Algometry with a clothes peg compared to an electronic pressure algometer: a randomized cross-sectional study in pain patients

Hypersensitivity of the central nervous system is widely present in pain patients and recognized as one of the determinants of chronic pain and disability. Electronic pressure algometry is often used to explore aspects of central hypersensitivity. We hypothesized that a simple pain provocation test with a clothes peg provides information on pain sensitivity that compares meaningfully to that obtained by a well-established electronic pressure algometer. "Clinically meaningful" was defined as a medium (r = 0.3-0.5) or high (r > 0.5) correlation coefficient according to Cohen's conventions.

PEG spacers of different length influence the biological profile of bombesin-based radiolabeled antagonists

INTRODUCTION The gastrin-releasing peptide receptor (GRPR) was shown to be expressed with high density on several types of cancers. Radiolabeled peptides for imaging and targeted radionuclide therapy have been developed. In this study, we evaluated the potential of statine-based bombesin antagonists, conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) through oligoethyleneglycol spacers, labeled with (177)Lu and we determined the effect of polyethyleneglycol (PEG) spacer length on in vitro and in vivo properties. METHODS The bombesin antagonists were synthesized on solid phase using Fmoc chemistry; the spacers Fmoc-dPEGx-OH (x=2, 4, 6 and 12) and the DOTA(tBu)3 were coupled using a standard procedure. The peptides were labeled with (177)Lu and evaluated in vitro (lipophilicity, serum stability, internalization and binding affinity assays). Biodistribution studies were performed in PC-3 tumor-bearing nude mice. RESULTS The solid-phase synthesis was straightforward with an overall yield ranging from 30% to 35% based on the first Fmoc cleavage. The hydrophilicity increased with spacer length (logD: -1.95 vs -2.22 of PEG2 and PEG12 analogs, respectively). There is a tendency of increased serum stability by increasing the spacer length (T1/2=246±4 and 584±20 for PEG2 and PEG6 analogs, respectively) which seems to reverse with the PEG12 analog. The IC50 values are similar with the only significant difference of the PEG12 analog. The (177)Lu-labeled PEG4 and PEG6 conjugates showed similar pharmacokinetic with high tumor uptake and excellent tumor-to-kidney ratios (7.8 and 9.7 at 4h for the PEG4 and PEG6 derivatives, respectively). The pancreas uptake was relatively high at 1h but it shows fast washout (0.46%±0.02% IA/g and 0.29%±0.08% IA/g already at 4h). CONCLUSION Among all the studied analogs the PEG4 and PEG6 showed significantly better properties. The very high tumor-to-non-target organ ratios, in particular tumor-to-kidney ratios, already at early time point will be important in regard to safety concerning kidney toxicity.

Mechanical Loading Promoted Discogenic Differentiation of Human Mesenchymal Stem Cells Incorporated in 3D-PEG Scaffolds with RhGDF5 and RGD

Hydrogels have been described as ideal scaffolds for cells of 3D tissue constructs and hold strong promises with respect to in vitro 3D-cell-culture, where cells are isolated from native extracellular matrix (ECM). Synthesized polyethyleneglycol (PEG) hydrogels are appealing with regard to potential for cell therapy or as vehicles for drug delivery or even to regenerate tissue with similar hydrogel-like properties such as the nucleus pulposus of the intervertebral disc (IVD). Here, we tested whether incorporation of RGD motive would hinder discogenic differentiation of primary bone marrow-derived human mesenchymal stem cells (hMSCs) but favor proliferation of undifferentiated hMSCs. HMSCs were embedded in +RGD containing or without RGD PEG hydrogel and pre-conditioned with or without growth and differentiation factor-5 (rhGDF-5) for 13 days. Afterwards, all hMSCs-PEG gels were subsequently cyclically loaded (15% strain, 1Hz) for 5 consecutive days in a bioreactor to generate an IVD-like phenotype. Higher metabolic activity (resazurin assay) was found in groups with rhGDF5 in both gel types with and without RGD. Cell viability and morphology measured by confocal laser microscopy and DNA content showed decreased values (~60%) after 18 days of culture. Real-time RT-PCR of an array of 15 key genes suspected to be distinctive for IVD cells revealed moderate response to rhGDF5 and mechanical loading as also shown by histology staining. Preconditioning and mechanical loading showed relatively moderate responses revealed from both RT-PCR and histology although hMSCs were demonstrated to be potent to differentiate into chondrocyte-progenitor cells in micro- mass and 3D alginate bead culture.

