Human Dermal Cells Research Articles

Expression Profile of CD157 Reveals Functional Heterogeneity of Capillaries in Human Dermal Skin

CD157 acts as a receptor, regulating leukocyte trafficking and the binding of extracellular matrix components. However, the expression pattern and the role of CD157 in human blood (BEC) and the lymphatic endothelial cells (LEC) of human dermal microvascular cells (HDMEC), remain elusive. We demonstrated constitutive expression of CD157 on BEC and LEC, in fetal and juvenile/adult skin, in situ, as well as in isolated HDMEC. Interestingly, CD157 epitopes were mostly localized on BEC, co-expressing high levels of CD31 (CD31High), as compared to CD31Low BEC, whereas the podoplanin expression level on LEC did not affect CD157. Cultured HDMEC exhibited significantly higher numbers of CD157-positive LEC, as compared to BEC. Interestingly, separated CD157− and CD157+ HDMEC demonstrated no significant differences in clonal expansion in vitro, but they showed distinct expression levels of cell adhesion molecules, before and after cytokine stimulation in vitro. In particular, we proved the enhanced and specific adherence of CD11b-expressing human blood myeloid cells to CD157+ HDMEC fraction, using an in vitro immune-binding assay. Indeed, CD157 was also involved in chemotaxis and adhesion of CD11b/c monocytes/neutrophils in prevascularized dermo–epidermal skin substitutes (vascDESS) in vivo. Thus, our data attribute specific roles to endothelial CD157, in the regulation of innate immunity during inflammation.

Tetramethacrylic benzylidene cyclopentanone dye for one- and two-photon photopolymerization

The crystalline one-component methacrylate containing photoinitiator 4MetBAC was synthesized and characterized by 1H NMR, 13C NMR, IR-, UV–Vis spectrum, and X-Ray analysis. The one- and two-photon absorption, photoluminescence, and electrochemical properties of 4MetBAC in solution were investigated. The polymerization of PETA was induced directly by 4MetBAC (2.5–11.5 mM) under irradiation with LED@380 (1PP) or focused beam of femtosecond laser with wavelength 780 nm (2PP). The polymer nanostructures with intricate architecture («sleepers-rails»-, «cell scaffold»- and cylindrical spiral structures) were fabricated by the DLW method. The polymerization threshold of the composition based on 4Met-BAC (11.5 mM) was 0.9 mW at the recording speed of 170 μm/s. The smallest stable structures with the linear fragment sizes of 72 ± 5 nm (less than λ/10) have been obtained by nanolithography. Due to the polymerizable groups, 4Met-BAC has the ability to link into the polymer networks. Experiments with human dermal fibroblasts and cells of the HEK 293 line have been demonstrated that the final polymers prepared by both one- and two-photon photopolymerization (cell scaffold) are biosafety. Hence we believe that the PETA composition based on one-component methacrylate-containing photoinitiator 4Met-BAC opens up wide capabilities for creating three-dimensional topological structures with high submicron resolution by the DLW method for optoelectronic and biological applications.

Growth kinetics and phenotypic markers expression in human dermal stem cells

Human dermal stem cells (DSCs) have generated significant interest in the field of regenerative medicine due to their prospects of autologous transplantation. The present study evaluated the growth kinetics and phenotypic markers expression in human DSCs. The primary cultures of DSCs (n=3) were established by explant culture and characterization of the cells was carried out by assessing morphology, viability, proliferation rate, population doubling time (PDT), cell cycle status and the expression of cell surface markers such as CD29, CD73, CD90 and CD166. The cells released from tissue explants showed spindleshaped fibroblast morphology with the mean percentage viability varying between 93.43% and 100% from passages 1 to 4. DSCs displayed a strong and steady proliferative potential with an average PDT of 42.55 hrs. Cell cycle profile of DSCs demonstrated the majority of cells (59.80% to 76.29%) at G0/G1 phase. Further, the phenotypic profile of markers confirmed the stromal origin of DSCs by exhibiting positivity for CD29, CD73, CD90 and CD166. In conclusion, the growth kinetics and expression of phenotypic markers are consistent with the notion that skin dermis contains a population of stem cells and can serve as a potential autologous source for therapeutic applications.

