Effect of fluoxetine and adenosine receptor NECA agonist on G alpha q/11 protein of C6 glioma cells.

OBJECTIVES: Trimeric G-proteins play a crucial role in the transmembrane signalling to intracellular pathways via effector phospholipase C (1,4,5 IP3) or adenylylcyclase (cAMP). G-protein modulation is considered to participate in the antidepressant mode of action by neurotransmitter G-protein coupled receptors (GPCR). Adenosine is naturally occured nucleoside and adenosine receptor belongs to GPCR family. Properties and functions of ubiquitous adenosine receptor were described with number of agonists and antagonists. METHODS: In C6 glioma cells, we studied acute administration of SSRI antidepressants - fluoxetine, sertraline and citalopram. We used immunochemical estimation (ELISA) of the main types of G-protein alpha subunits from isolated membranes of C6 glioma cells. We also estimated effect of NECA agonist on fluoxetine induced signalling via 1,4,5 IP3 and its levels. RESULTS: Results show involvement of the antidepressant drugs in the C6 glioma signal transduction cascades and their modulation in dependence on the antidepressant of SSRI type. We measured main G alpha protein profiles after fluoxetine, sertraline and citalopram administration. We found significant changes as following: decreased G alpha Gq/11 for fluoxetine, low G alpha s for sertraline and both high G alpha q/11 and high G alpha s for citalopram. Furthermore the NECA (5´-N-ethylcarboxamido- adenosine) agonist of adenosine receptor alone evoked high decrease of G alpha q/11 levels. Whereas fluoxetine influenced G alpha q/11 decline was abolished by NECA in concentration manner, especially at 10-8 and 10-9 M concentrations. These results support abolishion NECA effect on fluoxetin influenced 1,4,5 IP3 signalling via PLC. CONCLUSION: Main G alpha profiles are dependent on SSRI type antidepressant. Abolishing both fluoxetine evoked G alpha q/11 and and 1,4,5 IP3 signalling can indicate parallel interference between G-protein coupled receptors (GPCR) and the cell response. Presented data are first findings about adenosine receptor interaction with fluoxetine signalling. Thus in vitro studies contribute to the clarification of the molecular basis of antidepressant action.

Effect of fluoxetine or adenosine receptor NECA agonist on G-proteins of C6 glioma cells or NK immunocytes.

OBJECTIVE: Neurochemical approaches to antidepressant effects and depressive disorder are also focusing on G-protein coupled receptors (GPCR) and subsequent signalling. Trimeric G-proteins play a crucial role in transmembrane signalling, its amplification and processing. It is evident that immune system participates in antidepressant mode of action by neurotransmitter GPCR. METHODS: We studied the effect of acute administration of fluoxetine or NECA agonist of adenosine receptor (GPCR) on C6 glioma cells and natural killer (NK) cell line, innate immunity. We used immunochemical estimation (ELISA) of the main types of G-protein alpha subunits from isolated membranes of tested cells. RESULTS: Significant reduction of G alpha q/11 subunits after acute administration of fluoxetine or NECA agonist was found. In contrast, no significant influence of G alpha s or G alpha i1,2 subunit levels of C6 glioma cells were observed. Lowered Gq/11 signalling was in accordance with decreased 2nd messenger 1,4,5 IP3 formation by PLC. Acute effect of fluoxetine or NECA agonist on NK cell line resulted in significantly reduced G alpha q/11 levels without changes in G alpha s and G alpha i1,2. Furthermore, we determined that NECA agonist was able to abolish fluoxetine-evoked G alpha q/11 levels of NK cell line. CONCLUSIONS: Results show involvement of fluoxetine in the C6 glioma signal transduction and were comparable with NK cells. Similar inhibiton of G alpha q/11 by NECA agonist in both C6 glioma cells and NK cell line was determined. Furthermore NECA induced attenuation of fluoxetine evoked Galpha q/11 signalling can indicate parallel interference between GPCR and final response. Finally, we determined similarity in both interleukin 2, IL2 immunostimulator and fluoxetine evoked G q/11 levels in NK cell line and thus fluoxetine action could be related to signalling aspects of neuroimmunomodulatory activity.

