Human Skin Fibroblast Cultures Research Articles

Growth Suppressing Activity of Lhx2 in Human T Cell Acute Lymphoblastic Leukemia (T-ALL)-Derived Cells and Large-Scale Screening of a Lead Compounds Targeting T-ALL

Acute lymphoblastic leukemia (ALL) is one of the most frequently occurring cancers in infants and young children. For patients suffering from CD20-positive B-cell ALL (B-ALL) and Ph-positive ALL, overall survival rates have been greatly improved after clinical introduction of rituximab and imatinib, respectively. However, T-cell ALL (T-ALL) patients still exhibit poor prognosis, since there is no such an efficient molecular-targeted drug.It is known that LIM-only transcriptional co-factor LMO2 and its target gene HHEX are essential for self-renewal of T cell precursors and onset of T-ALL. LMO2 directly associates with LDB1 in a large DNA-containing nuclear complex and controls the transcription of T-ALL-related downstream genes. Recently, we reported that overexpression of LIM-homeodomain transcription factor Lhx2 results in liberation of Lmo2 protein from the Lmo2-Ldb1 complex followed by degradation by ubiquitin-proteasome system. We found that proliferation of 5 different human T-ALL-derived cell lines including CCRF-CEM was significantly suppressed by retroviral overexpression of Lhx2. In contrast, enforced overexpression of Lhx2 did not reduce the growth rate of B lymphoma-derived cell line Raji, oral cancer-derived cell line HSC-3, and osteosarcoma-derived cell line Saos-2. Majority of the Lhx2-transduced CCRF-CEM cells was arrested in G0 phase and subsequently underwent apoptosis. Expression of LMO2 protein and HHEX mRNA was repressed by the Lhx2 transduction. Importantly, the Lhx2-mediated growth inhibition was partially rescued by the simultaneous overexpression of Lmo2. However, both C-terminal LIM-domain and homeodomain of Lhx2 were required for the growth-suppressive activity. These data indicated that Lhx2 is capable to blocking proliferation of T-ALL-derived cells in LMO2-dependent and independent fashions. Lhx2 would be a useful molecular tool for designing a new type of anti-T-ALL drug.In order to develop a new drug that mimics the aforementioned activity of Lhx2, we performed large-scale screening of natural compound libraries to find out a compound that suppresses the proliferation of T-ALL cell line CCRF-CEM, but does not inhibit the growth of B lymphoma cell line Raji. Among 150,000 different compounds, we successfully identified 3 low-molecular-weight compounds (44D-L008, 31D-F005, 21D-D016) that fulfilled the above criteria. 44D-L008 and 31D-F005 possessed a common chemical structure. In the presence of 5μM of 44D-L008 and 31D-F005, proliferation of 5 human T-ALL cell lines including CCRF-CEM was severely blocked. On the other hand, Raji, HSC-3 and Saos-2 were completely resistant to both compounds in the same experimental settings. Intriguingly, 44D-L008 decreased viability of human skin fibroblasts in culture, whereas 31D-F005 displayed no such negative effects on skin fibroblasts, peripheral blood mononuclear cells, and peripheral blood T cells of human origin. These results indicated that small differences in the chemical structure between 44D-L008 and 31D-F005 are responsible for the side effect on normal cells. Finally, we collaborated with a hospital and found that 0.5 μM of 31D-F005 efficiently suppressed the in vitro growth of primary cancer cells of a T-ALL patient. Taken together, theses data demonstrated that 31D-F005 is a promising lead compound for a new anti-T-ALL drug. DisclosuresNo relevant conflicts of interest to declare.

