3T3 Mouse Fibroblast Cells Research Articles

Glutathione-triggered formation of molecular hydrogels for 3D cell culture

The development of three dimensional (3D) scaffolds that are suitable for cell encapsulation and proliferation is highly important for tissue engineering and regenerative medicine. We reported in this paper on several molecular hydrogels formed through glutathione (GSH) reduction, whose mechanical property and zeta potential could be regulated by concentration and structure of gelators in resulting gels, respectively. The hydrogels were characterized by several techniques including rheology, TEM and fluorescence. We found that, in our system, the mechanical property of hydrogels but not the zeta potential of self-assembled structures had big influences on mouse fibroblast 3T3 cells spreading and proliferation. Hydrogels with storage modulus (G′) of hundreds of pascals (Pa) were suitable for 3T3 cells spreading and proliferation. We believed that hydrogels reported in this study had big potential for applications in different fields, such as 3D cell culture and tissue engineering.

Insights into chitosan hydrogels on dentine bond strength and cytotoxicity

Contemporary dental adhesives show favorable immediate results in terms of bonding effectiveness. However, the durability of resin-dentin bonds is their major problem. Materials and Methods: Preparation of 3 chitosan-antioxidant hydrogels was achieved using modified hydrogel preparation method. Their effect on the bond strength to dentine both short term (after 24 hours) and long term (after 6 months) were evaluated using shear bond strength measurements using Instron Universal Testing Mascine). The SEM was used to study the surface of the hydrogels. The cell survival rate (cytotoxicity) of the antioxidants resveratrol, β-carotene and propolis towards Balb/c 3T3 mouse fibroblast cells was also assessed using the standard MTT assay. Results: It was found that chitosan-H treated dentine gives significantly (p β-carotene (92%) > propolis (68%) > resveratrol (33%). Conclusion: the antioxidant-chitosan hydrogels significantly improved bonding to dentine with or without phosphoric acid treatment. The pH of the growth medium had a high influence on the cell survival rate of Balb/c mouse 3T3 fibroblast cells. The release of the antioxidant β-carotene would not have an influence on the pulp cells. These materials might address the current perspectives for improving bond durability.

Induction of functional platelets from mouse and human fibroblasts by p45NF-E2/Maf

AbstractDeterminant factors leading from stem cells to megakaryocytes (MKs) and subsequently platelets have yet to be identified. We now report that a combination of nuclear factor erythroid–derived 2 p45 unit (p45NF-E2), Maf G, and Maf K can convert mouse fibroblast 3T3 cells and adult human dermal fibroblasts into MKs. To screen MK-inducing factors, gene expressions were compared between 3T3 cells that do not differentiate into MKs and 3T3-L1 cells known to differentiate into MKs. 3T3 cells transfected with candidate factors were cultured in a defined MK lineage induction medium. Among the tested factors, transfection with p45NF-E2/MafG/MafK lead to the highest frequency of CD41-positive cells. Adult human dermal fibroblasts transfected with these genes were cultured in MK lineage induction medium. Cultured cells had megakaryocytic features, including surface markers, ploidy, and morphology. More than 90% of MK-sized cells expressed CD41, designated induced MK (iMK). Infusion of these iMK cells into immunodeficient mice led to a time-dependent appearance of CD41-positive, platelet-sized particles. Blood samples from iMK-infused into thrombocytopenic immunodeficient mice were perfused on a collagen-coated chip, and human CD41-positive platelets were incorporated into thrombi on the chip, demonstrating their functionality. These findings demonstrate that a combination of p45NF-E2, Maf G, and Maf K is a key determinant of both megakaryopoiesis and thrombopoiesis.

