Search Results (451)

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

Although cold atmospheric plasma (CAP) has shown promise in facilitating wound repair due to its non-thermal and non-invasive properties, its dynamic effects on cellular response and metabolic regulation remain poorly characterized, and the mechanism is still unclear. In this study, we developed a microfluidic experimental system that integrates a CAP treatment module with multiparametric in situ sensing capabilities, along with precise environmental control of temperature, humidity, and CO₂ concentration. A stratified microfluidic chip was engineered to co-culture HaCaT keratinocytes and HSF fibroblasts. CAP treatment was applied within this platform, and the dynamic processes of cell migration, proliferation, and multiple metabolic markers were simultaneously monitored. The experimental results show that the system can not only achieve real-time observation in the healing process under plasma intervention, but also find that the healing process is closely related to the concentration of NO₂⁻. In addition, the study also found that keratin KRT14, which is thought to be closely related to wound healing, decreased significantly in the process of plasma-induced healing. The platform provides high-resolution experimental tools to elucidate the biological effects of CAP and has the potential for parameter optimization, material evaluation, and personalized therapeutic development to advance plasma research and clinical translational applications. Full article

Background/Objectives: Chronic wounds represent a significant clinical and public health challenge due to impaired tissue repair and high associated morbidity. This study investigates the therapeutic potential of the secretome derived from human mesenchymal stem cells obtained from the pulp of deciduous teeth (hDP-MSCs) in promoting skin wound healing. Methods: After confirming the mesenchymal identity and multipotent differentiation potential of hDP-MSCs by using flow cytometry and histological staining, the effects of the secretome on human keratinocyte (HaCaT) cultures were evaluated. Results: Scratch assays, performed under high- and low-glucose conditions, demonstrated that the secretome significantly promoted keratinocyte migration and wound closure without compromising cell viability. Additionally, the secretome modulated the expression of key genes involved in inflammation and tissue regeneration, including IL-1β, TNF-α, TGF-β1, and VEGF-α, in a time-dependent manner. Under inflammatory conditions induced by lipopolysaccharide, co-treatment with the secretome significantly reduced TNF-α expression and increased TGF-β1 expression, suggesting an anti-inflammatory effect. Conclusions: These findings indicate the potential of the hDP-MSC-derived secretome as a promising cell-free therapeutic strategy capable of accelerating skin regeneration and modulating the inflammatory response during the wound healing process. Full article

Exosomes, small extracellular vesicles secreted by various cell types, have gained significant attention in cancer investigations. Isolation and characterization of exosomes derived from DOK (dysplastic oral keratinocyte), SCC (squamous cell carcinoma) and HaCaT (normal skin keratinocyte) cell lines and microRNA profiling were conducted. Magnetic sorting was applied to obtain pure exosomes. Morphology and size were characterized by transmission electron microscopy and nanoparticle tracking analysis. Validation of membrane exosomal markers (CD9, CD63) was performed via Western blotting. MiR-21, miR-31, and miR-133 levels were analyzed in exosomes and parent cells by qPCR. Biological effects of the exosomes were tested by adding them to fibroblast cultures and determining the expression of relevant carcinogenesis markers by qPCR. Exosomes appeared as cup-shaped nano-sized particles, and there was no difference regarding particle diameter and concentration between the three types of exosomes. The oncogenic miR-21 was significantly upregulated both in SCC and SCC-derived exosomes compared to DOK and HaCaT cells and their respective exosomes. However, miR-31 unexpectedly showed the highest expression in normal cells and the lowest in HaCaT exosomes. MiR-133, the tumor suppressor miRNA, was downregulated in both SCC and DOK cells compared to normal (HaCaT) cells, while the opposite situation was observed in exosomes, with HaCaT cells showing the lowest levels of miR-133. The differences in exosome content were reflected in signaling pathway activation in exosome-treated fibroblasts, with SCC exosomes exerting the most potent effect on several cancer-related pathways, notably PIK3/AKT, PTEN, and NOTCH signaling cascades. 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

