Improving dermal fibroblast-to-epidermis communications and aging wound repair through extracellular vesicle-mediated delivery of Gstm2 mRNA.

Skin aging is characterized by the disruption of skin homeostasis and impaired skin injury repair. Treatment of aging skin has long been limited by the unclear intervention targets and delivery techniques. Engineering extracellular vesicles (EVs) as an upgraded version of natural EVs holds great potential in regenerative medicine. In this study, we found that the expression of the critical antioxidant and detoxification gene Gstm2 was significantly reduced in aging skin. Thus, we constructed the skin primary fibroblasts-derived EVs encapsulating Gstm2 mRNA (EVsGstm2), and found that EVsGstm2 could significantly improve skin homeostasis and accelerate wound healing in aged mice. Mechanistically, we found that EVsGstm2 alleviated oxidative stress damage of aging dermal fibroblasts by modulating mitochondrial oxidative phosphorylation, and promoted dermal fibroblasts to regulate skin epidermal cell function by paracrine secretion of Nascent Polypeptide-Associated Complex Alpha subunit (NACA). Furthermore, we confirmed that NACA is a novel skin epidermal cell protective molecule that regulates skin epidermal cell turnover through the ROS-ERK-ETS-Cyclin D pathway. Our findings demonstrate the feasibility and efficacy of EVs-mediated delivery of Gstm2 for aged skin treatment and unveil novel roles of GSTM2 and NACA for improving aging skin.

Hindering the biofilm of microbial pathogens and cancer cell lines development using silver nanoparticles synthesized by epidermal mucus proteins from Clarias gariepinus.

Scientists know very little about the mechanisms underlying fish skin mucus, despite the fact that it is a component of the immune system. Fish skin mucus is an important component of defence against invasive infections. Recently, Fish skin and its mucus are gaining interest among immunologists. Characterization was done on the obtained silver nanoparticles Ag combined with Clarias gariepinus catfish epidermal mucus proteins (EMP-Ag-NPs) through UV-vis, FTIR, XRD, TEM, and SEM. Ag-NPs ranged in size from 4 to 20 nm, spherical in form and the angles were 38.10°, 44.20°, 64.40°, and 77.20°, Where wavelength change after formation of EMP-Ag-NPs as indicate of dark brown, the broad band recorded at wavelength at 391 nm. Additionally, the antimicrobial, antibiofilm and anticancer activities of EMP-Ag-NPs was assessed. The present results demonstrate high activity against unicellular fungi C. albicans, followed by E. faecalis. Antibiofilm results showed strong activity against both S. aureus and P. aeruginosa pathogens in a dose-dependent manner, without affecting planktonic cell growth. Also, cytotoxicity effect was investigated against normal cells (Vero), breast cancer cells (Mcf7) and hepatic carcinoma (HepG2) cell lines at concentrations (200-6.25 µg/mL) and current results showed highly anticancer effect of Ag-NPs at concentrations 100, 5 and 25 µg/mL exhibited rounding, shrinkage, deformation and granulation of Mcf7 and HepG2 with IC50 19.34 and 31.16 µg/mL respectively while Vero cells appeared rounded at concentration 50 µg/mL and normal shape at concentration 25, 12.5 and 6.25 µg/ml with IC50 35.85 µg/mL. This study evidence the potential efficacy of biologically generated Ag-NPs as a substitute medicinal agent against harmful microorganisms. Furthermore, it highlights their inhibitory effect on cancer cell lines.

Comparative study of melasma in patients before and after treatment based on lipomics.

