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Topical retinoids are frequently applied for therapeutic and cosmeceutical reasons although their bioavailability is low due to their chemical and photochemical instability. Moreover, skin irritation is a common side effect. Therefore, proretinal nanoparticles (PRN) as a novel formulation of topical retinoids, which are based on chitosan grafted with retinal through reversible linkage, were developed and their skin penetration behavior was studied. As nanoparticles preferably penetrate into the hair follicles, the follicular penetration depths of PRN at different time points were investigated. Moreover, the release capacity of the nanoparticulate system was studied using fluorescein as a model drug. Additionally, the concentration of retinal in the stratum corneum and in the hair follicles was quantified after application in particulate and non-particulate form. The results showed that the nanocarriers reached the infundibular area of the hair follicles, irrespective of the incubation time. The nanoparticles were able to release their model drug within the hair follicle. The retinal concentration delivered to the stratum corneum and the hair follicles was significantly higher when retinal was applied in the particulate form. In conclusion, the presented proretinal nanoparticle system may help to overcome the main problems of topical retinoid therapy, which are skin irritation, chemical and photochemical instability and low bioavailability, thus improving the topical retinoid therapy.

Nanoparticles can be applied to the hair follicles, which can serve as reservoirs for triggered drug release. A valid measurement method for the determination of the pH within the hair follicle in vivo has not been shown yet. Here, melamine formaldehyde particles up to 9 µm in size were applied on 40 freshly plucked scalp hairs of eight individuals to determine the pH along the hair shaft down to the root area of the hair. For fluorescent pH indicators, pyranine and Nile blue were incorporated into the particles. Measurements were conducted using confocal laser scanning microscopy. A pH decay gradient could be found from the hair sheath towards the external hair shaft (p = 0.012) with pH values at the hair sheath of 6.63 ± 0.09, at the hair sheath end at 6.33 ± 0.11, and at the external hair shaft at 6.17 ± 0.09 (mean ± SE). The pH difference between the hair sheath end and the external hair shaft was found to be significant (p = 0.036). The results might be comparable with the pH within the hair follicle in vivo indicating a pH increase towards the hair root.

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 (approximate to 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 (approximate to 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.

Recent studies have shown that pollen proteins can penetrate the impaired skin barrier of atopic patients and exacerbate their disease. In the presented study the effect of a topically applied barrier-enhancing formulation was investigated for its preventive effect on the uptake of pollen allergens into CD1c+ epidermal cells. The pollen proteins were fluorescence labelled and applied on barrier-disrupted excised human skin. CD1c+ cells were selected after magnetic cell sorting and analysed using laser scanning microscopy. In untreated disrupted skin, 81% of the CD1c+ cells contained the fluorescence-labelled pollen allergens. In formulation-pretreated skin only 12% of the CD1c+ cells showed an uptake of pollen allergens. These results encourage the treatment of atopic patients with barrier-enhancing formulations to reduce the impact of pollen on air-exposed skin areas and hence the exacerbation of cutaneous symptoms.

Drug delivery through the skin barrier is a topic of high interest in cosmetology and dermatology. The disruption of the skin barrier is a common method to stimulate skin penetration. In the present chapter tissue tolerable plasma is used to damage the skin barrier, enabling topically applied substances to effectively pass the stratum corneum. Subsequently to plasma-tissue interaction the skin barrier recovers immediately. Topically applied drugs have to be applied before the plasma treatment onto the skin. In this case, the plasma chemical reaction of the drug has to be prevented by using plasma resistant nanocontainers.

Penetration studies are essential for the evaluation and optimization of topically applied substances. Stripping techniques are well suited for such penetration measurements. In this chapter the methods of tape stripping and cyanoacrylate biopsy are presented and discussed. While the tape stripping method is applied to analyze the penetration of topically applied substances into the stratum corneum, combined application of tape stripping and cyanoacrylate biopsy, which is known as differential stripping, permits to analyze the penetration of topically applied substances into the stratum corneum and into the hair follicles, separately. Although the described methods are relatively simple, their application requires adherence to standard protocols to avoid mistakes. The present chapter provides not only an overview of the scientific application methods for both tape stripping and differential stripping, but also useful recommendations for standardized work.

