Counting the Number of Glutamate Molecules in Single Synaptic Vesicles.

Analytical tools for quantitative measurements of glutamate, the principal excitatory neurotransmitter in the brain, are lacking. Here, we introduce a new enzyme-based amperometric sensor technique for the counting of glutamate molecules stored inside single synaptic vesicles. In this method, an ultra-fast enzyme-based glutamate sensor is placed into a solution of isolated synaptic vesicles, which stochastically rupture at the sensor surface in a potential-dependent manner at a constant negative potential. The continuous amperometric signals are sampled at high speed (10 kHz) to record sub-millisecond spikes, which represent glutamate release from single vesicles that burst open. Glutamate quantification is achieved by a calibration curve that is based on measurements of glutamate release from vesicles pre-filled with various glutamate concentrations. Our measurements show that an isolated single synaptic vesicle encapsulates about 8000 glutamate molecules and is comparable to the measured exocytotic quantal glutamate release in amperometric glutamate sensing in the nucleus accumbens of mouse brain tissue. Hence, this new methodology introduces the means to quantify ultra-small amounts of glutamate and to study synaptic vesicle physiology, pathogenesis, and drug treatments for neuronal disorders where glutamate is involved.

The effect of oligo(trimethylene carbonate) addition on the stiffness of acrylic bone cement.

With the increasing elderly population an increase in the number of bony fractures associated to age-related diseases such as osteoporosis also follows. The relatively high stiffness of the acrylic bone cements used in these patients has been suggested to give raise to a suboptimal load distribution surrounding the cement in vivo, and hence contribute to clinical complications, such as additional fractures. The aim of this study was to develop a low-modulus bone cement, based on currently used, commercially available poly(methyl methacrylate) (PMMA) cements for vertebroplasty. To this end, acrylate end-functionalized oligo(trimethylene carbonate) (oTMC) was incorporated into the cements, and the resulting compressive mechanical properties were evaluated, as well as the cytotoxic and handling properties of selected formulations. Sixteen wt%oTMC was needed in the vertebroplastic cement Osteopal V to achieve an elastic modulus of 1063 MPa (SD 74), which gave a corresponding compressive strength of 46.1 MPa (SD 1.9). Cement extracts taken at 1 and 12 hours gave a reduced MG-63 cell viability in most cases, while extracts taken at 24 hours had no significant effect on cell behavior. The modification also gave an increase in setting time, from 14.7 min (SD 1.7) to 18.0 min (SD 0.9), and a decrease in maximum polymerization temperature, from 41.5°C (SD 3.4) to 30.7°C (SD 1.4). While further evaluation of other relevant properties, such as injectability and in vivo biocompatibility, remains to be done, the results presented herein are promising in terms of approaching clinically applicable bone cements with a lower stiffness.

In vivo biomechanical stability of osseointegrating mesoporous TiO(2) implants.

Mesoporous materials are of high interest as implant coatings to receive an enhanced osseointegration. In this study, titanium implants coated with mesoporous TiO(2) thin films have been evaluated both in vitro and in vivo. Material characterization showed that, with partly crystalline TiO(2) (anatase), long-range-ordered hydrophilic mesoporous thin films with a pore size of 6nm were obtained. Evaluation of the mechanical resistance showed that the films were robust enough to withstand the standard implantation procedure. In vitro apatite formation was studied using simulated body fluids, showing that the pores are accessible for ions and that formation of apatite was increased due to the presence of the mesopores. An in vivo study using a rabbit model was executed in which the removal torque and histomorphometry were evaluated. The results show that the biomechanical stability of the TiO(2) coating was unaffected by the presence of mesopores and that osseointegration was achieved without any signs of inflammation.