Growth of vascular smooth muscle cells on collagen I exposed to RBL-2H3 mastocytoma cells.

Remodeling of the peripheral pulmonary vasculature during chronic hypoxia is characterized by accelerated collagenolysis and thickening of the vascular wall. Low molecular weight peptides, products of cleavage by interstitial collagenase and muscular layer in the peripheral pulmonary vessels, are typically present. The aim of this "in vitro" study was to verify that mast cells (RBL-2H3) as a potent source of a variety of biomolecules which can affect vessel wall remodeling are capable of splitting collagen and then facilitating the growth of vascular smooth muscle cells (VSMC). Collagen I was exposed to RBL-2H3 cells cultured 48 hours under normoxic or hypoxic (3% O(2)) conditions and then seeded with VSMC. The VSMC proliferated with the shortest doubling time and reached the highest cell population density on the collagen pre-modified with hypoxic RBL-2H3 cells. This increased growth activity of VSMC was probably due to the fragmentation of collagen by proteases released from RBL-2H3 cells. Absolute amount of collagen fragments was similar in samples exposed to normoxic and hypoxic RBL-2H3 cells, but the concentration of at least one collagen fragment was significantly higher under hypoxic conditions. Mast cells exposed to hypoxia are more capable to split collagen and facilitate the growth of VSMC.

Detection of choline transporter-like 1 protein CTL1 in neuroblastoma x glioma cells and in the CNS, and its role in choline uptake.

Choline is an essential nutrient necessary for synthesis of membrane phospholipids, cell signalling molecules and acetylcholine. The aim of this study was to detect and characterize the choline transporter-like 1 (CTL1/SLC44A1) protein in CNS tissues and the hybrid neuroblastoma x glioma cell line NG108-15, which synthesizes acetylcholine and has high affinity choline transport but does not express the cholinergic high affinity choline transporter 1. The presence of CTL1 protein in NG108-15 cells was confirmed using our antibody G103 which recognizes the C-terminal domain of human CTL1. Three different cognate small interfering RNAs were used to decrease CTL1 mRNA in NG108-15 cells, causing lowered CTL1 protein expression, choline uptake and cell growth. None of the small interfering RNAs influenced carnitine transport, demonstrating the absence of major non-specific effects. In parental C6 cells knockdown of CTL1 also reduced high affinity choline transport. Our results support the concept that CTL1 protein is necessary for the high affinity choline transport which supplies choline for cell growth. The presence of CTL1 protein in rat and human CNS regions, where it is found in neuronal, glial and endothelial cells, suggests that malfunction of this transporter could have important implications in nervous system development and repair following injury, and in neurodegenerative diseases.

Nanodiamond as promising material for bone tissue engineering.

The adhesion, growth and differentiation of human osteoblast-like MG 63 cells were investigated in cultures grown on nanostructured nanocrystalline diamond (NCD) films with either low surface roughness (rms of 8.2 nm) or hierarchically organized surfaces made of low roughness NCD films deposited on Si surfaces with the original microroughness (rms of 301.0 nm and 7.6 nm, respectively). The NCD films were grown using a microwave plasma-enhanced CVD method in an ellipsoidal cavity reactor. The films were treated in oxygen plasma to enhance the hydrophilic character of the diamond surface (water drop contact angle approx. 20 degrees). The samples were then sterilized by 70% ethanol, inserted into 12-well polystyrene multidishes (diameter 2.2 cm), seeded with human osteoblast-like MG 63 cells (40,000 cells/dish, 10,530 cells/cm2) and incubated in 2 ml of DMEM medium with 10% of fetal bovine serum. On day 3 after seeding, the cell numbers were significantly higher on the nanostructured NCD films (72,020 +/- 6540 cells/cm2) and also on the hierarchically micro- and nanostructured films (60200 +/- 6420 cells/cm2) than on the control polystyrene culture dish (40750 +/- 2,530 cells/cm2). The cells on hierarchically micro- and nanostructured diamond substrates also adhered over a significantly larger area (3730 +/- 180 microm2 compared to 2740 +/- 130 microm2 on polystyrene). The cell viability, measured by a LIVE/DEAD viability/cytotoxicity kit, reached 98% to 100% on both types of NCD films. The XTT test showed that the cells on both nanodiamond layers had significantly higher metabolic activity than those on the control polystyrene dish (approx. 2 to 3 times). Immunofluorescence staining of the cells on both NCD films revealed talin-containing focal adhesion plaques and beta-actin filaments, well apparent particularly at the cell periphery, as well as the presence of considerable amounts of osteocalcin, i.e., a marker of osteogenic cell differentiation. These results suggest that nanocrystalline diamond films give good support for adhesion, growth and differentiation of osteogenic cells and could be used for surface modification of bone implants in order to improve their integration with the surrounding bone tissue.

