Adhesion of human osteoblasts to titanium: A morpho-functional analysis with confocal microscopy.

Properties of surface affect the interactions between the implant and osteoblasts and direct the clinical osteointegrative outcome. The aim of this in vitro study was to describe the adhesion of living human osteoblasts to titanium disks with differently prepared surfaces: sand blasted with ZrO(2) particles and acid-etched (Soft-SLA, S-SLA) or with Al(2)O(3) particles and acid-etched (Hard-SLA, H-SLA), smooth surface (SS). Confocal microscopy was exploited to follow cell morpho-functional features either on living cells (cell shape with calcein-acethoxymethylester and mitochondria with tetramethylrhodamine methyl ester) or on fixed cells (immunocytochemistry of beta1-integrin and of actin) 6h or 24h after seeding. The underlying surface was visualized simultaneously on the same field. No cytotoxic effect was detected at any time and on any surface. At 6h after seeding, osteoblasts showed either a rounded or polygonal shape on both rough surfaces. Several features suggested that adhesion was faster with a higher level of organization on S-SLA than on H-SLA. Indeed osteoblasts grown on S-SLA were wider and with more extended protrusions than those on H-SLA. Active mitochondria on S-SLA occupied perinuclear areas and cellular prolongations, whereas on H-SLA they were mainly focused around nucleus. Organization of integrin beta1-subunit and actin, confirmed different kinetics of cell adhesion. At 6h integrin beta1-subunit was distributed along the periphery on the cell-biomaterial focal complexes in cells grown on S-SLA, whereas it was unevenly dispersed in membrane of cells cultured on H-SLA. Stress actin fibers were well defined in cells cultured on S-SLA, whereas they were scarcely evident on H-SLA. Osteoblasts seeded on smooth surface for 6h had morpho-functional features typical of adhesion, with some elements characterized by an elongated shape with an evident main longitudinal axis. At 24h osteoblasts were spread-out onto all surfaces. Nonetheless, different morphologies were shown in response to the different surfaces tested: polygonal cells prevailed on SLA surfaces, whereas almost all the cells on SS were long with two principal prolongations. At 24h number of cells adhered to the three kind of surfaces was similar, but during the following three days, cells seeded on S-SLA and on SS proliferated to a greater extent than those cultured on H-SLA. Analysis of morpho-functional parameters performed in living cells, and in particular the study of mitochondria organization, proved to be a valuable tool to follow cell-biomaterial adhesion. A higher level of spreading occurring in osteoblasts grown on S-SLA and SS at early times accounted for a faster subsequent cell proliferation. Nonetheless, these comparable activities were exerted by cells showing polygonal or elongated shapes when grown respectively on S-SLA or on SS. The former is typical of osteogenic cells, whereas the latter resumes a fibroblast-like morphology, that would result in an ineffective in vivo osteointegrative process.

Dose-dependent effects of platelet gel releasate on activities of human osteoblasts.

BACKGROUND: Platelet-rich plasma is used in oral and maxillofacial surgery; however, its real efficacy is debated. Also, the in vitro effects on bone-specific functions are contradictory. Understanding the mechanisms of action of platelet-derived factors could be the basis for their proper use in clinical applications. METHODS: The functional parameters of osteoblasts (proliferation, alkaline phosphatase, collagen synthesis, and calcium deposition) were analyzed in vitro for 14 days in the presence of different concentrations (100%, 33%, and 11%) of platelet gel releasate (PGR). RESULTS: Concentrations of 100% PGR and 33% PGR stimulated cells to proliferate more than 10% fetal calf serum. The effect on cell proliferation was dose dependent, and the addition of dexamethasone (dex) and beta-glycerophosphate (beta-GP) reduced the proliferative effects. Alkaline phosphatase activity was stimulated by 33% PGR and 11% PGR after 7 days and was induced further by dex and beta-GP. Also, collagen synthesis, measured on day 11, was stimulated by 33% PGR and 11% PGR. Calcium deposition, evaluated after 7 and 14 days, was greatest in cells treated with PGR supplemented with dex and GP. The mineralization process increased with time; on day 14, calcium aggregates were observed in all cultures treated with PGR (100%, 33%, and 11%). CONCLUSIONS: PGR stimulated osteoblast proliferation in a dose dependent manner and, when used at 33% and 11%, induced maximum levels of alkaline phosphatase and collagen synthesis. Moreover, in the presence of dex and beta-GP, PGR stimulated the end maturative status of cells as expressed by the deposition of calcium nodules.

