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.

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.

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.

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.

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.

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.

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.

Nanofibrous poly(lactide-co-glycolide) membranes loaded with diamond nanoparticles as promising substrates for bone tissue engineering.

BACKGROUND: Nanofibrous scaffolds loaded with bioactive nanoparticles are promising materials for bone tissue engineering. METHODS: In this study, composite nanofibrous membranes containing a copolymer of L-lactide and glycolide (PLGA) and diamond nanoparticles were fabricated by an electrospinning technique. PLGA was dissolved in a mixture of methylene chloride and dimethyl formamide (2:3) at a concentration of 2.3 wt%, and nanodiamond (ND) powder was added at a concentration of 0.7 wt% (about 23 wt% in dry PLGA). RESULTS: In the composite scaffolds, the ND particles were either arranged like beads in the central part of the fibers or formed clusters protruding from the fibers. In the PLGA-ND membranes, the fibers were thicker (diameter 270 ± 9 nm) than in pure PLGA meshes (diameter 218 ± 4 nm), but the areas of pores among these fibers were smaller than in pure PLGA samples (0.46 ± 0.02 µm(2) versus 1.28 ± 0.09 µm(2) in pure PLGA samples). The PLGA-ND membranes showed higher mechanical resistance, as demonstrated by rupture tests of load and deflection of rupture probe at failure. Both types of membranes enabled the attachment, spreading, and subsequent proliferation of human osteoblast-like MG-63 cells to a similar extent, although these values were usually lower than on polystyrene dishes. Nevertheless, the cells on both types of membranes were polygonal or spindle-like in shape, and were distributed homogeneously on the samples. From days 1-7 after seeding, their number rose continuously, and at the end of the experiment, these cells were able to create a confluent layer. At the same time, the cell viability, evaluated by a LIVE/DEAD viability/cytotoxicity kit, ranged from 92% to 97% on both types of membranes. In addition, on PLGA-ND membranes, the cells formed well developed talin-containing focal adhesion plaques. As estimated by the determination of tumor necrosis factor-alpha levels in the culture medium and concentration of intercellular adhesion molecule-1, MG-63 cells, and RAW 264.7 macrophages on these membranes did not show considerable inflammatory activity. CONCLUSION: This study shows that nanofibrous PLGA membranes loaded with diamond nanoparticles have interesting potential for use in bone tissue engineering.

A perivascular system releasing sirolimus prevented intimal hyperplasia in a rabbit model in a medium-term study.

The main complication of aortocoronary reconstruction with vein grafts is restenosis in the course of time. The aim was to assess the effect of a periadventitial polyester mesh releasing sirolimus on intimal hyperplasia of autologous grafts. We implanted v. jugularis ext. into a. carotis communis in rabbits. The vein graft was either intact, or was wrapped with a pure polyester mesh, or with a sirolimus-releasing mesh. Three and six weeks after surgery, the veins were subjected to standard histological staining and the thicknesses of the tunica intima, the media and the intima-media complex were measured. Wrapping the vein with a mesh releasing sirolimus or with a pure mesh decreased the thickness of the intima in comparison with a vein graft by 73 ± 11% or 73 ± 8% after 3 weeks, and by 73 ± 9% or 59 ± 12% after 6 weeks, respectively. Sirolimus-releasing meshes reduced the thickness of the media by 65 ± 9% and 20 ± 12% after 3 and 6 weeks. The thickness of the intima-media complex in grafts with sirolimus-releasing meshes decreased by 60 ± 6% and 30 ± 13% in comparison with pure PES meshes, after 3 and 6 weeks, respectively. A periadventitial polyester mesh releasing sirolimus has the potential to become an effective device in preventing vein graft restenosis.

Effect of different surface nanoroughness of titanium dioxide films on the growth of human osteoblast-like MG63 cells.

