Cultures Of Human Dermal Fibroblasts Research Articles

Arborvitae (Thuja plicata) essential oil significantly inhibited critical inflammation- and tissue remodeling-related proteins and genes in human dermal fibroblasts

Arborvitae (Thuja plicata) essential oil (AEO) is becoming increasingly popular in skincare, although its biological activity in human skin cells has not been investigated. Therefore, we sought to study AEO's effect on 17 important protein biomarkers that are closely related to inflammation and tissue remodeling by using a pre-inflamed human dermal fibroblast culture model. AEO significantly inhibited the expression of vascular cell adhesion molecule 1 (VCAM-1), intracellular cell adhesion molecule 1 (ICAM-1), interferon gamma-induced protein 10 (IP-10), interferon-inducible T-cell chemoattractant (I-TAC), monokine induced by interferon gamma (MIG), and macrophage colony-stimulating factor (M-CSF). It also showed significant antiproliferative activity and robustly inhibited collagen-I, collagen-III, plasminogen activator inhibitor-1 (PAI-1), and tissue inhibitor of metalloproteinase 1 and 2 (TIMP-1 and TIMP-2). The inhibitory effect of AEO on increased production of these protein biomarkers suggests it has anti-inflammatory property. We then studied the effect of AEO on the genome-wide expression of 21,224 genes in the same cell culture. AEO significantly and diversely modulated global gene expression. Ingenuity pathway analysis (IPA) showed that AEO robustly affected numerous critical genes and signaling pathways closely involved in inflammatory and tissue remodeling processes. The findings of this study provide the first evidence of the biological activity and beneficial action of AEO in human skin cells.

Essential oils diversely modulate genome-wide gene expression in human dermal fibroblasts

The increasing popularity of essential oils for skincare has led to investigation of their biological effects in human skin cells. In this study, we investigated the biological activities of three commercially available essential oils, i.e. rosemary oil, wild orange oil, and a blend (commercial name: Deep Blue) composed of oils from wintergreen, camphor, peppermint, blue tansy, German chamomile, Helichrysum, and Osmanthus, in a pre-inflamed human dermal fibroblast culture model, simulating chronic inflammation. The impact of essential oils on proteins associated with inflammation and tissue remodeling and on the genome-wide expression of 21,224 genes was investigated. The three essential oils diversely modulated global gene expression. Ingenuity Pathway Analysis showed that the oils affected numerous critical genes and signaling pathways. Specifically, rosemary oil influenced processes involved in cancer signaling and metabolism; orange oil affected processes related to cancer signaling, immunomodulation, and metabolism; the blend influenced inflammation, immunomodulation, and wound healing. These findings are largely consistent with the existing literature, supporting the beneficial biological activities of these essential oils. Our study provides the first evidence indicating how these essential oils affect genome-wide gene expression in human skin cells and establishes a basis for further research into their biological mechanisms of action.

L-lactide ring-opening polymerization on a magnesium catalyst based on acenaphthene-1,2-diimine ligand: Development of biocompatible materials for osteoplasty

Isotactic poly-L-lactide was prepared by L-lactide ring-opening polymerization in tetrahydrofuran solution in the presence of a magnesium complex derived from 1,2-bis-[(2,6-diisopropyl)phenylimino]acenaphthene (dpp-bian). Polymer specimens in the form of cylindrical blocks, prepared by hot molding, were tested in experiments with human dermal fibroblast cultures. The polymers prepared exhibit no cytotoxicity, which opens prospects for performing their preclinical animal tests.

Effect of silver/copper and copper oxide nanoparticle powder on growth of Gram-negative and Gram-positive bacteria and their toxicity against the normal human dermal fibroblasts

Engineered nanomaterials, especially metallic nanoparticles, are the most popular system applied in daily life products. The study of their biological and toxicity properties seems to be indispensable. In this paper, we present results of biological activity of Ag/Cu nanoparticles. These nanoparticles show more promising killing/inhibiting properties on Gram-negative bacteria than for Gram-positive ones. The Gram-negative bacteria show strong effect already at the concentration of 1 ppm after 15 min of incubation. Moreover, in vitro tests of toxicity made on normal human dermal fibroblast cultures showed that after 72 h of incubation with Ag/Cu nanoparticles, they are less toxic then Cu2O/CuO nanoparticles.