Acute and chronic effects of antidepressants on the G-protein alpha subunit profiles in vitro and in vivo.

OBJECTIVES: Neurochemical studies on the etiopathogenesis of depression are also focusing on the transduction system beyond receptors. Trimeric G-proteins play a crucial role in the transmembrane signalling, signal amplification and intracellular processing. Abnormalities of G-protein levels are observed in subjects with depression, G-protein modulation is considered to play a role in the antidepressant mode of action. METHODS: We studied acute or chronic administration of antidepressants from different pharmacological groups. We used immunochemical estimation (ELISA) of the main types of G-protein alpha subunits from isolated membranes of C6 glioma cells and rat brain tissue. RESULTS: Significant elevation of G alpha q/11 subunits after chronic administration of sertraline and significant reduction of G alpha s subunit levels following both acute and chronic administrations of sertraline were found. In contrast, no significant effects on G alpha subunit levels following acute desipramine and moclobemide administration were observed in vitro. Chronic moclobemide effect in vivo is causing significant elevation of Galpha s and Galpha i1,2 subunit levels. CONCLUSIONS: Results show involvement of antidepressant drugs in the C6 glioma signal transduction cascades modulation in dependence on the antidepressant class. Significant influence in the cAMP system modulation is observed after administration both SSRI and MAOA inhibitors. Astrocytoma cells - C6 glioma cells also can offer a model system of the glia where modulation of cell signalization cascades can influence cell functioning and production of neurotrophic factor molecules relevant to the antidepressant treatment and depression etiopathogenesis.

Improved adhesion, growth and maturation of vascular smooth muscle cells on polyethylene grafted with bioactive molecules and carbon particles.

High-density polyethylene (PE) foils were modified by an Ar(+) plasma discharge and subsequent grafting with biomolecules, namely glycine (Gly), polyethylene glycol (PEG), bovine serum albumin (BSA), colloidal carbon particles (C) or BSA and C (BSA + C). As revealed by atomic force microscopy (AFM), goniometry and Rutherford Backscattering Spectroscopy (RBS), the surface chemical structure and surface morphology of PE changed dramatically after plasma treatment. The contact angle decreased for the samples treated by plasma, mainly in relation to the formation of oxygen structures during plasma irradiation. A further decrease in the contact angle was obvious after glycine and PEG grafting. The increase in oxygen concentration after glycine and PEG grafting proved that the two molecules were chemically linked to the plasma-activated surface. Plasma treatment led to ablation of the PE surface layer, thus the surface morphology was changed and the surface roughness was increased. The materials were then seeded with vascular smooth muscle cells (VSMC) derived from rat aorta and incubated in a DMEM medium with fetal bovine serum. Generally, the cells adhered and grew better on modified rather than on unmodified PE samples. Immunofluorescence showed that focal adhesion plaques containing talin, vinculin and paxillin were most apparent in cells on PE grafted with PEG or BSA + C, and the fibres containing alpha-actin, beta-actin or SM1 and SM2 myosins were thicker, more numerous and more brightly stained in the cells on all modified PE samples than on pristine PE. An enzyme-linked immunosorbent assay (ELISA) revealed increased concentrations of focal adhesion proteins talin and vinculin and also a cytoskeletal protein beta-actin in cells on PE modified with BSA + C. A contractile protein alpha-actin was increased in cells on PE grafted with PEG or Gly. These results showed that PE activated with plasma and subsequently grafted with bioactive molecules and colloidal C particles, especially with PEG and BSA + C, promotes the adhesion, proliferation and phenotypic maturation of VSMC.