Life on the wire: on tensegrity and force balance in cells.

Since cell mechanics has attracted the attention of a growing number of researchers, several models have been proposed to explain cell mechanical behavior, among which tensegrity is certainly the most convincing one. Originally developed by the architect Buckminster Fuller, tensegrity structures are based on the presence of discontinuous compression elements that balance the force generated by continuous tension elements, thus reaching an equilibrium that is completely independent of gravity. This model is a useful tool to predict cell spreading, motility and especially mechanotransduction, i.e. the capability to transform mechanical stresses into biochemical responses, a key process in homeostasis of many tissues that must continuously withstand mechanical forces, like bone, but which is still poorly understood.

Sympathetic modulation by carvedilol and losartan reduces angiotensin II-mediated lipolysis in subcutaneous and visceral fat.

Advanced heart failure is characterized by increased activation of the renin-angiotensin system and the development of cachexia. Angiotensin II (Ang II) has been proposed as a lipid metabolism regulator. The effects of exogenous Ang II (osmotic minipump, 525 ng/kg/min for 12 d) on interstitial sc glycerol and norepinephrine levels, indexes of lipolysis, and sympathetic activation, respectively, were measured in Sprague Dawley rats by consecutive microdialysis performed in vivo in white adipose tissue. Higher sustained interstitial glycerol and norepinephrine levels were found after 7 and 12 d of Ang II infusion. Triglyceride to DNA content ratio and adipocyte diameter were reduced in sc and visceral (retroperitoneal and epididymal) fat tissues of Ang II-infused rats, whose body weight was lower and blood pressure higher. Losartan, an Ang II receptor 1 blocker, and carvedilol, an alpha1-nonselective-beta1,2,3-adrenergic blocker, but not doxazosin, an alpha1-selective-adrenergic blocker, lowered glycerol and norepinephrine levels, preventing lipolysis and weight loss. Our results indicate that Ang II stimulates lipolysis in sc and visceral adipocytes by sympathetic activation and beta-adrenergic-receptor stimulation. Nonselective-beta-adrenergic and Ang II-receptor1 blockade markedly attenuated the rise of norepinephrine, preventing catabolic effects. The metabolic benefits of carvedilol and losartan, in addition to recognized protective cardiovascular effects, may be relevant in cachectic patients with advanced heart failure.

Three-dimensional localization and dynamics of centromeres in mouse oocytes during folliculogenesis.

Very little is known about oocyte nuclear architecture during folliculogenesis. Using antibodies to reveal centromeres, Hoechst-staining to detect the AT-rich pericentromeric heterochromatin (chromocenters), combined with confocal microscopy for the three-dimensional analysis of the nucleus, we demonstrate that during mouse folliculogenesis the oocyte nuclear architecture undergoes dynamic changes. In oocytes isolated from primordial and primary follicles, centromeres and chromocenters were preferentially located at the periphery of the nucleus. During oocyte growth, centromeres and chromocenters were initially found spread within the nucleus and then progressively clustered around the periphery of the nucleolus. Our results indicate that the oocyte nuclear achitecture is developmentally regulated and they contribute to a further understanding of the role of nuclear organization in the regulation of genome functioning during differentiation and development.

Calcein-AM is a detector of intracellular oxidative activity.

