Imaging the distribution of skin lipids and topically applied compounds in human skin using mass spectrometry.

The barrier functions of skin against water loss, microbial invasion and penetration of xenobiotics rely, in part, on the spatial distribution of the biomolecular constituents in the skin structure, particularly its horny layer (stratum corneum). However, all skin layers are important to describe normal and dysfunctional skin conditions, and to develop adapted therapies or skin care products. In this work, time-of-flight secondary ion mass spectrometry (ToF-SIMS) combined with scanning electron microscopy (SEM) was used to image the spatial distribution of a variety of molecular species, from stratum corneum down to dermis, in cross-section samples of human abdominal skin. The results demonstrate the expected localization of ceramide and saturated long-chain fatty acids in stratum corneum (SC) and cholesterol sulfate in the upper part of the viable epidermis. The localization of exogenous compounds is demonstrated by the detection and imaging of carvacrol (a constituent of oregano or thyme essential oil) and ceramide, after topical application onto ex vivo human skin. Carvacrol showed pronounced accumulation to triglyceride-containing structures in the deeper parts of dermis. In contrast, the exogenous ceramide was found to be localized in SC. Furthermore, the complementary character of this approach with classical ex vivo skin absorption analysis methods is demonstrated.

Effects of intense pulsed light on the biological properties and ultrastructure of skin dermal fibroblasts: potential roles in photoaging.

OBJECTIVES: The aim of this study was to evaluate the effects of intense pulsed light (IPL) irradiation on skin fibroblasts in vitro, as well as to explore the biomolecular mechanisms and ultrastructure changes of the IPL effect. BACKGROUND: IPL is frequently used to improve telangiectasias, lentigos, and skin texture. Clinical studies have demonstrated that IPL has significant photorejuvenation effects on photoaged skin. However, the biomolecular mechanisms underlying the photorejuvenation of IPL treatment remain largely unknown. METHODS: Human skin fibroblasts cultured in vitro were irradiated with IPL via double pulses of 4 ms and 6 ms with a pulse interval of 20 ms and fluences of 18, 23, 28, and 33 J/cm(2). Twenty-four hours after irradiation, cell viability, cell cycle distribution, mRNA and protein levels of procollagen (types I and III), and changes of ultrastructure were evaluated using methyl thiazole tetrazolium (MTT) assay, flow cytometry, real-time reverse transcriptase polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assays (ELISA), and transmission electron microscopy, respectively. RESULTS: IPL irradiation resulted in the improvement of cell viability of skin fibroblasts in a dose-dependent manner. There were obvious changes in ultrastructure of fibroblasts, compared with the control group. The percentage of fibroblasts at the S and G(2)/M stage of the cell cycle was significantly higher, whereas the percentage at the G(0)/G(1) stage was lower. IPL irradiation increased the mRNA level of collagen I considerably, to 123%, 154%, 172%, and 141% of the control, and that of collagen III to 120%, 141%, 164%, and 132%. IPL irradiation was able to upregulate expression of collagen (types I and III) at the mRNA and protein level. CONCLUSIONS: The present study demonstrates that IPL irradiation imparts stimulatory effects on skin fibroblasts in vitro. This provides valuable evidence of the photorejuvenation effect of IPL in vivo.

Improving the reach of vaccines to low-resource regions, with a needle-free vaccine delivery device and long-term thermostabilization.

Dry-coated microprojections can deliver vaccine to abundant antigen-presenting cells in the skin and induce efficient immune responses and the dry-coated vaccines are expected to be thermostable at elevated temperatures. In this paper, we show that we have dramatically improved our previously reported gas-jet drying coating method and greatly increased the delivery efficiency of coating from patch to skin to from 6.5% to 32.5%, by both varying the coating parameters and removing the patch edge. Combined with our previous dose sparing report of influenza vaccine delivery in a mouse model, the results show that we now achieve equivalent protective immune responses as intramuscular injection (with the needle and syringe), but with only 1/30th of the actual dose. We also show that influenza vaccine coated microprojection patches are stable for at least 6 months at 23°C, inducing comparable immunogenicity with freshly coated patches. The dry-coated microprojection patches thus have key and unique attributes in ultimately meeting the medical need in certain low-resource regions with low vaccine affordability and difficulty in maintaining "cold-chain" for vaccine storage and transport.