New E-beam-initiated hyaluronan acrylate cryogels support growth and matrix deposition by dermal fibroblasts

Cryogels made of components of natural extracellular matrix components are potent biomaterials for bioengineering and regenerative medicine. Human dermal fibroblasts are key cells for tissue replacement during wound healing. Thus, any biomaterial for wound healing applications should enable growth, differentiation and matrix synthesis by these cells.Cryogels are highly porous scaffolds consisting of a network of interconnected pores. Here, we used a novel group of cryogels generated from acrylated hyaluronan where the polymerization was initiated by accelerated electrons (E-beam). This novel procedure omits any toxic polymerization initiators and results in sterile, highly elastic scaffolds with adjustable pore size, excellent swelling and low flow resistance properties.We show that these cryogels are effective 3D-substrates for long-term cultures of human dermal fibroblasts in vitro. The cells proliferate for at least 28days throughout the cryogels and deposit their own matrix in the pores. Moreover, key modulators of dermal fibroblasts during wound healing like TGFβ and PDGF efficiently stimulated the expression of wound healing-relevant genes.In conclusion, electron beam initiated cryogels of acrylated hyaluronan represent a functional and cell compatible biomaterial that could be adapted for special wound healing applications by further functionalization.

Plasma Rich in Growth Factors Inhibits Ultraviolet B Induced Photoageing of the Skin in Human Dermal Fibroblast Culture

Ultraviolet irradiation is able to deeply penetrate into the dermis and alter fibroblast structure and function, leading to a degradation of the dermal extracellular matrix. The regenerative effect of plasma rich in growth factors (PRGF) on skin ageing was investigated using UVB photo-stressed human dermal fibroblasts as an in vitro culture model. PRGF was assessed over the main indicative features of ultraviolet B irradiation, including ROS formation, cell viability and death detection, apoptosis/ necrosis analysis and biosynthetic activity measurement. Four different UV irradiation protocols were tested in order to analyze the beneficial effects of PRGF. Ultraviolet irradiation exhibited a dose dependent cytotoxicity and dose of 400mJ/cm2 was selected for subsequent experiments. PRGF increased the cell viability and decreased the cell death comparing to the non-treated group. The apoptosis and necrosis were significantly lower in PRGF treated fibroblasts. ROS production after UV irradiation was significantly reduced in the presence of PRGF. Procollagen type I, hyaluronic acid and TIMP-1 levels were higher in the when treated with PRGF. This preliminary in vitro study suggests that PRGF is able to prevent UVB derived photooxidative stress and to diminish the cell damage caused by ultraviolet irradiation.

Low, but not too low, oxygen tension and macromolecular crowding accelerate extracellular matrix deposition in human dermal fibroblast culture

A key challenge of in vitro organogenesis is the development in timely manner tissue equivalents. Herein, we assessed the simultaneous effect of oxygen tension (0.5%, 2% and 20%), foetal bovine serum concentration (0.5% and 10%) and macromolecular crowding (75μg/ml carrageenan) in human dermal fibroblast culture. Our data demonstrate that cells cultured at 2% oxygen tension, in the presence of carrageenan and at 0.5% serum concentration deposited within 3days in culture more extracellular matrix than cells grown for 14days, at 20% oxygen tension, 10% serum concentration and in the absence of carrageenan. These data suggest that optimal oxygen tension coupled with macromolecular crowding are important in vitro microenvironment modulators for accelerated development of tissue-like modules in vitro. To enable clinical translation and commercialisation of in vitro organogenesis therapies, we cultured human dermal fibroblast at 2% oxygen tension, under macromolecular crowding conditions (75μg/ml carrageenan) and at low foetal bovine serum concentration (0.5%). Within 3days in culture, more extracellular matrix was deposited under these conditions than cells grown for 14days, at 20% oxygen tension, 10% FBS concentration and in the absence of crowding agents. These data bring us closer to the development of more clinically relevant tissue-like modules.

