Guanosine enhances glutamate uptake and oxidation, preventing oxidative stress in mouse hippocampal slices submitted to high glutamate levels.

Glutamate (Glu) is the main mammalian brain neurotransmitter. Concerning the glutamatergic neurotransmission, excessive levels of glutamate in the synaptic cleft are extremally harmful. This phenomenon, named as excitotoxicity is involved in various acute and chronic brain diseases. Guanosine (GUO), an endogenous guanine nucleoside, possesses neuroprotective effects in several experimental models of glutamatergic excitotoxicity, an effect accompanied by an increase in astrocytic glutamate uptake. Therefore, the objective of this study was to investigate the involvement of an additional putative parameter, glutamate oxidation to CO2, involved in ex-vivo GUO neuroprotective effects in mouse hippocampal slices submitted to glutamatergic excitotoxicity. Mice were sacrificed by decapitation, the hippocampi were removed and sliced. The slices were incubated for various times and concentrations of Glu and GUO. First, the concentration of Glu that produced an increase in L-[14C(U)]-Glu oxidation to CO2 without cell injury was determined at different time points (between 0 and 90 min); 1000 µM Glu increased Glu oxidation between 30 and 60 min of incubation without cell injury. Under these conditions (Glu concentration and incubation time), 100 µM GUO increased Glu oxidation (35%). Additionally, 100 µM GUO increased L-[3,4-3H]-glutamate uptake (45%) in slices incubated with 1000 µM Glu (0-30 min). Furthermore, 1000  µM Glu increased reactive species levels, SOD activity, and decreased GPx activity, and GSH content after 30 and 60 min; 100 µM GUO prevented these effects. This is the first study demonstrating that GUO simultaneously promoted an increase in the uptake and utilization of Glu in excitotoxicity-like conditions preventing redox imbalance.

The chondrogenic differentiation potential of dental pulp stem cells.

Dental pulp stem cells (DPSCs) are particularly promising for tissue engineering (TE) due to the ease of their isolation procedure, great expansion potential and capability to differentiate towards several cell types of the mesodermal, ectodermal and endodermal lineages. Although several studies hint that DPSCs exhibit potential for cartilage tissue formation, the chondrogenic potential of DPSCs has only been marginally explored. Thus, the aim of the present study was to closely investigate the chondrogenic differentiation capacity of DPSCs for TE applications. More specifically, the potential of DPSCs for engineering hyaline and fibrous cartilage was determined. DPSCs obtained from 7 human molars were expanded and chondrogenically differentiated in a 3D pellet culture model. After 21 d of differentiation with chondrogenic stimuli, DPSCs displayed glycosaminoglycan, aggrecan and limited collagen type II deposition. Cells presented an elongated morphology and produced a collagen-rich extracellular matrix, with a predominance of collagen type I in most of the samples, a characteristic of fibrous cartilage tissue. Variations in the administration periods of several chondro-inductive growth factors, including transforming growth factor beta 3, bone morphogenetic protein-2, -6, -7 and insulin-like growth factor-1, did not increase glycosaminoglycan or collagen type II deposition, typical markers of hyaline cartilage tissue. Furthermore, DPSCs could not be stimulated to go into hypertrophic chondrogenesis. These results indicated that under a large variety of chondro-inductive culture conditions, DPSCs could form fibrocartilaginous tissues but not hyaline cartilage. Thus, DPSCs represent a valuable cell source for the regeneration of fibrocartilage in joints.

Complete regeneration of large bone defects in rats with commercially available fibrin loaded with BMP-2.

This study aimed at investigating in vitro and in vivo the efficiency of commercially available fibrin as a carrier for controlled and sustained bone morphogenetic protein-2 (BMP-2) release to induce bone formation and reduce the side effects of its use. In vitro release and activity of low-dose recombinant human BMP-2 (rhBMP-2) (37.5 µg/mL) embedded in commercially available fibrin were evaluated and, subsequently, critical-size femur defects in rats were grafted to study bone regeneration and vascularisation by micro-computed tomography (µCT) and histology. In vitro experiments showed a sustained BMP-2 release with a high BMP activity remaining after 28 d. In vivo, fibrin loaded with BMP-2 showed an extremely fast bone healing, with a large amount of new bone formation throughout the entire defect in the first 4 weeks and complete cortical repair and fusion after 8 weeks, with no ectopic bone formation. In contrast, the control fibrin group did not fuse after 12 weeks. Vascularisation was similar in both groups at 4 and 12 weeks after implantation. In conclusion, commercially available fibrin is a very efficient carrier for rhBMP-2 to graft critical-size cortical bone defects and might be a more optimal delivery vehicle for BMP-2-induced bone regeneration than currently available collagen sponges.

