Osteoblast cell behavior on the new beta-type Ti-25Ta-25Nb alloy.

Among metallic materials used as bone substitutes, ß titanium alloys gain an increasing importance because of their low modulus, high corrosion resistance and good biocompatibility. In this work, an investigation of the in vitro cytocompatibility of a recently new developed ß-type Ti-25Ta-25Nb alloy was carried out by evaluating the behavior of human osteoblasts. The metallic Ti-6Al-4V biomaterial, which is one of representative α+ß type titanium alloys for biomedical applications, and Tissue Culture Polystyrene (TCPS), were also investigated as reference Ti-based material and control substrate, respectively. Both metallic surfaces were analyzed by X-ray diffraction, atomic force microscopy and X-ray photoelectron spectroscopy. The cellular response was quantified by assessments of viability, cell attachment and spreading, cell morphology, production and extracellular organization of fibronectin and cell proliferation. Polished surfaces from both materials having an equiaxed grain microstructure and nanometre scale surface roughness elicited an essentially identical osteoblast response in terms of all analyzed cellular parameters. Thus, on both surfaces the cells displayed high survival rates, good cell adhesion and spreading, a dense and randomly dispersed fibronectin matrix and increasing cell proliferation rates over the incubation time. Furhermore, the enhanced biological performance of Ti-25Ta-25Nb was highly supported by the results obtained in comparison with TCPS. These findings, together with previously shown superelastic behavior, low Young's modulus and high corrosion resistance, recommend Ti-25Ta-25Nb as good candidate for applications in bone implantology.

In vitro effects of calcium fructoborate upon production of inflammatory mediators by LPS-stimulated RAW 264.7 macrophages.

The present study is supported by our previous findings suggesting that calcium fructoborate (CF) has anti-inflammatory and antioxidant properties. Thus, we investigated the effects of CF on a model for studying inflammatory disorders in vitro represented by lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7 cells. This investigation was performed by analyzing the levels of some mediators released during the inflammatory process: cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukins IL-1beta and IL-6 as well as cyclooxygenase-2 (COX-2), the main enzyme responsible for endotoxin/LPS-induced prostaglandin synthesis by macrophages. We also measured production of nitric oxide (NO) that plays an important role in the cytotoxicity activity of macrophages towards microbial pathogens. After CF treatment of LPS-stimulated macrophages we found an up-regulation of TNF-alpha protein level in culture medium, no significant changes in the level of COX-2 protein expression and a decrease in NO production as well as in IL-1beta and IL-6 release. Collectively, this series of experiments indicate that CF affect macrophage production of inflammatory mediators. However, further research is required in order to establish whether CF treatment can be beneficial in suppression of pro-inflammatory cytokine production and against progression of endotoxin-related diseases.

Comparative effects of boric acid and calcium fructoborate on breast cancer cells.

Recent studies suggested that boron has a chemo-preventive role in prostate cancer. In the present report, we investigated the effects of calcium fructoborate (CF) and boric acid (BA) on activation of the apoptotic pathway in MDA-MB-231 human breast cancer cells. Exposure to BA and CF inhibited the proliferation of breast cancer cells in a dose-dependent manner. Treatment with CF but not BA resulted in a decrease in p53 and bcl-2 protein levels. Furthermore, after the treatment with CF, augmentation of pro-caspase-3 protein expression, cytosolic cytochrome c level, and caspase-3 activity were observed, indicating apoptotic cell death induction. This was also demonstrated by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end-labeling assay. In conclusion, our data provide arguments to the fact that both BA and CF inhibited the growth of breast cancer cells, while only CF induced apoptosis. Additional studies will be needed to identify the underlying mechanism responsible for the observed cellular responses to these compounds and to determine if BA and CF may be further evaluated as chemotherapeutic agents for human cancer.

In vitro effects of calcium fructoborate on fMLP-stimulated human neutrophil granulocytes.