Calcein-acetoxymethylester (calcein-AM) is a non-fluorescent, cell permeant compound, which is converted by intracellular esterases into calcein, an anionic fluorescent form. It is used in microscopy and fluorometry and provides both morphological and functional information of viable cells. In this study we have tested the response of calcein-AM to oxidation. In cell-free fluorometric assays, H2O2 and xanthine-xanthine oxidase induced a dose-dependent emission of the AM form but had no effects on calcein. Fluorometric and confocal microscopy tests on human fibroblasts confirmed that the cell permeant AM form is the actual sensor since its removal from culture medium, and its consequent back-diffusion, made the system insensitive to oxidative stimuli. In time-lapse confocal microscopy, calcein-AM detected changes in the intracellular redox state following direct oxidation (H2O2, xanthine-xanthine oxidase) and phorbol ester treatment. Comparative tests showed that calcein-AM sensitivity to oxidation is about one order of magnitude higher than other fluorescein derivatives. The absence of leakage, due to the presence of the probe in the extracellular compartment, and its low toxicity allow to perform experiments for prolonged times following the response to the same or different stimuli repeatedly applied. We propose calcein-AM as a sensitive tool for intracellular ROS generation in living cells with useful applications for real-time imaging in confocal microscopy.

Methylmercury cytotoxicity in PC12 cells is mediated by primary glutathione depletion independent of excess reactive oxygen species generation.

Low doses, chronic exposure to mercurial organic compounds is a worldwide health concern and could be pathogenetically relevant as co-factor in several neurodegenerative diseases. In this in vitro study we wanted to further improve our knowledge on the mechanisms of toxicity of methylmercury hydroxide (MeHgOH) in the unprimed PC12 cell line. Cell viability, mitochondrial function, redox state, and cell morphology were recorded at different time points to sequence the events leading to cell death. The lowest cytotoxic concentration and EC50 were 0.3 and 1.3 microM, respectively. 5 microM MeHgOH was fatal for 80% of the cell population after 24 h; within 1 h it caused glutathione (GSH) depletion and a partial dissipation of Deltapsim. At this concentration, reactive oxygen species (ROS) generation was only slight and delayed. After 6h more than 50% of ATP was available and caspase 3 was active. Time-lapse confocal microscopy showed that only a fraction of the cells completed apoptosis while others turned toward necrosis (necrapoptosis). Pre-incubation with N-acetylcysteine (NAC) and GSH but not Cyclosporin A rescued over 80% of the cells. These results provide experimental evidence that, in this cell model, MeHgOH triggers cell death via a primary depletion of GSH but in the absence of ROS overproduction.

Different titanium surface treatment influences human mandibular osteoblast response.

BACKGROUND: Six titanium disks with six different surface treatments were examined: SS: smooth (polished) surface; TPS: plasma spray; C100: sand blasting by aluminum oxide (Al2O3) diameter 100 microm and acid etching; C150: sand blasting by Al2O3 diameter 150 microm and acid etching; B60: sand blasting by zirconium oxide (ZrO2) diameter 60 microm and acid etching; and B120: sand blasting by ZrO2 diameter 120 microm and acid etching. METHODS: The surface characteristics were determined by scanning electron microscopy (SEM) observation and a roughness tester. Raman spectroscopy was used to determine the presence of residual substances on the samples. Cells were seeded onto the disk and after 24 hours, 6 days, and 12 days were observed under SEM and growth curves generated with a cell counter. Some samples were used to determine alkaline phosphatase activity (ALP), using a colorimetric assay. RESULTS: SEM observation revealed drastic differences in surface microtopography, with a higher cell density on sand-blasted and acid-etched (SLA) samples than SS and TPS, and more regularly aligned cells on B60 and B120 surfaces than on the others. The growth curves showed a greater adhesion of cells on the etched/blasted surfaces compared to the SS and TPS surfaces. The number of cells increased on all the SLA samples, especially B60, throughout the experiment. At the same time, there was considerable ALP activity on the B60 sample, while it remained at extremely low levels on SS and TPS surfaces. Raman analyses revealed Al2O3 debris on C100 and C150, partly explaining the poorer performances of these two surface treatments, since this substance was shown to be toxic for cultured osteoblasts. CONCLUSIONS: Surface treatments influence the growth and the metabolic activity of cultured osteoblasts, and B60 seems to be the most favorable surface inducing a more pronounced proliferation of cells together with a high differentiation degree.

