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Analysis of the penetration of a caffeine containing shampoo into the hair follicles by in vivo laser scanning microscopy
Abstract
In previous in vitro investigations, it was demonstrated that caffeine is able to stimulate the hair growth. Therefore, a
penetration of caffeine into the hair follicle is necessary. In the present study, in vivo laser scanning microscopy (LSM)
was used to investigate the penetration and storage of a caffeine containing shampoo into the hair follicles. It was shown
that a 2-min contact time of the shampoo with the skin was enough to accumulate significant parts of the shampoo in the hair
follicles. A penetration of the shampoo up to a depth of approx. 200 μm could be detected, which represents the detection
limit of the LSM. At this depth, the close network of the blood capillaries surrounding the hair follicles commences. Even
after 24 h, the substance was still detectable in the hair follicles. This demonstrates the long-term reservoir function of
the hair follicles for topically applied substances such as caffeine.
... Caffeine follicular delivery has been evaluated previously [21][22][23] by a number of methods; we have used herein differential tape stripping [24,25] and cyanoacrylate biopsy [26,27] techniques with LC-MS/MS quantitation to demonstrate caffeine follicular delivery from this new shampoo technology, thereby delivering dual anti-oxidants (caffeine and piroctone olamine) to the hair follicle. ...
... As caffeine is also an effective anti-oxidant [18,20], we sought to determine whether the new shampoo formulation technology could effectively deliver it to the hair follicle as well. Caffeine follicular delivery has been demonstrated from a conventional shampoo previously [21][22][23]. ...
A key factor in the prevention of hair loss is the provision of optimal conditions on the scalp. In this regard, reduction of oxidative stress on the scalp is one critical requirement to support the hair follicles to function optimally. Recently, a novel shampoo formulation technology containing anti-oxidants such as piroctone olamine has been demonstrated to improve hair retention based on micellar degradation and coacervation effects. Caffeine has also been shown to exhibit anti-oxidant activity including the ability to inhibit lipid peroxidation. As with piroctone olamine, it is expected that follicular delivery of caffeine will enhance its anti-oxidant activity in a region that will be beneficial for hair retention. In this study, two shampoo formulations as well as a control formulation were applied to the calf area of n = 9 male participants. The technique of differential tape stripping was applied to obtain the caffeine penetrated to the stratum corneum and to the hair follicles. Isotope-dilution liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was performed to demonstrate caffeine follicular delivery from the shampoo formulas. The results showed that the percentage of caffeine recovered in the hair follicles was 8–9% of the caffeine absorbed into the skin and matched an existing caffeine-based shampoo. In conclusion, a novel shampoo formulation technology has been developed that effectively delivers beneficial anti-oxidants to improve hair retention. This new shampoo is expected to be especially useful in the goal of retaining hair during aging.
... Taken together, after topical application of caffeine-containing solutions or shampoos, caffeine was available in the skin from very shortly after application up to at least 24 h, even if the duration of the topical application was as short as 2 min. The penetration and storage behavior of caffeine-containing formulations in the skin has also been investigated using in vivo LSM [70,74]. Different formulations of a caffeine-containing shampoo with 10 mg/mL caffeine and a fluorescent marker (fluorescein, 2 μg/mL [70] or 1 μg/mL [74]) were applied to the scalp of 10 healthy female volunteers for 2 min. ...
... The penetration and storage behavior of caffeine-containing formulations in the skin has also been investigated using in vivo LSM [70,74]. Different formulations of a caffeine-containing shampoo with 10 mg/mL caffeine and a fluorescent marker (fluorescein, 2 μg/mL [70] or 1 μg/mL [74]) were applied to the scalp of 10 healthy female volunteers for 2 min. The caffeine-containing shampoo penetrated into the hair follicle up to 200 μm (which corresponds to the maximum measuring depth due to the experimental set up), where the marker was still detectable after 24 h, providing further evidence that the penetration of caffeine-containing formulations across the skin barrier is a fast and efficient process, with a reservoir formation for at least 1 day. ...
Caffeine, particularly after ingestion, is well known to exert various pharmacological effects. A growing body of evidence implicates the ingestion of caffeine with beneficial effects on several diseases. The easy penetration of caffeine across the skin barrier and into human skin makes caffeine an ideal compound for topical application. Hair loss is known to negatively affect the quality of life and predispose to depression and anxiety. Androgenetic alopecia (AGA) is the most common type of hair loss in both men and women. To date, only few approved drug-based treatments for AGA exist, and these are inevitably associated with side effects. Therefore, the development of topical treatments based on well-tolerated natural ingredients such as caffeine to alleviate hair loss may provide a much-needed alternative to drug-based approaches.
... Thus, caffeine was identified as a stimulator of human hair growth in vitro, which may have an important clinical impact on managing androgenetic alopecia [53]. Lademann et al. [54] and Teichmann et al. [55] demonstrated that shampoo containing caffeine efficiently penetrated into the hair follicles of the human scalp and affected hair growth. Thus, caffeine is used in hair care products that are claimed to reduce and slow down balding and also stimulate hair growth. ...
The aging process encompasses gradual and continuous changes at the cellular level that slowly accumulate with age. The signs of aging include many physiological changes in both skin and hair such as fine lines, wrinkles, age spots, hair thinning and hair loss. The aim of the current study was to investigate the anti-aging potential of coffee berry extract (CBE) on human dermal fibroblast (HDF) and hair follicle dermal papilla (HFDP) cells. Coffee berry was extracted by 50% ethanol and determined for chemical constituents by HPLC technique. Cytotoxicity of the extract was examined on both cells by MTT assay. Then, HDF cells were used to evaluate antioxidant properties by using superoxide dismutase activity (SOD) and nitric oxide inhibition as well as anti-collagenase inhibition assays. The effectiveness of anti-hair loss properties was investigated in HFDP cells by considering cell proliferation, 5α-reductase inhibition (5AR), and growth factor expression. The results showed that caffeine and chlorogenic acid were identified as major constituents in CBE. CBE had lower toxicity and cell proliferation than caffeine and chlorogenic acid on both cells. CBE showed SOD and nitric oxide inhibition activities that were higher than those of caffeine but lower than those of chlorogenic acid. Interestingly, CBE had the highest significant anti-collagenase activity, and its 5AR inhibition activity was comparable to that of chlorogenic acid, which was higher than caffeine. CBE also stimulated hair-related gene expression, especially insulin-like growth factor 1 (IGF-1), keratinocyte growth factor (KGF) and vascular endothelial growth factor (VEGF). The results confirmed that CBE provided anti-aging activity on both skin and hair cells and could be beneficial for applications in cosmeceuticals.
... Caffeine also stimulates the microcirculation of the capillaries to nourish the hair. Furthermore, some researchers showed that exposure to caffeine-induced hair-shampoos has been shown to decrease the risk of hair loss [4][5]. ...
Nowadays, Thai consumers are more alert and caring about their hair health. As a result, products that care and promote hair and scalp are in high demand. One of the most interesting products is the hair care line. One of the important substances that help in reducing hair loss and promoting hair regrowth is coffee extract. This research focuses on value creation of coffee products from the northern Thailand into a cosmeceutical product. Variety of coffee by-product are studied in terms of Caffeine content and antioxidant activity, which benefit the hair growth and scalp care function. In addition, product development technique has been implemented to develop the hair serum prototype that comply with customer’s requirement. The results show that the active ingredient can be extracted from a variety part of coffee by- product. The coffee bean extraction yields the highest amount of Caffeine, while the red coffee fruit extraction yields the highest amount of antioxidant activity. However, when compare in terms of performance/cost of material, the extraction of fall grade cherry coffee is the most cost effective source for both active ingredients for this serum development. In the meantime, the market demand was collected based on 252 sample size using online questionnaires. The results show that the sample group gave importance to the factor of no irritation to the scalp as the first concern, while the safety issue as the second and the properties of the product ranked the third on customer’s needs. Then the prototype was developed and verified in terms of active ingredient concentration and product stability. Furthermore, product was validated in terms of hair growth and skin irritation. Finally, the prototype was analyzed with 3 focus groups: (1) hair loss prevention group (2) hair loss solution group and (3) beauty service provider/ product supplier. According to these focus group evaluation, it shows that all three test participants were satisfied with prototype’s sensory. Some issues that could be improved are viscosity and stickiness. Additionally, marketing mix was evaluated on these group to estimate potential product-package design, selling price and place, as well as the promotion technique.
... to visualize the follicular penetration. 21 Moreover, the sodium fluorescein-labeled soot particulates could be efficiently analyzed using laser scanning microscopy. 37 ...
