Multi parametric biophysical assessment of treatment effects on xerotic skin.

Background: Topical moisturizing products are widely used to alleviate the problems associated with xerotic skin. Their use affects many properties of the stratum corneum (SC) in a complex and interrelated manner. The range of measurement techniques available to the researcher has increased in recent years. However, few studies have looked for correlations between the different techniques for assessing how aspects of xerotic skin change over time as a result of topical moisturizer usage. Objectives: A 3-week in vivo study using an oil-in-water based moisturizing product and an untreated site was conducted to determine the clinical significance of and any correlations between a range of different approaches for the measurement of skin lipid content and also skin hydration and visual grading of dry skin. Methods: A range of traditional and more recently developed skin measurement techniques have been used to examine a variety of SC properties in normal and xerotic skin during topical moisturizer usage. Results: In vivo confocal Raman spectroscopy and analysis of SC lipids from tape strips both showed an increase in SC lipid level and organization after 3 weeks of moisturizer usage on xerotic skin. Hydration, measured both optically and electrically, also increased and skin barrier function improved, with strong correlations between the different measures of dryness being observed. Conclusions: Strong correlations were observed between the skin measurements for lipid assessment and skin hydration with regard to the assessment of xerotic skin, providing valuable new information for future in vivo clinical research into dry and atopic skin. Keywords biophysical assessment, skin barrier, skin hydration, topical moisturizers, Xerosis.

UV reflectance photography of skin: what are you imaging?

OBJECTIVE: Photography can be a powerful tool for researching the skin. Moving outside the visible spectrum and into the ultra violet (UV) presents a unique set of challenges to the skin photographer because of the restrictions imposed by the equipment being used. This article discusses these challenges in relation to camera sensitivity, lens and filter transmission and lighting spectrum, with the aim of demystifying what is actually being captured when imaging skin. METHODS: In addition to a discussion of existing data on the subject of camera sensor sensitivity, filter transmission and flash spectral analysis, transmission in the UV of a variety of camera lenses using a new method has been carried out. RESULTS: Using the described approach, lens transmission between 280 and 420 nm of a range of lenses has been measured. Combining this with camera sensor sensitivity data and filter and light source characteristics, it has been possible to determine an overall, harmonized, spectral sensitivity curve for what is being imaged with a given setup. CONCLUSIONS: UV reflectance photography, while a powerful tool, is often misunderstood and misreported as to what is actually being imaged. By combining measurements on camera sensitivity, lens and filter transmission and light source spectra the researcher can more fully understand what is they are actually measuring, thereby enabling better communication with the consumer on what they are seeing and a more complete description for any claims support.

OBJECTIF: La photographie peut constituer un outil puissant pour effectuer des recherches sur la peau. Toutefois, qui veut dépasser le spectre visible et photographier l'ultraviolet (UV) doit faire face à un ensemble unique de défis dus aux limites imposées par l'équipement utilisé. Cet article présente ces défis, en lien avec la sensibilité de l'appareil photo, la/les lentille(s), le filtre de transmission et le spectre lumineux afin de mieux comprendre ce qui est enregistré par l'appareil lorsque l'on prend une photo de la peau. MÉTHODES: Après un examen des données existantes sur la sensibilité des capteurs d'appareil photo, les filtres de transmission et les analyses spectrales flash, une nouvelle méthode est essayée afin d'éprouver la transmission des UV via diverses lentilles photo. RÉSULTATS: À l'aide de l'approche décrite, des transmissions d'entre 280 et 420 nm ont été mesurées pour plusieurs lentilles. En combinant ces mesures avec les données de sensibilité du capteur de l'appareil photo ainsi que les caractéristiques des filtres et de la source lumineuse, on a pu déterminer une courbe harmonisée de la sensibilité spectrale pour ce qui est photographié dans des circonstances données. CONCLUSIONS: Si la photographie UV par réflectance constitue un outil puissant, elle demeure souvent mal connue, et on comprend mal ce qui est réellement pris en photo. En combinant les mesures de la sensibilité de l'appareil photo, de lentilles, de filtres de transmission et de spectres lumineux des sources, le chercheur peut mieux comprendre ce qu'il mesure réellement, ce qui permet une meilleure communication avec le client quant au(x) résultat(s) des photographies et une description plus exhaustive en cas de réclamation.

3D-printed Franz type diffusion cells.