Ex vivo characterization of tumor-derived melanoma antigen encoding gene-specific CD8+cells in patients with hepatocellular carcinoma.

BACKGROUND/AIMS: Members of the melanoma antigen encoding gene family are expressed in tumors of different histological types but not in normal tissue. For this reason, they are attractive targets for cancer immunotherapy. METHODS: In the present study, we analyzed the expression of MAGE-1 and -3 genes in the hepatocellular carcinoma (HCC) tissue as well as frequency, phenotype and function of circulating and tumor infiltrating CD8+ cells specific for HLA-A1 and -A2 restricted epitopes of MAGE-1 and -3. RESULTS: Our study shows for the first time the presence of MAGE/tetramer+ CD8 cells in the tumor tissue of patients with HCC. These cells are able to recognize the MAGE-1 sequence 161-169 and the MAGE-3 sequence 271-279. In a patient with a particularly high frequency of MAGE-1 sequence 161-169-specific T cells, phenotypic and functional analysis was performed showing a phenotype of recently-primed CD8 cells (CD28+CD27+CD45RA-CCR7). CONCLUSIONS: The observation of a spontaneous in vivo priming of a MAGE-specific T cell response in patients with HCC and the high frequency of MAGE antigens expression in this tumor, makes this antigen a potential candidate for a MAGE-specific immunotherapy in hepatocellular carcinoma.

Nuclear localization of NORs and centromeres in mouse oocytes during folliculogenesis.

Mouse oocytes at the germinal vesicle stage are characterized by one of two nuclear morphologies: surrounded nucleolus (SN), in which the nucleolus is surrounded by a rim of Hoechst positive chromatin and not surrounded nucleolus (NSN), in which this rim is essentially absent. This morphological difference has a biological relevance as NSN oocytes are transcriptionally active, yet incapable of development beyond the two-cell stage. Whereas SN oocytes, which are transcriptionally inactive, are capable of development to the blastocyst stage. To further our understanding of the nuclear organization of the mouse oocyte during folliculogenesis, we have conducted a series of investigations employing silver methods that stain nucleolus organizer region (NOR), centromeres, and heterochromatin, as well as, specific antibodies for centromeres. Results obtained by a variety of microscopic methods (light, electron, immunochemical, and confocal) demonstrate: (1) a changing pattern of NOR staining during folliculogenesis that is specific to follicular type, and (2) significant differences in the organization of NORs and centromeres of isolated, antral NSN, and SN oocytes. These observations suggest possible means by which, chromosomes of mature, germinal vesicle oocytes are organized with respect to the nucleolus.

Polydeoxyribonucleotide (PDRN) promotes human osteoblast proliferation: a new proposal for bone tissue repair.

Several researchers have recently shed new light upon the importance of extracellular nucleotides and nucleosides to stimulate cells growth. PDRN, a mixture of deoxyribonucleotides polymers of different lengths, has recently demonstrated to stimulate "in vitro" fibroblast proliferation and collagen production, probably stimulating the purinergic receptor system. In this work we evaluated the effects of PDRN on human cultured osteoblasts, focusing our attention on cell proliferation and alkaline phosphatase activity. PDRN at a concentration of 100 microg/ml induce an increase in osteoblasts growth after 6 days as compared to control (+21%). The addition of DMPX 50 microM and suramine (P2 inhibitor) 10 microM give different results: suramine has no significant effect, while DPMX reduce, even if partially, the PDRN induced cell growth. The alkaline phosphatase activity shows a gradual enhancement starting from day 0 to day 10, even if PDRN treated cells, examined at day 6, present a sensibly lower phosphatase activity when compared to controls. Our data demonstrate that PDRN acts as an osteoblast growth stimulator. Its action is partially due to a stimulation of the purinergic system mediated by A2 purinoreceptors, however we can not exclude the involvement of other mechanism like salvage pathway.

