In-vivo Human Skin Research Articles

In vivo characterization of laser-assisted delivery of hyaluronic acid using multiphoton fluorescence lifetime imaging.

Laser-assisted drug delivery (LADD) is a treatment method to enhance the penetration of pharmaceuticals through the skin. The aim of the present study is to track hyaluronic acid (HA) and analyse its effect on human skin invivo after ablative fractional laser (AFL) treatment. Healthy male and female subjects were recruited. Four areas were marked on their forearms of each volunteer, and each area was assigned to one of the following treatment options: AFL + HA, AFL only, HA only or untreated control. A carbon dioxide laser was used for the AFL treatment. Follow-up measurements were scheduled 30 min and 30 days after treatment using multiphoton tomography equipped with fluorescence lifetime imaging (MPT-FLIM). A total of 11 subjects completed the study. By detecting fluorescence lifetimes, the HA and the anaesthetic ointment were clearly distinguishable from surrounding tissue. After AFL treatment, HA could be visualized in all epidermal and upper dermal layers. In contrast, HA in intact skin was only detected in the superficial layers at distinctly lower levels. The applied HA gel seemed to have beneficial properties for the wound healing process after laser treatment. LADD has proven to be a fast and effective method to increase HA uptake into the skin, allowing for improved hydration and skin rejuvenation over time. Furthermore, LADD could be a beneficial treatment option in laser resurfacing. MPT-FLIM proved to be an appropriate diagnostic tool for drug delivery tracking and monitoring of treatment response for individualized therapy adjustment.

Optoacoustic Imaging Offers New Insights into In Vivo Human Skin Vascular Physiology.

Functional imaging with new photoacoustic tomography (PAT) offers improved spatial and temporal resolution quality in in vivo human skin vascular assessments. In the present study, we followed a suprasystolic reactive hyperemia (RH) maneuver with a multi-spectral optoacoustic tomography (MSOT) system. A convenience sample of ten participants, both sexes, mean age of 35.8 ± 13.3 years old, was selected. All procedures were in accordance with the principles of good clinical practice and approved by the institutional ethics committee. Images were obtained at baseline (resting), during occlusion, and immediately after pressure release. Observations of the RH by PAT identified superficial and deeper vascular structures parallel to the skin surface as part of the human skin vascular plexus. Furthermore, PAT revealed that the suprasystolic occlusion impacts both plexus differently, practically obliterating the superficial smaller vessels and evoking stasis at the deeper, larger structures in real-time (live) conditions. This dual effect of RH on the skin plexus has not been explored and is not considered in clinical settings. Thus, RH seems to represent much more than the local microvascular reperfusion as typically described, and PAT offers a vast potential for vascular clinical and preclinical research.

Topical Application of Metal Allergens Induces Changes to Lipid Composition of Human Skin.

Lipids are an important constituent of skin and are known to be modified in many skin diseases including psoriasis and atopic dermatitis. The direct effects of common metallic contact allergens on the lipid composition of skin has never been investigated, to the best of our knowledge. We describe skin lipid profiles in the stratum corneum and viable epidermis of ex vivo human skin from a female donor upon exposure to three metal allergens (nickel, cobalt and chromium) visualised using time-of-flight secondary ion mass spectrometry (ToF-SIMS), which allows for simultaneous visualisation of both the allergen and skin components such as lipids. Multivariate analysis using partial least squares discriminant analysis (PLS-DA) indicated that the lipid profile of metal-treated skin was different to non-treated skin. Analysis of individual ions led to the discovery that cobalt and chromium induced increases in the content of diacylglycerols (DAG) in stratum corneum. Cobalt also induced increases in cholesterol in both the stratum corneum and viable epidermis, as well as monoacylglycerols (MAG) in the viable epidermis. Chromium caused an increase in DAG in viable epidermis in addition to the stratum corneum. In contrast, nickel decreased MAG and DAG levels in viable epidermis. Our results indicate that skin lipid content is likely to be altered upon topical exposure to metals. This discovery has potential implications for the molecular mechanisms by which contact allergens cause skin sensitization.

Carotenoids in Human SkinIn Vivo: Antioxidant and Photo-Protectant Role against External and Internal Stressors.

