Rat Embryonic Fibroblast Cells Research Articles

Preparation and Characterization of an Electrospun Polycaprolactone/Polyurethane Scaffold for Soft Tissue Applications

In this study, a synthetic scaffold was prepared from polycaprolactone (PCL) and polyurethane (PU) blend, in a ratio of [2:1] [PCL: PU], using electrospinning technique. Electrospun scaffolds from native PCL and PU were also prepared for comparison, using the same polymer concentration 15% weight/ volume w/v. The detailed microstructure and other properties, like mechanical properties, porosity, and contact angle were investigated and compared between the three prepared scaffolds. Then, the survival, adhesion, proliferation and penetration of rat embryonic fibroblast (REF) cells were evaluated on these three prepared scaffolds after being in vitro cultured with these cells for 21 days, using scanning electron microscope (SEM) analysis and histological analysis. The results showed that, all the studied properties, including mechanical properties and contact angle were enhanced by combining PU with PCL in the [PCL: PU] scaffold. The average diameter of fiber and the average size of pore were suitable and proper for cell attachment, cell proliferation, and also the big average pore size in [PCL: PU] scaffold was enough for cell penetration to form a three- dimension 3-D structure, which is the aim of this study.

Cytotoxic Effect of flavonoids extracted from Conocarpus erectus leaves on SKG cell

The present study was conducted to investigate the cytotoxic activity flavonoids extracted from Conocarpus erectus L. leaves using ethyl acetate and chloroform, the results of the extracted flavonoids were detected by HPLC. The evaluation of its cytotoxic effect on cancerous SKG cell line Human Esophageal Cancer and non-cancerous REF (rat embryonic fibroblast) using MTT assay. HPLC analysis of both extracts showed 6 out of 10 flavonoids matched the standard peaks which were (Catechin, Rutin, Myrecetin, Quercetin, Apigenine and Kaempferol). The cytotoxicity of the ethyl acetate extract was greater than that of the chloroform extract against the SKG cell line, and the cytotoxic effect of both extracts increased with increasing concentration. At 200 μl/ml the cytotoxicity of the ethyl acetate and chloroform extracts were 71% and 64.6%, respectively, while the cytotoxicity of the extracts on REF cells was 10.5% and 11.2%. This implies that C.erectus extract has little negative effects on normal cells and is thus a safe and promising anticancer drug candidate.

Correlative Analysis of Intra‐ Versus Extracellular Cell Detachment Events via the Alignment of Optical Imaging and Detachment Force Quantification

AbstractIn recent decades, hybrid characterization systems have become pillars in the study of cellular biomechanics. Especially, Atomic Force Microscopy (AFM) is combined with a variety of optical microscopy techniques to discover new aspects of cell adhesion. AFM, however, is limited to the early‐stage of cell adhesion, so that the forces of mature cell contacts cannot be addressed. Even though the invention of Fluidic Force Microscopy (FluidFM) overcomes these limitations by combining the precise force‐control of AFM with microfluidics, the correlative investigation of detachment forces arising from spread mammalian cells has been barely achieved. Here, a novel multifunctional device integrating Fluorescence Microscopy (FL) into FluidFM technology (FL‐FluidFM) is introduced, enabling real‐time optical tracking of entire cell detachment processes in parallel to the undisturbed acquisition of force‐distance curves. This setup, thus, allows for entailing two pieces of information at once. As proof‐of‐principle experiment, this method is applied to fluorescently labeled rat embryonic fibroblast (REF52) cells, demonstrating a precise matching between identified force‐jumps and visualized cellular unbinding steps. This study, thus, presents a novel characterization tool for the correlated evaluation of mature cell adhesion, which has great relevance, for instance, in the development of biomaterials or the fight against diseases such as cancer.

