Application of biophysics and bioengineering to the assessment of skin barrier function

Atopic dermatitis (AD) is one of the most common inflammatory skin diseases. The cause of AD is multifactoral and it is affected by both genetic and environmental factors. Of all the causes of potential barrier defects, the lowered amino-acid derived natural moisturizing factor (NMF) in the stratum corneum (SC), especially associated with a known filaggrin mutation, shows the strongest link to AD. As a result, quantification of NMF in the SC in both healthy and compromised SC is the principal aim of this thesis. Because tape stripping is a key technique used to harvest the SC, a novel imaging method to measure the amount of SC per tape strip was validated. This method offers rapid, simple and reproducible SC quantification. It shows good correlation with existing gravimetric and infrared absorption methods and may provide a better standard method in the future. The tape-stripping extraction of NMF showed an abundant SC ‘reservoir’ of the constituents in healthy skin. Iontophoretic extraction of NMF was highly dependant upon molecular properties, particularly charge and concentration. In general, charged NMF constituents were easily extracted by reverse iontophoresis, whereas iontophoresis only offered modest enhancement of zwitterionic species. Quantification of NMF at different body sites, specifically forehead and forearm, showed similar profiles. However, forehead SC was thinner, and in general contained a lower total amount of NMF and less-ordered lipids. Forehead SC may therefore be considered a less competent barrier. A 3-week application of 0.1% w/v sodium lauryl sulphate (SLS) to healthy volunteers was used to model damaged skin similar to that in AD and chronic irritant contact dermatitis. The SC barrier post-treatment showed significantly reduced NMF, substantial lipid disordering, and the presence of immature corneocytes. The methods employed were sufficiently sensitive to detect these changes. In particular, the NMF components present at high levels in the SC may be useful, potential markers for skin ‘health’ and for its resistance to irritant chemicals.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

The scope and limitations of novel NMR techniques to characterise and quantify biologically active compounds in the 'Stratum corneum'

The accurate determination of biologically active compounds in the skin is of considerable importance when evaluating the penetration of skin health products through different layers of the skin. This thesisreportsonthecharacterisation andquantitation ofbiologically active compoundsinsimple model mixtures and complex mixtures which mimic that of a typical skin product, through the use of qNMR, pure shift NMR and DOSY techniques complemented by semi-automated software packages. Characterisation and quantitation conditions were acquired over several heterogeneous samples allowingfor analysisofhowdynamicrangeand complexity of different sample mixtures affect the Limits of Detection (LOD) and Limits of Quantitation (LOQ) of biologically active compounds. NMR is of particular value inthis task, as it is non-destructive, uses a primary ratio method for quantification, and tolerates a wide variety of hydrophilic and hydrophobic components within a given matrix. In this investigation we have attained a trueness level <10%, repeatability values of <1% and brought the limit of quantitation down to 100nM ( ̃limit of baseline range of several key biomarkers in the skin per litre seen in vivo), while commenting on the limitations observed, such as peakoverlap and sensitivity limits. Pure shift optimised sequences allow ustoreducepeakoverlapping, allowing further characterisation of individual compounds and the separation of complex mixtures using NMR. These validated methodologies are then all brought together to develop a new methodology for ex-vivo analysis of skin layers. This analysis allowed for characterisation and quantitation of natural moisturising factors (NMF), biomarkers of hydration and skin health alongside construction of permeation profiles for common topical components in different formulations. Data analysis demonstrates resultscorroboratewithresults seen in previous validated methodologies which havemore complex and time-consumingpreparation and data processingthan the proof of concept study presented here

Molecular dynamics modelling of skin and hair proteins

The binding free energy is one of the most important and desired thermodynamic properties in simulations of biological systems. The propensity of small molecules binding to macromolecules of human bio-substrates regulates their sub-cellular disposition. This subject is fundamental in transdermal permeation and hair absorption of cosmetic actives. Biomechanical and biophysical properties of hair and skin are related to keratin as their major constituent. A key challenge lies in predicting molecular and thermodynamic basis as the result of small molecules interacting with alpha helical keratin at the molecular level. In addition, elastic properties of human skin which are directly related to the interactions of keratin intermediate filaments remain a challenging subject. Molecular dynamics (MD) simulations provide a possibility of observing biological processes within atomistic resolution providing more detailed insight into experimental results. However, MD simulations are limited in terms of the achievable time scales. Hence, in this thesis MD simulations were employed in order to provide better understanding of the experimental results conducted in parallel and to overcome the main limiting factor of MD – the simulation time. For this purpose, thermodynamic and detailed structural basis have been delivered for small molecules interacting with keratin explaining and validating experimental data. On the top of this the fast free energy prediction tool has been built within all-atom force field by a use of steered molecular dynamics alone. Within the coarse grain approach, the force field was developed for the application of elastic properties of human skin enabling orders of magnitude faster than all-atom force fields simulations. The application of the coarser representation enabled assessing the influence of the natural moisturizing factor composed of small molecules on the elastic properties of the outermost human skin layer. In this work, MD results reached excellent agreement with the experimental data.Open Acces