OBJECTIVE: Franz cells are routinely used to measure in vitro skin permeation of actives and must be inert to the permeant under study. The aim of the present work was to develop and manufacture transparent Franz-type diffusion cells using 3D printing. Printouts were then tested using a range of model active compounds. The study also aims to identify the critical 3D-printing parameters necessary for the process, including object design, choice of printing resin, printout curing and post-curing settings and introduction of model coatings. METHODS: Transparent Franz cells were constructed using an online computer aided design program and reproduced with different stereolithography 3D printers. The two acrylate-based resins used for the fabrication process were a commercially available product and a polymer synthesised in-house. Comparative studies between glass and 3D-printed Franz cells were conducted with selected model actives: terbinafine hydrochloride (TBF), niacinamide (NIA), diclofenac free acid (DFA) and n-methyl paraben (MPB). In preliminary studies, MPB showed the lowest recovery when exposed to the receptor compartment of 3D printed cells. Consequently, in vitro permeation studies were carried out using only MPB with polydimethylsiloxane (PDMS) membrane. RESULTS: A decrease in the amounts of selected compounds was observed for transparent 3D-printed Franz cells compared to glass cells. MPB showed the lowest recovery (53.8 ± 13.1%) when compared with NIA (74.9 ± 4.0%), TBF (81.5 ± 12.0%) and DFA (90.2 ± 12.9%) after 72 h. Permeation studies conducted using 3D-printed transparent cells with PDMS membrane also showed a decrease in MPB recovery of 51.4 ± 3.7% for the commercial resin and 94.4 ± 3.5% for the polymer synthesised in-house, when compared to glass cells. Although hydrophobic coatings were subsequently applied to the 3D-printed cells, the same reduction in MPB concentration was observed in the receptor solution. CONCLUSION: Transparent Franz cells were successfully prepared using 3D printing and were observed to be robust and leak-proof. There are few resins currently available for preparation of transparent materials and incompatibilities between the actives investigated and the 3D-printed cells were evident. Hydrophobic coatings applied as barriers to the printed materials did not prevent these interactions.

Calibrating UVA reflectance photographs - standardisation using a low-cost method.

OBJECTIVE: While established methods for the calibration of visible light photographs are well defined, the use of these approaches in UVA reflectance photography is less well understood. A systematic, low-cost and simple method for the production of well-defined grey calibration standard targets for UVA reflectance photography, with a particular emphasis on low reflectivity surfaces, along with a comparison with standard visible light photographic standards is presented here. METHODS: Grey calibration standard targets suitable for use in the UVA region were produced, based on optimised methods from the literature. The standards were assessed using UV-Visible reflection spectroscopy, and visible and UVA light photography, and their behaviour compared with a commercially available visible light photographic calibration chart. RESULTS: Calibration standards with a relatively flat reflection response in the UVA region with a variety of reflectances between 2% and 35% were prepared. Imaging of the standards in both UV and visible light demonstrated the differences between these standards and the ones specifically designed for visible light photography. CONCLUSIONS: A low cost and simple method for the production of low reflectance UVA calibration targets, suitable for UVA reflectance photography has been described and tested against commercially available visual light calibration standards. These new UV suitable standards have potential for use in a wide range of applications such as forensics, biology and cosmetic science.

Understanding sunscreen SPF performance using cross-polarized UVA reflectance photography.

OBJECTIVES: Objective methods for understanding sunscreen behaviour in vitro before they are applied to the skin have failed to keep pace with the ever-increasing demands for higher SPF scores where the products are absorbing more and more similar levels of UV. A novel method for visualizing the spreading and location of SPF ingredients based on cross-polarized UVA reflectance photography is described here which gives new insights into the formation of final film morphology and how it correlates with in vivo SPF efficacy for a set of test products. METHODS: High-resolution UVA-based images of sunscreen films spread onto PMMA plates were captured using a modified commercial SLR camera in a custom imaging system. Visual grading and image analysis were used to describe the overall UVA absorbance and streakiness of the resultant films, and the data compared with both in vivo and calculated in vitro SPF scores for the products. RESULTS: Differences were observed between the products in terms of how they spread during application. A strong correlation was observed between the evenness of the resultant film as determined from the photographs and final in vivo SPF scores. CONCLUSIONS: Cross-polarized UVA reflectance photography has been demonstrated to be a valuable new method for assessing sunscreen distribution after spreading and to differentiate product based on film morphology, as well as strongly correlating with final in vivo behaviour.