Research possibility of using an enzymatic hydrolyzate of the lymphoid tissue of broiler chickens as an immunomodulator

Currently, various adverse factors can lead to disruption of the immune system. In order to restore the functional state of immunity, it is necessary to use immunomodulators. One such immunomodulators is an enzymatic hydrolyzate of the lymphoid tissue of broiler chickens . The aim of this work was to study the effect of an enzymatic hydrolyzate of the lymphoid tissue of broiler chickens on the morphofunctional state of normal and tumor cells. The object of the research was stem cell culture , human dermal fibroblasts and tumor cell cultures : human cervical carcinoma cells HeLa and adenocarcinoma MCF-7 human breast. The cells were incubated in 96-hole tripod in CO 2 incubator at 37 °C. These cultures were supplemented with dietary supplements in concentrations of 0.05%; 0.1%; 0.5%; 1%; 5%; 10%; 20%; 25%; 40% and 50%. After 48 h, the viability of the cells was examined using MTT-analysis. The experiment revealed cytotoxic effects on mesenchymal stem cells and on MCF-7 breast adenocarcinoma cells. An enzymatic hydrolyzate of the lymphoid tissue of broiler chickens did not have a pronounced effect on viability and did not show a cytotoxic effect in relation to human dermal fibroblasts. In the case of HeLa cells, dietary supplements stimulate their proliferative activity. The response from tumor and stem cells requires further study to identify possible action mechanisms of an enzymatic hydrolyzate of the lymphoid tissue of broiler chickens. • Hydrolysate in concentrations 0.05%; 0.1%; 0.5%; 1%; 5%; 10% it does not reduce the viability of mesenchymal cells. • High doses of hydrolysate 25% and 50% have a weak cytotoxic effect on mesenchymal cells. • Hydrolysate in concentrations of 10%, 20% and 40% has a significant dose-dependent cytotoxic effect on MCF-7 cell culture. • Hydrolysate in concentrations of 5%, 10% and 20% stimulates the proliferative activity of HeLa cells. • The reaction of tumor and stem cells requires further study to identify possible mechanisms of action of the hydrolysate.

Protective Effect of Conditioned Media of Human Fetal Dermal Mesenchymal Stem Cells Can Inhibit Burn-induced Microvascular Hyperpermeability.

Burns often cause loss of skin barrier protection, fluid exudation, and local tissue edema, which hinder functional recovery. Effectively improving the quality of deep burn wound healing, shortening the wound healing time, and reducing tissue fluid leakage are urgent problems in the medical field. Human mesenchymal stem cells (MSCs) can effectively stabilize vascular endothelial injury. Fetal dermal MSCs (FDMSCs) are a newly discovered source of MSCs derived from the skin of accidentally aborted fetuses. However, the effect of FDMSCs on vascular permeability remains poorly understood. In this study, conditioned media from FDMSCs (F-CM) extracted from fetal skin tissue was prepared. The effect of F-CM on vascular permeability was evaluated using the internal circulation method FITC-dextran in vivo, and several in vitro assays, including cell viability assay, transwell permeability test, immunofluorescence, and western blotting. Altogether, our results demonstrate that F-CM could inhibit burn-induced microvascular hyperpermeability by increasing the protein expression levels of occludin and VE-cadherin, while restoring the expression of endothelial F-actin, and providing the foundation of a novel therapy for the treatment of burns with F-CM.

Nicotinamide effects on the metabolism of human fibroblasts and keratinocytes assessed by quantitative, label-free fluorescence imaging.