The antioxidant system of the human body plays a crucial role in maintaining redox homeostasis and has an important protective function. Carotenoids have pronounced antioxidant properties in the neutralization of free radicals. In human skin, carotenoids have a high concentration in the stratum corneum (SC)—the horny outermost layer of the epidermis, where they accumulate within lipid lamellae. Resonance Raman spectroscopy and diffuse reflectance spectroscopy are optical methods that are used to non-invasively determine the carotenoid concentration in the human SC in vivo. It was shown by electron paramagnetic resonance spectroscopy that carotenoids support the entire antioxidant status of the human SC in vivo by neutralizing free radicals and thus, counteracting the development of oxidative stress. This review is devoted to assembling the kinetics of the carotenoids in the human SC in vivo using non-invasive optical and spectroscopic methods. Factors contributing to the changes of the carotenoid concentration in the human SC and their influence on the antioxidant status of the SC in vivo are summarized. The effect of chemotherapy on the carotenoid concentration of the SC in cancer patients is presented. A potential antioxidant-based pathomechanism of chemotherapy-induced hand-foot syndrome and a method to reduce its frequency and severity are discussed.

Diurnal Preference Contributes to Maximal UVB Sensitivity by the Hour of the Day in Human Skin In Vivo

Diurnal Preference Contributes to Maximal UVB Sensitivity by the Hour of the Day in Human Skin In Vivo

Perspectives on Cyclobutane Pyrimidine Dimers-Rise of the Dark Dimers.

Some early reports demonstrate that levels of cyclobutane pyrimidine dimers (CPD) may increase after UVR exposure had ended, although these observations were treated as artifacts. More recently, it has been shown unequivocally that CPD formation does occur post-irradiation, with maximal levels occurring after about 2-3 h. These lesions have been termed "dark CPD" (dCPD). Subsequent studies have confirmed their presence invitro, in mouse models and in human skin invivo. Melanin carbonyls have a role in the formation of dCPD, but they have also been observed in amelanotic systems, indicating other, unknown process(es) exist. In both cases, the formation of dCPD can be prevented by the presence of certain antioxidants. We lack data on the spectral dependence of dCPD, but it is unlikely to be the same as for incident CPD (iCPD), which are formed only during irradiation. There is evidence that iCPD and dCPD may have different repair kinetics, although this remains to be elucidated. It is also unknown whether iCPD and dCPD have different biological properties. The formation of dCPD in human skin invivo has implications for post solar exposure photoprotection, and skin carcinogenesis, with a need for this to be investigated further.

Characterization of Collagen I Fiber Thickness, Density, and Orientation in the Human Skin In Vivo Using Second-Harmonic Generation Imaging

The assessment of dermal alterations is necessary to monitor skin aging, cancer, and other skin diseases and alterations. The gold standard of morphologic diagnostics is still histopathology. Here, we proposed parameters to distinguish morphologically different collagen I structures in the extracellular matrix and to characterize varying collagen I structures in the skin with similar SAAID (SHG-to-AF Aging Index of Dermis, SHG—second-harmonic generation; AF—autofluorescence) values. Test datasets for the papillary and reticular extracellular matrix from images in 24 female subjects, 36 to 50 years of age, were generated. Parameters for SAAID, edge detection, and fast Fourier transformation directionality were determined. Additionally, textural analyses based on the grey level co-occurrence matrix (GLCM) were conducted. At first, changes in the GLCM parameters were determined in the native greyscale images and, furthermore, in the Hilbert-transformed images. Our results demonstrate a robust set of parameters for noninvasive in vivo classification for morphologically different collagen I structures in the skin, with similar and different SAAID values. We anticipate our method to enable an automated prevention and monitoring system with an age- and gender-specific algorithm.

In vivo measurement of the anisotropic mechanical properties of human skin by indentation test

A novel approach is presented to determine the anisotropic mechanical properties of human skin invivo by indentation method. In this paper, the skin is considered as an anisotropic elastomer due to fiber reinforcement with an additional property of limiting extensibility. The associated theory of indentation method is presented and specialized to the particular case of the constitutive behaviors of skin so as to establish the theory basis of this work. The indentation tests are performed on the inner forearm in the relaxant and uniaxially pre-stretched state, respectively. Different from the conventional indentation tests, a special optical system is used to capture the contact morphology in real time. The results demonstrate that the eccentricity of the elliptical contact region changes with the pre-stretch on the ground that the eccentricity has a dependence on the degree of anisotropy.

Increased Histone Acetylation and Decreased Expression of Specific Histone Deacetylases in Ultraviolet-Irradiated and Intrinsically Aged Human Skin In Vivo.