Low doses of nanodiamonds and silica nanoparticles have beneficial hormetic effects in normal human skin fibroblasts in culture

Nanodiamonds (ND) and silica nanoparticles (SiO2-NP) have been much investigated for their toxicity at high doses, little is known about their biological activity at low concentrations. Here we report the biphasic dose response of ND and SiO2-NP in modulating normal human facial skin fibroblasts (FSF1) in culture. ND and SiO2-NP at low concentration (up to 0.5 μg/ml) had beneficial effects on FSF1 in terms of increasing their proliferation and metabolic activity. Exposure of FSF1 cells to low levels of NP enhanced their wound healing ability in vitro and slowed down aging during serial passaging as measured by maintenance of youthful morphology, reduction in the rate of loss of telomeres, and the over all proliferative characteristics. Furthermore, NP treatment induced the activation of Nrf2- and FOXO3A-mediated cellular stress responses, including an increased expression of heme oxygenease (HO-1), sirtuin (SIRT1), and DNA methyltransferase II (DNMT2). These results imply that ND and SiO2-NP at low doses are potential hormetins, which exert mild stress-induced beneficial hormetic effects through improved survival, longevity, maintenance, repair and function of human cells.

Native Australian plant extracts differentially induce Collagen I and Collagen III in vitro and could be important targets for the development of new wound healing therapies

Australian native plants have a long history of therapeutic use in indigenous cultures, however, they have been poorly studied scientifically. We analysed the effects of 14 plant derived compounds from the species Pilidiostigma glabrum, Myoporum montanum, Geijera parviflora, and Rhodomyrtus psidioides for their potential wound healing properties by assessing their ability to induce or suppress Collagen I and Collagen III expression in human skin fibroblasts in culture. The compound 7-geranyloxycoumarin was able to significantly increase Collagen I (23.7%, p<0.0002) expression in comparison to control. Significant suppression of Collagen III was observed for the compounds flindersine (11.1%, p<0.02), and (N-acetoxymethyl) flindersine (27%, p<0.00005). The implications of these finding is that these compounds could potentially alter the expression of different collagens in the skin allowing for the potential development of new wound healing therapies and new approaches for treating various skin diseases as well as photo (sun) damaged, and aged skin.

Hyaluronic acid abrogates ethanol-dependent inhibition of collagen biosynthesis in cultured human fibroblasts.

IntroductionThe aim of the study was to evaluate the effect of ethanol on collagen biosynthesis in cultured human skin fibroblasts, and the role of hyaluronic acid (HA) in this process. Regarding the mechanism of ethanol action on human skin fibroblasts we investigated: expression of β1 integrin and insulin-like growth factor 1 receptor (IGF-IR), signaling pathway protein expression: mitogen-activated protein kinases (MAPKs), protein kinase B (Akt), nuclear factor kappa B (NF-κB) transcription factor, cytotoxicity assay and apoptosis, metalloproteinase activity, as well as the influence of HA on these processes.Materials and methodsCollagen biosynthesis, activity of prolidase, DNA biosynthesis, and cytotoxicity were measured in confluent human skin fibroblast cultures that have been treated with 25, 50, and 100 mM ethanol and with ethanol and 500 µg/mL HA. Western blot analysis and zymography were performed to evaluate expression of collagen type I, β1 integrin receptor, IGF-IR, NF-κB protein, phospho-Akt protein, kinase MAPK, caspase 9 activity, and matrix metalloproteinases (MMP-9 and MMP-2).ResultsEthanol in a dose-dependent manner lead to the impairment of collagen biosynthesis in fibroblast cultures through decreasing prolidase activity and expression of β1 integrin and IGF-IR. This was accompanied by an increased cytotoxicity, apoptosis and lowered expression of the signaling pathway proteins induced by β1 integrin and IGF-IR, that is, MAPK (ERK1/2) kinases. The lowered amount of synthesized collagen and prolidase activity disturbance may also be due to the activation of NF-κB transcription factor, which inhibits collagen gene expression. It suggests that the decrease in fibroblast collagen production may be caused by the disturbance in its biosynthesis but not degradation. The application of HA has a protective effect on disturbances caused by the examined substances. It seems that regulatory mechanism of ethanol-induced collagen aberration take place at the level of collagen biosynthesis, since no effect of ethanol and HA was found on process of collagen degradation by MMP-2 and MMP-9.ConclusionThis study provides evidence that ethanol impairs collagen metabolism in human skin fibroblasts, leading to a significant decrease in the amount of produced protein. This mechanism probably is due to downregulation of prolidase activity, expression of β1 integrin and IGF-IR receptors, and the signaling pathway proteins induced by these receptors.