Alterations in metabolism are central to the aging process. Therefore, understanding the subcellular functional and structural changes associated with metabolic aging is critical. Current established methods for exploring cell metabolism either require the use of exogenous agents or are destructive to the tissue or cells. Two-photon excited fluorescence (TPEF) imaging has emerged as a method for monitoring subtle metabolic changes non-invasively. In this study, we use TPEF imaging to acquire high-resolution fluorescence images from two coenzymes, NAD(P)H (reduced form of nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), within human fibroblasts and keratinocytes in response to B3 (a nicotinamide precursor) supplementation and/or UV irradiation, without addition of exogenous labels. In addition, multi-parametric analysis methods are used to extract functional information of cellular metabolism, including cellular redox state, NAD(P)H fluorescence lifetime, and mitochondrial organization. Our results demonstrate that such optical metabolic assessments can serve as sensitive, label-free, non-destructive reporters of known effects of B3 to maintain and in some cases even enhance the respiratory function of mitochondria, while lowering oxidative damage. Thus, TPEF imaging, supported by highly-quantitative analysis, can serve as a tool to understand aging-dependent metabolic changes as well as the effect of actives on human epidermal and dermal cells.

Efficacy of Chininum Sulphuricum 30C against Malaria: An in vitro and in vivo Study

Background: New effective, economical and safe antimalarial drugs are urgently needed due to the development of multi-drug-resistant strains of the parasite. Homeopathy uses ultra-diluted doses of various substances to stimulate autoregulatory and self-healing processes to cure various ailments. The aim of the study was to evaluate the in vitro and in vivo antimalarial efficacy of a homeopathic drug, Chininum sulphuricum 30C. Methods: In vitro antiplasmodial activity was screened against the P. falciparum chloroquine-sensitive (3D7) strain, and cell viability was assessed against normal human dermal fibroblasts and HepG2 cells. Suppressive, preventive and curative studies were carried out against P. berghei-infected mice in vivo. Results: Chininum sulphuricum (30C) revealed good antiplasmodial activity in vitro, with 92.79 ± 6.93% inhibition against the 3D7 strain. The cell viability was 83.6 ± 0.6% against normal human dermal fibroblasts and 95.22 ± 5.1% against HepG2 cells. It also exhibited suppressive efficacy with 95.56% chemosuppression on day 7 with no mortality throughout the follow-up period of 28 days. It also showed preventive activity against the disease. Drug treatment was also safe to the liver and kidney function of the host as evidenced by biochemical studies. Conclusion: Chininum sulphuricum 30C exhibited considerable antimalarial activity along with safety to the liver and kidney function of the host.

Asporin inhibits collagen matrix-mediated intercellular mechanocommunications between fibroblasts during keloid progression.

Keloids are fibrotic lesions that grow unceasingly and invasively and are driven by local mechanical stimuli. Unlike other fibrotic diseases and normal wound healing, keloids exhibit little transformation of dermal fibroblasts into α-SMA+ myofibroblasts. This study showed that asporin is the most strongly expressed gene in keloids and its gene-ontology terms relate strongly to ECM metabolism/organization. Experiments with human dermal cells (HDFs) showed that asporin overexpression/treatment abrogated the HDF ability to adopt a perpendicular orientation when subjected to stretching tension. It also induced calcification of the surrounding 3D collagen matrix. Asporin overexpression/treatment also prevented the HDFs from remodeling the surrounding 3D collagen matrix, leading to a disorganized network of thick, wavy collagen fibers that resembled keloid collagen architecture. This in turn impaired the ability of the HDFs to contract the collagen matrix. Asporin treatment also made the fibroblasts impervious to the fibrous collagen contraction of α-SMA+ myofibroblasts, which normally activates fibroblasts. Thus, by calcifying collagen, asporin prevents fibroblasts from linearly rearranging the surrounding collagen; this reduces both their mechanosensitivity and mechanosignaling to each other through the collagen network. This blocks fibroblast activation and differentiation into the mature myofibroblasts that efficiently remodel the extracellular matrix. Consequently, the fibroblasts remain immature, highly proliferative, and continue laying down abundant extracellular matrix, causing keloid growth and invasion. Notably, dermal injection of asporin-overexpressing HDFs into murine wounds recapitulated keloid collagen histopathological characteristics. Thus, disrupted interfibroblast mechanocommunication may promote keloid progression. Asporin may be a new diagnostic biomarker and therapeutic target for keloids.

