In vitro assessment of inflammatory skin potential of poly(methyl methacrylate) at non-cytotoxic concentrations.

Poly(methyl methacrylate) (PMMA) is considered an attractive substrate material for fabricating wearable skin sensors such as fitness bands and microfluidic devices. Despite its widespread use, inflammatory and allergic responses have been attributed to the use of this material. Therefore, the main objective of this study was to obtain a comprehensive understanding of potential biological effects triggered by PMMA at non-cytotoxic concentrations using in vitro models of NIH3T3 fibroblasts and reconstructed human epidermis (RhE). It was hypothesized that concentrations that do not reduce cell viability are sufficient to activate pathways of inflammatory processes in the skin. The study included cytotoxicity, cell metabolism, cytokine quantification, histopathological, and gene expression analyses. The NIH3T3 cell line was used as a testbed for screening cell toxicity levels associated with the concentration of PMMA with different molecular weights (MWs) (i.e., MW ~5,000 and ~15,000 g/mol). The lower MW of PMMA had a half-maximal inhibitory concentration (IC50 ) value of 5.7 mg/cm2 , indicating greater detrimental effects than the higher MW (IC50 = 14.0 mg/cm2 ). Non-cytotoxic concentrations of 3.0 mg/cm2 for MW ~15,000 g/mol and 0.9 mg/cm2 for MW ~5,000 g/mol) induced negative metabolic changes in NIH3T3 cells. Cell viability was severely reduced to 7% after the exposure to degradation by-products generated after thermal and photodegradation degradation of PMMA. PMMA at non-cytotoxic concentrations still induced overexpression of pro-inflammatory cytokines, chemokines, and growth factors (IL1B, CXCL10, CCL5, IL1R1, IL7, IL17A, VEGFA, FGF2, IFNG, IL15) on the RhE model. The inflammatory response was also supported by histopathological and gene expression analyses of PMMA-treated RhE, indicating tissue damage and gene overexpression. Results suggested that non-cytotoxic concentrations of PMMA (3.0 to 5.6 mg/cm2 for MW ~15,000 g/mol and 0.9 to 2.1 mg/cm2 for MW ~5,000 g/mol) were sufficient to negatively alter NIH3T3 cells metabolism and activate inflammatory events in the RhE skin.

Directional assessment of the skin barrier function in vivo.

INTRODUCTION: The fundamental function of the epidermis is to provide an inside-out barrier to water loss and an outside-in barrier to penetration of external irritants. Transepidermal water loss (TEWL) has been extensively used as a method of estimating the skin barrier quality, typically without any consideration of directionality. The validity of TEWL as an estimate of skin permeability to external substances has been controversial in vitro and in vivo. The aim of this work was to assess the relationship between TEWL and the penetration of a topically applied external marker (caffeine) in healthy skin in vivo before and following a challenge to the barrier. METHODS: The skin barrier was challenged by application of aqueous solutions of mild cleanser products under occlusion for 3 h on the forearms of nine human participants. Skin barrier quality was evaluated before and after the challenge by measuring the TEWL rate and the permeated amount of topically applied caffeine using in vivo confocal Raman microspectroscopy. RESULTS: No skin irritation was observed following the skin barrier challenge. TEWL rates and the caffeine penetrated amount in the stratum corneum after the challenge were not correlated. A weak correlation was observed when the changes were corrected to water-only treatment. TEWL values can be influenced by environmental conditions as well as the skin temperature and water content. CONCLUSIONS: Measuring TEWL rates is not always representative of the outside-in barrier. TEWL may be useful in differentiating large changes in skin barrier function (e.g., between healthy and compromised skin) but is less sensitive to small variations following topical application of mild cleansers.

Dermatoscopic assessment of treatment response in patients undergoing autologous non-cultured epidermal cell suspension for the treatment of stable vitiligo: A prospective study.

