Barrier function is the natural role of the skin. The lipid matrix present in the outermost layer of the skin, the stratum corneum is important for this function. Barrier impairment and altered... Show moreBarrier function is the natural role of the skin. The lipid matrix present in the outermost layer of the skin, the stratum corneum is important for this function. Barrier impairment and altered lipid composition are observed in several inflammatory skin diseases including atopic dermatitis and psoriasis. However, the relationship between the lipid properties and barrier function is not comprehended.In this project, a lipid model was prepared from synthetic lipids that closely resemble the stratum corneum lipid composition and organization. Subsequently, diseased skin models were developed to mimic various abnormalities in lipid composition observed in atopic dermatitis patients’ skin. Biophysical methods were used to monitor the changes in lipid organization in these models. Diffusion studies and trans-epidermal water loss measurements were performed to monitor the barrier function. This allowed the determination of the changes in lipid properties that were most instrumental in reducing the lipid barrier.This thesis further describes the use of simple skin lipid model membranes incorporating fewer components to provide a detailed insight into the relationship between lipid composition, lipid organization, and the skin barrier. The information gained in this project offers the opportunity to develop a new generation of formulations to treat these patients. Show less
The skin is our natural barrier and lipids are a key part of this barrier. In the outer skin layer, the stratum corneum (SC), lipids form a densely organized structure dependent on the composition... Show moreThe skin is our natural barrier and lipids are a key part of this barrier. In the outer skin layer, the stratum corneum (SC), lipids form a densely organized structure dependent on the composition of these lipids. Multiple skin diseases are characterized by alterations in SC lipid composition. These alterations are related to pathological barrier defects. This thesis describes the next steps towards a treatment modifying the lipid composition and thereby restoring this barrier. We developed a novel method to quantify a key SC lipid group called ceramides. This method was applied to compare SC regeneration of skin models to healthy volunteers. Regeneration in such an ex vivo skin model proved to be a potent model for formulation development. Ensuing, a clinical study was performed to determine the mechanistic effects of a formulation on barrier repair in healthy skin. The results warranted follow up analysis of the formulation in atopic dermatitis patients. This thesis also describes a detailed analysis of the ceramide fraction that is covalently attached to the cells in the SC. It was shown that a selected group of ceramides becomes bound. Further analysis showed that this group of ceramides was also affected in atopic dermatitis patients SC. Show less
The stratum corneum is the outermost skin layer and consists of dead cells embedded in a lipid matrix. The lipid matrix, consisting of ceramides, fatty acids, and cholesterol, is crucial for a... Show moreThe stratum corneum is the outermost skin layer and consists of dead cells embedded in a lipid matrix. The lipid matrix, consisting of ceramides, fatty acids, and cholesterol, is crucial for a proper skin barrier function. In inflammatory skin diseases the lipid composition and ordering is altered contributing to the impaired skin barrier. Vernix caseosa (VC) is the cheesy, white cream that covers the skin of the human fetus. Application of an in house developed synthetic VC enhanced skin barrier repair in mice. Currently, there are no suitable skin models available to study human skin barrier repair after application of a topical formulation. This thesis describes the development of a human skin barrier repair model and evaluates VC based formulations using this model. The results demonstrate that the barrier of this repair model mimics several aspects of inflammatory skin diseases. Additionally, it was shown that the lipid properties in this model were improved when a synthetic VC-based formulation was applied. Based on the outcome, clinical studies were performed. These studies showed that application on a disrupted human skin barrier in vivo enhanced the barrier repair. However, the effects of the formulation are limited when applied on atopic dermatitis skin Show less
The studies in this thesis describes the barrier defects in Atopic Dermatitis (AD) skin and various techniques to develop AD Human Skin Equivalents (HSEs) which can be used to better... Show more The studies in this thesis describes the barrier defects in Atopic Dermatitis (AD) skin and various techniques to develop AD Human Skin Equivalents (HSEs) which can be used to better understand the role of several factors in the pathogenesis of AD skin. The results described show that Inflammation plays a pivotal role in the development of epidermal and SC features of AD skin and that AD epidermal features can be maintained in vitro when AD skin biopsies are used to generate explant-HSEs. These AD-HSEs can also serve as a tool to screen potential therapeutics for AD and skin barrier repair. However, limitations exist in the complexity and full representation of all possible factors known to influence the development of AD e.g. FLG mutations, other aspects of inflammatory micro-environment, microbe colonization etc. Show less
