Full length articleA novel microstructural interpretation for the biomechanics of mouse skin derived from multiscale characterization
Graphical abstract
Introduction
The skin is a complex organ, with major physiological functions as regulation of the body temperature and hydration, and protection against physical aggressions and penetration of external agents. It is a multi-layered organ: starting from the outer surface, the skin comprises first the epidermis (a barrier made of epithelial cells), the dermis (a connective tissue that supports the epidermis) and finally the hypodermis (an adipose tissue). It is classically assumed that the dermis provides the main mechanical properties in stretching [1], [2], [3], although the other layers may be important for other loadings [4], [5], [6].
The dermis is a collagen-rich tissue, mostly composed of extracellular matrix with few cells inside. The matrix is a water solution of biomolecules, including proteoglycans, glycoproteins, and collagens, by far the most abundant protein. The collagen self-organizes into fibrils that further align into fibres in skin dermis. Dermal fibrils are heterotypic fibrils composed mainly of type I collagen, with other collagen types (as collagen V) as minor components. The dermis can then be viewed as a network of fibres (collagen as well as elastin) surrounded by an unorganized non-fibrillar matrix.
The macroscopic mechanical properties of the dermis are well-known [7], [8]. They are similar to the ones of other collagen-rich tissues, as tendon [9], cornea [10] or aorta [11]. First, they support large deformations (typically 50% for the skin, which is much larger than in other tissues) with a classical response in three parts. At low stretch, there is the toe-region, in which almost no force is recorded. Then, the force increases non-linearly in the heel-region, before reaching a linear part. At the end of this part, the tissue breaks. On top of this non-linear behaviour, the tissue exhibits viscous effects, not surprising for such hydrated material, with a modification of the force as a function of the loading rate, but also hysteresis loops if unloaded [12]. It may be noticed that repeated cyclic loadings lead also to a progressive shift of the response toward a limit cycle: it is the preconditioning effect [3], [13], [14], which can be compared to the Mullin’s effect in elastomers.
The relationship between these complex properties and the microstructure of the tissue has attracted a lot of theoretical attention, leading to the so-called “microstructural” models [2], [13], [15], [16]. Indeed, being able to predict the effects of an alteration of the microstructure on the macroscopic properties is of first interest for biological and medical applications. As examples, microstructural models have been used to predict the effects of surgical treatments [17] and the consequences of pathological or age-related alterations of the collagen [18].
The mechanical interpretation behind all these models is an alignment of the collagen fibres in the toe and heel regions, followed by the stretching of the fibres in the linear part [7], [19], [20]. However, only few papers have investigated experimentally this interpretation in skin, due to the difficulty to image the evolution of collagenous microstructure in this complex, disorganized medium. First qualitative observations were done with SEM [19], [20], using a new sample for each image. It is only recently that it has become possible to observe the collagenous microstructure on the same sample at all stretch levels [21]. This was permitted by the development of mechanical assays combined with multiphoton microscopy [9], [22], and in particular Second Harmonic Generation (SHG) microscopy. Thereby, we can now get three-dimensional images specific of the fibrillary collagen, without staining [23], [24], [25], [26]. This approach has been used very recently on skin [21], [27], but also on other tissues as aorta [22], [28], cornea [10], foetal membrane [29], [30] or liver capsule [31]. These observations have been challenging the classical interpretation of the link between microstructure and mechanical properties.
In this paper, we present a series of experimental results, probing the different aspects of the mechanical response of skin (uniaxial stretching, cyclic loadings, relaxation…), but also the reorganization of the microstructure during a stretching assay. We propose a new interpretation to explain all our observations in a single frame. We use this new interpretation in order to explain the consequences of an alteration of the collagen synthesis in genetically-modified mice.
Section snippets
Skin samples
The experiments were performed on ex vivo skin from one-month-old control (Wild Type, WT) mice (n = 25 under the multiphoton microscope and n = 7 outside) and from transgenic mice with a modified collagen V synthesis (n = 9 under the multiphoton microscope and n = 7 outside) from the same litters (referred to as K14-COL5A1).
All animal experiments were performed under animal care procedures and conducted in accordance with the guidelines set by the European Community Council Directives (86/609/EEC). All
Collagen network alignment
Microstructural observation of the collagen network in mouse dermis during a tensile test showed a progressive alignment of the collagen fibres over all the stretching. It is illustrated in Fig. 2, where we have plotted on the same graph the evolution of the stress versus stretch and the one of the OI, which is a quantitative scalar representing the fraction of collagen fibres aligned in the main direction. The OI behaviour versus stretch is very similar to the stress behaviour versus stretch,
Discussion
Classical microstructural explanation of the mechanical properties of the skin is based on an unfolding and alignment of the fibres in the heel region, followed by their stretching in the linear region [7]: each fibre brings an elastic linear stress/stretch response as soon as it is aligned in the direction of loading. For many years, only qualitative observations were possible [19], [20], and they indeed showed more aligned fibres for stretched skin than for resting ones.
Our observation of the
Conclusion
The complementary set of experiments carried out demonstrates that the classical model, in which the linear part of the J-shaped stress/stretch curve is attributed to the extension of aligned collagen fibres, is not appropriate for skin, although adequate for tendon-like connective tissues. In contrast, our multiscale experiments show that the fraction of fibres aligned in the direction of traction increases non-linearly with stretch and follows exactly the stress/stretch curve throughout the
Disclosures
We have no competing interest.
Acknowledgement
We thank Xavier Solinas, Jean-Marc Sintès and Vincent de Greef for technical implementation of the set-ups, Nadine Aguilera for breeding mice and maintaining transgenic strains (PBES, SFR Biosciences-Gerland, Lyon, France) and Isabelle Lamarre-Jouenne for technical support in skin handling. This work was supported by grants from Ecole Polytechnique (interdisciplinary project), from Agence Nationale de la Recherche – France (ANR-13-BS09-0004-02 and ANR-10-INBS-04).
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