Constructing human skin equivalents on porcine acellular peritoneum extracellular matrix for in vitro irritation testing

ObjectivesFibrosis is a complex pathophysiological process involving interplay between multiple cell types. Experimental modelling of fibrosis is essential for the understanding of its pathogenesis and for testing of putative antifibrotic drugs. However, most current models employ either phylogenetically distant species or rely on human cells cultured in an artificial environment. Here we evaluated the potential of vascularised in vitro human skin equivalents as a novel model of skin fibrosis and a platform for the evaluation of antifibrotic drugs.MethodsSkin equivalents were assembled on a three-dimensional extracellular matrix by sequential seeding of endothelial cells, fibroblasts and keratinocytes. Fibrotic transformation on exposure to transforming growth factor-β (TGFβ) and response to treatment with nintedanib as an established antifibrotic agent were evaluated by quantitative polymerase chain reaction (qPCR), capillary Western immunoassay, immunostaining and histology.ResultsSkin equivalents perfused at a physiological pressure formed a mature, polarised epidermis, a stratified dermis and a functional vessel system. Exposure of these models to TGFβ recapitulated key features of SSc skin with activation of TGFβ pathways, fibroblast to myofibroblast transition, increased release of collagen and excessive deposition of extracellular matrix. Treatment with the antifibrotic agent nintedanib ameliorated this fibrotic transformation.ConclusionOur data provide evidence that vascularised skin equivalents can replicate key features of fibrotic skin and may serve as a platform for evaluation of antifibrotic drugs in a pathophysiologically relevant human setting.

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OP0195 VASCULARIZED THREE-DIMENSIONAL MODELS OF HUMAN SKIN FIBROSIS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.3141 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 121.2-122

Author(s):

A. E. Matei ◽

C. W. Chen ◽

L. Kiesewetter ◽

A. H. Györfi ◽

Y. N. LI ◽

...

Keyword(s):

Extracellular Matrix ◽

Human Skin ◽

Three Dimensional ◽

Cell Polarization ◽

Skin Fibrosis ◽

Matrix Deposition ◽

Skin Equivalents ◽

Antifibrotic Drugs

Background:The complex pathophysiological processes that result in fibrotic tissue remodeling in systemic sclerosis involve interplay between multiple cell types (1). Experimental models of fibrosis are essential to provide a conceptual understanding of the pathogenesis of these diseases and to test antifibrotic drugs. Current models of fibrosis have important limitations: thein vivomodels rely on species that are phylogenetically distant, whereas thein vitromodels are oversimplified cultures of a single cell type in an artificial two-dimensional environment of excessive stiffness, which imposes an unphysiological cell polarization (2).Objectives:Here we evaluated the potential use of vascularized, three-dimensionalin vitrohuman skin equivalents as a novel model of skin fibrosis and a platform for the evaluation of antifibrotic drugs.Methods:Skin equivalents were generated by seeding human endothelial cells, fibroblasts and keratinocytes on a decellularized porcine extracellular matrix with perfusable vascular structure. The skin models were cultured for one month in a system that ensured perfusion of the vascular network at physiological pressure. Fibrotic transformation induced by TGFβ and response to nintedanib as an established antifibrotic drug was evaluated by capillary Western immunoassays, qPCR, histology and immunostaining.Results:The vascularized human skin equivalents formed the major skin structures relevant for the pathogenesis of fibrosis: a polarized, fully matured epidermis, a stratified dermis and a perfused vessel system with small capillaries. Exposure to TGFβ led to the fibrotic transformation of the skin equivalents, with activated TGFβ downstream pathways, increased fibroblast-to-myofibroblast transition and excessive deposition of extracellular matrix. Treatment of models exposed to TGFβ with nintedanib (a drug with proven antifibrotic effects) ameliorated the fibrotic transformation of skin equivalents with reduced TGFβ signaling, fibroblast-to-myofibroblast transition and decreased extracellular matrix deposition.Conclusion:Here we describe a novelin vitromodel of skin fibrosis. Our data show that vascularized skin equivalents can reproduce all skin layers affected by fibrosis, that, upon exposure to TGFβ, these models recapitulate key features of fibrotic skin and that these skin models can be used as a platform for evaluation of antifibrotic drugs in a setting with high relevance for human disease.References:[1]Distler JHW, Gyorfi AH, Ramanujam M, Whitfield ML, Konigshoff M, Lafyatis R. Shared and distinct mechanisms of fibrosis. Nature reviews Rheumatology. 2019;15(12):705-30.[2]Garrett SM, Baker Frost D, Feghali-Bostwick C. The mighty fibroblast and its utility in scleroderma research. Journal of scleroderma and related disorders. 2017;2(2):69-134.Disclosure of Interests:Alexandru-Emil Matei: None declared, Chih-Wei Chen: None declared, Lisa Kiesewetter: None declared, Andrea-Hermina Györfi: None declared, Yi-Nan Li: None declared, Thuong Trinh-Minh: None declared, Toin van Kuppevelt: None declared, Jan Hansmann: None declared, Astrid Juengel: None declared, Georg Schett Speakers bureau: AbbVie, BMS, Celgene, Janssen, Eli Lilly, Novartis, Roche and UCB, Florian Groeber-Becker: None declared, Jörg Distler Grant/research support from: Boehringer Ingelheim, Consultant of: Boehringer Ingelheim, Paid instructor for: Boehringer Ingelheim, Speakers bureau: Boehringer Ingelheim

