Review of the instrumental assessment of skin: Effects of

Transcription

1 J. Soc. Cosmet. Chem., 42, (July/August 1991) Review of the instrumental assessment of skin: Effects of cleansing products T. M. KAJS and V. GARTSTEIN, Procter & Gamble Co., Sharon Woods Technical Center, Reed Hartman Hwy, Cincinnati, OH 45241, and Miami Valley Laboratories, East Miami River Road, Ross, OH Received March 27, 199 I. Synopsis The role of biophysical instrumental techniques in assessing the effect of cleansing products on the skin is reviewed. Commercially available instruments can measure numerouskin characteristics: water holding, color, blood microcirculation, viscoelastic properties, surface profile, and desquamation. These noninvasive techniques can be used in concert with expert and consumer evaluations of visual and tactile changes. The use of multiple instruments is recommended to test the broad spectrum of surfactant and soap effects. Key requirements for accurate and reproducible measurements include a controlled environment, acclimation of subjects, standard measurement procedures, realistic product application protocols, and qualified operators. INTRODUCTION Biophysical instruments are objective and quantitative tools for characterization of various skin conditions. Instrumental techniques have been used to describe the effects on skin of cosmetic products, skin aging, solar exposure, and skin diseases. In the past, researchers often invented their own methods due to the lack of accepted methodologies and available commercial instruments. The result has been a diversity of techniques and protocols that has made comparison of data difficult. Today, many of the best technical approaches have been implemented in commercially available instruments, and attempts are beginning to be made to standardize their usage in investigative studies. This paper will review the noninvasive biophysical instruments used to assess changes in skin exposed to cleansing products. Particular emphasis will be given to commercially available techniques. Key aspects of instrumental protocols for measuring skin condi- tions will also be discussed. BACKGROUND Cleansing products are composed of soap and/or synthetic surfactants. Soap surfactants originate from animal or natural products; synthetic surfactants are either chemical 249

2 250 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS derivatives of these natural products or are derived from petrochemical sources. The cleansing action that makes a product hygienic--the removal of dirt, air pollutants, desquamating cells, cutaneousecretions, and pathogenic microorganisms, etc.--may also affect skin conditions (1). The mechanism of the effect of soap and synthetic formulas is not totally known; however, irritation may be evoked by modification of lipids and/or proteins in the stratum corneum or by penetration of surfactants through the stratum corneum to interact with the cells of the viable epidermis. With intensive use, surfactant exposure may induce skin redness, dryness, and roughness. Topical application methods for testing cleansing product effects vary from soap chamber models (2) to actual-use testing, e.g., forearm and split face wash tests (3-6). In early use tests, consumers and/or dermatologists would compare the product effects by evaluating redness, dryness, and smoothness. Instrumental techniques for quantitative evaluation of various skin parameters were developed to substantiate both consumer perceptions and expert gradings. Today product tests are often based on a three-pronged approach: consumer, expert, and instrumental assessment (7-10). Current noninvasive instrumental techniques allow assessment of many skin characteristics: water holding, color, blood microcirculation, viscoelastic properties, surface profile and appearance, thickness, and corneocyte desquamation. These methods can be used to objectively quantify expert and consumer assessment, such as erythema, dryness, roughness, scaliness, and stiffness or tightness. WATER FLUX (TEWL) Water is continually diffusing through the skin to the environment (except during submersion) due to the body's high water activity. Transcutaneous water flux depends on skin's permeability and the water activity difference of skin relative to the environment. The transepidermal water loss measurement (TEWL) is often taken as a measure of skin's intrinsic barrier properties. A high TEWL is indicative of an ineffective or damaged barrier function of the stratum corneum. Skin diseases (e.g., psoriasis) and chemicals (e.g., detergents) can cause high TEWL. The ServoMeal Evaporimeter (Figure 1) is the most widely used instrument for TEWL measurement (11). It consists of two humidity detectors placed vertically in an open tube above the skin surface at a fixed separation. These two sensors measure water vapor gradient above the skin surface. TEWL is calculated as the amount of water evaporated per unit of surface in an hour. The ServoMed Evaporimeter permits the skin surface to be exposed to ambient conditions during the period of measurement and offers high accuracy and sensitivity (12). Ambient relative humidity and temperature affect skin permeability and TEWL measurements and should be controlled during testing to allow comparative analyses (4,8, 11,13-16). Subject perspiration will also affect TEWL. To avoid inducing perspiration (and to provide general comfort), ambient temperatures from 19 ø to 22øC and relative humidity from 31% to 65% are generally recommended (17-20). An acclimation period of ten minutes to one hour is usually required and is dependent on ambient conditions and the subject's emotional state (4,14,19-22). During the acclimation period, it is important to ensure that the subject is emotionally calm to prevent sweating (4,19,22). The standardization group of the European Environmental and Contact

3 INSTRUMENTAL ASSESSMENT OF SKIN 251 Figure 1. The ServoMed Evaporimeter for measuring transepidermal water loss (TEWL) on skin. Dermatitis Society has developed preliminary guidelines to standardize instrumental evaluations of changes in the skin barrier function caused by disease or exposure to chemicals (23). Soap andsurfactant exposure. Most data on soap and surfactant effects on TEWL have been obtained with chamber test procedures (18,24-32). The current trend is to measure TEWL in exaggerated-use testing (e.g., on forearm and face), where prolonged (more than 30 seconds' soap exposure) and frequent washing cycles are involved. These studies have shown that an increase in TEWL correlates with increased visually assessed skin "dryness"(3-6). In many cases the change in TEWL precedes the visual dryness. The correlation between visual dryness and TEWL was not observed when the washing procedure excluded mechanical action (16). WATER CONTENT IN STRATUM CORNEUM The degree of hydration is most frequently determined by measuring electrical properties of skin. In addition, infrared and photoacoustic spectroscopy have also been used. ELECTRICAL METHODS Measurement of skin permeability to alternating electric current (impedance) reflects elec-

