scholarly journals Multitargeted Approach for the Optimization of Morphogenesis and Barrier Formation in Human Skin Equivalents

2021 ◽  
Vol 22 (11) ◽  
pp. 5790
Author(s):  
Arnout Mieremet ◽  
Richard W. J. Helder ◽  
Andreea Nadaban ◽  
Walter A. Boiten ◽  
Gert S. Gooris ◽  
...  
Keyword(s):  
Human Skin ◽  
Three Dimensional ◽  
Skin Barrier ◽  
Collagen Matrix ◽  
Optimization Approach ◽  
Skin Equivalents ◽  
Barrier Formation ◽  
Epidermal Morphogenesis

In vitro skin tissue engineering is challenging due to the manifold differences between the in vivo and in vitro conditions. Yet, three-dimensional (3D) human skin equivalents (HSEs) are able to mimic native human skin in many fundamental aspects. However, the epidermal lipid barrier formation, which is essential for the functionality of the skin barrier, remains compromised. Recently, HSEs with an improved lipid barrier formation were generated by (i) incorporating chitosan in the dermal collagen matrix, (ii) reducing the external oxygen level to 3%, and (iii) inhibiting the liver X receptor (LXR). In this study, we aimed to determine the synergic effects in full-thickness models (FTMs) with combinations of these factors as single-, double-, and triple-targeted optimization approaches. The collagen–chitosan FTM supplemented with the LXR inhibitor showed improved epidermal morphogenesis, an enhanced lipid composition, and a better lipid organization. Importantly, barrier functionality was improved in the corresponding approach. In conclusion, our leading optimization approach substantially improved the epidermal morphogenesis, barrier formation, and functionality in the FTM, which therefore better resembled native human skin.

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

2019 ◽  
Vol 20 (23) ◽  
pp. 6069 ◽  
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.

scholarly journals OP0195 VASCULARIZED THREE-DIMENSIONAL MODELS OF HUMAN SKIN FIBROSIS

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

scholarly journals Human skin equivalents cultured under hypoxia display enhanced epidermal morphogenesis and lipid barrier formation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Arnout Mieremet ◽  
Adela Vázquez García ◽  
Walter Boiten ◽  
Rianne van Dijk ◽  
Gert Gooris ◽  
...  
Keyword(s):  
Human Skin ◽  
Skin Equivalents ◽  
Barrier Formation ◽  
Epidermal Morphogenesis

scholarly journals Loss of S100A9 (MRP14) Results in Reduced Interleukin-8-Induced CD11b Surface Expression, a Polarized Microfilament System, and Diminished Responsiveness to Chemoattractants In Vitro

2003 ◽  
Vol 23 (3) ◽  
pp. 1034-1043 ◽  
Author(s):  
Marie-Pierre Manitz ◽  
Basil Horst ◽  
Stephan Seeliger ◽  
Anke Strey ◽  
Boris V. Skryabin ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Inflammatory Diseases ◽  
Three Dimensional ◽  
Collagen Matrix ◽  
Interleukin 8 ◽  
In Vivo Models ◽  
Migration Assay ◽  
Microfilament System

ABSTRACT The S100A9 (MRP14) protein is abundantly expressed in myeloid cells and has been associated with various inflammatory diseases. The S100A9-deficient mice described here were viable, fertile, and generally of healthy appearance. The myelopoietic potential of the S100A9-null bone marrow was normal. S100A8, the heterodimerization partner of S100A9 was not detectable in peripheral blood cells, suggesting that even a deficiency in both S100A8 and S100A9 proteins was compatible with viable and mature neutrophils. Surprisingly, the invasion of S100A9-deficient leukocytes into the peritoneum and into the skin in vivo was indistinguishable from that in wild-type mice. However, stimulation of S100A9-deficient neutrophils with interleukin-8 in vitro failed to provoke an up-regulation of CD11b. Migration upon a chemotactic stimulus through an endothelial monolayer was markedly diminished in S100A9-deficient neutrophils. Attenuated chemokinesis of the S100A9-deficient neutrophils was observed by using a three-dimensional collagen matrix migration assay. The altered migratory behavior was associated with a microfilament system that was highly polarized in unstimulated S100A9-deficient neutrophils. Our data suggest that loss of the calcium-binding S100A9 protein reduces the responsiveness of the neutrophils upon chemoattractant stimuli at least in vitro. Alternative pathways for neutrophil emigration may be responsible for the lack of any effect in the two in vivo models we have investigated so far.

scholarly journals In vitro rapid organization of endothelial cells into capillary-like networks is promoted by collagen matrices.

1983 ◽  
Vol 97 (5) ◽  
pp. 1648-1652 ◽  
Author(s):  
R Montesano ◽  
L Orci ◽  
P Vassalli
Keyword(s):  
Endothelial Cells ◽  
Three Dimensional ◽  
Collagen Matrix ◽  
Collagen Gels ◽  
Collagen Matrices ◽  
Narrow Lumen ◽  
Capillary Endothelial Cells ◽  
Vitro Model

We have studied the behavior of cloned capillary endothelial cells grown inside a three dimensional collagen matrix. Cell monolayers established on the surface of collagen gels were covered with a second layer of collagen. This induced the monolayers of endothelial cells to reorganize into a network of branching and anastomosing capillary-like tubes. As seen by electron microscopy, the tubes were formed by at least two cells (in transverse sections) delimiting a narrow lumen. In addition, distinct basal lamina material was present between the abluminal face of the endothelial cells and the collagen matrix. These results showed that capillary endothelial cells have the capacity to form vessel-like structures with well-oriented cell polarity in vitro. They also suggest that an appropriate topological relationship of endothelial cells with collagen matrices, similar to that occurring in vivo, has an inducive role on the expression of this potential. This culture system provides a simple in vitro model for studying the factors involved in the formation of new blood vessels (angiogenesis).