Histone deacetylases (HDACs) are conserved enzymes that remove acetyl groups from lysine side chains in histones and other proteins and play a crucial role in epigenetic regulation. Previously, we showed that histone acetylation is implicated in ultraviolet (UV)-induced inflammation and matrix impairment. To elucidate the histone acetylation status and specific HDACs involved in skin aging, we examined the changes in histone acetylation, global HDAC activity, and the expression of HDACs and sirtuins (SIRTs) in intrinsically aged and photoaged human skin as well as in UV-irradiated human skin in vivo. Following acute UV irradiation, the acetylated histone H3 (AcH3) level was increased, but HDAC activity and the expression levels of HDAC4, HDAC11, and SIRT4 were significantly decreased. In intrinsically aged skin, AcH3 levels were increased, but HDAC activity and the expression levels of HDAC4, HDAC5, HDAC10, HDAC11, SIRT6, and SIRT7 were significantly decreased. However, histone acetylation and HDAC expression in photoaged skin were not significantly different from those in intrinsically aged skin. Collectively, HDAC4 and HDAC11 were decreased in both UV-irradiated and intrinsically aged skin, suggesting that they may play a universal role in increased histone acetylation associated with skin aging.

A Weighted Average Phase Velocity Inversion Model for Depth-Resolved Elasticity Evaluation in Human Skin In-Vivo.

In current surface acoustic wave (SAW) elastography field, wavelength-depth inversion model is a straightforward and widely used inversion model for depth-resolved elasticity profile reconstruction. However, the elasticity directly evaluated from the wavelength-depth relationship is biased. Thus, a new inversion model, termed weighted average phase velocity (WAPV) inversion model, is proposed to provide depth-resolved Young's modulus estimate with better accuracy. The forward model for SAW phase velocity dispersion curve generation was derived from the numerical simulations of SAWs in layered materials, and inversion was implemented by matching the measured phase velocity dispersion curve to the one generated from the forward model using the least squares fitting. Three two-layer agar phantoms with different top-layer thicknesses and one three-layer agar phantom were tested to validate the proposed inversion model. Then the model was demonstrated on human skin at various sites (palm, forearm and back of hand) in-vivo. In multi-layered agar phantoms, depth-resolved elasticity estimates provided by the model have a maximal total inversion error of 15.2% per sample after inversion error compensation. In in-vivo human skin, the quantified bulk Young's moduli (palm: 212 ± 78 kPa; forearm: 32 ± 11 kPa and back of hand: 29 ± 8 kPa) are comparable to the reference values in the literature. The WAPV inversion model can provide accurate depth-resolved Young's modulus estimates in layered biological soft tissues. The proposed model can predict depth-resolved elasticity in layered biological soft tissues with a reasonable accuracy which traditional wavelength-depth inversion model cannot provide.

General method for classification of fiber families in fiber-reinforced materials: application to in-vivo human skin images

Fiber structures play a major role for the function of fiber-reinforced materials such as biological tissue. An objective classification of the fiber orientations into fiber families is crucial to understand its mechanical properties. We introduce the Fiber Image Network Evaluation Algorithm (FINE algorithm) to classify and quantify the number of fiber families in scientific images. Each fiber family is characterized by an amplitude, a mean orientation, and a dispersion. A new alignment index giving the averaged fraction of aligned fibers is defined. The FINE algorithm is validated by realistic grayscale Monte-Carlo fiber images. We apply the algorithm to an in-vivo depth scan of second harmonic generation images of dermal collagen in human skin. The derived alignment index exhibits a crossover at a critical depth where two fiber families with a perpendicular orientation around the main tension line arise. This strongly suggests the presence of a transition from the papillary to the reticular dermis. Hence, the FINE algorithm provides a valuable tool for a reliable classification and a meaningful interpretation of in-vivo collagen fiber networks and general fiber reinforced materials.

Harnessing the Secretome of Hair Follicle Fibroblasts to Accelerate Ex Vivo Healing of Human Skin Wounds

In skin homeostasis, dermal fibroblasts are responsible for coordinating the migration and differentiation of overlying epithelial keratinocytes. As hairy skin heals faster than nonhairy skin, we took bio-inspiration from the follicle and hypothesized that follicular fibroblasts would accelerate skin re-epithelialization after injury faster than interfollicular fibroblasts. Using both invitro and exvivo models of human skin wound closure, we found that hair follicle dermal papilla fibroblasts could accelerate closure of invitro scratch wounds by 1.8-fold and epithelial growth capacity by 1.5-fold compared with controls (P < 0.05). We used a cytokine array to determine how the dermal papilla fibroblasts were eliciting this effect and identified two cytokines, sAXL and CCL19, that are released at significantly higher levels by follicular fibroblasts than by interfollicular subtypes. Using sAXL and CCL19 individually, we found that they could also increase closure of epithelial cells in a scratch wound by 1.2- and 1.5-fold, respectively, compared with controls (P < 0.05). We performed an unbiased transcriptional analysis, combined with pathway analysis, and postulate that sAXL accelerates wound closure by promoting migration and inhibiting epithelial differentiation of skin keratinocytes. Long term, we believe these results can be exploited to accelerate wound closure of human skin invivo.