Inhibition of Staphylococcus aureus α‐Hemolysin Production Using Nanocurcumin Capped Au@ZnO Nanocomposite

Nanoparticles of gold with zinc oxide (Au@ZnO NPs) were prepared by laser ablation and then capped with curcumin nanoparticles (Cur-Au@ZnO NPs). The synthesized NPs were characterized using different techniques, including transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), UV-visible spectroscopy, and X-ray diffraction. In addition, the ability of NPs as a promising antibacterial agent was tested against Staphylococcus aureus through the agar well diffusion method and AO/EtBr staining assay. The results showed that the prepared nanoparticles (Cur-Au@ZnO) served as an antibacterial agent and can destroy the bacterial cells by losing the cell wall integrity and penetrating the cytoplasmic membrane. Moreover, the findings confirmed the role of the formed NPs in attenuation of the adherence and invasion of S. aureus to rat embryonic fibroblast (REF) cells. Furthermore, the activity of Cur-Au@ZnO NPs against the S. aureus α-hemolysin toxin was evaluated using the western blot technique, using human alveolar epithelial cells (A549), and through histopathology examination in a mouse model. In conclusion, the built Cur-Au@ZnO NPs can be used as a potential antibacterial agent and an inhibitor of α-hemolysin toxin secreted by S. aureus. These NPs may offer a new strategy in combating pathogen infections and in the future for biomedical and pharmaceutical applications.

Evaluation of mechanical properties and cytotoxicity of maxillofacial silicone material after incorporation of zirconia nanopowder

Abstract Resilient polymeric materials such as silicone elastomers are currently used for maxillofacial prostheses construction but the strength of these materials and their clinical performance need to be optimized with the addition of reinforcing fillers. This study investigates the effect of zirconia nanopowder addition on tear strength, tensile strength, elongation at break, Shore A hardness, surface roughness and cytotoxicity of VST-50 maxillofacial silicone. Silicone base was mixed with different amounts (1%, 2% and 3%) of zirconia nanopowder using a vacuum mixer. Silicone without filler was used as control for comparison. Scanning Electron Microscopy and Atomic Force Microscopy were utilized to assess the efficiency of high-shear vacuum mixing as filler dispersion method and the surface topography, respectively. Both SEM and AFM images showed that the zirconia nanopowder were distributed fairly well within the polymer. Statistically, highly significant increase in tear strength, tensile strength and hardness with non-significant decrease in elongation at break and non-significant increase in surface roughness were seen with 1% and 2% groups. Whereas with 3% group, there was significant improvement in tear strength, tensile strength and hardness but there was significant undesirable decrease in elongation and increase in roughness. Cytotoxicity test revealed that the addition of zirconia nanopowder was nontoxic to Rat Embryonic Fibroblast (REF) cells and there was non-significant change in the cell viability of all study groups after 24- and 72-hours incubation periods. In conclusion, the addition of 2% by weight nano zirconia to VST-50 maxillofacial silicone could be beneficial in enhancing its performance.

Impact of phosphate ester addition on the cytotoxicity of heat cured denture base material

This study aims to evaluate and compare the cytotoxicity and biocompatibility of a modified heat-cured acrylic denture base material containing 15% phosphoric acid 2-hydroxyethyl methacrylate ester (PA2HEME) with those of nonmodified PMMA. Discs with a diameter of 12 mm and a thickness of 2 mm were prepared using a heat-cured PMMA denture base material and divided into control and experimental groups. The experimental group was modified with 15% phosphoric acid 2-hydroxyethyl methacrylate ester (PA2HEME). The modified and nonmodified materials were tested via FTIR, and the effect of modification on surface roughness was evaluated with AFM. An in vitro test was conducted to examine the cytotoxicity and biocompatibility of heat-cured acrylic denture base materials (control and experimental groups) by using a rat embryonic fibroblast cell line (REF). Cell culture viability was estimated with a methyltetrazolium solution cytotoxicity assay. The surface roughness of PA2HEME-modified acrylic resin (mPMMA) was not significantly different from that of nonmodified PMMA acrylic resin. The cytotoxicity test on mPMMA demonstrated no significant change in the cell viability compared with that of the negative control. No deteriorating or inhibitory effect on cell growth was observed after 24 and 72 h. The modification of denture base materials with 15% PA2HEME has no cytotoxic or inhibitory effects on the growth of the studied fibroblast cell line.

Therapeutic Effects of Gallic Acid in Regulating Senescence and Diabetes; an In Vitro Study.