Background/Objectives: Drugs for human use require several studies for the assessment of their efficacy and safety. An important property is cytotoxicity, which should be tested in different environments and models in closer proximity to the final use of the drug, with greater reliability. Thus, we proposed to evaluate the toxicity of rifampicin, the only bactericidal drug in the anti-leprosy multidrug therapy, using skin cells and skin explant cultures. Methods: Cell viability was tested by the MTT method using primary keratinocytes and fibroblasts and immortalized skin cells (HaCaT and 3T3) at 24, 48, and 72 h of treatment. For the skin explant, we used the TTC assay to determine viability (24, 48, 72, and 96 h), hematoxylin and eosin staining to analyze the structure and architecture of the tissue, and TUNEL to assess apoptotic cells at 3, 6, 12, 24, 48, 72, and 96 h. Results: Regarding the toxicity of primary and immortalized cells, viability was above 70% up to a concentration of 50 μg/mL at 24, 48, and 72 h, and at the concentration of 200 μg/mL, all cells showed greater sensitivity, especially at 72 h. Tissue viability analysis revealed a high percentage (above 96%) of viable tissue at the concentrations of 100, 150, and 200 μg/mL at the time points studied. Histological analysis showed that tissue architecture was maintained, with no apoptotic cells being observed. Conclusions: Thus, our results showed the importance of evaluating drug toxicity using different cell types, with the ex vivo skin model proving to be an alternative to animal use. Full article

Skin cancer, including melanoma and non-melanoma cancers (basal and squamous cell carcinomas), is the most common type of cancer. UV radiation, family history, and genetic predisposition are the main risk factors. Although surgical excision is the standard treatment, essential oils are attracting growing interest for their anti-cancer effects. This study tested the effects of Juniperus excelsa M. Bieb. (Cupressaceae), Lavandula vera DC. (Lamiaceae), and Salvia fruticosa (Mill). (Lamiaceae) essential oils extracted from Middle Eastern medicinal plants on HaCaT (normal), A5 (benign), and II4 (low-grade malignant) keratinocytes. Essential oils were extracted from Juniperus excelsa, Lavandula vera, and Salvia libanotica using steam distillation and then were chemically analyzed. The oils were sterilized, dissolved in DMSO, and prepared at concentrations of 0.75, 0.5, and 0.25 mg/mL. Human keratinocyte (HaCaT), benign (A5), and malignant (II4) cell lines were cultured in DMEM and treated with the essential oils for 24 or 48 h. Cell viability was assessed using the Trypan Blue Exclusion Test, while cell proliferation was evaluated using the MTT assay. Statistical analysis was performed using ANOVA with appropriate post hoc tests, considering p < 0.05 as significant. The results show that J. excelsa is cytotoxic but lacks selectivity, limiting its efficacy. In contrast, L. vera and S. fruticosa preferentially target malignant cells, particularly at low concentrations, while sparing normal cells. These oils have dose-dependent anticancer effects, with L. vera efficacy increasing as the concentration increases. In conclusion, L. vera and S. fruticosa are promising candidates for the treatment of skin cancer, although further in vivo studies are required. 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

Accelerating healing is a clinical goal in both acute and chronic non-healing skin wounds. We leveraged the public Recon database, which seeks to aggregate all of the metabolic pathways in the human body, to uncover whether increasing the supply of specific metabolites can bolster cellular metabolism and, in turn, enhance wound healing. The database was reduced to a set of 357 reactions and 339 metabolites that were better suited for human cells in culture. Monte Carlo simulations were performed to identify the impact of 25 different inputs on the metabolic fluxes within the cellular biochemical network. Biomass and ATP production were used as surrogate markers for cell proliferation and cell migration (an energy-intensive process), respectively, both of which are critical to wound healing. The subset of simulations yielding the highest ATP production or biomass production were those where glycine and/or glutamine uptake was increased. Maximizing ATP and biomass also generally increased oxygen uptake. Due to its low availability in chronic wounds, another set of simulations was carried out in which oxygen uptake was held constant to mimic the effect of a limited oxygen supply. However, even with this constraint, glycine and glutamine remained the most promising interventions. The predictions were tested in vitro using immortalized human keratinocytes. Amino acid uptake was tentatively increased by supplementing the base culture media with additional glycine and/or glutamine, with valine supplementation with a similar nitrogen load as a control. Glycine supplementation significantly increased cellular proliferation above the base media and accelerated wound closure rate in wound scratch assay. However, glutamine and valine supplementation did not improve these parameters above base media, and glutamine even suppressed the benefit of glycine in cultures supplemented with both amino acids. In conclusion, glycine supplementation enhances cellular processes that are associated with wound healing. 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