Cell behavior depends strongly on the physical and chemical properties of the material surface, for example, its chemistry and topography. The authors have therefore assessed the influence of materials of different chemical composition (i.e., glass substrates with and without TiO(2) films in anatase form) and different surface roughness (R(a) = 0, 40, 100, or 170 nm) on the adhesion, proliferation, and osteogenic differentiation of human osteoblast-like MG63 cells. On day 1 after seeding, the largest cell spreading area was found on flat TiO(2) films (R(a) = 0 nm). On TiO(2) films with R(a) = 170 nm, the cell spreading area was larger and the number of initially adhering cells was higher than the values on the corresponding uncoated glass. On day 3 after seeding, the cell number was higher on the TiO(2) films (R(a) = 0 and 40 nm) than on the corresponding glass substrates and the standard polystyrene dishes. On day 7, all TiO(2) films contained higher cell numbers than the corresponding glass substrates, and the cells on the TiO(2) films with R(a) = 40 and 100 nm also contained a higher concentration of ß-actin. These results indicate that TiO(2) coating had a positive influence on the adhesion and subsequent proliferation of MG63 cells. In addition, on all investigated materials, the cell population density achieved on day 7 decreased with increasing surface roughness. The concentration of osteocalcin, measured per mg of protein, was significantly lower in the cells on rougher TiO(2) films (R(a) = 100 and 170 nm) than in the cells on the polystyrene dishes. Thus, it can be concluded that the adhesion, growth, and phenotypic maturation of MG63 cells were controlled by the interplay between the material chemistry and surface topography, and were usually better on smoother and TiO(2)-coated surfaces than on rougher and uncoated glass substrates.

Enhanced growth and osteogenic differentiation of human osteoblast-like cells on boron-doped nanocrystalline diamond thin films.

Intrinsic nanocrystalline diamond (NCD) films have been proven to be promising substrates for the adhesion, growth and osteogenic differentiation of bone-derived cells. To understand the role of various degrees of doping (semiconducting to metallic-like), the NCD films were deposited on silicon substrates by a microwave plasma-enhanced CVD process and their boron doping was achieved by adding trimethylboron to the CH(4):H(2) gas mixture, the B∶C ratio was 133, 1000 and 6700 ppm. The room temperature electrical resistivity of the films decreased from >10 MΩ (undoped films) to 55 kΩ, 0.6 kΩ, and 0.3 kΩ (doped films with 133, 1000 and 6700 ppm of B, respectively). The increase in the number of human osteoblast-like MG 63 cells in 7-day-old cultures on NCD films was most apparent on the NCD films doped with 133 and 1000 ppm of B (153,000 ± 14,000 and 152,000 ± 10,000 cells/cm(2), respectively, compared to 113,000 ± 10,000 cells/cm(2) on undoped NCD films). As measured by ELISA per mg of total protein, the cells on NCD with 133 and 1000 ppm of B also contained the highest concentrations of collagen I and alkaline phosphatase, respectively. On the NCD films with 6700 ppm of B, the cells contained the highest concentration of focal adhesion protein vinculin, and the highest amount of collagen I was adsorbed. The concentration of osteocalcin also increased with increasing level of B doping. The cell viability on all tested NCD films was almost 100%. Measurements of the concentration of ICAM-1, i.e. an immunoglobuline adhesion molecule binding inflammatory cells, suggested that the cells on the NCD films did not undergo significant immune activation. Thus, the potential of NCD films for bone tissue regeneration can be further enhanced and tailored by B doping and that B doping up to metallic-like levels is not detrimental for cells.

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.

Perivascular sirolimus-delivery system.

Autologous vein grafts are often used for treating damaged vessels, e.g. arteriovenous fistulas or arterial bypass conduits. Veins have a different histological structure from arteries, which often leads to intimal hyperplasia and graft restenosis. The aim of this study was to develop a perivascular sirolimus-delivery system that would release the antiproliferative drug sirolimus in a controlled manner. Polyester Mesh I was coated with purasorb, i.e. a copolymer of L-lactide and ɛ-caprolactone, with dissolved sirolimus; Mesh II was coated with two copolymer layers; the layer with dissolved sirolimus was overlaid with pure purasorb. This arrangement allowed sirolimus to be released for 6 and 4 weeks, for Mesh I and Mesh II, respectively. Mesh II released sirolimus more homogeneously, without the initial burst effect during the first week. However, the cumulative release curve was steeper at later time points than the curve for Mesh I. Both meshes inhibited proliferation of rat vascular smooth muscle cells during 14-day culture in vitro and preserved excellent cell viability. Newly developed sirolimus-releasing perivascular meshes are promising devices for preventing autologous graft restenosis.