Gallic acid (GA), a plant-derived ubiquitous secondary polyphenol metabolite, can be a useful dietary supplement. This in vitro study’s primary purpose was to assess the anti-aging properties of GA using rat embryonic fibroblast (REF) cells, antidiabetic effects via pancreatic islet cells, and finally, elucidating the molecular mechanisms of this natural compound. REF and islet cells were isolated from fetuses and pancreas of rats, respectively. Then, several senescence-associated molecular and biochemical parameters, along with antidiabetic markers, were investigated. GA caused a significant decrease in the β-galactosidase activity and reduced inflammatory cytokines and oxidative stress markers in REF cells. GA reduced the G0/G1 phase in senescent REF cells that led cells to G2/M. Besides, GA improved the function of the β cells. Flow cytometry and spectrophotometric analysis showed that it reduces apoptosis via inhibiting caspase-9 activity. Taken together, based on the present findings, this polyphenol metabolite at low doses regulates different pathways of senescence and diabetes through its antioxidative stress potential and modulation of mitochondrial complexes activities.

Polymer‐Based Porous Microcapsules as Bacterial Traps

AbstractExposure to live bacteria and accumulation of dead bacteria during bactericidal processes can cause bacterial infectious diseases, implant failure, and antibacterial surface deterioration. Microcapsules with asymmetrically distributed, funnel‐shaped pores, which are capable of capturing, retaining, and killing bacteria are developed, offering a solution to bacterial contamination in liquids. It is found that bacterial isolation inside microcapsules is mainly driven by the bacteria's own motility and the microcapsules' geometry. After entry into the microcapsule cavity, the bacteria are stably retained inside. The microcapsules shield surrounding cells from exposure to bacterial toxins, as demonstrated by the coculture of rat embryonic fibroblast cells with microcapsules loaded with live Escherichia coli. The microcapsules can be enhanced with a bactericidal coating covering only the interior cavity. This confines the bacteria‐killing process, thereby further increasing biocompatibility. The microcapsules may offer a viable bacteria combatant approach as a potentially advantageous method to eradicate bacterial contamination.

LAB-SCALE PRODUCTION OF RHAMNOLIPID BY PSEUDOMONAS AERUGINOSA A3 AND STUDY ITS SYNERGISTIC EFFECT WITH CERTAIN ANTIBIOTICS AGAINST SOME PATHOGENIC BACTERIA

This study was aimed to obtain the considerable amount of the biosurfactant (rhamnolipid) from local isolate of Pseudomonas aeruginosa on industrial-scale. A 7L laboratory bioreactor was appointed to accomplish this task and all conditions of production were set as previously by batch culture experiments. Bioreactor with 3.5 L working volume was conducted for 120 h to obtain 16 g/L at 30oC and 6.5 pH. The supernatant of production medium was used to reduce the surface tension of water from 72 to 27 mN/m, and the emulsification index was reached to 67%. The product was extracted with a mixture of solvents, purified by silica gel using a column of glass chromatography (3.5 × 30cm), and characterized by TLC and FTIR where it turn out that the biosurfactant is composed of both mono- and di-rhamnolipid. Mono-RL was tested for its antibacterial activity against some clinical isolates of Gram-positive and Gram-negative bacteria to obtain the MIC, which was about 250µg/ml, also at same concentration, the toxicity against Rat embryonic fibroblast (REF) cell line was reached to 23%. The combination effect of Mono-RL with some antibiotics was studied against pathogenic clinical isolates revealed that there is a synergistic effect with ampicillin against S. aureus and Serratia marcescens, with cefotaxime against E. coli and Kelbsiella pneumoniae, same as with tetracycline.