Intact vitreous humor as a potential extracellular matrix hydrogel for cartilage tissue engineering applications.

Decellularisation of tissues, utilising their biochemical cues, poses exciting tissue engineering (TE) opportunities. However, removing DNA from cartilage (dCart) requires harsh treatments due to its dense structure, causing loss of bioactivity and limiting its application as a cartilaginous extra cellular matrix (ECM). In this study, we demonstrate for the first time the successful application of vitreous humor (VH), a highly hydrated tissue closely resembling the glycosaminoglycan (GAG) and collagen composition of cartilage, as an ECM hydrogel to support chondrogenic differentiation. Equine VH was extracted followed by biochemical quantifications, histological examinations, cytotoxicity (human mesenchymal stromal cells, hMSCs and human articular chondrocytes, hACs) and U937 cell proliferation studies. VH was further seeded with hACs or hMSCs and cultured for 3-weeks to study chondrogenesis compared to scaffold-free micro-tissue pellet cultures and collagen-I hydrogels. Viability, metabolic activity, GAG and DNA content, chondrogenic gene expression (aggrecan, collagen I/II mRNA) and mechanical properties were quantified and matrix deposition was visualised using immunohistochemistry (Safranin-O, collagen I/II). VH was successfully extracted, exhibiting negligible amounts of DNA (0.4 ±â€¯0.4 µg/mg dry-weight) and notable preservation of ECM components. VH displayed neither cytotoxic responses nor proliferation of macrophage-like U937 cells, instead enhancing both hMSC and hAC proliferation. Interestingly, encapsulated cells self-assembled the VH-hydrogel into spheroids, resulting in uniform distribution of both GAGs and collagen type II with increased compressive mechanical properties, rendering VH a permissive native ECM source to fabricate cartilaginous hydrogels for potential TE applications. STATEMENT OF SIGNIFICANCE: Fabricating bioactive and cell-instructive cartilage extracellular matrix (ECM) derived biomaterials and hydrogels has over recent years proven to be a challenging task, often limited by poor retention of inherent environmental cues post decellularisation due to the dense and avascular nature of native cartilage. In this study, we present an alternative route to fabricate highly permissive and bioactive ECM hydrogels from vitreous humor (VH) tissue. This paper specifically reports the discovery of optimal VH extraction protocols and cell seeding strategy enabling fabrication of cartilaginous matrix components into a hydrogel support material for promoting chondrogenic differentiation. The work showcases a naturally intact and unmodified hydrogel design that improves cellular responses and may help guide the development of cell instructive and stimuli responsive hybrid biomaterials in a number of TERM applications.

The impact of immune response on endochondral bone regeneration.

Tissue engineered cartilage substitutes, which induce the process of endochondral ossification, represent a regenerative strategy for bone defect healing. Such constructs typically consist of multipotent mesenchymal stromal cells (MSCs) forming a cartilage template in vitro, which can be implanted to stimulate bone formation in vivo. The use of MSCs of allogeneic origin could potentially improve the clinical utility of the tissue engineered cartilage constructs in three ways. First, ready-to-use construct availability can speed up the treatment process. Second, MSCs derived and expanded from a single donor could be applied to treat several patients and thus the costs of the medical interventions would decrease. Finally, it would allow more control over the quality of the MSC chondrogenic differentiation. However, even though the envisaged clinical use of allogeneic cell sources for bone regeneration is advantageous, their immunogenicity poses a significant obstacle to their clinical application. The aim of this review is to increase the awareness of the role played by immune cells during endochondral ossification, and in particular during regenerative strategies when the immune response is altered by the presence of implanted biomaterials and/or cells. More specifically, we focus on how this balance between immune response and bone regeneration is affected by the implantation of a cartilaginous tissue engineered construct of allogeneic origin.