The Role of Impaired Epidermal Barrier Function in Atopic Dermatitis

Atopic dermatitis (AD) is a chronic, inflammatory, pruritic skin disease with increasing prevalence. The etiopathogenesis of atopic dermatitis is multifactorial and involves a complex interplay of environmental and genetic factors that induce derangements in the structure and function of the epidermal barrier and immune system. Due to great heterogeneity of etiopathogenesis, there is also great variability of clinical presentation, and diagnosis can sometimes be challenging and difficult. Diagnosis mostly relies on clinical features and laboratory tests, but morphology alone cannot reliably establish the diagnosis, so the spectrum of features associated with AD must be considered. Traditionally, patients with AD have been separated into two different subgroups, i.e. intrinsic and extrinsic. Today, most of authors prefer the outside to inside and back to outside hypothesis, suggesting that the primary disorder lies in epidermal structure and function, resulting in inflammation and immunological downstream activation which further provokes secondary barrier abnormalities. In this review, we discuss the structure and function of the epidermal barrier and the role of impaired barrier function in etiopathogenesis of atopic dermatitis.  </p

The skin barrier: Tape stripping studies

The skin, being an exposed organ, is an easy site to access for research. However, standard interventions, such as a biopsy, leave the skin scarred and are logistically cumbersome. Therefore, non-invasive methods are preferable providing reliable in vivo data can be obtained. The study of the skin barrier is particularly suited to non-invasive techniques, as the ability of the skin to allow molecules to permeate is a dynamic process best seen in living tissue. A widely utilized method is the application of adhesive tapes to the skin. These tapes, when removed, take with them a small amount of stratum corneum that is adherent: this can be studied further, as can the remaining skin at the stripping site. Typical studies performed in this way include studies of "drugs reservoirs" in the stratum corneum (where lipophilic compounds accumulate), studies of the skin barrier to water and other substances, studies of transdermal drug delivery and studies of wound healing and stratum corneum physiology. There is no standardized method for tape stripping, resulting in confusion over technique and difficulty in interpreting other workers' data. Recently there have been attempts to improve the methodology. One such improvement by this laboratory has been the development of a method to quantify the exact amount of stiatum comeum on each tape. This information is expected to advance the use of the tape stripping model considerably particularly because other variables can now be measured against stratum corneum mass and depth. It is the aim of this thesis to take that work forward. The aim of this thesis is to build on a new model of stratum corneum tape stripping and demonstrate its usefulness as a method of studying the stratum corneum, both physiologically and phannacologically. Each of the studies performed has advanced the existing database of knowledge in this field. Study 1 has demonstrated that a variety of tapes can be successfully and generally equivalently used to study skin water kinetics. It has provided a database of information on tape stripping for future investigators and re-calculated water kinetics using the new quantitative data. In addition, this study has identified that a proportion of the population do not display increased transepidermal water loss despite significant barrier damage. This implies that the sheer mass of stratum corneum alone cannot explain the skin barrier to water, but rather there must be other factors such as the proportion of different types of lipids that must be responsible for this. Study 2 has, for the first time, demonstrated a quantitative method to assess the efficacy of keratolytic drugs. In addition, it has refuted a commonly held premise that acids applied to the skin must be at a pH near their pKa in order to retain their efficacy. In fact, this thesis shows that such practice results in increased skin irritation but no increase in efficacy, compared to less acidic preparations of the same acid. This data will allow those who formulate such drugs to make less irritating preparations. Study 3 has, for the first time, localized the acid mantle in vivo in human skin and also confirmed that this acid mantle is distinct from skin surface acidity. Also, this study has discovered a zone in the stratum corneum where pH is almost stable for 1?m, consistently between subjects, suggesting that common metabolic processes are taking place at this site. This information is of use to those who formulate drugs for transdermal delivery, to the study of skin metabolism and to the study of diseases with altered skin pH, such as atopic dermatitis. (Abstract shortened by ProQuest.)