A new computer-based evaporimeter system for rapid and precise measurements of water diffusion through stratum corneum in vitro.

It is important to have reliable methods for evaluation of skin barrier function when questions such as barrier perturbing effects of different agents and occlusive effects of different formulations are to be elucidated. A wealth of clinical work relates to measurements of transepidermal water loss in vivo, a method much affected by ambient air relative humidity, temperature, skin irritation processes, psychologic status of the subject, etc., factors that cause the method to suffer from low precision (i.e., high random error). Relating to these obstacles, we have developed a closed in vitro system for measurements of water diffusion rate through pieces of isolated stratum corneum at steady-state conditions, where the relative humidity and temperature is held constant and data can be collected continuously. Our evaporimeter-based in vitro system has a more than 3-fold higher precision (lower random error) ( approximately 10%) than measurements of transepidermal water loss in vivo ( approximately 35%). The results of our study show that: (i) the corneocyte envelopes contribute to the barrier capacity of stratum corneum; (ii) removal of the lipid intercellular matrix results in approximately a 3-fold increase in the water diffusion rate through the isolated stratum corneum (n = 20; p < 0.05), not a 100-fold as has previously been suggested; (iii) exposure to sodium dodecyl sulfate in water does neither alter the water diffusion rate (n = 10; p > 0.05) nor the water holding capacity (n = 10; p > 0.05) of stratum corneum; (iv) exposure to 1 M CaCl2 in water yields an increased water diffusion rate through stratum corneum (n = 10; p < 0.05); and (v) when applied to the stratum corneum in excess concentrations, the penetration enhancer Azone has occlusive effects on water diffusion through the stratum corneum (n = 6; p < 0.05).

Calcium and sulfur location in human nail.

Analytical electron microscopy was performed in order to elucidate the importance of calcium and sulfur to the hardness of nails. The free surfaces adsorb a considerable quantity of calcium from the environment. It is suggested that such adsorption occurs due to ion exchange. This concept is supported by the fact that the distribution of sulfur does not vary over the nail cross-section. Analytical electron microscopy offers unique possibilities of elemental distribution analysis in tissue fragments or sections from keratinized tissues.

Inter- and intra-individual differences in human stratum corneum lipid content related to physical parameters of skin barrier function in vivo.

For a full understanding of the properties of the human skin barrier, physical macroscopic parameters of barrier function must be correlated to the structural organization of the barrier on a molecular level. This study was undertaken to relate differences in the relative composition of the three main lipid classes of human stratum corneum, i.e., free fatty acids, cholesterol, and ceramides, to differences in transepidermal water loss, stratum corneum electrical impedance, and corneometer value. A new high performance liquid chromatography/light scattering detection-based analysis method recently developed was used for collection of quantitative lipid data in conjunction with gas chromatography/mass spectrometry/flame ionization detection measurements on the free fatty acid fraction. After subtraction of contaminating lipid fractions we have estimated the molar ratio of the human skin barrier lipid composition to be, respectively, 15% cholesterol esters, 16% saturated long chain free fatty acids, 32% cholesterol, and 37% ceramides. The inter-individual difference in the relative amount of free fatty acids, cholesterol, and ceramides, respectively, can be >100% in the individual case. It was found that the relative amount of ceramides to cholesterol is larger in the wrist area, paralleled by a higher transepidermal water loss and corneometer value as well as different skin electrical impedance values as compared with the upper forearm area. We conclude that the site-dependent differences in the stratum corneum lipid composition are small compared with the large inter-individual variation. Interestingly, in the individual case, no correlation was registered between relative ceramide content and barrier properties.

X-ray microanalysis of psoriatic skin.

Electron probe x-ray microanalysis was used to study elemental distribution in uninvolved and involved skin from patients with psoriasis, and in skin from healthy controls. Significant differences were found between the involved and uninvolved psoriatic skin. In the involved skin, the concentrations of Mg, P, and K were higher in the stratum germinativum, spinosum, and granulosum, compared to the corresponding strata in uninvolved skin. Neither involved nor uninvolved psoriatic stratum germinativum differed markedly from nonpsoriatic control stratum germinativum. In uninvolved psoriatic skin only a lower level of K was noted. In comparison to uninvolved psoriatic skin, the elemental composition of the various strata of involved psoriatic skin shows a pattern typical for highly proliferative, nonneoplastic cells.