Electrospun nanostructured chitosan–poly(vinyl alcohol) scaffolds: a biomimetic extracellular matrix as dermal substitute

Electrospinning is a versatile technique to make biomimetic and nanostructured scaffolds for skin tissue engineering. In this study we have electrospun and characterized chitosan (C)–poly(vinyl alcohol) (PVA) blend nanofibers as dermal substitutes and compared with 2D C–PVA films. The in vitro characterization of the C–PVA nanofibers and 2D films were evaluated using mouse 3T3 fibroblast cells and our results demonstrated that the cells adhered and proliferated on the surface of C–PVA nanofibers. In our animal studies, the implantation of C–PVA nanofibers along with topical administration of growth factor R-Spondin 1 on full thickness wounds created on rats showed 98.6% wound closure after two weeks post-surgery. The catalase and superoxide dismutase activity of the healing tissue was significantly higher in the groups treated with topical administration of growth factor and C–PVA nanofibers (p < 0.05). Thus these C–PVA nanofibers along with novel growth factor are promising new biomaterials that could be used as dermal substitutes for accelerated wound healing.

Cytotoxicity of seven recent dentine bonding agents on mouse 3T3 fibroblast cells

Today it is generally accepted that most bonding agents are cytotoxic. In this study the relative cytotoxicity of seven recent dentine bonding agents on mouse 3T3 fibroblast cells were investigated. Materials and Methods. Near-confluent mouse 3T3 fibroblast cells were exposed to Dulbecco Modified Eagle’s Medium containing extractions from the seven different bonding agents. The cell survival rate was then determined using the standard MTT assay. Results. The cell survival rate ranking is: iBond (94%) < Gbond (78%) < Xeno V (71%) < Adper Easy Bond (63%) < Xeno V+ (61%) < Adper Scotchbond SE (33%) < XP Bond (32%). Part A of Adper Scotchbond SE had a survival rate of 35% and part B 38%. These two parts did not differ significantly. Adper Scotchbond SE and XP Bond do not differ significantly. While Xeno V+, Xeno V and Adper Easy Bond do not differ. (p < 5%; Tukey-Kramer Multiple-Comparison Test). Conclusion. All of the tested adhesive bonding agents were cytotoxic with survival rate of 3T3 cells between 94% to 31%. Of the 7 bonding agents tested iBond was found to be only slightly toxic and by far the least toxic. The two bonding agents (XP Bond and Adper Scotchbond SE) containing UDMA plus TEGDMA plus HEMA plus camphorquinone were found to be the most toxic.

Responsive Small Molecular Hydrogels Based on Adamantane–Peptides for Cell Culture

The development of responsive small molecular hydrogels that can be applied for recovery of cells postculture attract extensive interests for researchers in fields of cell biology, stem cell differentiation, and tissue engineering. We report in this study several responsive small molecular hydrogels based on adamantane-peptides whose gel to clear solution phase transition can be achieved by addition of β-cyclodextrin (β-CD) derivatives. The small molecular hydrogels are formed by our recently developed method of disulfide bond cleavage by glutathione (GSH). Mouse fibroblast 3T3 cells attach and grow well at the surface of hydrogels. Furthermore, 3T3 cells postculture can be recovered from the gels by the addition of a β-CD derivative due to formation of clear solutions by the adamantane-β-CD interaction. The culture on hydrogels and recovery process do not cause obvious effects on behaviors of 3T3 cells. The results shown in this study indicate that small molecular hydrogels based on adamantane-peptides have great potentials in research fields where further analysis of cells is needed.

Effects of Methacrylic Acid on Physical/Mechanical Properties and Biocompatibility of Urethane-Based Denture Biomaterials

