Titanium phosphate glass microspheres for bone tissue engineering

We have demonstrated the successful production of titanium phosphate glass microspheres in the size range of ~10-200 µm using an inexpensive, efficient, easily scalable process and assessed their use in bone tissue engineering applications. Glasses of the following compositions were prepared by melt-quench techniques: 0.5P2O5-0.4CaO-(0.1 - x)Na2O-xTiO2, where x = 0.03, 0.05 and 0.07 mol fraction (denoted as Ti3, Ti5 and Ti7 respectively). Several characterization studies such as differential thermal analysis, degradation (performed using a novel time lapse imaging technique) and pH and ion release measurements revealed significant densification of the glass structure with increased incorporation of TiO2 in the glass from 3 to 5 mol.%, although further TiO2 incorporation up to 7 mol.% did not affect the glass structure to the same extent. Cell culture studies performed using MG63 cells over a 7-day period clearly showed the ability of the microspheres to provide a stable surface for cell attachment, growth and proliferation. Taken together, the results confirm that 5 mol.% TiO2 glass microspheres, on account of their relative ease of preparation and favourable biocompatibility, are worthy candidates for use as substrate materials in bone tissue engineering applications.

Novel sol–gel preparation of (P2O5)0.4–(CaO)0.25–(Na2O)X–(TiO2)(0.35−X) bioresorbable glasses (X = 0.05, 0.1, and 0.15)

Quaternary phosphate-based glasses in the P2O5–CaO–Na2O–TiO2 system with a fixed P2O5 and CaO content of 40 and 25 mol% respectively have been successfully synthesised via sol–gel method and bulk, transparent samples were obtained. The structure, elemental proportion, and thermal properties of stabilised sol–gel glasses have been characterised using X-ray diffraction (XRD), energy dispersive X-ray spectroscopy (EDX), 31P nuclear magnetic resonance (31P NMR), titanium K-edge X-ray absorption near-edge structure (XANES), fourier transform infrared (FTIR) spectroscopy, and differential thermal analysis (DTA). The XRD results confirmed the amorphous nature for all stabilized sol–gel derived glasses. The EDX result shows the relatively low loss of phosphorus during the sol–gel process and Ti K-edge XANES confirmed titanium in the glass structure is in mainly six-fold coordination environment. The 31P NMR and FTIR results revealed that the glass structure consist of mainly Q1 and Q2 phosphate units and the Ti4+ cation was acting as a cross-linking between phosphate units. In addition DTA results confirmed a decrease in the glass transition and crystallisation temperature with increasing Na2O content. Ion release studies also demonstrated a decrease in degradation rates with increasing TiO2 content therefore supporting the use of these glasses for biomedical applications that require a degree of control over glass degradation. These sol–gel glasses also offer the potential to incorporate proactive molecules for drug delivery application due to the low synthesis temperature employed.

Release and diffusion of hydroxyl ion from calcium hydroxide-based medicaments

The release and diffusion of hydroxyl ions (OH-) of calcium hydroxide (Ca(OH)2)-based intracanal medications may be affected by the association with other substances. The aim of this study was to evaluate the diffusion of OH- ions through root dentin by the medications: G1, Ca(OH)2/saline; G2, Calen; G3, Calen/camphorated p-monochlorophenol (CMCP); and G4, Calen/0.4% chlorhexidine (CHX). Root canals from bovine teeth were prepared in a standardized manner. A cavity until dentin was prepared in the middle third of the root surface of each specimen. The external surface of the root was made impermeable using a layer of adhesive, except the prepared cavity. The root canals were filled with different medications, and teeth were individually stored in flasks containing 10 ml distilled water at 37 degrees C. The water pH was measured at 1, 3, 7, 14, 21, 30, and 60 days. Data obtained were subjected to anova and Tukeys tests. Increase in pH was observed at 3 days for Calen/CHX and from 7 to 14 days for the other mixtures. Calen paste promoted pH increase up to 21 days. Calen/CMCP had the highest pH up to 21 days, and all groups had similar results at 30 days. At 60 days, the greatest pH values were observed for Calen/CMCP and Calen alone. All different formulations of Ca(OH)2-based medications tested release hydroxyl ion that can diffuse through the dentin.