Novel nanosystems to enhance biological activity of hydroxyapatite against dental caries

This work aimed to study for the first time to our knowledge the influence of the structure of the dental flosses (DF) coated by hydroxyapatite nanoparticles (HAP NPs) on the biological performance of saliva probiotic bacteria (S. salivarius), and human dermal and osteoblast-like cells. We used three types of HAP@DF composites (based on two unwaxed dental flosses - “fluffy” and “smooth”, and one waxed “smooth”) with different morphologies. Obtained composites were characterized from the point of view of their structure, morphological characteristics, elemental and chemical composition.We observed that HAP NPs coated “smooth” dental flosses led to an increase of viability and proliferation of oral cavity probiotic bacteria (Streptococcus salivarius) and human cells (dermal fibroblasts and osteoblast-like). In contrast, the highest viability loss of probiotic bacteria (S. salivarius), fibroblasts, and osteoblast-like cells were observed for “fluffy” unwaxed dental flosses due to high cytotoxicity.Our studies showed that HAP NPs significantly improved the biological properties of “fluffy” dental floss. Pristine “smooth” DFs (waxed and unwaxed), as well as all HAP-coated DFs, induced acceptable biocompatibility toward selected human cells.

TOXICITY OF ZINC OXIDE NANOPARTICLES ON HUMAN SKIN DERMAL CELLS

Zinc oxide (ZnO) has special physical and chemical characteristics which enable it to be utilized in numerous applications including electronics, sunscreens, pigments, and most notably in biomedical applications. Nanoemulsions containing zinc oxide nanoparticles (ZnO NPs) are progressively sought-after as an active component in cosmetic formulations and are used in sunscreens, moisturizers, and antiaging products. Zinc paste bandages including Unna boot consist of open wove cotton gauze treated with ZnO paste are now common medicaments for leg ulcers. The damaged and broken skins are vulnerable to ZnO NPs uptake. This being the case, ZnO NPs on the skin surface can affect the functions of surrounding cells in numerous ways by penetrating into the skin cells. This could exert toxicity effects on the skin cells over time depending on the concentration and site of ZnO NPs exposure. This review brings together some findings regarding the toxicity of ZnO NPs on human skin dermal cells and thus in turn enlightens the safer usage of ZnO NPs in skin care applications.

Fatty acid potassium improves human dermal fibroblast viability and cytotoxicity, accelerating human epidermal keratinocyte wound healing in vitro and in human chronic wounds.

Effective cleaning of a wound promotes wound healing and favours wound care as it can prevent and control biofilms. The presence of biofilm is associated with prolonged wound healing, increased wound propensity to infection, and delayed wound closure. Anionic potassium salts of fatty acids are tested with commonly used anionic surfactants, such as sodium laureth sulphate (SLES) and sodium lauryl sulphate/sodium dodecyl sulphate (SLS/SDS). The normal human dermal cells demonstrated significantly greater viability in fatty acid potassium, including caprylic acid (C8), capric acid (C10), lauric acid (C12), oleic acid (C18:1), and linoleic acid (C18:2), than in SLES or SLS after a 24‐hour incubation. Cytotoxicity by LDH assay in a 5‐minute culture in fatty acid potassium was significantly lower than in SLES or SLS. in vitro wound healing of human epidermal keratinocytes during the scratch assay in 24‐hour culture was more significantly improved by fatty acid treatment than by SLES or SLS/SDS. In a live/dead assay of human epidermal keratinocytes, C8K and C18:1K demonstrated only green fluorescence, indicating live cells, whereas synthetic surfactants, SLES and SLS, demonstrated red fluorescence on staining with propidium iodide, indicating dead cells after SLES and SLS/SDS treatment. Potassium salts of fatty acids are useful wound cleaning detergents that do not interfere with wound healing, as observed in the scratch assay using human epidermal keratinocytes. As potassium salts of fatty acids are major components of natural soap, which are produced by natural oil and caustic potash using a saponification method, this may be clinically important in wound and peri‐wound skin cleaning. In human chronic wounds, natural soap containing fatty acid potassium increased tissue blood flow based on laser speckle flowgraphs after 2 weeks (P < .05), in addition to removing the eschars and debris. Wound cleansing by natural soap of fatty acid potassium is beneficial for wound healing.