3D screening device for the evaluation of cell response to different electrospun microtopographies.

Micro- and nano-topographies of scaffold surfaces play a pivotal role in tissue engineering applications, influencing cell behavior such as adhesion, orientation, alignment, morphology and proliferation. In this study, a novel microfabrication method based on the combination of soft-lithography and electrospinning for the production of micro-patterned electrospun scaffolds was proposed. Subsequently, a 3D screening device for electrospun meshes with different micro-topographies was designed, fabricated and biologically validated. Results indicated that the use of defined patterns could induce specific morphological variations in human mesenchymal stem cell cytoskeletal organization, which could be related to differential activity of signaling pathways. STATEMENT OF SIGNIFICANCE: We introduce a novel and time saving method to fabricate 3D micropatterns with controlled micro-architectures on electrospun meshes using a custom made collector and a PDMS mold with the desired topography. A possible application of this fabrication technique is represented by a 3D screening system for patterned electrospun meshes that allows the screening of different scaffold/electrospun parameters on cell activity. In addition, what we have developed in this study could be modularly applied to existing platforms. Considering the different patterned geometries, the cell morphological data indicated a change in the cytoskeletal organization with a close correspondence to the patterns, as shown by phenoplot and boxplot analysis, and might hint at the differential activity of cell signaling. The 3D screening system proposed in this study could be used to evaluate topographies favoring cell alignment, proliferation and functional performance, and has the potential to be upscaled for high-throughput.

Administration of Huperzia quadrifariata Extract, a Cholinesterase Inhibitory Alkaloid Mixture, has Neuroprotective Effects in a Rat Model of Cerebral Hypoxia-Ischemia.

Neonatal hypoxia-ischemia (HI) is an etiologic component of several neurologic pathologies associated to cognitive impairment. The mechanisms involved in HI-induced tissue damage start immediately after HI and extend for days. Acetylcholine is an important neurotransmitter in the central nervous system and exerts a protector effect on tissue damage by modulating inflammation, and cholinesterase inhibitors have shown neuroprotective properties and their action are often attributed to inhibition of the immune response. The administration of Huperzia quadrifariata alkaloid extract (HqAE), with potent and selective cholinesterase inhibitor properties, will reduce the HI induced behavioral deficits and tissue damage. A total of 84 newborn Wistar rat pups at post natal day 7 (PND7) were subjected to right carotid occlusion followed by 1 h of hypoxia (8% of O2) and i.p. injections of saline, vehicle or HqAE (10 mg/kg). Morris Water Maze and inhibitory avoidance tests were used to assess the cognitive function. Flow cytometry was performed at PND11. Histological analysis was performed at PND45. HqAE treatment was able to prevent the HI induced cognitive deficits in both tests and, at PND45, histological analysis showed that HqAE treatment reduced hippocampus tissue damage. Flow cytometry of the injured hippocampus revealed that the treatment was able to reduce cellular death and the number of infiltrating T cells. Altogether, these results show the therapeutic potential of the Huperzia quadrifariata alkaloid extract to prevent cognitive deficits and histological damage caused by neonatal hypoxia-ischemia, probably by reducing cellular death and T cell mobilization.

Triphasic scaffolds for the regeneration of the bone-ligament interface.

A triphasic scaffold (TPS) for the regeneration of the bone-ligament interface was fabricated combining a 3D fiber deposited polycaprolactone structure and a polylactic co-glycolic acid electrospun. The scaffold presented a gradient of physical and mechanical properties which elicited different biological responses from human mesenchymal stem cells. Biological test were performed on the whole TPS and on scaffolds comprised of each single part of the TPS, considered as the controls. The TPS showed an increase of the metabolic activity with culturing time that seemed to be an average of the controls at each time point. The importance of differentiation media for bone and ligament regeneration was further investigated. Metabolic activity analysis on the different areas of the TPS showed a similar trend after 7 days in both differentiation media. Total alkaline phosphatase (ALP) activity analysis showed a statistically higher activity of the TPS in mineralization medium compared to the controls. A different glycosaminoglycans amount between the TPS and its controls was detected, displaying a similar trend with respect to ALP activity. Results clearly indicated that the integration of electrospinning and additive manufacturing represents a promising approach for the fabrication of scaffolds for the regeneration of tissue interfaces, such as the bone-to-ligament one, because it allows mimicking the structural environment combining different biomaterials at different scales.