There is lack of literature on serial dermatoscopic assessment in patients undergoing non-cultured epidermal cell suspension (NCES) for treatment of stable vitiligo. This prospective study was conducted to evaluate the role of serial dermatoscopy in assessing disease stability and predicting repigmentation rates in vitiligo patients undergoing NCES. Dermatoscopic assessment of target lesions were done at baseline and post-NCES at week 4, 8, 12, 16, and 24. Patches obtaining >90% repigmentation at 24 weeks were categorized to have obtained excellent repigmentation. The dermatoscopic features of target lesions that showed clinical signs of disease activity anytime during the follow-up period were compared to those maintaining clinical stability throughout. Twenty-six vitiligo patients with 52 patches, clinically stable for atleast 1 year were recruited. At follow-up, six patches showed clinical signs of instability. Five patches in the unstable group developed satellite lesions by week 16, compared to none in the stable group (p < 0.05). Excellent repigmentation was achieved in 29 out of 52 patches. Appearance of normal reticular pigment network at 8 weeks was a positive predictor of excellent response (OR = 10.5, CI 1.2-89.7), whereas, altered pigment network at 12, 16, and 24 weeks and telangiectasias at 12 and 16 weeks significantly reduced the odds of excellent repigmentation.

Assessment of Non-cultured Autologous Epidermal Cell Grafting Resuspended in Hyaluronic Acid for Repigmenting Vitiligo and Piebaldism Lesions: A Randomized Clinical Trial.

The aim of this study was to assess the efficacy of non-cultured autologous epidermal cell grafting resuspended in hyaluronic acid, performed using a ready-to-use kit, compared with hyaluronic acid alone (neutral comparator) for repigmenting vitiligo and piebaldism lesions at 6 months. Two identified paired lesions per patient were randomized to be treated by either device. Devices with a ready-to-use kit were prepared by separate health professionals, to maintain blinding. A skin biopsy was digested using trypsin, and cells resuspended in hyaluronic acid solution. Among 38 patients screened, 36 (94.7%) patients, corresponding to 72 lesions, were analysed. For difficult-to-treat lesions, defined as those located on the wrist, elbow, and hands (n = 30), no repigmentation ≥ 50% was observed. For all other locations (n = 42), the success rate was significantly higher (p = 0.021) in the ready-to-use kit group (47.6% vs 9.5%) at 6 months and was maintained until 12 months. In conclusion, a single application of non-cultured epidermal cellular grafting using a ready-to-use kit was efficient at 6 months and at 1-year follow-up.

Direct assessment of individual skin barrier components by electrical impedance spectroscopy.

BACKGROUND: Expression of the tight junction proteins Cldn1 and 4 is altered in skin diseases such as atopic dermatitis, and Cldn1 deficiency affects skin barrier formation. Impedance spectroscopy (IS) has been proven to allow detection of alterations in the skin barrier but is currently unable to separate effects on viable epidermis (VE) and stratum corneum (SC). METHODS: Effects of siRNA-mediated Cldn1 and 4 knockdown in reconstructed human epidermis (RHE) on VE and SC barrier function were investigated with Ussing chamber-based IS. Barrier components were sequentially altered, employing iron oxide nanoparticles and EGTA, to identify their contribution to the impedance spectrum. Resistance changes due to apically applied hyperosmolar electrolyte were used to identify barrier defects non-invasively. RESULTS: IS of RHE yielded two relaxation frequencies, representing the barrier properties of the SC (~1000 Hz) and VE (~100 Hz). As proof of concept, it was shown that the Cldn1 knockdown-induced resistance drop arises from the impairment of both SC and VE, indicated by a shift of both relaxation frequencies. Hyperosmolar electrolyte penetration allowed non-invasive detection of Cldn1 knockdown via time-dependent frequency shifts. The absence of Cldn4 knockdown-induced changes revealed the weaknesses of transepithelial electrical resistance analysis. CONCLUSION: In conclusion, the present technique allows to separately measure the barrier properties of SC and VE and further evaluate the Cldn1 and 4 knockdown impact on the skin barrier. As the measurement with agarose-embedded electrolyte allowed non-invasive identification of the Cldn1 knockdown, this opens the way to detailed in vivo skin barrier assessment.

Isolation of Epidermal Keratinocytes from Human Skin: The Scratch-Wound Assay for Assessment of Epidermal Keratinocyte Migration.