Effects of PEG-Induced Water Deficit in Solanum nigrum on Zn and Ni Uptake and Translocation in Split Root Systems

Drought strongly influences root activities in crop plants and weeds. This paper is focused on the performance of the heavy metal accumulator Solanum nigrum, a plant which might be helpful for phytoremediation. The water potential in a split root system was decreased by the addition of polyethylene glycol (PEG 6000). Rubidium, strontium and radionuclides of heavy metals were used as markers to investigate the uptake into roots, the release to the shoot via the xylem, and finally the basipetal transport via the phloem to unlabeled roots. The uptake into the roots (total contents in the plant) was for most makers more severely decreased than the transport to the shoot or the export from the shoot to the unlabeled roots via the phloem. Regardless of the water potential in the labeling solution, 63Ni and 65Zn were selectively redistributed within the plant. From autoradiographs, it became evident that 65Zn accumulated in root tips, in the apical shoot meristem and in axillary buds, while 63Ni accumulated in young expanded leaves and roots but not in the meristems. Since both radionuclides are mobile in the phloem and are, therefore, well redistributed within the plant, the unequal transfer to shoot and root apical meristems is most likely caused by differences in the cell-to-cell transport in differentiation zones without functional phloem (immature sieve tubes).

Dual Role of Mesenchymal Stem Cells Allows for Microvascularized Bone Tissue-Like Environments in PEG Hydrogels.

In vitro engineered tissues which recapitulate functional and morphological properties of bone marrow and bone tissue will be desirable to study bone regeneration under fully controlled conditions. Among the key players in the initial phase of bone regeneration are mesenchymal stem cells (MSCs) and endothelial cells (ECs) that are in close contact in many tissues. Additionally, the generation of tissue constructs for in vivo transplantations has included the use of ECs since insufficient vascularization is one of the bottlenecks in (bone) tissue engineering. Here, 3D cocultures of human bone marrow derived MSCs (hBM-MSCs) and human umbilical vein endothelial cells (HUVECs) in synthetic biomimetic poly(ethylene glycol) (PEG)-based matrices are directed toward vascularized bone mimicking tissue constructs. In this environment, bone morphogenetic protein-2 (BMP-2) or fibroblast growth factor-2 (FGF-2) promotes the formation of vascular networks. However, while osteogenic differentiation is achieved with BMP-2, the treatment with FGF-2 suppressed osteogenic differentiation. Thus, this study shows that cocultures of hBM-MSCs and HUVECs in biological inert PEG matrices can be directed toward bone and bone marrow-like 3D tissue constructs.

One-Step (18)F-Labeling of Carbohydrate-Conjugated Octreotate-Derivatives Containing a Silicon-Fluoride-Acceptor (SiFA): In Vitro and in Vivo Evaluation as Tumor Imaging Agents for Positron Emission Tomography (PET)

