The Development of Human Ex Vivo Models of Inflammatory Skin Conditions.

Traditional research in inflammatory dermatoses has relied on animal models and reconstructed human epidermis to study these conditions. However, these models are limited in replicating the complexity of real human skin and reproducing the intricate pathological changes in skin barrier components and lipid profiles. To address this gap, we developed experimental models that mimic various human inflammatory skin phenotypes. Human ex vivo skins were stimulated with various triggers, creating models for inflammation-induced angiogenesis, irritation response, and chronic T-cell activation. We assessed the alterations in skin morphology, cellular infiltrates, cytokine production, and epidermal lipidomic profiles. In the pro-angiogenesis model, we observed increased mast cell degranulation and elevated levels of angiogenic growth factors. Both the irritant and chronic inflammation models exhibited severe epidermal disruption, along with macrophage infiltration, leukocyte exocytosis, and heightened cytokine levels. Lipidomic analysis revealed minor changes in the pro-angiogenesis model, whereas the chronic inflammation and irritant models exhibited significant decreases in barrier essential ceramide subclasses and a shift toward shorter acyl chain lengths (

Alteration to the Skin Ceramide Profile Following Broad-Spectrum UV Exposure.

The epidermal stratum corneum (SC) lipid matrix, principally consisting of an equimolar ratio of ceramides, free fatty acids, and cholesterol, plays a crucial role in maintaining proper skin barrier function. Conditions which impair barrier integrity, such as in atopic dermatitis, correlate with the alternation of key ceramide subclasses and reduced chain length of acyl moieties. However, there is limited knowledge about the impact of unprotected repeat sun exposure on the skin lipid composition, especially ceramide profiles.This study investigated the effects of ultraviolet (UV) radiation on the ceramide profile using both an ex vivo skin and a clinical model. Lipidomic analysis of UV-exposed skin showed shifts to the composition of ceramide subclasses essential in repairing and strengthening the SC barrier (including CER1[EOS], CER3[NP], and CER6[AP]) and reduced very long-chain acyl moieties. Gene expression analysis and immunohistochemical staining of key enzymes (aSMase, DES1, CerS5, CerS3) suggested that lipid alterations can be attributed to changes within the ceramide biosynthesis process. Topical application of ceramide-containing suncare products help maintain SC-essential ceramide subclasses and proper ceramide chain length, demonstrating the importance of proper photoprotection to maintain healthy skin barrier and ceramide quality during daily sun exposure. J Drugs Dermatol. 2022;21(1):77-85. doi:10.36849/JDD.6331.

ARTICLE: Alteration to the Skin Barrier Integrity Following Broad-Spectrum UV Exposure in an Ex Vivo Tissue Model.

Dynamic changes to the skin barrier’s molecular structure and ceramide profile are well-documented in skin conditions such as atopic dermatitis and psoriasis. Pathological and environmental factors have been shown to impair barrier integrity and demonstrate shifts in ceramide composition in the skin. However, the relationship between acute and prolonged sun exposure and its effects on skin barrier homeostasis is insufficiently investigated. This study aims to uncover new scientific evidence to elucidate the relationship of UV irradiation with the skin barrier using an ex vivo tissue model following simulated UVA/UVB exposure. Fresh ex vivo human skin pretreated either with or without a broad-spectrum sunscreen was exposed to either a physiological or elevated UV condition. Following eight days in culture, structural and molecular changes were evaluated. UV irradiated skin displayed epidermal cell death and altered expression of key barrier proteins. TEM analysis demonstrated disruption to adherens junctions and dissociation between tissue layers following both physiological and extensive UV exposures. An effective broad-spectrum sunscreen containing essential skin ceramides completely protected the skin from such changes. This is one of the first works demonstrating a clear correlation of altered skin barrier integrity using a physiologically relevant dose in an ex vivo tissue model. Our findings also further support the additional importance and benefits of sun protection among the consumers. J Drugs Dermatol. 20(4 Suppl):s23-28. doi:10.36849/JDD.S589D.

ARTICLE: Efficacy of Ceramide-Containing Formulations on UV-Induced Skin Surface Barrier Alterations.