Discovery of naturally occurring boron complexes with organic compounds containing hydroxyl groups, sugars, and polysaccharides, adenosine-5-phosphate, pyridoxine, riboflavin, dehydroascorbic acid, and pyridine nucleotides led to the reassessment of the biochemical role of boron. Boron's anti-inflammatory actions were claimed but not yet demonstrated. This study investigated the effects of calcium fructoborate (CF) on the human polymorphonuclear neutrophils (PMN) that play a central role in the inflammatory response. Our results demonstrated that CF exposure induced a dose-dependent decrease in cell viability. Treatment of PMN cells, for 24 h, with 22,500 microM CF led to a decrease in cell viability by 61.1%, an inhibition of respiratory burst by 92.9% in the case of fMLP-stimulated cells, a diminution of intracellular level of superoxide anion with 59.3%, and a stimulation of superoxide dismutase activity by 72% in unstimulated PMN cells. Altogether, these results suggest the antioxidant and anti-inflammatory properties of CF.

The biocompatibility of titanium in a buffer solution: compared effects of a thin film of TiO2 deposited by MOCVD and of collagen deposited from a gel.

This study aims at evaluating the biocompatibility of titanium surfaces modified according two different ways: (i) deposition of a bio-inert, thin film of rutile TiO(2) by chemical vapour deposition (MOCVD), and (ii) biochemical treatment with collagen gel, in order to obtain a bio-interactive coating. Behind the comparison is the idea that either the bio-inert or the bio-active coating has specific advantages when applied to implant treatment, such as the low price of the collagen treatment for instance. The stability in buffer solution was evaluated by open circuit potential (OCP) for medium time and cyclic voltametry. The OCP stabilized after 5.10(4) min for all the specimens except the collagen treated sample which presented a stable OCP from the first minutes. MOCVD treated samples stabilized to more electropositive values. Numeric results were statistically analysed to obtain the regression equations for long time predictable evolution. The corrosion parameters determined from cyclic curves revealed that the MOCVD treatment is an efficient way to improve corrosion resistance. Human dermal fibroblasts were selected for cell culture tests, taking into account that these cells are present in all bio-interfaces, being the main cellular type of connective tissue. The cells grew on either type of surface without phenotype modification. From the reduction of yellow, water-soluble 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT cytotoxicity test), MOCVD treated samples offer better viability than mechanically polished Ti and collagen treated samples as well. Cell spreading, as evaluated from microscope images processed by the program Sigma Scan, showed also enhancement upon surface modification. Depending on the experimental conditions, MOCVD deposited TiO(2) exhibits different nanostructures that may influence biological behaviour. The results demonstrate the capacity of integration in simulated physiologic liquids for an implant pretreated by either method.

In vitro evaluation of the antioxidant activity of calcium fructoborate.

Although increasing evidence shows the nutritional benefits of calcium fructoborate (CF) on animals and humans, its action mechanism has not been clearly identified. The present study aims to investigate the possible antioxidant function of CF. Based on its efficiency in skin wound healing, the authors tested whether CF possesses antioxidant properties on human keratinocytes cultures, in a complete serum-free medium (KMK-2; Sigma). The cells treated with CF (0-450 nmol/culture medium) were exposed to exogenous 100 micromol of hydrogen peroxide to mimic the oxidative stress. The changes in general cell oxidant production evaluated with dihydrorhodamine-123 showed that the intracellular reactive oxygen species (ROS) were markedly reduced by preincubation with CF. The maximum antioxidant activity was noticed at 90 nmol CF. To assess the reactivity of CF on ROS, we analyzed its ability to inhibit the superoxide- dependent auto-oxidation of pyrogallol. The CF inhibited the pyrogallol auto-oxidation depending on time and concentration, which suggests its possible role as a superoxide radical scavenger. Taken together, our results indicate that CF has antioxidant activity, which could have clinical significance in protecting cells from oxidant-induced injury. A hypothetic mechanism for the antioxidant activity of CF is proposed.