The synthesis, radiolabeling, and initial evaluation of new silicon-fluoride acceptor (SiFA) derivatized octreotate derivatives is reported. So far, the main drawback of the SiFA technology for the synthesis of PET-radiotracers is the high lipophilicity of the resulting radiopharmaceutical. Consequently, we synthesized new SiFA-octreotate analogues derivatized with Fmoc-NH-PEG-COOH, Fmoc-Asn(Ac?AcNH-?-Glc)-OH, and SiFA-aldehyde (SIFA-A). The substances could be labeled in high yields (38 ± 4%) and specific activities between 29 and 56 GBq/?mol in short synthesis times of less than 30 min (e.o.b.). The in vitro evaluation of the synthesized conjugates displayed a sst2 receptor affinity (IC?? = 3.3 ± 0.3 nM) comparable to that of somatostatin-28. As a measure of lipophilicity of the conjugates, the log P(ow) was determined and found to be 0.96 for SiFA-Asn(AcNH-?-Glc)-PEG-Tyr³-octreotate and 1.23 for SiFA-Asn(AcNH-?-Glc)-Tyr³-octreotate, which is considerably lower than for SiFA-Tyr³-octreotate (log P(ow) = 1.59). The initial in vivo evaluation of [¹?F]SiFA-Asn(AcNH-?-Glc)-PEG-Tyr³-octreotate revealed a significant uptake of radiotracer in the tumor tissue of AR42J tumor-bearing nude mice of 7.7% ID/g tissue weight. These results show that the high lipophilicity of the SiFA moiety can be compensated by applying hydrophilic moieties. Using this approach, a tumor-affine SiFA-containing peptide could successfully be used for receptor imaging for the first time in this proof of concept study.

Intracellular imaging of nanoparticles: is it an elemental mistake to believe what you see?

In order to understand how nanoparticles (NPs <100 nm) interact with cellular systems, potentially causing adverse effects, it is important to be able to detect and localize them within cells. Due to the small size of NPs, transmission electron microscopy (TEM) is an appropriate technique to use for visualizing NPs inside cells, since light microscopy fails to resolve them at a single particle level. However, the presence of other cellular and non-cellular nano-sized structures in TEM cell samples, which may resemble NPs in size, morphology and electron density, can obstruct the precise intracellular identification of NPs. Therefore, elemental analysis is recommended to confirm the presence of NPs inside the cell. The present study highlights the necessity to perform elemental analysis, specifically energy filtering TEM, to confirm intracellular NP localization using the example of quantum dots (QDs). Recently, QDs have gained increased attention due to their fluorescent characteristics, and possible applications for biomedical imaging have been suggested. Nevertheless, potential adverse effects cannot be excluded and some studies point to a correlation between intracellular particle localization and toxic effects. J774.A1 murine macrophage-like cells were exposed to NH2 polyethylene (PEG) QDs and elemental co-localization analysis of two elements present in the QDs (sulfur and cadmium) was performed on putative intracellular QDs with electron spectroscopic imaging (ESI). Both elements were shown on a single particle level and QDs were confirmed to be located inside intracellular vesicles. Nevertheless, ESI analysis showed that not all nano-sized structures, initially identified as QDs, were confirmed. This observation emphasizes the necessity to perform elemental analysis when investigating intracellular NP localization using TEM.

Quantum dot cytotoxicity in vitro: An investigation into the cytotoxic effects of a series of different surface chemistries and their core/shell materials

Abstract The aim of this study was to assess the effects of a series of different surface coated quantum dots (QDs) (organic, carboxylated [COOH] and amino [NH(2)] polytethylene glycol [PEG]) on J774.A1 macrophage cell viability and to further determine which part of the QDs cause such toxicity. Cytotoxic examination (MTT assay and LDH release) showed organic QDs to induce significant cytotoxicity up to 48 h, even at a low particle concentration (20 nM), whilst both COOH and NH(2) (PEG) QDs caused reduced cell viability and cell membrane permeability after 24 and 48 h exposure at 80 nM. Subsequent analysis of the elements that constitute the QD core, core/shell and (organic QD) surface coating showed that the surface coating drives QD toxicity. Elemental analysis (ICP-AES) after 48 h, however, also observed a release of Cd from organic QDs. In conclusion, both the specific surface coating and core material can have a significant impact on QD toxicity.