Fluoride release profile of a nanofilled resin-modified glass ionomer cement

The present study aimed to compare the fluoride (F-) release pattern of a nanofilled resin-modified glass ionomer cement (GIC) (Ketac N100 - KN) with available GICs used in dental practice (resin-modified GIC - Vitremer - V; conventional GIC - Ketac Molar - KM) and a nanofilled resin composite (Filtek Supreme - RC). Discs of each material (n=6) were placed into 4 mL of deionized water in sealed polyethylene vials and shaken, for 15 days. F- release (μg F-/cm²) was measured each day using a fluoride-ion specific electrode. Cumulative F- release means were statistically analyzed by linear regression analysis. In order to analyze the differences among materials and the influence of time in the daily F- release, 2-way ANOVA test was performed (α=0.05). The linear fits between the cumulative F- release profiles of RC and KM and time were weak. KN and V presented a strong relationship between cumulative F- release and time. There were significant differences between the daily F- release overtime up to the third day only for GICs materials. The daily F- release means for RC were similar overtime. The results indicate that the F- release profile of the nanofilled resin-modified GIC is comparable to the resin-modified GIC.

The inducing NO-vasodilation by chemical reduction of coordinated nitrite ion in cis-[Ru(NO(2))L(bpy)(2)](+) complex

The synthesis of [Ru(NO(2)) L(bpy)(2)](+) (bpy = 2,2'-bipyridine and L = pyridine (py) and pyrazine (pz)) can be accomplished by addition of [Ru(NO) L(bpy) 2](PF(6))(3) to aqueous solutions of physiological pH. The electrochemical processes of [Ru(NO2) L(bpy) 2]+ in aqueous solution were studied by cyclic voltammetry and differential pulse voltammetry. The anodic scan shows a peak around 1.00 V vs. Ag/AgCl attributed to the oxidation process centered on the metal ion. However, in the cathodic scan a second peak around-0.60 V vs. Ag/AgCl was observed and attributed to the reduction process centered on the nitrite ligand. The controlled reduction potential electrolysis at-0.80 V vs. Ag/AgCl shows NO release characteristics as judged by NO measurement with a NO-sensor. This assumption was confirmed by ESI/MS(+) and spectroelectrochemical experiment where cis-[Ru(bpy)(2)L(H(2)O)](2+) was obtained as a product of the reduction of cis-[Ru(II)(NO(2)) L(bpy)(2)](+). The vasorelaxation observed in denuded aortic rings pre-contracted with 0.1 mu mol L(-1) phenylephrine responded with relaxation in the presence of cis-[RuII(NO2) L(bpy) 2]+. The potential of rat aorta cells to metabolize cis-[RuII(NO(2)) L(bpy)(2)](+) was also followed by confocal analysis. The obtained results suggest that NO release happens by reduction of cis-[RuII(NO(2)) L(bpy)(2)](+) inside the cell. The maximum vasorelaxation was achieved with 1 x 10(-5) mol L(-1) of cis-[RuII(NO(2)) L(bpy)(2)](+) complex.

Activation of ion secretion via proteinase-activated receptor-2 in human colon

Proteinase-activated receptor (PAR) type 2 (PAR-2) has been shown to mediate ion secretion in cultured epithelial cells and rat jejunum. With the use of a microUssing chamber, we demonstrate the role of PAR-2 for ion transport in native human colonic mucosa obtained from 30 normal individuals and 11 cystic fibrosis (CF) patients. Trypsin induced Cl- secretion when added to the basolateral but not luminal side of normal epithelia. Activation of Cl- secretion by trypsin was inhibited by indomethacin and was further increased by cAMP in normal tissues but was not present in CF colon, indicating the requirement of luminal CF transmembrane conductance regulator. Effects of trypsin were largely reduced by low Cl-,by basolateral bumetanide, and in the presence of barium or clotrimazole, but not by tetrodotoxin. Furthermore, trypsin-induced secretion was inhibited by the Ca2+-ATPase inhibitor cyclopiazonic acid and in low-Ca2+ buffer. The effects of trypsin were almost abolished by trypsin inhibitor. Thrombin, an activator of PAR types 1, 3, and 4, had no effects on equivalent short-circuit currents. The presence of PAR-2 in human colon epithelium was confirmed by RT-PCR and additional experiments with PAR-2-activating peptide. PAR-2-mediated intestinal electrolyte secretion by release of mast cell tryptase and potentiation of PAR-2 expression by tumor necrosis factor-alpha may contribute to the hypersecretion observed in inflammatory processes such as chronic inflammatory bowel disease.