The effect of laser activation of 5,10,15,20-tetra-sulphophenyl-porphyrin loaded in K562 cells and human normal mononuclear cells.

Photodynamic therapy (PDT) is a relatively new type of treatment in cancer, based on a photosensitizer, visible light and molecular oxygen. Reactive oxygen species are generated, causing tumor cells death by apoptosis or necrosis. Significant nowadays research efforts are focused on finding new photosensitizers with antineoplastic activity and an acceptable toxicological profile. Although consistent information exists regarding PDT in solid tumors, relatively few data are available for PDT of blood cancers. Therefore, we carried out a comparative study on lymphoblastic K562 cells and human normal peripheral blood mononuclear cells (PBMC) treated at a density of 2 x 10(5) cells/mL with 5,10,15,20-tetra-sulphophenyl-porphyrin (TSPP) and then irradiated with He-Ne laser light (lamda = 632.8 nm). The following cell functions were investigated: viability, multiplication, RNA synthesis, total RNA levels and apoptosis. After irradiation, the viability of TSPP-loaded tumor cells decrease, the multiplication rate and the total RNA level are drastically reduced and cells undergo apoptosis. TSPP alone loaded into cells but not activated by irradiation, does not affect these cell parameters. Human normal PBMC subjected to TSPP loading and laser-irradiation develop a different cellular response, their viability and proliferative capacity not being altered by experimental PDT. Accordingly, it appears that TSPP is a non-aggressive compound for cellular physiology and becomes cytotoxic only by irradiation; moreover laser-activated TSPP affects only cells that have a tumoral pattern.

Phospholipase A2 modulates respiratory burst developed by neutrophils in patients with rheumatoid arthritis.

Activated by bacterial peptides, phorbol esters, calcium ionophores and other agonists, neutrophils (PMNs) release the proinflammatory mediator, arachidonic acid (AA) via the intervention of phospholipase A(2) (PLA(2)). AA may play an essential role in activation of NADPH-oxidase, which is involved in the generation of superoxide anion by neutrophils. The present study is focused on the involvement of PLA(2) in the respiratory burst developed by PMNs isolated from patients with rheumatoid arthritis (RA). PLA(2) exists in very high levels in diseases such as rheumatoid arthritis and may cause acute inflammatory and proliferative changes in synovial structures. The respiratory burst was evaluated as superoxide anion release, using an amplified chemiluminescence method. The assays were performed using PMNs untreated or treated with different doses of stimulatory reagents (phorbol 12-myristate-13-acetate (PMA), calcium ionophore (A23187)). Our data suggested that PMA stimulated the production of superoxide anion in a dose-response manner, as compared with A23187, which did not induce a significant release of superoxide anion in PMNs-RA. The exogenous addition of AA significantly amplified the superoxide anion release by PMNs-RA stimulated with PMA and to a lesser extent, by PMNs stimulated with A23187. AA has also reversed the inhibitory effect of arachidonyl-trifluorometylketone and E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)2H-pyran-2-one (BEL) on the superoxide anion release by PMNs-RA. In conclusion, the differential responses to these two agents suggested that different isoforms of PLA(2) were activated by A23187 or PMA, and support the idea that activation of these different PLA(2) served distinct functions of PMNs. Therefore, the inhibition of PLA(2) enzymes might be of great importance in the immunotherapy of rheumatoid arthritis.

Plasma membrane potential interferes with the respiratory burst of peripheral granulocytes.

Membrane potential is involved in the regulation of several immune functions developed by granulocytes. The Na(+)/K(+) gradient across the plasma membrane, mainly generated by the Na(+)/K(+) pump, plays a key role in the maintenance of membrane potential. This study is focused on the correlation between plasma membrane potential and the in vitro receptor - triggered respiratory burst of normal human peripheral granulocytes. The respiratory burst was measured as superoxide anion release by the cytochrome c reduction test and plasma membrane potential was modulated by experimental changes of the extracellular potassium concentration. Results show a differentiated cellular response, depending on the in vivo activation state and on the signals received in vitro by granulocytes via CR3 or FcgammaR. Alteration of the membrane potassium gradient modulates the respiratory burst of unstimulated and CR3-activated cells, whilst it does not seem to significantly interfere with the signals delivered by FcgammaR.