4 252 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS tromagnetic interaction with skin dipoles (proteins) and electrolytes (33). In vitro and in vivo studies have shown that a low resistance (high impedance) correlates with an increase in skin hydration (water content). The measurement is more influenced by changes in the nearby and lower impedance stratum corneum layer than is the moist underlying tissue (high impedance). These measurements have been used to investigate soaps or surfactants (4,6,9,27,34). In the past, the IBS Skin Surface Hygrometer was the instrument of choice for measuring conductance and capacitance (34-38). Lately the Skicon-100 or 200 hygrometer (conductance), the Corneometer (capacitance), and the Dermal Phase Meter (capacitance) have been used to measure skin hydration. These instruments are easy to use and are portable. The Corneometer and Dermal Phase Meter have a software option to facilitate data analysis. Figure 2 illustrates the Skicon. The dielectric constant probe (DCP, or microwave probe) uses a higher frequencyfocused electromagnetic field, the frequency range (> 1 GHz) being selected at a water absorption band (39,40). The DCP measurement is specific for the stratum corneum since the electromagnetic field can be focused to limit its penetration. In vitro tests have shown a linear relationship between the weight fraction of water and DCP output (39). Only a few focused microwave probe instruments were built, and the instrument is not commercially available today. A concern for electrical measurements in some cases is that a product film on the skin surface can influence instrumental responses (14,15). The measurement of skin electrical impedance at different frequencies may help to exclude the effects of product residue on skin (37); however, no commercial instrument currently implements this technique. SKICON-200 Figure 2. Skicon-200 for measuring water content of skin.

5 INSTRUMENTAL ASSESSMENT OF SKIN 253 SPECTROSCOPY Infrared (IR). Water strongly absorbs in the infrared region. It is possible to obtain an IR spectrum of skin by use of attenuated total reflectance (ATR) techniques (! 1,41). It has been shown that water absorbance peaks can be identified and measured. Near infrared (NIR) has recently been successfully employed to measure skin moisture (42). Also, ATR and Fourier Transform IR have been successfully used to measure Iipid order/disorder in stratum corneum, which may be related to the barrier function (43,44). IR methods have been little used because available instruments are expensive and not portable. Also, the measurement is less convenien than electrical techniques. Photoacoustic (PAS). Photoacoustic spectroscopy uses sound signals generated by pressure variation in skin. The pressure is produced by heating skin with periodic light radiation. The amount of water in the stratum corneum will affect heat dissipation and pressure waves propagated through the tissue. These waves can be measured with a sensitive acoustic microphone. Though the PAS technique has been used for qualitative measurements of hydration, it is not only difficult to use but occlusion of skin sites during the time-consuming experiments may lead to questionable results (11,45,46). Soap and surfactant exposure. Skin electrical measurement is the most widely used technique for measuring water content of skin after soap and surfactant exposure. The skin capacitance or conductance provides one of the earliest and most reliable indications of surfactant-induced changes to stratum corneum. A number of studies have shown a good correlation between water measurement and visual dryness as a result of skin exposure to cleansing products (4,6,9). Surfactant exposure (4% SDS in water) on legs resulted in significant decreases in capacitance (34). Also, a correlation between irritation and electrical impedance was found after occlusive patch treatment with surfactant in a Finn chamber study (27). SKIN SURFACE ph Skin surface ph is in actuality the ph value of skin water-soluble constituents. During the measurement, the ph meter electrode is coupled with the skin surface through an aqueous interface. Soluble components extracted into this liquid interface determine skin ph values. The normal ph of skin surface ranges from 4.5 to 6.0. The skin surface ph measurement is conducted with commercially available instruments using a flat- surface electrode. Soap exposure. Washing skin with alkaline soaps increases ph, while synthetic detergent bars have less effect on skin ph (47). The changes in skin surface ph caused by soap do not last long (<2 hr) and are not reliable predictors of soap harshness. XVhile alteration of skin surface ph is not likely to cause skin irritation, it may be indicative of changes in superficial skin layers (48). ERYTHEMA MEASUREMENTS Instrumental methods that have been used to evaluate erythema (redness) caused by

6 254 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS cleansing products include colorimetry, reflectance spectroscopy, laser Doppler ve- Iocimetry, and thermography. COLORIMETRY The introduction of the Minolta Chroma Meter (MCM) (Figure 3) and, recently, the Dia-Stron Erythema Meter (DEM) and the DermaSpectrometer (Cortex Technology), make skin color measurements a routine test. The MCM has been shown to be a reliable tool for assessment of erythema in patch tests and exaggerated wash tests (4,6,24). The MCM uses the three-dimensional color coordinate system recommended by the Commission Internationale de l'eclairage (CIE). The coordinates are L, a and b, where "L" represents the level of brightness from black to white, "a" the balance between red and green, and "b" the balance between yellow and blue. Redness is typically measured using the "a" scale (24). The DEM measureskin redness using a relative scale based on the difference in skin reflection in red and green light. Both instruments have a flexible, hand-held probe; the DEM's fiber optic probe is lightweight and designed for skin measurements. The instruments are portable and conveniento use. Readings can be taken in 15 seconds or less, preferably by the same operator to reduce the variation in contact pressure (a potential cause of blanching). In addition, the DEM has a software option designed to facilitate data analysis. Soap exposure. MCM measurements have been correlated with visual erythema and TEWL. Results from soap chamber testing showed significant differences between bar soap products (24). In an exaggerated use forearm wash test (a two-minute wash four Figure 3. The Minolta Chroma Meter for measuring redness of skin.