Two state-of-the-art microscopic optical methods, namely, confocal laser scanning microscopy in the fluorescence and reflectance regimes and multiphoton tomography in the autofluorescence and second harmonic generation regimes, are compared for porcine skin ex vivo and healthy human skin in vivo. All skin layers such as stratum corneum (SC), stratum spinosum (SS), stratum basale (SB), papillary dermis (PD) and reticular dermis (RD) as well as transition zones between these skin layers are measured noninvasively at a high resolution, using the above mentioned microscopic methods. In the case of confocal laser scanning microscopy (CLSM), measurements in the fluorescence regime were performed by using a fluorescent dye whose topical application on the surface is well suited for the investigation of superficial SC and characterisation of the skin barrier function. For investigations of deeply located skin layers, such as SS, SB and PD, the fluorescent dye must be injected into the skin, which markedly limits fluorescence measurements using CLSM. In the case of reflection CLSM measurements, the obtained results can be compared to the results of multiphoton tomography (MPT) for all skin layers excluding RD. CLSM cannot distinguish between dermal collagen and elastin measuring their superposition in the RD. By using MPT, it is possible to analyse the collagen and elastin structures separately, which is important for the investigation of anti-aging processes. The resolution of MPT is superior to CLSM. The advantages and limitations of both methods are discussed and the differences and similarities between human and porcine skin are highlighted.

Resonance Raman spectroscopy and multi-photon tomography were used in vivo to analyse the influence of sun exposure on the cutaneous carotenoids and collagen/elastin fibers. Comparing Berlin (low sun exposure) and Monegasque (high sun exposure) volunteers, it could be demonstrated that extended sun exposure significantly reduces the cutaneous carotenoids and collagen/elastin concentration (p < 0.05). The tendency towards correlation (R(2) = 0.41) between the dermal collagen/elastin (SAAID) and carotenoids confirms the important role of antioxidants in the protection against sun-induced negative effects. The application of sunscreen was shown to be effective, protecting cutaneous carotenoids and collagen/elastin from being damaged subsequent to sun exposure.

The aim of the present study was to solve the water insolubility limitation of the medically and cosmetically interesting substance Garcinia mangostana Linn (GML) extract by encapsulation, and to evaluate and investigate the penetration efficacy of free and encapsulated GML in two different vehicles (water and cream) in porcine ear skin. The follicular penetration depth was determined in 50 hair follicles for each of the four formulations by means of fluorescence microscopy. Tape stripping was used to compare the distribution properties of GML with all formulations on the stratum corneum. The results showed that encapsulated and free GML in the cream base penetrated deeper into hair follicles than if applied in an aqueous base. In addition, encapsulated GML could be distributed more homogeneously on the stratum corneum than the free GML. In conclusion, it was found that encapsulated GML in a cream base had the most effective penetration level in porcine ear skin.

Liposomes are frequently described as drug delivery systems for dermal and transdermal applications. Recently, it has been shown that particulate substances penetrate effectively into hair follicles and that the follicular penetration depth can be increased by massaging the skin, which simulates the in vivo movement of hairs in the hair follicles. In the present study, massage was applied to skin mounted to Franz diffusion cells. By means of confocal laser scanning microscopy, the influence of massage and occlusion on the follicular penetration depths of rigid and flexible liposomes loaded with a hydrophilic and lipophilic dye was investigated. The application of massage increased follicular penetration significantly. Occlusion resulted in an increased follicular penetration depth only for rigid liposomes, whereas invasomes did not penetrate more effectively if occlusion was applied. The results confirm that massage is an important tool for increasing follicular penetration in ex vivo studies using Franz diffusion cells. Occlusion may reduce the efficacy of follicular penetration depending on the specific liposomal preparation. Rigidity in particular appears to be a relevant parameter. (c) 2013 Elsevier B.V. All rights reserved.