The influence of sodium metavanadate on the process of diabetogenesis in BB rats.

Vanadium has been shown to be beneficial in the oral treatment of animal models of type 1 and type 2 diabetes. The aim of the study was to evaluate the short-term effects of sodium metavanadate in prediabetic BB-DP rats. To do this, 96 rats were divided into 4 equal groups. Groups V1, V2, V3 were treated with sodium metavanadate (0.1, 0.2 and 0.3 mg/ml respectively) and sodium chloride (0.5 mg/ml) in drinking water for 7 days. Group C received only sodium chloride (0.5 mg/ml). Blood glucose (BG), glycosuria, ketonuria, body weight and insulinemia were determined. The age of onset of diabetes was significantly higher for groups V2, V3 compared to group C, (p<0.05) and depends on the metavanadate concentration (V3 vs. V1, p=0.006). The incidence of diabetes was lower in the rats treated with metavanadate than in the control group, but this difference was not statistically significant. In diabetic rats, the BG at the onset was higher in group C than in groups V, p<0.05. Insulinemia, at the onset of the treatment as well as immediately after its cessation showed a drop in the treatment groups, proportionally to the dosage of vanadium, but later increased slowly and continuously until the end of the experiment. In conclusion, metavanadate delays the development of diabetes in BB-DP rats, but does not prevent its onset. A milder form of diabetes occurs in diabetic rats treated with metavanadate. The effects depend on the metavanadate concentration and 0.2 mg/ml is preferable.

Study of chemotactic activity developed by neutrophils from rheumatoid arthritis patients.

Neutrophils are the predominant cells accumulated in the synovial fluid (SF) of rheumatoid arthritis (RA) patients. Accumulation of neutrophils may be regarded as a possible way by which neutrophils exert cytotoxic functions. The aim of the present study was to analyze the chemotactic response of neutrophils (PMNs) isolated from the peripheral blood or SF of patients with RA by performing the chemotaxis assay, in which N-formyl-methionyl-leucyl-phenylalanine (FMLP) was used as chemotactic agent. Our results showed that FMLP induced response of peripheral blood neutrophils from 12 patients with RA was similar with the response of 15 healthy controls. A decreased chemotactic response to FMLP was, however, observed in PMNs isolated from the SF of RA patients as comlipared with peripheral blood cells. Therefore, this defective chemotactic ability of neutrophil, was inversely correlated with the number of infiltrating cells in SF. These results indicate that chemotactic ability of neutrophils may be reduced after migration to the SF. Because PMNs chemotaxis in vivo has likely occurred in the presence of serum or SF, we tried to simulate the same conditions in vitro. Therefore, we analyzed the effect of serum or SF on the RA-PMNs chemotaxis. Heat-inactivated serum produced a marked reduction of chemotactic activity developed by PMNs isolated from patients with RA. Notably, a significant increase of chemotactic activity was observed when FMLP and serum stimuli were used together, as compared with the same stimuli used alone. The results suggested that complement activation might interfere with neutrophils chemotaxis. SF amplifies the chemotactic activity of PMNs isolated from peripheral blood of RA patients, but does not affect the chemotaxis developed by PMNs isolated from SF. The data might suggest that several components of SF (IL-8, leukotrien B4, thrombin, platelet-activating factor, etc.) could serve as a potent stimulus for recruitment of neutrophils from periphery into the RA joint. In conclusion, serum or SF components seem to contribute to chemotaxis of neutrophils and play a role in differential killing of PMNs and incidence of infection.