Cytogenetic instability of chromosomal nucleolar organizer regions (NORs) in cloned mouse L929 fibroblasts.

Ribosomal DNA (rDNA) gene codes for 18S, 5.8S, and 28S rRNA form tandem repetitive clusters, which occupy distinct chromosomal loci called nucleolar organizer regions (NORs). The number and position of NORs on chromosomes are genetic characteristics of the species although within a cell, the NOR sizes can significantly vary due to loss or multiplication of rDNA copies. In the current study, we used mouse L929 fibroblasts, the aneuploid cells which differ in the FISH- and Ag-NOR numbers, to examine whether the parental NOR variability is inherited in clones. By statistical analysis, we showed that the cloned fibroblasts were able to restore the NOR numerical characteristics of the parental cells after long-term culturing. These results support the idea that mammalian cells may have mechanisms which control the number and activity of NORs at the population level. In L929 fibroblasts, we also regularly observed laterally asymmetry of FISH-NORs that evidenced in an unequal distribution of the mother rDNA copies between the daughter cells in mitosis.

Chemokine (C-X-C motif) ligand 14 contributes to lipopolysaccharide-induced fibrogenesis in mouse L929 fibroblasts via modulating PPM1A.

Herein, we found that serum chemokine ligand 14 (CXCL14) was significantly enhanced in patients with idiopathic pulmonary fibrosis (IPF). In our current study, mouse L929 fibroblasts were stimulated with lipopolysaccharide (LPS) (100 ng/mL). Cell proliferation, the levels of matrix metalloproteinase 2 (MMP2) and MMP9, as well as extracellular matrix (ECM) content were assessed to evaluate the fibrogenesis of L929 cells. Proliferating cell nuclear antigen and cell viability were assessed to evaluate cell proliferation. Hydroxyproline (Hyp), collagen I/III, connective tissue growth factor (CTGF), and phosphorylated Smad2/3 (p-Smad2/3) were assessed to evaluate ECM secretion and deposition. α-Smooth muscle actin (α-SMA) was used to measure the occurrence of differentiation from fibroblast toward myofibroblast. Our data suggested that knockdown of CXCL14 prevented LPS-induced fibrogenesis of L929 cells through inhibiting cell proliferation and decreasing the expression of MMP2/9, Hyp, collagen I/III, CTGF, p-Smad2/3, and α-SMA. Notably, upregulation of protein phosphatase magnesium-dependent 1A (PPM1A) was involved in this process. On the contrary, recombinant CXCL14 protein led to an opposite effect. We first suggested that overexpression of PPM1A ameliorated LPS-induced fibrogenesis. Furthermore, we substantiated that knockdown of CXCL14 exerted an antifibrotic effect in IPF in vitro probably via the upregulation of PPM1A. Besides, evidently enhanced CXCL14, yet reduced PPM1A, was found in bleomycin-induced rat pulmonary fibrosis, confirming the roles of CXCL14 and its potential association with PPM1A in IPF in vivo. In conclusion, CXCL14 could be considered as a therapeutic target for preventing fibrogenesis of mouse L929 fibroblasts.

Studies on the Synthesis, Photophysical and Biological Evaluation of Some Unsymmetrical Meso-Tetrasubstituted Phenyl Porphyrins.

Abstract: We designed three unsymmetrical meso-tetrasubstituted phenyl porphyrins for further development as theranostic agents for cancer photodynamic therapy (PDT): 5-(4-hydroxy-3-methoxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrin (P2.2), Zn(II)-5-(4-hydroxy-3-methoxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrin (Zn(II)2.2) and Cu(II)-5-(4-hydroxy-3-methoxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrin (Cu(II)2.2). The porphyrinic compounds were synthesized and their structures were confirmed by elemental analysis, FT-IR, UV-Vis, EPR and NMR. The compounds had a good solubility in polar/nonpolar media. P2.2 and, to a lesser extent, Zn(II)2.2 were fluorescent, albeit with low fluoresence quantum yields. P2.2 and Zn(II)2.2 exhibited PDT-acceptable values of singlet oxygen generation. A "dark" cytotoxicity study was performed using cells that are relevant for the tumor niche (HT-29 colon carcinoma cells and L929 fibroblasts) and for blood (peripheral mononuclear cells). Cellular uptake of fluorescent compounds, cell viability/proliferation and death were evaluated. P2.2 was highlighted as a promising theranostic agent for PDT in solid tumors considering that P2.2 generated PDT-acceptable singlet oxygen yields, accumulated into tumor cells and less in blood cells, exhibited good fluorescence within cells for imagistic detection, and had no significant cytotoxicity in vitro against tumor and normal cells. Complexing of P2.2 with Zn(II) or Cu(II) altered several of its PDT-relevant properties. These are consistent arguments for further developing P2.2 in animal models of solid tumors for in vivo PDT.