The requirements on nanoparticles for cosmetic and medical applications are very different. While nanoparticles applied in sunscreens shall remain on the skin surface or in the upper cell layers of the stratum corneum, nanoparticles for medical drug delivery shall penetrate through the skin barrier to the target structures in the living cells. Under the Collaborative Research Project 1112 various methods are used at the CCP to investigate the cutaneous penetration and storage of nanoparticles, hair follicles being in the focus of attention. Human hair follicles are ideal target structures for drug delivery. Hosting both the stem and dendritic cells, they are surrounded by a dense network of blood vessels. Investigating nanoparticles of different size and materials, particles of approximately 600 nm in diameter were found to penetrate best into the hair follicles, where they can be stored for maximally 10 days. Their retention time in the hair follicles exceeds that in the stratum corneum by almost one order of magnitude. Particles penetrate more efficiently into the hair follicles than non-particulate substances. For particles from 40 nm-1 µm in diameter, however, no follicular penetration has been detectable if the skin barrier was intact. This is plausible as the hair follicle has its own barrier. It will be demonstrated that the best way for drug delivery is the application of drug-loaded particulate carrier systems. In the hair follicles the particles may either dissolve and release the drug, or an external signal must trigger the drug release from the particle.

An improvement of the penetration efficiency combined with the controlled release of actives in the skin can facilitate the medical treatment of skin diseases immensely. Dexamethasone (Dx), a synthetic glucocorticoid, is frequently used for the treatment of inflammatory skin diseases. To investigate the penetration of nano-sized lipid particles (NLP) loaded with Dx in comparison to a commercially available base cream, different techniques were applied. Electron paramagnetic resonance (EPR) spectroscopy was used to monitor the penetration of Dx, which was covalently labeled with the spin probe 3-(Carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PCA). The penetration into hair follicles was studied using confocal laser scanning microscopy (CLSM) with curcumin-loaded NLP. The penetration of the vehicle was followed by confocal Raman microscopy (CRM). Penetration studies using excised porcine skin revealed a more than twofold higher penetration efficiency for DxPCA into the stratum corneum (SC) after 24h incubation compared to 4h incubation when loaded to the NLP, whereas when applied in the base cream, almost no further penetration was observed beyond 4h. The distribution of DxPCA within the SC was investigated by consecutive tape stripping. The release of DxPCA from the base cream after 24h in deeper SC layers and the viable epidermis was shown by EPR. For NLP, no release from the carrier was observed, although DxPCA was detectable in the skin after the complete SC was removed. This phenomenon can be explained by the penetration of the NLP into the hair follicles. However, penetration profiles measured by CRM indicate that NLP did not penetrate as deeply into the SC as the base cream formulation. In conclusion, NLP can improve the accumulation of Dx in the skin and provide a reservoir within the SC and in the follicular infundibula.

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.

The antimicrobial treatment of wounds is still a major problem. Tissue-tolerable electrical plasma (TTP) is a new approach for topical microbial disinfection of the skin surface. The aim of the present study was to investigate the influence of TTP on a carotenoid profile in relation to skin physiology parameters (epidermal barrier function, stratum corneum (SC) hydration, surface temperature and irritation parameters). We were interested in the interaction of TTP and the antioxidative network, as well as the consequences for skin physiology parameters. These parameters are also indicative of TTP safety in vivo. For plasma application, 'Kinpen 09' was used (surface exposure 30-43°C) for 3 s. Beta-carotene and water profiles were assessed by in vivo Raman microspectroscopy (skin composition analyzer 3510). Skin physiology parameters were measured with Tewameter TM 300, Corneometer CM 825, skin thermometer and Chromameter CR 300. All parameters were assessed non-invasively on seven healthy volunteers before and after plasma application in vivo. We could show that TTP application leads to a decrease in beta-carotene especially in the superficial SC. Skin-surface temperature increased by 1.74°C, while the transepidermal water loss (TEWL) increase indicated an impaired barrier function. SC hydration decreased as seen in water profile especially in the superficial layers and capacitance values. A slight increase in skin redness was measurable. The induction of reactive oxygen species is probably the major contributor of TTP efficacy in skin disinfection. Skin physiology parameters were influenced without damaging the skin or skin functions, indicating the safety of TTP under in vivo conditions.

The analysis of the skin barrier properties is important in various fields of medical treatment and cosmetology. The development and improvement of topically applied substances require an objective analysis of the skin barrier characteristics. Transepidermal water loss (TEWL) measurement is the standard method to characterize epidermal barrier function. The most important disadvantage of this method though, is that it can be affected by different exogenous and endogenous factors, e.g. water content of the applied formulation and room temperature. In the present study, TEWL measurements are compared to laser scanning microscopic (LSM) measurements, concerning the use of these two methods for the non-invasive in vivo characterization of the epidermal barrier function. The investigations were performed prior and subsequent to treatment of dry skin with a gel mixture, developed for skin treatment after radiotherapy for cancer. The present results indicate that in vivo laser scanning microscopy is an appropriate method for the characterization of the skin barrier structure without interference by external factors. (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

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.

