Search Results (134)

The Kindlin family of scaffold proteins plays key roles in integrin-mediated processes. Kindlin-1 and -2, encoded by the FERMT1 and FERMT2 genes, respectively, are expressed in the epidermis. Kindlin-1 plays protective roles against the development of cutaneous squamous cell carcinomas (cSCCs) in epidermal keratinocytes. However, the role of Kindlin-2 in transformed epidermal keratinocytes has remained virtually unexplored. In this study, we used siRNA approaches to generate Kindlin-2-depleted cells in three isogenic transformed keratinocyte lines. PM1, MET1, and MET4 cells model, respectively, a precancerous lesion, a primary cSCC, and a metastatic lesion of the latter. MET1 cells express both Kindlin-1 and -2. However, Kindlin-1 was not detectable in PM1 and MET4 cells. FERMT2 silencing in PM1 and MET4, but not in MET1 cells, reduced proliferation and the ability to adhere to culture surfaces and spreading. Furthermore, Kindlin-2-depleted PM1 and MET4, but not MET1 cells, exhibited decreased numbers of focal adhesions, as well as an altered F-actin and microtubule cytoskeletal organization. Significantly, FERMT2 silencing reduced the directional migration in all three cell types. These findings are consistent with the concept that, in the absence of other Kindlin orthologues, Kindlin-2 plays a prominent role in the modulation of the proliferation, spreading, focal adhesion assembly, and motility of transformed keratinocytes, as exemplified by PM1 and MET4 cells. Full article

Skin aging is characterized by compromised epidermal homeostasis and dermo-epidermal junction (DEJ) integrity, resulting in reduced stem cell potential and impaired tissue regeneration. This study investigated the effects of Andrographis paniculata extract (APE) on keratinocyte stem cells (KSCs) and DEJ composition in human skin. Using human skin explants and cell culture models, we demonstrated that APE treatment enhances DEJ composition by increasing Collagen IV and Laminin production while decreasing MMP-9 expression, without altering epidermal structure or differentiation. In the same model, APE preserved stemness potential by upregulating markers related to niche components (collagen XVII and β1-integrin), proliferation (Ki-67 and KRT15), and stem cell capacity (Survivin and LRIG1). In vitro studies revealed that APE selectively stimulated KSC proliferation without affecting transit amplifying cells and promoted Collagen IV and Laminin secretion, particularly in KSCs. Furthermore, in a co-culture model simulating a compromised DEJ (UVB-induced), APE increased Laminin production in KSCs, suggesting a protective effect against photo-damage. These findings indicate that APE enhances DEJ composition and preserves stem cell potential, highlighting its promise as a candidate for skin anti-aging strategies targeting stem cell maintenance and extracellular matrix stability to promote skin regeneration and repair. Full article

Plasma-derived fibrin hydrogels are widely used in tissue engineering because of their excellent biological properties. Specifically, human plasma-derived fibrin hydrogels serve as 3D matrices for autologous skin graft production, skeletal muscle repair, and bone regeneration. Nevertheless, for advanced applications such as in vitro skin equivalents and engineered grafts, the intrinsic limitations of native fibrin hydrogels in terms of long-term mechanical stability and resistance to degradation need to be addressed to enhance the usefulness and application of these hydrogels in tissue engineering. In this study, we chemically modified plasma-derived fibrin by incorporating succinimidyl alginate (SA), a version of alginate chemically modified to introduce reactive succinimidyl groups. These NHS ester groups (N-hydroxysuccinimide esters), attached to the alginate backbone, are highly reactive toward the primary amine groups present in plasma proteins such as fibrinogen. When mixed with plasma, the NHS groups covalently bond to the amine groups in fibrin, forming stable amide linkages that reinforce the fibrin network during hydrogel formation. This chemical modification improved mechanical properties, reduces contraction, and enhanced the stability of the resulting hydrogels. Hydrogels were prepared with a final fibrinogen concentration of 1.2 mg/mL and SA concentrations of 0.5, 1, 2, and 3 mg/mL. The objective was to evaluate whether this modification could create a more stable matrix suitable for supporting skin tissue development. The mechanical and microstructure properties of these new hydrogels were evaluated, as were their biocompatibility and potential to create 3D skin models in vitro. Dermo-epidermal skin cultures with primary human fibroblast and keratinocyte cells on these matrices showed improved dermal stability and better tissue structure, particularly SA concentrations of 0.5 and 1 mg/mL, as confirmed by H&E (Hematoxylin and Eosin) staining and immunostaining assays. Overall, these results suggest that SA-functionalized fibrin hydrogels are promising candidates for creating more stable in vitro skin models and engineered skin grafts, as well as for other types of engineered tissues, potentially. Full article