In vitro biocompatibility and wound healing properties of latex proteins dressing.

The chronification of ulcers or sores may result in a dramatic outcome such as amputation. Currently, the search for plant based treatments of various diseases/disorders, including complicated ones, is getting the attention of researchers worldwide. The soluble latex protein fraction (CpLP) obtained from Calotropis procera (Apocynaceae) was previously demonstrated to accelerate wound healing by topical application or when incorporated in a polyvinyl alcohol biomembrane (BioMemCpLP). Here, in vitro assays were performed to investigate and characterize the biocompatibility and bioactivity of latex proteins dressing. Macrophages (RAW 264.7), fibroblasts (L929) and keratinocytes (HaCaT) cell lines were used to evaluate the effect of CpLP. These cell lines were exposed to concentrations of CpLP comparable to those found in BioMemCpLP during 24-72 h. The cytotoxicity, proliferation, release of wound healing mediators (TGF-ß, VEGF, IL-10, IL-6, IL-1ß, TNF-α and NO) and migration of cells (E-cadherin and ß-catenin) incubated with CpLP was assessed and the cell adhesion to BioMemCpLP as well. The results showed that CpLP has no cytotoxic effects. It induced a suitable balance between pro- and anti-inflammatory mediators, enhanced proliferation and re-epithelialization in all cell lines, but the intensity of each effect was different at various doses in all cell strains. The BioMemCpLP stimulated cell adhesion to PVA substrate. The CpLP-PVA based biomembrane can be a good option for healing of different wounds.

Raman spectroscopy assisted biochemical evaluation of L929 fibroblast cells on differentially crosslinked gelatin hydrogels.

Biochemical evaluation of cell-matrix interaction using conventional labelling techniques often possesses limitations due to dye entrapment. In contrast, Raman spectroscopy guided approach offers label-free determination of cell-matrix biochemistry. Herein, gelatin (Gel) matrices modified with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide/ N-Hydroxysuccinimide (EDC/NHS) and glutaraldehyde (GTA) was used as standards for comparative evaluation. Raman spectroscopy was deployed as a label-free approach to investigate interaction of cells with Gel hydrogels. Raman-based approach assisted in evaluation of cell-matrix interactions by identifying key biomolecular signatures retrospecting the fact that L929 fibroblast cells portrayed excellent growth and proliferation kinetics in crosslinked Gel as compared to its bare counterpart. EDC crosslinked hydrogels exhibited superior cell proliferation than its GTA counterparts. Cell proliferation on differentially crosslinked gel was also confirmed using standard MTT Assay and Rhodamine-DAPI staining thus corroborating the fact that Raman spectroscopy can be deployed as a superior label-free alternative towards real-time determination of cell proliferation and growth.

Succinylation of Polyallylamine: Influence on Biological Efficacy and the Formation of Electrospun Fibers.

Succinylation of proteins is a commonly encountered reaction in biology and introduces negatively charged carboxylates on previously basic primary amine groups of amino acid residues. In analogy, this work investigates the succinylation of primary amines of the synthetic polyelectrolyte polyallylamine (PAA). It investigates the influence of the degree of succinylation on the cytotoxicity and antibacterial activity of the resulting polymers. Succinylation was performed in water with varying amounts of succinic anhydride and at different pH values. The PAA derivatives were analyzed in detail with respect to molecular structure using nuclear magnetic resonance and infrared absorbance spectroscopy. Polyelectrolyte and potentiometric charge titrations were used to elucidate charge ratios between primary amines and carboxylates in the polymers. The obtained materials were then evaluated with respect to their minimum inhibitory concentration against Staphylococcus aureus and Pseudomonas aeruginosa. The biocompatibility was assessed using mouse L929 fibroblasts. The degree of succinylation decreased cytotoxicity but more significantly reduced antibacterial efficacy, demonstrating the sensitivity of the fibroblast cells against this type of ampholytic polyelectrolytes. The obtained polymers were finally electrospun into microfiber webs in combination with neutral water-soluble polyvinyl alcohol. The resulting non-woven could have the potential to be used as wound dressing materials or coatings.

Cytotoxic and mutagenic potential of juglone: a comparison of free and nano-encapsulated form.