Hair follicles represent interesting target sites for topically applied substances such as topical vaccinations or agents used in the field of regenerative medicine. In recent years, it could be shown that particles penetrate very effectively into the hair follicles. In the present study, the influence of particle size on the follicular penetration depths was examined. The penetration depths of two different types of particles sized 122 to 1000 nm were determined in vitro on porcine skin. The results revealed that the particles of medium size (643 and 646 nm, respectively) penetrated deeper into the porcine hair follicles than smaller or larger particles. It was concluded that by varying the particle size, different sites within the porcine hair follicle can be targeted selectively. For the human terminal hair follicle, the situation can be expected to be similar due to a similar size ratio of the hair follicles.

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.

Background/purpose: Optical coherence tomography (OCT) is able to provide highly reproducible measurements of hair shaft thickness, including hair shaft diameter, cross-sectional surface area and hair shape, similar to histology but in vivo. Variations in the caliber of hair shafts have been described in patchy hair loss like alopecia areata (AA) using electron microscopy. The aim of this study was to evaluate whether OCT is useful for the evaluation of hair shaft abnormalities in AA. Methods: The measurements were performed on patients with AA (n=9), aged 2–66 years. Fifty hairs from the border of an alopecic area and 50 hairs from an unaffected area without hair loss were examined using the OCT technique. The hair parameters were characterized by the cross-section (CS) and the form factor. The ratio of the maximal and minimal diameters of the hair at a fixed measuring distance from the scalp surface determined the form factor (dmax/dmin). Results: In all cases, the CS of hairs from an AA patch was significantly lower compared with hairs of an unaffected area. However, the form factor did not indicate any disturbances in hair growth. Conclusions: The results demonstrate that structural abnormalities of hair shafts are found in active lesions of AA, but not in clinically unaffected hairs. The OCT technique is a promising method to gain more insight into the pathogenesis of AA in a non-invasive way.

Wound healing disorders frequently occur due to biofilm formation on wound surfaces requiring conscientious wound hygiene. Often, the application of conventional liquid antiseptics is not sufficient and sustainable as (1) the borders and the surrounding of chronic wounds frequently consist of sclerotic skin, impeding an effectual penetration of these products, and (2) the hair follicles representing the reservoir for bacterial recolonization of skin surfaces are not affected. Recently, it has been reported that tissue-tolerable plasma (TTP), which is used at a temperature range between 35 and 45°C, likewise has disinfecting properties. In the present study, the effectivity of TTP and a standard liquid antiseptic was compared in vitro on porcine skin. The results revealed that TTP was able to reduce the bacterial load by 94%, although the application of the liquid antiseptic remained superior as it reduced the bacteria by almost 99%. For in vivo application, however, TTP offers several advantages. On the one hand, TTP enables the treatment of sclerotic skin as well, and on the other hand, a sustainable disinfection can be realized as, obviously, also the follicular reservoir is affected by TTP.

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.

Recently, it was reported that a plasma-jet could be efficiently applied for the antisepsis of wounds. In this case, the discharge in an argon gas stream was used to produce a so-called ``cold plasma'' on the skin surface. The thermal action of the plasma on the skin was investigated in the present study by means of laser scanning microscopy (LSM) and by histological analysis. Consequently, the plasma beam was moved with a definite velocity at an optimal distance over the skin surface. The structural changes of the tissue were analyzed. It was found by LSM that a thermal damage could be detected only in the upper cell layers of the stratum corneum (SC) at moving velocities of the plasma beam, usually applied in clinical practice. Deeper parts of the SC were not damaged. The structural changes were so superficial that they could be detected only by LSM but not by analysis of the histological sections.

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.

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.

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.

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 μm. 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. A graph is presented. Typical Raman spectrum of untreated skin (forearm, 10 μm depth) under the excitation wavelength of 785 nm.

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 μm 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.