Fipronil (FIP) and imidacloprid (IMID) are two of the most commonly used ectoparasiticides to control parasites in pets. Compared with those of farm animals, their environmental risks have generally been considered low because of their limited use; however, the growing pet population and evolving treatment practices make this assumption challenging. To assess these risks, water samples were collected at an animal shelter in Italy to monitor the abundance of ectoparasiticides in aquatic environments. Additionally, laboratory-based ecotoxicological assays were carried out on a range of marine non-target species across different trophic levels (algae, copepods, and mussels). In vitro toxicity tests on human epithelial cell cultures were also implemented to examine potential cytotoxic effects at the levels of human exposure detectable in a domestic setting after pet treatment. Wastewater samples from the shelter contained 0.18 µg L⁻¹ of IMID, 0.50 µg L⁻¹ of FIP, and 0.20 µg L⁻¹ of FIP-sulfone, with these concentrations remaining stable for 60 days. Chronic exposure to FIP and IMID at 30.0 µg L⁻¹ impaired the mobility of the copepods. The EC₁₀ and EC₂₀ values were determined to be 1.7 (0.06–6.59) µg L⁻¹ and 2.8 (0.436–8.51) µg L⁻¹ for FIP and 2.6 (0.80–6.33) µg L⁻¹ and 7.6 (3.12–15.8) µg L⁻¹ for IMID, respectively. FIP and IMID exposure led to lipid peroxidation in the digestive glands and gills of mussels, whereas only IMID exposure increased acetylcholinesterase activity in the digestive glands at concentrations between 0.5 and 5.0 µg L⁻¹. Additionally, both fipronil and imidacloprid triggered the production of reactive oxygen species and lipid peroxidation and decreased the viability of human keratinocyte cells in a concentration-dependent manner. These findings highlight the persistence and potential risks of FIP and IMID, stressing the need for stricter regulations and further research on chronic environmental exposure to safeguard ecosystems and public health. Full article

Androgenetic alopecia is associated with testosterone-mediated anagen-to-catagen transition and matrix keratinocyte apoptosis in hair follicle cells. Activation of Nox isozymes is involved in testosterone-mediated keratinocyte apoptosis, leading to androgenetic alopecia. This indicates that Nox isozymes can serve as therapeutic targets for androgenetic alopecia. The isolated compounds from natural products were screened to evaluate their ROS-inhibition efficacy and it was found that 1′S-1′-acetoxychavicol acetate (ACA, 26), a natural compound isolated from Alpinia galanga (L.) Willd. (Zingiberaceae), exhibits inhibitory activity on Nox isozymes. Nox inhibition by ACA suppressed testosterone-dependent H₂O₂ generation and cell death in keratinocytes. Incubation with ACA in human hair follicle organ culture mitigated testosterone-dependent suppression of hair growth. We validated that ACA regulates androgenetic alopecia in a mouse model. Local application of ACA on the dorsal skin in an androgenetic alopecia model of C57BL/6 mice significantly suppressed testosterone-induced hair loss in a dose-dependent manner. Moreover, hair follicle length in ACA-treated mice was enhanced compared to that in control mice. These findings provide a molecular mechanism in which ACA inhibits Nox activity in hair follicle cells, indicating its potential as an effective treatment of AGA. Full article

Psoriasis pathogenesis involves dysregulated immune responses, yet the role of protein prenylation (particularly PGGT1B-mediated geranylgeranylation) in macrophage-driven inflammation remains poorly understood. This study aims to explore the role and molecular mechanism of protein geranylgeranyltransferase type I subunit beta (PGGT1B) in the development of psoriasis. Myeloid cell-specific PGGT1B gene knockout mice were generated, and a mouse psoriasis model was established with imiquimod to study the role and mechanism of PGGT1B gene downregulation-induced macrophage activation in the pathogenesis of psoriasis. Bone marrow-derived macrophages (BMDMs) from wild-type and PGGT1B knockout mice were cultured and stimulated with resiquimod (R848) to simulate the immune microenvironment of psoriasis. In addition, the differentially expressed genes induced by PGGT1B knockout were analyzed using RNA-seq, and bioinformatics analysis was carried out to study the possible biological process of PGGT1B regulation. Finally, PMA-THP-1 was co-cultured with HaCaT cells to study the effect of PGGT1B deletion in macrophages on the proliferation and differentiation of keratinocytes. Bone marrow PGGT1B deficiency aggravated the psoriasis-like lesions induced by imiquimod in mice. In BMDMs with PGGT1B deficiency, the NF-κB signaling pathway was over-activated by R848, and the expressions of proinflammatory cytokines IL-1β, IL-6, and TNF-α were significantly increased. Activation of cell division cycle 42 (CDC42) may mediate the activation of the NF-κB pathway in PGGT1B-deficient BMDMs. PGGT1B deletion can promote the proliferation and inhibit the differentiation of HaCaT cells. Reduced PGGT1B levels can increase the expression of CDC42, which further activates NLRP3 inflammation in macrophages through NF-κB signaling, further aggravating the inflammatory state of psoriasis. Psoriasis-like lesions induced by IMQ are aggravated when PGGT1B expression is reduced in mouse bone marrow cells. A possible mechanism for this is that PGGT1B-deficient macrophages migrate to the epidermis more easily during psoriasis, which leads to the activation of Cdc42, NF-κB signaling, and NLRP3 inflammatory corpuscles. 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