Quantitative evaluation of cellular uptake and trafficking of plain and polyethylene glycol-coated gold nanoparticles

This study addresses the cellular uptake and intracellular trafficking of 15-nm gold nanoparticles (NPs), either plain (i.e., stabilized with citrate) or coated with polyethylene glycol (PEG), exposed to human alveolar epithelial cells (A549) at the air-liquid interface for 1, 4, and 24 h. Quantitative analysis by stereology on transmission electron microscopy images reveals a significant, nonrandom intracellular distribution for both NP types. No particles are observed in the nucleus, mitochondria, endoplasmic reticulum, or golgi. The cytosol is not a preferred cellular compartment for both NP types, although significantly more PEG-coated than citrate-stabilized NPs are present there. The preferred particle localizations are vesicles of different sizes (<150, 150-1000, >1000 nm). This is observed for both NP types and indicates a predominant uptake by endocytosis. Subsequent inhibition of caveolin- and clathrin-mediated endocytosis by methyl-beta-cyclodextrin (MbetaCD) results in a significant reduction of intracellular NPs. The inhibition, however, is more pronounced for PEG-coated than citrate-stabilized NPs. The latter are mostly found in larger vesicles; therefore, they are potentially taken up by macropinocytosis, which is not inhibited by MbetaCD. With prolonged exposure times, both NPs are preferentially localized in larger-sized intracellular vesicles such as lysosomes, thus indicating intracellular particle trafficking. This quantitative evaluation reveals that NP surface coatings modulate endocytotic uptake pathways and cellular NP trafficking. Other nonendocytotic entry mechanisms are found to be involved as well, as indicated by localization of a minority of PEG-coated NPs in the cytosol.

Effect of enamel matrix derivative and parathyroid hormone on bone formation in standardized osseous defects: an experimental study in minipigs

Previous experimental studies have indicated that locally administered enamel matrix derivative (EMD) and parathyroid hormone (PTH) may have a stimulatory effect on bone formation. However, it is not clear if the positive effect of EMD is related to its effect on the periodontium as a whole or directly on the bone-forming cells. In addition, it is not known if the presentation of PTH by adding the amino acid sequence Arg-Gly-Asp (RGD) is essential for its osteopromotive effect. Local delivery of a bioactive substance at the right time and in the right concentration often constitutes a major challenge. Polyethylene glycol-based hydrogel (PEG) is a degradable vehicle developed for delivery of bioactive proteins. To enhance the mechanical stability of the PEG-bioactive substance complex, an osteoconductive bone substitute material is often needed.

Self-assembly of focal point oligo-catechol ethylene glycol dendrons on titanium oxide surfaces: adsorption kinetics, surface characterization, and nonfouling properties

This work covers the synthesis of second-generation, ethylene glycol dendrons covalently linked to a surface anchor that contains two, three, or four catechol groups, the molecular assembly in aqueous buffer on titanium oxide surfaces, and the evaluation of the resistance of the monomolecular adlayers against nonspecific protein adsorption in contact with full blood serum. The results were compared to those of a linear poly(ethylene glycol) (PEG) analogue with the same molecular weight. The adsorption kinetics as well as resulting surface coverages were monitored by ex situ spectroscopic ellipsometry (VASE), in situ optical waveguide lightmode spectroscopy (OWLS), and quartz crystal microbalance with dissipation (QCM-D) investigations. The expected compositions of the macromolecular films were verified by X-ray photoelectron spectroscopy (XPS). The results of the adsorption study, performed in a high ionic strength ("cloud-point") buffer at room temperature, demonstrate that the adsorption kinetics increase with increasing number of catechol binding moieties and exceed the values found for the linear PEG analogue. This is attributed to the comparatively smaller and more confined molecular volume of the dendritic macromolecules in solution, the improved presentation of the catechol anchor, and/or their much lower cloud-point in the chosen buffer (close to room temperature). Interestingly, in terms of mechanistic aspects of "nonfouling" surface properties, the dendron films were found to be much stiffer and considerably less hydrated in comparison to the linear PEG brush surface, closer in their physicochemical properties to oligo(ethylene glycol) alkanethiol self-assembled monolayers than to conventional brush surfaces. Despite these differences, both types of polymer architectures at saturation coverage proved to be highly resistant toward protein adsorption. Although associated with higher synthesis costs, dendritic macromolecules are considered to be an attractive alternative to linear polymers for surface (bio)functionalization in view of their spontaneous formation of ultrathin, confluent, and nonfouling monolayers at room temperature and their outstanding ability to present functional ligands (coupled to the termini of the dendritic structure) at high surface densities.