Effects of 915 nm laser irradiation on human osteoblasts: a preliminary in vitro study.

Photobiomodulation (PBM) is a non-invasive treatment that uses laser or led devices making its effects a response to light and not to heat. The possibility of accelerating dental implant osteointegration and orthodontic movements and the need to treat refractory bone lesions, such as bisphosphonate related osteonecrosis of the jaws, has led researchers to consider the effects of PBM on bone for dentistry purposes. The aim of our study was to investigate the effects of 915 nm light supplied with a GaAs diode laser on human osteoblasts in vitro. Osteoblasts were isolated from mandibular cortical bone of a young healthy donor. The irradiation parameters were as follows: doses = 5, 15 and 45 J/cm2; power densities = 0.12 and 1.25 W/cm2; and irradiation times = 41.7, 125 and 375 s. We performed one irradiation per day for 3 and 6 days to study proliferation and differentiation, respectively. Microscopic analysis showed a greater amount of bone nodules in samples treated with 5 J/cm2 and 0.12 W/cm2 compared to controls (56.00 ± 10.44 vs 19.67 ± 7.64, P = 0.0075). Cell growth and quantification of calcium deposition did not show any differences when comparing irradiated and non-irradiated samples. Photobiomodulation, with the parameters investigated in the present study, positively modulated the mineralization process in human osteoblasts, inducing the formation of a greater amount of bone nodules, but did not increase cell proliferation.

Effects of 915 nm GaAs diode laser on mitochondria of human dermal fibroblasts: analysis with confocal microscopy.

Low-level laser therapy (LLLT) is widely used in tissue regeneration and pain therapy. Mitochondria are supposed to be one of the main cellular targets, due to the presence of cytochrome C oxidase as photo-acceptor. Laser stimulation could influence mitochondria metabolism affecting mainly transmembrane mitochondrial potential (Δψm). The aim of our study is to evaluate "in vitro" the early mitochondrial response after irradiation with a 915 GaAs laser. Since some evidences suggest that cellular response to LLLT can be differently modulated by the mode of irradiation, we would like to evaluate whether there are changes in the mitochondrial potential linked to the use of the laser treatments applied with continuous wave (CW) in respect to those applied with pulsed wave (PW). In this study, we analyzed effects of irradiation with a 915-nm GaAs diode laser on human dermal fibroblast. We compared effects of irradiation applied with either CW or PW at different fluences 45-15-5 J/cm(2) on Δψm. Laser scanning microscopy (LSM) was used in living cells to detect ROS (reactive oxygen species) using calcein AM and real-time changes of and Δψm following distribution of the potentiometric probe tetramethylrhodamine methyl ester (TMRM). At higher doses (45-15 J/cm(2)), fibroblasts showed a dose-dependent decrement of Δψm in either the modalities employed, with higher amplitudes in CW-treated cells. This behavior is transient and not followed by any sign of toxicity, even if reactive oxygen species generation was observed. At 5 J/cm(2), CW irradiation determined a little decrease (5%) of the baseline level of Δψm, while opposite behavior was shown when cells were irradiated with PW, with a 10% increment. Our results suggest that different responses observed at cellular level with low doses of irradiation, could be at the basis of efficacy of LLLT in clinical application, performed with PW rather than CW modalities.

Polyphenon E(R), a standardized green tea extract, induces endoplasmic reticulum stress, leading to death of immortalized PNT1a cells by anoikis and tumorigenic PC3 by necroptosis.

Increasing doses of Polyphenon E®, a standardized green tea extract, were given to PNT1a and PC3 prostate epithelial cells mimicking initial and advanced stages of prostate cancer (PCa), respectively. Cell death occurred in both cell lines, with PNT1a being more sensitive [half-maximal inhibitory concentration (IC50) = 35 µg/ml] than PC3 (IC50 = 145 µg/ml) to Polyphenon E®. Cell cycle arrest occurred at G0/G1 checkpoint for PNT1a, and G2/M for PC3 cells. Endoplasmic reticulum stress (ERS) and unfolded protein response (UPR) occurred in both cell lines, with each exhibiting different timing in response to Polyphenon E®. Autophagy was transiently activated in PNT1a cells within 12 h after treatment as a survival response to overcome ERS; then activation of caspases and cleavage of poly (ADP ribose) polymerase 1 occurred, committing cells to anoikis death. Polyphenon E® induced severe ERS in PC3 cells, causing a dramatic enlargement of the ER; persistent activation of UPR produced strong upregulation of GADD153/CHOP, a key protein of ERS-mediated cell death. Thereafter, GADD153/CHOP activated Puma, a BH3-only protein, committing cells to necroptosis, a programmed caspase-independent mechanism of cell death. Our results provide a foundation for the identification of novel targets and strategies aimed at sensitizing apoptosis-resistant cells to alternative death pathways.

Sr, Mg cosubstituted HA porous macro-granules: potentialities as resorbable bone filler with antiosteoporotic functions.

Porous macro-granules of nanostructured apatite with Ca ions partially cosubstituted with Mg and Sr ions in different ratios (SrMgHAs), were synthesized at 37°C and compared with Mg and/or Sr free apatites (MgHAs and HA). Strontium improved the Mg substitution extent in the apatite and the chemical-physical and thermal stability of the resulting cosubstituted apatite. Porous macro-granules of 400-600 micron with selected composition were tested for the ionic release in synthetic body fluid and the data were related with the results of preliminary cell investigation in vitro. As compared to the corresponding Sr-free granulate, the SrMgHA could be exploited to prolong the beneficial Mg release during the bone regeneration process. In addition the contemporary in situ supply of Sr, an antiosteoporotic and anticarie ion, could influence the quality of new hard tissues. The ionic multirelease created a more favorable environment for human osteoblasts, demonstrated by a proliferative effect for each dose tested in the range 0.1-10 mg/mL.