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Interrelation of permeation and penetration parameters obtained from in vitro experiments with human skin and skin equivalents

Journal of Controlled Release ◽

10.1016/s0168-3659(01)00396-0 ◽

2001 ◽

Vol 75 (3) ◽

pp. 283-295 ◽

Cited By ~ 59

Author(s):

Heike Wagner ◽

Karl-Heinz Kostka ◽

Claus-Michael Lehr ◽

Ulrich F Schaefer

Keyword(s):

Human Skin ◽

In Vitro Experiments ◽

Skin Equivalents

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688 Epithelial precursor cell-conditioned media ameliorates UV irradiation-induced extracellular matrix damage in human skin equivalents

Journal of Investigative Dermatology ◽

10.1016/j.jid.2017.07.365 ◽

2017 ◽

Vol 137 (10) ◽

pp. S311

Author(s):

D. Kim ◽

H. Kim ◽

S. Sohn ◽

A. Kim ◽

S. Baek ◽

...

Keyword(s):

Extracellular Matrix ◽

Human Skin ◽

Uv Irradiation ◽

Precursor Cell ◽

Conditioned Media ◽

Skin Equivalents ◽

Matrix Damage

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Contribution of Palmitic Acid to Epidermal Morphogenesis and Lipid Barrier Formation in Human Skin Equivalents

International Journal of Molecular Sciences ◽

10.3390/ijms20236069 ◽

2019 ◽

Vol 20 (23) ◽

pp. 6069 ◽

Cited By ~ 1

Author(s):

Arnout Mieremet ◽

Richard Helder ◽

Andreea Nadaban ◽

Gert Gooris ◽

Walter Boiten ◽

...

Keyword(s):

