Olive Fruit Blends Modulate Lipid-Sensing Nuclear Receptor PPARγ, Cell Survival, and Oxidative Stress Response in Human Osteoblast Cells.

OBJECTIVE: The aim of the present study was to investigate how different extravirgin olive oils (EVOOs), obtained by blending Olea europea cultivars, could influence the cell growth, the response to inflammatory stimuli, and oxidative stress in a culture of the osteosarcoma cell line Saos-2. METHODS: Three different extravirgin olive oils were physicochemically characterized, determining the free acidity, the oxidation status, the polyphenols content, and the antioxidative activity. Moreover, the effects on Saos-2 cell culture were determined, studying the mRNA expression level by real-time polymerase chain reaction (PCR) assays and the antioxidative activity using fluorescent probes. RESULTS: The cultivars used in the south of Italy, yield extravirgin oils with different amount of fatty acids and polyphenols, which counteract induction of proinflammatory cytokines and regulate free radical production in hydrogen peroxide-stimulated cells. In vitro analysis using the human osteoblast cell line Saos-2 showed that the addition of oils to cell culture simulated a hypoxic stress followed by a reoxygenation period, during which the antioxidant activity of extravirgin olive oils protected cells from oxidative damages. On the other hand, the mRNA expression levels of factors involved in inflammatory processes, cell growth recovery, and antioxidant response, as heme oxygenase-1, were differently stimulated by EVOOs. Moreover, peroxisome proliferator activated receptor γ (PPARγ) was differently modulated by EVOOs. CONCLUSION: These findings show that the blending of different extravirgin olive oil can impact an osteoblast cell line, in particular regarding cell growth recovery and oxidative stress.

IKKα inibition by a glucosamine derivative enhances Maspin expression in osteosarcoma cell line.

Chronic inflammation has been associated to cancer development by the alteration of several inflammatory pathways, such as Nuclear Factor-κB pathway. In particular, IκB kinase α (IKKα), one of two catalytic subunit of IKK complex, has been described to be associated to cancer progression and metastasis in a number of cancers. The molecular mechanism by which IKKα affects cancer progression is not yet completely clarified, anyway an association between IKKα and the expression of Maspin (Mammary Serine Protease Inhibitor or SerpinB5), a tumor suppressor protein, has been described. IKKα shuttles between cytoplasm and nucleus, and when is localized into the nuclei, IKKα regulates the expression of several genes, among them Maspin gene, whose expression is repressed by high amount of nuclear IKKα. Considering that high levels of Maspin have been associated with reduced metastatic progression, it could be hypothesized that the repression of IKKα nuclear translocation could be associated with the repression of metastatic phenotype. The present study is aimed to explore the ability of a glucosamine derivative, 2-(N-Carbobenzyloxy)l-phenylalanylamido-2-deoxy-ß-d-glucose (NCPA), synthesized in our laboratory, to stimulate the production of Maspin in an osteosarcoma cell line, 143B. Immunofluorescence and Western blotting experiments showed that NCPA is able to inhibit IKKα nuclear translocation, and to stimulate Maspin production. Moreover, in association with stimulation of Maspin production we found the decrease of ß1 Integrin expression, the down-regulation of metalloproteases MMP-9 and MMP-13 production and cell migration inhibition. Taking in account that ß1 Integrin and MMP-9 and -13 have been correlated with the invasiveness of osteosarcoma, considering that NCPA affects the invasiveness of 143B cell line, we suggest that this molecule could affect the osteosarcoma metastatic ability.

Osseointegration is improved by coating titanium implants with a nanostructured thin film with titanium carbide and titanium oxides clustered around graphitic carbon.