The migration of epidermal keratinocytes is the basis for skin reepithelialization during wound healing. The in vitro scratch-wound assay using monolayers of primary human epidermal keratinocytes is a straightforward and effective method to assess their migratory capacity. The mechanical scratch of a confluent monolayer directly disrupts the adhesion of the keratinocytes to one another and to the underlying matrix, resembling the physical trauma of a wound in an in vitro assay. The keratinocytes will undergo an epithelial-to-mesenchymal transition, which will confer an ability to migrate toward each other to cover the gap by restructuring cell-cell and cell-extracellular matrix connections. However, a good scratch-wound method and protocol to ensure scratch reproducibility is essential, particularly when using primary cell cultures where donor variability may also impact on results.

A single-arm open-label clinical trial of autologous epidermal cell transplantation for stable vitiligo: A 30-month follow-up.

BACKGROUND: Recently, we introduced intralesional injection of autologous epidermal cells as a safe and feasible approach for transplantation in patients with stable vitiligo. This approach resulted in less pain during and after the procedure, no scarring or cobblestone formation at the recipient site, and was more feasible to perform on curved surfaces such as joints, lips, eyelids, ears, and face. OBJECTIVE: In this study, we aimed to investigate the long-term efficacy and safety of this transplantation technique. METHODS: In this open-label and single-arm clinical trial, we enrolled 300 patients with stable vitiligo. We obtained a partial thickness normo-pigmented skin specimen from the patients' thigh-buttock junction with an area of one tenth to one third of the recipient site area. The epidermal cell suspension was prepared by processing the autologous skin specimen. We injected the cell suspension into 1060 vitiligo patches in 300 patients. Patients did not use any adjuvant phototherapy during the study. An experienced dermatologist and patients respectively defined the repigmentation score and self-assessment score at regular follow-up visits for up to 30 months after treatment. The scores represented the repigmentation percentage as follows: 0 (0), I (1%-24%), II (25%-49%), III (50%-74%), and IV (75%-100%). RESULTS: The mean repigmentation score at 3 months post-transplantation was 1.12±0.73. A significant upward trend existed in the mean repigmentation score until 9 months after cell transplantation, when the mean repigmentation score reached to 1.98±1.20. At 9 months after treatment, repigmentation of >50% was obtained in 32.2% of treated patches. Acquired repigmentation remained stable in 79.3% of treated patches during the follow-up period. The number of received cells per cm2 positively influenced the repigmentation score. Patches located on face, neck and trunk showed significantly higher response to the treatment. CONCLUSION: The results of our study demonstrated efficacy and safety of autologus epidermal cell transplantation on repigmentation of vitiligo patches. The achieved repigmentation was stable in the majority of treated patches during the follow-up period.

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research.

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined "acufection", denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15ΔE7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15ΔE7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

Isolation and functional assessment of cutaneous stem cells.

The epidermis and associated appendages of the skin represent a multi-lineage tissue that is maintained by perpetual rounds of renewal. During homeostasis, turnover of epidermal lineages is achieved by input from regionalized keratinocytes stem or progenitor populations with little overlap from neighboring niches. Over the last decade, molecular markers selectively expressed by a number of these stem or progenitor pools have been identified, allowing for the isolation and functional assessment of stem cells and genetic lineage tracing analysis within intact skin. These advancements have led to many fundamental observations about epidermal stem cell function such as the identification of their progeny, their role in maintenance of skin homeostasis, or their contribution to wound healing. In this chapter, we provide a methodology to identify and isolate epidermal stem cells and to assess their functional role in their respective niche. Furthermore, recent evidence has shown that the microenvironment also plays a crucial role in stem cell function. Indeed, epidermal cells are under the influence of surrounding fibroblasts, adipocytes, and sensory neurons that provide extrinsic signals and mechanical cues to the niche and contribute to skin morphogenesis and homeostasis. A better understanding of these microenvironmental cues will help engineer in vitro experimental models with more relevance to in vivo skin biology. New approaches to address and study these environmental cues in vitro will also be addressed.

In vitro assessment of IL-4- or IL-13-mediated changes in the structural components of keratinocytes in mice and humans.

