Insulin and heparin co-immobilized 3D polyester fabrics for the cultivation of fibroblasts in low-serum media.

PubMed

Türkoğlu Saşmazel, Hilal; Aday, Sezin; Gümüşderelioğlu, Menemşe

2007-08-01

Insulin and/or heparin immobilized/co-immobilized non-woven polyester fabric (NWPF) discs were developed for the cultivation of L929 mouse fibroblasts in low-serum media. At first, NWPF discs were hydrolyzed to obtain a carboxylic acid group-introduced matrix (NWPF-hydrolyzed). Insulin and heparin co-immobilized NWPF (NWPF-insulin-heparin) was prepared by the grafting of PEO onto NWPF-hydrolyzed disc (NWPF-PEO), followed by the reaction first with insulin and then heparin. In the presence of spacer arm, PEO, the amount of immobilized insulin molecules significantly increased from 6.96 to 84.45 microg/cm(2). The amount of heparin bound to the NWPF-PEO (5.93 microg/cm(2)) was higher than that of the insulin immobilized surface (4.59 microg/cm(2)). Insulin and heparin immobilized NWPF discs were observed with fluorescence microscopy by labeling the insulin and heparin with 8-anilino-1-naphthalene sulfonic acid (ANS) or fluorescein isothiocyanate (FITC), respectively. L929 fibroblasts were used to check the cell adhesion and cell growth capabilities of modified NWPF discs in low-serum media (containing 5% fetal bovine serum). Optical photographs showed that after 2nd day of the culture, fibroblastic cells spread along the length of modified fibers, eventually filling the interfiber space. At the end of 6-day growth period, cell yield in the presence of immobilized heparin was a little bit higher than that of the immobilized insulin. Co-immobilized (insulin/heparin) NWPF discs did not accelerate the cell growth as well as insulin or heparin immobilized discs.

Different effects of a novel CaO-MgO-SiO₂-based multiphase glass-ceramic on cell behaviors of normal and cancer cells in vitro.

PubMed

Zhang, Mengjiao; Chen, Xianchun; Pu, Ximing; Liao, Xiaoming; Huang, Zhongbing; Yin, Guangfu

2014-04-01

The effects in vitro of a novel multiphase glass-ceramic (with nominal composition of 43.19% CaO, 7.68% MgO, and 49.13% SiO2 in weight percent) on cell adhesion, proliferation, differentiation and ultrastructure of human osteosarcoma cell line MG63, mouse fibroblasts L929, and human lung adenocarcinoma epithelial cell line A549 were investigated in this research. Scanning electron microscopy (SEM) micrographs revealed that the surface morphology of this glass-ceramic was beneficial to cell adhesion. The glass-ceramic extracts at certain concentrations could stimulate the proliferation and differentiation of MG63 and L929 cells, whereas inhibit A549 proliferation, which might be resulted from the released Si ions. In addition, when cultured with 0.1mg/mL glass-ceramic powder suspension, the cell ultrastructure of MG63 showed abundant organelles and L929 displayed the phenomena of cellular stress response. While more interestingly, A549 exhibited chromatin condensation, mitochondria swell and RER expansion, which was presumed to be early signs of apoptosis. These results suggest that this novel CaO-MgO-SiO2-based multiphase glass-ceramic has potential for bone regeneration and tissue engineering applications. Copyright © 2013 Elsevier B.V. All rights reserved.

Control of cell proliferation by a porous chitosan scaffold with multiple releasing capabilities

NASA Astrophysics Data System (ADS)

Cai, Shu-Jyun; Li, Ching-Wen; Weihs, Daphne; Wang, Gou-Jen

2017-12-01

The aim of this study was to develop a porous chitosan scaffold with long-acting drug release as an artificial dressing to promote skin wound healing. The dressing was fabricated by pre-freezing at different temperatures (-20 and -80 °C) for different periods of time, followed by freeze-drying to form porous chitosan scaffolds with different pore sizes. The chitosan scaffolds were then used to investigate the effect of the controlled release of fibroblast growth factor-basic (bFGF) and transforming growth factor-β1 (TGFβ1) on mouse fibroblast cells (L929) and bovine carotid endothelial cells (BEC). The biocompatibility of the prepared chitosan scaffold was confirmed with WST-1 proliferation and viability assay, which demonstrated that the material is suitable for cell growth. The results of this study show that the pore sizes of the porous scaffolds prepared by freeze-drying can change depending on the pre-freezing temperature and time via the formation of ice crystals. In this study, the scaffolds with the largest pore size were found to be 153 ± 32 μm and scaffolds with the smallest pores to be 34 ± 9 μm. Through cell culture analysis, it was found that the concentration that increased proliferation of L929 cells for bFGF was 0.005 to 0.1 ng/mL, and the concentration for TGFβ1 was 0.005 to 1 ng/mL. The cell culture of the chitosan scaffold and growth factors shows that 3.75 ng of bFGF in scaffolds with pore sizes of 153 ± 32 μm can promote L929 cell proliferation, while 400 pg of TGFβ1 in scaffolds with pore size of 34 ± 9 μm can enhance the proliferation of L929 cells, but also inhibit BEC proliferation. It is proposed that the prepared chitosan scaffolds can form a multi-drug (bFGF and TGFβ1) release dressing that has the ability to control wound healing via regulating the proliferation of different cell types.

Control of cell proliferation by a porous chitosan scaffold with multiple releasing capabilities

PubMed Central

Cai, Shu-Jyun; Li, Ching-Wen; Weihs, Daphne; Wang, Gou-Jen

2017-01-01

Abstract The aim of this study was to develop a porous chitosan scaffold with long-acting drug release as an artificial dressing to promote skin wound healing. The dressing was fabricated by pre-freezing at different temperatures (−20 and −80 °C) for different periods of time, followed by freeze-drying to form porous chitosan scaffolds with different pore sizes. The chitosan scaffolds were then used to investigate the effect of the controlled release of fibroblast growth factor-basic (bFGF) and transforming growth factor-β1 (TGFβ1) on mouse fibroblast cells (L929) and bovine carotid endothelial cells (BEC). The biocompatibility of the prepared chitosan scaffold was confirmed with WST-1 proliferation and viability assay, which demonstrated that the material is suitable for cell growth. The results of this study show that the pore sizes of the porous scaffolds prepared by freeze-drying can change depending on the pre-freezing temperature and time via the formation of ice crystals. In this study, the scaffolds with the largest pore size were found to be 153 ± 32 μm and scaffolds with the smallest pores to be 34 ± 9 μm. Through cell culture analysis, it was found that the concentration that increased proliferation of L929 cells for bFGF was 0.005 to 0.1 ng/mL, and the concentration for TGFβ1 was 0.005 to 1 ng/mL. The cell culture of the chitosan scaffold and growth factors shows that 3.75 ng of bFGF in scaffolds with pore sizes of 153 ± 32 μm can promote L929 cell proliferation, while 400 pg of TGFβ1 in scaffolds with pore size of 34 ± 9 μm can enhance the proliferation of L929 cells, but also inhibit BEC proliferation. It is proposed that the prepared chitosan scaffolds can form a multi-drug (bFGF and TGFβ1) release dressing that has the ability to control wound healing via regulating the proliferation of different cell types. PMID:29230255

Enhanced adherence of mouse fibroblast and vascular cells to plasma modified polyethylene.

PubMed

Reznickova, Alena; Novotna, Zdenka; Kolska, Zdenka; Kasalkova, Nikola Slepickova; Rimpelova, Silvie; Svorcik, Vaclav

2015-01-01

Since the last decade, tissue engineering has shown a sensational promise in providing more viable alternatives to surgical procedures for harvested tissues, implants and prostheses. Biomedical polymers, such as low-density polyethylene (LDPE), high-density polyethylene (HDPE) and ultra-high molecular weight polyethylene (UHMWPE), were activated by Ar plasma discharge. Degradation of polymer chains was examined by determination of the thickness of ablated layer. The amount of an ablated polymer layer was measured by gravimetry. Contact angle, measured by goniometry, was studied as a function of plasma exposure and post-exposure aging times. Chemical structure of modified polymers was characterized by angle resolved X-ray photoelectron spectroscopy. Surface chemistry and polarity of the samples were investigated by electrokinetic analysis. Changes in surface morphology were followed using atomic force microscopy. Cytocompatibility of plasma activated polyethylene foils was studied using two distinct model cell lines; VSMCs (vascular smooth muscle cells) as a model for vascular graft testing and connective tissue cells L929 (mouse fibroblasts) approved for standardized material cytotoxicity testing. Specifically, the cell number, morphology, and metabolic activity of the adhered and proliferated cells on the polyethylene matrices were studied in vitro. It was found that the plasma treatment caused ablation of the polymers, resulting in dramatic changes in their surface morphology and roughness. ARXPS and electrokinetic measurements revealed oxidation of the polymer surface. It was found that plasma activation has a positive effect on the adhesion and proliferation of VSMCs and L929 cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Biological Evaluation of the Copper/Low-density Polyethylene Nanocomposite Intrauterine Device

PubMed Central

Wang, Hong; Li, Jun; Xie, Changsheng; Duan, Zhuo; Sun, Li-Kui; Wang, Xin; Zhu, Changhong

2013-01-01

Devices and materials intended for clinical applications as medical and implant devices should be evaluated to determine their biocompatibility in physiological systems. This article presents results from cytotoxicity assay of L929 mouse fibroblasts culture, tests for skin irritation, intracutaneous reactivity and sensitization, and material implantation tests for the novel copper/low-density polyethylene nanocomposite intrauterine device (nano-Cu/LDPE IUD) with potential for future clinical utilization. Cytotoxicity test in vitro was conducted to evaluate the change in morphology, growth and proliferation of cultured L929 mouse fibroblasts, which in vivo examination for skin irritation (n = 6) and intracutaneous reactivity (n = 6) were carried out to explore the irritant behavior in New Zealand White rabbits. Skin sensitization was implemented to evaluate the potential skin sensitizing in Hartley guinea pigs (n = 35). The materials were implanted into the spinal muscle of rabbits (n = 9). The cytotoxicity grade of the nano-Cu/LDPE IUD was 0–1, suggested that the composite was nontoxic or mildly cytotoxic; no irritation reaction and skin sensitization were identified in any animals of specific extracts prepared from the material under test; similarly to the control sides, the inflammatory reaction was observed in the rabbits living tissue of the implanted material in intramuscular implantation assay. They indicated that the novel composite intrauterine device presented potential for this type of application because they meet the requirements of the standard practices recommended for evaluating the biological reactivity. The nano-Cu/LDPE IUD has good biocompatibility, which is biologically safe for the clinical research as a novel contraceptive device. PMID:24058521

Biological evaluation of the copper/low-density polyethylene nanocomposite intrauterine device.

PubMed

Hu, Li-Xia; He, Jing; Hou, Li; Wang, Hong; Li, Jun; Xie, Changsheng; Duan, Zhuo; Sun, Li-Kui; Wang, Xin; Zhu, Changhong

2013-01-01

Devices and materials intended for clinical applications as medical and implant devices should be evaluated to determine their biocompatibility in physiological systems. This article presents results from cytotoxicity assay of L929 mouse fibroblasts culture, tests for skin irritation, intracutaneous reactivity and sensitization, and material implantation tests for the novel copper/low-density polyethylene nanocomposite intrauterine device (nano-Cu/LDPE IUD) with potential for future clinical utilization. Cytotoxicity test in vitro was conducted to evaluate the change in morphology, growth and proliferation of cultured L929 mouse fibroblasts, which in vivo examination for skin irritation (n = 6) and intracutaneous reactivity (n = 6) were carried out to explore the irritant behavior in New Zealand White rabbits. Skin sensitization was implemented to evaluate the potential skin sensitizing in Hartley guinea pigs (n = 35). The materials were implanted into the spinal muscle of rabbits (n = 9). The cytotoxicity grade of the nano-Cu/LDPE IUD was 0-1, suggested that the composite was nontoxic or mildly cytotoxic; no irritation reaction and skin sensitization were identified in any animals of specific extracts prepared from the material under test; similarly to the control sides, the inflammatory reaction was observed in the rabbits living tissue of the implanted material in intramuscular implantation assay. They indicated that the novel composite intrauterine device presented potential for this type of application because they meet the requirements of the standard practices recommended for evaluating the biological reactivity. The nano-Cu/LDPE IUD has good biocompatibility, which is biologically safe for the clinical research as a novel contraceptive device.

Cytotoxic and Immunomodulatory Potential Activity of Physalis peruviana Fruit Extracts on Cervical Cancer (HeLa) and Fibroblast (L929) Cells.

PubMed

Mier-Giraldo, Helen; Díaz-Barrera, Luis Eduardo; Delgado-Murcia, Lucy Gabriela; Valero-Valdivieso, Manuel Fernando; Cáez-Ramírez, Gabriela

2017-10-01

It was purposed to evaluate the biological potential of ethanol and isopropanol crude extracts of ripe Physalis peruviana fruits. Cytotoxic and immunomodulatory effects of the expression of interleukin-6, interleukin-8, and monocyte chemoattractant protein-1 (MCP-1) were evaluated on human cervical cancer (HeLa) and murine fibroblast (L929) cells. The composition was evaluated by high-performance liquid chromatography diode-array detection and high-performance liquid chromatography ultraviolet/visible detection. The presence of ursolic acid and rosmarinic acid was found in both solvents. However, gallic acid, quercetin, and epicatechin were higher in isopropanol extracts ( P < .05). The results indicated a relationship among the total polyphenol content, antioxidant activity, and cytotoxic activity that was dependent on the solvent used. Isopropanol extracts presented a half-maximal inhibition concentration value (IC 50 ) of 60.48 ± 3.8 μg/mL for HeLa cells and 66.62 ± 2.67 μg/mL for L929 fibroblasts. The extracts reduced the release of interleukin-6, interleukin-8, and MCP-1 in a dose-dependent manner. Extracts showed anticancer and immunomodulatory potential for new complementary pharmaceutical products development.

Cytotoxic and Immunomodulatory Potential Activity of Physalis peruviana Fruit Extracts on Cervical Cancer (HeLa) and Fibroblast (L929) Cells

PubMed Central

Mier-Giraldo, Helen; Díaz-Barrera, Luis Eduardo; Delgado-Murcia, Lucy Gabriela; Valero-Valdivieso, Manuel Fernando; Cáez-Ramírez, Gabriela

2017-01-01

It was purposed to evaluate the biological potential of ethanol and isopropanol crude extracts of ripe Physalis peruviana fruits. Cytotoxic and immunomodulatory effects of the expression of interleukin-6, interleukin-8, and monocyte chemoattractant protein-1 (MCP-1) were evaluated on human cervical cancer (HeLa) and murine fibroblast (L929) cells. The composition was evaluated by high-performance liquid chromatography diode-array detection and high-performance liquid chromatography ultraviolet/visible detection. The presence of ursolic acid and rosmarinic acid was found in both solvents. However, gallic acid, quercetin, and epicatechin were higher in isopropanol extracts (P < .05). The results indicated a relationship among the total polyphenol content, antioxidant activity, and cytotoxic activity that was dependent on the solvent used. Isopropanol extracts presented a half-maximal inhibition concentration value (IC50) of 60.48 ± 3.8 μg/mL for HeLa cells and 66.62 ± 2.67 μg/mL for L929 fibroblasts. The extracts reduced the release of interleukin-6, interleukin-8, and MCP-1 in a dose-dependent manner. Extracts showed anticancer and immunomodulatory potential for new complementary pharmaceutical products development. PMID:28719984

Wound healing property of isolated compounds from Boesenbergia kingii rhizomes.

PubMed

Sudsai, Teeratad; Wattanapiromsakul, Chatchai; Tewtrakul, Supinya

2016-05-26

Boesenbergia kingii have been traditionally used in the treatment of inflammatory bowel disease, ulcerative colitis, aphthous ulcer, stomach discomfort, dysentery and abscess. Previously, we reported the B. kingii extract exert potential wound healing properties. Therefore the search of responsible constituents for wound healing property from these rhizomes is still relevant. This study was aimed to investigate for wound healing property of compounds from this plant in order to support its traditional uses. Wound healing activities were tested using in vitro assays including cell proliferation and migration assays, collagen production and H2O2-induced oxidative stress in mouse fibroblast L929 cells. The DPPH assay was also used to determine antioxidant activity. Fourteen compounds from the chloroform fraction possessed potent anti-oxidant and wound healing activities. Compound 11 exhibited the most potent anti-DPPH effect (IC50=21.0µM) and also active against 0.5mMH2O2-induced oxidative stress by increasing cell survival ability up to 60.3% at 10µM. In addition, compounds 3, 8 and 14 at 10µM significantly enhanced L929 viability with 119.2%, 122.7% and 113.7%, respectively. Compounds 2, 7, 8 and 14 markedly enhanced L929 migration on day 2 up to 60-76% at 10µM, whereas 7 and 14 strongly stimulated collagen production at 75.0 and 96.7µg/ml compared to the control group (57.5µg/ml), respectively. B. kingii is responsible for wound healing property via antioxidative effect, stimulation of fibroblast proliferation and migration as well as enhancement of collagen production. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Cytotoxicity and genotoxicity property of hydroxyapatite-mullite eluates.

PubMed

Kalmodia, Sushma; Sharma, Vyom; Pandey, Alok K; Dhawan, Alok; Basu, Bikramjit

2011-02-01

Long-term biomedical applications of implant materials may cause osteolysis, aseptic losing and toxicity. Therefore, we investigated the cytotoxic and genotoxic potential of hydroxyapatite (HA) mullite eluates in L929 mouse fibroblast cells. The spark plasma sintered HA-20% mullite biocomposite (HA20M) were ground using mortar and pestle as well as ball milling. The cells were exposed for 6 h to varying concentrations (10, 25, 50, 75 and 100%) of the eluates of HA-20% mullite (87 nm), HA (171 nm) and mullite (154 nm). The scanning electron microscopy and MTT assay revealed the concentration dependent toxicity of H20M eluate at and above 50%. The analysis of the DNA damaging potential of HA, mullite and HA20M eluates using Comet assay demonstrated a significant DNA damage by HA20M which was largely related to the presence of mullite. The results collectively demonstrate the cytotoxic and genotoxic potential of HA20M eluate in L929 cells is dependent on particle size, concentration and composition.

In vitro bioactivity of akermanite ceramics.

PubMed

Wu, Chengtie; Chang, Jiang; Ni, Siyu; Wang, Junying

2006-01-01

In this study, the bone-like apatite-formation ability of akermanite ceramics (Ca2MgSi2O7) in simulated body fluid (SBF) and the effects of ionic products from akermanite dissolution on osteoblasts and mouse fibroblasts (cell line L929) were investigated. In addition, osteoblast morphology and proliferation on the ceramics were evaluated. The results showed that akermanite ceramics possessed bone-like apatite-formation ability comparable with bioactive wollastonite ceramics (CaSiO3) after 20 days of soaking in SBF and the mechanism of bone-like apatite formation on akermanite ceramics is similar to that of wollastonite ceramics. The Ca, Si, and Mg ions from akermanite dissolution at certain ranges of concentration significantly stimulated osteoblast and L929 cell proliferation. Furthermore, osteoblasts spread well on the surface of akermanite ceramics, and proliferated with increasing the culture time. The results showed that akermanite ceramics possess bone-like apatite-formation ability and can release soluble ionic products to stimulate cell proliferation, which indicated good bioactivity. (c) 2005 Wiley Periodicals, Inc

Fibroblast responses and antibacterial activity of Cu and Zn co-doped TiO2 for percutaneous implants

NASA Astrophysics Data System (ADS)

Zhang, Lan; Guo, Jiaqi; Yan, Ting; Han, Yong

2018-03-01

In order to enhance skin integration and antibacterial activity of Ti percutaneous implants, microporous TiO2 coatings co-doped with different doses of Cu2+ and Zn2+ were directly fabricated on Ti via micro-arc oxidation (MAO). The structures of coatings were investigated; the behaviors of fibroblasts (L-929) as well as the response of Staphylococcus aureus (S. aureus) were evaluated. During the MAO process, a large number of micro-arc discharges forming on Ti performed as penetrating channels; O2-, Ca2+, Zn2+, Cu2+ and PO43- delivered via the channels, giving rise to the formation of doped TiO2. Surface characteristics including phase component, topography, surface roughness and wettability were almost the same for different coatings, whereas, the amount of Cu doped in TiO2 decreased with the increased Zn amount. Compared with Cu single-doped TiO2 (0.77 Wt% Cu), the co-doped with appropriate amounts of Cu and Zn, for example, 0.55 Wt% Cu and 2.53 Wt% Zn, further improved proliferation of L-929, facilitated fibroblasts to switch to fibrotic phenotype, and enhanced synthesis of collagen I as well as the extracellular collagen secretion; the antibacterial properties including contact-killing and release-killing were also enhanced. By analyzing the relationship of Cu/Zn amount in TiO2 and the behaviors of L-929 and S. aureus, it can be deduced that when the doped Zn is in a low dose (<1.79 Wt%), the behaviors of L-929 and S. aureus are sensitive to the reduced amount of Cu2+, whereas, Zn2+ plays a key role in accelerating fibroblast functions and reducing S. aureus when its dose obviously increases from 2.63 to 6.47 Wt%.

Dispersion fraction enhances cellular growth of carbon nanotube and aluminum oxide reinforced ultrahigh molecular weight polyethylene biocomposites.

PubMed

Patel, Anup Kumar; Balani, Kantesh

2015-01-01

Ultrahigh molecular weight polyethylene (UHMWPE) is widely used as bone-replacement material for articulating surfaces due to its excellent wear resistance and low coefficient of friction. But, the wear debris, generated during abrasion between mating surfaces, leads to aseptic loosening of implants. Thus, various reinforcing agents are generally utilized, which may alter the surface and biological properties of UHMWPE. In the current work, the cellular response of compression molded UHMWPE upon reinforcement of bioactive multiwalled carbon nanotubes (MWCNTs) and bioinert aluminum oxide (Al2O3) is investigated. The phase retention and stability were observed using X-ray diffraction, Raman spectroscopy and Fourier transform infrared (FTIR) spectroscopy. The reinforcement of MWCNTs and Al2O3 has shown to alter the wettability (from contact angle of ~88°±2° to ~118°±4°) and surface energy (from ~23.20 to ~17.75 mN/m) of composites with respect to UHMWPE, without eliciting any adverse effect on cytocompatibility for the L929 mouse fibroblast cell line. Interestingly, the cellular growth of the L929 mouse fibroblast cell line is observed to be dominated by the dispersion fraction of surface free energy (SFE). After 48 h of incubation period, a decrease in metabolic activity of MWCNT-Al2O3 reinforced composites is attributed to apatite formation that reduces the dispersion fraction of surface energy. The mineralized apatite during incubation was confirmed and quantified by energy dispersive spectroscopy and X-ray diffraction respectively. Thus, the dispersion fraction of surface free energy can be engineered to play an important role in achieving enhanced metabolic activity of the MWCNT-Al2O3 reinforced UHMWPE biopolymer composites. Copyright © 2014 Elsevier B.V. All rights reserved.

Synthesis of Thiolated Alginate and Evaluation of Mucoadhesiveness, Cytotoxicity and Release Retardant Properties

PubMed Central

Jindal, A. B.; Wasnik, M. N.; Nair, Hema A.

2010-01-01

Modification of polymers by covalent attachment of thiol bearing pendant groups is reported to impart many beneficial properties to them. Hence in the present study, sodium alginate–cysteine conjugate was synthesized by carbodiimide mediated coupling under varying reaction conditions and the derivatives characterized for thiol content. The thiolated alginate species synthesized had bound thiol content ranging from 247.8±11.03–324.54±10.107 ΅mol/g of polymer depending on the reaction conditions. Matrix tablets based on sodium alginate-cysteine conjugate and native sodium alginate containing tramadol hydrochloride as a model drug were prepared and mucoadhesive strength and in vitro drug release from the tablets were compared. Tablets containing 75 mg sodium alginate-cysteine conjugate could sustain release of 10 mg of model drug for 3 h, whereas 90% of the drug was released within 1 h from corresponding tablets prepared using native sodium alginate. An approximately 2-fold increase in the minimal detachment force of the tablets from an artificial mucin film was observed for sodium alginate–cysteine conjugate as compared to native sodium alginate. In vitro cytotoxicity studies in L-929 mouse fibroblast cells studied using an MTT assay revealed that at low concentrations of polymer, sodium alginate–cysteine conjugate was less toxic to L-929 mouse fibroblast cell line when compared to native sodium alginate. Hence, thiolation is found to be a simple route to improving polymer performance. The combination of improved controlled drug release and mucoadhesive properties coupled with the low toxicity of these new excipients builds up immense scope for the use of thiolated polymers in mucoadhesive drug delivery systems. PMID:21969750

Effect of artificial saliva with different pH levels on the cytotoxicity of soft denture lining materials.

PubMed

Akay, Canan; Tanış, Merve Ç; Sevim, Handan

2017-10-13

The aim of this study was to evaluate the cytotoxic effects of 9 different soft denture liners on the viability of L-929 mouse fibroblast cells at different incubation periods by storing them in artificial saliva (AS) with different pH levels. 96 disk samples from each lining material were prepared and divided into 4 groups: GI: No treatment; GII: Stored in artificial saliva with pH 3 for 21 days; Group III: Stored in artificial saliva with pH 7 for 21 days; and Group IV: Stored in artificial saliva with pH 14 for 21 days. The cytotoxicity of the extracts to cultured mouse fibroblasts (L-929) was measured by MTT (tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-dipHnyltetrazolium bromide) assay. Data were analyzed using 1-way analysis of variation (ANOVA). It was found that for the pH 3 values of New Truliner, Trusoft, Mollosil Plus, Dentusil, TDV, and HydroCast®; for the pH 7 values of Ufi Gel P and Elite plus; and for the pH 14 values of HydroCast®, there was a noncytotoxic effect during both the 24-hour and 48-hour incubation periods. In the control group 48-hour incubation period, HydroCast®, TDV, Mollosil, 24-hour incubation period Elite plus, for pH 3 values; Elite Plus 24-hour incubation period, for pH 7 values Trusoft 48-hour incubation period there was a moderately cytotoxic effect. This in vitro study revealed that storage in artificial saliva with different pH levels can affect the cytotoxicity of soft lining materials.

Evaluation of cytotoxicity, immune compatibility and antibacterial activity of biogenic silver nanoparticles.

PubMed

Składanowski, M; Golinska, P; Rudnicka, K; Dahm, H; Rai, M

2016-12-01

The study was focused on assessment of antibacterial activity, cytotoxicity and immune compatibility of biogenic silver nanoparticles (AgNPs) synthesized from Streptomyces sp. NH28 strain. Nanoparticles were biosynthesized and characterized by UV-Vis spectroscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, nanoparticle tracking analysis system and zeta potential. Antibacterial activity was tested against Gram-positive and Gram-negative bacteria; minimal inhibitory concentration was recorded. Cytotoxicity was estimated using L929 mouse fibroblasts via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test. Biocompatibility of AgNPs was performed using THP1-XBlue™ cells. Biogenic AgNPs presented high antibacterial activity against all tested bacteria. Minimum inhibitory concentration of AgNPs against bacterial cells was found to be in range of 1.25-10 μg/mL. Silver nanoparticles did not show any harmful interaction to mouse fibroblast cell line, and no activation of nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) cells was observed at concentration below 10 µg/mL. The half-maximal inhibitory concentration (IC 50 ) value was established at 64.5 μg/mL. Biological synthesis of silver can be used as an effective system for formation of metal nanoparticles. Biosynthesized AgNPs can be used as an antibacterial agent, which can be safe for eukaryotic cells.

In Vitro and In Vivo Toxicity Evaluation of Colloidal Silver Nanoparticles Used in Endodontic Treatments.

PubMed

Takamiya, Aline Satie; Monteiro, Douglas Roberto; Bernabé, Daniel Galera; Gorup, Luiz Fernando; Camargo, Emerson Rodrigues; Gomes-Filho, João Eduardo; Oliveira, Sandra Helena Penha; Barbosa, Debora Barros

2016-06-01

Silver nanoparticles have been used for different purposes in dentistry, including endodontic treatments. The aim of this study was to determine the cytotoxicity of different types of silver nanoparticles on mouse fibroblast cell line L929 and the reaction of subcutaneous connective tissue of Wistar rats to these nanoparticles. Silver nanoparticles of an average size of 5 nm were synthesized with ammonia (SNA) or polyvinylpyrrolidone (SNP). L929 was exposed to SNA and SNP (0.1-100 μg/mL), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and enzyme-linked immunosorbent assays were performed after 6, 24, and 48 hours. Culture medium was used as the control. Sixteen rats received, individually, 3 polyethylene tubes filled with a fibrin sponge embedded in 100 μL SNA or SNP (1 μg/mL). A fibrin sponge with no embedding was the control. Tissue reaction was performed qualitatively and quantitatively after 7, 15, 30, and 90 days of implantation in the dorsal connective tissue of Wistar rats. SNA and SNP were cytotoxic to L929 in higher concentrations, with SNA significantly more toxic than SNP. SNA and SNP did not induce significant interleukin-1β and interleukin-6 production. The release of stem cell factor by L929 increased 48 hours after the treatment with SNP at 5 μg/mL. Histologic examination showed that the inflammatory responses caused by SNA and SNP at 1 μg/mL were similar to the control in all experimental periods. It was concluded that SNA and SNP were not cytotoxic at 25 μg/mL or lower concentrations. However, for safe clinical use, further studies establishing others points of its toxicologic profile are recommended. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

In situ mineralization of hydroxyapatite on electrospun chitosan-based nanofibrous scaffolds.

PubMed

Yang, Dongzhi; Jin, Yu; Zhou, Yingshan; Ma, Guiping; Chen, Xiangmei; Lu, Fengmin; Nie, Jun

2008-03-10

A biocomposite of hydroxyapatite (HAp) with electrospun nanofibrous scaffolds was prepared by using chitosan/polyvinyl alcohol (CS/PVA) and N-carboxyethyl chitosan/PVA (CECS/PVA) electrospun membranes as organic matrix, and HAp was formed in supersaturated CaCl2 and KH2PO4 solution. The influences of carboxylic acid groups in CECS/PVA fibrous scaffold and polyanionic additive poly(acrylic acid) (PAA) in the incubation solution on the crystal distribution of the HAp were investigated. Field-emission scanning electron microscopy (FE-SEM), energy-dispersive spectroscopy (EDS), wide-angle X-ray diffraction (WAXD), and Fourier transform infrared (FTIR) were used to characterize the morphology and structure of the deposited mineral phase on the scaffolds. It was found that addition of PAA to the mineral solution and use of matrix with carboxylic acid groups promoted mineral growth and distribution of HAp. MTT testing and SEM imaging from mouse fibroblast (L929) cell culture revealed the attachment and growth of mouse fibroblast on the surface of biocomposite scaffold, and that the cell morphology and viability were satisfactory for the composite to be used in bioapplications.

Synthesis of a chitosan-based photo-sensitive hydrogel and its biocompatibility and biodegradability.

PubMed

He, Ming; Han, Baoqin; Jiang, Zhiwen; Yang, Yan; Peng, Yanfei; Liu, Wanshun

2017-06-15

Hydroxyethyl chitosan (HECTS) is one of the most important water soluble derivatives of chitosan. In this study, photo-sensitive azidehydroxyethyl chitosan (AZ-HECTS) was synthesized with grafting degree 3.4%, and its water solution resulted in an insoluble hydrogel by 254nm UV irradiation for 90s. AZ-HECTS hydrogels, with water absorption 86.21%, had little impact on growth of mouse fibroblast (L929) and presented good cell biocompatibility. Obvious sudden degradation stage, slow degradation stage and no apparent toxicity was observed after AZ-HECTS hydrogels implanted into rats, and tissue inflammation was slighter, which indicated favorable biological safety. Furthermore, AZ-HECTS hydrogels was loaded with heparin, and released 50% of heparin accumulated 14d. AZ-HECTS-heparin hydrogels showed inhibitory effects on L929, and pro-growth functions within vascular endothelial cells seeded on the hydrogels, meanwhile a positive influence on vascular endothelial growth factor expression. In conclusion, AZ-HECTS hydrogels possessed favorable biocompatibility and biodegradability and had good potential as drug carrier. Copyright © 2017 Elsevier Ltd. All rights reserved.

UV-shielding property, photocatalytic activity and photocytotoxicity of ceria colloid solutions.

PubMed

Zholobak, N M; Ivanov, V K; Shcherbakov, A B; Shaporev, A S; Polezhaeva, O S; Baranchikov, A Ye; Spivak, N Ya; Tretyakov, Yu D

2011-01-10

UV-shielding property, photocatalytic activity and cytotoxicity (including photocytotoxicity) of citrate-stabilized ceria colloid solutions were studied. It was established that UV-shielding property (namely, the sun protection factor, the critical absorption wavelength and the UVA/UVB-ratio) of ceria nanoparticles are as good as those of titanium dioxide and zinc oxide nanoparticles. It was further demonstrated that ceria nanoparticles possesses substantially lower photocatalytic activity, which additionally decreases upon decrease in ceria particle size. It was found that colloid ceria solutions are non-toxic to mouse fibroblasts (L929) and fibroblast-like cells of African Green monkey (VERO). Moreover, ceria nanoparticles are capable to protect these cells from UV-irradiation-induced damage. It was proposed that nanocrystalline ceria could be used not only as UV-blocking material, but also as prophylactic and even therapeutic compound for sunburns treatment. Copyright © 2010 Elsevier B.V. All rights reserved.

Optimized in vitro procedure for assessing the cytocompatibility of magnesium-based biomaterials.

PubMed

Jung, Ole; Smeets, Ralf; Porchetta, Dario; Kopp, Alexander; Ptock, Christoph; Müller, Ute; Heiland, Max; Schwade, Max; Behr, Björn; Kröger, Nadja; Kluwe, Lan; Hanken, Henning; Hartjen, Philip

2015-09-01

Magnesium (Mg) is a promising biomaterial for degradable implant applications that has been extensively studied in vitro and in vivo in recent years. In this study, we developed a procedure that allows an optimized and uniform in vitro assessment of the cytocompatibility of Mg-based materials while respecting the standard protocol DIN EN ISO 10993-5:2009. The mouse fibroblast line L-929 was chosen as the preferred assay cell line and MEM supplemented with 10% FCS, penicillin/streptomycin and 4mM l-glutamine as the favored assay medium. The procedure consists of (1) an indirect assessment of effects of soluble Mg corrosion products in material extracts and (2) a direct assessment of the surface compatibility in terms of cell attachment and cytotoxicity originating from active corrosion processes. The indirect assessment allows the quantification of cell-proliferation (BrdU-assay), viability (XTT-assay) as well as cytotoxicity (LDH-assay) of the mouse fibroblasts incubated with material extracts. Direct assessment visualizes cells attached to the test materials by means of live-dead staining. The colorimetric assays and the visual evaluation complement each other and the combination of both provides an optimized and simple procedure for assessing the cytocompatibility of Mg-based biomaterials in vitro. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Infection of nonphagocytic host cells by legionella.

PubMed

Day, Shandra R; Sifri, Costi D; Hoffman, Paul S

2013-01-01

Legionella pneumophila is an intracellular pathogen of free-living protozoa that can also infect alveolar macrophages, L929 fibroblast cells, and HeLa cells. Infection of nonphagocytic cells by L. pneumophila can be used to study invasion mechanisms, compare infectivity of different strains and identify factors important for virulence. Virulent strains of L. pneumophila exposed to monolayers of L929 cells are able to invade and form virus-like plaques, which can be enumerated as a measure of infectivity. Invasiveness of HeLa cells can also be used to evaluate relative infectivity and to study mechanisms of invasion and to track the development of cyst-like forms. The detailed methods of both the L929 plaque assay and HeLa cell invasion assay are described.

[Study on three kinds of gasoline oxygenates-induced DNA damage in mice fibroblasts].

PubMed

Song, Chonglin; Zhang, Zhifu; Chen, Xue; Zhang, Yanfeng; Wang, Chunhua; Liu, Keming

2002-10-01

To study DNA damage of three kinds of gasoline oxygenates. Single cell gel electrophoresis assay(Comet assay) was used to detect the damage effects of three gasoline oxygenates[methyl tertiary butyl ether(MTBE), ethanol anhydrous(EA) and dimethyl carbonate(DMC)] on DNA in L-929 mice fibroblasts. In certain concentation(37.500-150.000 mg/ml), MTBE could directly cause DNA damage of L-929 mice fibroblasts. There was obvious dose-effect relationship, i.e. when the concentration of MTBE was increased from 9.375 to 150.000 mg/ml, the comet rate also increased from 4% to 85%, and the length of comet tail changed correspondingly. The results of EA and DMC were negative. Under the condition of this experiment(150.000 mg/ml), MTBE could directly cause DNA damage while the effect of EA and DMC on DNA damage was not found.

In Vitro Cytotoxicity Assessment of an Orthodontic Composite Containing Titanium-dioxide Nano-particles.

PubMed

Heravi, Farzin; Ramezani, Mohammad; Poosti, Maryam; Hosseini, Mohsen; Shajiei, Arezoo; Ahrari, Farzaneh

2013-01-01

Background and aims. Incorporation of nano-particles to orthodontic bonding systems has been considered to prevent enamel demineralization around appliances. This study investigated cytotoxicity of Transbond XT adhesive containing 1 wt% titanium dioxide (TiO2) nano-particles. Materials and methods. Ten composite disks were prepared from each of the conventional and TiO2-containg composites and aged for 1, 3, 5, 7 and 14 days in Dulbecco's Modified Eagle's Medium (DMEM). The extracts were obtained and exposed to culture media of human gingival fibroblasts (HGF) and mouse L929 fibroblasts. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results. Both adhesives were moderately toxic for HGF cells on the first day of the experiment, but the TiO2-containing adhesive produced significantly lower toxicity than the pure adhesive (P<0.05). No significant differences were found in cell viability percentages between the two groups on the other days (P>0.05). There was a significant reduction in cell toxicity with increasing pre-incubation time (P<0.001). L929 cells showed similar toxicity trends, but lower sensitivity to detect cytotoxicity of dental composites. Conclusion. The orthodontic adhesive containing TiO2 nano-particles indicated comparable or even lower toxicity than its nano-particle-free counterpart, indicating that incorporation of 1 wt% TiO2 nano-particles to the composite structure does not result in additional health hazards compared to that occurring with the pure adhesive.

[Biocompatibility research of true bone ceramics].

PubMed

Qiao, Wei; Ren, Xiaoqi; Shi, Hao; Li, Jing; Yang, Ting; Ma, Shaoying; Zhao, Yaping; Su, Chengzhong; Li, Baoxing

2017-10-01

To investigate the biocompatibility of true bone ceramic (TBC) and provide experimental basis for clinic application. TBC was prepared from healthy adult bovine cancellous bone by deproteinization and high temperature calcinations. Mouse fibroblast cell line (L929 cells) were cultured with the leaching liquor of TBC in vitro , and the cytotoxicity was evaluated at 2nd, 4th, and 7th days. L929 cells were inoculated into the TBC and cultured for 4 days. The cell adhesion and proliferation on the surface of the TBC were observed by scanning electron microscopy, and evaluated the cell compatibility of TBC. Ten New Zealand white rabbits were divided into 2 groups, and drilled holes at the tibia of both hind limbs. TBC and hydroxyapatite (HA) were implanted into the left side (experimental group) and the right side (control group), respectively. And the biocompatibility of TBC was evaluated by general observation and histological observation at 4 and 26 weeks after implantation. Cytotoxicity test showed that the cytotoxicity level of leaching liquor of TBC was grade 0-1. Cell compatibility experiments showed that the L929 cells adhered well on the surface of TBC and migrated into the pores. The implantation test in vivo showed that experimental group and control group both had mild or moderate inflammatory response at 4 weeks, and new bone formation occurred. At 26 weeks, there was no inflammatory reaction observed in both groups, and new bone formation was observed in varying degrees. TBC have good biocompatibility and can be used to repair bone defect in clinic.

Akt Regulates TNFα Synthesis Downstream of RIP1 Kinase Activation during Necroptosis

PubMed Central

McNamara, Colleen R.; Ahuja, Ruchita; Osafo-Addo, Awo D.; Barrows, Douglas; Kettenbach, Arminja; Skidan, Igor; Teng, Xin; Cuny, Gregory D.; Gerber, Scott; Degterev, Alexei

2013-01-01

Necroptosis is a regulated form of necrotic cell death that has been implicated in the pathogenesis of various diseases including intestinal inflammation and systemic inflammatory response syndrome (SIRS). In this work, we investigated the signaling mechanisms controlled by the necroptosis mediator receptor interacting protein-1 (RIP1) kinase. We show that Akt kinase activity is critical for necroptosis in L929 cells and plays a key role in TNFα production. During necroptosis, Akt is activated in a RIP1 dependent fashion through its phosphorylation on Thr308. In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1. Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1). Akt activity, mediated in part through mTORC1, links RIP1 to JNK activation and autocrine production of TNFα. In other cell types, such as mouse lung fibroblasts and macrophages, Akt exhibited control over necroptosis-associated TNFα production without contributing to cell death. Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation. PMID:23469174

Osthole activates glucose uptake but blocks full activation in L929 fibroblast cells, and inhibits uptake in HCLE cells

PubMed Central

Alabi, Ola D.; Gunnink, Stephen M.; Kuiper, Benjamin D.; Kerk, Samuel A.; Braun, Emily; Louters, Larry L.

2016-01-01

Aims Osthole, a coumarin derivative, has been used in Chinese medicine and studies have suggested a potential use in treatment of diabetes and cancers. Therefore, we investigated the effects of osthole and other coumarins on GLUT1 activity in two cell lines that exclusively express GLUT1. Main Methods We measured the magnitude and time frame of the effects of osthole and related coumarins on glucose uptake in two cells lines; L929 fibroblast cells which have low GLUT1 expression levels and low basal glucose uptake and HCLE cells which have high GLUT1 concentrations and high basal uptake. We also explored the effects of these coumarins in combination with other GLUT1 activators. Key findings Osthole activates glucose uptake in L929 cells with a modest maximum 1.7-fold activation achieved by 50 µM with both activation and recovery occurring within minutes. However, osthole blocks full acute activation of glucose uptake by other, more robust activators. This behavior mimics the effects of other thiol reactive compounds and suggests that osthole is interacting with cysteine residues, possibly within GLUT1 itself. Coumarin, 7-hydroxycoumarin, and 7-methoxycoumarin, do not affect glucose uptake, which is consistent with the notion that the isoprenoid structure in osthole may be important to gain membrane access to GLUT1. In contrast to its effects in L929 cells, osthole inhibits basal glucose uptake in the more active HCLE cells. Significance The differential effects of osthole in L929 and HCLE cells indicated that regulation of GLUT1 varies, likely depending on its membrane concentration. PMID:24657891

Osthole activates glucose uptake but blocks full activation in L929 fibroblast cells, and inhibits uptake in HCLE cells.

PubMed

Alabi, Ola D; Gunnink, Stephen M; Kuiper, Benjamin D; Kerk, Samuel A; Braun, Emily; Louters, Larry L

2014-05-02

Osthole, a coumarin derivative, has been used in Chinese medicine and studies have suggested a potential use in treatment of diabetes and cancers. Therefore, we investigated the effects of osthole and other coumarins on GLUT1 activity in two cell lines that exclusively express GLUT1. We measured the magnitude and time frame of the effects of osthole and related coumarins on glucose uptake in two cells lines; L929 fibroblast cells which have low GLUT1 expression levels and low basal glucose uptake and HCLE cells which have high GLUT1 concentrations and high basal uptake. We also explored the effects of these coumarins in combination with other GLUT1 activators. Osthole activates glucose uptake in L929 cells with a modest maximum 1.7-fold activation achieved by 50 μM with both activation and recovery occurring within minutes. However, osthole blocks full acute activation of glucose uptake by other, more robust activators. This behavior mimics the effects of other thiol reactive compounds and suggests that osthole is interacting with cysteine residues, possibly within GLUT1 itself. Coumarin, 7-hydroxycoumarin, and 7-methoxycoumarin, do not affect glucose uptake, which is consistent with the notion that the isoprenoid structure in osthole may be important to gain membrane access to GLUT1. In contrast to its effects in L929 cells, osthole inhibits basal glucose uptake in the more active HCLE cells. The differential effects of osthole in L929 and HCLE cells indicated that regulation of GLUT1 varies, likely depending on its membrane concentration. Copyright © 2014 Elsevier Inc. All rights reserved.

Encapsulation of basic fibroblast growth factor by polyelectrolyte multilayer microcapsules and its controlled release for enhancing cell proliferation.

PubMed

She, Zhen; Wang, Chunxia; Li, Jun; Sukhorukov, Gleb B; Antipina, Maria N

2012-07-09

Basic fibroblast growth factor (FGF2) is an important protein for cellular activity and highly vulnerable to environmental conditions. FGF2 protected by heparin and bovine serum albumin was loaded into the microcapsules by a coprecipitation-based layer-by-layer encapsulation method. Low cytotoxic and biodegradable polyelectrolytes dextran sulfate and poly-L-arginine were used for capsule shell assembly. The shell thickness-dependent encapsulation efficiency was measured by enzyme-linked immunosorbent assay. A maximum encapsulation efficiency of 42% could be achieved by microcapsules with a shell thickness of 14 layers. The effects of microcapsule concentration and shell thickness on cytotoxicity, FGF2 release kinetics, and L929 cell proliferation were evaluated in vitro. The advantage of using microcapsules as the carrier for FGF2 controlled release for enhancing L929 cell proliferation was analyzed.

In Vitro Cytotoxicity Assessment of an Orthodontic Composite Containing Titanium-dioxide Nano-particles

PubMed Central

Heravi, Farzin; Ramezani, Mohammad; Poosti, Maryam; Hosseini, Mohsen; Shajiei, Arezoo; Ahrari, Farzaneh

2013-01-01

Background and aims. Incorporation of nano-particles to orthodontic bonding systems has been considered to prevent enamel demineralization around appliances. This study investigated cytotoxicity of Transbond XT adhesive containing 1 wt% titanium dioxide (TiO2) nano-particles. Materials and methods. Ten composite disks were prepared from each of the conventional and TiO2-containg composites and aged for 1, 3, 5, 7 and 14 days in Dulbecco’s Modified Eagle’s Medium (DMEM). The extracts were obtained and exposed to culture media of human gingival fibroblasts (HGF) and mouse L929 fibroblasts. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results. Both adhesives were moderately toxic for HGF cells on the first day of the experiment, but the TiO2-containing adhesive produced significantly lower toxicity than the pure adhesive (P<0.05). No significant differences were found in cell viability percentages between the two groups on the other days (P>0.05). There was a significant reduction in cell toxicity with increasing pre-incubation time (P<0.001). L929 cells showed similar toxicity trends, but lower sensitivity to detect cytotoxicity of dental composites. Conclusion. The orthodontic adhesive containing TiO2 nano-particles indicated comparable or even lower toxicity than its nano-particle-free counterpart, indicating that incorporation of 1 wt% TiO2 nano-particles to the composite structure does not result in additional health hazards compared to that occurring with the pure adhesive. PMID:24578816

Toxicity evaluation of ZnO nanostructures on L929 fibroblast cell line using MTS assay

NASA Astrophysics Data System (ADS)

Bakhori, Siti Khadijah Mohd; Mahmud, Shahrom; Ann, Ling Chuo; Mohamed, Azman Seeni; Saifuddin, Siti Nazmin; Masudi, Sam'an Malik; Mohamad, Dasmawati

2015-04-01

ZnO has wide applications in medical and dentistry apart from being used as optoelectronic devices such as solar cells, photodetectors, sensors and light emitting diodes (LEDs). Therefore, the toxicity evaluation is important to know the toxicity level on normal cell line. The toxicity of two grades ZnO nanostructures, ZnO-4 and ZnO-8 have been carried out using cytotoxicity test of MTS assay on L929 rat fibroblast cell line. Prior to that, ZnO-4 and ZnO-8 were characterized for its morphology, structure and optical properties using FESEM, X-ray diffraction, and Photoluminescence respectively. The two groups revealed difference in morphology and exhibit slightly shifted of near band edge emission of Photoluminescence other than having a similar calculated crystallite size of nanostructures. The viability of cells after 72h were obtained and the statistical significance value was calculated using SPSS v20. The p value is more than 0.05 between untreated and treated cell with ZnO. This insignificant value of p>0.05 can be summarized as a non-toxic level of ZnO-4 and ZnO-8 on the L929 cell line.

Toxicity evaluation of ZnO nanostructures on L929 fibroblast cell line using MTS assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bakhori, Siti Khadijah Mohd; Mahmud, Shahrom; Ann, Ling Chuo

2015-04-24

ZnO has wide applications in medical and dentistry apart from being used as optoelectronic devices such as solar cells, photodetectors, sensors and light emitting diodes (LEDs). Therefore, the toxicity evaluation is important to know the toxicity level on normal cell line. The toxicity of two grades ZnO nanostructures, ZnO-4 and ZnO-8 have been carried out using cytotoxicity test of MTS assay on L929 rat fibroblast cell line. Prior to that, ZnO-4 and ZnO-8 were characterized for its morphology, structure and optical properties using FESEM, X-ray diffraction, and Photoluminescence respectively. The two groups revealed difference in morphology and exhibit slightly shiftedmore » of near band edge emission of Photoluminescence other than having a similar calculated crystallite size of nanostructures. The viability of cells after 72h were obtained and the statistical significance value was calculated using SPSS v20. The p value is more than 0.05 between untreated and treated cell with ZnO. This insignificant value of p>0.05 can be summarized as a non-toxic level of ZnO-4 and ZnO-8 on the L929 cell line.« less

The improvement of fibroblast growth on hydrophobic biopolyesters by coating with polyhydroxyalkanoate granule binding protein PhaP fused with cell adhesion motif RGD.

PubMed

Dong, Ying; Li, Ping; Chen, Chong-bo; Wang, Zhi-hui; Ma, Ping; Chen, Guo-Qiang

2010-12-01

Polyhydroxyalkanoates (PHA), a family of biopolyesters, have been studied as tissue engineering biomaterials due to their adjustable mechanical properties, biodegradability and tissue compatibility. Amphiphilic PHA granule binding protein PhaP has been shown to be able to bind to hydrophobic surfaces of polymers, especially PHA, via strong hydrophobic interaction. Genes of PhaP and RGD peptides, which are a cell adhesion motif recognized by many cell surface receptors, were successfully expressed and obtained as a pure fusion protein PhaP-RGD in Escherichia coli DH5α. When films of poly(3-hydroxybutyrate-co-3-hydroxy- hexanoate) (PHBHHx), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and polylactic acid (PLA) were coated with PhaP-RGD, their surface hydrophilicities were all increased compared with their corresponding naked (non-coated) films, respectively. Among the three biopolyesters, PHBHHx demonstrated the strongest affinity to PhaP. In vitro study showed that mouse fibroblasts L929 and mouse embryonic fibroblasts NIH/3T3 attached better and grew faster on all three PhaP-RGD coated films compared with their related behaviors on PhaP coated and non-coated films, respectively. Both fibroblasts attached and grew very well on PhaP-RGD coated PHBHHx, PHBV and PLA, even in their serum-free medium, while the non-coated and PhaP coated biopolyesters poorly supported the cell growth if the two fibroblasts were incubated in their serum free medium. These results indicated that PhaP-RGD could be used as a coating material to improve cell growth on hydrophobic biopolyesters for implant tissue engineering purposes. Copyright © 2010 Elsevier Ltd. All rights reserved.

Cell death induced by hydroxyapatite on L929 fibroblast cells.

PubMed

Inayat-Hussain, S H; Rajab, N F; Roslie, H; Hussin, A A; Ali, A M; Annuar, B O

2004-05-01

Biomaterials intended for end-use application as bone-graft substitutes have to undergo safety evaluation. In this study, we investigated the in vitro cytotoxic effects especially to determine the mode of death of two hydroxyapatite compounds (HA2, HA3) which were synthesized locally. The methods used for cytotoxicity was the standard MTT assay whereas AO/PI staining was performed to determine the mode of cell death in HA treated L929 fibroblasts. Our results demonstrated that both HA2 and HA3 were not significantly cytotoxic as more than 75% cells after 72 hours treatment were viable. Furthermore, we found that the major mode of cell death in HA treated cells was apoptosis. In conclusion, our results demonstrated that these hydroxyapatite compounds are not cytotoxic where the mode of death was primarily via apoptosis.

Controlled release of mitomycin C from PHEMAH-Cu(II) cryogel membranes.

PubMed

Bakhshpour, Monireh; Yavuz, Handan; Denizli, Adil

2018-02-19

Molecular imprinting technique was used for the preparation of antibiotic and anti-neoplastic chemotherapy drug (mitomycin C) imprinted cryogel membranes (MMC-ICM). The membranes were synthezied by using metal ion coordination interactions with N-methacryloyl-(l)-histidine methyl ester (MAH) functional monomer and template molecules (i.e. MMC). The 2-hydroxyethyl methacrylate (HEMA) monomer and methylene bisacrylamide (MBAAm) crosslinker were used for the preparation of mitomycin C imprinted cryogel membranes by radical suspension polymerization technique. The imprinted cryogel membranes were characterized by scanning electron microscopy (SEM), Brunauer-Emmett-Teller (BET), Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR) and swelling degree measurements. Cytotoxicity of MMC-ICMs was investigated using mouse fibroblast cell line L929. Time-dependent release of MMC was demonstrated within 150 h from cryogel membranes. Cryogels demonstrated very high MMC loading efficiency (70-80%) and sustained MMC release over hours.

Biocompatibility of modified ultra-high-molecular-weight polyethylene

NASA Astrophysics Data System (ADS)

Novotná, Z.; Lacmanová, V.; Rimpelová, S.; Juřik, P.; Polívková, M.; Å vorčik, V.

2016-09-01

Ultra-high-molecular-weight polyethylene (UHMWPE, PE) is a synthetic polymer used for biomedical applications because of its high impact resistance, ductility and stability in contact with physiological fluids. Therefore this material is being used in human orthopedic implants such as total joint replacements. Surface modification of this material relates to changes of its surface hydrophilicity, energy, microstructure, roughness, and morphology, all influencing its biological response. In our recent work, PE was treated by an Ar+ plasma discharge and then grafted with biologically active polyethylene glycol in order to enhance adhesion and proliferation of mouse fibroblast (L929). The surface properties of pristine PE and its grafted counterparts were studied by goniometry (surface wettability). Furthermore, Atomic Force Microscopy was used to determine the surface morphology and roughness. The biological response of the L929 cell lines seeded on untreated and plasma treated PE matrices was quantified in terms of the cell adhesion, density, and metabolic activity. Plasma treatment leads to the ablation of the polymer surface layers. Plasma treatment and subsequent poly(ethylene glycol) grafting lead to dramatic changes in the polymer surface morphology and roughness. Biological tests, performed in vitro, show increased adhesion and proliferation of cells on modified polymers. Grafting with poly(ethylene glycol) increases cell proliferation compared to plasma treatment.

Biocompatible nano-gallium/hydroxyapatite nanocomposite with antimicrobial activity.

PubMed

Kurtjak, Mario; Vukomanović, Marija; Kramer, Lovro; Suvorov, Danilo

2016-11-01

Intensive research in the area of medical nanotechnology, especially to cope with the bacterial resistance against conventional antibiotics, has shown strong antimicrobial action of metallic and metal-oxide nanomaterials towards a wide variety of bacteria. However, the important remaining problem is that nanomaterials with highest antibacterial activity generally express also a high level of cytotoxicity for mammalian cells. Here we present gallium nanoparticles as a new solution to this problem. We developed a nanocomposite from bioactive hydroxyapatite nanorods (84 wt %) and antibacterial nanospheres of elemental gallium (16 wt %) with mode diameter of 22 ± 11 nm. In direct comparison, such nanocomposite with gallium nanoparticles exhibited better antibacterial properties against Pseudomonas aeruginosa and lower in-vitro cytotoxicity for human lung fibroblasts IMR-90 and mouse fibroblasts L929 (efficient antibacterial action and low toxicity from 0.1 to 1 g/L) than the nanocomposite of hydroxyapatite and silver nanoparticles (efficient antibacterial action and low toxicity from 0.2 to 0.25 g/L). This is the first report of a biomaterial composite with gallium nanoparticles. The observed strong antibacterial properties and low cytotoxicity make the investigated material promising for the prevention of implantation-induced infections that are frequently caused by P. aeruginosa.

Alkaline pH activates the transport activity of GLUT1 in L929 fibroblast cells.

PubMed

Gunnink, Stephen M; Kerk, Samuel A; Kuiper, Benjamin D; Alabi, Ola D; Kuipers, David P; Praamsma, Riemer C; Wrobel, Kathryn E; Louters, Larry L

2014-04-01

The widely expressed mammalian glucose transporter, GLUT1, can be acutely activated in L929 fibroblast cells by a variety of conditions, including glucose deprivation, or treatment with various respiration inhibitors. Known thiol reactive compounds including phenylarsine oxide and nitroxyl are the fastest acting stimulators of glucose uptake, implicating cysteine biochemistry as critical to the acute activation of GLUT1. In this study, we report that in L929 cells glucose uptake increases 6-fold as the pH of the uptake solution is increased from 6 to 9 with the half-maximal activation at pH 7.5; consistent with the pKa of cysteine residues. This pH effect is essentially blocked by the pretreatment of the cells with either iodoacetamide or cinnamaldehyde, compounds that form covalent adducts with reduced cysteine residues. In addition, the activation by alkaline pH is not additive at pH 8 with known thiol reactive activators such as phenylarsine oxide or hydroxylamine. Kinetic analysis in L929 cells at pH 7 and 8 indicate that alkaline conditions both increases the Vmax and decreases the Km of transport. This is consistent with the observation that pH activation is additive to methylene blue, which activates uptake by increasing the Vmax, as well as to berberine, which activates uptake by decreasing the Km. This suggests that cysteine biochemistry is utilized in both methylene blue and berberine activation of glucose uptake. In contrast a pH increase from 7 to 8 in HCLE cells does not further activate glucose uptake. HCLE cells have a 25-fold higher basal glucose uptake rate than L929 cells and the lack of a pH effect suggests that the cysteine biochemistry has already occurred in HCLE cells. The data are consistent with pH having a complex mechanism of action, but one likely mediated by cysteine biochemistry. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Viability and proliferation of L929, tumour and hybridoma cells in the culture media containing sericin protein as a supplement or serum substitute.

PubMed

Cao, Ting-Ting; Zhang, Yu-Qing

2015-09-01

Cell cultures often require the addition of animal serum and other supplements. In this study, silk sericin, a bioactive protein, recovered from the waste of silk floss production was hydrolysed into three pepsin-degraded sericin peptides with different ranges of molecular mass. Normal animal cells, tumour cells and hybridoma cells were cultured systematically in FBS culture media containing sericin as a supplement or serum substitute. The culture test and microscopic observation of L929 cells showed that the smaller molecular weight of the degraded sericin is most suitable for cell culture. The cell culture results showed that with the degradation of sericin, for normal mouse fibroblast L929 cells, addition of 0.75 % sericin into FBS culture medium yields cell viability that is superior to FBS culture medium alone. When all serum was replaced by sericin, cell viability in the sericin medium could reach about one half of that in FBS medium. When in a medium containing a mixture of FBS: sericin (6:4, v/v), the cell culture effect is about 80 %. For the cultures of four tumour and one hybridoma cells, regardless of the molecular weight range, these degraded sericin peptides could substitute all serum in FBS media. The cell viability and proliferation of these tumour and hybridoma cells are equivalent or superior to that in FBS medium. In other words, cell viability and proliferation of these tumour and hybridoma cells in sericin media are more preferable to serum media. The mechanism of the sericin protein to promote cell growth and proliferation will be further investigated later.

Cytotoxicity and biocompatibility of Zirconia (Y-TZP) posts with various dental cements

PubMed Central

Shin, Hyeongsoon; Ko, Hyunjung

2016-01-01

Objectives Endodontically treated teeth with insufficient tooth structure are often restored with esthetic restorations. This study evaluated the cytotoxicity and biological effects of yttria partially stabilized zirconia (Y-TZP) blocks in combination with several dental cements. Materials and Methods Pairs of zirconia cylinders with medium alone or cemented with three types of dental cement including RelyX U200 (3M ESPE), FujiCEM 2 (GC), and Panavia F 2.0 (Kuraray) were incubated in medium for 14 days. The cytotoxicity of each supernatant was determined using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays on L929 fibroblasts and MC3T3-E1 osteoblasts. The levels of interleukin-6 (IL-6) mRNA were evaluated by reverse transcription polymerase chain reaction (RT-PCR), and IL-6 protein was evaluated by enzyme-linked immunosorbent assays (ELISA). The data were analyzed using one-way ANOVA and Tukey post-hoc tests. A p < 0.05 was considered statistically significant. Results The MTT assays showed that MC3T3-E1 osteoblasts were more susceptible to dental cements than L929 fibroblasts. The resin based dental cements increased IL-6 expression in L929 cells, but reduced IL-6 expression in MC3T3-E1 cells. Conclusions Zirconia alone or blocks cemented with dental cement showed acceptable biocompatibilities. The results showed resin-modified glass-ionomer based cement less produced inflammatory cytokines than other self-adhesive resin-based cements. Furthermore, osteoblasts were more susceptible than fibroblasts to the biological effects of dental cement. PMID:27508157

Cytotoxicity and biocompatibility of Zirconia (Y-TZP) posts with various dental cements.

PubMed

Shin, Hyeongsoon; Ko, Hyunjung; Kim, Miri

2016-08-01

Endodontically treated teeth with insufficient tooth structure are often restored with esthetic restorations. This study evaluated the cytotoxicity and biological effects of yttria partially stabilized zirconia (Y-TZP) blocks in combination with several dental cements. Pairs of zirconia cylinders with medium alone or cemented with three types of dental cement including RelyX U200 (3M ESPE), FujiCEM 2 (GC), and Panavia F 2.0 (Kuraray) were incubated in medium for 14 days. The cytotoxicity of each supernatant was determined using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays on L929 fibroblasts and MC3T3-E1 osteoblasts. The levels of interleukin-6 (IL-6) mRNA were evaluated by reverse transcription polymerase chain reaction (RT-PCR), and IL-6 protein was evaluated by enzyme-linked immunosorbent assays (ELISA). The data were analyzed using one-way ANOVA and Tukey post-hoc tests. A p < 0.05 was considered statistically significant. The MTT assays showed that MC3T3-E1 osteoblasts were more susceptible to dental cements than L929 fibroblasts. The resin based dental cements increased IL-6 expression in L929 cells, but reduced IL-6 expression in MC3T3-E1 cells. Zirconia alone or blocks cemented with dental cement showed acceptable biocompatibilities. The results showed resin-modified glass-ionomer based cement less produced inflammatory cytokines than other self-adhesive resin-based cements. Furthermore, osteoblasts were more susceptible than fibroblasts to the biological effects of dental cement.

Comparative analysis of the effect of low-dimensional alumina structures on cell lines L929 and Neuro-2a

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fomenko, A. N., E-mail: alserova@ispms.tsc.ru; Korovin, M. S., E-mail: msk@ispms.tsc.ru

The paper presents the toxicity evaluation of nanostructures on the basis of alumina of different shape (nanofibers, nanoplates, nanosheets, nanosheet agglomerates) and with similar physical and chemical properties (particle size, specific surface area, phase composition, and zeta potential). The nanostructures were examined by transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), low-temperature nitrogen adsorption, and dynamic light scattering (DLS). The cytotoxicity of nanostructures was estimated using L929 fibroblast cells and Neuro-2a tumor cells. It has been found that the L929 cells are less subject to the influence of alumina nanoparticles than the Neuro-2a tumor cells. Probably, themore » differences in the proliferation activity of normal and tumor cells in contact with the synthesized nanostructures are due to a change in the pH of the cell microenvironment.« less

Comparative analysis of the effect of low-dimensional alumina structures on cell lines L929 and Neuro-2a

NASA Astrophysics Data System (ADS)

Fomenko, A. N.; Korovin, M. S.

2016-08-01

The paper presents the toxicity evaluation of nanostructures on the basis of alumina of different shape (nanofibers, nanoplates, nanosheets, nanosheet agglomerates) and with similar physical and chemical properties (particle size, specific surface area, phase composition, and zeta potential). The nanostructures were examined by transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), low-temperature nitrogen adsorption, and dynamic light scattering (DLS). The cytotoxicity of nanostructures was estimated using L929 fibroblast cells and Neuro-2a tumor cells. It has been found that the L929 cells are less subject to the influence of alumina nanoparticles than the Neuro-2a tumor cells. Probably, the differences in the proliferation activity of normal and tumor cells in contact with the synthesized nanostructures are due to a change in the pH of the cell microenvironment.

A promiscuous liaison between IL-15 receptor and Axl receptor tyrosine kinase in cell death control

PubMed Central

Budagian, Vadim; Bulanova, Elena; Orinska, Zane; Thon, Lutz; Mamat, Uwe; Bellosta, Paola; Basilico, Claudio; Adam, Dieter; Paus, Ralf; Bulfone-Paus, Silvia

2005-01-01

Discrimination between cytokine receptor and receptor tyrosine kinase (RTK) signaling pathways is a central paradigm in signal transduction research. Here, we report a ‘promiscuous liaison' between both receptors that enables interleukin (IL)-15 to transactivate the signaling pathway of a tyrosine kinase. IL-15 protects murine L929 fibroblasts from tumor necrosis factor α (TNFα)-induced cell death, but fails to rescue them upon targeted depletion of the RTK, Axl; however, Axl-overexpressing fibroblasts are TNFα-resistant. IL-15Rα and Axl colocalize on the cell membrane and co-immunoprecipitate even in the absence of IL-15, whereby the extracellular part of Axl proved to be essential for Axl/IL-15Rα interaction. Most strikingly, IL-15 treatment mimics stimulation by the Axl ligand, Gas6, resulting in a rapid tyrosine phosphorylation of both Axl and IL-15Rα, and activation of the phosphatidylinositol 3-kinase/Akt pathway. This is also seen in mouse embryonic fibroblasts from wild-type but not Axl−/− or IL-15Rα−/− mice. Thus, IL-15-induced protection from TNFα-mediated cell death involves a hitherto unknown IL-15 receptor complex, consisting of IL-15Rα and Axl RTK, and requires their reciprocal activation initiated by ligand-induced IL-15Rα. PMID:16308569

Green synthesis of silver nanoparticles by Ricinus communis var. carmencita leaf extract and its antibacterial study

NASA Astrophysics Data System (ADS)

Ojha, Sunita; Sett, Arghya; Bora, Utpal

2017-09-01

In this study, we report synthesis of silver nanoparticles (RcAgNPs) from silver nitrate solution using methanolic leaf extract of Ricinus communis var. carmencita. The polyphenols present in the leaves reduce Ag++ ions to Ag0 followed by a color change. Silver nanoparticle formation was ensured by surface plasmon resonance between 400 nm to 500 nm. Crystallinity of the synthesized nanoparticles was confirmed by UHRTEM, SAED and XRD analysis. The capping of phytochemicals and thermal stability of RcAgNPs were assessed by FTIR spectra and TGA analysis, respectively. It also showed antibacterial activity against both gram positive and gram negative strains. RcAgNPs were non-toxic against normal cell line (mouse fibroblast cell line L929) at lower concentrations (80 µg ml-1).

In vitro cytotoxicity evaluation of a 50.8% NiTi single crystal.

PubMed

Manceur, Aziza; Chellat, Fatiha; Merhi, Yahye; Chumlyakov, Yuriy; Yahia, L'Hocine

2003-11-01

To our knowledge, the biocompatibility of nickel-titanium (NiTi) single crystals has not been reported. Yet certain orientations of single crystals present several advantages over the polycrystalline form in terms of maximal strain, fatigue resistance, and temperature range of superelasticity. Therefore we tested the in vitro biocompatibility of 50.8% NiTi single crystals in the orientation <001> after four different heat treatments in a helium atmosphere followed by mechanical polishing. The study was performed on the material extracts after immersion of the specimens in cell culture medium (DMEM) for 7 days at 37 degrees C. Cytotoxicity studies were performed on L-929 mouse fibroblasts using the MTT assay. J-774 macrophages were used to assess the potential inflammatory effect of the extracts by IL1-beta and TNF-alpha dosages (sandwich ELISA method). Exposure of L-929 to material extracts did not affect cell viability. In addition, IL1-beta and TNF-alpha secretion was not stimulated after incubation with NiTi extracts compared to the negative controls. These results were predictable since atomic absorption spectroscopy did not detect nickel ions in the extracts with a resolution of 1 ppm. Within the limits of in vitro testing, our results demonstrate that the TiNi(50.8%) single crystals do not trigger a cytotoxic reaction. Copyright 2003 Wiley Periodicals, Inc.

Characterization and in vitro evaluation of electrospun chitosan/polycaprolactone blend fibrous mat for skin tissue engineering.

PubMed

Prasad, Tilak; Shabeena, E A; Vinod, D; Kumary, T V; Anil Kumar, P R

2015-01-01

The electrospinning technique allows engineering biomimetic scaffolds within micro to nanoscale range mimicking natural extracellular matrix (ECM). Chitosan (CS) and polycaprolactone (PCL) were dissolved in a modified solvent mixture consisting of formic acid and acetone (3:7) and mixed in different weight ratios to get chitosan-polycaprolactone [CS-PCL] blend solutions. The CS-PCL blend polymer was electrospun in the same solvent system and compared with PCL. The physicochemical characterization of the electrospun fibrous mats was done using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), tensile test, swelling properties, water contact angle (WCA) analysis, surface profilometry and thermo gravimetric analysis (TGA). The CS-PCL fibrous mat showed decreased hydrophobicity. The CS-PCL mats also showed improved swelling property, tensile strength, thermal stability and surface roughness. The cytocompatibility of the CS-PCL and PCL fibrous mats were examined using mouse fibroblast (L-929) cell line by direct contact and cellular activity with extract of materials confirmed non-cytotoxic nature. The potential of CS-PCL and PCL fibrous mats as skin tissue engineering scaffolds were assessed by cell adhesion, viability, proliferation and actin distribution using human keratinocytes (HaCaT) and L-929 cell lines. Results indicate that CS-PCL is a better scaffold for attachment and proliferation of keratinocytes and is a potential material for skin tissue engineering.

Phase assemblage study and cytocompatibility property of heat treated potassium magnesium phosphate-silicate ceramics.

PubMed

Kumar, Ravi; Kalmodia, Sushma; Nath, Shekhar; Singh, Dileep; Basu, Bikramjit

2009-08-01

This article reports the study on a new generation bioactive ceramic, based on MgKPO(4) (Magnesium Potassium Phosphate, abbreviated as MKP) for biomedical applications. A series of heat treatment experiments on the slip cast silica (SiO(2)) containing MKP ceramics were carried out at 900, 1,000 and 1,100 degrees C for 4 h in air. The density of the slip cast ceramic increases to 2.5 gm/cm(3) upon heat treatment at 900 degrees C. However, no significant change in density is measured upon heat treatment to higher temperature of 1,000 and 1,100 degrees C. On the basis of XRD results, the presence of K(2)MgSi(5)O(12) and dehydrated MgKPO(4) were confirmed and complementary information has also been obtained using FT-IR and Raman spectroscopy. In order to confirm the in vitro cytocompatibility property, the cell culture tests were carried out on selected samples and the results reveal good cell adhesion and spreading of L929 mouse fibroblast cells. MTT assay analysis with L929 cells confirmed non-cytotoxic behavior of MKP containing ceramics and the results are comparable with sintered HAp ceramics. It is expected that the newly developed MKP based materials could be a good substitute for hydroxyapatite (HAp or HA) based bioceramics.

Graphene oxide decorated electrospun gelatin nanofibers: Fabrication, properties and applications.

PubMed

Jalaja, K; Sreehari, V S; Kumar, P R Anil; Nirmala, R James

2016-07-01

Gelatin nanofiber fabricated by electrospinning process is found to mimic the complex structural and functional properties of natural extracellular matrix for tissue regeneration. In order to improve the physico-chemical and biological properties of the nanofibers, graphene oxide is incorporated in the gelatin to form graphene oxide decorated gelatin nanofibers. The current research effort is focussed on the fabrication and evaluation of physico-chemical and biological properties of graphene oxide-gelatin composite nanofibers. The presence of graphene oxide in the nanofibers was established by transmission electron microscopy (TEM). We report the effect of incorporation of graphene oxide on the mechanical, thermal and biological performance of the gelatin nanofibers. The tensile strength of gelatin nanofibers was increased from 8.29±0.53MPa to 21±2.03MPa after the incorporation of GO. In order to improve the water resistance of nanofibers, natural based cross-linking agent, namely, dextran aldehyde was employed. The cross-linked composite nanofibers showed further increase in the tensile strength up to 56.4±2.03MPa. Graphene oxide incorporated gelatin nanofibers are evaluated for bacterial activity against gram positive (Staphylococcus aureus) and gram negative (Escherichia coli) bacteria and cyto compatibility using mouse fibroblast cells (L-929 cells). The results indicate that the graphene oxide incorporated gelatin nanofibers do not prevent bacterial growth, nevertheless support the L-929 cell adhesion and proliferation. Copyright © 2016 Elsevier B.V. All rights reserved.

Sponge-Associated Bacteria Produce Non-cytotoxic Melanin Which Protects Animal Cells from Photo-Toxicity.

PubMed

Vijayan, Vijitha; Jasmin, Chekidhenkuzhiyil; Anas, Abdulaziz; Parakkaparambil Kuttan, Sreelakshmi; Vinothkumar, Saradavey; Perunninakulath Subrayan, Parameswaran; Nair, Shanta

2017-09-01

Melanin is a photo-protective polymer found in many organisms. Our research shows that the bacteria associated with darkly pigmented sponges (Haliclona pigmentifera, Sigmadocia pumila, Fasciospongia cavernosa, Spongia officinalis, and Callyspongia diffusa) secrete non-cytotoxic melanin, with antioxidant activity that protects animal cells from photo-toxicity. Out of 156 bacterial strains screened, 22 produced melanin and these melanin-producing bacteria (MPB) were identified as Vibrio spp., Providencia sp., Bacillus sp., Shewanella sp., Staphylococcus sp., Planococcus sp., Salinococcus sp., and Glutamicibacter sp. Maximum melanin production was exhibited by Vibrio alginolyticus Marine Microbial Reference Facility (MMRF) 534 (50 mg ml -1 ), followed by two isolates of Vibrio harveyi MMRF 535 (40 mg ml -1 ) and MMRF 546 (30 mg ml -1 ). Using pathway inhibition assay and FT-IR spectral analysis, we identified the melanin secreted into the culture medium of MPB as 1,8-dihydroxynaphthalene-melanin. The bacterial melanin was non-cytotoxic to mouse fibroblast L929 cells and brine shrimps up to a concentration of 200 and 500 ppm, respectively. Bacterial melanin showed antioxidant activity at very low concentration (IC 50 -9.0 ppm) and at 50 ppm, melanin protected L929 cells from UV-induced intracellular reactive oxygen stress. Our study proposes sponge-associated bacteria as a potential source of non-cytotoxic melanin with antioxidant potentials.

The construction of 3D-engineered tissues composed of cells and extracellular matrices by hydrogel template approach.

PubMed

Matsusaki, Michiya; Yoshida, Hiroaki; Akashi, Mitsuru

2007-06-01

The three-dimensional (3D)-engineered tissues composed of only cells and extracellular matrices (ECM) were constructed by the hydrogel template approach. The disulfide-crosslinked poly(gamma-glutamic acid) hydrogels were prepared as a template hydrogel. These template hydrogels were easily decomposed under physiological conditions using reductants such as cysteine, glutathione and dithiothreitol by cleavage of disulfide crosslinkage to thiol groups. The decomposed polymers are soluble in cell culture medium. The cleaving of disulfide bond was determined by UV-vis and FT-IR spectroscopies. We successfully prepared the 3D-engineered tissues (thickness/diameter, 2mm/1cm) composed of mouse L929 fibroblast cells and ECM by the decomposition of only the template hydrogel with cysteine after 10 days 3D-cell culture on/in the template hydrogel. The size and thickness of the 3D-engineered tissues was completely transferred from the template hydrogel. The cultured L929 cells viability in the obtained engineered tissues was confirmed by a culture test, WST-1 method and LIVE/DEAD staining assay. The engineered tissue was self-standing and highly dense composite of the cultured cells and collagen produced by the cells. This hydrogel template approach may be useful as a new class of soft-tissue engineering technology to substitute a synthetic polymer scaffold to the ECM scaffold produced from the cultured cells.

Prevention of peritendinous adhesions with electrospun ibuprofen-loaded poly(L-lactic acid)-polyethylene glycol fibrous membranes.

PubMed

Liu, Shen; Hu, Changmin; Li, Fengfeng; Li, Xu-jun; Cui, Wenguo; Fan, Cunyi

2013-02-01

Physical barriers are commonly used to reduce peritendinous adhesion after injury. However, the inflammatory response to surgery cannot be prevented. This study was designed to evaluate the ability of ibuprofen-loaded poly(l-lactic acid)-polyethylene glycol (PELA) diblock copolymer fibrous membranes in preventing adhesion formation and reduce inflammation. Electrospun PELA fibrous membranes underwent mechanical testing and were characterized by morphology, surface wettability, drug release, and degradation. Results of an in vitro drug release study showed that a burst release was followed by sustained release from fibrous membranes with high initial ibuprofen content. Fewer L929 mouse fibroblasts adhered to and proliferated on the ibuprofen-loaded PELA fibrous membrane compared with tissue culture plates or PELA fibrous membrane without ibuprofen. In a chicken model of flexor digitorum profundus tendon surgery, the ibuprofen-loaded PELA fibrous membranes prevented tissue adhesion and significantly reduced inflammation. Taken together, these results demonstrate that ibuprofen-loaded PELA fibrous membranes prevent peritendinous adhesion formation better than membranes that do not contain ibuprofen, through anti-adhesion and anti-inflammatory actions.

The effects of acoustic vibration on fibroblast cell migration.

PubMed

Mohammed, Taybia; Murphy, Mark F; Lilley, Francis; Burton, David R; Bezombes, Frederic

2016-12-01

Cells are known to interact and respond to external mechanical cues and recent work has shown that application of mechanical stimulation, delivered via acoustic vibration, can be used to control complex cell behaviours. Fibroblast cells are known to respond to physical cues generated in the extracellular matrix and it is thought that such cues are important regulators of the wound healing process. Many conditions are associated with poor wound healing, so there is need for treatments/interventions, which can help accelerate the wound healing process. The primary aim of this research was to investigate the effects of mechanical stimulation upon the migratory and morphological properties of two different fibroblast cells namely; human lung fibroblast cells (LL24) and subcutaneous areolar/adipose mouse fibroblast cells (L929). Using a speaker-based system, the effects of mechanical stimulation (0-1600Hz for 5min) on the mean cell migration distance (μm) and actin organisation was investigated. The results show that 100Hz acoustic vibration enhanced cell migration for both cell lines whereas acoustic vibration above 100Hz was found to decrease cell migration in a frequency dependent manner. Mechanical stimulation was also found to promote changes to the morphology of both cell lines, particularly the formation of lamellipodia and filopodia. Overall lamellipodia was the most prominent actin structure displayed by the lung cell (LL24), whereas filopodia was the most prominent actin feature displayed by the fibroblast derived from subcutaneous areolar/adipose tissue. Mechanical stimulation at all the frequencies used here was found not to affect cell viability. These results suggest that low-frequency acoustic vibration may be used as a tool to manipulate the mechanosensitivity of cells to promote cell migration. Copyright © 2016 Elsevier B.V. All rights reserved.

Green synthesis and characterization of silver nanoparticles using alcoholic flower extract of Nyctanthes arbortristis and in vitro investigation of their antibacterial and cytotoxic activities.

PubMed

Gogoi, Nayanmoni; Babu, Punuri Jayasekhar; Mahanta, Chandan; Bora, Utpal

2015-01-01

Here we report the synthesis of silver nanoparticles using ethanolic flower extract of Nyctanthes arbortristis, UVvisible spectra and TEM indicated the successful formation of silver nanoparticles. Crystalline nature of the silver nanoparticles was confirmed by X-ray diffraction. Fourier Transform Infra-Red Spectroscopy analysis established the capping of the synthesized silver nanoparticles with phytochemicals naturally occurring in the ethanolic flower extract of N. arbortristis. The synthesized silver nanoparticles showed antibacterial activity against the pathogenic strain of Escherichia coli MTCC 443. Furthermore, cytotoxicity of the silver nanoparticles was tested on mouse fibroblastic cell line (L929) and found to be non-toxic, which thus proved their biocompatibility. Antibacterial activity and cytotoxicity assay carried out in this study open up an important perspective of the synthesized silver nanoparticles. Copyright © 2014 Elsevier B.V. All rights reserved.

[Enhancement of wound healing by taspine and its effect on fibroblast].

PubMed

Dong, Yalin; He, Langchong; Chen, Fang

2005-07-01

To study the effect of taspine on enhancement of skin wound healing and its effect on fibroblast proliferation and autocrine. The plerosis effect of taspine on experimental skin wound was observed in vivo. Different concentrations of taspine were added in vitro and MTT technique was applied to observe its effect on fibroblast proliferation, the levels of transforming growth factor-beta1 (TGF-13P) and epidermal growth factor (EGF) were determined by ELISA. In vivo, exo-applied taspine 300 microg and 150 microg accelerated the recovery of skin wound. In vitro, 0.50-0.4 microg/ml taspine could increase autocrine of TGF-beta1and EGF by fibroblast, but it showed no effect on L929 fibroblast proliferation. Taspine enhances wound healing by increasing the autocrine of TGF-beta1 and EGF by fibroblast.

Stable cavitation induces increased cytoplasmic calcium in L929 fibroblasts exposed to 1-MHz pulsed ultrasound.

PubMed

Tsukamoto, Akira; Higashiyama, Satoru; Yoshida, Kenji; Watanabe, Yoshiaki; Furukawa, Katsuko S; Ushida, Takashi

2011-12-01

An increase in cytoplasmic calcium (Ca(2+) increase) is a second messenger that is often observed under ultrasound irradiation. We hypothesize that cavitation is a physical mechanism that underlies the increase in Ca(2+) in these experiments. To control the presence of cavitation, the wave type was controlled in a sonication chamber. One wave type largely contained a traveling wave (wave type A) while the other wave type largely contained a standing wave (wave type B). Fast Fourier transform (FFT) analysis of a sound field produced by the wave types ascertained that stable cavitation was present only under wave type A ultrasound irradiation. Under the two controlled wave types, the increase in Ca(2+) in L929 fibroblasts was observed with fluorescence imaging. Under wave type A ultrasound irradiation, an increase in Ca(2+) was observed; however, no increase in Ca(2+) was observed under wave type B ultrasound irradiation. We conclude that stable cavitation is involved in the increase of Ca(2+) in cells subjected to pulsed ultrasound. Copyright © 2011 Elsevier B.V. All rights reserved.

Development of gellan gum-based microparticles/hydrogel matrices for application in the intervertebral disc regeneration.

PubMed

Pereira, Diana Ribeiro; Silva-Correia, Joana; Caridade, Sofia Glória; Oliveira, Joao T; Sousa, Rui A; Salgado, Antonio J; Oliveira, Joaquim M; Mano, João F; Sousa, Nuno; Reis, Rui L

2011-10-01

Low back pain is one of the most reported medical conditions associated to intervertebral disc (IVD) degeneration. Nucleus pulposus (NP) is often regarded as the structure where IVD degeneration begins. Gellan gum (GG)-based hydrogels for acellular and cellular tissue engineering strategies have been developed for finding applications as NP substitutes. The innovative strategy is based on the reinforcement of the hydrogel matrix with biocompatible and biodegradable GG microparticles (MPs), which are expected to improve the mechanical properties, while allowing to tailor its degradation rate. In this study, several GG MP/hydrogel disc formulations were prepared by means of mixing high acyl GG (0.75% (w/v)) and low acyl GG (2% (w/v)) GG aqueous solutions at different ratios, namely, 75%:25% (v/v), 50%:50% (v/v), and 25%:75% (v/v), respectively. The GG MP size was measured using a stereo microscope, and their dispersion within the hydrogel matrix was evaluated by means of staining the MPs with Toluidine Blue-O. The developed GG MPs/hydrogel discs were physicochemically characterized by Fourier-transform infrared spectroscopy and (1)H-nuclear magnetic resonance spectroscopy. The swelling behavior and degradation rate were assessed by immersion in a phosphate buffer saline for 14 days. The morphology and mechanical behavior were investigated by scanning electron microscopy and dynamic mechanical analysis, respectively. The mechanical properties of the hydrogel disc were improved by mixing the gels with the MPs. In addition, the possible cytotoxicity of the leachables released by MPs/hydrogel discs was screened in vitro, using a mouse lung fibroblast cell line (L929 cells). To investigate the encapsulation efficacy of L929 cells into the GG MPs/hydrogel discs, cells were stained with DAPI blue/Texas Red-Phalloidin and observed by confocal microscopy, after 24, 48, and 72 h of culturing. A cell viability assay was also performed using Calcein AM staining. The cell culture studies demonstrated that MPs/hydrogel discs are noncytotoxic over L929 cells. It was also demonstrated that L929 cells can be successfully encapsulated into the GG MPs of different formulations, remaining viable after 72 h of culturing. This study showed that GG hydrogel matrices reinforced with cell-loaded MPs could be a candidate strategy for NP regeneration. © Mary Ann Liebert, Inc.

Protective effect of metoclopramide against organophosphate-induced apoptosis in the murine skin fibroblast L929.

PubMed

Jaber, Basem M; Petroianu, Georg A; Rizvi, Syed A; Borai, Anwar; Saleh, Nada A; Hala, Sharif M; Saleh, Ayman M

2018-03-01

This study was performed to evaluate the protective efficacy of metoclopramide (MCP) against the organophosphates paraoxon (POX)- and malathion (MLT)-induced apoptosis in the murine L929 skin fibroblasts. L929 cells were exposed to either POX (10 nm) or 1.0 μm MLT in the absence and presence of increased concentrations of MCP. The protective effect of MCP on these organophosphate-stimulated apoptotic events was evaluated by flow cytometry analysis after staining with annexin-V/propidium iodide, processing and activation of the executioner caspase-3, cleavage of the poly-ADP ribose polymerase, fragmentation of the nucleosomal DNA and disruption of the mitochondrial membrane potential (Δψ). Our results showed that increased doses of MCP alone (≥10 μm) did not induce apoptosis or activation of caspase-3. Pretreatment of the cells with MCP attenuated all the apoptotic events triggered by the organophosphate compounds in a dose-dependent manner reaching ~70-80% protection when they were preincubated at 1 and 5 μm of the drug before the addition of POX and MLT, respectively. Interestingly, MCP did not offer a significant protective effect against the cytotoxicity of tumor necrosis factor-α, cisplatinum, etoposide or paclitaxel, which stimulate apoptosis by various mechanisms, suggesting that the anti-apoptotic effect of the drug is specific to organophosphates. The strong and specific anti-apoptotic activity of subclinical doses of MCP against the cytotoxicity of organophosphate compounds suggests its potential clinical application in treating their poisoning. Copyright © 2017 John Wiley & Sons, Ltd.

In Vitro Evaluation of the Antioxidant Activity and Wound Healing Properties of Jaboticaba (Plinia peruviana) Fruit Peel Hydroalcoholic Extract.

PubMed

Pitz, Heloisa da S; Pereira, Aline; Blasius, Mayara B; Voytena, Ana Paula L; Affonso, Regina C L; Fanan, Simone; Trevisan, Adriana C D; Ribeiro-do-Valle, Rosa M; Maraschin, Marcelo

2016-01-01

Jaboticaba is a fruit from a native tree to Brazil, Plinia peruviana. Jaboticaba peels are an important source of antioxidant molecules such as phenolic compounds. This study aimed to evaluate in vitro the activity of a hydroalcoholic extract of jaboticaba fruit peels (HEJFP) in wound healing processes and antioxidant activity in murine fibroblasts (L929 cell line). HEJFP concentrations (0.5, 1, 5, 10, 25, 50, 100, and 200 µg/mL) were tested in MTT assay and cell proliferation was verified at 100 µg/mL after 24 h and at 25, 50, and 100 µg/mL after 48 h of extract exposure. Evaluation of antioxidant activity was performed at 0.5, 5, 25, 50, and 100 µg/mL HEJFP concentrations. Cell treatment with HEJFP at 25, 50, and 100 µg/mL for 24 h followed by H2O2 exposure for 3 h showed a strong cytoprotective effect. In vitro scratch wound healing assay indicated that none of tested HEJFP concentrations (0.5, 5, 25, 50, and 100 µg/mL) were capable of increasing migration rate after 12 h of incubation. These results demonstrate a positive effect of HEJFP on the wound healing process on L929 fibroblasts cell line, probably due to the antioxidant activity exhibited by phytochemicals in the extract.

Mechanisms underlying the wound healing potential of propolis based on its in vitro antioxidant activity.

PubMed

Cao, Xue-Ping; Chen, Yi-Fan; Zhang, Jiang-Lin; You, Meng-Meng; Wang, Kai; Hu, Fu-Liang

2017-10-15

Propolis is a resinous substance collected by honeybees, Apis mellifera, from various plant sources. Having various pharmacological and biological activities, it has been used in folk medicine and complementary therapies since ancient times. To evaluate the effects and underlying mechanism of the protective effects of the ethanol extract of Chinese propolis (EECP) on L929 cells injured by hydrogen peroxide (H 2 O 2 ). The wound healing activities of EECP in L929 cells with H 2 O 2 -induced damage were investigated. The main components of EECP were analyzed by RP-HPLC, and the free radical scavenging capacity and reducing power were also measured. The effects of EECP on the expression of antioxidant-related genes in fibroblast L929 cells were determined using qRT-PCR and western blotting. EECP had significant protective effects against cell death induced by H 2 O 2 and significantly inhibited the decline of collagen mRNA expression caused by H 2 O 2 in L929 cells. EECP induced the expression of antioxidant-related genes, such as HO-1, GCLM, and GCLC, which has great implications for the potential of propolis to alleviate oxidative stress in wound tissues. The protective effects of propolis have great implications for using propolis as a wound healing regent. Copyright © 2017 Elsevier GmbH. All rights reserved.

Proliferative, anti-apoptotic and immune-enhancing effects of L-arginine in culture of skin fibroblasts.

PubMed

Kocic, H; Arsic, I; Stankovic, M; Tiodorovic, D; Ciric, V; Kocic, G

2017-01-01

Semi-essential amino acid L-arginine may be of fundamental importance in various intracellular and intercellular pathways related to skin repair and wound healing. Our current study was aimed to explore the effect of L-arginine on skin fibroblast (L929) signaling pathways involved in cell proliferation (Akt-pAkt kinase, Erk/pErk1/2 kinase, JNK/pJNK kinase and pStat-1), apoptosis (Bcl2 and Bax) and immune defense (NF-κB and CD26). Significant upregulation of Erk (p<0.011), pErk (p<0.017) and JNK (p<0.002) was documented, while the rise was not significant for pJNK kinase. The Akt/pAkt signaling pathway did not change significantly for the above-mentioned time and dose, while pStat-1 was significantly down regulated (p<0.011). The exposure of skin fibroblasts to L-arginine increased anti-apoptotic Bcl2/Bax stoichiometry ratio (p<0.05), obtained by calculation of their individual quantities. L-arginine was able to elicit NF-κB signaling through the increase of p65 active subunit level (p<0.004), while CD26 surface antigen level was not significantly changed. In conclusion, the exposure of skin fibroblasts to L-arginine may help in maintaining and stimulating skin fibroblast proliferative, anti-apoptotic and immune defense function. Therefore, the proposed L-arginine dose may be used for tissue regeneration application, which would be of importance in regenerative medicine, skin rejuvenation approaches and wound healing.

Facile synthesis, cytotoxicity and bioimaging of Fe(3+) selective fluorescent chemosensor.

PubMed

Saleem, Muhammad; Abdullah, Razack; Ali, Anser; Park, Bong Joo; Choi, Eun Ha; Hong, In Seok; Lee, Ki Hwan

2014-04-01

The designing and development of fluorescent chemosensors have recently been intensively explored for sensitive and specific detection of environmentally and biologically relevant metal ions in aqueous solution and living cells. Herein, we report the photophysical results of alanine substituted rhodamine B derivative 3 having specific binding affinity toward Fe(3+) with micro molar concentration level. Through fluorescence titration at 599nm, we were confirmed that ligand 3 exhibited ratiometric fluorescence response with remarkable enhancement in emission intensity by complexation between 3 and Fe(3+) while it appeared no emission in case of the competitive ions (Sc(3+), Yb(3+), In(3+), Ce(3+), Sm(3+), Cr(3+), Sn(2+), Pb(2+), Ni(2+), Co(2+), Cu(2+), Ba(2+), Ca(2+), Mg(2+), Ag(+), Cs(+), Cu(+), K(+)) in aqueous/methanol (60:40, v/v) at neutral pH. However, the fluorescence as well as colorimetric response of ligand-iron complex solution was quenched by addition of KCN which snatches the Fe(3+) from complex and turn off the sensor confirming the recognition process was reversible. Furthermore, bioimaging studies against L-929 cells (mouse fibroblast cells) and BHK-21 (hamster kidney fibroblast), through confocal fluorescence microscopic experiment indicated that ligand showed good permeability and minimum toxicity against the tested cell lines. Copyright © 2014 Elsevier Ltd. All rights reserved.

Cellular proliferation, cellular viability, and biocompatibility of HA-ZnO composites.