Titanium implants coated with a 500nm nanostructured layer, deposited by the Ion Plating Plasma Assisted (IPPA) technology, composed of 60% graphitic carbon, 25% titanium oxides and 15% titanium carbide were implanted into rabbit femurs whilst into the controlateral femurs uncoated titanium implants were inserted as control. At four time points the animals were injected with calcein green, xylenol orange, oxytetracycline and alizarin. After 2, 4 and 8weeks femurs were removed and processed for histology and static and dynamic histomorphometry for undecalcified bone processing into methylmethacrylate, sectioned, thinned, polished and stained with Toluidine blue and Fast green. The overall bone-implant contacts rate (percentage of bone-implant contacts/weeks) of the TiC coated implant was 1.6 fold than that of the uncoated titanium implant. The histomorphometric analyses confirmed the histological evaluations. More precisely, higher Mineral Apposition Rate (MAR, µm/day) (p<0.005) and Bone Formation Rate (BFR, µm2/µm/day) (p<0.0005) as well as Bone Implant Contact (Bic) and Bone Ingrowth values (p<0.0005) were observed for the TiC coated implants compared to uncoated implants. In conclusion the hard nanostructured TiC layer protects the bulk titanium implant against the harsh conditions of biological tissues and in the same time, stimulating adhesion, proliferation and activity of osteoblasts, induces a better bone-implant contacts of the implant compared to the uncoated titanium implant.

Improving Osteoblast Response In Vitro by a Nanostructured Thin Film with Titanium Carbide and Titanium Oxides Clustered around Graphitic Carbon.

INTRODUCTION: Recently, we introduced a new deposition method, based on Ion Plating Plasma Assisted technology, to coat titanium implants with a thin but hard nanostructured layer composed of titanium carbide and titanium oxides, clustered around graphitic carbon. The nanostructured layer has a double effect: protects the bulk titanium against the harsh conditions of biological tissues and in the same time has a stimulating action on osteoblasts. RESULTS: The aim of this work is to describe the biological effects of this layer on osteoblasts cultured in vitro. We demonstrate that the nanostructured layer causes an overexpression of many early genes correlated to proteins involved in bone turnover and an increase in the number of surface receptors for α3ß1 integrin, talin, paxillin. Analyses at single-cell level, by scanning electron microscopy, atomic force microscopy, and single cell force spectroscopy, show how the proliferation, adhesion and spreading of cells cultured on coated titanium samples are higher than on uncoated titanium ones. Finally, the chemistry of the layer induces a better formation of blood clots and a higher number of adhered platelets, compared to the uncoated cases, and these are useful features to improve the speed of implant osseointegration. CONCLUSION: In summary, the nanostructured TiC film, due to its physical and chemical properties, can be used to protect the implants and to improve their acceptance by the bone.

The inflammatory circuitry of miR-149 as a pathological mechanism in osteoarthritis.

Osteoarthritis (OA) is a multifactorial degenerative pathology, whose progression is exacerbated by pro-inflammatory cytokines signaling. Among the changes triggered in chondrocytes during inflammation, modified expression of tiny epigenetic regulators as microRNAs was shown having deleterious implications for articular cartilage. Aim of the present study was to identify differentially expressed microRNAs in human OA cartilage and to determine their relevance to pathological progression. An OA model based on inflammatory stimulation of a chondrocytic human cell line was used to analyze microRNAs deregulation, and results revealed miR-149 severely down-regulated by IL1ß and TNFα. Real-time PCR analysis of miR-149 was exerted also in human primary chondrocytes isolated from cartilage of OA donors and postmortem from subjects with no known history of OA, confirming down-regulation in osteoarthritis. Moving on a functional study, miR-149 regulatory effect on tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL1ß) and interleukin 6 (IL6) 3'UTRs was evaluated by luciferase assays, and chondrocytes production of TNFα upon miR-149 transfection was measured by enzyme-linked immuno sorbent assay. We found that miR-149 is down-regulated in OA chondrocytes, and this decrease seems to be correlated to increased expression of pro-inflammatory cytokines such as TNFα, IL1ß and IL6. OA is a multifactorial disease and we think that our results give new insights for understanding the complex mechanisms of osteoarthritic pathogenesis.

L-carnitine enhances extracellular matrix synthesis in human primary chondrocytes.