Effect of vitex agnus castus fruits methanol extract against murine mammary adenocarcinoma cell line (amn3) and rat embryonic fibroblast normal cell line (ref)

Vitex agnus castus showed antiproliferative activity in several previous studies. Angiogenesis is one of the targets in the remediation of cancer. This study aimed to demonstrate the effect of methanol fruits extract of vitex agnus castus on mouse mammary gland adenocarcinoma cell line and rat embryonic fibroblast cell line. The cell lines used in this study were obtained from tissue culture unit/ Iraqi Center for Cancer and Medical Genetic Researches, Al-Mustansiriyah University was maintained in RPMI- 1640 tissue media after preparing from 10% fetal calf serum, antibiotics solution and other materials to make complete growth medium. Serial solutions of vitex agnus castus, methanol crude extract have been tested on 10*4 of AMN3 and REF in each well of 96 well plates. The results of the current study showed that the concentration that inhibits fifty percent of cell line after 72 hours of the experiment (IC50) was 129 ug/ml for AMN3 and 1324ug/ml for REF cell line. The antioxidant activity of Vitex agnus castus may indicate the proliferation inhibition activity of Vitex agnus castus methanol extract. The study concluded that this extract might be of benefit if used in combination with other anti-cancer drugs as adjuvant therapy.

α-Lipoic acid prevents senescence, cell cycle arrest, and inflammatory cues in fibroblasts by inhibiting oxidative stress

Senescence is a process characterized by an irreversible growth arrest in cells and induced by oxidative stress. In the current study, anti-aging potential of a well-known antioxidant, α-lipoic acid (α-LA), in rat embryonic fibroblast (REF) cells was assessed. In this regard, oxidative stress, inflammation, and apoptosis pathways were investigated on REF cells exposed to H2O2 as a senescence inducer and α-LA as a protective compound. In cells treated with α-LA and H2O2, level of β-galactosidase, as an aging marker, and oxidative stress biomarkers, were significantly lower than those exposed to H2O2 only. Furthermore, flow cytometry assay showed that α-LA caused a significant reduction in the number of apoptotic cells via the caspase-dependent pathway. In addition, it could neutralize the inflammatory effects of H2O2 and attenuated the concentration of inflammatory cytokines. In comparison to H2O2 group, a significant increase in G0/G1 arrest was observed during cell cycle analysis in cells exposed to H2O2 and α-LA. The results of this study show that α-LA has beneficial effects on H2O2-induced cellular senescence. α-LA works by attenuating the reactive oxygen species, subsiding inflammation, and affecting cell division.

Study on the regulation of focal adesions and cortical actin by matrix nanotopography in 3D environment

Matrix nanotopography plays an important role in regulating cell behaviors by providing spatial as well as mechanical cues for cells to sense. It has been proposed that nanoscale topography is possible to modulate the tensions which direct the formation of cytoskeleton and the organization of the membrane receptor within the cell, which in turn regulate intracellular mechanical and biochemical signaling. With current studies on this topic being performed mainly in 2D platforms, the question on how nanotopography can influence cell bahaviors in 3D environments has yet to be addressed. In this paper, we explored this question by placing cells in 3D hollow spherical polydimethylsiloxane scaffolds. After culturing rat embryonic fibroblast cells in two kinds of scaffold, one with smooth surface and the other with numerous nano-spikes, we observed that cells in the smooth scaffold have more anchoring sites and more focal adhesions than in the etched scaffold. Moreover, we found the presence of correlation between cortical actin, the important component for supporting cell attachment, and local cell geometry.

Cellular and molecular mechanisms of the protective effect of silybine on stress-induced premature senescence in rat embryonic fibroblast cells cells

Cellular and molecular mechanisms of the protective effect of silybine on stress-induced premature senescence in rat embryonic fibroblast cells cells

Molecular and biochemical evidence on the protective role of ellagic acid and silybin against oxidative stress-induced cellular aging.

Aging is a natural process in living organisms that is defined by some molecular and cellular changes with time. Various causes such as mitochondrial DNA aberrations, aggregation of proteins, telomere shortening, and oxidative stress have an influential role in aging of the cells. Natural antioxidants are compounds that are potent to protect the body from detrimental effects of molecules such as free radicals. The aim of this study was to evaluate the anti-aging properties of ellagic acid (EA) and silybin (SIL), as natural antioxidant compounds on rat embryonic fibroblast (REF) cells. These cells were pre-incubated with EA and SIL, thereafter were exposed to hydrogen peroxide (H2O2). Then, the cell viability, SA-β-GAL activity, distribution of cell cycle, NF-κB, and mitochondrial complex I, II/IV enzyme activity were measured. The results of this study revealed the protective effects of EA and SIL in H2O2-treated REF cells, which confirm the previous achieved data on antioxidant and anti-inflammatory characteristics of EA and SIL against H2O2 in the treated REF cells. However, more new in vivo experiments are required to discover the anti-aging effects and mechanism of action of such compounds.