Candida-associated denture stomatitis (CADS) is a significant clinical concern. We have demonstrated that urethane-based denture biomaterials with 10% methacrylic acid (MAA) could bind and then slowly release antifungal drug for months. Drugs on the resins could be repeatedly quenched/recharged, and in subsequent recharging, they could be changed/switched to more potent/effective ones. However, the physical/mechanical properties and biocompatibility of the new MAA-based resins are currently unknown. The objective of the current study is to evaluate the effects of copolymerization with MAA on physical/mechanical properties and biocompatibility of urethane-based denture resin materials. MAA and diurethane dimethacrylate (UDMA) were copolymerized using initiator azobisisobutyronitrile (AIBN). Water sorption and solubility were assessed with the specifications of ISO (International Standards Organization) test method 1567, flexural strength and modulus were measured according to ASTM D-790, and biocompatibility was preliminarily evaluated in cytotoxicity assay using mouse 3T3 fibroblast cells with the trypan blue method. The results demonstrated that copolymerization of UDMA with up to 10% MAA did not negatively affect water sorption/solubility, flexural strength/modulus, and biocompatibility. With 20% MAA, however, the mechanical properties of the resulting resins were significantly decreased. To sum up, UDMA-MAA copolymers with up to 10% MAA had adequate physical/mechanical properties for denture materials with no side effects on cell viability. The UDMA-MAA denture biomaterials have a good potential to be used clinically for managing CADS and other related infectious conditions.

Cis-Clerodane-Type Furanoditerpenoids from Tinospora crispa

Phytochemical and cytotoxicity investigations on organic solvent extracts of the aerial parts of Tinospora crispa have led to the isolation of 15 cis-clerodane-type furanoditerpenoids. Of these, nine compounds (1-9) were found to be new. Spectroscopic assignments of a previously reported compound, borapetoside A (13), were revised on the basis of HMQC and HMBC correlations. No discernible activity was observed when compounds 10-13 were subjected to evaluation in cytotoxicity assays against human prostate cancer (PC-3) and the normal mouse fibroblast (3T3) cell lines.

Simultaneous evaluation of toxicities using a mammalian cell array chip prepared by photocatalytic lithography

A prototype of a mammalian cell array chip was developed on a flat glass surface. A superhydrophilic (water contact angle = 5°)/highly hydrophobic (120°) pattern was prepared on a fluorinated polymer-coated glass surface by means of photocatalytic lithography, and A549 (a human alveolar epithelial cell line), Hep G2 (a human hepatoma cell line) and mouse fibroblast 3T3 cells were inoculated onto the superhydrophilic regions. The cell populations were confined in the superhydrophilic regions for at least 24 h and separated from each other for at least one week. Organ-specific toxicity of aflatoxin B 1 and non-specific toxicity of adriamycin were successfully detected by using the cell array chip.

Novel Pentablock Copolymers for Selective Gene Delivery to Cancer Cells

In this study, the novel poly(diethylaminoethylmethacrylate) (PDEAEM)/Pluronic F127 pentablock copolymers were found to be able to mediate high-efficiency transfection of human epithelial ovarian carcinoma (SKOV3) cell line while showing significantly lower efficacy in human epithelial retinal (ARPE-19) cell line and Swiss Mouse Fibroblast (3T3) cell line. The intracellular routes of polyplexes were investigated by confocal microscopy after appropriately labeling the polymer and DNA. It was found that lesser nuclear entry in the ARPE-19 cells may result in the lower efficiency of transfection. Since the SKOV3 proliferation rate was found to be much higher than that of the ARPE-19 cells, the nuclear entry of polyplexes was assumed to be correlated with the proliferation rate, and it was hypothesized that the novel pentablock copolymers could mediate gene delivery selectively in fast growing cells. The different intracellular barriers to gene transfer may also account for the observed difference of transfection efficacy. Although the validity of the hypothesis that our pentablock copolymer could selectively transfect hyperproliferative cells needs further examination, this present work provides a new perspective to design targeting vectors for cancer therapies based on different characteristics among specific cell types.

