In vivo confirmation of hydration-induced changes in human-skin thickness, roughness and interaction with the environment

The skin properties, structure, and performance can be inﬂuenced by many internal and extemal factors, such as age, gender, lifestyle, skin diseases, and a hydration level that can vary in relation to the environment. The aim of this work was to demonstrate the multifaceted inﬂuence of water on human skin through a combination of in vivo confocal Raman spectroscopy and images of volar—foream1 skin captured with the laser scanning confocal microscopy. By means of this pilot study, the authors have both qualitatively and quantitatively studied the inﬂuence of changing the depth-dependent hydration level of the stratum corneum (SC) on the real contact area, surface roughness, and the dimensions of the primary lines and presented a new method for characterizing the contact area for different states of the skin. The hydration level of the skin and the thickness of the SC increased signiﬁcantly due to uptake of moisture derived from liquid water or, to a much lesser extent, from humidity present in the environment. Hydrated skin was smoother and exhibited higher real contact area values. The highest rates of water uptake were observed for the upper few micrometers of skin and for short exposure times. © 2016 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http:// creativecommons.org/licenses/by/4.0/). [httpz//dx.doi.org/10.1116/1.4962547]

The skin properties, structure, and performance can be influenced by many internal and extemal factors, such as age, gender, lifestyle, skin diseases, and a hydration level that can vary in relation to the environment. The aim of this work was to demonstrate the multifaceted influence of water on human skin through a combination of in vivo confocal Raman spectroscopy and images of volar-foream1 skin captured with the laser scanning confocal microscopy. By means of this pilot study, the authors have both qualitatively and quantitatively studied the influence of changing the depth-dependent hydration level of the stratum corneum (SC) on the real contact area, surface roughness, and the dimensions of the primary lines and presented a new method for characterizing the contact area for different states of the skin. The hydration level of the skin and the thickness of the SC increased significantly due to uptake of moisture derived from liquid water or, to a much lesser extent, from humidity present in the environment. Hydrated skin was smoother and exhibited higher real contact area values. The highest rates of water uptake were observed for the upper few micrometers of skin and for short exposure times. © 2016 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http:// creativecommons.org/licenses/by/4.0/). [httpz//dx.doi.org/10.1116/1.4962547] l. INTRODUCTION 1 Skin is our protective armor in everyday life and our primary interface with the environment. It has an area of some 2m2, and is thus the largest single organ in the human body. Human skin is a multilayer structure, consisting of the ")Electronic mail: agnieszka.dabroWska@empa.ch epidermis, being the outer layer of the skin, mostly exposed to the extemal factors, dermis, responsible for, inter alia, flexibility and durability of the skin and subcutaneous tissue, acting as additional insulation and mechanical protection.2_4 One of the main functions of skin is to protect the body from extemal factors, such as mechanical injuries, extremes of temperature and radiation, as well as the transport of various substances. " The barrier function of skin is mostly provided by the stratum corneum ("horny layer," SC).7_9 This thin layer, reaching a thickness of 15-20 pm at the volar forearm, is the most external of the sublayers of epidermis. L7 Keratinocytes, comprising about 85% of the epidermis, migrate through the sublayers of the epidermis, gradually transfonning to horny cells by changing their size, shape, composition, and losing their nuclei. The SC consists of non-nucleated and flat cells named corneocytes.'O'1' According to the "brick and mortar" model, comeocytes are described as bricks surrounded by lipid bilayers as the mortars The hydration level of the SC can vary depending on environmental conditions, as comeocytes can take up water until the hydration level of the SC is in equilibrium with the environment." The hydration level of the SC is responsible for the physiology and homeostasis of the skin." The examples of the importance of hydration on the functions and properties of the skin are its influence on the mechanical toughness of skin, its barrier functions, and the regulation of enzyme activity.7'"H6 As suggested by Egawa, daily routines can lead to visible changes in the skin." Even a very short exposure to water, such as washing hands for 2min, is enough to hydrate the SC disjunctum while a bath can contribute to changes in the SC conjunctum, and thus, the influence of the environment on the properties of the skin are essential factors to be taken into account when studying skin-materials interactions.18'l9 Moreover, seasonal changes in humidity are an important factor influencing the skin.20 There is a variety of techniques that allow both in vivo and in vitro determination of the hydration level of skin, based on different principles, including the chemical analysis and electrical methods.2l_25 For the purposes of this paper, we have chosen confocal Raman spectroscopy as a noninvasive, depth-resolved method that provides quantitative information concerning the skin's hydration level.
The results extracted from the confocal Raman spectroscopy provide information on hydration and its variation with depth, the SC thickness, and also, in combination with information on the real contact area of skin with the Raman instrument, inferences about the interaction of skin with other objects. 9'15'26'3O Given that skin is able to take up water, it is reasonable to assume that water should also change its morphology and surface properties. In order to analyze these changes over time, we have employed 3D laser scanning microscopy, which allowed us to observe the surface of a skin replica under high magnification and provided 3D information. 3'_33 In this paper, we present a pilot study focused on global changes in the appearance and properties of human skin caused by exposure to water or humid conditions. In order to investigate the multifaceted response of skin to water we have examined the hydration level of the superficial stratum corneum (SSC), being the surface of skin, depth profiles of skin before and after exposure to external sources of water, the skin's water uptake abilities, the SC thickness, the real contact area against smooth CaF2, the skin's surface roughness, and the evolution of the dimensions of the primary lines. Biointerphases, Vol. 11, No. 3, September 2016 ll. EXPERIMENTAL SETUP AND METHODOLOGY A. Instrumentation