Background:
The decontamination of the skin is indispensable if airborne particulate contaminants deposit on the skin surface. Skin washing can have adverse effects as by skin rubbing the particles can be transferred deeply into the hair follicles, where they can be entrapped for a period of more than 10 days. Thus, alternative skin decontamination strategies are necessary.
Materials and methods:
For imaging the contaminants in the skin, sodium fluorescein-labeled soot particles of submicron size (≈600 nm) were visualized using laser scanning microscopy.
Results:
In the present ex vivo pilot study on porcine ear skin, it was shown that sodium fluorescein-labeled soot particles of submicron size (≈600 nm) could be efficiently removed from the skin with highly absorbent textile nanofiber material, whose efficacy could be further increased by spraying the contaminated skin area with the viscous fluid PEG-12 dimethicone before textile application.
Conclusion:
In case of skin contamination with particulates, the contact washing should be avoided due to rubbing particles deeply into the hair follicles, where they can accumulate for a long time and induce negative consequences. Efficient skin decontamination could include pretreatment of skin surface with the viscous fluid PEG-12 dimethicone and subsequent application of highly absorbent textile nanofiber material.
... Caffeine also arouses capillary vessel microcirculation in the head skin, thereby contributing to nurture hair bulbs. Teichmann et al. [55] and Lademann et al. [56] demonstrated that a 2-min contact of a shampoo with caffeine was sufficient for the formulation to penetrate deeply into the hair follicles and remain there for up to 48 h, even after washing the hair. Otberg et al. [57] showed that there is a quantitative distinction between follicular penetration and interfollicular diffusion of a formulation containing 2.5% caffeine applied to the chest of male Caucasian volunteers aged 26-39 with normal body mass indices. ...
Coffee silverskin, the major coffee-roasting by-product, is currently used as fuel and for soil fertilization. However, there are several studies reporting silverskin as a good source of bioactive compounds that can be extracted and further used by cosmetic industry. Its high antioxidant potential may be due to the synergistic interaction of chlorogenic acids (1-6%), caffeine (0.8-1.25%), and melanoidins (17-23%), among other antioxidant compounds. The bioactive compounds of silverskin can answer to the new fields of cosmetic industry on natural active ingredient resources that improve health skin appearance, counteract skin aging and related diseases, in an environmentally friendly approach. Skin aging is a complex process associated with oxidative metabolism and reactive oxygen species (ROS) generation. ROS production increase matrix metalloproteinases (MMPs), as well as pro-inflammatory mediators, resulting in consequent skin damage and aging. To counteract this process, cosmetic industry is looking for compounds able to increase MMP inhibitory activities, hyaluronidase inhibitory activity, expression of collagen and elastase inhibitory activity, as potential bioactive ingredients with anti-aging purposes. This review focuses on skin aging factors and the potential anti-aging, anti-inflammatory, antimicrobial, anti-cellulite and anti-hair loss activity, as well as protection against UV damage, of coffee silverskin and their bioactive compounds.
... Moreover, caffeine inhibits the activity of the 5a-reductase and leads to a significant stimulation of human hair follicle growth in vitro [37]. Teichmann et al. [150] and Lademann et al. [82] demonstrated that a 2-min contact between the shampoo with caffeine and the skin surface was sufficient for its penetration deeply into the hair follicles and remains there for up to 48 h, even after hair washing. With the exception of caffeine, most of the active components produced by plants are not able to pass through the skin barrier. ...
Currently available conventional therapies of hair loss using synthetic drugs are still imperfect and have a number of limitations. Their effectiveness as well as the safety of their use is often questioned. It has led to an increased interest in alternative treatments with fewer side-effects such as formulations containing herbs and/or their active constituents. For this purpose several electronic databases and hand-searched references were used to summarize current knowledge regarding topically used herbal products for the treatment of hair loss acquired on the basis of preclinical and clinical studies. Moreover, mechanism of their action, follicular penetration and possible adverse effect of herbal products will be also described.
... It strengthens and stimulates rapid growth by regularly providing nutrients with blood to the hair [46] . Teichmann et al. [48] and Lademann et al. [49] demonstrated that a 2-min contact of a shampoo with caffeine was sufficient for the formulation to penetrate deeply into the hair follicles and remain there for up to 48 h, even after washing of the hair. Otberg et al. [50] showed that there is a quantitative distinction between follicular penetration and interfollicular diffusion of a formulation containing 2.5% caffeine applied to the chest of male Caucasian volunteers aged 26-39 with normal body mass indices. ...
Caffeine is being increasingly used in cosmetics due to its high biological activity and ability to penetrate the skin barrier. This alkaloid is frequently used as a hydrophilic model substance in human and animal skin penetration as well as different synthetic membrane using Franz diffusion cell experiments. The commercially available topical formulations of caffeine normally contain 3% caffeine. As for a cosmetic purpose, caffeine is used as an active compound in anti-cellulite products because it prevents excessive accumulation of fat in cells. This alkaloid stimulates the degradation of fats during lipolysis through inhibition of the phosphodiesterase activity. Caffeine has potent antioxidant properties. It helps protect cells against the UV radiation and slows down the process of photoaging of the skin. Moreover, caffeine contained in cosmetics increases the microcirculation of blood in the skin and also stimulates the growth of hair through inhibition of the 5-alpha-reductase activity. Copyright (C) 2012 S. Karger AG, Basel
... cafeína in vitro estimula o crescimento de folículos pilosos humanos 7 e tem sido adicionada ao tratamento local da calvície,8 filhotes de ratas tratadas com cafeína apresentam alopecia decorrente da menor atividade proliferativa das células do folículo piloso.9 Essa diferença nos resultados pode ser explicada, uma vez que in vitro o modelo experimental é simplificado, e não é possível reproduzir um ambiente com todos os fatores e moléculas de sinalização que agem in vivo. ...
To evaluate the in vitro effetcs of caffeine on proliferation, apoptosis a nd gene transcripts expression of chondrogenic differentiation in growth cartilage.
THE CARTILAGINOUS EPIPHYSES OF FEMURS OF NEWBORN RATS, WHICH WERE DIVIDED INTO TWO SUBGROUPS: treated with caffeine and control group, both observed over the time periods of 0, 7, 14 and 21 days. The cartilaginous epiphyses of femurs of each subgroup and each time span were subjected to histomorphometric, immunohistochemical analysis, Tunel technique and RT-PCR in real time.
The decrease in proliferative activity and the increase of apoptotic chondroblasts at 21 days were found regardless of the subgroup. However, the decrease in cell proliferation caused by caffeine was lower than in the control group and significantly increased the expression of gene transcripts for chondrogenic differentiation, represented by Sox-9 and Runx-2. However, the in vitro culture with caffeine revealed antagonistic effects: despite the positive effect on chondroblasts proliferation and differentiation, caffeine increased apoptosis, characterized by increased expression of caspase 3 and of the number of cells undergoing apoptosis (p<0.05).
Caffeine presents antagonistic effects in vitro on growth cartilage, increasing the proliferation, differentiation and cell apoptosis. Experimental Study .
... So far, the hair follicle penetration could be analysed invasively by taking biopsies and subsequent histological analysis with the application of fluorescent dyes or radio-labelled compounds [13]. In vivo laser scanning microscopy permits, for the first time, the detection of fluorescent substances in the stratum corneum and in hair follicles of volunteers or patients, non-invasively [14,15]. However, the range of substances, which can be investigated by this method, is limited, as these must exhibit specific fluorescent properties. ...
Bacteria and fungi are located in the stratum corneum and the hair follicles. Therefore, the development and assessment of efficient drugs requires standard in vivo investigation methods permitting a differentiation between intercellular and follicular penetration and storage of topically applied anti-microbial substances. In the present study, the penetration and storage of Isoconazole nitrate in the stratum corneum and hair follicles was investigated by differential stripping after a 14-day topical application period and during a follow-up period of a further 21 days. One week after the application had terminated, Isoconazole nitrate could still be detected in concentrations above the minimal inhibition concentration in the stratum corneum and the hair follicles. In some subjects, Isoconazole nitrate could even be detected 14 days after the last application. No relevant changes in TEWL values were measured, indicating that the investigated compound did not induce an impairment of the barrier function. The study showed that differential stripping is suited to investigate the penetration and storage of topically applied substances into the stratum corneum and the hair follicles. Also, the hair follicles are a long-term reservoir for topically applied substances. This is of clinical importance, where a long-lasting therapeutic effect beyond the application time is required.
... However, human abdominal skin has a much lower follicular density than the scalp or forehead (Otberg et al., 2004). Hair follicles have been shown to contribute significantly to the penetration of shampoo formulations, even within 2-5 min after application (Lademann et al., 2010a;Otberg et al., 2008). Therefore, the weakness of the standard OECD protocol for shampoos, conditioners, and other products that are applied directly to the face or scalp suggests that the use of skin with greater follicular presence, such as pig ear skin (Lademann et al., 2010b) or human scalp skin, may be more appropriate for future studies of these products. ...