PubMed

Saha, Naresh; Dubey, Ashutosh K; Basu, Bikramjit

2012-01-01

One of the important issues in the development of hydroxyapatite (HA)-based biomaterials is the prosthetic infection, which limits wider use of monolithic HA despite superior cellular response. Recently, we reported that ZnO addition to HA can induce bactericidal property. It is therefore important to assess how ZnO addition influences the cytotoxicity property and cell adhesion/proliferation on HA-ZnO composite surfaces in vitro. In the above perspective, the objective of this study is to investigate the cell type and material composition dependent cellular proliferation and viability of pressureless sintered HA-ZnO composites. The combination of cell viability data as well as morphological observations of cultured human osteoblast-like SaOS2 cells and mouse fibroblast L929 cells suggests that HA-ZnO composites containing 10 Wt % or lower ZnO exhibit the ability to support cell adhesion and proliferation. Both SaOS2 and L929 cells exhibit extensive multidirectional network of actin cytoskeleton and cell flattening on the lower ZnO containing (≤10 Wt %) HA-ZnO composites. The in vitro results illustrate how variation in ZnO content can influence significantly the cell vitality, as evaluated using MTT biochemical assay. Also, the critical statistical analysis reveals that ZnO addition needs to be carefully tailored to ensure good in vitro cytocompatibility. The underlying reasons for difference in biological properties are analyzed. It is suggested that surface wettability as well as dissolution of ZnO, both contribute to the observed differences in cellular viability and proliferation. Copyright © 2011 Wiley Periodicals, Inc.

S-Nitrosothiol-Modified Nitric Oxide-Releasing Chitosan Oligosaccharides as Antibacterial Agents

PubMed Central

Lu, Yuan; Shah, Anand; Hunter, Rebecca A.; Soto, Robert J.; Schoenfisch, Mark H.

2017-01-01

S-nitrosothiol-modified chitosan oligosaccharides were synthesized by reaction with 2-iminothiolane hydrochloride and 3-acetamido-4,4-dimethylthietan-2-one, followed by the thiol nitrosation. The resulting nitric oxide (NO)-releasing chitosan oligosaccharides stored ~0.3 μmol NO/mg chitosan. Both the chemical structure of the nitrosothiol (i.e., primary and tertiary) and the use of ascorbic acid as a trigger for NO donor decomposition were used to control the NO-release kinetics. With ascorbic acid, the S-nitrosothiol-modified chitosan oligosaccharides elicited a 4-log reduction in Pseudomonas aeruginosa (P. aeruginosa) viability. Confocal microscopy indicated that the primary S-nitrosothiol-modified chitosan oligosaccharides associated more with the bacteria relative to the tertiary S-nitrosothiol system. The primary S-nitrosothiol-modified chitosan oligosaccharides elicited minimal toxicity towards L929 mouse fibroblast cells at the concentration necessary for a 4-log reduction in bacterial viability, further demonstrating the potential of S-nitrosothiol-modified chitosan oligosaccharides as NO-release therapeutics. PMID:25449913

Stability and Antimicrobial Activity of Nisin-Loaded Mesoporous Silica Nanoparticles: A Game-Changer in the War against Maleficent Microbes.

PubMed

Behzadi, Faezeh; Darouie, Sheyda; Alavi, S Mehdi; Shariati, Parvin; Singh, Gurvinder; Dolatshahi-Pirouz, Alireza; Arpanaei, Ayyoob

2018-04-25

Antimicrobial agents, such as nisin, are used extensively in the food industry. Here, we investigated various approaches to load nisin onto mesoporous silica nanoparticles (MSNs, 92 ± 10 nm in diameter), to enhance its stability and sustained release. The morphology, size, and surface charge of the as-prepared nanoparticles were analyzed using scanning transmission electron microscopy, dynamic light scattering, and ζ potential measurement. Nisin was either physically adsorbed or covalently attached to the variously functionalized MSNs, with high loading capacities (>600 mg of nisin g -1 of nanoparticles). The results of antibacterial activity analysis of nisin against Staphylococcus aureus showed that, despite the very low antibacterial activity of nisin covalently conjugated onto MSNs, the physical adsorption of nisin onto the unfunctionalized nanoparticles enhances its antimicrobial activities under various conditions, with no significant cytotoxicity effects on mouse fibroblast L929 cells. In conclusion, MSNs can be recommended as suitable carriers for nisin under various conditions.

Intrinsically radiopaque polyurethanes with chain extender 4,4'-isopropylidenebis [2-(2,6-diiodophenoxy)ethanol] for biomedical applications.

PubMed

Dawlee, S; Jayabalan, M

2015-05-01

Radiopaque polyurethanes are used for medical applications as it allows post-operative assessment of the biomaterial devices using X-ray. Inherently, radiopaque polyurethanes based on polytetramethylene glycol (PTMG), polypropylene glycol, 4,4'-methylenebis(phenyl isocyanate), and a new iodinated chain extender 4,4'-isopropylidenebis[2-(2,6-diiodophenoxy)ethanol] with flexible spacers were synthesized and characterized. The iodinated polyurethanes were clear, optically transparent, and had high molecular weights. The polyurethanes also possessed excellent radiopacity and high thermal stability. The biocompatibility of the most promising iodinated polyurethane was evaluated both in vitro (cytotoxicity evaluation by direct contact and MTT assay, using L929 mouse fibroblast cells) and in vivo (toxicology studies in rabbits and subcutaneous implantation in rats). The material was nontoxic and well tolerated by the animals. Thus, these radiopaque and transparent polyurethanes are expected to have potential for various biomedical applications. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Influence of Scaffold Size on Bactericidal Activity of Nitric Oxide Releasing Silica Nanoparticles

PubMed Central

Carpenter, Alexis W.; Slomberg, Danielle L.; Rao, Kavitha S.; Schoenfisch, Mark H.

2011-01-01

A reverse microemulsion synthesis was used to prepare amine functionalized silica nanoparticles of three distinct sizes (i.e., 50, 100, and 200 nm) with identical amine concentrations. The resulting hybrid nanoparticles, consisting of N-(6 aminohexyl) aminopropyltrimethoxysilane and tetraethoxysilane, were highly monodisperse in size. N-diazeniumdiolate nitric oxide (NO) donors were subsequently formed on secondary amines while controlling reaction conditions to keep the total amount of nitric oxide (NO) released constant for each particle size. The bactericidal efficacy of the NO releasing nanoparticles against Pseudomonas aeruginosa increased with decreasing particle size. Additionally, smaller diameter nanoparticles were found to associate with the bacteria at a faster rate and to a greater extent than larger particles. Neither control (non-NO-releasing) nor NO releasing particles exhibited toxicity towards L929 mouse fibroblasts at concentrations above their respective minimum bactericidal concentrations. This study represents the first investigation of the bactericidal efficacy of NO-releasing silica nanoparticles as a function of particle size. PMID:21842899

Novel levan and pNIPA temperature sensitive hydrogels for 5-ASA controlled release.

PubMed

Osman, Asila; Oner, Ebru Toksoy; Eroglu, Mehmet S

2017-06-01

Levan based cross-linker was successfully synthesized and used to prepare a series of more biocompatible and temperature responsive levan/N-isopropyl acrylamide (levan/pNIPA) hydrogels by redox polymerization at room temperature. Volume phase transition temperature (VPTT) of the hydrogels were precisely determined by derivative differential scanning calorimetry (DDSC). Incorporation of levan into the pNIPA hydrogel increased the VPTT from 32.8°C to 35.09°C, approaching to body temperature. Swelling behavior and 5-aminosalicylic acid (5-ASA) release of the hydrogels were found to vary significantly with temperature and composition. Moreover, a remarkable increase in thermal stability of levan within hydrogel with increase of pNIPA content was recorded. The biocompatibility of the hydrogels were tested against mouse fibroblast L929 cell line in phosphate buffer saline (PBS, pH 7.4). The hydrogels showed increasing biocompatibility with increasing levan ratio, indicating levan enhanced the hydrogel surface during swelling. Copyright © 2017 Elsevier Ltd. All rights reserved.

Iodinated glycidyl methacrylate copolymer as a radiopaque material for biomedical applications.

PubMed

Dawlee, S; Jayabalan, M

2013-07-01

Polymeric biomaterial was synthesized by copolymerizing 50:50 mol% of monomers, glycidyl methacrylate and methyl methacrylate. Iodine atoms were then grafted to the epoxide groups of glycidyl methacrylate units, rendering the copolymer radiopaque. The percentage weight of iodine in the present copolymer was found to be as high as 23%. The iodinated copolymer showed higher glass transition temperature and thermal stability in comparison with unmodified polymer. Radiographic analysis showed that the copolymer possessed excellent radiopacity. The iodinated copolymer was cytocompatible to L929 mouse fibroblast cells. The in vivo toxicological evaluation by intracutaneous reactivity test of the copolymer extracts has revealed that the material was nontoxic. Subcutaneous implantation of iodinated copolymer in rats has shown that the material was well tolerated. Upon explantation and histological examination, no hemorrhage, infection or necrosis was observed. The samples were found to be surrounded by a vascularized capsule consisting of connective tissue cells. The results indicate that the iodinated copolymer is biocompatible and may have suitable applications as implantable materials.

Quantitative phosphoproteomic analysis of RIP3-dependent protein phosphorylation in the course of TNF-induced necroptosis.

PubMed

Zhong, Chuan-Qi; Li, Yuanyue; Yang, Daowei; Zhang, Na; Xu, Xiaozheng; Wu, Yaying; Chen, Jinan; Han, Jiahuai

2014-03-01

Tumor necrosis factor (TNF) induced cell death in murine fibrosarcoma L929 cells is a model system in studying programed necrosis (also known as necroptosis). Receptor interacting protein 3 (RIP3), a serine-threonine kinase, is known to play an essential role in TNF-induced necroptosis; however, the phosphorylation events initiated by RIP3 activation in necroptotic process is still largely unknown. Here, we performed a quantitative MS based analysis to compare TNF-induced changes in the global phosphoproteome of wild-type (RIP3(+/+) ) and RIP3-knockdown L929 cells at different time points after TNF treatment. A total of 8058 phosphopeptides spanning 6892 phosphorylation sites in 2762 proteins were identified in the three experiments, in which cells were treated with TNF for 0.5, 2, and 4 h. By comparing the phosphorylation sites in wild-type and RIP3-knockdown L929 cells, 174, 167, and 177 distinct phosphorylation sites were revealed to be dependent on RIP3 at the 0.5, 2, and 4 h time points after TNF treatment, respectively. Notably, most of them were not detected in a previous phosphoproteomic analysis of RIP3-dependent phosphorylation in lipopolysaccharide-stimulated peritoneal macrophages and TNF-treated murine embryonic fibroblasts (MEFs), suggesting that the data presented in this report are highly relevant to the study of TNF-induced necroptosis of L929 cells. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Protective role of Nrf2 against mechanical-stretch-induced apoptosis in mouse fibroblasts: a potential therapeutic target of mechanical-trauma-induced stress urinary incontinence.

PubMed

Li, Qiannan; Li, Bingshu; Liu, Cheng; Wang, Linlin; Tang, Jianming; Hong, Li

2018-01-10

We investigated the protective effect and underlying molecular mechanism of nuclear factor-E2-related factor 2 (Nrf2) against mechanical-stretch-induced apoptosis in mouse fibroblasts. Normal cells, Nrf2 silencing cells, and Nrf2 overexpressing cells were respectively divided into two groups-nonintervention and cyclic mechanical strain (CMS)-subjected to CMS of 5333 μ (1.0 Hz for 4 h), six groups in total (control, CMS, shNfe212, shNfe212 + CMS, LV-shNfe212, and LV-shNfe212 + CMS). After treatment, cell apoptosis; cell-cycle distribution; expressions of Nrf2, Bax, Bcl-2, Cyt-C, caspase-3, caspase-9, cleaved-caspase-3, and cleaved-caspase-9; mitochondrial membrane potential (ΔΨm); reactive oxygen species (ROS); and malondialdehyde (MDA) levels were measured. Thirty virgin female C57BL/6 mice were divided into two groups: control (without intervention) and vaginal distension (VD) groups, which underwent VD for 1 h with an 8-mm dilator (0.3 ml saline). Leak-point pressure (LPP) was tested on day 7 after VD; Nrf2 expression, apoptosis, and MDA levels were then measured in urethra and anterior vaginal wall. Mechanical stretch decreased Nrf2 messenger RNA (mRNA) and protein expressions. Overexpression of Nrf2 alleviated mechanical-stretch-induced cell apoptosis; S-phase arrest of cell cycle; up-regulation of Bax, cytochrome C (Cyt-C), ROS, MDA, ratio of cleaved-caspase-3/caspase-3 and cleaved-caspase-9/caspase-9; and exacerbated the decrease of Bcl2 and ΔΨm in L929 cells. On the contrary, silencing of Nrf2 showed opposite effects. Besides, VD reduced LPP levels and Nrf2 expression and increased cell apoptosis and MDA generation in the urethra and anterior vaginal wall. Nrf2 exhibits a protective role against mechanical-stretch -induced apoptosis on mouse fibroblasts, which might indicate a potential therapeutic target of mechanical-trauma-induced stress urinary incontinence (SUI).

Spontaneous generation of germline characteristics in mouse fibrosarcoma cells

NASA Astrophysics Data System (ADS)

Ma, Zhan; Hu, Yao; Jiang, Guoying; Hou, Jun; Liu, Ruilai; Lu, Yuan; Liu, Chunfang

2012-10-01

Germline/embryonic-specific genes have been found to be activated in somatic tumors. In this study, we further showed that cells functioning as germline could be present in mouse fibrosarcoma cells (L929 cell line). Early germline-like cells spontaneously appeared in L929 cells and further differentiated into oocyte-like cells. These germline-like cells can, in turn, develop into blastocyst-like structures in vitro and cause teratocarcinomas in vivo, which is consistent with natural germ cells in function. Generation of germline-like cells from somatic tumors might provide a novel way to understand why somatic cancer cells have strong features of embryonic/germline development. It is thought that the germline traits of tumors are associated with the central characteristics of malignancy, such as immortalization, invasion, migration and immune evasion. Therefore, germline-like cells in tumors might provide potential targets to tumor biology, diagnosis and therapy.

Production of an aminoterminally truncated, stable type of bioactive mouse fibroblast growth factor 4 in Escherichia coli.

PubMed

Sugawara, Saiko; Ito, Toshihiko; Sato, Shiori; Sato, Yuki; Kasuga, Kano; Kojima, Ikuo; Kobayashi, Masayuki

2014-05-01

In mice, fibroblast growth factor 4 (Fgf4) is a crucial gene for the generation of trophectoderm, progenitor cells of the placenta. Therefore, exogenous FGF4 promotes the isolation and maintenance of trophoblast stem cells from preimplantation embryos. We previously produced a 6× histidine (His)-tagged, mouse FGF4 (Pro(31)-Leu(202)) without a secretory signal peptide at the amino-terminus, referred to as HismFGF4, in Escherichia coli. Here, we found that HismFGF4 was unstable, such as in phosphate-buffered saline. In these conditions, site-specific cleavage between Ser(50) and Leu(51) was identified. In order to generate stable mouse FGF4 derivatives, a 6× His-tagged mouse FGF4 (Leu(51)-Leu(202)), termed HismFGF4L, was expressed in E. coli. HismFGF4L could be purified from the supernatant of cell lysates by heparin column chromatography. In phosphate-buffered saline, HismFGF4L was relatively stable. HismFGF4L exerted significant mitogenic activities at concentrations as low as 0.01 nM (P < 0.01) in mouse embryonic fibroblast Balb/c 3T3 cells expressing FGF receptor 2. In the presence of PD173074, an FGF receptor inhibitor, the growth-promoting activity of HismFGF4L was abolished. Taken together, we suggest that aminoterminally truncated HismFGF4L is capable of promoting the proliferation of mouse-derived cells via an authentic FGF signaling pathway. We consider that HismFGF4L is useful as a derivative of mouse FGF4 protein for analyzing the effects of mouse FGF4 and for stimulating cell growth of mouse-derived cells, such as trophoblast stem cells. Our study provides a simple method for the production of a bioactive, stable mouse FGF4 derivative in E. coli. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Validation of in vitro assays in three-dimensional human dermal constructs.

PubMed

Idrees, Ayesha; Chiono, Valeria; Ciardelli, Gianluca; Shah, Siegfried; Viebahn, Richard; Zhang, Xiang; Salber, Jochen

2018-05-01

Three-dimensional cell culture systems are urgently needed for cytocompatibility testing of biomaterials. This work aimed at the development of three-dimensional in vitro dermal skin models and their optimization for cytocompatibility evaluation. Initially "murine in vitro dermal construct" based on L929 cells was generated, leading to the development of "human in vitro dermal construct" consisting of normal human dermal fibroblasts in rat tail tendon collagen type I. To assess the viability of the cells, different assays CellTiter-Blue ® , RealTime-Glo ™ MT, and CellTiter-Glo ® (Promega) were evaluated to optimize the best-suited assay to the respective cell type and three-dimensional system. Z-stack imaging (Live/Dead and Phalloidin/DAPI-Promokine) was performed to visualize normal human dermal fibroblasts inside matrix revealing filopodia-like morphology and a uniform distribution of normal human dermal fibroblasts in matrix. CellTiter-Glo was found to be the optimal cell viability assay among those analyzed. CellTiter-Blue reagent affected the cell morphology of normal human dermal fibroblasts (unlike L929), suggesting an interference with cell biological activity, resulting in less reliable viability data. On the other hand, RealTime-Glo provided a linear signal only with a very low cell density, which made this assay unsuitable for this system. CellTiter-Glo adapted to three-dimensional dermal construct by optimizing the "shaking time" to enhance the reagent penetration and maximum adenosine triphosphate release, indicating 2.4 times higher viability value by shaking for 60 min than for 5 min. In addition, viability results showed that cells were viable inside the matrix. This model would be further advanced with more layers of skin to make a full thickness model.

A comparison of the in vitro cytotoxicity of conventional and resin modified glass ionomer cements

PubMed Central

Selimović-Dragaš, Mediha; Huseinbegović, Amina; Kobašlija, Sedin; Hatibović-Kofman, Šahza

2012-01-01

To evaluate cytotoxicity of experimental conventional and resin modified glass-ionomer cements on UMR-106 osteoblast cell cultures and cell cultures of NIH3T3 mouse fibroblasts specimens were prepared for every experimental material and divided into: group 1. Conventional glass-ionomer cements: GC Fuji IX GP Fast, GC Fuji Triage and Ketac Silver; group 2. Resin modified glass-ionomer cements: GC Fuji II LC, GC Fuji Plus and Vitrebond; group 3. Positive control was presented by specimens of composite Vit-l-ecence® and negative control-group 4. was presented by α-minimum essential medium for UMR-106 – osteoblast-like cells and Dulbecco’s Modified Eagle’s Medium for NIH3T3 mouse fibroblast cells. Both cell cultures were exposed to 10% of eluate of each single specimen of each experimental material. Experimental dishes were incubated for 24 h. Cell metabolism was evaluated using methyltetrazolium assay. Kruskal-Wallis test and Tukey-Kramer post hoc test for the materials evaluated on NIH3T3 mouse fibroblast cells, as well as UMR-106 osteoblast-like cells showed significantly more cytotoxicity of RMGICs, predominantly Vitrebond to both GICs and composite-Vit-l-ecence®. The lowest influence on cell’s metabolism on UMR-106 osteoblas-like cells was shown by Ketac Silver and the lowest influence on cell’s metabolism on NIH3T3 mouse fibroblast cells was shown by Fuji IX GP Fast. Statistical evaluation of sensitivity of cell lines UMR-106 osteoblast-like cells and NIH3T3 mouse fibroblast cells, using Mann-Whitney test, showed that NIH3T3 mouse fibroblast cells were more sensitive for the evaluation of cytotoxicity of dental materials. PMID:23198945

MHC-I modulation due to changes in tumor cell metabolism regulates tumor sensitivity to CTL and NK cells

PubMed Central

Catalán, Elena; Charni, Seyma; Jaime, Paula; Aguiló, Juan Ignacio; Enríquez, José Antonio; Naval, Javier; Pardo, Julián; Villalba, Martín; Anel, Alberto

2015-01-01

Tumor cells have a tendency to use glucose fermentation to obtain energy instead of mitochondrial oxidative phosphorylation (OXPHOS). We demonstrated that this phenotype correlated with loss of ERK5 expression and with reduced MHC class I expression. Consequently, tumor cells could evade cytotoxic T lymphocyte (CTL)-mediated immune surveillance, but also increase their sensitivity to natural killer (NK) cells. These outcomes were evaluated using two cellular models: leukemic EL4 cells and L929 transformed fibroblasts and their derived ρ° cell lines, which lack mitochondrial DNA. We have also used a L929 cell sub-line that spontaneously lost matrix attachment (L929dt), reminiscent of metastasis generation, that also downregulated MHC-I and ERK5 expression. MHC-I expression is lower in ρ° cells than in the parental cell lines, but they were equally sensitive to CTL. On the contrary, ρ° cells were more sensitive to activated NK cells than parental cells. On the other hand, L929dt cells were resistant to CTL and NK cells, showed reduced viability when forced to perform OXPHOS, and surviving cells increased MHC-I expression and became sensitive to CTL. The present results suggest that when the reduction in MHC-I levels in tumor cells due to glycolytic metabolism is partial, the increase in sensitivity to NK cells seems to predominate. However, when tumor cells completely lose MHC-I expression, the combination of treatments that increase OXPHOS with CTL-mediated immunotherapy could be a promising therapeutic approach. PMID:25949869

MHC-I modulation due to changes in tumor cell metabolism regulates tumor sensitivity to CTL and NK cells.

PubMed

Catalán, Elena; Charni, Seyma; Jaime, Paula; Aguiló, Juan Ignacio; Enríquez, José Antonio; Naval, Javier; Pardo, Julián; Villalba, Martín; Anel, Alberto

2015-01-01

Tumor cells have a tendency to use glucose fermentation to obtain energy instead of mitochondrial oxidative phosphorylation (OXPHOS). We demonstrated that this phenotype correlated with loss of ERK5 expression and with reduced MHC class I expression. Consequently, tumor cells could evade cytotoxic T lymphocyte (CTL)-mediated immune surveillance, but also increase their sensitivity to natural killer (NK) cells. These outcomes were evaluated using two cellular models: leukemic EL4 cells and L929 transformed fibroblasts and their derived ρ° cell lines, which lack mitochondrial DNA. We have also used a L929 cell sub-line that spontaneously lost matrix attachment (L929dt), reminiscent of metastasis generation, that also downregulated MHC-I and ERK5 expression. MHC-I expression is lower in ρ° cells than in the parental cell lines, but they were equally sensitive to CTL. On the contrary, ρ° cells were more sensitive to activated NK cells than parental cells. On the other hand, L929dt cells were resistant to CTL and NK cells, showed reduced viability when forced to perform OXPHOS, and surviving cells increased MHC-I expression and became sensitive to CTL. The present results suggest that when the reduction in MHC-I levels in tumor cells due to glycolytic metabolism is partial, the increase in sensitivity to NK cells seems to predominate. However, when tumor cells completely lose MHC-I expression, the combination of treatments that increase OXPHOS with CTL-mediated immunotherapy could be a promising therapeutic approach.

In vitro cellular adhesion and antimicrobial property of SiO2-MgO-Al2O3-K2O-B2O3-F glass ceramic.

PubMed

Kalmodia, Sushma; Molla, Atiar Rahaman; Basu, Bikramjit

2010-04-01

The aim of the present study was to examine the cellular functionality and antimicrobial properties of SiO(2)-MgO-Al(2)O(3)-K(2)O-B(2)O(3)-F glass ceramics (GC) containing fluorophlogopite as major crystalline phase. The cellular morphology and cell adhesion study using human osteoblast-like Saos-2 cells and mouse fibroblast L929 cells reveals good in vitro cytocompatibility of GC. The potential use of the GC for biomedical application was also assessed by in vitro synthesis of the alkaline phosphatase (ALP) activity of Saos-2 cells. It is proposed that B(2)O(3) actively enhances the cell adhesion and supports osteoconduction process, whereas, fluorine component significantly influences cell viability. The Saos-2 and L929 cells on GC shows extensive multidirectional network of actin cytoskeleton. The in vitro results of this study illustrate how small variation in fluorine and boron in base glass composition influences significantly the biocompatibility and antimicrobial bactericidal property, as evaluated using a range of biochemical assays. Importantly, it shows that the cell viability and osteoconduction can be promoted in glass ceramics with lower fluorine content. The underlying reasons for difference in biological properties are analyzed and reported. It is suggested that oriented crystalline morphology in the lowest fluorine containing glass ceramic enhanced cellular spreading. Overall, the in vitro cell adhesion, cell flattening, cytocompatibility and antimicrobial study of the three different compositions of glass ceramic clearly reveals that microstructure and base glass composition play an important role in enhancing the cellular functionality and antimicrobial property.

DNA damage evaluation of hydroxyapatite on fibroblast cell L929 using the single cell gel electrophoresis assay.

PubMed

Rajab, N F; Yaakob, T A; Ong, B Y; Hamid, M; Ali, A M; Annuar, B O; Inayat-Hussain, S H

2004-05-01

Hydroxyapatite is the main component of the bone which is a potential biomaterial substance that can be applied in orthopaedics. In this study, the biocompatibility of this biomaterial was assessed using an in vitro technique. The cytotoxicity and genotoxicity effect of HA2 and HA3 against L929 fibroblast cell was evaluated using the MTT Assay and Alkaline Comet Assay respectively. Both HA2 and HA3 compound showed low cytotoxicity effect as determined using MTT Assay. Cells viability following 72 hours incubation at maximum concentration of both HA2 and HA3 (200 mg/ml) were 75.3 +/- 8.8% and 86.7 +/- 13.1% respectively. However, the cytotoxicity effect of ZnSO4.7H2O as a positive control showed an IC50 values of 46 mg/ml (160 microM). On the other hand, both HA2 and HA3 compound showed a slight genotoxicity effect as determined using the Alkaline Comet Assay following incubation at the concentration 200 mg/ml for 72 hours. This assay has been widely used in genetic toxicology to detect DNA strand breaks and alkali-labile site. The percentage of the cells with DNA damage for both substance was 27.7 +/- 1.3% and 15.6 +/- 1.0% for HA2 and HA3 respectively. Incubation of the cells for 24 hours with 38 microg/ml (IC25) of positive control showed an increase in percentage of cells with DNA damage (67.5 +/- 0.7%). In conclusion, our study indicated that both hydroxyapatite compounds showed a good biocompatibility in fibroblast cells.

Cytotoxic effects of resin-modified orthodontic band adhesives. Are they safe?

PubMed

Malkoc, Siddik; Corekci, Bayram; Botsali, Hayriye Esra; Yalçin, Muhammet; Sengun, Abdülkadir

2010-09-01

To evaluate the cytotoxic effects of three different resin-modified orthodontic band adhesives. Three resin-modified orthodontic band adhesives (Bisco Ortho Band Paste LC, Multi-Cure Glass Ionomer Band Cement, and Transbond Plus Light Cure Band Adhesive) were prepared and the samples were extracted in 3 mL of Basal Medium Eagle with 10% newborn calf serum for 24 hours. The L929 cells were plated (25,000 cells/mL) in wells of 96-well dishes and maintained in a humidified incubator for 24 hours at 37 degrees C, 5% CO(2), and 95% air. After 24-hour incubation of the cells, the incubation medium was replaced by the immersed medium in which the samples were stored. Then L929 cells were incubated in contact with eluates for 24 hours. The cell mitochondrial activity was evaluated by the methyltetrazolium test. Twelve wells were used for each specimen, and methyltetrazolium tests were applied two times. The data were statistically analyzed using one-way analysis of variance and Tukey Honestly Significantly Different tests. Results with L929 fibroblasts demonstrated that all freshly prepared resin-modified orthodontic band adhesive materials reduced vital cell numbers (P > .05), in comparison to the control group. Our data demonstrate that all materials showed significant cytotoxicity compared to the control group. The results indicate that all materials showed significant cytotoxicity compared to the control group, and further studies using different test methods are needed for all resin-modified orthodontic band adhesives.

The characteristics of bacterial nanocellulose gel releasing silk sericin for facial treatment.

PubMed

Aramwit, Pornanong; Bang, Nipaporn

2014-12-09

Recently, naturally derived facial masks with beneficial biological properties have received increasing interest. In this study, silk sericin-releasing bacterial nanocellulose gel was developed to be applied as a bioactive mask for facial treatment. The silk sericin-releasing bacterial nanocellulose gel produced at a pH of 4.5 had an ultrafine and extremely pure fiber network structure. The mechanical properties and moisture absorption ability of the gel were improved, compared to those of the commercially available paper mask. Silk sericin could be control-released from the gel. A peel test with porcine skin showed that the gel was less adhesive than the commercially available paper mask, which would be removed from the face more easily without pain. The in vitro cytotoxicity test showed that the gel was not toxic to L929 mouse fibroblast and HaCaT human keratinocyte cells. Furthermore, when implanted subcutaneously and evaluated according to ISO10993-6 standard, the gel was not irritant to tissue. The silk sericin-releasing bacterial nanocellulose gel had appropriate physical and biological properties and safety for the facial treatment application.

Magnetic iron oxide modified pyropheophorbide-a fluorescence nanoparticles as photosensitizers for photodynamic therapy against ovarian cancer (SKOV-3) cells.

PubMed

Tan, Guanghui; Li, Wenting; Cheng, Jianjun; Wang, Zhiqiang; Wei, Shuquan; Jin, Yingxue; Guo, Changhong; Qu, Fengyu

2016-11-30

Magnetic iron oxide modified pyropheophorbide-a fluorescence nanoparticles, Fe 3 O 4 @SiO 2 @APTES@PPa (FSAP), were designed as magnetically targeted photodynamic antineoplastic agents and prepared through continuous covalent chemical modification on the surface of Fe 3 O 4 nanoparticles. The properties of the intermediates and the final product were comprehensively characterized by transmission electron microscopy, powder X-ray diffraction analysis, Fourier transform infrared spectroscopy, vibrating sample magnetometry, zeta potential measurement, ultraviolet-visible absorption spectroscopy, fluorescence emission spectroscopy, and thermogravimetric analysis. In this work, we demonstrated the in vitro photodynamic therapy (PDT) of FSAP against ovarian cancer (SKOV-3) cells, which indicated that FSAP could be taken up successfully and showed low dark toxicity without irradiation, but remarkable phototoxicity after irradiation. Meanwhile, FSAP had showed good biocompatibility and low dark toxicity against normal cells in the biological experiments on mouse normal fibroblast cell lines (L929 cells). In addition, in the photochemical process of FSAP mediated photodynamic therapy, the Type-II photo-oxygenation process (generated singlet oxygen) played an important role in the induction of cell damage.

The Effect of Ag and Ag+N Ion Implantation on Cell Attachment Properties

DOE Office of Scientific and Technical Information (OSTI.GOV)

Urkac, Emel Sokullu; Oztarhan, Ahmet; Gurhan, Ismet Deliloglu

2009-03-10

Implanted biomedical prosthetic devices are intended to perform safely, reliably and effectively in the human body thus the materials used for orthopedic devices should have good biocompatibility. Ultra High Molecular Weight Poly Ethylene (UHMWPE) has been commonly used for total hip joint replacement because of its very good properties. In this work, UHMWPE samples were Ag and Ag+N ion implanted by using the Metal-Vapor Vacuum Arc (MEVVA) ion implantation technique. Samples were implanted with a fluency of 1017 ion/cm2 and extraction voltage of 30 kV. Rutherford Backscattering Spectrometry (RBS) was used for surface studies. RBS showed the presence of Agmore » and N on the surface. Cell attachment properties investigated with model cell lines (L929 mouse fibroblasts) to demonstrate that the effect of Ag and Ag+N ion implantation can favorably influence the surface of UHMWPE for biomedical applications. Scanning electron microscopy (SEM) was used to demonstrate the cell attachment on the surface. Study has shown that Ag+N ion implantation represents more effective cell attachment properties on the UHMWPE surfaces.« less

Fabrication, ultra-structure characterization and in vitro studies of RF magnetron sputter deposited nano-hydroxyapatite thin films for biomedical applications

NASA Astrophysics Data System (ADS)

Surmeneva, Maria A.; Surmenev, Roman A.; Nikonova, Yulia A.; Selezneva, Irina I.; Ivanova, Anna A.; Putlyaev, Valery I.; Prymak, Oleg; Epple, Matthias

2014-10-01

A series of nanostructured low-crystalline hydroxyapatite (HA) coatings averaging 170, 250, and 440 nm in thickness were deposited onto previously etched titanium substrates through radio-frequency (RF) magnetron sputtering. The HA coatings were analyzed using infrared spectroscopy (FTIR), X-ray diffraction (XRD), and scanning and transmission electron microscopy (SEM and TEM). Cross sections of the thin specimens were prepared by FIB to study the microstructure of the coatings by TEM. The deposition process formed nano-scale grains, generating an amorphous layer at the substrate/coating interface and inducing the growth of a columnar grain structure perpendicular to the substrate surface. A microstructural analysis of the film confirmed that the grain size and crystallinity increased when increasing the deposition time. The nanostructured HA coatings were not cytotoxic, as proven by in vitro assays using primary dental pulp stem cells and mouse fibroblast NCTC clone L929 cells. Low-crystallinity HA coatings with different thicknesses stimulated cells to attach, proliferate and form mineralized nodules on the surface better than uncoated titanium substrates.

Investigation of cell viability and morphology in 3D bio-printed alginate constructs with tunable stiffness.

PubMed

Shi, Pujiang; Laude, Augustinus; Yeong, Wai Yee

2017-04-01

In this article, mouse fibroblast cells (L929) were seeded on 2%, 5%, and 10% alginate hydrogels, and they were also bio-printed with 2%, 5%, and 10% alginate solutions individually to form constructs. The elastic and viscous moduli of alginate solutions, their interior structure and stiffness, interactions of cells and alginate, cell viability, migration and morphology were investigated by rheometer, MTT assay, scanning electron microscope (SEM), and fluorescent microscopy. The three types of bio-printed scaffolds of distinctive stiffness were prepared, and the seeded cells showed robust viability either on the alginate hydrogel surfaces or in the 3D bio-printed constructs. Majority of the proliferated cells in the 3D bio-printed constructs weakly attached to the surrounding alginate matrix. The concentration of alginate solution and hydrogel stiffness influenced cell migration and morphology, moreover the cells formed spheroids in the bio-printed 10% alginate hydrogel construct. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1009-1018, 2017. © 2017 Wiley Periodicals, Inc.

Comparative study of cytotoxicity of ferromagnetic nanoparticles and magnetitecontaining polyelectrolyte microcapsules

NASA Astrophysics Data System (ADS)

Minaeva, O. V.; Brodovskaya, E. P.; Pyataev, M. A.; Gerasimov, M. V.; Zharkov, M. N.; Yurlov, I. A.; Kulikov, O. A.; Kotlyarov, A. A.; Balykova, L. A.; Kokorev, A. V.; Zaborovskiy, A. V.; Pyataev, N. A.; Sukhorukov, G. B.

2017-01-01

The cytotoxicity of magnetite nanoparticles (MNP) stabilized with citrate acidand polyelectrolyte multilayer microcapsules containing these particles in the shell is analyzed. Microcapsules were prepared by co-precipitation of iron (II) and (III) chlorides. Polyelectrolyte microcapsules synthesized by the layer-by-layer method from biodegradable polymers polyarginine and dextran sulfate. Cytotoxicity of the synthesized objects was studied on the L929 cells culture and human leucocytes. It was also investigated the phagocytic activity of leukocytes for the MNP and magnetite containing polyelectrolyte microcapsules (MCPM). A set of tests (MTT assay, neutral red uptake assay, lactate dehydrogenase release assay) was used to study the cytotoxicity in vitro. All the tests have shown that the magnetic nanoparticles have a greater cytotoxicity in comparison with microcapsules containing an equivalent amount of magnetite. In contrast to the mouse fibroblast culture, human leukocytes were more resistant to the toxic effects of magnetite. At the concentrations used in our studies no significant reduction in the viability of leukocytes has been registered. Both MNP and MCPM undergo phagocytosis, however, the phagocytic activity of leukocytes for these particles was lower than for the standard objects (latex microparticles).

Caffeine inhibition of GLUT1 is dependent on the activation state of the transporter.

PubMed

Gunnink, Leesha K; Busscher, Brianna M; Wodarek, Jeremy A; Rosette, Kylee A; Strohbehn, Lauren E; Looyenga, Brendan D; Louters, Larry L

2017-06-01

Caffeine has been shown to be a robust uncompetitive inhibitor of glucose uptake in erythrocytes. It preferentially binds to the nucleotide-binding site on GLUT1 in its tetrameric form and mimics the inhibitory action of ATP. Here we demonstrate that caffeine is also a dose-dependent, uncompetitive inhibitor of 2-deoxyglucose (2DG) uptake in L929 fibroblasts. The inhibitory effect on 2DG uptake in these cells was reversible with a rapid onset and was additive to the competitive inhibitory effects of glucose itself, confirming that caffeine does not interfere with glucose binding. We also report for the first time that caffeine inhibition was additive to inhibition by curcumin, suggesting distinct binding sites for curcumin and caffeine. In contrast, caffeine inhibition was not additive to that of cytochalasin B, consistent with previous data that reported that these two inhibitors have overlapping binding sites. More importantly, we show that the magnitude of maximal caffeine inhibition in L929 cells is much lower than in erythrocytes (35% compared to 90%). Two epithelial cell lines, HCLE and HK2, have both higher concentrations of GLUT1 and increased basal 2DG uptake (3-4 fold) compared to L929 cells, and subsequently display greater maximal inhibition by caffeine (66-70%). Interestingly, activation of 2DG uptake (3-fold) in L929 cells by glucose deprivation shifted the responsiveness of these cells to caffeine inhibition (35%-70%) without a change in total GLUT1 concentration. These data indicate that the inhibition of caffeine is dependent on the activity state of GLUT1, not merely on the concentration. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Cytotoxicity Comparison of Harvard Zinc Phosphate Cement Versus Panavia F2 and Rely X Plus Resin Cements on Rat L929-fibroblasts.

PubMed

Mahasti, Sahabi; Sattari, Mandana; Romoozi, Elham; Akbar-Zadeh Baghban, Alireza

2011-01-01

Resin cements, regardless of their biocompatibility, have been widely used in restorative dentistry during the recent years. These cements contain hydroxy ethyl methacrylate (HEMA) molecules which are claimed to penetrate into dentinal tubules and may affect dental pulp. Since tooth preparation for metal ceramic restorations involves a large surface of the tooth, cytotoxicity of these cements would be more important in fixed prosthodontic treatments. The purpose of this study was to compare the cytotoxicity of two resin cements (Panavia F2 and Rely X Plus) versus zinc phosphate cement (Harvard) using rat L929-fibroblasts in vitro. In this experimental study, ninety hollow glass cylinders (internal diameter 5-mm, height 2-mm) were made and divided into three groups. Each group was filled with one of three experimental cements; Harvard Zinc Phosphate cement, Panavia F2 resin cement and Rely X Plus resin cement. L929- Fibroblast were passaged and subsequently cultured in 6-well plates of 5×10(5) cells each. The culture medium was RPMI_ 1640. All samples were incubated in CO2. Using enzyme-linked immune-sorbent assay (ELISA) and (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) (MTT) assay, the cytotoxicity of the cements was investigated at 1 hour, 24 hours and one week post exposure. Statistical analyses were performed via two-way ANOVA and honestly significant difference (HSD) Tukey tests. This study revealed significant differences between the three cements at the different time intervals. Harvard cement displayed the greatest cytotoxicity at all three intervals. After 1 hour Panavia F2 showed the next greatest cytotoxicity, but after 24-hours and oneweek intervals Rely X Plus showed the next greatest cytotoxicity. The results further showed that cytotoxicity decreased significantly in the Panavia F2 group with time (p<0.005), cytotoxicity increased significantly in the Rely X Plus group with time (p<0.001), and the Harvard cement group failed to showed no noticeable change in cytotoxicity with time. Although this study has limitations, it provides evidence that Harvard zinc phosphate cement is the most cytotoxic product and Panavia F2 appears to be the least cytotoxic cement over time.

Biosynthesis of silver nanoparticles using Caesalpinia ferrea (Tul.) Martius extract: physicochemical characterization, antifungal activity and cytotoxicity

PubMed Central

2018-01-01

Background Green synthesis is an ecological technique for the production of well characterized metallic nanoparticles using plants. This study investigated the synthesis of silver nanoparticles (AgNPs) using a Caesalpinia ferrea seed extract as a reducing agent. Methods The formation of AgNPs was identified by instrumental analysis, including ultraviolet–visible (UV–Vis) spectroscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD) of the AgNPs, and surface-enhanced Raman scattering (SERS) spectra of rhodamine-6G (R6G). We studied the physicochemical characterization of AgNPs, evaluated them as an antifungal agent against Candida albicans, Candida kruzei, Candida glabrata and Candida guilliermondii, and estimated their minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values. Lastly, this study evaluated the cytotoxicity of the AgNPs in murine L929 fibroblasts cells using an MTT assay. Results The UV–Vis spectroscopy, SERS, SEM and XRD results confirmed the rapid formation of spheroidal 30–50 nm AgNPs. The MIC and MFC values indicated the antifungal potential of AgNPs against most of the fungi studied and high cell viability in murine L929 fibroblasts. In addition, this study demonstrated that C. ferrea seed extracts may be used for the green synthesis of AgNPs at room temperature for the treatment of candidiasis. PMID:29576936

Cytotoxicity of a calcium aluminate cement in comparison with other dental cements and resin-based materials.

PubMed

Franz, Alexander; Konradsson, Katarina; König, Franz; Van Dijken, Jan W V; Schedle, Andreas

2006-02-01

The objective of this study was to compare the cytotoxic effects of a calcium aluminate cement with several currently used direct restorative materials. Specimens of three composites (QuiXfil, Tetric Ceram, Filtek Supreme), one zinc phosphate cement (Harvard Cement), one glass ionomer cement (Ketac Molar), and one calcium aluminate cement (DoxaDent), were used fresh or after 7-days' preincubation in cell culture medium at 37 degrees C, pH 7.2. PVC strips for ISO 10993-5 cytotoxicity test were used as positive control and glass specimens as negative control. L-929 fibroblasts (5-ml aliquots, containing 3 x 10(4) cells/ml), cultivated in DMEM with 10% FCS, 1% glutamine, and 1% penicillin/streptomycin at 37 degrees C/5% CO2 and trypsinized, were exposed to the specimens for 72 h. The cells were harvested, centrifuged, and resuspended in 500 microl DMEM and then counted in 500 microl DMEM for 30 s with a flow cytometer at 488 nm. The analysis of variance comparing the six materials showed different influences on L-929 fibroblast cytotoxicity (p <0.0001). The cytotoxicity of all specimens diminished with increasing preincubation time (p <0.0001). Fresh DoxaDent exhibited the lowest cytotoxicity, followed by QuiXfil. Ketac Molar showed the highest cytotoxicity. After 7 days of preincubation, Harvard Cement and Filtek Supreme demonstrated more cytotoxicity than the other materials (p <0.005).

Highly cytocompatible and flexible three-dimensional graphene/polydimethylsiloxane composite for culture and electrochemical detection of L929 fibroblast cells.

PubMed

Waiwijit, Uraiwan; Maturos, Thitima; Pakapongpan, Saithip; Phokharatkul, Ditsayut; Wisitsoraat, Anurat; Tuantranont, Adisorn

2016-08-01

Recently, three-dimensional graphene interconnected network has attracted great interest as a scaffold structure for tissue engineering due to its high biocompatibility, high electrical conductivity, high specific surface area and high porosity. However, free-standing three-dimensional graphene exhibits poor flexibility and stability due to ease of disintegration during processing. In this work, three-dimensional graphene is composited with polydimethylsiloxane to improve the structural flexibility and stability by a new simple two-step process comprising dip coating of polydimethylsiloxane on chemical vapor deposited graphene/Ni foam and wet etching of nickel foam. Structural characterizations confirmed an interconnected three-dimensional multi-layer graphene structure with thin polydimethylsiloxane scaffold. The composite was employed as a substrate for culture of L929 fibroblast cells and its cytocompatibility was evaluated by cell viability (Alamar blue assay), reactive oxygen species production and vinculin immunofluorescence imaging. The result revealed that cell viability on three-dimensional graphene/polydimethylsiloxane composite increased with increasing culture time and was slightly different from a polystyrene substrate (control). Moreover, cells cultured on three-dimensional graphene/polydimethylsiloxane composite generated less ROS than the control at culture times of 3-6 h. The results of immunofluorescence staining demonstrated that fibroblast cells expressed adhesion protein (vinculin) and adhered well on three-dimensional graphene/polydimethylsiloxane surface. Good cell adhesion could be attributed to suitable surface properties of three-dimensional graphene/polydimethylsiloxane with moderate contact angle and small negative zeta potential in culture solution. The results of electrochemical study by cyclic voltammetry showed that an oxidation current signal with no apparent peak was induced by fibroblast cells and the oxidation current at an oxidation potential of +0.9 V increased linearly with increasing cell number. Therefore, the three-dimensional graphene/polydimethylsiloxane composite exhibits high cytocompatibility and can potentially be used as a conductive substrate for cell-based electrochemical sensing. © The Author(s) 2016.

Synthesis, crystal structure and spectroscopy of bioactive Cd(II) polymeric complex of the non-steroidal anti-inflammatory drug diclofenac sodium: Antiproliferative and biological activity

NASA Astrophysics Data System (ADS)

Tabrizi, Leila; Chiniforoshan, Hossein; McArdle, Patrick

2015-02-01

The interaction of Cd(II) with the non-steroidal anti-inflammatory drug diclofenac sodium (Dic) leads to the formation of the complex [Cd2(L)41.5(MeOH)2(H2O)]n(L = Dic), 1, which has been isolated and structurally characterized by X-ray crystallography. Diclofenac sodium and its metal complex 1 have also been evaluated for antiproliferative activity in vitro against the cells of three human cancer cell lines, MCF-7 (breast cancer cell line), T24 (bladder cancer cell line), A-549 (non-small cell lung carcinoma), and a mouse fibroblast L-929 cell line. The results of cytotoxic activity in vitro expressed as IC50 values indicated the diclofenac sodium and cadmium chloride are non active or less active than the metal complex of diclofenac (1). Complex 1 was also found to be a more potent cytotoxic agent against T-24 and MCF-7 cancer cell lines than the prevalent benchmark metallodrug, cisplatin, under the same experimental conditions. The superoxide dismutase activity was measured by Fridovich test which showed that complex 1 shows a low value in comparison with Cu complexes. The binding properties of this complex to biomolecules, bovine or human serum albumin, are presented and evaluated. Antibacterial and growth inhibitory activity is also higher than that of the parent ligand compound.

Graphene/cobalt nanocarrier for hyperthermia therapy and MRI diagnosis.

PubMed

Hatamie, Shadie; Ahadian, Mohammad Mahdi; Ghiass, Mohammad Adel; Iraji Zad, Azam; Saber, Reza; Parseh, Benyamin; Oghabian, Mohammad Ali; Shanehsazzadeh, Saeed

2016-10-01

Graphene/cobalt nanocomposites are promising materials for theranostic nanomedicine applications, which are defined as the ability to diagnose, provide targeted therapy and monitor the response to the therapy. In this study, the composites were synthesized via chemical method, using graphene oxide as the source material and assembling cobalt nanoparticles of 15nm over the surface of graphene sheets. Various characterization techniques were then employed to reveal the morphology, size and structure of the nanocomposites, such as X-ray diffraction analysis, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, high resolution transmission electron microscopy and ultraviolet visible spectroscopy. Using ion-coupled plasma optical emission spectroscopy, cobalt concentration in the nanocomposites was found to be 80%. In addition, cytotoxicity of graphene/cobalt nanocomposites were evaluated using 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide or MTT assay. MTT viability assay exhibited biocompatibility to L929 mouse fibroblasts cells, under a high dose of 100μg/mL over 24h. Hyperthermia results showed the superior conversion of electromagnetic energy into heat at 350kHz frequency for 0.01 and 0.005g/L of the nanocomposites solution. The measured heat generation and energy transfer results were anticipated by the finite element analysis, conducted for the 3D structure. Magnetic resonance imaging characteristics also showed that negatively charge graphene/cobalt nanocomposites are suitable for T1-weighted imaging. Copyright © 2016 Elsevier B.V. All rights reserved.

[Isolation, purification and primary culture of adult mouse cardiac fibroblasts].

PubMed

Li, Rujun; Gong, Kaizheng; Zhang, Zhengang

2017-01-01

Objective To establish a method for primary culture of adult mouse cardiac fibroblasts. Methods Myocardial tissues from adult mice were digested with 1 g/L trypsin and 0.8 g/L collagenase IV by oscillating water bath for a short time repeatedly. Cardiac fibroblasts and myocardial cells were isolated with differential adhesion method. Immunofluorescence staining was used to assess the purity of cardiac fibroblasts. The cell morphology was observed under an inverted phase contrast microscope. The proliferation of cardiac fibroblasts was analyzed by growth curve and CCK-8 assay. The Smad2/3 phosphorylation induced by TGF-β1 was detected by Western blotting. Results After 90 minutes of differential adhesion, adherent fibroblasts formed spherical cell mass and after 3 days, cells were spindle-shaped and proliferated rapidly. Cells were confluent after 5 days and the growth curve presented nearly "S" shape. The positive expression rate of vimentin was 95%. CCK-8 assay showed that the optimal cell proliferating activity was found from day 3 to day 5. The level of phosphorylated Smad2/3 obviously increased at the second passage induced by TGF-β1. Conclusion This method is economical and stable to isolate cardiac fibroblasts with high activity and high purity from adult mice.

Remodeling of the fibroblast cytoskeletal architecture during the replication cycle of Ectromelia virus: A morphological in vitro study in a murine cell line.

PubMed

Szulc-Dabrowska, Lidia; Gregorczyk, Karolina P; Struzik, Justyna; Boratynska-Jasinska, Anna; Szczepanowska, Joanna; Wyzewski, Zbigniew; Toka, Felix N; Gierynska, Malgorzata; Ostrowska, Agnieszka; Niemialtowski, Marek G

2016-08-01

Ectromelia virus (ECTV, the causative agent of mousepox), which represents the same genus as variola virus (VARV, the agent responsible for smallpox in humans), has served for years as a model virus for studying mechanisms of poxvirus-induced disease. Despite increasing knowledge on the interaction between ECTV and its natural host-the mouse-surprisingly, still little is known about the cell biology of ECTV infection. Because pathogen interaction with the cytoskeleton is still a growing area of research in the virus-host cell interplay, the aim of the present study was to evaluate the consequences of ECTV infection on the cytoskeleton in a murine fibroblast cell line. The viral effect on the cytoskeleton was reflected by changes in migration of the cells and rearrangement of the architecture of tubulin, vimentin, and actin filaments. The virus-induced cytoskeletal rearrangements observed in these studies contributed to the efficient cell-to-cell spread of infection, which is an important feature of ECTV virulence. Additionally, during later stages of infection L929 cells produced two main types of actin-based cellular protrusions: short (actin tails and "dendrites") and long (cytoplasmic corridors). Due to diversity of filopodial extensions induced by the virus, we suggest that ECTV represents a valuable new model for studying processes and pathways that regulate the formation of cytoskeleton-based cellular structures. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

In vitro cytotoxicity evaluation of elemental ions released from different prosthodontic materials.

PubMed

Elshahawy, Waleed M; Watanabe, Ikuya; Kramer, Phillip

2009-12-01

This study investigated the cytotoxicity of elemental ions contained in four fixed prosthodontic materials (gold, nickel-chromium, stainless-steel alloys and CAD-CAM ceramics). According to the determination of elements released from prosthodontic materials by using inductively coupled plasma mass spectroscopy, similar amounts of elements Pd, Ag, Zn, Cu, Ni, Cr, Mo, Be, Fe, Al, and K were prepared as salt solutions. Wells with a tenfold higher concentration of the tested elements were used as positive controls, while a well without any tested element was used as a negative control. These salt solutions were tested for cytotoxicity by culturing mouse L-929 fibroblasts in the salt solutions for a 7-day period of incubation. Then, the percentage of viable cells for each element was measured using trypan blue exclusion assay. The data (n=5) were statistically analyzed by ANOVA/Tukey test (p<0.05). The results showed a statistically significant difference for the cytotoxic effect of the tested elements salt solutions. For the released element concentrations the lowest percentage of viable cells (mean+/-SD) was evident with Zn, Cu or Ni indicating that they are the highly toxic elements. Be and Ag were found to be intermediate in cytotoxic effect. Fe, Cr, Mo, Al, Pd or K were found to be the least cytotoxic elements. Zn and Cu released from gold alloys, and Ni released from nickel-chromium alloys, which are commonly used as fixed prosthodontic restorations, show evidence of a high cytotoxic effect on fibroblast cell cultures.

Effects of synthetic sphingosine-1-phosphate analogs on cytosolic phospholipase A2alpha-independent release of arachidonic acid and cell toxicity in L929 fibrosarcoma cells: the structure-activity relationship.

PubMed

Shimizu, Masaya; Muramatsu, Yuki; Tada, Eiko; Kurosawa, Takeshi; Yamaura, Erika; Nakamura, Hiroyuki; Fujino, Hiromichi; Houjyo, Yuuya; Miyasaka, Yuri; Koide, Yuuki; Nishida, Atsushi; Murayama, Toshihiko

2009-03-01

Sphingolipid metabolites including ceramide, sphingosine, and their phosphorylated products [sphingosine-1-phosphate (S1P) and ceramide-1-phosphate] regulate cell functions including arachidonic acid (AA) metabolism and cell death. The development of analogs of S1P may be useful for regulating these mediator-induced cellular responses. We synthesized new analogs of S1P and examined their effects on the release of AA and cell death in L929 mouse fibrosarcoma cells. Among the analogs tested, several compounds including DMB-mC11S [dimethyl (2S,3R)-2-tert-butoxycarbonylamino-3-hydroxy-3-(3'-undecyl)phenylpropyl phosphate] and DMB-mC9S [dimethyl (2S,3R)-2-tert-butoxycarbonylamino-3-hydroxy-3-(3'-nonyl)phenylpropyl phosphate] released AA within 1 h and caused cell death 6 h after treatment. The release of AA was observed in C12 cells [a L929 variant lacking a type alpha cytosolic phospholipase A(2) (cPLA(2)alpha)] and L929-cPLAalpha-siRNA cells (L929 cells treated with small interference RNA for cPLA(2)alpha). Treatment with pharmacological inhibitors of secretory and Ca(2+)-independent PLA(2)s decreased the DMB-mC11S-induced release of AA. The effect of the S1P analogs tested on the release of AA was comparable to that on cell death in L929 cells, and a high correlation coefficient was observed. Two analogs lacking a butoxycarbonyl moiety [DMAc-mC11S (dimethyl (2S,3R)-2-acetamino-3-hydroxy-3-(3'-undecyl)phenylpropyl phosphate] and DMAm-mC11S [dimethyl (2S,3R)-2-amino-3-hydroxy-3-(3'-undecyl)phenylpropyl phosphate)] had inhibitory effects on the release of AA and cell toxicity induced by DMB-mC11S. Synthetic phosphorylated lipid analogs may be useful for studying PLA(2) activity and its toxicity in cells. [Supplementary Fig. 1: available only at http://dx.doi.org/10.1254/jphs.08284FP].

Evaluation of the wound healing property of Boesenbergia longiflora rhizomes.

PubMed

Sudsai, Teeratad; Wattanapiromsakul, Chatchai; Nakpheng, Titpawan; Tewtrakul, Supinya

2013-10-28

The rhizomes of Boesenbergia longiflora (Wall.) Kuntze (Zingiberaceae) have been traditionally used for treatment of inflammatory bowel disease, ulcerative colitis, aphthous ulcer and abscess by decoction with alcohol. The rhizomes of Boesenbergia longiflora were carried out to investigate for anti-inflammatory and wound healing activities in order to support the traditional use. The ethanolic extract of Boesenbergia longiflora and its fractions were tested using relevant in vitro anti-inflammatory and wound healing assays. For the in vitro studies, murine macrophage RAW264.7 cells and mouse fibroblast L929 cells were assessed for anti-inflammatory and fibroblast stimulatory activities, respectively. In vivo anti-inflammatory activity was determined by carrageenan-induced rat paw edema model as well as acute toxicity estimated by the up-and-down method in mice. The present study has demonstrated that the ethanolic extract of Boesenbergia longiflora rhizomes possesses a potent anti-inflammatory and wound healing activities. Among the isolated fractions, the CHCl3 fraction showed potent anti-inflammatory effect through nitric oxide inhibitory activity (IC50=5.5 μg/ml) and reduction of carrageenan-induced rat paw edema (ED50=222.7 mg/kg), whereas this fraction exhibited wound healing property via fibroblast migration on both day 1 (77.3%) and day 2 (100%) as well as enhanced collagen production (187.5 μg/ml) at concentration of 3 μg/ml, compared to that of the controls, 39.4% for fibroblast and 60.8 μg/ml for collagen, respectively. The anti-inflammatory mechanism of the CHCl3 fraction is found to suppress the iNOS and COX-2 mRNA expression. The scientific investigation of wound healing activity of Boesenbergia longiflora rhizomes support the Thai traditional uses for treatment of inflammatory bowel disease, ulcerative colitis, aphthous ulcer and abscess. The EtOH extract and CHCl3 fraction exert potential wound healing property through NO inhibition, anti-oxidant effect and stimulation of fibroblast migration and collagen production. The phytochemical screening revealed that the CHCl3 fraction of Boesenbergia longiflora rhizomes contains diarylheptanoids, flavonoids and terpenes. The isolation of the compounds responsible for the wound healing effect is now in progress. © 2013 Elsevier Ireland Ltd. All rights reserved.

Comparison of the cytotoxicity of clinically relevant cobalt-chromium and alumina ceramic wear particles in vitro.

PubMed

Germain, M A; Hatton, A; Williams, S; Matthews, J B; Stone, M H; Fisher, J; Ingham, E

2003-02-01

Concern over polyethylene wear particle induced aseptic loosening of metal-on-polyethylene hip prostheses has led to renewed interest in alternative materials such as metal-on-metal and alumina ceramic-on-alumina ceramic for total hip replacement. This study compared the effects of clinically relevant cobalt-chromium and alumina ceramic wear particles on the viability of U937 histiocytes and L929 fibroblasts in vitro. Clinically relevant cobalt-chromium wear particles were generated using a flat pin-on-plate tribometer. The mean size of the clinically relevant metal particles was 29.5+/-6.3 nm (range 5-200 nm). Clinically relevant alumina ceramic particles were generated in the Leeds MkII anatomical hip simulator from a Mittelmieier prosthesis using micro-separation motion. This produced particles with a bimodal size distribution. The majority (98%) of the clinically relevant alumina ceramic wear debris was 5-20 nm in size. The cytotoxicity of the clinically relevant wear particles was compared to commercially available cobalt-chromium (9.87 microm+/-5.67) and alumina ceramic (0.503+/-0.19 microm) particles. The effects of the particles on the cells over a 5 day period at different particle volume (microm(3)) to cell number ratios were tested and viability determined using ATP-Lite(TM). Clinically relevant cobalt-chromium particles 50 and 5 microm(3) per cell reduced the viability of U937 cells by 97% and 42% and reduced the viability of L929 cells by 95% and 73%, respectively. At 50 microm(3) per cell, the clinically relevant ceramic particles reduced U937 cell viability by 18%. None of the other concentrations of the clinically relevant particles were toxic. The commercial cobalt-chromium and alumina particles did not affect the viability of either the U937 histiocytes or the L929 fibroblasts.Thus at equivalent particle volumes the clinically relevant cobalt-chromium particles were more toxic then the alumina ceramic particles. This study has emphasised the fact that the nature, size and volume of particles are important in assessing biological effects of wear debris on cells in vitro.

Marine macromolecules cross-linked hydrogel scaffolds as physiochemically and biologically favorable entities for tissue engineering applications.

PubMed

Sumayya, A S; Muraleedhara Kurup, G

2017-06-01

Marine biopolymer composite materials provide a technological platform for launching biomedical applications. Biomaterials demand good biocompatibility without the possibility of inflammation or foreign body reactions. In this study, we prepared two biocomposite hydrogels namely; HAC (hydroxyapatite, alginate & chitosan) and HACF (hydroxyapatite, alginate, chitosan & fucoidan) followed by calcium chloride cross linking. The prepared scaffolds were characterized by Fourier transform infrared spectroscopy and scanning electron microscopy. Porosity measurement, swelling, biodegradation, hemolysis, RBC aggregation, plasma protein adsorption and cytotoxicity studies were also done. The hydrogel scaffold HACF possessed a well-defined porous architecture, sufficient water holding capacity, better hemocompatibility and biodegradability. The biocompatibility was confirmed through in vitro cytotoxicity studies such as MTT assay, Neutral red uptake, DAPI staining, Trypan blue dye exclusion test and direct contact assay in L929 mouse fibroblast cells. In addition, immunomodulatory and anti-inflammatory properties of both of these scaffolds were revealed by the mRNA expressions of major inflammatory marker genes in cytotoxic condition such as TNF-α, IL-6 and NF-κB. The physiochemical characterization and biological responses of HACF hydrogel signifies its suitability for various tissue engineering applications.

Biomimetic Bone-like Hydroxyapatite by Mineralization on Supramolecular Porous Fiber Networks.

PubMed

Li, Bo; Kan, Lei; Zhang, Xinyue; Li, Jie; Li, Ruiting; Gui, Qinyuan; Qiu, Dengli; He, Fei; Ma, Ning; Wang, Yapei; Wei, Hao

2017-08-29

Hydroxyapatite (HA), the main inorganic component of bone tissue, is mineralized with collagen fibril scaffolds during bone formation. Inspired by the process, a self-assembled porous network architecture was designed and synthesized by using the 2-ureido-4[1H]-pyrimidone (UPy) modified glycerol molecule UPy-Gly, which was further utilized as a template for biomimetic mineralization. When incubated in simulated body fluid (SBF), the HA nucleus first formed in the holes of the template by the induction of hydroxyls on the surface, grew along the nanofibers, and fused with the template to fabricate hydroxyapatite composites (UPy-Gly/HA). Transmission electron microscopic observation demonstrates that the mineral clusters are accumulated by lamella-like nano hydroxyapatite and the elasticity modulus measured by atomic force microscopy is about 5.5 GPa, which is quite close to the natural cancellous bone tissue of human both in structure and in mechanical properties. The Cell Counting Kit 8 (CCK-8) assay of UPy-Gly and UPy-Gly/HA shows noncytotoxicity to mouse fibroblast L-929 cells. This bioinspired composite will be a promising material for potential use in bone tissue implantation and regeneration engineering.

Concurrent improvement in biocompatibility and bioinertness of diamond-like carbon films with nitrogen doping.

PubMed

Liao, Wen-Hsiang; Lin, Chii-Ruey; Wei, Da-Hua; Shen, You-Ruey; Li, Yi-Chieh; Lee, Jen-Ai; Liang, Chia-Yao

2012-11-01

The surfaces of implantable biomaterials improving biocompatibility and bioinertness are critical for new application of bioimplantable devices. Diamond-like carbon (DLC) film is a promising biomaterial with use for coating bioimplantable devices because of its good biocompatibility, bioinertness, and mechanical properties. In this study, concurrent improvement in biocompatibility and bioinertness of DLC films has been achieved using N-incorporation technique. The N doping degree was found to play an important role in affecting the biocompatibility and bioinertness of N-doped DLC films. The results indicated that the N-doped DLC films deposited at N(2) concentration of 5% could help to create suitable condition of surface/structure/adhesion combination of DLC films in the both affinity of the L929 mouse fibroblasts and electrochemical inertness in the Hank's balanced salt solutions (simulating human body fluids). N doping supports the attachment and proliferation of cells and prevents the permeation of electrolyte solutions, thereby simultaneity improved the biocompatibility and bioinertness of DLC films. This finding is useful for the fabrication and encapsulation of in vivo devices without induced immune response in the human body. Copyright © 2012 Wiley Periodicals, Inc.

Revealing the potential of squid chitosan-based structures for biomedical applications.

PubMed

Reys, L L; Silva, S S; Oliveira, J M; Caridade, S G; Mano, J F; Silva, T H; Reis, R L

2013-08-01

In recent years, much attention has been given to different marine organisms, namely as potential sources of valuable materials with a vast range of properties and characteristics. In this work, β-chitin was isolated from the endoskeleton of the giant squid Dosidicus gigas and further deacetylated to produce chitosan. Then, the squid chitosan was processed into membranes and scaffolds using solvent casting and freeze-drying, respectively, to assess their potential biomedical application. The developed membranes have shown to be stiffer and less hydrophobic than those obtained with commercial chitosan. On the other hand, the morphological characterization of the developed scaffolds, by SEM and micro-computed tomography, revealed that the matrices were formed with a lamellar structure. The findings also indicated that the treatment with ethanol prior to neutralization with sodium hydroxide caused the formation of larger pores and loss of some lamellar features. The in vitro cell culture study has shown that all chitosan scaffolds exhibited a non-cytotoxic effect over the mouse fibroblast-like cell line, L929 cells. Thus, chitosan produced from the endoskeletons of the giant squid Dosidicus gigas has proven to be a valuable alternative to existing commercial materials when considering its use as biomaterial.

Preparation and characterization of aminoethyl hydroxypropyl starch modified with collagen peptide.

PubMed

Wen, Huigao; Hu, Jin; Ge, Hongyu; Zou, Shengqiong; Xiao, Yao; Li, Ya; Feng, Han; Fan, Lihong

2017-08-01

The preparation of aminoethyl hydroxypropyl starch collagen peptide (AEHPS-COP) was via an enzyme-catalyzed reaction between amino groups in aminoethyl hydroxypropyl starch (AEHPS) and γ-carboxamide groups in collagen peptide (COP) by using microbial transglutaminase (MTGase) as biocatalyst. As an intermediate reactant, AEHPS was synthesized from hydroxypropyl starch (HPS) and 2-chloroethylamine hydrochloride (CEH). The chemical structures of HPS, AEHPS and AEHPS-COP were characterized by Fourier transform infrared spectroscopy (FT-IR) and 13 C nuclear magnetic resonance ( 13 C NMR). The reaction conditions that influenced the degree of substitution (DS) of AEHPS-COP were optimized, which included the reaction temperature, the reaction time, the mass ratio of collagen peptide to aminoethyl hydroxypropyl starch and the pH value. In addition, in vitro antioxidant activities of AEHPS-COP were evaluated through the scavenging activity of hydroxyl and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. Furthermore, the methylthiazol tetrazolium (MTT) assay was applied to investigate the cell viability of AEHPS-COP. The results indicated that the AEHPS-COP exhibited better cell viability to L929 mouse fibroblast cells. Therefore, the AEHPS-COP showed a promising potential application in cosmetic, biomedical and pharmaceutical fields. Copyright © 2017 Elsevier B.V. All rights reserved.

Scaffold percolative efficiency: in vitro evaluation of the structural criterion for electrospun mats.

PubMed

Heidarkhan Tehrani, Ashkan; Zadhoush, Ali; Karbasi, Saeed; Sadeghi-Aliabadi, Hojjat

2010-11-01

Fibrous scaffolds of engineered structures can be chosen as promising porous environments when an approved criterion validates their applicability for a specific medical purpose. For such biomaterials, this paper sought to investigate various structural characteristics in order to determine whether they are appropriate descriptors. A number of poly(3-hydroxybutyrate) scaffolds were electrospun; each of which possessed a distinguished architecture when their material and processing conditions were altered. Subsequent culture of mouse fibroblast cells (L929) was carried out to evaluate the cells viability on each scaffold after their attachment for 24 h and proliferation for 48 and 72 h. The scaffolds' porosity, pores number, pores size and distribution were quantified and none could establish a relationship with the viability results. Virtual reconstruction of the mats introduced an authentic criterion, "Scaffold Percolative Efficiency" (SPE), with which the above descriptors were addressed collectively. It was hypothesized to be able to quantify the efficacy of fibrous scaffolds by considering the integration of porosity and interconnectivity of the pores. There was a correlation of 80% as a good agreement between the SPE values and the spectrophotometer absorbance of viable cells; a viability of more than 350% in comparison to that of the controls.

Silk fibroin nanoparticles prepared by electrospray as controlled release carriers of cisplatin.

PubMed

Qu, Jing; Liu, Yu; Yu, Yanni; Li, Jing; Luo, Jingwan; Li, Mingzhong

2014-11-01

To maintain the anti-tumor activity of cis-dichlorodiamminoplatinum (CDDP) while avoiding its cytotoxicity and negative influence on normal tissue, CDDP-loaded silk fibroin nanoparticles approximately 59 nm in diameter were successfully prepared by electrospray without using organic solvent. CDDP was incorporated into nanoparticles through metal-polymer coordination bond exchange. In vitro release tests showed that the cisplatin in the nanoparticles could be slowly and sustainably released for more than 15 days. In vitro anti-cancer experiments and intracellular Pt content testing indicated that CDDP-loaded silk fibroin nanoparticles were easily internalized by A549 lung cancer cells, transferring CDDP into cancer cells and then triggering their apoptosis. In contrast, the particles were not easily internalized by L929 mouse fibroblast cells and hence showed weaker cell growth inhibition. The CDDP-loaded silk fibroin nanoparticles showed sustained and efficient killing of tumor cells but weaker inhibition of normal cells. In general, this study provides not only a novel method for preparing CDDP-loaded silk fibroin nanoparticles but also a new carrier system for clinical therapeutic drugs against lung cancers and other tumors. Copyright © 2014 Elsevier B.V. All rights reserved.

Polydopamine deposition with anodic oxidation for better connective tissue attachment to transmucosal implants.

PubMed

Teng, F; Chen, H; Xu, Y; Liu, Y; Ou, G

2018-04-01

Nowadays, most designs for the transmucosal surface of implants are machined-smooth. However, connective tissue adhered to the smooth surface of an implant has poor mechanical resistance, which can render separation of tissue from the implant interface and induce epithelial downgrowth. Modification of the transmucosal surface of implants, which can help form a good seal of connective tissue, is therefore desired. We hypothesized that anodic oxidation (AO) and polydopamine (PD) deposition could be used to enhance the attachment between an implant and peri-implant connective tissue. We tested this hypothesis in the mandibles of Beagle dogs. AO and PD were used to modify the transmucosal region of transmucosal implants (implant neck). The surface microstructure, surface roughness and elemental composition were investigated in vitro. L929 mouse fibroblasts were cultured to test the effect of PD on cell adhesion. Six Beagle dogs were used for the in vivo experiment (n = 6 dogs per group). Three months after building the edentulous animal model, four groups of implants (control, AO, PD and AO + PD) were inserted. After 4 months of healing, samples were harvested for histometric analyses. The surfaces of anodized implant necks were overlaid with densely distributed pores, 2-7 μm in size. On the PD-modified surfaces, N1s, the chemical bond of nitrogen in PD, was detected using X-ray photoelectron spectroscopy. L929 developed pseudopods more quickly on the PD-modified surfaces than on the surfaces of the control group. The in vivo experiment showed a longer connective tissue seal and a more coronally located peri-implant soft-tissue attachment in the AO + PD group than in the control group (P < .05). The modification of AO + PD on the implant neck yielded better attachment between the implant and peri-implant connective tissue. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

8,9-DIHYDROXY-8,9-DIHYDRODIBENZO[A,L]PYRENE IS A POTENT MORPHOLOGICAL CELL-TRANSFORMING AGENT IN C3H10T1/2C18 MOUSE EMBRYO FIBROBLASTS IN THE ABSENCE OF DETECTABLE STABLE COVALENT DNA ADDUCTS

EPA Science Inventory

The comparative genotoxic effects of racemic trans-8,9dihydroxy-8,9-dihydrodibenzo[a,l]pyrene (trans- DB[a,l]P8,9-diol), the metabolic K-region dihydrodiol of dibenzo[a,l] pyrene (DB[a,l]P) (dibenzo[def,p]chrysene) and DB[a,l]P in transformable mouse embryo C3HIOT1/2C18 (C3HIOT1/...

In vitro degradation and biocompatibility of Fe-Pd and Fe-Pt composites fabricated by spark plasma sintering.

PubMed

Huang, T; Cheng, J; Zheng, Y F

2014-02-01

In order to obtain biodegradable Fe-based materials with similar mechanical properties as 316L stainless steel and faster degradation rate than pure iron, Fe-5 wt.%Pd and Fe-5 wt.%Pt composites were prepared by spark plasma sintering with powders of pure Fe and Pd/Pt, respectively. The grain size of Fe-5 wt.%Pd and Fe-5 wt.%Pt composites was much smaller than that of as-cast pure iron. The metallic elements Pd and Pt were uniformly distributed in the matrix and the mechanical properties of these materials were improved. Uniform corrosion of Fe-Pd and Fe-Pt composites was observed in both electrochemical tests and immersion tests, and the degradation rates of Fe-Pd and Fe-Pt composites were much faster than that of pure iron. It was found that viabilities of mouse fibroblast L-929 cells and human umbilical vein endothelial cells (ECV304) cultured in extraction mediums of Fe-Pd and Fe-Pt composites were close to that of pure iron. After 4 days' culture, the viabilities of L-929 and ECV304 cells in extraction medium of experimental materials were about 80%. The result of direct contact cytotoxicity also indicated that experimental materials exhibited no inhibition on vascular endothelial process. Meanwhile, iron ions released from experimental materials could inhibit proliferation of vascular smooth muscle cells (VSMC), which may be beneficial for hindering vascular restenosis. Furthermore, compared with that of as-cast pure iron, the hemolysis rates of Fe-Pd and Fe-Pt composites were slightly higher, but still within the range of 5%, which is the criteria for good blood compatibility. The numbers of platelet adhered on the surface of Fe-Pd and Fe-Pt composites were lower than that of pure iron, and the morphology of platelets kept spherical. To sum up, the Fe-5 wt.%Pd and Fe-5 wt.%Pt composites exhibited good mechanical properties and degradation behavior, closely approaching the requirements for biodegradable metallic stents. © 2013.

Biocompatibility evaluation of cigarette and carbon papers used in repair of traumatic tympanic membrane perforations: experimental study.

PubMed

Altuntaş, Emine Elif; Sümer, Zeynep

2013-01-01

The purposes of this study were to investigate the biocompatibility of two different paper patches (carbon and cigarette papers) and compare the adhesion and proliferation features of L929 fibroblast cells by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT Test) test and scanning electron microscopy (SEM). In this study, time-dependent cytotoxic effects of cigarette and carbon papers used in repairing small traumatic TM perforations were investigated in vitro by using MTT test. And also adhesion and spreading of cells over disk surface were observed by SEM. Cytotoxicity test carried out by MTT analysis on leakage products collected from two types of paper patches at the end of 24 and 48 h revealed no cytotoxicity (P > 0.05). In SEM studies, it was observed that cells started to proliferate over disk surface as a result of 48-h incubation, and SEM revealed that the cell proliferation over cigarette paper was more compared to the one over carbon paper. We believe that this is the first study where biocompatibility and adhesion features of carbon and cigarette paper have been studied by using L929 fibroblast cell culture. As a result, biocompatibility of cigarette paper and also whether cigarette paper was superior to carbon paper in cell attachment and biocompatibility were studied. It was found, by MTT test and SEM test, that cigarette paper had a higher biocompatibility and cell attachment, and thus cigarette paper should be the patch to be preferred in cases where TM perforations are repaired by paper-patch method.

Structure, mechanical characteristics and in vitro degradation, cytotoxicity, genotoxicity and mutagenicity of novel biodegradable Zn-Mg alloys.

PubMed

Kubásek, J; Vojtěch, D; Jablonská, E; Pospíšilová, I; Lipov, J; Ruml, T

2016-01-01

Zn-(0-1.6)Mg (in wt.%) alloys were prepared by hot extrusion at 300 °C. The structure, mechanical properties and in vitro biocompatibility of the alloys were investigated. The hot-extruded magnesium-based WE43 alloy was used as a control. Mechanical properties were evaluated by hardness, compressive and tensile testing. The cytotoxicity, genotoxicity (comet assay) and mutagenicity (Ames test) of the alloy extracts and ZnCl2 solutions were evaluated with the use of murine fibroblasts L929 and human osteosarcoma cell line U-2 OS. The microstructure of the Zn alloys consisted of recrystallized Zn grains of 12 μm in size and fine Mg2Zn11 particles arranged parallel to the hot extrusion direction. Mechanical tests revealed that the hardness and strength increased with increasing Mg concentration. The Zn-0.8 Mg alloys showed the best combination of tensile mechanical properties (tensile yield strength of 203 MPa, ultimate tensile strength of 301 MPa and elongation of 15%). At higher Mg concentrations the plasticity of Zn-Mg alloys was deteriorated. Cytotoxicity tests with alloy extracts and ZnCl2 solutions proved the maximum safe Zn(2+) concentrations of 120 μM and 80 μM for the U-2 OS and L929 cell lines, respectively. Ames test with extracts of alloys indicated that the extracts were not mutagenic. The comet assay demonstrated that 1-day extracts of alloys were not genotoxic for U-2 OS and L929 cell lines after 1-day incubation. Copyright © 2015 Elsevier B.V. All rights reserved.

Evaluation of the In Vitro Cytotoxicity of Crosslinked Biomaterials

PubMed Central

Wang, Martha O.; Etheridge, Julie M.; Thompson, Joshua A.; Vorwald, Charlotte E.; Dean, David; Fisher, John P.

2013-01-01

This study evaluated the in vitro cytotoxicity of poly(propylene fumarate) (PPF). PPF is an aliphatic biodegradable polymer that has been well characterized for use in bone tissue engineering scaffolds. Four different cell types, human mesenchymal stem cells (hMSC), fibroblasts (L929), pre-osteoblasts (MC3T3), and canine mesenchymal stem cells (cMSC), were used to evaluate the cytotoxicity of PPF. These cell types represent the tissues that PPF would interact with in vivo as a bone tissue scaffold. The sol fraction of the PPF films was measured and then utilized to estimate crosslinking density. Cytotoxicity was evaluated using XTT assay and fluorescence imaging. Results showed that PPF supported similar cell metabolic activities of hMSC, L929, MC3T3 and cMSC compared to the non-cytotoxic control, high density polyethylene (HDPE) and were statistically different than those cultured with the cytotoxic control, a polyurethane film containing 0.1% zinc diethyldithiocarbamate (ZCF). Results showed differing cellular responses to ZCF, the cytotoxic control. The L929 cells had the lowest cell metabolic activity levels after exposure to ZCF compared to the cell metabolic activity levels of the MC3T3, hMSC or cMSC cells. Qualitative verification of the results using fluorescence imaging demonstrated no change in cell morphology, vacuolization, or detachment when cultured with PPF compared to HDPE or blank media cultures. Overall the cytotoxicity response of the cells to PPF was demonstrated to be similar to the cytotoxic response of cells to known non-cytotoxic materials (HDPE). PMID:23627804

Type I collagen gel protects murine fibrosarcoma L929 cells from TNFα-induced cell death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Hong-Ju; He, Wen-Qi; Chen, Ling

Murine fibrosarcoma L929 cells have been used to test efficacy of proinflammatory cytokine TNFα. In the present study, we reported on protective effect of type I collagen gel used as L929 cell culture. L929 cell grew and proliferated well on collagen gel. However, the L929 cells exhibited cobblestone-like morphology which was much different from the spread fusiform shape when cultured on conventional cell dishes as well as the cells tended to aggregate. On conventional cell culture dishes, the cells treated with TNFα became round in shape and eventually died in a necroptotic manner. The cells cultured on collagen gel, however,more » were completely unaffected. TNFα treatment was reported to induce autophagy in L929 cells on the plastic dish, and therefore we investigated the effect of collagen gel on induction of autophagy. The results indicated that autophagy induced by TNFα treatment was much reduced when the cells were cultured on collagen gel. In conclusion, type I collagen gel protected L929 cell from TNFα-induced cell death. - Highlights: • Collagen gel culture changed the morphology of L929 cells. • L929 cell cultured on collagen gel were resistant to TNFα-induced cell death. • Collagen gel culture inhibited TNFα-induced autophagy in L929 cells.« less

8,9-dihydroxy-8,9-dihydrodibenzo[a,l]pyrene is a potent morphological cell-transforming agent in C3H10T(1)/(2)Cl8 mouse embryo fibroblasts in the absence of detectable stable covalent DNA adducts.

PubMed

Nesnow, S; Davis, C; Padgett, W T; Adams, L; Yacopucci, M; King, L C

2000-06-01

The comparative genotoxic effects of racemic trans-8,9-dihydroxy-8, 9-dihydrodibenzo[a,l]pyrene (trans-DB[a,l]P-8,9-diol), the metabolic K-region dihydrodiol of dibenzo[a,l] pyrene (DB[a,l]P) (dibenzo[def, p]chrysene) and DB[a,l]P in transformable mouse embryo C3H10T(1)/(2)Cl8 (C3H10T(1)/(2)) fibroblasts was investigated. The C3H10T(1)/(2) mouse embryo morphological cell-transforming activities of these polycyclic aromatic hydrocarbons (PAHs) were assayed using concentration-response studies. At concentrations of 33 nM and above both trans-DB[a,l]P-8,9-diol and DB[a,l]P produced significant (and similar) numbers of type II and III foci per dish and numbers of dishes with type II and II foci. Concomitant cytotoxicity studies revealed a reduction in colony survival of approximately 25% up to 198 nM for both PAHs. DNA adducts of trans-DB[a,l]P-8,9-diol and DB[a,l]P in C3H10T(1)/(2) cells were analyzed by a (32)P-post-labeling TLC/HPLC method. No adducts were observed in the DNA of C3H10T(1)/(2) cells treated with trans-DB[a, l]P-8,9-diol at concentrations that induced morphological cell transformation. Under the same exposure and chromatographic conditions, DNA adducts of deoxyadenosine and deoxyguanosine derived from the fjord region anti-DB[a,l]P-11,12-diol-13,14-epoxide and syn-DB[a,l]P-11,12-diol-13,14-epoxide were observed in the DNA of DB[a,l]P-treated cells. These results indicate that trans-DB[a,l]P-8, 9-diol has intrinsic genotoxic activity equal to that of DB[a,l]P, based on morphological cell transformation of mouse embryo fibroblasts. The activity of trans-DB[a,l]P-8,9-diol is apparently not associated with the formation of observable stable covalent DNA adducts. These results suggest that under appropriate conditions, trans-DB[a,l]P-8,9-diol may serve as an intermediate in the genotoxicity of DB[a,l]P.

Cationic Copolymerization of 3,3-Bis(hydroxymethyl)oxetane and Glycidol: Biocompatible Hyperbranched Polyether Polyols with High Content of Primary Hydroxyl Groups.

PubMed

Christ, Eva-Maria; Hobernik, Dominika; Bros, Matthias; Wagner, Manfred; Frey, Holger

2015-10-12

The cationic ring-opening copolymerization of 3,3-bis(hydroxymethyl)oxetane (BHMO) with glycidol using different comonomer ratios (BHMO content from 25 to 90%) and BF3OEt2 as an initiator has been studied. Apparent molecular weights of the resulting hyperbranched polyether copolymers ranged from 1400 to 3300 g mol(-1) (PDI: 1.21-1.48; method: SEC, linear PEG standards). Incorporation of both comonomers is evidenced by MALDI-TOF mass spectroscopy. All hyperbranched polyether polyols with high content of primary hydroxyl groups portray good solubility in water, which correlates with an increasing content of glycerol units. Detailed NMR characterization was employed to elucidate the copolymer microstructures. Kinetic studies via FTIR demonstrated a weak gradient-type character of the copolymers. MTT assays of the copolymers (up to 100 μg mL(-1)) on HEK and fibroblast cell lines (3T3, L929, WEHI) as well as viability tests on the fibroblast cells were carried out to assess the biocompatibility of the materials, confirming excellent biocompatibility. Transfection efficiency characterization by flow cytometry and confocal laser microscopy demonstrated cellular uptake of the copolymers. Antiadhesive properties of the materials on surfaces were assessed by adhesion assays with fibroblast cells.

Biotransformation of aflatoxin B1 and aflatoxin G1 in peanut meal by anaerobic solid fermentation of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

PubMed

Chen, Yujie; Kong, Qing; Chi, Chen; Shan, Shihua; Guan, Bin

2015-10-15

The purpose of this study was to explore the ability of anaerobic solid fermentation of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus to biotransform aflatoxins in peanut meal. The pH of the peanut meal was adjusted above 10, and then heated for 10 min at 100 °C, 115 °C and 121 °C. The S. thermophilus and L. delbrueckii subsp. bulgaricus were precultured together in MRS broth for 48 h at 37 °C. The heated peanut meal was mixed with precultured MRS broth containing 7.0×10(8) CFU/mL of S. thermophilus and 3.0×10(3) CFU/mL of L. delbrueckii subsp. bulgaricus with the ratio of 1 to 1 (weight to volume) and incubated in anaerobic jars at 37 °C for 3 days. The aflatoxin content in the peanut meal samples was determined by HPLC. The results showed that the peanut meal contained mainly aflatoxin B1 (AFB1) (10.5±0.64 μg/kg) and aflatoxin G1 (AFG1) (18.7±0.55 μg/kg). When heat treatment was combined with anaerobic solid fermentation, the biotransformation rate of aflatoxins in peanut meal could attain 100%. The cytotoxicity of fermented peanut meal to L929 mouse connective tissue fibroblast cells was determined by MTT assay and no significant toxicity was observed in the fermented peanut meal. Furthermore, heat treatment and anaerobic solid fermentation did not change the amino acid concentrations and profile in peanut meal. Copyright © 2015 Elsevier B.V. All rights reserved.

Dynamized Preparations in Cell Culture

PubMed Central

Sunila, Ellanzhiyil Surendran; Preethi, Korengath Chandran; Kuttan, Girija

2009-01-01

Although reports on the efficacy of homeopathic medicines in animal models are limited, there are even fewer reports on the in vitro action of these dynamized preparations. We have evaluated the cytotoxic activity of 30C and 200C potencies of ten dynamized medicines against Dalton's Lymphoma Ascites, Ehrlich's Ascites Carcinoma, lung fibroblast (L929) and Chinese Hamster Ovary (CHO) cell lines and compared activity with their mother tinctures during short-term and long-term cell culture. The effect of dynamized medicines to induce apoptosis was also evaluated and we studied how dynamized medicines affected genes expressed during apoptosis. Mother tinctures as well as some dynamized medicines showed significant cytotoxicity to cells during short and long-term incubation. Potentiated alcohol control did not produce any cytotoxicity at concentrations studied. The dynamized medicines were found to inhibit CHO cell colony formation and thymidine uptake in L929 cells and those of Thuja, Hydrastis and Carcinosinum were found to induce apoptosis in DLA cells. Moreover, dynamized Carcinosinum was found to induce the expression of p53 while dynamized Thuja produced characteristic laddering pattern in agarose gel electrophoresis of DNA. These results indicate that dynamized medicines possess cytotoxic as well as apoptosis-inducing properties. PMID:18955237

Inhibition of matrix metalloproteinases and toxicity of gold and platinum nanoparticles in L929 fibroblast cells.

PubMed

Hashimoto, Masanori; Yamaguchi, Satoshi; Sasaki, Jun-Ichi; Kawai, Koji; Kawakami, Hayato; Iwasaki, Yasuhiko; Imazato, Satoshi

2016-02-01

This study evaluated the inhibition of matrix metalloproteases (MMPs) and cellular responses elicited by gold (Au) and platinum (Pt) nanoparticles (NPs). The interaction of MMP-1 and NPs was evaluated using an MMP assay kit. The cultured L929 cells were exposed to various concentrations of NPs. The cellular responses to NPs were examined using a cytotoxicity assay (that evaluated cell viability and lactic dehydrogenase production), real-time polymerase chain reaction (RT-qPCR), and transmission electron microscopy. Both types of NPs, when used at concentrations above 10 μg ml(-1), inhibited MMP-1 activity. No cytotoxic effects were found when the cells were exposed to AuNPs. In contrast, PtNPs, at both 100 and 400 μg ml(-1), induced cytotoxicity. No inflammatory responses (production of interleukin-6 and tumor necrosis factor-alpha) to NPs were identified by RT-qPCR. The negative surface charge of NPs (COOH(-)) binds to the Zn(2+) of the MMP active center by chelation, leading to MMP inhibition. Gold nanoparticles are plausible candidates for MMP inhibitors in resin-bonding materials because they effectively inhibit MMP-1 activity without cytotoxic or inflammatory effects. © 2015 Eur J Oral Sci.

KR-12-a5 is a non-cytotoxic agent with potent antimicrobial effects against oral pathogens.

PubMed

Caiaffa, Karina Sampaio; Massunari, Loiane; Danelon, Marcelle; Abuna, Gabriel Flores; Bedran, Telma Blanca Lombardo; Santos-Filho, Norival Alves; Spolidorio, Denise Madalena Palomari; Vizoto, Natalia Leal; Cilli, Eduardo Maffud; Duque, Cristiane

2017-11-01

This study evaluated the cytotoxicity and antimicrobial activity of analogs of cationic peptides against microorganisms associated with endodontic infections. L-929 fibroblasts were exposed to LL-37, KR-12-a5 and hBD-3-1C V and chlorhexidine (CHX, control), and cell metabolism was evaluated with MTT. The minimal inhibitory concentration (MIC) and the minimal bactericidal/fungicidal concentration (MBC/MFC) of the peptides and CHX were determined against oral pathogens associated with endodontic infections. Enterococcus faecalis and Streptococcus mutans biofilms were cultivated in bovine dentin blocks, exposed to different concentrations of the most efficient antimicrobial peptide and analyzed by confocal laser scanning microscopy. CHX and peptides affected the metabolism of L-929 at concentrations > 31.25 and 500 μg ml -1 , respectively. Among the peptides, KR-12-a5 inhibited growth of both the microorganisms tested with the lowest MIC/MBC/MFC values. In addition, KR-12-a5 significantly reduced E. faecalis and S. mutans biofilms inside dentin tubules. In conclusion, KR-12-a5 is a non-cytotoxic agent with potent antimicrobial and anti-biofilm activity against oral pathogens associated with endodontic infections.

Accelerated healing of full-thickness wounds by genipin-crosslinked silk sericin/PVA scaffolds.

PubMed

Aramwit, Pornanong; Siritienthong, Tippawan; Srichana, Teerapol; Ratanavaraporn, Juthamas

2013-01-01

Silk sericin has recently been studied for its advantageous biological properties, including its ability to promote wound healing. This study developed a delivery system to accelerate the healing of full-thickness wounds. Three-dimensional scaffolds were fabricated from poly(vinyl alcohol) (PVA), glycerin (as a plasticizer) and genipin (as a crosslinking agent), with or without sericin. The physical and biological properties of the genipin-crosslinked sericin/PVA scaffolds were investigated and compared with those of scaffolds without sericin. The genipin-crosslinked sericin/PVA scaffolds exhibited a higher compressive modulus and greater swelling in water than the scaffolds without sericin. Sericin also exhibited controlled release from the scaffolds. The genipin-crosslinked sericin/PVA scaffolds promoted the attachment and proliferation of L929 mouse fibroblasts. After application to full-thickness rat wounds, the wounds treated with genipin-crosslinked sericin/PVA scaffolds showed a significantly greater reduction in wound size, collagen formation and epithelialization compared with the control scaffolds without sericin but lower numbers of macrophages and multinucleated giant cells. These results indicate that the delivery of sericin from the novel genipin-crosslinked scaffolds efficiently healed the wound. Therefore, these genipin-crosslinked sericin/PVA scaffolds represent a promising candidate for the accelerated healing of full-thickness wounds. Copyright © 2013 S. Karger AG, Basel.

A comparative study of three cytotoxicity test methods for nanomaterials using sodium lauryl sulfate.

PubMed

Kwon, Jae-Sung; Kim, Kwang-Mahn; Kim, Kyoung-Nam

2014-10-01

The biocompatibility evaluation of nanomaterials is essential for their medical diagnostic and therapeutic usage, where a cytotoxicity test is the simplest form of biocompatibility evaluation. Three methods have been commonly used in previous studies for the cytotoxicity testing of nanomaterials: trypan blue exclusion, colorimetric assay using water soluble tetrazolium (WST), and imaging under a microscope following calcein AM/ethidium homodimer-1 staining. However, there has yet to be a study to compare each method. Therefore, in this study three methods were compared using the standard reference material of sodium lauryl sulfate (SLS). Each method of the cytotoxicity test was carried out using mouse fibroblasts of L-929 exposed to different concentrations of SLS. Compared to the gold standard trypan blue exclusion test, both colorimetric assay using water soluble tetrazolium (WST) and imaging under microscope with calcein AM/ethidium homodimer-1 staining showed results that were not statistically different. Also, each method exhibited various advantages and disadvantages, which included the need of equipment, time taken for the experiment, and provision of additional information such as cell morphology. Therefore, this study concludes that all three methods of cytotoxicity testing may be valid, though careful consideration will be needed when selecting tests with regard to time, finances, and the amount of information required by the researcher(s).

Impact of calcium ion on cytotoxic effect of the boroxine derivative, K2[B3O3F4OH].

PubMed

Ivankovic, Sinisa; Stojkovic, Ranko; Maksimovic, Milka; Galic, Borivoj; Milos, Mladen

2016-01-01

The effect of Ca 2+ ions on the cytotoxic ability of boron heterocyclic compound dipotassium-trioxohydroxytetrafluorotriborate (K 2 [B 3 O 3 F 4 OH]), on in vitro tumor cells (mammary adenocarcinoma 4T1, melanoma B16F10 and squamous cell carcinoma SCCVII) and non-tumoral fibroblast cells (mouse dermal L929 and hamster lung V79) was examined. At small concentrations of Ca 2+ ions (0.42 mM), K 2 [B 3 O 3 F 4 OH] (3.85 mM) has a very strong cytotoxic effect on all cancer cells tested (89.1, 85.6 and 84.6%) and significantly less effect on normal cells (19.5 and 24.2%), respectively. Applying larger concentrations of Ca 2+ ions (9.42-72.42 mM), at the same concentration of K 2 [B 3 O 3 F 4 OH], no significant cytotoxic effect was detected on cancer cells and normal cells investigated. The selective ability of K 2 [B 3 O 3 F 4 OH], in the medium with a low concentration of Ca 2+ ions has a strong cytotoxic effect on cancer cells and very weak effect in normal cells, opens up the possibility of its application in antitumor therapy.