Although in vitro skin absorption studies often detect small residues of applied test material in the epidermis/dermis, it is uncertain whether the residue is within the living skin. We studied the dermal absorption of a hair dye hydroxyanthraquinone-aminopropyl methyl morpholinium methosulphate (HAM) in human skin in vivo and in vitro. In vivo, skin (back and scalp) received 0.5% HAM in a commercial formulation at 20microg/cm2 After 0.5 or 48h, skin was tape stripped, followed by cyanoacrylate biopsies (CAB). Sebum from scalp sites was collected for 48h. In vitro, skin was treated with 20mg/cm2 dye for 0.5h, penetration determined after 24h. In vivo, at 0.5h, total recovery (back) was 0.67microg/cm2 (tape strips+CAB). Fluorescence microscopy showed HAM in the hair follicle openings (HFO). At 0.5h, scalp tape strips contained 1.80microg/cm2, HFO 0.82microg/cm2. At 48h, HFO contained 0.21microg/cm2, sebum 0.80microg/cm2. In vivo, skin residues were in the non-living skin and eliminated via desquamation and sebum secretion. In vitro, the SC contained 1.50microg/cm2, epidermis/dermis 0.86microg/cm2, receptor fluid

The uppermost layer of the skin – the stratum corneum – represents a barrier of the human organism to the environment. It prevents the penetration of substances coming into contact with the skin into the human body. In cases of strong exposure, the skin has to be protected additionally by barrier creams. In the present study, the influence of microparticles on the homogeneity of distribution of the protection cream on the skin was investigated by laser scanning microscopy. The protection cream contained antioxidant substances with a high radical protection factor for the prevention of palmar-plantar erythema often occurring during chemotherapy with doxorubicin. In this case, the chemotherapeutic substance comes out with the sweat onto the skin surface, from which it penetrates into the skin like topically applied. It was found that particles increase the homogeneity of distribution and, thus, the protection efficacy is significantly increased. The obtained results are important, not only for the prevention of side effects during chemotherapy, but also for the development and application of sunscreens and all types of topically applied drugs and cosmetics, which should form an efficient protection film on the skin.

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.

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.

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.

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.

Hair follicles have been assigned an increasingly significant role in dermatopharmacology. Previous studies have shown that the penetration of substances into the follicle depends on its activity. Telogen follicles that were not producing sebum were closed for follicular penetration. The present study uses optical coherence tomography (OCT) to show that the orifices of inactive follicles are blocked by plugs formed of corneocytes. The follicular penetration of a fluorescent dye was analyzed by cyanoacrylate surface biopsies in combination with laser scanning microscopy. Surface exfoliation and cyanoacrylate surface peels were used as a pre-treatment to remove the corneocyte plugs from the follicular orifices and therefore to open up closed follicles. It was shown that both procedures could increase the number of open hair follicles significantly. The present experiments show that OCT in combination with laser scanning microscopy is an excellent non-invasive in vivo method for the investigation of hair follicle properties and follicular penetration processes.

Topical glucocorticoids (GC) are known to induce changes in human skin with the potential to develop skin atrophy. Here, atrophogenic effects and subsequent structural changes in the skin after topical application of GC were investigated in vivo. Sixteen healthy volunteers were topically treated daily on the forearms with clobetasol propionate, betamethasone dipropionate, and the petrolatum vehicle for 4 weeks. All treated skin areas and a nontreated control area were examined by ultrasound, optical coherence tomography, confocal laser scanning microscopy, multiphoton tomography (MPT), and resonance Raman spectroscopy at baseline 1 day after last application and 1 week after last application. Investigated parameters included stratum corneum thickness, epidermal, and full skin thickness, keratinocyte size and density, keratinocyte nucleus-to-cytoplasm ratio, skin surface classification, relative collagen and elastin signal intensity, second-harmonic generation-to-autofluorescence aging index of dermis (SAAID), and the antioxidant status of the skin. A reduction in epidermal and dermal skin thickness was observed in GC treated as well as in vehicle-treated and untreated skin areas on the volar forearm. MPT analysis showed an increased epidermal cell density and reduced cell size and nucleus-to-cytoplasm ratio and a significant increase of SAAID after GC treatment indicating a restructuring or compression of collagen fibers clinically being observed as atrophic changes.