Environmental, genetic and cellular toxicity of tenuazonic acid isolated from Alternaira alternata

Alternaria alternata, an important pathogen of many plants, produces tenuazonic acid (TeA) with bioactivity to microbes, plants and animals. TeA is one of the main mycotoxin to humans and other organisms. Using Chlamydomonas reinhardtii, Vicia faba root tip and three mammalian normal cell lines as target materials, environmental toxicity, genetic toxicity and cytotoxicity of TeA were examined. The growth and chlorophyll concentration of C. reinhardtii were inhibited at above 100
g/ml concentration, with EC50(G) of 310.36
g/ml and EC50(Chl) of 294.27
g/ml. The micronuleus test results indicated that MCN‰ was >15‰ only at higher concentrations. TeA inhibited the proliferation and total protein contents of 3T3 mouse fibroblasts (3T3 cells), Chinese hamster lung cells (CHL cells) and human hepatocytes (L-O2 cells) at concentrations ranging from 12.5 - 400
g/ml. Of the three cell lines, 3T3 cells were the most sensitive to the toxin (EC50(24h) = 41.64
g/ml), followed by CHL cells (EC50(24h) = 59.33
g/ml), and L-O2 cells (EC50(24h) = 85.98
g/ml) had the lowest sensitivity.

Correlation of visual in vitro cytotoxicity ratings of biomaterials with quantitative in vitro cell viability measurements

Medical devices and implanted biomaterials are often assessed for biological reactivity using visual scores of cell-material interactions. In such testing, biomaterials are assigned cytotoxicity ratings based on visual evidence of morphological cellular changes, including cell lysis, rounding, spreading, and proliferation. For example, ISO 10993 cytotoxicity testing of medical devices allows the use of a visual grading scale. The present study compared visual in vitro cytotoxicity ratings to quantitative in vitro cytotoxicity measurements for biomaterials to determine the level of correlation between visual scoring and a quantitative cell viability assay. Biomaterials representing a spectrum of biological reactivity levels were evaluated, including organo-tin polyvinylchloride (PVC; a known cytotoxic material), ultra-high molecular weight polyethylene (a known non-cytotoxic material), and implantable tissue adhesives. Each material was incubated in direct contact with mouse 3T3 fibroblast cell cultures for 24 h. Visual scores were assigned to the materials using a 5-point rating scale; the scorer was blinded to the material identities. Quantitative measurements of cell viability were performed using a 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay; again, the assay operator was blinded to material identities. The investigation revealed a high degree of correlation between visual cytotoxicity ratings and quantitative cell viability measurements; a Pearson's correlation gave a correlation coefficient of 0.90 between the visual cytotoxicity score and the percent viable cells. An equation relating the visual cytotoxicity score and the percent viable cells was derived. The results of this study are significant for the design and interpretation of in vitro cytotoxicity studies of novel biomaterials.

Photophysical Properties and Photobiological Behavior of Amodiaquine, Primaquine and Chloroquine

This article describes the results of a coupled photophysical and photobiological study aimed at understanding the phototoxicity mechanism of the antimalarial drugs amodiaquine (AQ), primaquine (PQ) and chloroquine (CQ). Photophysical experiments were carried out in aqueous solutions by steady-state and time-resolved spectrometric techniques to obtain information on the different decay pathways of the excited states of the drugs and on the transient species formed upon laser irradiation. The results showed that all three drugs possess very low fluorescence quantum yields (10(-2)-10(-4)). Laser flash photolysis experiments proved the occurrence of photoionization processes leading to the formation of a radical cation in all three systems. In the case of AQ the lowest triplet state was also detected. Together with the photophysical properties the photobiological properties of the antimalarial drugs were investigated under UV irradiation, on various biological targets through a series of in vitro assays. Phototoxicity on mouse 3T3 fibroblast and human keratinocyte cell lines NCTC-2544 was detected for PQ and CQ but not for AQ. In particular, PQ- and CQ-induced apoptosis was revealed by the externalization of phosphatidylserine. Furthermore, upon UV irradiation, the drugs caused significant variations of the mitochondrial potential (Deltapsi(mt)) measured by flow cytometry. The photodamages produced by the drugs were also evaluated on proteins, lipids and DNA. The combined approaches were useful in understanding the mechanism of phototoxicity induced by these antimalarial drugs.