Strong-interaction shifts and widths of kaonic helium isotopes.

The kaonic 3He and 4He [Formula: see text] transitions in gaseous targets were observed by the SIDDHARTA experiment. The X-ray energies of these transitions were measured with large-area silicon-drift detectors using the timing information of the [Formula: see text] pairs produced by the DAΦNE [Formula: see text] collider. The strong-interaction shifts and widths both of the kaonic 3He and 4He 2p states were determined, which are much smaller than the results obtained by the previous experiments. The "kaonic helium puzzle" (a discrepancy between theory and experiment) was now resolved.

Preliminary study of kaonic deuterium X-rays by the SIDDHARTA experiment at DAΦNE.

The study of the [Formula: see text] system at very low energies plays a key role for the understanding of the strong interaction between hadrons in the strangeness sector. At the DAΦNE electron-positron collider of Laboratori Nazionali di Frascati we studied kaonic atoms with [Formula: see text] and [Formula: see text], taking advantage of the low-energy charged kaons from Φ-mesons decaying nearly at rest. The SIDDHARTA experiment used X-ray spectroscopy of the kaonic atoms to determine the transition yields and the strong interaction induced shift and width of the lowest experimentally accessible level (1s for H and D and 2p for He). Shift and width are connected to the real and imaginary part of the scattering length. To disentangle the isospin dependent scattering lengths of the antikaon-nucleon interaction, measurements of [Formula: see text] and of [Formula: see text] are needed. We report here on an exploratory deuterium measurement, from which a limit for the yield of the K-series transitions was derived: [Formula: see text] and [Formula: see text] (CL 90%). Also, the upcoming SIDDHARTA-2 kaonic deuterium experiment is introduced.

Evidences that maternal swimming exercise improves antioxidant defenses and induces mitochondrial biogenesis in the brain of young Wistar rats.

Physical exercise during pregnancy has been considered beneficial to mother and child. Recent studies showed that maternal swimming improves memory in the offspring, increases hippocampal neurogenesis and levels of neurotrophic factors. The objective of this work was to investigate the effect of maternal swimming during pregnancy on redox status and mitochondrial parameters in brain structures from the offspring. Adult female Wistar rats were submitted to five swimming sessions (30 min/day) prior to mating with adult male Wistar rats, and then trained during the pregnancy (five sessions of 30-min swimming/week). The litter was sacrificed when 7 days old, when cerebellum, parietal cortex, hippocampus, and striatum were dissected. We evaluated the production of reactive species and antioxidant status, measuring the activities of superoxide-dismutase (SOD), catalase (CAT) and glutathione-peroxidase (GPx), as well as non-enzymatic antioxidants. We also investigated a potential mitochondrial biogenesis regarding mitochondrion mass and membrane potential, through cytometric approaches. Our results showed that maternal swimming exercise promoted an increase in reactive species levels in cerebellum, parietal cortex, and hippocampus, demonstrated by an increase in dichlorofluorescein oxidation. Mitochondrial superoxide was reduced in cerebellum and parietal cortex, while nitrite levels were increased in cerebellum, parietal cortex, hippocampus, and striatum. Antioxidant status was improved in cerebellum, parietal cortex, and hippocampus. SOD activity was increased in parietal cortex, and was not altered in the remaining brain structures. CAT and GPx activities, as well as non-enzymatic antioxidant potential, were increased in cerebellum, parietal cortex, and hippocampus of rats whose mothers were exercised. Finally, we observed an increased mitochondrial mass and membrane potential, suggesting mitochondriogenesis, in cerebellum and parietal cortex of pups subjected to maternal swimming. In conclusion, maternal swimming exercise induced neurometabolic programing in the offspring that could be of benefit to the rats against future cerebral insults.

Measurements of the strong-interaction widths of the kaonic (3)He and (4)He 2p levels.

The kaonic (3)He and (4)He X-rays emitted in the [Formula: see text] transitions were measured in the SIDDHARTA experiment. The widths of the kaonic (3)He and (4)He 2p states were determined to be [Formula: see text], and [Formula: see text], respectively. Both results are consistent with the theoretical predictions. The width of kaonic (4)He is much smaller than the value of [Formula: see text] determined by the experiments performed in the 70's and 80's, while the width of kaonic (3)He was determined for the first time.

