This article describes a method of quantifying the dynamic drying behavior and mechanical properties of stratum corneum by measuring spatially resolved in-plane drying displacements of circular tissue samples adhered to an elastomer substrate. This technique can be used to measure how different chemical treatments alter drying and tissue mechanical properties.
Stratum corneum (SC) is the most superficial skin layer. Its contact with the external environment means that this tissue layer is subjected to both cleansing agents and daily variations in ambient moisture; both of which can alter the water content of the tissue. Reductions in water content from severe barrier dysfunction or low humidity environments can alter SC stiffness and cause a build-up of drying stresses. In extreme conditions, these factors can cause mechanical rupture of the tissue. We have established a high throughput method of quantifying dynamic changes in the mechanical properties of SC upon drying. This technique can be employed to quantify changes in the drying behavior and mechanical properties of SC with cosmetic cleanser and moisturizer treatments. This is achieved by measuring dynamic variations in spatially resolved in-plane drying displacements of circular tissue samples adhered to an elastomer substrate. In-plane radial displacements acquired during drying are azimuthally averaged and fitted with a profile based on a linear elastic contractility model. Dynamic changes in drying stress and SC elastic modulus can then be extracted from the fitted model profiles.
The outer most layer of the epidermis, or stratum corneum (SC) consists of cohesive corneocyte cells surrounded by a lipid rich matrix1,2. The composition and structural integrity of SC is essential for maintaining correct barrier functionality3, which prevents invasion from microorganisms and resists both mechanical forces and excessive water loss4. The capacity of personal care products to maintain or degrade skin barrier function is of great interest to skin healthcare and the cosmetic industry5. The daily application of personal care products is known to alter the mechanical properties of the SC6,7,8. For example, surfactants contained in cosmetic cleansers can cause significant increases in the elastic modulus and a build-up of drying stresses in SC, increasing the tissue's propensity to crack7,9. Glycerol contained in nearly all cosmetic moisturizers can soften SC and decrease the build-up of drying stresses8,10,11, reducing the likelihood of tissue rupture.
The method detailed in this article is capable of quantifying the dynamic drying behavior and mechanical properties of SC drying in controlled environments7,8. Previously, this technique has been demonstrated to be capable of elucidating the effect of different cosmetic products on changes in the dynamic drying behavior and mechanical properties of SC tissue. This is achieved by quantifying drying-induced shrinkage of human SC tissue adhered to a soft elastomer substrate, fitting drying displacements with a simple contractility model, and then extracting the elastic modulus and drying stress from the fitted profile. When testing of multiple SC samples is required, this method offers a more rapid alternative to uniaxial tensometry, utilizes significantly less tissue and provides more physiologically relevant drying by preventing evaporation from the sample underside.
An exempt approval (3002-13) to carry out research using de-identified tissue samples pursuant to the Department of Health and Human Services regulations, 45 CFR 46.101(b)(4) was granted. Full thickness skin is received from elective surgery. In this article, the tissue source is 66-year-old Caucasian female breast.
1. Preparation of Elastomer Coated Coverslips
2. Preparation of the Stratum Corneum
3. Sample Treatment and Deposition
4. Microscope Environmental Control
5. Imaging in Plane Drying Displacements
6. Substrate Preparation for Thickness Measurement
7. Imaging Thickness of SC
8. Quantifying and Modeling Tissue Deformation
Figure 1(a) shows a representative fluorescent image of an SC sample coated with fluorescent beads (section 3). The corresponding transmitted light image of the sample is shown in Figure 1(b) overlaid with a quiver plot of spatially resolved drying displacements that form after 16 h drying at 25% R.H. Due to the circular symmetry of the samples, these displacements can be azimuthally averaged. Figure 1(c) shows radial (ur, solid red line) and azimuthal (uθ, dashed blue line) displacement profiles plotted against the dimensionless radial position, r/R. Here, R denotes the mean SC sample radius, r/R = 0 denotes the sample center and r/R = 1 denotes the edge. Standard deviations at each radial position are denoted by the shaded regions around the mean. These variations are primarily caused by the structural heterogeneity of the SC3,7,12. Throughout drying, azimuthal displacements remain small. Radial displacement profiles however increase monotonically from center to edge and grow in magnitude until an equilibrium is reached.
Figure 1: Circular SC sample (6.2 mm diameter) adhered to an elastomer substrate with elastic modulus E = 16 ± 1 kPa after drying for 15 h in a 25 ± 1% R.H. environment. (a) Fluorescent image of the SC sample highlighting the deposited fluorescent marker beads used for tracking spatially resolved in-plane drying displacements. (b) Quiver plot of spatially resolved in-plane drying displacements overlaid on a transmitted light image of the SC sample. (c) Azimuthally averaged radial (ur, solid red line) and azimuthal (uθ, blue dashed line) displacements of the sample plotted against dimensionless radial position, r/R. Positive values of ur correspond to contractile displacements. Shaded regions surrounding the lines indicate the standard deviation about the mean at each radial position. Please click here to view a larger version of this figure.
Profiles recorded at 30 min intervals are plotted in Figure 2(a) and show the time evolution of in-plane displacements. The average SC thickness, hSC, is plotted in Figure 2(b). Decreases of SC during drying primarily occur over the first 2 h.