Alpha-hydroxy acids (AHAs), primarily glycolic and lactic acids, are widely used in cosmetics to alleviate dyspigmentation, photodamage, and other aging skin conditions and as pH adjusters. Glycolic acid reportedly enhances skin damage after repeated ultraviolet light exposure, e.g., increased sunburn cell formation. This study assessed potential in vitro skin penetration of lactic acid and malic acid incorporated into rinse-off personal care products, compared with rinse-off and leave-on exposures to glycolic acid (10%, pH 3.5) in a reference lotion. Radiolabeled AHA-fortified shampoo, conditioner, and lotion were evenly applied as single doses to human epidermal membranes mounted in static diffusion cells (not occluded). Exposures were 1-3 min (rinse-off) or 24 h (leave-on). Epidermal penetration of malic acid and lactic acid from the rinse-off shampoo and conditioner, respectively, was negligible, with >99% removed by rinsing, a negligible portion remaining in the stratum corneum (≤0.15%), and even less penetrating into the viable epidermis (≤0.04%). Glycolic acid penetration from the leave-on reference lotion was 1.42 μg equiv./cm2/h, with total absorbable dose recovery (receptor fluid plus epidermis) of 2.51%, compared to 0.009%, 0.003%, and 0.04% for the rinse-off reference lotion, shampoo (malic acid), and conditioner (lactic acid) exposures, respectively. Dermal penetration of AHAs into human skin is pH-, concentration-, and time-dependent. Alpha-hydroxy acids in rinse-off shampoos and conditioners are almost entirely removed from the skin within minutes by rinsing (resulting in negligible epidermal penetration). This suggests that ultraviolet radiation-induced skin effects of AHA-containing rinse-off products are negligible.
Caffeine is ubiquitous in our society—not only in the drinks consumed but also increasingly in dermatologic topicals. Given that coffee and caffeine are increasingly used for the production of many dermatologic anti-cancer topicals, sunscreens, and cosmetics, it is of imperative importance to review the basic science and clinical evidence for such claims. In this concise review, we outline the current evidence.
Caffeine is a bitter, white crystalline purine, a methylxanthine alkaloid, chemically related to the adenine and guanine bases of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Caffeine is found in different concentrations in seeds, leaves, and nuts of more than 60 different plant species. It acts as a naturally occurring pesticide since it can paralyze and kill predator insects feeding on the plant [1]. Humans consume caffeine as a stimulant, mainly in coffee beans, tea extracts, in products containing kola or guarana seeds, in the form of soft drinks or energy drinks, or even in everyday food. Caffeine is the world’s most widely consumed psychoactive compound or central nervous system (CNS) stimulant, and in the USA, more than 90% of adults consume an average of 2.4 mg/kg of caffeine daily [2].
This article discusses the mechanisms via topically applied products containing herbs and their active constituents affect the hair growth process. It was reported that the mechanisms involving (1) insulin-like growth factor-I (IGF-I), (2) vascular endothelial growth factor (VEGF), (3) epidermal growth factor (EGF), (4) fibroblast growth factor 2 (FGF-2), (5) endothelial nitric oxide synthase (eNOS), (6) Wnt/β-catenin signalling pathway, (7) prostaglandin E (PGE), (8) prostaglandin F (PGF) stimulate hair growth, whereas the mechanisms engaging (1) 5α-reductase and dihydrotestosterone (DHT), (2) transforming growth factor beta (TGF-β), (3) fibroblast growth factor 5 (FGF-5), (4) prostaglandin D2 (PGD2) inhibit hair growth. The knowledge summarized in the paper may be an inspiration to create new preparations for the treatment of hair loss.
Propylene glycol is one of the known substances added in cosmetic formulations as a penetration enhancer. Recently, nanocrystals have been employed also to increase the skin penetration of active components. Caffeine is a component with many applications and its penetration into the epidermis is controversially discussed in the literature. In the present study, the penetration ability of two components – caffeine nanocrystals and propylene glycol, applied topically on porcine ear skin in the form of a gel, was investigated ex vivo using two confocal Raman microscopes operated at different excitation wavelengths (785 nm and 633 nm).
Several depth profiles were acquired in the fingerprint region and different spectral ranges, i.e., 526–600 cm⁻¹ and 810–880 cm⁻¹ were chosen for independent analysis of caffeine and propylene glycol penetration into the skin, respectively. Multivariate statistical methods such as principal component analysis (PCA) and linear discriminant analysis (LDA) combined with Student’s t-test were employed to calculate the maximum penetration depths of each substance (caffeine and propylene glycol). The results show that propylene glycol penetrates significantly deeper than caffeine (20.7–22.0 μm versus 12.3–13.0 μm) without any penetration enhancement effect on caffeine. The results confirm that different substances, even if applied onto the skin as a mixture, can penetrate differently.
The penetration depths of caffeine and propylene glycol obtained using two different confocal Raman microscopes are comparable showing that both types of microscopes are well suited for such investigations and that multivariate statistical PCA–LDA methods combined with Student’s t-test are very useful for analyzing the penetration of different substances into the skin.
Schlüsselwörter Koffein · hautpenetration · cellulite · Mikrozirkulation · antioxidans Zusammenfassung Koffein findet in der Kosmetik zunehmend breite anwendung, da es hohe biologische aktivität zeigt und die hautbarriere zu durch-dringen vermag. Das alkaloid wird häufig als hydrophile Modell-substanz in Versuchen zur Penetration von Menschen-und tier-haut sowie verschiedenen synthetischen Membranen im rahmen von Versuchen mit Franz-Diffusionszellen verwendet. Die im han-del erhältlichen topischen Koffein-Formulierungen enthalten in der regel 3% Koffein. Für kosmetische Zwecke wird Koffein als Wirkstoff in Produkten gegen cellulite verwendet, weil es der über-schüssigen Fetteinlagerung in den Zellen entgegenwirkt. Das alka-loid stimuliert den abbau von Fetten im rahmen der Lipolyse, in-dem es die aktivität der Phosphodiesterase hemmt. Koffein hat ausgeprägte antioxidative eigenschaften. es schützt die Zellen vor UV-strahlen und verzögert die lichtbedingte hautalterung. Darü-ber hinaus regt das in Kosmetika enthaltene Koffein die Mikrozirku-lation in der haut an und stimuliert auch das haarwachstum, in-dem es die aktivität der 5-α-reduktase inhibiert.
Earlier investigations regarding the distribution of the bacterial flora on the human skin demonstrate that the hair follicle acts as a bacterial reservoir, providing a quick source for secondary recontamination. These findings highlight the importance of the hair follicle as a target for modern antiseptics. In the present study, we have assessed the follicular penetration of a curcumin-labeled particle-associated antiseptic into porcine skin by laser scanning microscopy. Therefore, the follicular penetration depth of the curcumin-labeled particle-associated antiseptic was compared to the follicular penetration depth of curcumin-labeled particles without antiseptic. The investigation was performed in vitro using porcine skin biopsies. By superposition of the images acquired in the transmission and the fluorescent modus, it was possible to visualize the distribution of the fluorescent dye inside the hair follicles. Quantitative and qualitative results showed that both dispersions penetrated efficiently into the hair follicles. The average penetration depth of the particles with attached antiseptic polihexanide was significantly higher than that of particles without the attached antiseptic. Also, whilst very little sample preparation was needed, laser scanning microscopy was found to be an efficient tool to visualize the skin relief and in particular the hair follicle shaft and localize fluorescent markers within the skin tissue and hair follicles.
Raman spectroscopy offers a powerful alternative analytical method for the detection and identification of lipids/oil in biological samples, such as algae and fish. Recent research in the authors' groups, and experimental data only very recently published by us and a few other groups suggest that Raman spectroscopy can be exploited in instances where fast and accurate determination of the iodine value (associated with the degree of lipid unsaturation) is required. Here the current status of Raman spectroscopy applications on algae is reviewed, and particular attention is given to the efforts of identifying and selecting oil-rich algal strains for the potential mass production of commercial biofuels and for utilization in the food industry. (© 2011 by Astro Ltd., Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA) (© 2011 by Astro Ltd., Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA)
A high number of treatments in dermatology are based on the penetration of topically applied drugs through the skin barrier. This process is predominantly inefficient, on account of the strong protection properties of the upper skin layer – the stratum corneum. If the skin barrier is damaged, the penetration efficiency of topically applied drugs increases. Therefore, different methods have been developed to influence the barrier properties of the skin.