Temperature Depth Profiles Induced in Human Skin In Vivo Using Pulsed 975 nm Irradiation.

The aim of this study was to determine the temperature depth profiles induced in human skin in vivo by using a pulsed 975 nm diode laser (with 5 ms pulse duration) and compare them with those induced by the more common 532 nm (KTP) and 1,064 nm (Nd:YAG) lasers. Quantitative assessment of the energy deposition characteristics in human skin at 975 nm should help design of safe and effective treatment protocols when using such lasers. Temperature depth profiles induced in the human skin by the three lasers were determined using pulsed photothermal radiometry (PPTR). This technique involves time-resolved measurement of mid-infrared emission from the irradiated test site and reconstruction of the laser-induced temperature profiles using an earlier developed optimization algorithm. Measurements were performed on volar sides of the forearms in seven volunteers with healthy skin. At irradiation spot diameters of 3-4 mm, the radiant exposures were 0.24, 0.36, and 5.7 J/cm2 for the 975, 532, and 1,064 nm lasers, respectively. Upon normalization to the same radiant exposure of 1 J/cm 2 , the assessed maximum temperature rise in the epidermis averaged 0.8 °C for the 975 nm laser, 7.4 °C for the 532 nm, and 0.6 °C for the 1,064 nm laser. The characteristic subsurface depth to which 50% of the absorbed laser energy was deposited was on average 0.31 mm at 975 nm irradiation, and slightly deeper at 1,064 nm, and 0.15 mm at 532 nm. The experimentally obtained relations were reproduced in a dedicated numerical simulation. The assessed energy deposition characteristics show that the pulsed 975 nm diode laser is very suitable for controlled heating of the upper dermis as required, for example, for nonablative skin rejuvenation. The risks of nonselective overheating of the epidermis and subcutis are significantly reduced in comparison with irradiation at 532 and 1,064 nm, respectively. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Effects of Different Types of Burn Wounds and its Dressings on Millimeter-Wave Images

Abstract Millimeter-wave imaging is a promising technology for diagnosing skin burns, that may make it easier to assess and determine the burn depth in the near future. However, up to now, it has not yet been brought to clinical use due to the lack of clinical trails on patients and a millimeter-wave-aided classification of skin burns. In this paper, in a preliminary step, ex-vivo burned porcine skin is utilized to visualize and quantify skin that has been burned in different ways, and to access its effect on millimeter-wave images. For the first time, a 24 hour study of in-vivo human skin visualizes the effect of wound dressings using a fast imaging system operating at frequencies from 70 to 80 GHz. For validation, the effective relative permittivity of the skin and the dressings are measured using a open-ended coaxial probe. An analytical model is applied to calculate the reflection coefficient which are compared to the intensity of the millimeter-wave images to validate the model.

In Vivo Human Skin Penetration Study of Sunscreens by Confocal Raman Spectroscopy.

This research work mainly deals with the application of confocal Raman spectroscopic technique to study in vivo human skin penetration of sunscreen products, as there are a lot of controversies associated with their skin penetration. Healthy human volunteers were tested for penetration of two commercial sunscreen products into their volar forearm skin for a period of 2h. Measurements were taken before and after application of these sunscreen products. All the confocal Raman spectra were pre-processed and then subjected to multivariate two-dimensional principal component analysis and classical least squares analysis to determine the skin penetration of these sunscreens in comparison to the "sunscreen product spectrum" which was considered as the control. Score plots of principal component analysis of confocal Raman spectra indicated clear separation between the spectra before and after application of sunscreen products. Loading plots showed the maximum differences in the spectral region from 1590 to 1626cm-1 where the characteristic peak of the pure sunscreen products was observed. Classical least squares analysis has shown a significant penetration to a depth of 10μm in the volar forearm skin of healthy human volunteers for both these sunscreen products. The results confirm that the penetration of these tested sunscreen products was restricted to stratum corneum and also prove that confocal Raman spectroscopy is a simple, fast, nondestructive, and noninvasive semi-quantitative analytical technique for these studies.

Broadband absorption and reduced scattering spectra of in-vivo skin can be noninvasively determined using δ-P1 approximation based spectral analysis.

