Study of the dermal-epidermal junction organization and mechanical properties by Atomic Force Microscopy
Nicolaÿ J-F, Valenti L, Markioli P-G, Runel G, Chlasta J. P-S4062
The dermal-epidermal junction (DEJ) is a complex and highly organized structure, which primary function is to anchor the epidermis to the dermis. The DEJ thus preserves skin integrity and participates to its mechanical properties. It also has an important metabolic role as it controls exchanges between epidermis and dermis (water, nutrients, growth factors,…). Atomic Force Microscopy (AFM) is an innovative technique that enables to determine DEJ molecular organization and mechanical properties (mechano organization al analysis). How some animals regenerate missing body parts is not well understood. Taking advantage of the zebrafish caudal fin model, we performed a global unbiased time-course transcriptomic analysis of fin regeneration. Biostatistics analyses identified extracellular matrix (ECM) as the most enriched gene sets. Basement membranes (BMs) are specialized ECM structures that provide tissues with structural cohesion and serve as a major extracellular signalling platform. While the embryonic formation of BM has been extensively investigated, its regeneration in adults remains poorly studied. We therefore focused on BM gene expression kinetics and showed that it recapitulates many aspects of development. As such, the re-expression of the embryonic col14a1a gene indicated that col14a1a is part of the regeneration-specific program. We showed that laminins and col14a1a genes display similar kinetics and that the corresponding proteins are spatially and temporally controlled during regeneration. Analysis of our CRISPR/Cas9-mediated col14a1a knockout fish showed that collagen XIV-A contributes to timely deposition of laminins. As changes in ECM organization can affect tissue mechanical properties, we analyzed the biomechanics of col14a1a-/- regenerative BM using atomic force microscopy (AFM). Our data revealed a thinner BM accompanied by a substantial increase of the stiffness when compared to controls. Further AFM 3D-reconstructions showed that BM is organized as a checkerboard made of alternation of soft and rigid regions that is compromised in mutants leading to a more compact structure. We conclude that collagen XIV-A transiently acts as a molecular spacer responsible for BM structure and biomechanics possibly by helping laminins integration within regenerative BM.
Exploration of biological and mechanical characteristics of a reconstructed dermal model
IFSCC (18-21 SEPTEMBER 2018)
Mariette V, Fernandez E, Runel G, Chlasta J, Marull-Tufeu S, Laperdrix C. P-S4048.
The 1970s saw the advent of cell culture methods, and the development of reconstructed dermal models, with the Bell’s model. Nowadays, full reconstructed skin models are highly developed. They reflect a large part of the biomechanical properties of the skin that are mainly explained by
the contractile power of fibroblasts (Bell et al, 1979). We interested in Bell’s model with fibroblasts conditioned by Transforming Growth Factor (TGFb1, 5 ng/ml). TGFb1 is known to stimulate cellular function and to improve contractile power of fibroblasts as it is implicated in healing process. Resistance and elasticity seem also to be improved but nothing was measured and described in literature. We decided to characterize mechanical properties of Bell’s model studying its behavior towards compression and decompression forces, representative of resistance and elasticity. Technics like indentation allowed us to measure the model hardness. Then, these physical results were conforted with microscopic measurements and observations using AFM technic. It highlighted cohesion forces between fibroblasts and the surrounding matrix, inside the dermal model. Finally, the study was completed by fibroblasts distribution observation within the matrix by two-photon microscopy.
Medaka fish as a good model to study skin aging by Atomic Force Microscopy
CENTRE DE RECHERCHE EN CANCÉROLOGIE DE LYON (CRCL)
Runel G, Lopez N, Cario-André M, Chlasta J, Masse I.
Studying skin aging in animal models, either to better understand age-related physiological changes or to find novel molecules that could act on aging, remains relatively long and costly. Fish could be appropriated models for this aim since skin structure is sufficiently similar to mammals one. Indeed, fish has been shown remarkably useful to investigate pigment biology (Schartl and Larue 2016). In particular, the Japanese Medaka, a small fish model with attractive experimental characteristics, could be of interest for studying aging since it accumulates over lifespan some biomarkers currently associated with aging features in others vertebrates. However, little is know about aging of the Medaka fish skin especially in its biomechanical properties and skin architecture. Our work aimed at studying Medaka skin aging at molecular, cellular and biomechanical levels.
Highlight the efficacy of your cosmetic products with Atomic Force Microscopy
Drillat A, Paillier C, Runel C, Chlasta J
Atomic Force Microscopy (AFM) is composed by a sharp tip attached on a flexible cantilever which is used to indent or to scan sample surface. When a known force is applied, the cantilever bends; this process is monitored with the use of a laser beam reflecting from the top of the cantilever into a photodetector. From the process mentioned above, we obtain force–displacement curves which give us accurate results about mechanical properties of the sample. It also provides probe displacement on the surface to generate high resolution imaging. By using high resolution surface imaging, traction force assay and Stiffness Tomography (ST), BioMeca has developing a non-invasive method to measure the mechanical properties of biological samples (skin, hair etc.. ): hydration, elasticity, ageing, UV radiation effects, blue light, etc..
Structural and biomechanical properties of a novel 3D microdermis model: the spheroid
Lorion C, Lopez-Gaydon A, Bonnet S, Drillat A, Milani P, Bechetoille N
Spheroids as microtissues are a powerful alternative to standard 2D cell culture for in vitro studies. 3D scaffold-free spheroids are formed within a few days from a cell suspension using hanging drop technology. The advantages of spheroids exclusively composed of fibroblasts rely on the physiological production of the extracellular matrix thanks to the aggregative capability of fibroblasts to self-assemble in a round tridimensional structure. The microdermis presents a complex tissue organization that closely mimics the architecture and composition of the human dermis in vivo. The aim of this study was to characterize structural and biomechanical properties of spheroids composed of normal human fibroblasts at 8 and 15 days of culture.
Application de la Microscopie à Force Atomique (AFM) à la biologie cutanée
Drillat A, Runel G, Chlasta J, Milani P
La peau constitue l’organe le plus étendu du corps humain. Elle joue un rôle de barrière, mais sert également à communiquer avec l’environnement extérieur. Elle est caractérisée par ses capacités d’élasticité et ses propriétés mécaniques qui lui sont propres et lui confèrent ses fonctions. La peau humaine est composée de plusieurs couches, chacune avec une structure et une fonction unique. Comprendre le comportement mécanique de ces différentes couches est important pour la recherche clinique et cosmétique, à titre d’exemple, pour le développement de produits de soins et la compréhension des affections cutanées.
Corneocytes nano-mechanical properties changes indicate alteration of the skin barrier