Recently, it could be demonstrated that a cold tissue tolerable plasma (TTP) produced by a plasma-jet can strongly enhance drug delivery through the skin. These investigations were performed by using a solution of fluorescent dye as a model drug. In the present study, these investigations were carried out using fluorescent silica particles at different sizes. The aim of the study was to investigate whether or not there is a limitation in size for topically applied substances to pass through the skin barrier after plasma treatment.
The stimulation of the penetration of topically applied substances into the skin is a topic of intensive dermatological and pharmacological research. In this context, it was found that in addition to the intercellular penetration, the follicular penetration also represents an efficient penetration pathway. The hair follicles act as a long-term reservoir for topically applied substances. They are surrounded by all important target structures, such as blood capillaries, stem and dendritic cells. Therefore, the hair follicles, as well as the skin, need to be protected from hazardous substances. The traditional method of decontamination after respective accidental contacts consists of an intensive washing of the skin. However, during this mechanical procedure, the substances can be pushed even deeper into the hair follicles.
In the present study, absorbent materials were applied to remove a fluorescent model substance from the skin without inducing mechanical stress. The results were compared to the decontamination effects obtained by intensive washing. Investigations were performed by means of in vivo laser scanning microscopy (LSM). The comparison revealed that decontamination with absorbent materials is more effective than decontamination with washing processes.
The application of tissue-tolerable electrical plasma (TTP) is highly efficient in skin antisepsis. However, the germs are not only located on the skin surface, but also in the hair follicles, from where they re-colonize the skin surface after antisepsis, e.g. The objective of the present study was to show that plasma is able to reach the follicular reservoir for antisepsis. For this purpose, a solution containing particulate chlorophyll dye had been applied onto porcine skin samples. The fluorescent properties of the dye changed during the plasma tissue interaction. The results demonstrate that TTP penetrates deep into the hair follicles, whereupon the hairs act as a conductor for the plasma. Therefore, it can be concluded that micro-organisms of the follicular reservoir are destroyed more efficiently by the plasma than by conventional liquid antiseptics. (© 2011 by Astro Ltd., Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA) (© 2011 by Astro Ltd., Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA)
Confocal laser scanning microscopy (CLSM) allows noninvasive visualization of human skin in vivo, without needing to fix or section the tissue. Melanocytes and pigmented keratinocytes at the level of the basal layer form bright dermal papillary rings which are readily amenable to identify in confocal images. Our purpose was to explore the role of dermal papillary rings in assessment of lesion location, the diagnosis, differential diagnosis of lesions and assessment of therapeutic efficacy by in vivo CLSM. Seventy-one patients were imaged with the VivaScope 1500 reflectance confocal microscope provided by Lucid, Inc. The results indicate that dermal papillary rings can assess the location of lesion; the application of dermal papillary rings can provide diagnostic support and differential diagnosis for vitiligo, nevus depigmentosus, tinea versicolor, halo nevus, common nevi, and assess the therapeutic efficacy of NBUVB phototherapy plus topical 0.1 percent tacrolimus ointment for vitiligo. In conclusion, our findings indicate that the dermal papillary rings play an important role in the assessment the location of lesion, diagnosis, differential diagnosis of lesions and assessment of therapeutic efficacy by in vivo CLSM. CLSM may be a promising tool for noninvasive examination in dermatology. However, larger studies are needed to expand the application of dermal papillary rings in dermatology.
The antioxidant β-carotene and lycopene substances were detected noninvasively, in vivo in human skin using Raman resonance spectrpscopy. Both substances were detected simultaneously. To distinguish between the substances, the Raman signals were excited at 488 nm and 514.5 nm simultaneously using a multiline Ar+ laser. The application of a fiber-based optical imaging system allowed the detection of β-carotene and lycopene on any skin area. The disturbance of the measurements because of nonhomogeneous skin pigmentation was avoided by using a measuring area of 28 mm2. The Raman spectroscopic method is well suited for the evaluation of the efficacy of topically or systemically applied amounts of β-carotene and lycopene.
The tape stripping technique is an experimental method frequently used for reconstruction of the in-depth distribution of
various topically administered substances within the horny layer of human skin, e.g., compounds contained in sunscreens. Titanium
dioxide (TiO2) nanoparticles (25–200 nm in diameter) are one such compound. Optical techniques which apply blue light are found to be suitable
for reconstruction. However, the presence of particles affects the light propagation within the skin and therefore causes
incorrect determination of strip thickness, leading to an improper reconstructed distribution of nanoparticle concentration
revealed from the experimental data. This study evaluates the errors emerging from the use of blue (400 nm) and NIR (800 nm)
radiation and finds the use of longer wavelength light more advantageous. Particles of different diameters are considered,
and it is revealed that the application of small particles (25–60 nm) results in the lowest rate of error.
Definitions of functional food vary but are essentially based on foods' ability to enhance the quality of life, or physical and mental performance, of regular consumers. The worldwide use of coffee for social engagement, leisure, enhancement of work performance and well-being is widely recognised. Depending on the quantities consumed, it can affect the intake of some minerals (K, Mg, Mn, Cr), niacin and antioxidant substances. Epidemiological and experimental studies have shown positive effects of regular coffee-drinking on various aspects of health, such as psychoactive responses (alertness, mood change), neurological (infant hyperactivity, Alzheimer's and Parkinson's diseases) and metabolic disorders (diabetes, gallstones, liver cirrhosis), and gonad and liver function. Despite this, most reviews do not mention coffee as fulfilling the criteria for a functional food. Unlike other functional foods that act on a defined population with a special effect, the wide use of coffee-drinking impacts a broad demographic (from children to the elderly), with a wide spectrum of health benefits. The present paper discusses coffee-drinking and health benefits that support the concept of coffee as a functional food.
The penetration of topically applied substances into the stratum corneum (SC) depends on several factors, e.g., the physicochemical properties of the vehicle used for application. The penetration of highly hydrophilic and lipophilic dyes into the skin was studied using a pure oil (o) or water (w) for the application compared to an o/w emulsion. The penetration and localization of both dyes, the lipophilic curcumin and the hydrophilic Patent blue V, was investigated in vivo using the method of tape stripping and microscopy. In addition, histological sections of biopsies, removed from porcine ear skin were studied using microscopy. Differences in the distribution and the localization of both dyes within the SC were observed. These differences depend on the physicochemical properties of both the vehicles and the dyes. The vehicle appears to affect, in particular, the pathways of penetration.
Nanoparticles represent an important drug carrier system. Recently, we have reported on the penetration and storage behavior of particular and non-particular substances revealing the superiority of particular substances in the range of 300-400 nm. In this regard, it was assumed that the rigid hair shaft acts as a geared pump, moving the particles deeper into the hair follicle. In the present investigation, the storage reservoir capacity of the stratum corneum and the hair follicle infundibulum and canal are compared. Interestingly, we could demonstrate a 10 times longer storage within the hair follicles. These results underscore the importance of the hair follicle for drug delivery purposes, mainly highlighting new possibilities for the future concerning retarded delivery, application frequency, and galenic design.
The horny layer functions both as a barrier against and as a reservoir for topically applied substances. Both functions are dependent on several parameters, e.g., the physicochemical properties of the applied active substance, the vehicle used for application, and the time after application. In the present study, the longterm reservoir of the horny layer was investigated in vivo using laser scanning microscopy. Water, propylene glycol, and paraffin oil were used as vehicles for the fluorescent dyes curcumin and sodium fluorescein. A dermatological laser scanning microscope was used to determine the distribution of the topically applied formulations in vivo. The fluorescence signals of curcumin and sodium fluorescein were measured at a wavelength above 600 nm after excitation at 488 nm at 5, 24, 48, 72, and 120 h after application. After treatment with sodium fluorescein in water, the fluorescence was detected both intra- and intercellular within the horny layer up to 120 h after application. The fluorescence of the lipophilic curcumin, applied in paraffin oil, was measured between the superficial horny-layer cells up to two days after application. The application of sodium fluorescein in propylene glycol led to intra- and intercellular fluorescence (up to five days), as well as fluorescence inside the follicular infundibulum (up to five days). These results indicate that the different localization of the reservoir, depending on the time after application, is determined by the physicochemical properties of both the dyes and the vehicles.
The dermatological laser scanning confocal microscope, Optiscan Stratum, was applied to in vivo investigations in skin physiology. The radiation of an Ar+ laser at 488 nm was used to excite the topically applied food dyes curcumin and sodium fluorescine. The radiation was transferred by an optical fiber to a hand piece set directly onto the skin surface. The structure of the different skin layers was analyzed. The distribution of topically applied substances in the stratum corneum and their penetration kinetics were investigated. It was demonstrated that laser scanning confocal microscopy is well suited to analyzing the penetration pathways of these substances into follicular orifices and sweat glands. The phenomena of active and passive follicles, concerning the penetration of topically applied substances, was investigated in vivo.
