Biomedical subjects

M Ponec

Publications and source records attributed to M Ponec.

At least 19 recordsLinked to original sources

PubMed2004

Human adipose tissue-derived cells delay re-epithelialization in comparison with skin fibroblasts in organotypic skin culture.

BACKGROUND: Wound healing of deep and extensive burns can induce hypertrophic scar formation. During the early steps of wound healing fibroblasts migrate into the wounded area. Fibroblastic cells present in tissues other than dermis may also migrate into the wounded area and participate in the wound healing process. OBJECTIVES: To examine the influence of human fibroblastic cells derived from subcutaneous fat or dermis on epidermal morphogenesis in vitro. METHODS: We prepared human skin equivalents (HSEs) made of a collagen type I matrix populated either with dermal fibroblasts or adipose tissue-derived cells (ADCs), on top of which keratinocytes were seeded and subsequently grown at the air-liquid interface. RESULTS: A fully differentiated epidermis was formed on matrices populated with ADCs. However, the HSE formed differed in a number of features from HSE generated with dermal fibroblasts. The major differences included: marked contraction of the dermal matrix, low lateral migration of keratinocytes, high keratin 17 expression indicating increased keratinocyte activation, delayed deposition of collagen IV at the epidermal/matrix junction, accumulation of alpha-smooth muscle actin-positive cells only underneath the epidermal compartment and positioning of these cells in a direction parallel to the epidermal compartment. The latter two phenomena have also been found in scar tissue. CONCLUSIONS: The possibility of generating HSEs with different cell types represents an attractive approach for in vitro studies focusing on the mechanism of wound healing.

Actins↗

PubMed2003

The phase behaviour of skin lipid mixtures based on synthetic ceramides.

The lipid lamellae present in the outermost layer of the skin, the stratum corneum (SC), form the main barrier for diffusion of molecules across the skin. The main lipid classes in SC are cholesterol (CHOL), free fatty acids (FFA) and at least nine classes of ceramides (CER), referred to as CER1 to CER9. In the present study the phase behaviour of four synthetic CER, either single or mixed with CHOL or CHOL and FFA, has been studied using small and wide angle X-ray diffraction. The lipid mixtures showed complex phase behaviour with coexistence of several phases. The results further revealed that the presence of synthetic CER1 as well as a proper composition of the other CER in the mixture were crucial for the formation of a phase with a long periodicity, characteristic for SC lipid phase behaviour. Only a mixture containing synthetic CER1 and CER3, CHOL and FFA showed similar phase behaviour to that of SC.

Ceramides↗

PubMed2002

Non-animal testing strategies for assessment of the skin corrosion and skin irritation potential of ingredients and finished products.

The dermatotoxicologist today is faced with a dilemma. Protection of workers and consumers from skin toxicities (irritation and allergy) associated with exposure to products, and the ingredients they contain, requires toxicological skin testing prior to manufacture, transport, or marketing. Testing for skin corrosion or irritation has traditionally been conducted in animals, particularly in rabbits via the long established Draize test method. However, this procedure, among others, has been subject to criticism, both for its limited predictive capacity for human toxicity, as well as for its use of animals. In fact, legislation is pending in the European Union which would ban the sale of cosmetic products, the ingredients of which have been tested in animals. These considerations, and advancements in both in vitro skin biology and clinical testing, have helped drive an intensive effort among skin scientists to develop alternative test methods based either on in vitro test systems (e.g. using rat, pig or human skin ex vivo, or reconstructed human skin models) or ethical clinical approaches (human volunteer studies). Tools are now in place today to enable a thorough skin corrosion and irritation assessment of new ingredients and products without the need to test in animals. Herein, we describe general testing strategies and new test methods for the assessment of skin corrosion and irritation. The methods described, and utilized within industry today, provide a framework for the practicing toxicologist to support new product development initiatives through the use of reliable skin safety testing and risk assessment tools and strategies.

Animal Testing Alternatives↗

PubMed2002

Proteomic analysis of skin irritation reveals the induction of HSP27 by sodium lauryl sulphate in human skin.