Nitric Oxide-Releasing Chitosan Oligosaccharides as Antibacterial Agents

PubMed Central

Lu, Yuan; Slomberg, Danielle L.; Schoenfisch, Mark H.

2014-01-01

Secondary amine-functionalized chitosan oligosaccharides of different molecular weights (i.e., ~2500, 5000, 10000) were synthesized by grafting 2-methyl aziridine from the primary amines on chitosan oligosaccharides, followed by reaction with nitric oxide (NO) gas under basic conditions to yield N-diazeniumdiolate NO donors. The total NO storage, maximum NO flux, and half-life of the resulting NO-releasing chitosan oligosaccharides were controlled by the molar ratio of 2-methyl aziridine to primary amines (e.g., 1:1, 2:1) and the functional group surrounding the N-diazeniumdiolates (e.g., polyethylene glycol (PEG) chains), respectively. The secondary amine-modified chitosan oligosaccharides greatly increased the NO payload over existing biodegradable macromolecular NO donors. In addition, the water-solubility of the chitosan oligosaccharides enabled their penetration across the extracellular polysaccharides matrix of Pseudomonas aeruginosa biofilms and association with embedded bacteria. The effectiveness of these chitosan oligosaccharides at biofilm eradication was shown to depend on both the molecular weight and ionic characteristics. Low molecular weight and cationic chitosan oligosaccharides exhibited rapid association with bacteria throughout the entire biofilm, leading to enhanced biofilm killing. At concentrations resulting in 5-log killing of bacteria in Pseudomonas aeruginosa biofilms, the NO-releasing and control chitosan oligosaccharides elicited no significant cytotoxicity to mouse fibroblast L929 cells in vitro. PMID:24268196

Cellulose acetate based 3-dimensional electrospun scaffolds for skin tissue engineering applications.

PubMed

Atila, Deniz; Keskin, Dilek; Tezcaner, Ayşen

2015-11-20

Skin defects that are not able to regenerate by themselves are among the major problems faced. Tissue engineering approach holds promise for treating such defects. Development of tissue-mimicking-scaffolds that can promote healing process receives an increasing interest in recent years. In this study, 3-dimensional electrospun cellulose acetate (CA) pullulan (PULL) scaffolds were developed for the first time. PULL was intentionally used to obtain 3D structures with adjustable height. It was removed from the electrospun mesh to increase the porosity and biostability. Different ratios of the polymers were electrospun and analyzed with respect to degradation, porosity, and mechanical properties. It has been observed that fiber diameter, thickness and porosity of scaffolds increased with increased PULL content, on the other hand this resulted with higher degradation of scaffolds. Mechanical strength of scaffolds was improved after PULL removal suggesting their suitability as cell carriers. Cell culture studies were performed with the selected scaffold group (CA/PULL: 50/50) using mouse fibroblastic cell line (L929). In vitro cell culture tests showed that cells adhered, proliferated and populated CA/PULL (50/50) scaffolds showing that they are cytocompatible. Results suggest that uncrosslinked CA/PULL (50/50) electrospun scaffolds hold potential for skin tissue engineering applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Proteomic analysis of the action of the Mycobacterium ulcerans toxin mycolactone: targeting host cells cytoskeleton and collagen.

PubMed

Gama, José B; Ohlmeier, Steffen; Martins, Teresa G; Fraga, Alexandra G; Sampaio-Marques, Belém; Carvalho, Maria A; Proença, Fernanda; Silva, Manuel T; Pedrosa, Jorge; Ludovico, Paula

2014-08-01

Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. The tissue damage characteristic of BU lesions is known to be driven by the secretion of the potent lipidic exotoxin mycolactone. However, the molecular action of mycolactone on host cell biology mediating cytopathogenesis is not fully understood. Here we applied two-dimensional electrophoresis (2-DE) to identify the mechanisms of mycolactone's cellular action in the L929 mouse fibroblast proteome. This revealed 20 changed spots corresponding to 18 proteins which were clustered mainly into cytoskeleton-related proteins (Dync1i2, Cfl1, Crmp2, Actg1, Stmn1) and collagen biosynthesis enzymes (Plod1, Plod3, P4ha1). In line with cytoskeleton conformational disarrangements that are observed by immunofluorescence, we found several regulators and constituents of both actin- and tubulin-cytoskeleton affected upon exposure to the toxin, providing a novel molecular basis for the effect of mycolactone. Consistent with these cytoskeleton-related alterations, accumulation of autophagosomes as well as an increased protein ubiquitination were observed in mycolactone-treated cells. In vivo analyses in a BU mouse model revealed mycolactone-dependent structural changes in collagen upon infection with M. ulcerans, associated with the reduction of dermal collagen content, which is in line with our proteomic finding of mycolactone-induced down-regulation of several collagen biosynthesis enzymes. Our results unveil the mechanisms of mycolactone-induced molecular cytopathogenesis on exposed host cells, with the toxin compromising cell structure and homeostasis by inducing cytoskeleton alterations, as well as disrupting tissue structure, by impairing the extracellular matrix biosynthesis.

The self-crosslinking smart hyaluronic acid hydrogels as injectable three-dimensional scaffolds for cells culture.

PubMed

Bian, Shaoquan; He, Mengmeng; Sui, Junhui; Cai, Hanxu; Sun, Yong; Liang, Jie; Fan, Yujiang; Zhang, Xingdong

2016-04-01

Although the disulfide bond crosslinked hyaluronic acid hydrogels have been reported by many research groups, the major researches were focused on effectively forming hydrogels. However, few researchers paid attention to the potential significance of controlling the hydrogel formation and degradation, improving biocompatibility, reducing the toxicity of exogenous and providing convenience to the clinical operations later on. In this research, the novel controllable self-crosslinking smart hydrogels with in-situ gelation property was prepared by a single component, the thiolated hyaluronic acid derivative (HA-SH), and applied as a three-dimensional scaffold to mimic native extracellular matrix (ECM) for the culture of fibroblasts cells (L929) and chondrocytes. A series of HA-SH hydrogels were prepared depending on different degrees of thiol substitution (ranging from 10 to 60%) and molecule weights of HA (0.1, 0.3 and 1.0 MDa). The gelation time, swelling property and smart degradation behavior of HA-SH hydrogel were evaluated. The results showed that the gelation and degradation time of hydrogels could be controlled by adjusting the component of HA-SH polymers. The storage modulus of HA-SH hydrogels obtained by dynamic modulus analysis (DMA) could be up to 44.6 kPa. In addition, HA-SH hydrogels were investigated as a three-dimensional scaffold for the culture of fibroblasts cells (L929) and chondrocytes cells in vitro and as an injectable hydrogel for delivering chondrocytes cells in vivo. These results illustrated that HA-SH hydrogels with controllable gelation process, intelligent degradation behavior, excellent biocompatibility and convenient operational characteristics supplied potential clinical application capacity for tissue engineering and regenerative medicine. Copyright © 2016 Elsevier B.V. All rights reserved.

Increased cellular uptake of lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles due to surface modification with folic acid.

PubMed

Feuser, Paulo Emilio; Arévalo, Juan Marcelo Carpio; Junior, Enio Lima; Rossi, Gustavo Rodrigues; da Silva Trindade, Edvaldo; Rocha, Maria Eliane Merlin; Jacques, Amanda Virtuoso; Ricci-Júnior, Eduardo; Santos-Silva, Maria Claudia; Sayer, Claudia; de Araújo, Pedro H Hermes

2016-12-01

Lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles surface modified with folic acid were synthesized by miniemulsion polymerization in just one step. In vitro biocompatibility and cytotoxicity assays on L929 (murine fibroblast), human red blood, and HeLa (uterine colon cancer) cells were performed. The effect of folic acid at the nanoparticles surface was evaluated through cellular uptake assays in HeLa cells. Results showed that the presence of folic acid did not affect substantially the polymer particle size (~120 nm), the superparamagnetic behavior, the encapsulation efficiency of lauryl gallate (~87 %), the Zeta potential (~38 mV) of the polymeric nanoparticles or the release profile of lauryl gallate. The release profile of lauryl gallate from superparamagnetic poly(methyl methacrylate) nanoparticles presented an initial burst effect (0-1 h) followed by a slow and sustained release, indicating a biphasic release system. Lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles with folic acid did not present cytotoxicity effects on L929 and human red blood cells. However, free lauryl gallate presented significant cytotoxic effects on L929 and human red blood cells at all tested concentrations. The presence of folic acid increased the cytotoxicity of lauryl gallate loaded in nanoparticles on HeLa cells due to a higher cellular uptake when HeLa cells were incubated at 37 °C. On the other hand, when the nanoparticles were incubated at low temperature (4 °C) cellular uptake was not observed, suggesting that the uptake occurred by folate receptor mediated energy-dependent endocytosis. Based on presented results our work suggests that this carrier system can be an excellent alternative in targeted drug delivery by folate receptor.

In vitro adhesion of fibroblastic cells to titanium alloy discs treated with sodium hydroxide.

PubMed

Al Mustafa, Maisa; Agis, Hermann; Müller, Heinz-Dieter; Watzek, Georg; Gruber, Reinhard

2015-01-01

Adhesion of osteogenic cells on titanium surfaces is a prerequisite for osseointegration. Alkali treatment can increase the hydrophilicity of titanium implant surfaces, thereby supporting the adhesion of blood components. However, it is unclear if alkali treatment also supports the adhesion of cells with a fibroblastic morphology to titanium. Here, we have used a titanium alloy (Ti-6AL-4V) processed by alkali treatment to demonstrate the impact of hydrophilicity on the adhesion of primary human gingival fibroblast and bone cells. Also included were the osteosarcoma and fibroblastoma cell lines, MG63 and L929, respectively. Cell adhesion was determined by scanning electron microscopy. We also measured viability, proliferation, and protein synthesis of the adherent cells. Alkali treatment increased the adhesion of gingival fibroblasts, bone cells, and the two cell lines when seeded onto the titanium alloy surface for 1 h. At 3 h, no significant changes in cell adhesion were observed. Cells grown for 1 day on the titanium alloy surfaces processed by alkali treatment behave similarly to untreated controls with regard to viability, proliferation, and protein synthesis. Based on these preliminary In vitro findings, we conclude that alkali treatment can support the early adhesion of cells with fibroblastic characteristics to a titanium alloy surface. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Saccharin and Cyclamate Inhibit Binding of Epidermal Growth Factor

NASA Astrophysics Data System (ADS)

Lee, L. S.

1981-02-01

The binding of 125I-labeled mouse epidermal growth factor (EGF) to 18 cell lines, including HeLa (human carcinoma), MDCK (dog kidney cells), HTC (rat hepatoma), K22 (rat liver), HF (human foreskin), GM17 (human skin fibroblasts), XP (human xeroderma pigmentosum fibroblasts), and 3T3-L1 (mouse fibroblasts), was inhibited by saccharin and cyclamate. The human cells were more sensitive to inhibition by these sweeteners than mouse or rat cells. EGF at doses far above the physiological levels reversed the inhibition in rodent cells but not in HeLa cells. In HeLa cells, the doses of saccharin and cyclamate needed for 50% inhibition were 3.5 and 9.3 mg/ml, respectively. Glucose, 2-deoxyglucose, sucrose, and xylitol did not inhibit EGF binding. Previous studies have shown that phorbol esters, strongly potent tumor promoters, also inhibit EGF binding to tissue culture cells. To explain the EGF binding inhibition by such greatly dissimilar molecules as phorbol esters, saccharin, and cyclamate, it is suggested that they operate through the activation of a hormone response control unit.

Attachment defect in mouse fibroblasts (L cells) persistently infected with Chlamydia psittaci.

PubMed Central

Moulder, J W; Levy, N J; Zeichner, S L; Lee, C K

1981-01-01

Almost all the cells in populations of mouse fibroblasts (L cells) persistently infected with the 6BC strain of Chlamydia psittaci were immune to superinfection with high multiplicities of C. psittaci, whether or not the L cells contained visible chlamydial inclusions. As ascertained by experiments with 14C-labeled C. psittaci, immunity to superinfection resulted from the failure of added chlamydiae to attach to persistently infected host cells. However, when exogenous C. psittaci was introduced into persistently infected L cells by centrifuging the inoculum onto host cell monolayers or by pretreating the monolayers with diethylaminoethyl-dextran, these chlamydiae produced expected numbers of infectious progeny. Persistently infected L cells were associated in an unknown way with a C. psittaci population that entered the host cells only with the aid of centrifugation or pretreatment with diethylaminoethyl-dextran. Inclusion-free, persistently infected L cells appeared to present at least two separate hindrances to chlamydial activity: blockage of the attachment of exogenous elementary bodies to persistently infected host cells and prevention of the initiation of chlamydial multiplication by means of a normal developmental cycle in the absence of added C. psittaci. Images PMID:7298188

Comparative Study of the Biological Activity of Allantoin and Aqueous Extract of the Comfrey Root.

PubMed

Savić, Vesna Lj; Nikolić, Vesna D; Arsić, Ivana A; Stanojević, Ljiljana P; Najman, Stevo J; Stojanović, Sanja; Mladenović-Ranisavljević, Ivana I

2015-08-01

This study investigates the biological activity of pure allantoin (PA) and aqueous extract of the comfrey (Symphytum officinale L.) root (AECR) standardized to the allantoin content. Cell viability and proliferation of epithelial (MDCK) and fibroblastic (L929) cell line were studied by using MTT test. Anti-irritant potential was determined by measuring electrical capacitance, erythema index (EI) and transepidermal water loss of artificially irritated skin of young healthy volunteers, 3 and 7 days after application of creams and gels with PA or AECR. Pure allantoin showed mild inhibitory effect on proliferation of both cell lines at concentrations 40 and 100 µg/ml, but more pronounced on MDCK cells. Aqueous extract of the comfrey root effect on cell proliferation in concentrations higher than 40 µg/ml was significantly stimulatory for L929 but inhibitory for MDCK cells. Pharmaceutical preparations that contained AECR showed better anti-irritant potential compared with PA. Creams showed better effect on hydration and EI compared with the gels that contained the same components. Our results indicate that the biological activity of the comfrey root extract cannot be attributed only to allantoin but is also likely the result of the interaction of different compounds present in AECR. Topical preparations that contain comfrey extract may have a great application in the treatment of skin irritation. Copyright © 2015 John Wiley & Sons, Ltd.

Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro.

PubMed

Cong, Shan; Cao, Guifang; Liu, Dongjun

2014-12-01

To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1-5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic fibroblast feeder layers of mouse and bovine embryonic fibroblast at different ratios and sources including mouse fibroblast cell lines. The bovine embryonic stem cell-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as OCT4, stage-specific embryonic antigen 1 and 4, alkaline phosphatase, SOX2, and NANOG mRNA levels. When feeder layers and cell growth factors were removed, the bovine embryonic stem cell-like cells formed embryoid bodies in a suspension culture. Furthermore, we compared the expression of the pluripotent markers during bovine embryonic stem cell-like cell in culture on mixed embryonic fibroblast feeder layers, including mouse fibroblast cell lines feeder layers and mouse embryonic fibroblast feeder layers by real-time quantitative polymerase chain reaction. Results suggested that mixed embryonic fibroblast and sources including mouse fibroblast cell lines feeder layers were more suitable for long-term culture and growth of bovine embryonic stem cell-like cells than mouse embryonic fibroblast feeder layers. The findings may provide useful experimental data for the establishment of an appropriate culture system for bovine embryonic stem cell lines.

Needle-free nasal delivery of glucagon for treatment of diabetes-related severe hypoglycemia: toxicology of polypropylene resin used in delivery device.

PubMed

Reno, Frederick E; Edwards, C Nicholas; Bendix Jensen, Morten; Török-Bathó, Magdolna; Esdaile, David J; Piché, Claude; Triest, Myriam; Carballo, Dolorès

2016-09-01

The intranasal route is a promising route of administration for several emergency rescue drugs including naloxone and glucagon. Glucagon nasal powder (GNP) is a novel, needle-free delivery system for intranasal administration of glucagon for the treatment of severe hypoglycemia, an infrequent but serious complication of insulin use in patients with diabetes. The GNP delivery device is a compact, highly portable, single-use nasal powder dosing device constructed of polypropylene that allows for simple, single-step administration. To evaluate the toxicological profile of the polypropylene resin used in the actuator part of the delivery device that will contact skin and nasal mucosal membranes of the patient, we performed an in vitro cytotoxicity study, a skin sensitization study and an irritation (intracutaneous reactivity) study in animal models. Extracts of the actuator of the GNP device were generated from HAM F12 medium with 10% fetal bovine serum, 0.9% sodium chloride (NaCl) or sesame oil. The in vitro cytotoxicity test was performed in cultured L929 mouse fibroblasts. Skin sensitization analysis was performed in 10 guinea pigs according to the Magnusson-Kligman method, using a maximization method with Freund's Complete Adjuvant. Irritation following intracutaneous/intradermal treatment with device extracts (NaCl and sesame oil extractants) was assessed in three New Zealand White rabbits. In vitro cytotoxicity test: Both undiluted and diluted extract showed no toxicity (i.e. no abnormal morphology, cell death or cell lysis) toward L929 fibroblasts (cytotoxicity grade 0). Sensitization test in guinea pigs: Challenge with device extracts did not evoke positive responses in test animals previously induced with device extracts. The net response value represented an incidence rate of 0% and a net dermal irritation score value of 0.00. Irritation (intracutaneous/intradermal) test in New Zealand White rabbits: Device extracts and corresponding vehicle controls caused similar irritation reactions. The difference between the mean scores for the device extracts and the corresponding vehicle controls was less than 1.0. Extracts of the polypropylene resin of the GNP delivery device are not cytotoxic, do not result in dermal sensitization and do not cause irritation when applied topically or intracutaneously. Given the infrequent use and very short duration of exposure to the nasal mucosa during administration of GNP, the polypropylene resin of the GNP device actuator will likely not cause adverse dermal sensitization effects or irritation effects in humans and can, therefore, be considered for use as a delivery device in clinical trials assessing the efficacy and safety of GNP for the treatment of insulin-using patients experiencing episodes of severe hypoglycemia.

Biologically active and biomimetic dual gelatin scaffolds for tissue engineering.

PubMed

Sánchez, P; Pedraz, J L; Orive, G

2017-05-01

We have designed, developed and optimized Genipin cross-linked 3D gelatin scaffolds that were biologically active and biomimetic, show a dual activity both for growth factor and cell delivery. Type B gelatin powder was dissolved in DI water. 100mg of genipin was dissolved in 10ml of DI water. Three genipin concentrations were prepared: 0.1%, 0.2% and 0.3% (w/v). Solutions were mixed at 40°C and under stirring and then left crosslinking for 72h. Scaffolds were obtained by punching 8 mm-cylinders into ethanol 70% solution for 10min and then freeze-drying. Scaffolds were biologically, biomechanically and morphologically evaluated. Cell adhesion and morphology of D1-Mesenchymal stem cells (MSCs) and L-929 fibroblast was studied. Vascular endothelial grwoth factor (VEGF) and Sonic hedgehog (SHH) were used as model proteins. Swelling ratio increased and younǵs module decreased along with the concentration of genipin. All scaffolds were biocompatible according to the toxicity test. MSC and L-929 cell adhesion improved in 0.2% of genipin, obtaining better results with MSCs. VEGF and SHH were released from the gels. This preliminary study suggest that the biologically active and dual gelatin scaffolds may be used for tissue engineering approaches like bone regeneration. Copyright © 2017 Elsevier B.V. All rights reserved.

pH and Glucose Dual-Responsive Injectable Hydrogels with Insulin and Fibroblasts as Bioactive Dressings for Diabetic Wound Healing.

PubMed

Zhao, Lingling; Niu, Lijing; Liang, Hongze; Tan, Hui; Liu, Chaozong; Zhu, Feiyan

2017-11-01

pH and glucose dual-responsive injectable hydrogels were prepared through the cross-linking of Schiff's base and phenylboronate ester using phenylboronic-modified chitosan, poly(vinyl alcohol) and benzaldehyde-capped poly(ethylene glycol). Protein drugs and live cells could be incorporated into the hydrogels during the in situ cross-linking, displaying sustained and pH/glucose-triggered drug release from the hydrogels and cell viability and proliferation in the three-dimensional hydrogel matrix as well. Hence, the hydrogels with insulin and fibroblasts were considered as bioactive dressings for diabetic wound healing. A streptozotocin-induced diabetic rat model was used to evaluate the efficacy of hydrogel dressings in wound repair. The results revealed that the incorporation of insulin and L929 in the hydrogels could promote neovascularization and collagen deposition and enhance the wound-healing process of diabetic wounds. Thus, the drug- and cell-loaded hydrogels have promising potential in wound healing as a medicated system for various therapeutic proteins and live cells.

Caprylic and Polygalacturonic Acid Combinations for Eradication of Microbial Organisms Embedded in Biofilm

PubMed Central

Rosenblatt, Joel; Reitzel, Ruth A.; Vargas-Cruz, Nylev; Chaftari, Anne-Marie; Hachem, Ray; Raad, Issam

2017-01-01

There is a need for non-antibiotic, antimicrobial compositions with low toxicity capable of broad-spectrum eradication of pathogenic biofilms in food preparation and healthcare settings. In this study we demonstrated complete biofilm eradication within 60 min with synergistic combinations of caprylic and polygalacturonic (PG) acids in an in vitro biofilm eradication model against representative hospital and foodborne infectious pathogen biofilms (methicillin-resistant Staphylococcus aureus, multidrug-resistant Pseudomonas aeruginosa, Candida albicans, Escherichia coli, and Salmonella enteritidis). Antimicrobial synergy against biofilms was demonstrated by quantifying viable organisms remaining in biofilms exposed to caprylic acid alone, PG acid alone, or combinations of the two. The combinations also synergistically inhibited growth of planktonic organisms. Toxicity of the combination was assessed in vitro on L929 fibroblasts incubated with extracts of caprylic and PG acid combinations using the Alamar Blue metabolic activity assay and the Trypan Blue exclusion cell viability assay. The extracts did not produce cytotoxic responses relative to untreated control fibroblasts. PMID:29093703

Evaluation of chitosan quaternary ammonium salt-modified resin denture base material.

PubMed

Song, Rong; Zhong, Zhaohua; Lin, Lexun

2016-04-01

Chitosan quaternary ammonium salt displays good antioxidant and antibacterial characteristics and it shows appreciable solubility in water. When added to the traditional denture material to form a resin base, it could promote good oral health by improving the oral environment. In this study, chitosan quaternary ammonium salt was added to the denture material following two different methods. After three months of immersion in artificial saliva, the specimens were tested for tensile strength and were scanned by electron microscope. The murine fibroblast cytotoxicity and antibacterial properties were also tested. The result showed no significant differences in the tensile strength and in the proliferation of murine L929 fibroblast cells. The two structures of chitosan quaternary ammonium salt-modified denture material had different degrees of corrosion resistance and antimicrobial properties. These results indicate that chitosan quaternary ammonium salt-modified resin denture base material has the potential to become a new generation oral denture composite material. Copyright © 2015 Elsevier B.V. All rights reserved.

Controlled Release of Chitosan and Sericin from the Microspheres-Embedded Wound Dressing for the Prolonged Anti-microbial and Wound Healing Efficacy.

PubMed

Aramwit, Pornanong; Yamdech, Rungnapha; Ampawong, Sumate

2016-05-01

One approach in wound dressing development is to incorporate active molecules or drugs in the dressing. In order to reduce the frequency of dressing changes as well as to prolong wound healing efficacy, wound dressings that can sustain the release of the active molecules should be developed. In our previous work, we developed chitosan/sericin (CH/SS) microspheres that released sericin in a controlled rate. However, the difficulty of applying the microspheres that easily diffuse and quickly degrade onto the wound was its limitations. In this study, we aimed to develop wound dressing materials which are easier to apply and to provide extended release of sericin. Different amounts of CH/SS microspheres were embedded into various compositions of polyvinyl alcohol/gelatin (PVA/G) scaffolds and fabricated using freeze-drying and glutaraldehyde crosslinking techniques. The obtained CH/SS microspheres-embedded scaffolds with appropriate design and formulation were introduced as a wound dressing material. Sericin was released from the microspheres and the scaffolds in a sustained manner. Furthermore, an optimized formation of the microspheres-embedded scaffolds (2PVA2G+2CHSS) was shown to possess an effective antimicrobial activity against both gram-positive and gram-negative bacteria. These microspheres-embedded scaffolds were not toxic to L929 mouse fibroblast cells, and they did not irritate the tissue when applied to the wound. Finally, probably by the sustained release of sericin, these microspheres-embedded scaffolds could promote wound healing as well as or slightly better than a clinically used wound dressing (Allevyn®) in a mouse model. The antimicrobial CH/SS microspheres-embedded PVA/G scaffolds with sustained release of sericin would appear to be a promising candidate for wound dressing application.

The cellular responses and antibacterial activities of silver nanoparticles stabilized by different polymers

NASA Astrophysics Data System (ADS)

Lin, Jiang-Jen; Lin, Wen-Chun; Dong, Rui-Xuan; Hsu, Shan-hui

2012-02-01

Silver nanoparticles (AgNPs) are known for their excellent antibacterial activities. The possible toxicity, however, is a major concern for their applications. Three types of AgNPs were prepared in this study by chemical processes. Each was stabilized by a polymer surfactant, which was expected to reduce the exposure of cells to AgNPs and therefore their cytotoxicity. The polymer stabilizers included poly(oxyethylene)-segmented imide (POEM), poly(styrene-co-maleic anhydride)-grafting poly(oxyalkylene) (SMA) and poly(vinyl alcohol) (PVA). The cytotoxicity of these chemically produced AgNPs to mouse skin fibroblasts (L929), human hepatocarcinoma cells (HepG2), and mouse monocyte macrophages (J774A1) was compared to that of physically produced AgNPs and gold nanoparticles (AuNPs) as well as the standard reference material RM8011 AuNPs. Results showed that SMA-AgNPs were the least cytotoxic among all materials, but cytotoxicity was still observed at higher silver concentrations (>30 ppm). Macrophages demonstrated the inflammatory response with cell size increase and viability decrease upon exposure to 10 ppm of the chemically produced AgNPs. SMA-AgNPs did not induce hemolysis at a silver concentration below 1.5 ppm. Regarding the antibacterial activity, POEM-AgNPs and SMA-AgNPs at 1 ppm silver content showed 99.9% and 99.3% growth inhibition against E. coli, while PVA-AgNPs at the same silver concentration displayed 79.1% inhibition. Overall, SMA-AgNPs demonstrated better safety in vitro and greater antibacterial effects than POEM-AgNPs and PVA-AgNPs. This study suggested that polymer stabilizers may play an important role in determining the toxicity of AgNPs.

Syntheses, spectral characterization, X-ray studies and in vitro cytotoxic activities of triorganotin(IV) derivatives of p-substituted N-methylbenzylaminedithiocarbamates

NASA Astrophysics Data System (ADS)

Khan, Naqeebullah; Farina, Yang; Mun, Lo Kong; Rajab, Nor Fadilah; Awang, Normah

2014-11-01

Two new organotin(IV) complexes of the type R3SnL, where (L = p-bromo-N-methylbenzylaminedithiocarbamate and p-fluoro-N-methylbenzylaminedithiocarbamate, and R = phenyl) have been synthesized in 1:1 molar ratio with good yields and isolated as crystalline solids. The newly synthesized compounds gave fairly sharp melting points indicating that the compounds were pure. A systematic investigation of the derivatives were carried out both in solid and in solution and were suitably characterized by elemental analysis, FT-IR, 1H, 13C, 119Sn NMR spectroscopies. The dithiocarbamate ligands chelated to the tin metal monodentately using only one sulfur atom showing a pair of bands due to ν(Cdbnd S) below 1000 cm-1. This phenomenon was supported by the occurrence of new medium to weak absorptions in the region 411-545, in the spectra of complexes, assigned to ν(Snsbnd S) and ν(Snsbnd C). The crystal structures of the two triorganotin(IV) complexes have been determined by X-ray crystallography. Both the complexes crystallized in the monoclinic, P2(1)/n space group. The spectral investigations and single crystal X-ray diffraction data illustrate that the two dithiocarbamato ligands in the triphenyltin(IV) derivatives 1 and 2 are monodentate and the geometry at tin is best described as a distorted tetrahedron. The in vitro antiproliferative tests of these two derivatives on three human cell lines, leukemic lymphoblastoma Jurkat cells, lymphoblastoma K-562 cells, hepatoblastoma HepG2 cells and one mouse fibroblast cells L929 show dose-dependent decrease of cell proliferation in all cell lines.

Susceptible cytotoxicity to ultraviolet B light in fibroblasts and keratinocytes cultured from autoimmune-prone MRL/Mp-lpr/lpr mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Furukawa, F.; Lyon, M.B.; Norris, D.A.

1989-09-01

The MRL/Mp-lpr/lpr (MRL/l) mouse is an autoimmune model of spontaneous lupus erythematosus (LE), in addition to lupus nephritis. In order to better understand the mechanisms of photosensitivity in LE, in vitro photocytotoxicity was examined by using fibroblasts and keratinocytes cultured from MRL/l mice, control MRL/Mp- +/+ (MRL/n) mice, and normal BALB/c mice. A colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and the acridine orange/ethidium bromide assay were used for determination of cytotoxicity. Fibroblasts cultured from newborn MRL/l mice showed higher susceptibility to single ultraviolet light B (UVB) light irradiation at a dose of 100-500 mJ than those from MRL/n, F1 hybrid ofmore » (MRL/l x MRL/n mice), and BALB/c mice. However, the susceptibility to UVB was not observed in young (1-month-old) and adult (4-month-old) MRL/l mice. UVA light irradiation was not cytotoxic. Keratinocytes cultured from MRL mice showed lower cytotoxicity to UVB irradiation than fibroblasts cultured. However, keratinocytes from newborn MRL/l mice showed higher cytotoxicity to 50 mJ UVB irradiation than cells from MRL/n mice. Syngeneic or allogeneic sera augmented UVB-induced cytotoxicity of fibroblasts cultured. UVB irradiation of spleen cells induced no significant difference of cytotoxicity between MRL/l and MRL/n mice. Based on the results of F1 hybrid of (MRL/l x MRL/n) mice, the susceptibility seemed to be associated with autoimmune traits and to be regulated by genetical background.« less

Surface Modification of Biodegradable Polymers towards Better Biocompatibility and Lower Thrombogenicity

PubMed Central

Rudolph, Andreas; Teske, Michael; Illner, Sabine; Kiefel, Volker; Sternberg, Katrin; Grabow, Niels; Wree, Andreas; Hovakimyan, Marina

2015-01-01

Purpose Drug-eluting stents (DES) based on permanent polymeric coating matrices have been introduced to overcome the in stent restenosis associated with bare metal stents (BMS). A further step was the development of DES with biodegradable polymeric coatings to address the risk of thrombosis associated with first-generation DES. In this study we evaluate the biocompatibility of biodegradable polymer materials for their potential use as coating matrices for DES or as materials for fully bioabsorbable vascular stents. Materials and Methods Five different polymers, poly(L-lactide) PLLA, poly(D,L-lactide) PDLLA, poly(L-lactide-co-glycolide) P(LLA-co-GA), poly(D,L-lactide-co-glycolide) P(DLLA-co-GA) and poly(L-lactide-co-ε-caprolactone), P(LLA-co-CL) were examined in vitro without and with surface modification. The surface modification of polymers was performed by means of wet-chemical (NaOH and ethylenediamine (EDA)) and plasma-chemical (O2 and NH3) processes. The biocompatibility studies were performed on three different cell types: immortalized mouse fibroblasts (cell line L929), human coronary artery endothelial cells (HCAEC) and human umbilical vein endothelial cells (HUVEC). The biocompatibility was examined quantitatively using in vitro cytotoxicity assay. Cells were investigated immunocytochemically for expression of specific markers, and morphology was visualized using confocal laser scanning (CLSM) and scanning electron (SEM) microscopy. Additionally, polymer surfaces were examined for their thrombogenicity using an established hemocompatibility test. Results Both endothelial cell types exhibited poor viability and adhesion on all five unmodified polymer surfaces. The biocompatibility of the polymers could be influenced positively by surface modifications. In particular, a reproducible effect was observed for NH3-plasma treatment, which enhanced the cell viability, adhesion and morphology on all five polymeric surfaces. Conclusion Surface modification of polymers can provide a useful approach to enhance their biocompatibility. For clinical application, attempts should be made to stabilize the plasma modification and use it for coupling of biomolecules to accelerate the re-endothelialization of stent surfaces in vivo. PMID:26641662

Vitamin E-loaded silk fibroin nanofibrous mats fabricated by green process for skin care application.

PubMed

Sheng, Xiaoyue; Fan, Linpeng; He, Chuanglong; Zhang, Kuihua; Mo, Xiumei; Wang, Hongsheng

2013-05-01

In the present study, we reported fabrication and skin benefit of a novel vitamin E (VE)-loaded silk fibroin (SF) nanofibrous mats. RRR-α-Tocopherol polyethylene glycol 1000 succinate (VE TPGS), a water-soluble derivative of VE, was incorporated into SF nanofiber successfully by aqua solution electrospinning for the first time. Morphology of the composite nanofibers changed with the different amount of VE TPGS: a ribbon-like shape for lower loading dose of VE TPGS, while a round shape for higher loading dose (more than 4% (wt/wt) based on the weight of SF). After treated with 75% (v/v) ethanol vapor, the composite nanofibrous mats showed an excellent water-resistant ability. In vitro study disclosed a sustained release behavior of VE TPGS disassociated from the nanofibrous mats. The mouse skin fibroblasts (L929 cells) cultured on the VE-loaded SF nanofibrous mats spread and proliferated much better than on cover slips. Moreover, the incorporation of VE TPGS was found strengthening the ability of SF nanofibrous mats on protecting the cells against oxidation stress induced by tert-butyl hydroperoxide. Our data presented impressive skin benefits of this VE-loaded SF nanofibrous mats, suggesting a promising applicative potential of this novel product on personal skin care, tissue regeneration and other related area. Copyright © 2013 Elsevier B.V. All rights reserved.

Development of core-shell coaxially electrospun composite PCL/chitosan scaffolds.

PubMed

Surucu, Seda; Turkoglu Sasmazel, Hilal

2016-11-01

This study was related to combining of synthetic Poly (ε-caprolactone) (PCL) and natural chitosan polymers to develop three dimensional (3D) PCL/chitosan core-shell scaffolds for tissue engineering applications. The scaffolds were fabricated with coaxial electrospinning technique and the characterizations of the samples were done by thickness and contact angle (CA) measurements, scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-Ray Photoelectron Spectroscopy (XPS) analyses, mechanical and PBS absorption and shrinkage tests. The average inter-fiber diameter values were calculated for PCL (0.717±0.001μm), chitosan (0.660±0.007μm) and PCL/chitosan core-shell scaffolds (0.412±0.003μm), also the average inter-fiber pore size values exhibited decreases of 66.91% and 61.90% for the PCL and chitosan scaffolds respectively, compared to PCL/chitosan core-shell ones. XPS analysis of the PCL/chitosan core-shell structures exhibited the characteristic peaks of PCL and chitosan polymers. The cell culture studies (MTT assay, Confocal Laser Scanning Microscope (CLSM) and SEM analyses) carried out with L929 ATCC CCL-1 mouse fibroblast cell line proved that the biocompatibility performance of the scaffolds. The obtained results showed that the created micro/nano fibrous structure of the PCL/chitosan core-shell scaffolds in this study increased the cell viability and proliferation on/within scaffolds. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of the incorporation of chitosan on the physico-chemical, mechanical properties and biological activity on a mixture of polycaprolactone and polyurethanes obtained from castor oil.

PubMed

Arévalo, Fabian; Uscategui, Yomaira L; Diaz, Luis; Cobo, Martha; Valero, Manuel F

2016-11-01

In the present study, polyurethane materials were obtained from castor oil, polycaprolactone and isophorone diisocyanate by incorporating different concentrations of chitosan (0.5, 1.0 and 2.0% w/w) as an additive to improve the mechanical properties and the biological activity of polyurethanes. The polyurethanes were characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis, scanning electron microscopy, stress/strain fracture tests and swelling analysis, and the hydrophilic character of the surface was determined by contact angle trials. The objectives of the study were to evaluate the effect of the incorporation of chitosan on the changes of the physico-chemical and mechanical properties and the in vitro biological activity of the polyurethanes. It was found that the incorporation of chitosan enhances the ultimate tensile strength of the polyurethanes and does not affect the strain at fracture in polyurethanes with 5% w/w of polycaprolactone and concentrations of chitosan ranging from 0 to 2% w/w. In addition, PCL5-Q-PU formulations and their degradation products did not affect cell viability of L929 mouse fibroblast and 3T3, respectively. Polyurethane formulations showed antibacterial activities against Staphylococcus aureus and Escherichia coli bacteria. The results of this study have highlighted the potential biomedical application of this polyurethanes related to soft and cardiovascular tissues. © The Author(s) 2016.

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture.

PubMed

Li, Yongsan; Zhang, Yaling; Wei, Yen; Tao, Lei

2017-09-29

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is prepared by mixing solutions of glycol chitosan with a synthesized benzaldehyde terminated polymer gelator, and hydrogels are efficiently obtained in several minutes at room temperature. By varying ratios between glycol chitosan, polymer gelator, and water contents, versatile hydrogels with different gelation times and stiffness are obtained. When damaged, the hydrogel can recover its appearances and modulus, due to the reversibility of the dynamic imine bonds as crosslinkages. This self-healable property enables the hydrogel to be injectable since it can be self-healed from squeezed pieces to an integral bulk hydrogel after the injection process. The hydrogel is also multi-responsive to many bio-active stimuli due to different equilibration statuses of the dynamic imine bonds. This hydrogel was confirmed as bio-compatible, and L929 mouse fibroblast cells were embedded following standard procedures and the cell proliferation was easily assessed by a 3D cell cultivation process. The hydrogel can offer an adjustable platform for different research where a physiological mimic of a 3D environment for cells is profited. Along with its multi-responsive, self-healable, and injectable properties, the hydrogels can potentially be applied as multiple carriers for drugs and cells in future bio-medical applications.

3D printable conducting hydrogels containing chemically converted graphene.

PubMed

Sayyar, Sepidar; Gambhir, Sanjeev; Chung, Johnson; Officer, David L; Wallace, Gordon G

2017-02-02

The development of conducting 3D structured biocompatible scaffolds for the growth of electroresponsive cells is critical in the field of tissue engineering. This work reports the synthesis and 3D processing of UV-crosslinkable conducting cytocompatible hydrogels that are prepared from methacrylated chitosan (ChiMA) containing graphenic nanosheets. The addition of chemically converted graphene resulted in mechanical and electrical properties of the composite that were significantly better than ChiMA itself, as well as improved adhesion, proliferation and spreading of L929 fibroblasts cells. The chemically converted graphene/ChiMA hydrogels were amenable to 3D printing and this was used to produce multilayer scaffolds with enhanced mechanical properties through UV-crosslinking.

Mind "De GaPP": in vitro efficacy of deferiprone and gallium-protoporphyrin against Staphylococcus aureus biofilms.

PubMed

Richter, Katharina; Ramezanpour, Mahnaz; Thomas, Nicky; Prestidge, Clive A; Wormald, Peter-John; Vreugde, Sarah

2016-07-01

Biofilms are clusters of bacteria embedded in a protective matrix that frequently cause failure of medical treatments and increase the risk of recurrent infections. In particular, Staphylococcus aureus biofilms are associated with a series of chronic and nosocomial infections that are increasingly resistant to antibiotics. This study proposes a novel intervention strategy targeting the essential iron metabolism for bacterial growth, survival and pathogenesis using the compounds deferiprone (Def) and gallium-protoporphyrin (GaPP). S. aureus biofilms were challenged with Def/GaPP as single and dual treatments. In vitro anti-biofilm efficacy was assessed by the AlamarBlue viability assay and confocal microscopy. In vitro cytotoxicity of the treatments was examined by the lactate dehydrogenase assay on mouse fibroblast (L929) and human bronchial epithelial cells (Nuli-1). Def (20 mM) and GaPP (200 μg/mL) monotherapy for 2 hours showed 35% and 74% biofilm removal, respectively, whereas simultaneous Def/GaPP administration showed 55% biofilm removal. In contrast, the consecutive treatment (2 hours Def followed by 2 hours GaPP) achieved 95% biofilm removal. Cytotoxicity studies indicated no cell hazard in all treatments. This study demonstrated the in vitro efficacy of a novel treatment combination against S. aureus biofilms targeting the bacterial iron metabolism. The consecutive Def/GaPP treatment showed significantly enhanced biofilm efficacy than the individual compounds, while being not toxic to 2 cell lines. This novel treatment combination is a promising approach to combat S. aureus-associated biofilm infections having high potential for future clinical application. © 2016 ARS-AAOA, LLC.

Wound Healing Activity of Extracts and Formulations of Aloe vera, Henna, Adiantum capillus-veneris, and Myrrh on Mouse Dermal Fibroblast Cells.

PubMed

Negahdari, Samira; Galehdari, Hamid; Kesmati, Mahnaz; Rezaie, Anahita; Shariati, Gholamreza

2017-01-01

Among the most important factors in wound healing pathways are transforming growth factor beta1 and vascular endothelial growth factor. Fibroblasts are the main cell in all phases wound closure. In this study, the extracts of plant materials such as Adiantum capillus-veneris , Commiphora molmol , Aloe vera , and henna and one mixture of them were used to treatment of normal mouse skin fibroblasts. Cytotoxic effects of each extract and their mixture were assessed on mouse skin fibroblasts cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. We performed migration assays to assess migration properties of mouse skin fibroblasts cells in response to the extracts. Changes in the gene expression of the Tgf β1 and Vegf-A genes were monitored by real-time polymerase chain reaction. A. capillus-veneris , C. molmol and henna extract improved the expression of Tgfβ1 gene. All used extracts upregulated the expression of Vegf-A gene and promoted the migration of mouse fibroblast cells in vitro . The present study demonstrated that the mentioned herbal extracts might be effective in wound healing, through the improvement in the migration of fibroblast cells and regulating the gene expression of Tgfβ1 and Vegf-A genes in fibroblast cells treated with extracts.

Centrifugation assay for measuring adhesion of serially passaged bovine chondrocytes to polystyrene surfaces.

PubMed

Kaplan, David S; Hitchins, Victoria M; Vegella, Thomas J; Malinauskas, Richard A; Ferlin, Kimberly M; Fisher, John P; Frondoza, Carmelita G

2012-07-01

A major obstacle in chondrocyte-based therapy for cartilage repair is the limited availability of cells that maintain their original phenotype. Propagation of chondrocytes as monolayer cultures on polystyrene surfaces is used extensively for amplifying cell numbers. However, chondrocytes undergo a phenotypic shift when propagated in this manner and display characteristics of more adherent fibroblastic cells. Little information is available about the effect of this phenotypic shift on cellular adhesion properties. We evaluated changes in adhesion property as bovine chondrocytes were serially propagated up to five passages in monolayer culture using a centrifugation cell adhesion assay, which was based on counting of cells before and after being exposed to centrifugal dislodgement forces of 120 and 350 g. Chondrocytes proliferated well in a monolayer culture with doubling times of 2-3 days, but they appeared more fibroblastic and exhibited elongated cell morphology with continued passage. The centrifugation cell adhesion assay showed that chondrocytes became more adhesive with passage as the percentage of adherent cells after centrifugation increased and was not statistically different from the adhesion of the fibroblast cell line, L929, starting at passage 3. This increased adhesiveness correlated with a shift to a fibroblastic morphology and increased collagen I mRNA expression starting at passage 2. Our findings indicate that the centrifugation cell adhesion assay may serve as a reproducible tool to track alterations in chondrocyte phenotype during their extended propagation in culture.

Preclinical study of mouse pluripotent parthenogenetic embryonic stem cell derivatives for the construction of tissue-engineered skin equivalent.

PubMed

Rao, Yang; Cui, Jihong; Yin, Lu; Liu, Wei; Liu, Wenguang; Sun, Mei; Yan, Xingrong; Wang, Ling; Chen, Fulin

2016-10-22

Embryonic stem cell (ESC) derivatives hold great promise for the construction of tissue-engineered skin equivalents (TESE). However, harvesting of ESCs destroys viable embryos and may lead to political and ethical concerns over their application. In the current study, we directed mouse parthenogenetic embryonic stem cells (pESCs) to differentiate into fibroblasts, constructed TESE, and evaluated its function in vivo. The stemness marker expression and the pluripotent differentiation ability of pESCs were tested. After embryoid body (EB) formation and adherence culture, mesenchymal stem cells (MSCs) were enriched and directed to differentiate into fibroblastic lineage. Characteristics of derived fibroblasts were assessed by quantitative real-time PCR and ELISA. Functional ability of the constructed TESE was tested by a mouse skin defects repair model. Mouse pESCs expressed stemness marker and could form teratoma containing three germ layers. MSCs could be enriched from outgrowths of EBs and directed to differentiate into fibroblastic lineage. These cells express a high level of growth factors including FGF, EGF, VEGF, TGF, PDGF, and IGF1, similar to those of ESC-derived fibroblasts and mouse fibroblasts. Seeded into collagen gels, the fibroblasts derived from pESCs could form TESE. Mouse skin defects could be successfully repaired 15 days after transplantation of TESE constructed by fibroblasts derived from pESCs. pESCs could be induced to differentiate into fibroblastic lineage, which could be applied to the construction of TESE and skin defect repair. Particularly, pESC derivatives avoid the limitations of political and ethical concerns, and provide a promising source for regenerative medicine.

Collagen gel protects L929 cells from TNFα-induced death by activating NF-κB.

PubMed

Wang, Hong-Ju; Li, Meng-Qi; Liu, Wei-Wei; Hayashi, Toshihiko; Fujisaki, Hitomi; Hattori, Shunji; Tashiro, Shin-Ichi; Onodera, Satoshi; Ikejima, Takashi

2017-09-01

Type I collagen is one of the most abundant components of extracellular matrix. We previously illustrated that murine fibrosarcoma L929 cells grew well on type I collagen gel and escaped from TNFα-induced cell death. In this study, we investigated the mechanism underlying the protective effect of collagen gel. We used western blot, confocal microscopy, MTT assay and flow cytometry by introducing fluorescence staining to determine the expression levels of nuclear factor kappa B (NF-κB), inhibitory ratio and autophagy. L929 cells on collagen gel showed higher expression of NF-κB in the nucleus. Inhibition of NF-κB with pyrrolidine dithiocarbamate hydrochloride (PDTC) or knockdown by NF-κB-siRNA canceled the protective effect of collagen gel on L929 cells from TNFα-induced death, suggesting for the role of NF-κB in the protection from cell death. We found a new aspect of the effect of PDTC on L929 cells cultured on collagen gel. PDTC alone without TNFα induced apoptosis in the L929 cells cultured on collagen gel but not the cells on plastic dish. The apoptosis induction of the L929 cells cultured on collagen gel with PDTC was repressed by inhibiting autophagy with chloroquine, an autophagy inhibitor, suggesting that autophagy contributes to the death induced by the treatment with PDTC. Possible underlying mechanism of this finding is discussed. NF-κB played an important role in protecting the L929 cells cultured on collagen gel from TNFα-induced death.

Effect of reduced exposure times on the cytotoxicity of resin luting cements cured by high-power led

PubMed Central

ERGUN, Gulfem; EGILMEZ, Ferhan; YILMAZ, Sukran

2011-01-01

Objective Applications of resin luting agents and high-power light-emitting diodes (LED) light-curing units (LCUs) have increased considerably over the last few years. However, it is not clear whether the effect of reduced exposure time on cytotoxicity of such products have adequate biocompatibility to meet clinical success. This study aimed at assessing the effect of reduced curing time of five resin luting cements (RLCs) polymerized by high-power LED curing unit on the viability of a cell of L-929 fibroblast cells. Material and Methods Disc-shaped samples were prepared in polytetrafluoroethylene moulds with cylindrical cavities. The samples were irradiated from the top through the ceramic discs and acetate strips using LED LCU for 20 s (50% of the manufacturer's recommended exposure time) and 40 s (100% exposure time). After curing, the samples were transferred into a culture medium for 24 h. The eluates were obtained and pipetted onto L-929 fibroblast cultures (3x104 per well) and incubated for evaluating after 24 h. Measurements were performed by dimethylthiazol diphenyltetrazolium assay. Statistical significance was determined by two-way ANOVA and two independent samples were compared by t-test. Results Results showed that eluates of most of the materials polymerized for 20 s (except Rely X Unicem and Illusion) reduced to a higher extent cell viability compared to samples of the same materials polymerized for 40 s. Illusion exhibited the least cytotoxicity for 20 s exposure time compared to the control (culture without samples) followed by Rely X Unicem and Rely X ARC (90.81%, 88.90%, and 83.11%, respectively). For Rely X ARC, Duolink and Lute-It 40 s exposure time was better (t=-1.262 p=0,276; t=-9.399 p=0.001; and t=-20.418 p<0.001, respectively). Conclusion The results of this study suggest that reduction of curing time significantly enhances the cytotoxicity of the studied resin cement materials, therefore compromising their clinical performance. PMID:21625748

Ankyrin-B is a PI3P effector that promotes polarized α5β1-integrin recycling via recruiting RabGAP1L to early endosomes

PubMed Central

Qu, Fangfei; Lorenzo, Damaris N; King, Samantha J; Brooks, Rebecca; Bear, James E; Bennett, Vann

2016-01-01

Endosomal membrane trafficking requires coordination between phosphoinositide lipids, Rab GTPases, and microtubule-based motors to dynamically determine endosome identity and promote long-range organelle transport. Here we report that ankyrin-B (AnkB), through integrating all three systems, functions as a critical node in the protein circuitry underlying polarized recycling of α5β1-integrin in mouse embryonic fibroblasts, which enables persistent fibroblast migration along fibronectin gradients. AnkB associates with phosphatidylinositol 3-phosphate (PI3P)-positive organelles in fibroblasts and binds dynactin to promote their long-range motility. We demonstrate that AnkB binds to Rab GTPase Activating Protein 1-Like (RabGAP1L) and recruits it to PI3P-positive organelles, where RabGAP1L inactivates Rab22A, and promotes polarized trafficking to the leading edge of migrating fibroblasts. We further determine that α5β1-integrin depends on an AnkB/RabGAP1L complex for polarized recycling. Our results reveal AnkB as an unexpected key element in coordinating polarized transport of α5β1-integrin and likely of other specialized endocytic cargos. DOI: http://dx.doi.org/10.7554/eLife.20417.001 PMID:27718357

In Vitro Corrosion and Cytocompatibility of ZK60 Magnesium Alloy Coated with Hydroxyapatite by a Simple Chemical Conversion Process for Orthopedic Applications

PubMed Central

Wang, Bing; Huang, Ping; Ou, Caiwen; Li, Kaikai; Yan, Biao; Lu, Wei

2013-01-01

Magnesium and its alloys—a new class of degradable metallic biomaterials—are being increasingly investigated as a promising alternative for medical implant and device applications due to their advantageous mechanical and biological properties. However, the high corrosion rate in physiological environments prevents the clinical application of Mg-based materials. Therefore, the objective of this study was to develop a hydroxyapatite (HA) coating on ZK60 magnesium alloy substrates to mediate the rapid degradation of Mg while improving its cytocompatibility for orthopedic applications. A simple chemical conversion process was applied to prepare HA coating on ZK60 magnesium alloy. Surface morphology, elemental compositions, and crystal structures were characterized using scanning electron microscopy, energy dispersive spectroscopy, and X-ray diffraction, respectively. The corrosion properties of samples were investigated by immersion test and electrochemical test. Murine fibroblast L-929 cells were harvested and cultured with coated and non-coated ZK60 samples to determine cytocompatibility. The degradation results suggested that the HA coatings decreased the degradation of ZK60 alloy. No significant deterioration in compression strength was observed for all the uncoated and coated samples after 2 and 4 weeks’ immersion in simulated body fluid (SBF). Cytotoxicity test indicated that the coatings, especially HA coating, improved cytocompatibility of ZK60 alloy for L929 cells. PMID:24300096

In vitro evaluation of wound healing and antimicrobial potential of ozone therapy.

PubMed

Borges, Gabriel Álvares; Elias, Silvia Taveira; da Silva, Sandra Márcia Mazutti; Magalhães, Pérola Oliveira; Macedo, Sergio Bruzadelli; Ribeiro, Ana Paula Dias; Guerra, Eliete Neves Silva

2017-03-01

Although ozone therapy is extensively applied when wound repair and antimicrobial effect are necessary, little is known about cellular mechanisms regarding this process. Thus, this study aimed to evaluate ozone cytotoxicity in fibroblasts (L929) and keratinocytes (HaCaT) cell lines, its effects on cell migration and its antimicrobial activity. Cells were treated with ozonated phosphate-buffered saline (8, 4, 2, 1, 0.5 and 0.25 μg/mL ozone), chlorhexidine 0.2% or buffered-solution, and cell viability was determined through MTT assay. The effect of ozone on cell migration was evaluated through scratch wound healing and transwell migration assays. The minimum inhibitory concentrations for Candida albicans and Staphylococcus aureus were determined. Ozone showed no cytotoxicity for the cell lines, while chlorhexidine markedly reduced cell viability. Although no significant difference between control and ozone-treated cells was observed in the scratch assay, a considerable increase in fibroblasts migration was noticed on cells treated with 8 μg/mL ozonated solution. Ozone alone did not inhibit growth of microorganisms; however, its association with chlorhexidine resulted in antimicrobial activity. This study confirms the wound healing and antimicrobial potential of ozone therapy and presents the need for studies to elucidate the molecular mechanisms through which it exerts such biological effects. Copyright © 2017 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

Oral administration of Lactobacillus plantarum HY7714 protects hairless mouse against ultraviolet B-induced photoaging.

PubMed

Kim, Hyun Mee; Lee, Dong Eun; Park, Soo Dong; Kim, Yong-Tae; Kim, Yu Jin; Jeong, Ji Woong; Jang, Sung Sik; Ahn, Young-Tae; Sim, Jae-Hun; Huh, Chul-Sung; Chung, Dae Kyun; Lee, Jung-Hee

2014-11-28

Ultraviolet (UV) irradiation alters multiple molecular pathways in the skin, thereby inducing skin damage, including photoaging. In recent years, probiotics have gained interest due to their beneficial effects on skin health, such as inhibiting atopic dermatitis and improving skin immunity or inflammation. However, little is known about the effects of probiotics on UVBinduced photoaging. In this study, we evaluated the effect of Lactobacillus plantarum HY7714 against UVB-induced photoaging in human dermal fibroblasts and hairless mice. The results showed that L. plantarum HY7714 treatment effectively rescued UVB-reduced procollagen expression through the inhibition of UVB-induced matrix metalloproteinase (MMP)-1 expression in human dermal fibroblasts. Data from a western blot showed that L. plantarum HY7714 inhibited the phosphorylation of Jun N-terminal kinase, thereby suppressing the UVB-induced phosphorylation and expression of c-Jun. Oral administration of L. plantarum HY7714 clearly inhibited the number, depth, and area of wrinkles in hairless mouse skin. Histological data showed that L. plantarum HY7714 significantly inhibited UVB-induced epidermal thickness in mice. Western blot and zymography data also revealed that L. plantarum HY7714 effectively inhibited MMP-13 expression as well as MMP-2 and -9 activities in dermal tissue. Collectively, these results provide further insight regarding the skin biological actions of L. plantarum HY7714, a potential skin anti-photoaging agent.

Isolation and Differentiation of Murine Macrophages.

PubMed

Rios, Francisco J; Touyz, Rhian M; Montezano, Augusto C

2017-01-01

Macrophages play a major role in inflammation, wound healing, and tissue repair. Infiltrated monocytes differentiate into different macrophage subtypes with protective or pathogenic activities in vascular lesions. In the heart and vascular tissues, pathological activation promotes cardiovascular inflammation and remodeling and there is increasing evidence that macrophages play important mechanisms in this environment. Primary murine macrophages can be obtained from: bone marrow by different treatments (granulocyte-macrophage colony-stimulating factor-GM-CSF, macrophage colony-stimulating factor-M-CSF or supernatant of murine fibroblast L929), peritoneal cavity (resident or thioglycolate elicit macrophages), from the lung (alveolar macrophages) or from adipose tissue. In this chapter we describe some protocols to obtain primary murine macrophages and how to identify a pure macrophage population or activation phenotypes using different markers.

Synthesis and characterization of biodegradable poly (ethylene glycol) and poly (caprolactone diol) end capped poly (propylene fumarate) cross linked amphiphilic hydrogel as tissue engineering scaffold material.

PubMed

Krishna, Lekshmi; Jayabalan, Muthu

2009-12-01

Biodegradable poly (caprolactone diol-co-propylene fumarate-co-ethylene glycol) amphiphilic polymer with poly (ethylene glycol) and poly (caprolactone diol) chain ends (PCL-PPF-PEG) was prepared. PCL-PPF-PEG undergoes fast setting with acrylamide (aqueous solution) by free radical polymerization and produces a crosslinked hydrogel. The cross linked and freeze-dried amphiphilic material has porous and interconnected network. It undergoes higher degree of swelling and water absorption to form hydrogel with hydrophilic and hydrophobic domains at the surface and appreciable tensile strength. The present hydrogel is compatible with L929 fibroblast cells. PCL-PPF-PEG/acrylamide hydrogel is a candidate scaffold material for tissue engineering applications.

Hsp70 as an indicator of stress in the cells after contact with nanoparticles

NASA Astrophysics Data System (ADS)

Hardilová, Šárka; Havrdová, Markéta; Panáček, Aleš; Kvítek, Libor; Zbořil, Radek

2015-05-01

In recent years, production of nanoparticles is increased and thus grows our contact with them too. Question of safety is closely related to the issue of use nanoparticles. There are a number of tests that monitor the viability, ROS production, the effect on the DNA and cell cycle, however, rarely encountered studies on stress in the cells after contact with nanoparticles. Heat shock proteins (HSP) are among the substances that can be used for monitoring stress in cells. HSP are structures with a chaperone activity. They are evolutionarily very old, conservative and they are found with a high degree of homology in prokaryotes and eukaryotes including humans. They exist at low concentrations under physiological conditions, while in the denaturing conditions e.g. high or low temperature, radiation, exposure to chemicals, heavy metals, or nanoparticles their expression is changed. HSPs are involved in maintaining homeostasis in the cell that the denatured protein conformations allow recovery to the original stage. One of the most common proteins from HSP family is Hsp70 - protein with a molecular weight of 70 kDa. The level of Hsp70 in a cell after exposure to the stress changes depending on the stress level to which the cell is exposed to and a time period during which lasted stressful conditions. Our research monitors stress levels of cells manifesting by Hsp70 production after contact with silver nanoparticles. Nanoparticles show different toxicity towards different types of target cells, which is reflected in the values of IC50 - concentration that kills 50% tested cells. Concentration of test substance toxic to one cell type may be innocuous to cells of another type. IC50 obtained from the MTT assay provides a suitable default data and if multiples of IC50 values are used, we can compare and generalize. Studies can be used to compare stress levels in cells that show different sensitivity to the tested nanoparticles compared with cells under optimal growth conditions. The study was done on two types of mouse fibroblasts NIH-3T3 and L929. While NIH-3T3 cells exhibit stress response proportional to the concentration of silver nanoparticles, for L929 cells this was not observed.

Gelatin promotes murine fibrosarcoma L929 cell detachment and protects the cells from TNFα-induced cytotoxicity.

PubMed

Wang, Hong-Ju; Li, Meng-Qi; Liu, Wei; Yao, Guo-Dong; Xia, Ming-Yu; Hayashi, Toshihiko; Fujisaki, Hitomi; Hattori, Shunji; Tashiro, Shin-Ichi; Onodera, Satoshi; Ikejima, Takashi

2016-07-01

Gelatin has been considered to exist as intermediate substance of collagen catabolism in tissue remodeling or under inflammatory conditions. We have initiated the study on possible biological functions of gelatin that can exist temporally and locally under the conditions of remodeling and inflammation Materials and methods: To this purpose, we investigated cell proliferation and survival on gelatin-coated dishes and the response to tumor necrosis factor α (TNFα)-induced cytotoxicity in L929 cells. Autophagy level, ATP level, and ROS generation are examined. L929 cells detached from the gelatin-coated dishes and formed multicellular aggregates. TNFα-induced cytotoxicity in L929 cells was inhibited by gelatin-coating culture. The cells on gelatin-coated dishes showed reduced cellular ATP levels and increased adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) phosphorylation, leading to increased ROS generation and autophagy. This study showed that gelatin-coated culture protected L929 cells from TNFα-induced cytotoxicity and suggested for a possible pathophysiological function of gelatin in regulating cellular functions.

Effect of the Heat-Treated Ti6Al4V Alloy on the Fibroblastic Cell Response

PubMed Central

Chávez-Díaz, Mercedes Paulina; Escudero-Rincón, María Lorenza; Arce-Estrada, Elsa Miriam; Cabrera-Sierra, Román

2017-01-01

Two heat treatments were carried out below (Ti6Al4V800) and above (Ti6Al4V1050) Ti6Al4V beta-phase transformation temperature (980 °C), with the purpose of studying the effect of microstructure on the adhesion and proliferation of fibroblast cells, as well as their electrochemical behavior. These alloys were seeded with 10,000 L929 fibroblast cells and immersed for 7 days in the cell culture at 37 °C, pH 7.40, 5% CO2 and 100% relative humidity. Cell adhesion was characterized by Scanning Electron Microscopy (SEM) and Electrochemical Impedance Spectroscopy (EIS) techniques. Polygonal and elongated cell morphology was observed independent of Ti6Al4V microstructure. Besides, C, O, P, S, Na and Cl signals were detected by Energy Dispersive X-Ray Spectroscopy (EDX), associated with the synthesis of organic compounds excreted by the cells, including protein adsorption from the medium. In certain areas on Ti6Al4V and Ti6Al4V800 alloys, cells were agglomerated (island type), likely related to the globular microstructure; meanwhile, larger cellular coverage is shown for Ti6Al4V1050 alloy, forming more than one layer on the surface, where only Ca was recorded. Impedance diagrams showed a similar passive behavior for the different Ti6Al4V alloys, mainly due to TiO2 overlaying the contribution of the organic compounds excreted by fibroblast cells. PMID:29301205

Immobilization of Ag nanoparticles/FGF-2 on a modified titanium implant surface and improved human gingival fibroblasts behavior.

PubMed

Ma, Qianli; Mei, Shenglin; Ji, Kun; Zhang, Yumei; Chu, Paul K

2011-08-01

The objective of this study was to form a rapid and firm soft tissue sealing around dental implants that resists bacterial invasion. We present a novel approach to modify Ti surface by immobilizing Ag nanoparticles/FGF-2 compound bioactive factors onto a titania nanotubular surface. The titanium samples were anodized to form vertically organized TiO(2) nanotube arrays and Ag nanoparticles were electrodeposited onto the nanotubular surface, on which FGF-2 was immobilized with repeated lyophilization. A uniform distribution of Ag nanoparticles/FGF-2 was observed on the TiO(2) nanotubular surface. The L929 cell line was used for cytotoxicity assessment. Human gingival fibroblasts (HGFs) were cultured on the modified surface for cytocompatibility determination. The Ag/FGF-2 immobilized samples displayed excellent cytocompatibility, negligible cytotoxicity, and enhanced HGF functions such as cell attachment, proliferation, and ECM-related gene expression. The Ag nanoparticles also exhibit some bioactivity. In conclusion, this modified TiO(2) nanotubular surface has a large potential for use in dental implant abutment. Copyright © 2011 Wiley Periodicals, Inc.

Cell-based capacitance sensor for analysis of EGFR expression on cell membrane

NASA Astrophysics Data System (ADS)

Shin, Dong-Myeong; Shin, Yong-Cheol; Ha, Ji Hye; Lee, Jong-Ho; Han, Dong-Wook; Kim, Jong-Man; Kim, Hyung Kook; Hwang, Yoon-Hwae

2013-02-01

Cancer cells have many kinds of cancer biomarkers. Among them, the epidermal growth factor (EGF) receptors can show a possibility for a cancer marker because the over-expression of EGF receptor is related with fibrous, colorectal, cervical and gastric tumorigenesis. We fabricated the capacitance sensor with a gap area of 50 μm × 200 μm by using photolithography and lift-off method. Using the capacitance sensor, we investigated the time dependent capacitance changes of different kinds of fibrous cells, such as HT1080 fibrosarcoma, L-929 fibroblast cell line and nHDF dermal fibroblast primary cell. We found that when we put the EGF, the capacitance decreased due to the immobilization of EGF to EGF receptor on the cell membrane. The quantitative determination of EGF receptor level for various fibrous cells was carried out and the results showed good correlation with conventional method. Based on our results, we suggest that the capacitance sensor can measure the expression level of the EGF receptor on cell membrane and be a good candidate as a cancer diagnosis.

Fluorescence lifetime imaging of induced pluripotent stem cells

NASA Astrophysics Data System (ADS)

Uchugonova, Aisada; Batista, Ana; König, Karsten

2014-02-01

The multiphoton FLIM tomograph MPTflex with its flexible scan head, articulated arm, and the tunable femtosecond laser source was employed to study cell monolayers and 3D cell clusters. FLIM was performed with 250 ps temporal resolution and submicron special resolution using time-correlated single photon counting. The autofluorescence based on NAD(P)H and flavins/flavoproteins has been measured in mouse embryonic fibroblasts, induced pluripotent stem cells (iPS cells) originated from mouse embryonic fibroblasts and non-proliferative mouse embryonic fibroblasts.

Synthesis, characterization, and assessment of cytotoxic, antiproliferative, and antiangiogenic effects of a novel procainamide hydrochloride-poly(maleic anhydride-co-styrene) conjugate.

PubMed

Karakus, Gulderen; Akin Polat, Zubeyde; Sahin Yaglıoglu, Ayse; Karahan, Mesut; Yenidunya, Ali Fazil

2013-01-01

Poly(maleic anhydride-co-styrene) (MAST) was synthesized by a free-radical polymerization reaction. A bioactive molecule, procainamide hydrochloride (PH), was then conjugated to MAST. The conjugation product was named as MAST/PH. Structural characterization of MAST and MAST/PH was carried out by Fourier Transform Infrared and Nuclear Magnetic Resonance spectroscopy. Their molecular weights were determined by size-exclusion chromatography. A mechanism was then suggested for the conjugation reaction. The results of the cytotoxicity assay, employing a mouse fibroblast cell line (L929), indicated that MAST/PH had no cytotoxicity at concentrations [Formula: see text] 62 μg mL(-1) (p > 0.05). Antiproliferative activities of MAST/PH and PH were determined by the BrdU cell proliferation ELISA assay, using C6 and HeLa cell lines. In the experiment, two anticancer chemotherapy drugs, cisplatin and 5-fluorouracil, were included as positive control. Antiproliferative activity results demonstrated that MAST/PH yielded the highest suppression profile (approximately 42%) at 20 μg/ml, while free PH exerted the same activity at 100 μg/ml. Interestingly, both MAST/PH and PH suppressed the proliferation of only one of the cell lines, C6 cells. Both cisplatin and 5-fluorouracil yielded approximately 60% antiproliferative activity on C6 cells at 20 and 100 μg/ml concentrations. Antiangiogenic capacity of both MAST and MAST/PH was also investigated by using the chicken chorioallantoic membrane assay. Results obtained indicated that while MAST/PH could be included into the category of good antiangiogenic substances, the activity score of MAST was within the weak category.

Preliminary Characterization of Genipin-Cross-Linked Silk Sericin/Poly(vinyl alcohol) Films as Two-Dimensional Wound Dressings for the Healing of Superficial Wounds

PubMed Central

Siritientong, Tippawan; Ratanavaraporn, Juthamas; Srichana, Teerapol; Aramwit, Pornanong

2013-01-01

The genipin-cross-linked silk sericin/poly(vinyl alcohol) (PVA) films were developed aiming to be applied as two-dimensional wound dressings for the treatment of superficial wounds. The effects of genipin cross-linking concentration on the physical and biological properties of the films were investigated. The genipin-cross-linked silk sericin/PVA films showed the increased surface density, tensile strength, and percentage of elongation, but decreased percentage of light transmission, water vapor transmission rate, and water swelling, compared to the non-cross-linked films. This explained that the cross-linking bonds between genipin and silk sericin would reduce the mobility of molecular chains within the films, resulting in the more rigid molecular structure. Silk sericin was released from the genipin-cross-linked films in a sustained manner. In addition, either L929 mouse fibroblast or HaCat keratinocyte cells showed high percentage of viability when cultured on the silk sericin/PVA films cross-linked with 0.075 and 0.1% w/v genipin. The in vivo safety test performed according to ISO 10993-6 confirmed that the genipin-cross-linked silk sericin/PVA films were safe for the medical usages. The efficacy of the films for the treatment of superficial skin wounds will be further investigated in vivo and clinically. The genipin-cross-linked silk sericin/PVA films would be promising choices of two-dimensional wound dressings for the treatment of superficial wounds. PMID:24106722

Preliminary characterization of genipin-cross-linked silk sericin/poly(vinyl alcohol) films as two-dimensional wound dressings for the healing of superficial wounds.

PubMed

Siritientong, Tippawan; Ratanavaraporn, Juthamas; Srichana, Teerapol; Aramwit, Pornanong

2013-01-01

The genipin-cross-linked silk sericin/poly(vinyl alcohol) (PVA) films were developed aiming to be applied as two-dimensional wound dressings for the treatment of superficial wounds. The effects of genipin cross-linking concentration on the physical and biological properties of the films were investigated. The genipin-cross-linked silk sericin/PVA films showed the increased surface density, tensile strength, and percentage of elongation, but decreased percentage of light transmission, water vapor transmission rate, and water swelling, compared to the non-cross-linked films. This explained that the cross-linking bonds between genipin and silk sericin would reduce the mobility of molecular chains within the films, resulting in the more rigid molecular structure. Silk sericin was released from the genipin-cross-linked films in a sustained manner. In addition, either L929 mouse fibroblast or HaCat keratinocyte cells showed high percentage of viability when cultured on the silk sericin/PVA films cross-linked with 0.075 and 0.1% w/v genipin. The in vivo safety test performed according to ISO 10993-6 confirmed that the genipin-cross-linked silk sericin/PVA films were safe for the medical usages. The efficacy of the films for the treatment of superficial skin wounds will be further investigated in vivo and clinically. The genipin-cross-linked silk sericin/PVA films would be promising choices of two-dimensional wound dressings for the treatment of superficial wounds.

A green salt-leaching technique to produce sericin/PVA/glycerin scaffolds with distinguished characteristics for wound-dressing applications.

PubMed

Aramwit, Pornanong; Ratanavaraporn, Juthamas; Ekgasit, Sanong; Tongsakul, Duangta; Bang, Nipaporn

2015-05-01

Sericin/PVA/glycerin scaffolds could be fabricated using the freeze-drying technique; they showed good physical and biological properties and can be applied as wound dressings. However, freeze-drying is an energy- and time-consuming process with a high associated cost. In this study, an alternative, solvent-free, energy- and time-saving, low-cost salt-leaching technique is introduced as a green technology to produce sericin/PVA/glycerin scaffolds. We found that sericin/PVA/glycerin scaffolds were successfully fabricated without any crosslinking using a salt-leaching technique. The salt-leached sericin/PVA/glycerin scaffolds had a porous structure with pore interconnectivity. The sericin in the salt-leached scaffolds had a crystallinity that was as high as that of the freeze-dried scaffolds. Compared to the freeze-dried scaffolds with the same composition, the salt-leached sericin/PVA/glycerin scaffolds has larger pores, a lower Young's modulus, and faster rates of biodegradation and sericin release. When cultured with L929 mouse fibroblast cells, a higher number of cells were found in the salt-leached scaffolds. Furthermore, the salt-leached scaffolds were less adhesive to the wound, which would reduce pain upon removal. Therefore, salt-leached sericin/PVA/glycerin scaffolds with distinguished characteristics were introduced as another choice of wound dressing, and their production process was simpler, more energy efficient, and saved time and money compared to the freeze-dried scaffolds. © 2014 Wiley Periodicals, Inc.

Functional characterization and novel rickettsiostatic effects of a Kunitz-type serine protease inhibitor from the tick Dermacentor variabilis.

PubMed

Ceraul, Shane M; Dreher-Lesnick, Sheila M; Mulenga, Albert; Rahman, M Sayeedur; Azad, Abdu F

2008-11-01

Here we report the novel bacteriostatic function of a five-domain Kunitz-type serine protease inhibitor (KPI) from the tick Dermacentor variabilis. As ticks feed, they release anticoagulants, anti-inflammatory and immunosuppressive molecules that mediate the formation of the feeding lesion on the mammalian host. A number of KPIs have been isolated and characterized from tick salivary gland extracts. Interestingly, we observe little D. variabilis KPI gene expression in the salivary gland and abundant expression in the midgut. However, our demonstration of D. variabilis KPI's anticoagulant properties indicates that D. variabilis KPI may be important for blood meal digestion in the midgut. In addition to facilitating long-term attachment and blood meal acquisition, gene expression studies of Drosophila, legumes, and ticks suggest that KPIs play some role in the response to microbial infection. Similarly, in this study, we show that challenge of D. variabilis with the spotted fever group rickettsia, Rickettsia montanensis, results in sustained D. variabilis KPI gene expression in the midgut. Furthermore, our in vitro studies show that D. variabilis KPI limits rickettsial colonization of L929 cells (mouse fibroblasts), implicating D. variabilis KPI as a bacteriostatic protein, a property that may be related to D. variabilis KPI's trypsin inhibitory capability. This work suggests that anticoagulants play some role in the midgut during feeding and that D. variabilis KPI may be involved as part of the tick's defense response to rickettsiae.

Functional Characterization and Novel Rickettsiostatic Effects of a Kunitz-Type Serine Protease Inhibitor from the Tick Dermacentor variabilis▿

PubMed Central

Ceraul, Shane M.; Dreher-Lesnick, Sheila M.; Mulenga, Albert; Rahman, M. Sayeedur; Azad, Abdu F.

2008-01-01

Here we report the novel bacteriostatic function of a five-domain Kunitz-type serine protease inhibitor (KPI) from the tick Dermacentor variabilis. As ticks feed, they release anticoagulants, anti-inflammatory and immunosuppressive molecules that mediate the formation of the feeding lesion on the mammalian host. A number of KPIs have been isolated and characterized from tick salivary gland extracts. Interestingly, we observe little D. variabilis KPI gene expression in the salivary gland and abundant expression in the midgut. However, our demonstration of D. variabilis KPI's anticoagulant properties indicates that D. variabilis KPI may be important for blood meal digestion in the midgut. In addition to facilitating long-term attachment and blood meal acquisition, gene expression studies of Drosophila, legumes, and ticks suggest that KPIs play some role in the response to microbial infection. Similarly, in this study, we show that challenge of D. variabilis with the spotted fever group rickettsia, Rickettsia montanensis, results in sustained D. variabilis KPI gene expression in the midgut. Furthermore, our in vitro studies show that D. variabilis KPI limits rickettsial colonization of L929 cells (mouse fibroblasts), implicating D. variabilis KPI as a bacteriostatic protein, a property that may be related to D. variabilis KPI's trypsin inhibitory capability. This work suggests that anticoagulants play some role in the midgut during feeding and that D. variabilis KPI may be involved as part of the tick's defense response to rickettsiae. PMID:18779339

Effects of sterilisation method on surface topography and in-vitro cell behaviour of electrostatically spun scaffolds.

PubMed

Andrews, Kirstie D; Hunt, John A; Black, Richard A

2007-02-01

Electrostatic spinning is a potentially significant technique for scaffold production within the field of tissue engineering; however, the effect of sterilisation upon these structures is not known. This research investigated the extent of any topographical alteration to electrostatically spun scaffolds post-production through sterilisation, and examined any subsequent effect on contacting cells. Scaffolds made from Tecoflex SG-80A polyurethane were sterilised using ethylene oxide and UV-ozone. Scaffold topography was characterized in terms of inter-fibre separation (ifs), fibre diameter (f.dia) and surface roughness. Cell culture was performed over 7 days with both mouse L929 and human embryonic lung fibroblasts, the results of which were assessed using SEM, image analysis and confocal microscopy. Sterilisation by UV-ozone and ethylene oxide decreased ifs and increased f.dia; surface roughness was decreased by UV-ozone but increased by ethylene oxide. Possible mechanisms to explain these observations are discussed, namely photo-oxidative degradation in the case of UV-ozone and process-induced changes in surface roughness. UV-ozone sterilised scaffolds showed greater cell coverage than those treated with ethylene oxide, but lower coverage than all the controls. Changes in cell attachment and morphology were thought to be due to the changes in topography brought about by the sterilisation process. We conclude that surface modification by sterilisation could prove to be a useful tool at the final stage of scaffold production to enhance cell contact, phenotype or function.

In-vitro assessment of oxidative stress generated by orthodontic archwires.

PubMed

Spalj, Stjepan; Mlacovic Zrinski, Magda; Tudor Spalj, Vedrana; Ivankovic Buljan, Zorana

2012-05-01

Several metals undergo redox cycling, producing free radicals and generating oxidative stress. The purpose of this study was to investigate in-vitro oxidative stress of orthodontic archwires made of various alloys. Mouse fibroblast cells L929 were exposed to 6 types of archwires, and the concentration of the oxidative stress marker 8-hydroxy-2'-deoxyguanosine in DNA was evaluated. Trypan blue dye was used in the determination of cell viability and numbers. Standard nickel-titanium archwires generated the highest oxidative stress, significantly higher than all other wires and the controls (P <0.05), and coated nickel-titanium, copper-nickel-titanium, and cobalt-chromium were lower than nickel-titanium (P <0.05), but higher than titanium-molybdenum and the negative and absolute controls (P <0.05). Titanium-molybdenum and stainless steel generated the lowest stress. Nickel-titanium induced the lowest viability, lower than the negative and absolute controls and all other wires (P <0.05) except titanium-molybdenum. Stainless steel showed the highest viability. Nickel-titanium produced the highest inhibition of cell growth, higher than all samples (P <0.05) except the positive control and cobalt-chromium. The lowest inhibition was observed in stainless steel and titanium-molybdenum, lower than nickel-titanium, cobalt-chromium, and the positive control (P <0.05). All orthodontic archwires generate oxidative stress in vitro. Stainless steel archwires have the highest and nickel-titanium the lowest biocompatibility. Copyright © 2012 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.

Development and evaluation of buccoadhesive tablet for selegiline hydrochloride based on thiolated polycarbophil.

PubMed

Wasnik, Mangesh N; Godse, Rutika D; Nair, Hema A

2014-05-01

Selegiline hydrochloride (SHCl), a monoamine oxidase B inhibitor, is used as an adjunct in the therapy of Parkinson's disease. This study is concerned with the preparation and evaluation of mucoadhesive buccal tablet for controlled systemic delivery of SHCl. Buccal absorption of selegiline can bypass its first-pass metabolism and improve bioavailability accompanied by greatly reduced metabolite formation, which is potentially of enhanced therapeutic value in patients with Parkinson's disease. Polycarbophil-cysteine (PCP-cys) conjugate, which is a thiolated derivative of the mucoadhesive polymer polycarbophil, was synthesized by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride-mediated amide bond coupling. Tablets of SHCl based on native and thiolated polycarbophil were prepared. The prepared tablets were evaluated for drug content, swelling behavior, mucoadhesive strength, in vitro drug release, ex vivo permeation and in vitro cytotoxicity. PCP-cys tablets showed enhanced mucoadhesion and retarded drug release compared to polycarbophil tablets. Permeation data of SHCl from matrices prepared using the PCP-cys polymer revealed a significantly higher value of apparent permeability in comparison to polycarbophil, which supported the information in literature that thiolation imparts permeation enhancing properties to mucoadhesive polymers. In vitro cytotoxicity studies on PCP-cys using L-929 mouse fibroblast cell line indicated that conjugation with cysteine does not impart any apparent toxicity to polycarbophil. The results from the study indicate that the buccal delivery of SHCl using thiolated polycarbophil tablet could provide a way for improved therapy of Parkinson's disease.

The interplay of plasma treatment and gold coating and ultra-high molecular weight polyethylene: On the cytocompatibility.

PubMed

Novotná, Zdenka; Rimpelová, Silvie; Juřík, Petr; Veselý, Martin; Kolská, Zdenka; Hubáček, Tomáš; Ruml, Tomáš; Švorčík, Václav

2017-02-01

We have investigated the application of Ar plasma for creation of nanostructured ultra high molecular weight polyethylene (PE) surface in order to enhance adhesion of mouse embryonic fibroblasts (L929). The aim of this study was to investigate the effect of the interface between plasma-treated and gold-coated PE on adhesion and spreading of cells. The surface properties of pristine samples and its modified counterparts were studied by different experimental techniques (gravimetry, goniometry and X-ray photoelectron spectroscopy (XPS), electrokinetic analysis), which were used for characterization of treated and sputtered layers, polarity and surface chemical structure, respectively. Further, atomic force microscopy (AFM) was employed to study the surface morphology and roughness. Biological responses of cells seeded on PE samples were evaluated in terms of cell adhesion, spreading, morphology and proliferation. Detailed cell morphology and intercellular connections were followed by scanning electron microscopy (SEM). As it was expected the thickness of a deposited gold film was an increasing function of the sputtering time. Despite the fact that plasma treatment proceeded in inert plasma, oxidized degradation products were formed on the PE surface which would contribute to increased hydrophilicity (wettability) of the plasma treated polymer. The XPS method showed a decrease in carbon concentration with increasing plasma treatment. Cell adhesion measured on the interface between plasma treated and gold coated PE was inversely proportional to the thickness of a gold layer on a sample. Copyright © 2016. Published by Elsevier B.V.

Annonalide and derivatives: Semisynthesis, cytotoxic activities and studies on interaction of annonalide with DNA.

PubMed

Marques, Ricardo A; Gomes, Akenaton O C V; de Brito, Maria V; Dos Santos, Ana L P; da Silva, Gladyane S; de Lima, Leandro B; Nunes, Fátima M; de Mattos, Marcos C; de Oliveira, Fátima C E; do Ó Pessoa, Cláudia; de Moraes, Manoel O; de Fátima, Ângelo; Franco, Lucas L; Silva, Marina de M; Dantas, Maria Dayanne de A; Santos, Josué C C; Figueiredo, Isis M; da Silva-Júnior, Edeíldo F; de Aquino, Thiago M; de Araújo-Júnior, João X; de Oliveira, Maria C F; Leslie Gunatilaka, A A

2018-02-01

The cytotoxic activity of the pimarane diterpene annonalide (1) and nine of its semisynthetic derivatives (2-10) was investigated against the human tumor cell lines HL-60 (leukemia), PC-3 (prostate adenocarcinoma), HepG2 (hepatocellular carcinoma), SF-295 (glioblastoma) and HCT-116 (colon cancer), and normal mouse fibroblast (L929) cells. The preparation of 2-10 involved derivatization of the side chain of 1 at C-13. Except for 2, all derivatives are being reported for the first time. Most of the tested compounds presented IC 50 s below 4.0 μM, being considered potential antitumor agents. The structures of all new compounds were elucidated by spectroscopic analyses including 2D NMR and HRMS. Additionally, the interaction of annonalide (1) with ctDNA was evaluated using spectroscopic techniques, and the formation of a supramolecular complex with the macromolecule was confirmed. Competition assays with fluorescent probes (Hoechst and ethidium bromide) and theoretical studies confirmed that 1 interacts preferentially via DNA intercalation with stoichiometric ratio of 1:1 (1:ctDNA). The ΔG value was calculated as -28.24 kJ mol -1 , and indicated that the interaction process occurs spontaneously. Docking studies revealed that van der Walls is the most important interaction in 1-DNA and EB-DNA complexes, and that both ligands (1 and EB) interact with the same DNA residues (DA6, DA17 and DT19). Copyright © 2018. Published by Elsevier B.V.

The effects of crosslinkers on physical, mechanical, and cytotoxic properties of gelatin sponge prepared via in-situ gas foaming method as a tissue engineering scaffold.

PubMed

Poursamar, S Ali; Lehner, Alexander N; Azami, Mahmoud; Ebrahimi-Barough, Somayeh; Samadikuchaksaraei, Ali; Antunes, A P M

2016-06-01

In this study porous gelatin scaffolds were prepared using in-situ gas foaming, and four crosslinking agents were used to determine a biocompatible and effective crosslinker that is suitable for such a method. Crosslinkers used in this study included: hexamethylene diisocyanate (HMDI), poly(ethylene glycol) diglycidyl ether (epoxy), glutaraldehyde (GTA), and genipin. The prepared porous structures were analyzed using Fourier Transform Infrared Spectroscopy (FT-IR), thermal and mechanical analysis as well as water absorption analysis. The microstructures of the prepared samples were analyzed using Scanning Electron Microscopy (SEM). The effects of the crosslinking agents were studied on the cytotoxicity of the porous structure indirectly using MTT analysis. The affinity of L929 mouse fibroblast cells for attachment on the scaffold surfaces was investigated by direct cell seeding and DAPI-staining technique. It was shown that while all of the studied crosslinking agents were capable of stabilizing prepared gelatin scaffolds, there are noticeable differences among physical and mechanical properties of samples based on the crosslinker type. Epoxy-crosslinked scaffolds showed a higher capacity for water absorption and more uniform microstructures than the rest of crosslinked samples, whereas genipin and GTA-crosslinked scaffolds demonstrated higher mechanical strength. Cytotoxicity analysis showed the superior biocompatibility of the naturally occurring genipin in comparison with other synthetic crosslinking agents, in particular relative to GTA-crosslinked samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Antibacterial nanosilver coated orthodontic bands with potential implications in dentistry.

PubMed

Prabha, Rahul Damodaran; Kandasamy, Rajasigamani; Sivaraman, U Sajeev; Nandkumar, Maya A; Nair, Prabha D

2016-10-01

Fixed orthodontic treatment, an indispensable procedure in orthodontics, necessitates insertion of dental bands. Insertion of band material could also introduce a site of plaque retention. It was hypothesized that band materials with slow-release antimicrobial properties could help in sustained infection control, prevention of dental plaque formation and further associated health risks. Considering the known antimicrobial proprieties of silver, a coating of silver nanoparticle (SNP) onto the stainless steel bands was done and characterized for its beneficial properties in the prevention of plaque accumulation. Coatings of SNPs on conventional stainless steel dental bands were prepared using thermal evaporation technology. The coated dental bands were characterized for their physicochemical properties and evaluated for antimicrobial activity and biocompatibility. The physiochemical characterization of band material both coated and uncoated was carried out using scanning electron microscope, energy dispersive spectroscopy, atomic force microscopyand contact angle test. Biocompatibility tests for coated band material were carried using L929 mouse fibroblast cell culture and MTT [3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay. Antimicrobial activity of coated band material against Gram-positive bacteria was tested. A stable and uniform coating of SNPs was obtained. The coated band materials were biocompatible as well as possessed distinct antimicrobial activity. The SNP coated dental bands could be potential antimicrobial dental bands for future clinical use. Further studies need to be done to validate the efficiency of coated band materials in oral environments.

Development of a biodegradable tissue adhesive based on functionalized 1,2-ethylene glycol bis(dilactic acid). II.

PubMed

Rohm, Henning W; Lurtz, Claudia; Wegmann, Juergen; Odermatt, Erich K; Behrend, Detlef; Schmitz, Klaus-Peter; Sternberg, Katrin

2011-04-01

In body regions where damage and bleeding must be avoided, a substitute for mechanical tissue fixation by sutures or staplers is needed. Since tissue adhesives provide easy and fast handling they are a promising alternative. The present study reports the development and analysis of a tissue adhesive that consists of two adhesive components: hexamethylene diisocyanate (HDI) functionalized 1,2-ethylene glycol bis(dilactic acid) (ELA-NCO) and chitosan chloride. This composition was chosen based on preliminary studies on several chain elongation agents. The present study evaluates this adhesive system by IR-spectroscopy, tensile tests, and gel point measurements in comparison to fibrin glue. The system's in vitro biocompatibility was tested with mouse fibroblasts (L929) according to ISO 10993-5. Furthermore, an implantation study was performed in SPF-Wistar rats. The adhesive strength of manually applied mixtures or mixtures applied by double chamber syringes with a mixing extruder was determined to be significantly higher than that of fibrin glue on bovine muscle tissue at 37°C. Tensile strength increased further when exposure time of the adhesive was increased from 10 min to 48 h. The rheological gel point determination showed that the mixture of ELA-NCO/DMSO and chitosan chloride offers a time window large enough to readjust the fused joint during surgery, as opposed to fibrin glue. Additionally, the in vitro and in vivo biocompatibility studies of the adhesive system revealed no toxic effects on the surrounding tissue. Copyright © 2010 Wiley Periodicals, Inc.

Thermo-responsive methylcellulose hydrogels as temporary substrate for cell sheet biofabrication.

PubMed

Altomare, Lina; Cochis, Andrea; Carletta, Andrea; Rimondini, Lia; Farè, Silvia

2016-05-01

Methylcellulose (MC), a water-soluble polymer derived from cellulose, was investigated as a possible temporary substrate having thermo-responsive properties favorable for cell culturing. MC-based hydrogels were prepared by a dispersion technique, mixing MC powder (2, 4, 6, 8, 10, 12 % w/v) with selected salts (sodium sulphate, Na2SO4), sodium phosphate, calcium chloride, or phosphate buffered saline, to evaluate the influence of different compositions on the thermo-responsive behavior. The inversion test was used to determine the gelation temperatures of the different hydrogel compositions; thermo-mechanical properties and thermo-reversibility of the MC hydrogels were investigated by rheological analysis. Gelation temperatures and rheological behavior depended on the MC concentration and type and concentration of salt used in hydrogel preparation. In vitro cytotoxicity tests, performed using L929 mouse fibroblasts, showed no toxic release from all the tested hydrogels. Among the investigated compositions, the hydrogel composed of 8 % w/v MC with 0.05 M Na2SO4 had a thermo-reversibility temperature at 37 °C. For that reason, this formulation was thus considered to verify the possibility of inducing in vitro spontaneous detachment of cells previously seeded on the hydrogel surface. A continuous cell layer (cell sheet) was allowed to grow and then detached from the hydrogel surface without the use of enzymes, thanks to the thermo-responsive behavior of the MC hydrogel. Immunofluorescence observation confirmed that the detached cell sheet was composed of closely interacting cells.

Influence of salinomycin treatment on division and movement of individual cancer cells cultured in normoxia or hypoxia evaluated with time-lapse digital holographic microscopy

PubMed Central

Kamlund, Sofia; Strand, Daniel; Janicke, Birgit; Alm, Kersti; Oredsson, Stina

2017-01-01

ABSTRACT Most studies on new cancer drugs are based on population-derived data, where the absence of response of a small population may pass unnoticed. Thus, individual longitudinal tracking of cells is important for the future development of efficient cancer treatments. We have used digital holographic microscopy to track individual JIMT-1 human breast cancer cells and L929 mouse fibroblast cultivated in normoxia or hypoxia. In addition, JIMT-1 cells were treated with salinomycin, a cancer stem cell targeting compound. Three-day time-lapse movies were captured and individual cells were analysed with respect to cell division (cell cycle length) and cell movement. Comparing population-doubling time derived from population-based growth curves and individual cell cycle time data from time-lapse movies show that the former hide a sub-population of dividing cells. Salinomycin treatment increased the motility of cells, however, this motility did not result in an increased distant migration i.e. the cells increased their local movement. MCF-7 breast cancer cells showed similar motility behaviour as salinomycin-treated JIMT-1 cells. We suggest that combining features, such as motility and migration, can be used to distinguish cancer cells with mesenchymal (JIMT-1) and epithelial (MCF-7) features. The data clearly emphasize the importance of longitudinal cell tracking to understand the biology of individual cells under different conditions. PMID:28933990

Conjugation of curcumin onto hyaluronic acid enhances its aqueous solubility and stability.

PubMed

Manju, S; Sreenivasan, K

2011-07-01

Polymer-drug conjugates have gained much attention largely to circumvent lower drug solubility and to enhance drug stability. Curcumin is widely known for its medicinal properties including its anticancer efficacy. One of the serious drawbacks of curcumin is its poor water solubility which leads to reduced bioavailability. With a view to address these issues, we synthesized hyaluronic acid-curcumin (HA-Cur) conjugate. The drug conjugate was characterized using FT-IR, NMR, Dynamic light scattering and TEM techniques. The conjugates, interestingly found to assembles as micelles in aqueous phase. The formation of micelles seems to improve the stability of the drug in physiological pH. We also assessed cytotoxicity of the conjugate using L929 fibroblast cells and quantified by MTT assay. Copyright © 2011 Elsevier Inc. All rights reserved.

Studies on biodegradable and crosslinkable poly(castor oil fumarate)/poly(propylene fumarate) composite adhesive as a potential injectable biomaterial.

PubMed

Mitha, M K; Jayabalan, M

2009-12-01

Biodegradable hydroxyl terminated-poly(castor oil fumarate) (HT-PCF) and poly(propylene fumarate) (HT-PPF) resins were synthesized as an injectable and in situ-cross linkable polyester resins for orthopedic applications. An injectable adhesive formulation containing this resin blend, N-vinyl pyrrolidone (NVP), hydroxy apatite, free radical initiator and accelerator was developed. The Composite adhesives containing the ratio of resin blend and NVP, 2.1:1.5, 2.1:1.2 and 2.1:1.0 set fast with tolerable exothermic temperature as a three dimensionally cross linked toughened material. Crosslink density and mechanical properties of the crosslinked composite increase with increase of NVP. The present crosslinked composite has hydrophilic character and cytocompatibility with L929 fibroblast cells.