BACKGROUND: Skin barrier alterations play a crucial function in melasma development. Past researches have demonstrated variations in lipid content between the epidermis of melasma lesions and normal tissues, along with the varied expression of lipid-related genes in melasma. This study aimed to analyze the lipidome profiles of skin surface lipids (SSL) in patients with melasma before and after treatment to understand associated abnormalities. METHODS: Melasma was treated with tranexamic acid orally and hydroquinone cream topically. Disease was assessed using the Melasma Area and Severity Index (MASI), and the impact to life was evaluated with Melasma Quality of Life (MELASQoL) score. Epidermal melanin particles were observed using reflection confocal microscopy (RCM), whereas epidermal pigment and blood vessel morphology were observed using dermoscopy, and SSL samples were collected. Specific information regarding alterations in lipid composition was obtained through multivariate analysis of the liquid chromatography-mass spectrometry data. RESULTS: After treatment, patients with melasma exhibited decreased MASI and MELASQoL scores (P < 0.001); RCM revealed reduced melanin content in the lesions, and dermoscopy revealed fewer blood vessels. Fifteen lipid subclasses and 382 lipid molecules were identified using lipidomic assays. The expression levels of total lipids, phosphatidylcholine, and phosphatidylethanolamine in the melasma lesions decreased after treatment (P < 0.05). CONCLUSION: This study revealed alterations in the SSL composition after effective melasma treatment, suggesting a compensatory role for lipids in melasma barrier function. The mechanism involving SSL and the lipid barrier, which influences melasma's occurrence, needs further elucidation.

JAK/STAT Inhibition Normalizes Lipid Composition in 3D Human Epidermal Equivalents Challenged with Th2 Cytokines.

Derangement of the epidermal barrier lipids and dysregulated immune responses are key pathogenic features of atopic dermatitis (AD). The Th2-type cytokines interleukin IL-4 and IL-13 play a prominent role in AD by activating the Janus Kinase/Signal Transduction and Activator of Transcription (JAK/STAT) intracellular signaling axis. This study aimed to investigate the role of JAK/STAT in the lipid perturbations induced by Th2 signaling in 3D epidermal equivalents. Tofacitinib, a low-molecular-mass JAK inhibitor, was used to screen for JAK/STAT-mediated deregulation of lipid metabolism. Th2 cytokines decreased the expression of elongases 1, 3, and 4 and serine-palmitoyl-transferase and increased that of sphingolipid delta(4)-desaturase and carbonic anhydrase 2. Th2 cytokines inhibited the synthesis of palmitoleic acid and caused depletion of triglycerides, in association with altered phosphatidylcholine profiles and fatty acid (FA) metabolism. Overall, the ceramide profiles were minimally affected. Except for most sphingolipids and very-long-chain FAs, the effects of Th2 on lipid pathways were reversed by co-treatment with tofacitinib. An increase in the mRNA levels of CPT1A and ACAT1, reduced by tofacitinib, suggests that Th2 cytokines promote FA beta-oxidation. In conclusion, pharmacological inhibition of JAK/STAT activation prevents the lipid disruption caused by the halted homeostasis of FA metabolism.

Three stepwise pH progressions in stratum corneum for homeostatic maintenance of the skin.

The stratum corneum is the outermost skin layer with a vital role in skin barrier function. It is comprised of dead keratinocytes (corneocytes) and is known to maintain its thickness by shedding cells, although, the precise mechanisms that safeguard stratum corneum maturation and homeostasis remain unclear. Previous ex vivo studies have suggested a neutral-to-acidic pH gradient in the stratum corneum. Here, we use intravital pH imaging at single-corneocyte resolution to demonstrate that corneocytes actually undergo differentiation to develop three distinct zones in the stratum corneum, each with a distinct pH value. We identified a moderately acidic lower, an acidic middle, and a pH-neutral upper layer in the stratum corneum, with tight junctions playing a key role in their development. The upper pH neutral zone can adjust its pH according to the external environment and has a neutral pH under steady-state conditions owing to the influence of skin microbiota. The middle acidic pH zone provides a defensive barrier against pathogens. With mathematical modeling, we demonstrate the controlled protease activation of kallikrein-related peptidases on the stratum corneum surface that results in proper corneocyte shedding in desquamation. This work adds crucial information to our understanding of how stratum corneum homeostasis is maintained.

Ultraviolet B irradiation increases the expression of cornulin and retepin in human skin xenotransplants.

