Development of an Experimental Ex Vivo Wound Model to Evaluate Antimicrobial Efficacy of Topical Formulations
Abstract
:1. Introduction
2. Results
2.1. Characteristics of Burn-Induced Ex Vivo Wounds
2.2. Effects of Antibacterial Treatment on Simulated S. aureus and P. aeruginosa Wound Infections
2.3. Longitudinal Evaluation of Antibacterial Treatment Using Bioluminescence Imaging
2.4. Visualization of Bacterial Infection in the Wound Tissue
3. Discussion
4. Materials and Methods
4.1. Bacteria and Growth Conditions
4.2. Chemicals
4.3. Pig Skin Collection and Storage
4.4. Wounding Method
4.5. Wound Infection
4.6. Antibacterial Treatment
4.7. Sampling and Viable Count Assay
4.8. Scanning Electron Microscopy (SEM)
4.9. Histology
4.10. Cryosectioning and Immunohistochemistry
4.11. Imaging of Infection
4.12. Statistical Analysis
Author Contributions
Funding
Institutional Review Board Statement
Acknowledgments
Conflicts of Interest
References
- Parnell, L.K.S.; Volk, S.W. The Evolution of Animal Models in Wound Healing Research: 1993–2017. Adv. Wound Care (New Rochelle) 2019, 8, 692–702. [Google Scholar] [CrossRef]
- Lebeaux, D.; Chauhan, A.; Rendueles, O.; Beloin, C. From in vitro to in vivo Models of Bacterial Biofilm-Related Infections. Pathogens 2013, 2, 288–356. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Dai, T.; Kharkwal, G.B.; Tanaka, M.; Huang, Y.Y.; Bil de Arce, V.J.; Hamblin, M.R. Animal models of external traumatic wound infections. Virulence 2011, 2, 296–315. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Abdullahi, A.; Amini-Nik, S.; Jeschke, M.G. Animal models in burn research. Cell. Mol. Life Sci. 2014, 71, 3241–3255. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Seaton, M.; Hocking, A.; Gibran, N.S. Porcine models of cutaneous wound healing. Ilar. J. 2015, 56, 127–138. [Google Scholar] [CrossRef] [PubMed]
- Lindblad, W.J. Considerations for selecting the correct animal model for dermal wound-healing studies. J. Biomater. Sci. Polym. Ed. 2008, 19, 1087–1096. [Google Scholar] [CrossRef] [PubMed]
- Davidson, J.M. Animal models for wound repair. Arch. Derm. Res. 1998, 290, S1–S11. [Google Scholar] [CrossRef] [PubMed]
- Gottrup, F.; Agren, M.S.; Karlsmark, T. Models for use in wound healing research: A survey focusing on in vitro and in vivo adult soft tissue. Wound Repair Regen. 2000, 8, 83–96. [Google Scholar] [CrossRef]
- Coolen, N.A.; Vlig, M.; van den Bogaerdt, A.J.; Middelkoop, E.; Ulrich, M.M. Development of an in vitro burn wound model. Wound Repair Regen. 2008, 16, 559–567. [Google Scholar] [CrossRef] [PubMed]
- Gurjala, A.N.; Geringer, M.R.; Seth, A.K.; Hong, S.J.; Smeltzer, M.S.; Galiano, R.D.; Leung, K.P.; Mustoe, T.A. Development of a novel, highly quantitative in vivo model for the study of biofilm-impaired cutaneous wound healing. Wound Repair Regen. 2011, 19, 400–410. [Google Scholar] [CrossRef]
- Perez, R.; Davis, S.C. Relevance of animal models for wound healing. Wounds 2008, 20, 3–8. [Google Scholar] [PubMed]
- Ganesh, K.; Sinha, M.; Mathew-Steiner, S.S.; Das, A.; Roy, S.; Sen, C.K. Chronic Wound Biofilm Model. Adv. Wound Care (New Rochelle) 2015, 4, 382–388. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Montagna, W.; Yun, J.S. The Skin of the Domestic Pig. J. Investig. Derm. 1964, 42, 11–21. [Google Scholar] [CrossRef] [Green Version]
- Bowler, P.G.; Duerden, B.I.; Armstrong, D.G. Wound microbiology and associated approaches to wound management. Clin. Microbiol. Rev. 2001, 14, 244–269. [Google Scholar] [CrossRef] [Green Version]