Primary mouse lung fibroblasts help macrophages to tackle Mycobacterium tuberculosis more efficiently and differentiate into myofibroblasts up on bacterial stimulation.

PubMed

Verma, Subash Chand; Agarwal, Pooja; Krishnan, Manju Y

2016-03-01

Keeping with their classical role in wound healing, fibroblasts of the lung take part in the resolution of tubercular granulomas. They are totally absent in nascent granulomas, but surround necrotizing granulomas, and are the majority of cells in healed granulomas. Lung fibroblasts may become infected with Mycobacterium tuberculosis (Mtb). Two previous studies suggested an immunomodulatory effect of fibroblasts on infected macrophages. In the present study, we looked at the role of primary mouse lung fibroblasts on naive or activated mouse bone marrow macrophages infected with Mtb and the effect of infection on fibroblast properties. We observed that with fibroblasts in the vicinity, infected naive macrophages restricted the bacterial growth, while activated macrophages turned more bactericidal with concomitant increase in nitrite production. Neutralizing IL-1α in fibroblast supernatant reduced the nitrite production by infected macrophages. Secretion of IL-6 and MCP-1 was down-regulated, while TNF-α was up-regulated in infected naive macrophages. In infected activated macrophages, the secretion of IL-6 was up-regulated, while that of MCP-1 and TNF-α was unaffected. The 'fibroblast effects' were enhanced when the fibroblasts too were infected. Mtb induced IL-1 secretion and pro-fibrotic responses by fibroblasts. Mtb-induced myofibroblast conversion was blocked by rapamycin suggesting cell signalling via mTOR. Copyright © 2015 Elsevier Ltd. All rights reserved.

Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dong, Yan; Hirane, Miku; Araki, Mutsumi

2014-04-04

Highlights: • LPA{sub 5} inhibits the cell growth and motile activities of 3T3 cells. • LPA{sub 5} suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA{sub 5} on the cell motile activities inhibited by LPA{sub 1} in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA{sub 5} in 3T3 cells. • LPA signaling via LPA{sub 5} acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA{sub 1}–LPA{sub 6}) mediates a variety of biological functions, including cellmore » migration. Recently, we have reported that LPA{sub 1} inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA{sub 5} in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA{sub 1} and LPA{sub 5} on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA{sub 5} may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA{sub 1}.« less

Superparamagnetic poly(methyl methacrylate) nanoparticles surface modified with folic acid presenting cell uptake mediated by endocytosis

NASA Astrophysics Data System (ADS)

Feuser, Paulo Emilio; Jacques, Amanda Virtuoso; Arévalo, Juan Marcelo Carpio; Rocha, Maria Eliane Merlin; dos Santos-Silva, Maria Claudia; Sayer, Claudia; de Araújo, Pedro H. Hermes

2016-04-01

The encapsulation of superparamagnetic nanoparticles (MNPs) in polymeric nanoparticles (NPs) with modified surfaces can improve targeted delivery and induce cell death by hyperthermia. The goals of this study were to synthesize and characterize surface modified superparamagnetic poly(methyl methacrylate) with folic acid (FA) prepared by miniemulsion polymerization (MNPsPMMA-FA) and to evaluate their in vitro cytotoxicity and cellular uptake in non-tumor cells, murine fibroblast (L929) cells and tumor cells that overexpressed folate receptor (FR) β, and chronic myeloid leukemia cells in blast crisis (K562). Lastly, hemolysis assays were performed on human red blood cells. MNPsPMMA-FA presented an average mean diameter of 135 nm and a saturation magnetization (Ms) value of 37 emu/g of iron oxide, as well as superparamagnetic behavior. The MNPsPMMA-FA did not present cytotoxicity in L929 and K562 cells. Cellular uptake assays showed a higher uptake of MNPsPMMA-FA than MNPsPMMA in K562 cells when incubated at 37 °C. On the other hand, MNPsPMMA-FA showed a low uptake when endocytosis mechanisms were blocked at low temperature (4 °C), suggesting that the MNPsPMMA-FA uptake was mediated by endocytosis. High concentrations of MNPsPMMA-FA showed hemocompatibility when incubated for 24 h in human red blood cells. Therefore, our results suggest that these carrier systems can be an excellent alternative in targeted drug delivery via FR.

In vitro cytotoxicity of maxillofacial silicone elastomers: effect of accelerated aging.

PubMed

Bal, Bilge Turhan; Yilmaz, Handan; Aydin, Cemal; Karakoca, Seçil; Yilmaz, Sükran

2009-04-01

The purpose of this in vitro study was to evaluate the cytotoxicity of three maxillofacial silicone elastomers at 24, 48, and 72 h on L-929 cells and to determine the effect of accelerated aging on the cytotoxicity of these silicone elastomers. Disc-shaped test samples of maxillofacial silicone elastomers (Cosmesil, Episil, Multisil) were fabricated according to manufacturers' instructions under aseptic conditions. Samples were then divided into three groups: (1) not aged; (2) aged for 150 h with an accelerated weathering tester; and (3) aged for 300 h. Then the samples were placed in Dulbecco's Modified Eagle Medium/Ham's F12 (DMEM/F12) for 24, 48, and 72 h. After the incubation periods, cytotoxicity of the extracts to cultured fibroblasts (L-929) was measured by MTT assay. The degree of cytotoxicity of each sample was determined according to the reference value represented by the cells with a control (culture without sample). Statistical significance was determined by repeated measurement ANOVA (p < 0.01) followed by Duncan's test (p < 0.05). All test materials in each group demonstrated high survival rates in MTT assay (Episil; 93.84%, Multisil; 88.30%, Cosmesil; 87.50%, respectively); however, in all groups, Episil material demonstrated significantly higher cell survival rate after each of the experimental incubation periods (p < 0.05). Accelerated aging for 150 and 300 h had no significant effect on the biocompatibility of maxillofacial silicone elastomers tested (p > 0.05).

Residual HEMA and TEGDMA Release and Cytotoxicity Evaluation of Resin-Modified Glass Ionomer Cement and Compomers Cured with Different Light Sources

PubMed Central

Botsali, Murat Selim; Kuşgöz, Adem; Altintaş, Subutay Han; Ülker, Hayriye Esra; Kiliç, Serdar; Başak, Feridun; Ülker, Mustafa

2014-01-01

The purpose of this study was first to evaluate the elution of 2-hydroxyethyl methacrylate (HEMA) and triethylene glycol dimethacrylate (TEGDMA) monomers from resin-modified glass ionomer cement (RMGIC) and compomers cured with halogen and light-emitting diode (LED) light-curing units (LCUs). The effect of cured materials on the viability of L929 fibroblast cells was also evaluated. One RMGIC (Ketac N100) and two compomers (Dyract Extra and Twinkystar) were tested. Materials were prepared in teflon disks and light-cured with LED or halogen LCUs. The residual monomers of resin materials in solution were identified using high-performance liquid chromatography. The fibroblast cells' viability was analyzed using MTT assay. The type of LCU did not have a significant effect on the elution of HEMA and TEGDMA. A greater amount of HEMA than TEGMDA was eluted. The amount of TEGDMA eluted from Twinkystar was greater than Dyract Extra (P < 0.05) when cured with a halogen LCU. All material-LCU combinations decreased the fibroblast cells' viability more than the control group (P < 0.01), except for Dyract Extra cured with a halogen LCU (P > 0.05). Curing with the LED LCU decreased the cells' viability more than curing with the halogen LCU for compomers. For Ketac N100, the halogen LCU decreased the cells' viability more than the LED LCU. PMID:24592149

Hydroxyaptite nanorods patterned ZrO2 bilayer coating on zirconium for the application of percutaneous implants.

PubMed

Zhang, Lan; Han, Yong; Tan, Guoxin

2015-03-01

Percutaneous implant requires a tight bond between the underlying dermis of skin and implant surface to prevent epithelial down-growth and infection, while fibroblasts play a key role in the skin-implant integration. In this work, nanorod-shaped hydroxyaptite (HA) with a mean diameter of 70 nm and length of 400 nm was hydrothermally grown on micro-arc oxidized (MAOed) Ca- and P-doped ZrO2 to form a bilayer coating. The hydrothermal formation mechanism of HA nanorods was explored, and the adsorption of total protein on the coating from α-MEM medium containing 10% fetal bovine serum was examined. Employing L-929 cells, the behaviors of fibroblasts on the bilayer coating, including adhesion and proliferation were evaluated together the polished Zr and as-MAOed ZrO2. The obtained results show that the HA nanorods nucleated on ZrO2 and grew at the expense of the doped Ca and P ions during the hydrothermal treatment (HT). The HA nanorods patterned coating enhanced protein absorption, and significantly improved the adhesion and proliferation of fibroblasts compared to the as-MAOed ZrO2 and polished Zr. It suggests that the HA nanorods/ZrO2 coated zirconium has a potential application for percutaneous implants to enhance the attachment of skin. Copyright © 2015 Elsevier B.V. All rights reserved.

Interactions of phosphatidylinositol kinase, GTPase-activating protein (GAP), and GAP-associated proteins with the colony-stimulating factor 1 receptor.

PubMed Central

Reedijk, M; Liu, X Q; Pawson, T

1990-01-01

The interactions of the macrophage colony-stimulating factor 1 (CSF-1) receptor with potential targets were investigated after ligand stimulation either of mouse macrophages or of fibroblasts that ectopically express mouse CSF-1 receptors. In Rat-2 cells expressing the mouse CSF-1 receptor, full activation of the receptor and cellular transformation require exogenous CSF-1, whereas NIH 3T3 cells expressing mouse c-fms are transformed by autocrine stimulation. Activated CSF-1 receptors physically associate with a phosphatidylinositol (PI) 3'-kinase. A mutant CSF-1 receptor with a deletion of the kinase insert region was deficient in its ability to bind functional PI 3'-kinase and to induce PI 3'-kinase activity precipitable with antiphosphotyrosine antibodies. In fibroblasts, CSF-1 stimulation also induced the phosphorylation of the GTPase-activating protein (GAP)-associated protein p62 on tyrosine, although GAP itself was a relatively poor substrate. In contrast to PI 3'-kinase association, phosphorylation of p62 and GAP was not markedly affected by deletion of the kinase insert region. These results indicate that the kinase insert region selectively enhances the CSF-1-dependent association of the CSF-1 receptor with active PI 3'-kinase. The insert deletion mutant retains considerable transforming activity in NIH 3T3 cells (G. Taylor, M. Reedijk, V. Rothwell, L. Rohrschneider, and T. Pawson, EMBO J. 8:2029-2037, 1989). This mutant was more seriously impaired in Rat-2 cell transformation, although mutant-expressing Rat-2 cells still formed small colonies in soft agar in the presence of CSF-1. Therefore, phosphorylation of GAP and p62 through activation of the CSF-1 receptor does not result in full fibroblast transformation. The interaction between the CSF-1 receptor and PI 3'-kinase may contribute to c-fms fibroblast transformation and play a role in CSF-1-stimulated macrophages. Images PMID:2172781

Functionalised nanoscale coatings using layer-by-layer assembly for imparting antibacterial properties to polylactide-co-glycolide surfaces.

PubMed

Gentile, Piergiorgio; Frongia, Maria E; Cardellach, Mar; Miller, Cheryl A; Stafford, Graham P; Leggett, Graham J; Hatton, Paul V

2015-07-01

In order to achieve high local biological activity and reduce the risk of side effects of antibiotics in the treatment of periodontal and bone infections, a localised and temporally controlled delivery system is desirable. The aim of this research was to develop a functionalised and resorbable surface to contact soft tissues to improve the antibacterial behaviour during the first week after its implantation in the treatment of periodontal and bone infections. Solvent-cast poly(d,l-lactide-co-glycolide acid) (PLGA) films were aminolysed and then modified by Layer-by-Layer technique to obtain a nano-layered coating using poly(sodium4-styrenesulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) as polyelectrolytes. The water-soluble antibiotic, metronidazole (MET), was incorporated from the ninth layer. Infrared spectroscopy showed that the PSS and PAH absorption bands increased with the layer number. The contact angle values had a regular alternate behaviour from the ninth layer. X-ray Photoelectron Spectroscopy evidenced two distinct peaks, N1s and S2p, indicating PAH and PSS had been introduced. Atomic Force Microscopy showed the presence of polyelectrolytes on the surface with a measured roughness about 10nm after 20 layers' deposition. The drug release was monitored by Ultraviolet-visible spectroscopy showing 80% loaded-drug delivery in 14 days. Finally, the biocompatibility was evaluated in vitro with L929 mouse fibroblasts and the antibacterial properties were demonstrated successfully against the keystone periodontal bacteria Porphyromonas gingivalis, which has an influence on implant failure, without compromising in vitro biocompatibility. In this study, PLGA was successfully modified to obtain a localised and temporally controlled drug delivery system, demonstrating the potential value of LbL as a coating technology for the manufacture of medical devices with advanced functional properties. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Alleviation of Ultraviolet B-Induced Photodamage by Coffea arabica Extract in Human Skin Fibroblasts and Hairless Mouse Skin

PubMed Central

Wu, Po-Yuan; Huang, Chi-Chang; Chu, Yin; Huang, Ya-Han; Lin, Ping; Liu, Yu-Han; Wen, Kuo-Ching; Lin, Chien-Yih; Hsu, Mei-Chich; Chiang, Hsiu-Mei

2017-01-01

Coffea arabica extract (CAE) containing 48.3 ± 0.4 mg/g of chlorogenic acid and a trace amount of caffeic acid was found to alleviate photoaging activity in human skin fibroblasts. In this study, polyphenol-rich CAE was investigated for its antioxidant and antiinflammatory properties, as well as for its capability to alleviate ultraviolet B (UVB)-induced photodamage in BALB/c hairless mice. The results indicated that 500 μg/mL of CAE exhibited a reducing power of 94.7%, ferrous ion chelating activity of 46.4%, and hydroxyl radical scavenging activity of 20.3%. The CAE dose dependently reduced UVB-induced reactive oxygen species (ROS) generation in fibroblasts. Furthermore, CAE inhibited the UVB-induced expression of cyclooxygenase-2 and p-inhibitor κB, and the translocation of nuclear factor-kappa B (NF-κB) to the nucleus of fibroblasts. In addition, CAE alleviated UVB-induced photoaging and photodamage in BALB/c hairless mice by restoring the collagen content and reduced UVB-induced epidermal hyperplasia. CAE also inhibited UVB-induced NF-κB, interleukin-6, and matrix metalloproteinase-1 expression in the hairless mouse skin. The results indicated that CAE exhibits antiphotodamage activity by inhibiting UV-induced oxidative stress and inflammation. Therefore, CAE is a candidate for use in antioxidant, antiinflammatory, and antiphotodamage products. PMID:28387707

Reprogramming fibroblasts into induced pluripotent stem cells with Bmi1

PubMed Central

Moon, Jai-Hee; Heo, June Seok; Kim, Jun Sung; Jun, Eun Kyoung; Lee, Jung Han; Kim, Aeree; Kim, Jonggun; Whang, Kwang Youn; Kang, Yong-Kook; Yeo, Seungeun; Lim, Hee-Joung; Han, Dong Wook; Kim, Dong-Wook; Oh, Sejong; Yoon, Byung Sun; Schöler, Hans R; You, Seungkwon

2011-01-01

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by the transcription factors Oct4, Sox2, and Klf4 in combination with c-Myc. Recently, Sox2 plus Oct4 was shown to reprogram fibroblasts and Oct4 alone was able to reprogram mouse and human neural stem cells (NSCs) into iPS cells. Here, we report that Bmi1 leads to the transdifferentiation of mouse fibroblasts into NSC-like cells, and, in combination with Oct4, can replace Sox2, Klf4 and c-Myc during the reprogramming of fibroblasts into iPS cells. Furthermore, activation of sonic hedgehog signaling (by Shh, purmorphamine, or oxysterol) compensates for the effects of Bmi1, and, in combination with Oct4, reprograms mouse embryonic and adult fibroblasts into iPS cells. One- and two-factor iPS cells are similar to mouse embryonic stem cells in their global gene expression profile, epigenetic status, and in vitro and in vivo differentiation into all three germ layers, as well as teratoma formation and germline transmission in vivo. These data support that converting fibroblasts with Bmi1 or activation of the sonic hedgehog pathway to an intermediate cell type that expresses Sox2, Klf4, and N-Myc allows iPS generation via the addition of Oct4. PMID:21709693

Depletion of stromal cells expressing fibroblast activation protein-α from skeletal muscle and bone marrow results in cachexia and anemia

PubMed Central

Roberts, Edward W.; Deonarine, Andrew; Jones, James O.; Denton, Alice E.; Feig, Christine; Lyons, Scott K.; Espeli, Marion; Kraman, Matthew; McKenna, Brendan; Wells, Richard J.B.; Zhao, Qi; Caballero, Otavia L.; Larder, Rachel; Coll, Anthony P.; O’Rahilly, Stephen; Brindle, Kevin M.; Teichmann, Sarah A.; Tuveson, David A.

2013-01-01

Fibroblast activation protein-α (FAP) identifies stromal cells of mesenchymal origin in human cancers and chronic inflammatory lesions. In mouse models of cancer, they have been shown to be immune suppressive, but studies of their occurrence and function in normal tissues have been limited. With a transgenic mouse line permitting the bioluminescent imaging of FAP+ cells, we find that they reside in most tissues of the adult mouse. FAP+ cells from three sites, skeletal muscle, adipose tissue, and pancreas, have highly similar transcriptomes, suggesting a shared lineage. FAP+ cells of skeletal muscle are the major local source of follistatin, and in bone marrow they express Cxcl12 and KitL. Experimental ablation of these cells causes loss of muscle mass and a reduction of B-lymphopoiesis and erythropoiesis, revealing their essential functions in maintaining normal muscle mass and hematopoiesis, respectively. Remarkably, these cells are altered at these sites in transplantable and spontaneous mouse models of cancer-induced cachexia and anemia. Thus, the FAP+ stromal cell may have roles in two adverse consequences of cancer: their acquisition by tumors may cause failure of immunosurveillance, and their alteration in normal tissues contributes to the paraneoplastic syndromes of cachexia and anemia. PMID:23712428

Toxicity of extracts from disposable chopsticks, toothpicks, and paper cups on L-929 cells.

PubMed

Li, Juntao; Chen, Sifan; Li, Wenxue; Yang, Guangyu; Zhu, Wei

2015-04-01

To evaluate the toxicity of extracts from disposable chopsticks, toothpicks, and paper cups on L-929 cells. We followed national standards to prepare the extracts from disposable chopsticks, toothpicks, and paper cups used for the cell culture media, and the morphology of L-929 cells was observed with an optical microscope. The loss rate for adherent cells was evaluated with the trypan blue exclusion method, and cell proliferation was determined using the WST-1 assay. Compared with the control group, the cells cultured in media containing the extracts showed signs of apoptosis and necrosis after culturing for 4 or 7 days, and the loss rate for adherent cells was significantly increased (P < 0.05). An obvious decrease in cell viability was also observed (P < 0.05). The extracts from disposable chopsticks, toothpicks, and paper cups can affect the growth and proliferation of L-929 cells and are potentially toxic to humans.

The effect of CO2 laser beam welded AISI 316L austenitic stainless steel on the viability of fibroblast cells, in vitro.

PubMed

Köse, Ceyhun; Kaçar, Ramazan; Zorba, Aslı Pınar; Bağırova, Melahat; Allahverdiyev, Adil M

2016-03-01

It has been determined by the literature research that there is no clinical study on the in vivo and in vitro interaction of the cells with the laser beam welded joints of AISI 316L biomaterial. It is used as a prosthesis and implant material and that has adequate mechanical properties and corrosion resistance characteristics. Therefore, the interaction of the CO2 laser beam welded samples and samples of the base metal of AISI 316L austenitic stainless steel with L929 fibroblast cells as an element of connective tissue under in vitro conditions has been studied. To study the effect of the base metal and the laser welded test specimens on the viability of the fibroblast cells that act as an element of connective tissues in the body, they were kept in DMEMF-12 medium for 7, 14, 28 days and 18 months. The viability study was experimentally studied using the MTT method for 7, 14, 28 days. In addition, the direct interaction of the fibroblast cells seeded on 6 different plates with the samples was examined with an inverted microscope. The MTT cell viability experiment was repeated on the cells that were in contact with the samples. The statistical relationship was analyzed using a Tukey test for the variance with the GraphPad statistics software. The data regarding metallic ion release were identified with the ICP-MS method after the laser welded and main material samples were kept in cell culture medium for 18 months. The cell viability of the laser welded sample has been detected to be higher than that of the base metal and the control based on 7th day data. However, the laser welded sample's viability of the fibroblast cells has diminished by time during the test period of 14 and 28 days and base metal shows better viability when compared to the laser welded samples. On the other hand, the base metal and the laser welded sample show better cell viability effect when compared to the control group. According to the ICP-MS results of the main material and laser welded samples which were kept in the cell culture medium for 18 months, it was determined that the Fe, Ni and Cr ion concentration released to the cell culture medium from the laser welded test sample was less than that of the main material. Copyright © 2015 Elsevier B.V. All rights reserved.

MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures.

PubMed

Muraoka, Naoto; Yamakawa, Hiroyuki; Miyamoto, Kazutaka; Sadahiro, Taketaro; Umei, Tomohiko; Isomi, Mari; Nakashima, Hanae; Akiyama, Mizuha; Wada, Rie; Inagawa, Kohei; Nishiyama, Takahiko; Kaneda, Ruri; Fukuda, Toru; Takeda, Shu; Tohyama, Shugo; Hashimoto, Hisayuki; Kawamura, Yoshifumi; Goshima, Naoki; Aeba, Ryo; Yamagishi, Hiroyuki; Fukuda, Keiichi; Ieda, Masaki

2014-07-17

Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors or microRNAs. However, induction of functional cardiomyocytes is inefficient, and molecular mechanisms of direct reprogramming remain undefined. Here, we demonstrate that addition of miR-133a (miR-133) to Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Mesp1 and Myocd improved cardiac reprogramming from mouse or human fibroblasts by directly repressing Snai1, a master regulator of epithelial-to-mesenchymal transition. MiR-133 overexpression with GMT generated sevenfold more beating iCMs from mouse embryonic fibroblasts and shortened the duration to induce beating cells from 30 to 10 days, compared to GMT alone. Snai1 knockdown suppressed fibroblast genes, upregulated cardiac gene expression, and induced more contracting iCMs with GMT transduction, recapitulating the effects of miR-133 overexpression. In contrast, overexpression of Snai1 in GMT/miR-133-transduced cells maintained fibroblast signatures and inhibited generation of beating iCMs. MiR-133-mediated Snai1 repression was also critical for cardiac reprogramming in adult mouse and human cardiac fibroblasts. Thus, silencing fibroblast signatures, mediated by miR-133/Snai1, is a key molecular roadblock during cardiac reprogramming. © 2014 The Authors.

MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures

PubMed Central

Muraoka, Naoto; Yamakawa, Hiroyuki; Miyamoto, Kazutaka; Sadahiro, Taketaro; Umei, Tomohiko; Isomi, Mari; Nakashima, Hanae; Akiyama, Mizuha; Wada, Rie; Inagawa, Kohei; Nishiyama, Takahiko; Kaneda, Ruri; Fukuda, Toru; Takeda, Shu; Tohyama, Shugo; Hashimoto, Hisayuki; Kawamura, Yoshifumi; Goshima, Naoki; Aeba, Ryo; Yamagishi, Hiroyuki; Fukuda, Keiichi; Ieda, Masaki

2014-01-01

Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors or microRNAs. However, induction of functional cardiomyocytes is inefficient, and molecular mechanisms of direct reprogramming remain undefined. Here, we demonstrate that addition of miR-133a (miR-133) to Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Mesp1 and Myocd improved cardiac reprogramming from mouse or human fibroblasts by directly repressing Snai1, a master regulator of epithelial-to-mesenchymal transition. MiR-133 overexpression with GMT generated sevenfold more beating iCMs from mouse embryonic fibroblasts and shortened the duration to induce beating cells from 30 to 10 days, compared to GMT alone. Snai1 knockdown suppressed fibroblast genes, upregulated cardiac gene expression, and induced more contracting iCMs with GMT transduction, recapitulating the effects of miR-133 overexpression. In contrast, overexpression of Snai1 in GMT/miR-133-transduced cells maintained fibroblast signatures and inhibited generation of beating iCMs. MiR-133-mediated Snai1 repression was also critical for cardiac reprogramming in adult mouse and human cardiac fibroblasts. Thus, silencing fibroblast signatures, mediated by miR-133/Snai1, is a key molecular roadblock during cardiac reprogramming. PMID:24920580

Biocompatibility of Titania Nanotube Coatings Enriched with Silver Nanograins by Chemical Vapor Deposition

PubMed Central

Piszczek, Piotr; Lewandowska, Żaneta; Radtke, Aleksandra; Kozak, Wiesław; Sadowska, Beata; Szubka, Magdalena; Talik, Ewa; Fiori, Fabrizio

2017-01-01

Bioactivity investigations of titania nanotube (TNT) coatings enriched with silver nanograins (TNT/Ag) have been carried out. TNT/Ag nanocomposite materials were produced by combining the electrochemical anodization and chemical vapor deposition methods. Fabricated coatings were characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and Raman spectroscopy. The release effect of silver ions from TNT/Ag composites immersed in bodily fluids, has been studied using inductively coupled plasma mass spectrometry (ICP-MS). The metabolic activity assay (MTT) was applied to determine the L929 murine fibroblasts adhesion and proliferation on the surface of TNT/Ag coatings. Moreover, the results of immunoassays (using peripheral blood mononuclear cells—PBMCs isolated from rats) allowed the estimation of the immunological activity of TNT/Ag surface materials. Antibacterial activity of TNT/Ag coatings with different morphological and structural features was estimated against two Staphylococcus aureus strains (ATCC 29213 and H9). The TNT/Ag nanocomposite layers produced revealed a good biocompatibility promoting the fibroblast adhesion and proliferation. A desirable anti-biofilm activity against the S. aureus reference strain was mainly noticed for these TiO2 nanotube coatings, which contain dispersed Ag nanograins deposited on their surface. PMID:28914821

Beclin 1 is involved in regulation of apoptosis and autophagy during replication of ectromelia virus in permissive L929 cells.

PubMed

Martyniszyn, Lech; Szulc, Lidia; Boratyńska, Anna; Niemiałtowski, Marek G

2011-12-01

Several reports have brought to light new and interesting findings on the involvement of autophagy and apoptosis in pathogenesis of viral and bacterial diseases, as well as presentation of foreign antigens. Our model studies focused on the involvement of apoptosis during replication of highly virulent Moscow strain of ectromelia virus (ECTV-MOS). Here, we show evidence that autophagy is induced during mousepox replication in a cell line. Fluorescence microscopy revealed increase of LC3 (microtubule-associated protein 1 light chain 3) aggregation in infected as opposed to non-infected control L929 cells. Furthermore, Western blot analysis showed that replication of ECTV-MOS in L929 cells led to the increase in LC3-II (marker of autophagic activity) expression. Beclin 1 strongly colocalized with extranuclear viral replication centers in infected cells, whereas expression of Bcl-2 decreased in those centers as shown by fluorescence microscopy. Loss of Beclin 1-Bcl-2 interaction may lead to autophagy in virus-infected L929 cells. To assess if Beclin 1 has a role in regulation of apoptosis during ECTV-MOS infection, we used small interfering RNA directed against beclin 1 following infection. Early and late apoptotic cells were analyzed by flow cytometry after AnnexinV and propidium iodide staining. Silencing of beclin 1 resulted in decreased percentage of early and late apoptotic cells in the late stage of ECTV-MOS infection in L929 cells. We conclude that Beclin 1 plays an important role in regulation of both, autophagy and apoptosis, during ECTV-MOS replication in L929 permissive cells.

Conditional knockout of N-WASP in mouse fibroblast caused keratinocyte hyper proliferation and enhanced wound closure

PubMed Central

Jain, Neeraj; Kalailingam, Pazhanichamy; Tan, Kai Wei; Tan, Hui Bing; Sng, Ming Keat; Chan, Jeremy Soon Kiat; Tan, Nguan Soon; Thanabalu, Thirumaran

2016-01-01

Neural-Wiskott Aldrich Syndrome Protein (N-WASP) is expressed ubiquitously, regulates actin polymerization and is essential during mouse development. We have previously shown that N-WASP is critical for cell-ECM adhesion in fibroblasts. To characterize the role of N-WASP in fibroblast for skin development, we generated a conditional knockout mouse model in which fibroblast N-WASP was ablated using the Cre recombinase driven by Fibroblast Specific Protein promoter (Fsp-Cre). N-WASPFKO (N-WASPfl/fl; Fsp-cre) were born following Mendelian genetics, survived without any visible abnormalities for more than 1 year and were sexually reproductive, suggesting that expression of N-WASP in fibroblast is not critical for survival under laboratory conditions. Histological sections of N-WASPFKO mice skin (13 weeks old) showed thicker epidermis with higher percentage of cells staining for proliferation marker (PCNA), suggesting that N-WASP deficient fibroblasts promote keratinocyte proliferation. N-WASPFKO mice skin had elevated collagen content, elevated expression of FGF7 (keratinocyte growth factor) and TGFβ signaling proteins. Wound healing was faster in N-WASPFKO mice compared to control mice and N-WASP deficient fibroblasts were found to have enhanced collagen gel contraction properties. These results suggest that N-WASP deficiency in fibroblasts improves wound healing by growth factor-mediated enhancement of keratinocyte proliferation and increased wound contraction in mice. PMID:27909303

HPLC-DAD phenolic profile, cytotoxic and anti-kinetoplastidae activity of Melissa officinalis.

PubMed

Cunha, Francisco; Tintino, Saulo R; Figueredo, Fernando; Barros, Luiz; Duarte, Antonia E; Vega Gomez, Maria Celeste; Coronel, Cathia Cecilia; Rolón, Mírian; Leite, Nadghia; Sobral-Souza, Celestina E; Brito, S V; Waczuc, Emily Pansera; Boligon, Aline Augusti; Athayde, Margareth; Kamdem, Jean Paul; Coutinho, Henrique Douglas Melo; Franco, Jéferson

2016-09-01

Context Melissa officinalis subsp. inodora Bornm. (Lamiaceae) has been used since ancient times in folk medicine against various diseases, but it has not been investigated against protozoa. Objective To evaluate the activities of M. officinalis against Leishmania braziliensis, Leishmania infantum and Trypanosoma cruzi as well as its cytotoxicity in fibroblast cell line. Materials and methods The fresh leaves were chopped into 1 cm(2) pieces, washed and macerated with 99.9% of ethanol for 72 h at room temperature. Antiparasitic activity of M. officinalis was accessed by direct counting of cells after serial dilution, while the cytotoxicity of M. officinalis was evaluated in fibroblast cell line (NCTC929) by measuring the reduction of resazurin. The test duration was 24 h. High-performance liquid chromatography (HPLC) was used to characterise the extract. Results The extract at concentrations of 250 and 125 μg/mL inhibited 80.39 and 54.27% of promastigote (LC50  value = 105.78 μg/mL) form of L. infantum, 80.59 and 68.61% of L. brasiliensis (LC50 value  = 110.69 μg/mL) and against epimastigote (LC50 value  = 245.23 μg/mL) forms of T. cruzi with an inhibition of 54.45 and 22.26%, respectively, was observed. The maximum toxicity was noted at 500 μg/mL with 95.41% (LC50  value = 141.01 μg/mL). The HPLC analysis identified caffeic acid and rutin as the major compounds. Discussion The inhibition of the parasites is considered clinically relevant (< 500 μg/mL). Rutin and caffeic acids may be responsible for the antiprotozoal effect of the extract. Conclusion The ethanol extract of M. officinalis can be considered a potential alternative source of natural products with antileishmania and antitrypanosoma activities.

Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line

PubMed Central

Shafaghat, Farzaneh; Abbasi-Kenarsari, Hajar; Majidi, Jafar; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

2015-01-01

Purpose: Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs). We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. Methods: CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively) as shown by flow cytometry. Conclusion: Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems. PMID:25789221

Development of ethyl alcohol-precipitated silk sericin/polyvinyl alcohol scaffolds for accelerated healing of full-thickness wounds.

PubMed

Siritienthong, Tippawan; Ratanavaraporn, Juthamas; Aramwit, Pornanong

2012-12-15

Silk sericin has been recently reported for its advantageous biological properties to promote wound healing. In this study, we established that the ethyl alcohol (EtOH) could be used to precipitate sericin and form the stable sericin/polyvinyl alcohol (PVA) scaffolds without the crosslinking. The sericin/PVA scaffolds were fabricated via freeze-drying and subsequently precipitating in various concentrations of EtOH. The EtOH-precipitated sericin/PVA scaffolds showed denser structure, higher compressive modulus, but lower water swelling ability than the non-precipitated scaffolds. Sericin could be released from the EtOH-precipitated sericin/PVA scaffolds in a sustained manner. After cultured with L929 mouse fibroblasts, the 70 vol% EtOH-precipitated sericin/PVA scaffolds showed the highest potential to promote cell proliferation. After applied to the full-thickness wounds of rats, the 70 vol% EtOH-precipitated sericin/PVA scaffolds showed significantly higher percentage of wound size reduction and higher extent of type III collagen formation and epithelialization, compared with the control scaffolds without sericin. The accelerated wound healing by the 70 vol% EtOH-precipitated sericin/PVA scaffolds was possibly due to (1) the bioactivity of sericin itself to promote wound healing, (2) the sustained release of precipitated sericin from the scaffolds, and (3) the activation and recruitment of wound healing-macrophages by sericin to the wounds. This finding suggested that the EtOH-precipitated sericin/PVA scaffolds were more effective for the wound healing, comparing with the EtOH-precipitated PVA scaffolds without sericin. Copyright © 2012 Elsevier B.V. All rights reserved.

Synthesis, characterization and antioxidant activity of a novel electroactive and biodegradable polyurethane for cardiac tissue engineering application.

PubMed

Baheiraei, Nafiseh; Yeganeh, Hamid; Ai, Jafar; Gharibi, Reza; Azami, Mahmoud; Faghihi, Faezeh

2014-11-01

There has been a growing trend towards applying conducting polymers for electrically excitable cells to increase electrical signal propagation within the cell-loaded substrates. A novel biodegradable electroactive polyurethane containing aniline pentamer (AP-PU) was synthesized and fully characterized by spectroscopic methods. To tune the physico-chemical properties and biocompatibility, the AP-PU was blended with polycaprolactone (PCL). The presence of electroactive moieties and the electroactivity behavior of the prepared films were confirmed by UV-visible spectroscopy and cyclic voltammetry. A conventional four probe analysis demonstrated the electrical conductivity of the films in the semiconductor range (~10(-5)S/cm). MTT assays using L929 mouse fibroblast and human umbilical vein endothelial cells (HUVECs) showed that the prepared blend (PB) displayed more cytocompatibility compared with AP-PU due to the introduction of a biocompatible PCL moiety. The in vitro cell culture also confirmed that PB was as supportive as tissue culture plate. The antioxidant activity of the AP-PU was proved using 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay by employing UV-vis spectroscopy. In vitro degradation tests conducted in phosphate-buffered saline, pH7.4 and pH5.5, proved that the films were also biodegradable. The results of this study have highlighted the potential application of this bioelectroactive polyurethane as a platform substrate to study the effect of electrical signals on cell activities and to direct desirable cell function for tissue engineering applications. Copyright © 2014 Elsevier B.V. All rights reserved.

Cytotoxicity of dental glass ionomers evaluated using dimethylthiazol diphenyltetrazolium and neutral red tests.

PubMed

Lönnroth, E C; Dahl, J E

2001-02-01

The purpose of this study was to assess the cytotoxicity of some commonly used glass ionomers. Three chemically cured glass ionomers (Fuji II, Lining cement, and Ketac Silver) and one light-cured (Fuji II LC) were tested. Extracts of mixed non-polymerized materials and polymerized specimens were prepared in accordance with ISO standard 10993-12. The polymerized specimens were cured and placed either directly in the medium (freshly cured), left for 24 h (aged), or aged plus ground before being placed in the medium. The cytotoxicity of extracts was evaluated on mouse fibroblasts (L, 929), using dimethylthiazol diphenyltetrazolium (MTT) and neutral red (NR) assays. Further, the concentrations of aluminum, arsenic and lead were analyzed in aqueous extracts from freshly cured and aged samples, and the fluoride levels analyzed in aqueous extracts from freshly cured samples. All extracts except that of non-polymerized Ketac Silver were rated as severely cytotoxic in both assays. Extracts of polymerized material were significantly more cytotoxic than extracts of non-polymerized material. All freshly cured glass ionomers released aluminum and fluoride concentrations far above what is considered cytotoxic (aluminum >0.2 ppm and fluoride >20 ppm). Extracts from freshly cured Lining Cement contained the highest concentrations of aluminum and fluoride (215 ppm and 112 ppm). Extracts from freshly cured Ketac Silver had the lowest concentrations of aluminum and fluoride but the highest of lead (100 ppm). It can be concluded that all extracts from non-cured, freshly cured, and aged glass ionomers contained cytotoxic levels of substances. Curing did not reduce the toxicity significantly.

Glycogen-gold nanohybrid escalates the potency of silymarin.

PubMed

Kandimalla, Raghuram; Dash, Suvakanta; Bhowal, Ashim Chandra; Kalita, Sanjeeb; Talukdar, Narayan Chandra; Kundu, Sarathi; Kotoky, Jibon

2017-01-01

In this study, a glycogen-gold nanohybrid was fabricated to enhance the potency of a promising hepatoprotective agent silymarin (Sly) by improving its solubility and gut permeation. By utilizing a facile green chemistry approach, biogenic gold nanoparticles were synthesized from Annona reticulata leaf phytoconstituents in combination with Sly (SGNPs). Further, the SGNPs were aggregated in glycogen biopolymer to yield the therapeutic nanohybrids (GSGNPs). Transmission electron microscopy, UV-Vis spectroscopy, X-ray diffraction, and Fourier transform infrared spectroscopy analysis confirmed the successful formation and conjugation of both SGNPs and GSGNPs. The fabricated nanohybrids showed significant protection against CCl 4 -induced hepatic injury in Wistar rats and maintained natural antioxidant (superoxide dismutase and catalase) levels. Animals treated with GSGNPs (10 mg/kg) and SGNPs (20 mg/kg) retained usual hepatic functions with routine levels of hepatobiliary enzymes (aspartate transferase, alanine transaminase, alkaline phosphatase, and lactate dehydrogenase) and inflammatory markers (interleukin-1β and tumor necrosis factor-α) with minimal lipid peroxidation, whereas those treated with 100 mg/kg of Sly showed the similar effect. These results were also supported by histopathology of the livers where pronounced hepatoprotection with normal hepatic physiology and negligible inflammatory infiltrate were observed. Significant higher plasma C max supported the enhanced bioavailability of Sly upon GSGNPs treatment compared to SGNPs and free Sly. Graphite furnace atomic absorption spectrophotometry analysis also substantiated the efficient delivery of GSGNPs over SGNPs. The fabricated therapeutic nanohybrids were also found to be biocompatible toward human erythrocytes and L929 mouse fibroblast cells. Overall, due to increased solubility, bioavailability and profuse gut absorption; GSGNPs demonstrated tenfold enhanced potency compared to free Sly.

Synthesis, characterization and in vitro anticancer evaluations of two novel derivatives of deferasirox iron chelator.

PubMed

Salehi, Samie; Saljooghi, Amir Sh; Shiri, Ali

2016-06-15

Iron (Fe) chelation therapy was initially designed to alleviate the toxic effects of excess Fe evident in Fe-overload diseases. However, the novel toxicological properties of some Fe chelator-metal complexes have shifted significant attention to their application in cancer chemotherapy. The present study investigates the new role of deferasirox as an anticancer agent due to its ability to chelate with iron. Because of aminoacids antioxidant effect, deferasirox and its two novel amino acid derivatives have been synthesized through the treatment of deferasirox with DCC as well as glycine or phenylalanine methyl ester. All new compounds have been characterized by elemental analysis, FT-IR NMR and mass spectrometry. Therefore, the cytotoxicity of these compounds was screened for antitumor activity against some cell lines using cisplatin as a comparative standard by MTT assay and Flow cytometry. The impact of iron in the intracellular generation of reactive oxygen species was assessed on HT29 and MDA-MB-231 cells. The potential of the synthesized iron chelators for their efficacy to protect cells against model oxidative injury induced was compared. The reactive oxygen species intracellular fluorescence intensity were measured and the result showed that the reactive oxygen species intensity after iron incubation increased while after chelators incubation the reactive oxygen species intensity were decreased significantly. Besides, the effect of the synthesized compounds on mouse fibroblast cell line (L929) was simultaneously evaluated as control. The pharmacological results showed that deferasirox and its two novel aminoacid derivatives were potent anticancer agents. Copyright © 2016 Elsevier B.V. All rights reserved.

Relationship between microstructure, cytotoxicity and corrosion properties of a Cu-Al-Ni shape memory alloy.

PubMed

Colić, Miodrag; Rudolf, Rebeka; Stamenković, Dragoslav; Anzel, Ivan; Vucević, Dragana; Jenko, Monika; Lazić, Vojkan; Lojen, Gorazd

2010-01-01

Cu-Al-Ni shape memory alloys (SMAs) have been investigated as materials for medical devices, but their biomedical application is still limited. The aim of this work was to compare the microstructure, corrosion and cytotoxicity in vitro of a Cu-Al-Ni SMA. Rapidly solidified (RS) thin ribbons, manufactured via melt spinning, were used for the tests. The control alloy was a permanent mould casting of the same composition, but without shape memory effect. The results show that RS ribbons are significantly more resistant to corrosion compared with the control alloy, as judged by the lesser release of Cu and Ni into the conditioning medium. These results correlate with the finding that RS ribbons were not cytotoxic to L929 mouse fibroblasts and rat thymocytes. In addition, the RS ribbon conditioning medium inhibited cellular proliferation and IL-2 production by activated rat splenocytes to a much lesser extent. The inhibitory effects were almost completely abolished by conditioning the RS ribbons in culture medium for 4 weeks. Microstructural analysis showed that RS ribbons are martensitic, with boron particles as a minor phase. In contrast, the control Cu-Al-Ni alloy had a complex multiphase microstructure. Examination of the alloy surfaces after conditioning by energy dispersive X-ray and Auger electron spectroscopy showed the formation of Cu and Al oxide layers and confirmed that the metals in RS ribbons are less susceptible to oxidation and corrosion compared with the control alloy. In conclusion, these results suggest that rapid solidification significantly improves the corrosion stability and biocompatibility in vitro of Cu-Al-Ni SMA ribbons.

Densification, phase stability and in vitro biocompatibility property of hydroxyapatite-10 wt% silver composites.

PubMed

Nath, Shekhar; Kalmodia, Sushma; Basu, Bikramjit

2010-04-01

In this paper, we demonstrate how a simple fabrication route, i.e., pressureless sintering of mechanically mixed powders can be employed to develop hydroxyapatite (HAp, Ca(10)(PO(4))(6)(OH)(2))-silver (Ag) bioceramic composites with superior combination of physical (hardness, toughness), non-cytotoxicity, cytocompatiblity and anti-microbial property. The densification results show that such composites can be sintered at 1200 degrees C for 2 h near to theoretical density (>98% rho(th).) An important observation is that the dissociation of HAp phase can be prevented during sintering up to 1300 degrees C for 2 h in HAp-10 wt% Ag composites. The stability of HAp in presence of silver is discussed in reference to the results obtained using XRD, FTIR and Raman spectroscopy. The hardness values of the composites are comparable (approximately 6.5 GPa) to that of pure HAp, despite of the presence of softer Ag particles. The sintered composites exhibit modest crack growth resistance property and their toughness varies in the range of 0.9-1.2 MPa m(0.5), depending on sintering temperature. For selected samples, the in vitro characterization was performed using mouse fibroblast (L929) and human osteosarcoma (MG63) cell lines. The combination of biochemical assays (MTT, ALP and osteocalcin) confirm that HAp-10 wt% Ag biocomposites have comparable or even better cellular viability, osteogenic differentiation and bone mineralization as well as osteoinduction property. Antibacterial experiments involving gram-negative bacteria, Escherichia coli confirm excellent bactericidal property of HAp-10 wt% Ag composites, sintered using mechanically mixed powders.

Comparative evaluation of cytotoxicity of a glucosamine-TBA conjugate and a chitosan-TBA conjugate.

PubMed

Guggi, Davide; Langoth, Nina; Hoffer, Martin H; Wirth, Michael; Bernkop-Schnürch, Andreas

2004-07-08

D-glucosamine and chitosan were modified by the immobilization of thiol groups utilizing 2-iminothiolane. The toxicity profile of the resulting D-glucosamine-TBA (4-thiobutylamidine) conjugate, of chitosan-TBA conjugate and of the corresponding unmodified controls was evaluated in vitro. On the one hand, the cell membrane damaging effect of 0.025% solutions of the test compounds was investigated via red blood cell lysis test. On the other hand, the cytotoxity of 0.025, 0.25 and 0.5% solutions of the test compounds was evaluated on L-929 mouse fibroblast cells utilizing two different bioassays: the MTT assay (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide), which assess the mitochondrial metabolic activity of the cells, and the BrdU-based enzyme-linked immunosorbent assay, which measures the incorporation in the DNA of 5-bromo-2'-deoxyuridine and consequently the cell proliferation. Results of the red blood cell lysis test showed that both thiolated compounds displayed a lower membrane damaging effect causing a significantly lower haemoglobine release than the unmodified compounds. Data obtained by the MTT assay and the BrdU assay revealed a concentration dependent relative cytotoxicity for all tested compounds. The covalent linkage of the TBA-substructure to D-glucosamine did not cause a significant increase in cytotoxicity, whereas at higher concentrations a slightly enhanced cytotoxic effect was caused by the derivatisation of chitosan. In conclusion, the -TBA derivatives show a comparable toxicity profile to the corresponding unmodified compounds, which should not compromise their future use as save pharmaceutical excipients.

Laser cutting: influence on morphological and physicochemical properties of polyhydroxybutyrate.

PubMed

Lootz, D; Behrend, D; Kramer, S; Freier, T; Haubold, A; Benkiesser, G; Schmitz, K P; Becher, B

2001-09-01

Polyhydroxybutyrate (PHB) is a biocompatible and resorbable implant material. For these reasons, it has been used for the fabrication of temporary stents, bone plates, nails and screws (Peng et al. Biomaterials 1996;17:685). In some cases, the brittle mechanical properties of PHB homopolymer limit its application. A typical plasticizer, triethylcitrate (TEC), was used to overcome such limitations by making the material more pliable. In the past few years, CO2-laser cutting of PHB was used in the manufacturing of small medical devices such as stents. Embrittlement of plasticized PHB tubes has been observed, after laser machining. Consequently, the physicochemical and morphological properties of laser-processed surfaces and cut edges of plasticized polymer samples were examined to determine the extent of changes in polymer properties as a result of laser machining. These studies included determination of the depth of the laser-induced heat affected zone by polariscopy of thin polymer sections. Molecular weight changes and changes in the TEC content as a function of distance from the laser-cut edge were determined. In a preliminary test, the cellular response to the processed material was investigated by cell culture study of L929 mouse fibroblasts on laser-machined surfaces. The heat-affected zone was readily classified into four different regions with a total depth of about 60 to 100 microm (Klamp, Master Thesis, University of Rostock, 1998). These results correspond well with the chemical analysis and molecular weight measurements. Furthermore, it was found that cells grew preferentially on the laser-machined area. These findings have significant implications for the manufacture of medical implants from PHB by laser machining.

Antimicrobial Efficacy of Two Surface Barrier Discharges with Air Plasma against In Vitro Biofilms

PubMed Central

Matthes, Rutger; Bender, Claudia; Schlüter, Rabea; Koban, Ina; Bussiahn, René; Reuter, Stephan; Lademann, Jürgen; Weltmann, Klaus-Dieter; Kramer, Axel

2013-01-01

The treatment of infected wounds is one possible therapeutic aspect of plasma medicine. Chronic wounds are often associated with microbial biofilms which limit the efficacy of antiseptics. The present study investigates two different surface barrier discharges with air plasma to compare their efficacy against microbial biofilms with chlorhexidine digluconate solution (CHX) as representative of an important antibiofilm antiseptic. Pseudomonas aeruginosa SG81 and Staphylococcus epidermidis RP62A were cultivated on polycarbonate discs. The biofilms were treated for 30, 60, 150, 300 or 600 s with plasma or for 600 s with 0.1% CHX, respectively. After treatment, biofilms were dispensed by ultrasound and the antimicrobial effects were determined as difference in the number of the colony forming units by microbial culture. A high antimicrobial efficacy on biofilms of both plasma sources in comparison to CHX treatment was shown. The efficacy differs between the used strains and plasma sources. For illustration, the biofilms were examined under a scanning electron microscope before and after treatment. Additionally, cytotoxicity was determined by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay with L929 mouse fibroblast cell line. The cell toxicity of the used plasma limits its applicability on human tissue to maximally 150 s. The emitted UV irradiance was measured to estimate whether UV could limit the application on human tissue at the given parameters. It was found that the UV emission is negligibly low. In conclusion, the results support the assumption that air plasma could be an option for therapy of chronic wounds. PMID:23894661

Evaluation of a bioceramic-based nanocomposite material for controlled delivery of a non-steroidal anti-inflammatory drug.

PubMed

Hesaraki, S; Moztarzadeh, F; Nezafati, N

2009-12-01

In this study, nanocomposite of 50wt% calcium sulfate and 50wt% nanocrystalline apatite was produced and its biocompatibility, physical and structural properties were compared with pure calcium sulfate (CS) cement. Indomethacin (IM), a non-steroidal anti-inflammatory drug, was also loaded on both CS and nanocomposite cements and its in vitro release was evaluated over a period of time. The effect of the loaded IM on basic properties of the cements was also investigated. Biocompatibility tests showed a partial cytotoxicity in CS cement due to the reduced number of viable mouse fibroblast L929 cells in contact with the samples as well as spherical morphologies of the cells. However, no cytotoxic effect was observed for nanocomposite cement and no significant difference was found between the number of the cells seeded in contact with this specimens and culture plate as control. Other results showed that the setting time and injectability of the nanocomposite cement was much higher than those of CS cement, whereas reverse result obtained for compressive strength. In addition, incorporation of IM into compositions slightly increased the initial setting time and injectability of the cements and did not change their compressive strength. While a fast IM release was observed from CS cement in which about 97% of the loaded drug was released during 48h, nanocomposite cement showed a sustained release behavior in which 80% of the loaded IM was liberated after 144h. Thus, the nanocomposite can be a more appropriate carrier than CS for controlled release of IM in bone defect treatments.

Chemical composition of the essential oil from basil (Ocimum basilicum Linn.) and its in vitro cytotoxicity against HeLa and HEp-2 human cancer cell lines and NIH 3T3 mouse embryonic fibroblasts.

PubMed

Kathirvel, Poonkodi; Ravi, Subban

2012-01-01

This study examines the chemical composition and in vitro anticancer activity of the essential oil from Ocimum basilicum Linn. (Lamiaceae), cultivated in the Western Ghats of South India. The chemical compositions of basil fresh leaves were identified by GC-MS: 11 components were identified. The major constituents were found to be methyl cinnamate (70.1%), linalool (17.5%), β-elemene (2.6%) and camphor (1.52%). The results revealed that this plant may belong to the methyl cinnamate and linalool chemotype. A methyl thiazol tetrazolium assay was used for in vitro cytotoxicity screening against the human cervical cancer cell line (HeLa), human laryngeal epithelial carcinoma cell line (HEp-2) and NIH 3T3 mouse embryonic fibroblasts. The IC(50) values obtained were 90.5 and 96.3 µg mL(-1), respectively, and the results revealed that basil oil has potent cytotoxicity.

Dual pharmacological inhibition of glutathione and thioredoxin systems synergizes to kill colorectal carcinoma stem cells.

PubMed

Tanaka, Genki; Inoue, Ken-Ichi; Shimizu, Takayuki; Akimoto, Kazumi; Kubota, Keiichi

2016-09-01

NRF2 stabilizes redox potential through genes for glutathione and thioredoxin antioxidant systems. Whether blockade of glutathione and thioredoxin is useful in eliminating cancer stem cells remain unknown. We used xenografts derived from colorectal carcinoma patients to investigate the pharmacological inhibition of glutathione and thioredoxin systems. Higher expression of five glutathione S-transferase isoforms (GSTA1, A2, M4, O2, and P1) was observed in xenograft-derived spheroids than in fibroblasts. Piperlongumine (2.5-10 μmol/L) and auranofin (0.25-4 μmol/L) were used to inhibit glutathione S-transferase π and thioredoxin reductase, respectively. Piperlongumine or auranofin alone up-regulated the expression of NRF2 target genes, but not TP53 targets. While piperlongumine showed modest cancer-specific cell killing (IC50 difference between cancer spheroids and fibroblasts: P = 0.052), auranofin appeared more toxic to fibroblasts (IC50 difference between cancer spheroids and fibroblasts: P = 0.002). The synergism of dual inhibition was evaluated by determining the Combination Index, based on the number of surviving cells with combination treatments. Molar ratios indicated synergism in cancer spheroids, but not in fibroblasts: (auranofin:piperlongumine) = 2:5, 1:5, 1:10, and 1:20. Cancer-specific cell killing was achieved at the following drug concentrations (auranofin:piperlongumine): 0.25:2.5 μmol/L, 0.5:2.5 μmol/L, or 0.25:5 μmol/L. The dual inhibition successfully decreased CD44v9 surface presentation and delayed tumor emergence in nude mouse. However, a small subpopulation persistently survived and accumulated phosphorylated histone H2A. Such "persisters" still retained lesser but significant tumorigenicity. Thus, dual inhibition of glutathione S-transferase π and thioredoxin reductase could be a feasible option for decreasing the tumor mass and CD44v9-positive fraction by disrupting redox regulation. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Photobiomodulation of wound healing via visible and infrared laser irradiation.

PubMed

Solmaz, Hakan; Ulgen, Yekta; Gulsoy, Murat

2017-05-01

Fibroblast cells are known to be one of the key elements in wound healing process, which has been under the scope of research for decades. However, the exact mechanism of photobiomodulation on wound healing is not fully understood yet. Photobiomodulation of 635 and 809 nm laser irradiation at two different energy densities were investigated with two independent experiments; first, in vitro cell proliferation and then in vivo wound healing. L929 mouse fibroblast cell suspensions were exposed with 635 and 809 nm laser irradiations of 1 and 3 J/cm 2 energy densities at 50 mW output power separately for the investigation of photobiomodulation in vitro. Viabilities of cells were examined by means of MTT assays performed at the 24th, 48th, and 72nd hours following the laser irradiations. Following the in vitro experiments, 1 cm long cutaneous incisional skin wounds on Wistar albino rats (n = 24) were exposed with the same laser sources and doses in vivo. Wound samples were examined on 3rd, 5th, and 7th days of healing by means of mechanical tensile strength tests and histological examinations. MTT assay results showed that 635 nm laser irradiation of both energy densities after 24 h were found to be proliferative. One joule per square centimeter laser irradiation results also had positive effect on cell proliferation after 72 h. However, 809 nm laser irradiation at both energy densities had neither positive nor negative affects on cell viability. In vivo experiment results showed that, 635 nm laser irradiation of both energy densities stimulated wound healing in terms of tensile strength, whereas 809 nm laser stimulation did not cause any stimulative effect. The results of mechanical tests were compatible with the histological evaluations. In this study, it is observed that 635 nm laser irradiations of low energy densities had stimulative effects in terms of cell proliferation in vitro and mechanical strength of incisions in vivo. However, 809 nm laser irradiations at the same doses did not have any positive effect.

Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells.

PubMed

Unger, Christian; Felldin, Ulrika; Rodin, Sergey; Nordenskjöld, Agneta; Dilber, Sirac; Hovatta, Outi

2016-02-03

After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures for research-grade human skin fibroblast lines. We also describe how to make feeder layers for hESCs using these fibroblasts. Copyright © 2016 John Wiley & Sons, Inc.

Mitotic Defects Lead to Pervasive Aneuploidy and Accompany Loss of RB1 Activity in Mouse LmnaDhe Dermal Fibroblasts

PubMed Central

Pratt, C. Herbert; Curtain, Michelle; Donahue, Leah Rae; Shopland, Lindsay S.

2011-01-01

Background Lamin A (LMNA) is a component of the nuclear lamina and is mutated in several human diseases, including Emery-Dreifuss muscular dystrophy (EDMD; OMIM ID# 181350) and the premature aging syndrome Hutchinson-Gilford progeria syndrome (HGPS; OMIM ID# 176670). Cells from progeria patients exhibit cell cycle defects in both interphase and mitosis. Mouse models with loss of LMNA function have reduced Retinoblastoma protein (RB1) activity, leading to aberrant cell cycle control in interphase, but how mitosis is affected by LMNA is not well understood. Results We examined the cell cycle and structural phenotypes of cells from mice with the Lmna allele, Disheveled hair and ears (LmnaDhe). We found that dermal fibroblasts from heterozygous LmnaDhe (LmnaDhe/+) mice exhibit many phenotypes of human laminopathy cells. These include severe perturbations to the nuclear shape and lamina, increased DNA damage, and slow growth rates due to mitotic delay. Interestingly, LmnaDhe/+ fibroblasts also had reduced levels of hypophosphorylated RB1 and the non-SMC condensin II-subunit D3 (NCAP-D3), a mitosis specific centromere condensin subunit that depends on RB1 activity. Mitotic check point control by mitotic arrest deficient-like 1 (MAD2L1) also was perturbed in LmnaDhe /+ cells. LmnaDhe /+ fibroblasts were consistently aneuploid and had higher levels of micronuclei and anaphase bridges than normal fibroblasts, consistent with chromosome segregation defects. Conclusions These data indicate that RB1 may be a key regulator of cellular phenotype in laminopathy-related cells, and suggest that the effects of LMNA on RB1 include both interphase and mitotic cell cycle control. PMID:21464947

The effect of hirudin modification of silk fibroin on cell growth and antithrombogenicity.

PubMed

Wang, Qiongyu; Tu, Fangfang; Liu, Yunfei; Zhang, Yujin; Li, Helei; Kang, Zhao; Yin, Yin; Wang, Jiannan

2017-06-01

Thrombus formation remains a particular challenge for small-diameter vascular grafts. In this study, the direct thrombin inhibitor hirudin (Hir) was used to modify silk fibroin films in an attempt to enhance its antithrombogenic properties. Hir was successfully attached to silk fibroin and uniformly distributed in the regenerative material. Hir-modified films showed good cytocompatibility, and supported adhesion and proliferation of fibroblasts (L929), human umbilical vascular endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs). Proliferation of HAVSMCs was inhibited by increasing Hir concentration. Activated partial thrombin time (APTT), prothrombin time (PT) and thrombin time (TT) of Hir-modified silk fibroin tubular scaffolds (SFTSs) were all increased markedly compared with fresh rabbit blood, ethanol-treated SFTS and unmodified SFTS, demonstrating the improved antithrombogenicity of SFTSs following modification with Hir. Copyright © 2017 Elsevier B.V. All rights reserved.

Reactive hydroxyapatite fillers for pectin biocomposites.

PubMed

Munarin, Fabiola; Petrini, Paola; Barcellona, Giulia; Roversi, Tommaso; Piazza, Laura; Visai, Livia; Tanzi, Maria Cristina

2014-12-01

In this work, a novel injectable biocomposite hydrogel is produced by internal gelation, using pectin as organic matrix and hydroxyapatite either as crosslinking agent and inorganic reinforcement. Tunable gelling kinetics and rheological properties are obtained varying the hydrogels' composition, with the final aim of developing systems for cell immobilization. The reversibility by dissolution of pectin-hydroxyapatite hydrogels is achieved with saline solutions, to possibly accelerate the release of the cells or active agents immobilized. Texture analysis confirms the possibility of extruding the biocomposites from needles with diameters from 20 G to 30 G, indicating that they can be implanted with minimally-invasive approaches, minimizing the pain during injection and the side effects of the open surgery. L929 fibroblasts entrapped in the hydrogels survive to the immobilization procedure and exhibit high cell viability. On the overall, these systems result to be suitable supports for the immobilization of cells for tissue regeneration applications. Copyright © 2014 Elsevier B.V. All rights reserved.

Comparative evaluation of the three different surface treatments - conventional, laser and Nano technology methods in enhancing the surface characteristics of commercially pure titanium discs and their effects on cell adhesion: An in vitro study.

PubMed

Vignesh; Nayar, Sanjna; Bhuminathan; Mahadevan; Santhosh, S

2015-04-01

The surface area of the titanium dental implant materials can be increased by surface treatments without altering their shape and form, thereby increasing the biologic properties of the biomaterial. A good biomaterial helps in early cell adhesion and cell signaling. In this study, the commercially pure titanium surfaces were prepared to enable machined surfaces to form a control material and to be compared with sandblasted and acid-etched surfaces, laser treated surfaces and titanium dioxide (20 nm) Nano-particle coated surfaces. The surface elements were characterized. The biocompatibility was evaluated by cell culture in vitro using L929 fibroblasts. The results suggested that the titanium dioxide Nano-particle coated surfaces had good osteoconductivity and can be used as a potential method for coating the biomaterial.

Characterization of decellularized scaffold derived from porcine meniscus for tissue engineering applications

NASA Astrophysics Data System (ADS)

Gao, Shuang; Yuan, Zhiguo; Xi, Tingfei; Wei, Xiaojuan; Guo, Quanyi

2016-06-01

Menisci are fundamental fibrocartilaginous organs in knee joints. The injury in meniscus can impair normal knee function and predisposes patients to osteoarthritis. This study prepared decellularized meniscus scaffolds using a 1% (w/w) sodium dodecyl sulfate solution and sufficient rinsing steps. Complete cell removal was verified by hematoxylin and eosin staining and DNA content assay. Decellularized menisci had accordant tension properties to intact ones, but with declined compression properties. This occurred because the collagen fiber was not damaged but glycosaminoglycans was significantly lost during the decellularization process, which was confirmed by biochemical assay and histology staining. In vitro cytotoxicity assay demonstrated that decellularized meniscus scaffolds have no toxicity on L929 murine fibroblasts and porcine chondrocytes. Further experiment showed that porcine chondrocytes could adhere and proliferate on the scaffold surface, and some cells even could infiltrate into the scaffold. All results showed the potential of this decellularized meniscus to be the scaffolds in tissue engineering.

Development of segmented polyurethane elastomers with low iodine content exhibiting radiopacity and blood compatibility.

PubMed

Dawlee, S; Jayabalan, Muthu

2011-10-01

Biofunctionally active and inherently radiopaque polymers are the emerging need for biomedical applications. Novel segmented polyurethane elastomer with inherent radiopacity was prepared using aliphatic chain extender 2,3-diiodo-2-butene-1,4-diol, polyol polytetramethylene glycol and 4,4'-methylenebis(phenyl isocyanate) (MDI) for blood compatible applications. Aliphatic polyurethane was also prepared using hexamethylene diisocyanate for comparison. X-ray analysis of the polyurethanes revealed good radiopacity even at a relatively low concentration of 3% iodine in aromatic polyurethane and 10% in aliphatic polyurethane. The polyurethanes also possessed excellent thermal stability. MDI-based polyurethane showed considerably higher tensile strength than the analogous HDI-based polyurethane. MDI-based aromatic polyurethane exhibited a dynamic surface morphology in aqueous medium, resulting in the segregation of hydrophilic domains which was more conducive to anti-thrombogenic properties. The polyurethane was cytocompatible with L929 fibroblast cells, non-hemolytic, and possessed good blood compatibility.

Fabrication of chitin-chitosan/nano TiO2-composite scaffolds for tissue engineering applications.

PubMed

Jayakumar, R; Ramachandran, Roshni; Divyarani, V V; Chennazhi, K P; Tamura, H; Nair, S V

2011-03-01

In this study, we prepared chitin-chitosan/nano TiO(2) composite scaffolds using lyophilization technique for bone tissue engineering. The prepared composite scaffold was characterized using SEM, XRD, FTIR and TGA. In addition, swelling, degradation and biomineralization capability of the composite scaffolds were evaluated. The developed composite scaffold showed controlled swelling and degradation when compared to the control scaffold. Cytocompatibility of the scaffold was assessed by MTT assay and cell attachment studies using osteoblast-like cells (MG-63), fibroblast cells (L929) and human mesenchymal stem cells (hMSCs). Results indicated no sign of toxicity and cells were found attached to the pore walls within the scaffolds. These results suggested that the developed composite scaffold possess the prerequisites for tissue engineering scaffolds and it can be used for tissue engineering applications. Copyright © 2010 Elsevier B.V. All rights reserved.

Genetic mapping of xenotropic murine leukemia virus-inducing loci in five mouse strains

PubMed Central

1980-01-01

A single mendelian gene was identified for induction of the endogenous xenotropic murine leukemia virus in five mouse strains (C57BL/10, C57L, C57BR, AKR, and BALB/c). This locus, designated Bxv-1, mapped to the same site on chromosome 1 in all strains: Id-1-Pep-3-[Bxv-1-Lp]. Thus, inducibility loci for xenotropic virus are more limited in number and chromosomal distribution than ecotropic inducibility loci. Virus expression in mice with Bxv-1 was induced by treatment of fibroblasts with 5-iododeoxyuridine or by exposure of spleen cells to a B cell mitogen, bacterial lipopolysaccharide. An analysis of the hamster X mouse somatic cell hybrids indicated that chromosome 1, alone, was sufficient for virus induction. PMID:6249881

Genetic mapping of xenotropic murine leukemia virus-inducing loci in five mouse strains.

PubMed

Kozak, C A; Rowe, W P

1980-07-01

A single mendelian gene was identified for induction of the endogenous xenotropic murine leukemia virus in five mouse strains (C57BL/10, C57L, C57BR, AKR, and BALB/c). This locus, designated Bxv-1, mapped to the same site on chromosome 1 in all strains: Id-1-Pep-3-[Bxv-1-Lp]. Thus, inducibility loci for xenotropic virus are more limited in number and chromosomal distribution than ecotropic inducibility loci. Virus expression in mice with Bxv-1 was induced by treatment of fibroblasts with 5-iododeoxyuridine or by exposure of spleen cells to a B cell mitogen, bacterial lipopolysaccharide. An analysis of the hamster X mouse somatic cell hybrids indicated that chromosome 1, alone, was sufficient for virus induction.

A mouse model of a human congenital disorder of glycosylation caused by loss of PMM2

PubMed Central

Chan, Barden; Clasquin, Michelle; Smolen, Gromoslaw A.; Histen, Gavin; Powe, Josh; Chen, Yue; Lin, Zhizhong; Lu, Chenming; Liu, Yan; Cang, Yong; Yan, Zhonghua; Xia, Yuanfeng; Thompson, Ryan; Singleton, Chris; Dorsch, Marion; Silverman, Lee; Su, Shin-San Michael; Freeze, Hudson H.; Jin, Shengfang

2016-01-01

The most common congenital disorder of glycosylation (CDG), phosphomannomutase 2 (PMM2)-CDG, is caused by mutations in PMM2 that limit availability of mannose precursors required for protein N-glycosylation. The disorder has no therapy and there are no models to test new treatments. We generated compound heterozygous mice with the R137H and F115L mutations in Pmm2 that correspond to the most prevalent alleles found in patients with PMM2-CDG. Many Pmm2R137H/F115L mice died prenatally, while survivors had significantly stunted growth. These animals and cells derived from them showed protein glycosylation deficiencies similar to those found in patients with PMM2-CDG. Growth-related glycoproteins insulin-like growth factor (IGF) 1, IGF binding protein-3 and acid-labile subunit, along with antithrombin III, were all deficient in Pmm2R137H/F115L mice, but their levels in heterozygous mice were comparable to wild-type (WT) littermates. These imbalances, resulting from defective glycosylation, are likely the cause of the stunted growth seen both in our model and in PMM2-CDG patients. Both Pmm2R137H/F115L mouse and PMM2-CDG patient-derived fibroblasts displayed reductions in PMM activity, guanosine diphosphate mannose, lipid-linked oligosaccharide precursor and total cellular protein glycosylation, along with hypoglycosylation of a new endogenous biomarker, glycoprotein 130 (gp130). Over-expression of WT-PMM2 in patient-derived fibroblasts rescued all these defects, showing that restoration of mutant PMM2 activity is a viable therapeutic strategy. This functional mouse model of PMM2-CDG, in vitro assays and identification of the novel gp130 biomarker all shed light on the human disease, and moreover, provide the essential tools to test potential therapeutics for this untreatable disease. PMID:27053713

[Observation of the L929 cell membrane after infrasound exposure with atomic force microscope].

PubMed

Wang, Bing-shui; Chen, Jing-zao; Liu, Bin; Li, Ling; Yi, Nan; Liu, Jing; Zhang, Sa

2005-12-01

To observe the changes of L929 cell membrane with atomic force microscope (AFM) after infrasound exposure and to explore the mechanisms of effect of infrasound on cell membrane. After primary culture, the L929 cells were exposed to infrasound with intensity output of 130 dB and frequency of 16 Hz 2 hours each day for 3 days. The subsequent changes in the membrane of the control cells and the cells exposed to the infrasound were determined by nano-scale scanning with AFM. After infrasound exposure, the normal prominence of the membrane became short and the dent became shallow in the 7.5 microm x 7.5 microm and 4.0 microm x 4.0 microm photographs. The prominence appeared as cobblestones. The surface of the membrane became smooth. The membrane structure of the L929 cells can be changed by infrasound exposure with intensity of 130 dB and frequency of 16 Hz. The change might be one of the characteristics of effect of infrasound on cell membrane.

Studies on wound healing potential of polyherbal formulation using in vitro and in vivo assays.

PubMed

Talekar, Yogesh P; Apte, Kishori G; Paygude, Shubhangi V; Tondare, Prasad R; Parab, Pradeep B

The use of herbal plant extracts in wound healing is known through decades, but it is necessary to provide scientific data through reverse pharmacology. The aim of the present study is to find the mechanism behind the healing of wounds using in vitro and in vivo assays. The study was designed to determine proliferation and mobilization of fibroblast and keratinocytes at the site of injury, angiogenesis at the site of healing and reduction in oxidative stress while healing. In our earlier studies it was observed that herbal extract of Vitex negundo L. (VN), Emblica officinalis Gaertn (EO), and Tridax procumbens L. (TP) showed rapid regeneration of skin, wound contraction and collagen synthesis at the site of injury in excision wound model. In the present study the cell mobilization was monitored in the scratch assay on L929 fibroblastic cell line and HaCaT keratinocytes cell line under the influence of aqueous plant extracts and its formulation. This formulation was also assessed for its angiogenic potential using CAM assay. Study was carried out to probe synergistic effect of polyherbal formulation using excision model in rat. The formulation was found to contain high amount of flavonoids, tannins and phenols which facilitate wound healing. At 20 μg/ml concentration of formulation, significant increase in tertiary and quaternary vessels were observed due to angiogenic potential of formulation. Formulation at the concentration of 3 μg/ml and 5 μg/ml showed significant mobilization of keratinocytes and fibroblasts respectively at the site of injury. Polyherbal formulation showed rapid regeneration of skin and wound contraction. Biochemical parameters like hydroxyproline, hexosamine and collagen turnover was increased in test drug treated animals as compared to untreated, whereas antioxidants such as catalase and GSH were increased significantly and decreased amount of tissue MDA was observed. Polyherbal formulation prepared from the plant extracts accelerates wound healing process by proliferation and mobilization of fibroblast and keratinocytes, and angiogenesis at the site of injury. It also shows fast contraction of wound with its beneficial improvement in tissue biochemical and antioxidant parameters. Copyright © 2017 Transdisciplinary University, Bangalore and World Ayurveda Foundation. Published by Elsevier B.V. All rights reserved.

On the mechanism for PPAR agonists to enhance ABCA1 gene expression

PubMed Central

Ogata, Masaki; Tsujita, Maki; Hossain, Mohammad Anwar; Akita, Nobukatsu; Gonzalez, Frank J.; Staels, Bart; Suzuki, Shogo; Fukutomi, Tatsuya; Kimura, Genjiro; Yokoyama, Shinji

2009-01-01

Expression of ATP binding cassette transporter A1 (ABCA1), a major regulator of high density lipoprotein (HDL) biogenesis, is known to be up-regulated by the transcription factor liver X receptor (LXR) α, and expression is further enhanced by activation of the peroxisome proliferator activated receptors (PPARs). We investigated this complex regulatory network using specific PPAR agonists: four fibrates (fenofibrate, bezafibrate, gemfibrozil and LY518674), a PPAR δ agonist (GW501516) and a PPAR γ agonist (pioglitazone). All of these compounds increased the expression of LXRs, PPARs and ABCA1 mRNAs, and associated apoA-I-mediated lipid release in THP-1 macrophage, WI38 fibroblast and mouse fibroblast. When mouse fibroblasts lacking expression of PPAR α were examined, the effects of fenofibrate and LY518674 were markedly diminished while induction by other ligands were retained. The PPAR α promoter was activated by all of these compounds in an LXR α-dependent manner, and partially in a PPAR α-dependent manner, in mouse fibroblast. The LXR responsive element (LXRE)-luciferase activity was enhanced by all the compounds in an LXR α-dependent manner in mouse fibroblast. This activation was exclusively PPAR α-dependent by fenofibrate and LY518674, but nonexclusively by the others. We conclude that PPARs and LXRs are involved in the regulation of ABCA1 expression and HDL biogenesis in a cooperative signal transduction pathway. PMID:19201410

Ectromelia Virus Affects Mitochondrial Network Morphology, Distribution, and Physiology in Murine Fibroblasts and Macrophage Cell Line

PubMed Central

Gregorczyk, Karolina P.; Wyżewski, Zbigniew; Szczepanowska, Joanna; Mielcarska, Matylda B.; Bossowska-Nowicka, Magdalena; Gieryńska, Małgorzata; Boratyńska-Jasińska, Anna; Niemiałtowski, Marek G.

2018-01-01

Mitochondria are multifunctional organelles that participate in numerous processes in response to viral infection, but they are also a target for viruses. The aim of this study was to define subcellular events leading to alterations in mitochondrial morphology and function during infection with ectromelia virus (ECTV). We used two different cell lines and a combination of immunofluorescence techniques, confocal and electron microscopy, and flow cytometry to address subcellular changes following infection. Early in infection of L929 fibroblasts and RAW 264.7 macrophages, mitochondria gathered around viral factories. Later, the mitochondrial network became fragmented, forming punctate mitochondria that co-localized with the progeny virions. ECTV-co-localized mitochondria associated with the cytoskeleton components. Mitochondrial membrane potential, mitochondrial fission–fusion, mitochondrial mass, and generation of reactive oxygen species (ROS) were severely altered later in ECTV infection leading to damage of mitochondria. These results suggest an important role of mitochondria in supplying energy for virus replication and morphogenesis. Presumably, mitochondria participate in transport of viral particles inside and outside of the cell and/or they are a source of membranes for viral envelope formation. We speculate that the observed changes in the mitochondrial network organization and physiology in ECTV-infected cells provide suitable conditions for viral replication and morphogenesis. PMID:29772718

Ectromelia Virus Affects Mitochondrial Network Morphology, Distribution, and Physiology in Murine Fibroblasts and Macrophage Cell Line.

PubMed

Gregorczyk, Karolina P; Wyżewski, Zbigniew; Szczepanowska, Joanna; Toka, Felix N; Mielcarska, Matylda B; Bossowska-Nowicka, Magdalena; Gieryńska, Małgorzata; Boratyńska-Jasińska, Anna; Struzik, Justyna; Niemiałtowski, Marek G; Szulc-Dąbrowska, Lidia

2018-05-16

Mitochondria are multifunctional organelles that participate in numerous processes in response to viral infection, but they are also a target for viruses. The aim of this study was to define subcellular events leading to alterations in mitochondrial morphology and function during infection with ectromelia virus (ECTV). We used two different cell lines and a combination of immunofluorescence techniques, confocal and electron microscopy, and flow cytometry to address subcellular changes following infection. Early in infection of L929 fibroblasts and RAW 264.7 macrophages, mitochondria gathered around viral factories. Later, the mitochondrial network became fragmented, forming punctate mitochondria that co-localized with the progeny virions. ECTV-co-localized mitochondria associated with the cytoskeleton components. Mitochondrial membrane potential, mitochondrial fission⁻fusion, mitochondrial mass, and generation of reactive oxygen species (ROS) were severely altered later in ECTV infection leading to damage of mitochondria. These results suggest an important role of mitochondria in supplying energy for virus replication and morphogenesis. Presumably, mitochondria participate in transport of viral particles inside and outside of the cell and/or they are a source of membranes for viral envelope formation. We speculate that the observed changes in the mitochondrial network organization and physiology in ECTV-infected cells provide suitable conditions for viral replication and morphogenesis.

Adventitial fibroblasts are activated in the early stages of atherosclerosis in the apolipoprotein E knockout mouse

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu Fang; Ji Jian; Li Li

2007-01-19

The role of the adventitia in vascular function and vascular lesion formation has been largely ignored. This study observed the activation of the adventitia and specifically the fibroblasts in the development of atherosclerosis in the apoE(-/-) mouse. The results showed a gradual increase in expression of collagen types I and III after 2, 4, and 8 weeks of hyperlipidic diet. The earliest expression of monocyte chemoattractant protein-1 (MCP-1) protein and mRNA was detected in the adventitial fibroblast before the formation of intimal lesions. Proliferation, too, was first found in the adventitial fibroblasts. We hypothesize that the adventitial fibroblast is activatedmore » in the early stage of atherosclerosis. Adventitial inflammation may be an early event in the development of atherosclerotic lesions.« less

Effects of leukemia inhibitory factor and basic fibroblast growth factor on free radicals and endogenous stem cell proliferation in a mouse model of cerebral infarction.

PubMed

Huang, Weihui; Li, Yadan; Lin, Yufeng; Ye, Xue; Zang, Dawei

2012-07-05

The present study established a mouse model of cerebral infarction by middle cerebral artery occlusion, and monitored the effect of 25 μg/kg leukemia inhibitory factor and (or) basic fibroblast growth factor administration 2 hours after model establishment. Results showed that following administration, the number of endogenous neural stem cells in the infarct area significantly increased, malondialdehyde content in brain tissue homogenates significantly decreased, nitric oxide content, glutathione peroxidase and superoxide dismutase activity significantly elevated, and mouse motor function significantly improved as confirmed by the rotarod and bar grab tests. In particular, the effect of leukemia inhibitory factor in combination with basic fibroblast growth factor was the most significant. Results indicate that leukemia inhibitory factor and basic fibroblast growth factor can improve the microenvironment after cerebral infarction by altering free radical levels, improving the quantity of endogenous neural stem cells, and promoting neurological function of mice with cerebral infarction.

mtDNA lineage analysis of mouse L-cell lines reveals the accumulation of multiple mtDNA mutants and intermolecular recombination

PubMed Central

Fan, Weiwei; Lin, Chun Shi; Potluri, Prasanth; Procaccio, Vincent; Wallace, Douglas C.

2012-01-01

The role of mitochondrial DNA (mtDNA) mutations and mtDNA recombination in cancer cell proliferation and developmental biology remains controversial. While analyzing the mtDNAs of several mouse L cell lines, we discovered that every cell line harbored multiple mtDNA mutants. These included four missense mutations, two frameshift mutations, and one tRNA homopolymer expansion. The LA9 cell lines lacked wild-type mtDNAs but harbored a heteroplasmic mixture of mtDNAs, each with a different combination of these variants. We isolated each of the mtDNAs in a separate cybrid cell line. This permitted determination of the linkage phase of each mtDNA and its physiological characteristics. All of the polypeptide mutations inhibited their oxidative phosphorylation (OXPHOS) complexes. However, they also increased mitochondrial reactive oxygen species (ROS) production, and the level of ROS production was proportional to the cellular proliferation rate. By comparing the mtDNA haplotypes of the different cell lines, we were able to reconstruct the mtDNA mutational history of the L–L929 cell line. This revealed that every heteroplasmic L-cell line harbored a mtDNA that had been generated by intracellular mtDNA homologous recombination. Therefore, deleterious mtDNA mutations that increase ROS production can provide a proliferative advantage to cancer or stem cells, and optimal combinations of mutant loci can be generated through recombination. PMID:22345519

Activity of nitric oxide-generating compounds against encephalomyocarditis virus.

PubMed Central

Guillemard, E; Geniteau-Legendre, M; Kergot, R; Lemaire, G; Petit, J F; Labarre, C; Quero, A M

1996-01-01

Nitric oxide (NO) generated by two NO donors (sodium nitroprusside or S-nitroso-L-glutathione) was shown to exert a dose-dependent inhibition of encephalomyocarditis virus growth in L-929 cells. This activity was not due to the cytotoxic or direct virucidal effects of NO donors. L-929 cells were shown to produce NO endogenously, but this low level of production did not counter encephalomyocarditis virus replication. PMID:8849231

Evaluation of antioxidant and cytoprotective activities of Arnica montana L. and Artemisia absinthium L. ethanolic extracts

PubMed Central

2012-01-01

Background Arnica montana L. and Artemisia absinthium L. (Asteraceae) are medicinal plants native to temperate regions of Europe, including Romania, traditionally used for treatment of skin wounds, bruises and contusions. In the present study, A. montana and A. absinthium ethanolic extracts were evaluated for their chemical composition, antioxidant activity and protective effect against H2O2-induced oxidative stress in a mouse fibroblast-like NCTC cell line. Results A. absinthium extract showed a higher antioxidant capacity than A. montana extract as Trolox equivalent antioxidant capacity, Oxygen radical absorbance capacity and 2,2-diphenyl-1-picrylhydrazyl free radical-scavenging activity, in correlation with its flavonoids and phenolic acids content. Both plant extracts had significant effects on the growth of NCTC cells in the range of 10–100 mg/L A. montana and 10–500 mg/L A. absinthium. They also protected fibroblast cells against hydrogen peroxide-induced oxidative damage, at the same doses. The best protection was observed in cell pre-treatment with 10 mg/L A. montana and 10–300 mg/L A. absinthium, respectively, as determined by Neutral red and lactate dehydrogenase assays. In addition, cell pre-treatment with plant extracts, at these concentrations, prevented morphological changes induced by hydrogen peroxide. Flow-cytometry analysis showed that pre-treatment with A. montana and A. absinthium extracts restored the proportion of cells in each phase of the cell cycle. Conclusions A. montana and A. absinthium extracts, rich in flavonoids and phenolic acids, showed a good antioxidant activity and cytoprotective effect against oxidative damage in fibroblast-like cells. These results provide scientific support for the traditional use of A. montana and A. absinthium in treatment of skin disorders. PMID:22958433

Effect of leaching residual methyl methacrylate concentrations on in vitro cytotoxicity of heat polymerized denture base acrylic resin processed with different polymerization cycles

PubMed Central

BURAL, Canan; AKTAŞ, Esin; DENIZ, Günnur; ÜNLÜÇERÇI, Yeşim; BAYRAKTAR, Gülsen

2011-01-01

Objectives Residual methyl methacrylate (MMA) may leach from the acrylic resin denture bases and have adverse effects on the oral mucosa. This in vitro study evaluated and correlated the effect of the leaching residual MMA concentrations ([MMA]r) on in vitro cytotoxicity of L-929 fibroblasts. Material and Methods A total of 144 heat-polymerized acrylic resin specimens were fabricated using 4 different polymerization cycles: (1) at 74ºC for 9 h, (2) at 74ºC for 9 h and terminal boiling (at 100ºC) for 30 min, (3) at 74ºC for 9 h and terminal boiling for 3 h, (4) at 74ºC for 30 min and terminal boiling for 30 min. Specimens were eluted in a complete cell culture medium at 37ºC for 1, 2, 5 and 7 days. [MMA]r in eluates was measured using high-performance liquid chromatography. In vitro cytotoxicity of eluates on L-929 fibroblasts was evaluated by means of cell proliferation using a tetrazolium salt XTT (sodium 3´-[1-phenyl-aminocarbonyl)-3,4-tetrazolium]bis(4-methoxy-6-nitro)benzenesulphonic acid) assay. Differences in [MMA]r of eluates and cell proliferation values between polymerization cycles were statistically analyzed by Kruskal-Wallis, Friedman and Dunn's multiple comparison tests. The correlation between [MMA]r of eluates and cell proliferation was analyzed by Pearson's correlation test (p<0.05). Results [MMA]r was significantly (p≤0.001) higher in eluates of specimens polymerized with cycle without terminal boiling after elution of 1 and 2 days. Cell proliferation values for all cycles were significantly (p<0.01) lower in eluates of 1 day than those of 2 days. The correlation between [MMA]r and cell proliferation values was negative after all elution periods, showing significance (p<0.05) for elution of 1 and 2 days. MMA continued to leach from acrylic resin throughout 7 days and leaching concentrations markedly reduced after elution of 1 and 2 days. Conclusion Due to reduction of leaching residual MMA concentrations, use of terminal boiling in the polymerization process for at least 30 min and water storage of the heat-polymerized denture bases for at least 1 to 2 days before denture delivery is clinically recommended for minimizing the residual MMA and possible cytotoxic effects. PMID:21956586

The development of non-toxic ionic-crosslinked chitosan-based microspheres as carriers for the controlled release of silk sericin.

PubMed

Aramwit, Pornanong; Ekasit, Sanong; Yamdech, Rungnapha

2015-10-01

Silk sericin is recently shown to possess various biological activities for biomedical applications. While various sericin carriers were developed for drug delivery system, very few researches considered sericin as a bioactive molecule itself. In this study, sericin incorporated in the chitosan-based microspheres was introduced as a bioactive molecule and bioactive carrier at the same time. The chitosan/sericin (CH/SS) microspheres at different composition (80/20, 70/30, 60/40, and 50/50) were successfully fabricated using anhydroustri-polyphosphate (TPP) as a polyanionic crosslinker. The microspheres with an average size of 1-4 μm and narrow size distribution were obtained. From FT-IR spectra, the presence of both chitosan and sericin in the microspheres confirmed the occurrence of ionic interaction that crosslink them within the microspheres. We also found that the CH/SS microspheres prepared at 50/50 could encapsulate sericin at the highest percentage (37.28%) and release sericin in the most sustained behavior, possibly due to the strong ionic interaction of the positively charged chitosan and the negatively charged sericin. On the other hand, the composition of CH/SS had no effect on the degradation rate of microspheres. All microspheres continuously degraded and remained around 20% after 14 days of enzymatic degradation. This explained that the ionic crosslinkings between chitosan and sericin could be demolished by the enzyme and hydrolysis. Furthermore, we have verified that all CH/SS microspheres at any concentrations showed non-toxicity to L929 mouse fibroblast cells. Therefore, we suggested that the non-toxic ionic-crosslinked CH/SS microspheres could be incorporated in wound dressing material to achieve the sustained release of sericin for accelerated wound healing.

In Vitro Cytotoxicity and Setting Time Assessment of Calcium-Enriched Mixture Cement, Retro Mineral Trioxide Aggregate and Mineral Trioxide Aggregate

PubMed Central

Pornamazeh, Tahereh; Yadegari, Zahra; Ghasemi, Amir; Sheykh-al-Eslamian, Seyedeh Mahsa; Shojaeian, Shiva

2017-01-01

Introduction: The present study sought to evaluate and compare biocompatibility and setting time of Retro mineral trioxide aggregate (MTA), calcium-enriched mixture (CEM) and Angelus MTA. Methods and Materials: CEM cement, Angelus MTA and Retro MTA were assessed in set and fresh states. Extracts transformed to each cavity of three 24-well plates in which 1×104 cell were seeded into each well 24 h earlier. All specimens were incubated in a humidified incubator with 5% CO2 at 37°C. Mosmann’s tetrazolium toxicity (MTT) assay was used to determine in vitro cytotoxicity on L929 mouse fibroblast cell line. Cell viability was determined at 1, 24, and 72 h after exposure. The initial setting time was measured by 113.4 g Gilmore needle testing. Then, final setting times were assessed by the 456.5 g Gilmore needle. Data comparisons were performed using the analysis of variance (ANOVA) and Tukey's post hoc test (α=0.05). Results: All groups in both forms indicated higher cell vitality compared to positive control group (P<0.001). After 24 h, the set Retro MTA showed better biocompatibility compared to set CEM and set Angelus MTA (P<0.001). Retro MTA showed significantly lower initial and final setting time compared to CEM and Angelus MTA (P<0.001). Conclusion: Our results indicated the good cell viability values of Retro MTA and relatively short period of setting time. It seems a promising alternative material in clinical situations where accelerated setting is required. However, more clinical and in vivo investigations are needed for a clear decision making. PMID:29225646

Synthesis and cytotoxicity study of magnesium ferrite-gold core-shell nanoparticles.

PubMed

Nonkumwong, Jeeranan; Pakawanit, Phakkhananan; Wipatanawin, Angkana; Jantaratana, Pongsakorn; Ananta, Supon; Srisombat, Laongnuan

2016-04-01

In this work, the core-magnesium ferrite (MgFe2O4) nanoparticles were prepared by hydrothermal technique. Completed gold (Au) shell coating on the surfaces of MgFe2O4 nanoparticles was obtained by varying core/shell ratios via a reduction method. Phase identification, morphological evolution, optical properties, magnetic properties and cytotoxicity to mammalian cells of these MgFe2O4 core coated with Au nanoparticles were examined by using a combination of X-ray diffraction, scanning electron microscopy, transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy, UV-visible spectroscopy (UV-vis), vibrating sample magnetometry and resazurin microplate assay techniques. In general, TEM images revealed different sizes of the core-shell nanoparticles generated from various core/shell ratios and confirmed the completed Au shell coating on MgFe2O4 core nanoparticles via suitable core/shell ratio with particle size less than 100 nm. The core-shell nanoparticle size and the quality of coating influence the optical properties of the products. The UV-vis spectra of complete coated MgFe2O4-Au core-shell nanoparticles exhibit the absorption bands in the near-Infrared (NIR) region indicating high potential for therapeutic applications. Based on the magnetic property measurement, it was found that the obtained MgFe2O4-Au core-shell nanoparticles still exhibit superparamagnetism with lower saturation magnetization value, compared with MgFe2O4 core. Both of MgFe2O4 and MgFe2O4-Au core-shell also showed in vitro non-cytotoxicity to mouse areola fibroblast (L-929) cell line. Copyright © 2015 Elsevier B.V. All rights reserved.

Facile modulation of cell adhesion to a poly(ethylene glycol) diacrylate film with incorporation of polystyrene nano-spheres.

PubMed

Yang, Wenguang; Yu, Haibo; Li, Gongxin; Wang, Yuechao; Liu, Lianqing

2016-12-01

Poly(ethylene glycol) diacrylate (PEGDA) is a common hydrogel that has been actively investigated for various tissue engineering applications owing to its biocompatibility and excellent mechanical properties. However, the native PEGDA films are known for their bio-inertness which can hinder cell adhesion, thereby limiting their applications in tissue engineering and biomedicine. Recently, nano composite technology has become a particularly hot topic, and has led to the development of new methods for delivering desired properties to nanomaterials. In this study, we added polystyrene nano-spheres (PS) into a PEGDA solution to synthesize a nano-composite film and evaluated its characteristics. The experimental results showed that addition of the nanospheres to the PEGDA film not only resulted in modification of the mechanical properties and surface morphology but further improved the adhesion of cells on the film. The tensile modulus showed clear dependence on the addition of PS, which enhanced the mechanical properties of the PEGDA-PS film. We attribute the high stiffness of the hybrid hydrogel to the formation of additional cross-links between polymeric chains and the nano-sphere surface in the network. The effect of PS on cell adhesion and proliferation was evaluated in L929 mouse fibroblast cells that were seeded on the surface of various PEGDA-PS films. Cells density increased with a larger PS concentration, and the cells displayed a spreading morphology on the hybrid films, which promoted cell proliferation. Impressively, cellular stiffness could also be modulated simply by tuning the concentration of nano-spheres. Our results indicate that the addition of PS can effectively tailor the physical and biological properties of PEGDA as well as the mechanical properties of cells, with benefits for biomedical and biotechnological applications.

In vitro evaluation of novel antimicrobial coatings for surgical sutures using octenidine.

PubMed

Obermeier, A; Schneider, J; Föhr, P; Wehner, S; Kühn, K-D; Stemberger, A; Schieker, M; Burgkart, R

2015-09-24

Sutures colonized by bacteria represent a challenge in surgery due to their potential to cause surgical site infections. In order to reduce these type of infections antimicrobially coated surgical sutures are currently under development. In this study, we investigated the antimicrobial drug octenidine as a coating agent for surgical sutures. To achieve high antimicrobial efficacy and required biocompatibility for medical devices, we focused on optimizing octenidine coatings based on fatty acids. For this purpose, antimicrobial sutures were prepared with either octenidine-laurate or octenidine-palmitate at 11, 22, and 33 μg/cm drug concentration normalized per length of sutures. Octenidine containing sutures were compared to the commercial triclosan-coated suture Vicryl® Plus. The release of octenidine into aqueous solution was analyzed and long-term antimicrobial efficacy was assessed via agar diffusion tests using Staphylococcus aureus. For determining biocompatibility, cytotoxicity assays (WST-1) were performed using L-929 mouse fibroblasts. In a 7 days elution experiment, octenidine-palmitate coated sutures demonstrated much slower drug release (11 μg/cm: 7%; 22 μg/cm: 5%; 33 μg/cm: 33%) than octenidine-laurate sutures (11 μg/cm: 82%; 22 μg/cm: 88%; 33 μg/cm: 87%). Furthermore sutures at 11 μg/cm drug content were associated with acceptable cytotoxicity according to ISO 10993-5 standard and showed, similar to Vicryl® Plus, relevant efficacy to inhibit surrounding bacterial growth for up to 9 days. Octenidine coated sutures with a concentration of 11 μg/cm revealed high antimicrobial efficacy and biocompatibility. Due to their delayed release, palmitate carriers should be preferred. Such coatings are candidates for clinical testing in regard to their safety and efficacy.