Cornulin (CRNN) and repetin (RPTN) belong to the fused-type S100 protein family. Although these proteins have been reported to be expressed in the granular layer of the epidermis and have been suggested to be associated with barrier formation in the epidermis, their exact function remains unclear. This study examined the effects of ultraviolet B (UVB) irradiation on CRNN and RPTN expression in human skin xenotransplantation. The CRNN expression increased in the granular layer of UVB-irradiated skin 2 days after UVB irradiation compared to that in sham-irradiated skin. Interestingly, CRNN signals were observed not only in the cytoplasm, but also in the peripheral regions of granular keratinocytes. In contrast, RPTN was rarely expressed in sham-irradiated skin; however, RPTN signals were markedly increased in the granular layer of the UVB-irradiated skin. In addition, activation of ERK1/2 and STAT3 was observed in UVB-irradiated skin. Accordingly, the present study demonstrated that CRNN and RPTN are novel proteins whose expression can be increased by UVB irradiation. The activation of ERK1/2 and STAT3 may be associated with the regeneration of a UVB-damaged epidermis, and CRNN and RPTN may be induced to repair any dysfunction in the epidermal barrier during this regeneration process.

Cellular structure of dinosaur scales reveals retention of reptile-type skin during the evolutionary transition to feathers.

Fossil feathers have transformed our understanding of integumentary evolution in vertebrates. The evolution of feathers is associated with novel skin ultrastructures, but the fossil record of these changes is poor and thus the critical transition from scaled to feathered skin is poorly understood. Here we shed light on this issue using preserved skin in the non-avian feathered dinosaur Psittacosaurus. Skin in the non-feathered, scaled torso is three-dimensionally replicated in silica and preserves epidermal layers, corneocytes and melanosomes. The morphology of the preserved stratum corneum is consistent with an original composition rich in corneous beta proteins, rather than (alpha-) keratins as in the feathered skin of birds. The stratum corneum is relatively thin in the ventral torso compared to extant quadrupedal reptiles, reflecting a reduced demand for mechanical protection in an elevated bipedal stance. The distribution of the melanosomes in the fossil skin is consistent with melanin-based colouration in extant crocodilians. Collectively, the fossil evidence supports partitioning of skin development in Psittacosaurus: a reptile-type condition in non-feathered regions and an avian-like condition in feathered regions. Retention of reptile-type skin in non-feathered regions would have ensured essential skin functions during the early, experimental stages of feather evolution.

Unraveling the Mechanisms Involved in the Beneficial Effects of Magnesium Treatment on Skin Wound Healing.

The skin wound healing process consists of hemostatic, inflammatory, proliferative, and maturation phases, with a complex cellular response by multiple cell types in the epidermis, dermis, and immune system. Magnesium is a mineral essential for life, and although magnesium treatment promotes cutaneous wound healing, the molecular mechanism and timing of action of the healing process are unknown. This study, using human epidermal-derived HaCaT cells and human normal epidermal keratinocyte cells, was performed to investigate the mechanism involved in the effect of magnesium on wound healing. The expression levels of epidermal differentiation-promoting factors were reduced by MgCl2, suggesting an inhibitory effect on epidermal differentiation in the remodeling stage of the late wound healing process. On the other hand, MgCl2 treatment increased the expression of matrix metalloproteinase-7 (MMP7), a cell migration-promoting factor, and enhanced cell migration via the MEK/ERK pathway activation. The enhancement of cell migration by MgCl2 was inhibited by MMP7 knockdown, suggesting that MgCl2 enhances cell migration which is mediated by increased MMP7 expression. Our results revealed that MgCl2 inhibits epidermal differentiation but promotes cell migration, suggesting that applying magnesium to the early wound healing process could be beneficial.

PRSS3/mesotrypsin as a putative regulator of the biophysical characteristics of epidermal keratinocytes in superficial layers.

