The aim of this study was to characterize an in vitro modulating effect of three commensal Lactobacillus strains on cellular differentiation of non-transformed crypt-like rat small intestinal cell line IEC-18. IEC-18 was grown on extracellular matrix, with or without presence of Lactobacillus strains. Gene expression of IEC-18 bacterial detection system - such as Toll-like receptors TLR-2, TLR-4, signal adapter MyD88, cytoplasmic NOD2 receptor, inflammatory cytokines IL-18, IL-1beta, chemokine IL-8 and enzyme caspase-1 - was evaluated using real-time PCR. Expression and localization of TLR-2, TLR-4, IL-18 and caspase-1 proteins was demonstrated by Western blotting and immunofluorescent staining. Secretion of IL-18 to apical and basolateral surfaces was assayed by ELISA. Our results suggested that L. casei LOCK0919 accelerated differentiation of IEC-18 by stimulating TLR-2, TLR-4, MyD88, IL-18, caspase-1 mRNAs and proteins. L. casei LOCK0919 increased expression and transfer of villin and beta-catenin from cytoplasm to cell membrane. Presence of L. rhamnosus LOCK0900 resulted in detachment of IEC-18 layer from extracellular matrix leading to induction of IL-1beta, of TLR-2 and IL-8 mRNAs and stimulation of MyD88, caspase-1 and cytosolic receptor NOD2 mRNAs. L. rhamnosus LOCK0908 was not recognized by TLR-2 or TLR-4 receptors. Lactobacilli-IEC-18 crosstalk enhanced immune and barrier mucosal functions.
- MeSH
- beta-katenin biosyntéza MeSH
- buněčná diferenciace účinky léků MeSH
- cytokiny biosyntéza MeSH
- epitelové buňky účinky léků MeSH
- interleukin-18 biosyntéza MeSH
- kaspasa 1 biosyntéza MeSH
- krysa rodu rattus MeSH
- Lacticaseibacillus rhamnosus * MeSH
- Lactobacillus casei * MeSH
- messenger RNA biosyntéza MeSH
- mikrofilamentové proteiny biosyntéza MeSH
- probiotika farmakologie MeSH
- regulace genové exprese účinky léků MeSH
- střevní sliznice cytologie účinky léků MeSH
- subcelulární frakce metabolismus MeSH
- toll-like receptory biosyntéza účinky léků MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
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.
- MeSH
- buněčná adheze MeSH
- buněčná diferenciace MeSH
- buněčné linie MeSH
- endoteliální buňky cytologie MeSH
- fibrin chemie MeSH
- kolagen chemie MeSH
- laminin chemie MeSH
- skot MeSH
- tkáňové podpůrné struktury chemie MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- cytoskeletální proteiny metabolismus MeSH
- fyziologie buňky účinky léků MeSH
- hydroxyapatit chemie farmakologie MeSH
- lidé MeSH
- mikrosféry MeSH
- nádorové buněčné linie MeSH
- nanočástice chemie MeSH
- osteoblasty MeSH
- osteokalcin metabolismus MeSH
- osteopontin metabolismus MeSH
- rozpustnost MeSH
- spektrometrie rentgenová emisní MeSH
- velikost částic * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- buněčná adheze MeSH
- buněčná diferenciace * MeSH
- buněčné linie MeSH
- hydroxyapatit chemie MeSH
- kostní náhrady MeSH
- lidé MeSH
- nanovlákna chemie MeSH
- osteoblasty cytologie metabolismus MeSH
- osteokalcin biosyntéza MeSH
- polyestery chemie MeSH
- tkáňové inženýrství metody MeSH
- viabilita buněk MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- biologické markery metabolismus MeSH
- buněčná adheze účinky léků MeSH
- buněčná diferenciace účinky léků MeSH
- buněčné linie MeSH
- kolagen typu I metabolismus MeSH
- lidé MeSH
- lipopolysacharidy farmakologie MeSH
- makrofágy cytologie účinky léků metabolismus MeSH
- myši MeSH
- osteoblasty cytologie účinky léků metabolismus MeSH
- osteokalcin metabolismus MeSH
- oxidace-redukce MeSH
- povrchové vlastnosti MeSH
- proliferace buněk účinky léků MeSH
- slitiny chemie farmakologie MeSH
- statická elektřina MeSH
- tkáňové podpůrné struktury * MeSH
- TNF-alfa farmakologie MeSH
- vysoká teplota MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- aorta cytologie účinky léků MeSH
- biokompatibilní materiály chemie farmakologie MeSH
- buněčná adheze účinky léků MeSH
- glycin chemie farmakologie MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- myocyty hladké svaloviny cytologie účinky léků MeSH