Confocal Raman spectroscopy
The hydration level of skin was determined from in vivo Raman spectra that were acquired using an inverted confocal Raman spectrometer equipped with a 60>< oil immersion objective, skin composition analyzer (SCA), model 3510 (RiverD, Rotterdam, the Netherlands). The depth profiles were measured in 2 pm steps, from the surface of the skin to a depth of 60 /.im. Laser excitation with a wavelength of 67lnm (laser power 19.5 i 1.8 mW) was used for ls in order to obtain spectra in the region of 2550-4000 cm7l, providing information about the amount of water and proteins in the skin.16 '34'35 The z-resolution of the optical setup was determined to be 4.7 pm by placing a water droplet on the CaF2 window of the inverted microscope and fitting the slope of the Raman signal at the CaF2/water interface with a Lorentz function, taking the full width at half maximum. To account for the known discrepancy between the true confocal depth and mechanical displacement of the optical table,36 all depth profiles were corrected with the depth-correction factor fdepm : 1.06. fdepth was determined by comparing the confocal thickness of a NIST polystyrene standard film with its true thickness. Therefore, the film was placed onto the CaF2 window with an additional water contact layer of approx. Five micrometers and the Raman signal of polystyrene between 3040 and 3076 cm71 was followed in steps of 0.5 um. The true thickness of the polystyrene film was determined to be 52.7 um as calculated from the infrared transmission interference pattern in the 3200-3600 cm71 wavenumber region (refractive index npolystyrene : 1.59).

Three dimensional laser scanning confocal microscopy
The surface morphology of polyvinylsiloxane (Profil novo light type 3, Heraeus Kulzer GmbH, Hanau, Germany) skin replicas was observed by means of a 3D Laser Scanning Confocal Microscope, model VK-X250 (Keyence, Osaka, Japan), using a violet laser with a wavelength of 408 nm (maximum laser power: 0.95 mW).