Laser scanning microscopy in combination with cyanoacrylate skin surface biopsy was used to observe follicular penetration of topically applied curcumin emulsion into the hair follicle. The dye was found in most of the hair follicles, although some follicles were obviously closed to penetration. Reasons for these different penetration characteristics were found in follicle physiology. A combination of various tape stripping and staining methods made it possible to measure hair growth and sebum excretion of every single hair follicle in the defined skin area. A correlation between the penetration properties, hair growth activity, and sebum production was found.
The hair follicles play an important role in vivo for the penetration of topically applied substances. However, it is still an open question as to whether the follicular penetration contributes to the results obtained using the in vitro model of porcine skin in Franz diffusion cells. In the present study, the availability of follicular penetration in in vitro experiments using porcine ear skin was determined qualitatively using optical methods. The fluorescent dye sodium fluorescein was topically applied onto porcine skin mounted in Franz diffusion cells and in the acceptor compartment beneath the dermis. Sections of removed biopsies were studied using light and fluorescence microscopy. After topical application, fluorescence was detected on the surface, within the horny layer, and in most of the follicles. This in vitro penetration into the porcine hair follicles might be due to penetration processes similar to those on humans in vivo. After application of the dye in the acceptor fluid, fluorescence was detected within the dermis, the viable epidermis, and the SC in all sections. This might be caused by hydration of the tissue by the acceptor fluid containing the dye, as was observed previously in vitro on human skin. The obtained results confirm the use of readily available porcine skin instead of human skin for in vitro studies.
The defense mechanism of the human body is based on the action of antioxidant substances such as carotenoids. Beta carotene and lycopene represent more than 70% of the carotenoids in the human organism. The topical or systemic application of beta carotene and lycopene are general strategies for improving the defense system of the human body. For the evaluation and optimization of this treatment, it is necessary to measure the beta carotene and lycopene concentrations in tissue and especially in human skin, which is the barrier to the environment. We have used resonance Raman scattering as a fast and noninvasive optical method for measuring the absolute concentrations of beta carotene and lycopene in living human skin. Beta carotene and lycopene have different absorption values at 488 and 514.5 nm. As a result, the Raman lines for beta carotene and lycopene have different scattering efficiencies at the 488- and 514.5-nm excitations. These differences were used for the determination of the concentrations of beta carotene and lycopene. Using multiline Ar + laser excitation, clearly distinguishable Raman spectra of carotenoids are obtained, which are then superimposed on a large fluorescence background. The Raman signals are characterized by two prominent Stokes lines at 1160 and 1525 cm-1, which have nearly identical relative intensities. Both substances were detected simultaneously. The Raman spectra were obtained rapidly, i.e., within about 10 seconds, and the required laser-light-exposure level is well within safety standards. Any disturbance of the measurements by nonhomogeneous skin pigmentation was avoided by using a relatively large measuring area of 33 mm2.
During the last years, an increased misuse of doping substances in sport has been observed. The action of doping substances characterized by the stimulation of blood flow and metabolic processes is also reflected in the hair structure. In the present study it was demonstrated that optical coherent tomography is well suited for the analysis of hair parameters influenced by doping.
Analyzing 20 patients, systemically treated with steroids which also represent doping substances, it was found that in all cases a significant increase in the cross-section of the hairs could be detected. The results obtained in the study are not only important for the screening of doping substances but also for medical diagnostics and control of compliance of patients.
Fluorescence confocal scanning laser microscopy (CLSM) using a handheld scanner, was performed to visualize the microscopic architecture of stratum corneum (SC) of the three reconstructed human epidermal (RHE) models: EpiDermTM (MatTek Corporation, Ashland, MA), EPISKIN® (EPISKIN SNC, Lyon, France) and SkinEthic® (SkinEthic Laboratories, Nice, France). To compare the differences between the SC structure of the RHE models and human SC, experiments were also performed on normal human epidermis in vivo. Sodium fluorescein stained skin cultures and human skin were imaged continuously using the confocal laser microscope Stratum, Optiscan. Fluorescein was excited at 488 nm and the fluorescent emission was detected at >505 nm. In each experiment, a series of representative images of each visualized layer of the RHE models and human SC was collected. Our early observations confirmed that the reconstructed human skin models closely resemble human SC. After improving the experimental conditions, the method might be used for studying the effects of topically applied compounds e.g. pharmaceuticals and cosmetic to the SC. (© 2007 by Astro Ltd., Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA)
Tape stripping in combination with spectroscopic measurements is a suitable method for the non-invasive determination of the dermatopharmacokinetics of topically applied substances. The penetration profiles obtained by these methods represent a cut to the stratum corneum, where the distribution of the topically applied substances is shown. For the penetration of these penetration profiles knowledge regarding the penetration pathways is necessary. Laser scanning microscopy is a well-suited method for the analysis of the penetration pathways. In the present paper, the results obtained by the combined tape stripping method and laser scanning microscopy were compared for the interpretation of penetration profiles concerning the penetration pathways of the topically applied substances. (© 2007 by Astro Ltd., Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA)
Laser scanning microscopy and tape stripping, in combination with optical methods, were used to analyze the distribution and penetration of a barrier cream into the horny layer (stratum corneum) of the human skin under in vivo conditions. The barrier cream contained microparticles of 10 – 100 μ m loaded with antioxidant substances. The cream was designed for protection of the skin surface against the destructive action of free radicals, produced by systemically applied chemotherapeutic agents reaching the skin surface via the sweat. Both methods were able to demonstrate that the barrier cream was distributed homogeneously on the skin surface forming a protection film. A penetration into deeper parts of the stratum corneum (SC) was not observed. (© 2008 by Astro Ltd., Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA)
In the present study, the distribution of the carotenoids as a marker for the complete antioxidative potential in human skin was investigated before and after the topical application of carotenoids by in vivo Raman spectroscopy with an excitation wavelength of 785 nm. The carotenoid profile was assessed after a short term topical application in 4 healthy volunteers.
In the untreated skin, the highest concentration of natural carotenoids was detected in different layers of the stratum corneum (SC) close to the skin surface. After topical application of carotenoids, an increase in the antioxidative potential in the skin could be observed. Topically applied carotenoids penetrate deep into the epidermis down to approximately 24 ?m.
This study supports the hypothesis that antioxidative substances are secreted via eccrine sweat glands and/or sebaceous glands to the skin surface. Subsequently they penetrate into the different layers of the SC.
In the present study, the ability of a shampoo formulation containing caffeine as well as the fluorescent dye fluorescein to penetrate into hair follicles was investigated by in vivo laser scanning microscopy. A contact time of two minutes between the shampoo and the skin surface was sufficient for the formulation to penetrate deeply into the hair follicles and to remain there for up to 48 hours, even after washing. As hair follicles are surrounded by a close network of capillaries, dendritic cells and stem cells, they represent an important target for drug delivery. The results of the present study demonstrated that in vivo laser scanning microscopy is an efficient tool for the investigation of the dermatopharmacokinetics of topically applied molecules and their penetration pathways, as the method yields space- and timeresolved measurements. (© 2007 by Astro Ltd., Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA)
Contact allergies to textile dyes are common and can cause severe eczema. In the present study, we investigated the penetration of a fluorescent textile dye, dissolved from a black pullover, into the skin of one volunteer during perspiration and nonperspiration. Previously, wearing this pullover had induced a severe contact dermatitis in an 82-year old woman, who was not aware of her sensitization to textile dyes. The investigations were carried out by in vivo laser scanning microscopy. It could be demonstrated that the dye was eluted from the textile material by sweat. Afterwards, the dye penetrated into the stratum corneum and into the hair follicles. Inside the hair follicles, the fluorescent signal was still detectable after 24 h, whereas it was not verifiable anymore in the stratum corneum, Laser scanning microscopy represents an efficient tool for in vivo investigation of the penetration and storage of topically applied substances and allergens into the human skin and reveals useful hints for the development and optimization of protection strategies. (© 2009 by Astro Ltd., Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA)
Basal cell carcinoma (BCC) is the most common cutaneous malignancy with increasing incidence rates worldwide. A number of established treatments are available, including surgical excision. The emergence of new non-invasive treatment modalities has prompted the development of non-invasive optical devices for therapeutic monitoring and evaluating treatment efficacy.