Normalization of epidermal calcium distribution profile in reconstructed human epidermis is related to improvement of terminal differentiation and stratum corneum barrier formation.

Calcium plays an important role in the regulation of cellular differentiation and desquamation of epidermal keratinocytes. In this study, we examined the calcium distribution in reconstructed epidermis in an attempt to understand the physiology of keratinocyte differentiation and desquamation in vitro. Ion capture cytochemistry (the potassium oxalate-pyroantimonate method) was employed to localize ionic calcium in reconstructed epidermis generated under three different culture conditions (in serum-containing medium, serum-free medium, and serum-free medium supplemented with retinoic acid), allowing a comparison of the physiology of incompletely and well-differentiated keratinocytes. The reconstructed epidermis generated in serum-containing medium showed features of incomplete differentiation, and compared with the native skin, a high calcium content within incompletely differentiated cells in the stratum corneum. Use of serum-free medium containing vitamin and lipid supplements led to a marked improvement of the stratum corneum ultrastructure and penetration pathway across the stratum corneum, indicating improved barrier formation of the reconstructed epidermis. In parallel, the calcium distribution pattern was normalized showing the highest levels of calcium in the stratum granulosum and low levels in the inner stratum corneum. Addition of retinoic acid to the serum-free medium resulted in an altered keratinocyte differentiation and re-appearance of large quantities of calcium precipitates in the stratum corneum. Proton probe X-ray microanalysis was applied to investigate the calcium distribution quantitatively in native and reconstructed epidermis generated in serum-free medium, and verified the calcium distribution demonstrated by the precipitation technique. Regardless of the presence or absence of calcium in the stratum corneum, all examined culture systems exhibited insufficient desquamation, which correlates with the finding that stratum corneum chymotryptic enzyme was present predominantly as an inactive precursor. This study demonstrates that improvement of the stratum corneum barrier properties in vitro is concurrent with the normalization of the epidermal calcium gradient, whereas deregulation of terminal differentiation correlates with an accumulation of calcium ions within incompletely differentiated corneocytes.

A novel approach to the understanding of human skin barrier function.

The basis for externally caused skin disorders is penetration of the skin barrier. A recent model for the skin barrier, the domain mosaic model, based on current knowledge of the physics of lipid bilayer organization gave tentative explanations for several aspects of function. It is demonstrated here that a development of the model explains how the requirements are met for a water-tight structure that will still allow a controlled, minute loss of water, the perspiratio insensibilis, necessary for maintaining plasticity of the keratin. A major advantage of the extended model is that it allows an interpretation of the changes imposed on the structure when in contact with detergents and/or penetration enhancers.

Aspects on the physiology of human skin: studies using particle probe analysis.