No evidence for inhibition of ENaC through CFTR-mediated release of ATP

Both purinergic stimulation and activation of cystic fibrosis transmembrane conductance regulator (CFTR) increases Cl- secretion and inhibit amiloride-sensitive Na+ transport. CFTR has been suggested to conduct adenosine 5'-triphosphate (ATP) or to control ATP release to the luminal side of epithelial tissues. Therefore, a possible mechanism on how CFTR controls the activity of epithelial Na+ channels (ENaC) could be by release of ATP or uridine 5'-triphosphate (UTP), which would then bind to P2Y receptors and inhibit ENaC. We examined this question in native tissues from airways and colon and in Xenopus oocytes. Inhibition of amiloride-sensitive transport by both CFTR and extracellular nucleotides was observed in colon and trachea. However, nucleotides did not inhibit ENaC in Xenopus oocytes, even after coexpression of P2Y(2) receptors. Using different tools such as hexokinase, the P2Y inhibitor suramin or the Cl- channel blocker 4,4'diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), we did not detect any role of a putative ATP secretion in activation of Cl- transport or inhibition of amiloride sensitive short circuit currents by CFTR. In addition, N-2,2'-O-dibutyrylguanosine 3',5-cyclic monophosphate (cGMP) and protein kinase G (PKG)-dependent phosphorylation or the nucleoside diphosphate kinase (NDPK) do not seem to play a role for the inhibition of ENaC by CFTR, which, however, requires the presence of extracellular Cl-. (C) 2002 Elsevier Science B.V. All rights reserved.

Development of a poly(L-Lactic acid) based drug release system

Dissertação de mestrado em Biofísica e Bionanossistemas

What do we know about gliotransmitter release from astrocytes?

Astrocytes participate in information processing by actively modulating synaptic properties via gliotransmitter release. Various mechanisms of astrocytic release have been reported, including release from storage organelles via exocytosis and release from the cytosol via plasma membrane ion channels and pumps. It is still not fully clear which mechanisms operate under which conditions, but some of them, being Ca(2+)-regulated, may be physiologically relevant. The properties of Ca(2+)-dependent transmitter release via exocytosis or via ion channels are different and expected to produce different extracellular transmitter concentrations over time and to have distinct functional consequences. The molecular aspects of these two release pathways are still under active investigation. Here, we discuss the existing morphological and functional evidence in support of either of them. Transgenic mouse models, specific antagonists and localization studies have provided insight into regulated exocytosis, albeit not in a systematic fashion. Even more remains to be uncovered about the details of channel-mediated release. Better functional tools and improved ultrastructural approaches are needed in order fully to define specific modalities and effects of astrocytic gliotransmitter release pathways.

Antimalarial drugs disrupt ion homeostasis in malarial parasites

Plasmodium chabaudi malaria parasite organelles are major elements for ion homeostasis and cellular signaling and also target for antimalarial drugs. By using confocal imaging of intraerythrocytic parasites we demonstrated that the dye acridine orange (AO) is accumulated into P. chabaudi subcellular compartments. The AO could be released from the parasite organelles by collapsing the pH gradient with the K+/H+ ionophore nigericin (20 µM), or by inhibiting the H+-pump with bafilomycin (4 µM). Similarly, in isolated parasites loaded with calcium indicator Fluo 3-AM, bafilomycin caused calcium mobilization of the acidic calcium pool that could also be release with nigericin. Interestingly after complete release of the acidic compartments, addition of thapsigargin at 10 µM was still effective in releasing parasite intracellular calcium stores in parasites at trophozoite stage. The addition of antimalarial drugs chloroquine and artemisinin resulted in AO release from acidic compartments and also affected maintenance of calcium in ER store by using different drug concentrations.

Calcium entry via TRPV1 but not ASICs induces neuropeptide release from sensory neurons

Inflammatory mediators induce neuropeptide release from nociceptive nerve endings and cell bodies, causing increased local blood flow and vascular leakage resulting in edema. Neuropeptide release from sensory neurons depends on an increase in intracellular Ca2+ concentration. In this study we investigated the role of two types of pH sensors in acid-induced Ca2+ entry and neuropeptide release from dorsal root ganglion (DRG) neurons. The transient receptor potential vanilloid 1 channel (TRPV1) and acid-sensing ion channels (ASICs) are both H+-activated ion channels present in these neurons, and are therefore potential pH sensors for this process. We demonstrate with in situ hybridization and immunocytochemistry that TRPV1 and several ASIC subunits are co-expressed with neuropeptides in DRG neurons. Activation of ASICs and of TRPV1 led to an increase in intracellular Ca2+ concentration. While TRPV1 has a high Ca2+ permeability and allows direct Ca2+ entry when activated, we show here that ASICs of DRG neurons mediate Ca2+ entry mostly by depolarization-induced activation of voltage-gated Ca2+ channels and only to a small extent via the pore of Ca2+-permeable ASICs. Extracellular acidification led to release of the neuropeptide calcitonin gene-related peptide from DRG neurons. The pH dependence and the pharmacological profile indicated that TRPV1, but not ASICs, induced neuropeptide secretion. In conclusion, this study shows that although both TRPV1 and ASICs mediate Ca2+ influx, TRPV1 is the principal sensor for acid-induced neuropeptide secretion from sensory neurons.