Effect of aged garlic extract on wound healing: a new frontier in wound management.

Successful wound healing depends upon angiogenesis, and impaired angiogenesis is a hallmark of the chronic wounds encountered with diabetes and venous or arterial insufficiency. To intervene and improve wound closure, it is essential to investigate the effects of different natural remedies in wound healing. The chicken dorsum skin excisional wound assay was used to investigate the influence of different concentrations of aged garlic solution (AGS) on wound healing. Gross, histopathology, scanning electron microscopy (SEM) and computer-based three-dimensional (3D) image-probing techniques were utilized to determine the effects of AGS on wound closure, re-epithelialization, dermal matrix regeneration, and angiogenesis. Ninety chicks, aged 1 week and divided in 6 groups, were topically exposed to different concentrations of AGS for 6 days: control (group A), 1% (group B), 5% (group C), 10% (group D), 15% (group E), and skin lotion (group F). Different patterns, ranging from incomplete to almost complete wound closure, were observed among different groups with highly significant results (P < 0.001) in group E. Histological investigations revealed a positive augment in the re-epithelialization of all AGS exposed wounds. An increase in the number of new loosely packed collagen and maturation of collagen bundles was observed in all treated wounds at days 4 and 6 post-wounding, respectively. Similar results were achieved through SEM of treated wounds. Histological investigations revealed the profuse dose-dependent neovascularization among AGS-treated wounds. Abbott curve, angular spectrum, and different parameters of 3D surface roughness of wounds were also measured for the precise quantification of angiogenesis. A very highly significant (P < 0.001) increase in angiogenesis was observed among all treated groups. No significant change was observed among control and skin lotion-treated groups. These observations substantiate the beneficial use of AGS in the treatment of wounds. Additional studies are needed to study the specific wound-healing mechanisms of chemical, or group of chemicals, present in AGS.

Clinical, biometric and structural evaluation of the long-term effects of a topical treatment with ascorbic acid and madecassoside in photoaged human skin.

Skin ageing is a complex process determined by the genetic endowment of individual and environmental factors, such as sun exposure. The effects of skin ageing are mostly encountered in the superficial dermis and in the epidermis. We have previously demonstrated in vivo the beneficial effect of a topically applied formula of 5% vitamin C in the treatment of skin ageing. Another active compound, madecassoside extracted from Centella asiatica, known to induce collagen expression and/or to modulate inflammatory mediators, might thus prevent and correct some signs of ageing. A randomized double-blind study was carried out on photoaged skin of 20 female volunteers to investigate the effects of topically applied 5% vitamin C and 0.1% madecassoside on the clinical, biophysical and structural skin properties. After 6 months of treatment, we observed a significant improvement of the clinical score for deep and superficial wrinkles, suppleness, firmness, roughness and skin hydration. These results were corroborated by measurements of skin elasticity and semi-quantitative histological assessment of the elastic fibre network in the papillary dermis. Two-thirds of the subjects showed an improvement. The re-appearance of a normally structured elastic fibre network was observed. Our results revealed a functional and structural remodelling of chronically sun-damaged skin.

Skin rejuvenation in Asian skin: the analysis of clinical effects and basic mechanisms of intense pulsed light.

BACKGROUND: Skin aging consists of photoaging and intrinsic aging. It is characterized clinically not only by rhytides, but also by pigmentary alterations and facial telangiectasias. There continues to be a growing interest in the efficacy of intense pulsed light (IPL) devices in the treatment of skin aging, as well as further defining its mechanism of action. OBJECTIVES: The objective of this clinical trial was to evaluate the effects and the mechanism of action of an IPL by comparing clinical photographs and biopsy results before and after treatment. METHODS: A total of 58 patients were treated using a new IPL device. Clinical photographs were taken before treatment and compared to those taken 3 weeks after the treatment. Also, 4 cases had pathological analyses of tissues that were stained by haematoxylin-eosin and Uana orcein. Immunohistology of human collagen of types 1 and 3 and quantitative analyses of elastin and collagen were performed by a poly-functional digital image light microscope; a transmission electron microscope was used for 2 of the cases to look for additional changes. RESULTS: After 3 treatments, 62.1% of the patients showed improvement in wrinkles and skin texture. Pigmentation improved in 84.6% of the patients, and a reduction in telangiectasis was seen in 81.25% of the patients. Pathological examination showed that both type 1 and type 3 collagens increased following treatment, but elastin content decreased; however, the elastin fibers were arranged more neatly. In the transmission electron microscope study, the amount of fibroblast activity increased, the fibroblasts were more active, and there were more collagen fibers neatly rearranged within the stroma. CONCLUSION: Clinical and pathological studies demonstrated that the IPL was effective in improving wrinkles and skin texture. The mechanism of action may be through the increasing activity of the fibroblasts, hyperplasia of the fibroblasts, and rearrangement of both collagen and elastin within the stroma.