STAT6-Dependent Collagen Synthesis in Human Fibroblasts Is Induced by Bovine Milk.

Since the domestication of the urus, 10.000 years ago, mankind utilizes bovine milk for different purposes. Besides usage as a nutrient also the external application of milk on skin has a long tradition going back to at least the ancient Aegypt with Cleopatra VII as a great exponent. In order to test whether milk has impact on skin physiology, cultures of human skin fibroblasts were exposed to commercial bovine milk. Our data show significant induction of proliferation by milk (max. 2,3-fold, EC50: 2,5% milk) without toxic effects. Surprisingly, bovine milk was identified as strong inducer of collagen 1A1 synthesis at both, the protein (4-fold, EC50: 0,09% milk) and promoter level. Regarding the underlying molecular pathways, we show functional activation of STAT6 in a p44/42 and p38-dependent manner. More upstream, we identified IGF-1 and insulin as key factors responsible for milk-induced collagen synthesis. These findings show that bovine milk contains bioactive molecules that act on human skin cells. Therefore, it is tempting to test the herein introduced concept in treatment of atrophic skin conditions induced e.g. by UV light or corticosteroids.

Influence of caffeine and hyaluronic acid on collagen biosynthesis in human skin fibroblasts.

AimThe aim of this study was to evaluate the effect of caffeine on collagen biosynthesis in human skin fibroblasts and the influence of hyaluronic acid (HA) on this process.Materials and methodsCollagen, [3H]-thymidine incorporation, and prolidase activity were measured in confluent human skin fibroblast cultures that had been treated with 1, 2, and 5 mM caffeine and with caffeine and 500 μg/mL HA. Western immunoblot analysis was performed to evaluate expression of β1-integrin receptor, insulin-like growth factor receptor phospho-Akt protein and mitogen-activated protein kinase (phospho-extracellular signal-regulated kinase).ResultsCaffeine inhibited collagen biosynthesis in a dose-dependent manner. The mechanism of this process was found at the level of prolidase activity. Caffeine significantly inhibited the enzyme activity. The addition of HA had no effect on collagen biosynthesis or prolidase activity in fibroblasts incubated with caffeine. Caffeine also had an inhibitory effect on DNA biosynthesis. HA, however, did not have any significant effect on this process. The inhibition of the expression of β1-integrin and insulin-like growth factor receptor in fibroblasts incubated with the caffeine indicates a possible mechanism of inhibition of collagen biosynthesis.ConclusionCaffeine reduces collagen synthesis in human cultured skin fibroblasts. HA did not have any significant protective effect on this process. This is the first study to our knowledge that reports caffeine-induced inhibition of collagen synthesis in human skin fibroblasts.

Physical Properties and Biocompatibility of Oligochitosan Membrane Film as Wound Dressing

The physical and biological characteristics of oligochitosan (O-C) film, including its barrier and mechanical properties, in vitro cytotoxicity and in vivo biocompatibility, were studied to assess its potential use as a wound dressing. Membrane films were prepared from water-soluble O-C solution blended with various concentrations of glycerol to modify the physical properties of the films. In vitro and in vivo biocompatibility evaluations were performed using primary human skin fibroblast cultures and subcutaneous implantation in a rat model, respectively. Addition of glycerol significantly influenced the barrier and mechanical properties of the films. Water absorption capacity was in the range of 80%-160%, whereas water vapor transmission rate varied from 1,180 to 1,618 g/m2 per day. Both properties increased with increasing glycerol concentration. Tensile strength decreased while elongation at break increased with the addition of glycerol. O-C films were found to be noncytotoxic to human fibroblast cultures and histological examination proved that films are biocompatible. These results indicate that the membrane film from O-C has potential application as a wound-dressing material.