Understanding the effects of topography on skin moisturization measurement via two-dimensional capacitance imaging.

OBJECTIVE: Methods which assess skin moisturization based on changes in its electrical properties are widely used in both cosmetic and medical research industries. However, the devices themselves often give results which are significantly different to each other. Recently two-dimensional imaging moisturization systems have become commercially available, which have the capability to provide a more detailed assessment of what is contributing to measured skin moisturization. Presented here is a new in vitro method for preparing textured model test substrates for use with these devices, and results of their use to provide a clearer insight into the devices operation. METHODS: A variety of different textured model test substrates were measured using a commercially available skin moisturization measurement device, the Epsilon. The response of the Epsilon was also tested against conventional skin moisturization devices. RESULTS: Surface morphology of model test substrates was found to have a significant influence on the measurement of its electrical properties with both the conventional and two-dimensional skin moisturization measurement devices. Through modification of the areas of the image being assessed for the two-dimensional moisturization mapping device, the parts of the model test substrate in contact with the device were indentified and analysed separately to areas not in contact with the sensor. This provided a more robust assessment of the electrical properties of substrate itself, rather than being influenced by texture like the conventional skin moisturization measurement devices. CONCLUSIONS: While the two-dimensional moisturization mapping systems can be used like a conventional electrical skin measurement device giving a simple overall reading of skin moisturization for the test area, their true value over existing electrical measures comes from its ability to isolate the skin itself from areas which are not in contact with the sensor.

Chemical ultraviolet absorbers topically applied in a skin barrier mimetic formulation remain in the outer stratum corneum of porcine skin.

The objective of the present study was to evaluate the fate of three chemical sunscreens, isoamyl p-methoxycinnamate (IPMC), diethylamino hydroxybenzoyl hexyl benzoate (DHHB), and bis-ethylhexylphenol methoxyphenyl triazine (BEMT), topically applied to mammalian skin from a skin barrier mimetic oil-in-water formulation. High Performance Liquid Chromatography (HPLC) methods were developed for the analysis of each molecule and validated. Franz cell permeation studies were conducted following application of finite doses of the formulations to excised porcine skin. A vehicle formulation containing no sunscreens was evaluated as a control. Permeation studies were conducted for 12h after which full mass balance studies were carried out. Analysis of individual UV sunscreens was achieved with HPLC following application of the formulation to the skin with no interference from the vehicle components. No skin permeation of any of the chemical sunscreens was evident after 12h. While sunscreens were detected in up to 12 tape strips taken from the SC, 87% or more of the applied doses recovered in the first 5 tape strips. When corrected for the amount of protein removed per tape strip this corresponded to a penetration depth in porcine stratum corneum of ∼1.7µm. Mass balance studies indicated total recovery values were within accepted guidelines for cosmetic formulations. Overall, only superficial penetration into the SC was observed for each compound. These findings are consistent with the physicochemical properties of the selected UV absorbing molecules and their formulation into an ordered biomimetic barrier formulation thus support their intended use in topical consumer formulations designed to protect from UV exposure. To our knowledge this is the first report of depth profiling of chemical sunscreens in the SC that combines tape stripping and protein determination following in vitro Franz cell studies.

Understanding effects of topical ingredients on electrical measurement of skin hydration.

OBJECTIVE: Methods that assess skin hydration based on changes in its electrical properties are widely used in both cosmetic and medical research. However, the devices themselves often give results which are significantly different to each other. Although some work has previously been carried out to try and understand what these devices are actually reading, it was based on a technique for measuring the devices' responses to filter discs impregnated with different liquids, which could in itself be influencing the measurements. Presented here is a new method for measuring the devices' direct responses to different materials and solutions which removes any other confounding effects, thereby providing a clearer insight into their operation. METHODS: The responses of a variety of different liquids and solutions were measured using the Corneometer® and Skicon® . A new method is presented, based on the use of a custom-designed PTFE block to hold the liquids, allowing their measurement without using a filter paper. This method was developed and tested against the existing filter paper-based approach. RESULTS: Differences were observed in results between filter paper- and PTFE block-based approach, indicating that the filter paper itself is capable of influencing the measurements and as such is not to be recommended for assessing how different liquids impact on results from the devices. A positive correlation was observed between Corneometer® and Skicon® readings for certain solutions and under certain conditions. A large influence of salt concentration was noted for the Skicon® device with no or minimal impact from the actual water itself, humectants and emollients. Salts, emollients, water and humectants were observed to have an effect on Corneometer® readings. CONCLUSIONS: Both the Corneometer® and Skicon® were influenced to different extents by chemicals other than water and therefore cannot be seen purely as measures of skin 'hydration'. Although there is strong evidence that the devices do correlate with expert assessment of skin dryness, the level of water in the skin is only part of the story when it comes to understanding the benefits of topical moisturizing products applied to the skin. An alternative approach would be to consider skin 'moisturization' as a property which is influenced by water, salts and other materials such as humectants and emollients, which is more consistent with how the stratum corneum itself helps to maintain its plasticity and flexibility. In the work presented here, the Corneometer® was more suited to providing a measurement which reflects the impact of multiple different components.