Constitutive expression of IL-18 and IL-18R in differentiated IEC-6 cells: effect of TNF-alpha and IFN-gamma treatment.

The multifunctional cytokine interleukin-18 (IL-18) is an important mediator in intestinal inflammatory processes. The aim of this study was to evaluate the constitutive expression of IL-18 and its receptors (IL-18Ralpha and IL-18Rbeta) in intestinal epithelial cells (IEC) stimulated by tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). In addition, cellular proliferation and evaluation of brush border enzymes as differentiation markers were studied. Nontransformed rat intestinal epithelial IEC-6 cells were grown on an extracellular matrix (ECM) in medium with or without TNF-alpha, IFN-gamma, or a combination of both. Gene expression of IL-18, its receptors and apoptotic markers was evaluated using real-time PCR. Expression of IL-18Ralpha protein was demonstrated by flow cytometry and Western blot. Enzymatic activities of brush border enzymes and caspase-1 were determined. The constitutive expression of IL-18, IL-18Ralpha and IL-18Rbeta mRNAs and proteins were detected in IEC-6 cells. The biologically active form of IL-18 was released in response to TNF-alpha and IFN-gamma treatment. Exogenous IL-18 had no effect on cellular proliferation, brush border enzyme activities, and gene expression of apoptotic markers. However, the addition of IL-18 stimulated production and release of the chemokine IL-8. These data suggest that IEC-6 cells may be not only a source of IL-18 but also a target for its action.

Expression and enzymatic activity of dipeptidyl peptidase-IV in human astrocytic tumours are associated with tumour grade.

Alterations in dipeptidyl peptidase-IV (DPP-IV) enzymatic activity are characteristic of malignant transformation. Through its well-characterized functionality in regulating the activity of bioactive peptides by removal of the N-terminal dipeptide, DPP-IV activity may have profound effects upon metastatic potential and cell growth. Although DPP-IV/CD26 (EC 3.4.14.5) is the canonical representative of the group, a number of other proteins including DPP-7, 8, 9, and seprase/fibroblast activation protein-alpha (FAP-alpha) have been shown to have similar enzymatic activity. This study was set up to address the relative representation and enzymatic activity of plasma membrane localized DPP-IV/CD26 and FAP-alpha in human brain and astrocytic tumours. In parallel, expression of CXCR4, receptor for glioma cell growth stimulator chemokine SDF-1alpha known to be a DPP-IV substrate, was investigated. This is the first report showing that non-malignant brain tissue contains a DPP-IV-like enzymatic activity attributable mostly to DPP-8/9, while the substantial part of the activity in glioma is due to increased DPP-IV/CD26, localized in both the vascular and parenchymal compartments. DPP-IV enzymatic activity increased dramatically with tumour grade severity. A grade-related increase in CXCR4 receptor paralleled the rise in DPP-IV expression and activity. These data might support a role for DPP-IV regulation of the CXCR4-SDF-1alpha axis in glioma development.

Docosahexaenoic acid supports cell growth and expression of choline acetyltransferase and muscarinic receptors in NG108-15 cell line.