Resveratrol inhibits cell growth and induces apoptosis of rat C6 glioma cells

Resveratrol (Res) has been reported to inhibit tumor initiation, promotion, and progression in a variety of cell culture systems depending on the specific cell type and cellular environment. In the present study, we determined the effect of Res on the cell growth and apoptosis of rat glioma C6 cell line as well as mouse fibroblast 3T3 cell line, in vitro. Concurrently, we investigated whether caspase-3 is involved in the Res-induced apoptosis of rat glioma cells. Exposure to Res exhibits a significant anti-proliferative effect and induces an increase in the population of apoptotic cells on C6 cells in a concentration- and time-dependent manner, but not for normal 3T3 fibroblast cells, as measured by methyl thiazolyl tetrazolium assay and flow cytometer. Distinguished increase of C6 cells in S phase is observed after the treatment of Res as compared to insignificant change in cell cycle distribution of 3T3 cells. TdT-mediated dUTP nick end labeling fluorescence staining, HE staining, and scanning electron microscope revealed abnormal morphology and ultrastructure in C6 cells treated with Res. Our data showed that Res can increase the expression and induced the activation of caspase-3 in rat glioma C6 cells. These results suggest that Res has significant apoptosis-inducing effect on C6 glioma cells other than normal fibroblast 3T3 cells in vitro and caspase-3 may act as a potential mediator in the process.

Applications of Supercritical Fluid in Alloplastic Bone Graft: A Novel Method and in Vitro Tests

In this study, supercritical fluid (SCF) technology is discussed in relation to biomaterial processing, especially the fabrication and processing of alloplastic bone graft. SCF offers excellent extraction properties for some compounds because of its favorable diffusivity, viscosity, zero surface tension, liquidlike density and solvating power, and other physical properties. The most desirable SCF solvent for extraction is supercritical carbon dioxide (SCCO 2 ), which is nontoxic, nonflammable, inexpensive, friendly to mankind, and environmentally benign and has mild supercritical conditions (T c = 31.2 °C, P c = 7.386 MPa). For the porous structure of porcine vertebra with its content of lipids, proteins, and inorganic substances in the cells and intercellular matrix, SCCO 2 combined with ethanol or glutaraldehyde (GA) is used to prepare novel porcine-derived bone grafts in three supercritical settings and one subcritical condition for control comparison. The biocompatibility of these novel bone grafts was tested with human MG63 osteoblast-like cells and mouse fibroblast 3T3 cells by MTT assay. The results revealed that materials processed by SCCO 2 combined with both ethanol and GA were not cytotoxic and allowed the differentiation and proliferation of test cells. The good performances of these novel bone grafts can be explained by the characteristics of SCF. Applications of SCF in biomaterials are very promising for bone regeneration and tissue engineering. Additional advanced in vitro and in vivo studies must be performed in the future for optimization.

Interferonα Activates NF-κB in JAK1-deficient Cells through aTYK2-dependentPathway

In addition to activating members of the STAT transcription factor family, interferon alpha/beta (IFNalpha/beta) activates the NF-kappaB transcription factor. To determine the role of the Janus tyrosine kinase (JAK)-STAT pathway in NF-kappaB activation by IFN, we examined NF-kappaB activation in JAK1-deficient mutant human fibrosarcoma cells. In wild-type fibrosarcoma cells (2fTGH), IFN activates STAT1, STAT2, and STAT3, as well as NF-kappaB complexes comprised of p50 and p65. In contrast, in JAK1-deficient cells, IFN induces NF-kappaB activation and NF-kappaB dependent gene transcription but does not activate these STAT proteins and has no effect on STAT-dependent gene transcription. Expression of a catalytically inactive TYK2 tyrosine kinase in JAK1-deficient cells, as well as in the highly IFN-sensitive Daudi lymphoblastoid cell line, abrogates NF-kappaB activation by IFN. Moreover, IFN does not promote NF-kappaB activation in TYK2-deficient mutant fibrosarcoma cells. Our results demonstrate a dichotomy between the classical JAK-STAT pathway and the NF-kappaB signaling pathway. In the IFN signaling pathway leading to STAT activation, both JAK1 and TYK2 are essential, whereas NF-kappaB activation requires only TYK2.