Indocyanine Green-Loaded PLGA Nanoparticles Conjugated with Hyaluronic Acid Improve Target Specificity in Cervical Cancer Tumors.

PurposeIndocyanine green (ICG) is a promising agent for intraoperative visualization of tumor tissues and sentinel lymph nodes in early-stage gynecological cancer. However, it has some limitations, including a short half-life and poor solubility in aqueous solutions. This study aimed to enhance the efficacy of near-infrared (NIR) fluorescence imaging by overcoming the shortcomings of ICG using a nano-drug delivery system and improve target specificity in cervical cancer.Materials and MethodsICG and poly(lactic-co-glycolic acid) (PLGA) conjugated with polyethylenimine (PEI) were assembled to enhance stability. Hyaluronic acid (HA) was coated on PEI-PLGA-ICG nanoparticles to target CD44-positive cancer cells. The manufactured HA-ICG-PLGA nanoparticles (HINPs) were evaluated in vitro and in vivo on cervical cancer cells (SiHa; CD44+) and human dermal cells (ccd986sk; CD44-), respectively, using NIR imaging to compare intracellular uptake and to quantify the fluorescence intensities of cells and tumors.ResultsHINPs were confirmed to have a mean size of 200 nm and a zeta-potential of 33 mV using dynamic light scattering. The stability of the HINPs was confirmed at pH 5.0–8.0. Cytotoxicity assays, intracellular uptake assays, and cervical cancer xenograft models revealed that, compared to free ICG, the HINPs had significantly higher internalization by cervical cancer cells than normal cells (p<0.001) and significantly higher accumulation in tumors (p<0.001) via CD44 receptor-mediated endocytosis.ConclusionThis study demonstrated the successful application of HINPs as nanocarriers for delivering ICG to CD44-positive cervical cancer, with improved efficacy in NIR fluorescence imaging.

Decoction of Heat-Clearing, Detoxifying and Blood Stasis Removing Relieves Acute Soft Tissue Injury via Modulating MiR-26b-5p/COX2 Axis to Inhibit Inflammation.

Traditional Chinese medicine (TCM), such as Huanglian-Jie-Du-Tang, a heat-clearing and detoxifying decoction is beneficial for alleviation of inflammation-related diseases. The objective of the present study is to uncover the effect and mechanism of heat-clearing, detoxifying and blood stasis removing decoction (HDBD) on the treatment of acute soft tissue injury (STI) which is characterized with excessive inflammatory cascade at the onset. Male Sprague–Dawley (SD) rats with hammer beating served as the in vivo models of acute STI. Hematoxylin–Eosin (HE) staining was used for histopathology assessment. The levels of inflammatory factors, including prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), interleukin (IL)-1t and IL-6 were measured by enzyme-linked immunosorbent assay (ELISA). Human dermal microvascular endothelium cell line HMEC-1 and rat vascular endothelium cell line RAOEC were used to explore the mechanism in vitro. Luciferase gene reporter assay was applied to determine the relationship between miR-26b-5p and Cyclo-oxygenase 2 (COX2). The results showed that HDBD intervention significantly reduced the temperature difference between the healthy side and affected side of rats with hammer beating, together with the decreased levels of COX2, PGE2, TNF-α, IL-6 and IL-1β, and the increased level of miR-26b-5p. In mechanism, miR-26b-5p targeted COX2 and decreased its expression, leading to significant decreases in the levels of PGE2, TNF-α and IL-6 in RAOEC and HMEC-1 cells. In addition, miR-26b-5p inhibition impaired the effects of HDBD on the suppression of PGE2, TNF-α, IL-6 and IL-1β in vitro. In conclusion, the present study revealed that HDBD relieved acute STI via modulating miR-26b-5p/COX2 axis to inhibit inflammation.

Human Fetal Scalp Dermal Papilla Enriched Genes and the Role of R-Spondin-1 in the Restoration of Hair Neogenesis in Adult Mouse Cells.