A large body of evidence indicates that adequate intake of polyunsaturated fatty acids is essential for brain development in early ontogenesis and positively impacts various pathological states connected with aging, as well as other neurodegenerative diseases (Jump, 2002; Bazan, 2003; Ruxton et al., 2004). In the present experiments, we investigated the possible effects of polyunsaturated docosahexanoic acid (DHA [22:6, n = 3]) on the expression of cholinergic phenotype-represented by choline acetyltransferase (ChAT) activity and a number of surface muscarinic receptors-as well as on cell growth in the cholinergic cell line NG108-15(Hamprecht, 1977; Hamprecht et al., 1985). However, chemical composition of different batches of sera is neither stable nor defined, and this fact complicates investigations on in vitro effects of substances that are natural constituents of serum. To avoid this restraint we employed defined medium in which fatty acid-free bovine albumin as a carrier of DHA replaced serum. Growth of most cell lines, as well as cells in primary cultures, depends strictly on the presence of serum in growth medium. As expected, withdrawal of serum resulted in growth arrest exemplified by a decrease in protein content compared with control cells grown in the presence of serum and also caused a decrease in ChAT activity (Fig. 1, lower left). DHA, at a concentration of 10 mumol/L, largely prevented both growth arrest in defined medium with fatty acid-free bovine albumin as a carrier of DHA and the attenuation of ChAT activity. DHA at concentrations 10 times higher had no further effect. At a concentration of 100 mumol/L, DHA also significantly increased the number of surface muscarinic receptors compared with cells grown in serum-containing as well as serum-free medium (Fig. 1, upper right). These data demonstrate the ability of DHA at low micromolar concentrations to support cell growth and expression of ChAT activity. Although it is not possible to stipulate a mechanism of action on the expression of ChAT and muscarinic receptors, a plausible explanation could be prevention of apoptosis, evidenced by a sharp decrease in executive caspase-3 activity (Fig. 1, lower right). Apoptosis is a process with a high requirement for energy. An improved metabolic state of cells consequent to suppression of apoptosis might thus better fulfill requirements for protein synthesis and targeting.

Chronic treatment with amyloid beta(1-42) inhibits non-cholinergic high-affinity choline transport in NG108-15 cells through protein kinase C signaling.

We investigated the influence of the amyloid-beta-peptide(1-42) on hemicholinum-3-sensitive high-affinity choline uptake in NG108-15 cells. RT-PCR analysis revealed the presence of mRNA for a choline transporter-like protein but not for cholinergic high-affinity choline transporter. Differentiation of cells increased both hemicholinum-3-sensitive choline uptake and high-affinity hemicholinium-3 binding. This transport was not influenced by tenfold excess of carnitine. Continuous presence of submicromolar concentrations of amyloid-beta-peptide(1-42) during differentiation resulted in a decrease of both choline uptake and hemicholinium-3 binding. These effects were not present when amyloid-beta-peptide(1-42) was added 5 min prior to measurements. Neither differentiation nor amyloid-beta-peptide(1-42) treatment changed levels of choline transporter-like protein mRNA. Protein kinase C inhibition by staurosporine or its inactivation by continuous presence of tetradecanoyl phorbol acetate prevented the inhibitory effect of amyloid-beta-peptide(1-42) treatment on choline uptake. Activation of protein kinase C by tetradecanoyl phorbol acetate during measurement had inhibitory effect on choline uptake in control but not amyloid-beta-peptide(1-42)-treated cells. The concentration of amyloid-beta-peptide(1-42) maximally effective on hemicholinium-3-sensitive choline uptake had no effect on cell growth, oxidative activity, membrane integrity, number of surface muscarinic receptors, caspase-3 and -8 activities, or uptake of deoxyglucose. Results demonstrate that long-term treatment with non-toxic concentrations of amyloid-beta-peptide(1-42) downregulates choline uptake presumably mediated by a choline transporter-like protein through activation of protein kinase C signaling. The decrease of choline uptake may have relevance to the pathogenesis of Alzheimer's disease.

Ultraviolet light-irradiated collagen III modulates expression of cytoskeletal and surface adhesion molecules in rat aortic smooth muscle cells in vitro.

Systemic and pulmonary hypertension is characterised by structural reconstruction of the vascular wall which includes hypertrophy and hyperplasia of vascular smooth muscle cells (VSMCs) and fibroproduction. We hypothesise that these changes are stimulated by non-enzymatic modification of collagen molecules in the injured vascular wall by radicals. We exposed collagen III to ultraviolet (UV) light irradiation which, as indicated by fluorescence and electrophoretic analyses, resulted in its fragmentation. Both irradiated and control unmodified collagen were adsorbed on culture dishes and seeded with VSMCs derived from the rat thoracic aorta. During the first week after seeding, the cells on the modified collagen attained significantly higher population density (by 15-83%), higher mitotic index (by 31-135%) and higher BrdU labelling index (by 32%). However, these cells were less resistant to spontaneous and trypsin-mediated detachment from the growth support. As revealed using enzyme-linked immunosorbent assay in 3-day-old cultures, the cells growing on the irradiated collagen exhibited a lower concentration of beta-1 integrins (-10%, measured per milligram of protein), vinculin (-18%), talin (-6%) and vimentin (-15%). Immunofluorescence staining showed that these molecules were distributed more diffusely and less organised into focal adhesion plaques or cytoskeletal fibres. The concentration of two adhesion molecules of immunoglobulin type, ICAM-1 and VCAM-1, was increased by 11% and 16%, respectively. The concentration of alpha-v integrins and alpha-actin was unchanged; the latter, however, formed fewer distinct microfilament bundles in cells on the modified collagen. Our results suggest that the VSMCs growing on UV-modified collagen are more prone to escape the growth control mediated by cell-extracellular matrix contact and can bind the cells of the immune system.