B. Measurements
The single-person pilot study was performed on a healthy, left-handed Caucasian woman aged 26 with a body mass index of 21. For at least 48 h before the measurements, the skin was not treated with moisturizers and heavy exercises were avoided. All measurements were performed on the left arm. For simplicity, the skin before water/humidity exposure is tenned "dry," whereas the skin exposed to an external source of water is termed "hydrated." After exposure to water/humidity, the forearm was immediately placed on the CaF2 acquisition window of the Raman instrument and maintained in this position throughout the entire measurement. After each depth profile, the lateral position of the laser was changed between 0.2 and 2.0 mm and another = = ; 031015-3 Dabrowska et aI.: In vivo confirmation of hydration-induced changes in human-skin thickness 031015-3 Fro. 1. (a) Forearm exposed to water with the use of patch test chambers.
(b) Forearm inside the humidity box.
depth profile was collected. The measurements, consisting of ten depth profiles collected at ten different positions on the volar forearm, were repeated three times for each exposure time/set of conditions.

Influence of water on skin hydration
Hydration levels of skin under atmospheric conditions were measured at four different points that were equally distributed along a line from about 7 cm from the wrist up to the elbow [ Fig. 1(a)]. Then, the defined measuring points were exposed to water using patch-test chambers (Van der Bend, Brielle, the Netherlands) filled with 20 /.tl of distilled water. Exposure time was varied from 2 to 60 min. After unsticking the patch, excess water was removed with a paper towel.

Influence of water vapor on skin hydration
Hydration levels of skin under atmospheric conditions as well as after exposure to air at a defined humidity were measured midway between the wrist and the elbow. To expose skin to an atmosphere at a specified humidity, the left arm was placed in a purpose-built PMMA humidity chamber (Bi -' --ar ean o r ma zednenst y l a. u. 2600 2a00 3000 3200 3400 3600 3a00 2600 2000 0000 3200 3400 3600 aaoo with the dimensions of 60 >< 50 >< 30cm for 1 h [ Fig. 1(b)]. Saturated NaCl solution or a travel air humidifier, both assisted by a fan to homogenize the air inside the chamber, were used in order to obtain a relative humidity (RH) of 70% or 90%, respective1y.37'38

Surface morphology
Polyvinylsiloxane replicas of the skin taken before and after 2-60 min of exposure to water (according to the same procedures as explained above) were prepared in triplicate. The change in surface morphology due to exposure to water was investigated by means of a 3D laser scanning confocal microscope. Each replica was analyzed in three different spots using the 20>< objective lens.
C. Data processing 1. Hydration level Hydration levels of skin were automatically determined from the Raman spectra using SKIN TOOLS 2.0 software (RiverD, Rotterdam, the Netherlands), where the water content is calculated relative to keratin, based on the integrals of OH-vibration signals (W) in the range of 3350-3550 cm_1 and the integrals of the relevant CH-vibration signals (2910-2966 cm_l), P),l6'2l' 34'39 using the relation hydration level (%) : >< 100% according to Caspers et al.,2l where R = 2 is a proportionality constant that was obtained by calibrating against protein solutions. To determine the integrals W and P, Raman spectra were baseline corrected with a first-order polynomial fitted through the spectral regions 2580-2620 and 3780-3820 cm71, see also Fig. 2. Both the absolute hydration level and the water uptake, understood as the difference between the hydration level before and after exposure to water, were taken into consideration in further investigations.
(b) as % ar ean o r ma zed ' nt ens 0; l a. u.
Raman shift I cm'1 Fro. 2. Typical baseline corrected Raman spectra and hydration levels of the superficial stratum comeum at 0 ,um (a) and of the viable epidermis at 40 ,um (b) captured before (dry, red) and after lh exposure to water (wet, blue), Hydration levels were determined based on the ratio of protein and water vibrations (shaded areas). The difference of 3% in (b) reflects the uncertainty of skin hydration due to local variation of the hydration level.  Biexponential fitting was applied to the data of timedependent change in the hydration level of superficial stratum corneum according to where hl(t) and hlo are the respective hydration levels at time t and at t: 0 min. The variables hf and D are the hydration factors in percentage and the hydration rate coefficient in min_1 for the two exponential functions A and B, see also Fig. 3.