scholarly journals Characterization of Fibroblasts with Son of Sevenless-1 Mutation

2006 ◽  
Vol 85 (11) ◽  
pp. 1050-1055 ◽  
Author(s):  
E.J. Lee ◽  
S.I. Jang ◽  
D. Pallos ◽  
J. Kather ◽  
T.C. Hart
Keyword(s):  
Cell Proliferation ◽  
Three Dimensional ◽  
Collagen Matrix ◽  
Histological Assessment ◽  
M Phase ◽  
Son Of Sevenless ◽  
Gingival Fibromatosis

Although non-syndromic hereditary gingival fibromatosis (HGF) is genetically heterogeneous, etiologic mutations have been identified only in the Son of Sevenless-1 gene ( SOS1). To test evidence of increased cell proliferation, we studied histological, morphological, and proliferation characteristics in monolayer and three-dimensional cultures of fibroblasts with the SOS1 g.126,142–126,143insC mutation. Histological assessment of HGF gingiva indicated increased numbers of fibroblasts (30%) and increased collagen (10%). Cell proliferation studies demonstrated increased growth rates and 5-bromo-2-deoxyuridine incorporation for HGF fibroblasts. Flow cytometry showed greater proportions of HGF fibroblasts in the G2/M phase. Attachment of HGF fibroblasts to different extracellular matrix surfaces demonstrated increased formation of protrusions with lamellipodia. HGF fibroblasts in three-dimensional culture showed greater cell proliferation, higher cell density, and alteration of surrounding collagen matrix. These findings revealed that increased fibroblast numbers and collagen matrix changes are associated with mutation of the SOS1 gene in vitro and in vivo.

Intracellular diaphragmed fenestrae in cultured capillary endothelial cells

1988 ◽  
Vol 89 (3) ◽  
pp. 441-447 ◽  
Author(s):  
R. Montesano ◽  
L. Orci
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Three Dimensional ◽  
Collagen Matrix ◽  
Cell Cytoplasm ◽  
Experimental Conditions ◽  
Intracellular Location ◽  
Capillary Endothelial Cells

The endothelium of visceral capillaries is characterized by the occurrence of numerous fenestrae, which are usually bridged by a thin, single-layered diaphragm. Both in vivo and in vitro, diaphragmed fenestrae perforate the endothelial cell cytoplasm in the most attenuated regions of the cell. We report here that in capillary endothelial cells grown under experimental conditions promoting the development of intracellular lumina (for example, suspension within a three-dimensional collagen matrix), diaphragmed fenestrae can form in a unique, previously undescribed intracellular location - that is, within thin cytoplasmic septa separating contiguous luminal compartments.

Ultrastructural characterization of an in vitro model of human epidermis: its potential for sulfur mustard study

1995 ◽  
Vol 53 ◽  
pp. 1004-1005
Author(s):  
J.P. Petrali ◽  
P.T. Lopolito ◽  
T.A. Hamilton ◽  
S.B. Oglesby-Megee ◽  
L.S. Rhoads
Keyword(s):  
Human Skin ◽  
Sulfur Mustard ◽  
Cellular Models ◽  
Skin Equivalents ◽  
Information Gap ◽  
Ultrastructural Characterization ◽  
Vitro Model ◽  
Membrane Components

Morphological correlates of sulfur mustard toxicity continue to be studied in in vivo models such as the haired and hairless guinea pig and the domestic weanling pig. These animal models provide useful evidence for the mechanisms of sulfur mustard-induced lesions, however the correlation with human skin is not exact and the availability of a continuing supply of any animal model is problematic. In vitro studies have largely been performed with cultured monotypic human cells such keratinocytes and lymphocytes. Although monotypic cellular models have added data to the mechanisms of the observed cytopathology, the acclimatized nature of media-nourished cells may present a different phenotype for study than in vivo. To bridge the information gap between monolayers of cells and in vivo studies, in vitro skin equivalents have been proposed as suitable models. We have used several commercially available human skin equivalents in the past, however the lack of basement membrane components, the absence or paucity of hemidesmosomes, general problems with replication and inconsistences of availability, question the usefulness of commercial preparations at this time.

Reconstructed Human Epidermis In Vitro: An Alternative to Animal Testing

1995 ◽  
Vol 23 (1) ◽  
pp. 97-110 ◽  
Author(s):  
Maria Ponec
Keyword(s):  
Human Skin ◽  
Three Dimensional ◽  
Structural Features ◽  
Animal Testing ◽  
Normal Human Skin ◽  
Normal Human ◽  
Biochemical Mechanisms ◽  
Human Skin Models

Various three-dimensional human skin models, in which the epidermis exhibits in vivo-like morphological and functional characteristics, have recently been developed. Such models are currently being used to study the development and physiology of the skin, the processes involved in wound healing, and the reactivity of skin to environmental and chemical insults. Since these models reproduce to a large extent the barrier function properties of normal human skin, they can be used for screening potential skin irritants. These substances can be applied topically and their irritant potential can be evaluated using various endpoints, such as the induction of tissue damage or the release of various pro-inflammatory mediators. Studies with human skin equivalents can therefore contribute to our knowledge of the basic biochemical mechanisms underlying irritant reactions, and can be used to understand the structural features of molecules which may be responsible for eliciting an irritant reaction. In addition”, the generation of epidermal equivalents populated with melanocytes, as well as keratinocytes, makes it possible to study the regulation of melanogenesis, melanocyte–keratinocyte interactions, and how these are affected by UV irradiation. Such a model can also be used for testing the phototoxic or photoprotective potentials of various compounds and sunscreens.

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