- Church, D.; Elsayed, S.; Reid, O.; Winston, B.; Lindsay, R. Burn wound infections. Clin. Microbiol. Rev. 2006, 19, 403–434. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Korber, A.; Schmid, E.N.; Buer, J.; Klode, J.; Schadendorf, D.; Dissemond, J. Bacterial colonization of chronic leg ulcers: Current results compared with data 5 years ago in a specialized dermatology department. J. Eur. Acad. Derm. Venereol. 2010, 24, 1017–1025. [Google Scholar] [CrossRef]
- Jeschke, M.G.; van Baar, M.E.; Choudhry, M.A.; Chung, K.K.; Gibran, N.S.; Logsetty, S. Burn injury. Nat. Rev. Dis. Primers 2020, 6, 11. [Google Scholar] [CrossRef]
- Jabeen, S.; Clough, E.C.S.; Thomlinson, A.M.; Chadwick, S.L.; Ferguson, M.W.J.; Shah, M. Partial thickness wound: Does mechanism of injury influence healing? Burns 2019, 45, 531–542. [Google Scholar] [CrossRef] [PubMed]
- Zulkowski, K. Wound terms and definitions. WCET J. 2015, 35, 83–94. [Google Scholar]
- Saleh, K.; Schmidtchen, A. Surgical site infections in dermatologic surgery: Etiology, pathogenesis, and current preventative measures. Derm. Surg. 2015, 41, 537–549. [Google Scholar] [CrossRef]
- Saleh, K.; Sonesson, A.; Persson, B.; Riesbeck, K.; Schmidtchen, A. A descriptive study of bacterial load of full-thickness surgical wounds in dermatologic surgery. Derm. Surg. 2011, 37, 1014–1022. [Google Scholar] [CrossRef]
- Wolcott, R.D.; Hanson, J.D.; Rees, E.J.; Koenig, L.D.; Phillips, C.D.; Wolcott, R.A.; Cox, S.B.; White, J.S. Analysis of the chronic wound microbiota of 2963 patients by 16S rDNA pyrosequencing. Wound Repair Regen. 2016, 24, 163–174. [Google Scholar] [CrossRef]
- Horrocks, A. Prontosan wound irrigation and gel: Management of chronic wounds. Br. J. Nurs. 2006, 15, 1222–1228. [Google Scholar] [CrossRef] [PubMed]
- Hirsch, T.; Koerber, A.; Jacobsen, F.; Dissemond, J.; Steinau, H.U.; Gatermann, S.; Al-Benna, S.; Kesting, M.; Seipp, H.M.; Steinstraesser, L. Evaluation of toxic side effects of clinically used skin antiseptics in vitro. J. Surg. Res. 2010, 164, 344–350. [Google Scholar] [CrossRef] [PubMed]
- Langtry, H.D.; Lamb, H.M. Levofloxacin. Its use in infections of the respiratory tract, skin, soft tissues and urinary tract. Drugs 1998, 56, 487–515. [Google Scholar] [CrossRef]
- Sakamoto, H.; Sakamoto, M.; Hata, Y.; Kubota, T.; Ishibashi, T. Aqueous and vitreous penetration of levofloxacin after topical and/or oral administration. Eur. J. Ophthalmol. 2007, 17, 372–376. [Google Scholar] [CrossRef]
- Shepherd, J.; Douglas, I.; Rimmer, S.; Swanson, L.; MacNeil, S. Development of three-dimensional tissue-engineered models of bacterial infected human skin wounds. Tissue Eng. Part C Methods 2009, 15, 475–484. [Google Scholar] [CrossRef] [PubMed]
- Serra, R.; Grande, R.; Butrico, L.; Rossi, A.; Settimio, U.F.; Caroleo, B.; Amato, B.; Gallelli, L.; de Franciscis, S. Chronic wound infections: The role of Pseudomonas aeruginosa and Staphylococcus aureus. Expert Rev. Anti Infect. 2015, 13, 605–613. [Google Scholar] [CrossRef] [PubMed]
- Fazli, M.; Bjarnsholt, T.; Kirketerp-Moller, K.; Jorgensen, B.; Andersen, A.S.; Krogfelt, K.A.; Givskov, M.; Tolker-Nielsen, T. Nonrandom distribution of Pseudomonas aeruginosa and Staphylococcus aureus in chronic wounds. J. Clin. Microbiol. 2009, 47, 4084–4089. [Google Scholar] [CrossRef] [Green Version]
- Sacha, M.; Faucon, L.; Hamon, E.; Ly, I.; Haltner-Ukomadu, E. Ex vivo transdermal absorption of a liposome formulation of diclofenac. Biomed. Pharm. 2019, 111, 785–790. [Google Scholar] [CrossRef] [PubMed]