Systemic sclerosis (SSc) is an autoimmune fibrotic disorder affecting the skin and internal organs, categorized as either limited cutaneous SSc, where distal areas of skin are involved, or diffuse cutaneous SSc, where more extensive proximal skin involvement is seen. Vascular remodelling and internal organ involvement are frequent complications in both subsets. Multiple pathogenic mechanisms have been demonstrated, including production of disease-specific autoantibodies, endothelial cell damage at an early stage, infiltration of involved tissues by immune cells, as well as environmental factors triggering the onset such as solvents and viruses. Although not strongly familial, susceptibility to SSc is associated with multiple single nucleotide polymorphisms in immunoregulatory genes relevant to antigen presentation, T cell signalling and adaptive immunity, as well as innate immunity. In addition, several lines of evidence demonstrate abnormalities within the epithelial cell layer in SSc. Macroscopically, the SSc epidermis is pigmented, thickened and stiff and strongly promotes myofibroblasts in co-culture. Moreover, multiple activating factors and pathways have been implicated in the disease epidermis, including wound healing responses, induction of damage associated molecular patterns (DAMPS) and the release of pro-fibrotic growth factors and cytokines. Similar to SSc, data from studies of cutaneous wound healing indicate a major role for epidermal keratinocytes in regulating local fibroblast responses during repair of the wound defect. Since the epithelium is strongly exposed to environmental factors and richly populated with protective immune cells, it is possible that disease-initiating mechanisms in SSc involve dysregulated immunity and tissue repair within this cell layer. Treatments designed to restore epithelial homeostasis or else disrupt epithelial–fibroblast cross-talk could be of benefit in this severe and resistant disease. Accordingly, single cell analysis has confirmed an active signature in SSc keratinocytes, which was partially reversed following a period of JAK inhibitor therapy. Full article

Cultured epidermal autografts (CEAs) serve as an alternative permanent skin replacement, though high costs often limit their use in resource-constrained settings and to life-saving cases. This case report presents the first documented cosmetic application of a modified CEA technique in a bodybuilder, demonstrating favorable aesthetic outcomes. A 28-year-old Black male with a 20% total body surface area burn sustained in a domestic fire exhibited superficial and deep partial-thickness burns to the face, arms, torso, and feet. Refusing grafts from visible donor sites, treatment using a low-cost modified CEA approach was employed to minimize donor site morbidity. Keratinocytes harvested from a groin biopsy were cultured on Cutimed Sorbact^® (Essity AB, BSN Medical (Pty) Ltd., Pinetown, RSA) dressings with autogenous plasma and hydrogel supplementation and incubated at 37 °C for two weeks. Xenografts provided temporary coverage before CEA transplantation. Graft take was 85%, with minor (15%) loss at 21 days, requiring small autograft coverage. At two months, the Vancouver Scar Scale score was 4, indicating optimal pigmentation, smoother texture, and minimal scarring. These findings align with limited studies on CEAs for cosmetic applications, suggesting this cost-effective technique may broaden the scope of CEAs beyond life-saving interventions to include aesthetic reconstruction, reducing both donor site morbidity and scarring. Full article

Severe skin damage poses a significant clinical challenge, as limited availability of skin donors, postoperative skin defects, and scarring often impair skin function. Traditional two-dimensional (2D) nanofibers exhibit small pore sizes that hinder cellular infiltration, unable to simulate the three-dimensional (3D) structure of the skin. To address these issues, we developed 3D porous nanofiber scaffolds composed of polycaprolactone–polylactic acid–mussel adhesive protein (PLGA-PCL-MAP) using low-temperature electrospinning combined with nano-spray technology. Meanwhile, this 3D scaffold features high porosity, enhanced water absorption, and improved air permeability. The incorporation of mussel adhesive protein (MAP) further increased the scaffold’s adhesive properties and biocompatibility. In vitro experiments demonstrated that the 3D nanofiber scaffolds significantly promoted the adhesion, proliferation, and migration of epidermal keratinocytes (HaCaTs) and human fibroblasts (HFBs), while providing ample space for inward cellular growth. Successful co-culture of HaCaT and HFBs within the scaffold revealed key functional outcomes: HaCaTs expressed keratinocyte differentiation markers CK10 and CK14, while HFBs actively secreted extracellular matrix components critical for wound healing, including collagen I, collagen III, and fibronectin. This skin substitute with a composite structure of epidermis and dermis based on three-dimensional nanofiber scaffolds can be used as an ideal skin replacement and is expected to be applied in wound repair in the future. Full article