Chum salmon egg extracts induce upregulation of collagen type I and exert antioxidative effects on human dermal fibroblast cultures.

Components of fish roe possess antioxidant and antiaging activities, making them potentially very beneficial natural resources. Here, we investigated chum salmon eggs (CSEs) as a source of active ingredients, including vitamins, unsaturated fatty acids, and proteins. We incubated human dermal fibroblast cultures for 48 hours with high and low concentrations of CSE extracts and analyzed changes in gene expression. Cells treated with CSE extract showed concentration-dependent upregulation of collagen type I genes and of multiple antioxidative genes, including OXR1, TXNRD1, and PRDX family genes. We further conducted in silico phylogenetic footprinting analysis of promoter regions. These results suggested that transcription factors such as acute myeloid leukemia-1a and cyclic adenosine monophosphate response element-binding protein may be involved in the observed upregulation of antioxidative genes. Our results support the idea that CSEs are strong candidate sources of antioxidant materials and cosmeceutically effective ingredients.

Platelet-rich plasma stimulates human dermal fibroblast proliferation via a Ras-dependent extracellular signal-regulated kinase 1/2 pathway

Platelet-rich plasma (PRP) contains a high concentration of several growth factors and contributes to soft-tissue engineering and wound healing. However, the effect of PRP on human dermal fibroblast proliferation and responses is unknown. This was investigated in the present study using PRP prepared from the whole human blood using the double-spin method. Human dermal fibroblast cultures were established from skin samples collected during plastic surgery. Platelet concentration and growth factor levels in PRP were estimated, and a cell proliferation assay was carried out after PRP treatment. The role of Ras-dependent extracellular signal-regulated kinase (ERK)1/2 in the effects of PRP was investigated in human dermal fibroblasts by suppressing ERK1/2 expression with an inhibitor or by short interfering (si)RNA-mediated knockdown, and assessing ERK1/2 phosphorylation by western blotting as well as proliferation in PRP-treated cells. We found that PRP stimulated human dermal fibroblast proliferation, which was suppressed by ERK1/2 inhibitor treatment (P<0.01). ERK1/2 phosphorylation was increased in the presence of PRP, while siRNA-mediated knockdown of ERK1/2 blocked cell proliferation normally induced by PRP treatment (P<0.01). These results demonstrate that PRP induces human dermal fibroblast proliferation via activation of ERK1/2 signaling. Our findings provide a basis for the development of agents that can promote wound healing and can be applied to soft-tissue engineering.

Specific functional pathologies of Cx43 mutations associated with oculodentodigital dysplasia.

Oculodentodigital dysplasia (ODDD) is a rare genetic disease that affects the development of multiple organs in the human body. More than 70 mutations in the gap junction connexin43 (Cx43) gene, GJA1, are associated with ODDD, most of which are inherited in an autosomal dominant manner. Many patients exhibit similar clinical presentations. However, there is high intrafamilial and interfamilial phenotypic variability. To better understand this variability, we established primary human dermal fibroblast cultures from several ODDD patients and unaffected controls. In the present study, we characterized three fibroblast lines expressing heterozygous p.L7V, p.G138R, and p.G143S Cx43 variants. All ODDD fibroblasts exhibited slower growth, reduced migration, and defective cell polarization, traits common to all ODDD fibroblasts studied so far. However, we found striking differences in overall expression levels, with p.L7V down-regulated at the mRNA and protein level. Although all of the Cx43 variants could traffic to the cell surface, there were stark differences in gap junction plaque formation, gap junctional intercellular communication, Cx43 phosphorylation, and hemichannel activity among Cx43 variants, as well as subtle differences in myofibroblast differentiation. Together these findings enabled us to discover mutation-specific pathologies that may help to predict future clinical outcomes.

Combined effects of low-level laser therapy and human bone marrow mesenchymal stem cell conditioned medium on viability of human dermal fibroblasts cultured in a high-glucose medium.