Nuclear microscopy: a tool for imaging elemental distribution and percutaneous absorption in vivo.

Nuclear microscopy is a technique based on a focused beam of accelerated particles that has the ability of imaging the morphology of the tissue in vivo and of producing the correspondent elemental maps, whether in major, minor, or trace concentrations. These characteristics constitute a strong advantage in studying the morphology of human skin, its elemental distributions and the permeation mechanisms of chemical compounds. In this study, nuclear microscopy techniques such as scanning transmission ion microscopy and particle induced X-ray emission were applied simultaneously, to cryopreserved human skin samples with the purpose of obtaining high-resolution images of cells and tissue morphology. In addition, quantitative elemental profiling and mapping of phosphorus, calcium, chlorine, and potassium in skin cross-sections were obtained. This procedure accurately distinguishes the epidermal strata and dermis by overlapping in real time the elemental information with density images obtained from the transmitted beam. A validation procedure for elemental distributions in human skin based on differential density of epidermal strata and dermis was established. As demonstrated, this procedure can be used in future studies as a tool for the in vivo examination of trans-epidermal and -dermal delivery of products.

In vivo drug screening in human skin using femtosecond laser multiphoton tomography.

The novel femtosecond laser multiphoton imaging system DermaInspect forin vivotomography of human skin was used to study the diffusion and intradermal accumulation of topically applied cosmetic and pharmaceutical components. Near-infrared 80 MHz picojoule femtosecond laser pulses were employed to excite endogenous fluorophores and fluorescent components of a variety of ointments via a two-photon excitation process. In addition, collagen was imaged by second harmonic generation. A high submicron spatial resolution and 50 ps temporal resolution was achieved using galvoscan mirrors and piezodriven focusing optics together with a time-correlated single-photon counting module with a fast microchannel plate detector. Individual intratissue cells, intracellular mitochondria, melanosomes, and the morphology of the nuclei as well as extracellular matrix elements were clearly visualized due to NAD(P)H, melanin, elastin, and collagen imaging and the calculation of fluorescence lifetime images. Nanoparticles and intratissue drugs were detected by two-photon-excited fluorescence. In addition, hydration effects and UV effects were studied by monitoring modifications of cellular morphology and autofluorescence. The system was used to observe the diffusion through the stratum corneum and the accumulation and release of functionalized nanoparticles along hair shafts and epidermal ridges. The novel noninvasive 4-D multiphoton tomography tool provides high-resolution optical biopsies with subcellular resolution, and offers for the first time the possibility to study in situ the diffusion through the skin barrier, long-term pharmacokinetics, and cellular response to cosmetic and pharmaceutical products.

Biomimetic bilayered gelatin-chondroitin 6 sulfate-hyaluronic acid biopolymer as a scaffold for skin equivalent tissue engineering.

In order to develop an adequate scaffold for skin tissue engineering, a bilayered gelatin-chondroitin 6 sulfate-hyaluronic acid membrane with a different pore size on either side was prepared. A rete ridges-like topographic microporous structure, which provided the paracrine crosstalk in the epithelial-mesenchymal interactions, was formed. Chondroitin-6-sulfate and hyaluronic acid were incorporated within the gelatin membrane to mimic skin composition and create an appropriate microenvironment for cell proliferation, differentiation, and migration. In the study, the lower layer of the membrane (pore size: 150 microm) was seeded with dermal fibroblasts and acted as the feeder layer for keratinocyte inoculation. Meanwhile, the upper layer (pore size: 20-50 microm) was seeded with keratinocytes for epidermalization. The dermal fibroblasts were dynamically seeded in a self-designed spinner flask for more even cell distribution. The keratinocytes were cultured in submerged conditions for 5 days and then in an air-liquid interface condition for further differentiation. After being cultured for 21 days, the upper layer, seeded with keratinocytes, developed into an epidermis-like structure while the lower part, which was seeded with dermal fibroblasts developed into a dermis-like structure. A histological examination and immunostain were used to prove that keratinocytes maintain their phenotype and stratified epidermis layers were formed within 21 days. In brief, the bilayered skin substitute with biological dermal analog and epidermal structure was successfully fabricated. From this study, we can suggest that the culture model is suitable for autologous skin equivalent preparation.