7 INSTRUMENTAL ASSESSMENT OF SKIN 255 times daily for four consecutive days with an additional two washings on the fifth day), MCM detected differences in the erythema produced by a soap and synthetic bar. These results correlated with visual erythema assessments. The same authors found that the MCM has less variability in measuring erythema than the laser Doppler velocimeter (4,6). Because skin color can affect results, it is important to manage color differences between and within subjects. Also, panelistshould not be exposed to sun during the test period to avoid tanning and sunburning. REFLECTANCE SPECTROSCOPY Reflectance spectroscopy is based on measuring the spectrum of light ( nm) remitted from skin. Usually, it requires an integrated sphere to collect all backscattering light. The main disadvantages of existing reflectance spectrophotometers are possible skin blanching from the heavy probe and the cost of the instrument (49,50). Also, it does not appear to have an advantage over colorimetric techniques in measuring ery- thema. Soap exposure. Research on the use of reflectance spectrophotometry in measuring soap product effects on the skin is minimal. One study investigated erythema resulting from exposure to soap products in a chamber test; however, the results were inconclusive (51). LASER DOPPLER VELOCIMETRY Laser Doppler Velocimetry (LDV), also known as laser Doppler flow (LDF) or skin blood flow (SBF), is a technique that can be used to measure cutaneous blood flow and thereby provide information on erythema. LDV has been used for the past ten years to measure skin irritation and contact dermatitis. LDV measures blood flow by detecting a shift in scattered light frequency. This shift in monochromatic laser light results from scattering from moving blood cells (Doppler effect) (52). Light (for example, from a 2-mW helium-neon laser) is guided by an optical fiber to the skin surface, where it penetrates to a depth of about 1 mm. The backscattered light is collected by optical fibers and brought to photodetectors. The low frequency signal produced by the photodetectors is proportional to the Doppler frequency shift. The instrumental output is expressed in relative units and corresponds to blood flow values (53,54). Total blood volume can also be measured by some instruments. The LDV method is used to measure subclinical irritation when redness is not visible. Microcirculation is easily influenced by the subject's physiological condition, which can result in high variability of LDV measurements. For this reason, stringent screening of panelists (to prevent interference from food and medication) and a long acclimation period (to reduce emotional or physical activity) are necessary. Soap and surfactant exposure. LDV has been used primarily in the measurement of skin irritation caused by surfactants using the occlusive chamber test. It has been shown that blood flow depends on concentration of surfactant (sodium lauryl sulphate) (54-57). Also, LDV blood flow increases with increased visual erythema, TEWL, and skin thickness (25,27,54, 56,58,59).

8 256 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS The only reported measurements of soap irritation by LDV compares a soap bar and synthetic bar in an exaggerated forearm wash study (4,6). A significant difference in blood flow was found; the data paralleled TEWL results. THERMOGRAPHY Contact and infrared thermography, which measure changes in skin temperature (60,61), have been proposed for assessment of skin irritation (erythema). Because skin temperature is influenced by many factors (moisture evaporation, tissue heat transfer, inflammation, etc.), we have found it is difficult to establish a definitive relationship between temperature changes and visual erythema. Soap and surfactant exposure. Contact thermography has been used to measur effects of experimental irritants on skin. The different irritants caused specific temperature changes (SLS reduced and croton oil increased the skin temperature) (62). The use of a liquid crystal thermometer provided a pictorial representation of skin temperature gradient. In a separate study, infrared thermography was used to assess the temperature changes caused by cleansing products (63). No significant differences were reported; however, the authors demonstrated high-resolution images of skin temperature profiles. VISCOELASTICITY MEASUREMENTS Multiple skin layers (stratum corneum, epidermis, and dermis) are involved in skin's viscoelastic properties. Viscoelasticity of stratum corneum is a function of skin hydration, while dermal mechanical propertiestrongly depend on skin age (64). Instrumental measurements, therefore, may depend on both skin hydration and age (65,66), particularly if contributions from different layers of skin cannot be isolated. At one time, the pinch test of Hollingworth was used to measure elasticity (67), but today a number of more sophisticated methods are available. Skin elasticity techniques can be divided into two classes (65): 1) skin extension perpendicular to the surface--for example, indentation, leverometry (68), suction, and ballistometry (65,69) 2) skin extension parallel to the surface for example, stretching, torsion, and sound propagation. Elastic properties are responsible for skin's returning to normal shape after deformation. The higher the stress-strain ratio, the more force is required to produce deformation. Four approaches can be used to acquire skin stress-strain curves: Type 1: single stretching a fixed length of skin (Young modulus) Type 2: multiple stretching of a fixed length of skin (dynamic modulus) Type 3: single stretching of unimpeded skin Type 4: multiple stretching of unimpeded skin (dynamic modulus) MEASUREMENTS OF ELASTICITY PERPENDICULAR TO THE SKIN SURFACE Methods of indentation, suction, and elevation or leverometry (68) involve either pull-

9 ... INSTRUMENTAL ASSESSMENT OF SKIN 257 ing or compressing the skin with a known force and measuring displacement. The commercial instruments are the Cutometer (70) and Dermarlex A (71), which are based on the Type 2 approach. The Cutometer (Figures 4a and 4b) measures viscoelasticity by drawing the skin into a low-pressure aperture of the probe where skin penetration is determined by a non-contact optical measuring system. [The Dermarlex A (Cortex Technology) has a similar design; however, skin penetration is measured with an electrical sensor.] The resulting displacement versus time curves from the Cutometer are :.' ½<', ' "/)v ':--½.'..:!.."-., i ::! i½: "-': :, : :': :i : : : :: e i.-':.½..-?' :i.*.': P? ;¾' ¾ ¾...4;?*./3.' :?.??&:: :!: : :!:!: : :3:i:!: :i!.'.::i i:!:i :i:!:i:3:i: :3:i:i:.:: :3. : :-':.; :'.:.':::::-...4.x...({.::::?&,,:-:-:-: :-:-:-:-; :.:-:- :-:. :-:-:-:-:-:.-:-:-:.:.:-:-:-:.:!-'!.'I if. ============================= '".. :::::::::::::::::::::::: :.:-:.:-:-:-:, ;:i:i:3e: t.:.:.-:..:. ::' :!::!i.:.:.:.:.:...:.:.:.:..:..* i:i:i: ::i i:::]:i:: : : : :: 4- '-" :.. ;;:$: :;;;;;:;:::::: :::::2:::: measuring >- $-<½,-',,-. ß 4..;q?..:,:8:3 :i:i:i:i:i:i : :i:i:!:!:!:..%.-:i: :!:Mei:!: i:i:i:..%!(.:... low vaccum light beam? " ' ' ::i i :: ::: '2'' ß '" " :::::::::::::::::::::: i:: :.::?.. '-'-': :,.-:..: :::: '+'::;:::-::-::::;::::::" ::::::'::-: ::::-:i - -:-:::. -;.;. :;; ':-... e.' (0-500 mbar)... ' I!::i::i::i::i::!::i fi::i:. i:?:4...;:;!::?:i::iii?':..-':,.:...:. '...":' ;:;: : :;:;: :;: :;: :;2;: 2.`.;2:::2:2;:;2;:;:;:;:;:;:;:.:.:.:.:..``:.: :.:.:.:.`..: :.:..: :.:.:.:4.: :`:.: :.: : :.:.:.:... :.:.x.x-x.:.:.:-:.x.x.x.:.:.:.:.:-:.:.:.:.;.:.:.x.:.:.:.:.:.:.:.:.:.-..:.x.:.:... :-:.x.:.:.:.:.x.:.: :ø.'aø.:-.'. '....,,,,,,,,,....>>:... Figure 4. a. The Cutometer for measuring elasticity of skin. b. Schematic diagram of the Cutometer probe.

