Chiono, Valeria

Ciardelli, Gianluca

Idrees, Ayesha

Marlinghaus, Lennart

Salber, Jochen

Schmitz, Inge

Viebahn, Richard

English

Ayesha Idrees

Inge Schmitz

Lennart Marlinghaus

Gianluca Ciardelli

Richard Viebahn

Valeria Chiono

Jochen Salber

PORTO@iris (Publications Open Repository TOrino - Politecnico di Torino)

04 August 2020POLITECNICO DI TORINORepository ISTITUZIONALEDevelopment of 3D skin model and 3D skin infection model, as advanced testing tools for the bio-evaluation ofantimicrobial biomaterials for wound healing / Idrees, Ayesha; Schmitz, Inge; Marlinghaus, Lennart; Ciardelli, Gianluca;Viebahn, Richard; Chiono, Valeria; Salber, Jochen. - (2019). ((Intervento presentato al convegno Centro 3R - 2nd AnnualMeeting tenutosi a Genova, Italy nel 20-21 June, 2019.OriginalDevelopment of 3D skin model and 3D skin infection model, as advanced testing tools for the bio-evaluation of antimicrobial biomaterials for wound healing.Publisher:PublishedDOI:Terms of use:openAccessPublisher copyright(Article begins on next page)This article is made available under terms and conditions as specified in the  corresponding bibliographic description inthe repositoryAvailability:This version is available at: 11583/2737993 since: 2019-06-28T01:53:10Zjournal Biomedical Science and Engineering - PagePressAyesha Idrees1,2,3, Inge Schmitz4, Lennart Marlinghaus5, Gianluca Ciardelli1, Richard Viebahn2, Valeria Chiono1, Jochen Salber2,31Department of Mechanical and Aerospace Engineering (DIMEAS), Politecnico di Torino, Turin, Italy2UMC Knappschaftskrankenhaus, Department of Surgery, Hospital of the RUHR-University Bochum, Bochum, Germany3Department of Experimental Surgery, Centre for Clinical Research, RUHR-University Bochum, Germany4Institute of Pathology, RUHR-University Bochum, Germany5Department of Medical Microbiology, Institute for Hygiene and Microbiology, RUHR-University Bochum, GermanyAuthors sharing last author position: valeria.chiono@polito.it, jochen.salber@hotmail.comA bacterial colonized human skin equivalent (c-HSE)The aim of this study was the development of a human skin model and human skinwound infection model for the bio-evaluation of antimicrobial biomaterialsintended for wound healing purposes. The three-dimensional in vitro models willrepresent advanced and complex systems to perform more reliable preclinicalstudies. These models will be employed for in vitro screening of both antibacterialactivity as well as cytocompatibility of new biomaterials and wound dressings tooptimize their in vivo performances. In the study, the 3D systems were developedand their structure was characterized. The antibacterial activity and cytotoxicity of amodel wound dressing releasing Ag+ was analyzed in the models.020406080100Percent Viability of Skin Equivalent  (%)Decreasing concentration of Ag+ 020406080100Ag+ MBC Ag+ wounddressingPercent Reduction of S. aureus in Liquid Broth CFU assay (%)77.47.88.28.699.4Ag+ MBC Ag+ wounddressingUntreatedLog CFU per S. aureus-colonized Skin Equivalent (Log) 3D Dermal Fibroblast Model020406080100Ag+ wounddressingWounddressingcontrol(WithoutAg+)UntreatedcontrolPercent Viability of Skin Equivalent  (%)Development of 3D skin model and 3D skin infection model, as advanced testing tools for the bio-evaluation of antimicrobial biomaterials for wound healingOptimizing the dermal part of human skin:Z-stacked fluorescence