First measurement of kaonic helium-3 X-rays.

The first observation of the kaonic (3)He 3d→2p transition was made, using slow K- mesons stopped in a gaseous (3)He target. The kaonic atom X-rays were detected with large-area silicon drift detectors using the timing information of the K+K- pairs of ϕ-meson decays produced by the DAΦNE e+e- collider. The strong interaction shift of the kaonic (3)He 2p state was determined to be -2±2(stat)±4(syst) eV.

The DRAGO gamma camera.

In this work, we present the results of the experimental characterization of the DRAGO (DRift detector Array-based Gamma camera for Oncology), a detection system developed for high-spatial resolution gamma-ray imaging. This camera is based on a monolithic array of 77 silicon drift detectors (SDDs), with a total active area of 6.7 cm(2), coupled to a single 5-mm-thick CsI(Tl) scintillator crystal. The use of an array of SDDs provides a high quantum efficiency for the detection of the scintillation light together with a very low electronics noise. A very compact detection module based on the use of integrated readout circuits was developed. The performances achieved in gamma-ray imaging using this camera are reported here. When imaging a 0.2 mm collimated (57)Co source (122 keV) over different points of the active area, a spatial resolution ranging from 0.25 to 0.5 mm was measured. The depth-of-interaction capability of the detector, thanks to the use of a Maximum Likelihood reconstruction algorithm, was also investigated by imaging a collimated beam tilted to an angle of 45 degrees with respect to the scintillator surface. Finally, the imager was characterized with in vivo measurements on mice, in a real preclinical environment.

Elemental mapping and microimaging by x-ray capillary optics.

Recently, many experiments have highlighted the advantage of using polycapillary optics for x-ray fluorescence studies. We have developed a special confocal scheme for micro x-ray fluorescence measurements that enables us to obtain not only elemental mapping of the sample but also simultaneously its own x-ray imaging. We have designed the prototype of a compact x-ray spectrometer characterized by a spatial resolution of less than 100 microm for fluorescence and less than 10 microm for imaging. A couple of polycapillary lenses in a confocal configuration together with a silicon drift detector allow elemental studies of extended samples (approximately 3 mm) to be performed, while a CCD camera makes it possible to record an image of the same samples with 6 microm spatial resolution, which is limited only by the pixel size of the camera. By inserting a compound refractive lens between the sample and the CCD camera, we hope to develop an x-ray microscope for more enlarged images of the samples under test.

[The palliative surgical treatment of paralysis of the hand]. / Il trattamento palliativo chirurgico delle paralisi della mano.

Restorative tendon transfer in upper limb palsy should be regarded as an important tool in hand surgery. An adequate planning and patient selection must be required, as well as several techniques can be used. Particularly, associated articular lesions must be detected and cured in the same time. Wrist extension palsies should be treated surgically with PT or LS transfer, whereas flexion palsies can be corrected with opportune balance in movements. Finger palsies can be treated with Zancolli's methods, including MP plasties and "lazos". Thumb palsies can be considered for extensor muscles transposition or reflexionplasties.

Determinants of fibrinogen in an Italian population suffering from claudication. Lower fibrinogen in the south compared to middle and north of Italy. The ADEP Group.

BACKGROUND AND OBJECTIVE: Prospective studies have shown that high plasma levels of fibrinogen are independently associated with the risk of cardiovascular complications. In patients suffering from peripheral vascular disease (PVD) fibrinogen has been shown to be an independent predictor of cardiovascular disease but its determinants have never been examined in this clinical setting. DESIGN AND METHODS: Fibrinogen levels were related to clinical and laboratory variables in 2,111 patients suffering from PVD. We also analyzed whether there was a regional distribution of risk factors. RESULTS: The median values of fibrinogen was 312 mg/dL. The clinical variables examined did not differentiate patients with elevated or normal fibrinogen levels. In particular, patients with ankle/arm pressure ratio < 0.8 did not show a higher prevalence of fibrinogen > 312 mg/dL. Conversely, white blood cell (WBC) count and serum cholesterol levels were significantly associated with high fibrinogen levels (p < 0.0001). Multiple logistic regression analysis demonstrated that areas of Italy were differently associated with high plasma fibrinogen levels (p < 0.03): subjects in the north and middle of Italy having significantly higher values of fibrinogen than subjects in the south of Italy (p < 0.01). A similar regional distribution was observed for WBC count and serum cholesterol levels. INTERPRETATION AND CONCLUSIONS: The regional distribution of risk factors raises the question as to whether the already reported large variability of cardiovascular events so in PVD may be attributed to a non homogeneous distribution of risk factors.

