Introduction

Human skin has a stratified and very complex structure undergoing constant renewal. Each layer of the skin has different components. The dermis forms the thickest layer (from 50μm to 1,2mm) with a complex and a dense network of collagen and elastin fibers [1,2]. The various components of the skin have specific optical properties, which allows them to be distinguished. These properties also provide access to the composition of the tissue and its biochemical properties. The absorption and diffusion properties of the skin

depend on the scale of observation of these properties. These properties are not the same for the skin layers as for the elements it contains at the cellular level. The skin is known for its nonhomogeneous behavior due to the large number of cellular-scale elements it contains. For this reason, it is often considered that the optical properties of skin layers are those of optically homogeneous elements [3]. The epidermis has an absorption coefficient, which varies between 35 and 66cm^-1, and a diffusion coefficient, which varies between 450 and 800cm ^-1 for a wavelength between 415 and 633nm. The dermis is made up of a dense network of collagen and elastin fibers. Accordingly, diffusion of the dermis is described as multiple diffusion of these fibers. It varies between 187 and 320cm ^-1 for a wavelength between 415 and 633nm. However, the absorption coefficient varies between 1.9 and 4.7cm^-1 for a wavelength between 415 and 700nm [4].

Thanks to the optical properties of the skin (absorption, refraction and diffusion), which ensure its protective function against UV rays, and which provide information on the properties of its different layers, it is possible to characterize and identify the biochemical components of the skin, to quantify and locate them. The identification of these components makes it possible to understand their contribution to the structuring of the skin tissue, as well as the modifications they may undergo with age. Consequently, the modifications of the mechanical properties, which result from it. Optical techniques and measuring instruments have been developed for this such as Raman spectroscopy, diffuse reflectance, fluorescence [5], etc. In this study, we are interested in diffuse reflectance spectroscopy, which uses the scattering of light in the skin. It allows the analysis of backscattered light in a wide spectral range extending from 200 to 1000nm, and it provides information on fluorescence. This technique makes it possible to macroscopically characterize the tissues at a depth that varies from 2 to 5mm [5]. The skin has endogenous fluorescence due to the presence of fluorophore molecules such as aromatic amino acid rings or the presence of Advanced Glycosylation End-products (AGEs) [6,7]. These AGEs are markers of skin aging. Previous studies carried out on mouse skin and human dermis ex vivo [6,8,9] show, using the technique of diffuse reflectance spectroscopy, that the amplitude of the fluorescence of the advanced glycation end products increases with age. Interestingly, the accumulation of AGEs in human skin in vivo, related to age and body area, has not yet been addressed in the literature.

The present work proposes an experimental characterization of the biochemical and mechanical properties of the human skin in vivo for two body areas (the forearm and the thigh) with statistical analysis. The biochemical test is diffuse reflectance spectroscopy that allows measuring the fluorescence intensity of the AGEs. The mechanical experimental tests are non-contact impact tests performed to measure the Rayleigh wave propagation speed and to calculate the Young’s modulus. These methods are described and detailed in the section 2. The experimental results are presented in section 3, discussed, and analyzed in section 4. We finally conclude on the results obtained.

Materials and Methods

Volunteers

The tests were performed on the right forearms (12 cm above the wrist) and the left thighs (12cm above the knee) of 42 Caucasian French women volunteers divided into two groups: a young group [20-30] years old (21 volunteers) and an elderly group [45-55] years old (21 volunteers). Volunteers were non-smokers, in good health and they had healthy skin in the zones studied of the forearm and thigh without scars and tattoos. The volunteers had: a Body Mass Index (BMI) between 18.5 and 27 kg/m^², the cellulite index < 2 for the thigh, and the phototype between I and III.

The volunteers did not apply any cosmetic products on the body on the test day. After an acclimatization period of at least 10 minutes in an air-conditioned room (T = 21 ± 2 °C and H = 50 ± 10%), the tests were carried out in another room under the same conditions. The volunteer sat in an armchair (dentist type), her legs extended, uncrossed, and slightly bent, and her right arm resting on an armrest, the palm of her hand upwards. The volunteer was asked not to move for the duration of the measurements to ensure the homogeneity of the records and to reduce the level of noise in the data recorded as much as possible. All the volunteers participated after giving informed consent and all the procedures adhered to the latest revision of the Declaration of Helsinki.

Biochemical Properties of Skin: Diffuse Reflectance Spectroscopy (DRS)

In this study, we are interested to evaluate the biochemical modifications that collagen and elastin fibers can undergo with age due to the accumulation of AGEs in the fibers. In particular, the AGEs used as markers of skin aging studied here are Pepsin-Digestible Collagen Cross-Links (PDCCL), Collagenase-Digestible Collagen Cross-Links (CDCCL), and Elastin Cross-Links (ECL). We therefore use the fluorescence of the accumulation of these AGEs as a marker of the rate of aging and the rate of degradation of the fibers properties.

The study of the fluorescence of AGEs is carried out by diffuse reflectance spectroscopy, which is a quantitative method using the scattering of light in the skin. The spectrofluorometric used in this study is Jobin Yvon Fluorolog^® (17-FRA-QOT1389GFL3-LR) from HORIBA Scientific, France. It makes it possible to analyze backscattered light in a wide spectral range extending from 200 to 1000nm and to provide information on endogenous fluorescence in vivo on human skin. It is made up of three essential elements: An excitation source: 450 W continuous Xenon lamp white light (ozone-free), a distribution system: multimode optical fiber, and a signal acquisition and processing system: the R928 photomultiplier. A computer and SynerJY software controls the whole system. In vivo diffuse reflectance spectroscopy is performed by placing the fiber optic probe in contact with the skin area of interest (Figure 1). The acquisition of fluorescence intensity spectra of the skin can be done by recording the excitation spectrum or by recording the emission spectrum. The excitation spectrum is made by fixing the fluorescence emission length and sweeping the excitation wavelength. Whereas for the emission spectrum, it is made by fixing the excitation wavelength and sweeping the fluorescence emission wavelength. In this study, the excitation spectrum is the chosen method to measure skin fluorescence in vivo. Indeed, the excitation spectra are like the absorption spectra and the bands tend to be narrower than in the acquisition of emissions. This facilitates the identification of individual fluorophores in a complex spectrum as shown by Stamatas et al. [6]. We referred to the literature to identify the emission and excitation parameters suitable for carrying out our measurements [6,9]. The parameters used are shown in Table 1.

Figure 1: DRS measurement probe.

Table 1: Acquisition parameters of the fluorescence spectra where λ Excitation and λ Emission are respectively the excitation and emission wavelengths.

Mechanical Properties of Skin: Non-Contact Impact Test

The mechanical properties of skin were determined by the non-contact impact test. This test was performed using the WaveSkin ^® device developed in the Laboratory of Tribology and Dynamics of Systems (LTDS, Lyon, France) and described in Ayadh et al. [10]. Its principle is to mechanically generate an air flow and apply it onto the skin surface, then to measure the resulting displacement of the skin using a laser profilometer (l = 7 mm, 800 sensors, wavelength = 405nm). The pressure applied generates the propagation of a Rayleigh wave in the skin. The propagation speed of the wave is calculated by the method described in Ayadh et al. [10] and synthetized here. The minima of the displacement curve for each sensor and the time at which they are reached are identified. Therefore, it is possible to calculate the propagation speed of wave V such that: V = Δ

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