BACKGROUND: There is an increasing need for screening of mild irritants in vitro to reduce animal testing. OBJECTIVES: Proteomics were used to search for new markers of which the expression changes after mild irritation. METHODS: Sodium lauryl sulphate (SLS) was applied topically on excised human skin. Epidermal proteins were isolated from SLS-treated skin specimens that showed hardly any morphological changes. The proteins were analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and proteins that significantly increased or decreased after SLS treatment in a dose-dependent way were characterized by mass spectrometry. Subsequently, immunohistochemistry was performed on skin samples treated with SLS in vivo and nonanoic acid (NAA) or benzalkonium chloride (BC) in vitro to evaluate one of the identified proteins for its predictive value. RESULTS: We identified seven proteins as potentially new epidermal markers for skin irritation. Among these seven proteins, the 27 kDa heat shock protein (HSP27) was identified as the most prominently upregulated protein. A strong nuclear HSP27 staining was seen in the SLS-treated skin, whereas in the vehicle controls only cytoplasmic staining was observed. Moreover, nuclear staining was also observed after topical application of SLS in vivo and after exposure to NAA and BC in vitro. CONCLUSIONS: Our findings suggest that HSP27 may serve as a sensitive marker of skin irritation and eventually as a novel tool in clinics for testing the sensitivity of the patient for a panel of irritants.

Animal Testing Alternatives↗

PubMed2002

Effect of fibroblasts on epidermal regeneration.

BACKGROUND: There is little information on specific interactions between dermal fibroblasts and epidermal keratinocytes. The use of engineered skin equivalents consisting of organotypic cocultures of keratinocytes and fibroblasts offers an attractive approach for such studies. OBJECTIVES: To examine the role fibroblasts play in generation and maintenance of reconstructed epidermis. METHODS: Human keratinocytes were seeded on collagen matrices populated with increasing numbers of fibroblasts and cultured for 2 weeks at the air-liquid interface. RESULTS: In the absence of fibroblasts, stratified epidermis with only three or four viable cell layers was formed. In the presence of fibroblasts, keratinocyte proliferation was stimulated and epidermal morphology was improved. Epidermal morphogenesis was also markedly improved in epidermis generated in organotypic keratinocyte monocultures grown in medium derived from dermal equivalents or from organotypic keratinocyte-fibroblast cocultures. These observations clearly indicate the proliferation-stimulating activity of soluble factors released from fibroblasts. Under all experimental conditions, onset of keratinocyte differentiation was shown by the expression of keratin 10 in all suprabasal cell layers. With increasing numbers of fibroblasts incorporated into the collagen matrix, the expression of markers associated with keratinocyte activation, e.g. keratins 6, 16 and 17 and the cornified envelope precursor SKALP decreased, and involucrin localization shifted toward the granulosum layer. This fibroblast-mediated effect was even more pronounced when the fibroblasts were precultured in the collagen matrices for 1 week instead of overnight. The basement membrane proteins collagen VII and laminin 5 were present at the epithelial-matrix border. The expression of integrin alpha 6 beta 4 and of E-cadherin was comparable with that seen in native skin and was not significantly modulated by fibroblasts. Under all experimental conditions the expression of integrin subunits alpha 2, alpha 3 and beta 1 was upregulated, indicating keratinocyte activation. CONCLUSIONS: Our results illustrate that numbers of fibroblasts in the collagen matrix and their functional state is a critical factor for establishment of normal epidermal morphogenesis.

Biomarkers↗

PubMed2001

Determination of stratum corneum lipid profile by tape stripping in combination with high-performance thin-layer chromatography.

Intercellular lipids in the stratum corneum (SC) are responsible for the barrier function of mammalian skin. The main components of the SC lipids are ceramides, cholesterol, and free fatty acids, as established by thin-layer chromatographic analysis of lipids extracted from the human and mammalian SC. Up to now, for lipid analysis the extracts of the entire SC has been used and information on whether the lipid composition changes with the depth in the SC is scarce. Tape stripping is a technique which removes corneocyte layers step by step with an adhesive film. The use of this technique for lipid analysis was hampered by the contamination of lipid extracts with compounds co-extracted from the tape with organic solvents used for the extraction of SC lipids. The aim of the present study was to establish a suitable analytical method for the determination of the local SC lipid composition. For this purpose, the SC samples were collected by sequential stripping with Leukoplex tape in five healthy volunteers. The lipids were extracted with ethyl acetate:methanol mixture (20:80) and separated by means of HPTLC. The results of this study revealed that the free fatty acid level is highest and the cholesterol and ceramide levels lowest in the uppermost SC layers (about 4 strippings). The levels remained unchanged in the underlying SC layers. In these layers, the ceramide level was about 60 wt% and the free fatty acid and cholesterol levels were about 20 wt% each. Ceramides could be separated into seven different fractions and the relative amounts of individual ceramide fractions did not significantly change with the SC depth. Cholesterol sulfate levels were about 5% of total cholesterol and did not change with the SC depth, except for the for the first strip where the level was about 1%. The method developed makes it possible to study the differences in the SC lipid profile in healthy and diseased human skin with relation to the SC lipid organization and to the skin barrier function in vivo.