Despite its evidenced beneficial herbicidal, antibacterial, antiviral, antifungal, and antioxidant effects, the application of juglone (5-hydroxy-1,4,-naphthoquinone) is limited due to its low water solubility and allelopathic and toxic effects. In recent years, research has aimed to overcome these limitations by increasing its solubility and controlling its release through nanoparticular systems. This is the first study to have synthesised and characterised juglone-loaded polymeric nanoparticles and compared them with free juglone for cytotoxicity in mouse (L929 fibroblasts) and alfalfa cells and for mutagenic potential in Salmonella typhimurium TA98/100. Mouse and plant cells treated with free and nano-encapsulated juglone showed a decrease in cell viability in a dose and time-dependent manner, but this effect was significantly lower with the nano-encapsulated form at lower doses. In the TA98 strain with S9, nano-encapsulated juglone did not exhibit mutagenic effects, unlike the free form. Since all results show that juglone encapsulation with polymeric nanoparticles reduced the toxic and mutagenic effects, it has a promising potential to be applied in medicine, food safety, and agriculture.

Conformity of dextran-coated fullerene C70 with L929 fibroblast cells.

Fullerene C70 with symmetric nanostructure and unique properties that open up leeway for both material science and healthcare applications. Poor water dispersity and limited knowledge about its associated toxicity hinders the biomedical scope of C70. These restraining factors need to be addressed. Dextran, natural and water-soluble polymer was used to improve the dispersity of C70 in water. Dextran coating on C70 successfully yielded stable dispersion of C70 in water with remarkable cytocompatibility with L929 fibroblast cells. The dextran-coated C70 was characterized using different characterization techniques such as fourier transformed-infrared spectroscopy (FT-IR), transition electron microscopy (TEM), dynamic light scattering (DLS) and zeta potential. The cell viability assays suggested that the L929 cells retained more than 80% cell viability after 24 h treatment with dextran-coated C70. The mitochontrial membrane potential of the treated cells were found to be uncompromised. Fluorescent imaging techniques done with the aim of evaluating the integrity of lysosomes detected no potential toxicity in L929 cells treated with dextran-coated C70. Actin filaments showed intact organelles when viewed using Rhodamine- phalloidin staining after 24 h post-treatment. DAPI staining also revealed the integrity of nucleus after exposure to dextran-coated C70. The calcein AM/PI flow cytometry analysis further confirmed that dextran-coated C70 kept cell viability of treated cells above 80% for all concentrations. The results points out the scope of dextran-coated C70 for various health care applications.

The variable toxicity of silver ions in cell culture media.

The elevated interest in silver ions (Ag+) as a broad spectrum antimicrobial for use on medical devices has increased the number and importance of in vitro biocompatibility testing, however little consideration is given to the culture environment in which the assessments are performed. The current investigation assessed the viability of mouse fibroblasts (L929) exposed to different concentrations of Ag+ in both Dulbecco's modified Eagle's medium (DMEM) and minimal essential medium Eagle, alpha modification (αMEM). We identified a significant increase in the EC50 of L929 cells exposed to Ag+ in αMEM compared to DMEM, which was matched by a corresponding decrease in Ag+ availability in αMEM at concentrations ≤400 µM, as detected by inductively coupled plasma mass spectrometry (ICP-MS). The reduced availability was not observed for Ag+ > 400 µM, the concentration above which caused in vitro cytotoxicity in L929 cells in αMEM; while linear quantification of Ag+ was observed in DMEM. Equilibration of the chloride and glucose components between media did not affect cytotoxicity on primary test cells; mesenchymal stromal cells (MSCs). Overall, our results present evidence of the importance of culture conditions on the in vitro evaluation of silver, with DMEM providing a reliable basal media in which to conduct assessments.

Photodamage attenuating potential of Nectandra hihua against UVB-induced oxidative stress in L929 fibroblasts.