Zinc and propolis reduces cytotoxicity and proliferation in skin fibroblast cell culture: total polyphenol content and antioxidant capacity of propolis.

It has been demonstrated that zinc exerts its beneficial influence on skin fibroblasts. Propolis, a complex mixture of plant-derived and bees’ products, was reported to stimulate cicatrization processes in skin and prevent infections. The aim of this study was to find out how zinc and propolis influence human skin fibroblasts in cell culture and to compare the effect of individual compounds to the effect of a mixture of zinc and propolis. In this study, zinc, as zinc aspartate, at a concentration of 16 μM, increased human fibroblasts proliferation in cell culture, whereas propolis at a concentration of 0.01 % (w/v) revealed antiproliferative and cytotoxic action followed by mild cell necrosis. In culture, zinc was effectively transported into fibroblasts, and propolis inhibited the amount of zinc incorporated into the cells. An addition of propolis to the medium caused a decrease in the Zn(II) amount incorporated into fibroblasts. The obtained results also indicate an appreciable antioxidant property of propolis and revealed its potential as a supplement when applied at doses lower than 0.01 % (w/v). In conclusion, the present study showed that zinc had a protective effect on human cultured fibroblasts’ viability, although propolis revealed its antiproliferative action and caused mild necrosis.

A three-dimensional dual-layer nano/microfibrous structure of electrospun chitosan/poly(d,l-lactide) membrane for the improvement of cytocompatibility

In tissue engineering, structure control of biodegradable membrane scaffolds to shape developing tissues is critical for balanced organogenesis requirements. It is currently challenging to control a three-dimensional (3-D) membrane scaffold with beneficial growth of skin cells during wound-healing process. Here we report a new dual-layer fibrous structure made of electrospun chitosan/poly(d,l-lactic acid) (PDLLA) membrane for human skin fibroblast culture. In this work, the electrospinning process incorporated with rotating control was introduced to prepare the random PDLLA microfibrous matrix covered with aligned chitosan nanofibers, providing an increased specific interface area to promote cell attachment, migration and infiltration. The effects of electrospining parameters on the reduction of beads and the uniformity of fibers were determined. The morphology, proliferation and penetration of cells seeded on each electrospun fibrous membrane were observed using laser scanning confocal microscope (LSCM). A dual-layer fibrous membrane prepared with a highly uniform matrix layer of PDLLA microfibers and a well-defined cover layer of chitosan nanofibers without fibrous beads result in aligned migration and directional penetration of skin fibroblasts into 3-D domain. The chitosan nanofibers which covered on PDLLA microfibers promote the skin cells growth and improve their infiltration into the target scaffolds in vitro. Wound-healing test demonstrated that the dual-layer structure significantly improves the rat tissue regeneration in vivo. The results suggest that the dual-layer chitosan/PDLLA structure can mimic the interface of natural extracellular matrices and has the potential for 3-D tissue-engineering applications.

The performance of an orthosilicic acid-releasing silica gel fiber fleece in wound healing

In the present work, we have examined the impact of an inorganic orthosilicic acid-releasing spun fiber fleece (SIFIB) on wound closure in a porcine wound model in vivo as well as on wound healing-relevant parameters in vitro. In vivo SIFIB was completely bio-degradable and had no negative effects on wound closure or the wound healing process. In the in vitro experiments, SIFIB had no negative effects on proliferation of human skin fibroblast (FB) and endothelial cell (EC) cultures but strongly retarded the growth of the human monocyte cell line THP-1, and effectively inhibited human skin keratinocyte (KC) proliferation, which based on significantly enhanced KC differentiation. Furthermore, SIFIB exhibited strong anti-inflammatory properties, which based on SIFIB-dependent inhibition of expression and activity of NF-кB and/or concomitant enhanced expression of IкB, a NF-кB-inhibiting protein. Additionally, SIFIB significantly inhibited TGFβ-induced fibroblast differentiation and collagen synthesis as well as effectively reduced TGF-β synthesis of activated fibroblasts. We have demonstrated wound healing-relevant biological activities of a silica-based bio-degradable inorganic material, which might represent a new therapeutic tool in the treatment of chronic wounds.