10 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS plotted on the monitor, displaying skin penetration in the probe and skin recovery during the test cycle. Multiple parameters can be measured from the same data (stressstrain ratios, recovery, displacement, etc.) A set of apertures is provided with the instrument (2-, 4-, 6-, and 8-mm). The smaller apertures help to reduce the influence of the dermal layer to the measurement. MEASUREMENTS OF ELASTICITY PARALLEL TO THE SKIN SURFACE Measurement of constrained skin is typically conducted by extensiometry or twistometry (72-74). Commercially available instruments based on this principle are the Dermal Torque Meter (DTM) and Rheometer (Dia-Stron, Inc.). The DTM has a probe that allows rotation of a small skin surface area with a given torque (Figures 5a and 5b). Both angles of rotation and recovery are measured. Either the Type 1 or the Type 2 approach can be used with the instrument. The torque/angle ratio can be calculated to characterize skin elastic properties. This ratio reflects the resistance of the skin to stretching and is a function of skin hydration. Depending on the amplitude of the applied torque and the geometrical arrangement of the device (gap between stationary and rotational discs), the properties of either the stratum comeum or stratum comeum and underlying tissue can be studied. The gas-bearing electrodynamometer (GBE) method utilizes a free-standing probe that measures the dynamic elastic modulus (Type 4 approach). The probe consists of a small etating Element -Housing.,.- j " " "'"'"... :';" Spacing --"alltlllltl1111tlll from 1 ' 5 mm to Figure 5. a. The Dermal Torque Meter for measuring elasticity of skin. b. Schematic diagram of the Dermal Torque Meter probe.

11 INSTRUMENTAL ASSESSMENT OF SKIN 259 tab attached to the skin surface. Displacement of the probe by a sinusoidal driving force is measured (65,75). The slope of the stress-strain curve, a measure of the viscous component, has been related to skin hydration. (Usually greater displacement equates with greater consumer-perceived softness.) (7,74,76-78). Sound propagation methods have been investigated to measure skin elasticity of the stratum corneum (9,79). The sound transmission relates to the elastic modulus and density of the tissue. Improvement in skin condition, as indicated by this technique, has been correlated to consumer perception of smoothness and expert graders. Body sites for measurement. The forearm is the typical body site used for viscoelastic measurements. Some instruments can be used on the face, hand, or leg (calif. Soap exposure. A noncommercial twistometer has been used to discriminate the effects of a synthetic versus a soap bar (4,6). The studies indicated that skin treated with a synthetic bar had a lower torque/angle ratio (less stift and was more resilient than skin treated with a soap bar. Cleansing bars with little formula variation produced no significant differences. Sound propagation measurements made on skin treated with glycerin-added soaps were found to correlate with consumer-perceived skin condition (9). FRICTION Skin friction plays an important role in both subjective and objective evaluations of many skin attributes (e.g., texture, suppleness, softness, smoothness, dryness, and oiliness) (80-83). It has been shown that skin friction increases with skin hydration (80,84). Skin friction techniques measur either the static or the dynamic coefficient of friction. Techniques for determining the dynamic coefficient of friction are based on either a wheel or a rotational probe. The rotational probe can be vertical or horizontal. The only available commercial instrument is the Newcastle Friction meter, which utilizes the horizontal probe (wheel) pressed against the skin with a constant pressure. For this instrument, friction depends on the probe material and skin elasticity, with softer skin resulting in a higher contact area. Friction studies reported in the literature have dealt mainly with effects of moisturizers (83,85). Soap exposure. Effects of soap and emollient on skin friction were investigated by Prall (86). He found that washing with soap caused an immediate large decrease (>70%) in skin friction. SKIN SURFACE Profilometry is the commonly used method to quantitate skin surface topography. Optical and scanning electron microscopy as well as photography can be used to visualize the skin surface. Image analysis techniques may be used to quantify both profilometric and visual data.

12 260 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS PROFILOMETRY Profilometry measures the height of skin surface relief from replicas (81,87-89) or directly from the skin (17). The measurements may consist of single profiles or threedimensional representation of the skin surface (89). Normal skin is generally characterized by smooth-surfaced plateau separated by furrows. In dry skin, uplifted scales result in a jagged surface, which obscures the plateau and furrow pattern (87,88). A new profilometric technique utilizing a laser focusing probe is commercially available (Rodenstock Precision Optics, Inc.). Laser profilometry can provide three-dimensional images of skin surface replicas (Figure 6). This technique provides faster and more accurate measurement and visualization of skin surface changes caused by cleansing products (90). Profilometry has been used for measuring the effects of emollients, glycerol, occlusion, and antiwrinkle creams (17,87,91-94). IMAGE ANALYSIS Image analysis techniques can be used to quantify skin surface using profilometric, shadow, microscopic, and photographic images. The typical use of image analysis for surface measurements includes a low-angle illumination to visualize surface roughness X[m] 2.4S LI Figure 6 Profilometric three-dimensional image of the volar forearm

13 INSTRUMENTAL ASSESSMENT OF SKIN 261 from skin replicas. Images of shadows can be quantitatively analyzed, and results are directly related to surface relief (95,96). Image analysis techniques require specialized equipment: image digitizer (TV camera), video processor, computer, and software. More researchers are using image analysis for skin surface evaluation as complete systems become commercially available (91,97-100). Recently developed optical instruments (Scopeman, Microwatcher, Nikon) collect and store electronic images directly from the skin surface (101). High-quality images can be evaluated by expert graders and quantified by image analysis techniques (Figure 7). Various magnification lenses are provided to optimize the resolution of skin surface topography; however, the depth of field is limited. Scanning electron microscopy provides microscopic images of skin surface replicas with greater depth of field than light microscopy. The images are suitable for subjective and objective evaluations (13,17,82,88,102). These images can be quantified using image analysis techniques. Soap exposure. Quantification of skin surface topography for measuring soap and surfactant effects is a new area. Research to date indicates that surface roughness increases after soap exposure. The use of image analysis provides multiple ways to quantify changes in skin microtopography (97,103); however, no standard image analysis technique currently exists. THICKNESS MEASUREMENTS Skin thickness measurements are useful to characterize skin disorders, e.g., psoriasis and eczema, and skin conditions, e.g., edema and epidermal hyperplasia. Ultrasound is the commonly used technique for measuring skin thickness. Surfactant exposure. Ultrasound thickness measurements were conducted after patch tests (Finn chambers) with SLS. Higher concentrations resulted in increased skin thickness as well as increased visual irritation, TEWL, and LDV (27). Figure 7. a. Scopeman for documenung images of skin at various magnifications. b. Image collected by Scopeman.