This study was aimed to evaluate the clinical applicability of a fluorescence confocal laser scanning microscope (CFLSM) for non-invasive therapeutic monitoring of basal cell carcinoma treated with Imiquimod (Aldara®) as topical immune-response modifier. Eight participants with a diagnosis of basal cell carcinoma (BCC) were enrolled in this investigation. Sequential evaluation during treatment with Imiquimod showed progressive normalization of the confocal histomorphologic parameters in correlation with normal skin.
Confocal laser scanning microscopy was able to identify characteristic features of BCC and allowed the visualization of therapeutic effects over time. Thus our results indicate the clinical applicability of CFLSM imaging to evaluate treatment efficacy in vivo and non-invasively.
Much research in medicine is focused on the development of antitumoral therapies. The encapsulation of the active single chemotherapy agent doxorubicin in highly stable Stealth ® liposomes can cause dominant and dose-limiting mucocutaneous reactions, such as the hand-foot syndrome. The aim of the present study was to determine the kinetic and localization of doxorubicin or its metabolites within the skin in vivo. Therefore, a laser scanning microscope was used to excite and detect the fluorescence of these compounds. The approach was tested on porcine skin after topical application of the agent prior to measurement. Subsequently, the laser scanning microscope was used to study skin areas mainly affected by the hand-foot syndrome on a patient who had undergone a therapy with the encapsulated doxorubicin (Caelyx ®). Fluorescence was detected on the skin surface of the axilla, on the forehead inside furrows and the sebaceous glands (1 h after commencement of the treatment) and within the skin of the sole and the palm (2 h after beginning the injection). These signals were determined for up to 3.5 h. The results obtained indicate that both the sweat glands and sebaceous glands function as pathways for the release of doxorubicin or its metabolites. The described method opens the possibility for further studies to develop and test prevention strategies against the hand-foot syndrome. (© 2004 by HMS Consultants. Inc. Published exclusively by WILEY-VCH Verlag GmbH & Co.KGaA)
In the present paper the application of optical noninvasive methods in dermatology and cosmetology is discussed. Laser scanning microscopy (LSM) and optical coherent tomography (OCT) are the most promising methods for this application. Using these methods, the analysis of different skin parameters like dryness and oiliness of the skin, the barrier function and the structure of furrows and wrinkles are discussed. Additionally the homogeneity of distribution of topically applied creams, as well as their penetration into the skin were investigated. It is shown that these methods are highly valuable in dermatology for diagnostic and therapy control and for basic research, for instance in the field of structure analysis of hair follicles and sweat glands. The vertical images of the tissue produced by OCT can be easily compared with histological sections. Unfortunately, the resolution of the OCT technique is not high enough to carry out measurements on a cellular level, as is possible by LSM. LSM has the advantage that it can be used for the investigation of penetration and storage processes of topically applied substances, if these substances have fluorescent properties or if they are fluorescentlabelled. (© 2008 by Astro Ltd., Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA)
The occurrence of skin irritation and contact dermatitis is a common problem in various occupational groups. The use of barrier creams represents a frequently utilized prophylactic measure for the protection of the skin, even if their effectiveness is hotly debated. Up to now, a number of in-vivo and in-vitro methods are in existence for the evaluation of barrier creams (BC), which have shown different results regarding the effectiveness of BC. The aim of the present study was the application of the in-vivo laser scanning microscopy for the evaluation of barrier creams. Therefore, sodium fluorescein was applied topically to track its penetration. Three different barrier creams were investigated (Vaseline (B) and 2 different commercially available barrier creams (C and D)) and compared to a reference area (A). It was shown that Vaseline represents a 100% protection against sodium fluorescein as no fluorescence could be detected in the stratum corneum. For barrier cream C, only a weak fluorescent signal was detectable, while in the case of barrier cream D, the fluorescent signal was comparable to that of the reference skin area. The present study revealed that the laser scanning microscopy represents an adequate, non-invasive, quick in-vivo technique for the evaluation of BC that even tracks low differences of effectiveness, which enables the ranking of BC. (© 2007 by Astro, Ltd. Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA)
Normal skin barrier function is an essential aspect of skin homeostasis and regeneration. Dynamic inflammatory, proliferative and neoplastic skin processes such as wound healing, psoriasis and contact dermatitis are associated with a significant disruption of the skin barrier. In recent years, there has been increasing interest in evaluating cosmetic and pharmacologic products for their ability to restore these protective properties. The gold standard for characterization of barrier function has been the measurement of the transepidermal water loss, however the disadvantage of this method is its interference with several endogenous and exogenous factors such as hydration, perspiration and topically applied substances. This study was aimed to test the clinical applicability of a fluorescence confocal laser scanning microscope (LSM) for a systematic morphologic analysis of the structure, integrity and thickness of the stratum corneum in 10 otherwise healthy volunteers. The influence of skin treatment with commercial moisturizing cream on skin barrier function was evaluated in serial non-invasive examinations. Our findings showed that in vivo LSM may represent a simple and efficient method for the characterization of skin barrier properties, such as the thickness and hydration of the stratum corneum. (© 2008 by Astro Ltd., Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA)
The aim of this study was to determine whether Laser scanning confocal microscopy (LSM) is able to visualize differences in melanin content and distribution in different Skin Phototypes. The investigations were carried out on six healthy volunteers with Skin Phototypes II, IV, and VI. Representative skin samples of Skin Phototypes II, V, and VI were obtained for histological analysis from remaining tissue of skin grafts and were used for LSM-pathologic correlation.
LSM evaluation showed significant differences in melanin distribution in Skin Phototypes II, IV, and VI, respectively. Based on the differences in overall reflectivity and image brightness, a visual evaluation scheme showed increasing brightness of the basal and suprabasal layers with increasing Skin Phototypes. The findings correlated well with histological analysis. The results demonstrate that LSM may serve as a promising adjunctive tool for real time assessment of melanin content and distribution in human skin, with numerous clinical applications and therapeutic and preventive implications.
Fiber-based confocal laser scanning microscopy affords a vast field of application in medical research and clinical practice. The application of fluorescent dye allows real-time imaging of yeasts of the genus Malassezia on human skin in vivo. An Ar⁺-laser is used to excite the fluorescent food dye sodium fluorescein at 488 nm. Its emission is simultaneously detected in the spectral region from 500–600 nm. Topically applied fluorescein labels fungal microstructures in native habitat. Cumulative intradermal injection of the same dye enables a subsurface view of the underlying cutaneous area.
In the present paper, we report the ability of the combination of in vivo confocal microscopy and sodium fluorescein application to produce real-time and high-resolution images of fungal structures on human skin. The obtained confocal images demonstrate the micro architecture of superficial Malassezia yeasts and corneocytes in horizontal plane. Taxonomic classification into different Malassezia species and observation of their colonisation patterns in native milieu brings new conclusions to fungal skin behaviour and diseases. We demonstrate advantages in clinical investigation practice over traditional laboratory routine using this non-invasive imaging tool. The described method opens new promising prospects and the possibility for further studies in fungal research.
It is well known from the literature that carotenoid antioxidant substances decrease after the irradiation of the skin with
UV light. Available literature has shown no information about the response time and total dynamics of degradation of carotenoid
antioxidants after UV irradiation. The measurements were made with the HPLC method, which is time-consuming and does not give
relevant information about the dynamics of degradation of carotenoids in the skin after UV irradiation. With the introduction
of new noninvasive spectroscopic methods, it became possible to measure in vivo the behavior of carotenoid antioxidant substances,
such as betacarotene and lycopene in the skin after UV light exposure. In the present study, the resonance Raman spectroscopy
method was used as a fast and noninvasive optical method to measure the dynamics of degradation of beta-carotene and lycopene
in living human skin after UV exposure. It was found that the beta-carotene and lycopene concentration in the skin does not
decrease immediately after UV irradiation. There is a time delay, which varies from 30 up to 90 min for beta-carotene and
from 0 up to 30 min for lycopene. A strong nonlinear correlation between the individual antioxidant level of volunteers and
the magnitude of destruction of antioxidants in the skin was found.
Confocal scanning laser microscopy (CSLM) constitutes an optical, noninvasive method providing visualization of tissue architecture
with resolution similar to that of light microscopy. In dermatology, confocal imaging enables in vivo measurements of surface
and subsurface skin microstructures. Skin annexes, as well as cutaneous cells from different epidermal layers, can be easily
distinguished; their change in morphology from skin surface to the papillary dermis can be observed. Therefore, CSLM possesses
a high potential for diagnostical purposes and dermatological research. The aspect of normal skin in contrast to the pathogenic
state can be exposed. In our studies, we used in vivo fluorescence CSLM for morphometric analysis of healthy human skin and
for imaging a number of clinically relevant inflammatory, proliferative, and neoplastic skin disorders. We report the ability
to produce high-resolution histoimages of normal and pathological epidermis using this nondestructive visualization technique.