The cellular part of the skin, the epidermis, is a very thin structure, approximately 120 microns thick, a fact which has hindered the exploration of the physiology of the epidermis in normal and pathological conditions. An additional complication is the fact that the epidermis contains layers of cells at different stages of differentiation. Therefore, conventional physiological capillary probes cannot, with any satisfactory precision, be located within a specified cell of a specified layer of the skin in vivo. Hence, alternative ways for the exploration of skin physiology have been sought for. In the past, analysis of the elemental content of skin was done was done as bulk measurements, and surprisingly wide ranges of elemental content were recorded. The width of these ranges was most certainly due to the sampling methods used rather than the sensitivity of the chosen method of analysis. Also, these older measurements did not discriminate between the different strata, and therefore the information provided little if any substance for a functional analysis of processes involved in normal and pathological differentiation of the epidermis. Particle probes, however, have been able to overcome such methodological problems. Over a period of 15 years we have studied normal human skin, normal-looking, paralesional skin from psoriatics, and skin from persons suffering from atopic dermatitis using PIXE analysis. In recent years, trace elements have been shown to work as secondary messengers or regulatory substances. As an example, calcium (Ca2+) has proven to be a very important signalling substance in a great variety of cellular systems. Studies with the transmission electron microscope (TEM) as well as histochemical methods have allowed an understanding of the role of Ca2+ in the differentiation process of the epidermis. Ca2+ has also been shown to play an important role in apoptosis (programmed cell death), which is currently a hot subject for the obvious reason that the final differentiation step between the stratum granulosum level and the stratum corneum represents a particular aspect of programmed cell death. The importance of the balance between calcium and zinc in apoptosis has been clearly demonstrated in a number of cellular systems, but we have still to clarify the validity of topical treatment with Zn ointments in different skin conditions. Substantial iron (Fe) losses via psoriatic lesions were demonstrated more than two decades ago, and these data were given new meaning when we found that a more discrete loss occurs in clinically normal-looking psoriatic skin. Obviously, such findings stress the importance of understanding the relation between the elemental content and normal and abnormal physiology. The ultimate goal of particle probe studies is to provide an understanding of the formation of a mature stratum corneum with a functional barrier reflected in physiological/biochemical mechanisms behind the properties of changed skin in patients afflicted with skin disorders of genetic or constitutional origin. This paper aims to give an overview of the state of the art in skin physiology made possible through the use of particle probes.

Human hair form. Morphology revealed by light and scanning electron microscopy and computer aided three-dimensional reconstruction.

The alleged relationship between the cross sectional shape of the hair shaft and the form of the hair, eg, curly or straight hair, has been challenged. By serial sections of human hair follicles from ten patients representing the three biological races, the relation between the follicle form and the hair form was studied. Using three-dimensional computer-aided reconstruction it was demonstrated that the follicle form determines the hair form, eg, the Negroid follicle has a helical form, whereas that of the Oriental follicle is completely straight. The caucasoid follicle represent variation between these extremes. However, even a straight caucasoid follicle may produce a hair shaft that has an oval cross section.

Stratum corneum swelling. Biophysical and computer assisted quantitative assessments.

The aim of this study was to characterize the swelling behaviour of the stratum corneum. Stratum corneum pieces isolated from the breast region of 20 different females were incubated in distilled water at two different temperatures (20 degrees C and 45 degrees C) for 90 min and 24 h, respectively. Half of the stratum corneum pieces were previously extracted with chloroform-methanol (2:1). The area-enlargement was photographically recorded. The thickness enlargement was determined using a confocal laser scanning microscope. The average swelling (99% confidence interval) in the area dimension at 20 degrees C was 8.4% +/- 1.4% (n = 20), which corresponded to an average swelling in the length (lateral) dimension of approximately 4.1%. The swelling in the thickness dimension was 26.3% +/- 16.3% (n = 8). The swelling was most pronounced in the thickness dimension and was complete after 90 min of water immersion (P < 0.01, n = 5). In addition, the removal of the intercellular lipids with chloroform/methanol (2:1) induced a decreased swelling in the samples (P < 0.01, n = 20). An increase in temperature of the water from 20 degrees C to 45 degrees C resulted in an increase in swelling (P < 0.01, n = 20). Taken together our results support the idea that the mechanism of stratum corneum swelling is linked to the intercellular lipid structure and hence to skin barrier function.

A new HPLC-based method for the quantitative analysis of inner stratum corneum lipids with special reference to the free fatty acid fraction.

The inner stratum corneum is likely to represent the location of the intact skin barrier, unperturbed by degradation processes. In our studies of the physical skin barrier a new high-performance liquid chromatography (HPLC)-based method was developed for the quantitative analysis of lipids of the inner stratum corneum. All main lipid classes were separated and quantitated by HPLC/light scattering detection (LSD) and the free fatty acid fraction was further analysed by gas-liquid chromatography (GLC). Mass spectrometry (MS) was used for peak identification and flame ionization detection (FID) for quantitation. Special attention was paid to the free fatty acid fraction since unsaturated free fatty acids may exert a key function in the regulation of the skin barrier properties by shifting the physical equilibrium of the multilamellar lipid bilayer system towards a noncrystalline state. Our results indicated that the endogenous free fatty acid fraction of the stratum corneum barrier lipids in essence exclusively consisted of saturated long-chain free fatty acids. This fraction was characterized as a very stable population (low interindividual peak variation) dominated by saturated lignoceric acid (C24:0, 39 molar%) and hexacosanoic acid (C26:0, 23 molar%). In addition, trace amounts of very long-chain (C32-C36) saturated and monounsaturated free fatty acids were detected in human forearm inner stratum corneum. Our analysis method gives highly accurate and precise quantitative information on the relative composition of all major lipid species present in the skin barrier. Such data will eventually permit skin barrier model systems to be created which will allow a more detailed analysis of the physical nature of the human skin barrier.