Modification of skin composition by conjugated linoleic acid alone or with combination of other fatty acids in mice.

The effects of conjugated linoleic acid (CLA), gamma-linolenic acid (GLA), linoleic acid (LA), and their combinations, on skin composition in mice were investigated. Mice (8 weeks old) were orally administered with either LA, GLA, CLA, LA + GLA, LA + CLA, or CLA + GLA for 4 weeks. Then, the skin was analysed for triacylglycerol content, fatty acid composition and collagen content. Additionally, thicknesses of the dermis layer and subcutaneous tissue layer, and the size and number of adipocytes were measured histologically. The skin fatty acid composition was modified depending upon the fatty acid composition of supplemented oils. In each oil-alone group, skin triacylglycerol content was the highest in LA, followed by GLA and CLA treatments. Combinations with CLA had a similar triacylglycerol content compared with the CLA-alone group. No significant changes in collagen content were observed among any treatments. The effects on subcutaneous thickness were similar to the results obtained in the triacylglycerol contents, where groups supplemented with CLA alone or other fatty acids had significantly thinner subcutaneous tissue compared with the LA-alone group. However, no significant difference was detected in the thickness of the dermis layers. The number of adipocytes was highest in the LA + GLA group and tended to be reduced by CLA with or without the other fatty acids. These results suggest that CLA alone or in combination with other fatty acids strongly modifies skin composition in mice.

The relation between the acupoint structures and the clinical therapeutic effects.

We performed a histological study upon the acupuncture points and their effectiveness of clinical treatment; the background was the clinic evidence that a multitude of points are used in treating a disease, but only some of which may have an efficacy, since the others did not. After a comparative histological and anatomic study, it comes out that those points, which are more effective from a structural point of view, identify a neural fibrillar concentration, a well developed capillary network and an increased mucopolysaccharides (MPS) concentration, in particular, acid mucopolysaccharides. The present paper presents histological data, which demonstrate the difference in the structure of the acupuncture points, postulating their specific influence on clinical treatment.

Ultrastructural changes elicited by a non-ablative wrinkle reduction laser.

BACKGROUND AND OBJECTIVES: Cosmeceuticals, chemical peels and collagen injections are used to rejuvenate skin, but none of these methods is effective or permanent. Recently, laser resurfacing has been found to be effective, but the incidence of side effects is relatively high. Two years ago, the non-ablative wrinkle reduction laser (N-Lite, ICN Photonics, UK) was developed, and there have been several reports about its clinical effectiveness. In this study, we have investigated ultrastructural changes elicited by exposure to the N-Lite laser. STUDY DESIGN/MATERIALS AND METHODS: Eight adult volunteers were recruited for this study. They were treated with the N-Lite laser and 3-mm skin punch biopsies were obtained 3 hours, 1 day, 3 days, 1 week, 2 weeks, 4 weeks and 5 weeks after the laser exposure. These specimens were examined by electron microscopy. RESULTS: Three hours after the laser therapy, the capillaries showed endothelial cell edema with hemostasis and marked edema was observed around them. Neutrophils, monocytes and mast cells were observed in the extravascular dermis. These acute dermal inflammatory changes were observed until 1 week after the laser treatment. Two weeks after the laser treatment, the capillaries showed an almost normal structure, and dermal edema was not observed around them. New elastic fibers and collagen fibers had increased around the capillaries. Four weeks after the laser treatment, interstitial fibrosis was observed around the capillaries. CONCLUSIONS: N-Lite laser irradiation leads to interstitial fibrosis, especially around the capillaries, 4 weeks after the laser irradiation.