergman21\/Mean-Intensity-Fluorescence-Macro-for-Immunofluorescence-Images-in-Fiji-Isabelle-Bergman-.git."},{"key":"ref_35","first-page":"469","article-title":"American Society of Clinical Oncology, 2010 Annual Meeting and Rose Bengal: From a Wool Dye to a Cancer Therapy","volume":"35","author":"Alexander","year":"2010","journal-title":"Pharm. Ther."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2135","DOI":"10.1245\/s10434-014-4169-5","article-title":"Phase 2 Study of Intralesional PV-10 in Refractory Metastatic Melanoma","volume":"22","author":"Thompson","year":"2015","journal-title":"Ann. Surg. Oncol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"353","DOI":"10.1007\/978-3-319-54090-0_14","article-title":"Macrophage Differentiation in Normal and Accelerated Wound Healing","volume":"62","author":"Kotwal","year":"2017","journal-title":"Results Probl. Cell Differ."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1089\/wound.2014.0608","article-title":"The Innate Immune System in Acute and Chronic Wounds","volume":"5","author":"MacLeod","year":"2016","journal-title":"Adv. Wound Care"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"101686","DOI":"10.1016\/j.mtbio.2025.101686","article-title":"Bioactive multifunctional hydrogels accelerate burn wound healing via M2 macrophage-polarization, antioxidant and anti-inflammatory","volume":"32","author":"Zhao","year":"2025","journal-title":"Mater. Today Bio"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"66","DOI":"10.1046\/j.1524-475x.2001.00066.x","article-title":"The Pig as a Model for Human Wound Healing","volume":"9","author":"Sullivan","year":"2001","journal-title":"Wound Repair Regen."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1534","DOI":"10.1038\/s42003-024-07219-w","article-title":"Cellular and molecular roles of reactive oxygen species in wound healing","volume":"7","author":"Hunt","year":"2024","journal-title":"Commun. Biol."},{"key":"ref_42","first-page":"383","article-title":"Biomedical Applications of Photo- and Sono-Activated Rose Bengal: A Review","volume":"37","author":"Vanerio","year":"2019","journal-title":"Photobiomodul. Photomed. Laser Surg."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"150653","DOI":"10.1155\/2013\/150653","article-title":"Innovative Strategies to Overcome Biofilm Resistance","volume":"2013","author":"Taraszkiewicz","year":"2013","journal-title":"BioMed Res. Int."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"104575","DOI":"10.1016\/j.pdpdt.2025.104575","article-title":"Rose Bengal photodynamic antimicrobial therapy as an adjunct treatment for Pseudomonas aeruginosa infectious necrotizing scleritis","volume":"53","author":"Merikansky","year":"2025","journal-title":"Photodiagn. Photodyn. Ther."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1438","DOI":"10.1021\/acsbiomaterials.0c01588","article-title":"Light-Triggered Antibacterial Hydrogels Containing Recombinant Growth Factor for Treatment of Bacterial Infections and Improved Wound Healing","volume":"7","author":"Wang","year":"2021","journal-title":"ACS Biomater. Sci. Eng."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"758","DOI":"10.1111\/wrr.13202","article-title":"Photoactivated rose bengal mitigates a fibrotic phenotype and improves cutaneous wound healing in full-thickness injuries","volume":"32","author":"Jay","year":"2024","journal-title":"Wound Repair Regen."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1615","DOI":"10.1038\/cdd.2013.138","article-title":"Cell death and diseases related to oxidative stress:4-hydroxynonenal (HNE) in the balance","volume":"20","author":"Dalleau","year":"2013","journal-title":"Cell Death Differ."},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Ansari, M., and Darvishi, A. (2024). A review of the current state of natural biomaterials in wound healing applications. Front. Bioeng. Biotechnol., 12.","DOI":"10.3389\/fbioe.2024.1309541"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"122232","DOI":"10.1016\/j.lfs.2023.122232","article-title":"Functional hydrogel-based wound dressings: A review on biocompatibility and therapeutic efficacy","volume":"334","author":"Nasra","year":"2023","journal-title":"Life Sci."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"299","DOI":"10.1016\/j.jamcollsurg.2009.11.008","article-title":"Verified Centers, Nonverified Centers, or Other Facilities: A National Analysis of Burn Patient Treatment Location","volume":"210","author":"Zonies","year":"2010","journal-title":"J. Am. Coll. Surg."}],"container-title":["Medicina"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1648-9144\/62\/4\/629\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,3,26]],"date-time":"2026-03-26T14:13:57Z","timestamp":1774534437000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1648-9144\/62\/4\/629"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3,26]]},"references-count":50,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2026,4]]}},"alternative-id":["medicina62040629"],"URL":"https:\/\/doi.org\/10.3390\/medicina62040629","relation":{},"ISSN":["1648-9144"],"issn-type":[{"value":"1648-9144","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,3,26]]}}}