Low-level laser therapy (LLLT) exhibited biostimulatory effects on fibroblasts viability. Secretomes can be administered to culture mediums by using bone marrow mesenchymal stem cells conditioned medium (BM-MSCs CM). This study investigated the combined effects of LLLT and human bone marrow mesenchymal stem cell conditioned medium (hBM-MSCs CM) on the cellular viability of human dermal fibroblasts (HDFs), which was cultured in a high-glucose (HG) concentration medium. The HDFs were cultured either in a concentration of physiologic (normal) glucose (NG; 5.5 mM/l) or in HG media (15 mM/l) for 4 days. LLLT was performed with a continuous-wave helium-neon laser (632.8 nm, power density of 0.00185 W/cm(2) and energy densities of 0.5, 1, and 2 J/cm(2)). About 10% of hBM-MSCs CM was added to the HG HDF culture medium. The viability of HDFs was evaluated using dimethylthiazol-diphenyltetrazolium bromide (MTT) assay. A significantly higher cell viability was observed when laser of either 0.5 or 1 J/cm(2) was used to treat HG HDFs, compared to the control groups. The cellular viability of HG-treated HDFs was significantly lower compared to the LLLT + HG HDFs, hBM-MSCs CM-treated HG HDFs, and LLLT + hBM-MSCs CM-treated HG HDFs. In conclusion, hBM-MSCs CM or LLLT alone increased the survival of HG HDFs cells. However, the combination of hBM-MSCs CM and LLLT improved these results in comparison to the conditioned medium.

Human dermal fibroblasts cultured on direct-spinning silk fibroin sheet scaffolds: free cells seeding

Nonwoven silk fibroin membranes were directly prepared by spinning Thai race silkworms and used as scaffolds for culturing of human dermal fibroblasts (HDFs). The silk fibroin scaffolds (SFS) were characterized using SEM, and a microtester was used to detect the mechanical properties. The HDFs were seeded on the scaffolds to investigate cellular biocompatibility. The SFS showed good compatibility with the HDFs as the cells showed cell attachment, viability, and growth up to the last day of the culture period (day 28). SFS could be used for HDFs culture.

A three-dimensional skin equivalent reflecting some aspects of in vivo aged skin.

Human skin undergoes morphological, biochemical and functional modifications during the ageing process. This study was designed to produce a 3-dimensional (3D) skin equivalent in vitro reflecting some aspects of in vivo aged skin. Reconstructed skin was generated by co-culturing skin fibroblasts and keratinocytes on a collagen-glycosaminoglycan-chitosan scaffold, and ageing was induced by the exposition of fibroblasts to Mitomycin-C (MMC). Recently published data showed that MMC treatment resulted in a drug-induced accelerated senescence (DIAS) in human dermal fibroblast cultures. Next to established ageing markers, histological changes were analysed in comparison with in vivo aged skin. In aged epidermis, the filaggrin expression is reduced in vivo and in vitro. Furthermore, in dermal tissue, the amount of elastin and collagen is lowered in aged skin in vivo as well as after the treatment of 3D skin equivalents with MMC in vitro. Our results show histological signs and some aspects of ageing in a 3D skin equivalent in vitro, which mimics aged skin in vivo.

Synthetic ligands of the elastin receptor induce elastogenesis in human dermal fibroblasts via activation of their IGF-1 receptors