Novel poly(ε-caprolactone)/gelatin wound dressings prepared by emulsion electrospinning with controlled release capacity of Ketoprofen anti-inflammatory drug.

PubMed

Basar, A O; Castro, S; Torres-Giner, S; Lagaron, J M; Turkoglu Sasmazel, H

2017-12-01

In the present study, a single and binary Ketoprofen-loaded mats of ultrathin fibers were developed by electrospinning and their physical properties and drug release capacity was analyzed. The single mat was prepared by solution electrospinning of poly(ε-caprolactone) (PCL) with Ketoprofen at a weight ratio of 5wt%. This Ketoprofen-containing PCL solution was also used as the oil phase in a 7:3 (wt/wt) emulsion with gelatin dissolved in acidified water. The resultant stable oil-in-water (O/W) emulsion of PCL-in-gelatin, also containing Ketoprofen at 5wt%, was electrospun to produce the binary mat. Cross-linking process was performed by means of glutaraldehyde vapor on the electrospun binary mat to prevent dissolution of the hydrophilic gelatin phase. The performed characterization indicated that Ketoprofen was successfully embedded in the single and binary electrospun mats, i.e. PCL and PCL/gelatin, and both mats showed high hydrophobicity but poor thermal resistance. In vitro release studies interestingly revealed that, in comparison to the single PCL electrospun mat, the binary PCL/gelatin mat significantly hindered Ketoprofen burst release and exhibited a sustained release capacity of the drug for up to 4days. In addition, the electrospun Ketoprofen-loaded mats showed enhanced attachment and proliferation of L929 mouse fibroblast cells, presenting the binary mat the highest cell growth yield due to its improved porosity. The here-developed electrospun materials clearly show a great deal of potential as novel wound dressings with an outstanding controlled capacity to release drugs. Copyright © 2017 Elsevier B.V. All rights reserved.

Chiral Platinum(II) Complexes Featuring Phosphine and Chloroquine Ligands as Cytotoxic and Monofunctional DNA-Binding Agents.

PubMed

Villarreal, Wilmer; Colina-Vegas, Legna; Rodrigues de Oliveira, Clayton; Tenorio, Juan C; Ellena, Javier; Gozzo, Fábio C; Cominetti, Marcia Regina; Ferreira, Antonio G; Ferreira, Marco Antonio Barbosa; Navarro, Maribel; Batista, Alzir A

2015-12-21

Chiral molecules in nature are involved in many biological events; their selectivity and specificity make them of great interest for understanding the behavior of bioactive molecules, by providing information about the chiral discrimination. Inspired by these conformational properties, we present the design and synthesis of novel chiral platinum(II) complexes featuring phosphine and chloroquine ligands with the general formula [PtCl(P)2(CQ)]PF6 (where (P)2 = triphenylphosphine (PPh3) (5), 1,3-bis(diphenylphosphine)propane (dppp) (6), 1,4-bis(diphenylphosphine)butane (dppb) (7), 1,1'-bis(diphenylphosphine)ferrocene (dppf) (8), and CQ = chloroquine] and their precursors of the type [PtCl2(P)2] are described. The complexes were characterized by elemental analysis, absorption spectroscopy in the infrared and ultraviolet-visible (UV-vis) regions, multinuclear ((1)H, (13)C, (31)P, (15)N, and (195)Pt) NMR spectroscopy, cyclic voltammetry, and mass spectrometry (in the case of chloroquine complexes). The interactions of the new platinum-chloroquine complexes with both albumin (BSA), using fluorescence spectroscopy, and DNA, by four widely reported methods were also evaluated. These experiments showed that these Pt-CQ complexes interact strongly with DNA and have high affinities for BSA, in contrast to CQ and CQDP (chloroquine diphosphate), which interact weakly with these biomolecules. Additional assays were performed in order to investigate the cytotoxicity of the platinum complexes against two healthy cell lines (mouse fibroblasts (L929) and the Chinese hamster lung (V79-4)) and four tumor cell lines (human breast (MDA-MB-231 and MCF-7), human lung (A549), and human prostate (DU-145)). The results suggest that the Pt-CQ complexes are generally more cytotoxic than the free CQ, showing that they are promising as anticancer drugs.

Construction and characterization of Gal-chitosan graft methoxy poly (ethylene glycol) (Gal-CS-mPEG) nanoparticles as efficient gene carrier

NASA Astrophysics Data System (ADS)

Jin, Jiting; Fu, Wandong; Liao, Miaofei; Han, Baoqin; Chang, Jing; Yang, Yan

2017-10-01

In the present study, galactosylated chitosan (Gal-CS) was conjugated with methoxy poly(ethylene glycol) (mPEG) as a hydrophilic group. The structure of Gal-CS-mPEG polymer was characterized and the nanoparticles (NPs) were prepared using ironic gelation method. The study was designed to investigate the characteristics and functions of Gal-CS-mPEG NPs. The morphology of Gal-CS-mPEG NPs was observed by SEM and it was a compact and spherical shape. The size of the NPs was approximately 200 nm in diameter under the ideal process parameters. The interaction between Gal-CS-mPEG NPs and pDNA, and the protection of pDNA against DNase I and serum degradation by Gal-CS-mPEG NPs were evaluated. Agarose gel electrophoresis results showed that Gal-CS-mPEG NPs had strong interaction with pDNA at the weight ratio of 12:1, 4:1 and 2:1 and could protect pDNA from DNase I and serum degradation. Gal-CS-mPEG NPs exhibited high loading efficiency and sustainable in vitro release. The blood compatibility studies demonstrated that Gal-CS-mPEG NPs had superior compatibility with erythrocytes in terms of aggregation degree and hemolysis level. Gal-CS-mPEG NPs showed no cytotoxicity on L929 cells, which is a normal mouse connective tissue fibroblast, but showed inhibitory effects on the proliferation of Bel-7402 cells, which is a liver cancer cell line. In conclusion, Gal-CS-mPEG NP is a bio-safe and efficient gene carrier with potential application in gene delivery.

Primary Mouse Myoblast Purification using Magnetic Cell Separation.

PubMed

Sincennes, Marie Claude; Wang, Yu Xin; Rudnicki, Michael A

2017-01-01

Primary myoblasts can be isolated from mouse muscle cell extracts and cultured in vitro. Muscle cells are usually dissociated manually by mincing with razor blades or scissors in a collagenase/dispase solution. Primary myoblasts are then gradually enriched by pre-plating on collagen-coated plates, based on the observation that mouse fibroblasts attach quickly to collagen-coated plates, and are less adherent. Here, we describe an automated muscle dissociation protocol. We also propose an alternative to pre-plating using magnetic bead separation of primary myoblasts, which improve myoblast purity by minimizing fibroblast contamination.

Peroxotitanates for Biodelivery of Metals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hobbs, David; Elvington, M.

2009-02-11

Metal-based drugs are largely undeveloped in pharmacology. One limiting factor is the systemic toxicity of metal-based compounds. A solid-phase, sequestratable delivery agent for local delivery of metals could reduce systemic toxicity, facilitating new drug development in this nascent area. Amorphous peroxotitanates (APT) are ion exchange materials with high affinity for several heavy metal ions, and have been proposed to deliver or sequester metal ions in biological contexts. In the current study, we tested a hypothesis that APT are able to deliver metals or metal compounds to cells. We exposed fibroblasts (L929) or monocytes (THP1) to metal-APT materials for 72 hmore » in vitro, then measured cellular mitochondrial activity (SDH-MTT method) to assess the biological impact of the metal-APT materials vs. metals or APT alone. APT alone did not significantly affect cellular mitochondrial activity, but all metal-APT materials suppressed the mitochondrial activity of fibroblasts (by 30-65% of controls). The concentration of metal-APT materials required to suppress cellular mitochondrial activity was below that required for metals alone, suggesting that simple extracellular release of the metals from the metal-APT materials was not the primary mechanism of mitochondrial suppression. In contrast to fibroblasts, no metal-APT material had a measurable effect on THP1 monocyte mitochondrial activity, despite potent suppression by metals alone. This latter result suggested that 'biodelivery' by metal-APT materials may be cell type-specific. Therefore, it appears that APT are plausible solid phase delivery agents of metals or metal compounds to some types of cells for potential therapeutic effect.« less

On the influence of various physicochemical properties of the CNTs based implantable devices on the fibroblasts' reaction in vitro.

PubMed

Benko, Aleksandra; Frączek-Szczypta, Aneta; Menaszek, Elżbieta; Wyrwa, Jan; Nocuń, Marek; Błażewicz, Marta

2015-11-01

Coating the material with a layer of carbon nanotubes (CNTs) has been a subject of particular interest for the development of new biomaterials. Such coatings, made of properly selected CNTs, may constitute an implantable electronic device that facilitates tissue regeneration both by specific surface properties and an ability to electrically stimulate the cells. The goal of the presented study was to produce, evaluate physicochemical properties and test the applicability of highly conductible material designed as an implantable electronic device. Two types of CNTs with varying level of oxidation were chosen. The process of coating involved suspension of the material of choice in the diluent followed by the electrophoretic deposition to fabricate layers on the surface of a highly biocompatible metal-titanium. Presented study includes an assessment of the physicochemical properties of the material's surface along with an electrochemical evaluation and in vitro biocompatibility, cytotoxicity and apoptosis studies in contact with the murine fibroblasts (L929) in attempt to answer the question how the chemical composition and CNTs distribution in the layer alters the electrical properties of the sample and whether any of these properties have influenced the overall biocompatibility and stimulated adhesion of fibroblasts. The results indicate that higher level of oxidation of CNTs yielded materials more conductive than the metal they are deposited on. In vitro study revealed that both materials were biocompatible and that the cells were not affected by the amount of the functional group and the morphology of the surface they adhered to.

Peptide-enhanced mRNA transfection in cultured mouse cardiac fibroblasts and direct reprogramming towards cardiomyocyte-like cells

PubMed Central

Lee, Kunwoo; Yu, Pengzhi; Lingampalli, Nithya; Kim, Hyun Jin; Tang, Richard; Murthy, Niren

2015-01-01

The treatment of myocardial infarction is a major challenge in medicine due to the inability of heart tissue to regenerate. Direct reprogramming of endogenous cardiac fibroblasts into functional cardiomyocytes via the delivery of transcription factor mRNAs has the potential to regenerate cardiac tissue and to treat heart failure. Even though mRNA delivery to cardiac fibroblasts has the therapeutic potential, mRNA transfection in cardiac fibroblasts has been challenging. Herein, we develop an efficient mRNA transfection in cultured mouse cardiac fibroblasts via a polyarginine-fused heart-targeting peptide and lipofectamine complex, termed C-Lipo and demonstrate the partial direct reprogramming of cardiac fibroblasts towards cardiomyocyte cells. C-Lipo enabled the mRNA-induced direct cardiac reprogramming due to its efficient transfection with low toxicity, which allowed for multiple transfections of Gata4, Mef2c, and Tbx5 (GMT) mRNAs for a period of 2 weeks. The induced cardiomyocyte-like cells had α-MHC promoter-driven GFP expression and striated cardiac muscle structure from α-actinin immunohistochemistry. GMT mRNA transfection of cultured mouse cardiac fibroblasts via C-Lipo significantly increased expression of the cardiomyocyte marker genes, Actc1, Actn2, Gja1, Hand2, and Tnnt2, after 2 weeks of transfection. Moreover, this study provides the first direct evidence that the stoichiometry of the GMT reprogramming factors influence the expression of cardiomyocyte marker genes. Our results demonstrate that mRNA delivery is a potential approach for cardiomyocyte generation. PMID:25834424

Androgen actions in mouse wound healing: Minimal in vivo effects of local antiandrogen delivery.

PubMed

Wang, Yiwei; Simanainen, Ulla; Cheer, Kenny; Suarez, Francia G; Gao, Yan Ru; Li, Zhe; Handelsman, David; Maitz, Peter

2016-05-01

The aims of this work were to define the role of androgens in female wound healing and to develop and characterize a novel wound dressing with antiandrogens. Androgens retard wound healing in males, but their role in female wound healing has not been established. To understand androgen receptor (AR)-mediated androgen actions in male and female wound healing, we utilized the global AR knockout (ARKO) mouse model, with a mutated AR deleting the second zinc finger to disrupt DNA binding and transcriptional activation. AR inactivation enhanced wound healing rate in males by increasing re-epithelialization and collagen deposition even when wound contraction was eliminated. Cell proliferation and migration in ARKO male fibroblasts was significantly increased compared with wild-type (WT) fibroblasts. However, ARKO females showed a similar healing rate compared to WT females. To exploit local antiandrogen effects in wound healing, while minimizing off-target systemic effects, we developed a novel electrospun polycaprolactone (PCL) scaffold wound dressing material for sustained local antiandrogen delivery. Using the antiandrogen hydroxyl flutamide (HF) at 1, 5, and 10 mg/mL in PCL scaffolds, controlled HF delivery over 21 days significantly enhanced in vitro cell proliferation of human dermal fibroblasts and human keratinocytes. HF-PCL scaffolds also promoted in vivo wound healing in mice compared with open wounds but not to PCL scaffolds. © 2016 by the Wound Healing Society.

Properties of the recombinant TNF-binding proteins from variola, monkeypox, and cowpox viruses are different.

PubMed

Gileva, Irina P; Nepomnyashchikh, Tatiana S; Antonets, Denis V; Lebedev, Leonid R; Kochneva, Galina V; Grazhdantseva, Antonina V; Shchelkunov, Sergei N

2006-11-01

Tumor necrosis factor (TNF), a potent proinflammatory and antiviral cytokine, is a critical extracellular immune regulator targeted by poxviruses through the activity of virus-encoded family of TNF-binding proteins (CrmB, CrmC, CrmD, and CrmE). The only TNF-binding protein from variola virus (VARV), the causative agent of smallpox, infecting exclusively humans, is CrmB. Here we have aligned the amino acid sequences of CrmB proteins from 10 VARV, 14 cowpox virus (CPXV), and 22 monkeypox virus (MPXV) strains. Sequence analyses demonstrated a high homology of these proteins. The regions homologous to cd00185 domain of the TNF receptor family, determining the specificity of ligand-receptor binding, were found in the sequences of CrmB proteins. In addition, a comparative analysis of the C-terminal SECRET domain sequences of CrmB proteins was performed. The differences in the amino acid sequences of these domains characteristic of each particular orthopoxvirus species were detected. It was assumed that the species-specific distinctions between the CrmB proteins might underlie the differences in these physicochemical and biological properties. The individual recombinant proteins VARV-CrmB, MPXV-CrmB, and CPXV-CrmB were synthesized in a baculovirus expression system in insect cells and isolated. Purified VARV-CrmB was detectable as a dimer with a molecular weight of 90 kDa, while MPXV- and CPXV-CrmBs, as monomers when fractioned by non-reducing SDS-PAGE. The CrmB proteins of VARV, MPXV, and CPXV differed in the efficiencies of inhibition of the cytotoxic effects of human, mouse, or rabbit TNFs in L929 mouse fibroblast cell line. Testing of CrmBs in the experimental model of LPS-induced shock using SPF BALB/c mice detected a pronounced protective effect of VARV-CrmB. Thus, our data demonstrated the difference in anti-TNF activities of VARV-, MPXV-, and CPXV-CrmBs and efficiency of VARV-CrmB rather than CPXV- or MPXV-CrmBs against LPS-induced mortality in mice.

A high-throughput screen for inhibitors of the prolyl isomerase, Pin1, identifies a seaweed polyphenol that reduces adipose cell differentiation.

PubMed

Mori, Tadashi; Hidaka, Masafumi; Ikuji, Hiroko; Yoshizawa, Ibuki; Toyohara, Haruhiko; Okuda, Toru; Uchida, Chiyoko; Asano, Tomoichiro; Yotsu-Yamashita, Mari; Uchida, Takafumi

2014-01-01

The peptidyl prolyl cis/trans isomerase Pin1 enhances the uptake of triglycerides and the differentiation of fibroblasts into adipose cells in response to insulin stimulation. Pin1 downregulation could be a potential approach to prevent and treat obesity-related disorders. In order to identify an inhibitor of Pin1 that exhibited minimal cytotoxicity, we established a high-throughput screen for Pin1 inhibitors and used this method to identify an inhibitor from 1,056 crude fractions of two natural product libraries. The candidate, a phlorotannin called 974-B, was isolated from the seaweed, Ecklonia kurome. 974-B inhibited the differentiation of mouse embryonic fibroblasts and 3T3-L1 cells into adipose cells without inducing cytotoxicity. We discovered the Pin1 inhibitor, 974-B, from the seaweed, E. kurome, and showed that it blocks the differentiation of fibroblasts into adipose cells, suggesting that 974-B could be a lead drug candidate for obesity-related disorders.

EVALUATION OF BENZO[C]CHRYSENE DIHYDRODIOLS IN THE MORPHOLOGICAL CELL TRANSFORMATION OF MOUSE EMBRYO FIBROBLAST C3H10T1/2CL8 CELLS

EPA Science Inventory

EVALUATION OF BENZO[c]CHRYSENE DIHYDRODIOLS IN THE MORPHOLOGICAL CELL TRANSFORMATION OF MOUSE EMBRYO FIBROBLAST C3H10T?CL8 CELLS

Abstract The morphological cell transforming activities of three dihydrodiols of benzo[c]chrysene (B[c]C), trans-B[c]C-7,8-diol, trans-B[c]C-9...

CELLULAR TOXICITY IN CHINESE HAMSTER OVARY CELL CULTURES. 2. A STATISTICAL APPRAISAL OF SENSITIVITY WITH THE RABBIT ALVEOLAR MACROPHAGE, SYRIAN HAMSTER EMBRYO, BALB 3T3 MOUSE, AND HUMAN NEONATAL FIBROBLAST CELL SYSTEMS

EPA Science Inventory

Chinese hamster ovary, rabbit alveolar macrophage, Syrian Hamster embryo, mouse, and human neonatal fibroblast cells were employed in a statistical evaluation of the relative sensitivity of the cells to toxic substances. The cells were exposed to 1,2,4-trichlorobenzene, 2,4-dimet...

New Hydrogels Enriched with Antioxidants from Saffron Crocus Can Find Applications in Wound Treatment and/or Beautification.

PubMed

Zeka, Keti; Ruparelia, Ketan C; Sansone, Claudia; Macchiarelli, Guido; Continenza, Maria Adelaide; Arroo, Randolph R J

2018-01-01

Saffron extracts have a long history of application as skin protectant, possibly due to their ability to scavenge free radicals. In this work, the performance of a hydrogel enriched with antioxidant compounds isolated from saffron crocus (Crocus sativus L.) petals was tested. These hydrogels could be considered as new drug delivery system. Hydrogels are crosslinked polymer networks that absorb large quantities of water but retain the properties of a solid, thus making ideal dressings for sensitive skin. We tested antioxidant-enriched hydrogels on primary mouse fibroblasts. Hydrogels enriched with kaempferol and crocin extracted from saffron petals showed good biocompatibility with in vitro cultured fibroblasts. These new types of hydrogels may find applications in wound treatment and/or beautification. © 2018 S. Karger AG, Basel.

Nucleotide excision repair modulates the cytotoxic and mutagenic effects of N-n-butyl-N-nitrosourea in cultured mammalian cells as well as in mouse splenocytes in vivo.

PubMed

Bol, S A; van Steeg, H; van Oostrom, C T; Tates, A D; Vrieling, H; de Groot, A J; Mullenders, L H; van Zeeland, A A; Jansen, J G

1999-05-01

The butylating agent N-n-butyl-N-nitrosourea (BNU) was employed to study the role of nucleotide excision repair (NER) in protecting mammalian cells against the genotoxic effects of monofunctional alkylating agents. The direct acting agent BNU was found to be mutagenic in normal and XPA mouse splenocytes after a single i.p. treatment in vivo. After 25 and 35 mg/kg BNU, but not after 75 mg/ kg, 2- to 3-fold more hprt mutants were detected in splenocytes from XPA mice than from normal mice. Using O6-alkylguanine-DNA alkyltransferase (AGT)-deficient hamster cells, it was found that NER-deficient CHO UV5 cells carrying a mutation in the ERCC-2 gene were 40% more mutable towards lesions induced by BNU when compared with parental NER-proficient CHO AA8 cells. UV5 cells were 1.4-fold more sensitive to the cytotoxic effects of BNU compared with AA8 cells. To investigate whether this increased sensitivity of NER-deficient cells is modulated by AGT activity, cell survival studies were performed in human and mouse primary fibroblasts as well. BNU was 2.7-fold more toxic for mouse XPA fibroblasts compared with normal mouse fibroblasts. Comparable results were found for human fibroblasts. Taken together these data indicate that the role of NER in protecting rodent cells against the mutagenic and cytotoxic effects of the alkylating agent BNU depends on AGT.

Silibinin negatively contributes to primary cilia length via autophagy regulated by histone deacetylase 6 in confluent mouse embryo fibroblast 3T3-L1 cells.

PubMed

Xu, Qian; Liu, Wei; Liu, Xiaoling; Liu, Weiwei; Wang, Hongju; Yao, Guodong; Zang, Linghe; Hayashi, Toshihiko; Tashiro, Shin-Ichi; Onodera, Satoshi; Ikejima, Takashi

2016-09-01

Primary cilium is a cellular antenna, signalling as a sensory organelle. Numerous pathological manifestation is associated with change of its length. Although the interaction between autophagy and primary cilia has been suggested, the role of autophagy in primary cilia length is largely unknown. In this study the primary cilia were immunostained and observed by using confocal fluorescence microscopy, and we found that silibinin, a natural flavonoid, shortened the length of primary cilia, meanwhile it also induced autophagy in 3T3-L1 cells. This study was designed to investigate the significance of silibinin-induced autophagy in primary ciliary structure in confluent mouse embryo fibroblast 3T3-L1 cells. Either blocking the autophagic flux with pre-treatment with the autophagy inhibitor, 3-methyladenine (3-MA), or transfection of siRNA targeting LC3 inhibited the reduction of cilia length caused by silibinin exposure. Autophagy induced by silibinin decreased expressions of the cilia-associated proteins, such as IFT88, KIF3a and Ac-tubulin, while 3-MA restored it, indicating that autophagy induced by silibinin led to a reduction of primary cilia length. Histone deacetylase 6 (HDAC6), which was suggested as a mediator of autophagy, was up-regulated by silibinin in a time-dependent manner. In addition, 3T3-L1 cells treated with siRNA against HDAC6 had a reduced autophagic level and were protected from silibinin-induced cilia shortening. Taken together, we conclude that the HDAC6-mediated autophagy negatively regulates primary cilia length during silibinin treatment and has the potential to serve as a therapeutic target for primary cilia-associated ciliopathies. These findings thus provide new information about the potential link between autophagy and primary cilia.

The D prostanoid receptor agonist BW245C [(4S)-(3-[(3R,S)-3-cyclohexyl-3-hydroxypropyl]-2,5-dioxo)-4-imidazolidineheptanoic acid] inhibits fibroblast proliferation and bleomycin-induced lung fibrosis in mice.

PubMed

van den Brule, Sybille; Wallemme, Laurent; Uwambayinema, Francine; Huaux, François; Lison, Dominique

2010-11-01

Prostaglandin (PG) D(2) exerts contrasting activities in the inflamed lung via two receptors, the D prostanoid receptor (DP) and the chemoattractant receptor-homologous molecule expressed on T helper 2 lymphocytes. DP activation is known mainly to inhibit proinflammatory cell functions. We tested the effect of a DP-specific agonist, (4S)-(3-[(3R,S)-3-cyclohexyl-3-hydroxypropyl]-2,5-dioxo)-4-imidazolidineheptanoic acid (BW245C), on pulmonary fibroblast functions in vitro and in a mouse model of lung fibrosis induced by bleomycin. DP mRNA expression was detected in cultured mouse lung primary fibroblasts and human fetal lung fibroblasts and found to be up- and down-regulated by interleukin-13 and transforming growth factor (TGF)-β, respectively. Although micromolar concentrations of BW245C and PGD(2) did not affect mouse fibroblast collagen synthesis or differentiation in myofibroblasts, they both inhibited fibroblast basal and TGF-β-induced proliferation in vitro. The repeated administration of BW245C (500 nmol/kg body weight instilled transorally in the lungs 2 days before and three times per week for 3 weeks) in bleomycin-treated mice significantly decreased both inflammatory cell recruitment and collagen accumulation in the lung (21 days). Our results indicate that BW245C can reduce lung fibrosis in part via its activity on fibroblast proliferation and suggest that DP activation should be considered as a new therapeutic target in fibroproliferative lung diseases.

Interferon induction by two 2'-modified double-helical RNAs, poly(2'-fluoro-2'-deoxyinosinic acid) x poly(cytidylic acid) and poly(2'-chloro-2'-deoxyinosinic acid) x poly(cytidylic acid).

PubMed

De Clercq, E; Stollar, B D; Hobbs, J; Fukui, T; Kakiuchi, N; Ikehara, M

1980-01-01

In addition to the 2'-azido analogue of (I)n x (C)n, (dIn3)n x (C)n, we have found two other (I)n x (C)n analogues, (dIfl)n x (C)n and (dIcl)n x (C)n, in which the 2'-hydroxyls of the (I)n strand are replaced by either fluorine or chlorine, to be highly effective in inducing interferon. This contrasted with the lack of interferon-inducing activity noted for various other 2'-halogeno analogues of (I)n x (C)n and (A)n x (U)n, i.e. (I)n x (dCcl)n, (dAfl)n x (U)n, (dAcl)n x (U)n, (A)n x (dUfl)n and (A)n x (dUcl)n. In most assay systems, viz. primary rabbit kidney cells, human diploid fibroblasts, HeLa cells, interferon-primed mouse L-929 cells, and intact rabbits, (dIfl)n x (C)n and (dIcl)n x (C)n induced interferon levels that were comparable to those induced by (I)n x (C)n. There was one particular system (L-929 cells treated with DEAE-dextran), however, in which (dIfl)n x (C)n and (dIcl)n x (C)n, unlike (I)n x (C)n, failed to stimulate interferon production. As monitored by both radiochemical and biological means, (dIfl)n x (C)n and, to a lesser extent, (dIcl)n x (C)n were more resistant to degradation by ribonuclease A, T1 and human serum nucleases than was (I)n x (C)n. In their reactivity towards antibodies to double-stranded RNA (dIfl)n x (C)n and (dIcl)n x (C)n conformed more closely to (I)n x (C)n than did other 2'-substituted (e.g. 2'-O-methyl or 2'-O-ethyl) analogues of (I)n x (C)n. The high interferon-inducing potency of (dIfl)n x (C)n and (dIcl)n x (C)n has both theoretical and practical implications. While our findings suggest that (dIfl)n x (C)n and (dIcl)n x (C)n should be further explored for their therapeutic potentials, they also strengthen the notion that the interferon-inducing capacity, and possibly other biological functions of double-stranded RNAs is dependent on the recognition of the overall conformation of the polynucleotide rather than on the binding of specific functional groups such as the 2'-hydroxyl group.

Caspase inhibition augmented oridonin-induced cell death in murine fibrosarcoma l929 by enhancing reactive oxygen species generation.

PubMed

Wu, Jin-Nan; Huang, Jian; Yang, Jia; Tashiro, Shin-Ichi; Onodera, Satoshi; Ikejima, Takashi

2008-09-01

Oridonin, a diterpenoid isolated from Rabdosia rubescences, has been reported to have antitumor effects. In this study, the growth-inhibitory activity of oridonin for L929 cells was exerted in a time-and dose-dependent manner. After treatment with oridonin for 24 h, L929 cells underwent both apoptosis and necrosis as measured by an lactate dehydrogenase (LDH) activity-based assay. A rapid generation of reactive oxygen species (ROS) was triggered by oridonin, and subsequently up-regulation of phospho-p53 (ser 15) expression and an increased expression ratio of Bax/Bcl-2 was observed. Furthermore, there was a significant fall in mitochondrial membrane potential (MMP) and increase in caspase-3 activity after exposure to oridonin for 24 h. Surprisingly, the pan-caspase inhibitor z-VAD-fmk and caspase3 inhibitor z-DEVD-fmk rendered L929 cells more sensitive to oridonin, rather than preventing oridonin-induced cell death. Oridonin and z-VAD-fmk co-treatment not only resulted in an even higher ROS production, but also made a more significant reduction in the MMP. Pretreatment of ROS scavenger N-acetylcysteine (NAC) led to a complete inhibition of oridonin-induced cell death, intracellular ROS generation, and MMP collapse. NAC treatment also reversed the potentiation of cell death by the pan-caspase inhibitor z-VAD-fmk. Taken together, these observations showed that oridonin-induced cell death in L929 cells involved intracellular ROS generation, activation of phospho-p53 (ser 15), and up-regulation of the Bax/Bcl-2 ratio; and the augmented cell death by z-VAD-fmk was dependent on an increased ROS production.

Neutrality of the canonical NF-kappaB-dependent pathway for human and murine cytomegalovirus transcription and replication in vitro.

PubMed

Benedict, Chris A; Angulo, Ana; Patterson, Ginelle; Ha, Sukwon; Huang, Huang; Messerle, Martin; Ware, Carl F; Ghazal, Peter

2004-01-01

Cytomegalovirus (CMV) is known to rapidly induce activation of nuclear factor kappaB (NF-kappaB) after infection of fibroblast and macrophage cells. NF-kappaB response elements are present in the enhancer region of the CMV major immediate-early promoter (MIEP), and activity of the MIEP is strongly upregulated by NF-kappaB in transient-transfection assays. Here we investigate whether the NF-kappaB-dependent pathway is required for initiating or potentiating human and murine CMV replication in vitro. We show that expression of a dominant negative mutant of the inhibitor of NF-kappaB-alpha (IkappaBalphaM) does not alter the replication kinetics of human or mouse CMV in cultured cells. In addition, mouse embryo fibroblasts genetically deficient for p65/RelA actually showed elevated levels of MCMV replication. Mutation of all NF-kappaB response elements within the enhancer of the MIEP in a recombinant mouse CMV containing the human MIEP (hMCMV-ES), which we have previously shown to replicate in murine fibroblasts with kinetics equivalent to that of wild-type mouse CMV, did not negatively affect replication in fibroblasts. Taken together, these data show that, for CMV replication in cultured fibroblasts activation of the canonical NF-kappaB pathway and binding of NF-kappaB to the MIEP are dispensable, and in the case of p65 may even interfere, thus uncovering a previously unrecognized level of complexity in the host regulatory network governing MIE gene expression in the context of a viral infection.

Inhibiting aerobic glycolysis suppresses renal interstitial fibroblast activation and renal fibrosis.

PubMed

Ding, Hao; Jiang, Lei; Xu, Jing; Bai, Feng; Zhou, Yang; Yuan, Qi; Luo, Jing; Zen, Ke; Yang, Junwei

2017-09-01

Chronic kidney diseases generally lead to renal fibrosis. Despite great progress having been made in identifying molecular mediators of fibrosis, the mechanism that governs renal fibrosis remains unclear, and so far no effective therapeutic antifibrosis strategy is available. Here we demonstrated that a switch of metabolism from oxidative phosphorylation to aerobic glycolysis (Warburg effect) in renal fibroblasts was the primary feature of fibroblast activation during renal fibrosis and that suppressing renal fibroblast aerobic glycolysis could significantly reduce renal fibrosis. Both gene and protein assay showed that the expression of glycolysis enzymes was upregulated in mouse kidneys with unilateral ureter obstruction (UUO) surgery or in transforming growth factor-β1 (TGF-β1)-treated renal interstitial fibroblasts. Aerobic glycolysis flux, indicated by glucose uptake and lactate production, was increased in mouse kidney with UUO nephropathy or TGF-β1-treated renal interstitial fibroblasts and positively correlated with fibrosis process. In line with this, we found that increasing aerobic glycolysis can remarkably induce myofibroblast activation while aerobic glycolysis inhibitors shikonin and 2-deoxyglucose attenuate UUO-induced mouse renal fibrosis and TGF-β1-stimulated myofibroblast activation. Furthermore, mechanistic study indicated that shikonin inhibits renal aerobic glycolysis via reducing phosphorylation of pyruvate kinase type M2, a rate-limiting glycolytic enzyme associated with cell reliance on aerobic glycolysis. In conclusion, our findings demonstrate the critical role of aerobic glycolysis in renal fibrosis and support treatment with aerobic glycolysis inhibitors as a potential antifibrotic strategy. Copyright © 2017 the American Physiological Society.

OX40L blockade protects against inflammation-driven fibrosis

PubMed Central

Elhai, Muriel; Avouac, Jérôme; Hoffmann-Vold, Anna Maria; Ruzehaji, Nadira; Amiar, Olivia; Ruiz, Barbara; Brahiti, Hassina; Ponsoye, Matthieu; Fréchet, Maxime; Burgevin, Anne; Pezet, Sonia; Sadoine, Jérémy; Guilbert, Thomas; Nicco, Carole; Akiba, Hisaya; Heissmeyer, Vigo; Subramaniam, Arun; Resnick, Robert; Molberg, Øyvind; Kahan, André; Chiocchia, Gilles; Allanore, Yannick

2016-01-01

Treatment for fibrosis represents a critical unmet need, because fibrosis is the leading cause of death in industrialized countries, and there is no effective therapy to counteract the fibrotic process. The development of fibrosis relates to the interplay between vessel injury, immune cell activation, and fibroblast stimulation, which can occur in various tissues. Immunotherapies have provided a breakthrough in the treatment of immune diseases. The glycoprotein OX40–OX40 ligand (OX40L) axis offers the advantage of a targeted approach to costimulatory signals with limited impact on the whole immune response. Using systemic sclerosis (SSc) as a prototypic disease, we report compelling evidence that blockade of OX40L is a promising strategy for the treatment of inflammation-driven fibrosis. OX40L is overexpressed in the fibrotic skin and serum of patients with SSc, particularly in patients with diffuse cutaneous forms. Soluble OX40L was identified as a promising serum biomarker to predict the worsening of lung and skin fibrosis, highlighting the role of this pathway in fibrosis. In vivo, OX40L blockade prevents inflammation-driven skin, lung, and vessel fibrosis and induces the regression of established dermal fibrosis in different complementary mouse models. OX40L exerts potent profibrotic effects by promoting the infiltration of inflammatory cells into lesional tissues and therefore the release of proinflammatory mediators, thereafter leading to fibroblast activation. PMID:27298374

Endothelial Dysfunction Exacerbates Renal Interstitial Fibrosis through Enhancing Fibroblast Smad3 Linker Phosphorylation in the Mouse Obstructed Kidney

PubMed Central

Sun, Yu Bo Yang; Qu, Xinli; Li, Xueling; Nikolic-Paterson, David J.; Li, Jinhua

2013-01-01

Endothelial dysfunction and enhanced transforming growth factor-β (TGF-β)/Smad3 signalling are common features of progressive renal fibrosis. This study investigated a potential link between these mechanisms. In unilateral ureteric obstruction (UUO) we observed an acute (6 hr) down-regulation of nitric oxide synthase 3 (NOS3/eNOS) levels and increased phosphorylation of the linker region of Smad3 at T179 and S208 in Smad3/JNK complexes. These events preceded Smad3 C-terminal domain phosphorylation and the induction of myofibroblast proliferation at 48 hrs. Mice deficient in NOS3 showed enhanced myofibroblast proliferation and collagen accumulation compared to wild type mice in a 7 day UUO model. This was associated with enhanced phosphorylation of Smad3 T179 and S208 by 92% and 88%, respectively, whereas Smad3-C-terminal phosphorylation was not affected. Resolvin D1 (RvD1) can suppress renal fibrosis in the UUO model, and further analysis herein showed that RvD1 protected against endothelial dysfunction and suppressed Smad3/JNK complex formation with a consequent reduction in phosphorylation of Smad3 T179 and S208 by 78% and 65%, respectively, while Smad3 C-terminal phosphorylation was unaltered. In vitro, conditioned media from mouse microvascular endothelial cells (MMEC) treated with a general inhibitor of nitric oxide synthase (L-NAME) augmented the proliferation and collagen production of renal fibroblasts (NRK49F cells) compared to control MMEC media and this was associated with increased phosphorylation of JNK and Smad3 T179 and S208, whereas Smad3-C-terminal domain phosphorylation was unaffected. The addition of RvD1 to L-NAME treated MMEC abrogated these effects of the conditioned media on renal fibroblasts. Finally, Smad3 T179/V and S208/A mutations significantly inhibit TGF-β1 induced up-regulation collagen I promoter. In conclusion, these data suggest that endothelial dysfunction can exacerbate renal interstitial fibrosis through increased fibroblast proliferation and collagen production via enhanced Smad3 linker phosphorylation. PMID:24391884

Endothelial dysfunction exacerbates renal interstitial fibrosis through enhancing fibroblast Smad3 linker phosphorylation in the mouse obstructed kidney.

PubMed

Sun, Yu Bo Yang; Qu, Xinli; Li, Xueling; Nikolic-Paterson, David J; Li, Jinhua

2013-01-01

Endothelial dysfunction and enhanced transforming growth factor-β (TGF-β)/Smad3 signalling are common features of progressive renal fibrosis. This study investigated a potential link between these mechanisms. In unilateral ureteric obstruction (UUO) we observed an acute (6 hr) down-regulation of nitric oxide synthase 3 (NOS3/eNOS) levels and increased phosphorylation of the linker region of Smad3 at T179 and S208 in Smad3/JNK complexes. These events preceded Smad3 C-terminal domain phosphorylation and the induction of myofibroblast proliferation at 48 hrs. Mice deficient in NOS3 showed enhanced myofibroblast proliferation and collagen accumulation compared to wild type mice in a 7 day UUO model. This was associated with enhanced phosphorylation of Smad3 T179 and S208 by 92% and 88%, respectively, whereas Smad3-C-terminal phosphorylation was not affected. Resolvin D1 (RvD1) can suppress renal fibrosis in the UUO model, and further analysis herein showed that RvD1 protected against endothelial dysfunction and suppressed Smad3/JNK complex formation with a consequent reduction in phosphorylation of Smad3 T179 and S208 by 78% and 65%, respectively, while Smad3 C-terminal phosphorylation was unaltered. In vitro, conditioned media from mouse microvascular endothelial cells (MMEC) treated with a general inhibitor of nitric oxide synthase (L-NAME) augmented the proliferation and collagen production of renal fibroblasts (NRK49F cells) compared to control MMEC media and this was associated with increased phosphorylation of JNK and Smad3 T179 and S208, whereas Smad3-C-terminal domain phosphorylation was unaffected. The addition of RvD1 to L-NAME treated MMEC abrogated these effects of the conditioned media on renal fibroblasts. Finally, Smad3 T179/V and S208/A mutations significantly inhibit TGF-β1 induced up-regulation collagen I promoter. In conclusion, these data suggest that endothelial dysfunction can exacerbate renal interstitial fibrosis through increased fibroblast proliferation and collagen production via enhanced Smad3 linker phosphorylation.

Enhanced cloning efficiency of mouse bone marrow macrophage progenitors correlates with increased content of CSF-1 receptor of their progeny at low oxygen tension.

PubMed

Flamant, Stéphane; Lebastard, Maï; Pescher, Pascale; Besmond, Claude; Milon, Geneviève; Marchal, Gilles

2003-10-01

Mononuclear phagocytes are located in every tissue of metazoan organisms. In this extravascular space, they are designated as macrophages and are known to sense and process many signals including the local oxygen tension (PO2), which ranges from 150 mmHg at the lung apices to around 40 mmHg in mixed venous blood and most organs, and to less than 10 mmHg in tissues where long-term and dynamic remodeling processes occur. Most tissue macrophages survive and maintain their differentiated status within an environment bathed by colony-stimulating factor (CSF)-1 through the CSF-1 receptor, encoded by the Csf1r gene. In order to investigate the mRNA expression profile of macrophages as a function of PO2, we developed an in vitro model in which monocyte-derived macrophages were generated from mouse bone marrow progenitor cells grown and maintained under low (36 mmHg) or atmospheric (142 mmHg) PO2, in the presence of L929-conditioned medium (L-CM) as a source of CSF-1. We show that CSF-1-reactive C57BL/6 bone marrow cells displayed an increased cloning efficiency under a PO2 of 36, compared with 142 mmHg. Furthermore, we provide evidence of the overexpression of both CSF-1 receptor protein and mRNA by mouse monocyte-derived macrophages generated from bone marrow under low PO2.

Effect of Air-Polishing on Titanium Surfaces, Biofilm Removal, and Biocompatibility: A Pilot Study

PubMed Central

Bennani, Vincent; Hwang, Linda; Tawse-Smith, Andrew; Dias, George J.; Cannon, Richard D.

2015-01-01

Purpose. The aims of this in vitro study were to evaluate morphological changes induced by glycine powder air-polishing on titanium surfaces, biofilm removal, and biocompatibility. Material and Methods. Titanium grade IV discs were allocated into two groups: (1) discs without biofilm and (2) discs for Streptococcus mutans biofilm formation. Discs in each group were further subdivided into (a) no treatment and (b) air-polishing treatment with glycine powder. Discs were characterized by scanning electron microscopy (SEM), electron-dispersive spectroscopy (EDS), and confocal microscopy. Bacterial biofilms were quantified using a crystal violet dye-binding assay. Biocompatibility was evaluated by measuring the coverage and viability of L929 fibroblast cells cultured on the discs. Results. Air-polishing increased the roughness of treated discs (P < 0.05). EDS analysis did not show significant differences in the chemical composition of treated and nontreated discs. The amount of residual biofilm on treated discs was 8.6-fold lower than untreated controls (P < 0.05). Coverage of treated discs by fibroblasts was half that of untreated discs (P < 0.05) although both groups had the same cell viability. Conclusions. Air-polishing removed a significant amount of biofilm from titanium surfaces. The “polishing” was accompanied by increased surface roughness, but there were no changes in chemical and elemental compositions, nor the biocompatibility. PMID:26881198

In vitro and in vivo photoprotective/photochemopreventive potential of Garcinia brasiliensis epicarp extract.

PubMed

Figueiredo, Sônia Aparecida; Vilela, Fernanda Maria Pinto; da Silva, Claudinei Alves; Cunha, Thiago Mattar; Dos Santos, Marcelo Henrique; Fonseca, Maria José Vieira

2014-02-05

The damaging effects of sunlight to the skin has triggered studies that involve the synthesis and extraction of organic compounds from natural sources that can absorb UV radiation, and studies on polyphenolic compounds with antioxidant and anti-inflammatory properties that can be used as photochemopreventive agents for reducing skin damage. We investigated the in vitro and in vivo photoprotective/photochemopreventive potential of Garcinia brasiliensis epicarp extract (GbEE). We evaluated the cell viability of L929 fibroblasts after UVB exposure using a quartz plate containing the extract solution or the GbEE formulation. The in vivo photoprotective effect of the GbEE formulation was evaluated by measuring the UVB damage-induced decrease in endogenous reduced glutathione (GSH), the increase in myeloperoxidase (MPO) activity and secretion of cytokines IL-1β and TNF-α. The in vitro methodology using fibroblasts showed that the photoprotective properties of the GbEE solutions and 10% GbEE formulation were similar to the commercial sunscreen (SPF-15). In vivo results demonstrated of the GbEE formulation in decreasing UVB induced-damage such as GSH depletion, an increased in MPO activity and secretion of cytokines IL-1β and TNF-α. The results showed that the extract has great potential for use as a sunscreen in topical formulations in addition to UV filters. Copyright © 2014 Elsevier B.V. All rights reserved.

In vitro study on silk fibroin textile structure for anterior cruciate ligament regeneration.

PubMed

Farè, Silvia; Torricelli, Paola; Giavaresi, Gianluca; Bertoldi, Serena; Alessandrino, Antonio; Villa, Tomaso; Fini, Milena; Tanzi, Maria Cristina; Freddi, Giuliano

2013-10-01

A novel hierarchical textile structure made of silk fibroin from Bombyx mori capable of matching the mechanical performance requirements of anterior cruciate ligament (ACL) and in vitro cell ingrowth is described. This sericin-free, Silk Fibroin Knitted Sheath with Braided Core (SF-KSBC) structure was fabricated using available textile technologies. Micro-CT analysis confirmed that the core was highly porous and had a higher degree of interconnectivity than that observed for the sheath. The in vivo cell colonization of the scaffolds is thus expected to penetrate even the internal parts of the structure. Tensile mechanical tests demonstrated a maximum load of 1212.4±56.4 N (under hydrated conditions), confirming the scaffold's suitability for ACL reconstruction. The absence of cytotoxic substances in the extracts of the SF-KSBC structure in culture medium was verified by in vitro tests with L929 fibroblasts. In terms of extracellular matrix production, Human Periodontal Ligament Fibroblasts (HPdLFs) cultured in direct contact with SF-KSBC, compared to control samples, demonstrated an increased secretion of aggrecan (PG) and fibronectin (FBN) at 3 and 7 days of culture, and no change in IL-6 and TNF-α secretion. Altogether, the outcomes of this investigation confirm the significant utility of this novel scaffold for ACL tissue regeneration. Copyright © 2013 Elsevier B.V. All rights reserved.

Deficiency of Suppressor Enhancer Lin12 1 Like (SEL1L) in Mice Leads to Systemic Endoplasmic Reticulum Stress and Embryonic Lethality*

PubMed Central

Francisco, Adam B.; Singh, Rajni; Li, Shuai; Vani, Anish K.; Yang, Liu; Munroe, Robert J.; Diaferia, Giuseppe; Cardano, Marina; Biunno, Ida; Qi, Ling; Schimenti, John C.; Long, Qiaoming

2010-01-01

Stress in the endoplasmic reticulum (ER) plays an important causal role in the pathogenesis of several chronic diseases such as Alzheimer, Parkinson, and diabetes mellitus. Insight into the genetic determinants responsible for ER homeostasis will greatly facilitate the development of therapeutic strategies for the treatment of these debilitating diseases. Suppressor enhancer Lin12 1 like (SEL1L) is an ER membrane protein and was thought to be involved in the quality control of secreted proteins. Here we show that the mice homozygous mutant for SEL1L were embryonic lethal. Electron microscopy studies revealed a severely dilated ER in the fetal liver of mutant embryos, indicative of alteration in ER homeostasis. Consistent with this, several ER stress responsive genes were significantly up-regulated in the mutant embryos. Mouse embryonic fibroblast cells deficient in SEL1L exhibited activated unfolded protein response at the basal state, impaired ER-associated protein degradation, and reduced protein secretion. Furthermore, markedly increased apoptosis was observed in the forebrain and dorsal root ganglions of mutant embryos. Taken together, our results demonstrate an essential role for SEL1L in protein quality control during mouse embryonic development. PMID:20197277

Deficiency of suppressor enhancer Lin12 1 like (SEL1L) in mice leads to systemic endoplasmic reticulum stress and embryonic lethality.

PubMed

Francisco, Adam B; Singh, Rajni; Li, Shuai; Vani, Anish K; Yang, Liu; Munroe, Robert J; Diaferia, Giuseppe; Cardano, Marina; Biunno, Ida; Qi, Ling; Schimenti, John C; Long, Qiaoming

2010-04-30

Stress in the endoplasmic reticulum (ER) plays an important causal role in the pathogenesis of several chronic diseases such as Alzheimer, Parkinson, and diabetes mellitus. Insight into the genetic determinants responsible for ER homeostasis will greatly facilitate the development of therapeutic strategies for the treatment of these debilitating diseases. Suppressor enhancer Lin12 1 like (SEL1L) is an ER membrane protein and was thought to be involved in the quality control of secreted proteins. Here we show that the mice homozygous mutant for SEL1L were embryonic lethal. Electron microscopy studies revealed a severely dilated ER in the fetal liver of mutant embryos, indicative of alteration in ER homeostasis. Consistent with this, several ER stress responsive genes were significantly up-regulated in the mutant embryos. Mouse embryonic fibroblast cells deficient in SEL1L exhibited activated unfolded protein response at the basal state, impaired ER-associated protein degradation, and reduced protein secretion. Furthermore, markedly increased apoptosis was observed in the forebrain and dorsal root ganglions of mutant embryos. Taken together, our results demonstrate an essential role for SEL1L in protein quality control during mouse embryonic development.

A novel p53 mutational hotspot in skin tumors from UV-irradiated Xpc mutant mice alters transactivation functions.

PubMed

Inga, Alberto; Nahari, Dorit; Velasco-Miguel, Susana; Friedberg, Errol C; Resnick, Michael A

2002-08-22

A mutation in codon 122 of the mouse p53 gene resulting in a T to L amino acid substitution (T122-->L) is frequently associated with skin cancer in UV-irradiated mice that are both homozygous mutant for the nucleotide excision repair (NER) gene Xpc (Xpc(-/-)) and hemizygous mutant for the p53 gene. We investigated the functional consequences of the mouse T122-->L mutation when expressed either in mammalian cells or in the yeast Saccharomyces cerevisiae. Similar to a non-functional allele, high expression of the T122-->L allele in p53(-/-) mouse embryo fibroblasts and human Saos-2 cells failed to suppress growth. However, the T122-->L mutant p53 showed wild-type transactivation levels with Bax and MDM2 promoters when expressed in either cell type and retained transactivation of the p21 and the c-Fos promoters in one cell line. Using a recently developed rheostatable p53 induction system in yeast we assessed the T122-->L transactivation capacity at low levels of protein expression using 12 different p53 response elements (REs). Compared to wild-type p53 the T122-->L protein manifested an unusual transactivation pattern comprising reduced and enhanced activity with specific REs. The high incidence of the T122-->L mutant allele in the Xpc(-/-) background suggests that both genetic and epigenetic conditions may facilitate the emergence of particular functional p53 mutations. Furthermore, the approach that we have taken also provides for the dissection of functions that may be retained in many p53 tumor alleles.

Studies on Typhus and Spotted Fever.

DTIC Science & Technology

1980-02-01

prowazekii-infected human somatic (fibroblast, endothelia)), but not chick, mouse or monkey , cells in culture: (a) intracellular antirickettsial action...that of the controls. No such effect on growth was apparent in CE cells, Nu E % o0 M Ŕ ZOO - .0 E 00 (1 CI - 4D W = .) C ~ o r- -!NBI Go !N 21501,,o o...human origin transformed or malignant cells, monkey primary or diploid and primary mouse embryo fibroblasts will permit expression of these effects to

Cellulose-ethylenediaminetetraacetic acid conjugates protect mammalian cells from bacterial cells.

PubMed

Luo, Jie; Lv, Wei; Deng, Ying; Sun, Yuyu

2013-04-08

Cellulose-ethylenediaminetetraacetic acid (EDTA) conjugates were synthesized by the esterification of cellulose with ethylenediaminetetraacetic dianhydride (EDTAD). The new materials provided potent antimicrobial activities against Staphylococcus aureus (S. aureus, Gram-positive bacteria) and Pseudomonas aeruginosa (P. aeruginosa, Gram-negative bacteria), and inhibited the formation of bacterial biofilms. The biocompatibility of the new cellulose-EDTA conjugates was evaluated with mouse skin fibroblasts for up to 14 days. SEM observation and DNA content analysis suggested that the new materials sustained the viability of fibroblast cells. Moreover, in mouse skin fibroblast-bacteria co-culture systems, the new cellulose-EDTA conjugates prevented bacterial biofilm formation and protected the mammalian cells from the bacterial cells for at least one day.

Recombinant porcine epidermal growth factor-secreting Lactococcus lactis promotes the growth performance of early-weaned piglets

PubMed Central

2014-01-01

Background Epidermal growth factor (EGF) is an important growth factor in regulation of cell proliferation, differentiation, survival and apoptosis. Studies showed that food-grade Lactococcus lactis (L. lactis) and NICE expression system have superior performance in exogenous protein expression. This study aimed to construct and express porcine EGF (pEGF), and use L. lactis as vehicle for producing and delivering pEGF. Furthermore, investigating biological activity of pEGF and exploring applications feasibility of combination effects of L. lactis and pEGF on early weaned piglets’ production. Results A recombinant Lactococcus lactis which produced and secreted pEGF at 1000 ng/ml in culture supernatant was generated. Secreted pEGF was a fully biologically active protein, as demonstrated by its capacity to stimulate L929 mouse fibroblast cell line proliferation in vitro. For in vivo study, forty piglets were randomly allocated to control, antibiotic control, empty vector-expressing L. lactis (LL-EV) and pEGF-secreting L. lactis (LL-pEGF). After 14 d of rearing, final body weight and average daily gain in LL-pEGF were greater (P < 0.05, 8.95 vs. 8.37 kg, 206.1 vs. 157.7 g/day, respectively) than those in control, but no significant differences between LL-pEGF, LL-EV and antibiotic control. Overall period average daily feed intake was higher in LL-pEGF, LL-EV and antibiotic control than in control (P < 0.05, 252.9, 255.6, 250.0, 207.3 g/day, respectively). No significant difference was observed on ADFI/ADG. LL-pEGF increased villous height in the duodenum, jejunum and ileum than in control and LL-EV (P < 0.05). Sucrase in the 3 intestinal segments, aminopeptidase A in the duodenum and Jejunum, aminopeptidase N and dipeptidase IV in the duodenum in LL-pEGF were higher than those in control (P < 0.05). Furthermore, Escherichia coli and Enterococcus counts decreased in the ileum and Lactobacillus increased in the ileum and cecum digesta in LL-pEGF compare with the control (P < 0.05). Lactobacillus increased in the cecum in LL-EV compared with control and antibiotic control (P < 0.05). Conclusion We have generated a recombinant Lactococcus lactis which produced and secreted fully biologically active porcine EGF. Oral administration of pEGF-secreting L. lactis had beneficial effects on intestinal health and performance of early-weaned piglets. PMID:25142032

On the in vitro biocompatibility of Elgiloy, a co-based alloy, compared to two titanium alloys.

PubMed

Es-Souni, Martha; Fischer-Brandies, Helge; Es-Souni, Mohammed

2003-01-01

The aim of the present investigation was to contribute to an understanding of the effects of surface topography and chemical composition on the corrosion behavior and thus the biocompatibility of Elgiloy (RMO, Denver, CO, USA), a common Co-based alloy. The results are compared with those obtained for a binary NiTi alloy, Neo Sentalloy (GAC, Central Islip, NY, USA) and a beta-III-Ti alloy, TMA (Ormco, Glendora, CA, USA). In the present study, the surface topography and the chemical composition of two different grades of Elgiloy, Blue Elgiloy (soft) and Yellow Elgiloy (ductile), were examined by means of scanning electron microscopy (SEM) and energy-dispersive spectroscopy analysis (EDS). Their corrosion behavior in half-strength Ringer solution and in an artificial saliva solution according to Barrett [1] was investigated using potentiodynamic corrosion testing (PDC). The photometry-based PAN method was used to quantify the released Ni and Co ions. The in vitro biocompatibility of the two grades of Elgiloy was tested in three different cell cultures: in L929, a commercially available mouse fibroblast cell line, and in primary human epithelial cells and fibroblasts. The results of the corrosion testing showed satisfactorily high pitting corrosion potentials but lower repassivation potentials and a strong increase in current density once pitting had occurred. The photometric results revealed the release of Ni and Co ions in both tested electrolytes. The tested native surfaces exhibited numerous grinding and polishing grooves, inclusions and inhomogeneities of the microstructure. After corrosion testing the same surfaces displayed numerous signs of corrosion, especially in areas with microstructural inhomogeneities. In vitro biocompatibility testing showed a substantially reduced dehydrogenase activity in the presence of Elgiloy. The reduced quality of surface finish resulting from the manufacturing process led in the case of the tested Elgiloy types to decreased corrosion resistance with consequently reduced in vitro biocompatibility. In this context it is also conceivable that patients with a proven allergy to nickel, cobalt or chromium may react sensitively to the deployment of this alloy, at least in the surface quality tested by us. From this aspect, the introduction of a binding standard for the surface quality of materials used in orthodontic appliances is urgently recommended.

Microwave based synthesis and spectral characterization of thermo-sensitive poly(N,N-diethylacrylamide) grafted pectin copolymer.

PubMed

Işıklan, Nuran; Tokmak, Şeyma

2018-07-01

The functionalization of polysaccharides with synthetic polymers has attracted great attention owing to its application in many industrial fields. The aim of this work was to study the impact of pectin functionalization with N,N-diethylacrylamide (DEAAm). Pectin was modified via microwave-induced graft copolymerization of DEAAm using ceric ammonium nitrate (CAN) and N,N,N',N'-tetramethylethylenediamine (TEMED). FTIR, 13 C NMR, DSC/TGA, XRD, and SEM techniques were used to verify the structure of graft copolymers. Various reaction conditions such as microwave irradiation time, temperature, microwave power, monomer, initiator, and TEMED concentrations were investigated to get a maximum grafting yield of 192%. Lower critical solution temperatures (LCST) of graft copolymers were determined by UV spectroscopy. Graft copolymers were found to be thermo-sensitive, with LCST of 31°C and high thermal resistance. Biocompatibility test of copolymers showed that copolymers were not cytotoxic to L929 fibroblasts cells and can be used as a biomaterial. Copyright © 2018 Elsevier B.V. All rights reserved.

Conjugation of curcumin onto alginate enhances aqueous solubility and stability of curcumin.

PubMed

Dey, Soma; Sreenivasan, K

2014-01-01

Curcumin is a potential drug for various diseases including cancer. Prime limitations associated with curcumin are low water solubility, rapid hydrolytic degradation and poor bioavailability. In order to redress these issues we developed Alginate-Curcumin (Alg-Ccm) conjugate which was characterized by FTIR and (1)H NMR spectroscopy. The conjugate self-assembled in aqueous solution forming micelles with an average hydrodynamic diameter of 459 ± 0.32 nm and negative zeta potential. The spherical micelles were visualized by TEM. The critical micelle concentration (CMC) of Alg-Ccm conjugate was determined. A significant enhancement in the aqueous solubility of curcumin was observed upon conjugation with alginate. Formation of micelles improved the stability of curcumin in water at physiological pH. The cytotoxic activity of Alg-Ccm was quantified by MTT assay using L-929 fibroblast cells and it was found to be potentially cytotoxic. Hence, Alg-Ccm could be a promising drug conjugate as well as a nanosized delivery vehicle. Copyright © 2013 Elsevier Ltd. All rights reserved.

Quantitative in vitro assessment of Mg65 Zn30 Ca5 degradation and its effect on cell viability.

PubMed

Cao, Jake D; Martens, Penny; Laws, Kevin J; Boughton, Philip; Ferry, Michael

2013-01-01

A bulk metallic glass (BMG) of composition Mg(65) Zn(30) Ca(5) was cast directly from the melt and explored as a potential bioresorbable metallic material. The in vitro degradation behavior of the amorphous alloy and its associated effects on cellular activities were assessed against pure crystalline magnesium. Biocorrosion tests using potentiodynamic polarization showed that the amorphous alloy corroded at a much slower rate than the crystalline Mg. Analysis of the exchanged media using inductively coupled plasma optical emission spectrometry revealed that the dissolution rate of Mg ions in the BMG was 446 μg/cm(2)/day, approximately half the rate of crystalline Mg (859 μg/cm(2)/day). A cytotoxicity study, using L929 murine fibroblasts, revealed that both the BMG and pure Mg are capable of supporting cellular activities. However, direct contact with the samples created regions of minimal cell growth around both amorphous and crystalline samples, and no cell attachment was observed. Copyright © 2012 Wiley Periodicals, Inc.

Characterization of surface modified polyester fabric.

PubMed

Joseph, Roy; Shelma, R; Rajeev, A; Muraleedharan, C V

2009-12-01

Woven polyethylene terephthalate (PET) fabric has been used in the construction of vascular grafts and sewing ring of prosthetic heart valves. In an effort to improve haemocompatibility and tissue response to PET fabric, a fluoropolymer, polyvinylidine fluoride (PVDF), was coated on PET fabric by dip coating technique. The coating was found to be uniform and no significant changes occurred on physical properties such as water permeability and burst strength. Cell culture cytotoxicity studies showed that coated PET was non-cytotoxic to L929 fibroblast cell lines. In vitro studies revealed that coating improved haemocompatibility of PET fabric material. Coating reduced platelet consumption of PET fabric by 50%. Upon surface modification leukocyte consumption of PET was reduced by 24%. About 60% reduction in partial thromboplastin time (PTT) observed when PET was coated with PVDF. Results of endothelial cell proliferation studies showed that surface coating did not have any substantial impact on cell proliferation. Overall results indicate that coating has potential to improve haemocompatibility of PET fabric without affecting its mechanical performance.

Evaluation of gallic acid loaded zein sub-micron electrospun fibre mats as novel active packaging materials.

PubMed

Neo, Yun Ping; Swift, Simon; Ray, Sudip; Gizdavic-Nikolaidis, Marija; Jin, Jianyong; Perera, Conrad O

2013-12-01

The applicability of gallic acid loaded zein (Ze-GA) electrospun fibre mats towards potential active food packaging material was evaluated. The surface chemistry of the electrospun fibre mats was determined using X-ray photon spectroscopy (XPS). The electrospun fibre mats showed low water activity and whitish colour. Thermogravimetric analysis (TGA) and Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy revealed the stability of the fibre mats over time. The Ze-GA fibre mats displayed similar rapid release profiles, with Ze-GA 20% exhibiting the fastest release rate in water as compared to the others. Gallic acid diffuses from the electrospun fibres in a Fickian diffusion manner and the data obtained exhibited a better fit to Higuchi model. L929 fibroblast cells were cultured on the electrospun fibres to demonstrate the absence of cytotoxicity. Overall, the Ze-GA fibre mats demonstrated antibacterial activity and properties consistent with those considered desirable for active packaging material in the food industry. Copyright © 2013 Elsevier Ltd. All rights reserved.

Manufacturing of biodegradable polyurethane scaffolds based on polycaprolactone using a phase separation method: physical properties and in vitro assay

PubMed Central

Asefnejad, Azadeh; Khorasani, Mohammad Taghi; Behnamghader, Aliasghar; Farsadzadeh, Babak; Bonakdar, Shahin

2011-01-01

Background Biodegradable polyurethanes have found widespread use in soft tissue engineering due to their suitable mechanical properties and biocompatibility. Methods In this study, polyurethane samples were synthesized from polycaprolactone, hexamethylene diisocyanate, and a copolymer of 1,4-butanediol as a chain extender. Polyurethane scaffolds were fabricated by a combination of liquid–liquid phase separation and salt leaching techniques. The effect of the NCO:OH ratio on porosity content and pore morphology was investigated. Results Scanning electron micrographs demonstrated that the scaffolds had a regular distribution of interconnected pores, with pore diameters of 50–300 μm, and porosities of 64%–83%. It was observed that, by increasing the NCO:OH ratio, the average pore size, compressive strength, and compressive modulus increased. L929 fibroblast and chondrocytes were cultured on the scaffolds, and all samples exhibited suitable cell attachment and growth, with a high level of biocompatibility. Conclusion These biodegradable polyurethane scaffolds demonstrate potential for soft tissue engineering applications. PMID:22072874

Design and characterization of a composite material based on Sr(II)-loaded clay nanotubes included within a biopolymer matrix.

PubMed

Del Buffa, Stefano; Bonini, Massimo; Ridi, Francesca; Severi, Mirko; Losi, Paola; Volpi, Silvia; Al Kayal, Tamer; Soldani, Giorgio; Baglioni, Piero

2015-06-15

This paper reports on the preparation, characterization, and cytotoxicity of a hybrid nanocomposite material made of Sr(II)-loaded Halloysite nanotubes included within a biopolymer (3-polyhydroxybutyrate-co-3-hydroxyvalerate) matrix. The Sr(II)-loaded inorganic scaffold is intended to provide mechanical resistance, multi-scale porosity, and to favor the in-situ regeneration of bone tissue thanks to its biocompatibility and bioactivity. The interaction of the hybrid system with the physiological environment is mediated by the biopolymer coating, which acts as a binder, as well as a diffusional barrier to the Sr(II) release. The degradation of the polymer progressively leads to the exposure of the Sr(II)-loaded Halloysite scaffold, tuning its interaction with osteogenic cells. The in vitro biocompatibility of the composite was demonstrated by cytotoxicity tests on L929 fibroblast cells. The results indicate that this composite material could be of interest for multiple strategies in the field of bone tissue engineering. Copyright © 2015 Elsevier Inc. All rights reserved.

Green synthesis of gold nanoparticles using aqueous extract of Dillenia indica

NASA Astrophysics Data System (ADS)

Sett, Arghya; Gadewar, Manoj; Sharma, Pragya; Deka, Manab; Bora, Utpal

2016-06-01

In this study, we report a novel method of gold nanoparticle (AuNP) synthesis using aqueous fruit extract of Dillenia indica. The phytochemicals present in the fruit extract act as an effective reducing and capping agent to synthesize AuNPs. The synthesized AuNPs were characterized by spectrophotometry, transmission electron microscopy (TEM), x-ray diffraction (XRD), and Fourier transform infrared (FTIR) spectroscopy. TEM studies revealed the particles of various sizes and mainly spherical in shape. Selected-area electron diffraction (SAED) patterns and high-resolution transmission electron microscopy (HRTEM) images confirmed the crystallinity of the particles. The XRD patterns showed peaks at (111), (200), (220) which exhibited preferential orientation of the AuNPs as face-centered cubic crystal. FTIR measurements confirmed the coating of phenolic compounds on the AuNPs indicating a possible role of biomolecules for the capping and efficient stabilization of the AuNPs. The synthesized AuNPs did not show any form of cytotoxicity in the normal fibroblast cell line L929.

Modafinil inhibits K(Ca)3.1 currents and muscle contraction via a cAMP-dependent mechanism.

PubMed

Choi, Shinkyu; Kim, Moon Young; Joo, Ka Young; Park, Seonghee; Kim, Ji Aee; Jung, Jae-Chul; Oh, Seikwan; Suh, Suk Hyo

2012-07-01

Modafinil has been used as a psychostimulant for the treatment of narcolepsy. However, its primary mechanism of action remains elusive. Therefore, we examined the effects of modafinil on K(Ca)3.1 channels and vascular smooth muscle contraction. K(Ca)3.1 currents and channel activity were measured using a voltage-clamp technique and inside-out patches in mouse embryonic fibroblast cell line, NIH-3T3 fibroblasts. Intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration was measured, and the phosphorylation of K(Ca)3.1 channel protein was examined using western blotting in NIH-3T3 fibroblasts and/or primary cultured mouse aortic smooth muscle cells (SMCs). Muscle contractions were recorded from mouse aorta and rat pulmonary artery by using a myograph developed in-house. Modafinil was found to inhibit K(Ca)3.1 currents in a concentration-dependent manner, and the half-maximal inhibition (IC(50)) of modafinil for the current inhibition was 6.8 ± 0.7 nM. The protein kinase A (PKA) activator forskolin also inhibited K(Ca)3.1 currents. The inhibitory effects of modafinil and forskolin on K(Ca)3.1 currents were blocked by the PKA inhibitors PKI(14-22) or H-89. In addition, modafinil relaxed blood vessels (mouse aorta and rat pulmonary artery) in a concentration-dependent manner. Modafinil increased cAMP concentrations in NIH-3T3 fibroblasts or primary cultured mouse aortic SMCs and phosphorylated K(Ca)3.1 channel protein in NIH-3T3 fibroblasts. However, open probability and single-channel current amplitudes of K(Ca)3.1 channels were not changed by modafinil. From these results, we conclude that modafinil inhibits K(Ca)3.1 channels and vascular smooth muscle contraction by cAMP-dependent phosphorylation, suggesting that modafinil can be used as a cAMP-dependent K(Ca)3.1 channel blocker and vasodilator. Copyright © 2012 Elsevier Ltd. All rights reserved.

Roughness threshold for cell attachment and proliferation on plasma micro-nanotextured polymeric surfaces: the case of primary human skin fibroblasts and mouse immortalized 3T3 fibroblasts

NASA Astrophysics Data System (ADS)

Bourkoula, A.; Constantoudis, V.; Kontziampasis, D.; Petrou, P. S.; Kakabakos, S. E.; Tserepi, A.; Gogolides, E.

2016-08-01

Poly(methyl methacrylate) surfaces have been micro-nanotextured in oxygen plasmas with increasing ion energy, leading to micro-nanotopography characterized by increased root mean square roughness, correlation length and fractal dimension. Primary human skin fibroblasts and mouse immortalized 3T3 fibroblasts were cultured on these surfaces and the number of adhering cells, their proliferation rate and morphology (cytoplasm and nucleus area) were evaluated as a function of roughness height, correlation length, and fractal dimension. A roughness threshold behavior was observed for both types of cells leading to dramatic cell number decrease above this threshold, which is almost similar for the two types of cells, despite their differences in size and stiffness. The results are discussed based on two theoretical models, which are reconciled and unified when the elastic moduli and the size of the cells are taken into account.

Biocompatibility evaluation of sputtered zirconium-based thin film metallic glass-coated steels.

PubMed

Subramanian, Balasubramanian; Maruthamuthu, Sundaram; Rajan, Senthilperumal Thanka

2015-01-01

Thin film metallic glasses comprised of Zr48Cu36Al8Ag8 (at.%) of approximately 1.5 μm and 3 μm in thickness were prepared using magnetron sputtering onto medical grade 316L stainless steel. Their structural and mechanical properties, in vitro corrosion, and antimicrobial activity were analyzed. The amorphous thin film metallic glasses consisted of a single glassy phase, with an absence of any detectable peaks corresponding to crystalline phases. Elemental composition close to the target alloy was noted from EDAX analysis of the thin film. The surface morphology of the film showed a smooth surface on scanning electron microscopy and atomic force microscopy. In vitro electrochemical corrosion studies indicated that the zirconium-based metallic glass could withstand body fluid, showing superior resistance to corrosion and electrochemical stability. Interactions between the coated surface and bacteria were investigated by agar diffusion, solution suspension, and wet interfacial contact methods. The results indicated a clear zone of inhibition against the growth of microorganisms such as Escherichia coli and Staphylococcus aureus, confirming the antimicrobial activity of the thin film metallic glasses. Cytotoxicity studies using L929 fibroblast cells showed these coatings to be noncytotoxic in nature.

Biocompatibility evaluation of sputtered zirconium-based thin film metallic glass-coated steels

PubMed Central

Subramanian, Balasubramanian; Maruthamuthu, Sundaram; Rajan, Senthilperumal Thanka

2015-01-01

Thin film metallic glasses comprised of Zr48Cu36Al8Ag8 (at.%) of approximately 1.5 μm and 3 μm in thickness were prepared using magnetron sputtering onto medical grade 316L stainless steel. Their structural and mechanical properties, in vitro corrosion, and antimicrobial activity were analyzed. The amorphous thin film metallic glasses consisted of a single glassy phase, with an absence of any detectable peaks corresponding to crystalline phases. Elemental composition close to the target alloy was noted from EDAX analysis of the thin film. The surface morphology of the film showed a smooth surface on scanning electron microscopy and atomic force microscopy. In vitro electrochemical corrosion studies indicated that the zirconium-based metallic glass could withstand body fluid, showing superior resistance to corrosion and electrochemical stability. Interactions between the coated surface and bacteria were investigated by agar diffusion, solution suspension, and wet interfacial contact methods. The results indicated a clear zone of inhibition against the growth of microorganisms such as Escherichia coli and Staphylococcus aureus, confirming the antimicrobial activity of the thin film metallic glasses. Cytotoxicity studies using L929 fibroblast cells showed these coatings to be noncytotoxic in nature. PMID:26491304

Sustained-release of naproxen sodium from electrospun-aligned PLLA-PCL scaffolds.

PubMed

Lui, Yuan Siang; Lewis, Mark P; Loo, Say Chye Joachim

2017-04-01

Spontaneous tendon healing may result in reduced tissue functionality. In view of this, tissue engineering (TE) emerges as a promising approach in promoting proper tendon regeneration. However, unfavourable post-surgical adhesion formations restrict adequate tendon healing through the TE approach. Naproxen sodium (NPS), a non-steroidal anti-inflammatory drug (NSAID), has been demonstrated to prevent adhesions by inhibiting the inflammatory response. Therefore, in this study, various factors, such as polymer composition, i.e. different poly-l-lactic acid (PLLA):polycaprolactone (PCL) ratios, and percentage of water:hexafluoroisopropanol (HFIP; as co-solvent) ratios, were investigated to understand how these can influence the release of NPS from electrospun scaffolds. By adjusting the amount of water as the co-solvent, NPS could be released sustainably for as long as 2 weeks. Scaffold breaking strength was also enhanced with the addition of water as the co-solvent. This NPS-loaded scaffold showed no significant cytotoxicity, and L929 murine fibroblasts cultured on the scaffolds were able to proliferate and align along the fibre orientation. These scaffolds with desirable tendon TE characteristics would be promising candidates in achieving better tendon regeneration in vivo. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Factors affecting the electrofusion of mouse and ferret oocytes with ferret somatic cells.

PubMed

Li, Ziyi; Sun, Xingshen; Chen, Juan; Leno, Gregory H; Engelhardt, John F

2005-09-01

The domestic ferret, Mustela putorius furos, holds great promise as a genetic model for human lung disease, provided that key technologies for somatic cell nuclear transfer (SCNT) are developed. In this report, we extend our understanding of SCNT in this species by defining conditions for efficient cell fusion by electrical pulse. Two experimental systems were employed in this study. First, in vivo-matured mouse oocytes and ferret somatic cells were used to establish general parameters for fusion. One fibroblast, or cumulus cell, was agglutinated to nucleate, zona pellucida-free, mouse oocytes, and subjected to an electrical pulse. Similar electrical pulse conditions were also tested with 1 or 2 somatic cells inserted into the perivitelline space (PVS) of intact mouse oocytes. The fusion rate for a single fibroblast with a zona-free oocyte was 80.2%, significantly higher (P < 0.05) than that observed for 1, or 2, fibroblasts placed in the PVS (52.0% and 63.8%, respectively). The fusion rate (44.1%) following insertion of two cumulus cells was significantly higher (P < 0.05) than that following insertion of one cumulus cell (25.1%). Second, in vitro-matured ferret oocytes were enucleated, and one to three fibroblasts or cumulus cells were inserted into the PVS. Zona pellucida-free ferret oocytes were fragile and excluded from the study. The fusion rates with two or three fibroblasts were 71.4% and 76.8%, respectively; significantly higher (P < 0.05) than that for one fibroblast (48.6%). This cell number-dependent difference in fusion efficiency was also observed with cumulus cells. Fusion-derived (ferret-ferret) NT embryos cleaved, formed blastocysts in vitro, and underwent early-stage fetal development following embryo transfer. The rate of development was cell type-independent, in contrast to the cell type-dependent differences observed in fusion efficiency. In conclusion, fibroblasts fused more efficiently than cumulus cells and the efficiency of single cell fusions was improved when two or more cells were inserted into the PVS. These studies define conditions for efficient cell fusion with ferret oocytes and should facilitate SCNT and the development of genetically defined animal models in this species.

Gallic Acid Induces a Reactive Oxygen Species-Provoked c-Jun NH2-Terminal Kinase-Dependent Apoptosis in Lung Fibroblasts

PubMed Central

Chen, Chiu-Yuan; Chen, Kun-Chieh; Yang, Tsung-Ying; Liu, Hsiang-Chun; Hsu, Shih-Lan

2013-01-01

Idiopathic pulmonary fibrosis is a chronic lung disorder characterized by fibroblasts proliferation and extracellular matrix accumulation. Induction of fibroblast apoptosis therefore plays a crucial role in the resolution of this disease. Gallic acid (3,4,5-trihydroxybenzoic acid), a common botanic phenolic compound, has been reported to induce apoptosis in tumor cell lines and renal fibroblasts. The present study was undertaken to examine the role of mitogen-activated protein kinases (MAPKs) in lung fibroblasts apoptosis induced by gallic acid. We found that treatment with gallic acid resulted in activation of c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and protein kinase B (PKB, Akt), but not p38MAPK, in mouse lung fibroblasts. Inhibition of JNK using pharmacologic inhibitor (SP600125) and genetic knockdown (JNK specific siRNA) significantly inhibited p53 accumulation, reduced PUMA and Fas expression, and abolished apoptosis induced by gallic acid. Moreover, treatment with antioxidants (vitamin C, N-acetyl cysteine, and catalase) effectively diminished gallic acid-induced hydrogen peroxide production, JNK and p53 activation, and cell death. These observations imply that gallic acid-mediated hydrogen peroxide formation acts as an initiator of JNK signaling pathways, leading to p53 activation and apoptosis in mouse lung fibroblasts. PMID:23533505

In vitro cellular uptake of evodiamine and rutaecarpine using a microemulsion

PubMed Central

Zhang, Yong-Tai; Huang, Zhe-Bin; Zhang, Su-Juan; Zhao, Ji-Hui; Wang, Zhi; Liu, Ying; Feng, Nian-Ping

2012-01-01

Objective To investigate the cellular uptake of evodiamine and rutaecarpine in a microemulsion in comparison with aqueous suspensions and tinctures. Materials and methods A microemulsion was prepared using the dropwise addition method. Mouse skin fibroblasts were cultured in vitro to investigate the optimal conditions for evodiamine and rutaecarpine uptake with different drug concentrations and administration times. Under optimal conditions, the cellular uptake of microemulsified drugs was assayed and compared to tinctures and aqueous suspensions. Rhodamine B labeling and laser scanning confocal microscopy (LSCM) were used to explore the distribution of fluorochrome transferred with the microemulsion in fibroblasts. Cellular morphology was also investigated, using optical microscopy to evaluate microemulsion-induced cellular toxicity. Results The maximum cellular drug uptake amounts were obtained with a 20% concentration (v/v) of microemulsion and an 8 hour administration time. Drug uptake by mouse skin fibroblasts was lowest when the drugs were loaded in microemulsion. After incubation with rhodamine B-labeled microemulsion for 8 hours, the highest fluorescence intensity was achieved, and the fluorochrome was primarily distributed in the cytochylema. No obvious cellular morphologic changes were observed with the administration of either the microemulsion or the aqueous suspension; for the tincture group, however, massive cellular necrocytosis was observed. Conclusion The lower cellular uptake with microemulsion may be due to the fact that most of the drug loaded in the microemulsion vehicle was transported via the intercellular space, while a small quantity of free drug (released from the vehicle) was ingested through transmembrane transport. Mouse skin fibroblasts rarely endocytosed evodiamine and rutaecarpine with a microemulsion as the vehicle. The microemulsion had no obvious effect on cellular morphology, suggesting there is little or no cellular toxicity associated with the administration of microemulsion on mouse skin fibroblasts. PMID:22679361

In vitro cellular uptake of evodiamine and rutaecarpine using a microemulsion.

PubMed

Zhang, Yong-Tai; Huang, Zhe-Bin; Zhang, Su-Juan; Zhao, Ji-Hui; Wang, Zhi; Liu, Ying; Feng, Nian-Ping

2012-01-01

To investigate the cellular uptake of evodiamine and rutaecarpine in a microemulsion in comparison with aqueous suspensions and tinctures. A microemulsion was prepared using the dropwise addition method. Mouse skin fibroblasts were cultured in vitro to investigate the optimal conditions for evodiamine and rutaecarpine uptake with different drug concentrations and administration times. Under optimal conditions, the cellular uptake of microemulsified drugs was assayed and compared to tinctures and aqueous suspensions. Rhodamine B labeling and laser scanning confocal microscopy (LSCM) were used to explore the distribution of fluorochrome transferred with the microemulsion in fibroblasts. Cellular morphology was also investigated, using optical microscopy to evaluate microemulsion-induced cellular toxicity. The maximum cellular drug uptake amounts were obtained with a 20% concentration (v/v) of microemulsion and an 8 hour administration time. Drug uptake by mouse skin fibroblasts was lowest when the drugs were loaded in microemulsion. After incubation with rhodamine B-labeled microemulsion for 8 hours, the highest fluorescence intensity was achieved, and the fluorochrome was primarily distributed in the cytochylema. No obvious cellular morphologic changes were observed with the administration of either the microemulsion or the aqueous suspension; for the tincture group, however, massive cellular necrocytosis was observed. The lower cellular uptake with microemulsion may be due to the fact that most of the drug loaded in the microemulsion vehicle was transported via the intercellular space, while a small quantity of free drug (released from the vehicle) was ingested through transmembrane transport. Mouse skin fibroblasts rarely endocytosed evodiamine and rutaecarpine with a microemulsion as the vehicle. The microemulsion had no obvious effect on cellular morphology, suggesting there is little or no cellular toxicity associated with the administration of microemulsion on mouse skin fibroblasts.

Revisiting Cardiac Cellular Composition

PubMed Central

Pinto, Alexander R.; Ilinykh, Alexei; Ivey, Malina J.; Kuwabara, Jill T.; D'Antoni, Michelle L.; Debuque, Ryan; Chandran, Anjana; Wang, Lina; Arora, Komal; Rosenthal, Nadia; Tallquist, Michelle D.

2015-01-01

Rationale Accurate knowledge of the cellular composition of the heart is essential to fully understand the changes that occur during pathogenesis and to devise strategies for tissue engineering and regeneration. Objective To examine the relative frequency of cardiac endothelial cells, hematopoietic-derived cells and fibroblasts in the mouse and human heart. Methods and Results Using a combination of genetic tools and cellular markers, we examined the occurrence of the most prominent cell types in the adult mouse heart. Immunohistochemistry revealed that endothelial cells constitute over 60%, hematopoietic-derived cells 5–10%, and fibroblasts under 20% of the non-myocytes in the heart. A refined cell isolation protocol and an improved flow cytometry approach provided an independent means of determining the relative abundance of non-myocytes. High dimensional analysis and unsupervised clustering of cell populations confirmed that endothelial cells are the most abundant cell population. Interestingly, fibroblast numbers are smaller than previously estimated, and two commonly assigned fibroblast markers, Sca-1 and CD90, underrepresent fibroblast numbers. We also describe an alternative fibroblast surface marker that more accurately identifies the resident cardiac fibroblast population. Conclusions This new perspective on the abundance of different cell types in the heart demonstrates that fibroblasts comprise a relatively minor population. By contrast, endothelial cells constitute the majority of non-cardiomyocytes and are likely to play a greater role in physiologic function and response to injury than previously appreciated. PMID:26635390

The proinflammatory cytokines IL-1beta and TNF-alpha induce the expression of Synoviolin, an E3 ubiquitin ligase, in mouse synovial fibroblasts via the Erk1/2-ETS1 pathway.

PubMed

Gao, Beixue; Calhoun, Karen; Fang, Deyu

2006-01-01

The overgrowth of synovial tissues is critical in the pathogenesis of rheumatoid arthritis (RA). The expression of Synoviolin (SYN), an E3 ubiquitin ligase, is upregulated in arthritic synovial fibroblasts and is involved in the overgrowth of synovial cells during RA. However, the molecular mechanisms involved in the elevated SYN expression are not known. Here, we found that SYN expression is elevated in the synovial fibroblasts from mice with collagen-induced arthritis (CIA). The proinflammatory cytokines interleukin (IL)-1beta and tumor necrosis factor-alpha (TNF-alpha) induce SYN expression in mouse synovial fibroblasts. Cultivation of mouse synovial fibroblasts with IL-1beta activates mitogen-activated protein kinases, including extra-cellular signal-regulated kinase (Erk), JNK (c-Jun N-terminal kinase), and p38, while only Erk-specific inhibitor blocks IL-1beta-induced SYN expression. Expression of transcription factor ETS1 further enhances IL-1beta-induced SYN expression. The dominant negative ETS1 mutant lacking the transcription activation domain inhibits SYN expression in a dose-dependent manner. The activation of both Erk1/2 and ETS1 is increased in the CIA synovial fibroblasts. Inhibition of Erk activation reduces ETS1 phosphorylation and SYN expression. Our data indicate that the proinflammatory cytokines IL-1beta and TNF-alpha induce the overgrowth of synovial cells by upregulating SYN expression via the Erk1/-ETS1 pathway. These molecules or pathways could therefore be potential targets for the treatment of RA.

The proinflammatory cytokines IL-1β and TNF-α induce the expression of Synoviolin, an E3 ubiquitin ligase, in mouse synovial fibroblasts via the Erk1/2-ETS1 pathway

PubMed Central

Gao, Beixue; Calhoun, Karen; Fang, Deyu

2006-01-01

The overgrowth of synovial tissues is critical in the pathogenesis of rheumatoid arthritis (RA). The expression of Synoviolin (SYN), an E3 ubiquitin ligase, is upregulated in arthritic synovial fibroblasts and is involved in the overgrowth of synovial cells during RA. However, the molecular mechanisms involved in the elevated SYN expression are not known. Here, we found that SYN expression is elevated in the synovial fibroblasts from mice with collagen-induced arthritis (CIA). The proinflammatory cytokines interleukin (IL)-1β and tumor necrosis factor-α (TNF-α) induce SYN expression in mouse synovial fibroblasts. Cultivation of mouse synovial fibroblasts with IL-1β activates mitogen-activated protein kinases, including extra-cellular signal-regulated kinase (Erk), JNK (c-Jun N-terminal kinase), and p38, while only Erk-specific inhibitor blocks IL-1β-induced SYN expression. Expression of transcription factor ETS1 further enhances IL-1β-induced SYN expression. The dominant negative ETS1 mutant lacking the transcription activation domain inhibits SYN expression in a dose-dependent manner. The activation of both Erk1/2 and ETS1 is increased in the CIA synovial fibroblasts. Inhibition of Erk activation reduces ETS1 phosphorylation and SYN expression. Our data indicate that the proinflammatory cytokines IL-1β and TNF-α induce the overgrowth of synovial cells by upregulating SYN expression via the Erk1/-ETS1 pathway. These molecules or pathways could therefore be potential targets for the treatment of RA. PMID:17105652

Further Insights into the Ciliary Gene and Protein KIZ and Its Murine Ortholog PLK1S1 Mutated in Rod-Cone Dystrophy

PubMed Central

Méjécase, Cécile; Bertelli, Matteo; Terray, Angélique; Michiels, Christelle; Condroyer, Christel; Fouquet, Stéphane; Sadoun, Maxime; Clérin, Emmanuelle; Liu, Binqian; Léveillard, Thierry; Goureau, Olivier; Sahel, José-Alain; Audo, Isabelle

2017-01-01

We identified herein additional patients with rod-cone dystrophy (RCD) displaying mutations in KIZ, encoding the ciliary centrosomal protein kizuna and performed functional characterization of the respective protein in human fibroblasts and of its mouse ortholog PLK1S1 in the retina. Mutation screening was done by targeted next generation sequencing and subsequent Sanger sequencing validation. KIZ mRNA levels were assessed on blood and serum-deprived human fibroblasts from a control individual and a patient, compound heterozygous for the c.52G>T (p.Glu18*) and c.119_122del (p.Lys40Ilefs*14) mutations in KIZ. KIZ localization, documentation of cilium length and immunoblotting were performed in these two fibroblast cell lines. In addition, PLK1S1 immunolocalization was conducted in mouse retinal cryosections and isolated rod photoreceptors. Analyses of additional RCD patients enabled the identification of two homozygous mutations in KIZ, the known c.226C>T (p.Arg76*) mutation and a novel variant, the c.3G>A (p.Met1?) mutation. Albeit the expression levels of KIZ were three-times lower in the patient than controls in whole blood cells, further analyses in control- and mutant KIZ patient-derived fibroblasts unexpectedly revealed no significant difference between the two genotypes. Furthermore, the averaged monocilia length in the two fibroblast cell lines was similar, consistent with the preserved immunolocalization of KIZ at the basal body of the primary cilia. Analyses in mouse retina and isolated rod photoreceptors showed PLK1S1 localization at the base of the photoreceptor connecting cilium. In conclusion, two additional patients with mutations in KIZ were identified, further supporting that defects in KIZ/PLK1S1, detected at the basal body of the primary cilia in fibroblasts, and the photoreceptor connecting cilium in mouse, respectively, are involved in RCD. However, albeit the mutations were predicted to lead to nonsense mediated mRNA decay, we could not detect changes upon expression levels, protein localization or cilia length in KIZ-mutated fibroblast cells. Together, our findings unveil the limitations of fibroblasts as a cellular model for RCD and call for other models such as induced pluripotent stem cells to shed light on retinal pathogenic mechanisms of KIZ mutations. PMID:29057815

Anti-sessile bacterial and cytocompatibility properties of CHX-loaded nanohydroxyapatite.

PubMed

Barros, J; Grenho, L; Fernandes, M H; Manuel, C M; Melo, L F; Nunes, O C; Monteiro, F J; Ferraz, M P

2015-06-01

Nanohydroxyapatite possesses exceptional biocompatibility and bioactivity regarding bone cells and tissues, justifying its use as a coating material or as a bone substitute. Unfortunately, this feature may also encourage bacterial adhesion and biofilm formation. Surface functionalization with antimicrobials is a promising strategy to reduce the likelihood of bacterial infestation and colonization on medical devices. Chlorhexidine digluconate is a common and effective antimicrobial agent used for a wide range of medical applications. The purpose of this work was the development of a nanoHA biomaterial loaded with CHX to prevent surface bacterial accumulation and, simultaneously, with good cytocompatibility, for application in the medical field. CHX (5-1500 mg/L) was loaded onto nanoHA discs and the materials were evaluated for CHX adsorption and release profile, physic-chemical features, antibacterial activity against Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis, and cytocompatibility toward L929 fibroblasts. Results showed that the adsorption of CHX on nanoHA surface occurred by electrostatic interactions between the cationic group of CHX and the phosphate group of nanoHA. The release of CHX from CHX-loaded nanoHA showed a fast initial rate followed by a slower kinetics release, due to constraints caused by dilution and diffusion-limiting processes. NanoHA.50 to nanoHA.1500 showed strong anti-sessile activity, inhibiting bacterial adhesion and the biofilm formation. CHX-nanoHA caused a dose- and time-dependent inhibitory effect on the proliferation of fibroblasts for nanoHA.100 to nanoHA.1500. Cellular behavior on nanoHA.5 and nanoHA.50 was similar to control. Therefore, CHX-loaded nanoHA surfaces appear as a promising alternative to prevention of devices-related infections. Copyright © 2015 Elsevier B.V. All rights reserved.

PPARγ ligand production is tightly linked to clonal expansion during initiation of adipocyte differentiation[S

PubMed Central

Hallenborg, Philip; Petersen, Rasmus Koefoed; Feddersen, Søren; Sundekilde, Ulrik; Hansen, Jacob B.; Blagoev, Blagoy; Madsen, Lise; Kristiansen, Karsten

2014-01-01

Adipocyte differentiation is orchestrated by the ligand-activated nuclear receptor PPARγ. Endogenous ligands comprise oxidized derivatives of arachidonic acid and structurally similar PUFAs. Although expression of PPARγ peaks in mature adipocytes, ligands are produced primarily at the onset of differentiation. Concomitant with agonist production, murine fibroblasts undergo two rounds of mitosis referred to as mitotic clonal expansion. Here we show that mouse embryonic fibroblasts deficient in either of two cell cycle inhibitors, the transcription factor p53 or its target gene encoding the cyclin-dependent kinase inhibitor p21, exhibit increased adipogenic potential. The antiadipogenic effect of p53 relied on its transcriptional activity and p21 expression but was circumvented by administration of an exogenous PPARγ agonist suggesting a linkage between cell cycling and PPARγ ligand production. Indeed, cell cycle inhibitory compounds decreased PPARγ ligand production in differentiating 3T3-L1 preadipocytes. Furthermore, these inhibitors abolished the release of arachidonic acid induced by the hormonal cocktail initiating adipogenesis. Collectively, our results suggest that murine fibroblasts require clonal expansion for PPARγ ligand production at the onset of adipocyte differentiation. PMID:25312885

A monoclonal IgM smooth muscle antibody reactive with fibroblast stress fibres produced by immunization with Treponema pallidum.