Much remains unknown about the regulatory networks which govern the dermal papilla’s (DP) ability to induce hair follicle neogenesis, a capacity which decreases greatly with age. To further define the core genes which characterize the DP cell and to identify pathways prominent in DP cells with greater hair inductive capacity, comparative transcriptome analyses of human fetal and adult dermal follicular cells were performed. 121 genes were significantly upregulated in fetal DP cells in comparison to both fetal dermal sheath cup (DSC) cells and interfollicular dermal (IFD) populations. Comparison of the set of enriched human fetal DP genes with human adult DP, newborn mouse DP, and embryonic mouse dermal condensation (DC) cells revealed differences in the expression of Wnt/β-catenin, Shh, FGF, BMP, and Notch signaling pathways. We chose R-spondin-1, a Wnt agonist, for functional verification and show that exogenous administration restores hair follicle neogenesis from adult mouse cells in skin reconstitution assays. To explore upstream regulators of fetal DP gene expression, we identified twenty-nine transcription factors which are upregulated in human fetal DP cells compared to adult DP cells. Of these, seven transcription factor binding motifs were significantly enriched in the candidate promoter regions of genes differentially expressed between fetal and adult DP cells, suggesting a potential role in the regulatory network which confers the fetal DP phenotype and a possible relationship to the induction of follicle neogenesis.

Nanogel Encapsulated Hydrogels As Advanced Wound Dressings for the Controlled Delivery of Antibiotics

AbstractBiocompatible and degradable dual‐delivery gel systems based on hyperbranched dendritic−linear−dendritic copolymers (HBDLDs) is herein conceptualized and accomplished via thiol‐ene click chemistry. The elasticity of the hydrogels is tunable by varying the lengths of PEG (2, 6, 10 kDa) or the dry weight percentages (20, 30, 40 wt%), and are found to range from 2–14.7 kPa, comparable to human skin. The co‐delivery of antibiotics is achieved, where the hydrophilic drug novobiocin sodium salt (NB) is entrapped within the hydrophilic hydrogel, while the hydrophobic antibiotic ciprofloxacin (CIP) is encapsulated within the dendritic nanogels (DNGs) with hydrophobic cores (DNGs‐CIP). The DNGs‐CIP with drug loading capacity of 2.83 wt% are then physically entrapped within the hybrid hydrogels through UV curing. The hybrid hydrogels enable the quick release of NB and prolonged released of CIP. In vitro cell infection assays showed that the antibiotic‐loaded hybrid hydrogels are able to treat bacterial infections with significant bacterial reduction. Hybrid hydrogel band aids are fabricated and exhibited better antibacterial activity compared with commercial antimicrobial band aids. Remarkably, most hydrogels and hybrid hydrogels show enhanced human dermal cell proliferation and could be degraded into non‐toxic constituents, showing great promise as wound dressing materials.

Generation of Human Induced Pluripotent Stem Cells and Differentiation into Cardiomyocytes.

Failure to regenerate myocardium after injury is a major cause of mortality and morbidity in humans. Direct differentiation of human induced pluripotent stem cells (iPSCs) into cardiomyocytes provides an invaluable resource to pursue cardiac regeneration based on cellular transplantation. Beyond the potential for clinical therapies, iPSC technology also enables the generation of cardiomyocytes to recapitulate patient-specific phenotypes, thus presenting a powerful in vitro cell-based model to understand disease pathology and guide precision medicine. Here, we describe protocols for reprogramming of human dermal fibroblasts and blood cells into iPSCs using the non-integrative Sendai virus system and for the monolayer differentiation of iPSCs to cardiomyocytes using chemically defined media.

Small Extracellular Vesicles from Human Fetal Dermal Cells and Their MicroRNA Cargo: KEGG Signaling Pathways Associated with Angiogenesis and Wound Healing.