Ceramides↗

PubMed2001

New aspects of the skin barrier organization.

In the superficial layer of the skin, the stratum corneum (SC), the lipids form two crystalline lamellar phases with periodicities of 6.4 and 13.4 nm (long-periodicity phase). The main lipid classes in SC are ceramides, free fatty acids and cholesterol. Studies with mixtures prepared with isolated ceramides revealed that cholesterol and ceramides are very important for the formation of the lamellar phases, and the presence of ceramide 1 is crucial for the formation of the long-periodicity phase. This observation and the broad-narrow-broad sequence of lipid layers in the 13.4-nm phase led us to propose a molecular model for this phase. This consists of one narrow central lipid layer with fluid domains on both sides of a broad layer with a crystalline structure. This model is referred to as 'the sandwich model'. While the presence of free fatty acids does not substantially affect the lipid lamellar organization, it is crucial for the formation of the orthorhombic sublattice, since the addition of free fatty acids to cholesterol/ceramide mixtures results in transition from a hexagonal to a crystalline lipid phase. Studies examining lipid organization in SC derived from dry or lamellar X-linked ichthyosis skin revealed that in native tissue the role of ceramide 1 and free fatty acids is similar to that observed with mixtures prepared with isolated SC lipids. From this we conclude that the results obtained with lipid mixtures can be used to predict the SC lipid organization in native tissue.

Animals↗

PubMed2001

Barrier function in reconstructed epidermis and its resemblance to native human skin.

One of the prerequisites for the use of human skin equivalents for scientific and screening purposes is that their barrier function is similar to that of native skin. Using human epidermis reconstructed on de-epidermized dermis we demonstrated that the formation of the stratum corneum (SC) barrier in vitro proceeds similarly as in vivo as judged from the extensive production of lamellar bodies, their complete extrusion at the stratum granulosum/SC interface, and the formation of multiple broad lamellar structures in the intercorneocyte space. The presence of well-ordered lipid lamellar phases was confirmed by small-angle X-ray diffraction. Although the long periodicity lamellar phase was present in both the native and the reconstructed epidermis, the short periodicity lamellar phase was present only in native tissue. In addition, the SC lipids predominantly formed the hexagonal sublattice. Analysis of lipid composition revealed that all SC lipids are synthesized in vitro. Differences in SC lipid organization in reconstructed epidermis may be ascribed to the differences in fatty acid content and profile indicating that further improvement in culture conditions is required for generation of in vitro reconstructed epidermis with stratum barrier properties of the native tissue.

Animals↗

PubMed2001

Phase behavior of lipid mixtures based on human ceramides: coexistence of crystalline and liquid phases.

The lipid regions in the outermost layer of the skin (stratum corneum) form the main barrier for diffusion of substances through the skin. In this layer the main lipid classes are ceramides, cholesterol (CHOL), and FFA. Previous studies revealed a coexistence of two crystalline lamellar phases with periodicities of approximately 13 nm (referred to as long periodicity phase) and 6 nm (short periodicity phase). Additional studies showed that lipid mixtures prepared with isolated pig ceramides (pigCER) mimic lipid phase behavior in stratum corneum closely. Because the molecular structure of pigCER differs in some important aspects from that of human ceramides (HCER), in the present study the phase behavior of mixtures prepared with HCER has been examined. Phase behavior studies of mixtures based on HCER revealed that in CHOL:HCER mixtures the long periodicity phase dominates. In the absence of HCER1 the short periodicity phase is dominant. Addition of FFA promotes the formation of the short periodicity phase and induces a transition from a hexagonal sublattice to an orthorhombic sublattice. Furthermore, the presence of FFA promotes the formation of a liquid phase. Finally, cholesterol sulfate, a minor but important lipid in the stratum corneum, reduces the amount of cholesterol that phase separates in crystalline domains. From these observations it can be concluded that the phase behavior of mixtures prepared from HCER differs in some important aspects from that prepared from pigCER. The most prevalent differences are the following: i) the addition of FFA promotes the formation of the short periodicity phase; and ii) liquid lateral packing is obviously present in CHOL:HCER:FFA mixtures. These changes in phase behavior might be due to a larger amount of linoleic acid moiety in HCER mixtures compared with that in pigCER mixtures.