imagesreveal filopodia-like protrusions,elongated morphology anduniform distribution of humanfibroblasts at different planeswithin a Collagen-I (Col-I) matrix.Images show cell nuclei stainedwith DAPI and cytoskeletal F-actin filaments stained withPhalloidin. Scale bar=100 µmH&E stained cross section of in vitroHSE model:- Epidermal layer (1)- Dermal layer (2)The epidermal layer (1) of the HSEhas the characteristic structure:Stratum corneum, St. granulosum,St. spinosum and St. basale.HSE: Histological AnalysisImmunohistochemistry of the 3D Human Skin Model   Ultrastructure Analysis   S. aureus-colonized HSE    TEM image:- Collagen-I fibres (Col I)- Epidermal-dermal junction (DEJ).The DEJ is represented  by two very thin layers, called lamina lucida (LL) and lamina densa (LD), mimicking exactly the DE-barrier of native human skin.Antibacterial Analysis Cytocompatibility Analysis Inoculated bacteria (blue) adhereto dermal structures, migrate,proliferate, and colonize toestablish spots of biofilms.Big arrows: Bacteria located within a biofilm matrix inside the dermis;Small arrows: Bacteria surrounding keratinocytes within the epidermis.100 µmDAPI/K10bDAPI/Ki67/βTubulin100 µmcDAPI/Lam 550 µmd100 µmDAPI/K14a(a) Primary human cellsviability analysis applyingthe 3D-HSE. The modelwas exposed to a range ofsilver ion concentrations(Ag+) for a period of 24hours.(b) A clinically appliedAg+-releasing wounddressing served as modelmaterial and was testedin the 3D-HSE along withits control material(without Ag+).(a) The graph compares theefficacy of S. aureusreduction by Ag+ at MBC(recorded as per CLSIguidelines) and a clinicallyapplied Ag+-dressing intryptic soy broth (TSB). 100%reduction of bacteria wasobserved.(b) The graph shows theinefficacy of the sameantimicrobial Ag+ at morerealistic condition given by a3D c-HSE.ConclusionWith an increasing need for reliable in vitro test systems, we were successfully able to verify our advanced 3Dmodels, to serve as a risk assessment platform for cytocompatibility and antibacterial properties.Tissue culture models indicated an ’environmental effect’ on cytotoxicity, with decreased sensitivity to Ag+cytotoxicity for cells in 3D with respect to cells in 2D cultures.The considerable variation in antibacterial activity was observed under different antimicrobial evaluationsystems indicated that antibacterial ability of Ag+ was highly dependent on wound extracellular micro-environment, that could affect Ag+ availability.Moreover, these results also suggested the critical considerations to be taken into account while deciding theuse of Ag and Ag-dressings for wound care strategies.HSE and c-HSE have great potential to develop even more complex skin models for testing skin treatmentstrategies, as these models allow the study of skin-pathogen interactions, and of novel targets for designing newantibacterial agents.DEJLLCol ILD(a) (b)(a) (b)IHC verification of theHuman Skin Equivalent(HSE):(a) Keratin 14 (K14) red;(b) Keratin 10 (K10) green;(c) Ki67 (arrows) green;(d) Laminin5 (Lam5) green;Cell nuclei are shown inblue stained with DAPI.Laminin 5 is used as adermal-epidermal junction(DEJ) marker andappears as a thin green-stained line.12AcknowledgementThis work was supported by the European Union’s Horizon 2020 research and innovation program under theMarie Sklodowska-Curie Grant agreement No. 643050 (HyMedPoly Project).