Aging, marked by a decline in cellular function and increased risk of diseases, involves the accumulation of senescent cells. This study aims to develop and characterize 3D senescent skin models to understand cellular senescence mechanisms’ implications in cutaneous aging. Normal human epidermal keratinocytes (NHEKs) were cultured from early to late passages (p2 to p7) to induce replicative senescence or sourced from both young and aged donors to reconstruct 3D models. Histological analyses assessed tissue morphology and integrity, while permeability assays evaluated epidermal barrier function. Analyses using immunostaining, RT-PCR, Affymetrix™ GeneChip™ Microarrays identified key markers of cellular senescence, epidermal homeostasis, and other related processes. Results showed that NHEKs at p5 and beyond, and those from aged donors, exhibited significant morphological disruptions, decreased expression of differentiation-associated genes, and impaired barrier function. Increased p16ink4a-positive cells indicated enhanced senescence. Transcriptome analysis revealed significant changes in keratinocyte differentiation, cell–cell interaction, cell cycle regulation, extracellular matrix homeostasis, and inflammation. These findings underscore the relevance of addressing cellular senescence for enhancing skin health and promoting skin longevity. These 3D senescent skin models, validated by consistent results from both passage-induced senescence and aged donor keratinocytes, are valuable for understanding skin aging and developing anti-aging treatments, positioning them as essential tools in the pursuit of skin longevity-focused innovations. Full article

Hair follicle stem cells, located in the bulge region of the outer root sheath, are multipotent epithelial stem cells capable of differentiating into epidermal, sebaceous gland, and hair shaft cells. Efficient culturing of these cells is crucial for advancements in dermatology, regenerative medicine, and skin model development. This investigation aimed to develop a protocol for isolating enriched bulge-derived epithelial cells from scalp specimens to produce tissue-engineered substitutes. The epithelium, including hair follicles, was separated from the dermis using thermolysin, followed by microdissection of the bulge region. Epithelial stem cells were isolated using enzymatic dissociation to create a single-cell suspension and compared with the direct explant culture and a benchmark method which isolates cells from the epidermis and pilosebaceous units. After 8 days of culture, the enzymatic digestion of microdissected bulges yielded 5.3 times more epithelial cells compared to explant cultures and proliferated faster than the benchmark method. Cells cultured from all methods exhibited comparable morphology and growth rates. The fully stratified epidermis of tissue-engineered skin was similar, indicating comparable differentiation potential. This enzymatic digestion method improved early-stage cell recovery and expansion while maintaining keratinocyte functionality, offering an efficient hair bulge cell-extraction technique for tissue engineering and regenerative medicine applications. Full article

Activated keratinocytes play a crucial role in skin inflammation through the production of multiple inflammatory mediators; however, little is known about cytokine secretion by activated keratinocytes in dogs. This study aimed to investigate the effects of the Th1 and Th2 types of cytokines on the production of keratinocyte-derived inflammatory mediators. Canine progenitor epidermal keratinocytes (CPEKs) were incubated with canine recombinant IL-4, IL-13, an IL4/IL13 mixture, IFN-γ, TNF-α, or an IFN-γ/TNF-α mixture for 24 h following 100% confluency. Culture supernatants were analyzed for cytokine concentration, including chemokine ligand (CXCL) 8, IL-10, IL-6, IL-1ß, IL-12, and chemokine ligand 2 (CCL2) by enzyme-linked immunosorbent assay. CPEKs incubated with the IFN-γ/TNF-α mixture showed significantly increased IL-6 concentration. In addition, significantly increased concentrations of CXCL8 were detected in CPEKs incubated with TNF-α and with the IFN-γ/TNF-α mixture. CCL2 concentrations increased in cells incubated with IFN-γ, TNF-α, and the IFN-γ/TNF-α mixture. The IFN-γ/TNF-α mixture synergistically enhanced CCL2 production. Dose-dependent elevations were also observed in IL-6 in response to the IFN-γ/TNF-α mixture, and in CCL2 in response to IFN-γ, TNF-α, and the IFN-γ/TNF-α mixture. These findings indicate that IFN-γ and TNF-α synergistically increase pro-inflammatory cytokines and chemokines secreted by canine keratinocytes. This in vitro culture system could be useful to investigate cytokine-mediated crosstalk between keratinocytes and immune cells and new therapeutic strategies for keratinocyte-mediated inflammatory skin diseases. Full article