Animals↗

PubMed2000

Lipid and ultrastructural characterization of reconstructed skin models.

The study aimed at evaluating tissue architecture and quality of the permeability barrier in commercially available reconstructed human skin models; EpiDerm, SkinEthic and Episkin in comparison to native tissue. For this purpose, tissue architecture was examined by electron microscopy and epidermal lipid composition was analyzed by HPTLC. Stratum corneum lipid organization was investigated by electron microscopy in combination with RuO(4) post-fixation and by SAXD. Ultrastructurally, the overall tissue architecture showed high similarities with native epidermis. In the stratum corneum extracellular space, lipid lamellae consisting of multiple alternating electron-dense and electron-lucent bands were present. This regular pattern was not seen throughout the whole stratum corneum probably due to the observed irregular lamellar body extrusion in some areas. Lipid analyses revealed the presence of all major epidermal lipid classes. Compared with native epidermis the content of polar ceramides 5 and 6 was lower, ceramide 7 was absent, and the content of free fatty acids was very low. These differences in lipid composition may account for differences observed in SAXD pattern of Episkin and EpiDerm penetration models. In the latter only the long-distance periodicity unit of about 12 nm was observed and the short periodicity unit was missing. In conclusion, all three skin models provide a promising means for studying the effects of topically applied chemicals, although the observed deviations in tissue homeostasis and barrier properties need to be optimized.

Basement Membrane↗

PubMed2000

HPV-DNA is not detectable in outgrowing cells from explant cultures of skin lesions established at the air-liquid-interface.

Keratinocyte cultures established from HPV containing skin cancers were described earlier to lose their HPV DNA after passaging in vitro. A different approach was therefore used in this study. Explant cultures were generated by depositing small pieces of various benign and (pre)malignant skin specimens of renal transplant recipients and non-immunosuppressed patients on fibroblast-populated collagen lattices or on de-epidermized dermis. Subsequently, the cultures were maintained at the air-liquid interface. At various time points, samples were collected for both HPV analysis, using a nested PCR approach, and morphology. The outgrowing keratinocytes developed into multilayered epithelial structures showing terminal differentiation. No histological differences were observed between cultures established from HPV positive and negative lesions. Eighteen biopsy specimens were tested for their HPV content before and after culture. Before culture 11 out of these skin specimens contained DNA of the Epidermodysplasia Verruciformis-related HPV types (EV-HPV). Comparison of the HPV types detected in two different parts of the same skin specimen before culture was strongly suggestive for a non-homogeneous distribution of EV-HPV in the lesions. From the explant cultures derived from the 11 HPV-positive biopsies, 31 samples from the originally explanted pieces of tissue and 38 samples from the outgrowing multilayered epithelial sections were collected. HPV DNA was detected in 10 of the 31 and in 3 of the 38 samples (Chi-square test, P = 0.01), respectively. These results indicate that EV-HPV positive keratinocytes do not efficiently proliferate or lose their HPV DNA in this culture system or EV-HPV DNA is present in only a few basal cells, making it improbable that these cells are located at the outgrowing margins.

Adult↗

PubMed2000

Intrinsic regulation of differentiation markers in human epidermis, hard palate and buccal mucosa.

Different epithelia show extensive variation in differentiation. Epidermis and epithelium from the hard palate are both typical examples of orthokeratinized epithelia whereas buccal mucosa is an example of a non-keratinized epithelium. Each of these tissues can be distinguished morphologically and also by the expression of a number of structural proteins. Tissue explants derived from epidermis, hard palate or buccal mucosa were cultured at the air-liquid interface on collagen gels containing human dermal fibroblasts. Reconstructed epithelia that retained many of the morphological and immunohistochemical characteristics of the original tissue were formed. Cultures derived from epidermis and the hard palate both had a well-defined stratum basale, stratum spinosum, stratum granulosum and stratum corneum whereas cultures derived from buccal mucosa had no stratum granulosum or corneum and the cells retained their nuclei. Significantly more living cell layers were observed in both types of epithelia obtained from the mouth than in epidermis. The specific localization of proliferation and differentiation markers (Ki67, loricrin, involucrin, SPRR2, SPRR3 and keratin 10) closely resembled that of the tissue from which the cultures were derived. As identical three-dimensional culture models were used here, it is concluded that the differences observed between these epithelia were due to intrinsic properties of the keratinocytes.