The total phenolic content (TP) and antioxidant activity of the ethanolic extract and fractions that were obtained from the leaves of Nectandra hihua were assessed using different methods. The ethanolic extract (EE) and ethyl acetate fraction (EAF) had the best antioxidant capacity, which was comparable to butylated hydroxytoluene and quercetin (ABTS+ 2.55 ±â€¯0.06, 3.54 ±â€¯0.03 mmol TE/g; DPPH IC50 10.27 ±â€¯0.05, 9.88 ±â€¯0.02 µg/mL; FRAP 2.17 ±â€¯0.08, 2.38 ±â€¯0.04 mmol TE/g; ORAC 5.16 ±â€¯0.08, 5.35 ±â€¯0.07 mmol TE/g; TP 568.05 ±â€¯18.15, 397.20 ±â€¯17.88 mg GAE/g, respectively). The cytoprotective effect, reactive oxygen species (ROS) and lipid peroxidation inhibitions on L929 fibroblasts irradiated with UVB (600 mJ/cm2) in pre- and post-treatments with EE and EAF were determined. These plant materials demonstrated high ROS scavenging activity and lipid peroxidation inhibition on L929 fibroblasts in both treatments, especially with pre-treatment (EE 38.47 ±â€¯1.95% and EAF 40.20 ±â€¯4.5% inhibition of ROS production, and EE 39.03 ±â€¯3.33% and EAF 41.67 ±â€¯7.6% of lipid peroxidation inhibition), indicating the best cytoprotection with pre-treatment (13.52 ±â€¯1.66% and 13.34 ±â€¯2.61% increases in cell viability). The antioxidant flavonoids quercitrin, avicularin, juglalin, afzelin and astragalin were isolated from EAF. The results obtained indicate that EE and EAF present photodamage attenuating potential against UVB-induced oxidative stress and can be useful as a starting point for developing dermatological products to prevent oxidative skin damage.

Polydopamine-collagen complex to enhance the biocompatibility of polydimethylsiloxane substrates for sustaining long-term culture of L929 fibroblasts and tendon stem cells.

Polydimethylsiloxane (PDMS) is a commercialized polymer extensively used in the fabrication of versatile microfluidic microdevices for studies in cell biology and tissue engineering. However, the inherent surface hydrophobicity of PDMS is not optimal for cell culture and thus restrains its applications for investigation of long-term behaviors of fibroblasts and stem cells. To improve the surface biocompatibility of PDMS, a facile technique was developed by modifying the PDMS surface with polydopamine-collagen (COL/PDA) complex. The successful synthesis of COL/PDA was verified through proton nuclear magnetic resonance spectroscopy. Compared to surface coating solely with COL or PDA, the surface wettability was significantly improved on COL/PDA-modified PDMS substrates based on water contact angle characterizations. The modified PDMS surface remarkably enhanced the initial adhesion and long-term proliferation of L929 fibroblasts and tendon stem cells (TSCs). Additionally, the effects of COL/PDA coating on cell viability and apoptosis were further investigated under prolonged incubation. We found that the COL/PDA coating on PDMS resulted in a substantial increase of cell viability compared to native PDMS, and the cell apoptosis was considerably impeded on the modified PDMS. This study demonstrated that COL/PDA coating can effectively enhance the surface biocompatibility of PDMS as verified by the enhanced adhesion and long-term proliferation of L929 fibroblasts and TSCs. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 408-418, 2018.

Macroporous modified poly (vinyl alcohol) hydrogels with charged groups for tissue engineering: Preparation and in vitro evaluation.

Poly(vinyl alcohol) (PVA) hydrogels are widely employed for various biomedical applications, including tissue engineering, due to their biocompatibility, high water solubility, low protein adsorption, and chemical stability. However, non-charged surface of PVA-based hydrogels is not optimal for cell adhesion and spreading. Here, cross-linked macroporous hydrogels based on low molecular weight acrylated PVA (Acr-PVA) was synthesized by modification of the pendant alcohol groups on the PVA with glycidyl methacrylate (GMA). To enhance cell affinity, charged groups were introduced to the hydrogel composition. For this purpose, Acr-PVA was copolymerized with either negatively charged acrylic acid (AA) or positively charged 2-(diethylamino) ethyl methacrylate (DEAEMA) monomers. A surface charge of the obtained hydrogels was found to be in function of the co-monomer type and content. Confocal microscopy observations confirmed that adhesion and spreading of both mouse fibroblasts (L929) and human mesenchymal stem cells (hMSC) on the modified Acr-PVA-AA and Acr-PVA-DEAEMA hydrogels were better than those on the non-modified Acr-PVA hydrogel. The increase of DEAEMA monomer content from 5 to 15mol% resulted in the enhancement of cell viability which was 1.5-fold higher for Acr-PVA-DEAEMA-15 hydrogel than that of the non-modified Acr-PVA hydrogel sample.

Strontium doped poly-ε-caprolactone composite scaffolds made by reactive foaming.