The use of cell secreted factors in clinical settings could be an alternative to conventional cell therapy, with the advantage of limiting concerns generally associated with traditional cell transplantation, such as tumorigenicity, immunoreactivity, and carrying of infections. Based on our published data, we predict a potential role for extracellular vesicles (EVs) in contributing to the proangiogenic activity of human fetal dermal cell secretome. Depletion of nanosized EVs from secretome significantly impaired its ability to induce formation of mesh-like structures in vitro. The isolated EVs were characterized for size and concentration by nanoparticle tracking analysis, and for protein markers (Rab5+, Alix+, CD63+, and calnexin−). The microRNA profile of EVs revealed 87 microRNAs significantly upregulated (≥15-fold increase) in fetal compared to adult dermal cell-derived EVs. Interestingly, these upregulated microRNAs included microRNAs with a validated role in angiogenesis according to literature. Moreover, the DIANA-TarBase v7.0 analysis confirmed enrichment in the KEGG signaling pathways associated with angiogenesis and wound healing, with the identification of putative target genes including thrombospondin 1. To validate the in silico data, EVs were also characterized for total protein contents. When tested in in vitro angiogenesis, fetal dermal cell-derived EVs were more effective than their adult counterpart in inducing formation of complete mesh-like structures. Furthermore, treatment of fibroblasts with fetal dermal-derived EVs determined a 4-fold increase of thrombospondin 1 protein amounts compared with the untreated fibroblasts. Finally, visualization of CSFE-labeled EVs in the cytosol of target cells suggested a successful uptake of these particles at 4-8 hours of incubation. We conclude that EVs are important contributors of the proangiogenic effect of fetal dermal cell secretome. Hence, EVs could also serve as vehicle for a successful delivery of microRNAs or other molecules of therapeutic interest to target cells.

Hair Regeneration Potential of Human Dermal Sheath Cells Cultured Under Physiological Oxygen.

We investigated the effect of oxygen tension on the proliferation and hair-inductive capacity of human dermal papilla cells (DPCs) and dermal sheath cells (DSCs). DPCs and DSCs were separately obtained from human hair follicles and each cultured under atmospheric/hyperoxic (20% O2), physiological/normoxic (6% O2), or hypoxic (1% O2) conditions. Proliferation of DPCs and DSCs was highest under normoxia. Compared with hyperoxia, hypoxia inhibited proliferation of DPCs, but enhanced that of DSCs. In DPCs, hypoxia downregulated the expression of hair-inductive capacity-related genes, including BMP4, LEF1, SOX2, and VCAN. In DSCs, both normoxia and hypoxia upregulated SOX2 expression, whereas hypoxia downregulated BMP4 expression. Microarray analysis revealed that normoxia increased the expression of pluripotency-related genes, including SPRY, NR0B1, MSX2, IFITM1, and DAZL, compared with hyperoxia. In an in vivo hair follicle reconstitution assay, cultured DPCs and DSCs were transplanted with newborn mouse epidermal keratinocytes into nude mice using a chamber method. In this experiment, normoxia resulted in the most efficient induction of DPC hair follicles, whereas hypoxia caused the most efficient induction and maturation of DSC hair follicles. These results suggest that application of physiological/hypoxic oxygen tension to cultured human DSCs enhances proliferation and maintenance of hair inductivity for skin engineering and clinical applications. Impact statement Dermal sheath cells (DSCs) and dermal papilla cells (DPCs) are useful cell sources for cell-based regenerative therapy. This is the first report to describe that low-oxygen conditions are better for DSCs. Normoxic and hypoxic culture of DSCs is beneficial for expanding these hair follicular cells and advancing development of cell-based therapy for both wound healing and hair regeneration. The current study supports that optimized oxygen tension can be applied to use expanded human DPCs and DSCs for skin engineering and clinical applications.