14 262 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS There are no reports to indicate changes in skin thickness after soap exposure under actual use conditions; however, surfactant may induce epidermal hyperplasia (104). BIOLOGICAL METHODS Quantitative methods exist to measure skin structures and physiological changes caused by application of topical products. The goal of noninvasive biological assessment is to directly measure the condition of the viable epidermalayers. EXFOLIATIVE CYTOLOGY Noninvasive techniques are used to investigate the structure of the stratum corneum. Skin surface layers are removed and examined to measure characteristics of either individual corneocytes cell aggregates. Cells are obtained from the superficial skin layers by using adhesive materials [e.g., sticky slides, D'Squames by CuDerm (Figure 8), etc.] (103) or detergent scrubs (105). Histological markers of "dry skin" include retention of nuclei, cell size, scaling, diffuse cell borders, and changes in staining properties (97). Nucleated cells. The presence of nucleated cells (parakeratoticorneocytes) in skin superficial layers is a sign of ongoing epidermal hyperplasia. A high turnover rate in the epidermis results in immature corneocytes on the skin surface. Appropriate staining procedures allow quantification of nucleated cells (106). Size. The projected area of corneocytes can be quantitated by image analysis (73). The Figure 8. The use of D'Squames for removing dry skin flakes from skin

15 INSTRUMENTAL ASSESSMENT OF SKIN 263 Coulter counter can measure thickness of corneocytes, which is inversely related to the projected area (107). The size of corneocytes provides an indirect assessment of epidermal conditions (73) and is related to skin barrier function; for example, permeability increases as cell area decreases (108,109). Amount of scaling. The number and size of cell aggregrates determines the degree of visible scaling. The Squameter was the first commercial instrument developed to measure cell aggregrates (110). Recently image analysis has been used to measure size and amount of scales (97,99,111). The differences in normal and dry skin can be easily visualized and quantified (Figure 9). DESQUAMATION Visual scales are a clinical manifestation of abnormal epidermal turnover (92). The scales are probably due to higher than normal intercorneocyte adhesion (l 12). Multiple methods have been developed to quantify the rate of desquamation, corneocyte adhesion, and structure of desquamating layers. Rate. The rate of desquamation can be measured by disappearance of dansyl chloridestained stratum corneum (13, 113). Other methods involve collection (104,114)and counting of corneocytes (115) from the skin surface. Cohesion. Intercorneocyte binding force can be measured in vivo using a "cohesograph" (116). A gradual loss of cohesion has been found toward the skin surface (117). Morphology. The morphology of desquamating corneocyte layers can be assessed if an Scales Area = 7.3 sq. mm Scales Area = 48.4 sq. mm Figure 9. Image analysis of D'Squames. a. Normal skin. b. Dry skin.

16 264 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS intact portion of stratum corneum is removed with cyano-acrylate glue (surfometry). The unevenness of the stratum corneum inner surface and the amount of removed material relate to both the internal structure and the strength of intracorneal adhesion. The method has been used to distinguish between normal and diseased skin condition (e.g., psoriasis, ichthyosis). It was also demonstrated that the structure of stratum corneum relates to skin hyration (118). Soap and surfactant exposure. Biological methods have been successfully used for evaluating skin moisturizers (103); however, their application in investigating soap or surfac- tant effects has been limited. BIOCHEMICAL ANALYSES OF THE SKIN Visual and instrumental changes caused by cleansing products are a result of skin biochemical alterations. For example, surfactant-induced abnormal stratum corneum structure could be the result of chemical changes in skin lipids and proteins (119). These changes have been shown to correlate with visible redness and dryness and instrumental measurements (electrical impedance, TEWL, etc.) (34, ). Most biochemical researchas been conducted on skin biopsies. A novel approach to allow assessment of biochemical characteristics using noninvasive collection techniques have been investigated. For example, acid phosphatase activity in the stratum corneum has been measured using tape-stripping samples (123,124). The development of noninvasive biochemical collection techniques is a new area for understanding effects of skin care products. Biological markers may indicate the mechanism and kinetics of adverse reactions and provide insight to sensory, visual, and instrumental assessments. DISCUSSION MULTIPLE INSTRUMENTAL APPROACH We recommend the use of multiple instruments in measuring the effect of cleansing products on skin to characterize the broad spectrum of surfactant effects. Also, a multiinstrumental approach can aid in interpretation of test results. Single methods may produce ambiguous data; for example, electrical impedance measurements may be influenced by product components (14,15). An additional measurement of skin viscoelastic properties, which are not as sensitive to surface residues, can aid in clarification of skin response. Researchers have used the multiple instrumental approach for cleansing products (3,4,6,9) and found that the instrumental measurements are consistent and correlate to subjective visual assessments. INSTRUMENT SELECTION This review indicates that instrumental methods can provide a definitive assessment of cleansing product effects on skin. The majority of studies include measurement of skin TEWL and electrical properties; therefore, it would be advantageous to include these assessments in future studies to facilitate comparisons and provide continuity. Other