Method for quantification of oils and sebum levels on skin using the Sebumeter(®).

OBJECTIVE: The Sebumeter(®) is widely used in both cosmetic and medical research, for measuring changes in sebum levels on skin. It is commonly reported that the units correlated to a mass of sebum on the skin in µg cm(-2) ; however, validation for this has not been published. Also, its use for assessing the presence of other oily materials which are widely utilized in topical skincare products on skin has not been widely discussed. Determining a calibration scale and whether the response of the device is linear with the level of oils present enables quantification of the output of the device, and would validate the device for claims substantiation. METHODS: Different doses of a variety of oily materials (paraffin oil, white soft paraffin, capric-caprylic triglyceride, 350cSt silicone fluid and synthetic sebum) were applied to skin, and the Sebumeter(®) used to collect and quantify them. The mass per square centimetre of the oily material delivered to the skin was then compared to the Sebumeter(®) output to develop calibration curves for the different materials. Measurements were carried out on a single volunteer as this work was to verify the concept of quantitative oil assessment using the device. RESULTS: A linear correlation between the mass of the oily material and the Sebumeter(®) output was seen for all the materials tested. However, the absolute response of the device was different for each material, and the output values did not directly give the mass of material on the skin in µg cm(-2) . As part of the calibration, it was also demonstrated that to remove all the oily material from a given area of the skin required multiple 30-s applications of the Sebumeter(®) cartridge. CONCLUSIONS: The Sebumeter(®) is a precise analytical instrument capable of quantitative measurement of deposition of oily materials onto skin from topical products (down to the µg cm(-2) level), as well as its traditional use of measuring sebum levels. However, the output values do not directly correlate with the mass of oil present, and generation of a calibration curve is necessary for any ingredient of interest to produce quantitative data for claim support and formulation development.

Influence of niacinamide containing formulations on the molecular and biophysical properties of the stratum corneum.

Niacinamide-containing moisturisers are known be efficacious in alleviating dry skin conditions and improving stratum corneum (SC) barrier function. However, the mechanisms of action of niacinamide at the molecular level in the SC are still not well understood. Previously, we have reported the development of novel methods to probe SC barrier properties in vivo. The aim of the present study was to characterise changes in Trans Epidermal Water Loss (TEWL), corneocyte surface area and maturity, selected protease activities and SC thickness after repeated application of a simple vehicle containing niacinamide. A commercial formulation was also included as a reference. The left and right mid-volar forearms of 20 healthy volunteers were used as study sites, to which topical formulations were applied twice daily for 28 days. After successive tape-stripping, corneocyte maturity and surface area were assessed. In addition, activity of the desquamatory kallikrein (KLK) protease enzymes KLK5 and KLK7, and tryptase and plasmin (implicated in inflammatory process) were measured using a fluorogenic probe assay. The amount of protein removed and TEWL were also recorded. SC thickness before and after treatment was determined using Confocal Raman Spectroscopy (CRS). Overall (i) corneocyte maturity and surface area decreased with increasing number of tape strips, (ii) activity of both the desquamatory and inflammatory enzymes was highest in the outer layers of the SC and decreased with depth (iii) TEWL increased as more SC layers were removed. Furthermore, areas treated with formulations containing niacinamide were significantly different to pre-treatment baseline and untreated/vehicle-control treated sites, with larger and more mature corneocytes, decreased inflammatory activity, decreased TEWL and increased SC thickness. These data (a) confirm the utility of measures and metrics developed previously for the non-invasive assay of SC barrier function, (b) present an holistic picture of a SC compartment managing barrier function through dynamic optimisation of pathlength and quality of building materials used, and (c) shed new light on niacinamide as a topical formulation adjunct with unique SC barrier-augmentation properties.