- polyethylen chemie farmakologie MeSH
- polyethylenglykoly chemie farmakologie MeSH
- povrchové vlastnosti MeSH
- proliferace buněk účinky léků MeSH
- sérový albumin hovězí chemie farmakologie MeSH
- svaly hladké cévní cytologie účinky léků MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
γ-Glutamyltranspeptidase (GGT, syn. γ-Glutamyltransferase) and dipeptidylpeptidase-IV (DPP-IV) activity participates in metabolic and growth control of normal and tumor cells by processing biologically active peptides. Here, we report on up-regulation of these enzymes in human brain gliomas determined by catalytic enzyme histochemistry and immunocytochemistry. Higher activity of GGT was found in 50%, 68% and 81% of WHO grade II, III and IV tumors, respectively. The process started at/near the microvasculature, from where it spread to the parenchyma. On average, the enzyme activity in grade II, III and IV gliomas exceeded controls 2.0, 3.0 and 3.5-fold, respectively. Up-regulation of DPP-IV-like activity also started at the microvasculature, but mainly in pericytes and mononuclear-like cells around the vessels and dispersed in the parenchyma. Marked elevation of this enzyme activity, comprising also tumor parenchyma, occurred only in grade IV glioblastomas (65% patients; 3.6 times above controls) which can, therefore, help in their differentiation from grade III gliomas. The increase of total DPP-IV-like activity also included its two enzymatic homologs, the canonical DPP-IV/CD26 and FAP-1α. The increase in GGT is supposed to be a tumor grade dependent response of microvasculature and tumor astrocytes to stress induced by tissue hypoxia and/or the metabolic aberrancies. The increase in DPP-IV-like activity in high-grade tumors can be attributed to inflammatory/scavenging processes performed by the mononuclear-like cells and, in glioblastomas, also to regressive changes in the structure and function of the microvasculature and tumor parenchyma, including astrocyte stress response. The inverse relationship between DPP-IV-like activity and Ki67 in most glioblastomas and shorter survival time of patients with low activity of this enzyme also suggest its anti-oncogenic effects.
- MeSH
- dipeptidylpeptidasa 4 biosyntéza MeSH
- dospělí MeSH
- gama-glutamyltransferasa biosyntéza MeSH
- gliom enzymologie patologie MeSH
- imunohistochemie MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- nádorové biomarkery analýza MeSH
- nádory mozku enzymologie patologie MeSH
- prognóza MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- stupeň nádoru MeSH
- upregulace MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- buněčná adheze MeSH
- buněčné linie MeSH
- diamant chemie MeSH
- kostní náhrady chemie MeSH
- kyselina mléčná chemie MeSH
- kyselina polyglykolová chemie MeSH
- lidé MeSH
- mikrofilamenta metabolismus MeSH
- mikroskopie elektronová rastrovací MeSH
- myši MeSH
- nanočástice chemie ultrastruktura MeSH
- nanomedicína MeSH
- nanovlákna chemie ultrastruktura MeSH
- osteoblasty cytologie imunologie fyziologie MeSH
- proliferace buněk MeSH
- testování materiálů MeSH
- tkáňové inženýrství metody MeSH
- tkáňové podpůrné struktury chemie MeSH
- transmisní elektronová mikroskopie MeSH
- viabilita buněk MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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 β-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.
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.
- MeSH
- adenosin-5'-(N-ethylkarboxamid) farmakologie MeSH
- agonisté purinergního receptoru P1 farmakologie MeSH
- citalopram farmakologie MeSH
- deprese farmakoterapie metabolismus MeSH
- fluoxetin farmakologie MeSH
- gliom MeSH
- krysa rodu rattus MeSH
- nádorové buněčné linie MeSH
- nádory mozku MeSH
- neurony cytologie účinky léků metabolismus MeSH
- proteiny vázající GTP - alfa-podjednotky Gq-G11 metabolismus MeSH
- selektivní inhibitory zpětného vychytávání serotoninu farmakologie MeSH
- sertralin farmakologie MeSH
- signální transdukce účinky léků fyziologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