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.

Expression of dipeptidyl peptidase-IV activity and/or structure homologs in human meningiomas.

Meningiomas are tumors derived from arachnoid cap cells that represent approximately 30% of all intracranial tumors. In this study, we investigated 22 human meningiomas for the expression of dipeptidyl peptidase (DPP)-IV activity and/or structure homologs (DASH), including canonical DPP-IV/CD26, fibroblast activation protein-alpha (FAPalpha), DPP8 and DPP9. DPP-IV-like enzymatic activity, including all enzymatically-active DASH molecules, was found in all 18 benign meningiomas WHO grade I and IV atypical meningiomas WHO grade II by continuous rate fluorimetric assay in tissue homogenates and catalytic enzyme histochemistry in situ. In atypical meningiomas, this activity was significantly higher and was associated with higher cell proliferation as detected by Ki67 antigen immunohistochemistry. The expression of DPP-IV/CD26 and FAPalpha demonstrated by real-time RT-PCR and immunohistochemistry was low. As shown histochemically, it occurred most often on the surface of fibrous bundles and whorls rich in extracellular matrix. Compared to DPP-IV/CD26 and FAPalpha, the expression of DPP8 and DPP9 was higher and, in addition, it was present also in the cells inside these structures. Expression of CXCR4, the receptor of pro-proliferative chemokine stromal cell-derived factor-1alpha (SDF-1alpha), DPP-IV substrate, was found in all tumors, suggesting higher values in atypical grade II samples. This is the first report on the expression status of dipeptidyl peptidase-IV and related molecules in meningiomas. It shows that DPP8 and DPP9 prevail over canonical DPP-IV/CD26 and FAPalpha in all examined patients. In addition, the study suggests an increase of DPP-IV-like enzymatic activity in these tumors of WHO grade II.

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.

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.

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.

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.

Nanocomposite Ti/hydrocarbon plasma polymer films from reactive magnetron sputtering as growth support for osteoblast-like and endothelial cells.

Nanocomposite Ti/hydrocarbon plasma polymer (Ti/ppCH) films were deposited by DC magnetron sputtering of titanium target in n-hexane, argon, or a mixture of these two gases. The resultant films were heterogeneous, with inorganic regions of nanometer scale distributed within a plasma polymer matrix. The titanium content was controlled by adjusting the argon/n-hexane ratio in the working gas. In the pure n-hexane atmosphere, the Ti concentration was found to be below 1 at %, whereas in pure argon it reached 20 at %, as measured by Rutherford backscattering spectroscopy and elastic recoil detection analysis (RBS/ERDA). A high level of titanium oxidation is detected with TiO(2), substoichiometric titania, and titanium carbide, composing an inorganic phase of the composite films. In addition, high hydrogen content is detected in films rich with titanium. Ti-deficient and Ti-rich films proved equally good substrates for adhesion and growth of cultured human osteoblast-like MG 63 cells. In these cells, the population densities on days 1, 3, and 7 after seeding, spreading area on day 1, formation of talin-containing focal adhesion plaques as well as concentrations of talin and osteocalcin (per mg of protein) were comparable to the values obtained in cells on the reference cell culture materials, represented by microscopic glass coverslips or a polystyrene dish. An interesting finding was made when the Ti/ppCH films were seeded with calf pulmonary artery endothelial cells of the line CPAE. The cell population densities, the spreading area and also the concentration of von Willebrand factor, a marker of endothelial cell maturation, were significantly higher on Ti-rich than on Ti-deficient films. On Ti-rich films, these parameters were also higher or similar in comparison with the reference cell culture materials. Thus, both types of films could be used for coating bone implants, of which the Ti-rich film remains effective in enhancing the endothelialization of blood contacting artificial materials.