Scalp imaging techniques are necessary tools for the trichological practice and for visualization of permeation, penetration and absorption processes into and through the scalp and for the research on drug delivery and toxicology. The present letter reviews different scalp imaging techniques and discusses their utility. Moreover, two different studies on scalp imaging techniques are presented in this letter: (1) scalp imaging with phototrichograms in combination with laser scanning microscopy, and (2) follicular measurements with cyanoacrylate surface replicas and light microscopy in combination with laser scanning microscopy. The experiments compare different methods for the determination of hair density on the scalp and different follicular measures. An average terminal hair density of 132 hairs cm−2 was found in 6 Caucasian volunteers and 135 hairs cm−2 in 6 Asian volunteers. The area of the follicular orifices accounts to 16.3% of the skin surface on average measured with laser scanning microscopy images. The potential volume of the follicular infundibulum was calculated based on the laser scanning measurements and is found to be 4.63 mm3 per cm2 skin on average. The experiments show that hair follicles are quantitatively relevant pathways and potential reservoirs for topically applied drugs and cosmetics.

Most treatments in dermatology and cosmetology are based on the penetration of topically applied drugs into the skin or through the skin barrier to the target structure in the living tissue. In the case of healthy skin, scarcely 1% of the applied drugs pass the skin barrier, depending on their chemical properties. Therefore, different physical and chemical methods have been developed to stimulate the penetration process. All these methods are based on the partial destruction of the barrier. In this study, an electrical tissue-tolerable plasma (TTP) was used to increase the penetration of a topically applied model drug (fluorescent dye) through the skin barrier. Using laser scanning microscopy, the distribution of the model drug in different depths of the skin was investigated. It was found that the plasma treatment of the skin is a very efficient process to deliver topically applied substances into the living tissue. In the case of the non-plasma-treated skin, it was found that the fluorescent dye could be detected exclusively on the skin surface. If the dye was applied to the TTP-treated skin, it could be observed in high concentration also in deeper parts of the skin extending down to the stratum basale and the papillary structure.

The skin represents the largest organ of our organism and is the barrier to the environment. UV sun radiation and environmental hazards damage our skin. Therefore, it is the aim of skin care products to protect the skin from damage and stimulate the regeneration. The efficacy of skin care products for barrier improvement is usually analyzed by transepidermal water loss measurements (TEWL). This method is suitable for the investigation of the untreated skin. Topically applied products can influence the TEWL measurements. In vivo laser scanning microscopy is a new approach for analyzing the barrier function of the skin. This method is not disturbed by topically applied formulation. In the present paper, laser scanning microscopy was used in comparison to TEWL measurements for the characterization of the skin care product Bedan-Gesichtscreme®

The knowledge of penetration pathways in and through the skin is a prerequisite for the development and optimization of topically applied drugs and cosmetics. Skin penetration has been assumed to occur via diffusion though the lipid domains of the stratum corneum. Recent studies show that the skin appendages, especially the hair follicles, play an important role in skin penetration processes. Topically applied substances cannot enter all follicles. We discuss the reasons for the phenomenon of open and closed hair follicles.

Skin antisepsis is a key element for the prevention of surgical site infections, as well as for infections after injection and punctures. Recent investigations have shown that about 25% of the resident bacterial flora of the human skin resides within the hair follicle. These findings strongly suggest that the skin appendages play the role of a bacterial reservoir. The bacteria within the hair follicles therefore may be the cause of endogenous germ repopulation after skin antisepsis, highlighting the need for new antiseptic formulations that can sufficiently penetrate into the hair follicles. Various experiments have found that nano-sized particles as well as oil-in-water emulsions are efficient carriers for substances into the hair follicles. In the present study, we investigated the in vivo antiseptic potential of the particle-associated and aqueous polihexanide on the human skin by monitoring bacterial growth after antisepsis over a period of 2.5h. The experiments suggest that the use of a particle-bound antiseptic can achieve a better and longer lasting antisepsis of the human skin than in non-particulate form.