PubMed Central

Strugnell, R A; Underwood, J R; Clarke, F M; Pedersen, J S; Chalmers, P J; Faine, S; Toh, B H

1983-01-01

A monoclonal IgM smooth muscle antibody secreted by a hybrid (MMI-1) of mouse plasmacytoma NS-1 with spleen cells from mouse immunized with Treponema pallidum was detected by indirect immunofluorescence tests on frozen tissue sections and on acetone fixed monolayers of rat and human fibroblasts. The antibody did not react with acetone fixed smears of T. pallidum but reacted with smooth muscle fibres and with striations of skeletal and cardiac muscle. In non-muscle cells, the antibody stained liver in a 'polygonal' pattern, thymus with accentuated staining of the thymic medulla, renal glomeruli and the brush border and peritubular fibrils of renal tubules. In fibroblast monolayers, the antibody stained stress fibres in an interrupted pattern. Immunoblotting with muscle proteins and the antibody showed labelling of a 100K molecule. The cellular distribution of the mouse monoclonal antibody is similar to that obtained with anti-actin antibody suggesting that the corresponding antigen may be an actin binding protein. Images Fig. 3 PMID:6347470

Cytotoxicity of Doxycycline Effluent Generated by the Fenton Process

PubMed Central

Borghi, Alexandre Augusto; Stephano, Marco Antônio; Monteiro de Souza, Paula; Alves Palma, Mauri Sérgio

2014-01-01

This study aims at determining the Minimum Inhibitory Concentration with Escherichia coli ATCC 25922 and cytotoxicity to L929 cells (ATCC CCL-1) of the waste generated by doxycycline degradation by the Fenton process. This process has shown promise in this treatment thanks mainly to the fact that the waste did not show any relevant inhibitory effect on the test organism and no cytotoxicity to L-929 cells, thus demonstrating that the antibiotic properties were inactivated. PMID:25379532

Aspirin suppresses cardiac fibroblast proliferation and collagen formation through downregulation of angiotensin type 1 receptor transcription

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Xianwei, E-mail: XWang2@UAMS.edu; Lu, Jingjun; Khaidakov, Magomed

Aspirin (acetyl salicylic acid, ASA) is a common drug used for its analgesic and antipyretic effects. Recent studies show that ASA not only blocks cyclooxygenase, but also inhibits NADPH oxidase and resultant reactive oxygen species (ROS) generation, a pathway that underlies pathogenesis of several ailments, including hypertension and tissue remodeling after injury. In these disease states, angiotensin II (Ang II) activates NADPH oxidase via its type 1 receptor (AT1R) and leads to fibroblast growth and collagen synthesis. In this study, we examined if ASA would inhibit NADPH oxidase activation, upregulation of AT1R transcription, and subsequent collagen generation in mouse cardiacmore » fibroblasts challenged with Ang II. Mouse heart fibroblasts were isolated and treated with Ang II with or without ASA. As expected, Ang II induced AT1R expression, and stimulated cardiac fibroblast growth and collagen synthesis. The AT1R blocker losartan attenuated these effects of Ang II. Similarly to losartan, ASA, and its SA moiety suppressed Ang II-mediated AT1R transcription and fibroblast proliferation as well as expression of collagens and MMPs. ASA also suppressed the expression of NADPH oxidase subunits (p22{sup phox}, p47{sup phox}, p67{sup phox}, NOX2 and NOX4) and ROS generation. ASA did not affect total NF-κB p65, but inhibited its phosphorylation and activation. These observations suggest that ASA inhibits Ang II-induced NADPH oxidase expression, NF-κB activation and AT1R transcription in cardiac fibroblasts, and fibroblast proliferation and collagen expression. The critical role of NADPH oxidase activity in stimulation of AT1R transcription became apparent in experiments where ASA also inhibited AT1R transcription in cardiac fibroblasts challenged with H{sub 2}O{sub 2}. Since SA had similar effect as ASA on AT1R expression, we suggest that ASA's effect is mediated by its SA moiety. -- Highlights: ► Aspirin in therapeutic concentrations decreases mouse cardiac fibroblast growth and collagen formation. ► Aspirin decreases the transcription of angiotensin II type 1 receptor by inhibiting NADPH oxidase–NF-κB pathway. ► The inhibition of angiotensin II type 1 receptor expression may be the basis for reduction in fibroblast growth and collagen formation. ► The effects of aspirin appear to be mediated via its salicylate moiety.« less

The Induction of Growth Inhibition and Apoptosis in HeLa and MCF-7 Cells by Teucrium sandrasicum, Having Effective Antioxidant Properties.

PubMed

Tarhan, Leman; Nakipoğlu, Mahmure; Kavakcıoğlu, Berna; Tongul, Burcu; Nalbantsoy, Ayşe

2016-03-01

The hidromethanolic (Met/W), ethyl acetate (EA(EA/W)), and water (W(EA/W)) extracts from Teucrium sandrasicum leaves (L) and flowers (F) were investigated for antioxidant properties and antiproliferative effects on HeLa, MCF-7, and L929. The highest DPPH scavenging, metal chelating capacities, and total phenolic and flavonoid contents were observed in Met/WL. The highest hydroxyl scavenging and reducing power capacities were found in EA(EA/W)L. Met/WL, EA(EA/W)L and EA(EA/W)F inhibited cancer cell growths, while they did not show significant cytotoxicity on L929. While the reactive oxygen species (ROS) levels were generally close to controls in HeLa, they were induced in MCF-7 with the treatment of Met/WL, EA(EA/W)L, and EA(EA/W)F and acted as antioxidant for L929. The highest apoptosis inductions were observed in Met/WL-treated HeLa and EA(EA/W)L-treated MCF-7, which were supported with the changes in mitochondrial membrane potentials. The highest caspase-9 activities were found in Met/WL-treated HeLa and EA(EA/W)F-treated MCF-7. Caspase-3 activity was only induced in EA(EA/W)F-treated HeLa.

Depletion of RIPK3 or MLKL blocks TNF-driven necroptosis and switches towards a delayed RIPK1 kinase-dependent apoptosis

PubMed Central

Remijsen, Q; Goossens, V; Grootjans, S; Van den Haute, C; Vanlangenakker, N; Dondelinger, Y; Roelandt, R; Bruggeman, I; Goncalves, A; Bertrand, M J M; Baekelandt, V; Takahashi, N; Berghe, T V; Vandenabeele, P

2014-01-01

In human cells, the RIPK1–RIPK3–MLKL–PGAM5–Drp1 axis drives tumor necrosis factor (TNF)-induced necroptosis through mitochondrial fission, but whether this pathway is conserved among mammals is not known. To answer this question, we analyzed the presence and functionality of the reported necroptotic axis in mice. As in humans, knockdown of receptor-interacting kinase-3 (RIPK3) or mixed lineage kinase domain like (MLKL) blocks TNF-induced necroptosis in L929 fibrosarcoma cells. However, repression of either of these proteins did not protect the cells from death, but instead induced a switch from TNF-induced necroptosis to receptor-interacting kinase-1 (RIPK1) kinase-dependent apoptosis. In addition, although mitochondrial fission also occurs during TNF-induced necroptosis in L929 cells, we found that knockdown of phosphoglycerate mutase 5 (PGAM5) and dynamin 1 like protein (Drp1) did not markedly protect the cells from TNF-induced necroptosis. Depletion of Pink1, a reported interactor of both PGAM5 and Drp1, did not affect TNF-induced necroptosis. These results indicate that in these murine cells mitochondrial fission and Pink1 dependent processes, including Pink-Parkin dependent mitophagy, apparently do not promote necroptosis. Our data demonstrate that the core components of the necrosome (RIPK1, RIPK3 and MLKL) are crucial to induce TNF-dependent necroptosis both in human and in mouse cells, but the associated mechanisms may differ between the two species or cell types. PMID:24434512

Fate of the surface protein gp70 during entry of retrovirus into mouse fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Andersen, K.B.

1985-04-15

The kinetics of the viral surface protein gp70 and the viral core proteins p30 and p15C were followed during retrovirus entry into mouse fibroblasts. All three proteins were internalized, but whereas essentially all the gp70 was degraded, approximately one-third of the core proteins remained stable in the cells. These diverging routes of the different proteins are in agreement with the proposed route, that retrovirus enters the cells by endocytosis followed by a membrane fusion between the virus membrane and the vesicle membrane.

Lafoensia pacari A. St.-Hil.: Wound healing activity and mechanism of action of standardized hydroethanolic leaves extract.

PubMed

Pereira, Lucas Olivo Martins; Vilegas, Wagner; Tangerina, Marcelo Marucci Pereira; Arunachalam, Karuppusamy; Balogun, Sikiru Olaitan; Orlandi-Mattos, Paulo Eduardo; Colodel, Edson Moleta; Martins, Domingos Tabajara de Oliveira

2018-06-12

Lafoensia pacari A. St.-Hil., (Lythraceae) is a native tree of Brazilian Cerrado and commonly known in Brazil as "mangava-brava". Its leaves are used in Brazilian folk medicine in wound healing, cutaneous mycoses, and in the treatment of gastritis and ulcers. The present study was designed to evaluate the wound healing activity and mechanism of action of the hydroethanolic extract of Lafoensia pacari A. St.-Hil. leaves (HELp), and to advance in its chemical profiling. HELp was prepared by maceration in 70% hydroethanolic solution (1:10, w/v). The phytochemical analyses were investigated using colorimetry and electrospray ionization/mass spectrometric detection (ESI-MS n ). Its in vitro cytotoxicity was evaluated in CHO-K1 and L929 cells, while the in vivo acute toxicity was performed in mice. The potential in vivo wound healing activity was assessed using excision and incision rat models and histopathology of the wounded skin (excision model) was carried out. The in vitro wound healing activity of HELp was demonstrated by scratch assay in L-929 cells, by measuring proliferation/migration rate and p-ERK 1/2 protein expression using western blot analysis. HELp's in vivo anti-inflammatory activity was evaluated by lipopolysaccharide (LPS) induced peritonitis in mice, along with the determination of nitric oxide (NO) and cytokines (TNF-α and IL-10) in the peritoneal lavages. Its potential in vitro antibacterial activity was performed using microbroth dilution assay, while in vitro antioxidant activities was by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, and ferric reducing antioxidant power (FRAP) assays. The phytochemical analysis of HELp revealed the presence of polyphenols with ellagic acid, punicalagin, punicalin, kaempferol, quercetin-3-O-xylopyranoside and quercetin-3-O-rhamnopyranoside being the most prominent. HELp showed no toxicity on CHO-k1 and L929 cell lines. Topical treatment with HELp (10 and 30 mg/g of gel) presented increased rates of wound contraction at all the days evaluated with complete wound re-epithelialization at 22.0 ± 1.5 (p < 0.05) and 21.7 ± 1.6 (p < 0.01) days, respectively. Topical application of HELp (10, 30 or 100 mg/g of gel) in incised wounds caused an increase in tensile break strength at all concentrations resulting in moderate re-epithelialization and neovascularization, increased cell proliferation an accelerated remodeling phase of the wound, in a manner comparable to standard drug (Madecassol ® , 10 mg/g). In the scratch assay with L929 cells, HELp (0.1 and 0.03 mg/mL) and PDGF (5 ng/mL) resulted in the increased proliferation/migration rate of fibroblasts and higher expression of p-ERK 1/2 protein. In LPS-induced peritonitis, HELp (100 and 200 mg/kg p.o.) decreased total leukocyte migration, comparable to the dexamethasone (0.5 mg/kg p.o.). In RAW 264.7 macrophages activated by LPS, HELp produced anti-inflammatory activity dependent on increased concentrations of IL-10, reduction in NO production, without altering the TNF-α levels. HELp also presented potent antioxidant activity in the DPPH and FRAP, but lacks in vitro antibacterial activity. The present study results support the popular use of the leaves of L. pacari in the treatment of wounds. Its wound healing activity is multi-targeted and involves inhibition of the proliferative and anti-inflammatory phases, antioxidant and positive modulation of the remodeling phase that might be involved different secondary metabolites, with emphasis on the ellagic acid, punicalagin, punicalin, kaempferol, quercetin-3-O-xylopyranoside and quercetin-3-O-rhamnopyranoside. Copyright © 2018 Elsevier B.V. All rights reserved.

CARFMAP: A Curated Pathway Map of Cardiac Fibroblasts.

PubMed

Nim, Hieu T; Furtado, Milena B; Costa, Mauro W; Kitano, Hiroaki; Rosenthal, Nadia A; Boyd, Sarah E

2015-01-01

The adult mammalian heart contains multiple cell types that work in unison under tightly regulated conditions to maintain homeostasis. Cardiac fibroblasts are a significant and unique population of non-muscle cells in the heart that have recently gained substantial interest in the cardiac biology community. To better understand this renaissance cell, it is essential to systematically survey what has been known in the literature about the cellular and molecular processes involved. We have built CARFMAP (http://visionet.erc.monash.edu.au/CARFMAP), an interactive cardiac fibroblast pathway map derived from the biomedical literature using a software-assisted manual data collection approach. CARFMAP is an information-rich interactive tool that enables cardiac biologists to explore the large body of literature in various creative ways. There is surprisingly little overlap between the cardiac fibroblast pathway map, a foreskin fibroblast pathway map, and a whole mouse organism signalling pathway map from the REACTOME database. Among the use cases of CARFMAP is a common task in our cardiac biology laboratory of identifying new genes that are (1) relevant to cardiac literature, and (2) differentially regulated in high-throughput assays. From the expression profiles of mouse cardiac and tail fibroblasts, we employed CARFMAP to characterise cardiac fibroblast pathways. Using CARFMAP in conjunction with transcriptomic data, we generated a stringent list of six genes that would not have been singled out using bioinformatics analyses alone. Experimental validation showed that five genes (Mmp3, Il6, Edn1, Pdgfc and Fgf10) are differentially regulated in the cardiac fibroblast. CARFMAP is a powerful tool for systems analyses of cardiac fibroblasts, facilitating systems-level cardiovascular research.

Anti-proliferative activities of finasteride in benign prostate epithelial cells require stromal fibroblasts and c-Jun gene.

PubMed

Wang, Kai; Jin, Song; Fan, Dongdong; Wang, Mingshuai; Xing, Nianzeng; Niu, Yinong

2017-01-01

This study aimed to identify the role of mouse fibroblast-mediated c-Jun and IGF-1 signaling in the therapeutic effect of finasteride on benign prostatic epithelial cells. BPH-1 cells, alone or with fibroblasts (c-Jun+/+ or c-Jun-/-), were implanted subcutaneously in male nude mice who were then treated with finasteride. The degrees of cell proliferation, apoptosis, and sizes of the xenografts were determined. BPH-1 cells were grown alone or co-cultured with mouse fibroblasts in the presence of finasteride and the level of IGF-1 secreted into the medium by the fibroblasts was determined. The proliferation-associated signaling pathway in BPH-1 cells was also evaluated. Fibroblasts and c-Jun promoted xenograft growth, stimulated Ki-67 expression, and inhibited BPH-1 apoptosis. Finasteride did not induce the shrinkage of xenografts in the combined-grafted groups despite repressing Ki-67 expression and inducing cell apoptosis. The addition of c-Jun-/- fibroblasts did not promote xenograft growth. In the absence of c-Jun and fibroblasts, finasteride did not alter xenograft growth, Ki-67 expression, or cell apoptosis. The in vitro results demonstrated that when BPH-1 cells were grown in monoculture, treatment with finasteride did not induce cell death and stimulated the expression of pro-proliferative signaling molecules, while in the presence of fibroblasts containing c-Jun, finasteride treatment repressed epithelial cell proliferation, the level of IGF-1 in the medium, and the activation of downstream pro-proliferative signaling pathways. Taken together, our results suggest that fibroblasts, c-Jun, and IGF-1 play key roles in mediating stromal-epithelial interactions that are required for the therapeutic effects of finasteride in benign prostate epithelial cells.

Anti-proliferative activities of finasteride in benign prostate epithelial cells require stromal fibroblasts and c-Jun gene

PubMed Central

Fan, Dongdong; Wang, Mingshuai; Xing, Nianzeng; Niu, Yinong

2017-01-01

This study aimed to identify the role of mouse fibroblast-mediated c-Jun and IGF-1 signaling in the therapeutic effect of finasteride on benign prostatic epithelial cells. BPH-1 cells, alone or with fibroblasts (c-Jun+/+ or c-Jun-/-), were implanted subcutaneously in male nude mice who were then treated with finasteride. The degrees of cell proliferation, apoptosis, and sizes of the xenografts were determined. BPH-1 cells were grown alone or co-cultured with mouse fibroblasts in the presence of finasteride and the level of IGF-1 secreted into the medium by the fibroblasts was determined. The proliferation-associated signaling pathway in BPH-1 cells was also evaluated. Fibroblasts and c-Jun promoted xenograft growth, stimulated Ki-67 expression, and inhibited BPH-1 apoptosis. Finasteride did not induce the shrinkage of xenografts in the combined-grafted groups despite repressing Ki-67 expression and inducing cell apoptosis. The addition of c-Jun-/- fibroblasts did not promote xenograft growth. In the absence of c-Jun and fibroblasts, finasteride did not alter xenograft growth, Ki-67 expression, or cell apoptosis. The in vitro results demonstrated that when BPH-1 cells were grown in monoculture, treatment with finasteride did not induce cell death and stimulated the expression of pro-proliferative signaling molecules, while in the presence of fibroblasts containing c-Jun, finasteride treatment repressed epithelial cell proliferation, the level of IGF-1 in the medium, and the activation of downstream pro-proliferative signaling pathways. Taken together, our results suggest that fibroblasts, c-Jun, and IGF-1 play key roles in mediating stromal-epithelial interactions that are required for the therapeutic effects of finasteride in benign prostate epithelial cells. PMID:28196103

Aldehyde dehydrogenase inhibition blocks mucosal fibrosis in human and mouse ocular scarring

PubMed Central

Ahadome, Sarah D.; Abraham, David J.; Rayapureddi, Suryanarayana; Saw, Valerie P.; Saban, Daniel R.; Calder, Virginia L.; Norman, Jill T.; Ponticos, Markella; Daniels, Julie T.; Dart, John K.

2016-01-01

Mucous membrane pemphigoid (MMP) is a systemic mucosal scarring disease, commonly causing blindness, for which there is no antifibrotic therapy. Aldehyde dehydrogenase family 1 (ALDH1) is upregulated in both ocular MMP (OMMP) conjunctiva and cultured fibroblasts. Application of the ALDH metabolite, retinoic acid (RA), to normal human conjunctival fibroblasts in vitro induced a diseased phenotype. Conversely, application of ALDH inhibitors, including disulfiram, to OMMP fibroblasts in vitro restored their functionality to that of normal controls. ALDH1 is also upregulated in the mucosa of the mouse model of scarring allergic eye disease (AED), used here as a surrogate for OMMP, in which topical application of disulfiram decreased fibrosis in vivo. These data suggest that progressive scarring in OMMP results from ALDH/RA fibroblast autoregulation, that the ALDH1 subfamily has a central role in immune-mediated ocular mucosal scarring, and that ALDH inhibition with disulfiram is a potential and readily translatable antifibrotic therapy. PMID:27699226

Akt1/protein kinase B enhances transcriptional reprogramming of fibroblasts to functional cardiomyocytes

PubMed Central

Zhou, Huanyu; Dickson, Matthew E.; Kim, Min Soo; Bassel-Duby, Rhonda; Olson, Eric N.

2015-01-01

Conversion of fibroblasts to functional cardiomyocytes represents a potential approach for restoring cardiac function after myocardial injury, but the technique thus far has been slow and inefficient. To improve the efficiency of reprogramming fibroblasts to cardiac-like myocytes (iCMs) by cardiac transcription factors [Gata4, Hand2, Mef2c, and Tbx5 (GHMT)], we screened 192 protein kinases and discovered that Akt/protein kinase B dramatically accelerates and amplifies this process in three different types of fibroblasts (mouse embryo, adult cardiac, and tail tip). Approximately 50% of reprogrammed mouse embryo fibroblasts displayed spontaneous beating after 3 wk of induction by Akt plus GHMT. Furthermore, addition of Akt1 to GHMT evoked a more mature cardiac phenotype for iCMs, as seen by enhanced polynucleation, cellular hypertrophy, gene expression, and metabolic reprogramming. Insulin-like growth factor 1 (IGF1) and phosphoinositol 3-kinase (PI3K) acted upstream of Akt whereas the mitochondrial target of rapamycin complex 1 (mTORC1) and forkhead box o3 (Foxo3a) acted downstream of Akt to influence fibroblast-to-cardiomyocyte reprogramming. These findings provide insights into the molecular basis of cardiac reprogramming and represent an important step toward further application of this technique. PMID:26354121

A novel grapheme oxide-modified collagen-chitosan bio-film for controlled growth factor release in wound healing applications.

PubMed

Liu, Ting; Dan, Weihua; Dan, Nianhua; Liu, Xinhua; Liu, Xuexu; Peng, Xu

2017-08-01

Collagen-chitosan composite film modified with grapheme oxide (GO) and 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), termed CC-G-E film, was loaded with basic fibroblast growth factor (bFGF) as the development of an efficacious wound healing device. In this study we report a novel drug delivery system that prevents the initial burst release and loss of bioactivity of drugs in vitro and in vivo applications. The results showed that CC-G-E film possessed improved thermal stability and a higher rate of crosslinking with increased mechanical properties when the dosage of GO was between 0.03% and 0.07%. It was shown that the in vitro release of bFGF from CC-G-E film continued for more than 28d. Furthermore, the CC-G-E films demonstrated excellent in vitro biocompatibility following culture with L929 fibroblasts in terms of cell adhesion and proliferation. CC-G-E films were implanted into Sprague-Dawley rats to characterize their ability to repair full-thickness skin wounds. Results showed that the CC-G-E film accelerated the wound healing process compared with the blank control. Based on all the results, it was concluded that CC-G-E film operates as a novel drug delivery system and due to its performance in wound remodeling, has potential to be developed as a wound dressing material. Copyright © 2017 Elsevier B.V. All rights reserved.

Promotion of mouse fibroblast collagen gene expression by mast cells stimulated via the Fc epsilon RI. Role for mast cell-derived transforming growth factor beta and tumor necrosis factor alpha

PubMed Central

1994-01-01

Chronic allergic diseases and other disorders associated with mast cell activation can also be associated with tissue fibrosis, but a direct link between mast cell mediator release and fibroblast collagen gene expression has not been established. Using in situ hybridization, we show that the elicitation of an IgE-dependent passive cutaneous anaphylaxis (PCA) reaction in mice results in a transient, but marked augmentation of steady state levels of type alpha-1 (I) collagen mRNA in the dermis. While peak levels of collagen mRNA expression in the skin are observed 16-24 h after mast cell activation, substantial numbers of dermal cells are strongly positive for collagen mRNA at 1 and 2 h after antigen challenge, before circulating inflammatory cells are recruited into the tissues. Furthermore, experiments in mast cell- reconstituted or genetically mast cell-deficient WBB6F1-W/Wv mice demonstrate that the increased expression of collagen mRNA at sites of PCA reactions is entirely mast cell dependent. In vitro studies show that the supernatants of mouse serosal mast cells activated via the Fc epsilon RI markedly increase type alpha-1 (I) collagen mRNA levels in mouse embryonic skin fibroblasts, and also upregulate collagen secretion by these cells. The ability of mast cell supernatants to induce increased steady state levels of collagen mRNA in mouse skin fibroblasts is markedly diminished by absorption with antibodies specific for either of two mast cell-derived cytokines, transforming growth factor beta (TGF-beta 1) or tumor necrosis factor alpha (TNF- alpha), and is eliminated entirely by absorption with antibodies against both cytokines. Taken together, these findings demonstrate that IgE-dependent mouse mast cell activation can induce a transient and marked increase in steady state levels of type alpha-1 (I) collagen mRNA in dermal fibroblasts and that mast cell-derived TGF-beta 1 and TNF-alpha importantly contribute to this effect. PMID:7964480

Transcription factor Nrf2 mediates an adaptive response to sulforaphane that protects fibroblasts in vitro against the cytotoxic effects of electrophiles, peroxides and redox-cycling agents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Higgins, Larry G.; Kelleher, Michael O.; Eggleston, Ian M.

2009-06-15

Sulforaphane can stimulate cellular adaptation to redox stressors through transcription factor Nrf2. Using mouse embryonic fibroblasts (MEFs) as a model, we show herein that the normal homeostatic level of glutathione in Nrf2{sup -/-} MEFs was only 20% of that in their wild-type counterparts. Furthermore, the rate of glutathione synthesis following its acute depletion upon treatment with 3 {mu}mol/l sulforaphane was very substantially lower in Nrf2{sup -/-} MEFs than in wild-type cells, and the rebound leading to a {approx} 1.9-fold increase in glutathione that occurred 12-24 h after Nrf2{sup +/+} MEFs were treated with sulforaphane was not observed in Nrf2{sup -/-}more » fibroblasts. Wild-type MEFs that had been pre-treated for 24 h with 3 {mu}mol/l sulforaphane exhibited between 1.4- and 3.2-fold resistance against thiol-reactive electrophiles, including isothiocyanates, {alpha},{beta}-unsaturated carbonyl compounds (e.g. acrolein), aryl halides and alkene epoxides. Pre-treatment of Nrf2{sup +/+} MEFs with sulforaphane also protected against hydroperoxides (e.g. cumene hydroperoxide, CuOOH), free radical-generating compounds (e.g. menadione), and genotoxic electrophiles (e.g. chlorambucil). By contrast, Nrf2{sup -/-} MEFs were typically {approx} 50% less tolerant of these agents than wild-type fibroblasts, and sulforaphane pre-treatment did not protect the mutant cells against xenobiotics. To test whether Nrf2-mediated up-regulation of glutathione represents the major cytoprotective mechanism stimulated by sulforaphane, 5 {mu}mol/l buthionine sulfoximine (BSO) was used to inhibit glutathione synthesis. In Nrf2{sup +/+} MEFs pre-treated with sulforaphane, BSO diminished intrinsic resistance and abolished inducible resistance to acrolein, CuOOH and chlorambucil, but not menadione. Thus Nrf2-dependent up-regulation of GSH is the principal mechanism by which sulforaphane pre-treatment induced resistance to acrolein, CuOOH and chlorambucil, but not menadione.« less

Osthole regulates TGF-β1 and MMP-2/9 expressions via activation of PPARα/γ in cultured mouse cardiac fibroblasts stimulated with angiotensin II.

PubMed

Chen, Rong; Xue, Jie; Xie, Meilin

2013-01-01

Our previous studies have demonstrated that osthole, an active constituent isolated from the fruit of Cnidium monnieri (L.) Cusson, can prevent isoprenaline-induced myocardial fibrosis in mice, but the underlying mechanism is still unclear. The mouse cardiac fibroblasts (CFs) stimulated with angiotensin II (Ang II) were cultured and treated with different concentrations of osthole. The mRNA expressions of peroxisome proliferator-activated receptor (PPAR) α/γ, transforming growth factor β1 (TGF-β1), and matrix metalloproteinase (MMP)-2/9 were detected by reverse transcription polymerase chain reaction method, and the protein expressions of nuclear factor-κB (NF-κB) and TGF-β1 were detected by Western blot method, respectively. Following treatment of cells with osthole at 2.5, 5, 10 and 20 μg/mL, the NF-κB and TGF-β1 expressions were dose-dependently decreased, while the expressions of PPARα/γ and MMP-2/9 were dose-dependently increased. After the cells were preincubated with PPARα antagonist (MK886) or/and PPARγ antagonist (GW9662), the inhibitions of osthole on the NF-κB and TGF-β1 expressions were decreased or completely halted and the increment of osthole on the MMP-2/9 expressions were also decreased or completely cancelled. Osthole could inhibit the NF-κB and TGF-β1 expressions by activation of PPARα/γ, and subsequently enhance the MMP-2/9 expressions in cultured CFs, and these effects of osthole may play the beneficial roles in the prevention and treatment of myocardial fibrosis.

Direct In Vivo Reprogramming with Sendai Virus Vectors Improves Cardiac Function after Myocardial Infarction.

PubMed

Miyamoto, Kazutaka; Akiyama, Mizuha; Tamura, Fumiya; Isomi, Mari; Yamakawa, Hiroyuki; Sadahiro, Taketaro; Muraoka, Naoto; Kojima, Hidenori; Haginiwa, Sho; Kurotsu, Shota; Tani, Hidenori; Wang, Li; Qian, Li; Inoue, Makoto; Ide, Yoshinori; Kurokawa, Junko; Yamamoto, Tsunehisa; Seki, Tomohisa; Aeba, Ryo; Yamagishi, Hiroyuki; Fukuda, Keiichi; Ieda, Masaki

2018-01-04

Direct cardiac reprogramming holds great promise for regenerative medicine. We previously generated directly reprogrammed induced cardiomyocyte-like cells (iCMs) by overexpression of Gata4, Mef2c, and Tbx5 (GMT) using retrovirus vectors. However, integrating vectors pose risks associated with insertional mutagenesis and disruption of gene expression and are inefficient. Here, we show that Sendai virus (SeV) vectors expressing cardiac reprogramming factors efficiently and rapidly reprogram both mouse and human fibroblasts into integration-free iCMs via robust transgene expression. SeV-GMT generated 100-fold more beating iCMs than retroviral-GMT and shortened the duration to induce beating cells from 30 to 10 days in mouse fibroblasts. In vivo lineage tracing revealed that the gene transfer of SeV-GMT was more efficient than retroviral-GMT in reprogramming resident cardiac fibroblasts into iCMs in mouse infarct hearts. Moreover, SeV-GMT improved cardiac function and reduced fibrosis after myocardial infarction. Thus, efficient, non-integrating SeV vectors may serve as a powerful system for cardiac regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

Alternative dominance of the parental genomes in hybrid cells generated through the fusion of mouse embryonic stem cells with fibroblasts.

PubMed

Matveeva, Natalia M; Fishman, Veniamin S; Zakharova, Irina S; Shevchenko, Alexander I; Pristyazhnyuk, Inna E; Menzorov, Aleksei G; Serov, Oleg L

2017-12-22

For the first time, two types of hybrid cells with embryonic stem (ES) cell-like and fibroblast-like phenotypes were produced through the fusion of mouse ES cells with fibroblasts. Transcriptome analysis of 2,848 genes differentially expressed in the parental cells demonstrated that 34-43% of these genes are expressed in hybrid cells, consistent with their phenotypes; 25-29% of these genes display intermediate levels of expression, and 12-16% of these genes maintained expression at the parental cell level, inconsistent with the phenotype of the hybrid cell. Approximately 20% of the analyzed genes displayed unexpected expression patterns that differ from both parents. An unusual phenomenon was observed, namely, the illegitimate activation of Xist expression and the inactivation of one of two X-chromosomes in the near-tetraploid fibroblast-like hybrid cells, whereas both Xs were active before and after in vitro differentiation of the ES cell-like hybrid cells. These results and previous data obtained on heterokaryons suggest that the appearance of hybrid cells with a fibroblast-like phenotype reflects the reprogramming, rather than the induced differentiation, of the ES cell genome under the influence of a somatic partner.

Dissecting the Functions of Autophagy in Breast Cancer Associated Fibroblasts

DTIC Science & Technology

2014-10-01

compound transgenic mouse model of mammary cancer (MMTV-PyMT) harboring genetic deletion of Atg12 in stromal fibroblasts using the fibroblast specific...Cre;MMTV-PyMT mice (months 2-18). Using the breeding strategy outlined in Figure 1, we have successfully generated these quadruple transgenic mice...could then use for generating lysate and interrogation by Western blot (Fig. 7). However, our data suggest that the autophagy incompetent MMFs (from

Inhibition of α-SMA by the Ectodomain of FGFR2c Attenuates Lung Fibrosis

PubMed Central

Ju, Wang; Zhihong, Yu; Zhiyou, Zhou; Qin, Huang; Dingding, Wang; Li, Sun; Baowei, Zhu; Xing, Wei; Ying, He; An, Hong

2012-01-01

The soluble ectodomain of fibroblast growth factor receptor-IIIc (sFGFR2c) is able to bind to fibroblast growth factor (FGF) ligands and block the activation of the FGF-signaling pathway. In this study, sFGFR2c inhibited lung fibrosis dramatically in vitro and in vivo. The upregulation of α-smooth muscle actin (α-SMA) in fibroblasts by transforming growth factor-β1 (TGF-β1) is an important step in the process of lung fibrosis, in which FGF-2, released by TGF-β1, is involved. sFGFR2c inhibited α-SMA induction by TGF-β1 via both the extracellular signal-regulated kinase 1/2 (ERK1/2) and Smad3 pathways in primary mouse lung fibroblasts and the proliferation of mouse lung fibroblasts. In a mouse model of bleomycin (BLM)-induced lung fibrosis, mice were treated with sFGFR2c from d 3 or d 10 to 31 after BLM administration. Then we used hematoxylin and eosin staining, Masson staining and immunohistochemical staining to evaluate the inhibitory effects of sFGFR2c on lung fibrosis. The treatment with sFGFR2c resulted in significant attenuation of the lung fibrosis score and collagen deposition. The expression levels of α-SMA, p-FGFRs, p-ERK1/2 and p-Smad3 in the lungs of sFGFR2c-treated mice were markedly lower. sFGFR2c may have potential for the treatment of lung fibrosis as an FGF-2 antagonist. PMID:22451267

The safety and efficacy of bacterial nanocellulose wound dressing incorporating sericin and polyhexamethylene biguanide: in vitro, in vivo and clinical studies.

PubMed

Napavichayanun, Supamas; Yamdech, Rungnapha; Aramwit, Pornanong

2016-03-01

In our previous work, we have attempted to develop a novel bacterial nanocellulose wound dressing which composed of both polyhexamethylene biguanide (PHMB) as an antimicrobial agent and sericin as an accelerative wound healing component. The loading sequence and concentration of PHMB and sericin were optimized to provide the wound dressing with the most effective antimicrobial activity and enhanced collagen production. In this study, further in vitro, in vivo, and clinical studies of this novel wound dressing were performed to evaluate its safety, efficacy, and applicability. For the in vitro cytotoxic test with L929 mouse fibroblast cells, our novel dressing was not toxic to the cells and also promoted cell migration as good as the commercially available dressing, possibly due to the component of sericin released. When implanted subcutaneously in rats, the lower inflammation response was observed for the novel dressing implanted, comparing to the commercially available dressing. This might be that the antimicrobial PHMB component of the novel dressing played a role to reduce infection and inflammation reaction. The clinical trial patch test was performed on the normal skin of healthy volunteers to evaluate the irritation effect of the dressing. Our novel dressing did not irritate the skin of any volunteers, as characterized by the normal levels of erythema and melanin and the absence of edema, papule, vesicle, and bullae. Then, the novel dressing was applied for the treatment of full-thickness wounds in rats. The wounds treated with our novel dressing showed significantly lower percentage of wound size and higher extent of collagen formation mainly due to the activity of sericin. We concluded that our novel bacterial nanocellulose incorporating PHMB and sericin was a safe and efficient wound dressing material for further investigation in the wound healing efficacy in clinic.

Surface Functionalization of Orthopedic Titanium Implants with Bone Sialoprotein

PubMed Central

Ritz, Ulrike; Ackermann, Angelika; Anthonissen, Joris; Kaufmann, Kerstin B.; Brendel, Christian; Götz, Hermann; Rommens, Pol M.; Hofmann, Alexander

2016-01-01

Orthopedic implant failure due to aseptic loosening and mechanical instability remains a major problem in total joint replacement. Improving osseointegration at the bone-implant interface may reduce micromotion and loosening. Bone sialoprotein (BSP) has been shown to enhance bone formation when coated onto titanium femoral implants and in rat calvarial defect models. However, the most appropriate method of BSP coating, the necessary level of BSP coating, and the effect of BSP coating on cell behavior remain largely unknown. In this study, BSP was covalently coupled to titanium surfaces via an aminosilane linker (APTES), and its properties were compared to BSP applied to titanium via physisorption and untreated titanium. Cell functions were examined using primary human osteoblasts (hOBs) and L929 mouse fibroblasts. Gene expression of specific bone turnover markers at the RNA level was detected at different intervals. Cell adhesion to titanium surfaces treated with BSP via physisorption was not significantly different from that of untreated titanium at any time point, whereas BSP application via covalent coupling caused reduced cell adhesion during the first few hours in culture. Cell migration was increased on titanium disks that were treated with higher concentrations of BSP solution, independent of the coating method. During the early phases of hOB proliferation, a suppressive effect of BSP was observed independent of its concentration, particularly when BSP was applied to the titanium surface via physisorption. Although alkaline phosphatase activity was reduced in the BSP-coated titanium groups after 4 days in culture, increased calcium deposition was observed after 21 days. In particular, the gene expression level of RUNX2 was upregulated by BSP. The increase in calcium deposition and the stimulation of cell differentiation induced by BSP highlight its potential as a surface modifier that could enhance the osseointegration of orthopedic implants. Both physisorption and covalent coupling of BSP are similarly effective, feasible methods, although a higher BSP concentration is recommended. PMID:27111551

Lanthanide-Connecting and Lone-Electron-Pair Active Trigonal-Pyramidal-AsO3 Inducing Nanosized Poly(polyoxotungstate) Aggregates and Their Anticancer Activities

NASA Astrophysics Data System (ADS)

Zhao, Jun-Wei; Li, Hai-Lou; Ma, Xing; Xie, Zhigang; Chen, Li-Juan; Zhu, Yongsheng

2016-05-01

By virtue of the stereochemical effect of the lone-electron pair located on the trigonal-pyramidal-AsO3 groups and the one-pot self-assembly strategy in the conventional aqueous solution, a series of novel lanthanide-bridging and lone-electron-pair active trigonal-pyramidal-AsO3 inducing nanosized poly(polyoxotungstate) aggregates [H2N(CH3)2]6 Na24H16{[Ln10W16(H2O)30O50](B-α-AsW9O33)8}·97H2O [Ln = EuIII (1), SmIII (2), GdIII (3), TbIII (4), DyIII (5), HoIII (6), ErIII (7), TmIII (8)] were prepared and further characterized by elemental analyses, IR spectra, UV spectra, thermogravimetric (TG) analyses and single-crystal X-ray diffraction. The most remarkable structural feature is that the polyanionic skeleton of {[Ln10W16(H2O)30O50](B-α-AsW9O33)8}46- is constructed from eight trivacant Keggin [B-α-AsW9O33]9- fragments through ten Ln centers and sixteen bridging W atoms in the participation of fifty extraneous oxygen atoms. Notably, 4 and 8 can be stable in the aqueous solution not only for eight days but also in the range of pH = 3.9-7.5. Moreover, the cytotoxicity tests of 4 and 8 toward human cervical cancer (HeLa) cells, human breast cancer (MCF-7) cells and mouse fibroblast (L929) cells were performed by the 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and the cell apoptosis processes were characterized by calcein AM/PI staining experiments, annexin V-FITC/PI staining experiments and morphological changes.

Targeting Lysine Deacetylases (KDACs) in Parasites

PubMed Central

Wang, Qi; Rosa, Bruce A.; Nare, Bakela; Powell, Kerrie; Valente, Sergio; Rotili, Dante; Mai, Antonello; Marshall, Garland R.; Mitreva, Makedonka

2015-01-01

Due to an increasing problem of drug resistance among almost all parasites species ranging from protists to worms, there is an urgent need to explore new drug targets and their inhibitors to provide new and effective parasitic therapeutics. In this regard, there is growing interest in exploring known drug leads of human epigenetic enzymes as potential starting points to develop novel treatments for parasitic diseases. This approach of repurposing (starting with validated targets and inhibitors) is quite attractive since it has the potential to reduce the expense of drug development and accelerate the process of developing novel drug candidates for parasite control. Lysine deacetylases (KDACs) are among the most studied epigenetic drug targets of humans, and a broad range of small-molecule inhibitors for these enzymes have been reported. In this work, we identify the KDAC protein families in representative species across important classes of parasites, screen a compound library of 23 hydroxamate- or benzamide-based small molecules KDAC inhibitors, and report their activities against a range of parasitic species, including the pathogen of malaria (Plasmodium falciparum), kinetoplastids (Trypanosoma brucei and Leishmania donovani), and nematodes (Brugia malayi, Dirofilaria immitis and Haemonchus contortus). Compound activity against parasites is compared to that observed against the mammalian cell line (L929 mouse fibroblast) in order to determine potential parasite-versus-host selectivity). The compounds showed nanomolar to sub-nanomolar potency against various parasites, and some selectivity was observed within the small panel of compounds tested. The possible binding modes of the active compounds at the different protein target sites within different species were explored by docking to homology models to help guide the discovery of more selective, parasite-specific inhibitors. This current work supports previous studies that explored the use of KDAC inhibitors in targeting Plasmodium to develop new anti-malarial treatments, and also pioneers experiments with these KDAC inhibitors as potential new anthelminthics. The selectivity observed begins to address the challenges of targeting specific parasitic diseases while limiting host toxicity. PMID:26402733

Bioactive and thermally compatible glass coating on zirconia dental implants.

PubMed

Kirsten, A; Hausmann, A; Weber, M; Fischer, J; Fischer, H

2015-02-01

The healing time of zirconia implants may be reduced by the use of bioactive glass coatings. Unfortunately, existing glasses are either bioactive like Bioglass 45S5 but thermally incompatible with the zirconia substrate, or they are thermally compatible but exhibit only a very low level of bioactivity. In this study, we hypothesized that a tailored substitution of alkaline earth metals and alkaline metals in 45S5 can lead to a glass composition that is both bioactive and thermally compatible with zirconia implants. A novel glass composition was analyzed using x-ray fluorescence spectroscopy, dilatometry, differential scanning calorimetry, and heating microscopy to investigate its chemical, physical, and thermal properties. Bioactivity was tested in vitro using simulated body fluid (SBF). Smooth and microstructured glass coatings were applied using a tailored spray technique with subsequent thermal treatment. Coating adhesion was tested on implants that were inserted in bovine ribs. The cytocompatibility of the coating was analyzed using L929 mouse fibroblasts. The coefficient of thermal expansion of the novel glass was shown to be slightly lower (11.58 · 10(-6) K(-1)) than that of the zirconia (11.67 · 10(-6) K(-1)). After storage in SBF, the glass showed reaction layers almost identical to the bioactive glass gold standard, 45S5. A process window between 800 °C and 910 °C was found to result in densely sintered and amorphous coatings. Microstructured glass coatings on zirconia implants survived a minimum insertion torque of 60 Ncm in the in vitro experiment on bovine ribs. Proliferation and cytotoxicity of the glass coatings was comparable with the controls. The novel glass composition showed a strong adhesion to the zirconia substrate and a significant bioactive behavior in the SBF in vitro experiments. Therefore, it holds great potential to significantly reduce the healing time of zirconia dental implants. © International & American Associations for Dental Research 2014.

Glucose response of near-infrared alginate-based microsphere sensors under dynamic reversible conditions.

PubMed

Chaudhary, Ayesha; Harma, Harri; Hanninen, Pekka; McShane, Michael J; Srivastava, Rohit

2011-08-01

Minimally invasive optical glucose biosensors with increased functional longevity form one of the most promising techniques for continuous glucose monitoring, because of their long-term stability, reversibility, repeatability, specificity, and high sensitivity. They are based on the principle of competitive binding and fluorescence resonance energy transfer. Moving to the near-infrared region of the spectrum has the potential to improve signal throughput for implanted sensors, but requires a change in dye chemistry that could alter response sensitivity, range, and toxicity profiles. The near-infrared dissolved-core alginate microsphere sensors were fabricated by emulsion followed by surface coating by layer-by-layer self-assembly. The particles were characterized for sensor stability, sensor response, and reversibility in deionized water and simulated interstitial fluid. The sensor response to step changes in bulk glucose concentrations was also evaluated under dynamic conditions using a microflow cell unit. Finally, in vitro cytotoxicity assays were performed with L929 mouse fibroblast cell lines to demonstrate preliminary biocompatibility of the sensors. The glucose sensitivity under controlled and dynamic conditions was observed to be 0.86%/mM glucose with an analytical response range of 0-30 mM glucose, covering both the physiological and pathophysiological range. The sensor demonstrated a repeatable, reversible, and reproducible response, with a maximum response time of 120 s. In vitro cytotoxicity assays revealed nearly 95% viability of cells, thereby suggesting that the alginate microsphere sensor system does not exhibit cytotoxicity. The incorporation of near-infrared dyes shows promise in improving sensor response because of their high sensitivity and improved tissue penetration of infrared light. The sensitivity for the sensors was approximately 1.5 times greater than that observed for visible dye sensors, and the new dye chemistry did not significantly alter the biocompatibility of the materials. These findings provide additional support for the potential application of alginate microspheres and similar systems such as "smart-tattoo" glucose sensors.

The in vitro biological properties of Mg-Zn-Sr alloy and superiority for preparation of biodegradable intestinal anastomosis rings

PubMed Central

Liu, Ling; Li, Nianfeng; Lei, Ting; Li, Kaimo; Zhang, Yangde

2014-01-01

Background Magnesium (Mg) alloy is a metal-based biodegradable material that has received increasing attention in the field of clinical surgery, but it is currently seldom used in intestinal anastomosis. This study was conducted to comprehensively assess a ternary magnesium (Mg)-zinc (Zn)-strontium (Sr) alloy’s biological superiorities as a preparation material for intestinal anastomosis ring. Material/Methods Mouse L-929 fibroblasts were cultured with Mg-Zn-Sr alloy extract and compared with both positive (0.64% phenol) and negative (original broth culture) controls. The cell morphology of different groups was examined using microscopy, and a cytotoxicity assessment was performed. Fresh anticoagulated human blood was mixed with Mg-Zn-Sr alloy extract and compared with both positive (distilled water) and negative (normal saline) controls. The absorbance of each sample at 570 nm was used to calculate the Mg-Zn-Sr alloy hemolysis ratio in order to test the Mg alloy’s blood compatibility. Bacterial cultures of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were added to Mg-Zn-Sr alloy block samples and compared with positive (Ceftazidime), negative (316LSS stainless steel), and blank controls. The broth cultures were sampled to compare their bacterial colony counts so as to evaluate the antibacterial properties of the Mg-Zn-Sr alloy. The Mg-Zn-Sr alloy was surface-coated with a layer of poly(lactic-co-glycolic acid) carrying everolimus. The surface morphology and degradability of the coating were examined so as to demonstrate feasibility of coating, which can release the drug evenly. Results The experiments proved that Mg-Zn-Sr alloy has good biocompatible, antibacterial, and drug-loaded coating performances, which are lacking in existing intestinal anastomosis devices/materials. Conclusions The Mg-Zn-Sr alloy increases biocompatibility, and yields a safer and better therapeutic effect; therefore, it is a novel biomaterial that is feasible for use when preparing biodegradable intestinal anastomosis rings. PMID:24957079

Bioactive and Thermally Compatible Glass Coating on Zirconia Dental Implants

PubMed Central

Kirsten, A.; Hausmann, A.; Weber, M.; Fischer, J.

2015-01-01

The healing time of zirconia implants may be reduced by the use of bioactive glass coatings. Unfortunately, existing glasses are either bioactive like Bioglass 45S5 but thermally incompatible with the zirconia substrate, or they are thermally compatible but exhibit only a very low level of bioactivity. In this study, we hypothesized that a tailored substitution of alkaline earth metals and alkaline metals in 45S5 can lead to a glass composition that is both bioactive and thermally compatible with zirconia implants. A novel glass composition was analyzed using x-ray fluorescence spectroscopy, dilatometry, differential scanning calorimetry, and heating microscopy to investigate its chemical, physical, and thermal properties. Bioactivity was tested in vitro using simulated body fluid (SBF). Smooth and microstructured glass coatings were applied using a tailored spray technique with subsequent thermal treatment. Coating adhesion was tested on implants that were inserted in bovine ribs. The cytocompatibility of the coating was analyzed using L929 mouse fibroblasts. The coefficient of thermal expansion of the novel glass was shown to be slightly lower (11.58·10–6 K–1) than that of the zirconia (11.67·10–6 K–1). After storage in SBF, the glass showed reaction layers almost identical to the bioactive glass gold standard, 45S5. A process window between 800 °C and 910 °C was found to result in densely sintered and amorphous coatings. Microstructured glass coatings on zirconia implants survived a minimum insertion torque of 60 Ncm in the in vitro experiment on bovine ribs. Proliferation and cytotoxicity of the glass coatings was comparable with the controls. The novel glass composition showed a strong adhesion to the zirconia substrate and a significant bioactive behavior in the SBF in vitro experiments. Therefore, it holds great potential to significantly reduce the healing time of zirconia dental implants. PMID:25421839

Coaxial nozzle-assisted 3D bioprinting with built-in microchannels for nutrients delivery.

PubMed

Gao, Qing; He, Yong; Fu, Jian-zhong; Liu, An; Ma, Liang

2015-08-01

This study offers a novel 3D bioprinting method based on hollow calcium alginate filaments by using a coaxial nozzle, in which high strength cell-laden hydrogel 3D structures with built-in microchannels can be fabricated by controlling the crosslinking time to realize fusion of adjacent hollow filaments. A 3D bioprinting system with a Z-shape platform was used to realize layer-by-layer fabrication of cell-laden hydrogel structures. Curving, straight, stretched or fractured filaments can be formed by changes to the filament extrusion speed or the platform movement speed. To print a 3D structure, we first adjusted the concentration and flow rate of the sodium alginate and calcium chloride solution in the crosslinking process to get partially crosslinked filaments. Next, a motorized XY stages with the coaxial nozzle attached was used to control adjacent hollow filament deposition in the precise location for fusion. Then the Z stage attached with a Z-shape platform moved down sequentially to print layers of structure. And the printing process always kept the top two layers fusing and the below layers solidifying. Finally, the Z stage moved down to keep the printed structure immersed in the CaCl2 solution for complete crosslinking. The mechanical properties of the resulting fused structures were investigated. High-strength structures can be formed using higher concentrations of sodium alginate solution with smaller distance between adjacent hollow filaments. In addition, cell viability of this method was investigated, and the findings show that the viability of L929 mouse fibroblasts in the hollow constructs was higher than that in alginate structures without built-in microchannels. Compared with other bioprinting methods, this study is an important technique to allow easy fabrication of lager-scale organs with built-in microchannels. Copyright © 2015 Elsevier Ltd. All rights reserved.

Isolation and Characterization of Orientia tsutsugamushi from Rodents Captured following a Scrub Typhus Outbreak at a Military Training Base, Bothong District, Chonburi Province, Central Thailand

DTIC Science & Technology

2011-01-01

pended in M199 medium (Grand Island Biological Company [GIBCO), Grand Island, NY), and subsequently exposed to 3,000-rad irradiation in 60Co gamma... irradiator (Atomic Energy of Canada, Ltd., Ottawa, Canada). Irradiated L-929 cells were dispensed into three 50-cm2 flasks to develop a monolayer...monolayers of irradiated L-929 cells. Inoculated cells were incubated at room temperature for 1 hour on a shaker and then the BHI supernatant was dis

Identification of the Gene for Scleroderma in the Tsk/2 Mouse Strain: Implications for Human Scleroderma Pathogenesis and Subset Distinctions

DTIC Science & Technology

2015-09-01

skin)and)used)as) a)marker)of)fibrosis)[3,)4]),)we)assessed)both) protein )and)mRNA)levels)in)fibroblasts)that)received)DNA)from)a) plasmid...containing)a)single)allele)of)a)single)Col3a1%gene.)In)three)independent)experiments,)COL1A1) protein ) was)significantly)elevated)after)48)hours)of)transfection...Mouse& Col3a1eKO& fibroblasts& transfected& with& a& plasmid& bearing&the&mutant&Col3a1Tsk2&express&34%&more&COL1A1& protein &than& Col3a1WT

Polyethyleneimine modified poly(Hyaluronic acid) particles with controllable antimicrobial and anticancer effects.

PubMed

Sahiner, Nurettin; Sagbas, Selin; Sahiner, Mehtap; Ayyala, Ramesh S

2017-03-01

Poly(hyaluronic acid) (p(HA)) particles with sizes from few hundred nm to few tens of micrometer were synthesized by using epoxy groups containing crosslinker glycerol diglycidyl ether (GDE) with high yield, 94±5%. P(HA) particles were oxidized by treatment with sodium periodate and then reacted with cationic polyethyleneimine (PEI) at 1:0.5, 1:1, and 1:2 wt ratio of p(HA):PEI to obtain p(HA)-PEI particles. From zeta potential measurements, isoelectronic points of bare p(HA) particles increased to pH 8.7 from 2.7 after modification with cationic PEI. New properties, such as antibacterial property, were attained for p(HA)-PEI after modification. The highest minimum bactericidal concentration (MBC) values were 0.5, 1, and 0.5mg/mL against Escherichia coli, Staphylococcus aureus, and Bacillus subtilis species for 1:0.5 ratio of p(HA)-PEI at 72h incubation time. Moreover, the p(HA)-PEI particles were found to be biocompatible with L929 fibroblast cells, and interestingly, p(HA)-PEI particles were found to inhibit MDA-MB-231 breast and H1299 cancer cell growth depending on amount of PEI in p(HA)-PEI particles. Copyright © 2016 Elsevier Ltd. All rights reserved.

Pseudolaric Acid B Induced Cell Cycle Arrest, Autophagy and Senescence in Murine Fibrosarcoma L929 Cell

PubMed Central

hua Yu, Jing; yu Liu, Chun; bin Zheng, Gui; Zhang, Li Ying; hui Yan, Ming; yan Zhang, Wen; ying Meng, Xian; fang Yu, Xiao

2013-01-01

Objective: PAB induced various cancer cell apoptosis, cell cycle arrest and senescence. But in cell line murine fibrosarcoma L929, PAB did not induce apoptosis, but autophagy, therefore it was thought by us as a good model to research the relationship of cell cycle arrest, autophagy and senescence bypass apoptosis. Methods: Inhibitory ratio was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. Phase contrast microscopy visualized cell morphology. Hoechst 33258 staining for nuclear change, propidium iodode (PI) staining for cell cycle, monodansylcadaverine (MDC) staining for autophagy, and rodanmine 123 staining for mitochondrial membrane potential (MMP) were measured by fluorescence microscopy or flowcytometry. Apoptosis was determined by DNA ladder test. Protein kinase C (PKC) activity was detected by PKC assay kit. SA-β-galactosidase assay was used to detect senescence. Protein expression was examined by western blot. Results: PAB inhibited L929 cell growth in time-and dose-dependent manner. At 12 h, 80 μmol/L PAB induced obvious mitotic arrest; at 24 h, PAB began to induce autophagy; at 36 h, cell-treated with PAB slip into G1 cell cycle; and 3 d PAB induced senescence. In time sequence PAB induced firstly cell cycle arrest, then autophagy, then slippage into G1 phase, lastly senescence. Senescent cells had high level of autophagy, inhibiting autophagy led to apoptosis, and no senescence. PAB activated PKC activity to induce cell cycle arrest, autophagy and senescence, inhibiting PKC activity suppressed cell cycle arrest, autophagy and senescence. Conclusion: PAB induced cell cycle arrest, autophagy and senescence in murine fibrosarcoma L929 cell through PKC. PMID:23630435

Fabrication of thermoplastic ductile films of chitin butyrate/poly(ɛ-caprolactone) blends and their cytocompatibility.

PubMed

Hashiwaki, Hiroki; Teramoto, Yoshikuni; Nishio, Yoshiyuki

2014-12-19

We fabricate thermoplastic films of chitin burtyrate (ChB)/poly(ɛ-caprolactone) (PCL) blends with different degree of miscibility (miscible (M), partially miscible (PM), and immiscible (IM)), and examined the feasibility as a cell scaffold system through evaluating mechanical properties and cytocompatibility. We found a remediation of the brittleness and an increase in ductility of ChB by blending PCL for the M and PM blends. The blend films were subjected to alkaline hydrolysis (2-M NaOH/37°C/48 h) with expectation of the improvement of the surface hydrophilicity and cell accessibility. ATR-FTIR spectroscopy of the alkaline-treated PM and IM films revealed that PCL component and ester side-chains of acyl chitin were selectively removed from the surface domain. L929 fibroblast cells well adhered and proliferated on these films. Therefore, the materials possess a great potential for the utilization as a thermoplastic cell scaffold in tissue engineering by adequate selection of the degree of miscibility and post treatment. Copyright © 2014 Elsevier Ltd. All rights reserved.

Development of Biomimetic Hybrid Porous Scaffold of Chitosan/Polyvinyl Alcohol/Carboxymethyl Cellulose by Freeze-Dried and Salt Leached Technique.

PubMed

Kanimozhi, K; Basha, S Khaleel; Kumari, V Sugantha; Kaviyarasu, K

2018-07-01

Freeze drying and salt leaching methods were applied to fabricate Chitosan/Poly(vinyl alcohol)/Carboxymethyl cellulose (CPCMC) biomimetic porous scaffolds for soft tissue engineering. The properties of these scaffolds were investigated and compared to those by freeze drying and salt leaching methods respectively. The salt-leached CS/PVA/CMC scaffolds were easily formed into desired shapes with a uniformly distributed and interconnected pore structure with an average pore size. The mechanical strength of the scaffolds increased with the porosity, and were easily modulated by the addition of carboxymethyl cellulose. The morphology of the porous scaffolds observed using a SEM exhibited good porosity and interconnectivity of pores. MTT assay using L929 fibroblast cells demonstrated that the cell viability of the porous scaffold was good. Scaffolds prepared by salt leached method show larger swelling capacity, and mechanical strength, potent antibacterial activity and more cell viability than freeze dried method. It is found that salt leaching method has distinguished characteristics of simple, efficient, feasible and less economic than freeze dried scaffolds.

In vitro corrosion study by EIS of a nickel-free stainless steel for orthopaedic applications.

PubMed

Rondelli, G; Torricelli, P; Fini, M; Giardino, R

2005-03-01

The electrochemical impedance spectroscopy (EIS) technique was used for the study of the electrochemical behaviour of Ni-free austenitic stainless steel for orthopaedic applications. Experiments were carried out using four different test solutions: (i) phosphate-buffered saline (PBS), (ii) minimum essential medium (MEM), (iii) MEM + 10% fetal calf serum (FCS), (iv) MEM + 10% fetal calf serum + L929 fibroblast cell line (Cell). Bode-phase spectra showed the presence of two maxima and were fitted with an equivalent circuit characterized by two parallel combinations (Resistance, Constant Phase Element). The (R(1), CPE(1)) branch was assigned to the inner compact passive film and the (R(2), CPE(2)) branch to the external porous film. The resistance of the inner film R(1), here directly related to the material's uniform corrosion resistance, raised with the immersion time and increased in the following order: PBS

Integrated antibacterial and antifouling surfaces via cross-linking chitosan-g-eugenol/zwitterionic copolymer on electrospun membranes.

PubMed

Li, Zhenguang; Hu, Wenhong; Zhao, Yunhui; Ren, Lixia; Yuan, Xiaoyan

2018-04-27

Integrated antibacterial and antifouling surfaces in favor of avoiding implant-related infections are necessarily required for biomaterials when they contact with the body fluid. In this work, an antibacterial and antifouling membrane was developed via cross-linking chitosan-g-eugenol and the zwitterionic copolymer poly(sulfobetaine methylacrylate-co-2-aminoethyl methacrylate) on the electrospun polycarbonate urethane substrate using genipin as a cross-linker. Antibacterial assays demonstrated that the prepared membranes had efficient antibacterial activity with 92.8 ± 2.5% and 95.2 ± 1.3% growth inhibition rates against Escherichia coli and Staphylococcus aureus, respectively. The investigations on antifouling activity and hemocompatibility of the membranes showed significant resistances to bacterial attachment, non-specific protein adsorption and platelet adhesion, and presented lower hemolytic activity and good anticoagulant activity as well. Moreover, cell culture assays indicated that the prepared membranes exerted no obvious cytotoxicity with more than 80% of relative L929 fibroblast viability. Therefore, the membranes with integrated antibacterial and antifouling properties could be potentially applied in promising indwelling devices. Copyright © 2018 Elsevier B.V. All rights reserved.

Effects of mitomycin-C on normal dermal fibroblasts.

PubMed

Chen, Theodore; Kunnavatana, Shaun S; Koch, R James

2006-04-01

To evaluate the effects of mitomycin-C on the growth and autocrine growth factor production of human dermal fibroblasts from the face. In vitro study using normal adult dermal fibroblast cell lines in a serum-free model. Cell cultures were exposed to 4 mg/mL, 0.4 mg/mL, 0.04 mg/mL, 0.004 mg/mL, and 0.0004 mg/mL concentrations of mitomycin-C solution. Cell counts were performed, and the cell-free supernatants were collected at 0, 1, 3, and 5 days after the initial exposure. Population doubling times were calculated and supernatants were quantitatively assayed for basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-beta1. Continuous exposure to mitomycin-C caused fibroblast cell death by day 7 at all tested concentrations. A 4 minute exposure to mitomycin-C at 4 mg/mL caused rapid fibroblast cell death. A 4-minute exposure to mitomycin-C at either 0.4 mg/mL or 0.04 mg/mL resulted in decreased fibroblast proliferation. A 4 minute exposure to mitomycin-C at 0.4 mg/mL resulted in a marked increase in the production of both bFGF and TGF-beta1. A clinically ideal concentration of mitomycin-C would slow fibroblast proliferation yet not cause cell death to allow for a wound healing response. Mitomycin-C 0.4 mg/mL for 4 minutes satisfies the above criteria in vitro.

Bioactivities of anastasia black (Russian sweet pepper).

PubMed

Shirataki, Yoshiaki; Kawase, Masami; Sakagami, Hiroshi; Nakashima, Hideki; Tani, Satoru; Tanaka, Toru; Sohara, Yoshitaka; Schelz, Zsuzsanna; Molnar, Joseph; Motohashi, Noboru

2005-01-01

Anastasia Black (Russian sweet pepper) of Capsicum annuum L. var. angulosum Mill. (Solanaceae) was successively extracted with hexane, acetone, methanol and 70% methanol, and the extracts were further separated into a total of twenty-three fractions by silica gel or octadecylsilane (ODS; C18) column chromatography. These extracts and fractions were investigated for their cytotoxicity, anti-human immunodeficiency virus (HIV), anti-Helicobacter pylori (H. pylori), urease inhibition and multidrug resistance (MDR) reversal activity. Some fractions of hexane and acetone extracts showed higher cytotoxic activity against three human oral tumor cell lines (squamous cell carcinoma HSC-2, HSC-3, submandibular gland carcinoma HSG) than against three normal human oral cells (gingival fibroblast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF), suggesting a tumor-specific cytotoxic activity. No fractions displayed anti-HIV activity, but some hydrophobic fractions showed higher anti-H. pylori activity, urease inhibition activity and MDR reversal activity. The higher MDR activity of these fractions against MDR gene-transfected L5178 mouse lymphoma T cells may possibly be due to their higher content of carotene or polyphenol. These data suggest that Anastasia Black should be further investigated as a potent supplement for cancer chemotherapy.

Augmented sphingosine 1 phosphate receptor-1 signaling in cardiac fibroblasts induces cardiac hypertrophy and fibrosis through angiotensin II and interleukin-6

PubMed Central

Ohkura, Sei-ichiro; Takashima, Shin-ichiro; Yoshioka, Kazuaki; Okamoto, Yasuo; Inagaki, Yutaka; Sugimoto, Naotoshi; Kitano, Teppei; Takamura, Masayuki; Wada, Takashi; Kaneko, Shuichi; Takuwa, Yoh

2017-01-01

Background: Cardiac fibroblasts, together with cardiomyocytes, occupy the majority of cells in the myocardium and are involved in myocardial remodeling. The lysophospholipid mediator sphigosine-1-phosphate (S1P) regulates functions of cardiovascular cells through multiple receptors including S1PR1–S1PR3. S1PR1 but not other S1P receptors was upregulated in angiotensin II-induced hypertrophic hearts. Therefore, we investigated a role of S1PR1 in fibroblasts for cardiac remodeling by employing transgenic mice that overexpressed S1PR1 under the control of α-smooth muscle actin promoter. In S1PR1-transgenic mouse heart, fibroblasts and/or myofibroblasts were hyperplastic, and those cells as well as vascular smooth muscle cells overexpressed S1PR1. Transgenic mice developed bi-ventricular hypertrophy by 12-week-old and diffuse interstitial fibrosis by 24-week-old without hemodynamic stress. Cardiac remodeling in transgenic mice was associated with greater ERK phosphorylation, upregulation of fetal genes, and systolic dysfunction. Transgenic mouse heart showed increased mRNA expression of angiotensin-converting enzyme and interleukin-6 (IL-6). Isolated fibroblasts from transgenic mice exhibited enhanced generation of angiotensin II, which in turn stimulated IL-6 release. Either an AT1 blocker or angiotensin-converting enzyme inhibitor prevented development of cardiac hypertrophy and fibrosis, systolic dysfunction and increased IL-6 expression in transgenic mice. Finally, administration of anti-IL-6 antibody abolished an increase in tyrosine phosphorylation of STAT3, a major signaling molecule downstream of IL-6, in the transgenic mouse heart and prevented development of cardiac hypertrophy in transgenic mice. These results demonstrate a promoting role of S1PR1 in cardiac fibroblasts for cardiac remodeling, in which angiotensin II—AT1 and IL-6 are involved. PMID:28771545

Yin yang 1 is a novel regulator of pulmonary fibrosis.

PubMed

Lin, Xin; Sime, Patricia J; Xu, Haodong; Williams, Marc A; LaRussa, Larry; Georas, Steve N; Guo, Jia

2011-06-15

The differentiation of fibroblasts into myofibroblasts is a cardinal feature of idiopathic pulmonary fibrosis (IPF). The transcription factor Yin Yang 1 (YY1) plays a role in the proliferation and differentiation of diverse cell types, but its role in fibrotic lung diseases is not known. To elucidate the mechanism by which YY1 regulates fibroblast differentiation and lung fibrosis. Lung fibroblasts were cultured with transforming growth factor (TGF)-β or tumor necrosis factor-α. Nuclear factor (NF)-κB, YY1, and α-smooth muscle actin (SMA) were determined in protein, mRNA, and promoter reporter level. Lung fibroblasts and lung fibrosis were assessed in a partial YY1-deficient mouse and a YY1(f/f) conditional knockout mouse after being exposed to silica or bleomycin. TGF-β and tumor necrosis factor-α up-regulated YY1 expression in lung fibroblasts. TGF-β-induced YY1 expression was dramatically decreased by an inhibitor of NF-κB, which blocked I-κB degradation. YY1 is significantly overexpressed in both human IPF and murine models of lung fibrosis, including in the aggregated pulmonary fibroblasts of fibrotic foci. Furthermore, the mechanism of fibrogenesis is that YY1 can up-regulate α-SMA expression in pulmonary fibroblasts. YY1-deficient (YY1(+/-)) mice were significantly protected from lung fibrosis, which was associated with attenuated α-SMA and collagen expression. Finally, decreasing YY1 expression through instilled adenovirus-cre in floxed-YY1(f/f) mice reduced lung fibrosis. YY1 is overexpressed in fibroblasts in both human IPF and murine models in a NF-κB-dependent manner, and YY1 regulates fibrogenesis at least in part by increasing α-SMA and collagen expression. Decreasing YY1 expression may provide a new therapeutic strategy for pulmonary fibrosis.

Altered prostate epithelial development in mice lacking the androgen receptor in stromal fibroblasts.

PubMed

Yu, Shengqiang; Yeh, Chiuan-Ren; Niu, Yuanjie; Chang, Hong-Chiang; Tsai, Yu-Chieh; Moses, Harold L; Shyr, Chih-Rong; Chang, Chawnshang; Yeh, Shuyuan

2012-03-01

Androgens and the androgen receptor (AR) play important roles in the development of male urogenital organs. We previously found that mice with total AR knockout (ARKO) and epithelial ARKO failed to develop normal prostate with loss of differentiation. We have recently knocked out AR gene in smooth muscle cells and found the reduced luminal infolding and IGF-1 production in the mouse prostate. However, AR roles of stromal fibroblasts in prostate development remain unclear. To further probe the stromal fibroblast AR roles in prostate development, we generated tissue-selective knockout mice with the AR gene deleted in stromal fibroblasts (FSP-ARKO). We also used primary culture stromal cells to confirm the in vivo data and investigate mechanisms related to prostate development. The results showed cellular alterations in the FSP-ARKO mouse prostate with decreased epithelial proliferation, increased apoptosis, and decreased collagen composition. Further mechanistic studies demonstrated that FSP-ARKO mice have defects in the expression of prostate stromal growth factors. To further confirm these in vivo findings, we prepared primary cultured mouse prostate stromal cells and found knocking down the stromal AR could result in growth retardation of prostate stromal cells and co-cultured prostate epithelial cells, as well as decrease of some stromal growth factors. Our FSP-ARKO mice not only provide the first in vivo evidence in Cre-loxP knockout system for the requirement of stromal fibroblast AR to maintain the normal development of the prostate, but may also suggest the selective knockdown of stromal AR might become a potential therapeutic approach to battle prostate hyperplasia and cancer. Copyright © 2011 Wiley Periodicals, Inc.

The Influence of Primary Microenvironment on Prostate Cancer Osteoblastic Bone Lesion Development

DTIC Science & Technology

2015-09-01

for inhibiting PCa bone lesion development: 3a. Basic fibroblast growth factor (bFGF) in PC3 bone metastasis: bFGF was identified by cytokine...II receptor (TβRII) knockout (Tgfbr2 KO) mouse models. Col1creERT/Tgfbr2 KO (Col/Tgfbr2 KO), which have TGF-β signaling specific KO in fibroblasts ... fibroblasts and osteoblasts in the bone by Colcre/Tgfbr2 KO, or in the myeloid lineage cells, including osteoclasts in the bone by LysMcre/Tgfbr2 KO

Photodynamic therapy mediates innate immune responses via fibroblast-macrophage interactions.

PubMed

Zulaziz, N; Azhim, A; Himeno, N; Tanaka, M; Satoh, Y; Kinoshita, M; Miyazaki, H; Saitoh, D; Shinomiya, N; Morimoto, Y

2015-10-01

Antibacterial photodynamic therapy (PDT) has come to attract attention as an alternative therapy for drug-resistant bacteria. Recent reports revealed that antibacterial PDT induces innate immune response and stimulates abundant cytokine secretion as a part of inflammatory responses. However, the underlying mechanism how antibacterial PDT interacts with immune cells responsible for cytokine secretion has not been well outlined. In this study, we aimed to clarify the difference in gene expression and cytokine secretion between combined culture of fibroblasts and macrophages and their independent cultures. SCRC-1008, mouse fibroblast cell line and J774, mouse macrophage-like cell line were co-cultured and PDT treatments with different parameters were carried out. After various incubation periods (1-24 h), cells and culture medium were collected, and mRNA and protein levels for cytokines were measured using real-time PCR and ELISA, respectively. Our results showed that fibroblasts and macrophages interact with each other to mediate the immune response. We propose that fibroblasts initially respond to PDT by expressing Hspa1b, which regulates the NF-κB pathway via Tlr2 and Tlr4. Activation of the NF-κB pathway then results in an enhanced secretion of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) and neutrophil chemoattractant MIP-2 and KC from macrophages.

Reprogrammed mouse astrocytes retain a "memory" of tissue origin and possess more tendencies for neuronal differentiation than reprogrammed mouse embryonic fibroblasts.

PubMed

Tian, Changhai; Wang, Yongxiang; Sun, Lijun; Ma, Kangmu; Zheng, Jialin C

2011-02-01

Direct reprogramming of a variety of somatic cells with the transcription factors Oct4 (also called Pou5f1), Sox2 with either Klf4 and Myc or Lin28 and Nanog generates the induced pluripotent stem cells (iPSCs) with marker similarity to embryonic stem cells. However, the difference between iPSCs derived from different origins is unclear. In this study, we hypothesized that reprogrammed cells retain a "memory" of their origins and possess additional potential of related tissue differentiation. We reprogrammed primary mouse astrocytes via ectopic retroviral expression of OCT3/4, Sox2, Klf4 and Myc and found the iPSCs from mouse astrocytes expressed stem cell markers and formed teratomas in SCID mice containing derivatives of all three germ layers similar to mouse embryonic stem cells besides semblable morphologies. To test our hypothesis, we compared embryonic bodies (EBs) formation and neuronal differentiation between iPSCs from mouse embryonic fibroblasts (MEFsiPSCs) and iPSCs from mouse astrocytes (mAsiPSCs). We found that mAsiPSCs grew slower and possessed more potential for neuronal differentiation compared to MEFsiPSCs. Our results suggest that mAsiPSCs retain a "memory" of the central nervous system, which confers additional potential upon neuronal differentiation.

Cytotoxic effects in 3T3-L1 mouse and WI-38 human fibroblasts following 72 hour and 7 day exposures to commercial silica nanoparticles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stępnik, Maciej, E-mail: mstep@imp.lodz.pl; Arkusz, Joanna; Smok-Pieniążek, Anna

The potential toxic effects in murine (3T3-L1) and human (WI-38) fibroblast cell lines of commercially available silica nanoparticles (NPs), Ludox CL (nominal size 21 nm) and CL-X (nominal size of 30 nm) were investigated with particular attention to the effect over long exposure times (the tests were run after 72 h exposure up to 7 days). These two formulations differed in physico-chemical properties and showed different stabilities in the cell culture medium used for the experiments. Ludox CL silica NPs were found to be cytotoxic only at the higher concentrations to the WI-38 cells (WST-1 and LDH assays) but notmore » to the 3T3-L1 cells, whereas the Ludox CL-X silica NPs, which were less stable over the 72 h exposure, were cytotoxic to both cell lines in both assays. In the clonogenic assay both silica NPs induced a concentration dependent decrease in the surviving fraction of 3T3-L1 cells, with the Ludox CL-X silica NPs being more cytotoxic. Cell cycle analysis showed a trend indicating alterations in both cell lines at different phases with both silica NPs tested. Buthionine sulfoximine (γ-glutamylcysteine synthetase inhibitor) combined with Ludox CL-X was found to induce a strong decrease in 3T3-L1 cell viability which was not observed for the WI-38 cell line. This study clearly indicates that longer exposure studies may give important insights on the impact of nanomaterials on cells. However, and especially when investigating nanoparticle effects after such long exposure, it is fundamental to include a detailed physico-chemical characterization of the nanoparticles and their dispersions over the time scale of the experiment, in order to be able to interpret eventual impacts on cells. -- Highlights: ► Ludox CL silica NPs are cytotoxic to WI-38 fibroblasts but not to 3T3-L1 fibroblasts. ► Ludox CL-X silica NPs are cytotoxic to both cell lines. ► In clonogenic assay both silica NPs induce cytotoxicity, higher for CL-X silica. ► Cell cycle analysis shows alterations in both cell lines with both silica NP tested. ► Buthionine sulfoximine enhances cytotoxicity of Ludox CL-X in 3T3-L1 cells.« less

miR-199a-5p Is Upregulated during Fibrogenic Response to Tissue Injury and Mediates TGFbeta-Induced Lung Fibroblast Activation by Targeting Caveolin-1

PubMed Central

Courcot, Elisabeth; Roderburg, Christoph; Cauffiez, Christelle; Aubert, Sébastien; Copin, Marie-Christine; Wallaert, Benoit; Glowacki, François; Dewaeles, Edmone; Milosevic, Jadranka; Maurizio, Julien; Tedrow, John; Marcet, Brice; Lo-Guidice, Jean-Marc; Kaminski, Naftali; Barbry, Pascal; Luedde, Tom; Perrais, Michael

2013-01-01

As miRNAs are associated with normal cellular processes, deregulation of miRNAs is thought to play a causative role in many complex diseases. Nevertheless, the precise contribution of miRNAs in fibrotic lung diseases, especially the idiopathic form (IPF), remains poorly understood. Given the poor response rate of IPF patients to current therapy, new insights into the pathogenic mechanisms controlling lung fibroblasts activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies for this devastating disease. To identify miRNAs with potential roles in lung fibrogenesis, we performed a genome-wide assessment of miRNA expression in lungs from two different mouse strains known for their distinct susceptibility to develop lung fibrosis after bleomycin exposure. This led to the identification of miR-199a-5p as the best miRNA candidate associated with bleomycin response. Importantly, miR-199a-5p pulmonary expression was also significantly increased in IPF patients (94 IPF versus 83 controls). In particular, levels of miR-199a-5p were selectively increased in myofibroblasts from injured mouse lungs and fibroblastic foci, a histologic feature associated with IPF. Therefore, miR-199a-5p profibrotic effects were further investigated in cultured lung fibroblasts: miR-199a-5p expression was induced upon TGFβ exposure, and ectopic expression of miR-199a-5p was sufficient to promote the pathogenic activation of pulmonary fibroblasts including proliferation, migration, invasion, and differentiation into myofibroblasts. In addition, we demonstrated that miR-199a-5p is a key effector of TGFβ signaling in lung fibroblasts by regulating CAV1, a critical mediator of pulmonary fibrosis. Remarkably, aberrant expression of miR-199a-5p was also found in unilateral ureteral obstruction mouse model of kidney fibrosis, as well as in both bile duct ligation and CCl4-induced mouse models of liver fibrosis, suggesting that dysregulation of miR-199a-5p represents a general mechanism contributing to the fibrotic process. MiR-199a-5p thus behaves as a major regulator of tissue fibrosis with therapeutic potency to treat fibroproliferative diseases. PMID:23459460

LXA{sub 4} actions direct fibroblast function and wound closure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Herrera, Bruno S.; Microbiology Branch, US Army Dental and Trauma Research Detachment, Institute of Surgical Research, JBSA Fort Sam Houston, TX; Kantarci, Alpdogan

Timely resolution of inflammation is crucial for normal wound healing. Resolution of inflammation is an active biological process regulated by specialized lipid mediators including the lipoxins and resolvins. Failure of resolution activity has a major negative impact on wound healing in chronic inflammatory diseases that is manifest as excess fibrosis and scarring. Lipoxins, including Lipoxin A{sub 4} (LXA{sub 4}), have known anti-fibrotic and anti-scarring properties. The goal of this study was to elucidate the impact of LXA{sub 4} on fibroblast function. Mouse fibroblasts (3T3 Mus musculus Swiss) were cultured for 72 h in the presence of TGF-β1, to induce fibroblast activation.more » The impact of exogenous TGF-β1 (1 ng/mL) on LXA{sub 4} receptor expression (ALX/FPR2) was determined by flow cytometry. Fibroblast proliferation was measured by bromodeoxyuridine (BrdU) labeling and migration in a “scratch” assay wound model. Expression of α-smooth muscle actin (α-SMA), and collagen types I and III were measured by Western blot. We observed that TGF-β1 up-regulates LXA{sub 4} receptor expression, enhances fibroblast proliferation, migration and scratch wound closure. α-SMA levels and Collagen type I and III deposition were also enhanced. LXA{sub 4} slowed fibroblast migration and scratch wound closure at early time points (24 h), but wound closure was equal to TGF-β1 alone at 48 and 72 h. LXA{sub 4} tended to slow fibroblast proliferation at both concentrations, but had no impact on α-SMA or collagen production by TGF-β1 stimulated fibroblasts. The generalizability of the actions of resolution molecules was examined in experiments repeated with resolvin D2 (RvD2) as the agonist. The activity of RvD2 mimicked the actions of LXA{sub 4} in all assays, through an as yet unidentified receptor. The results suggest that mediators of resolution of inflammation enhance wound healing and limit fibrosis in part by modulating fibroblast function. - Highlights: • TGF-β1 up-regulates LXA{sub 4} receptor (ALX/FPR2) expression on fibroblast. • LXA{sub 4} regulates fibroblast migration and proliferation induced by TGF-β1. • SPMs have no impact on α-SMA, collagen type-I and III expression by fibroblast. • RvD2 regulates TGF-β1-induced fibroblast proliferation and scratch wound closure.« less

Oxidative tyrosylation of high density lipoprotein by peroxidase enhances cholesterol removal from cultured fibroblasts and macrophage foam cells.

PubMed Central

Francis, G A; Mendez, A J; Bierman, E L; Heinecke, J W

1993-01-01

Lipoprotein oxidation is thought to play a pivotal role in atherogenesis, yet the underlying reaction mechanisms remain poorly understood. We have explored the possibility that high density lipoprotein (HDL) might be oxidized by peroxidase-generated tyrosyl radical. Exposure of HDL to L-tyrosine, H2O2, and horseradish peroxidase crosslinked its apolipoproteins and strikingly increased protein-associated fluorescence. The reaction required L-tyrosine but was independent of free metal ions; it was blocked by either catalase or the heme poison aminotriazole. Dityrosine and other tyrosine oxidation products were detected in the apolipoproteins of HDL modified by the peroxidase/L-tyrosine/H2O2 system, implicating tyrosyl radical in the reaction pathway. Further evidence suggests that tyrosylated HDL removes cholesterol from cultured cells more effectively than does HDL. Tyrosylated HDL was more potent than HDL at inhibiting cholesterol esterification by the acyl-CoA:cholesterol acyltransferase reaction, stimulating the incorporation of [14C]acetate into [14C]cholesterol, and depleting cholesteryl ester stores in human skin fibroblasts. Moreover, exposure of mouse macrophage foam cells to tyrosylated HDL markedly diminished cholesteryl ester and free cholesterol mass. We have recently found that myeloperoxidase, a heme protein secreted by activated phagocytes, can also convert L-tyrosine to o,o'-dityrosine. This raises the possibility that myeloperoxidase-generated tyrosyl radical may modify HDL, enabling the lipoprotein to protect the artery wall against pathological cholesterol accumulation. Images Fig. 1 PMID:8341680

Physicochemical and in vitro biological evaluations of furazolidone-based β-cyclodextrin complexes in Leishmania amazonensis.

PubMed

Carvalho, Suzana Gonçalves; Siqueira, Larissa Ataíde; Zanini, Marcos Santos; Dos Santos Matos, Ana Paula; Quaresma, Carla Holandino; da Silva, Luisa Mota; de Andrade, Sérgio Faloni; Severi, Juliana Aparecida; Villanova, Janaina Cecília Oliveira

2018-06-15

Recently, there have been numerous cases of leishmaniasis reported in different Brazilian states. The use of furazolidone (FZD) to treat leishmaniasis has been previously described; however, the drug is associated with adverse effects such as anorexia, weight loss, incoordination, and fatigue in dogs. Thus, in the present study, we prepared and evaluated inclusion complexes between FZD and β-cyclodextrin (β-CD) to guarantee increased drug solubility and reduce the toxicity associated with high doses. The FZD:β-CD complexes were prepared by two different techniques (kneading and lyophilization) prior to incorporation in an oral pharmaceutical dosage form. Formation of the complexes was confirmed using appropriate physicochemical methods. Antileishmanial activity against L. amazonensis was tested in vitro via a microplate assay using resazurin dye and cytotoxicity was determined using the fibroblast L929 lineage. Solubility studies showed the formation of complexes with complexation efficiencies lower than 100%. Physicochemical analysis revealed that FZD was inserted into the β-CD cavity after complexation by both methods. Biological in vitro evaluations demonstrated that free FZD and the FZD:β-CD complexes presented significant leishmanicidal activity against L. amazonensis with IC 50 values of 6.16 μg/mL and 1.83 μg/mL for the complexes prepared by kneading and lyophilization, respectively. The data showed that these complexes reduced the survival of promastigotes and presented no toxicity for tested cells. Our results indicate that the new compounds could be a cost-effective alternative for use in the pharmacotherapy of leishmaniasis in dogs infected with L. amazonensis. Copyright © 2018 Elsevier Ltd. All rights reserved.

Osthole inhibits the expressions of collagen I and III through Smad signaling pathway after treatment with TGF-β1 in mouse cardiac fibroblasts.

PubMed

Liu, Jin-Cheng; Wang, Feng; Xie, Mei-Lin; Cheng, Zong-Qi; Qin, Qiong; Chen, Lin; Chen, Rong

2017-02-01

Osthole, a natural coumarin and bioactive compound isolated from the fruit of Cnidium monnieri (L.) Cusson, was reported to prevent isoprenaline-induced myocardial fibrosis in mice by inhibiting the transforming growth factor-β1 (TGF-β1) expression, but the underlying mechanism is still unclear. The aim of this study is to illuminate whether the mechanism of osthole inhibiting collagen I and III expressions is associated with Smad signaling pathway in mouse cardiac fibroblasts (CFs) treated with TGF-β1. The mouse CFs stimulated with TGF-β1 were cultured and treated with osthole 1.25-5μg/ml for 24h. The expressions of α-SMA, collagen I, collagen III, TGF-β receptor I (TβRI), Smad2/3, phospho-Smad2/3 (P-Smad2/3), Smad4 and Smad7 were detected by real-time PCR method and western blot method, respectively. After treatment with TGF-β1 and osthole in CFs, the levels of α-SMA expression and collagen I and III were reduced by osthole treatment. Accordingly, the ratio of collagen I/III had a similar changing trend. Besides, the levels of TβRI, Smad2/3, P-Smad2/3 and Smad4 expressions were decreased, while the level of Smad7 expression was increased after treatment with osthole. The present results demonstrated that osthole could inhibit the collagen I and III expressions and their ratio in CFs treated with TGF-β1 via Smad signaling pathway, which might be one of its anti-fibrotic action mechanisms. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Fibroblast heterogeneity: implications for human disease.

PubMed

Lynch, Magnus D; Watt, Fiona M

2018-01-02

Fibroblasts synthesize the extracellular matrix of connective tissue and play an essential role in maintaining the structural integrity of most tissues. Researchers have long suspected that fibroblasts exhibit functional specialization according to their organ of origin, body site, and spatial location. In recent years, a number of approaches have revealed the existence of fibroblast subtypes in mice. Here, we discuss fibroblast heterogeneity with a focus on the mammalian dermis, which has proven an accessible and tractable system for the dissection of these relationships. We begin by considering differences in fibroblast identity according to anatomical site of origin. Subsequently, we discuss new results relating to the existence of multiple fibroblast subtypes within the mouse dermis. We consider the developmental origin of fibroblasts and how this influences heterogeneity and lineage restriction. We discuss the mechanisms by which fibroblast heterogeneity arises, including intrinsic specification by transcriptional regulatory networks and epigenetic factors in combination with extrinsic effects of the spatial context within tissue. Finally, we discuss how fibroblast heterogeneity may provide insights into pathological states including wound healing, fibrotic diseases, and aging. Our evolving understanding suggests that ex vivo expansion or in vivo inhibition of specific fibroblast subtypes may have important therapeutic applications.

Utility of Survival Motor Neuron ELISA for Spinal Muscular Atrophy Clinical and Preclinical Analyses

PubMed Central

Kobayashi, Dione T.; Olson, Rory J.; Sly, Laurel; Swanson, Chad J.; Chung, Brett; Naryshkin, Nikolai; Narasimhan, Jana; Bhattacharyya, Anuradha; Mullenix, Michael; Chen, Karen S.

2011-01-01

Objectives Genetic defects leading to the reduction of the survival motor neuron protein (SMN) are a causal factor for Spinal Muscular Atrophy (SMA). While there are a number of therapies under evaluation as potential treatments for SMA, there is a critical lack of a biomarker method for assessing efficacy of therapeutic interventions, particularly those targeting upregulation of SMN protein levels. Towards this end we have engaged in developing an immunoassay capable of accurately measuring SMN protein levels in blood, specifically in peripheral blood mononuclear cells (PBMCs), as a tool for validating SMN protein as a biomarker in SMA. Methods A sandwich enzyme-linked immunosorbent assay (ELISA) was developed and validated for measuring SMN protein in human PBMCs and other cell lysates. Protocols for detection and extraction of SMN from transgenic SMA mouse tissues were also developed. Results The assay sensitivity for human SMN is 50 pg/mL. Initial analysis reveals that PBMCs yield enough SMN to analyze from blood volumes of less than 1 mL, and SMA Type I patients' PBMCs show ∼90% reduction of SMN protein compared to normal adults. The ELISA can reliably quantify SMN protein in human and mouse PBMCs and muscle, as well as brain, and spinal cord from a mouse model of severe SMA. Conclusions This SMN ELISA assay enables the reliable, quantitative and rapid measurement of SMN in healthy human and SMA patient PBMCs, muscle and fibroblasts. SMN was also detected in several tissues in a mouse model of SMA, as well as in wildtype mouse tissues. This SMN ELISA has general translational applicability to both preclinical and clinical research efforts. PMID:21904622

The anti-fibrotic agent pirfenidone synergizes with cisplatin in killing tumor cells and cancer-associated fibroblasts.

PubMed

Mediavilla-Varela, Melanie; Boateng, Kingsley; Noyes, David; Antonia, Scott J

2016-03-02

Anti-fibrotic drugs such as pirfenidone have been developed for the treatment of idiopathic pulmonary fibrosis. Because activated fibroblasts in inflammatory conditions have similar characteristics as cancer-associated fibroblasts (CAFs) and CAFs contribute actively to the malignant phenotype, we believe that anti-fibrotic drugs have the potential to be repurposed as anti-cancer drugs. The effects of pirfenidone alone and in combination with cisplatin on human patient-derived CAF cell lines and non-small cell lung cancer (NSCLC) cell lines were examined. The impact on cell death in vitro as well as tumor growth in a mouse model was determined. Annexin V/PI staining and Western blot analysis were used to characterize cell death. Synergy was assessed with the combination index method using Calcusyn software. Pirfenidone alone induced apoptotic cell death in lung CAFs at a high concentration (1.5 mg/mL). However, co-culture in vitro experiments and co-implantation in vivo experiments showed that the combination of low doses of cisplatin (10 μM) and low doses of pirfenidone (0.5 mg/mL), in both CAFs and tumors, lead to increased cell death and decreased tumor progression, respectively. Furthermore, the combination of cisplatin and pirfenidone in NSCLC cells (A549 and H157 cells) leads to increased apoptosis and synergistic cell death. Our studies reveal for the first time that the combination of cisplatin and pirfenidone is active in preclinical models of NSCLC and therefore may be a new therapeutic approach in this disease.

Stromal loss of TGFβ drives cancer growth in the epithelium via inflammation | Center for Cancer Research

Cancer.gov

Interactions between epithelial and stromal cells play an important role in cancer development and progression. Epithelial cancers develop when changes occur to tumor suppressor genes in stromal fibroblast cells. For example, loss of tumor suppressor, p53, in stromal fibroblasts leads to p53 inactivation in the epithelium in a prostate cancer model, and disruption of the transforming growth factor-b receptor II (TGF-βRII) in stromal fibroblasts results in intraepithelial dysplasia in prostate cancer and invasive squamous cell carcinoma (SCC) in mouse forestomach.

Dexras1 mediates glucocorticoid-associated adipogenesis and diet-induced obesity

PubMed Central

Cha, Jiyoung Y.; Kim, Hyo Jung; Yu, Jung Hwan; Xu, Jing; Kim, Daham; Paul, Bindu D.; Choi, Hyeonjin; Kim, Seyun; Lee, Yoo Jeong; Ho, Gary P.; Rao, Feng; Snyder, Solomon H.; Kim, Jae-woo

2013-01-01

Adipogenesis, the conversion of precursor cells into adipocytes, is associated with obesity and is mediated by glucocorticoids acting via hitherto poorly characterized mechanisms. Dexras1 is a small G protein of the Ras family discovered on the basis of its marked induction by the synthetic glucocorticoid dexamethasone. We show that Dexras1 mediates adipogenesis and diet-induced obesity. Adipogenic differentiation of 3T3-L1 cells is abolished with Dexras1 depletion, whereas overexpression of Dexras1 elicits adipogenesis. Adipogenesis is markedly reduced in mouse embryonic fibroblasts from Dexras1-deleted mice, whereas adiposity and diet-induced weight gain are diminished in the mutant mice. PMID:24297897

Nickel Inhibits Mitochondrial Fatty Acid Oxidation

PubMed Central

Uppala, Radha; McKinney, Richard W.; Brant, Kelly A.; Fabisiak, James P.; Goetzman, Eric S.

2015-01-01

Nickel exposure is associated with changes in cellular energy metabolism which may contribute to its carcinogenic properties. Here, we demonstrate that nickel strongly represses mitochondrial fatty acid oxidation—the pathway by which fatty acids are catabolized for energy—in both primary human lung fibroblasts and mouse embryonic fibroblasts. At the concentrations used, nickel suppresses fatty acid oxidation without globally suppressing mitochondrial function as evidenced by increased glucose oxidation to CO2. Pre-treatment with L-carnitine, previously shown to prevent nickel-induced mitochondrial dysfunction in neuroblastoma cells, did not prevent the inhibition of fatty acid oxidation. The effect of nickel on fatty acid oxidation occurred only with prolonged exposure (>5 hr), suggesting that direct inhibition of the active sites of metabolic enzymes is not the mechanism of action. Nickel is a known hypoxia-mimetic that activates hypoxia inducible factor-1α (HIF1α). Nickel-induced inhibition of fatty acid oxidation was blunted in HIF1α knockout fibroblasts, implicating HIF1α as one contributor to the mechanism. Additionally, nickel down-regulated the protein levels of the key fatty acid oxidation enzyme very long-chain acyl-CoA dehydrogenase (VLCAD) in a dose-dependent fashion. In conclusion, inhibition of fatty acid oxidation by nickel, concurrent with increased glucose metabolism, represents a form of metabolic reprogramming that may contribute to nickel-induced carcinogenesis. PMID:26051273

Tissue-engineered skin preserving the potential of epithelial cells to differentiate into hair after grafting.

PubMed

Larouche, Danielle; Cuffley, Kristine; Paquet, Claudie; Germain, Lucie

2011-03-01

The aim of this study was to evaluate whether tissue-engineered skin produced in vitro was able to sustain growth of hair follicles in vitro and after grafting. Different tissues were designed. Dissociated newborn mouse keratinocytes or newborn mouse hair buds (HBs) were added onto dermal constructs consisting of a tissue-engineered cell-derived matrix elaborated from either newborn mouse or adult human fibroblasts cultured with ascorbic acid. After 7-21 days of maturation at the air-liquid interface, no hair was noticed in vitro. Epidermal differentiation was observed in all tissue-engineered skin. However, human fibroblast-derived tissue-engineered dermis (hD) promoted a thicker epidermis than mouse fibroblast-derived tissue-engineered dermis (mD). In association with mD, HBs developed epithelial cyst-like inclusions presenting outer root sheath-like attributes. In contrast, epidermoid cyst-like inclusions lined by a stratified squamous epithelium were present in tissues composed of HBs and hD. After grafting, pilo-sebaceous units formed and hair grew in skin elaborated from HBs cultured 10-26 days submerged in culture medium in association with mD. However, the number of normal hair follicles decreased with longer culture time. This hair-forming capacity after grafting was not observed in tissues composed of hD overlaid with HBs. These results demonstrate that epithelial stem cells can be kept in vitro in a permissive tissue-engineered dermal environment without losing their potential to induce hair growth after grafting.

Effect of a feeder layer composed of mouse embryonic and human foreskin fibroblasts on the proliferation of human embryonic stem cells.

PubMed

Yang, Hua; Qiu, Ying; Zeng, Xianghui; Ding, Yan; Zeng, Jianye; Lu, Kehuan; Li, Dongsheng

2016-06-01

The aim of the present study was to investigate the effects of feeder layers composed of various ratios of mouse embryonic fibroblasts (MEFs) and human foreskin fibroblasts (hFFs) on the growth of human embryonic stem cells (hESCs). In addition, the secretion levels of basic fibroblast growth factor (bFGF) by the feeder layers was detected. MEFs and hFFs were treated with mitomycin C and seeded onto gelatin-coated plates at a density of 1×10 8 cells/l. The hFFs and MEFs were combined and plated at the following ratios: 0:1, 1:2, 1:1, 2:1 and 1:0. The secretion of bFGF by the various hFF/MEF ratio feeder layers was detected using an enzyme-linked immunosorbent assay. Subsequently, hESCs were cultured on top of the various feeder layers. The differences in the cellular morphology of the hESCs were observed using microscopy, and the expression levels alkaline phosphatase (AKP) and octamer-binding transcription factor 4 (OCT-4) were detected using immunohistochemical analysis as indicators of differentiation status. The results showed that the hFFs secreted substantial quantities of bFGF, while no bFGF was secreted by the MEFs. The clones of hESC growing on the feeder layer containing MEF or hFF alone were flat. By contrast, hESC clones grown on a mixed feeder layer containing hFFs + MEFs at a ratio of 1:1 exhibited an accumulated growth with a clear edge, as compared with the other ratios. In addition, hESCs growing on the feeder layer were positive for the expression of AKP and OCT-4. In summary, feeder layer hFFs secreted bFGF, while MEFs did not, indicating that bFGF is not the only factor that supports the growth and differentiation of hESCs. The optimal growth of hESCs was achieved using a mixed feeder layer composed of hFFs + MEFs at a ratio of 1:1.

