Platelet-Rich Plasma Promotes the Proliferation of Human Keratinocytes via a Progression of the Cell Cycle. A Role of Prolidase

Although the role of platelet-rich plasma (PRP) in tissue regeneration has been confirmed in many studies, the mechanism of this process is still not fully understood. Human keratinocytes (HaCaT) cells were used as an experimental model for studies on the effects of PRP on cell proliferation, migration, collagen biosynthesis, prolidase activity, and its expression and anabolic signaling. The activation of epidermal growth factor receptor (EGFR), β₁-integrin, and insulin-like growth factor-1 receptor (IGF-1R) by PRP were investigated by western blot and immunocytochemistry. It has been found that PRP induced keratinocytes migration and proliferation through activation of cell cycle progression and EGFR downstream signaling. Similar biological effects were achieved by an addition to the culture medium of prolidase (PEPD), a ligand of EGFR (PRP is a rich source of PEPD–2 ng/mL). PRP-dependent stimulation of collagen biosynthesis was accompanied by an increase in the expression of NF-κβ, IGF-1R-downstream signaling proteins, and PEPD activity. The data suggest that PRP activates a complex of growth factors and adhesion receptors that stimulate cell proliferation, migration, and collagen biosynthesis. PRP induces PEPD-dependent human keratinocyte proliferation through activation of the EGFR receptor. Our study provides a novel mechanism of PRP-dependent wound healing.     

Melanin Distribution in Human Skin: Influence of Cytoskeletal,Polarity, and Centrosome-Related Machinery of Stratum basale Keratinocytes

Melanin granules cluster within supra-nuclear caps in basal keratinocytes (KCs) of the human epidermis, where they protect KC genomic DNA against ultraviolet radiation (UVR) damage. While much is known about melanogenesis in melanocytes (MCs) and a moderate amount about melanin transfer from MC to KC, we know little about the fate of melanin once inside KCs. We recently reported that melanin fate in progenitor KCs is regulated by rare asymmetric organelle movement during mitosis. Here, we explore the role of actin, microtubules, and centrosome-associated machinery in distributing melanin within KCs. Short-term cultures of human skin explants were treated with cytochalasin-B and nocodazole to target actin filaments and microtubules, respectively. Treatment effects on melanin distribution were assessed by the Warthin–Starry stain, on centrosome-associated proteins by immunofluorescence microscopy, and on co-localisation with melanin granules by brightfield microscopy. Cytochalasin-B treatment disassembled supra-nuclear melanin caps, while nocodazole treatment moved melanin from the apical to basal KC domain. Centrosome and centriolar satellite-associated proteins showed a high degree of co-localisation with melanin. Thus, once melanin granules are transferred to KCs, their preferred apical distribution appears to be facilitated by coordinated movement of centrosomes and centriolar satellites. This mechanism may control melanin’s strategic position within UVR-exposed KCs.     

Cold Atmospheric Plasma Jet Treatment Improves Human Keratinocyte Migration and Wound Closure Capacity without Causing Cellular Oxidative Stress

Cold Atmospheric Plasma (CAP) is an emerging technology with great potential for biomedical applications such as sterilizing equipment and antitumor strategies. CAP has also been shown to improve skin wound healing in vivo, but the biological mechanisms involved are not well known. Our study assessed a possible effect of a direct helium jet CAP treatment on keratinocytes, in both the immortalized N/TERT-1 human cell line and primary keratinocytes obtained from human skin samples. The cells were covered with 200 µL of phosphate buffered saline and exposed to the helium plasma jet for 10–120 s. In our experimental conditions, micromolar concentrations of hydrogen peroxide, nitrite and nitrate were produced. We showed that long-time CAP treatments (≥60 s) were cytotoxic, reduced keratinocyte migration, upregulated the expression of heat shock protein 27 (HSP27) and induced oxidative cell stress. In contrast, short-term CAP treatments (<60 s) were not cytotoxic, did not affect keratinocyte proliferation and differentiation, and did not induce any changes in mitochondria, but they did accelerate wound closure in vitro by improving keratinocyte migration. In conclusion, these results suggest that helium-based CAP treatments improve wound healing by stimulating keratinocyte migration. The study confirms that CAP could be a novel therapeutic method to treat recalcitrant wounds.     