Mesotrypsin, encoded by the PRSS3 gene, is a distinctive trypsin isoform renowned for its exceptional resistance to traditional trypsin inhibitors and unique substrate specificity. Within the skin epidermis, this protein primarily expresses in the upper layers of the stratified epidermis and plays a crucial role in processing pro-filaggrin (Pro-FLG). Although prior studies have partially elucidated its functions using primary cultured keratinocytes, challenges persist due to these cells' differentiation-activated cell death program. In the present study, HaCaT keratinocytes, characterized by minimal endogenous mesotrypsin expression and sustained proliferation in differentiated states, were utilized to further scrutinize the function of mesotrypsin. Despite the ready degradation of the intact form of active mesotrypsin in these cells, fusion with Venus, flanked by a peptide linker, enables evasion from the protein elimination machinery, thus facilitating activation of the Pro-FLG processing system. Inducing Venus-mesotrypsin expression in the cells resulted in a flattened phenotype and reduced proliferative capacity. Moreover, these cells displayed altered F-actin assembly, enhanced E-cadherin adhesive activity, and facilitated tight junction formation without overtly influencing epidermal differentiation. These findings underscore mesotrypsin's potentially pivotal role in shaping the characteristic cellular morphology of upper epidermal layers.

(S)-(-)-blebbistatin O-benzoate has the potential to improve atopic dermatitis symptoms in NC/Nga mice by upregulating epidermal barrier function and inhibiting type 2 alarmin cytokine induction.

Atopic dermatitis is a multi-pathogenic disease characterized by chronic skin inflammation and barrier dysfunction. Therefore, improving the skin's ability to form an epidermal barrier and suppressing the production of cytokines that induce type 2 inflammatory responses are important for controlling atopic dermatitis symptoms. (-)-Blebbistatin, a non-muscle myosin II inhibitor, has been suggested to improve pulmonary endothelial barrier function and control inflammation by suppressing immune cell migration; however, its efficacy in atopic dermatitis is unknown. In this study, we investigated whether (S)-(-)-blebbistatin O-benzoate, a derivative of (-)-blebbistatin, improves dermatitis symptoms in a mite antigen-induced atopic dermatitis model using NC/Nga mice. The efficacy of the compound was confirmed using dermatitis scores, ear thickness measurements, serum IgE levels, histological analysis of lesions, and filaggrin expression analysis, which is important for barrier function. (S)-(-)-Blebbistatin O-benzoate treatment significantly reduced the dermatitis score and serum IgE levels compared to those in the vehicle group (p < 0.05). Furthermore, the histological analysis revealed enhanced filaggrin production and a decreased number of mast cells (p < 0.05), indicating that (S)-(-)-blebbistatin O-benzoate improved atopic dermatitis symptoms in a pathological model. In vitro analysis using cultured keratinocytes revealed increased expression of filaggrin, loricrin, involucrin, and ceramide production pathway-related genes, suggesting that (S)-(-)-blebbistatin O-benzoate promotes epidermal barrier formation. Furthermore, the effect of (S)-(-)-blebbistatin O-benzoate on type 2 alarmin cytokines, which are secreted from epidermal cells upon scratching or allergen stimulation and are involved in the pathogenesis of atopic dermatitis, was evaluated using antigens derived from mite feces. The results showed that (S)-(-)-blebbistatin O-benzoate inhibited the upregulation of these cytokines. Based on the above, (S)-(-)-blebbistatin O-benzoate has the potential to be developed as an atopic dermatitis treatment option that controls dermatitis symptoms by suppressing inflammation and improving barrier function by acting on multiple aspects of the pathogenesis of atopic dermatitis.

CLDN1 knock out keratinocytes as a model to investigate multiple skin disorders.