Repeated exposure of mouse dermal fibroblasts at a sub-cytotoxic dose of UVB leads to premature senescence: a robust model of cellular photoaging.

PubMed

Zeng, Ji-ping; Bi, Bo; Chen, Liang; Yang, Ping; Guo, Yu; Zhou, Yi-qun; Liu, Tian-yi

2014-01-01

Photoaging skin is due to accumulative effect of UV irradiation that mainly imposes its damage on dermal fibroblasts. To mimic the specific cellular responses invoked by long term effect of UVB, it is preferable to develop a photo-damaged model in vitro based on repeated UVB exposure instead of a single exposure. To develop a photo-damaged model of fibroblasts by repeated UVB exposure allowing for investigation of molecular mechanism underlying premature senescence and testing of potential anti-photoaging compounds. Mouse dermal fibroblasts (MDFs) at early passages (passages 1-3) were exposed to a series of 4 sub-cytotoxic dose of UVB. The senescent phenotypes were detected at 24 or 48h after the last irradiation including cell viability, ROS generation, mitochondrial membrane potential, cell cycle, production and degradation of extracellular matrix. Repeated exposure of UVB resulted in remarkable features of senescence. It effectively avoided the disadvantages of single dose such as induction of cell death rather than senescence, inadequate stress resulting in cellular self-rehabilitation. Our work confirms the possibility of detecting cellular machinery that mediates UVB damage to fibroblasts in vitro by repeated exposure, while the potential molecular mechanisms including cell surface receptors, protein kinase signal transduction pathways, and transcription factors remain to be further evaluated. Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Inefficient reprogramming of fibroblasts into cardiomyocytes using Gata4, Mef2c, Tbx5

PubMed Central

Chen, J.X.; Krane, M.; Deutsch, M. A.; Wang, L.; Rav-Acha, M.; Gregoire, S.; Engels, M. C.; Rajarajan, K.; Karra, R.; Abel, E. D.; Wu, J. C.; Milan, D.; Wu, S. M.

2012-01-01

Rationale Direct reprogramming of fibroblasts into cardiomyocytes is a novel strategy for cardiac regeneration. However, the key determinants involved in this process are unknown. Objective To assess the efficiency of direct fibroblast reprogramming via viral overexpression of GATA4, Mef2c, and Tbx5 (GMT). Methods and Results We induced GMT overexpression in murine tail tip fibroblasts (TTFs) and cardiac fibroblasts (CFs) from multiple lines of transgenic mice carrying different cardiomyocyte lineage reporters. We found that the induction of GMT overexpression in TTFs and CFs is inefficient at inducing molecular and electrophysiological phenotypes of mature cardiomyocytes. In addition, transplantation of GMT infected CFs into injured mouse hearts resulted in decreased cell survival with minimal induction of cardiomyocyte genes. Conclusions Significant challenges remain in our ability to convert fibroblasts into cardiomyocyte-like cells and a greater understanding of cardiovascular epigenetics is needed to increase the translational potential of this strategy. PMID:22581928

Reduction of apoptosis through the mitochondrial pathway by the administration of acetyl-L-carnitine to mouse fibroblasts in culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pillich, Rudolf Tito; Dipartimento di Genetica e Biologia Molecolare, Universita di Roma 'La Sapienza', P.le A. Moro, 5-00185 Rome; Scarsella, Gianfranco

It is shown in literature that stress, such as deprivation of trophic factors and hypoxia, induces apoptosis in cultured cells and in tissues. In light of these results, we explored the possibility of protecting cells from programmed death by improving the metabolism of the mitochondrion. To this end, acetyl-L-carnitine was administered at various concentrations under conditions of serum deprivation. The choice of this drug was based on the accepted notion that acetyl-L-carnitine is able to stabilize mitochondrial membranes and to increase the supply of energy to the organelle. The results presented here indicate that the drug protects cells from apoptoticmore » death: this is demonstrated by a lower positivity to the TUNEL reaction and by a strong reduction of the apoptotic DNA ladder in serum-deprived cells. The involvement of the mitochondrial apoptotic pathway was assessed by cytochrome C release and immunoreactivity to caspase 3. Moreover, acetyl-L-carnitine stimulates cell proliferation.« less

The Locomotion of Mouse Fibroblasts in Tissue Culture

PubMed Central

Gail, Mitchell H.; Boone, Charles W.

1970-01-01

Time-lapse cinematography was used to investigate the motion of mouse fibroblasts in tissue culture. Observations over successive short time intervals revealed a tendency for the cells to persist in their direction of motion from one 2.5 hr time interval to the next. Over 5.0-hr time intervals, however, the direction of motion appeared random. This fact suggested that D, the diffusion constant of a random walk model, might serve to characterize cellular motility if suitably long observation times were used. We therefore investigated the effect of “persistence” on the pure random walk model, and we found theoretically and confirmed experimentally that the motility of a persisting cell could indeed be characterized by an augmented diffusion constant, D*. A method for determining confidence limits on D* was also developed. Thus a random walk model, modified to comprehend the persistence effect, was found to describe the motion of fibroblasts in tissue culture and to provide a numerical measure of cellular motility. PMID:5531614

Reprogramming of somatic cells induced by fusion of embryonic stem cells using hemagglutinating virus of Japan envelope (HVJ-E)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yue, Xiao-shan; Department of Biomolecular Engineering, Graduate School of Bioscience and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501; Fujishiro, Masako

In this research, hemagglutinating virus of Japan envelope (HVJ-E) was used to reprogram somatic cells by fusion with mouse embryonic stem (ES) cells. Neomycin-resistant mouse embryonic fibroblasts (MEFs) were used as somatic cells. Nanog-overexpressing puromycin-resistant EB3 cells were used as mouse ES cells. These two cells were fused by exposing to HVJ-E and the generated fusion cells were selected by puromycin and G418 to get the stable fusion cell line. The fusion cells form colonies in feeder-free culture system. Microsatellite analysis of the fusion cells showed that they possessed genes from both ES cells and fibroblasts. The fusion cells weremore » tetraploid, had alkali phosphatase activity, and expressed stem cell marker genes such as Pou5f1, Nanog, and Sox2, but not the fibroblast cell marker genes such as Col1a1 and Col1a2. The pluripotency of fusion cells was confirmed by their expression of marker genes for all the three germ layers after differentiation induction, and by their ability to form teratoma which contained all the three primary layers. Our results show that HVJ-E can be used as a fusion reagent for reprogramming of somatic cells.« less

The Stil protein regulates centrosome integrity and mitosis through suppression of Chfr

PubMed Central

Castiel, Asher; Danieli, Michal Mark; David, Ahuvit; Moshkovitz, Sharon; Aplan, Peter D.; Kirsch, Ilan R.; Brandeis, Michael; Krämer, Alwin; Izraeli, Shai

2011-01-01

Stil (Sil, SCL/TAL1 interrupting locus) is a cytosolic and centrosomal protein expressed in proliferating cells that is required for mouse and zebrafish neural development and is mutated in familial microcephaly. Recently the Drosophila melanogaster ortholog of Stil was found to be important for centriole duplication. Consistent with this finding, we report here that mouse embryonic fibroblasts lacking Stil are characterized by slow growth, low mitotic index and absence of clear centrosomes. We hypothesized that Stil regulates mitosis through the tumor suppressor Chfr, an E3 ligase that blocks mitotic entry in response to mitotic stress. Mouse fibroblasts lacking Stil by genomic or RNA interference approaches, as well as E9.5 Stil−/− embryos, express high levels of the Chfr protein and reduced levels of the Chfr substrate Plk1. Exogenous expression of Stil, knockdown of Chfr or overexpression of Plk1 reverse the abnormal mitotic phenotypes of fibroblasts lacking Stil. We further demonstrate that Stil increases Chfr auto-ubiquitination and reduces its protein stability. Thus, Stil is required for centrosome organization, entry into mitosis and cell proliferation, and these functions are at least partially mediated by Chfr and its targets. This is the first identification of a negative regulator of the Chfr mitotic checkpoint. PMID:21245198

Astragaloside IV suppresses transforming growth factor-β1 induced fibrosis of cultured mouse renal fibroblasts via inhibition of the MAPK and NF-κB signaling pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Che, Xiajing; Wang, Qin; Xie, Yuanyuan

Renal fibrosis, a progressive process characterized by the accumulation of extracellular matrix (ECM) leading to organ dysfunction, is a characteristic of chronic kidney diseases. Among fibrogenic factors known to regulate the renal fibrotic process, transforming growth factor-β (TGF-β) plays a central role. In the present study, we examined the effect of Astragaloside IV (AS-IV), a component of the traditional Chinese medicinal plant Astragalus membranaceus, on the processes associated with renal fibrosis in cultured mouse renal fibroblasts treated with TGF-β1. RT-PCR, western blotting, immunofluorescence staining and collagen assays showed that AS-IV suppressed TGF-β1 induced fibroblast proliferation, transdifferentiation, and ECM production inmore » a dose-dependent manner. Examination of the underlying mechanisms showed that the effect of AS-IV on the inhibition of fibroblast differentiation and ECM formation were mediated by its modulation of the activity of the MAPK and NF-κB signaling pathways. Taken together, our results indicate that AS-IV alleviates renal interstitial fibrosis via a mechanism involving the MAPK and NF-κB signaling pathways and demonstrate the therapeutic potential of AS-IV for the treatment of chronic kidney diseases. - Highlights: • AS-IV suppressed TGF-β1 induced renal fibroblast proliferation. • AS-IV suppressed TGF-β1 induced renal fibroblast transdifferentiation. • AS-IV suppressed TGF-β1 induced ECM production. • AS-IV alleviates renal fibrosis via the MAPK and NF-κB signaling pathways.« less

Silk fibroin-Thelebolan matrix: A promising chemopreventive scaffold for soft tissue cancer.

PubMed

Mukhopadhyay, Sourav K; Naskar, Deboki; Bhattacharjee, Promita; Mishra, Abheepsa; Kundu, Subhas C; Dey, Satyahari

2017-07-01

Research of improved functional bio-mimetic matrix for regenerative medicine is currently one of the rapidly growing fields in tissue engineering and medical sciences. This study reports a novel bio-polymeric matrix, which is fabricated using silk protein fibroin from Bombyx mori silkworm and fungal exopolysaccharide Thelebolan from Antarctic fungus Thelebolus sp. IITKGP-BT12 by solvent evaporation and freeze drying method. Natural cross linker genipin is used to imprison the Thelebolan within the fibroin network. Different cross-linked and non-cross-linked fibroin/Thelebolan matrices are fabricated and biophysically characterized. Cross-linked thin films show robustness, good mechanical strength and high temperature stability in comparison to non-cross-linked and pure matrices. The 3D sponge matrices demonstrate good cytocompatibility. Interestingly, sustained release of the Thelebolan from the cross-linked matrices induce apoptosis in colon cancer cell line (HT-29) in time dependent manner while it is nontoxic to the normal fibroblast cells (L929).The findings indicate that the cross-linked fibroin/Thelebolan matrices can be used as potential topical chemopreventive scaffold for preclusion of soft tissue carcinoma. Copyright © 2017 Elsevier B.V. All rights reserved.

A new synthesis route to high surface area sol gel bioactive glass through alcohol washing

PubMed Central

M. Mukundan, Lakshmi; Nirmal, Remya; Vaikkath, Dhanesh; Nair, Prabha D.

2013-01-01

Bioactive glass is one of the widely used bone repair material due to its unique properties like osteoconductivity, osteoinductivity and biodegradability. In this study bioactive glass is prepared by the sol gel process and stabilized by a novel method that involves a solvent instead of the conventional calcinations process. This study represents the first attempt to use this method for the stabilization of bioactive glass. The bioactive glass stabilized by this ethanol washing process was characterized for its physicochemical and biomimetic property in comparison with similar composition of calcined bioactive glass. The compositional similarity of the two stabilized glass powders was confirmed by spectroscopic and thermogravimetric analysis. Other physicochemical characterizations together with the cell culture studies with L929 fibroblast cells and bone marrow mesenchymal stem cells proved that the stabilization was achieved with the retention of its inherent bioactive potential. However an increase in the surface area of the glass powder was obtained as a result of this ethanol washing process and this add up to the success of the study. Hence the present study exhibits a promising route for high surface area bioactive glass for increasing biomimicity. PMID:23512012

Pectin-chitosan-PVA nanofibrous scaffold made by electrospinning and its potential use as a skin tissue scaffold.

PubMed

Lin, Hsin-Yi; Chen, Hsin-Hung; Chang, Shih-Hsin; Ni, Tsung-Sheng

2013-01-01

Scaffolds made of chitosan nanofibers are often too mechanically weak for their application and often their manufacturing processes involve the use of harmful and flammable organic solvents. In the attempt to improve the mechanical properties of nanofibrous scaffolds made of chitosan without the use of harmful chemicals, pectin, an anionic polymer was blended with chitosan, a cationic polymer, to form a polyelectrolyte complex and electrospun into nanofibers for the first time. The electrospun chitosan-pectin scaffolds, when compared to electrospun chitosan scaffolds, had a 58% larger diameter, a 21% higher Young's modulus, a 162% larger strain at break, and a 104% higher ultimate tensile strength. Compared to the chitosan scaffolds, the chitosan-pectin scaffolds' swelling ratios decreased by 55% after 60 min in a saline solution and more quickly released the preloaded tetracycline HCl. The L929 fibroblast cells proliferated slightly slower on the chitosan-pectin scaffolds than on the chitosan scaffolds. Nonetheless, cells on both materials deposited similar levels of extracellular type I collagen on a per DNA basis. In conclusion, a novel chitosan-pectin nanofibrous scaffold with superior mechanical properties than a chitosan nanofibrous scaffold was successfully made without the use of harmful solvents.

Evaluation of Fibrin-Based Interpenetrating Polymer Networks as Potential Biomaterials for Tissue Engineering.

PubMed

Gsib, Olfat; Duval, Jean-Luc; Goczkowski, Mathieu; Deneufchatel, Marie; Fichet, Odile; Larreta-Garde, Véronique; Bencherif, Sidi Ahmed; Egles, Christophe

2017-12-10

Interpenetrating polymer networks (IPNs) have gained great attention for a number of biomedical applications due to their improved properties compared to individual components alone. In this study, we investigated the capacity of newly-developed naturally-derived IPNs as potential biomaterials for tissue engineering. These IPNs combine the biologic properties of a fibrous fibrin network polymerized at the nanoscale and the mechanical stability of polyethylene oxide (PEO). First, we assessed their cytotoxicity in vitro on L929 fibroblasts. We further evaluated their biocompatibility ex vivo with a chick embryo organotypic culture model. Subcutaneous implantations of the matrices were subsequently conducted on nude mice to investigate their biocompatibility in vivo. Our preliminary data highlighted that our biomaterials were non-cytotoxic (viability above 90%). The organotypic culture showed that the IPN matrices induced higher cell adhesion (across all the explanted organ tissues) and migration (skin, intestine) than the control groups, suggesting the advantages of using a biomimetic, yet mechanically-reinforced IPN-based matrix. We observed no major inflammatory response up to 12 weeks post implantation. All together, these data suggest that these fibrin-based IPNs are promising biomaterials for tissue engineering.

Evaluation of Fibrin-Based Interpenetrating Polymer Networks as Potential Biomaterials for Tissue Engineering

PubMed Central

Gsib, Olfat; Duval, Jean-Luc; Goczkowski, Mathieu; Deneufchatel, Marie; Fichet, Odile; Larreta-Garde, Véronique

2017-01-01

Interpenetrating polymer networks (IPNs) have gained great attention for a number of biomedical applications due to their improved properties compared to individual components alone. In this study, we investigated the capacity of newly-developed naturally-derived IPNs as potential biomaterials for tissue engineering. These IPNs combine the biologic properties of a fibrous fibrin network polymerized at the nanoscale and the mechanical stability of polyethylene oxide (PEO). First, we assessed their cytotoxicity in vitro on L929 fibroblasts. We further evaluated their biocompatibility ex vivo with a chick embryo organotypic culture model. Subcutaneous implantations of the matrices were subsequently conducted on nude mice to investigate their biocompatibility in vivo. Our preliminary data highlighted that our biomaterials were non-cytotoxic (viability above 90%). The organotypic culture showed that the IPN matrices induced higher cell adhesion (across all the explanted organ tissues) and migration (skin, intestine) than the control groups, suggesting the advantages of using a biomimetic, yet mechanically-reinforced IPN-based matrix. We observed no major inflammatory response up to 12 weeks post implantation. All together, these data suggest that these fibrin-based IPNs are promising biomaterials for tissue engineering. PMID:29232876

Chitosan functionalized poly-ε-caprolactone electrospun fibers and 3D printed scaffolds as antibacterial materials for tissue engineering applications.

PubMed

Tardajos, Myriam G; Cama, Giuseppe; Dash, Mamoni; Misseeuw, Lara; Gheysens, Tom; Gorzelanny, Christian; Coenye, Tom; Dubruel, Peter

2018-07-01

Tissue engineering (TE) approaches often employ polymer-based scaffolds to provide support with a view to the improved regeneration of damaged tissues. The aim of this research was to develop a surface modification method for introducing chitosan as an antibacterial agent in both electrospun membranes and 3D printed poly-ε-caprolactone (PCL) scaffolds. The scaffolds were functionalized by grafting methacrylic acid N-hydroxysuccinimide ester (NHSMA) onto the surface after Ar-plasma/air activation. Subsequently, the newly-introduced NHS groups were used to couple with chitosan of various molecular weights (Mw). High Mw chitosan exhibited a better coverage of the surface as indicated by the higher N% detected by X-ray photoelectron spectroscopy (XPS) and the observations with either scanning electron microscopy (SEM)(for fibers) or Coomassie blue staining (for 3D-printed scaffolds). A lactate dehydrogenase assay (LDH) using L929 fibroblasts demonstrated the cell-adhesion and cell-viability capacity of the modified samples. The antibacterial properties against S. aureus ATCC 6538 and S. epidermidis ET13 revealed a slower bacterial growth rate on the surface of the chitosan modified scaffolds, regardless the chitosan Mw. Copyright © 2018 Elsevier Ltd. All rights reserved.

Correlating cytotoxicity to elution behaviors of composite resins in term of curing kinetic.

PubMed

Meng, Junquan; Yang, Huichuan; Cao, Man; Li, Lei; Cai, Qing

2017-09-01

Cytotoxicity of photocurable composite resins is a key issue for their safe use in dental restoration. Curing kinetic and elution behaviors of the composite resin would have decisive effects on its cytotoxicity. In this study, composite resins composed of bisphenol-glycidyl dimethacrylate (Bis-GMA), triethyleneglycol dimethacrylate (TEGDMA), camphorquinone (CQ), N,N-dimethylaminoethyl methacrylate (DMAEMA) and barium glass powders were prepared by setting the photoinitiators CQ/DMAEMA at 0.5wt%, 1wt% or 3wt% of the total weight of Bis-GMA/TEGDMA. The ratio of Bis-GMA/TEGDMA was 6:4, the ratio of CQ/DMAEMA was 1:1, and the incorporated inorganic powder was 75wt%. Then, curing kinetics were studied by using real-time Fourier transform infrared spectroscopy (FTIR) and photo-DSC (differential scanning calorimeter). Elution behaviors in both ethanol solution and deionized water were monitored by using liquid chromatogram/mass spectrometry (LC/MS). Cytotoxicity was evaluated by in vitro culture of L929 fibroblasts. Finally, they were all analyzed and correlated in terms of initiator contents. It was found that the commonly used 0.5wt% of photoinitiators was somewhat insufficient in obtaining composite resin with low cytotoxicity. Copyright © 2017. Published by Elsevier B.V.

Novel bilayer wound dressing based on electrospun gelatin/keratin nanofibrous mats for skin wound repair.

PubMed

Yao, Chun-Hsu; Lee, Chia-Yu; Huang, Chiung-Hua; Chen, Yueh-Sheng; Chen, Kuo-Yu

2017-10-01

A bilayer membrane (GKU) with a commercial polyurethane wound dressing as an outer layer and electrospun gelatin/keratin nanofibrous mat as an inner layer was fabricated as a novel wound dressing. Scanning electron micrographs showed that gelatin/keratin nanofibers had a uniform morphology and bead-free structure with average fiber diameter of 160.4nm. 3-(4,5-Dimethylthiazolyl)-2,5-diphenyltetrazolium bromide assay using L929 fibroblast cells indicated that the residues released from the gelatin/keratin composite nanofibrous mat accelerated cell proliferation. Cell attachment experiments revealed that adhered cells spread better and migrated deeper into the gelatin/keratin nanofibrous mat than that into the gelatin nanofibrous mat. In animal studies, compared with the bilayer membrane without keratin, gauze and commercial wound dressing, Comfeel®, GKU membrane gave much more number of blood vessels and a greater reduction in wound area at 4days, and better wound repair at 14days with a thicker epidermis and larger number of newly formed hair follicles. GKU membrane, thus, could be a good candidate for wound dressing applications. Copyright © 2017 Elsevier B.V. All rights reserved.

In vitro and in vivo acute response towards injectable thermosensitive chitosan/TEMPO-oxidized cellulose nanofiber hydrogel.

PubMed

Nguyen, Trang Ho Minh; Abueva, Celine; Ho, Hai Van; Lee, Sun-Young; Lee, Byong-Taek

2018-01-15

TEMPO-oxidized cellulose nanofiber (TOCNF) is a natural material with many promising properties, including biocompatibility and degradability. In this study, we integrated TOCNF at different concentrations (0.2, 0.4, 0.6, 0.8% w/v) with chitosan (CS) and created a thermosensitive injectable hydrogel intended for biomedical applications. These hydrogels can undergo sol-gel transition at body temperature through interactions between chitosan and β-glycerophosphate. The addition of TOCNF resulted in faster gelation time and increased porosity. These hydrogels with TOCNF showed improved biocompatibility both in vitro and in vivo compared to CS hydrogel. Both MC3T3-E1 pre-osteoblast cells and L929 fibroblast cells showed biocompatibility towards CS/TOCNF 0.4. After 7days of implantation, initial inflammatory response to CS/TOCNF 0.4 was found. Such response was significantly subsided within 14days. Cell infiltration within the hydrogel was also prominent, showing anti-inflammatory or wound healing (M2) macrophage at 14days after implantation. These results showed that the addition of TOCNF could significantly improve the biocompatibility of CS hydrogel as a biomaterial for biomedical application. Copyright © 2017 Elsevier Ltd. All rights reserved.

Biocompatibility of Bespoke 3D-Printed Titanium Alloy Plates for Treating Acetabular Fractures.

PubMed

Lin, Xuezhi; Xiao, Xingling; Wang, Yimeng; Gu, Cheng; Wang, Canbin; Chen, Jiahui; Liu, Han; Luo, Juan; Li, Tao; Wang, Di; Fan, Shicai

2018-01-01

Treatment of acetabular fractures is challenging, not only because of its complicated anatomy but also because of the lack of fitting plates. Personalized titanium alloy plates can be fabricated by selective laser melting (SLM) but the biocompatibility of these three-dimensional printing (3D-printed) plates remains unknown. Plates were manufactured by SLM and their cytocompatibility was assessed by observing the metabolism of L929 fibroblasts incubated with culture medium extracts using a CCK-8 assay and their morphology by light microscopy. Allergenicity was tested using a guinea pig maximization test. In addition, acute systemic toxicity of the 3D-printed plates was determined by injecting extracts from the implants into the tail veins of mice. Finally, the histocompatibility of the plates was investigated by implanting them into the dorsal muscles of rabbits. The in vitro results suggested that cytocompatibility of the 3D-printed plates was similar to that of conventional plates. The in vivo data also demonstrated histocompatibility that was comparable between the two manufacturing techniques. In conclusion, both in vivo and in vitro experiments suggested favorable biocompatibility of 3D-printed titanium alloy plates, indicating that it is a promising option for treatment of acetabular fractures.

Biocompatibility of Bespoke 3D-Printed Titanium Alloy Plates for Treating Acetabular Fractures

PubMed Central

Xiao, Xingling; Wang, Yimeng; Gu, Cheng; Wang, Canbin; Chen, Jiahui; Liu, Han; Luo, Juan; Li, Tao

2018-01-01

Treatment of acetabular fractures is challenging, not only because of its complicated anatomy but also because of the lack of fitting plates. Personalized titanium alloy plates can be fabricated by selective laser melting (SLM) but the biocompatibility of these three-dimensional printing (3D-printed) plates remains unknown. Plates were manufactured by SLM and their cytocompatibility was assessed by observing the metabolism of L929 fibroblasts incubated with culture medium extracts using a CCK-8 assay and their morphology by light microscopy. Allergenicity was tested using a guinea pig maximization test. In addition, acute systemic toxicity of the 3D-printed plates was determined by injecting extracts from the implants into the tail veins of mice. Finally, the histocompatibility of the plates was investigated by implanting them into the dorsal muscles of rabbits. The in vitro results suggested that cytocompatibility of the 3D-printed plates was similar to that of conventional plates. The in vivo data also demonstrated histocompatibility that was comparable between the two manufacturing techniques. In conclusion, both in vivo and in vitro experiments suggested favorable biocompatibility of 3D-printed titanium alloy plates, indicating that it is a promising option for treatment of acetabular fractures. PMID:29682523

Regulatory role of NADPH oxidase in glycated LDL-induced upregulation of plasminogen activator inhibitor-1 and heat shock factor-1 in mouse embryo fibroblasts and diabetic mice.

PubMed

Zhao, Ruozhi; Le, Khuong; Moghadasian, Mohammed H; Shen, Garry X

2013-08-01

Cardiovascular disease is the predominant cause of death in diabetic patients. Fibroblasts are one of the major types of cells in the heart or vascular wall. Increased levels of glycated low-density lipoprotein (glyLDL) were detected in diabetic patients. Previous studies in our group demonstrated that oxidized LDL increased the amounts of NADPH oxidase (NOX), plasminogen activator inhibitor-1 (PAI-1), and heat shock factor-1 (HSF1) in fibroblasts. This study examined the expression of NOX, PAI-1, and HSF1 in glyLDL-treated wild-type or HSF1-deficient mouse embryo fibroblasts (MEFs) and in leptin receptor-knockout (db/db) diabetic mice. Treatment with physiologically relevant levels of glyLDL increased superoxide and H2O2 release and the levels of NOX4 and p22phox (an essential component of multiple NOX complexes) in wild-type or HSF1-deficient MEFs. The levels of HSF1 and PAI-1 were increased by glyLDL in wild-type MEFs, but not in HSF1-deficient MEFs. Diphenyleneiodonium (a nonspecific NOX inhibitor) or small interfering RNA for p22phox prevented glyLDL-induced increases in the levels of NOX4, HSF1, or PAI-1 in MEFs. The amounts of NOX4, HSF1, and PAI-1 were elevated in hearts of db/db diabetic mice compared to wild-type mice. The results suggest that glyLDL increased the abundance of NOX4 or p22phox via an HSF1-independent pathway, but that of PAI-1 via an HSF1-dependent manner. NOX4 plays a crucial role in glyLDL-induced expression of HSF1 and PAI-1 in mouse fibroblasts. Increased expression of NOX4, HSF1, and PAI-1 was detected in cardiovascular tissue of diabetic mice. Copyright © 2013 Elsevier Inc. All rights reserved.

Inhibition and recovery of the replication of depurinated parvovirus DNA in mouse fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vos, J.M.; Avalosse, B.; Su, Z.Z.

Apurinic sites were introduced in the single-stranded DNA of parvovirus minute-virus-of-mice (MVM) and their effect on viral DNA synthesis was measured in mouse fibroblasts. Approximately one apurinic site per viral genome, is sufficient to block its replication in untreated cells. The exposure of host cells to a sublethal dose of UV-light 15 hours prior to virus infection, enhances their ability to support the replication of depurinated MVM. Cell preirradiation induces the apparent overcome of 10-15% of viral DNA replication blocks. These results indicate that apurinic sites prevent mammalian cells from replicating single-stranded DNA unless a recovery process is activated bymore » cell UV-irradiation.« less

Modest increased sensitivity to radiation oncogenesis in ATM heterozygous versus wild-type mammalian cells

NASA Technical Reports Server (NTRS)

Smilenov, L. B.; Brenner, D. J.; Hall, E. J.

2001-01-01

Subpopulations that are genetically predisposed to radiation-induced cancer could have significant public health consequences. Individuals homozygous for null mutations at the ataxia telangiectasia gene are indeed highly radiosensitive, but their numbers are very small. Ataxia Telangiectasia heterozygotes (1-2% of the population) have been associated with somewhat increased radiosensitivity for some end points, but none directly related to carcinogenesis. Here, intralitter comparisons between wild-type mouse embryo fibroblasts and mouse embryo fibroblasts carrying ataxia telangiectasia mutated (ATM) null mutation indicate that the heterozygous cells are more sensitive to radiation oncogenesis than their normal, litter-matched, counterparts. From these data we suggest that Ataxia Telangiectasia heterozygotes could indeed represent a societally-significant radiosensitive human subpopulation.

Biocompatibility effects of biologically synthesized graphene in primary mouse embryonic fibroblast cells

PubMed Central

2013-01-01

Due to unique properties and unlimited possible applications, graphene has attracted abundant interest in the areas of nanobiotechnology. Recently, much work has focused on the synthesis and properties of graphene. Here we show that a successful reduction of graphene oxide (GO) using spinach leaf extract (SLE) as a simultaneous reducing and stabilizing agent. The as-prepared SLE-reduced graphene oxide (S-rGO) was characterized by ultraviolet–visible spectroscopy and Fourier transform infrared spectroscopy. Dynamic light scattering technique was used to determine the average size of GO and S-rGO. Scanning electron microscopy and atomic force microscopy images provide clear surface morphological evidence for the formation of graphene. The resulting S-rGO has a mostly single-layer structure, is stable, and has significant water solubility. In addition, the biocompatibility of graphene was investigated using cell viability, leakage of lactate dehydrogenase and alkaline phosphatase activity in primary mouse embryonic fibroblast (PMEFs) cells. The results suggest that the biologically synthesized graphene has significant biocompatibility with PMEF cells, even at a higher concentration of 100 μg/mL. This method uses a ‘green’, natural reductant and is free of additional stabilizing reagents; therefore, it is an environmentally friendly, simple, and cost-effective method for the fabrication of soluble graphene. This study could open up a promising view for substitution of hydrazine by a safe, biocompatible, and powerful reduction for the efficient deoxygenation of GO, especially in large-scale production and potential biomedical applications. PMID:24059222

Biocompatibility effects of biologically synthesized graphene in primary mouse embryonic fibroblast cells

NASA Astrophysics Data System (ADS)

Gurunathan, Sangiliyandi; Han, Jae Woong; Eppakayala, Vasuki; Dayem, Ahmed Abdal; Kwon, Deug-Nam; Kim, Jin-Hoi

2013-09-01

Due to unique properties and unlimited possible applications, graphene has attracted abundant interest in the areas of nanobiotechnology. Recently, much work has focused on the synthesis and properties of graphene. Here we show that a successful reduction of graphene oxide (GO) using spinach leaf extract (SLE) as a simultaneous reducing and stabilizing agent. The as-prepared SLE-reduced graphene oxide (S-rGO) was characterized by ultraviolet-visible spectroscopy and Fourier transform infrared spectroscopy. Dynamic light scattering technique was used to determine the average size of GO and S-rGO. Scanning electron microscopy and atomic force microscopy images provide clear surface morphological evidence for the formation of graphene. The resulting S-rGO has a mostly single-layer structure, is stable, and has significant water solubility. In addition, the biocompatibility of graphene was investigated using cell viability, leakage of lactate dehydrogenase and alkaline phosphatase activity in primary mouse embryonic fibroblast (PMEFs) cells. The results suggest that the biologically synthesized graphene has significant biocompatibility with PMEF cells, even at a higher concentration of 100 μg/mL. This method uses a `green', natural reductant and is free of additional stabilizing reagents; therefore, it is an environmentally friendly, simple, and cost-effective method for the fabrication of soluble graphene. This study could open up a promising view for substitution of hydrazine by a safe, biocompatible, and powerful reduction for the efficient deoxygenation of GO, especially in large-scale production and potential biomedical applications.

Quantitative and semi-quantitative immunoassay of growth factors and cytokines in the conditioned medium of STO and CF-1 mouse feeder cells

USDA-ARS?s Scientific Manuscript database

Feeder-cells of irradiated mouse fibroblasts are commonly used for, and are generally necessary for, the in vitro maintenance and growth of many fastidious cell types, particularly embryonic stem cells or induced pluripotent stem cells. Quantitative and semi-quantitative immunoassays were performed...

Electron microscopy and in vitro deneddylation reveal similar architectures and biochemistry of isolated human and Flag-mouse COP9 signalosome complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rockel, Beate; Schmaler, Tilo; Huang, Xiaohua

2014-07-25

Highlights: • Deneddylation rates of human erythrocyte and mouse fibroblast CSN are very similar. • 3D models of native human and mouse CSN reveal common architectures. • The cryo-structure of native mammalian CSN shows a horseshoe subunit arrangement. - Abstract: The COP9 signalosome (CSN) is a regulator of the ubiquitin (Ub) proteasome system (UPS). In the UPS, proteins are Ub-labeled for degradation by Ub ligases conferring substrate specificity. The CSN controls a large family of Ub ligases called cullin-RING ligases (CRLs), which ubiquitinate cell cycle regulators, transcription factors and DNA damage response proteins. The CSN possesses structural similarities with themore » 26S proteasome Lid complex and the translation initiation complex 3 (eIF3) indicating similar ancestry and function. Initial structures were obtained 14 years ago by 2D electron microscopy (EM). Recently, first 3D molecular models of the CSN were created on the basis of negative-stain EM and single-particle analysis, mostly with recombinant complexes. Here, we compare deneddylating activity and structural features of CSN complexes purified in an elaborate procedure from human erythrocytes and efficiently pulled down from mouse Flag-CSN2 B8 fibroblasts. In an in vitro deneddylation assay both the human and the mouse CSN complexes deneddylated Nedd8-Cul1 with comparable rates. 3D structural models of the erythrocyte CSN as well as of the mouse Flag-CSN were generated by negative stain EM and by cryo-EM. Both complexes show a central U-shaped segment from which several arms emanate. This structure, called the horseshoe, is formed by the PCI domain subunits. CSN5 and CSN6 point away from the horseshoe. Compared to 3D models of negatively stained CSN complexes, densities assigned to CSN2 and CSN4 are better defined in the cryo-map. Because biochemical and structural results obtained with CSN complexes isolated from human erythrocytes and purified by Flag-CSN pulldown from mouse B8 fibroblasts are very similar, Flag-CSN pulldowns are a proper alternative to CSN preparation from erythrocytes.« less

Scleral fibroblast response to experimental glaucoma in mice

PubMed Central

Tezel, Gülgün; Cone-Kimball, Elizabeth; Steinhart, Matthew R.; Jefferys, Joan; Pease, Mary E.; Quigley, Harry A.

2016-01-01

Purpose To study the detailed cellular and molecular changes in the mouse sclera subjected to experimental glaucoma. Methods Three strains of mice underwent experimental bead-injection glaucoma and were euthanized at 3 days and 1, 3, and 6 weeks. Scleral protein expression was analyzed with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using 16O/18O labeling for quantification in 1- and 6-week tissues. Sclera protein samples were also analyzed with immunoblotting with specific antibodies to selected proteins. The proportion of proliferating scleral fibroblasts was quantified with Ki67 and 4’,6-diamidino-2-phenylindole (DAPI) labeling, and selected proteins were studied with immunohistochemistry. Results Proteomic analysis showed increases in molecules involved in integrin-linked kinase signaling and actin cytoskeleton signaling pathways at 1 and 6 weeks after experimental glaucoma. The peripapillary scleral region had more fibroblasts than equatorial sclera (p=0.001, n=217, multivariable regression models). There was a sixfold increase in proliferating fibroblasts in the experimental glaucoma sclera at 1 week and a threefold rise at 3 and 6 weeks (p=0.0005, univariate regression). Immunoblots confirmed increases for myosin, spectrin, and actinin at 1 week after glaucoma. Thrombospondin-1 (TSP-1), HINT1, vimentin, actinin, and α-smooth muscle actin were increased according to immunohistochemistry. Conclusions Scleral fibroblasts in experimental mouse glaucoma show increases in actin cytoskeleton and integrin-related signaling, increases in cell division, and features compatible with myofibroblast transition. PMID:26900327

Vinpocetine Attenuates Pathological Cardiac Remodeling by Inhibiting Cardiac Hypertrophy and Fibrosis

PubMed Central

Wu, Mei-ping; Zhang, Yi-shuai; Xu, Xiangbin; Zhou, Qian

2017-01-01

Purpose Pathological cardiac remodeling, characterized by cardiac hypertrophy and fibrosis, is a pathological feature of many cardiac disorders that leads to heart failure and cardiac arrest. Vinpocetine, a derivative of the alkaloid vincamine, has been used for enhancing cerebral blood flow to treat cognitive impairment. However, its role in pathological cardiac remodeling remains unknown. The aim of this study is to examine the effect of vinpocetine on pathological cardiac remodeling induced by chronic stimulation with angiotensin II (Ang II). Methods Mice received Ang II infusion via osmotic pumps in the presence of vehicle or vinpocetine. Cardiac hypertrophy and fibrosis were assessed by morphological, histological, and biochemical analyses. Mechanistic studies were carried out in vitro with isolated mouse adult cardiac myocytes and fibroblasts. Results We showed that chronic Ang II infusion caused cardiac hypertrophy and fibrosis, which were all significantly attenuated by systemic administration of vinpocetine. In isolated adult mouse cardiomyocytes, vinpocetine suppressed Ang II-stimulated myocyte hypertrophic growth. In cultured cardiac fibroblasts, vinpocetine suppressed TGFβ-induced fibroblast activation and matrix gene expression, consistent with its effect in attenuating cardiac fibrosis. The effects of vinpocetine on cardiac myocyte hypertrophy and fibroblast activation are likely mediated by targeting cyclic nucleotide phosphodiesterase 1 (PDE1). Conclusions Our results reveal a novel protective effect of vinpocetine in attenuating pathological cardiac remodeling through suppressing cardiac myocyte hypertrophic growth and fibroblast activation and fibrotic gene expression. These studies may also shed light on developing novel therapeutic agents for antagonizing pathological cardiac remodeling. PMID:28321644

Vinpocetine Attenuates Pathological Cardiac Remodeling by Inhibiting Cardiac Hypertrophy and Fibrosis.

PubMed

Wu, Mei-Ping; Zhang, Yi-Shuai; Xu, Xiangbin; Zhou, Qian; Li, Jian-Dong; Yan, Chen

2017-04-01

Pathological cardiac remodeling, characterized by cardiac hypertrophy and fibrosis, is a pathological feature of many cardiac disorders that leads to heart failure and cardiac arrest. Vinpocetine, a derivative of the alkaloid vincamine, has been used for enhancing cerebral blood flow to treat cognitive impairment. However, its role in pathological cardiac remodeling remains unknown. The aim of this study is to examine the effect of vinpocetine on pathological cardiac remodeling induced by chronic stimulation with angiotensin II (Ang II). Mice received Ang II infusion via osmotic pumps in the presence of vehicle or vinpocetine. Cardiac hypertrophy and fibrosis were assessed by morphological, histological, and biochemical analyses. Mechanistic studies were carried out in vitro with isolated mouse adult cardiac myocytes and fibroblasts. We showed that chronic Ang II infusion caused cardiac hypertrophy and fibrosis, which were all significantly attenuated by systemic administration of vinpocetine. In isolated adult mouse cardiomyocytes, vinpocetine suppressed Ang II-stimulated myocyte hypertrophic growth. In cultured cardiac fibroblasts, vinpocetine suppressed TGFβ-induced fibroblast activation and matrix gene expression, consistent with its effect in attenuating cardiac fibrosis. The effects of vinpocetine on cardiac myocyte hypertrophy and fibroblast activation are likely mediated by targeting cyclic nucleotide phosphodiesterase 1 (PDE1). Our results reveal a novel protective effect of vinpocetine in attenuating pathological cardiac remodeling through suppressing cardiac myocyte hypertrophic growth and fibroblast activation and fibrotic gene expression. These studies may also shed light on developing novel therapeutic agents for antagonizing pathological cardiac remodeling.

PDGFRα plays a crucial role in connective tissue remodeling.

PubMed

Horikawa, Shinjiro; Ishii, Yoko; Hamashima, Takeru; Yamamoto, Seiji; Mori, Hisashi; Fujimori, Toshihiko; Shen, Jie; Inoue, Ran; Nishizono, Hirofumi; Itoh, Hiroshi; Majima, Masataka; Abraham, David; Miyawaki, Toshio; Sasahara, Masakiyo

2015-12-07

Platelet derived growth factor (PDGF) plays a pivotal role in the remodeling of connective tissues. Emerging data indicate the distinctive role of PDGF receptor-α (PDGFRα) in this process. In the present study, the Pdgfra gene was systemically inactivated in adult mouse (α-KO mouse), and the role of PDGFRα was examined in the subcutaneously implanted sponge matrices. PDGFRα expressed in the fibroblasts of Pdgfra-preserving control mice (Flox mice), was significantly reduced in the sponges in α-KO mice. Neovascularized areas were largely suppressed in the α-KO mice than in the Flox mice, whereas the other parameters related to the blood vessels and endothelial cells were similar. The deposition of collagen and fibronectin and the expression of collagen 1a1 and 3a1 genes were significantly reduced in α-KO mice. There was a significantly decrease in the number and dividing fibroblasts in the α-KO mice, and those of macrophages were similar between the two genotypes. Hepatocyte growth factor (Hgf) gene expression was suppressed in Pdgfra-inactivated fibroblasts and connective tissue. The findings implicate the role of PDGFRα-dependent ECM and HGF production in fibroblasts that promotes the remodeling of connective tissue and suggest that PDGFRα may be a relevant target to regulate connective tissue remodeling.

PDGFRα plays a crucial role in connective tissue remodeling

PubMed Central

Horikawa, Shinjiro; Ishii, Yoko; Hamashima, Takeru; Yamamoto, Seiji; Mori, Hisashi; Fujimori, Toshihiko; Shen, Jie; Inoue, Ran; Nishizono, Hirofumi; Itoh, Hiroshi; Majima, Masataka; Abraham, David; Miyawaki, Toshio; Sasahara, Masakiyo

2015-01-01

Platelet derived growth factor (PDGF) plays a pivotal role in the remodeling of connective tissues. Emerging data indicate the distinctive role of PDGF receptor-α (PDGFRα) in this process. In the present study, the Pdgfra gene was systemically inactivated in adult mouse (α-KO mouse), and the role of PDGFRα was examined in the subcutaneously implanted sponge matrices. PDGFRα expressed in the fibroblasts of Pdgfra-preserving control mice (Flox mice), was significantly reduced in the sponges in α-KO mice. Neovascularized areas were largely suppressed in the α-KO mice than in the Flox mice, whereas the other parameters related to the blood vessels and endothelial cells were similar. The deposition of collagen and fibronectin and the expression of collagen 1a1 and 3a1 genes were significantly reduced in α-KO mice. There was a significantly decrease in the number and dividing fibroblasts in the α-KO mice, and those of macrophages were similar between the two genotypes. Hepatocyte growth factor (Hgf) gene expression was suppressed in Pdgfra-inactivated fibroblasts and connective tissue. The findings implicate the role of PDGFRα-dependent ECM and HGF production in fibroblasts that promotes the remodeling of connective tissue and suggest that PDGFRα may be a relevant target to regulate connective tissue remodeling. PMID:26639755

Medicinal flowers. XXVII. New flavanone and chalcone glycosides, arenariumosides I, II, III, and IV, and tumor necrosis factor-alpha inhibitors from everlasting, flowers of Helichrysum arenarium.

PubMed

Morikawa, Toshio; Wang, Li-Bo; Nakamura, Seikou; Ninomiya, Kiyofumi; Yokoyama, Eri; Matsuda, Hisashi; Muraoka, Osamu; Wu, Li-Jun; Yoshikawa, Masayuki

2009-04-01

The methanolic extract from the flowers of Helichrysum arenarium L. MOENCH was found to show inhibitory effect on tumor necrosis factor-alpha (TNF-alpha, 1 ng/ml)-induced cytotoxicity in L929 cells. From the methanolic extract, 50 constituents including four new flavanone and chalcone glycosides named arenariumosides I (1), II (2), III (3), and IV (4) were isolated. The stereostructures of 1-4 were elucidated on the basis of chemical and physicochemical evidence. Among the constituents, naringenin 7-O-beta-D-glucopyranoside (7), apigenin 7-O-beta-D-glucopyranoside (14), apigenin 7-O-gentiobioside (16), and apigenin 7,4'-di-O-beta-D-glucopyranoside (17) significantly inhibited TNF-alpha-induced cytotoxicity in L929 cells at 30 microM.

Limited mutagenicity of electronic cigarettes in mouse or human cells in vitro.

PubMed

Tommasi, Stella; Bates, Steven E; Behar, Rachel Z; Talbot, Prue; Besaratinia, Ahmad

2017-10-01

Electronic cigarettes (e-cig), which are promoted as safe alternatives to tobacco cigarettes or as aides to smoking cessation, are becoming increasingly popular among adult chronic smokers and adolescents experimenting with tobacco products. Despite the known presence of toxicants and carcinogens in e-cig liquid and vapor, the possible carcinogenic effects of e-cig use in humans are unknown. We have utilized two validated in vitro model systems to investigate whether e-cig vapor induces mutation in mouse or human cells. We have exposed transgenic mouse fibroblasts in vitro to e-cig vapor extracts prepared from three popular brands, and determined the induction of mutagenesis in a reporter gene, the cII transgene. Furthermore, we have treated the pSP189 plasmid with e-cig vapor extract, transfected human fibroblast cells with the e-cig-treated plasmid, and screened for the induced mutations in the supF gene. We observed no statistically significant increases in relative mutant frequency in the cII transgene or supF gene in the e-cig treated mouse or human cells, respectively. Our data indicate that e-cig vapor extracts from the selected brands and at concentrations tested in this study have limited mutagenicity in both mouse and human cells in vitro. Copyright © 2017 Elsevier B.V. All rights reserved.

Merging bioreactor technology with 3D hepatocyte-fibroblast culturing approaches: Improved in vitro models for toxicological applications.

PubMed

Leite, Sofia B; Teixeira, Ana P; Miranda, Joana P; Tostões, Rui M; Clemente, João J; Sousa, Marcos F; Carrondo, Manuel J T; Alves, Paula M

2011-06-01

During the last years an increasing number of in vitro models have been developed for drug screening and toxicity testing. Primary cultures of hepatocytes are, by far, the model of choice for those high-throughput studies but their spontaneous dedifferentiation after some time in culture hinders long-term studies. Thus, novel cell culture systems allowing extended hepatocyte maintenance and more predictive long term in vitro studies are required. It has been shown that hepatocytes functionality can be improved and extended in time when cultured as 3D-cell aggregates in environmental controlled stirred bioreactors. In this work, aiming at further improving hepatocytes functionality in such 3D cellular structures, co-cultures with fibroblasts were performed. An inoculum concentration of 1.2×10(5) cell/mL and a 1:2 hepatocyte:mouse embryonic fibroblast ratio allowed to improve significantly the albumin secretion rate and both ECOD (phase I) and UGT (phase II) enzymatic activities in 3D co-cultures, as compared to the routinely used 2D hepatocyte monocultures. Significant improvements were also observed in relation to 3D monocultures of hepatocytes. Furthermore, hepatocytes were able to respond to the addition of beta-Naphtoflavone by increasing ECOD activity showing CYP1A inducibility. The dependence of CYP activity on oxygen concentration was also observed. In summary, the improved hepatocyte specific functions during long term incubation of 3D co-cultures of hepatocytes with fibroblasts indicate that this system is a promising in vitro model for long term toxicological studies. Copyright © 2011 Elsevier Ltd. All rights reserved.

Bioglass Activated Skin Tissue Engineering Constructs for Wound Healing.

PubMed

Yu, Hongfei; Peng, Jinliang; Xu, Yuhong; Chang, Jiang; Li, Haiyan

2016-01-13

Wound healing is a complicated process, and fibroblast is a major cell type that participates in the process. Recent studies have shown that bioglass (BG) can stimulate fibroblasts to secrete a multitude of growth factors that are critical for wound healing. Therefore, we hypothesize that BG can stimulate fibroblasts to have a higher bioactivity by secreting more bioactive growth factors and proteins as compared to untreated fibroblasts, and we aim to construct a bioactive skin tissue engineering graft for wound healing by using BG activated fibroblast sheet. Thus, the effects of BG on fibroblast behaviors were studied, and the bioactive skin tissue engineering grafts containing BG activated fibroblasts were applied to repair the full skin lesions on nude mouse. Results showed that BG stimulated fibroblasts to express some critical growth factors and important proteins including vascular endothelial growth factor, basic fibroblast growth factor, epidermal growth factor, collagen I, and fibronectin. In vivo results revealed that fibroblasts in the bioactive skin tissue engineering grafts migrated into wound bed, and the migration ability of fibroblasts was stimulated by BG. In addition, the bioactive BG activated fibroblast skin tissue engineering grafts could largely increase the blood vessel formation, enhance the production of collagen I, and stimulate the differentiation of fibroblasts into myofibroblasts in the wound site, which would finally accelerate wound healing. This study demonstrates that the BG activated skin tissue engineering grafts contain more critical growth factors and extracellular matrix proteins that are beneficial for wound healing as compared to untreated fibroblast cell sheets.

Generation of neural progenitor cells by chemical cocktails and hypoxia

PubMed Central

Cheng, Lin; Hu, Wenxiang; Qiu, Binlong; Zhao, Jian; Yu, Yongchun; Guan, Wuqiang; Wang, Min; Yang, Wuzhou; Pei, Gang

2014-01-01

Neural progenitor cells (NPCs) can be induced from somatic cells by defined factors. Here we report that NPCs can be generated from mouse embryonic fibroblasts by a chemical cocktail, namely VCR (V, VPA, an inhibitor of HDACs; C, CHIR99021, an inhibitor of GSK-3 kinases and R, Repsox, an inhibitor of TGF-β pathways), under a physiological hypoxic condition. These chemical-induced NPCs (ciNPCs) resemble mouse brain-derived NPCs regarding their proliferative and self-renewing abilities, gene expression profiles, and multipotency for different neuroectodermal lineages in vitro and in vivo. Further experiments reveal that alternative cocktails with inhibitors of histone deacetylation, glycogen synthase kinase, and TGF-β pathways show similar efficacies for ciNPC induction. Moreover, ciNPCs can also be induced from mouse tail-tip fibroblasts and human urinary cells with the same chemical cocktail VCR. Thus our study demonstrates that lineage-specific conversion of somatic cells to NPCs could be achieved by chemical cocktails without introducing exogenous factors. PMID:24638034

Role of FGF/FGFR signaling in skeletal development and homeostasis: learning from mouse models

PubMed Central

Su, Nan; Jin, Min; Chen, Lin

2014-01-01

Fibroblast growth factor (FGF)/fibroblast growth factor receptor (FGFR) signaling plays essential roles in bone development and diseases. Missense mutations in FGFs and FGFRs in humans can cause various congenital bone diseases, including chondrodysplasia syndromes, craniosynostosis syndromes and syndromes with dysregulated phosphate metabolism. FGF/FGFR signaling is also an important pathway involved in the maintenance of adult bone homeostasis. Multiple kinds of mouse models, mimicking human skeleton diseases caused by missense mutations in FGFs and FGFRs, have been established by knock-in/out and transgenic technologies. These genetically modified mice provide good models for studying the role of FGF/FGFR signaling in skeleton development and homeostasis. In this review, we summarize the mouse models of FGF signaling-related skeleton diseases and recent progresses regarding the molecular mechanisms, underlying the role of FGFs/FGFRs in the regulation of bone development and homeostasis. This review also provides a perspective view on future works to explore the roles of FGF signaling in skeletal development and homeostasis. PMID:26273516

Fibroblast-matrix interplay: Nintedanib and pirfenidone modulate the effect of IPF fibroblast-conditioned matrix on normal fibroblast phenotype.

PubMed

Epstein Shochet, Gali; Wollin, Lutz; Shitrit, David

2018-03-12

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. Activated fibroblasts are the key effector cells in fibrosis, producing excessive amounts of collagen and extracellular matrix (ECM) proteins. Whether the ECM conditioned by IPF fibroblasts determines the phenotype of naïve fibroblasts is difficult to explore. IPF-derived primary fibroblasts were cultured on Matrigel and then cleared using ammonium hydroxide, creating an IPF-conditioned matrix (CM). Normal fibroblast CM served as control. Normal fibroblasts were cultured on both types of CM, and cell count, cell distribution and markers of myofibroblast differentiation; transforming growth factor beta (TGFβ) signalling; and ECM expression were assessed. The effects of the anti-fibrotic drugs nintedanib and pirfenidone at physiologically relevant concentrations were also explored. Normal fibroblasts cultured on IPF-CM arranged in large aggregates as a result of increased proliferation and migration. Moreover, increased levels of pSmad3, pSTAT3 (phospho signal transducer and activator of transcription 3), alpha smooth muscle actin (αSMA) and Collagen1a were found, suggesting a differentiation towards a myofibroblast-like phenotype. SB505124 (10 μmol/L) partially reversed these alterations, suggesting a TGFβ contribution. Furthermore, nintedanib at 100 nmol/L and, to a lesser extent, pirfenidone at 100 μmol/L prevented the IPF-CM-induced fibroblast phenotype alterations, suggesting an attenuation of the ECM-fibroblast interplay. IPF fibroblasts alter the ECM, thus creating a CM that further propagates an IPF-like phenotype in normal fibroblasts. This assay demonstrated differences in drug activities for approved IPF drugs at clinically relevant concentrations. Thus, the matrix-fibroblast phenotype interplay might be a relevant assay to explore drug candidates for IPF treatment. © 2018 Asian Pacific Society of Respirology.

Cell-Cell Communication Between Fibroblast and 3T3-L1 Cells Under Co-culturing in Oxidative Stress Condition Induced by H2O2.

PubMed

Subramaniyan, Sivakumar Allur; Kim, Sidong; Hwang, Inho

2016-10-01

The present study was carried out to understand the interaction between fibroblast and 3T3-L1 preadipocyte cells under H 2 O 2 -induced oxidative stress condition. H 2 O 2 (40 μM) was added in co-culture and monoculture of fibroblast and 3T3-L1 cell. The cells in the lower well were harvested for analysis and the process was carried out for both cells. The cell growth, oxidative stress markers, and antioxidant enzymes were analyzed. Additionally, the mRNA expressions of caspase-3 and caspase-7 were selected for analysis of apoptotic pathways and TNF-α and NF-κB were analyzed for inflammatory pathways. The adipogenic marker such as adiponectin and PPAR-γ and collagen synthesis markers such as LOX and BMP-1 were analyzed in the co-culture of fibroblast and 3T3-L1 cells. Cell viability and antioxidant enzymes were significantly increased in the co-culture compared to the monoculture under stress condition. The apoptotic, inflammatory, adipogenic, and collagen-synthesized markers were significantly altered in H 2 O 2 -induced co-culture of fibroblast and 3T3-L1 cells when compared with the monoculture of H 2 O 2 -induced fibroblast and 3T3-L1 cells. In addition, the confocal microscopical investigation indicated that the co-culture of H 2 O 2 -induced 3T3-L1 and fibroblast cells increases collagen type I and type III expression. From our results, we suggested that co-culture of fat cell (3T3-L1) and fibroblast cells may influence/regulate each other and made the cells able to withstand against oxidative stress and aging. It is conceivable that the same mechanism might have been occurring from cell to cell while animals are stressed by various environmental conditions.

Therapeutic potential of trichostatin A to control inflammatory and fibrogenic disorders of the ocular surface.

PubMed

Kitano, Ai; Okada, Yuka; Yamanka, Osamu; Shirai, Kumi; Mohan, Rajiv R; Saika, Shizuya

2010-12-31

To examine the effects of a histone deacetylase inhibitor, Trichostatin A (TSA), on the behavior of macrophages and subconjunctival fibroblasts in vitro and on ocular surface inflammation and scarring in vivo using an alkali burn wound healing model. Effects of TSA on expression of inflammation-related growth factors or collagen I were examined by real-time RT-PCR or immunoassay in mouse macrophages or human subconjunctival fibroblasts. Effects of TSA on trans forming growth factor β (TGFβ)/Smad signaling were evaluated with western blotting and/or immunocytochemistry. Alkali-burn injuries on the eyes of mice were performed with three µl of 0.5 N NaOH under general and topical anesthesia. TSA (600 µg/Kg daily) or vehicle was administered to animals via intraperitoneal (i.p.) injection. Histology and real-time RT-PCR investigations evaluated the effects of TSA on the healing process of the cornea. TSA inhibited TGFβ 1 and vascular endothelial growth factor (VEGF) expression in macrophages, and TGFβ1 and collagen I in ocular fibroblasts. It elevated the expression of 5'-TG-3'-interacting factor (TGIF) and Smad7 in fibroblasts and blocked nuclear translocation of phospho-Smad2. Real-time PCR and immunocytochemistry studies showed that systemic administration of TSA suppressed the inflammation and fibrotic response in the stroma and accelerated epithelial healing in the alkali-burned mouse cornea. Systemic administration of TSA reduces inflammatory and fibrotic responses in the alkali-burned mouse ocular surface in vivo. The mechanisms of action involve attenuation of Smad signal in mesenchymal cells and reduction in the activation and recruitment of macrophages. TSA has the potential to treat corneal scarring in vivo.

Absence of DNA damage after 60-Hz electromagnetic field exposure combined with ionizing radiation, hydrogen peroxide, or c-Myc overexpression.

PubMed

Jin, Yeung Bae; Choi, Seo-Hyun; Lee, Jae Seon; Kim, Jae-Kyung; Lee, Ju-Woon; Hong, Seung-Cheol; Myung, Sung Ho; Lee, Yun-Sil

2014-03-01

The principal objective of this study was to assess the DNA damage in a normal cell line system after exposure to 60 Hz of extremely low frequency magnetic field (ELF-MF) and particularly in combination with various external factors, via comet assays. NIH3T3 mouse fibroblast cells, WI-38 human lung fibroblast cells, L132 human lung epithelial cells, and MCF10A human mammary gland epithelial cells were exposed for 4 or 16 h to a 60-Hz, 1 mT uniform magnetic field in the presence or absence of ionizing radiation (IR, 1 Gy), H(2)O(2) (50 μM), or c-Myc oncogenic activation. The results obtained showed no significant differences between the cells exposed to ELF-MF alone and the unexposed cells. Moreover, no synergistic or additive effects were observed after 4 or 16 h of pre-exposure to 1 mT ELF-MF or simultaneous exposure to ELF-MF combined with IR, H(2)O(2), or c-Myc activation.

N-(3-Oxo-acyl)-homoserine lactone induces apoptosis primarily through a mitochondrial pathway in fibroblasts.

PubMed

Neely, Aaron M; Zhao, Guoping; Schwarzer, Christian; Stivers, Nicole S; Whitt, Aaron G; Meng, Shuhan; Burlison, Joseph A; Machen, Terry E; Li, Chi

2018-01-01

N-(3-Oxododecanoyl)-l-homoserine lactone (C12) is produced by Pseudomonas aeruginosa to function as a quorum-sensing molecule for bacteria-bacteria communication. C12 is also known to influence many aspects of human host cell physiology, including induction of cell death. However, the signalling pathway(s) leading to C12-triggered cell death is (are) still not completely known. To clarify cell death signalling induced by C12, we examined mouse embryonic fibroblasts deficient in "initiator" caspases or "effector" caspases. Our data indicate that C12 selectively induces the mitochondria-dependent intrinsic apoptotic pathway by quickly triggering mitochondrial outer membrane permeabilisation. Importantly, the activities of C12 to permeabilise mitochondria are independent of activation of both "initiator" and "effector" caspases. Furthermore, C12 directly induces mitochondrial outer membrane permeabilisation in vitro. Overall, our study suggests a mitochondrial apoptotic signalling pathway triggered by C12, in which C12 or its metabolite(s) acts on mitochondria to permeabilise mitochondria, leading to activation of apoptosis. © 2017 John Wiley & Sons Ltd.

A new inhibitor of synovial phospholipase A2 from fermentations of Penicillium sp. 62-92.

PubMed

Witter, L; Anke, T; Sterner, O

1998-01-01

Penidiamide, a new tripetide containing dehydrotryptamine, glycine and anthranilic acid linked together by two amide bonds, and oxindole were isolated from submerged cultures of Penicillium sp. 62-92. Both compounds preferentially inhibited human synovial phospholipase A2, penidiamide with an IC50 of 30 microM and oxindole of 380 microM. With the exception of U 937 cells (leukemia, human), no cytotoxic activities were detected against HL-60- (leukemia, human), HeLa S3- (epitheloid carcinoma, human), BHK 21- (kidney fibroblasts, hamster), and L1210-cells (leukemia, mouse). No antimicrobial activity was detected for oxindole, and only weak antibacterial activity for penidiamide. The structure of penidiamide was elucidated by spectroscopic methods.

Idiopathic pulmonary fibrosis fibroblasts become resistant to Fas ligand-dependent apoptosis via the alteration of decoy receptor 3.

PubMed

Im, Jintaek; Kim, Kyutae; Hergert, Polla; Nho, Richard Seonghun

2016-09-01

Idiopathic pulmonary fibrosis (IPF) is an irreversible lethal lung disease with an unknown etiology. IPF patients' lung fibroblasts express inappropriately high Akt activity, protecting them in response to an apoptosis-inducing type I collagen matrix. FasL, a ligand for Fas, is known to be increased in the lung tissues of patients with IPF, implicated with the progression of IPF. Expression of Decoy Receptor3 (DcR3), which binds to FasL, thereby subsequently suppressing the FasL-Fas-dependent apoptotic pathway, is frequently altered in various human disease. However, the role of DcR3 in IPF fibroblasts in regulating their viability has not been examined. We found that enhanced DcR3 expression exists in the majority of IPF fibroblasts on collagen matrices, resulting in the protection of IPF fibroblasts from FasL-induced apoptosis. Abnormally high Akt activity suppresses GSK-3β function, thereby accumulating the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) in the nucleus, increasing DcR3 expression in IPF fibroblasts. This alteration protects IPF cells from FasL-induced apoptosis on collagen. However, the inhibition of Akt or NFATc1 decreases DcR3 mRNA and protein levels, which sensitizes IPF fibroblasts to FasL-mediated apoptosis. Furthermore, enhanced DcR3 and NFATc1 expression is mainly present in myofibroblasts in the fibroblastic foci of lung tissues derived from IPF patients. Our results showed that when IPF cells interact with collagen matrix, aberrantly activated Akt increases DcR3 expression via GSK-3β-NFATc1 and protects IPF cells from the FasL-dependent apoptotic pathway. These findings suggest that the inhibition of DcR3 function may be an effective approach for sensitizing IPF fibroblasts in response to FasL, limiting the progression of lung fibrosis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Use of Disposable Micro Tissue Culture Plates for Antiviral and Interferon Induction Studies

PubMed Central

Sidwell, Robert W.; Huffman, John H.

1971-01-01

A reproducible test system requiring small amounts of test compound was developed for evaluating antiviral and interferon-inducing activity. In the antiviral experiments, KB cells were grown in disposable polystyrene microplates covered with a standard domestic plastic wrap. Viruses used in the system were types 1 and 2 herpes simplex virus, vaccinia virus, type 3 adenovirus, myxoma virus, pseudorabies virus, type 3 parainfluenza virus, types 1A and 13 rhinovirus, vesicular stomatitis virus, coxsackievirus B, and type 2 poliovirus. Inhibition of viral cytopathogenic effect was the primary criterion of evaluation of antiviral activity. Reduction in cell and supernatant fluid virus titers was used as a secondary means of evaluation. The microplate system was adaptable for determining prophylactic, therapeutic, and inactivating effects against viruses. Mouse L-929 cells were used for the interferon induction studies, with vesicular stomatitis virus utilized as the indicator of interferon activity. Known active compounds evaluated in this microplate system had activity similar to that seen in macro in vitro systems. PMID:4332040

Cross-species functional analysis of cancer-associated fibroblasts identifies a critical role for CLCF1 and IL6 in non-small cell lung cancer in vivo

PubMed Central

Vicent, Silvestre; Sayles, Leanne C.; Vaka, Dedeepya; Khatri, Purvesh; Gevaert, Olivier; Chen, Ron; Zheng, Yanyan; Gillespie, Anna K.; Clarke, Nicole; Xu, Yue; Shrager, Joseph; Hoang, Chuong D.; Plevritis, Sylvia; Butte, Atul J.; Sweet-Cordero, E. Alejandro

2013-01-01

Cancer-associated fibroblasts (CAFs) have been reported to support tumor progression by a variety of mechanisms. However, their role in the progression of non-small cell lung cancer (NSCLC) remains poorly defined. In addition, the extent to which specific proteins secreted by CAFs contribute directly to tumor growth is unclear. To study the role of CAFs in NSCLC, a cross-species functional characterization of mouse and human lung CAFs was performed. CAFs supported the growth of lung cancer cells in vivo by secretion of soluble factors that directly stimulate the growth of tumor cells. Gene expression analysis comparing normal mouse lung fibroblasts (NFs) and mouse lung CAFs identified multiple genes that correlate with the CAF phenotype. A gene signature of secreted genes upregulated in CAFs was an independent marker of poor survival in NSCLC patients. This secreted gene signature was upregulated in NFs after long-term exposure to tumor cells, demonstrating that NFs are “educated” by tumor cells to acquire a CAF-like phenotype. Functional studies identified important roles for CLCF1-CNTFR and IL6-IL6R signaling, in promoting growth of NSCLC cells. This study identifies novel soluble factors contributing to the CAF protumorigenic phenotype in NSCLC and suggests new avenues for the development of therapeutic strategies. PMID:22962265

Tyrosine kinase inhibitor BIBF1120 ameliorates inflammation, angiogenesis and fibrosis in CCl4-induced liver fibrogenesis mouse model

PubMed Central

Öztürk Akcora, Büsra; Storm, Gert; Prakash, Jai; Bansal, Ruchi

2017-01-01

Hepatic fibrosis, a progressive chronic disease mainly caused by hepatitis viral infections, alcohol abuse or metabolic syndrome leading to liver dysfunction and is the growing cause of mortality worldwide. Tyrosine kinase inhibitor BIBF1120 (Nintedanib) has been evaluated in clinical trials for idiopathic pulmonary fibrosis and advanced Hepatocellular carcinoma, but has not been explored for liver fibrosis yet. In this study, we aimed to investigate the therapeutic effects and mechanism of BIBF1120 in liver fibrogenesis. The effects of BIBF1120 were evaluated in TGFβ-activated mouse 3T3 fibroblasts, LX2 cells, primary human hepatic stellate cells (HSCs) and CCl4-induced liver fibrogenesis mouse model. Fibroblasts-conditioned medium studies were performed to assess the paracrine effects on macrophages and endothelial cells. In-vitro in TGFβ-activated fibroblasts, BIBF1120 significantly inhibited expression of major fibrotic parameters, wound-healing and contractility. In vivo in CCl4-induced acute liver injury model, post-disease BIBF1120 administration significantly attenuated collagen accumulation and HSC activation. Interestingly, BIBF1120 drastically inhibited intrahepatic inflammation and angiogenesis. To further elucidate the mechanism of action, 3T3-conditioned medium studies demonstrated increased 3T3-mediated macrophage chemotaxis and endothelial cells tube formation and activation, which was significantly decreased by BIBF1120. These results suggests that BIBF1120 can be a potential therapeutic approach for the treatment of liver fibrosis. PMID:28291245

Effects of exposure to DAMPS and GSM signals on ornithine decarboxylase (ODC) activity: II. SH-SY5Y human neuroblastoma cells.

PubMed

Billaudel, Bernard; Taxile, Murielle; Poulletier de Gannes, Florence; Ruffie, Gilles; Lagroye, Isabelle; Veyret, Bernard

2009-06-01

An increase in Ornithine Decarboxylase (ODC) activity was reported in L929 murine fibroblast cells after exposure to a digital cellular telephone signal. This result was not confirmed by several other studies, including the one reported in a companion paper. As a partner in the Perform-B programme, we extended this study to human neuroblastoma cells (SH-SY5Y), using well-defined waveguide systems to imitate exposure to radiofrequency radiation (RFR): Digital Advanced Mobile Phone System (DAMPS) or Global System for Mobile communications (GSM) signals emitted by mobile phones. Human neuroblastoma cells (SH-SY5Y) were exposed at various Specific Absorption Rates (SAR) to DAMPS or GSM signals using different set-ups. Cell ODC activities were assayed using 14CO2 generation from 14C-labeled L-ornithine. SH-SY5Y cells were incubated for 20 hours, and were blindly exposed to 50 Hz-modulated DAMPS-835 or 217 Hz-modulated GSM-1800 for 8 or 24 h using Information Technologies in Society (IT'IS) waveguides equipped with fans. After cell lysis, ODC activity was determined using 14C-labeled L-ornithine. ODC activity was estimated by the 14CO2 generated from 14C-labeled L-ornithine, as generated d.p.m. 14CO2/h/mg protein. The results showed that, irrespective of the signal used (835 MHz/DAMPS, or 1800 MHz/GSM) and exposure conditions (duration and SAR), human SH-SY5Y neuroblastoma cells did not exhibit any alteration in ODC enzyme activity. This work did not show a significant effect of mobile phone RFR exposure on ODC activity in neuroblastoma cells (SH-SY5Y).

Bactobolin resistance is conferred by mutations in the L2 ribosomal protein.

PubMed

Chandler, Josephine R; Truong, Thao T; Silva, Patricia M; Seyedsayamdost, Mohammad R; Carr, Gavin; Radey, Matthew; Jacobs, Michael A; Sims, Elizabeth H; Clardy, Jon; Greenberg, E Peter

2012-12-18

Burkholderia thailandensis produces a family of polyketide-peptide molecules called bactobolins, some of which are potent antibiotics. We found that growth of B. thailandensis at 30°C versus that at 37°C resulted in increased production of bactobolins. We purified the three most abundant bactobolins and determined their activities against a battery of bacteria and mouse fibroblasts. Two of the three compounds showed strong activities against both bacteria and fibroblasts. The third analog was much less potent in both assays. These results suggested that the target of bactobolins might be conserved across bacteria and mammalian cells. To learn about the mechanism of bactobolin activity, we isolated four spontaneous bactobolin-resistant Bacillus subtilis mutants. We used genomic sequencing technology to show that each of the four resistant variants had mutations in rplB, which codes for the 50S ribosome-associated L2 protein. Ectopic expression of a mutant rplB gene in wild-type B. subtilis conferred bactobolin resistance. Finally, the L2 mutations did not confer resistance to other antibiotics known to interfere with ribosome function. Our data indicate that bactobolins target the L2 protein or a nearby site and that this is not the target of other antibiotics. We presume that the mammalian target of bactobolins involves the eukaryotic homolog of L2 (L8e). Currently available antibiotics target surprisingly few cellular functions, and there is a need to identify novel antibiotic targets. We have been interested in the Burkholderia thailandensis bactobolins, and we sought to learn about the target of bactobolin activity by mapping spontaneous resistance mutations in the bactobolin-sensitive Bacillus subtilis. Our results indicate that the bactobolin target is the 50S ribosome-associated L2 protein or a region of the ribosome affected by L2. Bactobolin-resistant mutants are not resistant to other known ribosome inhibitors. Our evidence indicates that bactobolins interact with a novel antibiotic target.

Nickel inhibits mitochondrial fatty acid oxidation.

PubMed

Uppala, Radha; McKinney, Richard W; Brant, Kelly A; Fabisiak, James P; Goetzman, Eric S

2015-08-07

Nickel exposure is associated with changes in cellular energy metabolism which may contribute to its carcinogenic properties. Here, we demonstrate that nickel strongly represses mitochondrial fatty acid oxidation-the pathway by which fatty acids are catabolized for energy-in both primary human lung fibroblasts and mouse embryonic fibroblasts. At the concentrations used, nickel suppresses fatty acid oxidation without globally suppressing mitochondrial function as evidenced by increased glucose oxidation to CO2. Pre-treatment with l-carnitine, previously shown to prevent nickel-induced mitochondrial dysfunction in neuroblastoma cells, did not prevent the inhibition of fatty acid oxidation. The effect of nickel on fatty acid oxidation occurred only with prolonged exposure (>5 h), suggesting that direct inhibition of the active sites of metabolic enzymes is not the mechanism of action. Nickel is a known hypoxia-mimetic that activates hypoxia inducible factor-1α (HIF1α). Nickel-induced inhibition of fatty acid oxidation was blunted in HIF1α knockout fibroblasts, implicating HIF1α as one contributor to the mechanism. Additionally, nickel down-regulated the protein levels of the key fatty acid oxidation enzyme very long-chain acyl-CoA dehydrogenase (VLCAD) in a dose-dependent fashion. In conclusion, inhibition of fatty acid oxidation by nickel, concurrent with increased glucose metabolism, represents a form of metabolic reprogramming that may contribute to nickel-induced carcinogenesis. Copyright © 2015 Elsevier Inc. All rights reserved.

Role of mannose-6-phosphate receptors in herpes simplex virus entry into cells and cell-to-cell transmission.

PubMed Central

Brunetti, C R; Burke, R L; Hoflack, B; Ludwig, T; Dingwell, K S; Johnson, D C

1995-01-01

Herpes simplex virus (HSV) glycoprotein D (gD) is essential for virus entry into cells, is modified with mannose-6-phosphate (M-6-P), and binds to both the 275-kDa M-6-P receptor (MPR) and the 46-kDa MPR (C. R. Brunetti, R. L. Burke, S. Kornfeld, W. Gregory, K. S. Dingwell, F. Masiarz, and D. C. Johnson, J. Biol. Chem. 269:17067-17074, 1994). Since MPRs are found on the surfaces of mammalian cells, we tested the hypothesis that MPRs could serve as receptors for HSV during virus entry into cells. A soluble form of the 275-kDa MPR, derived from fetal bovine serum, inhibited HSV plaques on monkey Vero cells, as did polyclonal rabbit anti-MPR antibodies. In addition, the number and size of HSV plaques were reduced when cells were treated with bovine serum albumin conjugated with pentamannose-phosphate (PM-PO4-BSA), a bulky ligand which can serve as a high-affinity ligand for MPRs. These data imply that HSV can use MPRs to enter cells; however, other molecules must also serve as receptors for HSV because a reasonable fraction of virus could enter cells treated with even the highest concentrations of these inhibitors. Consistent with the possibility that there are other receptors, HSV produced the same number of plaques on MPR-deficient mouse fibroblasts as were produced on normal mouse fibroblasts, but there was no inhibition with PM-PO4-BSA with either of these embryonic mouse cells. Together, these results demonstrate that HSV does not rely solely on MPRs to enter cells, although MPRs apparently play some role in virus entry into some cell types and, perhaps, act as one of a number of cell surface molecules that can facilitate entry. We also found that HSV produced small plaques on human fibroblasts derived from patients with pseudo-Hurler's polydystrophy, cells in which glycoproteins are not modified with M-6-P residues and yet production of infectious HSV particles was not altered in the pseudo-Hurler cells. In addition, HSV plaque size was reduced by PM-PO4-BSA; therefore, it appears that M-6-P residues and MPRs are required for efficient transmission of HSV between cells, a process which differs in some respects from entry of exogenous virus particles. PMID:7745699

Ligand-induced adhesion to activated endothelium and to vascular cell adhesion molecule-1 in lymphocytes transfected with the N-formyl peptide receptor.

PubMed

Honda, S; Campbell, J J; Andrew, D P; Engelhardt, B; Butcher, B A; Warnock, R A; Ye, R D; Butcher, E C

1994-04-15

Binding of FMLP to the neutrophil N-formyl peptide receptor (FPR) transmits signals through pertussis toxin-sensitive G proteins triggering Ca2+ flux, superoxide production, granule exocytosis, and neutrophil aggregation and adhesion involving the beta 2 (CD18) integrins. Expression of the FPR in mouse fibroblasts or human kidney cells has been shown to confer an N-formyl peptide-inducible Ca2+ flux in transfectants. Here we demonstrate that the transfected receptor can also support ligand-induced alterations in cellular adhesion. We established stable transfectants of mouse L1-2 pre-B cells with cDNA for human FPR (L1-2 FPR cells). The transfectants bind N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein with 1.4 x 10(5) sites per cell and a dissociation constant of 3.3 nM. Stimulation with FMLP induces a transient Ca2+ flux. FMLP also triggers adhesion of L1-2 FPR cells to TNF-alpha- or LPS-activated bEnd3 cells (mouse brain-derived endothelial cells) and to purified mouse VCAM-1. Binding is inhibited by Abs to VCAM-1 and to the alpha-chain of its lymphocyte receptor (the alpha 4 beta 1 integrin, VLA-4). Stimulation with FMLP does not induce a change in cell surface expression of alpha 4. Induced adhesion to VCAM-1 is rapid, detectable at the earliest times measurable (30 to 60 s after FMLP addition), and is inhibited by pertussis toxin. We conclude that FPR can mediate integrin activation not only in neutrophils but also in lymphocytes, and can trigger rapid adhesion via lymphocyte alpha 4 beta 1. The adhesion of lymphocytes is critical to their migration and targeting; our results suggest the possibility of manipulating adhesive responses through expression of chemoattractant receptors in lymphoid cells engineered for cellular therapy, allowing targeted adhesion and potentially migration in response to locally administered ligands.

Evaluation of effect of galvanic corrosion between nickel-chromium metal and titanium on ion release and cell toxicity

PubMed Central

Choi, Jung-Yun

2015-01-01

PURPOSE The purpose of this study was to evaluate cell toxicity due to ion release caused by galvanic corrosion as a result of contact between base metal and titanium. MATERIALS AND METHODS It was hypothesized that Nickel (Ni)-Chromium (Cr) alloys with different compositions possess different corrosion resistances when contacted with titanium abutment, and therefore in this study, specimens (10×10×1.5 mm) were fabricated using commercial pure titanium and 3 different types of Ni-Cr alloys (T3, Tilite, Bella bond plus) commonly used for metal ceramic restorations. The specimens were divided into 6 groups according to the composition of Ni-Cr alloy and contact with titanium. The experimental groups were in direct contact with titanium and the control groups were not. After the samples were immersed in the culture medium - Dulbecco's modified Eagle's medium[DMEM] for 48 hours, the released metal ions were detected using inductively coupled plasma mass spectrometer (ICP-MS) and analyzed by the Kruskal-Wallis and Mann-Whitney test (P<.05). Mouse L-929 fibroblast cells were used for cell toxicity evaluation. The cell toxicity of specimens was measured by the 3-{4,5-dimethylthiazol-2yl}-2,5-diphenyltetrazolium bromide (MTT) test. Results of MTT assay were statistically analyzed by the two-way ANOVA test (P<.05). Post-hoc multiple comparisons were conducted using Tukey's tests. RESULTS The amount of metal ions released by galvanic corrosion due to contact between the base metal alloy and titanium was increased in all of the specimens. In the cytotoxicity test, the two-way ANOVA showed a significant effect of the alloy type and galvanic corrosion for cytotoxicity (P<.001). The relative cell growth rate (RGR) was decreased further on the groups in contact with titanium (P<.05). CONCLUSION The release of metal ions was increased by galvanic corrosion due to contact between base metal and titanium, and it can cause adverse effects on the tissue around the implant by inducing cytotoxicity. PMID:25932317

Effects of diamines on ornithine decarboxylase activity in control and virally transformed mouse fibroblasts.

PubMed Central

Bethell, D R; Pegg, A E

1979-01-01

1. The induction of ornithine decarboxylase activity in mouse 3T3 fibroblasts or an SV-40 transformed 3T3 cell line by serum was prevented by addition of the naturally occurring polyamines putrescine (butane-1,4-diamine) and spermidine. Much higher concentrations of these amines were required to fully suppress ornithine decarboxylase activity in the transformed SV-3T3 cells than in the 3T3 fibroblasts. 2. Synthetic alpha omega-diamines with 3--12 carbon atoms also prevented the increase in ornithine decarboxylase activity induced by serum in these cells. The longer chain diamines were somewhat more potent than propane-1,3-diamine in this effect, but the synthetic diamines were less active than putrescine in the 3T3 cells. There was little difference between the responses of 3T3 and SV-3T3 cells to the synthetic diamines propane-1,3-diamine and heptane-1,7-diamine. 3. These results are discussed in relation to the control of polyamine synthesis in mammalian cells. PMID:486108

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Xiao-Qing; Zhang, Dao-Liang; Zhang, Ming-Jian

Aims: Atrial fibroblasts and macrophages have long been thought to participate in atrial fibrillation (AF). However, which specific mediator may regulate the interaction between them remains unclear. Methods and results: We provided the evidence for the involvement of Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF), an important inflammation-related molecule, in the pathophysiology of AF. Patients with AF showed higher levels of angiotensin II (AngII) and TRIF expression and larger number of macrophages infiltration in left atria appendage than individuals with sinus rhythm (SR). In the cell study, AngII induced chemokines expressions in mouse atrial fibroblasts and AngII-stimulated atrial fibroblasts inducedmore » the chemotaxis of macrophages, which were reduced by losartan and TRIF siRNA. Meanwhile, AngII-stimulated atrial fibroblasts proliferation was enhanced by macrophages. Conclusions: Our data demonstrated that TRIF may be a crucial factor promoting the interaction between atrial fibroblasts and macrophages, leading to atrial fibrosis. - Highlights: • Compared with SR, AF showed higher TRIF expression in left atrial appendage. • TRIF siRNA reversed macrophage chemotaxis induced by AngII-treated fibroblast. • TRIF siRNA reversed chemokines expressions induced by AngII in fibroblast. • AngII-stimulated atrial fibroblast proliferation was enhanced by macrophage.« less

Feasibility study of a biocompatible pneumatic dispensing system using mouse 3T3-J2 fibroblasts

NASA Astrophysics Data System (ADS)

Lee, Sangmin; Kim, Hojin; Kim, Joonwon

2017-12-01

This paper presents results for dispensing living cells using a pneumatic dispensing system to verify the feasibility of using this system to fabricate biomaterials. Living cells (i.e., mouse 3T3-J2 fibroblast) were dispensed with different dispensing pressures in order to evaluate the effect of dispensing process on cell viability and proliferation. Based on the results of a live-dead assay, more than 80% of cell viability has been confirmed which was reasonably similar to that in the control group. Furthermore, measurement of cell metabolic activity after dispensing confirmed that the dispensed cell proliferated at a rate comparable to that of the control group. These results demonstrate that the pneumatic dispensing system is a promising tool for fabrication of biomaterials.

Neuronal expression of fibroblast growth factor receptors in zebrafish.

PubMed

Rohs, Patricia; Ebert, Alicia M; Zuba, Ania; McFarlane, Sarah

2013-12-01

Fibroblast growth factor (FGF) signaling is important for a host of developmental processes such as proliferation, differentiation, tissue patterning, and morphogenesis. In vertebrates, FGFs signal through a family of four fibroblast growth factor receptors (FGFR 1-4), one of which is duplicated in zebrafish (FGFR1). Here we report the mRNA expression of the five known zebrafish fibroblast growth factor receptors at five developmental time points (24, 36, 48, 60, and 72h postfertilization), focusing on expression within the central nervous system. We show that the receptors have distinct and dynamic expression in the developing zebrafish brain, eye, inner ear, lateral line, and pharynx. In many cases, the expression patterns are similar to those of homologous FGFRs in mouse, chicken, amphibians, and other teleosts. Copyright © 2013 Elsevier B.V. All rights reserved.

Interference of a synthetic C18 juvenile hormone with mammalian cells in vitro, I. Effects on growth and morphology.

PubMed

Zielińska, Z M; Laskowska-Bozek, H; Jastreboff, P

1978-01-01

Some of structural and functional analogs of juvenile hormones are now under field examinations as growth inhibitors of some pest-insect populations. So far however very little is known about the possible interference of these compounds with mammalian cells or organisms. In this research the interference of a synthetic preparation of the insect C18 juvenile hormone with mouse embryo fibroblasts (ME-cells) and mouse cells of an established line (L-cells) was studied. Aliquots of juvenile hormone solution or those of the solvent (DMSO plus ethanol, 9:1) were included into the culture medium and after defined times of contact the cells were tested for their morphology, pattern of growth, proliferation rate and viability. The data for the parameters under examination were evaluated by means of the analysis of variance and checked by the Tuckey test. The sensitivity of ME-cells and L-cells to the agent tested was compared by means of the analysis of variance of the data for mitotic indices of these cells and by evaluation of the number of dead cells in cultures under the particular conditions of the experiments. The main findings can be summarized as follows: 1. Cells of both types are evidently more sensitive to juvenile hormone than to the solvent. 2. ME-cells are more sensitive to both agents than are L-cells. 3. The concentrations of the hormone in the medium required to evoked the cytocidal effect on the mouse cells similarly as those affecting some insect non-target cells were far above concentrations found in insect blood, but they were of the same order of magnitude as those used in physiological experiments with insect organs in vitro.

Comparative analysis of lysyl oxidase (like) family members in pulmonary fibrosis.

PubMed

Aumiller, Verena; Strobel, Benjamin; Romeike, Merrit; Schuler, Michael; Stierstorfer, Birgit E; Kreuz, Sebastian

2017-03-10

Extracellular matrix (ECM) composition and stiffness are major driving forces for the development and persistence of fibrotic diseases. Lysyl oxidase (LOX) and LOX-like (LOXL) proteins play crucial roles in ECM remodeling due to their collagen crosslinking and intracellular functions. Here, we systematically investigated LOX/L expression in primary fibroblasts and epithelial cells under fibrotic conditions, Bleomycin (BLM) induced lung fibrosis and in human IPF tissue. Basal expression of all LOX/L family members was detected in epithelial cells and at higher levels in fibroblasts. Various pro-fibrotic stimuli broadly induced LOX/L expression in fibroblasts, whereas specific induction of LOXL2 and partially LOX was observed in epithelial cells. Immunohistochemical analysis of lung tissue from 14 IPF patients and healthy donors revealed strong induction of LOX and LOXL2 in bronchial and alveolar epithelium as well as fibroblastic foci. Using siRNA experiments we observed that LOXL2 and LOXL3 were crucial for fibroblast-to-myofibroblast transition (FMT). As FMT could only be reconstituted with an enzymatically active LOXL2 variant, we conclude that LOXL2 enzymatic function is crucial for fibroblast transdifferentiation. In summary, our study provides a comprehensive analysis of the LOX/L family in fibrotic lung disease and indicates prominent roles for LOXL2/3 in fibroblast activation and LOX/LOXL2 in IPF.

Apoptosis gene expression and death receptor signaling in mitomycin-C-treated human tenon capsule fibroblasts.

PubMed

Crowston, Jonathan G; Chang, Lydia H; Constable, Peter H; Daniels, Julie T; Akbar, Arne N; Khaw, Peng T

2002-03-01

To examine the effect of mitomycin-C on the expression of apoptosis genes in human Tenon capsule fibroblasts and to evaluate whether death receptor signaling modulates mitomycin-C cytotoxicity. Bcl-2, Bax, Bcl-x, Fas (CD95) and tumor necrosis factor (TNF) receptor expression was determined by flow cytometry in control and mitomycin-C-treated Tenon fibroblasts. Fibroblast death was quantified using a lactate dehydrogenase release assay. The effect of Fas and TNF-receptor signaling was evaluated using Fas-specific antibodies and soluble TNF-alpha. Tenon fibroblasts constitutively express Bcl-2, Bax, and Bcl-x in culture. Mitomycin-C (0.4 mg/mL) induced a small but consistent increase in the expression of all three proteins. Tenon fibroblasts express low levels of Fas but are resistant to the effects of Fas-receptor ligation. Mitomycin-C (0.01-1.0 mg/mL) led to a significant increase in Fas expression at all concentrations tested (P < 0.01). Pretreatment with mitomycin-C (0.4 mg/mL) rendered fibroblasts susceptible to agonistic anti-Fas monoclonal IgM antibodies (50-500 ng/mL) and led to a further 50% reduction in viable fibroblasts at 48 hours, compared with mitomycin-C alone (P < 0.05). Antibodies that block the Fas receptor did not inhibit mitomycin-C-induced apoptosis. Mitomycin-C alters apoptosis gene expression and primes fibroblasts to the effects of Fas receptor ligation. Factors other than the level of Fas receptor expression modulate the response to Fas receptor signaling. Determining the signals that regulate fibroblast apoptosis may help to refine therapeutic strategies for switching off the subconjunctival healing response and maintaining intraocular pressure control.

Cilostazol ameliorates metabolic abnormalities with suppression of proinflammatory markers in a db/db mouse model of type 2 diabetes via activation of peroxisome proliferator-activated receptor gamma transcription.

PubMed

Park, So Youn; Shin, Hwa Kyoung; Lee, Jeong Hyun; Kim, Chi Dae; Lee, Won Suk; Rhim, Byung Yong; Hong, Ki Whan

2009-05-01

In a previous study, cilostazol promoted differentiation of 3T3-L1 fibroblasts into adipocytes and improved insulin sensitivity by stimulating peroxisome proliferator-activated receptor (PPAR) gamma transcription. This study evaluated the in vivo efficacy of cilostazol to protect a db/db mouse model of type 2 diabetes against altered metabolic abnormalities and proinflammatory markers via activation of PPARgamma transcription. Eight-week-old db/db mice were treated with cilostazol or rosiglitazone for 12 days. Cilostazol significantly decreased plasma glucose and triglyceride levels, as did rosiglitazone, a PPARgamma agonist. Elevated plasma insulin and resistin levels were significantly decreased by cilostazol, and decreased adiponectin mRNA expression was elevated along with increased plasma adiponectin. Cilostazol significantly increased both adipocyte fatty acid binding protein and fatty acid transport protein-1 mRNA expressions with increased glucose transport 4 in the adipose tissue. Cilostazol and rosiglitazone significantly suppressed proinflammatory markers (superoxide, tumor necrosis factor-alpha, and vascular cell adhesion molecule-1) in the carotid artery of db/db mice. In an in vitro study with 3T3-L1 fibroblasts, cilostazol significantly increased PPARgamma transcription activity, as did rosiglitazone. The transcription activity stimulated by cilostazol was attenuated by KT5720 [(9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9, 12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo [3,4-I][1,6]-benzodiazocine-10-carboxylic acid hexyl ester], a cAMP-dependent protein kinase inhibitor, and GW9662 (2-chloro-5-nitrobenzanilide), an antagonist of PPARgamma activity, indicative of implication of the phosphatidylinositol 3-kinase/Akt signal pathway. These results suggest that cilostazol may improve insulin sensitivity along with anti-inflammatory effects in type 2 diabetic patients via activation of both cAMP-dependent protein kinase and PPARgamma transcription.

Pan-PPAR agonist IVA337 is effective in experimental lung fibrosis and pulmonary hypertension.

PubMed

Avouac, Jerome; Konstantinova, Irena; Guignabert, Christophe; Pezet, Sonia; Sadoine, Jeremy; Guilbert, Thomas; Cauvet, Anne; Tu, Ly; Luccarini, Jean-Michel; Junien, Jean-Louis; Broqua, Pierre; Allanore, Yannick

2017-11-01

To evaluate the antifibrotic effects of the pan-peroxisome proliferator-activated receptor (PPAR) agonist IVA337 in preclinical mouse models of pulmonary fibrosis and related pulmonary hypertension (PH). IVA337 has been evaluated in the mouse model of bleomycin-induced pulmonary fibrosis and in Fra-2 transgenic mice, this latter being characterised by non-specific interstitial pneumonia and severe vascular remodelling of pulmonary arteries leading to PH. Mice received two doses of IVA337 (30 mg/kg or 100 mg/kg) or vehicle administered by daily oral gavage up to 4 weeks. IVA337 demonstrated at a dose of 100 mg/kg a marked protection from the development of lung fibrosis in both mouse models compared with mice receiving 30 mg/kg of IVA337 or vehicle. Histological score was markedly reduced by 61% in the bleomycin model and by 50% in Fra-2 transgenic mice, and total lung hydroxyproline concentrations decreased by 28% and 48%, respectively, as compared with vehicle-treated mice. IVA337 at 100 mg/kg also significantly decreased levels of fibrogenic markers in lesional lungs of both mouse models. In addition, IVA337 substantially alleviated PH in Fra-2 transgenic mice by improving haemodynamic measurements and vascular remodelling. In primary human lung fibroblasts, IVA337 inhibited in a dose-dependent manner fibroblast to myofibroblasts transition induced by TGF-β and fibroblast proliferation mediated by PDGF. We demonstrate that treatment with 100 mg/kg IVA337 prevents lung fibrosis in two complementary animal models and substantially attenuates PH in the Fra-2 mouse model. These findings confirm that the pan-PPAR agonist IVA337 is an appealing therapeutic candidate for these cardiopulmonary involvements. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Effects of interleukins on connective tissue type mast cells co-cultured with fibroblasts.