Osteoarthritis (OA) is one of the most common degenerative joint disease for which there is no cure. It is treated mainly with non-steroidal anti-inflammatory drugs to control the symptoms and some supplements, such as glucosamine and chondroitin sulphate in order to obtain structure-modifying effects. Aim of this study is to investigate the effects of L-carnitine, a molecule with a role in cellular energy metabolism, on extracellular matrix synthesis in human primary chondrocytes (HPCs). Dose-dependent effect of L-carnitine on cartilage matrix production, cell proliferation and ATP synthesis was examined by incubating HPCs with various amounts of molecule in monolayer (2D) and in hydromatrix scaffold (3D). L-Carnitine affected extracellular matrix synthesis in 3D in a dose-dependent manner; moreover, L-carnitine was very effective to stimulate cell proliferation and to induce ATP synthesis, mainly in 3D culture condition. In conclusion, L-carnitine enhances cartilage matrix glycosaminoglycan component production and cell proliferation, suggesting that this molecule could be useful in the treatment of pathologies where extracellular matrix is degraded, such as OA. To our knowledge, this is the first study where the effects of L-carnitine are evaluated in HPCs.

A peptidyl-glucosamine derivative affects IKKalpha kinase activity in human chondrocytes.

INTRODUCTION: Nuclear factor-kappaB (NF-kappaB) transcription factor regulates several cell signaling pathways, such as differentiation and inflammation, which are both altered in osteoarthritis. Inhibitor kappaB kinase (IKK)alpha and IKKbeta are kinases involved in the activation of the NF-kappaB transcription factor. The aim of the present study was to determine the effects of glucosamine (GlcN), which is administered in the treatment of osteoarthritis, and of its 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-beta-D-glucose (NAPA) derivative on IKK kinases and, consequently, on NF-kappaB activation in human chondrocytes. METHODS: The human chondrosarcoma cell line HTB-94 and human primary chondrocytes were stimulated with tumor necrosis factor (TNF)alpha after pre-treatment with GlcN or NAPA. Gene mRNA expression level was evaluated by real-time PCR. Inhibitor kappaB protein (IkappaB)alpha phosphorylation and p65 nuclear re-localization were analyzed by Western blotting; IKKalpha nuclear re-localization was also investigated by immunocytochemistry and Western blotting. IKK kinase activity was studied by in vitro kinase assay. RESULTS: After TNFalpha stimulation, the mRNA expression level of some of the genes under NF-kappaB control, such as interleukin (IL)-6 and IL-8, increased, while treatment with GlcN and NAPA reverted the effect. We investigated the possibility that GlcN and NAPA inhibit IKK kinase activity and found that NAPA inhibits the IKKalpha kinase activity, whereas GlcN does not. Interestingly, both GlcN and NAPA inhibit IKKalpha nuclear re-localization. CONCLUSIONS: Our results demonstrate that glucosamine and its peptidyl derivative can interfere with NF-kappaB signaling pathway by inhibiting IKKalpha activity in human chondrocytes. However, the mechanism of action of the two molecules is not completely overlapping. While NAPA can both specifically inhibit the IKKalpha kinase activity and IKKalpha nuclear re-localization, GlcN only acts on IKKalpha nuclear re-localization.

pH-, temperature- and ion-dependent oligomerization of Sulfolobus solfataricus recombinant amidase: a study with site-specific mutants.

Recombinant amidase from Sulfolobus solfataricus occurred as a dimer of 110 kDa comprising identical subunits. Only dimers were present at pHs above 7.0, but with decreasing pH, dimers associated into octamers, with complete oligomerization occurring at pH 3.0. Oligomerization showed reversible temperature-dependence, with octamer formation increasing with temperature from 36 degrees C to between 70 and 80 degrees C. Increasing salt concentrations, favored dissociation of the octamers. Among the three investigated factors affecting the dimer-octamer equilibrium, the most important was pH. Among four mutants obtained by site-specific mutagenesis and selection for pH and temperature sensitivity, the T319I and D487N mutant amidases, like that of the native Sulfolobus solfataricus, responded to changes in pH and temperature with a conformational change affecting the dimer-octamer equilibrium. The Y41C and L34P mutant amidases were unaffected by pH and temperature, remaining always in the dimeric state. The differences among mutants in protein conformation must be related to the position of the introduced mutation. Although the L34P and Y41C mutations are located in the helical region 33-48 (LLKLQLESYERLDSLP), which is close to the amino-terminal segment of the protein, the T319I mutation is located in a strand on the surface of the protein, which is far from, and opposite to, the amino-terminal segment. The D487N mutation is located in the center of the protein, far distant from the 33-48 segment. These observations suggest that the segment of the protein closest to the amino-terminus plays a key role in the association of dimers into octamers.