BackgroundWe have previously reported that a mixture of peptides obtained after chemical or enzymatic degradation of bovine elastin, induced new elastogenesis in human skin. ObjectiveNow, we investigated the elastogenic potential of synthetic peptides mimicking the elastin-derived, VGVAPG sequence, IGVAPG sequence that we found in the rice bran, and a similar peptide, VGVTAG that we identified in the IGF-1-binding protein-1 (IGFBP-1). ResultsWe now demonstrate that treatment with each of these xGVxxG peptides (recognizable by the anti-elastin antibody), up-regulated the levels of elastin-encoding mRNA, tropoelastin protein, and the deposition of new elastic fibers in cultures of human dermal fibroblasts and in cultured explants of human skin. Importantly, we found that such induction of new elastogenesis may involve two parallel signaling pathways triggered after activation of IGF-1 receptor. In the first one, the xGVxxG peptides interact with the cell surface elastin receptor, thereby causing the downstream activation of the c-Src kinase and a consequent cross-activation of the adjacent IGF-1R, even in the absence of its principal ligand. In the second pathway their hydrophobic association with the N-terminal domain (VGVTAG) of the serum-derived IGFBP-1 induces conformational changes of this IGF-1 chaperone allowing for the release of its cargo and a consequent ligand-specific phosphorylation of IGF-1R. ConclusionWe present a novel, clinically relevant mechanism in which products of partial degradation of dermal elastin may stimulate production of new elastic fibers by dermal fibroblasts. Our findings particularly encourage the use of biologically safe synthetic xGVxxG peptides for regeneration of the injured or aged human skin.

Hydrocellular foam dressing increases the leptin level in wound fluid.

Hydrocellular foam dressing (HCF) absorbs excessive wound fluid, which contains various cytokines and growth factors, and ensures a moist environment to promote wound healing. However, the molecular mechanisms underlying the wound fluid component changes induced by HCF are poorly understood. In the present study, we examined the effect of HCF on wound healing and the associated regulatory mechanisms in relation to variations in cytokine levels in the wound fluid. We created full-thickness wounds on the dorsolateral skin of rats and collected the resulting wound fluid samples. HCF was immersed in a plate containing the wound fluids. HCF was then removed and the excess wound fluid remaining in the plate was examined by cytokine array and enzyme-linked immunosorbent assay. We also used a rat model and human dermal fibroblast cultures to examine the effect of wound fluid component changes during the wound healing process. Upon treatment with HCF, leptin levels were upregulated in the wound fluid. Fibroblast proliferation was enhanced and the effect was suppressed in the presence of leptin antagonist. In our in vivo model, HCF increased wound contraction compared with film dressings and this positive effect of HCF was suppressed by addition of leptin antagonist. Our results suggest that dermal fibroblast proliferation is upregulated by HCF due to increased leptin level at the wound surface, and these effects promote wound healing. We believe that the present study contributes to furthering the understanding of the mechanisms underlying the effects of HCF-induced wound healing.

Regeneration of elastic fibers by three-dimensional culture on a collagen scaffold and the addition of latent TGF-β binding protein 4 to improve elastic matrix deposition

The objective of this study was to investigate the effects of latent TGF-β binding protein 4 (LTBP-4) on elastic fiber regeneration in three-dimensional cultures of human dermal fibroblasts (HDFs). Appropriate collagen scaffold for elastic fiber regeneration was also examined. Collagen sponges cross-linked at 120 °C and composed of small pores (25 μm on average) was favorable for elastic fiber regeneration by HDFs. Addition of LTBP-4, followed by culture for 21 days, accelerated elastic fiber accumulation within the scaffolds. Conditioned scaffolds containing either HDFs or LTBP-4-built mature elastic fibers were implanted between the dermis and the cutaneous muscle of mice. The combined use of HDFs and LTBP-4 resulted in thicker tissues containing elastic fibers. These results indicate that weakly cross-linked collagen sponges can be used as scaffolds for regenerating elastic fibers both in vitro and in vivo, and that the addition of LTBP-4 accelerates the deposition of both elastin and fibrillin-1, and increases cell proliferation. These techniques may be useful for generating cutaneous or cardiovascular tissue equivalents; furthermore, they may serve as a useful method for the three-dimensional analyses of drugs used to treat skin diseases or to examine the microstructure of elastin networks.

Controlling the Crystallinity of Thermoresponsive Poly(2-oxazoline)-Based Nanolayers to Cell Adhesion and Detachment.