Endothelin receptor antagonists: effects on extracellular matrix synthesis in primary cultures of skin fibroblasts from systemic sclerosis patients

Endothelin-1 (ET-1) seems to enhance the pro-fibrotic protein synthesis by skin fibroblasts and its effects are mediated by endothelin-A and B (ETA and ETB) receptors. This study aimed to investigate the effects of ETA and ETB receptor antagonists (ETARA-sitaxsentan and ETA/BRA-bosentan) on type I collagen (COL-1), fibronectin (FN) and fibrillin-1 (FBL-1) synthesis in primary cultures of skin fibroblasts from systemic sclerosis patients. Primary cultures of fibroblasts were obtained from skin biopsies of 6 female systemic sclerosis patients and were treated with ET-1 (100 nM) for 24 and 48 hrs with or without pre-treatment (1 hr) with ETARA (2 μM) or ETA/BRA (10 μM). Primary culture of human scleroderma skin fibroblasts not treated with ET-1 or ET receptor antagonists (ETARA and ETA/BRA) were used as controls. COL-1, FN and FBL-1 synthesis was evaluated by immunocytochemistry and Western blot analysis. Immunocytochemistry and Western blot analysis showed that ET-1 significantly increased COL-1 and FN synthesis at 24 and 48 hrs and FBL-1 synthesis at 48 hrs vs untreated cells. ETARA significantly contrasted the ET-1-mediated increase in COL-1 and FN at 24 hrs as well as COL-1 and FBL-1 at 48 hrs, but not FN synthesis vs ET-1-treated fibroblasts. Conversely, ETA/BRA significantly antagonized the ET-1-mediated overproduction of COL-1 and FN both at 24 and 48 hrs and the FBL-1 synthesis at 48 hrs vs ET-1-treated cells. The single ETARA treatment seems to contrast significantly the increase in COL-1 synthesis, whereas the dual ETA/BRA treatment seems active in significantly antagonizing both COL-1 and FN overproduction induced by ET-1. In conclusion, ET-1 antagonism might have positive effects in contrasting the profibrotic activity of systemic sclerosis skin fibroblasts.

RTTN Mutations Link Primary Cilia Function to Organization of the Human Cerebral Cortex

Polymicrogyria is a malformation of the developing cerebral cortex caused by abnormal organization and characterized by many small gyri and fusion of the outer molecular layer. We have identified autosomal-recessive mutations in RTTN, encoding Rotatin, in individuals with bilateral diffuse polymicrogyria from two separate families. Rotatin determines early embryonic axial rotation, as well as anteroposterior and dorsoventral patterning in the mouse. Human Rotatin has recently been identified as a centrosome-associated protein. The Drosophila melanogaster homolog of Rotatin, Ana3, is needed for structural integrity of centrioles and basal bodies and maintenance of sensory neurons. We show that Rotatin colocalizes with the basal bodies at the primary cilium. Cultured fibroblasts from affected individuals have structural abnormalities of the cilia and exhibit downregulation of BMP4, WNT5A, and WNT2B, which are key regulators of cortical patterning and are expressed at the cortical hem, the cortex-organizing center that gives rise to Cajal-Retzius (CR) neurons. Interestingly, we have shown that in mouse embryos, Rotatin colocalizes with CR neurons at the subpial marginal zone. Knockdown experiments in human fibroblasts and neural stem cells confirm a role for RTTN in cilia structure and function. RTTN mutations therefore link aberrant ciliary function to abnormal development and organization of the cortex in human individuals.