In the reconstruction and regeneration of bone tissue, a primary goal is to initiate bone growth and to stabilize the surrounding bone. In this regard, a potentially useful component in biomaterials for bone tissue engineering is strontium, which acts as cationic active agent, triggering certain intracellular pathways and acting as so called dual action bone agent which inhibits bone resorption while stimulating bone regeneration. In this study we established a novel processing for the foaming of a polymer (poly-ε-caprolactone) and simultaneous chemical reaction of a mixture of calcium and strontium hydroxides to the respective carbonates using supercritical carbon dioxide. The resultant porous composite scaffold was optimized in composition and strontium content and was characterized via different spectroscopic (infrared and Raman spectroscopy, energy dispersive X-ray spectroscopy), imaging (SEM, µCT), mechanical testing and in vitro methods (fluorescence vital staining, MTT-assay). As a result, the composite scaffold showed good in vitro biocompatibility with partly open pore structure and the expected chemistry. First mechanical testing results indicate sufficient mechanical stability to support future in vivo applications.

Optimization of nanostructured lipid carriers loaded with methotrexate: A tool for inflammatory and cancer therapy.

The aim of this study was to optimize and assess the potential of nanostructured lipid carriers (NLC), prepared by the hot ultrasonication method, as carrier for methotrexate (MTX), highlighting the application of factorial design. Preliminary screening drug/lipid solubility, allowed us to select Witepsol(®) E85 as the solid lipid and Mygliol(®) 812 as liquid lipid for the NLC loaded with MTX. Then, a 3-level, 3-factor Box-Behnken design and validated by ANOVA analysis; the correspondence between the predicted values and those measured experimentally confirmed the robustness of the design. Properties of optimized MTX-loaded NLCs such as morphology, size, zeta potential, entrapment efficiency, storage stability, in vitro drug release and cytotoxicity were investigated. NLCs loaded with MTX exhibited spherical shape with 252-nm, a polydispersity of 0.06±0.02, zeta potential of -14 mV and an entrapment efficiency of 87%. In vitro release studies revealed a fast initial release followed by a prolonged release of MTX from the NLC up to 24-h. The release kinetics of the optimized NLC best fitted the Peppas-Korsmeyer model for physiological and inflammatory environments and the Hixson-Crowell model skin simulation conditions. No toxicity was observed in fibroblasts. Thus, the optimized MTX-loaded NLC have the potential to be exploited as delivery system.

Polymer films with surfaces unmodified and modified by non-thermal plasma as new substrates for cell adhesion.

The surface properties of biomaterials, such as wettability, polar group distribution, and topography, play important roles in the behavior of cell adhesion and proliferation. Gaseous plasma discharges are among the most common means to modify the surface of a polymer without affecting its properties. Herein, we describe the surface modification of poly(styrene) (PS) and poly(methyl methacrylate) (PMMA) films using atmospheric pressure plasma processing through exposure to a dielectric barrier discharge (DBD). After treatment the film surface showed significant changes from hydrophobic to hydrophilic as the water contact angle decreasing from 95° to 37°. All plasma-treated films developed more hydrophilic surfaces compared to untreated films, although the reasons for the change in the surface properties of PS and PMMA differed, that is, the PS showed chemical changes and in the case of PMMA they were topographical. Excellent adhesion and cell proliferation were observed in all films. In vitro studies employing flow cytometry showed that the proliferation of L929 cells was higher in the film formed by a 1:1 mixture of PS/PMMA, which is consistent with the results of a previous study. These findings suggest better adhesion of L929 onto the 1:1 PS/PMMA modified film, indicating that this system is a new candidate biomaterial for tissue engineering.

Response of MC3T3-E1 osteoblasts, L929 fibroblasts, and J774 macrophages to fluoride surface-modified AZ31 magnesium alloy.

The present work evaluates the biocompatibility of a fluoride surface-modified AZ31 magnesium alloy (AZ31HF) with different cell lines that coexist in the implant environment to test its potential use as a biodegradable and absorbable biomaterial for bone repair. A clear stimulation of cell proliferation and an enhancement of the mitochondrial respiratory activity were observed when mouse osteoblasts (MC3T3-E1), fibroblasts (L929), and macrophages (J774) cell lines were cultured with the modified alloy. No significant change in apoptosis or viability rates was observed when osteoblasts and fibroblasts cultures were grown in the presence of this alloy. A proteomic analysis of the MC3T3-E1 cell extracts cultured in the presence of AZ31HF showed an overexpression of proteins related with the mineralization process, which is a necessary step for bone repair. An increase in the lactate dehydrogenase activity was observed in the MC3T3-E1 and J774 cell cultures that could be a response of the oxidative stress produced by the presence of the material. This stress could be related to the increase observed in the respiratory mitochondrial activity or respiratory burst measured in theses cultures that indicate damage in the cell membranes and subsequently some cell death. Results reported here, for and against AZ31HF, should be taken into account when considering the potential use of this modified alloy in bone repair applications.