Semicrystalline, thermoresponsive poly(2-isopropyl-2-oxazoline) (PIPOx) layers covalently bonded to glass or silica wafers were obtained via the surface-termination of the living polymer chains. Polymer solutions in acetonitrile were exposed to 50 °C for various time periods and were poured onto the functionalized solid wafers. Fibrillar crystallites formed in polymerization solutions settled down onto the wafers next to the amorphous polymer. The amount of crystallites adsorbed on thermoresponsive polymer layers depended on the annealing time of the PIPOx solution. The wettability of PIPOx layers decreased with the increasing amount of crystallites. The higher content of crystallites weakened the temperature response of the layer, as evidenced by the philicity and thickness measurements. Semicrystalline thermoresponsive PIPOx layers were used as biomaterials for human dermal fibroblasts (HDFs) culture and detachment. The presence of crystallites on the PIPOx layers promoted the proliferation of HDFs. Changes in the physicochemical properties of the layer, caused by the temperature response of the polymer, led to the change in the cells shape from a spindle-like to an ellipsoidal shape, which resulted in their detachment. A supporting membrane was used to assist the detachment of the cells from PIPOx biosurfaces and to prevent the rolling of the sheet.

A New Natural Extract with Anti‐Aging and Regenerative Properties for Skin

Objective:The skin, like other organs, is subject to chronological ageing. In addition, exposure to ultraviolet radiation can lead to a reduction in collagen and an increased expression of Matrix metalloproteinases (MMPs).The aim of this study is to determine the anti‐aging and regenerative effect of BIX003, a natural extract from porcine cartilage with Cosmetic and Nutricosmetic potential. BIX003 is a natural product rich in bioactive substances due to the mild conditions used in the manufacturing process.Methods:The anti‐aging action was determined using a culture of human dermal fibroblasts (HDF). The potential to increase skin elasticity was determined by quantifying elastin levels and the effect on MMP‐1 production was quantified by ELISA. Effects on proliferation of HDF were also tested.Results:BIX003 induced a significant increase of HDF proliferation. In addition, the extract showed a potent induction of elastin synthesis up to 12%. Moreover, it was a potent inhibitor of the MMP‐1 production at all tested concentrations in a dose‐dependent manner. The same effect was observed in MMP‐1 activity (up to 70% reduction). Finally, a mild hydrating action was observed (12% increase at 500 µg/mL).ConclusionsThese data demonstrate that BIX003 exerts its anti‐ageing benefits not only via enhanced fibroblast proliferation, but also through an increase in elastin synthesis and a potent inhibition of production and activity of MMP‐1. All together, these results suggest that this natural extract may be useful for Skin Care, specially for mature skin in which there is a reduction in skin firmness due to the increased MMP synthesis.Supported by BIOIBERICA S.A., Barcelona, Spain.

Modulation of expression of genes involved in glycosaminoglycan metabolism and lysosome biogenesis by flavonoids.

Flavonoids were found previously to modulate efficiency of synthesis of glycosaminoglycans (GAGs), compounds which are accumulated in cells of patients suffering from mucopolysaccharidoses (MPSs). The aim of this work was to determine effects of different flavonoids (genistein, kaempferol, daidzein) used alone or in combinations, on expression of genes coding for proteins involved in GAG metabolism. Analyses with DNA microarray, followed by real-time qRT-PCR revealed that genistein, kaempferol and combination of these two compounds induced dose- and time-dependent remarkable alterations in transcript profiles of GAG metabolism genes in cultures of wild-type human dermal fibroblasts (HDFa). Interestingly, effects of the mixture of genistein and kaempferol were stronger than those revealed by any of these compounds used alone. Similarly, the most effective reduction in levels of GAG production, in both HDFa and MPS II cells, was observed in the presence of genistein, keampferol and combination of these compounds. Forty five genes were chosen for further verification not only in HDFa, but also in MPS II fibroblasts by using real-time qRT-PCR. Despite effects on GAG metabolism-related genes, we found that genistein, kaempferol and mixture of these compounds significantly stimulated expression of TFEB. Additionally, a decrease in MTOR transcript level was observed at these conditions.

Reciprocity in breast cancer progression.

Reciprocity in breast cancer progression.