Effect of cellular differentiation on 11beta-hydroxysteroid dehydrogenase activity in the intestine.

11beta-Hydroxysteroid dehydrogenase (11betaHSD) converts endogenous glucocorticoids to their biologically inactive 11-dehydro derivatives and is therefore able to determine, at least in part, the biological action of glucocorticoids. Type 1 11betaHSD has both oxidase and reductase activities interconverting corticosterone and 11-dehydrocorticosterone, whereas type 2 11betaHSD has only oxidase activity converting corticosterone to 11-dehydrocorticosterone. Since 11betaHSD expression is regulated during development and by hormones in a tissue-specific manner and since glucocorticoids play an important role in postnatal intestinal maturation, we investigated the role of corticosteroids and cytodifferentiation in the regulation of intestinal 11betaHSD. Using rat intestinal organ cultures and epithelial cell lines derived from rat small intestine (IEC-6, IEC-18) and from human colon adenocarcinoma (Caco-2, HT-29), we analyzed the effect of corticosteroids and cytodifferentiation on 11betaHSD. Screening of the clonal cell lines showed that Caco-2 cells expressed by far the greatest 11betaHSD2 oxidase activity, lower activity was observed in HT-29 cells, and lowest activity was seen in IEC cells. Treatment with dexamethasone (50 nM) increased the activity of 11betaHSD2 in IEC-6 cells (+59%) and HT-29 cells (+31%), whereas aldosterone (50 nM) stimulated 11betaHSD2 in IEC-6 cells only (+31%). Caco-2 cells and IEC-18 cells did not respond to corticosteroids. Growth of IEC-6 cells on Matrigel, treatment of HT-29 cells with butyrate, and postconfluency of Caco-2 cells increased not only the markers of cytodifferentiation, such as alkaline phosphatase and sucrase, but also the activity of 11betaHSD2 in all of these cell lines (IEC-6, +96%; HT-29, +139%; Caco-2, +95%). Addition of corticosteroids to these more differentiated cell cultures did not enhance 11betaHSD2 activity. In intestinal organ cultures of suckling rat small intestine, dexamethasone and aldosterone stimulated 11betaHSD by more than 300%. We conclude that corticosteroids markedly and differentially regulate intestinal 11betaHSD2 and that cytodifferentiation of intestinal epithelial cells is associated with upregulation of 11betaHSD2 activity that is independent of corticosteroids.

Quinolinic acid induces NMDA receptor-mediated lipid peroxidation in rat brain microvessels.

Quinolinic acid increased the generation of lipid peroxidation products by isolated rat brain microvessels in vitro. The effect was inhibited both by a specific NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid and by reduced glutathione (GSH). Furthermore, quinolinic acid displaced specific binding of [(3)H]-L-glutamate by cerebral microvessel membranes, particularly in the presence of NMDA receptor co-agonist (glycine) and modulator (spermidine). We conclude that quinolinic acid can cause potentially cytotoxic lipid peroxidation in brain microvessels via an NMDA receptor mediated mechanism.

Interaction of human osteoblast-like Saos-2 and MG-63 cells with thermally oxidized surfaces of a titanium-niobium alloy.