Changes in keratinocyte size, shape, and morphology, as well as changes in the distribution pattern of the fluorescent emission
of the dye, can be detected. Furthermore, novel fiber optic elements support a flexible handling of the rigid microscopic
gadgetry. Four clinical examples of implementation were elected and instanced for demonstration.
Hair follicles represent a long-term storage of topically applied drugs and cosmetics in the skin. Analyzing the penetration
of particle-and nonparticle-containing formulations by laser scanning microscopy, it was found, surprisingly, that particles
at a size similar to the thickness of the keratin cells of the hair penetrate more efficiently into the hair follicles. These
results were obtained from in vitro and in vivo investigations. It seems that the moving hairs in the follicles act as a geared
pump because of the zigzag structure of the surface of the hairs. This pumping effect probably pushes particles with the corresponding
size deep into the hair follicles.
We report on a double-blind, vehicle-controlled, single-center confirmatory study with random assignment. The purpose of the study was to investigate the topical bioavailability of different topical corticosteroid formulations in healthy human beings focussing on desoximetasone (DM).
Two DM 0.25% formulations [ointment (DM-o) and fatty ointment (DM-fo, water-free); class III corticosteroids], the corresponding active ingredient-free vehicles and three comparators of different strength [clobetasol propionate 0.05% (CP 0.05%), fatty ointment, class IV; hydrocortisone (HC) 1%, fatty ointment, class I, and betamethasone (BM) 0.05%, fatty ointment, class III] were tested using the vasoconstriction assay. The degree of vasoconstriction (blanching) in the treatment field was compared to the one found in untreated control fields using chromametric measurements and clinical assessment.
DM-o 0.25%, DM-fo 0.25% and BM 0.05% showed similar vasoconstrictive potential, i.e., clear blanching. In fact, both DM preparations were proven to be noninferior to BM 0.05%, while CP 0.05% was found a little less active. HC 1.0% and the DM vehicles showed no clear-cut vasoconstrictive effect. No adverse events related to the study medications were observed. Good topical bioavailability of both DM formulations was detected by chromametric measurement and clinical assessment.
In the past, it was assumed that the intercellular route was the only relevant penetration pathway for topically applied substances. Recent results on follicular penetration obtained at the Center for Experimental and Applied Cutaneous Physiology, Charité - Universitätsmedizin Berlin, Germany, emphasize that the hair follicles represent a highly relevant and efficient penetration pathway and reservoir for topically applied substances.
Cutaneous irritation presents a major health problem with serious social and occupational impact. The interaction between an irritant and the human skin depends on multiple factors: the intrinsic properties and the nature of the irritant itself, and specific individual- and environment-related variables. The main pathological mechanisms of irritancy include skin barrier disruption, induction of a cytokine cascade and involvement of the oxidative stress network; all of them resulting in a visible or subclinical inflammatory reaction. In vivo, different non-invasive parameters for the evaluation of skin irritation and irritant potential of compounds and their specific formulations have been introduced, such as epidermal barrier function, skin hydration, surface pH, lipid composition, skin colour and skin blood flow. The diverse physiological changes caused by irritating agents require implementation of a multiparametric approach in the evaluation of cutaneous irritancy.
Follicular drug delivery is the prerequisite for an effective treatment of androgenetic alopecia or other reasons of premature hair loss.
The follicular penetration of caffeine, applied topically in a shampoo formulation for 2 min, was measured with highly sensitive surface ionization in combination with mass spectroscopy, a selective method for the detection of very small quantities of transcutaneously absorbed substances in the blood. An experimental protocol, developed to selectively block the follicular pathway within the test area, was used. Based on this principle, a clear distinction between interfollicular and follicular penetration of topically applied caffeine was feasible.
After 2 min, caffeine penetrated via the hair follicles and stratum corneum.
It was found that the penetration via hair follicles was faster and higher compared with the interfollicular route and that hair follicles are the only pathway for fast caffeine absorption during the first 20 min after application.
The fascinating topic of skin barrier continues to engage researchers from diverse disciplines both in academia and industry. Much of the information on the basic biology of barrier formation, its ontogeny as well as repair and homeostasis comes from studies on animal models. A smaller number of human studies have validated the usefulness of animal models, while highlighting some essential differences. We submit that the human skin barrier is unique in several ways, as much due to our adaptive ability as our control over the environment (macro and micro) that none of the other species have exerted. The human skin is not only exposed to the greatest variations of environment due to our phenomenal mobility but also to the largest number of xenobiotics, both chemical and microbial, resulting from human activity. In this overview, we attempt to evaluate the interdependent relation of skin barriers to environmental stressors hoping to raise interest in some of the lesser known or neglected aspects of human skin barriers as they relate to skin health and dysfunctions.
The stratum corneum, the outermost layer of the skin, regulates the passive loss of water to the environment. Furthermore, it is well accepted that drug penetration is influenced by skin hydration, which may be manipulated by the application of moisturizing or oleaginous vehicles. Measurements of transepidermal water loss (TEWL), and of skin hydration using a corneometer, were used to assess the effect of different vehicles on stratum corneum barrier function in vivo in human volunteers. A microemulsion significantly increased skin hydration relative to a reference vehicle based on medium chain triglycerides; in contrast, Transcutol(R) lowered skin hydration. TEWL measurements confirmed these observations.
The effect of anatomic site on the in vivo relationship between the total penetration of four compounds and the amount of the compounds present in the stratum corneum at the end of application was studied in humans. For each anatomic site, 1,000 nmol of 14C-radiolabeled benzoic acid, benzoic acid sodium salt, caffeine, or acetylsalicylic acid was applied to 1-cm2 area of skin of male Caucasian patients aged 28 +/- 2 years (groups of 6-8). For each molecule and each site, a first application on the right-hand side of the body allowed total absorption to be determined by measuring the amount excreted in the urine. A second application, performed 48 h later on the contralateral site, enabled the total amount of substance present in the stratum corneum at the end of application (30 min) to be assessed after cellophane-tape stripping of the treated area. The results showed that skin permeability varied substantially, depending both on the physicochemical nature of the molecule and on the anatomical location. In general, the rank order in skin permeability of the studied areas appears to be as follows: arm less than or equal to abdomen less than postauricular less than forehead. Whatever the compound applied, the forehead was approximately 2 times as permeable as the arm or abdomen. Independent of the origin of the differences in permeability observed among sites, there exists a linear correlation (r = 0.97, p less than 0.001) between the amounts of substance present in the stratum corneum at the end of application (30 min) and the total amounts which penetrated within a 4-d period.(ABSTRACT TRUNCATED AT 250 WORDS)
Percutaneous absorption-enhancing effects on the skin of hairless mice of 11 monoterpenes [1, (+)-limonene; 2, (-)-menthone; 3, (+)-terpinen-4-ol; 4, alpha-terpineol; 5, 1,8-cineole; 6, (+)-carvone; 7, (-)-verbenone; 8, (-)-fenchone; 9, p-cymene; 10, (+)-neomenthol; and 11, geraniol] were investigated using three different model drugs (caffeine, hydrocortisone, triamcinolone acetonide [TA]) with varying lipophilicities. Terpenes were applied at 0.4 M in propylene glycol (PG) to mouse skin. The model drugs were applied as suspensions in PG 1 hr following enhancer pretreatment. The combination of terpenes in PG provided significant enhancement of the permeation of caffeine through mouse skin. The most active compounds 10 and 11 increased permeation by between 13-fold and 16-fold. The terpenes also enhanced the delivery of hydrocortisone, but not to as great an extent. The most active compounds 3 and 4 increased permeation between 3.9-fold and 5-fold. The compounds examined did not significantly increase the delivery of TA. The most active compound 4 only increased delivery 2.5-fold, while the next most active compound 6 only increased delivery 1.7-fold. Overall, these results indicate that the combination of terpenes with PG can significantly increase the transdermal penetration of the hydrophilic drug caffeine and the polar steroid hydrocortisone.
New and emerging capillary blood sampling technologies for self-monitoring of blood glucose (SMBG) are reviewed for their impact on factors pertaining to users such as pain, and from the standpoint of skin physiology and technical feasibility. Innovative blood sampling techniques based on lancets for skin penetration on nonfinger (alternate) sites such as the forearm seem to be virtually painless, convenient and cost effective as compared to other methods such as laser-based perforation of fingertips. Alternate site blood sampling with new lancet devices appears not only medically sound but also technically practical and user-friendly. It is anticipated that alternate site blood sampling techniques would improve compliance rate and, consequently, outcome of treatment for patients with diabetes.