Development of 3D skin model and 3D skin infection model, as advanced testing tools for the bio-evaluation of antimicrobial biomaterials for wound healing.

04 August 2020POLITECNICO DI TORINORepository ISTITUZIONALEDevelopment of 3D skin model and 3D skin infection model, as advanced testing tools for the bio-evaluation ofantimicrobial biomaterials for wound healing - Young Scientist Award 2019 / Idrees, Ayesha; Schmitz, Inge; Marlinghaus,Lennart; Ciardelli, Gianluca; Viebahn, Richard; Chiono, Valeria; Salber, Jochen. - (2019). ((Intervento presentato alconvegno Centro 3R - 2nd Annual Meeting tenutosi a Genova, Italy nel 20-21 June, 2019.OriginalDevelopment of 3D skin model and 3D skin infection model, as advanced testing tools for the bio-evaluation of antimicrobial biomaterials for wound healing - Young Scientist AwardPublisher:PublishedDOI:Terms of use:openAccessPublisher copyright(Article begins on next page)This article is made available under terms and conditions as specified in the  corresponding bibliographic description inthe repositoryAvailability:This version is available at: 11583/2737992 since: 2019-06-28T01:47:50Zjournal Biomedical Science and Engineering - PagePressDevelopment of 3D skin model and 3D skin infection model, as advanced testing tools for the bio-evaluation of antimicrobial biomaterials for wound healing Ayesha Idrees1,2,3, Inge Schmitz4, Lennart Marlinghaus5, Gianluca Ciardelli1, Richard Viebahn2, Valeria Chiono1, Jochen Salber2,3 1Department of Mechanical and Aerospace Engineering (DIMEAS), Politecnico di Torino, Turin, Italy 2UMC Knappschaftskrankenhaus Bochum, Clinic of Surgery, Hospital of the RUHR University Bochum, Bochum, Germany 3Department of Experimental Surgery, Centre for Clinical Research, RUHR University Bochum, Germany 4Institute of Pathology, RUHR University Bochum, Germany 5Department of Medical Microbiology, Institute for Hygiene and Microbiology, RUHR University Bochum, Germany Authors sharing last author position. valeria.chiono@polito.it, jochen.salber@hotmail.com Introduction: EU 7th amendment (Dir. 2003/15/EC) of the “Cosmetics Directive” (76/768/EEC) has required the replacement of animal trials with reliable in vitro tests for assessing cutaneous resorption since 2009. In vitro bio-evaluation would be more accurate using biomimetic 3D testing systems respect to 2D cell cultures. In this work, in vitro 3D human skin equivalent (HSE) and wound infection model (c-HSE) were developed and used for testing commercially available Ag-dressings, then compared to 2D cultures.  Materials and methods: Initially murine in vitro dermal construct (MDC) and human in vitro dermal construct (HDC) were generated, and different viability assays were optimized for 3D system [1]. Then, in-house reconstructed human epidermis (RHE) and reconstructed murine epidermis (RME) [2] were developed with the aim to understand the relevance of outcomes obtained from human- and animal-based systems reducing the animal trials. The human skin equivalent (HSE) having both dermal and epidermal compartments was obtained by optimizing 3D cell culture conditions based on serum/animal component-free and fully-defined media to obtain epidermal differentiation mimicking as closely as possible native human skin (NHS). Skin infection model (c-HSE) was created by full-thickness incision and colonization with S. aureus. To validate the 3D systems, Ag+ and several commercially available Ag containing antimicrobial wound dressings were bio-evaluated using HSE and c-HSE. On the other hand, cell monolayer cultures (based on primary cells and cell lines) were used as 2D cytocompatibility evaluation systems. These materials were also evaluated for their antibacterial activity against several clinically relevant pathogens under different growth culture conditions including simulated wound fluid (SWF). Moreover, additional efforts were addressed to the bio-evaluation of novel drug-free biomaterials intended for infected wound healing applications. Results: Histological results showed characteristic well-differentiated layers of epidermis. Immunohistochemistry for proteins’ tissue distribution analysis showed Ki-67 & K14 positive basal keratinocytes, K10 positive supra-basal keratinocytes, filaggrin & loricin positive sub-corneal & corneal keratinocytes, and laminin 5 positive basement membrane at dermal-epidermal junction. TEM revealed the ultrastructure of basement membrane demonstrating lamina lucida, lamina densa, regular hemidesmosomes, and anchoring fibres. The epidermal layers showed abundant intracellular keratin filaments, desmosomes, and tight junction between keratinocytes. SEM revealed the interwoven network and architecture of ECM with embedded dermal fibroblasts. The contact angle of 82.5° ± 8.9° demonstrated the barrier function of HSE with respect to 90.0° ± 5.1° for NHS. This model represented a fundamental construct with highly resembling features to that of NHS. Histology of c-HSE demonstrated bacterial aggregations and early biofilm formation while TEM revealed interesting features including S. aureus induced ECM degradation, fibroblasts/keratinocytes cell lysis, and bacterial internalization by keratinocytes. SEM revealed grape-like bacterial clusters covered within extracellular polymeric substance at wound site in dermis and epidermis. The c-HDC results suggested that co-existence of S. aureus and P. aeruginosa may be significant for both bacterial colonization and pathogenicity in wounds. The bio-evaluation outcomes were different in 2D monolayer vs. 3D HSE based cell culture systems, and conventionally used microbial methods vs. advanced c-HSE system.  Conclusions: With an increasing need for reliable in vitro testing systems, we were successfully able to establish closely mimicking and verify our advanced 3D models, to serve as a risk assessment platform for cytocompatibility and antibacterial properties.  References:  1. Idrees, A., et al., Validation of in vitro assays in three-dimensional human dermal constructs. The International journal of artificial organs, 2018: p. 0391398818775519. 2. Segrelles, C., et al., Establishment of a murine epidermal cell line suitable for in vitro and in vivo skin modelling. BMC dermatology, 2011. 11(1): p. 9. Figure: H & E stained histological image of A) HSE and B) c-HSE. 

Development of 3D skin model and 3D skin infection model, as advanced testing tools for the bio-evaluation of antimicrobial biomaterials for wound healing - Young Scientist Award 2019

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Development of 3D skin model and 3D skin infection model, as advanced testing tools for the bio-evaluation of antimicrobial biomaterials for wound healing - Young Scientist Award 2019

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