Drug delivery across the skin is a challenging task because of the skin barrier. The skin barrier underlies in the outermost layer of the skin, the stratum corneum (SC). The lipids play a crucial... Show moreDrug delivery across the skin is a challenging task because of the skin barrier. The skin barrier underlies in the outermost layer of the skin, the stratum corneum (SC). The lipids play a crucial role in this barrier function. The focus of this PhD project was to elucidate the molecular structure of the lipid matrix present in the SC and to relate this structure with the barrier function. The lipid compositions selected for these studies were particularly chosen to understand the changes in barrier function in dry and diseased skin, i.e.,atopic eczema, Netherton syndrome compared to healthy skin. A variety of biophysical and analytical methods such as X-ray diffraction, neutron diffraction, infra-red spectroscopy, microscopy and LC/MS were combined to unravel the molecular structure. Diffusion studies and trans-epidermal water loss measurements were carried out to relate lipid organization with the lipid barrier. All the diffraction studies were performed in Grenoble, France at the ESRF (X-rays) and ILL (neutron). Neutron diffraction studies are in collaboration with King's College, University of London (Prof. J. Lawrence, Dr. D. Barlow). Show less
The skin barrier function strongly relies on the outermost layer of the skin, the stratum corneum (SC), which consists of dead corneocytes embedded in a highly organized extracellular lipid matrix.... Show moreThe skin barrier function strongly relies on the outermost layer of the skin, the stratum corneum (SC), which consists of dead corneocytes embedded in a highly organized extracellular lipid matrix. The lipids are thought to play a crucial role in the skin barrier function. This lipid matrix consists mainly of ceramides, cholesterol and free fatty acids in an approximately equimolar ratio. Atopic eczema (AE) is a chronic relapsing inflammatory skin disease that is characterized by dryness, erythema and pruritus. AE patients have a decreased skin barrier function as monitored with transepidermal water loss. The main objective of this thesis is to determine the SC lipid composition, lipid organization and lipid/protein ratio in AE patients and control subjects and to determine how these changes are associated with the impaired skin barrier function and disease severity of AE patients. The studies demonstrate that there is an altered lipid composition and lipid/protein ratio in non-lesional as well as lesional SC of AE patients. The changes in lipid composition result in an altered lipid organization that is associated with an impaired skin barrier function in AE patients Show less
Human skin equivalents (HSEs) are generated from isolated skin cells. As the primary function of the skin is to form a barrier, in this thesis the barrier properties of three HSEs were assessed and... Show moreHuman skin equivalents (HSEs) are generated from isolated skin cells. As the primary function of the skin is to form a barrier, in this thesis the barrier properties of three HSEs were assessed and compared with native human skin. The results show that all HSEs have a decreased skin barrier function compared to native human skin. Lipids in the outermost layer of the skin, the stratum corneum (SC), play a key role in this barrier function. The lipids in the HSEs are arranged in lipid lamellae, similarly as in human skin, but form a less crystalline organization. Investigation of the lipid composition reveals that all HSEs have an increased presence of mono-unsaturated fatty acids and reduced total fatty acid content compared to human SC, which most likely is responsible for the reduced density in lipid organization. Another group of lipids, the ceramides, show a comparable composition, although the HSEs have increa sed levels of acylceramides compared to native human SC. As we show that the culture conditions are of crucial importance for the SC lipid properties of HSEs, a future change is to optimize the culture conditions to improve epidermal lipid metabolism in HSEs, resulting in improved SC barrier properties Show less
The stratum corneum (SC), the thin uppermost layer of the skin, consists of dead flattened skin cells (corneocytes) embedded in a lipid matrix. The lipid matrix is considered to play a crucial role... Show moreThe stratum corneum (SC), the thin uppermost layer of the skin, consists of dead flattened skin cells (corneocytes) embedded in a lipid matrix. The lipid matrix is considered to play a crucial role in the skin barrier function. It consists of ceramides (CER), cholesterol (CHOL) and free fatty acids (FFA) forming crystalline lipid lamellae. From studies with native SC and SC lipid models much information has been gained on the phase behavior of the SC lipid matrix. However, little is known about the correlation between SC lipid organization and the permeability of the SC. This is difficult to investigate using native SC, due to its complex structure. Therefore SC lipids were casted on a porous membrane, resulting in a lipid organization and lamellar orientation similar to that in SC. This lipid membrane is referred to as the stratum corneum substitute (SCS). The SCS can be used to perform diffusion studies. Therefore, when modifying the lipid composition and thus the lipid organization in the SCS, it is possible to study the relationship between lipid organization and permeability. The main objectives of this thesis are 1) to investigate the influence of lipid organization on the barrier function in the SCS and 2) to obtain insights in the molecular organization within the unit cell of the lamellar phases in SC. Show less
In this thesis, the mode of action of stratum corneum moisturizers is studied using a variety of techniques: cryo-scanning electron microscopy, freeze fracture transmission electron microscopy,... Show moreIn this thesis, the mode of action of stratum corneum moisturizers is studied using a variety of techniques: cryo-scanning electron microscopy, freeze fracture transmission electron microscopy, small angle X-ray diffraction and Fourier transform infrared spectroscopy. Show less
