Structural and Biomechanical Characterization of a Scaffold-Free Skin Equivalent Model via Biophysical Methods.

AIMS: Among in vitro skin models, the scaffold-free skin equivalent (SFSE), without exogenous material, is interesting for pharmacotoxicological studies. Our aim was to adapt in vivo biophysical methods to study the structure, thickness, and extracellular matrix of our in vitro model without any chemical fixation needed as for histology. METHODS: We evaluated 3 batches of SFSE and characterized them by histology, transmission electron microscopy (TEM), and immunofluorescence. In parallel, we investigated 3 biophysical methods classically used for in vivo evaluation, optical coherence tomography (OCT), and laser scanning microscopy (LSM) imaging devices as well as the cutometer suction to study the biomechanical properties. RESULTS: OCT allowed the evaluation of SFSE total thickness and its different compartments. LSM has a greater resolution enabling an evaluation at the cell scale and the orientation of collagen fibers. The viscoelasticity measurement by cutometry was possible on our thin skin model and might be linked with mature collagen bundles visible in TEM and LSM and with elastic fibers seen in immunofluorescence. CONCLUSION: Our data demonstrated the simplicity and sensitivity of these different in vivo biophysical devices on our thin skin model. These noninvasive tools allow to study the morphology and the biomechanics of in vitro models.

Measurement of the biomechanical function and structure of ex vivo drying skin using raman spectral analysis and its modulation with emollient mixtures.

An important aspect of the biomechanical behaviour of the stratum corneum (SC) is the drying stresses that develop with water loss. These stresses act as a driving force for damage in the form of chapping and cracking. Betasitosterol is a plant sterol with a structure similar to cholesterol, a key component in the intercellular lipids of the outermost layer of human skin, the SC. Cholesterol plays an important role in stabilizing the SC lipid structure, and altered levels of cholesterol have been linked with SC barrier abnormalities. Betasitosterol is currently applied topically to skin for treatment of wounds and burns. However, it is unknown what effect betasitosterol has on the biomechanical barrier function of skin. Here, by analysing the drying stress profile of SC generated during a kinetics of dehydration, we show that betasitosterol, in combination with two emollient molecules, isocetyl stearoyl stearate (ISS) and glyceryl tri-2-ethylhexanoate (GTEH), causes a significant modulation of the drying stress behaviour of the SC by reducing both the maximal peak stress height and average plateau of the drying stress profile. Raman spectra analyses demonstrate that the combination of betasitosterol with the two emollients, ISS and GTEH, allows a high water retention capacity within the SC, while the lipid conformational order by increasing the amount of trans conformers. Our study highlights the advantage of combining a biomechanical approach together with Raman spectroscopy in engineering a suitable combination of molecules for alleviating dryness and dry skin damage.

The relationship between water loss, mechanical stress, and molecular structure of human stratum corneum ex vivo.

Proper hydration of the stratum corneum (SC) is important for maintaining skin's vital functions. Water loss causes development of drying stresses, which can be perceived as 'tightness', and plays an important role in dry skin damage processes. However, molecular structure modifications arising from water loss and the subsequent development of stress has not been established. We investigated the drying stress mechanism by studying, ex vivo, the behaviors of the SC components during water desorption from initially fully hydrated samples using Raman spectroscopy. Simultaneously, we measure the SC mechanical stress with a substrate curvature instrument. Very good correlations of water loss to the mechanical stress of the stratum corneum were obtained, and the latter was found to depend mainly on the unbound water fraction. In addition to that, the water loss is accompanied with an increase of lipids matrix compactness characterized by lower chain freedom, while protein structure showed an increase in amount of α-helices, a decline in α-sheets, and an increase in folding in the tertiary structure of keratin. The drying process of SC involves a complex interplay of water binding, molecular modifications, and mechanical stress. This article provides a better understanding of the molecular mechanism associated to SC mechanics.

Effects of atmospheric relative humidity on Stratum Corneum structure at the molecular level: ex vivo Raman spectroscopy analysis.