Palmitic Acid ◽

Human Skin ◽

De Novo ◽

Skin Equivalents ◽

Barrier Formation ◽

Lipid Organization ◽

Epidermal Morphogenesis ◽

Lateral Organization

The outermost barrier layer of the skin is the stratum corneum (SC), which consists of corneocytes embedded in a lipid matrix. Biosynthesis of barrier lipids occurs de novo in the epidermis or is performed with externally derived lipids. Hence, in vitro developed human skin equivalents (HSEs) are developed with culture medium that is supplemented with free fatty acids (FFAs). Nevertheless, the lipid barrier formation in HSEs remains altered compared to native human skin (NHS). The aim of this study is to decipher the role of medium supplemented saturated FFA palmitic acid (PA) on morphogenesis and lipid barrier formation in HSEs. Therefore, HSEs were developed with 100% (25 μM), 10%, or 1% PA. In HSEs supplemented with reduced PA level, the early differentiation was delayed and epidermal activation was increased. Nevertheless, a similar SC lipid composition in all HSEs was detected. Additionally, the lipid organization was comparable for lamellar and lateral organization, irrespective of PA concentration. As compared to NHS, the level of monounsaturated lipids was increased and the FFA to ceramide ratio was drastically reduced in HSEs. This study describes the crucial role of PA in epidermal morphogenesis and elucidates the role of PA in lipid barrier formation of HSEs.

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Evaluation of Two In Vitro Human Skin Equivalents (Epiderm™ and Skin[sup(2)]™ Model ZK1300) for Assessing the Skin Irritation Potential of Personal Care Products and Chemicals

Advances In Animal Alternatives For Safety And Efficacy Testing ◽

10.1201/9781439805817-40 ◽

1997 ◽

pp. 305-312

Keyword(s):

Human Skin ◽

Skin Irritation ◽

Personal Care Products ◽

Personal Care ◽

Skin Equivalents

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Apremilast interferes with the TGFβ1-induced transition of human skin fibroblasts into profibrotic myofibroblasts: in vitro study

Rheumatology ◽

10.1093/rheumatology/keaa249 ◽

2020 ◽

Vol 59 (12) ◽

pp. 3927-3938

Author(s):

Maurizio Cutolo ◽

Stefano Soldano ◽

Paola Montagna ◽

Giulia Martinelli ◽

Samuele Tardito ◽

...

Keyword(s):

Extracellular Matrix ◽

Protein Synthesis ◽

Human Skin ◽

Skin Fibroblasts ◽

Intracellular Signalling ◽

Pde4 Inhibitor ◽

Signalling Pathways ◽

Human Skin Fibroblasts ◽

Intracellular Signalling Pathways

Abstract Objectives Fibroblast-to-myofibroblast transition and extracellular matrix overproduction represent progressive events in chronic inflammatory and fibrotic diseases, in which TGFβ1 is one of the key mediators. Phosphodiesterase 4 (PDE4) acts as a proinflammatory enzyme through the degradation of cyclic adenosine monophosphate and it is overexpressed in skin fibroblasts. The study investigated how apremilast (a PDE4 inhibitor) interferes with the intracellular signalling pathways responsible for the TGFβ1-induced fibroblast-to-myofibroblast transition and profibrotic extracellular matrix protein synthesis. Methods Cultured human skin fibroblasts were stimulated with TGFβ1 (10 ng/ml) alone or combined with apremilast (1 and 10 μM) for 4, 16 and 24 h. Other aliquots of the same cells were previously stimulated with TGFβ1 and then treated with apremilast (1 and 10 μM) for 4, 16 and 24 h, always under stimulation with TGFβ1. Gene and protein expression of αSMA, type I collagen (COL1) and fibronectin were evaluated, together with the activation of small mothers against decapentaplegic 2 and 3 (Smad2/3) and extracellular signal-regulated kinase (Erk1/2) proteins. Results Apremilast reduced the TGFβ1-induced increase in αSMA, COL1 and fibronectin gene expression at 4 and 16 h, and protein synthesis at 24 h of treatment in cultured fibroblasts, even for cells already differentiated into myofibroblasts by way of a previous stimulation with TGFβ1. Apremilast inhibited the TGFβ1-induced Smad2/3 and Erk1/2 phosphorylation at 15 and 30 min. Conclusion Apremilast seems to inhibit in vitro the fibroblast-to-myofibroblast transition and the profibrotic activity induced by TGFβ1 in cultured human skin fibroblasts by downregulating Smad2/3 and Erk1/2 intracellular signalling pathways.

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