Effects of intra-articular administration of glucosamine and a peptidyl-glucosamine derivative in a rabbit model of experimental osteoarthritis: a pilot study.

The aim of this pilot study was to analyze the effects of glucosamine (GlcN) and its N-acetyl-phenylalanine derivative (NAPA) in Vitamin A model of osteoarthritis (OA) in rabbits. GlcN or NAPA or saline solution was intra-articularly administered in rabbit OA knees. Histological analysis revealed that treatment with GlcN or NAPA was associated with more homogeneous chondrocyte cellularity, absence of fissures and fragmentation and more intense staining of the matrix with Alcian Blue compared to the articular surfaces of the knees treated with saline solution. Comparative in vitro study performed on rabbit primary chondrocytes revealed that GlcN and NAPA were also able to counteract the IL-1beta-upregulation of genes coding for metalloproteases and inflammatory cytokines. Our preliminary in vivo and in vitro studies suggest that GlcN and NAPA could play a disease-modifying protective role in OA by an anti-catabolic effect and an anti-inflammatory activity on chondrocytes.

Glucosamine affects intracellular signalling through inhibition of mitogen-activated protein kinase phosphorylation in human chondrocytes.

The aim of this study was to determine the effects of glucosamine on matrix metalloprotease (MMP) production, on mitogen-activated protein kinase (MAPK) phosphorylation, and on activator protein (AP)-1 transcription factor activation in human chondrocytes. The human immortalized cell line lbpva55 and healthy human chondrocytes (obtained from healthy donors) were subjected to challenge with 10 ng/ml IL-1beta after pretreatment with 2.5 or 10 mmol/l glucosamine. MMP mRNA expression levels were evaluated using quantitative real-time PCR, and MMP protein production levels were evaluated in the culture supernatant using ELISA. MAPK phosphorylation was evaluated using Western blotting. AP-1 transcription factor activation was evaluated by measuring AP-1 DNA-binding activity. After IL-1beta stimulation, levels of MMP-1, MMP-3 and MMP-13 production were markedly increased. Treatment with 2.5 and 10 mmol/l glucosamine reduced expression of these metalloproteases. MMP expression is regulated by transcription factors such as the AP-1 complex, which is activated by phosphorylated MAPKs. IL-1beta stimulated phosphorylation of c-jun amino-terminal kinase, p38 MAPK and extracellular signal-regulated kinase-1/2. Glucosamine inhibited c-jun amino-terminal kinase and p38 phosphorylation, and consequently c-jun binding activity. These findings demonstrate, for the first time, that glucosamine inhibits IL-1beta-stimulated MMP production in human chondrocytes by affecting MAPK phosphorylation.

Cadmium induces mitogenic signaling in breast cancer cell by an ERalpha-dependent mechanism.

Breast cancer (BC) is linked to estrogen exposure. Estradiol (E2) stimulates BC cells proliferation by binding the estrogen receptor (ER). Hormone-related cancers have been linked to estrogenic environmental contaminants. Cadmium (Cd) a toxic pollutant, acts as estrogens in BC cells. Purpose of our study was to evaluate whether Cd regulates MCF-7 cell proliferation by activating ERK1/2, Akt and PDGFRalpha kinases. Cd increased cell proliferation and the ER-antagonist ICI 182,780 blunted it. To characterize an ER-dependent mechanism, ERalpha/beta expression was evaluated. Cd decreased ERalpha expression, but not ERbeta. Cd also increased ERK1/2, Akt and PDGFRalpha phosphorylation while ICI blocked it. Since stimulation of phosphorylation was slower than expected, c-fos and c-jun proto-oncogenes, and PDGFA were analyzed. Cd rapidly increased c-jun, c-fos and PDGFA expression. Cells were also co-incubated with the Cd and specific kinases inhibitors, which blocked the Cd-stimulated proliferation. In conclusion, our results indicate that Cd increases BC cell proliferation in vitro by stimulating Akt, ERK1/2 and PDGFRalpha kinases activity likely by activating c-fos, c-jun and PDGFA by an ERalpha-dependent mechanism.

Effect of titanium carbide coating on the osseointegration response in vitro and in vivo.