Sunitinib-eluting beads for chemoembolization: methods for in vitro evaluation of drug release.

Drug-eluting microspheres are used for embolization of hypervascular tumors and allow for local controlled drug release. Although the drug release from the microspheres relies on fast ion-exchange, so far only slow-releasing in vitro dissolution methods have been correlated to in vivo data. Three in vitro release methods are assessed in this study for their potential to predict slow in vivo release of sunitinib from chemoembolization spheres to the plasma, and fast local in vivo release obtained in an earlier study in rabbits. Release in an orbital shaker was slow (t50%=4.5h, 84% release) compared to fast release in USP 4 flow-through implant cells (t50%=1h, 100% release). Sunitinib release in saline from microspheres enclosed in dialysis inserts was prolonged and incomplete (t50%=9 days, 68% release) due to low drug diffusion through the dialysis membrane. The slow-release profile fitted best to low sunitinib plasma AUC following injection of sunitinib-eluting spheres. Although limited by lack of standardization, release in the orbital shaker fitted best to local in vivo sunitinib concentrations. Drug release in USP flow-through implant cells was too fast to correlate with local concentrations, although this method is preferred to discriminate between different sphere types.

Counter-ion effect on surfactant-DNA gel particles as controlled DNA delivery systems

The ability to entrap drugs within vehicles and subsequently release them has led to new treatments for a number of diseases. Based on an associative phase separation and interfacial diffusion approach, we developed a way to prepare DNA gel particles without adding any kind of cross-linker or organic solvent. Among the various agents studied, cationic surfactants offered particularly efficient control for encapsulation and DNA release from these DNA gel particles. The driving force for this strong association is the electrostatic interaction between the two components, as induced by the entropic increase due to the release of the respective counter-ions. However, little is known about the influence of the respective counter-ions on this surfactant-DNA interaction. Here we examined the effect of different counter-ions on the formation and properties of the DNA gel particles by mixing DNA (either single- (ssDNA) or double-stranded (dsDNA)) with the single chain surfactant dodecyltrimethylammonium (DTA). In particular, we used as counter-ions of this surfactant the hydrogen sulfate and trifluoromethane sulfonate anions and the two halides, chloride and bromide. Effects on the morphology of the particles obtained, the encapsulation of DNA and its release, as well as the haemocompatibility of these particles, are presented, using the counter-ion structure and the DNA conformation as controlling parameters. Analysis of the data indicates that the degree of counter-ion dissociation from the surfactant micelles and the polar/hydrophobic character of the counter-ion are important parameters in the final properties of the particles. The stronger interaction with amphiphiles for ssDNA than for dsDNA suggests the important role of hydrophobic interactions in DNA.