Background The skin represents a potent barrier to the environment, which can be enhanced by the topical application of skin care products, such as oil and oil-based formulations by moisturizing the skin. Methods The aim of this study was the investigation of the penetration behaviour of four vegetable oils and of paraffin oil into the stratum corneum by laser scanning microscopy. In addition, the occlusion capacity of these substances was assessed by transepidermal water loss (TEWL) measurements. Petrolatum served as a positive control for skin occlusion. The study was conducted in vivo and included six healthy volunteers. Results Paraffin oil, as well as the vegetable oils, penetrated only into the first upper layers of the stratum corneum. TEWL measurements indicated that the application of the vegetable oils (except jojoba oil) as well as paraffin oil, led to a similar occlusion of the skin surface. The most effective occlusion was found for petrolatum. Conclusion For the investigated oils, a deeper penetration than into the first upper layers of the stratum corneum could be excluded. The decreased TEWL values indicate that the application of the oils leads to a semi-occlusion of the skin surface as it is intended by the use of oils to retain moisture in skin.

Variations in hair shaft morphology reflect ethnical diversity, but may also indicate internal diseases, nutritional deficiency, or hair and scalp disorders. The measurement and the follow-up of the hair shaft thickness over a defined period of time would be a valuable diagnostic tool in clinical practice. Standard light microscopy (LM) measurements require the epilation of hair shafts and frequently yield inaccurate values caused by the elliptic geometry of human hair shafts. Optical coherence tomography (OCT) is a noninvasive investigation method based on the principles of Michelson interferometry with a detection depth of approximately 1 mm in human skin. Two-dimensional images of the cross sections of tissue samples at a resolution of approximately 10 μm are produced, which allows convenient calculation of hair shaft thickness. To evaluate this new methodology for hair shaft thickness measurements, hair shafts taken from 28 healthy volunteers were analyzed by in vivo OCT and compared to standard in vitro LM measurements of hair shaft thickness. OCT yielded highly reproducible measurements of hair shaft thickness with a distinctly reduced variation compared to standard LM. This technique offers a unique opportunity for in vivo measurement and a follow-up of the kinetics of hair shaft thickness in humans during medical therapy.

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.

The hair follicles could be a reservoir for topically applied substances. They are not only surrounded by a close network of blood capillaries, which makes them interesting for drug delivery, but they are also the host of dendritic cells, which are important for immunomodulation. Previously, pollen allergens were shown to penetrate into the hair follicles. The application of barrier-enhancing formulations might represent an effective strategy to prevent pollen protein penetration into the hair follicle. In the present study, porcine skin areas were pretreated with 4 barrier-enhancing emulsions. One skin area served as control and remained without pretreatment. Afterwards, fluorescein-isothiocyanate-labeled grass pollen proteins were applied to the porcine skin samples, and their penetration was investigated via fluorescent laser scanning microscopy. It was shown that the barrier-enhancing formulations were able to significantly reduce the penetration of exogenous proteins into the hair follicles, the extent of such reduction depending on the formulation.

The exact qualitative and quantitative analysis of wound healing processes is a decisive prerequisite for optimizing wound care and for therapy control. Transepidermal water loss (TEWL) measurements are considered to be the standard procedure for assessing the progress of epidermal wound healing. The damage to the stratum corneum correlates with an increased loss of water through the skin barrier. This method is highly susceptible to failure by environmental factors, in particular by temperature and moisture. This study was aimed at comparing TEWL measurements and in vivo laser scanning microscopy (LSM) for the characterization of the epidermal wound healing process. LSM is a high-resolution in vivo method permitting to analyze the kinetics and dynamics of wound healing at a cellular level. While the TEWL values for the individual volunteers showed a wide scattering, LSM permitted the wound healing process to be clearly characterized at the cellular level. However, a comparison between the two methods was very difficult, because the results provided by LSM were images and not numerical. Therefore, a scoring system was set up which evaluates the stages of wound healing. Thus, the healing process could be numerically described. This method is independent of any environmental factors. Providing morphologically qualitative and numerically quantitative analyses of the wound healing process and being far less vulnerable to failure, LSM is advantageous over TEWL.

Stimulation of the penetration of topically applied substances into the skin is a topic of intensive dermatological and pharmacological research. Next to intercellular penetration, i.e. a penetration inside the lipid layers around the corneocytes, 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 or contain several important target structures, such as blood capillaries, stem cells and dendritic cells. Therefore, the hair follicles have to be well protected from hazardous substances coming into contact with the skin. The traditional method of decontamination of the skin involves an intensive washing procedure. However, this process represents a massage, which pushes the hazardous substances even deeper into the hair follicles. In the present study, the application of absorbing materials for decontamination of the skin was investigated after the application of a model substance utilizing the tape-stripping procedure. It was found that absorbing materials are better suited than the washing process for decontamination of the skin.

