A novel microstructural interpretation for the biomechanics of mouse skin derived from multiscale characterization

Abstract

Skin is a complex, multi-layered organ, with important functions in the protection of the body. The dermis provides structural support to the epidermal barrier, and thus has attracted a large number of mechanical studies. As the dermis is made of a mixture of stiff fibres embedded in a soft non-fibrillar matrix, it is classically considered that its mechanical response is based on an initial alignment of the fibres, followed by the stretching of the aligned fibres. Using a recently developed set-up combining multiphoton microscopy with mechanical assay, we imaged the fibres network evolution during dermis stretching. These observations, combined with a wide set of mechanical tests, allowed us to challenge the classical microstructural interpretation of the mechanical properties of the dermis: we observed a continuous alignment of the collagen fibres along the stretching. All our results can be explained if each fibre contributes by a given stress to the global response. This plastic response is likely due to inner sliding inside each fibre. The non-linear mechanical response is due to structural effects of the fibres network in interaction with the surrounding non-linear matrix. This multiscale interpretation explains our results on genetically-modified mice with a simple alteration of the dermis microstructure.

Statement of Significance

Soft tissues, as skin, tendon or aorta, are made of extra-cellular matrix, with very few cells embedded inside. The matrix is a mixture of water and biomolecules, which include the collagen fibre network. The role of the collagen is fundamental since the network is supposed to control the tissue mechanical properties and remodeling: the cells attach to the collagen fibres and feel the network deformations. This paper challenges the classical link between fibres organization and mechanical properties. To do so, it uses multiscale observations combined to a large set of mechanical loading. It thus appears that the behaviour at low stretches is mostly controlled by the network structural response, while, at large stretches, the fibre inner-sliding dominate.

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.