Previously, we revealed that a linear gradient line source illumination (LGLSI) geometry could work with advanced diffusion models to recover the sample optical properties at wavelengths where sample absorption and reduced scattering were comparable. In this study, we employed the LGLSI geometry with a broadband light source and utilized the spectral analysis to determine the broadband absorption and scattering spectra of turbid samples in the wavelength range from 650 to 1350 nm. The performance of the LGLSI δ-P1 diffusion model based spectral analysis was evaluated using liquid phantoms, and it was found that the sample optical properties could be properly recovered even at wavelengths above 1000 nm where μs' to μa ratios were in the range between 1 to 20. Finally, we will demonstrate the use of our system for recovering the 650 to 1350 nm absorption and scattering spectra of in-vivo human skin. We expect this system can be applied to study deep vessel dilation induced hemoglobin concentration variation and determine the water and lipid concentrations of in-vivo skin in clinical settings in the future.

Investigation of in-vivo skin autofluorescence lifetimes under long-term cw optical excitation

The main results obtained during the last five years in the field of laser-excited in-vivo human skin photobleaching effects are presented. The main achievements and results obtained, as well as methods and experimental devices are briefly described. In addition, the impact of long-term cw low-power laser excitation on the skin autofluorescence lifetime is experimentally investigated.

High-Resolution Ultrasound Imaging of Human Skin In Vivo by Using Three-Dimensional Ultrasound Microscopy

Observing the morphology of human skin is important in the diagnosis of skin cancer and inflammation and in the assessment of skin aging. High-frequency ultrasound imaging provides high spatial resolution of the deep layers of the skin, which cannot be visualized by optical methods. The objectives of the present study were to develop a three-dimensional (3-D) ultrasound microscope and to observe the morphology of normal human skin in vivo. A concave polyvinylidene fluoride transducer with a central frequency of 120 MHz was excited using an electric pulse generated by semiconductor switching. The transducer was scanned two-dimensionally by using two linear motors on the region-of-interest and the ultrasonic reflection was digitized with 2-GHz sampling. Consecutive B-mode images perpendicular to the skin surface were reconstructed to generate multiplanar reconstructed images and 3-D volume-rendering images clearly showing microstructures such as sebaceous glands and hair follicles. The 3-D ultrasound microscope could be used to successfully image the morphology of human skin noninvasively and may provide important information on skin structure.

UVA1 Induces Cyclobutane Pyrimidine Dimers but Not 6-4 Photoproducts in Human Skin In Vivo

UVB readily induces cyclobutane pyrimidine dimers, mainly thymine dimers (TTs), and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) in DNA. These lesions result in "UVB signature mutations" found in skin cancers. We have investigated the induction of TTs and 6-4PPs in human skin in vivo by broadband UVA1, and have compared this with comparable erythemal doses of monochromatic UVB (300 nm). In vitro and ex vivo studies have shown the production of TTs, without 6-4PPs, by UVA1. We show that UVA1 induces TTs, without 6-4PPs, in the epidermis of healthy volunteers in vivo, whereas UVB induced both photoproducts. UVB induced more TTs than UVA1 for the same level of erythema. The level of UVA1-induced TTs increased with epidermal depth in contrast to a decrease that was seen with UVB. UVA1- and UVB-induced TTs were repaired in epidermal cells at a similar rate. The mechanism by which UVA1 induces TTs is unknown, but a lack of intra-individual correlation between our subjects' UVB and UVA1 minimal erythema doses implies that UVA1 and UVB erythema occur by different mechanisms. Our data suggest that UVA1 may be more carcinogenic than has previously been thought.

Determining elastic properties of skin by measuring surface waves from an impulse mechanical stimulus using phase-sensitive optical coherence tomography

The mechanical properties of skin are important tissue parameters that are useful for understanding skin patho-physiology, which can aid disease diagnosis and treatment. This paper presents an innovative method that employs phase-sensitive spectral-domain optical coherence tomography (PhS-OCT) to characterize the biomechanical properties of skin by measuring surface waves induced by short impulses from a home-made shaker. Experiments are carried out on single and double-layer agar-agar phantoms, of different concentrations and thickness, and on in vivo human skin, at the forearm and the palm. For each experiment, the surface wave phase-velocity dispersion curves were calculated, from which the elasticity of each layer of the sample was determined. It is demonstrated that the experimental results agree well with previous work. This study provides a novel combination of PhS-OCT technology with a simple and an inexpensive mechanical impulse surface wave stimulation that can be used to non-invasively evaluate the mechanical properties of skin in vivo, and may offer potential use in clinical situations.