- Tazrart, A.; Bolzinger, M.A.; Moureau, A.; Molina, T.; Coudert, S.; Angulo, J.F.; Briancon, S.; Griffiths, N.M. Penetration and decontamination of americium-241 ex vivo using fresh and frozen pig skin. Chem. Biol. Interact. 2017, 267, 40–47. [Google Scholar] [CrossRef] [PubMed]
- Donlan, R.M.; Costerton, J.W. Biofilms: Survival mechanisms of clinically relevant microorganisms. Clin. Microbiol. Rev. 2002, 15, 167–193. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Zhao, G.; Usui, M.L.; Lippman, S.I.; James, G.A.; Stewart, P.S.; Fleckman, P.; Olerud, J.E. Biofilms and Inflammation in Chronic Wounds. Adv. Wound Care (New Rochelle) 2013, 2, 389–399. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Wu, Y.K.; Cheng, N.C.; Cheng, C.M. Biofilms in Chronic Wounds: Pathogenesis and Diagnosis. Trends Biotechnol. 2019, 37, 505–517. [Google Scholar] [CrossRef]
- Tiwari, V.K. Burn wound: How it differs from other wounds? Indian J. Plast. Surg. 2012, 45, 364–373. [Google Scholar] [CrossRef]
- Weaver, A.J., Jr.; Brandenburg, K.S.; Smith, B.W.; Leung, K.P. Comparative Analysis of the Host Response in a Rat Model of Deep-Partial and Full-Thickness Burn Wounds with Pseudomonas aeruginosa Infection. Front. Cell. Infect. Microbiol. 2019, 9, 466. [Google Scholar] [CrossRef] [PubMed]
- Pruitt, B.A., Jr.; McManus, A.T.; Kim, S.H.; Goodwin, C.W. Burn wound infections: Current status. World J. Surg. 1998, 22, 135–145. [Google Scholar] [CrossRef]
- Azeredo, J.; Azevedo, N.F.; Briandet, R.; Cerca, N.; Coenye, T.; Costa, A.R.; Desvaux, M.; Di Bonaventura, G.; Hebraud, M.; Jaglic, Z.; et al. Critical review on biofilm methods. Crit. Rev. Microbiol. 2017, 43, 313–351. [Google Scholar] [CrossRef] [Green Version]
- Brackman, G.; Coenye, T. In Vitro and In Vivo Biofilm Wound Models and Their Application. Adv. Exp. Med. Biol. 2016, 897, 15–32. [Google Scholar] [CrossRef]
- Wilkinson, H.N.; McBain, A.J.; Stephenson, C.; Hardman, M.J. Comparing the Effectiveness of Polymer Debriding Devices Using a Porcine Wound Biofilm Model. Adv. Wound Care (New Rochelle) 2016, 5, 475–485. [Google Scholar] [CrossRef]
- Rabin, N.; Zheng, Y.; Opoku-Temeng, C.; Du, Y.; Bonsu, E.; Sintim, H.O. Biofilm formation mechanisms and targets for developing antibiofilm agents. Future Med. Chem. 2015, 7, 493–512. [Google Scholar] [CrossRef] [PubMed]
- Yang, S.Y.; Liu, Y.; Mao, J.; Wu, Y.B.; Deng, Y.L.; Qi, S.C.; Zhou, Y.C.; Gong, S.Q. The anti-biofilm and collagen-stabilizing effects of proanthocyanidin as an auxiliary endodontic irrigant. Int. Endod. J. 2020. [Google Scholar] [CrossRef] [PubMed]
- Schmidtchen, A.; Pasupuleti, M.; Morgelin, M.; Davoudi, M.; Alenfall, J.; Chalupka, A.; Malmsten, M. Boosting antimicrobial peptides by hydrophobic oligopeptide end tags. J. Biol. Chem. 2009, 284, 17584–17594. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Myhrman, E.; Hakansson, J.; Lindgren, K.; Bjorn, C.; Sjostrand, V.; Mahlapuu, M. The novel antimicrobial peptide PXL150 in the local treatment of skin and soft tissue infections. Appl. Microbiol. Biotechnol. 2013, 97, 3085–3096. [Google Scholar] [CrossRef] [Green Version]
- McDonnell, G.; Haines, K.; Klein, D.; Rippon, M.; Walmsley, R.; Pretzer, D. Clinical correlation of a skin antisepsis model. J. Microbiol. Methods 1999, 35, 31–35. [Google Scholar] [CrossRef]
- Phillips, P.L.; Yang, Q.; Davis, S.; Sampson, E.M.; Azeke, J.I.; Hamad, A.; Schultz, G.S. Antimicrobial dressing efficacy against mature Pseudomonas aeruginosa biofilm on porcine skin explants. Int. Wound J. 2015, 12, 469–483. [Google Scholar] [CrossRef]