Current Knowledge on Interactions of Plant Materials Traditionally Used in Skin Diseases in Poland and Ukraine with Human Skin Microbiota

Skin disorders of different etiology, such as dermatitis, atopic dermatitis, eczema, psoriasis, wounds, burns, and others, are widely spread in the population. In severe cases, they require the topical application of drugs, such as antibiotics, steroids, and calcineurin inhibitors. With milder symptoms, which do not require acute pharmacological interventions, medications, dietary supplements, and cosmetic products of plant material origin are gaining greater popularity among professionals and patients. They are applied in various pharmaceutical forms, such as raw infusions, tinctures, creams, and ointments. Although plant-based formulations have been used by humankind since ancient times, it is often unclear what the mechanisms of the observed beneficial effects are. Recent advances in the contribution of the skin microbiota in maintaining skin homeostasis can shed new light on understanding the activity of topically applied plant-based products. Although the influence of various plants on skin-related ailments are well documented in vivo and in vitro, little is known about the interaction with the network of the skin microbial ecosystem. The review aims to summarize the hitherto scientific data on plant-based topical preparations used in Poland and Ukraine and indicate future directions of the studies respecting recent developments in understanding the etiology of skin diseases. The current knowledge on investigations of interactions of plant materials/extracts with skin microbiome was reviewed for the first time.     

Hypercholesterolemia in Cancer and in Anorexia Nervosa: A Hypothesis for a Crosstalk

The relationship between cholesterol and cancer has been widely demonstrated. Clinical studies have shown changes in blood cholesterol levels in cancer patients. In parallel, basic research studies have shown that cholesterol is involved in the mechanisms of onset and progression of the disease. On the other hand, anorexic patients have high cholesterol levels and a high susceptibility to cancer. In this review, we first present a brief background on the relations among nutrition, eating disorders and cancer. Using several notable examples, we then illustrate the changes in cholesterol in cancer and in anorexia nervosa, providing evidence for their important relationship. Finally, we show a new possible link between cholesterol disorder in cancer and in anorexia nervosa.     

Application of Coffee Silverskin in cosmetic formulations: physical/antioxidant stability studies and cytotoxicity effects

Context: Currently, there is an increasing interest of cosmetic industry on natural extracts. The inclusion of antioxidants in topical formulations can contribute to minimize oxidative stress in the skin, which has been associated with aging. Also, questions of sustainability are leading to the study of new cosmetic ingredients obtained from food by-products. Coffee Silverskin (CS) is a food by-product with established antioxidant activity that has not yet been incorporated into a topical formulation.

Objective: The aim of this study was to evaluate the physical and microbiological stabilities and antioxidant activity of a hand cream formulation containing 2.5% (w/w) of CS extract upon production and after 6 months of shelf-life and in vitro safety/cytotoxicity on skin cell lines after production.

Materials and methods: The in vitro cytotoxicity was evaluated with MTS and LDH assays, at different concentrations, in HaCaT and HFF-1 cells. Formulations were stored at 25?°C/65% RH and 40?°C/75% RH. Physical, microbiological, and antioxidant stabilities were evaluated by centrifugation, viscosity, total colony count, DPPH and total phenolic content (TPC).

Results: The hand cream containing 2.5% (w/w) of CS extract showed stable physical characteristics independently of the storage conditions. The DPPH activity and TPC of the CS formulation were significantly higher compared with those of the base formulation. However, during storage, the antioxidant activity decreases slightly. Microbiological quality was also confirmed. No cytotoxic effects were observed.

Conclusion: It is possible to suggest that this formulation is stable under extreme conditions and safe for topical use.     

Platelet-Released Growth Factors Influence Wound Healing-Associated Genes in Human Keratinocytes and Ex Vivo Skin Explants