The human skin, particularly the stratum corneum, serves as a protective barrier against exogenous factors, including ultraviolet radiation (UVR) and pathogen invasions. The impact of UVR on skin cancer and photoaging has been extensively studied. However, the direct impact of UVR on skin barrier integrity under clinical settings remains poorly explored. Due to their benefits in reducing inflammation and promoting skin barrier repair, ceramide-containing formulations can provide added photoprotection benefits. In this study, the efficacy of a ceramide-containing sunscreen and moisturizer were evaluated in preventing UV-induced skin surface barrier changes. Expert grading, instrumental, and tape-stripping assessments demonstrated that UVR induced erythema and hyperpigmentation and caused changes in skin cells surface morphological organization and maturation. Treatment with a ceramide-containing sunscreen and moisturizing cream routine reduced erythema and hyperpigmentation, improved skin hydration, and maintained normal superficial skin cells morphology and turnover after UVR. Our results indicate that barrier-enforcing lipids formulations can provide additional benefits in patient’s daily routine by strengthening the barrier and improving skin health overall against chronic sun exposure. J Drugs Dermatol. 20(4 Suppl):s29-35. doi:10.36849/JDD.S589E.

Impact of Iron-Oxide Containing Formulations Against Visible Light-Induced Skin Pigmentation in Skin of Color Individuals.

Visible light (400-700nm), which contributes to 45% of solar radiation, contributes to skin darkening and worsening of dyschromias, particularly in individuals with Fitzpatrick skin phototypes III and higher. Currently, sunscreens provide limited protection against that spectrum. Due to their capabilities in absorbing, scattering, and reflecting visible light, topical products containing pigments and/or metal oxides can provide additional photoprotection. In this study, the efficacy of two formulations containing iron oxide was evaluated in preventing visible light-induced pigmentation compared with a non-tinted mineral SPF 50+ sunscreen. Expert grading and colorimetry demonstrated that the iron-oxide containing formulations significantly protected against visible light-induced pigmentation compared to untreated skin or mineral SPF 50+ sunscreen in Fitzpatrick IV individuals. These results highlight that iron-oxide containing formulas in a foundation format have dual functions and can provide additional benefits in patients' daily routine by masking existing pigmentation and preventing the development of pigmentation triggered by sunlight exposure, extending protection beyond UV spectrum. J Drugs Dermatol. 2020;19(7): doi:10.36849/JDD.2020.5032 THIS ARTICLE HAD BEEN MADE AVAILABLE FREE OF CHARGE. PLEASE SCROLL DOWN TO ACCESS THE FULL TEXT OF THIS ARTICLE WITHOUT LOGGING IN. NO PURCHASE NECESSARY. PLEASE CONTACT THE PUBLISHER WITH ANY QUESTIONS.

Long-Term Benefits of Daily Photo-Protection With a Broad-Spectrum Sunscreen in United States Hispanic Female Population.

aThe Vitiligo and Pigmentation Institute of Southern California, Los Angeles, CA bDepartment of Dermatology, Howard University, Washington, DC cL’Oreal Research and Innovation, Paris, France dL’Oreal Research and Innovation, Clark, NJ.

Photoaging and Photoprotection in United States Hispanic Population

Photoaging is a complex and chronic process that induces structural and functional changes in sun-exposed skin, including coarse wrinkles, laxity, dyschromia, telangiectasias, and potential precancerous lesions. Pigmented skin presents different structure and physiology that contribute to distinctive photoaging process. The skin of color population is reported to "age better" than their Caucasian counterparts in general, with fewer wrinkles and better skin texture. However, pigmentary disorders and sun-exposure related dyschromia are highly prevalent in skin of color. Hispanics are the fastest growing population in the U.S. and represents a heterogenous group of people with different skin tones and Fitzpatrick phototypes. They demonstrate large diversity and heterogeneity in skin physiology, pigmentary disorders, and photoaging-related skin color shifting. Specific concerns around hyperpigmentation, skin tone evenness, and texture or roughness are very common among Hispanics, demanding targeted medical and cosmeceutical solutions. Customized daily routines including sufficient photo-protection are essential to address such needs. This mini review identifies some of the specific skin concerns of Hispanics in America and emphasizes the needs for long-term sunscreen use and education. J Drugs Dermatol. 2019;18(3 Suppl):s121-123.

Activation of toll-like receptors alters the microRNA expression profile of keratinocytes.