17 INSTRUMENTAL ASSESSMENT OF SKIN 265 Table I Instrumental Evaluation of Effects of Soap and Surfactant on Skin Assessed skin condition Measurement method Instrument ErythemaJredness Dryness Roughness/scaliness Stiffness/not soft Biological changes Optical Water holding Surface topography Viscoelasticity Cytological analyses Chromameter (Minolta), Dermal Erythema Meter (Dia-Stron), DermaSpectrometer (Cortex Technology) Laser Doppler Velocimeter (PeriMed, TSI) Evaporimeter (ServoMed), Skicon (lbs), Corneometer (Courage& Khazaka), Dermal Phase Meter (NOVA) Scopeman (Moritex) Microwatcher (Mitsubishi Kasei) Skin replicas (Silflo) Laser profilometry (Rodenstock) Dermal Torque Meter (Dia-Stron), Cutometer (Courage& Khazaka) Dermarlex A (Cortex Technology) D-Squames (CuDerm) instrumental techniques are useful for further characterization of skin changes (Table I). These methods include measurements of erythema, surface topography, elasticity, and cytology. STANDARDIZATION OF INSTRUMENTS When procuring a new commercial instrument, we would encourage the adoption of an existing operating procedure that is appropriate for the study design. The recent publication of TEWL measurement guidelines (23) is an example of efforts to standardize an application. Further standardization of instrumental techniques in the future can be expected. INSTRUMENTAL PROTOCOL CONSIDERATIONS We have found that the quality of instrumental measurements depends upon a controlled environment (temperature and humidity), acclimation of subjects, qualified operators, consistent measurement site, and a fixed measurement schedule after application. In addition, the accuracy can be increased by taking a baseline measurement, having a control group to monitor environmental changes during the study, and paired comparison evaluations to reduce the impact of subject-to-subject variability. Key methodological differences in published study protocols include product application procedures, time interval after application for measurement, study duration, and types of instruments used. For example, some procedures which involve intensive mechanical agitation during the wash may exaggerate skin erythema and remove skin scales. Protocols that involve gentle rubbing produce less erythema and more pronounced skin

18 266 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS dryness. Protocol differences must be recognized when results of various studies are compared. NEW INSTRUMENTATION Continuous progress in understanding skin and instrumental techniques promotes the development of novel testing methodologies. A new generation of instruments will be hand-held, computer-controlledevice suitable for various body sites. Immediate data acquisition and analysis will simplify testing procedures. New instruments should be less influenced by, or corrected for, the ambient environment while preserving high sensitivity. They should assess multiple, relevant consumer characteristics. Future instrumentation should not neglect dermatological needs to identify subclinical changes (invisible dermatology) in patients with subtle skin conditions (e.g., sensitive skin, aging skin). Further advancement in skin assessment can be achieved through development of multiprobe instrumental diagnostic tools and artificial intelligence capabilities. New instrumental approaches are also needed to understand the etiology of surfactantinduced effects. Understanding the biological mechanism and the sequence of events of skin alterations by cleansing products is important for developing milder cleansing products. Currently, few methods provide dermatologically relevant insights, and many lack sensitivity necessary for biological understanding. Noninvasive biological methods (cytological or biochemical techniques) appear to be potential candidates for discovering biochemical mediators of surfactant-induced effects in elucidating skin alterations. The instrumentation can increase the efficiency of biological methods and transform them into routine assessment techniques. SUMMARY Many methods exist for measuring changes in skin exposed to soaps or surfactants. The commercially available instruments that can be used in their clinical evaluation are listed in Table I. Instrumental measurements are becoming an industry standard for objective assessment of cleansing products. This literature review emphasizes commercial techniques that are useful in evaluating cleansing products. We expect the appearance of new instruments that will increase our current capabilities in the near future. REFERENCES (1) R. R. Suskind, Cutaneous effects of soaps and synthetic detergents, J. Am. Med Assoc., 163, (1957). (2) P. J. Frosch and A.M. Kligman, The soap chamber test: A new method for assessing the irritancy of soaps, J. Am. Acad. Dermatol., 1(1), (1979). (3) M. F. Lukacovic, F. E. Dunlap, S. E. Michaels, M. O. Visscher, and D. D. Watson, Forearm wash test to evaluate the clinical mildness of cleansing products, J. Soc. Cosmet. Chem., 39, (1988). (4) G. L. Grove, J. J. Leyden, P. T. Sharko, D. D. Strube, and K. L. Van Dyk, Correlation between instrumental and clinical methods for assessing the relative harshness of personal washing products, 48th Annual Meeting of the American Academy of Dermatology (abstr.)(1989).

19 INSTRUMENTAL ASSESSMENT OF SKIN 267 (5) M. B. Finkey and D. M. Crowe, The use ofevaporimetry to evaluate soap induced irritation on the face, Bioeng. Skin, 4, (1988). (6) P. T. Sharko, R. I. Murahata, J. J. Leyden, and G. L. Grove, Arm wash with instrumental evaluation--a sensitive technique for differentiating the irritation potential of personal washing products, 48th Annual Meeting of the American Academy of Dermatology (abstr.)(1989). (7) J. Nucci, Measurement of skin-surface viscoelasticity for claim support and product development, Cosmet. Toiletr., 104(3), (1989). (8) D. L. Miller, In vivo instrumentation in clinical dermal testing, Cosmet. Toiletr., 101, (1986). (9) R. M. Dahlgren, M. F. Lukacovic, S. E. Michaels, and M. O. Visscher, "Effects of Bar Soap Constituents on Product Mildness," in Proceedings of the Second World Conference Detergents, Montreux, Switzerland, A. R. Baldwin, Ed. (American Oil Chemists Society, 1987), pp (10) G. L. Grove, Design of studies to measure skin care product performance, Bioeng. Skin, 3(4), (1987). (11) R. O. Potts, Stratum corneum hydration: Experimental techniques and interpretations of results. J. Soc. Cosmet. Chem,, 37, 9-33 (1986). (12) G. E. Nilsson, Measurement of water exchange through skin. Med. Biol. Eng. Cornput., 15, , (1977). (13) E. P. Pittz, Non-destructive methods for evaluation of cutaneous irritancy, Cosmet. Toiletr., 98, 51-65, (1983). (14) R. I. Murahata, S. A. O. Hing, H. I. Maibach, and J. R. Roheim, The use of a microwave probe to evaluate the hydration of human stratum corneum in vivo, Bioeng. Skin, 2, (1986). (15) D. L. Miller, Impedance and water loss measurements, Biophysical Methods for Skin Characterisation Symposium, New York (abstr.)(1986). (16) D. Wilson, E. Berardesca, and H. I. Maibach, In vivo transepidermal water loss and skin surface hydration in assessment of moisturization and soap effects, Int. J. Cosmet. Sci,, 10(5), (1988). (17) M.D. Batt and E. Fairhurst, Hydration of the stratum corneum, Int. J. Cosmet. Sci., 8(6), (1986). (18) R. A. Tupker, J. Pinnagoda, P. J. Coenraads, and J.P. Nater, The influence of repeated exposure to surfactants on the human skin as determined by transepidermal water loss and visual scoring, Contact Dermatitis, 20, (1989). (19) M. Wu, D. J. Yee, and M. E. Sullivan, Effect of a skin moisturizer on the water distribution on human stratum corneum, J. Invest. Dermatol., 81, (1983). (20) J. L. Leveque, J. C. Garson, and J. de Rigal, Transepidermal water loss from dry and normal skin. J. Soc. Cosmet. Chem., 30, (1979). (21) J. Pinnagoda, R. A. Tupker, P. J. Coenraads, and J. P. Nater, Comparability and reproducibility of the results of water loss measurements: A study of 4 evaporimeters, Contact Dermatitis, 20(4), (1989). (22) D. R. Wilson and H. Maibach, "Transepidermal Water Loss: A Review," in Cutaneous Investigation in Health and Disease. Noninvasive Methods and Instrumentation, J. L. Leveque, Ed. (Marcel Dekker, New York and Basel, 1989), pp (23) J. Pinnagoda, R. A. Tupker, T. Agner, and J. Serup, Guidelines for transepidermal water loss (TWEL) measurement. A report from the standardization group of the European Society of Contact Dermatitis, Contact Dermatitis, 22, (1990). (24) S. W. Babulak, L. D. Rhein, D. D. Scala, F. A. Simion, and G. L. Grove, Quantitation of erythema in a soap chamber test using the Minolta Chroma (Reflectance) Meter: Comparison of instrumental results with visual assessments, J. Soc. Cosmet. Chem., 37, (1986). (25) S. Freeman and H. Maibach, Study of irritant contact dermatitis produced by repeat patch test with sodium lauryl sulfate and assessed by visual methods, transepidermal water loss, and laser Doppler velocimetry, J, Am. Acad. Dermatol., 19(3), (1988). (26) J. H. Hassing, J. P. Nater, and E. Bleumink, Irritancy of low concentrations of soap and synthetic detergents as measured by skin water loss, Dermatologica, 164, (1982). (27) T. Agner and J. Serup, Skin reactions to irritants assessed by non-invasive bioengineering methods, Contact Dermatitis, 20, (1989). (28) P. G. M. Van der Valk, J. P. Nater, and E. Bleumink, Skin irritancy of surfactants as assessed by water vapor loss measurements, J. Invest. Dermatol., 82, (1984). (29) R. A. Tupker, J. Pinnagoda, P. J. Coenraads, H. Kerstholt, and J. P. Nater, Evaluation of hand