Michelia alba extract attenuates UVB-induced expression of matrix metalloproteinases via MAP kinase pathway in human dermal fibroblasts

Solar ultraviolet (UV) radiation can cause skin photoaging by inducing secretion of matrix metalloproteinases (MMPs). It has been reported that MMPs, especially MMP-1, -3 and -9, reduce elasticity of the dermis by degrading collagen. Polyphenols are a group of compounds that exist mainly in glycosides in the plants and they may transform to aglycone after hydrolysis. Polyphenols can inhibit MMP expression and elastase activity. In this study, we investigated the effects of Michelia alba extract (MAE) on expression and activity of MMPs in human skin fibroblast cultures after UVB exposure. The results showed that MAE and its hydrolysates (MAH) inhibited collagenase and elastase activities. In addition, MAE exhibited antioxidant activity, elevated hyaluronic acid content and inhibited UVB-induced MMP-1, MMP-3 and MMP-9 expression. In addition, the zymography assay revealed that MAE also inhibited MMP-9 activity. We also found that MAE inhibited UVB-induced ERK and JNK kinase but not p38 kinase expression, suggesting that MAE may regulate the UVB-induced expression of MMP-1, MMP-3 and MMP-9 via the ERK and JNK kinase pathway. MAE could restore total collagen synthesis reduced by UVB. The results also suggest that MAE treatment may prevent UVB-induced extracellular matrix damage by inhibiting the expression of MMP-1, MMP-3 and MMP-9 through the MAP kinase pathway. Our findings imply that MAE is an effective agent against UVB-induced photodamage.

Intracellular Proton-mediated Activation of TRPV3 Channels Accounts for the Exfoliation Effect of α-Hydroxyl Acids on Keratinocytes

α-Hydroxyl acids (AHAs) from natural sources act as proton donors and topical compounds that penetrate skin and are well known in the cosmetic industry for their use in chemical peels and improvement of the skin. However, little is known about how AHAs cause exfoliation to expose fresh skin cells. Here we report that the transient receptor potential vanilloid 3 (TRPV3) channel in keratinocytes is potently activated by intracellular acidification induced by glycolic acid. Patch clamp recordings and cell death assay of both human keratinocyte HaCaT cells and TRPV3-expressing HEK-293 cells confirmed that intracellular acidification led to direct activation of TRPV3 and promoted cell death. Site-directed mutagenesis revealed that an N-terminal histidine residue, His-426, known to be involved in 2-aminoethyl diphenylborinate-mediated TRPV3 activation, is critical for sensing intracellular proton levels. Taken together, our findings suggest that intracellular protons can strongly activate TRPV3, and TRPV3-mediated proton sensing and cell death in keratinocytes may serve as a molecular basis for the cosmetic use of AHAs and their therapeutic potential in acidic pH-related skin disorders.

Bioactive reagents used in mesotherapy for skin rejuvenation in vivo induce diverse physiological processes in human skin fibroblasts in vitro– a pilot study

The promise of mesotherapy is maintenance and/or recovery of a youthful skin with a firm, bright and moisturized texture. Currently applied medications employ microinjections of hyaluronic acid, vitamins, minerals and amino acids into the superficial layer of the skin. However, the molecular and cellular processes underlying mesotherapy are still elusive. Here we analysed the effect of five distinct medication formulas on pivotal parameters involved in skin ageing, that is collagen expression, cell proliferation and morphological changes using normal human skin fibroblast cultures in vitro. Whereas in the presence of hyaluronic acid, NCTF135(®) and NCTF135HA(®) , cell proliferation was comparable to control cultures; however, with higher expression of collagen type-1, matrix metalloproteinase-1 and tissue inhibitor of matrix metalloproteinase-1, addition of Soluvit(®) N and Meso-BK led to apoptosis and/or necrosis of human fibroblasts. The data indicate that bioactive reagents currently applied for skin rejuvenation elicit strikingly divergent physiological processes in human skin fibroblast in vitro.