Keratinocytes recognize invading pathogens by various receptors, among them Toll-like receptors (TLRs), and provide the first line of defense in skin immunity. The role of microRNAs in this important defense mechanism has not been explored yet. Our aim was to identify microRNAs involved in the innate immune response of keratinocytes. MicroRNA expression profiling revealed that the TLR2 ligand zymosan, the TLR3 ligand poly(I:C) or the TLR5 ligand flagellin significantly altered the microRNA expression in keratinocytes. The regulation of microRNAs was concentration-dependent and it could be neutralized by siRNAs specific for TLR2, TLR3 and TLR5, respectively, confirming the specificity of the TLR response. Interestingly, one microRNA, miR-146a, was strongly induced by all studied TLR ligands, while other microRNAs were regulated in a TLR- or time point-specific manner. These findings suggest an important role for microRNAs in the innate immune response of keratinocytes and provide a basis for further investigations.

The application of a multi-component reaction peptide as a model regenerative active to enhance skin wound-healing postlaser procedure in a double-blinded placebo-controlled clinical trial.

INTRODUCTION: Esthetic procedures are currently among the most effective options for consumers seeking to correct aging signs such as fine lines, wrinkles, and skin tone unevenness. Currently, there is a scientific need for an adjunct active to be paired with esthetic procedures to encourage wound recovery and address postprocedure pigmentation concerns. OBJECTIVE: Toward that goal, this study assessed the efficacy of a peptide created from a multi-component reaction (multi-component peptide, MCP) as a model active for postprocedure care and evaluated its ability to promote skin healing in an ablative laser-induced wound model on the forearm. METHODS: The mechanism of action of MCP was investigated using tubo assays, 2D melanocyte, and fibroblast cultures, reconstructed skin equivalents, and ex vivo skin explants. The MCP formula and the clinical benchmark formula of Aquaphor were assessed head-to-head by applying the products topically in an ablative laser-induced wound model (n = 20 subjects). The promotion of wound healing was evaluated by the investigator assessment of epithelial confluence, crusting or scabbing, general wound appearance, erythema, and edema. RESULTS: MCP was determined to be beneficial to postprocedure skin recovery and healing by four main mechanisms of action: barrier repair as determined in an ex vivo tape-stripping model, reduction of inflammation and postinflammatory hyperpigmentation, reduction of elastase activity, and stimulation of fibroblast through the mTOR pathway. The formula containing 10% MCP enhanced the kinetics of epithelial confluence and improvement of the crusting or scabbing appearance of the laser-generated wounds in a laser-induced mini-zone wound healing study on the forearm. CONCLUSION: This study demonstrates the use of MCP as a proof of concept regenerative active that when incorporated into an optimized postprocedure skincare formula can improve skin healing and enhance the appearance of skin after injury with relevance to ablative aesthetic procedures.

TGFß signaling regulates lipogenesis in human sebaceous glands cells.

BACKGROUND: Sebaceous glands are components of the skin essential for its normal lubrication by the production of sebum. This contributes to skin health and more importantly is crucial for the skin barrier function. A mechanistic understanding of sebaceous gland cells growth and differentiation has lagged behind that for keratinocytes, partly because of a lack of an in vitro model that can be used for experimental manipulation. METHODS: We have developed an in vitro culture model to isolate and grow primary human sebocytes without transformation that display functional characteristics of sebocytes. We used this novel method to probe the effect of Transforming Growth Factor ß (TGFß) signaling on sebocyte differentiation, by examining the expression of genes involved in lipogenesis upon treatment with TGFß1. We also repressed TGFß signaling through knockdown of the TGFß Receptor II to address if the effect of TGFß activation is mediated via canonical Smad signal transduction. RESULTS: We find that activation of the TGFß signaling pathway is necessary and sufficient for maintaining sebocytes in an undifferentiated state. The presence of TGFß ligand triggered decreased expression in genes required for the production of characteristics sebaceous lipids and for sebocyte differentiation such as FADS2 and PPARγ, thereby decreasing lipid accumulation through the TGFß RII-Smad2 dependent pathway. CONCLUSION: TGFß signaling plays an essential role in sebaceous gland regulation by maintaining sebocytes in an undifferentiated state. This data was generated using a novel method for human sebocyte culture, which is likely to prove generally useful in investigations of sebaceous gland growth and differentiation. These findings open a new paradigm in human skin biology with important implications for skin therapies.