Skin hydration plays an important role in the optimal physical properties and physiological functions of the skin. Despite the advancements in the last decade, dry skin remains the most common characteristic of human skin disorders. Thus, it is important to understand the effect of hydration on Stratum Corneum (SC) components. In this respect, our interest consists in correlating the variations of unbound and bound water content in the SC with structural and organizational changes in lipids and proteins using a non-invasive technique: Raman spectroscopy. Raman spectra were acquired on human SC at different relative humidity (RH) levels (4-75%). The content of different types of water, bound and free, was measured using the second derivative and curve fitting of the Raman bands in the range of 3100-3700 cm(-1). Changes in lipidic order were evaluated using νC-C and νC-H. To analyze the effect of RH on the protein structure, we examined in the Amide I region, the Fermi doublet of tyrosine, and the νasymCH3 vibration. The contributions of totally bound water were found not to vary with humidity, while partially bound water varied with three different rates. Unbound water increased greatly when all sites for bound water were saturated. Lipid organization as well as protein deployment was found to be optimal at intermediate RH values (around 60%), which correspond to the maximum of SC water binding capacity. This analysis highlights the relationship between bound water, the SC barrier state and the protein structure and elucidates the optimal conditions. Moreover, our results showed that increased content of unbound water in the SC induces disorder in the structures of lipids and proteins.

Clinical and biometrological efficacy of a hyaluronic acid-based mesotherapy product: a randomised controlled study.

Data demonstrating the efficacy of hyaluronic acid (HA)-based mesotherapy for skin rejuvenation are scarce. The aim of the study is to assess the efficacy of non-reticulated HA-based mesotherapy on skin elasticity and complexion radiance. 55 women with cutaneous ageing signs included in the Full Analysis Set (FAS) population blindly received intradermal micro-injections (50 × 0.02 mL) of non-cross-linked HA filler with mannitol (Glytone 1, HA concentration: 14 mg/g) in one cheek and saline physiological solution in the other according to hemifacial randomisation in 3 monthly sessions. Elasticity (E1 and E2 stiffness parameters) and dermis thickness were measured by cutometry and 20 MHz echography, before (D0) treatment and 1 (1M) and 3 months (3M) after the last injection. A trained panel blindly scored skin complexion radiance from standardised and calibrated photographs, using 100 mm analogue scales. In the FAS population, only HA filler significantly decreased E1 at 1M (-10.9 %, p = 0.026) and 3M (-10.5 %, p = 0.035) compared with D0; its effect versus the control tended to be more persistent, with a difference between treatments at 3M close to significance (p = 0.063). E2 also decreased at 1M (-8.2 %, p = 0.027 in the per protocol population, n = 53) and 3M after HA-treatment only. Dermis thickness significantly increased after HA-treatment at 1M (+3.4 %, p = 0.028) and 3M (+4 %, p = 0.008), and after control-treatment at 1M only (+2.5 %, p = 0.015). The HA filler significantly improved complexion radiance at 3M compared with the control (p = 0.012) and for 51 % of subjects, their skin status. Non-reticulated HA-based mesotherapy significantly and sustainably improves skin elasticity and complexion radiance.

Association between collagen production and mechanical stretching in dermal extracellular matrix: in vivo effect of cross-linked hyaluronic acid filler. A randomised, placebo-controlled study.

BACKGROUND: The effects of hyaluronic acid (HA) injection on tissue collagen anabolism are suggested to be related to the induction of mechanical stress, causing biochemical changes in skin physiology. OBJECTIVES: To ascertain the association between dermal mechanics modulated by a hyaluronic acid-based filler effect and metabolism. METHODS: Sixty females were randomised to receive a 0.5mL injection of HA gel or isotonic sodium chloride (control) in the arm. Skin biopsies were taken at baseline and after 1, 3 and 6 months. Protein and gene expression of procollagen, matrix metalloproteinases (MMP) and MMP tissue inhibitors (TIMP1) were measured blind by ELISA and qPCR, respectively. Injected volumes were measured by high-frequency ultrasound and radiofrequency analysis. Skin layer effects of injections were analysed by finite element digital modelling. RESULTS: One month after injection, the filler induced an increase in procollagen (p=0.0016) and TIMP-1 (p=0.0485) levels and relative gene expression of procollagen III and I isoforms compared with the controls. After 3 months, procollagen levels remained greater than in the controls (p=0.0005), whereas procollagen expression and TIMP-1 and MMP content were no longer different. Forty-three percent of the injected filler volume was found at 1 month, 26% after 3 months and 20% after 6 months. LIMITATIONS: The ultrasound imaging technique limited the scope of the investigation and precluded an evaluation of the action of the filler at the hypodermic level. CONCLUSIONS: Integrating both mechanical and biological aspects, our results suggest that mechanical stress generated by cross-linked HA plays a role in dermal cell biochemical response.