The transmembrane protein claudin-1 is critical for formation of the epidermal barrier structure called tight junctions (TJ) and has been shown to be important in multiple disease states. These include neonatal ichthyosis and sclerosing cholangitis syndrome, atopic dermatitis and various viral infections. To develop a model to investigate the role of claudin-1 in different disease settings, we used CRISPR/Cas9 to generate human immortalized keratinocyte (KC) lines lacking claudin-1 (CLDN1 KO). We then determined whether loss of claudin-1 expression affects epidermal barrier formation/function and KC differentiation/stratification. The absence of claudin-1 resulted in significantly reduced barrier function in both monolayer and organotypic cultures. CLDN1 KO cells demonstrated decreases in gene transcripts encoding the barrier protein filaggrin and the differentiation marker cytokeratin-10. Marked morphological differences were also observed in CLDN1 KO organotypic cultures including diminished stratification and reduced formation of the stratum granulosum. We also detected increased proliferative KC in the basale layer of CLDN1 KO organotypic cultures. These results further support the role of claudin-1 in epidermal barrier and suggest an additional role of this protein in appropriate stratification of the epidermis.

Hyaluronic Acid as a LYVE-1 Receptor Ligand in the Lymphatic System of Healthy Human Skin.

Immunohistochemical detection of the LYVE-1 marker in healthy human full-thickness skin (the epidermis and the dermis) was carried out. LYVE-1 expression was found in the endothelium of lymphatic capillaries located in the papillary dermis, in the endothelium of larger lymphatic vessels of the reticular dermis, and in fibroblasts, which indicates their joint participation in hyaluronan metabolism. LYVE-1+ staining detected for the first time in cells of the stratum basale, the stratum spinosum, and the stratum granulosum of healthy human epidermis indicates their participation in hyaluronan metabolism and allows us to consider the spaces between epidermis cells as prelimphatics.

Uridine phosphorylase-1 promotes cell viability and cell-cycle progression in human epidermal keratinocytes via the glycolytic pathway.

Glycolysis is vital for the excessive proliferation of keratinocytes in psoriasis, and uridine phosphorylase-1 (UPP1) functions as an enhancer of cancer cell proliferation. However, little is known about whether UPP1 promotes keratinocyte proliferation and accelerates psoriasis development. This study revealed that UPP1 facilitates cell viability and cell-cycle progression in human epidermal keratinocytes (HEKs) by modulating the glycolytic pathway. Bioinformatics analysis of UPP1 gene expression and its correlation with the Reactome revealed that UPP1 mRNA expression, cell-cycle progression, the interleukin-6 (IL-6)/Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway and glycolysis were positively associated with psoriasis. Cell proliferation, the cell cycle and glycolysis were evaluated after UPP1 was silenced or overexpressed. The results showed that UPP1 overexpression increased cell proliferation, cell-cycle progression and glycolysis, which was contrary to the effects of UPP1 silencing. However, the STAT3 inhibitor diminished UPP1 expression because STAT3 can bind to the UPP1 promoter. In conclusion, UPP1 was significantly activated by the IL-6/STAT3 pathway and could modulate glycolysis to regulate cell proliferation and cell-cycle progression in keratinocytes during the development of psoriasis.

Cutaneous inflammasome driving ASC / gasdermin-D activation and IL-1ß-secreting macrophages in severe atopic dermatitis.

Atopic dermatitis (AD) is an inflammatory skin disease with intense pruritus, and chronic skin colonization by Staphylococcus aureus. To understand the inflammatory status in AD, we investigated the inflammasome complex, that activates ASC (Apoptosis-associated speck-like protein containing a CARD), caspase-1 and GSDMD (gasdermin-D), and production of IL-1ß and IL-18. We aimed to evaluate the expression of the inflammasome pathway in the skin of adults with AD. Thirty patients with moderate to severe AD and 20 healthy controls were enrolled in the study. We performed the analysis of the inflammasome components NLRP1, NLRP3, AIM-2, IL-1ß, IL-18, Caspase-1, ASC, GSDMD, and CD68 expression (macrophage marker) by immunohistochemistry and immunofluorescence. The main findings included increased expression of NLRP3, NLRP1 and AIM-2 at dermal level of severe AD; augmented IL-18 and IL-1ß expression at epidermis of moderate and severe patients, and in the dermis of severe AD; augmented expression of ASC, caspase-1 and GSDMD in both epidermis and dermis of moderate and severe AD. We detected positive correlation between caspase-1, GSDMD and IL-1ß (epidermis) and caspase-1 (dermis) and AD severity; NLRP3, AIM-2 and IL-1ß, and NLRP3 with IL-18 in the epidermis; ASC, GSDMD and IL-1ß, and NLRP3, AIM-2, caspase-1, and IL-18 in the dermis. We also evidenced the presence of CD68+ macrophages secreting GSDMD, ASC and IL-1ß in moderate and severe AD. Cutaneous macrophages, early detected in moderate AD, have its role in the disease inflammatory mechanisms. Our study indicates a canonical activation pathway of inflammasomes, reinforced by the chronic status of inflammation in AD. The analysis of the inflammasome complex evidenced an imbalance in its regulation, with increased expression of the evaluated components, which is remarkably in severe AD, emphasizing its relevance as potential disease biomarkers and targets for immunomodulatory interventions.