T helper type 2 (Th2) cytokines, IL-4 and IL-13, attenuate the expression of genes that regulate epidermal cellular structures and the barrier function at the terminal stage of keratinocyte differentiation. However, whether these Th2 cytokines act at earlier stages remains unknown. We investigated the roles of cytokines in expression levels of mRNAs and/or proteins in primary mouse keratinocytes and human keratinocyte HaCaT cells at earlier stages. We showed that IL-4 downregulated the expression levels of Krt1, Krt10, Dsg1, and Dsc1 via IL-4Rα- and signal transducer and activator of transcription factor 6 (STAT6)-dependent mechanisms in differentiating mouse keratinocytes at early stages. As the expression levels of keratin-1 and -10 in the keratinocytes transiently expressing an active form of STAT6 were not downregulated, STAT6 and other IL-4-induced molecules may synergistically regulate this expression. The restoration of the downregulated expression levels of Krt1 and Krt10 induced by IL-4 with the MEK (mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase) inhibitor U0126 indicated the involvement of the p44/42 MAPK signaling pathway in the attenuated expression. IL-13 also downregulated the expression of the four genes. Furthermore, IL-4 or IL-13 caused the downregulation of these genes in HaCaT cells and promoted the fragmentation of cell sheets with mechanical stress. Our results showed that IL-4 or IL-13 acted on differentiating keratinocytes in vitro at early stages to attenuate the gene expression.

Statistical validation of the acceleration of the differentiation at the expense of the proliferation in human epidermal cells exposed to extremely low frequency electric fields.

An acceleration of differentiation at the expense of proliferation is observed in our previous publications and in the literature after exposure of various biological models to low frequency and low-amplitude electric and electromagnetic fields. This observation is related with a significant modification of genes expression. We observed and compared over time this modification. This study use microarray data obtained on epidermis cultures harvested from human abdominoplasty exposed to ELF electric fields. This protocol is repeated with samples collected on three different healthy patients. The sampling over time allows comparison of the effect of the stimulus at a given time with the evolution of control group. After 4 days, we observed a significant difference of the genes expression between control (D4C) and stimulated (D4S) (p < 0.05). On the control between day 4 and 7, we observed another group of genes with significant difference (p < 0.05) in their expression. We identify the common genes between these two groups and we select from them those expressing no difference between stimulate at 4 days (D4S) and control after 7 days (D7C). The same analysis was performed with D4S-D4C-D12C and D7S-D7C-D12C. The lists of genes which follow this pattern show acceleration in their expressions under stimulation appearing on control at a later time. In this list, genes such as DKK1, SPRR3, NDRG4, and CHEK1 are involved in cell proliferation or differentiation. Numerous other genes are also playing a function in mitosis, cell cycle or in the DNA replication transcription and translation.

Assessment of the human epidermal model LabCyte EPI-MODEL for In vitro skin corrosion testing according to the OECD test guideline 431.

A new OECD test guideline 431 (TG431) for in vitro skin corrosion tests using human reconstructed skin models was adopted by OECD in 2004. TG431 defines the criteria for the general function and performance of applicable skin models. In order to confirm that the new reconstructed human epidermal model, LabCyte EPI-MODEL is applicable for the skin corrosion test according to TG431, the predictability and repeatability of the model for the skin corrosion test was evaluated. The test was performed according to the test protocol described in TG431. Based on the knowledge that LabCyte EPI-MODEL is an epidermal model as well as EpiDerm, we decided to adopt the the Epiderm prediction model of skin corrosion for the LabCyte EPI-MODEL, using twenty test chemicals (10 corrosive chemicals and 10 non-corrosive chemicals) in the 1(st) stage. The prediction model results showed that the distinction of non-corrosion to corrosion corresponded perfectly. Therefore, it was judged that the prediction model of EpiDerm could be applied to the LabCyte EPI-MODEL. In the 2(nd) stage, the repeatability of this test protocol with the LabCyte EPI-MODEL was examined using twelve chemicals (6 corrosive chemicals and 6 non-corrosive chemicals) that are described in TG431, and these results recognized a high repeatability and accurate predictability. It was concluded that LabCyte EPI-MODEL is applicable for the skin corrosive test protocol according to TG431.

Assessment of the variation of skin barrier function with anatomic site, age, gender and ethnicity.

The skin is the largest organ of the human body and its functions include protection, thermoregulation sensation and secretion. Significant advances in our understanding of how the morphology and physiology of the skin contribute to the skin's barrier role have been achieved in recent years. The aim of this review is to summarize the principal approaches which have been used to assess variation in skin barrier function with anatomic site, age, gender, and ethnicity. The methods discussed include trans-epidermal water loss (TEWL) measurement, assessment of corneocyte size, response to vasoactive compounds and attenuated total reflectance Fourier transform infrared (ATR-FTIR) interrogation of skin. The utility of the various methods is considered and the most important findings in the literature to date are highlighted.