The analysis of wound healing is important for the therapy control and for the development of drugs stimulating the healing process. Wounds cause damage to the skin barrier. A damaged stratum corneum leads to an increased water loss through the skin barrier. The standard measuring procedure for characterization of wound healing is the measurement of transepidermal water loss (TEWL). The disadvantage of this method is that it can be easily disturbed by the perspiration of the volunteers and by topically applied substances, for instance wound healing creams. In the study presented, in vivo laser scanning microscopy and optical coherent tomography were compared concerning the application for their analysis of wound healing processes. The laser scanning microscopy allows the analysis of the healing process on a cellular level. The course of wound healing determined by laser scanning microscopy was correlated with numerical values, allowing the numerical characterization of the wound healing process.

Epidermal wound healing is a complex and dy- namic regenerative process necessary to reestablish skin integrity. Fluorescence confocal laser scanning micros- copy FLSM is a noninvasive imaging technique that has previously been used for evaluation of inflammatory and neoplastic skin disorders in vivo and at high resolution. We employed FLSM to investigate the evolution of epider- mal wound healing noninvasively over time and in vivo. Two suction blisters were induced on the volar forearms of the study participants, followed by removal of the epider- mis. To study the impact of wound ointment on the pro- cess of reepithelization, test sites were divided into two groups, of which one test site was left untreated as a nega- tive control. FLSM was used for serial/consecutive evalu- ations up to 8 days. FLSM was able to visualize the devel- opment of thin keratinocyte layers developing near the wound edge and around hair follicles until the entire epi- dermis has been reestablished. Wounds treated with the wound ointment were found to heal significantly faster than untreated wounds. This technique allows monitoring of the kinetics of wound healing noninvasively and over time, while offering new insights into the potential effects of topically applied drugs on the process of tissue repair. ©

Background: The cutaneous antioxidants form an efficient protection system against the destructive potential of free radicals, produced by environmental factors, such as UV-sun irradiation, hazardous substances and lifestyle habits. Most of the antioxidants cannot be produced by the human organism. Thus, they have to be incorporated by food and beverages. Material and Methods: In the present manuscript, the distribution of carotenoids as a marker for antioxidative potential in human skin was investigated with two different in vivo Raman spectroscopy methods with an excitation wavelength of 785 nm (Skin Analyzer) and at 488 nm (resonance Raman spectroscopy). The carotenoid profile was assessed at three different anatomical locations (palm, forehead and volar forearm) in 12 healthy volunteers. Results: In untreated skin, the major fraction of the carotenoids is located in the upper part of the stratum corneum (SC). The amount of carotenoid is lower in the upper part of the SC on the forearm compared to forehead and palm shown with both methods. Both methods detect similar distinction patterns of carotenoid levels for the three anatomical locations. Conclusion: The present study supports the hypothesis that antioxidative substances; here carotenoids, are secreted via eccrine sweat glands and/or sebaceous glands to the skin surface. Raman spectroscopic methods are an efficient tool to analyze the distribution of carotenoids in the human skin over time and with the Skin Analyzer over different layers of the epidermis. Resonance Raman spectroscopy is suited to analyze deeper parts of the skin.

It has recently been demonstrated by the utilization of different techniques, such as differential stripping and laser scanning microscopy, that the hair follicles represent an efficient long-term reservoir for topically applied substances. In the present paper, the different in vivo techniques are compared to those of in vitro analysis. It was found that in vitro measurements on pig ear skin are highly superior for the analysis of follicular penetration, as compared to in vitro investigations on excised human skin, mainly due to the fact that the human skin contracts after removal. Restretching of the skin to its original size mainly stretches the interfollicular fibres, whereas the fibres around the hair follicles remain contracted. In contrast to excised human skin, pig ear tissue does not contract when the cartilage is not removed. Moreover, it has an intact barrier on both sides of the ear. Regardless of the fact that the hair follicles on pig ear skin are larger than those of humans, the porcine ear skin represents a more suitable in vitro model for the analysis of the penetration and storage of topically applied substances in the hair follicles than excised human skin.