High-Resolution High-Count-Rate X-ray Spectroscopy with State-of-the-Art Silicon Detectors.

For the European X-ray multi-mirror (XMM) satellite mission and the German X-ray satellite ABRIXAS, fully depleted pn-CCDs have been fabricated, enabling high-speed low-noise position-resolving X-ray spectroscopy. The detector was designed and fabricated with a homogeneously sensitive area of 36 cm(2). At 150 K it has a noise of 4 e(-) r.m.s., with a readout time of the total focal plane array of 4 ms. The maximum count rate for single-photon counting was 10(5) counts s(-1) under flat-field conditions. In the integration mode more than 10(9) counts s(-1) can be detected at 6 keV. Its position resolution is of the order of 100 micro m. The quantum efficiency is higher than 90% from carbon K X-rays (277 eV) up to 10 keV. New cylindrical silicon drift detectors have been designed, fabricated and tested. They comprise an integrated on-chip amplifier system with continuous reset, on-chip voltage divider, electron accumulation layer stabilizer, large area, homogeneous radiation entrance window and a drain for surface-generated leakage current. At count rates as high as 2 x 10(6) counts cm(-2) s(-1), they still show excellent spectroscopic behaviour at room-temperature operation in single-photon detection mode. The energy resolution at room temperature is 220 eV at 6 keV X-ray energy and 140 eV at 253 K, being achieved with Peltier coolers. These systems were operated at synchrotron light sources (ESRF, HASYLAB and NLS) as X-ray fluorescence spectrometers in scanning electron microscopes and as ultra low noise photodiodes. The operation of a multi-channel silicon drift detector system is already foreseen at synchrotron light sources for X-ray holography experiments. All systems are fabricated in planar technology having the detector and amplifiers monolithically integrated on high-resistivity silicon.

Silicon drift detectors with on-chip electronics for x-ray spectroscopy.

The silicon drift detector (SDD) is a semiconductor device based on high resistivity silicon fully depleted through junctions implanted on both sides of the semiconductor wafer. The electrons generated by the ionizing radiation are driven by means of a suitable electric field from the point of interaction toward a collecting anode of small capacitance, independent of the active area of the detector. A suitably designed front-end JFET has been directly integrated on the detector chip close to the anode region, in order to obtain a nearly ideal capacitive matching between detector and transistor and to minimize the stray capacitances of the connections. This feature allows it to reach high energy resolution also at high count rates and near room temperature. The present work describes the structure and the performance of SDDs specially designed for high resolution spectroscopy with soft x rays at high detection rate. Experimental results of SDDs used in spectroscopy applications are also reported.

Effects of picotamide, an antiplatelet agent, on cardiovascular, events in 438 claudicant patients with diabetes: a retrospective analysis of the ADEP study.

Picotamide is an antiplatelet drug which inhibits thromboxane A2 (TxA2) synthase and antagonizes TxA2 receptors. In the ADEP (Atherosclerotic Disease Evolution by Picotamide) trial, 2304 patients with peripheral obstructive arterial disease (POAD) were studied in a double-blind, placebo-controlled, 18-month, multicentre trial. In this study, 151 events (13.1%) occurred on placebo and 122 (10.6%) on picotamide (900 mg day-1). The relative risk reduction was 19%, (P = 0.056). This paper reports a post-hoc analysis in a subgroup of 438 diabetic patients (picotamide = 230; placebo = 208). There were 32 vascular events on placebo (15%) and 18 on picotamide (8%) (relative risk reduction: 48%; 95% CI = 26, 76; P = 0.022). The results of this retrospective analysis suggest that a prospective study to investigate events in claudicant patients with diabetes mellitus is warranted.