An investigation was made of the adhesion, growth and differentiation of osteoblast-like MG-63 and Saos-2 cells on titanium (Ti) and niobium (Nb) supports and on TiNb alloy with surfaces oxidized at 165°C under hydrothermal conditions and at 600°C in a stream of air. The oxidation mode and the chemical composition of the samples tuned the morphology, topography and distribution of the charge on their surfaces, which enabled us to evaluate the importance of these material characteristics in the interaction of the cells with the sample surface. Numbers of adhered MG-63 and Saos-2 cells correlated with the number of positively-charged (related with the Nb2O5 phase) and negatively-charged sites (related with the TiO2 phase) on the alloy surface. Proliferation of these cells is correlated with the presence of positively-charged (i.e. basic) sites of the Nb2O5 alloy phase, while cell differentiation is correlated with negatively-charged (acidic) sites of the TiO2 alloy phase. The number of charged sites and adhered cells was substantially higher on the alloy sample oxidized at 600°C than on the hydrothermally treated sample at 165°C. The expression values of osteoblast differentiation markers (collagen type I and osteocalcin) were higher for cells grown on the Ti samples than for those grown on the TiNb samples. This was more particularly apparent in the samples treated at 165°C. No considerable immune activation of murine macrophage-like RAW 264.7 cells on the tested samples was found. The secretion of TNF-α by these cells into the cell culture media was much lower than for either cells grown in the presence of bacterial lipopolysaccharide, or untreated control samples. Thus, oxidized Ti and TiNb are both promising materials for bone implantation; TiNb for applications where bone cell proliferation is desirable, and Ti for induction of osteogenic cell differentiation.

Support for the initial attachment, growth and differentiation of MG-63 cells: a comparison between nano-size hydroxyapatite and micro-size hydroxyapatite in composites.

Hydroxyapatite (HA) is considered to be a bioactive material that favorably influences the adhesion, growth, and osteogenic differentiation of osteoblasts. To optimize the cell response on the hydroxyapatite composite, it is desirable to assess the optimum concentration and also the optimum particle size. The aim of our study was to prepare composite materials made of polydimethylsiloxane, polyamide, and nano-sized (N) or micro-sized (M) HA, with an HA content of 0%, 2%, 5%, 10%, 15%, 20%, 25% (v/v) (referred to as N0-N25 or M0-M25), and to evaluate them in vitro in cultures with human osteoblast-like MG-63 cells. For clinical applications, fast osseointegration of the implant into the bone is essential. We observed the greatest initial cell adhesion on composites M10 and N5. Nano-sized HA supported cell growth, especially during the first 3 days of culture. On composites with micro-size HA (2%-15%), MG-63 cells reached the highest densities on day 7. Samples M20 and M25, however, were toxic for MG-63 cells, although these composites supported the production of osteocalcin in these cells. On N2, a higher concentration of osteopontin was found in MG-63 cells. For biomedical applications, the concentration range of 5%-15% (v/v) nano-size or micro-size HA seems to be optimum.

Improved adhesion and differentiation of endothelial cells on surface-attached fibrin structures containing extracellular matrix proteins.

Currently used vascular prostheses are hydrophobic and do not allow endothelial cell (EC) adhesion and growth. The aim of this study was to prepare fibrin (Fb)-based two-dimensional (2D) and three-dimensional (3D) assemblies coated with extracellular matrix (ECM) proteins and to evaluate the EC adhesion, proliferation and differentiation on these assemblies in vitro. Coating of Fb with collagen, laminin (LM), and fibronectin (FN) was proved using the surface plasmon resonance technique. On all Fb assemblies, ECs reached higher cell densities than on polystyrene after 3 and 7 days of culture. Immunoflurescence staining showed better assembly of talin and vinculin into focal adhesion plaques, and also more apparent staining of vascular endothelial cadherin on surface-attached 3D Fb and protein-coated Fb assemblies. On these samples, ECs also contained a lower concentration of intercellular adhesion molecule-1, measured by enzyme-linked immunosorbent assay. Higher concentrations of CD31 (platelet-endothelial cell adhesion molecule-1) were found on 3D Fb coated with LM, and higher concentrations of von Willebrand factor were found on 3D Fb coated with type I collagen or LM in comparison to 2D Fb layers. The results indicate that ECM protein-coated 2D and 3D Fb assemblies can be used for versatile applications in various tissue replacements where endothelialization is desirable, for example, vascular prostheses and heart valves.