Hericenone C attenuates the second phase of formalin-induced nociceptive behavior by suppressing the accumulation of CD11c-positive cells in the paw epidermis via phosphorylated P65.

Hericenone C is one of the most abundant secondary metabolites derived from Hericium erinaceus, under investigation for medicinal properties. Here, we report that Hericenone C inhibits the second phase of formalin-induced nociceptive behavior in mice. As the second phase is involved in inflammation, in a mechanistic analysis on cultured cells targeting NF-κB response element (NRE): luciferase (Luc)-expressing cells, lipopolysaccharide (LPS)-induced NRE::Luc luciferase activity was found to be significantly inhibited by Hericenone C. Phosphorylation of p65, which is involved in the inflammatory responses of the NF-κB signaling pathway, was also induced by LPS and significantly reduced by Hericenone C. Additionally, in mice, the number of CD11c-positive cells increased in the paw during the peak of the second phase of the formalin test, which decreased upon Hericenone C intake. Our findings confirm the possibility of Hericenone C as a novel therapeutic target for pain-associated inflammation.

Spectral and mass characterization of kinetic conversion from retinoids to retinoic acid in an in vitro 3-D human skin equivalent model.

To investigate the effect of retinoids, such as retinol (ROL), retinal (RAL), and retinyl palmitate (RP), on epidermal integrity, skin deposition, and bioconversion to retinoic acid (RA). 3-D human skin equivalent model (EpiDermFT™) was used. Epidermal cellular integrity measured by TEER values was significantly higher for a topical treatment of ROL and RAL than RP (p < 0.05). The skin deposition (µM) of ROL and RAL was approximately 269.54 ± 73.94 and 211.35 ± 20.96, respectively, greater than that of RP (63.70 ± 37.97) over 2 h incubation. Spectral changes were revealed that the CO maximum absorbance occurred between 1600∼1800 cm-1 and was greater from ROL than that from RAL and RP, indicating conjugation of R-OH to R-CHO or R-COOH could strongly occur after ROL treatment. Subsequently, a metabolite from the bioconversion of ROL and RAL was identified as RA, which has a product ion of m/z 283.06, by using liquid a chromatography-mass spectrometry (LC-MS) - total ion chromatogram (TIC). The amount of bioconversion from ROL and RAL to RA in artificial skin was 0.68 ± 0.13 and 0.70 ± 0.10 µM at 2 h and 0.60 ± 0.04 and 0.57 ± 0.06 µM at 24 h, respectively. RA was not detected in the skin and the receiver compartment after RP treatment. ROL could be a useful dermatological ingredient to maintain epidermal integrity more effectively, more stably deposit on the skin, and more steadily metabolize to RA than other retinoids such as RAL and RP.

Radiofrequency Treatment Attenuates Age-Related Changes in Dermal-Epidermal Junctions of Animal Skin.