A multi-study analysis enables identification of potential microbial features associated with skin aging signs.

Introduction: During adulthood, the skin microbiota can be relatively stable if environmental conditions are also stable, yet physiological changes of the skin with age may affect the skin microbiome and its function. The microbiome is an important factor to consider in aging since it constitutes most of the genes that are expressed on the human body. However, severity of specific aging signs (one of the parameters used to measure "apparent" age) and skin surface quality (e.g., texture, hydration, pH, sebum, etc.) may not be indicative of chronological age. For example, older individuals can have young looking skin (young apparent age) and young individuals can be of older apparent age. Methods: Here we aim to identify microbial taxa of interest associated to skin quality/aging signs using a multi-study analysis of 13 microbiome datasets consisting of 16S rRNA amplicon sequence data and paired skin clinical data from the face. Results: We show that there is a negative relationship between microbiome diversity and transepidermal water loss, and a positive association between microbiome diversity and age. Aligned with a tight link between age and wrinkles, we report a global positive association between microbiome diversity and Crow's feet wrinkles, but with this relationship varying significantly by sub-study. Finally, we identify taxa potentially associated with wrinkles, TEWL and corneometer measures. Discussion: These findings represent a key step towards understanding the implication of the skin microbiota in skin aging signs.

Utilization of ex vivo tissue model to study skin regeneration following microneedle stimuli.

Microneedling is a popular skin resurfacing and rejuvenation procedure. In order to develop better adjunct products for consumers, there is a scientific need to establish greater understanding of the mechanism in which microneedling stimulates regeneration within skin. The purpose of this study is to develop a physiologically relevant ex vivo tissue model which closely mimics the actual microneedling procedure to elucidate its mechanism of action. In this study, human ex vivo skin was subjected to microneedling treatment and cultured for 6 days. Histological analysis demonstrated that the ex vivo skin was able to heal from microneedling injury throughout the culture period. Microneedling treatment stimulated proliferation and barrier renewal of the skin. The procedure also increased the levels of inflammatory cytokines and angiogenic growth factors in a dynamic and time dependent fashion. The tissue demonstrated hallmark signs of epidermal regeneration through morphological and molecular changes after the treatment. This is one of the first works to date that utilizes microneedled ex vivo skin to demonstrate its regenerative behavior. Our model recapitulates the main features of the microneedling treatment and enables the evaluation of future cosmetic active ingredients used in conjunction with microneedling.

Oligomeric α-Synuclein induces skin degeneration in reconstructed human epidermis.

Aged and photoaged skin exhibit fine wrinkles that are signs of epidermal inflammation and degeneration. It has been shown that healthy elderly skin expresses amyloidogenic proteins, including α-Synuclein, which are known to oligomerize and trigger inflammation and neurodegeneration. However, little is known about their putative role in skin physiology and sensitivity. To unravel this possible role, we investigated the impact of oligomeric α-Synuclein (Oα-Syn) in 2D and 3D keratinocyte human models. Exogenous Oα-Syn caused degeneration of reconstructed human epidermis (RHE) by diminishing proliferation and thickness of the stratum basale. Oα-Syn also increased NF-kB nuclear translocation in keratinocytes and triggered inflammation in the RHE, by increasing expression of interleukin-1ß and tumor necrosis factor-alpha, and the release of tumor necrosis factor-alpha in a time-dependent manner. Dexamethasone and an IL-1ß inhibitor partially diminished RHE degeneration caused by Oα-Syn. These findings suggest that Oα-Syn induces epidermal inflammation and decreases keratinocyte proliferation, and therefore might contribute to epidermal degeneration observed in human skin aging.

SkinEthic™ RHE for in vitro evaluation of skin irritation of medical device extracts.