Molecular evidence on the protective effect of ellagic acid on phosalone-induced senescence in rat embryonic fibroblast cells

Salient evidence testifies the link between organophosphorus (OPs) exposure and the formation of free radical oxidants; and it is well accepted that free radicals are one of the basic concerns of senescence. To show the oxidative features of phosalone (PLN) as a key member of OPs, to induce senescence in rat embryonic fibroblast (REF) cells and to demonstrate the beneficial effects of the known antioxidant ellagic acid (EA) in diminishing the PLN-induced toxic effects, the levels of cell viability, oxidative stress markers, inflammatory cytokines, telomerase activity, and the expression of the genes related to senescence were investigated. Our results lend support to the hypothesis that PLN enhances the entire premature senescence parameters of REF cells. This accounts for the mechanistic approval of the role of OPs in induction of senescence in rat fibroblasts. Moreover, incorporation of EA diminished PLN toxicity mainly through suppression of p38 and p53 at gene and protein levels, and tempered the inflammation factors (TNF-α, IL-1β, IL-6 and NF-κB), which further affected cell division. Analysis of cell cycle showed that the percentage of G0/G1 arrest, in REF cells treated by EA was elevated as compared to control and PLN treated cells.

Cellular and molecular mechanisms of aging and oxidative stress-induced senescence in rat embryonic fibroblast cells by phosalone and hydrogen peroxide

Cellular and molecular mechanisms of aging and oxidative stress-induced senescence in rat embryonic fibroblast cells by phosalone and hydrogen peroxide

In vitro isolation and molecular identification of reptarenavirus in Malaysia.

Boid inclusion body disease (BIBD) is a viral disease of boids caused by reptarenavirus. In this study, tissue from naturally infected boid snakes were homogenized and propagated in African Monkey kidney (Vero) and rat embryonic fibroblast (REF) cells. Virus replication was determined by the presence of cytopathic effect, while viral morphology was observed using transmission electron microscopy. Viral RNA was amplified using RT-PCR with primers specific for the L-segment of reptarenavirus; similarly, quantification of viral replication was done using qPCR at 24-144h postinfection. Viral cytopathology was characterized by cell rounding and detachment in both Vero and REF cells. The viral morphology showed round-to-pleomorphic particles ranging from 105 to 150nm which had sand-like granules. Sanger sequencing identified four closely associated reptarenavirus species from 15 (37.5%) of the total samples tested, and these were named as follows: reptarenavirus UPM-MY 01, 02, 03, and 04. These isolates were phylogenetically closely related to the University Helsinki virus (UHV), Boa Arenavirus NL (ROUTV; BAV), and unidentified reptarenavirus L20 (URAV-L20). Comparison of deduced amino acid sequences further confirmed identities to L-protein of UHV, L-polymerase of BAV and RNA-dependent RNA polymerase of URAV-L20. Viral replication in Vero cells increased steadily from 24 to 72h and peaked at 144h. This is the first study in South East Asia to isolate and characterize reptarenavirus in boid snakes with BIBD.