PubMed Central

Levi-Schaffer, F; Segal, V; Shalit, M

1991-01-01

We investigated the effects of interleukin-2 (IL-2), interleukin-3 (IL-3) and interleukin-4 (IL-4) on mouse and rat peritoneal mast cells (MC) co-cultured with 3T3 fibroblasts (MC/3T3). The continuous presence of these cytokines for 7-9 days in the culture media was neither toxic nor caused proliferation of MC, as determined by the stability of MC numbers in culture. Long-term incubation of mouse MC/3T3 with IL-2 (100 U/ml), IL-3 (50 U/ml), IL-4 (50 U/ml) or a mixture of IL-3 and IL-4 (25 U/ml) induced an increase in basal histamine release of 79.3 +/- 19.0%, 41.0 +/- 17.3%, 25.2 +/- 10.4% and 30.2 +/- 3.2%, respectively, over control cells incubated with medium alone. When rat MC/3T3 were incubated for 7 days with the various interleukins an enhancement in histamine release similar to that observed with mouse MC/3T3 was found. Preincubation (1 hr) of rat MC/3T3 with interleukins prior to immunological activation with anti-IgE antibodies enhanced histamine release. The highest effect was observed with IL-3 + IL-4 (60.4 +/- 10.8% increase) followed by IL-2 (51.5 +/- 4.5%), IL-4 (28.6 +/- 10.3%) and IL-3 (13.2 +/- 4.2%). This study demonstrates that when mouse and rat peritoneal MC are cultured with fibroblasts in the presence of interleukins they do not proliferate, suggesting that they preserve their connective tissue type MC phenotype. Moreover, interleukins display a pro-inflammatory effect on these cells by enhancing both basal and anti-IgE-mediated histamine release. PMID:2016117

Induction of cardiomyocyte-like cells in infarct hearts by gene transfer of Gata4, Mef2c, and Tbx5.

PubMed

Inagawa, Kohei; Miyamoto, Kazutaka; Yamakawa, Hiroyuki; Muraoka, Naoto; Sadahiro, Taketaro; Umei, Tomohiko; Wada, Rie; Katsumata, Yoshinori; Kaneda, Ruri; Nakade, Koji; Kurihara, Chitose; Obata, Yuichi; Miyake, Koichi; Fukuda, Keiichi; Ieda, Masaki

2012-10-12

After myocardial infarction (MI), massive cell death in the myocardium initiates fibrosis and scar formation, leading to heart failure. We recently found that a combination of 3 cardiac transcription factors, Gata4, Mef2c, and Tbx5 (GMT), reprograms fibroblasts directly into functional cardiomyocytes in vitro. To investigate whether viral gene transfer of GMT into infarcted hearts induces cardiomyocyte generation. Coronary artery ligation was used to generate MI in the mouse. In vitro transduction of GMT retrovirus converted cardiac fibroblasts from the infarct region into cardiomyocyte-like cells with cardiac-specific gene expression and sarcomeric structures. Injection of the green fluorescent protein (GFP) retrovirus into mouse hearts, immediately after MI, infected only proliferating noncardiomyocytes, mainly fibroblasts, in the infarct region. The GFP expression diminished after 2 weeks in immunocompetent mice but remained stable for 3 months in immunosuppressed mice, in which cardiac induction did not occur. In contrast, injection of GMT retrovirus into α-myosin heavy chain (αMHC)-GFP transgenic mouse hearts induced the expression of αMHC-GFP, a marker of cardiomyocytes, in 3% of virus-infected cells after 1 week. A pooled GMT injection into the immunosuppressed mouse hearts induced cardiac marker expression in retrovirus-infected cells within 2 weeks, although few cells showed striated muscle structures. To transduce GMT efficiently in vivo, we generated a polycistronic retrovirus expressing GMT separated by 2A "self-cleaving" peptides (3F2A). The 3F2A-induced cardiomyocyte-like cells in fibrotic tissue expressed sarcomeric α-actinin and cardiac troponin T and had clear cross striations. Quantitative RT-PCR also demonstrated that FACS-sorted 3F2A-transduced cells expressed cardiac-specific genes. GMT gene transfer induced cardiomyocyte-like cells in infarcted hearts.

The interaction in vitro of Mycoplasma pulmonis with mouse peritoneal macrophages and L-cells.

PubMed

Jones, T C; Hirsch, J G

1971-02-01

Methods have been devised for establishing infection in vitro of mouse macrophages and fibroblasts with Mycoplasma pulmonis. The mycoplasmas attached to the cells and under appropriate cultural conditions grew into a lawn of microorganisms covering most of the cell surface. The mycoplasmas grew abundantly on fibroblasts cultured in minimal essential medium containing 20% fetal calf serum; supplementation of this medium with heart infusion broth was necessary to obtain similar growth on macrophages. The infection of these cells appeared to be essentially an extracellular process; only rarely were partially degraded mycoplasmas seen with phagocytic vacuoles. The addition to heavily infected macrophage cultures of low concentrations of anti-mycoplasma antibody stimulated rapid, massive phagocytosis of the surface microorganisms. In sharp contrast, the same antiserum had no discernable effect on the mycoplasma-fibroblast relationship. The antibody effect in the macrophage system was apparently a direct opsonic one rather than an indirect result of microbial killing, since the mycoplasmas in macrophage or fibroblast cultures incorporated labelled thymidine into DNA after the addition of antiserum to the medium. The phagocytic event and the subsequent fate of the mycoplasmas were studied in detail after the addition of antibody to the macrophage cultures. Phase-contrast cinemicrophotography revealed membrane ruffles surrounding the surface mycoplasmas and disappearance from view of the organisms; 10-30 min later translucent grapelike clusters were seen in large phagocytic vacuoles. On electronmicroscopic study the surface mycoplasmas were surrounded by pincers-like projections of the macrophage. Numerous mycoplasmas were seen in phagocytic vacuoles; in the early minutes after the addition of antibody the intracellular mycoplasmas appeared normal, but within 2 hr they appeared partially degraded with a central electron-lucent area and electron-opaque deposits at the microbial cell margin. 24 hr after the addition of antiserum, digestion of the mycoplasmas was nearly complete; the cells appeared normal except for large residual bodies composed of amorphous moderately dense material and increased lipid deposits. Degradation of mycoplasmas within macrophages was also studied using infected cultures in which the mycoplasmas, but not the macrophages, had incorporated tritiated thymidine into DNA. The appearance of large amounts of acid-soluble radiolabel after phagocytosis stimulated by antibody confirmed the degradation of the intracellular mycoplasmas.

Cyclic AMP and alkaline pH downregulate carbonic anhydrase 2 in mouse fibroblasts.

PubMed

Mardones, Pablo; Chang, Jung Chin; Oude Elferink, Ronald P J

2014-06-01

The hydration of CO2 catalyzed by the ubiquitous carbonic anhydrase 2 (Ca2) is central for bicarbonate transport, bone metabolism and acid-base homeostasis in metazoans. There is evidence that in some tissues Ca2 expression can be acutely induced by cAMP, whereas in other cell types it is unresponsive to cAMP-mediated transcriptional activation. We isolated fibroblasts from wild type and mice lacking the ubiquitous chloride/bicarbonate exchanger (Ae2a,b(-/-) mice). In these cells the regulation of carbonic anhydrase 2 by cAMP was studied. We show that Ca2 expression is strongly inhibited by chronic incubation with dibutyryl-cAMP, forskolin or alkaline pH in cultured mouse fibroblasts. Furthermore, fibroblasts obtained from anion exchanger 2 deficient (Ae2a,b(-/-)) mice, which display intracellular alkalosis and increased cAMP production, express less than 10% of control Ca2 mRNA and protein. Surprisingly, inhibition of the bicarbonate-sensitive soluble adenylyl cyclase (sAC) was found to reduce CA2 expression instead of increasing it. CA2 expression is strongly regulated by intracellular pH and by cAMP, suggesting a role for soluble adenylyl cyclase. Regulation occurs in opposite directions which may be explained by an incoherent feedforward loop consisting of activation by pCREB and repression by ICER. Copyright © 2013 Elsevier B.V. All rights reserved.

Impact of transforming growth factor-beta1 on atrioventricular node conduction modification by injected autologous fibroblasts in the canine heart.

PubMed

Bunch, T Jared; Mahapatra, Srijoy; Bruce, G Keith; Johnson, Susan B; Miller, Dylan V; Horne, Benjamin D; Wang, Xiao-Li; Lee, Hon-Chi; Caplice, Noel M; Packer, Douglas L

2006-05-30

Atrioventricular (AV) nodal ablation for management of atrial fibrillation (AF) is irreversible and requires permanent pacemaker implantation. We hypothesized that as an alternative, implantation of autologous fibroblasts in the perinodal region would focally modify AV nodal conduction and that this modulation would be enhanced by pretreatment with transforming growth factor-beta1 (TGF-beta1), a stimulant of fibroblasts. Skin biopsies were taken from 12 mongrel dogs, and derived fibroblasts were dissociated and grown in culture for 2 weeks. Multiple injections (0.25 mL) were made through an 8F NOGA catheter along the fast/slow AV nodal pathways as guided by an electroanatomic mapping system. Seven dogs received fibroblasts alone (1x10(6) cells/mL), 7 dogs received TGF-beta1 (5 microg), 4 dogs received fibroblasts and TGF-beta1 (1x10(6) cells/mL+5 microg), and 4 dogs received saline only. AV node function was assessed at baseline and after 4 weeks. Saline (80 mL) with assigned therapy (0.25 mL per injection) was injected into the peri-AV nodal region in each dog. At baseline, the AH interval (66+/-3 ms) and the average RR interval (331+/-17 ms) in pacing-induced AF were similar in each cohort. The increase in AH interval in normal sinus rhythm was longer after fibroblast (23+/-4 versus 5+/-5 ms; P=0.05) and fibroblast plus TGF-beta1 (50+/-5 versus 5+/-5 ms; P<0.001) injections than with saline alone, with similar findings during high right atrium and distal coronary sinus pacing. The AH interval was not significantly increased after TGF-beta1 injections. The AH interval was significantly longer after fibroblast plus TGF-beta1 injections than with either therapy (TGF-beta1 or fibroblasts) alone. The RR interval during AF was increased in dogs that received fibroblasts alone (110+/-36 versus -41+/-34 ms) and to a greater extent with the addition of TGF-beta1 (294+/-108 versus -41+/-34 ms). No AV block was seen in any cohort at 4 weeks. Labeled fibroblasts that expressed vimentin were identified in all dogs that received cell injections at 4 weeks. AV nodal modification can be achieved with injected fibroblasts without the creation of AV block. The effect on AV node conduction is substantially enhanced by pretreatment of fibroblasts with TGF-beta1. These data have therapeutic potential for the management of rapid ventricular rate during AF without pacemaker implantation.

Neurite outgrowth stimulatory effects of culinary-medicinal mushrooms and their toxicity assessment using differentiating Neuro-2a and embryonic fibroblast BALB/3T3.

PubMed

Phan, Chia-Wei; David, Pamela; Naidu, Murali; Wong, Kah-Hui; Sabaratnam, Vikineswary

2013-10-11

Mushrooms are not only regarded as gourmet cuisine but also as therapeutic agent to promote cognition health. However, little toxicological information is available regarding their safety. Therefore, the aim of this study was to screen selected ethno-pharmacologically important mushrooms for stimulatory effects on neurite outgrowth and to test for any cytotoxicity. The stimulatory effect of mushrooms on neurite outgrowth was assessed in differentiating mouse neuroblastoma (N2a) cells. Neurite length was measured using Image-Pro Insight processor system. Neuritogenesis activity was further validated by fluorescence immunocytochemical staining of neurofilaments. In vitro cytotoxicity was investigated by using mouse embryonic fibroblast (BALB/3T3) and N2a cells for any embryo- and neuro-toxic effects; respectively. Aqueous extracts of Ganoderma lucidum, Lignosus rhinocerotis, Pleurotus giganteus and Grifola frondosa; as well as an ethanol extract of Cordyceps militaris significantly (p < 0.05) promoted the neurite outgrowth in N2a cells by 38.4 ± 4.2%, 38.1 ± 2.6%, 33.4 ± 4.6%, 33.7 ± 1.5%, and 35.8 ± 3.4%; respectively. The IC50 values obtained from tetrazolium (MTT), neutral red uptake (NRU) and lactate dehydrogenase (LDH) release assays showed no toxic effects following 24 h exposure of N2a and 3T3 cells to mushroom extracts. Our results indicate that G. lucidum, L. rhinocerotis, P. giganteus, G. frondosa and C. militaris may be developed as safe and healthy dietary supplements for brain and cognitive health.

Neurite outgrowth stimulatory effects of culinary-medicinal mushrooms and their toxicity assessment using differentiating Neuro-2a and embryonic fibroblast BALB/3T3

PubMed Central

2013-01-01

Background Mushrooms are not only regarded as gourmet cuisine but also as therapeutic agent to promote cognition health. However, little toxicological information is available regarding their safety. Therefore, the aim of this study was to screen selected ethno-pharmacologically important mushrooms for stimulatory effects on neurite outgrowth and to test for any cytotoxicity. Methods The stimulatory effect of mushrooms on neurite outgrowth was assessed in differentiating mouse neuroblastoma (N2a) cells. Neurite length was measured using Image-Pro Insight processor system. Neuritogenesis activity was further validated by fluorescence immunocytochemical staining of neurofilaments. In vitro cytotoxicity was investigated by using mouse embryonic fibroblast (BALB/3T3) and N2a cells for any embryo- and neuro-toxic effects; respectively. Results Aqueous extracts of Ganoderma lucidum, Lignosus rhinocerotis, Pleurotus giganteus and Grifola frondosa; as well as an ethanol extract of Cordyceps militaris significantly (p < 0.05) promoted the neurite outgrowth in N2a cells by 38.4 ± 4.2%, 38.1 ± 2.6%, 33.4 ± 4.6%, 33.7 ± 1.5%, and 35.8 ± 3.4%; respectively. The IC50 values obtained from tetrazolium (MTT), neutral red uptake (NRU) and lactate dehydrogenase (LDH) release assays showed no toxic effects following 24 h exposure of N2a and 3T3 cells to mushroom extracts. Conclusion Our results indicate that G. lucidum, L. rhinocerotis, P. giganteus, G. frondosa and C. militaris may be developed as safe and healthy dietary supplements for brain and cognitive health. PMID:24119256

A Novel Role of Peripheral Corticotropin-Releasing Hormone (CRH) on Dermal Fibroblasts

PubMed Central

Rassouli, Olga; Liapakis, George; Lazaridis, Iakovos; Sakellaris, George; Gkountelias, Kostas; Gravanis, Achille; Margioris, Andrew N.

2011-01-01

Corticotropin-releasing hormone, or factor, (CRH or CRF) exerts important biological effects in multiple peripheral tissues via paracrine/autocrine actions. The aim of our study was to assess the effects of endogenous CRH in the biology of mouse and human skin fibroblasts, the primary cell type involved in wound healing. We show expression of CRH and its receptors in primary fibroblasts, and we demonstrate the functionality of fibroblast CRH receptors by induction of cAMP. Fibroblasts genetically deficient in Crh (Crh−/−) had higher proliferation and migration rates and compromised production of IL-6 and TGF-β1 compared to the wildtype (Crh+/+) cells. Human primary cultures of foreskin fibroblasts exposed to the CRF1 antagonist antalarmin recapitulated the findings in the Crh−/− cells, exhibiting altered proliferative and migratory behavior and suppressed production of IL-6. In conclusion, our findings show an important role of fibroblast-expressed CRH in the proliferation, migration, and cytokine production of these cells, processes associated with the skin response to injury. Our data suggest that the immunomodulatory effects of CRH may include an important, albeit not explored yet, role in epidermal tissue remodeling and regeneration and maintenance of tissue homeostasis. PMID:21765902

[The growth behavior of mouse fibroblasts on intraocular lens surface of various silicone and PMMA materials].

PubMed

Kammann, J; Kreiner, C F; Kaden, P

1994-08-01

Experience with intraocular lenses (IOL) made of PMMA dates back ca. 40 years, while silicone IOLs have been in use for only about 10 years. The biocompatibility of PMMA and silicone caoutchouc was tested in a comparative study investigating the growth of mouse fibroblasts on different IOL materials. Spectrophotometric determination of protein synthesis and liquid scintillation counting of DNA synthesis were carried out. The spreading of cells was planimetrically determined, and the DNA synthesis of individual cells in direct contact with the test sample was tested. The results showed that the biocompatibility of silicone lenses made of purified caoutchouc is comparable with that of PMMA lenses; there is no statistically significant difference. However, impurities arising during material synthesis result in a statistically significant inhibition of cell growth on the IOL surfaces.

Quantitative genetic-interaction mapping in mammalian cells

PubMed Central

Roguev, Assen; Talbot, Dale; Negri, Gian Luca; Shales, Michael; Cagney, Gerard; Bandyopadhyay, Sourav; Panning, Barbara; Krogan, Nevan J

2013-01-01

Mapping genetic interactions (GIs) by simultaneously perturbing pairs of genes is a powerful tool for understanding complex biological phenomena. Here we describe an experimental platform for generating quantitative GI maps in mammalian cells using a combinatorial RNA interference strategy. We performed ~11,000 pairwise knockdowns in mouse fibroblasts, focusing on 130 factors involved in chromatin regulation to create a GI map. Comparison of the GI and protein-protein interaction (PPI) data revealed that pairs of genes exhibiting positive GIs and/or similar genetic profiles were predictive of the corresponding proteins being physically associated. The mammalian GI map identified pathways and complexes but also resolved functionally distinct submodules within larger protein complexes. By integrating GI and PPI data, we created a functional map of chromatin complexes in mouse fibroblasts, revealing that the PAF complex is a central player in the mammalian chromatin landscape. PMID:23407553

EGR-1 regulates Ho-1 expression induced by cigarette smoke

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Huaqun, E-mail: chenhuaqun@njnu.edu.cn; Wang, Lijuan; Gong, Tao

2010-05-28

As an anti-oxidant molecule, heme oxygenase-1 (HO-1) has been implicated in the protection of lung injury by cigarette smoke (CS). The mechanisms regulating its expression have not been defined. In this report, the role of early growth response 1 (EGR-1) in the regulation of Ho-1 expression was investigated. In C57BL/6 mice with CS exposure, HO-1 was greatly increased in bronchial epithelial cells and alveolar inflammatory cells. In primary cultured mouse lung fibroblasts and RAW264.7 cells exposed to cigarette smoke water extract (CSE), an increase in HO-1 protein level was detected. In addition, CSE induced HO-1 expression was decreased in Egr-1more » deficient mouse embryo fibroblasts (Egr-1{sup -/-} MEFs). Nuclear localization of EGR-1 was examined in mouse lung fibroblasts after exposure to CSE. Luciferase reporter activity assays showed that the enhancer region of the Ho-1 gene containing a proposed EGR-1 binding site was responsible for the induction of HO-1. A higher increase of alveolar mean linear intercept (Lm) was observed in lung tissues, and a larger increase in the number of total cells and monocytes/macrophages from bronchial alveolar lavage fluid was found in CS-exposed mice by loss of function of EGR-1 treatment. In summary, the present data demonstrate that EGR-1 plays a critical role in HO-1 production induced by CS.« less

The Role of the MHV Receptor and Related Glycoproteins in Murine Hepatitis Virus Infection of Murine Cell Lines

DTIC Science & Technology

1995-04-13

rhodamine-coupled goat anti -mouse antibody . A rare , fused Cl 1 D giant cell was selected to show (Al, while extensive fusion was common throughout the...mouse anti - MHV-AS9 antiserum. To quantify the lev el of susceptibility of cells to MHV infection , ten randomly selected fields for each sample...named CealO) was discovered and found to be co-expressed with MHVR in the CI 1 D and F40 lines of mouse fibroblasts. A monoclonal anti - MHVR

Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo

Highlights: Black-Right-Pointing-Pointer We identify a function of the YIGSR peptide to enhance collagen synthesis in Hs27. Black-Right-Pointing-Pointer YIGSR peptide enhanced collagen type 1 synthesis both of gene and protein levels. Black-Right-Pointing-Pointer There were no changes in cell proliferation and MMP-1 level in YIGSR treatment. Black-Right-Pointing-Pointer The YIGSR effect on collagen synthesis mediated activation of FAK, pyk2 and ERK. Black-Right-Pointing-Pointer The YIGSR-induced FAK and ERK activation was modulated by FAK and MEK inhibitors. -- Abstract: The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin,more » respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929-933 sequence of the {beta}1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67 kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate peptide for the treatment of skin aging and wrinkles.« less

The RNA-Editing Enzyme ADAR1 Controls Innate Immune Responses to RNA

PubMed Central

Mannion, Niamh M.; Greenwood, Sam M.; Young, Robert; Cox, Sarah; Brindle, James; Read, David; Nellåker, Christoffer; Vesely, Cornelia; Ponting, Chris P.; McLaughlin, Paul J.; Jantsch, Michael F.; Dorin, Julia; Adams, Ian R.; Scadden, A.D.J.; Öhman, Marie; Keegan, Liam P.; O’Connell, Mary A.

2014-01-01

Summary The ADAR RNA-editing enzymes deaminate adenosine bases to inosines in cellular RNAs. Aberrant interferon expression occurs in patients in whom ADAR1 mutations cause Aicardi-Goutières syndrome (AGS) or dystonia arising from striatal neurodegeneration. Adar1 mutant mouse embryos show aberrant interferon induction and die by embryonic day E12.5. We demonstrate that Adar1 embryonic lethality is rescued to live birth in Adar1; Mavs double mutants in which the antiviral interferon induction response to cytoplasmic double-stranded RNA (dsRNA) is prevented. Aberrant immune responses in Adar1 mutant mouse embryo fibroblasts are dramatically reduced by restoring the expression of editing-active cytoplasmic ADARs. We propose that inosine in cellular RNA inhibits antiviral inflammatory and interferon responses by altering RLR interactions. Transfecting dsRNA oligonucleotides containing inosine-uracil base pairs into Adar1 mutant mouse embryo fibroblasts reduces the aberrant innate immune response. ADAR1 mutations causing AGS affect the activity of the interferon-inducible cytoplasmic isoform more severely than the nuclear isoform. PMID:25456137

The RNA-editing enzyme ADAR1 controls innate immune responses to RNA.

PubMed

Mannion, Niamh M; Greenwood, Sam M; Young, Robert; Cox, Sarah; Brindle, James; Read, David; Nellåker, Christoffer; Vesely, Cornelia; Ponting, Chris P; McLaughlin, Paul J; Jantsch, Michael F; Dorin, Julia; Adams, Ian R; Scadden, A D J; Ohman, Marie; Keegan, Liam P; O'Connell, Mary A

2014-11-20

The ADAR RNA-editing enzymes deaminate adenosine bases to inosines in cellular RNAs. Aberrant interferon expression occurs in patients in whom ADAR1 mutations cause Aicardi-Goutières syndrome (AGS) or dystonia arising from striatal neurodegeneration. Adar1 mutant mouse embryos show aberrant interferon induction and die by embryonic day E12.5. We demonstrate that Adar1 embryonic lethality is rescued to live birth in Adar1; Mavs double mutants in which the antiviral interferon induction response to cytoplasmic double-stranded RNA (dsRNA) is prevented. Aberrant immune responses in Adar1 mutant mouse embryo fibroblasts are dramatically reduced by restoring the expression of editing-active cytoplasmic ADARs. We propose that inosine in cellular RNA inhibits antiviral inflammatory and interferon responses by altering RLR interactions. Transfecting dsRNA oligonucleotides containing inosine-uracil base pairs into Adar1 mutant mouse embryo fibroblasts reduces the aberrant innate immune response. ADAR1 mutations causing AGS affect the activity of the interferon-inducible cytoplasmic isoform more severely than the nuclear isoform. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Fibrosis in connective tissue disease: the role of the myofibroblast and fibroblast-epithelial cell interactions

PubMed Central

Krieg, Thomas; Abraham, David; Lafyatis, Robert

2007-01-01

Fibrosis, characterized by excessive extracellular matrix accumulation, is a common feature of many connective tissue diseases, notably scleroderma (systemic sclerosis). Experimental studies suggest that a complex network of intercellular interactions involving endothelial cells, epithelial cells, fibroblasts and immune cells, using an array of molecular mediators, drives the pathogenic events that lead to fibrosis. Transforming growth factor-β and endothelin-1, which are part of a cytokine hierarchy with connective tissue growth factor, are key mediators of fibrogenesis and are primarily responsible for the differentiation of fibroblasts toward a myofibroblast phenotype. The tight skin mouse (Tsk-1) model of cutaneous fibrosis suggests that numerous other genes may also be important. PMID:17767742

Mesenchymal stem cell-derived inflammatory fibroblasts mediate interstitial fibrosis in the aging heart.

PubMed

Trial, JoAnn; Entman, Mark L; Cieslik, Katarzyna A

2016-02-01

Pathologic fibrosis in the aging mouse heart is associated with dysregulated resident mesenchymal stem cells (MSC) arising from reduced stemness and aberrant differentiation into dysfunctional inflammatory fibroblasts. Fibroblasts derived from aging MSC secrete higher levels of 1) collagen type 1 (Col1) that directly contributes to fibrosis, 2) monocyte chemoattractant protein-1 (MCP-1) that attracts leukocytes from the blood and 3) interleukin-6 (IL-6) that facilitates transition of monocytes into myeloid fibroblasts. The transcriptional activation of these proteins is controlled via the farnesyltransferase (FTase)-Ras-Erk pathway. The intrinsic change in the MSC phenotype acquired by advanced age is specific for the heart since MSC originating from bone wall (BW-MSC) or fibroblasts derived from them were free of these defects. The potential therapeutic interventions other than clinically approved strategies based on findings presented in this review are discussed as well. This article is a part of a Special Issue entitled "Fibrosis and Myocardial Remodeling". Copyright © 2015 Elsevier Ltd. All rights reserved.

The in vivo anti-fibrotic function of calcium sensitive receptor (CaSR) modulating poly(p-dioxanone-co-l-phenylalanine) prodrug.

PubMed

Wang, Bing; Wen, Aiping; Feng, Chengmin; Niu, Lijing; Xiao, Xin; Luo, Le; Shen, Chengyi; Zhu, Jiang; Lei, Jun; Zhang, Xiaoming

2018-06-01

In present study, the apoptosis induction and proliferation suppression effects of l-phenylalanine (l-Phe) on fibroblasts were confirmed. The action sites of l-Phe on fibroblasts suppression were deduced to be calcium sensitive receptor (CaSR) which could cause the release of endoplasmic reticulum (ER) Ca 2+ stores; disruption of intracellular Ca 2+ homeostasis triggers cell apoptosis via the ER or mitochondrial pathways. The down-regulation of CaSR were observed after the application of l-Phe, and the results those l-Phe triggered the increasing of intracellular Ca 2+ concentration and calcineurin expression, and then the apoptosis and increasing G1 fraction of fibroblasts have verified our deduction. Hence, l-Phe could be seen as a kind of anti-fibrotic drugs for the crucial participation of fibroblast in the occurrence of fibrosis. And then, poly(p-dioxanone-co-l-phenylalanine) (PDPA) which could prolong the in-vivo anti-fibrotic effect of l-Phe for the sustained release of l-Phe during its degradation could be treated as anti-fibrotic polymer prodrugs. Based on the above, the in vivo anti-fibrotic function of PDPA was evaluated in rabbit ear scarring, rat peritoneum lipopolysaccharide, and rat sidewall defect/cecum abrasion models. PDPA reduced skin scarring and suppressed peritoneal fibrosis and post operation adhesion as well as secretion of transforming growth factor-β1 in injured tissue. These results indicate that PDPA is an effective agent for preventing fibrosis following tissue injury. We have previously demonstrated that poly(p-dioxanone-co-l-phenylalanine) (PDPA) could induce apoptosis to fibroblast and deduced that the inhibitory effect comes from l-phenylalanine. In present study, the inhibition mechanism of l-phenylalanine on fibroblast proliferation was demonstrated. The calcium sensitive receptor (CaSR) was found to be the action site. The CaSR was downregulated after the application of l-phenylalanine, and then the ER Ca 2+ stores were released. The released Ca 2+ can simultaneously activate Ca 2+ /calcineurin and then trigger apoptosis and G1 arrest of fibroblast. Hence, l-phenylalanine could be seen as anti-fibrosis drug and PDPA which conjugate l-phenylalanine by hydrolytic covalent bonds could be seen as l-phenylalanine polymer prodrug. Based above, the in vivo anti-fibrotic function of PDPA were verified in three different animal models. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer. | Office of Cancer Genomics

Cancer.gov

Pancreatic stellate cells (PSCs) differentiate into cancer-associated fibroblasts (CAFs) that produce desmoplastic stroma, thereby modulating disease progression and therapeutic response in pancreatic ductal adenocarcinoma (PDA). However, it is unknown whether CAFs uniformly carry out these tasks or if subtypes of CAFs with distinct phenotypes in PDA exist. We identified a CAF subpopulation with elevated expression of α-smooth muscle actin (αSMA) located immediately adjacent to neoplastic cells in mouse and human PDA tissue.

Wound-healing activity of a proteolytic fraction from Carica candamarcensis on experimentally induced burn.

PubMed

Gomes, Flávia S L; Spínola, Cássia de V; Ribeiro, Henrique A; Lopes, Miriam T P; Cassali, Geovanni D; Salas, Carlos E

2010-03-01

Carica candamarcensis is a species from the Caricaceae family whose immature fruit contains latex with large amounts of cysteine proteinases. In prior studies, we isolated two of these enzymes displaying mitogenic activity when incubated with L929 fibroblastic cells. One of the fractions containing these enzymes (P1G10) was shown to enhance wound healing of skin and to accelerate healing of chemically induced gastric ulcer. In this study we evaluate the effect of P1G10 on heat-induced, third-degree burn using a rodent model. The results show that 0.1% P1G10 accelerates epithelisation while the effect of 1% or 0.01% P1G10 is not significantly different to 1% silver sulphadiazine, 2% papain or the hydrosoluble vehicle used as control. In a double-blind randomised experiment comparing the healing response of 0.1%, 1% and the vehicle alone, we confirmed the enhanced healing property of P1G10. Histological analysis of burn-tissue sections following treatment with P1G10 support these observations. These results extend the healing properties of these groups of enzymes to a different type of trauma and open the way to future clinical applications. Copyright (c) 2009 Elsevier Ltd and ISBI. All rights reserved.

Screening on binary Ti alloy with excellent mechanical property and castability for dental prosthesis application

PubMed Central

Li, H. F.; Qiu, K. J.; Yuan, W.; Zhou, F. Y.; Wang, B. L.; Li, L.; Zheng, Y. F.; Liu, Y. H.

2016-01-01

In the present study, the microstructure, mechanical property, castability, corrosion behavior and in vitro cytocompatibility of binary Ti–2X alloys with various alloying elements, including Ag, Bi, Ga, Ge, Hf, In, Mo, Nb, Sn and Zr, were systematically investigated, in order to assess their potential applications in dental field. The experimental results showed that all binary Ti‒2X alloys consisted entirely α–Ti phase. The tensile strength and microhardness of Ti were improved by adding alloying elements. The castability of Ti was significantly improved by separately adding 2 wt.% Bi, Ga, Hf, Mo, Nb, Sn and Zr. The corrosion resistance of Ti in both normal artificial saliva solution (AS) and extreme artificial saliva solution (ASFL, AS with 0.2 wt.% NaF and 0.3 wt.% lactic acid) has been improved by separately adding alloying elements. In addition, the extracts of studied Ti‒2X alloys produced no significant deleterious effect to both fibroblasts L929 cells and osteoblast-like MG63 cells, indicating a good in vitro cytocompatibility, at the same level as pure Ti. The combination of enhanced mechanical properties, castability, corrosion behavior, and in vitro cytocompatibility make the developed Ti‒2X alloys have great potential for future stomatological applications. PMID:27874034

Cytotoxicity of peracetic acid: evaluation of effects on metabolism, structure and cell death.

PubMed

Viola, K S; Rodrigues, E M; Tanomaru-Filho, M; Carlos, I Z; Ramos, S G; Guerreiro-Tanomaru, J M; Faria, G

2017-01-30

To evaluate the cytotoxicity and the mechanism of cell aggression of peracetic acid (PA) in comparison with sodium hypochlorite (NaOCl). L929 fibroblasts were exposed to 1% PA and 2.5% NaOCl, at several dilutions for 10 min. The following parameters were evaluated: cell metabolism by methylthiazol tetrazolium assay, external morphology by scanning electron microscopy, ultrastructure by transmission electron microscopy, the cytoskeleton by means of actin and α-tubulin labelling, and the type of cell death by flow cytometry (apoptosis/necrosis). The data were analysed by two-way anova and the Bonferroni post-test (α = 0.05). The PA group had lower cell viability and a higher percentage of necrotic cells than the NaOCl group (P < 0.05). Both solutions diminished cell metabolism, led to destructuring of the cytoskeleton, created changes in the external morphology, resulted in the accumulation of proteins in the rough endoplasmic reticulum and induced cell death predominantly by necrosis. However, these changes were observed in lower doses of PA when compared with NaOCl. Although they had the same mechanism of cytotoxicity, 1% PA had greater cytotoxic potential than 2.5% NaOCl. © 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.

The Bioactivity and Photocatalytic Properties of Titania Nanotube Coatings Produced with the Use of the Low-Potential Anodization of Ti6Al4V Alloy Surface

PubMed Central

Radtke, Aleksandra; Kozak, Wiesław; Sadowska, Beata; Więckowska-Szakiel, Marzena; Szubka, Magdalena; Talik, Ewa; Pleth Nielsen, Lars; Piszczek, Piotr

2017-01-01

Titania nanotube (TNT) coatings were produced using low-potential anodic oxidation of Ti6Al4V substrates in the potential range 3–20 V. They were analysed by X-ray diffraction (XRD), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM). The wettability was estimated by measuring the contact angle when applying water droplets. The bioactivity of the TNT coatings was established on the basis of the biointegration assay (L929 murine fibroblasts adhesion and proliferation) and antibacterial tests against Staphylococcus aureus (ATCC 29213). The photocatalytic efficiency of the TNT films was studied by the degradation of methylene blue under UV irradiation. Among the studied coatings, the TiO2 nanotubes obtained with the use of 5 V potential (TNT5) were found to be the most appropriate for medical applications. The TNT5 sample possessed antibiofilm properties without enriching it by additional antimicrobial agent. Furthermore, it was characterized by optimal biocompatibility, performing better than pure Ti6Al4V alloy. Moreover, the same sample was the most photocatalytically active and exhibited the potential for the sterilization of implants with the use of UV light and for other environmental applications. PMID:28933732

Biosynthetic hydrogels--studies on chemical and physical characteristics on long-term cellular response for tissue engineering.

PubMed

Thankam, Finosh Gnanaprakasam; Muthu, Jayabalan

2014-07-01

Biosynthetic hydrogels can meet the drawbacks caused by natural and synthetic ones for biomedical applications. In the current article we present a novel biosynthetic alginate-poly(propylene fumarate) copolymer based chemically crosslinked hydrogel scaffolds for cardiac tissue engineering applications. Partially crosslinked PA hydrogel and fully cross linked PA-A hydrogel scaffolds were prepared. The influence of chemical and physical (morphology and architecture of hydrogel) characteristics on the long term cellular response was studied. Both these hydrogels were cytocompatible and showed no genotoxicity upon contact with fibroblast cells. Both PA and PA-A were able to resist deleterious effects of reactive oxygen species and sustain the viability of L929 cells. The hydrogel incubated oxidative stress induced cells were capable of maintaining the intra cellular reduced glutathione (GSH) expression to the normal level confirmed their protective effect. Relatively the PA hydrogel was found to be unstable in the cell culture medium. The PA-A hydrogel was able to withstand appreciable cyclic stretching. The cyclic stretching introduced complex macro and microarchitectural features with interconnected pores and more structured bound water which would provide long-term viability of around 250% after the 24th day of culture. All these qualities make PA-A hydrogel form a potent candidate for cardiac tissue engineering. © 2013 Wiley Periodicals, Inc.

HDPE-Al2O3-HAp composites for biomedical applications: processing and characterizations.

PubMed

Nath, Shekhar; Bodhak, Subhadip; Basu, Bikramjit

2009-01-01

The objective of this work is to demonstrate how the stiffness, hardness, as well as the biocompatibility property, of bioinert high-density polyethylene (HDPE) can be significantly improved by the combined addition of both bioinert and bioactive ceramic fillers. For this purpose, different volume fractions of hydroxyapatite and alumina, limited to a total of 40 vol %, have been incorporated in HDPE matrix. All the hybrid composites and monolithic HDPE were developed under optimized hot pressing condition (130 degrees C, 0.5 h, 92 MPa pressure). The results of the mechanical property characterization reveal that higher elastic modulus (6.2 GPa) and improved hardness (226.5 MPa) could be obtained in the developed HDPE-20 vol %-HAp-20 vol % Al(2)O(3) composite. Under the selected fretting conditions against various counterbody materials (steel, Al(2)O(3), and ZrO(2)), an extremely low COF of (0.07-0.11) and higher wear resistance (order of 10(-6) mm(3)/Nm) are obtained with the HDPE/20 vol % HAp/20 vol % Al(2)O(3) composite in both air and simulated body fluid environment. Importantly, in-vitro cell culture study using L929 fibroblast cells confirms favorable cell adhesion properties in the developed hybrid composite. (c) 2008 Wiley Periodicals, Inc.

Preparation and characterization of magnetic core-shell iron oxide@glycyrrhizic acid nanoparticles in ethanol-water mixed solvent

NASA Astrophysics Data System (ADS)

Saeedi, Mostafa; Vahidi, Omid

2018-06-01

In this research, we used the co-precipitation method to synthesize glycyrrhizic acid coated iron oxide magnetic nanoparticles. The aim of this study is to investigate how different amounts of glycyrrhizic acid affect the structural, biological, magnetic, and hyperthermic characteristics of the synthesized magnetic nanoparticles. The synthesis was conducted under different glycyrrhizic acid concentrations in water with the presence of ethanol to generate coated nanoparticles with different amounts of coating agent. The characteristics of the synthesized nanoparticles were examined by several devices including X-ray diffractometer, transmission electron microscope, field-emission scanning electron microscope, vibrating sample magnetometer, Fourier transform infrared spectra, and thermal gravimetric analyzer. The cytotoxicity of synthesized nanoparticles was examined by MTT assay using L929 fibroblast cell line. The results indicated the enhanced biocompatibility of the coated iron oxide nanoparticles due to the presence of glycyrrhizic acid. The comparison of the coated samples shows that the samples with higher amounts of coating agent were more biocompatible. The possibility of using the synthesized magnetic nanoparticles for medical hyperthermia was examined by performing hyperthermia process on a nanofluid made up of the nanoparticles dispersed in water using a high-frequency alternating magnetic field generator and the results confirm the effectiveness of the synthesized nanoparticles in the elevation of the solutions temperature.

Nano-hydroxyapatite/chitosan-starch nanocomposite as a novel bone construct: Synthesis and in vitro studies.

PubMed

Shakir, Mohammad; Jolly, Reshma; Khan, Mohd Shoeb; Iram, Noor e; Khan, Haris M

2015-09-01

A novel ternary nanocomposite system incorporating hydroxyapatite, chitosan and starch (n-HA/CS-ST) has been synthesized by co-precipitation method at room temperature, addressing the issues of biocompatibility, mechanical strength and cytotoxicity required for bone tissue engineering. The interactions, crystallite size, surface morphology and thermal stability against n-HA/CS nanocomposite have been obtained by comparing the results of FTIR, SEM, TEM, DLS, XRD and TGA/DTA. A comparative study of bioactivity and thermal stability of n-HA/CS and n-HA/CS-ST nanocomposites revealed that the incorporation of starch as templating agent enhanced these properties in n-HA/CS-ST nanocomposite. A lower swelling rate of n-HA/CS-ST relative to n-HA/CS indicates a higher mechanical strength supportive of bone tissue ingrowths. The MTT assay on murine fibroblast L929 and human osteoblasts-like MG-63 cells and in vitro bioactivity of n-HA/CS-ST matrix referred superior non-toxic nature of n-HA/CS-ST nanocomposite and greater possibility of osteointegration in vivo respectively. Furthermore n-HA/CS-ST exhibited improved antibacterial property against both Gram-positive and Gram-negative bacteria relative to n-HA/CS. Copyright © 2015. Published by Elsevier B.V.

A poly(glycerol sebacate) based photo/thermo dual curable biodegradable and biocompatible polymer for biomedical applications.

PubMed

Wang, Min; Lei, Dong; Liu, Zenghe; Chen, Shuo; Sun, Lijie; Lv, Ziying; Huang, Peng; Jiang, Zhongxing; You, Zhengwei

2017-10-01

Due to its biomimetic mechanical properties to soft tissues, excellent biocompatibility and biodegradability, poly (glycerol sebacate) (PGS) has emerged as a representative bioelastomer and been widely used in biomedical engineering. However, the typical curing of PGS needs high temperature (>120 °C), high vacuum (>1 Torr), and long duration (>12 h), which limit its further applications. Accordingly, we designed, synthesized and characterized a photo/thermo dual curable polymer based on PGS. Treatment of PGS with 2-isocyanatoethyl methacrylate without additional reagents readily produced a methacrylated PGS (PGS-IM). Photo-curing of PGS-IM for 10 min at room temperature using salt leaching method efficiently produced porous scaffolds with a thickness up to 1 mm. PGS-IM was adapt to thermo-curing as well. The combination of photo and thermo curing provided a further way to modulate the properties of resultant porous scaffolds. Interestingly, photo-cured scaffolds exhibited hierarchical porous structures carrying extensive micropores with a diameter from several to hundreds micrometers. All the scaffolds showed good elasticity and biodegradability. In addition, PGS-IM exhibited good compatibility with L929 fibroblast cells. We expect this new PGS based biomaterial will have a wide range of biomedical applications.

In vitro evaluation of cytotoxicity of silver-containing borate bioactive glass.

PubMed

Luo, Shi-Hua; Xiao, Wei; Wei, Xiao-Juan; Jia, Wei-Tao; Zhang, Chang-Qing; Huang, Wen-Hai; Jin, Dong-Xu; Rahaman, Mohamed N; Day, Delbert E

2010-11-01

The cytotoxicity of silver-containing borate bioactive glass was evaluated in vitro from the response of osteoblastic and fibroblastic cells in media containing the dissolution products of the glass. Glass frits containing 0-2 weight percent (wt %) Ag were prepared by a conventional melting and quenching process. The amount of Ag dissolved from the glass into a simulated body fluid (SBF), measured using atomic emission spectroscopy, increased rapidly within the first 48 h, but slowed considerably at longer times. Structural and microchemical analysis showed that the formation of a hydroxyapatite-like layer on the glass surface within 14 days of immersion in the SBF. The response of MC3T3-E1 and L929 cells to the dissolution products of the glass was evaluated using SEM observation of cell morphology, and assays of MTT hydrolysis, lactate dehydrogenase release, and alkaline phosphatase activity after incubation for up to 48 h. Cytotoxic effects were found for the borate glass containing 2 wt % Ag, but not for 0.75 and 1 wt % Ag. This borate glass containing up to ∼1 wt % Ag could provide a coating material for bacterial inhibition and enhanced bioactivity of orthopaedic implant materials such as titanium. © 2010 Wiley Periodicals, Inc.

[Neoplastic transformation of mouse fibroblasts under the influence of high-energy protons and gamma-rays].

PubMed

Voskanian, K Sh

2004-01-01

Oncoginic transformations of mouse fibroblasts C3H10T1/2 after exposure to proton energies 150 and 584 MeV were compared with fibroblast effects of gamma-radiation. Prior to exposure, cell populations (2.7 x 10(3) cells/cm2) were inoculated in plastic vials with the surface area of 75 cm2 and cultivated 11 days. Survivability was determined by comparing the number of cell colonies in irradiated and non-irradiated (control) vials. Transformation rate was calculated by dividing the total transformation focus number by the number of survived cells in a vial. Rate of oncogenic transformations after gamma- and proton (584 MeV) irradiation was essentially identical, i.e. the parameter grew rapidly at the doses < 1 Gy and slowed down at the doses > 1 Gy. In the dose interval between 1 and 5 Gy, transformation rate for proton energy 150 MeV was found low compared with gamma-radiation and proton energy 584 MeV. It is hypothesized that the different transformation rate after exposure to proton energy 150 MeV is linked with the high linear energy transfer as compared with the proton energy of 584 MeV and gamma-radiation.

Cardiac fibroblast GSK-3β regulates ventricular remodeling and dysfunction in ischemic heart

PubMed Central

Lal, Hind; Ahmad, Firdos; Zhou, Jibin; Yu, Justine E.; Vagnozzi, Ronald J.; Guo, Yuanjun; Yu, Daohai; Tsai, Emily J.; Woodgett, James; Gao, Erhe; Force, Thomas

2014-01-01

Background Myocardial infarction-induced remodeling includes chamber dilatation, contractile dysfunction, and fibrosis. Of these, fibrosis is the least understood. Following MI, activated cardiac fibroblasts (CFs) deposit extracellular matrix. Current therapies to prevent fibrosis are inadequate and new molecular targets are needed. Methods and Results Herein we report that GSK-3β is phosphorylated (inhibited) in fibrotic tissues from ischemic human and mouse heart. Using two fibroblast-specific GSK-3β knockout mouse models, we show that deletion of GSK-3β in CFs leads to fibrogenesis, left ventricular dysfunction and excessive scarring in the ischemic heart. Deletion of GSK-3β induces a pro-fibrotic myofibroblast phenotype in isolated CFs, in post-MI hearts, and in MEFs deleted for GSK-3β. Mechanistically, GSK-3β inhibits pro-fibrotic TGF-β1-SMAD-3 signaling via interactions with SMAD-3. Moreover, deletion of GSK-3β resulted in the suppression of SMAD-3 transcriptional activity. This pathway is central to the pathology since a small molecule inhibitor of SMAD-3 largely prevented fibrosis and limited LV remodeling. Conclusion These studies support targeting GSK-3β in myocardial fibrotic disorders and establish critical roles of CFs in remodeling and ventricular dysfunction. PMID:24899689

Cardiac Niche Influences the Direct Reprogramming of Canine Fibroblasts into Cardiomyocyte-Like Cells

PubMed Central

Palazzolo, Giacomo; Quattrocelli, Mattia; Toelen, Jaan; Dominici, Roberto; Tettamenti, Guido; Barthelemy, Inès; Blot, Stephane; Gijsbers, Rik; Cassano, Marco

2016-01-01

The Duchenne and Becker muscular dystrophies are caused by mutation of dystrophin gene and primarily affect skeletal and cardiac muscles. Cardiac involvement in dystrophic GRMD dogs has been demonstrated by electrocardiographic studies with the onset of a progressive cardiomyopathy similar to the cardiac disease in DMD patients. In this respect, GRMD is a useful model to explore cardiac and skeletal muscle pathogenesis and for developing new therapeutic protocols. Here we describe a protocol to convert GRMD canine fibroblasts isolated from heart and skin into induced cardiac-like myocytes (ciCLMs). We used a mix of transcription factors (GATA4, HAND2, TBX5, and MEF2C), known to be able to differentiate mouse and human somatic cells into ciCLMs. Exogenous gene expression was obtained using four lentiviral vectors carrying transcription factor genes and different resistance genes. Our data demonstrate a direct switch from fibroblast into ciCLMs with no activation of early cardiac genes. ciCLMs were unable to contract spontaneously, suggesting, differently from mouse and human cells, an incomplete differentiation process. However, when transplanted in neonatal hearts of SCID/Beige mice, ciCLMs participate in cardiac myogenesis. PMID:26681949

Cardiac Niche Influences the Direct Reprogramming of Canine Fibroblasts into Cardiomyocyte-Like Cells.

PubMed

Palazzolo, Giacomo; Quattrocelli, Mattia; Toelen, Jaan; Dominici, Roberto; Anastasia, Luigi; Tettamenti, Guido; Barthelemy, Inès; Blot, Stephane; Gijsbers, Rik; Cassano, Marco; Sampaolesi, Maurilio

2016-01-01

The Duchenne and Becker muscular dystrophies are caused by mutation of dystrophin gene and primarily affect skeletal and cardiac muscles. Cardiac involvement in dystrophic GRMD dogs has been demonstrated by electrocardiographic studies with the onset of a progressive cardiomyopathy similar to the cardiac disease in DMD patients. In this respect, GRMD is a useful model to explore cardiac and skeletal muscle pathogenesis and for developing new therapeutic protocols. Here we describe a protocol to convert GRMD canine fibroblasts isolated from heart and skin into induced cardiac-like myocytes (ciCLMs). We used a mix of transcription factors (GATA4, HAND2, TBX5, and MEF2C), known to be able to differentiate mouse and human somatic cells into ciCLMs. Exogenous gene expression was obtained using four lentiviral vectors carrying transcription factor genes and different resistance genes. Our data demonstrate a direct switch from fibroblast into ciCLMs with no activation of early cardiac genes. ciCLMs were unable to contract spontaneously, suggesting, differently from mouse and human cells, an incomplete differentiation process. However, when transplanted in neonatal hearts of SCID/Beige mice, ciCLMs participate in cardiac myogenesis.

Overexpression of SerpinE2/protease nexin-1 Contribute to Pathological Cardiac Fibrosis via increasing Collagen Deposition

PubMed Central

Li, Xuelian; Zhao, Dandan; Guo, Zhenfeng; Li, Tianshi; Qili, Muge; Xu, Bozhi; Qian, Ming; Liang, Haihai; E, Xiaoqiang; Chege Gitau, Samuel; Wang, Lu; Huangfu, Longtao; Wu, Qiuxia; Xu, Chaoqian; Shan, Hongli

2016-01-01

Although increases in cardiovascular load (pressure overload) are known to elicit ventricular remodeling including cardiomyocyte hypertrophy and interstitial fibrosis, the molecular mechanisms of pressure overload or AngII -induced cardiac interstitial fibrosis remain elusive. In this study, serpinE2/protease nexin-1 was over-expressed in a cardiac fibrosis model induced by pressure-overloaded via transverse aortic constriction (TAC) in mouse. Knockdown of serpinE2 attenuates cardiac fibrosis in a mouse model of TAC. At meantime, the results showed that serpinE2 significantly were increased with collagen accumulations induced by AngII or TGF-β stimulation in vitro. Intriguingly, extracellular collagen in myocardial fibroblast was reduced by knockdown of serpinE2 compared with the control in vitro. In stark contrast, the addition of exogenous PN-1 up-regulated the content of collagen in myocardial fibroblast. The MEK1/2- ERK1/2 signaling probably promoted the expression of serpinE2 via transcription factors Elk1 in myocardial fibroblast. In conclusion, stress-induced the ERK1/2 signaling pathway activation up-regulated serpinE2 expression, consequently led accumulation of collagen protein, and contributed to cardiac fibrosis. PMID:27876880

Optimization of Direct Fibroblast Reprogramming to Cardiomyocytes Using Calcium Activity as a Functional Measure of Success

PubMed Central

Addis, Russell C.; Ifkovits, Jamie L.; Pinto, Filipa; Kellam, Lori D.; Esteso, Paul; Rentschler, Stacey; Christoforou, Nicolas; Epstein, Jonathan A.; Gearhart, John D.

2013-01-01

Direct conversion of fibroblasts to induced cardiomyocytes (iCMs) has great potential for regenerative medicine. Recent publications have reported significant progress, but the evaluation of reprogramming has relied upon non-functional measures such as flow cytometry for cardiomyocyte markers or GFP expression driven by a cardiomyocyte-specific promoter. The issue is one of practicality: the most stringent measures - electrophysiology to detect cell excitation and the presence of spontaneously contracting myocytes - are not readily quantifiable in the large numbers of cells screened in reprogramming experiments. However, excitation and contraction are linked by a third functional characteristic of cardiomyocytes: the rhythmic oscillation of intracellular calcium levels. We set out to optimize direct conversion of fibroblasts to iCMs with a quantifiable calcium reporter to rapidly assess functional transdifferentiation. We constructed a reporter system in which the calcium indicator GCaMP is driven by the cardiomyocyte-specific Troponin T promoter. Using calcium activity as our primary outcome measure, we compared several published combinations of transcription factors along with novel combinations in mouse embryonic fibroblasts. The most effective combination consisted of Hand2, Nkx2.5, Gata4, Mef2c, and Tbx5 (HNGMT). This combination is >50-fold more efficient than GMT alone and produces iCMs with cardiomyocyte marker expression, robust calcium oscillation, and spontaneous beating that persists for weeks following inactivation of reprogramming factors. HNGMT is also significantly more effective than previously published factor combinations for the transdifferentiation of adult mouse cardiac fibroblasts to iCMs. Quantification of calcium function is a convenient and effective means for the identification and evaluation of cardiomyocytes generated by direct reprogramming. Using this stringent outcome measure, we conclude that HNGMT produces iCMs more efficiently than previously published methods. PMID:23591016

Syndecan-2 Attenuates Radiation-induced Pulmonary Fibrosis and Inhibits Fibroblast Activation by Regulating PI3K/Akt/ROCK Pathway via CD148.

PubMed

Tsoyi, Konstantin; Chu, Sarah G; Patino-Jaramillo, Nasly G; Wilder, Julie; Villalba, Julian; Doyle-Eisele, Melanie; McDonald, Jacob; Liu, Xiaoli; El-Chemaly, Souheil; Perrella, Mark A; Rosas, Ivan O

2018-02-01

Radiation-induced pulmonary fibrosis is a severe complication of patients treated with thoracic irradiation. We have previously shown that syndecan-2 reduces fibrosis by exerting alveolar epithelial cytoprotective effects. Here, we investigate whether syndecan-2 attenuates radiation-induced pulmonary fibrosis by inhibiting fibroblast activation. C57BL/6 wild-type mice and transgenic mice that overexpress human syndecan-2 in alveolar macrophages were exposed to 14 Gy whole-thoracic radiation. At 24 weeks after irradiation, lungs were collected for histological, protein, and mRNA evaluation of pulmonary fibrosis, profibrotic gene expression, and α-smooth muscle actin (α-SMA) expression. Mouse lung fibroblasts were activated with transforming growth factor (TGF)-β1 in the presence or absence of syndecan-2. Cell proliferation, migration, and gel contraction were assessed at different time points. Irradiation resulted in significantly increased mortality and pulmonary fibrosis in wild-type mice that was associated with elevated lung expression of TGF-β1 downstream target genes and cell death compared with irradiated syndecan-2 transgenic mice. In mouse lung fibroblasts, syndecan-2 inhibited α-SMA expression, cell contraction, proliferation, and migration induced by TGF-β1. Syndecan-2 attenuated phosphoinositide 3-kinase/serine/threonine kinase/Rho-associated coiled-coil kinase signaling and serum response factor binding to the α-SMA promoter. Syndecan-2 attenuates pulmonary fibrosis in mice exposed to radiation and inhibits TGF-β1-induced fibroblast-myofibroblast differentiation, migration, and proliferation by down-regulating phosphoinositide 3-kinase/serine/threonine kinase/Rho-associated coiled-coil kinase signaling and blocking serum response factor binding to the α-SMA promoter via CD148. These findings suggest that syndecan-2 has potential as an antifibrotic therapy in radiation-induced lung fibrosis.

Identification of sirtuin 1 as a promising therapeutic target for hypertrophic scars

PubMed Central

Bai, Xiao‐Zhi; Liu, Jia‐Qi; Yang, Long‐Long; Fan, Lei; He, Ting; Su, Lin‐Lin; Shi, Ji‐Hong; Tang, Chao‐Wu

2016-01-01

Background and Purpose Sirtuin1 (SIRT1), the founding member of mammalian class III histone deacetylases, is reported to be a drug target involved in fibrotic diseases. However, whether it is an effective drug target in hypertrophic scar treatment is still not known. Experimental Approach In the present study, we observed that SIRT1 localized to both the epidermis and the dermis of skin tissues by immunohistochemistry. After knock‐down of SIRT1 by shRNA or up‐regulating SIRT1 by resveratrol, the expression of α‐SMA, Col1 and Col3 in fibroblasts were detected by western blots. A mouse excision wound healing model was used to observe the changes in collagen fibre associated with the different expression levels of SIRT1. Key Results SIRT1 expression was inhibited in hypertrophic scar tissue. The down‐regulation of SIRT1 resulted in an increased expression of α‐SMA, Col1 and Col3 in hypertrophic scar‐derived fibroblasts. In contrast, the up‐regulation of SIRT1 not only inhibited the expression of α‐SMA, Col1 and Col3 in hypertrophic scar‐derived fibroblasts but also blocked the activation of TGFβ1‐induced normal skin‐derived fibroblasts. In the mouse model of wound healing, the deletion of SIRT1 resulted in denser collagen fibres and a more disordered structure, whereas resveratrol treatment led to a more organized and thinner collagen fibre, which was similar to that observed during normal wound healing. Conclusions and Implications The results revealed that SIRT1 negatively regulates TGFβ1‐induced fibroblast activation and inhibits excessive scar formation and is, therefore, a promising drug target for hypertrophic scar formation. PMID:26891034

Epicardially-derived Fibroblasts Preferentially Contribute to the Parietal Leaflets of the Atrioventricular Valves in the Murine Heart

PubMed Central

Wessels, Andy; van den Hoff, Maurice J. B.; Adamo, Richard F.; Phelps, Aimee L.; Lockhart, Marie M.; Sauls, Kimberly; Briggs, Laura E.; Norris, Russell A.; van Wijk, Bram; Perez-Pomares, Jose M.; Dettman, Robert W.; Burch, John B. E.

2012-01-01

The importance of the epicardium for myocardial and valvuloseptal development has been well established; perturbation of epicardial development results in cardiac abnormalities, including thinning of the ventricular myocardial wall and malformations of the atrioventricular valvuloseptal complex. To determine the spatiotemporal contribution of epicardially derived cells to the developing fibroblast population in the heart we have used a mWt1/IRES/GFP-Cre mouse to trace the fate of EPDCs from embryonic day (ED)10 until birth. EPDCs begin to populate the compact ventricular myocardium around ED12. The migration of epicardially-derived fibroblasts toward the interface between compact and trabecular myocardium is completed around ED14. Remarkably, epicardially-derived fibroblasts do not migrate into the trabecular myocardium until after ED17. Migration of EPDCs into the atrioventricular cushion mesenchyme commences around ED12. As development progresses, the number of EPDCs increases significantly, specifically in the leaflets which derive from the lateral atrioventricular cushions. In these developing leaflets the epicardially-derived fibroblasts eventually largely replace the endocardially-derived cells. Importantly, the contribution of EPDCs to the leaflets derived from the major AV cushions is very limited. The differential contribution of EPDCs to the various leaflets of the atrioventricular valves provides a new paradigm in valve development and could lead to new insights into the pathogenesis of abnormalities that preferentially affect individual components of this region of the heart. The notion that there is a significant difference in the contribution of epicardially and endocardially derived cells to the individual leaflets of the atrioventricular valves has also important pragmatic consequences for the use of endocardial and epicardial cre-mouse models in studies of heart development. PMID:22546693

Activation and overexpression of Sirt1 attenuates lung fibrosis via P300.

PubMed

Zeng, Zhilin; Cheng, Sheng; Chen, Huilong; Li, Qinghai; Hu, Yinan; Wang, Qi; Zhu, Xianying; Wang, Jun

2017-05-13

Persistent fibroblast activation is a predominant feature of idiopathic pulmonary fibrosis (IPF), but the transcriptional and epigenetic mechanisms controlling this process are not well understood. Silent information regulator type-1 (Sirt1) is a member of class Ⅲ histone deacetylase with important regulatory roles in a variety of pathophysiologic processes, but its role in fibrotic lung diseases is not clearly elucidated. Sirt1 expression in lung tissues of IPF patients and in a mouse model of bleomycin (BLM)-induced lung fibrosis were evaluated by immunofluorescence. The function of Sirt1 in BLM-induced lung fibrosis in the mouse model or transforming growth factor β1 (TGF-β1)-mediated lung fibroblast cellular model was investigated by Sirt1 activation, overexpression and knockdown of Sirt1. Finally, the involvement of p300 signaling pathways was assessed. In this study, we found up-regulation of Sirt1 in BLM-induced lung fibrosis, as well as in the lungs of IPF patients, including in the aggregated pulmonary fibroblasts of fibrotic foci. Activation or overexpression of Sirt1 attenuated TGF-β1-mediated lung fibroblast differentiation and activation and diminished the severity of experimental lung fibrosis in mice. Whereas knockdown of Sirt1 promoted the pro-fibrogenic activity of TGF-β1 in lung fibroblasts. A potential mechanism for the role of Sirt1 in lung fibrosis was through regulating the expression of p300. Thus, we characterized Sirt1 as an important regulator of lung fibrosis and provides a proof of principle for activation or overexpression of Sirt1 as a potential novel therapeutic strategy for IPF. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression of XBP1s in fibroblasts is critical for TiAl6 V4 particle-induced RANKL expression and osteolysis.

PubMed

Wang, Zhenheng; Liu, Naicheng; Zhou, Gang; Shi, Tongguo; Wang, Zhenzhen; Gan, Jingjing; Wang, Rui; Qian, Hongbo; Bao, Nirong; Guo, Ting; Zhao, Jianning

2017-04-01

Wear particle-induced osteolysis is a major cause of aseptic loosening, which is one of the most common reasons for total hip arthroplasty (THA) failure. Previous studies have shown that the expression of Receptor activation of nuclear factor (NF)-kB (RANKL) by fibroblasts in periprosthetic membrane played a crucial role in wear particle-induced osteolysis. However, the underlying mechanism of RANKL expression remains largely unknown. In the present study, we investigated the effect of TiAl 6 V 4 particle (TiPs)-induced XBP1s (spliced form of X-box binding protein 1) on RANKL expression and osteoclastogenesis both in vitro and in vivo. The levels of XBP1s in peri-implant membrane, animal models, and TiPs-stimulated fibroblasts were determined by western blots. To assess the effect of XBP1s on RANKL expression, fibroblasts were treated with both a small interfering RNA (siRNA) and an inhibitor of XBP1 prior to exposure to TiPs. The effect of XBP1s on osteoclasts formation was determined by tartrate-resistant acid phosphatase (TRAP) staining in vitro osteoclastogenesis assay and in animal models. The resorption of bone was assessed by micro-computed tomography (micro-CT) with three-dimensional reconstruction. Our results demonstrated that XBP1s was activated in periprosthetic membrane, mouse calvaria models, and TiPs-stimulated human synovial fibroblasts. Further, inhibition of XBP1s decreased the expression of RANKL and osteoclasts formation in vitro. In mouse calvaria models, both of the osteoclastogenesis and osteolysis were inhibited XBP1s inhibitor. Our results suggested that XBP1s mediated TiPs-induced of RANKL expression in fibroblasts, and down regulating XBP1s may represent a potential therapy for wear particle-induced osteolysis. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:752-759, 2017. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

The Initiator Methionine tRNA Drives Secretion of Type II Collagen from Stromal Fibroblasts to Promote Tumor Growth and Angiogenesis

PubMed Central

Clarke, Cassie J.; Berg, Tracy J.; Birch, Joanna; Ennis, Darren; Mitchell, Louise; Cloix, Catherine; Campbell, Andrew; Sumpton, David; Nixon, Colin; Campbell, Kirsteen; Bridgeman, Victoria L.; Vermeulen, Peter B.; Foo, Shane; Kostaras, Eleftherios; Jones, J. Louise; Haywood, Linda; Pulleine, Ellie; Yin, Huabing; Strathdee, Douglas; Sansom, Owen; Blyth, Karen; McNeish, Iain; Zanivan, Sara; Reynolds, Andrew R.; Norman, Jim C.

2016-01-01

Summary Expression of the initiator methionine tRNA (tRNAiMet) is deregulated in cancer. Despite this fact, it is not currently known how tRNAiMet expression levels influence tumor progression. We have found that tRNAiMet expression is increased in carcinoma-associated fibroblasts, implicating deregulated expression of tRNAiMet in the tumor stroma as a possible contributor to tumor progression. To investigate how elevated stromal tRNAiMet contributes to tumor progression, we generated a mouse expressing additional copies of the tRNAiMet gene (2+tRNAiMet mouse). Growth and vascularization of subcutaneous tumor allografts was enhanced in 2+tRNAiMet mice compared with wild-type littermate controls. Extracellular matrix (ECM) deposited by fibroblasts from 2+tRNAiMet mice supported enhanced endothelial cell and fibroblast migration. SILAC mass spectrometry indicated that elevated expression of tRNAiMet significantly increased synthesis and secretion of certain types of collagen, in particular type II collagen. Suppression of type II collagen opposed the ability of tRNAiMet-overexpressing fibroblasts to deposit pro-migratory ECM. We used the prolyl hydroxylase inhibitor ethyl-3,4-dihydroxybenzoate (DHB) to determine whether collagen synthesis contributes to the tRNAiMet-driven pro-tumorigenic stroma in vivo. DHB had no effect on the growth of syngeneic allografts in wild-type mice but opposed the ability of 2+tRNAiMet mice to support increased angiogenesis and tumor growth. Finally, collagen II expression predicts poor prognosis in high-grade serous ovarian carcinoma. Taken together, these data indicate that increased tRNAiMet levels contribute to tumor progression by enhancing the ability of stromal fibroblasts to synthesize and secrete a type II collagen-rich ECM that supports endothelial cell migration and angiogenesis. PMID:26948875

The Initiator Methionine tRNA Drives Secretion of Type II Collagen from Stromal Fibroblasts to Promote Tumor Growth and Angiogenesis.

PubMed

Clarke, Cassie J; Berg, Tracy J; Birch, Joanna; Ennis, Darren; Mitchell, Louise; Cloix, Catherine; Campbell, Andrew; Sumpton, David; Nixon, Colin; Campbell, Kirsteen; Bridgeman, Victoria L; Vermeulen, Peter B; Foo, Shane; Kostaras, Eleftherios; Jones, J Louise; Haywood, Linda; Pulleine, Ellie; Yin, Huabing; Strathdee, Douglas; Sansom, Owen; Blyth, Karen; McNeish, Iain; Zanivan, Sara; Reynolds, Andrew R; Norman, Jim C

2016-03-21

Expression of the initiator methionine tRNA (tRNAi(Met)) is deregulated in cancer. Despite this fact, it is not currently known how tRNAi(Met) expression levels influence tumor progression. We have found that tRNAi(Met) expression is increased in carcinoma-associated fibroblasts, implicating deregulated expression of tRNAi(Met) in the tumor stroma as a possible contributor to tumor progression. To investigate how elevated stromal tRNAi(Met) contributes to tumor progression, we generated a mouse expressing additional copies of the tRNAi(Met) gene (2+tRNAi(Met) mouse). Growth and vascularization of subcutaneous tumor allografts was enhanced in 2+tRNAi(Met) mice compared with wild-type littermate controls. Extracellular matrix (ECM) deposited by fibroblasts from 2+tRNAi(Met) mice supported enhanced endothelial cell and fibroblast migration. SILAC mass spectrometry indicated that elevated expression of tRNAi(Met) significantly increased synthesis and secretion of certain types of collagen, in particular type II collagen. Suppression of type II collagen opposed the ability of tRNAi(Met)-overexpressing fibroblasts to deposit pro-migratory ECM. We used the prolyl hydroxylase inhibitor ethyl-3,4-dihydroxybenzoate (DHB) to determine whether collagen synthesis contributes to the tRNAi(Met)-driven pro-tumorigenic stroma in vivo. DHB had no effect on the growth of syngeneic allografts in wild-type mice but opposed the ability of 2+tRNAi(Met) mice to support increased angiogenesis and tumor growth. Finally, collagen II expression predicts poor prognosis in high-grade serous ovarian carcinoma. Taken together, these data indicate that increased tRNAi(Met) levels contribute to tumor progression by enhancing the ability of stromal fibroblasts to synthesize and secrete a type II collagen-rich ECM that supports endothelial cell migration and angiogenesis. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

An interaction between L-prostaglandin D synthase and arrestin increases PGD2 production.

PubMed

Mathurin, Karine; Gallant, Maxime A; Germain, Pascale; Allard-Chamard, Hugues; Brisson, Jessy; Iorio-Morin, Christian; de Brum Fernandes, Artur; Caron, Marc G; Laporte, Stéphane A; Parent, Jean-Luc

2011-01-28

L-type prostaglandin synthase (L-PGDS) produces PGD(2), a lipid mediator involved in neuromodulation and inflammation. Here, we show that L-PGDS and arrestin-3 (Arr3) interact directly and can be co-immunoprecipitated endogenously from MG-63 osteoblasts. Perinuclear L-PGDS/Arr3 co-localization is observed in PGD(2)-producing MG-63 cells and is induced by the addition of the L-PGDS substrate or co-expression of COX-2 in HEK293 cells. Inhibition of L-PGDS activity in MG-63 cells triggers redistribution of Arr3 and L-PGDS to the cytoplasm. Perinuclear localization of L-PGDS is detected in wild-type mouse embryonic fibroblasts (MEFs) but is more diffused in MEFs-arr-2(-/-)-arr-3(-/-). Arrestin-3 promotes PGD(2) production by L-PGDS in vitro. IL-1β-induced PGD(2) production is significantly lower in MEFs-arr-2(-/-)-arr-3(-/-) than in wild-type MEFs but can be rescued by expressing Arr2 or Arr3. A peptide corresponding to amino acids 86-100 of arrestin-3 derived from its L-PGDS binding domain stimulates L-PGDS-mediated PGD(2) production in vitro and in MG-63 cells. We report the first characterization of an interactor/modulator of a PGD(2) synthase and the identification of a new function for arrestin, which may open new opportunities for improving therapies for the treatment of inflammatory diseases.

The sesquiterpene (-)-α-bisabolol is active against the causative agents of Old World cutaneous leishmaniasis through the induction of mitochondrial-dependent apoptosis.

PubMed

Corpas-López, V; Merino-Espinosa, G; Díaz-Sáez, V; Morillas-Márquez, F; Navarro-Moll, M C; Martín-Sánchez, J

2016-10-01

Cutaneous leishmaniasis treatment remains challenging due to the absence of a satisfactory treatment. The screening of natural compounds is a valuable strategy in the search of new drugs against leishmaniasis. The sesquiterpene (-)-α-bisabolol is effective in vivo against visceral leishmaniasis due to Leishmania infantum, but its mechanism of action remains elusive. The aim of this study is to validate this promising compound against the causative species of Old World cutaneous leishmaniasis and to get an insight into its antileishmanial mode of action. The compound was evaluated on L. tropica promastigotes and intracellular amastigotes using bone marrow-derived macrophages and its cytotoxicity was evaluated on L929 fibroblasts. The reactive oxygen species generation was evaluated using a sensitive probe. Mitochondrial depolarization was assessed evaluating the fluorescence due to rhodamine 123 in a flow cytometer. Apoptosis was investigated by measuring the fluorescence due to annexin V and propidium iodide in a flow cytometer. The ultrastructure of treated promastigotes and intracellular amastigotes was analysed through transmission electron microscopy. (-)-α-Bisabolol was active against L. tropica intracellular amastigotes displaying an inhibitory concentration 50 % of 25.2 µM and showing low cytotoxicity. This compound induced time and dose-dependent oxidative stress, mitochondrial depolarization and phosphatidilserine externalization (a marker of apoptosis). These effects were noticed at a low concentration and short exposure time. In the ultrastructural analyses, the treated parasites showed mitochondrial disruption, presence of electron-dense structures and chromatin condensation. These results suggest that this natural compound induces oxidative stress and mitochondrial-dependent apoptosis on Leishmania without disturbing the plasma membrane.

Connective tissue growth factor/CCN2-null mouse embryonic fibroblasts retain intact transforming growth factor-{beta} responsiveness

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mori, Yasuji; Hinchcliff, Monique; Wu, Minghua

2008-03-10

Background: The matricellular protein connective tissue growth factor (CCN2) has been implicated in pathological fibrosis, but its physiologic role remains elusive. In vitro, transforming growth factor-{beta} (TGF-{beta}) induces CCN2 expression in mesenchymal cells. Because CCN2 can enhance profibrotic responses elicited by TGF-{beta}, it has been proposed that CCN2 functions as an essential downstream signaling mediator for TGF-{beta}. To explore this notion, we characterized TGF-{beta}-induced activation of fibroblasts from CCN2-null (CCN2{sup -/-}) mouse embryos. Methods: The regulation of CCN2 expression was examined in vivo in a model of fibrosis induced by bleomycin. Cellular TGF-{beta} signal transduction and regulation of collagen genemore » expression were examined in CCN2{sup -/-} MEFs by immunohistochemistry, Northern, Western and RT-PCR analysis, immunocytochemistry and transient transfection assays. Results: Bleomycin-induced skin fibrosis in the mouse was associated with substantial CCN2 up-regulation in lesional fibroblasts. Whereas in vitro proliferation rate of CCN2{sup -/-} MEFs was markedly reduced compared to wild type MEFs, TGF-{beta}-induced activation of the Smad pathways, including Smad2 phosphorylation, Smad2/3 and Smad4 nuclear accumulation and Smad-dependent transcriptional responses, were unaffected by loss of CCN2. The stimulation of COL1A2 and fibronectin mRNA expression and promoter activity, and of corresponding protein levels, showed comparable time and dose-response in wild type and CCN2{sup -/-} MEFs, whereas stimulation of alpha smooth muscle actin and myofibroblast transdifferentiation showed subtle impairment in MEFs lacking CCN2. Conclusion: Whereas endogenous CCN2 plays a role in regulation of proliferation and TGF-{beta}-induced myofibroblast transdifferentiation, it appears to be dispensable for Smad-dependent stimulation of collagen and extracellular matrix synthesis in murine embryonic fibroblasts.« less

Knock-in reporter mice demonstrate that DNA repair by non-homologous end joining declines with age.

PubMed

Vaidya, Amita; Mao, Zhiyong; Tian, Xiao; Spencer, Brianna; Seluanov, Andrei; Gorbunova, Vera

2014-07-01

Accumulation of genome rearrangements is a characteristic of aged tissues. Since genome rearrangements result from faulty repair of DNA double strand breaks (DSBs), we hypothesized that DNA DSB repair becomes less efficient with age. The Non-Homologous End Joining (NHEJ) pathway repairs a majority of DSBs in vertebrates. To examine age-associated changes in NHEJ, we have generated an R26NHEJ mouse model in which a GFP-based NHEJ reporter cassette is knocked-in to the ROSA26 locus. In this model, NHEJ repair of DSBs generated by the site-specific endonuclease, I-SceI, reconstitutes a functional GFP gene. In this system NHEJ efficiency can be compared across tissues of the same mouse and in mice of different age. Using R26NHEJ mice, we found that NHEJ efficiency was higher in the skin, lung, and kidney fibroblasts, and lower in the heart fibroblasts and brain astrocytes. Furthermore, we observed that NHEJ efficiency declined with age. In the 24-month old animals compared to the 5-month old animals, NHEJ efficiency declined 1.8 to 3.8-fold, depending on the tissue, with the strongest decline observed in the skin fibroblasts. The sequence analysis of 300 independent NHEJ repair events showed that, regardless of age, mice utilize microhomology sequences at a significantly higher frequency than expected by chance. Furthermore, the frequency of microhomology-mediated end joining (MMEJ) events increased in the heart and lung fibroblasts of old mice, suggesting that NHEJ becomes more mutagenic with age. In summary, our study provides a versatile mouse model for the analysis of NHEJ in a wide range of tissues and demonstrates that DNA repair by NHEJ declines with age in mice, which could provide a mechanism for age-related genomic instability and increased cancer incidence with age.

Reprogramming mouse fibroblasts into engraftable myeloerythroid and lymphoid progenitors

PubMed Central

Cheng, Hui; Ang, Heather Yin-Kuan; A. EL Farran, Chadi; Li, Pin; Fang, Hai Tong; Liu, Tong Ming; Kong, Say Li; Chin, Michael Lingzi; Ling, Wei Yin; Lim, Edwin Kok Hao; Li, Hu; Huber, Tara; Loh, Kyle M.; Loh, Yuin-Han; Lim, Bing

2016-01-01

Recent efforts have attempted to convert non-blood cells into hematopoietic stem cells (HSCs) with the goal of generating blood lineages de novo. Here we show that hematopoietic transcription factors Scl, Lmo2, Runx1 and Bmi1 can convert a developmentally distant lineage (fibroblasts) into ‘induced hematopoietic progenitors' (iHPs). Functionally, iHPs generate acetylcholinesterase+ megakaryocytes and phagocytic myeloid cells in vitro and can also engraft immunodeficient mice, generating myeloerythoid and B-lymphoid cells for up to 4 months in vivo. Molecularly, iHPs transcriptionally resemble native Kit+ hematopoietic progenitors. Mechanistically, reprogramming factor Lmo2 implements a hematopoietic programme in fibroblasts by rapidly binding to and upregulating the Hhex and Gfi1 genes within days. Moreover the reprogramming transcription factors also require extracellular BMP and MEK signalling to cooperatively effectuate reprogramming. Thus, the transcription factors that orchestrate embryonic hematopoiesis can artificially reconstitute this programme in developmentally distant fibroblasts, converting them into engraftable blood progenitors. PMID:27869129

Heart repair by reprogramming non-myocytes with cardiac transcription factors

PubMed Central

Song, Kunhua; Nam, Young-Jae; Luo, Xiang; Qi, Xiaoxia; Tan, Wei; Huang, Guo N.; Acharya, Asha; Smith, Christopher L.; Tallquist, Michelle D.; Neilson, Eric G.; Hill, Joseph A.; Bassel-Duby, Rhonda; Olson, Eric N.

2012-01-01

The adult mammalian heart possesses little regenerative potential following injury. Fibrosis due to activation of cardiac fibroblasts impedes cardiac regeneration and contributes to loss of contractile function, pathological remodeling and susceptibility to arrhythmias. Cardiac fibroblasts account for a majority of cells in the heart and represent a potential cellular source for restoration of cardiac function following injury through phenotypic reprogramming to a myocardial cell fate. Here we show that four transcription factors, GATA4, Hand2, MEF2C and Tbx5 can cooperatively reprogram adult mouse tail-tip and cardiac fibroblasts into beating cardiac-like myocytes in vitro. Forced expression of these factors in dividing non-cardiomyocytes in mice reprograms these cells into functional cardiac-like myocytes, improves cardiac function and reduces adverse ventricular remodeling following myocardial infarction. Our results suggest a strategy for cardiac repair through reprogramming fibroblasts resident in the heart with cardiogenic transcription factors or other molecules. PMID:22660318

IGFBP-3, hypoxia and TNF-{alpha} inhibit adiponectin transcription

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zappala, Giovanna, E-mail: zappalag@mail.nih.gov; Rechler, Matthew M.; Clinical Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD

2009-05-15

The thiazolidinedione rosiglitazone, an agonist ligand for the nuclear receptor PPAR-{gamma}, improves insulin sensitivity in part by stimulating transcription of the insulin-sensitizing adipokine adiponectin. It activates PPAR-{gamma}-RXR-{alpha} heterodimers bound to PPAR-{gamma} response elements in the adiponectin promoter. Rosiglitazone-stimulated adiponectin protein synthesis in 3T3-L1 mouse adipocytes has been shown to be inhibited by IGFBP-3, which can be induced by hypoxia and the proinflammatory cytokine, TNF-{alpha}, two inhibitors of adiponectin transcription. The present study demonstrates that IGFBP-3, the hypoxia-mimetic agent cobalt chloride, and TNF-{alpha} inhibit rosiglitazone-induced adiponectin transcription in mouse embryo fibroblasts that stably express PPAR-{gamma}2. Native IGFBP-3 can bind RXR-{alpha} andmore » inhibited rosiglitazone stimulated promoter activity, whereas an IGFBP-3 mutant that does not bind RXR-{alpha} did not. These results suggest that IGFBP-3 may mediate the inhibition of adiponectin transcription by hypoxia and TNF-{alpha}, and that IGFBP-3 binding to RXR-{alpha} may be required for the observed inhibition.« less

Spectroellipsometric, AFM and XPS probing of stainless steel surfaces subjected to biological influences

NASA Astrophysics Data System (ADS)

Vinnichenko, M.; Chevolleau, Th; Pham, M. T.; Poperenko, L.; Maitz, M. F.

2002-11-01

Surface modification of austenitic stainless steel (SS) 316L after incubation in growing cell cultures and cell-free media as control has been studied. The following treatments were applied: mouse fibrosarcoma cells L929 for 3 and 7 days, polymorphonuclear neutrophils for 3 and 7 days and human osteosarcoma cells SAOS-2 for 7 and 14 days. Cells were enzymatically removed in all cases. The modified surfaces were probed in comparison with untreated ones by means of spectroscopic ellipsometry (SE), X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). XPS shows the appearance of the peak of bonded nitrogen at 400.5 eV characteristic for adsorbed proteins on the surface for each type of cells and for the cell-free medium. Migration of Ni in the adsorbed layer is observed in all cases for samples after the cell cultures. The protein layer thickness is ellipsometrically determined to be within 2.5-6.0 nm for all treated samples with parameterization of its optical constants in Cauchy approach. The study showed that for such biological treatments of the SS the protein layer adsorption is the dominating process in the first 2 weeks, which could play a role in the process of corrosion by complex forming properties with metal ions.

Endosomal accumulation of Toll-like receptor 4 causes constitutive secretion of cytokines and activation of signal transducers and activators of transcription in Niemann-Pick disease type C (NPC) fibroblasts: a potential basis for glial cell activation in the NPC brain.

PubMed

Suzuki, Michitaka; Sugimoto, Yuko; Ohsaki, Yuki; Ueno, Makoto; Kato, Shinsuke; Kitamura, Yukisato; Hosokawa, Hiroshi; Davies, Joanna P; Ioannou, Yiannis A; Vanier, Marie T; Ohno, Kousaku; Ninomiya, Haruaki

2007-02-21

Niemann-Pick disease type C (NPC) is an inherited lipid storage disorder caused by mutations in NPC1 or NPC2 genes. Loss of function of either protein results in the endosomal accumulation of cholesterol and other lipids, progressive neurodegeneration, and robust glial cell activation. Here, we report that cultured human NPC fibroblasts secrete interferon-beta, interleukin-6 (IL-6), and IL-8, and contain increased levels of signal transducers and activators of transcription (STATs). These cells also contained increased levels of Toll-like receptor 4 (TLR4) that accumulated in cholesterol-enriched endosomes/lysosomes, and small interfering RNA knockdown of this receptor reduced cytokine secretion. In the NPC1-/- mouse brain, glial cells expressed TLR4 and IL-6, whereas both glial and neuronal cells expressed STATs. Genetic deletion of TLR4 in NPC1-/- mice reduced IL-6 secretion by cultured fibroblasts but failed to alter STAT levels or glial cell activation in the brain. In contrast, genetic deletion of IL-6 normalized STAT levels and suppressed glial cell activation. These findings indicate that constitutive cytokine secretion leads to activation of STATs in NPC fibroblasts and that this secretion is partly caused by an endosomal accumulation of TLR4. These results also suggest that similar signaling events may underlie glial cell activation in the NPC1-/- mouse brain.

Prolongation of the survival of breast cancer-bearing mice immunized with GM-CSF-secreting syngeneic/allogeneic fibroblasts transfected with a cDNA expression library from breast cancer cells.

PubMed

Kim, Tae S; Jung, Mi Y; Cho, Daeho; Cohen, Edward P

2006-10-30

Breast cancer cells, like other types of neoplastic cells, form weakly immunogenic tumor-associated antigens. The antigenic properties of the tumor-associated antigens can be enhanced if they are expressed by highly immunogenic cells. In this study, a cancer vaccine was prepared by transfer of a cDNA expression library from SB5b breast carcinoma into mouse fibroblast cells of C3H/He mouse origin (H-2(k)), that had been previously modified to secrete GM-CSF and to express allogeneic class I-determinants (H-2(b)). The transfected syngeneic/allogeneic fibroblasts secreting GM-CSF were used as a vaccine in C3H/He mice. Robust cell-mediated immunity toward the breast cancer cells was generated in mice immunized with the cDNA-based vaccine. The immunity, mediated predominantly by CD8(+) T lymphocytes, was directed toward the breast cancer cells, but not against either of two other non-cross-reactive neoplasms of C3H/He mice. The immunity was sufficient to prolong the survival of mice with established breast cancer. Among other advantages, preparation of the vaccine by cDNA-transfer into a fibroblast cell line enabled the recipient cells to be modified in advance of DNA-transfer to augment their immunogenic properties. As the transferred DNA is replicated as the transfected cells divide, the vaccine could be prepared from microgram quantities of tumor tissue.

RickA Expression Is Not Sufficient to Promote Actin-Based Motility of Rickettsia raoultii

PubMed Central

Balraj, Premanand; Karkouri, Khalid El; Vestris, Guy; Espinosa, Leon; Raoult, Didier; Renesto, Patricia

2008-01-01

Background Rickettsia raoultii is a novel Rickettsia species recently isolated from Dermacentor ticks and classified within the spotted fever group (SFG). The inability of R. raoultii to spread within L929 cells suggests that this bacterium is unable to polymerize host cell actin, a property exhibited by all SFG rickettsiae except R. peacocki. This result led us to investigate if RickA, the protein thought to generate actin nucleation, was expressed within this rickettsia species. Methodology/Principal Findings Amplification and sequencing of R. raoultii rickA showed that this gene encoded a putative 565 amino acid protein highly homologous to those found in other rickettsiae. Using immunofluorescence assays, we determined that the motility pattern (i.e. microcolonies or cell-to-cell spreading) of R. raoultii was different depending on the host cell line in which the bacteria replicated. In contrast, under the same experimental conditions, R. conorii shares the same phenotype both in L929 and in Vero cells. Transmission electron microscopy analysis of infected cells showed that non-motile bacteria were free in the cytosol instead of enclosed in a vacuole. Moreover, western-blot analysis demonstrated that the defect of R. raoultii actin-based motility within L929 cells was not related to lower expression of RickA. Conclusion/Significance These results, together with previously published data about R. typhi, strongly suggest that another factor, apart from RickA, may be involved with be responsible for actin-based motility in bacteria from the Rickettsia genus. PMID:18612416

Rhodamine B conjugates of triterpenoic acids are cytotoxic mitocans even at nanomolar concentrations.

PubMed

Sommerwerk, Sven; Heller, Lucie; Kerzig, Christoph; Kramell, Annemarie E; Csuk, René

2017-02-15

Triterpenoic acids 1-6 exhibited very low or no cytotoxicity at all, but their corresponding 2,3-di-O-acetyl-piperazinyl amides 13-18 showed low EC 50 values for several human tumor cell lines. Their cytotoxicity, however, was also high for the non-malignant mouse fibroblasts NIH 3T3. A significant improvement was achieved by preparing the rhodamine B derivatives 19-24. While rhodamine B is not cytotoxic (up to a concentration of 30μM - cut-off of the assay), the triterpenoid piperazine-spacered rhodamine B derivatives were cytotoxic in nano-molar concentration. Compound 24 (a diacetylated maslinic acid derivative) was most toxic for several human tumor cell lines but less toxic for mouse fibroblasts NIH 3T3. Staining and double-staining experiments revealed 24 to act as a mitocan. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Fertile offspring from sterile sex chromosome trisomic mice§

PubMed Central

Hirota, Takayuki; Ohta, Hiroshi; Powell, Benjamin E.; Mahadevaiah, Shantha K.; Ojarikre, Obah A.; Saitou, Mitinori; Turner, James M. A.

2017-01-01

Having the correct number of chromosomes is vital for normal development and health. Sex chromosome trisomy (SCT) affects 0.1% of the human population and is associated with infertility. We show that during reprogramming to induced pluripotent stem cells (iPSC), fibroblasts from sterile trisomic XXY and XYY mice lose the extra sex chromosome, by a phenomenon we term trisomy-biased chromosome loss (TCL). Resulting euploid XY iPSCs can be differentiated into the male germ cell lineage and functional sperm that can be used in intracytoplasmic sperm injection to produce chromosomally normal, fertile offspring. Sex chromosome loss is comparatively infrequent during mouse XX and XY iPSC generation. TCL also applies to other chromosomes, generating euploid iPSCs from cells of a Down syndrome mouse model. It can also create euploid iPSCs from human trisomic patient fibroblasts. The findings have relevance to overcoming infertility and other trisomic phenotypes. PMID:28818972

Validation of the Glaucoma Filtration Surgical Mouse Model for Antifibrotic Drug Evaluation

PubMed Central

Seet, Li-Fong; Lee, Wing Sum; Su, Roseline; Finger, Sharon N; Crowston, Jonathan G; Wong, Tina T

2011-01-01

Glaucoma is a progressive optic neuropathy, which, if left untreated, leads to blindness. The most common and most modifiable risk factor in glaucoma is elevated intraocular pressure (IOP), which can be managed surgically by filtration surgery. The postoperative subconjunctival scarring response, however, remains the major obstacle to achieving long-term surgical success. Antiproliferatives such as mitomycin C are commonly used to prevent postoperative scarring. Efficacy of these agents has been tested extensively on monkey and rabbit models of glaucoma filtration surgery. As these models have inherent limitations, we have developed a model of glaucoma filtration surgery in the mouse. We show, for the first time, that the mouse model typically scarred within 14 d, but when augmented with mitomycin C, more animals maintained lower intraocular pressures for a longer period of time concomitant with prolonged bleb survival to beyond 28 d. The morphology of the blebs following mitomycin C treatment also resembled well-documented clinical observations, thus confirming the validity and clinical relevance of this model. We demonstrate that the antiscarring response to mitomycin C is likely to be due to its effects on conjunctival fibroblast proliferation, apoptosis and collagen deposition and the suppression of inflammation. Indeed, we verified some of these properties on mouse conjunctival fibroblasts cultured in vitro. These data support the suitability of this mouse model for studying the wound healing response in glaucoma filtration surgery, and as a potentially useful tool for the in vivo evaluation of antifibrotic therapeutics in the eye. PMID:21229189

Derivation of cat embryonic stem-like cells from in vitro-produced blastocysts on homologous and heterologous feeder cells.

PubMed

Gómez, M C; Serrano, M A; Pope, C Earle; Jenkins, J A; Biancardi, M N; López, M; Dumas, C; Galiguis, J; Dresser, B L

2010-09-01

The domestic cat is a focal mammalian species that is used as a model for developing assisted reproductive technologies for preserving endangered cats and for studying human diseases. The generation of stable characterized cat embryonic stem cells (ESC) lines to use as donor nuclei may help to improve the efficiency of interspecies somatic cell nuclear transfer for preserving endangered cats and allow the creation of knockout cell lines to generate knockout cats for studying function of specific genes related to human diseases. It will also enable the possibility of producing gametes in vitro from ESC of endangered cats. In the present study, we report the generation of cat embryonic stem-like (cESL) cells from blastocysts derived entirely in vitro. We generated 32 cESL cell lines from 331 in vitro derived blastocysts from which inner cell masses were isolated by immunosurgery or by a mechanical method. Inhibition of cat dermal fibroblast (CDF) proliferation after exposure to mitomycin-C was both dose and time dependent, where doses of 30 to 40 microg/mL for 5 h were most efficient. These dosages were higher than that required to inhibit cell proliferation of mouse fetal fibroblasts (MFF; 10 microg/mL for 2.5 h). Mitomycin-C did not significantly increase necrosis of cells from either species, and had an anti-proliferative effect at concentrations below cytotoxicity. A clear species-specific relationship between feeder layers and derivation of cESL cell lines was observed, where higher numbers of cESL cell lines were generated on homologous cat feeder layers (n = 26) than from those derived on heterologous mouse feeder layers (n = 6). Three cESL cell lines generated from immunosurgery and cultured on CDF maintained self-renewal and were morphologically undifferentiated for nine and twelve passages (69-102 days). These lines showed a tightly packed dome shaped morphology, exhibited alkaline phosphatase activity and immuno-expression of the pluripotent marker OCT-4 and surface marker SSEA-1. Primary colonies at P0 to P3 and cat blastocysts expressed transcription factors OCT-4, NANOG and SOX-2 and the proto-oncogene C-MYC. However, expression was at levels significantly lower than in vitro produced blastocysts. During culture, cESL colonies spontaneously differentiated into fibroblasts, cardiomyocytes, and embryoid bodies. Development of techniques to prevent differentiation of cESL cells will be essential for maintaining defined cell lines. Copyright 2010 Elsevier Inc. All rights reserved.

Isthmin is a novel secreted protein expressed as part of the Fgf-8 synexpression group in the Xenopus midbrain-hindbrain organizer.

PubMed

Pera, Edgar M; Kim, James I; Martinez, Sarah L; Brechner, Mariel; Li, Su Yu; Wessely, Oliver; De Robertis, E M

2002-08-01

Patterning of the central nervous system is regulated by a signaling center located at the midbrain-hindbrain boundary (MHB), or isthmus organizer. Fibroblast growth factors secreted from the MHB are required and sufficient to direct the ordered growth and regionalization of the midbrain and anterior hindbrain. In an unbiased secretion cloning screen of Xenopus gastrula embryos we identified a novel gene, which we designated as Isthmin (xIsm) due to its prominent expression at the MHB. xIsm encodes a secreted protein of 449 amino acids containing one copy of the thrombospondin type 1 repeat (TSR). We also found orthologous Isthmin genes in human (hIsm) and mouse (mIsm), as well as a gene encoding an Isthmin-like human unknown protein (hIsm-l). The conservation of a unique carboxy-terminal region between hIsm and hIsm-l suggests that Isthmin is the founding member of a new family of secreted proteins. xIsm was strongly expressed maternally in the Xenopus egg and showed zygotic expression in the ventral blastopore lip, notochord, and MHB. Additional expression domains were detected in neural crest, ear vesicle, and developing blood islands. Interestingly, xIsm was co-expressed with Fibroblast growth factor-8 (xFgf-8) at multiple sites including the MHB, indicating that these two genes are part of a synexpression group which also includes sprouty and sef homologs.