20 268 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS cleansers: Assessment of composition, skin compatibility by transepidermal water loss measurements, and cleansing power, J. Soc. Cosmet. Chem., 40, (1989). (30) P. G. M. Van der Valk, M. C. Crijns, J. P. Nater, and E. Bleumink, Skin irritancy of commercially available soap and detergent bars as measured by water vapour loss, Dermatosen, 32, (1984). (31) R. I. Murahata, D. M. Crowe, and J. R. Roheim, The use of transepidermal water loss to measure and predict the irritation response to surfactants, Int. J. Cosmet. Sci., 8, (1986). (32) P. G. M. Van der Valk, M. H. Kruis-de Vries, J.P. Nater, E. Bleumink, and M. C. J. M. De Jong, Eczematous (irritant and allergic) reactions of the skin and barrier function as determined by water vapour loss, Clin. Exp. Dermatol., 10, (1985). (33) J. L. Leveque and J. De Rigal, Impedance methods for studying skin moisturization, J. Soc. Cosmet. Chem., 34, (1983). (34) A. W. Fulmer and G. J. Kramer, Stratum corneum lipid abnormalities in surfactant-induced dry scaly skin, J. Invest. Dermatol., 86(5), (1986). (35) H. Tagami, Y. Kanamaru, K. Inoue, S. Suehisa, F. Inoue, K. Iwatsuki, K. Yoshikuni, and M. Yamada, Water sorption-desorption test of the skin in vivo for functional assessment of the stratum corneum, J. Invest. Dermatol., 78, (1982). (36) H. Tagami, "Impedance Measurement for Evaluation of the Hydration State of the Skin Surface," in Cutaneous Investigation in Health and Disease. Noninvasive Methods and Instrumentation, J. L. Leveque, Ed. (Marcel Dekker, New York and Basel, 1989), pp (37) D. C. Salter, "Quantifying Skin Disease and Healing In Vivo Using Electrical Impedance Measurements," in Non-Invasive Physiological Measurements, P. Rolfe, Ed. (Academic Press, New York, 1979), Vol I., pp (38) G. Imokawa, S. Akasaki, A. Kawamata, S. Yano, and N. Takaishi, Water-retaining function in the stratum corneum and its recovery properties by synthetic pseudoceramides, J. Soc. Cosmet. Chem., 40, (1989). (39) S. L. Jacques, A linear measurement of the water content of the stratum corneum of human skin using a microwave probe, IEEE Eng. Med. Biol. Soc. Conf., (1979). (40) S. L. Jacques, D. Yeung, S. Nacht, I. H. Blank, and S. DeGirolamo, In vivo changes in skin surface water content, transepidermal water loss and mechanical properties with environmental relative humidity, Bioeng. Skin, 1(3), 260 (abstr.)(1984). (41) R. O. Potts, In vivo measurement of water content of the stratum corneum using infrared spectroscopy: A review, Cosmet. Toiletr., 100(10), (1985). (42) P. Walling, Principal component analysis of moisture in the skin by near infrared reflectance analysis, Seventeenth Annual Meeting of the Federation of Analytical Chemistry and Spectroscopy Societies, Cleveland, Ohio (abstr.) (1990). (43) V. H. W. Mak, R. O. Potts, and R. H. Guy, Oleic acid concentration and effect in human stratum corneum: Non-invasive determination by attenuated total reflectance infrared spectroscopy in vivo, J. Controlled Release, 12, (1990). (44) T. Kai, V. H. W. Mak, R. O. Potts, and R. H. Guy, Mechanism of percutaneous penetration enhancement: Effect of n-alkanols on the permeability barrier of hairless mouse skin, J. Controlled Release, 12, (1990). (45) A. Rosencwaig, Photoacoustic spectroscopy of biological materials, Science, 181, (1973). (46) M. A. Steinmetz and T. Adams, "Epidermal Water and Electrolyte Content and the Thermal, Electrical and Mechanical Properties of Skin," in Bioengineering and the Skin, R. Marks and P. A. Payne, Eds. (MTP Press Limited, Lancaster, UK, 1981), pp (47) H. C. Korting, M. Kober, M. Meuller, and O. Braun-Falco, Influence of repeated washing with soap and synthetic detergent on ph and resident flora of forehead and forearm, Acta Derm. Venereol. (Stockh), 67, (1987). (48) R. R. Wickett and C. M. Trobaugh, Personal care products: Effects on skin surface ph, Cosmet. Toilerr., 105, (1990). (49) E. Berardesca and H. I. Maibach, Bioengineering and the patch test, Contact Dermatitis, 18, 3-9 (1988). (50) P. Agache, I. Girardot, and J. C. Bernengo, "Optical Properties of the Skin," in Cutaneous Investigation in Health and Disease. Noninvasive Methods and Instrumentation, J. L. Leveque, Ed. (Marcel Dekker, New York and Basel, 1989), pp (51) D. M. Crowe, M. S. Willard, and R. I. Murahata, Quantitation of erythema by reflectance spectroscopy, J. Soc, Cosmet. Chem., 38, (1987). (52) G. E. Nilsson, E.G. Salerud, T. Tenland, P. A. Oberg, and J. E. Wahlberg, Laser Doppler