An a priori shading correction technique for contact imaging devices.

Digital imaging devices are increasingly used for color measurements in fields such as forensics, the food industry, and ecological and medical research. Especially concerning the skin, in the follow-up of benign or malignant lesions, it is crucial to ensure that the measured colors are accurate and reproducible. Several color calibration methods having been presented over the last few years. However, the choice of illuminant used remains a major source of color misinterpretation, thus, much effort is being spent in trying to evaluate this a posteriori. The device presented overcomes this problem by integrating its own light source, although corrections in lighting heterogeneity are still required. In this paper, we present a lighting modelling technique used for shading correction which improves color consistency (as assessed by ∆E evaluation versus colorimeter), noise filtering, computation time, and memory consumption for this type of device.

Mechanical skin thinning-to-thickening transition observed in vivo through 2D high frequency elastography.

This study was based on two dimensional (2D) high frequency elastography to describe quantitatively the mechanical behavior of the human dermis in vivo. The study was conducted on the forearm skin and elastographic tests were performed using a combination of two devices: an extensiometer developed for the in vivo study of the mechanical behavior of the skin using uniaxial stretching stress, and a 20MHz real time sonographer (Dermcup 2020™) for ultrasound skin imaging. The staggered strain estimation algorithm (SSE) was used to produce elastograms. A temporal cumulative technique was applied to improve elastogram quality and to monitor variations in skin strain during stretching. The influence of the natural skin tension controlled by arm bending was studied and distinctive mechanical behavior was observed for low and high mechanical stress levels. In a preliminary analysis, the reproducibility of measurements was assessed by means of coefficient of variation (CV) in 5 selected healthy volunteers.Finally, two hypotheses linked to the geometrical and structural properties of the dermis are proposed to account for the new findings described in this study.

Characterization of the mechanical properties of skin by inverse analysis combined with the indentation test.

This study proposes a new method to determine the mechanical properties of human skin by the use of the indentation test [Pailler-Mattei, 2004. Caractérisation mécanique et tribologique de la peau humaine in vivo, Ph.D. Thesis, ECL-no. 2004-31; Pailler-Mattei, Zahouani, 2004. Journal of Adhesion Science and Technology 18, 1739-1758]. The principle of the measurements consists in applying an in vivo compressive stress [Zhang et al., 1994. Proceedings of the Institution of Mechanical Engineers 208, 217-222; Bosboom et al., 2001. Journal of Biomechanics 34, 1365-1368; Oomens et al., 1984. Selected Proceedings of Meetings of European Society of Biomechanics, pp. 227-232; Oomens et al., 1987. Journal of Biomechanics 20(9), 877-885] on the skin tissue of an individual's forearm. These measurements show an increase in the normal contact force as a function of the indentation depth. The interpretation of such results usually requires a long and tedious phenomenological study. We propose a new method to determine the mechanical parameters which control the response of skin tissue. This method is threefold: experimental, numerical, and comparative. It consists combining experimental results with a numerical finite elements model in order to find out the required parameters. This process uses a scheme of extended Kalman filters (EKF) [Gu et al., 2003. Materials Science and Engineering A345, 223-233; Nakamura et al., 2000. Acta Mater 48, 4293-4306; Leustean and Rosu, 2003. Certifying Kalman filters. RIACS Technical Report 03.02, 27pp. http://gureni.cs.uiuc.edu/~grosu/download/luta + leo.pdf; Welch and Bishop, An introduction to Kalman filter, University of North Carolina at Chapel Hill, 16p. http://www.cs.unc.edu/~welch/kalman/]. The first results presented in this study correspond to a simplified numerical modeling of the global system. The skin is assumed to be a semi-infinite layer with an isotropic linear elastic mechanical behavior [Zhang et al., 1994. Proceedings of the Institution of Mechanical Engineers 208, 217-222] This analysis will be extended to more realistic models in further works.