Platelet-released growth factors (PRGFs) or other thrombocyte concentrate products, e.g., Platelet-Rich Fibrin (PRF), have become efficient tools of regenerative medicine in many medical disciplines. In the context of wound healing, it has been demonstrated that treatment of chronic or complicated wounds with PRGF or PRF improves wound healing in the majority of treated patients. Nevertheless, the underlying cellular and molecular mechanism are still poorly understood. Therefore, we aimed to analyze if PRGF-treatment of human keratinocytes caused the induction of genes encoding paracrine factors associated with successful wound healing. The investigated genes were Semaphorin 7A (SEMA7A), Angiopoietin-like 4 (ANGPLT4), Fibroblast Growth Factor-2 (FGF-2), Interleukin-32 (IL-32), the CC-chemokine-ligand 20 (CCL20), the matrix-metalloproteinase-2 (MMP-2), the chemokine C-X-C motif chemokine ligand 10 (CXCL10) and the subunit B of the Platelet-Derived Growth Factor (PDGFB). We observed a significant gene induction of SEMA7A, ANGPLT4, FGF-2, IL-32, MMP-2 and PDGFB in human keratinocytes after PRGF treatment. The CCL20- and CXCL10 gene expressions were significantly inhibited by PRGF therapy. Signal transduction analyses revealed that the PRGF-mediated gene induction of SEMA7A, ANGPLT4, IL-32 and MMP-2 in human keratinocytes was transduced via the IL-6 receptor pathway. In contrast, EGF receptor signaling was not involved in the PRGF-mediated gene expression of analyzed genes in human keratinocytes. Additionally, treatment of ex vivo skin explants with PRGF confirmed a significant gene induction of SEMA7A, ANGPLT4, MMP-2 and PDGFB. Taken together, these results describe a new mechanism that could be responsible for the beneficial wound healing properties of PRGF or related thrombocytes concentrate products such as PRF.     

Evaluation of Cell Migration and Cytokines Expression Changes under the Radiofrequency Electromagnetic Field on Wound Healing In Vitro Model

Wound healing (WH) proceeds through four distinct phases: hemostasis, inflammation, proliferation, and remodeling. Impaired WH may be the consequence of the alteration of one of these phases and represents a significant health and economic burden to millions of individuals. Thus, new therapeutic strategies are the topics of intense research worldwide. Although radiofrequency electromagnetic field (RF-EMF) has many medical applications in rehabilitation, pain associated with musculoskeletal disorders, and degenerative joint disorders, its impact on WH is not fully understood. The process of WH begins just after injury and continues during the inflammatory and proliferative phases. A thorough understanding of the mechanisms by which RF-EMF can improve WH is required before it can be used as a non-invasive, inexpensive, and easily self-applicable therapeutic strategy. Thus, the aim of this study is to explore the therapeutic potential of different exposure setups of RF-EMF to drive faster healing, evaluating the keratinocytes migration, cytokines, and matrix metalloproteinases (MMPs) expression. The results showed that RF-EMF treatment promotes keratinocytes’ migration and regulates the expression of genes involved in healing, such as MMPs, tissue inhibitors of metalloproteinases, and pro/anti-inflammatory cytokines, to improve WH.     

Microgravity Modifies the Phenotype of Fibroblast and Promotes Remodeling of the Fibroblast–Keratinocyte Interaction in a 3D Co-Culture Model

Microgravity impairs tissue organization and critical pathways involved in the cell–microenvironment interplay, where fibroblasts have a critical role. We exposed dermal fibroblasts to simulated microgravity by means of a Random Positioning Machine (RPM), a device that reproduces conditions of weightlessness. Molecular and structural changes were analyzed and compared to control samples growing in a normal gravity field. Simulated microgravity impairs fibroblast conversion into myofibroblast and inhibits their migratory properties. Consequently, the normal interplay between fibroblasts and keratinocytes were remarkably altered in 3D co-culture experiments, giving rise to several ultra-structural abnormalities. Such phenotypic changes are associated with down-regulation of α-SMA that translocate in the nucleoplasm, altogether with the concomitant modification of the actin-vinculin apparatus. Noticeably, the stress associated with weightlessness induced oxidative damage, which seemed to concur with such modifications. These findings disclose new opportunities to establish antioxidant strategies that counteract the microgravity-induced disruptive effects on fibroblasts and tissue organization.     

Optimized protocol for the biocompatibility testing of compression stockings and similar products with close skin contact in vitro

Biocompatibility of six different compression stockings and cytotoxic effects were determined using HaCaT keratinocytes, L929 mouse fibroblasts, primary adult and juvenile keratinocytes Cells were quantified using a luminometric ATP assay and the photometric BCA test. Cytotoxic effects were determined by LDH release. An area-based extraction ratio of 1.25 cm²:mL could be shown to be superior to the weight-based extraction of test material. Extraction medium should be an acidic sweat solution as this helps to recreate in vivo conditions. Monolayer cultures of HaCaT keratinocytes or L929 mouse fibroblasts should be used for testing. Primary adult keratinocytes or primary juvenile keratinocytes can also be used. For the latter, testing under DMEM with FCS is recommended to achieve comparable results. It was found that the compression stockings tested exhibited no negative influence on cell viability in vitro and no direct cytotoxic effects measured as release of LDH. Hence, good biocompatibility could be asserted.