- Wilkinson, H.N.; Iveson, S.; Catherall, P.; Hardman, M.J. A Novel Silver Bioactive Glass Elicits Antimicrobial Efficacy against Pseudomonas aeruginosa and Staphylococcus aureus in an ex Vivo Skin Wound Biofilm Model. Front. Microbiol. 2018, 9, 1450. [Google Scholar] [CrossRef] [Green Version]
- Rizzo, A.E.; Beckett, L.A.; Baier, B.S.; Isseroff, R.R. The linear excisional wound: An improved model for human ex vivo wound epithelialization studies. Ski. Res. Technol. 2012, 18, 125–132. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Guggenheim, M.; Thurnheer, T.; Gmur, R.; Giovanoli, P.; Guggenheim, B. Validation of the Zurich burn-biofilm model. Burns 2011, 37, 1125–1133. [Google Scholar] [CrossRef] [PubMed]
- Thet, N.T.; Alves, D.R.; Bean, J.E.; Booth, S.; Nzakizwanayo, J.; Young, A.E.; Jones, B.V.; Jenkins, A.T. Prototype Development of the Intelligent Hydrogel Wound Dressing and Its Efficacy in the Detection of Model Pathogenic Wound Biofilms. ACS Appl. Mater. Interfaces 2016, 8, 14909–14919. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Bager, C.L.; Gudmann, N.; Willumsen, N.; Leeming, D.J.; Karsdal, M.A.; Bay-Jensen, A.C.; Hogdall, E.; Balslev, I.; He, Y. Quantification of fibronectin as a method to assess ex vivo extracellular matrix remodeling. Biochem. Biophys. Res. Commun. 2016, 478, 586–591. [Google Scholar] [CrossRef] [PubMed]
- Werthen, M.; Davoudi, M.; Sonesson, A.; Nitsche, D.P.; Morgelin, M.; Blom, K.; Schmidtchen, A. Pseudomonas aeruginosa-induced infection and degradation of human wound fluid and skin proteins ex vivo are eradicated by a synthetic cationic polymer. J. Antimicrob. Chemother. 2004, 54, 772–779. [Google Scholar] [CrossRef] [Green Version]
- Zeitlinger, M.A.; Dehghanyar, P.; Mayer, B.X.; Schenk, B.S.; Neckel, U.; Heinz, G.; Georgopoulos, A.; Muller, M.; Joukhadar, C. Relevance of soft-tissue penetration by levofloxacin for target site bacterial killing in patients with sepsis. Antimicrob. Agents Chemother. 2003, 47, 3548–3553. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Puthia, M.; Butrym, M.; Petrlova, J.; Stromdahl, A.C.; Andersson, M.A.; Kjellstrom, S.; Schmidtchen, A. A dual-action peptide-containing hydrogel targets wound infection and inflammation. Sci. Transl. Med. 2020, 12. [Google Scholar] [CrossRef] [PubMed]
- Rueden, C.T.; Schindelin, J.; Hiner, M.C.; DeZonia, B.E.; Walter, A.E.; Arena, E.T.; Eliceiri, K.W. ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinform. 2017, 18, 529. [Google Scholar] [CrossRef] [PubMed]
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Andersson, M.Å.; Madsen, L.B.; Schmidtchen, A.; Puthia, M. Development of an Experimental Ex Vivo Wound Model to Evaluate Antimicrobial Efficacy of Topical Formulations. Int. J. Mol. Sci. 2021, 22, 5045. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22095045
Andersson MÅ, Madsen LB, Schmidtchen A, Puthia M. Development of an Experimental Ex Vivo Wound Model to Evaluate Antimicrobial Efficacy of Topical Formulations. International Journal of Molecular Sciences. 2021; 22(9):5045. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22095045
Chicago/Turabian StyleAndersson, Madelene Å, Lone Bruhn Madsen, Artur Schmidtchen, and Manoj Puthia. 2021. "Development of an Experimental Ex Vivo Wound Model to Evaluate Antimicrobial Efficacy of Topical Formulations" International Journal of Molecular Sciences 22, no. 9: 5045. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22095045