Polylactide nanofibers with hydroxyapatite as growth substrates for osteoblast-like cells.

Various types of nanofibers are increasingly used in tissue engineering, mainly for their ability to mimic the architecture of tissue at the nanoscale. We evaluated the adhesion, growth, viability, and differentiation of human osteoblast-like MG 63 cells on polylactide (PLA) nanofibers prepared by needle-less electrospinning and loaded with 5 or 15 wt % of hydroxyapatite (HA) nanoparticles. On day 7 after seeding, the cell number was the highest on samples with 15 wt % of HA. This result was confirmed by the XTT test, especially after dynamic cultivation, when the number of metabolically active cells on these samples was even higher than on control polystyrene. Staining with a live/dead kit showed that the viability of cells on all nanofibrous scaffolds was very high and comparable to that on control polystyrene dishes. An enzyme-linked immunosorbent assay revealed that the concentration of osteocalcin was also higher in cells on samples with 15 wt % of HA. There was no immune activation of cells (measured by production of TNF-alpha), associated with the incorporation of HA. Moreover, the addition of HA suppressed the creep behavior of the scaffolds in their dry state. Thus, nanofibrous PLA scaffolds have potential for bone tissue engineering, particularly those with 15 wt % of HA.

Adhesion and Growth of Vascular Smooth Muscle Cells on Nanostructured and Biofunctionalized Polyethylene.

Cell colonization of synthetic polymers can be regulated by physical and chemical modifications of the polymer surface. High-density and low-density polyethylene (HDPE and LDPE) were therefore activated with Ar⁺ plasma and grafted with fibronectin (Fn) or bovine serum albumin (BSA). The water drop contact angle usually decreased on the plasma-treated samples, due to the formation of oxidized groups, and this decrease was inversely related to the plasma exposure time (50-300 s). The presence of nitrogen and sulfur on the polymer surface, revealed by X-ray photoelectron spectroscopy (XPS), and also by immunofluorescence staining, showed that Fn and BSA were bound to this surface, particularly to HDPE. Plasma modification and grafting with Fn and BSA increased the nanoscale surface roughness of the polymer. This was mainly manifested on HDPE. Plasma treatment and grafting with Fn or BSA improved the adhesion and growth of vascular smooth muscle cells in a serum-supplemented medium. The final cell population densities on day 6 after seeding were on an average higher on LDPE than on HDPE. In a serum-free medium, BSA grafted to the polymer surface hampered cell adhesion. Thus, the cell behavior on polyethylene can be modulated by its type, intensity of plasma modification, grafting with biomolecules, and composition of the culture medium.

Adhesion, growth, and maturation of vascular smooth muscle cells on low-density polyethylene grafted with bioactive substances.

The attractiveness of synthetic polymers for cell colonization can be affected by physical, chemical, and biological modification of the polymer surface. In this study, low-density polyethylene (LDPE) was treated by an Ar(+) plasma discharge and then grafted with biologically active substances, namely, glycine (Gly), polyethylene glycol (PEG), bovine serum albumin (BSA), colloidal carbon particles (C), or BSA+C. All modifications increased the oxygen content, the wettability, and the surface free energy of the materials compared to the pristine LDPE, but these changes were most pronounced in LDPE with Gly or PEG, where all the three values were higher than in the only plasma-treated samples. When seeded with vascular smooth muscle cells (VSMCs), the Gly- or PEG-grafted samples increased mainly the spreading and concentration of focal adhesion proteins talin and vinculin in these cells. LDPE grafted with BSA or BSA+C showed a similar oxygen content and similar wettability, as the samples only treated with plasma, but the nano- and submicron-scale irregularities on their surface were more pronounced and of a different shape. These samples promoted predominantly the growth, the formation of a confluent layer, and phenotypic maturation of VSMC, demonstrated by higher concentrations of contractile proteins alpha-actin and SM1 and SM2 myosins. Thus, the behavior of VSMC on LDPE can be regulated by the type of bioactive substances that are grafted.