Contact area
We propose a new noninvasive in vivo method to measure the influence of hydration on the contact area between skin and other objects. For each spectrum, an image of the contact between the skin and the CaF2 acquisition window of the SCA was taken. Based on the images corresponding to each spectrum, the contact area could be calculated by means of CORELDRAW X6 software, supported with the Getarea macro.

Thickness of the stratum corneum
As proposed by Crowther, the thickness of the SC can be determined from each water profile by fitting with a Weibull curve. 16'34'40 This was performed with MATLAB (The Mathworks, Natick, MA).

Surface morphology
The surface-roughness parameters, Sa, Sz, and the characteristic dimension of the profiles, were extracted with VK-HlXMEI VK-X A1-Analyzer software, each measurement being the average of three profiles with the interval of 20 um of replicas. Each image was inverted, and artifacts as well as characteristic features, such as sweat glands, hair follicles, etc., were not considered in further data processing. Biointerphases, Vol. 11, No. 3, September 2016 lll. RESULTS A. Raman spectra of the stratum corneum and viable epidermis before and after exposure to water Raman spectra of skin show characteristic features depending on the sampling depth and the preconditioning of the skin (Fig. 2). For the dry skin, the Raman spectrum captured at the SSC (0 pm depth) level shows strong CHvibration signals that are characteristic of proteins (2930cmTl) and lipids (2850 and 2880 cmil), as well as weak OH-vibration signals (3350-3550 cm_l) characteristic of water [ Fig. 2(a)]. This is in contrast to the spectrum captured in the VE (40 um depth) [ Fig. 2(b)], where the strong lipid signals are absent, and the OH-signals are stronger, corresponding to a higher water content. After lh exposure to water, the spectrum captured at the SSC level shows a strong water peak [ Fig. 2(a)] while no such significant change was observed in the VE-level spectrum [ Fig. 2(b)].
B. Environmentally dependent changes in the structure and properties of human skin 1. Hydration level of the stratum corneum Figure 3 shows the influence of the environment on the hydration level at the surface of the skin, at 0,um depth (SSC). It can be clearly seen that water exposure influenced the hydration level of the SSC to a far higher extent than was observed for the relative humidity up to 90%. The level of hydration gradually increased from 26.2 i 3.4% to 60.2 i 7.0% after 60 min of water exposure. The extent of the forced hydration was significant, especially for short exposure time (up to 5 min).
The increase in RH from 40% to 90% contributed toward an increase in the hydration level of the SSC from 24.7 i 3.5% to 27.6 i 3.8% after 60 min. In comparison, a significantly higher hydration level (35.2 i 5.0%) was observed after just 2min exposure to liquid water. Consistent with the abovementioned phenomenon, it was also clear from the depth profiles of water content [ Fig. 4(a)] that the hydration level in the SSC increased most significantly after exposure to water. In addition to the results presented in Fig. 3, the depth profiles show that the largest change in hydration level caused by external factors could be observed within the first 10/rm. The hydration level of the dry skin varied with depth from 26.2 i 3.4% in the SSC to 70.3 i 2.9% in the VE at a depth of 50 um. The depth profiles before and after exposure to the external source of water coincide for deeper layers of the skin. In order to show this effect even more clearly, the hydration levels measured at different depths of the skin from 0 to 10 um were plotted as a function of the exposure time to water [ Fig. 4(b)]. As presented in the graph, the influence of the exposure time to water on the hydration level was more marked for the (initially drier) outer layers of the skin. Water uptake, being the difference between the hydration level of the skin before and after water exposure for different depths of the skin, is presented in Fig. 5. Confirming the effect visible on Fig. 4, Fig.  5 presents the uptake of water at different depths of the skin when the skin was exposed to water for 2, 30, and 60 min. It is clearly visible that the deeper the layer of skin that was investigated, the lower was the uptake of water. The hydration rate also decreased with the exposure time. Considering the surface of the skin, the uptake of water after 2min of exposure was 9.03 i 6.03%, changing to 26.53 i 7.47% after 30 min and 34.02 1" 8.95% after 60 min.