Morphometric evaluation of collagen fibril dimensions in expanded human breast skin.

A great innovation in plastic surgery in recent years has been skin expansion, which has provided the discipline with new possibilities for skin reconstruction. At present, little is known about the biology of skin expansion although it is clear that cell proliferation occurs both in the epidermis and the dermis. During previous morphological investigations of skin under expansion we recorded a number of signs comparable with those seen in wound healing. In the present study, the collagen fibril diameter of of skin before and under expansion has been recorded using an IBAS computer-based morphometric system. Preferentially, we have studied the papillary dermis where the most conspicuous morphological events occur.

The skin barrier: analysis of physiologically important elements and trace elements.

Changes in the properties of the skin barrier should have correlates in the physiological status of the differentiating epidermal cells. However, the quantitative distributions of physiologically important elements and trace elements of the skin has been a neglected area of research for lack of tools to investigate this highly differentiated tissue. With the event of the particle probes, the electron microprobe and the scanning proton microprobe, it has become possible to investigate different aspects of normal skin physiology as well as pathophysiological processes. In addition penetration profiles of allergenic metals can be demonstrated with the trace element sensitive proton probe. Future approaches to the study of skin physiology in normal and pathological conditions should incorporate other techniques including immunological and biochemical tagging of particular cells to achieve a broad basis for interpretations of data.

The morphology of chromium allergic skin reactions at electron microscopic resolution: studies in man and guinea pig.

Previous studies on clinical patch test reactions have been expanded to short-term studies in humans hypersensitive to chromium. Preliminary results are discussed in relation to experiments in normal and sensitized guinea pigs.

Fibrin gels as biological filters and interfaces.

We present data which show that fibrin gels are ordered network structures, the porosity of which is determined by the amount of thrombin being present during the activation step preceding gelation. On activation of fibrinogen, fibrinopeptides are released and simultaneously polymers are formed, obeying apparent first order kinetics. The pore size of the network is inversely related to the rate of polymer formation. It is proposed that at the time of gelation the polymers form an ordered lattice structure, which in the organism may serve as a biological interface.

The use of PIXE in experimental studies of the physiology of human skin epidermis.

In order to understand the normal and pathological physiologies of the epidermal cells, the simultaneous determination of several elements in the different cellular strata is of crucial importance. In recent years the electron microprobe (EMP) has become an established technique in this field. Its high spatial resolution, in principle, allows measurements of various cell organelles. However, the limited (intrinsic) sensitivity of the EMP represents a serious drawback to the technique. The introduction of the proton microprobe (PMP) has significantly improved the sensitivity, although the ultimate spatial resolution of the PMP is much less than that of the EMP.When studying the elemental profiles in skin epidermis, it is possible to use skin sections with a thickness of the order of 10 µm, then the spatial resolution of the PMP is equal to or better than that of the EMP since the electrons are scattered to a significant degree in the sample. The characteristics of the two methods have been compared by analysis of parallel duplicate freeze-dried sections of normal human skin. The distributions of the elements P, S, Cl, and K, obtained with the two techniques, were in good agreement. In addition, the PMP provided distributions of the important elements Ca, Fe, and Zn.In a recently started study, the useful features of the PMP will be used for studying how efficient a barrier the skin is to nickel and chromate ions. A preliminary experiment has been performed by exposing cadaverous skin, not older than 24-h postmortem, to solutions of the two ions. After an 18-h exposure, samples were prepared by shock-freezing and sectioning. The first results from PMP analysis of these samples demonstrate the presence of a nickel and chromium gradient in the outer strata in the epidermis (mainly stratum corneum).A third experiment deals with the physiology of psoriatic skin. Calcium is an important element in the differentiation. Hence, the higher sensitivity of the PMP has been used in analysis of sections from psoriatic skin epidermis. Preliminary results are presented.