For the evaluation and quantification of follicular penetration processes, the knowledge of variations of hair follicle parameters in different body sites is basic. Characteristics of follicle sizes and potential follicular reservoir were determined in cyanoacrylate skin surface biopsies, taken from seven different skin areas (lateral forehead, back, thorax, upper arm, forearm, thigh, and calf region). The highest hair follicle density and percentage of follicular orifices on the skin surface and infundibular surface were found on the forehead, whereas the highest average size of the follicular orifices was measured in the calf region. The highest infundibular volume and therefore a potential follicular reservoir was calculated for the forehead and for the calf region, although the calf region showed the lowest hair follicle density. The calculated follicular volume of these two skin areas was as high as the estimated reservoir of the stratum corneum. The lowest values for every other parameter were found on the forearm. The present investigation clearly contradicts former hypothesis that the amount of appendages of the total skin surface represents not more than 0.1%. Every body region disposes its own hair follicle characteristics, which, in the future, should lead us to a differential evaluation of skin penetration processes and a completely different understanding of penetration of topically applied drugs and cosmetics.
In the past, intercellular penetration was assumed to be the most important penetration pathway of topically applied substances. First hints that follicular penetration needs to be taken into consideration were confirmed by recent investigations, presented during the workshop "Follicular Penetration and Targeting" at the 4th Intercontinental Meeting of Hair Research Societies", in Berlin 2004. Hair follicles represent an efficient reservoir for the penetration of topically applied substances with subsequent targeting of distinct cell populations, e.g., nestin-expressing follicular bulge cells. The volume of this reservoir can be determined by differential stripping technology. The follicular penetration processes are significantly influenced by the state of the follicular infundibulum; recent experimental investigations could demonstrate that it is essential to distinguish between open and closed hair follicles. Topically applied substances can only penetrate into open hair follicle. Knowledge of follicular penetration is of high clinical relevance for functional targeting of distinct follicular regions. Human hair follicles show a hair-cycle-dependent variation of the dense neuronal and vascular network. Moreover, during hair follicle cycling with initiation of anagen, newly formed vessels occur. Thus, the potential of nestin-expressing hair follicle stem cells to form neurons and blood vessels was investigated.
To determine the effect of skin thickness on the percutaneous penetration and distribution of test compounds with varying physicochemical properties using in vitro systems. Studies were carried out in accordance with OECD guidelines on skin absorption tests.
Percutaneous penetration of caffeine (log P -0.01), testosterone (log P 3.32), propoxur (log P 1.52) (finite dose in ethanol to water vehicle ratio) and butoxyethanol (log P 0.83) (undiluted finite dose or as an infinite dose 50% [v/v] aqueous solution) through skin of varying thicknesses under occluded conditions was measured using flow through cells for 8-24 h. Saline (adjusted to pH 7.4) was used as receptor fluid, with BSA added for studies with testosterone and propoxur. Following exposure, the remaining surface dose was removed by swabbing and the skin digested prior to scintillation counting.
The maximum flux of caffeine was increased with decreasing skin thickness, although these differences were found to be non-significant. The presence of caffeine in the skin membrane was not altered by skin thickness. Maximum flux and cumulative dose absorbed of testosterone and butoxyethanol (in both finite and infinite doses) were markedly reduced with full thickness (about 1 mm thick) skin compared with split thickness skin (about 0.5 mm). Maximum flux of propoxur (dissolved in 60% ethanol) was clearly higher through skin of 0.71 mm than through skin of 1.36 mm, but no difference was found between 0.56 and 0.71 mm. The proportion of propoxur present in the membrane after 24 h increased significantly over the complete range of thicknesses tested (0.56-1.36 mm).
A complex relationship exists between skin thickness, lipophilicity and percutaneous penetration and distribution. This has implications for risk assessment studies and for the validation of models with data from different sources.
Androgenetic alopecia (AGA) is a common problem in men of all ages, affecting approximately 50% at 50 years of age. The underlying cause is an androgen-dependent miniaturization of genetically predetermined hair follicles. Here, the hair organ culture model was used to investigate the effects of testosterone and caffeine; the latter being a promising candidate for hair growth stimulation.
Hair follicles from 14 biopsies, taken from the vertex areas from male AGA patients, were cultivated for 120-192 h in vitro with normal William's E medium (control) or William's E medium containing different concentrations of testosterone and/or caffeine. Hair shaft elongation was measured daily and at the end of cultivation, cryosections of follicles were stained with Ki-67 to evaluate the degree and localization of keratinocyte proliferation.
Significant growth suppression was found in hair follicles treated with 5 microg/ml testosterone. This was counteracted by caffeine in concentrations of 0.001% and 0.005%. Moreover, caffeine alone led to a significant stimulation of hair follicle growth. These results were confirmed immunohistochemically by Ki-67 staining.
Androgen-dependent growth inhibition of ex vivo hair follicles from patients suffering from AGA was present in the human hair organ culture model, a constellation which may serve for future studies to screen new substances against androgen-dependent hair loss. Caffeine was identified as a stimulator of human hair growth in vitro; a fact which may have important clinical impact in the management of AGA.
This influence of skin stretching and hair follicle sealing on the delivery of retinyl ascorbate (RA-AsA) to the epidermis was probed in vitro. Porcine ear skin was subjected to stretching by 2 and 4 mm (3.3 and 6.7%, respectively); the hair follicles of other skin sections were located and painstakingly sealed using adhesive. After mounting in Franz cells the skin was dosed with 100 microl of 2.5 mM RA-AsA in methanol/PBS with water as receptor phase. After 24 h the diffused areas were subjected to tape stripping, and the amount of RA-AsA was determined in 5 groups of 9 strips. Statistical analysis of the resulting depth profiles showed that there was no statistical difference between unstretched skin and the skin that had the follicles sealed across the 5 depth bands. Between 0 and 2 mm stretch there were generally significant differences; between 0 and 4 mm p < 0.001 was obtained at each depth. The data from this limited exercise suggest that in native skin the follicular route does not contribute to dermal absorption, but when disturbed (as when stretched) follicular delivery can substantially increase drug delivery into the skin by up to approximately 20-40%. Skin stretching becomes difficult beyond about 7%.
What is already known about this subject:
* In recent years, it has been suggested that hair follicles represent important shunt routes into the skin for drugs and chemicals [1-3]. * In vitro studies have shown the importance of skin appendages for skin penetration by hydrophilic compounds [4]. Investigation of follicular penetration in vivo has been difficult due to the absence of appropriate analytical methods or suitable animal model systems. * Recently, a new method was described that quantifies follicular penetration in vivo by using selective closure of hair follicles [5]. * Caffeine is frequently used in skin penetration experiments as a model for highly water-soluble compounds. Occlusion [6] and skin thickness [7] seem to have little influence on the penetration of caffeine. However, percutaneous absorption rates for caffeine exhibit regional skin differences in humans in vivo[1].
What this study adds:
* The results of the present study demonstrate that a fast drug delivery of caffeine occurs through shunt routes. Therefore, hair follicles are considerable weak spots in our protective sheath against penetration into the body by hydrophilic substances. * We showed that there is a quantitative distinction between follicular penetration and interfollicular diffusion of caffeine in vivo. * These findings are of importance for the development and optimization of topically applied drugs and cosmetics. In addition, such properties must be considered in the development of skin protection measures.
Aims:
The skin and its appendages are our protective shield against the environment and are necessary for the maintenance of homeostasis. Hypotheses concerning the penetration of substances into the skin have assumed diffusion through the lipid domains of the stratum corneum. It is believed that while hair follicles represent a weakness in the shield, they play a subordinate role in the percutaneous penetration processes. Previous investigation of follicular penetration has mostly addressed methodical and technical problems. Our study utilized a selective closure technique of hair follicle orifices in vivo, for the comparison of interfollicular and follicular absorption rates of caffeine in humans.
Methods:
Every single hair follicle within a delimited area of skin was blocked with a microdrop of a special varnish-wax-mixture in vivo. Caffeine in solution was topically applied and transcutaneous absorption into the blood was measured by a new surface ionization mass spectrometry (SI/MS) technique, which enabled a clear distinction to be made between interfollicular and follicular penetration of a topically applied substance.
Results:
Caffeine (3.75 ng ml(-1)) was detected in blood samples, 5 min after topical application, when the follicles remained open. When the follicles were blocked, caffeine was detectable after 20 min (2.45 ng ml(-1)). Highest values (11.75 ng caffeine ml(-1)) were found 1 h after application when the follicles were open.
Conclusions:
Our findings demonstrate that hair follicles are considerable weak spots in our protective sheath against certain hydrophilic drugs and may allow a fast delivery of topically applied substances.
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