According to ISO 10993 standards for biocompatibility of medical devices, skin irritation is one of the three toxicological endpoints to be always addressed in a biological risk assessment. This work presents a new protocol to assess this endpoint in vitro rather than in vivo. The protocol was adapted to medical devices extracts from the OECD TG 439 with the SkinEthic™ RHE model as test system. It was challenged with irritant chemicals, Sodium Dodecyl Sulfate, Lactic Acid and Heptanoic Acid spiked in polar solvents, sodium chloride solution or phosphate buffer saline and non-polar solvent, Sesame Oil. Cell viability measured by MTT reduction after 24 h exposure was used as readout. Quantification of IL-1α release as secondary readout did not increased performance. Samples of heat-pressed polyvinyl chloride (PVC) and silicone sheets infused with or without known irritant (4% Genapol-X80, 6% Genapol-X100 and 15% SDS) were tested after extraction in polar and non-polar solvents. Medical device extracts are classified irritant when the cell viability is inferior or equal to 50%, compared to the negative controls tissues, in at least one extraction solvent. The correct classification of all the samples confirmed the good performance of this new protocol for in vitro skin irritation of medical devices extracts with the SkinEthic™ RHE model. Seven naïve laboratories were trained in prevision of the Round Robin Study to evaluate Reconstructed Human Epidermis (RhE) models as in vitro skin irritation test for detection of irritant potential in medical device extracts.

The lysyl oxidase LOX is absent in basal and squamous cell carcinomas and its knockdown induces an invading phenotype in a skin equivalent model.

Lysyl oxidase initiates the enzymatic stage of collagen and elastin cross-linking. Among five isoforms comprising the lysyl oxidase family, LOX is the better studied. LOX is associated to an antitumor activity in ras-transformed fibroblasts, and its expression is down-regulated in many carcinomas. The aim of this work was to shed light on LOX functions within the epidermis by studying its expression in human basal and squamous cell carcinomas and analyzing the effect of its enzymatic activity inhibition and protein absence on human keratinocytes behavior in a skin equivalent. In both carcinomas, LOX expression by epidermal tumor cells was lacking, while it was up-regulated around invading tumor cells in association with the stromal reaction. Lysyl oxidase activity inhibition using beta-aminoproprionitrile in a skin equivalent model prepared with both primary human keratinocytes and HaCaT cell line affected keratin 10 and filaggrin expression and disorganized the collagen network and the basement membrane. In spite of all these changes, no invasion phenotype was observed. Modelization of the invasive phenotype was only noticed in the skin equivalent developed with LOX antisense HaCaT cell line, where the protein LOX is specifically absent. Our results clearly indicate that lysyl oxidase enzymatic activity is essential not only for the integrity maintenance of the dermis but also for the homeostasis of the epidermis. Moreover, LOX protein plays a role in the skin carcinomas and invasion but not through its enzymatic activity.

Epidermal and dermal characteristics in skin equivalent after systemic and topical application of skin care ingredients.

Effects of active ingredients from topical and systemic skincare products on structure and organization of epidermis, dermal-epidermal junction (DEJ), and dermis were examined using an in vitro reconstructed skin equivalent (SE). Imedeen Time Perfection (ITP) ingredients (a mixture of BioMarine Complex, grape seed extract, tomato extract, vitamin C) were supplemented systemically into culture medium. Kinetin, an active ingredient from Imedeen Expression Line Control Serum, was applied topically. Both treatments were tested separately or combined. In epidermis, all treatments stimulated keratinocyte proliferation, showing a significant increase of Ki67-positive keratinocytes (P < 0.05). Kinetin showed a twofold increase of Ki67-positive cells, ITP resulted in a fivefold, and ITP+kinetin showed a nine-fold increase. Differentiation of keratinocytes was influenced only by kinetin since filaggrin was found only in kinetin and kinetin+ITP samples. At the DEJ, laminin 5 was slightly increased by all treatments. In dermis, only ITP increased the amount of collagen type I. Both kinetin and ITP stimulated formation of fibrillin-1 and elastin deposition. The effect of kinetin was seen in upper dermis. It stimulated not only the amount of deposited fibrillin-1 and elastin fibers but also their organization perpendicularly to the DEJ. ITP stimulated formation of fibrillin-1 in deeper dermis. In summary, the combination of topical treatment with kinetin and systemic treatment with ITP had complementary beneficial effects in the formation and development of epidermis and dermis.

Evolutive skin reconstructions: from the dermal collagen-glycosaminoglycan-chitosane substrate to an immunocompetent reconstructed skin.