Evaluation of a new X-ray fluorescent analysis technique for the creation of a Nordic hair database: elemental distributions within the root and the virgin segment of hair fibers.

A new, non-destructive X-ray fluorescence technique for quantitative estimation of elemental content in biological tissues has been developed. Technical and instrumental characteristics of the ITRAX X-ray spectrometer have been evaluated in relation to the properties of biological samples, i.e., human hair fibers. Thus, attenuation variations of the fluorescent X-rays in the hair bulk mass were demonstrated by analysis of sulfur, calcium, and zinc in a virgin part near the root of one hair fiber with elliptical cross section. By rotation of the hair fiber and successive analyses made of the same part of the hair fiber, the results showed that concentrations of elements varied as functions of the diameter of the analyzed hair volume. Other sources of errors are also discussed. The ITRAX instrument allows for precise, fast, non-destructive, simultaneous, quantitative recording of the detected elements and trace elements down to levels of 1 ppm (microg/g). It was used for assessment of normal values of physiologically important elements present in hair in a cohort of normal, healthy Swedish, Caucasian individuals. The database constructed from data retrieved from a conceivably homogeneous ethnic set of individuals represents, to our knowledge, the first of its kind.

Dimensions of capsular collagen fibrils: image analysis of rapid compared with slow tissue expansion for breast reconstruction.

Thirty women who were to undergo breast reconstruction by tissue expansion were randomly divided into two groups. Those in the first group were to undergo expansion once a day (rapid expansion) and the second group once a week (slow expansion). When the expanders were replaced by permanent prostheses, biopsy specimens were taken from the capsules around the expanders, and were examined by transmission electron microscopy at a magnification x 22,000. The diameters of the collagen fibrils in the capsules were analysed by an interactive image analysis system and measured. An analysis of variance was performed on a test series to optimize the sample. Ninety fibrils from each patient (two patients were excluded), were analysed and there were no significant differences in collagen fibrillar diameters (about 50 nm) between those who had undergone rapid or slow expansion, or between patients who had also undergone radiotherapy to the chest wall and those who had not. These results indicate that the collagen fibrils may still be in a transitional stage, and that further longer term studies are desirable. It is difficult, however, to see how they could be justified ethically in patients who are otherwise well.

A comparison of the capsules around smooth and textured silicone prostheses used for breast reconstruction. A light and electron microscopic study.

A total of 18 women who had undergone modified radical mastectomy and tissue expansion for breast reconstruction were studied. When the expanders were replaced, nine patients had received smooth, gel-filled permanent prostheses and nine had received textured, gel-filled permanent prostheses. For medical reasons, e.g. capsular contraction, incorrectly placed or sized implant, an additional operation was performed, and then biopsy specimens from the capsules around the prostheses were taken and subsequently examined with the aid of the light and transmission electron microscope (TEM). A histologist was able to classify blindly 11 capsules out of 13 which were investigated by light microscopy in the correct groups and several differences between the capsules were found. Capsules around the smooth implants had a clear line of separation between the inner surface and the prosthesis, and formed a single collageneous layer. They were sparse in fibroblasts, which were long and thin. Capsules around textured implants consisted of two layers, the outer layer being compact with long, slender fibroblasts, and the inner one looking rugged with wavy bundles of collagen often splitting from each other, and with shorter and more rounded fibroblasts. The overall thickness seemed greater compared to the capsules around the smooth prostheses, which, on the other hand, showed a greater variation in thickness. To analyze collagen fibril diameters, sample sections were photographed at magnification 22,000 x in the TEM. The fibril diameters were measured with an interactive image analysis system (IBAS). The mean diameter of the collagen fibrils was 47.2 nm in the capsules around the smooth prostheses and 51.7 nm in the capsules around the textured prostheses.(ABSTRACT TRUNCATED AT 250 WORDS)