Techniques for time-efficient isolation of human skin dendritic cell subsets and assessment of their antigen uptake capacity.

Dendritic cells (DCs) residing in skin are important sentinels for foreign antigens. Methods to facilitate studies of subsets of skin DCs are important to increase the understanding of various pathogens, allergens, topical treatments or vaccine components targeting the skin. In this study, we developed a new DC purification method using a skin graft mesher, clinically used for expansion of skin grafts, to accelerate processing of skin into nets that allowed efficient enzymatic disruption and single cell isolation. The reduction in processing time using the skin graft mesher enabled processing of larger skin samples and also limited the ex vivo handling of the specimens which is associated with maturation of DCs. In addition, a skin explant model to functionally monitor early events of antigen uptake by DC subsets in situ was developed. DCs isolated from epidermis represented a uniform CD1a(+) HLA-DR(+) CD11c(+) Langerin(+) DC-SIGN(-) DC-LAMP(int) DEC-205(int) Langerhans cell (LC) population whereas three subtypes of HLA-DR(+) CD11c(+) DCs were isolated from dermis based on their varying expression of CD1a. Epidermal LCs showed a significantly higher antigen uptake capacity of fluorescently-labelled ovalbumin (OVA) and dextran as compared to any of the dermal DC (dDC) subsets. In contrast, injection of antigen directly into skin explants followed by in situ imaging revealed that the majority of DCs with internalized antigen were localized in the dermis, likely as a consequence of the anatomical site for antigen delivery. These methods offer potency for various applications addressing antigen uptake, microbial DC interactions or other antigenic stimulation targeting the skin and can enhance our knowledge of basic DC biology in human skin.

Optical fluence distribution study in tissue in dark-field confocal photoacoustic microscopy using a modified Monte Carlo convolution method.

We have modified the existing convolution method of the Monte Carlo simulation for finite photon beams with both translational and rotational invariance. The modified convolution method was applied to simulate the optical fluence distribution in tissue in dark-field confocal photoacoustic microscopy. We studied the influence of the size of the dark field and the illumination incident angle on the depth position of the effective optical focus (the region with the highest fluence) and the fluence ratio (the ratio of the optical fluence at the effective optical focus inside the tissue to the optical fluence on the tissue surface along the ultrasonic axis). Within the reach of diffuse photons, the depth position of the effective optical focus increases with the size of the dark field and is much less sensitive to the incident angle. The findings show that, while the fluence at the effective optical focus decreases, the fluence ratio increases with the size of the dark field. The incident angle has a weaker influence on the fluence ratio than the size of the dark field does. An incident angle between 30 and 50 degrees gives the highest fluence at the effective optical focus.

The Göttingen minipig for assessment of retinoid efficacy in the skin: comparison of results from topically treated animals with results from organ-cultured skin.

Göttingen minipigs were treated topically for 6 d with a novel retinoid (MDI 301) at concentrations ranging from 0.3% to 30% in cream vehicle. Treatment of the minipigs did not adversely affect their health (hematological and necropsy parameters) or produce changes in the skin suggestive of retinoid-induced skin irritation. After killing the animals, skin samples from each treatment site were excised and maintained in organ culture for 6 d. In addition, untreated skin was also maintained in organ culture and treated with MDI 301 (0.1-5 microg/ml). After 3 d, the culture supernatants were collected and analyzed for levels of collagen type I and for matrix metalloproteinases (MMPs). Both skin samples treated in vivo and skin samples exposed to MDI 301 in culture demonstrated increased collagen production. Only slight changes in levels of MMP-2 (gelatinase A) or MMP-9 (gelatinase B) were seen. After 6 d, the organ-cultured skin was fixed in formalin and prepared for histology. The organ-cultured skin was compared to skin that was fixed at killing after in vivo treatment. Epidermal hyperplasia was quantified at various MDI 301 concentrations. In vivo and in vitro treatments showed similar results-although the thickness was not substantially changed on average, there were focal areas of hyperplasia at higher retinoid concentrations. Taken together, these data suggest that MDI 301 enhances collagen production in minipig skin, without irritation. Furthermore, these studies suggest that minipig skin exposed to the retinoid in organ culture is equally predictive as topically treated skin. The in vitro organ culture approach may provide a cost-effective alternative model to that of the intact animal for skin retinoid testing.