Toxicity of Some Phenolic Derivatives—In Vitro Studies

Cytotoxicity of 5 phenol derivatives (phenol, catechol, resorcinol, hydroquinone and phloroglucinol) was tested using a mouse 3T3 fibroblast cell line. Its relationships with structural and physicochemical properties were investigated.Linear regression analysis and Pearson’s correlation coefficient were used to characterise the relationship between cytotoxicity (expressed by IC50 values) and parameters of compounds or their toxicity in vivo expressed by LD 50 values. The studies showed that physicochemical properties of compounds seemed to have less influence on their cytotoxic potency than structural properties. Cytotoxicity of the compounds probably depends on the number of-OH groups and their location in the aromatic ring more than on physicochemical properties of compounds.The best correlation was obtained for IC50 values and LD 50values determined following rabbit skin administration and experimental skin irritation score.

Biocompatibility of sorbitol-containing polyesters. Part I: Synthesis, surface analysis and cell response in vitro

A series of sorbitol-containing polyesters were synthesized via a one-pot lipase-catalyzed condensation polymerization. Thin films were prepared by spin coating on silicon wafers and surfaces were analyzed by tapping mode atomic force microscopy and contact angle measurements. Surface morphologies and surface energies across the series of polyester films, including a poly( ε-caprolactone) (PCL) control were nearly indistinguishable. Biocompatibility of the sorbitol-containing polyester series was evaluated against a PCL control by measuring cell spreading and proliferation of a mouse fibroblast 3T3 cell line in vitro. Results confirmed that the sorbitol-containing polyester surfaces elicited cell behavior similar to the PCL control. These results establish the sorbitol-containing polyester series as a promising material for tissue engineering research and development.

The biocompatibility of crosslinkable copolymer coatings containing sulfobetaines and phosphobetaines

The comparison of copolymers containing sulfobetaine or phosphobetaine moieties for use as potential biocompatible coatings has been investigated. Two statistical copolymers were produced by a free radical polymerisation technique, one based on a sulfobetaine and the other on a phosphobetaine, both with a silyl group component to allow thermal crosslinking after coating. PMMA and glass discs were dip-coated with the polymers and their properties were compared to the uncoated controls. Bacterial adhesion to these coated materials was assessed using Staphylococcus epidermidis, Staphylococcus aureus and Pseudomonas aeruginosa. Human macrophages and granulocytes were used to assess the adhesion and activation of inflammatory cells whilst mouse 3T3 fibroblast cells were used to assess the propensity for the materials to support fibroblast cell adhesion. In all cases the polymer coatings reduced cell adhesion with respect to the base materials. The phosphobetaine-based copolymer coatings were shown to be markedly superior to the sulfobetaine-based copolymer coatings.

Cell adhesion on polytetrafluoroethylene modified by UV-irradiation in an ammonia atmosphere.

We report on the modification of polytetrafluoroethylene (PTFE) by exposure to the ultraviolet (UV) light of a Xe(2)*-excimer lamp at a wavelength of 172 nm in an ammonia atmosphere. Typical treatment times were up to 30 min. Subsequently, the samples were grafted with the amino acid alanine from an aqueous solution. The samples were characterized by means of optical transmission spectroscopy, laser-induced fluorescence and contact-angle measurements. We studied the adhesion of rat aortic smooth muscle cells (SMC) and mouse fibroblasts (3T3 cells) to the modified polymer samples using an in vitro technique, where the population density and spread of adhering cells is determined 24 h after seeding by image analysis. For both cell types the exposure of PTFE to UV-light in an ammonia atmosphere resulted in a significant increase in the number of adhering cells and in the size of their spreading area. The grafting with alanine enhanced this effect. Additional experiments with human endothelial cells (HEC) also demonstrated improved adhesion to modified PTFE. Thus, PTFE modified by our method appears to be a promising material for fabrication of artificial vascular prostheses and implants or for cultivation of skin substitutes.