235 The importance of sirtuins in skin and new findings about sirt2 and its link to mechanobiology

Sirtuins (SIRTs) are a family of NAD+-dependent histone deacetylases that play a critical role in many diverse cellular processes including transcriptional signaling, gene silencing, metabolism, genomic stability, inflammation, energy, stress response and aging. The objectives of this study were to define the temporal role of SIRTs in skin by kinetically quantifying SIRT expression and to assess environmental impact by examining the response to environmental stressors via measuring the effect on energy (ATP) production and oxidative damage (ROS). Additionally, the effect on mechanical properties such as collagen production were measured. Finally, we demonstrate the importance of SIRT2 and its effect on age-induced changes in cell shape. Sirtuin expression levels by Normal Human Epidermal Keratinocytes (NHEK) were measured by RT-PCR. NHEK were exposed to low doses of UVB (10mJ/cm2). Cellular energy (ATP) production as well as oxidative damage (H2O2) were measured. Finally, the effects of SIRTs on pro-collagen type I production in Normal Human Dermal Fibroblasts (NHDF) and cell spreading by aged NHDF were measured. Temporal differences in sirtuin expression levels were observed over time. Furthermore, SIRTs were impacted by environmental stressors such as UVB exposure and ozone, resulting in differences in ATP and H2O2 production. These data show that SIRTs 1,2,3 and 6 support cellular activity necessary for skin mechanical properties such as boosting collagen production and through SIRT-2 increasing cellular-spreading in aging skin cells. SIRTs exhibit temporal expression and are sensitive to environmental stressors. We show that SIRTs are beneficial for both cellular integrity and repair activity. Therefore, supporting their activity is imperative for maintaining optimal skin cell activity.

Development of dynamic cell and organotypic skin models, for the investigation of a novel visco-elastic burns treatment using molecular and cellular approaches

BackgroundBurn injuries are a major cause of morbidity and mortality worldwide. Despite advances in therapeutic strategies for the management of patients with severe burns, the sequelae are pathophysiologically profound, up to the systemic and metabolic levels. Management of patients with a severe burn injury is a long-term, complex process, with treatment dependent on the degree and location of the burn and total body surface area (TBSA) affected. In adverse conditions with limited resources, efficient triage, stabilisation, and rapid transfer to a specialised intensive care burn centre is necessary to provide optimal outcomes. This initial lag time and the form of primary treatment initiated, from injury to specialist care, is crucial for the burn patient. This study aims to investigate the efficacy of a novel visco-elastic burn dressing with a proprietary bio-stimulatory marine mineral complex (MXC) as a primary care treatment to initiate a healthy healing process prior to specialist care. MethodsA new versatile emergency burn dressing saturated in a >90% translucent water-based, sterile, oil-free gel and carrying a unique bio-stimulatory marine mineral complex (MXC) was developed. This dressing was tested using LabSkin as a burn model platform. LabSkin a novel cellular 3D-dermal organotypic full thickness human skin equivalent, incorporating fully-differentiated dermal and epidermal components that functionally models skin. Cell and molecular analysis was carried out by in vitro Real-Time Cellular Analysis (RTCA), thermal analysis, and focused transcriptomic array profiling for quantitative gene expression analysis, interrogating both wound healing and fibrosis/scarring molecular pathways. In vivo analysis was also performed to assess the bio-mechanical and physiological effects of this novel dressing on human skin. ResultsThis hybrid emergency burn dressing (EBD) with MXC was hypoallergenic, and improved the barrier function of skin resulting in increased hydration up to 24 h. It was demonstrated to effectively initiate cooling upon application, limiting the continuous burn effect and preventing local tissue from damage and necrosis. xCELLigence RTCA® on primary human dermal cells (keratinocyte, fibroblast and micro-vascular endothelial) demonstrated improved cellular function with respect to tensegrity, migration, proliferation and cell-cell contact (barrier formation) [1]. Quantitative gene profiling supported the physiological and cellular function finding. A beneficial quid pro quo regulation of genes involved in wound healing and fibrosis formation was observed at 24 and 48 h time points. ConclusionUtilisation of this EBD + MXC as a primary treatment is an effective and easily applicable treatment in cases of burn injury, proving both a cooling and hydrating environment for the wound. It regulates inflammation and promotes healing in preparation for specialised secondary burn wound management. Moreover, it promotes a healthy remodelling phenotype that may potentially mitigate scarring. Based on our findings, this EBD + MXC is ideal for use in all pre-hospital, pre-surgical and resource limited settings.