Figure 2: (a) Overlay of radial displacement profiles (ur, solid red lines) at 30 min intervals over a 15 h drying period in 25% R.H. conditions plotted against dimensionless radial position r/R for a typical SC sample. Positive values of ur correspond to contractile displacements. (b) Average SC sample thickness, (hSC, n=3), plotted against drying time. (c) Radial displacement profiles from (a) overlaid with minimum least squares fits (blue dashed line) of Equation (1) for the first and last recorded radial displacement profile. Please click here to view a larger version of this figure.
Fitting displacement profiles with the linear elastic contractility model described by Equation (1) provides further insight into the mechanical properties of drying SC. Displacement profiles at each time step are fitted with the model using a minimum least squares approach, as shown in Figure 2(c). The contractile drying stress, PSC, and elastic modulus, ESC, are subsequently extracted from the model at each time step. Average changes in these parameters (based on 3 individual SC samples) are shown respectively in Figures 3(a) and 3(b). Both parameters increase rapidly over the first 2 h drying period and reach a plateau within 5 h.
Figure 3: (a) Averaged SC elastic modulus, ESC, plotted against drying time over a 15 h period. (b) Average contractile drying stress, PSC, plotted against drying time over a 15 h period. Please click here to view a larger version of this figure.
In this article, we describe a technique that can be used to measure the dynamic drying behavior and mechanical properties of human SC. Previous studies have demonstrated that this technique can be used to quantify the effects of environmental conditions and chemical products commonly used in cosmetic cleansers and moisturizers on the dynamic drying behavior of SC7,8. There are a number of key steps in the protocol. Firstly, SC swells notably with water content; therefore, measurements of SC thickness as well as in-plane displacements are essential for accurately predicting the elastic modulus and drying stress magnitude. Secondly, samples need to be fully adhered to the substrate. Incomplete adhesion, non-radially symmetric samples or samples with small tears or holes should be avoided because they will significantly impact the distribution of drying deformations and the radial displacement profiles used for model fitting.
The technique can be used if a humidity control system is unavailable. Without environmental control, tissue samples will dry in laboratory conditions12. As such, the laboratory environment should be continuously monitored and maintained, as drying behavior and the repeatability of results will be impacted by both diurnal and seasonal variations in temperature and humidity.
Currently, the technique is limited only to samples that can adhere to the substrate and induce deformations within the elastomer film. While the technique can be readily adapted to test samples that undergo smaller in-plane displacements, by reducing the substrate elastic modulus12, results from samples that simply slip over the substrate will lack meaning.
Numerous in-vivo and ex-vivo techniques that can assess the drying behavior and mechanical properties of SC have been reported3,8,9,10,18,19,20,21. However, in-vivo techniques cannot fully distinguish mechanical changes in SC from the underlying epidermal and dermal layers. Moreover, ex-vivo techniques can typically only assess one sample per experiment. The method we report in this article allows up to 6 SC samples to be assessed per experiment. The size of the substrate and environmental chamber however could be scaled up to allow more samples to be assessed simultaneously. We estimate for n=6 SC samples, a timescale of ~13 h is required for preparation and testing, excluding substrate curing and tissue equilibration. In comparison, we estimate uniaxial tensometry testing would require more than twice this period. Significantly less SC tissue is also required per individual sample (0.28 cm2) in comparison with those required for tensometry9 (2.5 cm2). This technique further enables more physiologically relevant drying by preventing evaporation from the underside of the SC tissue. In addition to assessing drying behavior and mechanics in SC, we believe this technique could also be applied to studies of polymeric or colloidal systems that form a cohesive film upon drying.
The authors have nothing to disclose.
The authors have no acknowledgements.
Silicone elastomer base | Dow-Corning | 1064291 |
Silicone elastomer Curing Agent | Dow-Corning | 1015311 |
FluoSpheres Carboxylate 0.1 µm yellow green fluorescent 505/515 | Thermo Fisher | F8803 |
FluoSpheres Carboxylate 1 µm yellow green fluorescent 505/515 | Thermo Fisher | F8823 |
FluoSpheres Carboxylate 1 µm nile red fluorescent 535/575 | Thermo Fisher | F8819 |
Trypsin from porcine pancreas | Sigma-Aldrich | T6567 |
Trypsin inhibitor type II-s | Sigma-Aldrich | T9128 |
(3-aminopropyl)triethoxysilane | Sigma-Aldrich | 440140 |
Sodium tetraborate | Sigma-Aldrich | 221732 |
Boric acid | Sigma-Aldrich | B0294 |
Phosphate buffered saline | Sigma-Aldrich | P7059 |
N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride | Sigma-Aldrich | E7750 |
Vortexer mixer | VWR | 58816-123 |
6mm diameter hole punch | Sigma-Aldrich | Z708860 |
SOLA 6-LCR-SB | Lummencor light engine | No.3526 |
Cfi Plan Achro Uw 1x Objective | Nikon Plan UW | MRL00012 |
CFI Plan Fluor 40x Oil Objective 1.3 na – 0.20mm wd | Nikon Plan Fluor | MRH01401 |
Nikon Eclipse Ti-U inverted microscope | Nikon | MEA53200 |
Clara-E Camera | Andor | DR-328G-C02-SIL |
Remote Focus Attachment E-RFA Ergo Design | Nikon | 99888 |
Ti-S-E Motorized Stage | Nikon | MEC56110 |