Human osteoblast behavior on as-synthesized SiO(4) and B-CO(3) co-substituted apatite.

The functional behavior of synthetic apatite, commonly used as fillers or scaffolds, depends on physical and chemical parameters, which vary in response to chemical substitutions and to thermal treatments. The effect of silicon co-substituting with carbonate ions in the apatite lattice on the properties of the as-synthesized powder and finally on human osteoblast in vitro behavior was investigated. Dose-response curves of Si-free and Si-substituted carbonated apatites (namely CHA and SiCHA-1 and SiCHA-2 with 0.88 and 0.55 wt % of Si, respectively) showed that SiCHA-1 had toxic effect, whereas CHA and SiCHA-2, at worst, hindered osteoblast proliferation, but no toxicity occurred. Subsequent experiments compared the effects of CHA and SiCHA-2 used at the doses of 0.3 and 1 mg/mL. After 7 days of treatment, both the powders stimulated cell proliferation and protein content and inhibited alkaline phosphatase activity. However, SiCHA-2 slightly stimulated osteoblast differentiation, as shown by higher calcium deposition, compared with CHA. The cell behaviors were linked to the peculiar powder characteristics. The as-synthesized powder represents the most critical system in terms of reactivity toward cells and can inform on the limits for positively exploiting the characteristics of SiCHA powders in making bone fillers or scaffolds, using no thermal treatments. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

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.

Polydeoxyribonucleotide promotes cyclobutane pyrimidine dimer repair in UVB-exposed dermal fibroblasts.

BACKGROUND: DNA is the main cellular chromophore for ultraviolet B (UVB). Its absorption leads to the generation of typical photoproducts. The most frequent types (about 80%) are cyclobutane pyrimidine dimers (CPDs). Several studies have suggested that treatment with deoxyribonucleosides can protect some cell types from DNA damage. The aim of this work was to evaluate the ability of the polydeoxyribonucleotide (PDRN) to protect human dermal fibroblasts from UVB-induced DNA damage. METHODS: Human dermal fibroblasts were irradiated with 600 mJ/cm(2) of UVB radiation. Cells were analyzed at increasing time points from irradiation to study the recovery from UVB-induced DNA photodamage. Damage repair was subsequently assessed by immunocytochemical analysis of CPDs levels and by measurement of p53 protein expression. RESULTS: The extracellular addition of 100 microg/ml PDRN immediately after irradiation caused a strong activation of p53 protein in the first 24 h. This signal was accompanied by an increase in CPDs repair rates at early time points of recovery. CONCLUSIONS: The addition of PDRN to the culture medium supports CPDs repair probably providing a faster supply of precursors for the deoxyribonucleotide triphosphates pool necessary to UVB-damaged cells. This condition could promote the action of the salvage pathway, thereby accelerating DNA repair, but other inducible responses linked to increased p53 expression could be involved.

Inhibition of glutamine synthetase triggers apoptosis in asparaginase-resistant cells.

The resistance to L-asparaginase (ASNase) has been associated to the overexpression of asparagine synthetase (AS), although the role played by other metabolic adaptations has not been yet defined. Both in ASNase-sensitive Jensen rat sarcoma cells and in ARJ cells, their ASNase-resistant counterparts endowed with a five-fold increased AS activity, ASNase treatment rapidly depletes intracellular asparagine. Under these conditions, cell glutamine is also severely reduced and the activity of glutamine synthetase (GS) is very low. After 24 h of treatment, while sensitive cells have undergone massive apoptosis, ARJ cells exhibit a marked increase in GS activity, associated with overexpression of GS protein but not of GS mRNA, and a partial restoration of glutamine and asparagine. However, when ARJ cells are treated with both ASNase and L-methionine-sulfoximine (MSO), an inhibitor of GS, no restoration of cell amino acids occurs and the cell population undergoes a typical apoptosis. No toxicity is observed upon MSO treatment in the absence of ASNase. The effects of MSO are not referable to depletion of cell glutathione or inhibition of AS. These findings indicate that, in the presence of ASNase, the inhibition of GS triggers apoptosis. GS may thus constitute a target for the suppression of ASNase-resistant phenotypes.

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.

Parenteral exposure to high HIV viremia leads to virus-specific T cell priming without evidence of infection.

Previous studies on CTL responses in HIV-exposed uninfected individuals assumed that the patients were exposed to replicating HIV, but the possibility that the immune responses detected were primed by exposure to a defective virus or viral antigen could not be excluded. Epidemiological and laboratory analysis of a nosocomial outbreak of acute hepatitis B unequivocally allowed the identification of an HIV-1- and HBV-co-infected patient with high plasma levels of both viruses, as the source case of the epidemics. This clinical setting provided a natural model for testing the HIV-specific T cell response in patients exposed to blood from a patient with highly replicating HIV. Parenteral exposure to both viruses led to acute hepatitis B in five subjects without evidence of HIV-1 infection. Cryopreserved lymphocytes derived from three exposed patients were tested ex vivo in an ELISPOT assay for IFN-gamma release upon stimulation with peptides from structural and non-structural HIV proteins; one of the patients was also tested with four HLA/class I tetramers. Circulating HIV-specific CD8 cells were detected by tetramer staining and a high frequency of T cells were able to release IFN-gamma upon stimulation with HIV peptides, showing in vivo T cell priming by HIV. These results unequivocally demonstrate a HIV-specific cell-mediated immune response in the absence of infection after exposure to highly replicating HIV.

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.

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.

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.