Surface chemical immobilization of parylene C with thermosensitive block copolymer brushes based onN‐isopropylacrylamide andN‐tert‐butylacrylamide: Synthesis, characterization, and cell adhesion/detachment

Poly(N-isopropylacrylamide) (pNIPAM), poly(N-tert-butylacrylamide) (pNTBAM), and their copolymer brushes were covalently immobilized onto parylene C (PC) surfaces via surface initiated atom transfer radical polymerization (ATRP). Contact angle measurement between 13 and 40°C showed that the hydrophobicity of the modified PC surfaces was thermally sensitive. Among these samples, PC grafted with pNIPAM (PC-NI), PC grafted with pNTBAM (PC-NT) and PC grafted with copolymer brushes containing pNTBAM and pNIPAM (PC-NT-NI) exhibited the lower critical solution temperature (LCST) at 29, 22, and 24°C, respectively. Cytocompatibility study for the modified surfaces was performed by 5 days human skin fibroblast culture at 37°C. Data showed that only a very small amount of cells adhered on the PC and PC-NI surfaces, while a significantly higher amount of cell adhesion and growth was observed on PC-NT and PC-NT-NI surfaces. Furthermore, cell detachment at the temperatures of 24 and 6°C were studied after the substrates were cultured with cells at 37°C for 24 h. The results showed that the cells on PC-NI formed the aggregations and loosely attached on the substrate after 30-min culture at 24°C, while no significant cell detachment was observed for PC-NT and PC-NT-NI samples at this temperature. By continuing the cell culture for additional 100 min at 6°C for PC-NT and PC-NT-NI, about 10 and 35% of the cells were found detached respectively, and the unattached cells aggregated on the substrate. In comparison, cells cultured on the tissue culture petri dish (TCP) exhibited no quantity and morphology changes at the culture temperatures of 37, 24, and 6°C. This study showed that: (1) immobilization of PC with nonthermal sensitive pNTBAM could provide PC surface thermal sensitive hydrophilicity; (2) the chlorines on the polymer brushes of PC-NT could be used to further initiate the ATRP pNIPAM and form block copolymer brushes; (3) the incorporation of pNTBAM into pNIPAM on PC-NT-NI could change the surface thermal hydrophilicity property, and be further applied to decrease the LCST of the modified PC surface; (4) grafted pNIPAM brushes on PC-NI by ATRP showed very low cell adhesion and proliferation in 5 days fibroblast culture at 37°C, and cell detached at 24°C; (5) the incorporation of pNTBAM into pNIPAM on PC-NT-NI decreased the thermal sensitivity of cell adhesion/detachment, cell detached at 6°C, but the cell adhesion and proliferation were significantly improved at a wide temperature range.

On the Thermus thermophilus HB8 potential pathogenicity triggered from rhamnolipids secretion: morphological alterations and cytotoxicity induced on fibroblastic cell line

A limited number of bacterial strains usually grown under nutrient limitation secrete rhamnolipids (RLs), which are recorded as virulence factors that are implicated in the pathogenicity of a microorganism. The non-pathogenic T. thermophilus HB8 produces extracellular rhamnolipids (TthRLs) under defined cultivation conditions using sunflower seed oil and sodium gluconate as carbon sources. In particular, the secreted TthRLs have been isolated, purified and identified with ATR-FTIR. Their effects on the cells' viability were examined when they were supplemented in a culture of human skin fibroblasts. Purified TthRLs triggered a sequence of rapid and pronounced morphological alterations characterized by transformation of fibroblast shape from polygonal to fusiform; retraction with cytoplasm condensation, rounding up, distortion of nuclei and loss of lamellar processes, and finally disruption of membrane. The addition of TthRLs in the cultured fibroblasts caused cytotoxicity, in contrast to that of rhamnose that stimulated viability, as it was assessed by MTT test. These results revealed that among the constituents of RLs that are implicated in the cytotoxicity, it has to be attributed to the lipidic chain variation and not to the carbohydrate part. TthRLs cytotoxicity on fibroblasts is comparable, and provoked similar effects, to that caused by saponin white, a known surfactant. TthRLs secretion might be a crucial point for the transformation of a non-pathogenic bacterium to a pathogenic one under certain environmental conditions favoring their secretion. RLs secretion in the microorganism's world might be a general route for the passage in the pathogenicity to ensure their survival under nutrient limitation conditions.