Human skin responses to UV radiation: pigment in the upper epidermis protects against DNA damage in the lower epidermis and facilitates apoptosis.

Melanin plays an important role in protecting the skin against UV radiation, and melanomas and basal/squamous cell carcinomas occur more frequently in individuals with fair/light skin. We previously reported that levels of melanin correlate inversely with amounts of DNA damage induced by UV in normal human skin of different racial/ethnic groups. We have now separately examined DNA damage in the upper and lower epidermal layers in various types of skin before and after exposure to UV and have measured subsequent apoptosis and phosphorylation of p53. The results show that two major mechanisms underlie the increased photocarcinogenesis in fair/light skin. First, UV-induced DNA damage in the lower epidermis (including keratinocyte stem cells and melanocytes) is more effectively prevented in darker skin, suggesting that the pigmented epidermis is an efficient UV filter. Second, UV-induced apoptosis is significantly greater in darker skin, which suggests that UV-damaged cells may be removed more efficiently in pigmented epidermis. The combination of decreased DNA damage and more efficient removal of UV-damaged cells may play a critical role in the decreased photocarcinogenesis seen in individuals with darker skin.

Assessment of epidermal cell viability by near infrared multi-photon microscopy following ballistic delivery of gold micro-particles.

The use of gene guns in ballistically delivering DNA vaccine coated gold micro-particles to skin can potentially damage targeted cells, therefore influencing transfection efficiencies. In this paper, we assess cell death in the viable epidermis by non-invasive near infrared two-photon microscopy following micro-particle bombardment of murine skin. We show that the ballistic delivery of micro-particles to the viable epidermis can result in localised cell death. Furthermore, experimental results show the degree of cell death is dependant on the number of micro-particles delivered per unit of tissue surface area. Micro-particles densities of 0.16+/-0.27 (mean+/-S.D.), 1.35+/-0.285 and 2.72+/-0.47 per 1000 microm(2) resulted in percent deaths of 3.96+/-5.22, 45.91+/-10.89, 90.52+/-12.28, respectively. These results suggest that optimization of transfection by genes administered with gene guns is - among other effects - a compromise of micro-particle payload and cell death.

Skin compatibility of cyclodextrins and their derivatives: a comparative assessment using a corneoxenometry bioassay.

Few studies have been performed to assess the risk of skin damage by cyclodextrins (CD) and they have yielded contradictory results. The present study was conducted using the corneoxenometry bioassay on human stratum corneum to compare the skin compatibility of CD currently used in pharmaceutical preparations (betaCD, gammaCD, Rameb, Dimeb, Trimeb, HP-betaCD and HP-gammaCD) and that of new amphiphilic CD derivatives, namely, the phospholipidyl-CD (DMPE-Dimeb and DMPE-Trimeb). All the tested CD were well tolerated by the stratum corneum at a concentration of 5%. However, inter-individual reactivity was larger for DMPE-Dimeb, suggesting a more aggressive trend for this compound. Cutaneous Index of Mildness values obtained confirm that Dimeb is able to extract some skin components and shows that DMPE-Dimeb performs similarly.

Drug delivery routes in skin: a novel approach.

The role of hair follicles in transdermal delivery remains difficult to elucidate due partly to animal model complications. This paper explores a novel technique employing two human skin membranes to differentiate shunt route delivery from bulk transepidermal input. The method monitors penetration through epidermal membranes and compares this with delivery through a sandwich of stratum corneum and epidermis, with the corneum forming a top membrane. As orifices of shunts occupy only 0.1% of the area, there is negligible chance that shunts in the membranes will superimpose. The top layer blocks shunts available in the bottom layer. If shunts are important, delivery through sandwiches will be much reduced compared with that through epidermis, allowing for increased double membrane thickness. Experiments with penetrants under passive, iontophoretic and electroporation conditions illustrated the value of the method. A Monte Carlo simulation suggested that any failure of membrane adherence would not affect conclusions drawn.