Titanium has limitations in its clinical performance in dental and orthopaedic applications. This study describes a coating process using pulsed laser deposition (PLD) technology to produce surfaces of titanium carbide (TiC) on titanium substrates and evaluates the biological response both in vitro and in vivo. X-ray photoelectron spectroscopy (XPS) analysis revealed the presence of 18.6-21.5% TiC in the surface layer, accompanied by oxides of titanium 78.5-81.4% in the following concentrations: 11.1-13.0% Ti(2)O(3), 50.8-55.8% TiO(2), 14.5-14.7% TiO. Expression of genes central to osteoblast differentiation (alkaline phosphatase, A2 pro-collagen type 1, osteocalcin, BMP-4, TGFbeta and Cbfa-1) were up-regulated in all cell lines (primary human osteoblasts, hFOB1.19 and ROS.MER#14) grown on TiC compared with uncoated titanium when measured by semiquantitative PCR and real time-PCR, whilst genes involved in modulation of osteoclastogenesis and osteoclast activity (IL-6 and M-CSF) were unchanged. Bone density was shown to be greater around TiC-coated implants after 2 and 4 weeks in sheep and both 4 and 8 weeks in rabbits compared to uncoated titanium. Rapid bone deposition was demonstrated after only 2 weeks in the rabbit model when visualized with intravital staining. It is concluded that coating with TiC will, in comparison to uncoated titanium, improve implant hardness, biocompatibility through surface stability and osseointegration through improved bone growth.

Determination of Cd, Cu, Pb and Zn in neoplastic kidneys and in renal tissue of fetuses, newborns and corpses.

The incidence of kidney tumors in USA and Europe (in particular, Central Europe and Italy) has been dramatically increasing since the 1970s, possibly as a consequence of ongoing environmental pollution. Environmental factors have been considered responsible for at least 80% of the incidence of neoplastic diseases. To shed some light on this issue, the amounts of Cd and Pb were measured in neoplastic tissue and adjacent normal part of kidney excised for carcinoma and compared with those in renal tissues of fetuses, newborns and subjects that died of non-neoplastic diseases. Cd and Pb were determined by Inductively Coupled Plasma Atomic Emission Spectrometry and Atomic Absorption Spectrometry with Electrothermal Atomization. Metallothionein immunoperoxidase staining technique was used to localize the accumulation of Cd and Zn in the nephrons. Content of Cd and Pb in kidneys of fetuses and newborns was extremely low. However, it was significantly increased in adjacent-normal tissues of kidneys with carcinomas, and significantly higher compared to kidneys of individuals that died of non-neoplastic diseases. In tumoral tissues of the excised kidneys, Cd content was very low, while that of Pb significantly elevated. High amounts of Cd and Pb in the adjacent-normal parts of kidneys with carcinomas are suggestive of possible, individual or synergistic, effects of these pollutants on enzymatic systems, priming an oncogenic pathway. Detection of metallothioneins, primary ligands of Cd, exclusively in the cells of proximal tubuli, i.e. wherein renal carcinoma develops in over 80% of cases, strongly supports the assumption that Cd exerts a carcinogenic effect.

Oligomerization of Sulfolobus solfataricus signature amidase is promoted by acidic pH and high temperature.

The recombinant amidase from the hyperthermophylic archaeon Sulfolobus solfataricus (SSAM) a signature amidase, was cloned, purified and characterized. The enzyme is active on a large number of aliphatic and aromatic amides over the temperature range 60-95 degrees C and at pH values between 4.0 and 9.5, with an optimum at pH 5.0. The recombinant enzyme is in the form of a dimer of about 110 kD that reversibly associates into an octamer in a pH-dependent reaction. The pH dependence of the state of association was studied using gel permeation chromatography, analytical ultracentrifugation and dynamic light scattering techniques. At pH 7.0 all three techniques show the presence of two species, in about equal amounts, which is compatible with the existence of a dimeric and an octameric form. In decreasing pH, the dimers formed the octameric species and in increasing pH, the octameric species was converted to dimers. Above pH 8.0, only dimers were present, below pH 3.0 only octamers were present. The association of dimers into octamers decreased in non-polar solvents and increased with temperature. A mutant (Y41C) was obtained that did not show this behavior.