Honeycomb Self‐Assembled Peptide Scaffolds by the Breath Figure Method

The self-assembly of molecules into desired architectures is currently a challenging subject for the development of supramolecular chemistry. Here we present a facile "breath figure" assembly process through the use of the self-assembled peptide building block diphenylalanine (L-Phe-L-Phe, FF). Macroporous honeycomb scaffolds were fabricated, and average pore size could be regulated, from (1.00±0.18) μm to (2.12±0.47) μm, through the use of different air speeds. It is indicated that the honeycomb formation is humidity-, solvent-, concentration-, and substrate-dependent. Moreover, water molecules introduced from "breath figure" intervene in the formation of hydrogen bonds during FF molecular self-assembly, which results in a hydrogen bond configuration transition from antiparallel β sheet to parallel β sheet. Meanwhile, as a result of the higher polarity of water molecules, the FF molecular array is transformed from laminar stacking into a hexagonal structure. These findings not only elucidate the FF molecule self-assembly process, but also strongly support the mechanism of breath figure array formation. Finally, human embryo skin fibroblast (ESF) culture experiments suggest that FF honeycomb scaffolds are an attractive biomaterial for growth of adherent cells with great potential applications in tissue engineering.

Influence of endothelin-1 receptors antagonists on extracellular matrix protein synthesis in culture of human scleroderma skin fibroblasts

Background and objectivesSkin fibrosis is an important pathological event in the systemic sclerosis (SSc) and is characterised by excessive synthesis and deposition of extracellular matrix (ECM) proteins by activated scleroderma...

Modulation of collagen synthesis and its gene expression in human skin fibroblasts by tocotrienol-rich fraction

IntroductionSkin aging may occur as a result of increased free radicals in the body. Vitamin E, the major chain-breaking antioxidant, prevents propagation of oxidative stress, especially in biological membranes. In this study, the molecular mechanism of tocotrienol-rich fraction (TRF) in preventing oxidative stress-induced skin aging was evaluated by determining the rate of total collagen synthesis and its gene expression in human skin fibroblasts.Material and methodsPrimary culture of human skin fibroblasts was derived from circumcision foreskin of 9 to 12 year-old boys. Fibroblast cells were divided into 5 different treatment groups: untreated control, hydrogen peroxide (H2O2)-induced oxidative stress (20 µM H2O2 exposure for 2 weeks), TRF treatment, and pre- and post-treatment of TRF to H2O2-induced oxidative stress.ResultsOur results showed that H2O2-induced oxidative stress decreased the rate of total collagen synthesis and down-regulated COL I and COL III in skin fibroblasts. Pre-treatment of TRF protected against H2O2-induced oxidative stress as shown by increase in total collagen synthesis and up-regulation of COL I and COL III (p<0.05) genes. However, similar protective effects against H2O2-induced oxidative stress were not observed in the post-treated fibroblasts.ConclusionsTocotrienol-rich fraction protects against H2O2-induced oxidative stress in human skin fibroblast culture by modulating the expression of COL I and COL III genes with concomitant increase in the rate of total collagen synthesis. These findings may indicate TRF protection against oxidative stress-induced skin aging.