The development of human skin models that have the same properties as genuine human skin is of particular significance. Very promising skin models are the three-dimensional artificial skin constructs, which, similar to genuine skin, consist of an epidermis of differentiated keratinocytes and a dermis. A skin equivalent based on a collagen-glycosaminoglycan-chitosan dermal substrate has been developed to meet the growing demand in tissue engineered skin equivalents. We used this model to investigate whether CD34-generated Langerhans/dendritic cell precursors could be integrated into this skin equivalent model and pursue their differentiation without addition of cytokine and growth factor. To address the issue of dendritic cell (DC) differentiation, an endothelialized skin equivalent coculture model was used to study the behaviour of haematopoietic progenitor cells (HPC) in epidermal and dermal environments. CD34(+) HPC were cultured for 6 days with GM-CSF, TGFbeta1 and TNFalpha and seeded in the endothelialized skin equivalent at different time points to favour dermal or epidermal integration. This integration (after keratinocyte seeding, only and in absence of exogenous GM-CSF, TNFalpha, TGFbeta1) gave rise both cutaneous DC, i.e. epidermal Langerhans cells (CD1a(+), HLA-DR(+)) and dermal DC (DC-SIGN(+), HLA-DR(+)) while endothelial cells are sufficiently activated to acquire HLA-DR expression. For the first time, the presence of a living dermal equivalent could provide a more complex environment integrating vascular components to study the differentiation of interstitial DC in a dermis equivalent. Such sophisticated skin equivalent may clarify some intriguing aspects of the numerous regulatory mechanisms controlling skin homeostasis.

Optimization and characterization of an engineered human skin equivalent.

Skin equivalents (SEs) have been designed to meet both basic and applied research needs. The successful application of tissue-engineered SEs requires that the reconstituted tissues be endowed with the correct organization and function. A large body of experimental evidence now supports the notion that the inducing effects of mesenchymal tissue on epithelial cell morphogenesis are mediated, at least in part, by extracellular matrix components in addition to cell-cell interactions. A coculture model including both fibroblasts and keratinocytes was used to study the effects of progressive serum reduction on epidermal differentiation, quality of dermal and dermal-epidermal junctions, and expression of extracellular matrix proteins. The cells were successively added to a dermal substrate composed of collagen, glycosaminoglycans, and chitosan. The main aim of this study was to optimize this model for pharmacotoxicological trials. Control skin equivalents were cultured with medium containing 10% serum throughout the production process. Serum content was reduced to 1 and 0% at the air-liquid interface and compared with control skin equivalents. First, we demonstrated that serum deprivation at the air-liquid interface improves keratinocyte terminal differentiation. Second, we showed that, in the absence of serum, the specific characteristics of the SE are maintained, including epidermal and dermal ultrastructure, the expression of major dermal extracellular matrix components (human collagen types I, III, and V, fibronectin, elastin, and fibrillin 1), and the dermal-epidermal junction (laminin, human type IV collagen, alpha6 integrin). Furthermore, our results indicate that coculture models using keratinocytes and fibroblasts have both morphological and functional properties required for biologically useful tissues.

Lysyl oxidase-like and lysyl oxidase are present in the dermis and epidermis of a skin equivalent and in human skin and are associated to elastic fibers.

Elastic fiber formation involves the secretion of tropoelastin which is converted to insoluble elastin by cross-linking, initiated by the oxidative deamination of lysine residues by lysyl oxidase. Five lysyl oxidase genes have been discovered. This study deals with the expression of two isoforms, LOX and LOX-like (LOXL), in human foreskin and in a human skin-equivalent (SE) model that allows the formation of elastic fibers. In this model, keratinocytes are added to a dermal equivalent made of fibroblasts grown on a chitosan-cross-linked collagen-GAG matrix. LOX and LOXL were detected by immunohistochemistry in the dermis and the epidermis of both normal skin and in a SE. This expression was confirmed by in situ hybridization on the SE. LOX and LOXL expression patterns were confirmed in human skin. The ultrastructural localization of LOXL was indicative of its association with elastin-positive materials within the SE and human skin, though interaction with collagen could not be discarded. LOX was found on collagen fibers and could be associated with elastin-positive materials in the SE and human skin. LOXL and LOX were detected in keratinocytes where LOX was mainly expressed by differentiating keratinocytes, in contrast to LOXL that can be found in both proliferating and differentiating fibroblasts. These data favor a role for LOXL in elastic fiber formation, together with LOX, and within the epidermis where both enzymes should play a role in post-translational modification of yet unknown substrates.