Cytotoxic and apoptotic effects of ceranib-2 offering potential for a new antineoplastic agent in the treatment of cancer cells

Fibrocarcinomas are malignant tumours that originate in mesenchymal cells. The tumours typically form in the presence of connective tissue and are of key consideration when assessing research priorities for future healthcare management. Ceramidase inhibitors, such as ceranib-2, have demonstrated capacity to interfere with cellular DNA functionality, initiating apoptosis in many cancer cell lines. The enzyme ceramidase can regulate cellular levels of sphingosine and sphingosine-1-phosphate by controlling hydrolysis of ceramide. The present study investigates the antigrowth effects of ceranib-2 on mouse embryonic fibroblast cells (NIH/3T3 normal cell line) and rat embryonic fibroblast cells (5RP7 cancer cell line). Research was conducted using colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, confocal microscopy, and transmission electron microscopy. The results indicate that in vitro fibrocarcinomas have the potential to undergo apoptotic death when influenced by the antigrowth behaviour of ceranib-2. This is true even of cells exposed only to minimal doses of ceranib-2. Indicators of apoptotic death, specifically presentation of a pyknotic nucleus, were observed by confocal micrographs in treated cells double-stained with acridine orange and Alexa Fluor-488 Phalloidin. It is theorised that ceranib-2 will function in accordance with the antiproliferative activity of other acid ceramidases, ultimately resulting in the molecule's classification as a new antitumour agent.

Licofelone abolishes survival of carcinogenic fibroblasts by inducing apoptosis

Dual inhibitors of cyclooxygenase (COX) and lipoxygenase (LOX) pathways of arachidonic acid metabolism prevent cancer development and induce apoptosis. One of the most promising compounds that blocks both of these pathways is licofelone. We questioned whether licofelone affects the survival and/or promotes apoptosis of H-ras transformed rat embryonic fibroblast (5RP7) cells in vitro. Using 5-fluorouracil (5-FU) and colchicine as positive controls, we determined cell viability with 3-3-(4,5-D-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, thyazolyl blue (MTT), apoptosis with flow cytometry and activity of caspase enzyme with real-time reverse transcription polymerase chain reaction (PCR). Compared to the control, all used six doses (10, 50, 100, 150, 250 and 250 µM) of 5-FU, colchicine and licofelone, which were cytotoxic and reduced the number of H-Ras transformed 5RP7 cells by as much as 78, 72 and 92%, respectively. In addition, we found that 150, 200 and 250 µM of licofelone induced apoptosis and necrosis of H-Ras transformed 5RP7 cells in a dose- and time-dependent manner. Each three tested drugs at 250 µM also increased the level of caspase-3 enzyme up to 5-fold. Although colchicine was effective in inducing early apoptosis, licofelone had much more capacity to induce the total of early plus late apoptosis by approximately 96% in cells after 48 hours. The present study reveals the possibility that licofelone posseses strong dose- and time-dependent anticancer and apoptotic properties on carcinogenic fibroblasts.

Surface Properties of Nanostructured Bio-Active Interfaces: Impacts of Surface Stiffness and Topography on Cell-Surface Interactions.

Due to their ability to confer key functions of the native extracellular matrix (ECM) poly(ethylene glycol) (PEG)-based and PEG-modified materials have been extensively used as biocompatible and biofunctionalized substrate systems to study the influence of environmental parameters on cell adhesion in vitro. Given wide-ranging recent evidence that ECM compliance influences a variety of cell functions, the detailed determination and characterization of the specific PEG surface characteristics including topography, stiffness and chemistry is required. Here, we studied two frequently used bio-active interfaces - PEG-based and PEG-modified surfaces - to elucidate the differences between the physical surface properties, which cells can sense and respond to. For this purpose, two sets of surfaces were synthesized: the first set consisted of nanopatterned glass surfaces containing cRGD-functionalized gold nanoparticles surrounded by a passivated PEG-silane layer and the second set consisted of PEG-diacrylate (PEG-DA) hydrogels decorated with cRGD-functionalized gold nanoparticlesAlthough the two sets of nanostructured materials compared here were highly similar in terms of density and geometrical distribution of the presented bio-ligands as well as in terms of mechanical bulk properties, the topography and mechanical properties of the surfaces were found to be substantially different and are described in detail. In comparison to very stiff and ultrasmooth surface properties of the PEG-passivated glasses, the mechanical properties of PEG-DA surfaces in the biologically relevant stiffness range, together with the increased surface roughness at micro- and nanoscale levels have the potential to affect cell behavior. This potential was verified by studying the adhesive behavior of hematopoietic KG-1a and rat embryonic fibroblast (REF52) cells on both surfaces.