The dermal-epidermal junction (DEJ) is essential for maintaining skin structural integrity and regulating cell survival and proliferation. Thus, DEJ rejuvenation is key for skin revitalization, particularly in age-related DEJ deterioration. Radiofrequency (RF) treatment, known for its ability to enhance collagen fiber production through thermal mechanisms and increase heat shock protein (HSP) expression, has emerged as a promising method for skin rejuvenation. Additionally, RF activates Piezo1, an ion channel implicated in macrophage polarization toward an M2 phenotype and enhanced TGF-ß production. This study investigated the impact of RF treatment on HSP47 and HSP90 expression, known stimulators of DEJ protein expression. Furthermore, using in vitro and aged animal skin models, we assessed whether RF-induced Piezo1 activation and the subsequent M2 polarization could counter age-related DEJ changes. The RF treatment of H2O2-induced senescent keratinocytes upregulated the expression of HSP47, HSP90, TGF-ß, and DEJ proteins, including collagen XVII. Similarly, the RF treatment of senescent macrophages increased Piezo1 and CD206 (M2 marker) expression. Conditioned media from RF-treated senescent macrophages enhanced the expression of TGF-ß and DEJ proteins, such as nidogen and collagen IV, in senescent fibroblasts. In aged animal skin, RF treatment increased the expression of HSP47, HSP90, Piezo1, markers associated with M2 polarization, IL-10, and TGF-ß. Additionally, RF treatment enhanced DEJ protein expression. Moreover, RF reduced lamina densa replication, disrupted lesions, promoted hemidesmosome formation, and increased epidermal thickness. Overall, RF treatment effectively enhanced DEJ protein expression and mitigated age-related DEJ structural changes by increasing HSP levels and activating Piezo1.

Exploring the Influence of Cold Plasma on Epidermal Melanogenesis In Situ and In Vitro.

Epidermal melanin synthesis determines an individual's skin color. In humans, melanin is formed by melanocytes within the epidermis. The process of melanin synthesis strongly depends on a range of cellular factors, including the fine-tuned interplay with reactive oxygen species (ROS). In this context, a role of cold atmospheric plasma (CAP) on melanin synthesis was proposed due to its tunable ROS generation. Herein, the argon-driven plasma jet kINPen® MED was employed, and its impact on melanin synthesis was evaluated by comparison with known stimulants such as the phosphodiesterase inhibitor IBMX and UV radiation. Different available model systems were employed, and the melanin content of both cultured human melanocytes (in vitro) and full-thickness human skin biopsies (in situ) were analyzed. A histochemical method detected melanin in skin tissue. Cellular melanin was measured by NIR autofluorescence using flow cytometry, and a highly sensitive HPLC-MS method was applied, which enabled the differentiation of eu- and pheomelanin by their degradation products. The melanin content in full-thickness human skin biopsies increased after repeated CAP exposure, while there were only minor effects in cultured melanocytes compared to UV radiation and IBMX treatment. Based on these findings, CAP does not appear to be a useful option for treating skin pigmentation disorders. On the other hand, the risk of hyperpigmentation as an adverse effect of CAP application for wound healing or other dermatological diseases seems to be neglectable.

Noninvasive evaluation of the skin barrier in reconstructed human epidermis using speckle analysis: Correlation with Raman microspectroscopy.

BACKGROUND: Reconstructed epidermis models, obtained from 3D keratinocytes culture, have gained significant prominence as prototypes for safety and efficacy testing in skin research. To effectively evaluate these models, it is essential to perform molecular and functional characterization. The skin's barrier function is one of the essential aspects of the epidermis that needs to be assessed. A noninvasive method is thus required for the evaluation of the skin barrier in these models. With this perspective, the aim of this feasibility study is to apply the speckle technique for the assessment of the skin barrier in the Reconstructed Human Epidermis (RHE). MATERIALS AND METHODS: Speckle analysis as well as Raman microspectroscopy were performed on RHE samples at two maturation days, D17 and D20. RESULTS: Between D17 and D20, our study showed an increase in various Raman parameters, including stratum corneum percentage, lateral lipid packing, lipid-to-protein ratio, and protein secondary structure. Furthermore, the degree of light polarization and the speckle grain size also increased over this period. CONCLUSION: The speckle technique proved to be effective for evaluating the skin barrier in Reconstructed Human Epidermis (RHE) models. Comparison with Raman validates this approach and provides comprehensive molecular and functional characterization of reconstructive skin models.