The human skin is a remarkable organ capable of extensive regeneration, especially after severe injuries such as burns and related wounds. The de-epidermized dermis (DED) model has become a valuable in vitro tool for skin regeneration studies, particularly for testing the mechanism of action and the efficacy of clinical cutaneous cell therapies. To further improve the quality and robustness of these applications, our study focused on optimizing and standardizing DED tissue preparation and storage, enhancing its effectiveness for clinical testing. Therefore, we optimized the air-liquid interfacial culture medium composition by simplifying the historical formulation without compromising keratinocyte (therapeutic cell model) viability or proliferation. Furthermore, we investigated the impacts of adding burn wound exudates in the model by focusing on cell behavior for enhanced translational significance. The results revealed notable differences in keratinocyte adhesion and proliferation between burn wound exudates collected at the early stages and late stages of acute patient treatment, providing new information on a possible therapeutic window to apply cell therapies on burn patients. Generally, this study reported a robust method for the preclinical in vitro assessment of keratinocyte-based cutaneous cell therapies using DED models. Overall, the study underscored the importance of using in vitro models with enhanced translational relevance to better predict the clinical effects of cutaneous cell therapies in burn patient populations. Full article

Background: Photodynamic therapy (PDT) is a treatment modality that uses light to activate a photosensitizing agent, destroying target cells. The growing awareness of the necessity to reduce or eliminate the use of mammals in research has prompted the search for safer toxicity testing models aligned with the new global guidelines and compliant with the relevant regulations. Objective: The objective of this study was to assess the impact of PDT on alternative models to mammals, including in vitro three-dimensional (3D) cultures and in vivo, in invertebrate animals, utilizing a potent photosensitizer, 2-hydroxychalcone. Methods: Cytotoxicity was assessed in two cellular models: monolayer (2D) and 3D. For this purpose, spheroids of two cell lines, primary dermal fibroblasts (HDFa) and adult human epidermal cell keratinocytes (HaCat), were developed and characterized following criteria on cell viability, shape, diameter, and number of cells. The survival percentages of Caenorhabditis elegans and Galleria mellonella were evaluated at 1 and 7 days, respectively. Results: The findings indicated that all the assessed platforms are appropriate for investigating PDT toxicity. Furthermore, 2-hydroxychalcone demonstrated low toxicity in the absence of light and when mediated by PDT across a range of in vitro (2D and 3D cultures) and in vivo (invertebrate animal models, including G. mellonella and C. elegans) models. Conclusion: There was a strong correlation between the in vitro and in vivo tests, with similar toxicity results, particularly in the 3D models and C. elegans, where the concentration for 50% viability was approximately 100 µg/mL. Full article

Difamilast, a phosphodiesterase 4 (PDE4) inhibitor, has been shown to be effective in the treatment of atopic dermatitis (AD), although the mechanism involved remains unclear. Since IL-33 plays an important role in the pathogenesis of AD, we investigated the effect of difamilast on IL-33 activity. Since an in vitro model of cultured normal human epidermal keratinocytes (NHEKs) has been utilized to evaluate the pharmacological potential of adjunctive treatment of AD, we treated NHEKs with difamilast and analyzed the expression of the suppression of tumorigenicity 2 protein (ST2), an IL-33 receptor with transmembrane (ST2L) and soluble (sST2) isoforms. Difamilast treatment increased mRNA and protein levels of sST2, a decoy receptor suppressing IL-33 signal transduction, without affecting ST2L expression. Furthermore, supernatants from difamilast-treated NHEKs inhibited IL-33-induced upregulation of TNF-α, IL-5, and IL-13 in KU812 cells, a basophil cell line sensitive to IL-33. We also found that difamilast activated the aryl hydrocarbon receptor (AHR)–nuclear factor erythroid 2-related factor 2 (NRF2) axis. Additionally, the knockdown of AHR or NRF2 abolished the difamilast-induced sST2 production. These results indicate that difamilast treatment produces sST2 via the AHR–NRF2 axis, contributing to improving AD symptoms by inhibiting IL-33 activity. Full article