Measuring the effects of topical moisturizers on changes in stratum corneum thickness, water gradients and hydration in vivo.

BACKGROUND: Moisturizers are the most commonly used topically applied product for the treatment of dry skin conditions. They affect many properties and functions of the stratum corneum but some moisturizers have been reported to be detrimental to barrier function. Stratum corneum barrier function is a composite of its total structure and thickness but few studies have taken this into account. As a biosensor, the stratum corneum (SC) will change its structure in response to treatment and a swelling effect has been clearly demonstrated by skin hydration. Recently several moisturizing agents have been shown to have an effect on SC swelling behaviour with conflicting results. However, there is a paucity of data reported for measuring the effects of long-term usage of moisturizers on SC thickness in vivo as, until recently, traditional techniques did not have the resolution to measure the effects of moisturizers on nonpalmoplantar body sites. The development of confocal Raman spectroscopy for use in human subjects provides noninvasive, real-time, in vivo measurement of SC water concentration profiles and we have also used this state of the art equipment to measure the effect of the long-term use of moisturizers on SC thickness for the first time. OBJECTIVES: To validate the use of confocal Raman spectroscopy (CRS) to measure SC thickness and then use it to investigate the short- and long-term effects of moisturizers (one of which is known to improve SC barrier function) on SC thickness, water gradients and hydration. METHODS: Two studies were conducted: (i) to validate the use of CRS for measuring SC thickness through comparison with optical coherence tomography (OCT); and (ii) once validated to use CRS to measure the long-term effects of three commercially available moisturizers (A, B, C) on SC thickness and water gradients, together with total hydration, over a 3-week period (2 weeks of treatment and 1 week regression) and compare the spectroscopy-derived hydration value with instrumentally derived capacitance hydration values. RESULTS: (i) A strong, positive correlation in SC thickness was obtained between CRS and OCT (OCT-derived thickness = 0.96 x CRS-derived thickness, r(2) = 0.93; P <0.0001). OCT was shown, however, to have a lower resolution than CRS in distinguishing SC thickness on thinner nonpalmoplantar body sites. Using the CRS method, differences in SC thickness were readily apparent on different body sites (cheek 12.8 +/- 0.9 microm, volar forearm 18.0 +/- 3.9 microm, leg 22.0 +/- 6.9 microm). (ii) Examining the effects of moisturizers in a blinded, randomized 3-week study in human volunteers (n = 14) demonstrated that only one commercially available formulation (A) changed SC water gradients, thickness and hydration as measured by CRS. These hydration data did not directly correlate with capacitance hydration values. CONCLUSIONS: (i) In vivo CRS was validated as a technique to measure SC thickness on both palmoplantar and, particularly, on nonpalmoplantar skin sites. (ii) Moisturizers improve skin moisturization but in this study only formulation A improved SC thickness, water gradients and hydration as measured by CRS. We hypothesize that this was due to compositional differences between the products. We believe that niacinamide (nicotinamide, vitamin B(3)) is probably contributing significantly to this effect, as it has been proven to increase epidermal lipogenesis and SC barrier function in other studies. These results show that by using CRS, we were able for the first time to determine the effect of moisturizer on multiple SC barrier endpoints including SC thickness, and water content as a function of depth and total SC water content.

On the interpretation of event and sub-event rainfall chemistry.

Variations in precipitation chemistry between and within rain events have been examined in order to identify possible relationships with synoptic, mesoscale and micrometeorological processes. A microprocessor-based acid rain monitor was used to provide high resolution meteorological and rain chemistry data from which two case study events have been selected to illustrate event and sub-event rainfall chemistry characteristics. Event rainfall chemistry is strongly influenced by the history of the prevailing air mass and the synoptic situation. From back trajectories calculated at the 950 mbar level it is clear that air mass history can change markedly within a few hours. These observations emphasise the value of high resolution rainfall chemistry measurements. Pollutant concentrations in rainwater have been shown to fluctuate markedly within the course of individual events as a result of both advective and scavenging processes. Advective effects may result from: (a) air mass discontinuities at frontal zones; and/or (b) variable rainfall interception of the air mass prior to arrival at the site. A simple mathematical model has been developed to describe the scavenging mechanisms and it shows good agreement with field observations. Theoretical considerations suggest that in-cloud processes give rise to most of the observed decline in concentrations.