Evolution of the contact area
The real contact area, which corresponds to the direct contact between skin and the CaF2 acquisition window, can be recognized as dark areas in Figs. 6(a)-6(d), which were captured within the first few seconds after the forearm was placed on the CaF2 window. The rapid acquisition was necessary in order to avoid the influence of sweating and relaxation process and to show the clear influence of the external source Biointerphases, Vol. 11, No.3, September 2016 of water on the contact area. Apparent contact area is defined as the area of the apparent contact between the skin and the CaF2 window, which itself had an area of 70 734 ,um2.
From the very low real contact area values, it is clear that dry skin has little direct contact area with the CaF2 window values [ Fig. 6(a)]. This is due to its roughness and limited elasticity. After only 2min exposure to water, there was a significant increase in the real contact area [ Fig. 6(b)]. Longer exposures, such as 30 [ Fig. 6(0)] or 60 min [ Fig. 6(d)], did not lead to a significantly greater real contact area value.
The real/apparent contact area ratio for the dry skin (hydration level: 26.2 i 3.4%, as shown in Fig. 2) had a value in the range of 36% [Figs. 7(a) and 7(b)]. Once the skin was exposed to water, the contact area significantly increased, and the real/apparent contact area ratio (hydration level 35.2 i 5.0%) reached 73% after 2 min exposure. The values of the real contact area and the real/apparent contact area ratio for skin exposed to water for 2-60 min were comparable.
In agreement with other analyses, the influence of humidity on the contact area was lower than it was measured for water. Fro. 6. Evolution of the skin/CaF2 acquisition window contact area before (a) and after 2 (b), 30 (c), and 60 min (d) of water exposure.
The real/apparent contact area ratio gradually increased from 35% for 40% RH to 48% for 90% RH.

Thickness of the stratum corneum
As presented above, the hydration measurements showed that human skin absorbs water from the environment. To confinn this phenomenon, the measurements of the change in the structure and morphology of human skin were performed. Figure 8 presents the influence of water absorption on the thickness of the SC. When the skin was exposed to water, the thickness of the SC, determined based on the Raman spectra, increased linearly with increasing exposure time. The SC thickness increased from 17.5 i 2.5 um measured for skin before water exposure to 21.2 i 3.0 um after 60 min exposure to water. Exposure to humidity influenced the SC thickness to a smaller extent, causing an increase from 17.2 i 2.0 um for 40% RH to 18.5 i 2.4 um measured for the skin exposed to 90% RH for 60 min. Rea /a 0 Skin exposed to water 10 -ISkin exposed to humidity  after 60 min water exposure [ Fig. 10(c)]. The depth of the primary lines also decreased, reducing from 44.5 i 9.8 pm for the dry skin to 20.5 i 10.2 um for skin exposed to water [ Fig. 10(d)].