Zika virus (ZIKV), a mosquito-borne flavivirus, is prominently associated with microcephaly in babies born to infected mothers as well as Guillain-Barré Syndrome in adults. Each cell type infected by ZIKV—neuronal cells (radial glial cells, neuronal progenitor cells, astrocytes, microglia cells, and glioblastoma stem cells) and non-neuronal cells (primary fibroblasts, epidermal keratinocytes, dendritic cells, monocytes, macrophages, and Sertoli cells)—displays its own characteristic changes to their cell physiology and has various impacts on disease. Here, we provide an in-depth review of the ZIKV life cycle and its cellular targets, and discuss the current knowledge of how infections cause neuropathologies, as well as what approaches researchers are currently taking to further advance such knowledge. A key aspect of ZIKV neuropathogenesis is virus-induced neuronal apoptosis via numerous mechanisms including cell cycle dysregulation, mitochondrial fragmentation, ER stress, and the unfolded protein response. These, in turn, result in the activation of p53-mediated intrinsic cell death pathways. A full spectrum of infection models including stem cells and co-cultures, transwells to simulate blood–tissue barriers, brain-region-specific organoids, and animal models have been developed for ZIKV research. Full article

Determination of the hypericin–photodynamic (HY–PDT) effect on the secretion of cytokines secreted by the skin cells, may be the basis for using the immunomodulatory effect of photodynamic action in the treatment of inflammatory skin diseases. The study aimed to evaluate the cytotoxic and immunomodulatory effects of hypericin (HY) in photodynamic therapy (PDT) performed in vitro on cultures of selected skin cell lines. The study used two human cell lines, primary dermal fibroblast (HDFa) and primary epidermal keratinocytes (HEKa). The MTT test was used to define the metabolic activity of treated cells. Cell supernatants subjected to sublethal PDT were assessed to determine the interleukins: IL-2, IL-8, IL-10, IL-11, IL-19, IL-22, and metalloproteinase 1 (MMP-1). The results confirm the destructive effect of HY–PDT and the immunomodulatory effects of sublethal doses on the selected skin cells, depending on the concentration of HY and the light doses. No statistically significant differences were noted in IL-2 and IL-10 concentration after HY–PDT for HEKa and HDFa lines. After using HY–PDT, the concentration of IL-8, MMP-1, IL-22, and IL-11 significantly decreased in the HEKa line. Moreover, the concentration of IL-19 and MMP-1 significantly decreased in the HDFa line. The concentration of IL-11 in the HDFa line after using only the HY, without the light, increased but decreased after HY–PDT. Our experiment confirmed that HY–PDT has not only a cytotoxic effect but, used in sublethal doses, also presents immunomodulatory properties. These may be an advantage of HY–PDT when used in the treatment of persistent skin inflammation, connected with the release of pro-inflammatory cytokines resistant to conventional treatment methods. Full article

Skin yellowness is a hallmark of dull or unhealthy skin, particularly among Asians. Previous research has indicated a link between skin glycation and skin yellowness. However, the specific glycated chemicals contributing to yellowish skin appearance have not been identified yet. Using HPLC-PDA-HRMS coupled with native and artificially glycated human epidermal explant skin, we identified intensely yellow colored glycated chromophores “(1R, 8aR) and (1S, 8aR)-4-(2-furyl)-7-[(2-furyl)-methylidene]-2-hydroxy-2H,7H,8AH-pyrano-[2,3-B]-pyran-3-one” (abbreviated as AGEY) from human skin samples for the first time. The abundance of AGEY was strongly correlated with skin yellowness in the multiple skin explant tissues. We further confirmed the presence of AGEY in cultured human keratinocytes and 3D reconstructed human epidermal (RHE) models. Additionally, we demonstrated that a combination of four cosmetic compounds with anti-glycation properties can inhibit the formation of AGEY and reduce yellowness in the RHE models. In conclusion, we have identified specific advanced glycation end products with an intense yellow color, namely AGEY, in human skin tissues for the first time. The series of study results highlighted the significant contribution of AGEY to the yellow appearance of the skin. Furthermore, we have identified a potential cosmetic solution to mitigate AGEY formation, leading to a reduction in yellowness in the in vitro RHE models. Full article