Time course assessment of methylmercury effects on C6 glioma cells: submicromolar concentrations induce oxidative DNA damage and apoptosis.

Organic mercury is a well-known neurotoxicant although its mechanism of action has not been fully clarified. In addition to a direct effect on neurons, much experimental evidence supports an involvement of the glial component. We assessed methylmercury hydroxide (MeHgOH) toxicity in a glial model, C6 glioma cells, exposed in the 10(-5)-10(-8) M range. The time course of the effects was studied by time-lapse confocal microscopy and supplemented with biochemical data. We have monitored cell viability and proliferation rate, reactive oxygen species (ROS), mitochondrial transmembrane potential, DNA oxidation, energetic metabolism and modalities of cell death. The earliest effect was a measurable ROS generation followed by oxidative DNA damage paralleled by a partial mitochondrial depolarization. The effect on cell viability was dose dependent. TUNEL, caspase activity and real-time morphological observation of calcein-loaded cells showed that apoptosis was the only detectable mode of cell death within this concentration range. N-acetyl-cysteine (NAC) or reduced glutathione (GSH) completely prevent the apoptotic effect of MeHgOH. The lowest effective MeHgOH concentration was 10(-7) M for ROS and DNA OH-adducts generation. The effect of submicromolar concentrations of MeHgOH on C6 cells could be relevant in the developmental neurotoxicity caused by low dose, long-term exposures, such as those of food origin. In addition, we have shown that the same concentrations are effective in the induction of DNA oxidative damage, with further potential pathogenetic implications.

The analysis of chromatin organisation allows selection of mouse antral oocytes competent for development to blastocyst.

Mouse antral oocytes can be classified in two different types termed SN or NSN oocytes, depending on the presence or absence, respectively, of a ring of Hoechst 33342-positive chromatin surrounding the nucleolus. The aim of the present study was to test the developmental competence to blastocyst of the two types of oocytes. Here we show that following isolation, classification and culture of cumulus-free antral oocytes, 14.7% and 74.5% of NSN and SN oocytes, respectively, reached the metaphase II stage. When fertilised and further cultured none of the metaphase II NSN oocytes developed beyond the 2-cell stage whilst 47.4% of the metaphase II SN oocytes reached the 4-cell stage and 18.4% developed to blastocyst. The findings reported in this paper may contribute to improved procedures of female gamete selection for in vitro fertilisation of humans and farm animals. Furthermore, the selection of oocytes with better developmental potential may be of interest for studies on nuclear/cytoplasm interaction, particularly in nuclear-transfer experiments.

Mouse Xist expression begins at zygotic genome activation and is timed by a zygotic clock.

The imprinted mouse Xist (X-inactive specific transcript) gene is involved in the initiation of X-chromosome inactivation. Only the paternal Xist is expressed in preimplantation development beginning from the 4-cell stage, preceding and in correlation with paternal X-inactivation in the extraembryonic lineage of the blastocyst. To better understand the mechanisms regulating Xist expression in early development, we investigated the precise timing of its onset. We set up a single-cell RT-PCR for the simultaneous analysis on single embryos of Xist and Hprt (internal control) cDNAs and a Y-chromosome specific DNA sequence, Zfy (for embryo sexing). Applying this procedure, we demonstrate that Xist expression begins at the G2-phase of 2-cell female embryos, earlier than previously reported and at the same time of the major wave of zygotic genome activation (ZGA). We then examined, if Xist expression at the 2-cell stage is dependent on the lapse of time spent since fertilization, as previously reported for zygotic genes. One-cell embryos at the G2-phase of the first cell-cycle were cultured with cytochalasin D (inhibitor of cytokinesis but not of DNA synthesis or nuclear progression) for a time equivalent to the 4-cell stage in control, untreated embryos. We show that Xist activation occurs at a scheduled time following fertilization despite the embryos being blocked at the 1-cell stage, suggesting the existence of a zygotic clock involved in the regulation of the transcription of this imprinted gene.