IV. DISCUSSION
In the present study, we were able to demonstrate how hydration conditions influence human skin on several levels. Under the dry conditions, human skin can be considered as a rough material} Dry SC is characterized by high values of Young's modulus, reaching into the GPa range.4'_44 Therefore, as a rough and not easily deformable material, characterized by wide and deep primary lines," human skin in a dry state shows limited real contact area with the Cal-72 window. The hydration level of the dry skin increased with the depth of the measurement, exhibiting the lowest value for the SC, consisting of the dead and shriveled corneocytess and the highest value for VE. 16'35'40'45 The natural variation of the water content at different depths of the skin is the explanation for a clear difference in the ratio between protein and water peaks in Raman spectra for the SC and VE of the dry skin.
The hydration state has a major influence on the performance of the skin. As can be seen in the Raman spectra, Biointerphases, Vol. 11, No. 3, September 2016 when the skin was exposed to water for 60 min, the ratio between the protein and water peaks for the SSC changed drastically due to water uptake. However, the exposure to water did not influence the VE.35 The depth profiles also confirmed that water can only influence the surface of the skin and showed that, below a certain depth, there was no difference between the dry and hydrated skin. This behavior can be explained by the barrier function of the SC, as the threshold depth corresponds to the location of the lower SC, known to act as a barrier layer for water.4649 Another threshold can be observed, suggesting that the skin was more accessible to penetration of water, and that it occurred faster at depths for the first few micrometers of the SC. The time dependent change of the hydration level of the SSC (Fig. 3) fits well to a biexponential model with a fast and slow hydration rate coefficient DA = 0.31 min" and DB = 0.52 >< 10* min".
This could indicate at least two different water diffusion mechanisms as expressed in the literature by multilayer or multicompartment skin models and the finding of true fonnation of water pools within the SC.5O_52 This observation is consistent with a statement by Loth that the transport of water within the SC disjunctum takes place through spreading into the intercellular space due to the capillary forces, whereas the much slower and less straightforward water transport within the SC  conjunctum is based only upon diffusion. l8'53'55 This also explains the obseryation that the closer to the surface of the skin, the faster and more significant is the water uptake as well as the fact that even a short exposure to water (2 min) caused appreciable changes in the SSC. As the SC becomes hydrated, it is no longer stiff and rough. Due to the plasticizing effect of water, the Young's modulus of the SC may decrease by as much as 3 orders of magnitude.42 '44 The dimensions of the primary lines decrease, making the surface of the skin smoother. The softer and smoother skin results in a significantly higher real contact area with the CaF2 window. The uptake of water by corneocytes not only makes the main furrows shallower but also leads to an increasing thickness of the SC.'6'35 Water diffusion requires space and therefore leads to physical expansion, i.e., swel1ing.51 '52 For all investigated parameters, an increase in relative humidity had a minor influence on the skin compared to the direct contact with water. Skin hydrated through the exposure to humid conditions followed the same tendencies as the skin hydrated through the direct exposure to water, but to a much lesser extent. In the case of our experiments, the humid conditions can be compared with the amount of water in the air equal to 13.6 g/m3 for the RH = 70% and 19.6 g/m3 rsi the RH = 90%."-5* The observed effects caused by water on the skin are summarized in Fig. 11.

V. CONCLUSIONS
In conclusion, although still consisting of the same cells and chemical components, the hydrated skin can be perceived as a material with significantly different properties than skin in its usual dry state.
Our study shows that as a result of exposure to water, corneocytes take up the liquid, resulting not only in increased hydration on the SC, but also, due to swelling, in an increased SC thickness and a smoother surface. Moreover, plasticized SC exhibits a lower modulus, i.e., is more easily deformable, leading to a higher real contact area with the CaF2 window and presumably other objects. This will clearly have tribological consequences. Biointerphases, Vol. 11, No. 3, September 2016 Improved understanding of the influence of environmental conditions on the properties of human skin is important for various research areas. The barrier function of human skin is the focus of research useful for the drug delivery.59 It was proven that hydration of the skin (and, consequently, environmental conditions) has a major impact on dermatological issues. Proper hydration is a requirement for the flawless wound healing process.6°Various skin diseases are caused by skin dryness. Dermatological treatment of, for example, Xerosis cutis or eczema, could be supported with monitoring and modification of the hydration level of skin.61 Hydration of skin plays a key role also in aging prevention." The presented knowledge can also be useful for developing skin models or understanding the skin-friction mechanisms, as skin friction is directly related to the skin hydration and environmental conditions and, as a consequence, skin roughness and real contact area with counter surfaces.26 This could also contribute in the prevention of the decubitus ulcers, as the creation of ulcers depends on the friction between skin and the bedsheet.63