ROLES OF ACID-SENSING ION CHANNELS (ASICS) IN PERIPHERAL NEUROPEPTIDE SECRETION AND PAIN

Acid-sensing ion channels (ASICs) are non-voltage-gated sodium channels activated by an extracellular acidification. They are widely expressed in neurons of the central and peripheral nervous system. ASICs have a role in learning, the expression of fear, in neuronal death after cerebral ischemia, and in pain sensation. Tissue damage leads to the release of inflammatory mediators. There is a subpopulation of sensory neurons which are able to release the neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP). Neurogenic inflammation refers to the process whereby peripheral release of the neuropeptides CGRP and SP induces vasodilation and extravasation of plasma proteins, respectively. Our laboratory has previously shown that calcium-permeable homomeric ASIC1a channels are present in a majority of CGRP- or SP-expressing small diameter sensory neurons. In the first part of my thesis, we tested the hypothesis that a local acidification can produce an ASIC-mediated calcium-dependant neuropeptide secretion. We have first verified the co-expression of ASICs and CGRP/SP using immunochemistry and in-situ hybridization on dissociated rat dorsal root ganglion (DRG) neurons. We found that most CGRP/SP-positive neurons also expressed ASIC1a and ASIC3 subunits. Calcium imaging experiments with Fura-2 dye showed that an extracellular acidification can induce an increase of intracellular Ca2+ concentration, which is essential for secretion. This increase of intracellular Ca2+ concentration is, at least in some cells, ASIC-dependent, as it can be prevented by amiloride, an ASIC antagonist, and by Psalmotoxin (PcTx1), a specific ASIC1a antagonist. We identified a sub-population of neurons whose acid-induced Ca2+ entry was completely abolished by amiloride, an amiloride-resistant population which does not express ASICs, but rather another acid-sensing channel, possibly transient receptor potential vanilloïde 1 (TRPV1), and a population expressing both H+-gated channel types. Voltage-gated calcium channels (Cavs) may also mediate Ca2+ entry. Co-application of the Cavs inhibitors (ω-conotoxin MVIIC, Mibefradil and Nifedipine) reduced the Ca2+ increase in neurons expressing ASICs during an acidification to pH 6. This indicates that ASICs can depolarise the neuron and activate Cavs. Homomeric ASIC1a are Ca2+-permeable and allow a direct entry of Ca2+ into the cell; other ASICs mediate an indirect entry of Ca2+ by inducing a membrane depolarisation that activates Cavs. We showed with a secretion assay that CGRP secretion can be induced by extracellular acidification in cultured rat DRG neurons. Amiloride and PcTx1 were not able to inhibit the secretion at acidic pH, but BCTC, a TRPV1 inhibitor was able to decrease the secretion induced by an extracellular acidification in our in vitro secretion assay. In conclusion, these results show that in DRG neurons a mild extracellular acidification can induce a calcium-dependent neuropeptide secretion. Even if our data show that ASICs can mediate an increase of intracellular Ca2+ concentration, this appears not to be sufficient to trigger neuropeptide secretion. TRPV1, a calcium channel whose activation induces a sustained current - in contrary of ASICs - played in our experimental conditions a predominant role in neurosecretion. In the second part of my thesis, we focused on the role of ASICs in neuropathic pain. We used the spared nerve injury (SNI) model which consists in a nerve injury that induces symptoms of neuropathic pain such as mechanical allodynia. We have previously shown that the SNI model modifies ASIC currents in dissociated rat DRG neurons. We hypothesized that ASICs could play a role in the development of mechanical allodynia. The SNI model was performed on ASIC1a, -2, and -3 knock-out mice and wild type littermates. We measured mechanical allodynia on these mice with calibrated von Frey filaments. There were no differences between the wild-type and the ASIC1, or ASIC2 knockout mice. ASIC3 null mice were less sensitive than wild type mice at 21 day after SNI, indicating a role for ASIC3. Finally, to investigate other possible roles of ASICs in the perception of the environment, we measured the baseline heat responses. We used two different models; the tail flick model and the hot plate model. ASIC1a null mice showed increased thermal allodynia behaviour in the hot plate test at three different temperatures (49, 52, 55°C) compared to their wild type littermates. On the contrary, ASIC2 null mice showed reduced thermal allodynia behaviour in the hot plate test compared to their wild type littermates at the three same temperatures. We conclude that ASIC1a and ASIC2 in mice can play a role in temperature sensing. It is currently not understood how ASICs are involved in temperature sensing and what the reason for the opposed effects in the two knockout models is.

The Role of Diffusion in ISOL Targets for the Production of Radioactive Ion Beams

On line isotope separation techniques (ISOL) for production of ion beams of short-lived radionuclides require fast separation of nuclear reaction products from irradiated target materials followed by a transfer into an ion source. As a first step in this transport chain the release of nuclear reaction products from refractory metals has been studied systematically and will be reviewed. High-energy protons (500 - 1000 MeV) produce a large number of radionuclides in irradiated materials via the nuclear reactions spallation, fission and fragmentation. Foils and powders of Re, W, Ta, Hf, Mo, Nb, Zr, Y, Ti and C were irradiated with protons (600 - 1000 MeV) at the Dubna synchrocyclotron, the CERN synchrocyclotron and at the CERN PS-booster to produce different nuclear reaction products. The main topic of the paper is the determination of diffusion coefficients of the nuclear reaction products in the target matrix, data evaluation and a systematic interpretation of the data. The influence of the ionic radius of the diffusing species and the lattice type of the host material used as matrix or target on the diffusion will be evaluated from these systematics. Special attention was directed to the release of group I, II and III-elements. Arrhenius plots lead to activation energies of the diffusion process.