Biomarkers↗

PubMed2000

Microdialysis technique as a method to study the percutaneous penetration of methyl nicotinate through excised human skin, reconstructed epidermis, and human skin in vivo.

PURPOSE: The aim was to assess the feasibility of cutaneous microdialysis as a method to study percutaneous penetration of methyl nicotinate through human skin in vitro and in vivo. METHODS: Microdialysis was applied in vitro in excised human skin, in isolated dermis, in reconstructed human epidermis and in vivo in the volar forearm skin of volunteers using methyl nicotinate (MN) as a model compound. After topical application of MN, aliquots of the perfusate were collected and analyzed for the presence of MN spectrophotometrically and by HPLC. In vivo, visual scoring and laser Doppler perfusion imaging (LDPI) were used to monitor the effects on skin blood flow. RESULTS: In vitro, MN was detected in the dialysate after a 1 min exposure of excised skin to concentrations as low as 25 mM. Higher concentrations up to 500 mM showed increased levels. Prolongation of the application time to 60 min resulted in increased levels of MN in the perfusate as the duration of application increased. Reconstructed epidermis and isolated dermis showed an almost 2- and 20-fold higher penetration compared to excised skin, respectively. In vivo, LDPI measurements showed a rapid increase in skin blood flow after application of 25 to 100 mM MN for 1 min. MN was only detectable in the microdialysate after application of 100 mM for 10 min (two of three subjects). CONCLUSIONS: Cutaneous microdialysis may be a tool for comparative studies linking responses in human skin in vivo to in vitro data using the same technique and endpoint.

Administration, Cutaneous↗

PubMed2000

Melanosome capping of keratinocytes in pigmented reconstructed epidermis--effect of ultraviolet radiation and 3-isobutyl-1-methyl-xanthine on melanogenesis.

Reconstructed pigmented epidermis was established by co-seeding autologous melanocytes and keratinocytes onto a dermal substrate and culturing for up to 6 weeks at the air-liquid interface. Inspection of the tissue architecture revealed that melanocytes are regularly interspersed only in the basal layer and transfer melanosomes to the keratinocytes. We report for the first time, the in vitro formation of supranuclear melanin caps above the keratinocyte nuclei. The formation and abundance of these melanin caps could be enhanced by pigment modifiers such as ultraviolet light and 3-isobutyl-1-methyl-xanthine (IBMX). In untreated cultures, the capping was observed in the spinous layers after 6 weeks of culture, whereas after irradiation or supplementation of the culture medium with IBMX, the capping occurred already in the basal layer 2 weeks after initiation of the stimulus. In this study, we show that IBMX and ultraviolet irradiation stimulate pigmentation via different mechanisms. After supplementation of the culture medium with IBMX the increase in pigmentation was entirely due to the increase in melanocyte activity as observed by increased dendrite formation, melanin production and transport to the keratinocytes and was not due to an increase in melanocyte proliferation. In contrast, after UV irradiation, the increase in pigmentation was also accompanied with an increase in melanocyte proliferation as well as an increase in melanocyte activity. In conclusion, we describe the establishment of pigmented reconstructed epidermis with autologous keratinocytes and melanocytes that can be kept in culture for a period of at least 6 weeks. The complete program of melanogenesis occurs: melanosome synthesis, melanosome transport to keratinocytes, supranuclear capping of keratinocyte nuclei and tanning of the epidermis. This enables sustained application of pigment stimulators over a prolonged period of time and also repeated application of pigment stimulators to be studied.

1-Methyl-3-isobutylxanthine↗

PubMed2000

Differentiation-dependent alternative splicing and expression of the extracellular matrix protein 1 gene in human keratinocytes.