21 INSTRUMENTAL ASSESSMENT OF SKIN 269 flowmetry, a new technique for noninvasive assessment of skin blood flow, Cosmet. Toiletr., 99, (1984). (53) G. L. Grove, M. J. Grove, C. R. Zerweck, J. J. Leyden, Determination of topical tretinoin effects on cutaneous microcirculation in photoaged skin by laser Doppler velocimetry, J. Cut. Aging Cosmet. Derwtatol., 1(1), (1988). (54) G. E. Nilsson, U. Otto, and J. E. Wahlberg, Assessment of skin irritancy in man by laser Doppler flowmetry, Contact Dermatitis, 8, (1982). (55) B. Staberg and J. Serup, Allergic and irritant skin reactions evaluated by laser Doppler flowmetry, Contact Derwtatitis, 18, (1988). (56) R. Blanken, P. G. M. van der Valk, and J. P. Nater, Laser-Doppler flowmetry in the investigation of irritant compounds on human skin, Dermatosen, 34, 5-9 (1986). (57) D. van Neste, M. Masmoudi, B. Leroy, G. Mahmoud, and J. M. Lachapelle, Regression patterns of transepidermal water loss and cutaneous blood flow values in sodium lauryl sulfate induced irritation: A human model of rough dermatitic skin, Bioeng. Skin, 2(2), (1986). (58) C. M. Willis, C. J. M. Stephens, and J. D. Wilkinson, Assessment of erythema in irritant contact dermatitis. Comparison between visual scoring and laser Doppler flowmetry, Contact Dermatitis, 18, (1988). (59) M. Engelhart and J. K. Kristensen, Evaluation of cutaneous blood flow responses by 33Xenon washout and a laser-doppler flowmeter, J. Invest. Dermatol., 80, (1983). (60) S. R. Schwartz, The use of thermography as a means of evaluating topically applied products, 7th International Symposium of Bioengineering and the Skin, Milwaukee, WI (abstr.)(1988). (61) P. F. Millington and R. Wilkinson, "Mechanical, Thermal and Electrical Properties," in Skin (Cambridge University Press, Cambridge, 1983), pp (62) T. Agner and J. Serup, Contact thermography for assessment of skin damage due to experimental irritants, Acta Derm. Venereol. (Stockh), 68, (1988). (63) R. I. Murahata, J. G. Barrows, P. T. Sharko, and S. R. Schwartz, Visualization of surfactantinduced irritation by infrared thermography, Derwtal Clinical Evaluation Society, Elizabeth, NJ (abstr.) (1990). (64) J. L. Leveque, J. de Rigal, P. G. Agache, and C. Monneur, Influence of ageing on the in vivo extensibility of human skin at a low stress, Arch. Derwtatol. Res., 269, (1980). (65) J. L. Leveque, In vivo methods for measuring the viscoelastic properties of the skin, Bioeng. Skin, 3, (1987). (66) P. F. Millington and R. Wilkinson, "Mechanical Properties of Skin," in Skin (Cambridge University Press, Cambridge, 1983), pp (67) R. W. Shanahan and H. Schwartz, Parameters for assessing the efficacy of skin care products, Drug Cosmet. Ind., 140(1), (1987). (68) S. Dikstein and A. Hartzshtark, "In Vivo Measurement of Some Elastic Properties of Human Skin," in Bioengineering and the Skin, R. Marks and P. A. Payne, Eds. (MTP Press Limited, Lancaster, UK, 1981), pp (69) C. W. Hargens, C. G. Fthenakis, D. Maes, K. D. Marenus, and W. P. Smith, The ballistometer revisisted, applications to skin testing, 7th International Symposium of Bioengineering and the Skin, Milwaukee, WI (abstr.) (1988). (70) P. Eisner, D. Wilhelm, and H. I. Maibach, Mechanical properties of human forearm and vulvar skin, Br. J. Derwtatol., 122, (1990). (71) G. Jemec, B. Jemec, and J. Serup, The effect of superficial hydration on the rheology of human skin in-vivo, 7th International Symposium of Bioengineering and the Skin, Milwaukee, WI (abstr.) (1988). (72) J. L. Leveque, G. Grove, J. de Rigal, P. Corcuff, A.M. Kligman, and D. Saint Leger, Biophysical characterization of dry facial skin, J. Soc. Cosmet. Chem., 82, (1987). (73) J. L. Leveque, G. Porte, J. de Rigal, P. Corcuff, A.M. Francois, and D. Saint-Leger, Influence of chronic sun exposure on some biophysical parameters of the human skin: An in vivo study. J. Cut. Aging Cosmet. Dermatol., 1(2), (1988/89). (74) J. de Rigal and J. L. Leveque, In vivo measurement of the stratum corneum elasticity, Bioeng. Skin, 1, (1985). (75) C. W. Hatgens, "The Gas-Bearing Electrodynamometer (GBE) Applied to Measuring Mechanical Changes in Skin and Other Tissues," in Bioengineering and the Skin, R. Marks and P. A. Payne, Eds. (MTP Press Limited, Lancaster, UK, 1981), pp