The human extracellular matrix protein 1 (Ecm1) gene is located at chromosome band 1q21 close to the epidermal differentiation complex and is transcribed in two discrete mRNAs: a full length Ecm1a and a shorter, alternatively spliced, Ecm1b transcript, the expression of which is restricted to tonsils and skin. The chromosomal localization and the Ecm1b expression in skin prompted us to investigate the role of Ecm1 in keratinocyte differentiation. In this study, we provide evidence for the existence of a relationship between keratinocyte differentiation and expression of the Ecm1b transcript. Cultures of subconfluent undifferentiated normal human keratinocytes express only Ecm1a. Upon reaching confluence, the cells start to differentiate, as measured by keratin K10 mRNA expression. Concomitantly Ecm1b mRNA expression is induced, although expression of Ecm1a mRNA remains unchanged. In addition, treatment of undifferentiated normal human keratinocyte cells with 12-O-tetradecanoyl-phorbol-13-acetate strongly induces the expression of Ecm1b mRNA. Expression of Ecm1b can also be induced by coculturing normal human keratinocytes with lethally irradiated feeder cells and by a diffusible factor secreted by stromal cells. In adult human skin, Ecm1a mRNA is expressed throughout the epidermis with the strongest expression in the basal and first suprabasal cell layers, whereas expression of Ecm1b mRNA is predominantly found in spinous and granular cell layers. Immunohistochemically, Ecm1a expression is almost completely restricted to the basal cell layer, whereas Ecm1b is detected in the suprabasal layers. These results are strongly suggestive of a role for Ecm1b in terminal keratinocyte differentiation, which is also supported by the localization of the Ecm1 gene at 1q21. Refinement of its genomic localization, however, placed Ecm1 centromeric of the epidermal differentiation complex.

Adult↗

PubMed2000

The lipid organisation in the skin barrier.

The main function of the skin is to protect the body against exogenous substances. The skin barrier is located in the outermost layer of the skin, the stratum corneum. This layer consists of keratin enriched cells embedded in lipid lamellae. These lamellae form the main barrier for diffusion of substances through the skin. In diseased skin the barrier function is often impaired. For a full understanding of the properties of the human skin barrier, insight in the stratum corneum lipid organisation is of great importance. In this paper a short description of the lipid organisation in normal human stratum corneum will be given, after which the role the main lipid classes play in the stratum corneum lipid organisation will be described. In addition the effect of cholesterol sulfate and calcium on the lipid organisation will be discussed. Finally a new model, the "sandwich model", will be proposed that describe the localisation of the fluid phases in the stratum corneum.

Calcium↗

PubMed2000

Skin equivalent: an attractive model to evaluate early melanoma metastasis.

The growth patterns and morphological phenotype of four human melanoma cell lines with different metastatic potentials were investigated in submerged and in air-exposed (skin equivalent) keratinocyte-melanoma cell co-cultures. In contrast to the submerged co-cultures, all four cell lines formed sharply demarcated tumour cell nests within the epidermal compartment of the skin equivalent model, with the morphology highly mimicking the in vivo situation. Differences among the melanoma cell lines tested were observed with respect to the number of clusters formed and the ability to exhibit invasive growth. Only the two metastatic cell lines were able to invade the dermal compartment. Screening of cellular adhesion molecules revealed that the expression patterns in different cell lines were heterogeneous and remained unchanged during the whole culture period, irrespective of whether the melanoma cells were located in the epidermal or dermal compartment. A correlation was found between expression of a lower number of different cellular adhesion molecules and the ability to acquire invasive growth capability. Our results indicate that melanoma cells exhibit a heterogeneous growth behaviour when co-cultured with human keratinocytes, and the air-exposed skin equivalent model was shown to be suitable for studying differences in growth patterns and potential invasive behaviour.

Air↗

PubMed2000

Phase behaviour of skin barrier model membranes at pH 7.4.

The main function of the skin is to protect the body against exogenous substances. The skin barrier is located in the outermost layer of the skin, the stratum corneum (SC). This layer consists of keratin enriched cells embedded in lipid lamellae that form the main barrier for diffusion of substances through the skin. The main lipid classes in this barrier are ceramides, cholesterol and free fatty acids. Cholesterol sulfate and calcium are also present in SC. Furthermore it has been suggested that a pH gradient exists. In a previous paper the effect of cholesterol sulfate and calcium on the lipid phase behaviour of mixtures prepared from cholesterol, ceramides and free fatty acids at pH 5 was reported (approximate pH at the skin surface). In the present study the phase behaviour of mixtures prepared from cholesterol, ceramides and free fatty acids prepared at pH 7.4 (the pH of viable cells) has been examined between 25 and 95 degrees C. Our studies reveal that a reversed hexagonal phase has been formed at elevated temperatures. Addition of calcium inhibits the formation of the reversed hexagonal phase, while cholesterol sulfate promotes the presence of the reversed hexagonal phase at increased temperatures. From our results we can conclude that the lipid mixtures prepared at pH 5 resemble more closely the lipid phase behaviour in intact SC than the lipid mixtures prepared at pH 7.4.

Animals↗