Confocal Raman Microspectroscopy : Applications in Cartilage Tissue Engineering

Weefseltechniek, ofwel ‘tissue engineering’, van kraakbeen behelst het gebruik van driedimensionale dragermaterialen (‘scaffolds’) in combinatie met primaire chondrocyten of gedifferentieerde mesenchymale stamcellen om een bio-artificieel construct te creëren voor klinische toepassing. Over het algemeen worden hiervoor, omwille van hun gunstige biomechanische eigenschappen, polymeer ‘scaffolds’ gebruikt die zodanig zijn ontworpen dat zij de proliferatie en differentiatie van chondrocyten en/of progenitorcellen ondersteunen, de productie van extracellulaire matrix stimuleren en – wanneer ze in vivo worden toegepast – kraakbeenherstel bevorderen. Studies naar de compositie en kwaliteit van ‘tissue-engineered’ kraakbeen maken over het algemeen gebruik van destructieve methoden als immuunhistochemie, biochemie en elektronenmicroscopie. Niet-invasieve methoden die het mogelijk maken om op een niet-destructieve manier het gedrag van cellen in combinatie met biomaterialen in de tijd te volgen kunnen nieuwe inzichten geven in een breed spectrum van nutriënten, extracellulaire matrix en cellulaire componenten, alsmede intracellulaire veranderingen: stuk voor stuk belangrijke parameters voor het creëren van een bio-artificieel kraakbeen construct. Deze thesis heeft als doel relevante parameters in kraakbeen ‘tissue engineering’ te bestuderen met behulp van niet-destructieve biochemische hoge resolutie microscopie: confocale Raman microspectroscopie. Dit is een label-vrije microscopische techniek voor de analyse en visualisatie van de biochemische compositie van individuele cellen en de omliggende extracellulaire matrix in weefsels, celkweken en ‘tissue-engineered’ constructen

Oxidation and Release of Ruthenium from White Inclusions

In this paper the laboratory test results on oxidation and release of ruthenium as a fission product element are summarised. The ruthenium appears in the nuclear fuel pellets of pressurized water reactors as one of the fission product elements during burnup. In case of severe accident when the air can contact the degraded hot fuel, the ruthenium oxidises and its gaseous oxides, especially the RuO4, release rapidly from the pellets to the environment. Because of high radio- and chemotoxicity of ruthenium tetra-oxide further experimental study of oxidation and release is essential. It is well known that ruthenium in the irradiated fuel UO2 fuel appears in small metallic alloy precipitations together with fission product elements as Mo, Rh, Pd and Tc. The precipitations are seen in the metallographic pictures as white inclusions. This separate effect study focused on the differences in the release rate of gaseous ruthenium oxides when pure ruthenium or Mo-Ru-Rh-Pd metallic alloy is present in the simulated nuclear fuel. The oxidation and release were studied at constant reaction temperatures of 1000 or 1100 Celsius. The tests showed that during high-temperature oxidation of the Mo-Ru-Rh-Pd alloy in air flow the release rate of gaseous ruthenium oxides is reduced to 60-80% compared to the value measured in case of oxidation of pure metallic ruthenium powder in the same thermal-hydraulic conditions. Furthermore, if additional elements and chemical compounds representing other fission products were added in the alloy, a time delay of 30 to 60 min appeared in the release of gaseous ruthenium to the room-temperature environment. One of the main results was that in the outlet air flow reaching the environment the partial pressure of RuO4 was far above what could be expected for room-temperature equilibrium conditions. It was pointed out that the highly volatile RuO4 can decompose in solid, non-volatile RuO2 and O2. The X-ray fluorescence analysis results showed that some ruthenium compounds deposited on the colder circuit walls of the test facility. This suggests RuO4 is not fully airstable, i.e., its stability in air can be limited in time.JRC.F.4-Nuclear design safet

Oxidation and Release of Ruthenium from Short Fuel Rods above 1500oC

The fission product ruthenium is radiotoxic and in oxidized form as ruthenium-tetroxide (RuO4) also chemical toxic. During fuel element change or in case of a leakage in fuel storages, air flows in the containment or fuel storage. If the circulation pump fails, the fuel elements are heated-up, and at high temperatures gaseous ruthenium oxides are formed and rapidly released from the fuel. Due to the significant higher volatility of RuO4 in the Chernobyl Catastrophe the measured concentration of the Ru-isotopes in the fall-outs was comparable to those of iodine and caesium. In order to get more insight in the chemical and physical behaviour of this fission product under severe accident conditions, the Ruthenium release was studied in a series of Separate Effect Tests (RUSET). In the test short fuel rod segments were used and exposed to air and steam atmosphere at high temperatures. The experiments in air atmosphere showed, that the partial pressures of the released Ru-oxides was two orders of magnitudes lower compared to those measured in previous tests with Ru-powder diluted in a ZrO2 matrix. It was found that ruthenium was not released in steam atmosphere in the examined temperature range. Furthermore, the temperature dependence of the deposition was detected by XRF analysis.JRC.F.4-Nuclear design safet

Raman microspectroscopy: A non-invasive analysis tool for monitoring of collagen-containing extracellular matrix formation in a medium-throughput culture system

The three-dimensional environment is known to play an important role in promoting cell–matrix interactions. We have investigated the possibility of using Raman microspectroscopy—which has the great advantage of noninvasive sensing—for in vitro monitoring of extracellular matrix (ECM) formation in a medium-throughput pellet (3D) culture system with soft-litography, agarose-microwell arrays. Chondrocytes were seeded in the agarose microwells in basic or chondrocyte medium. After 3, 7, and 14 days of culture, samples were analyzed for ECM formation by Raman microspectroscopy, histology, and immunofluorescence. ECM formation in the chondrocyte medium-cultured samples was detected by histology and immunofluorescence, and also noninvasively by Raman microspectroscopy. The Raman band of collagen found at 937 cm−1 can be used as a Raman marker for collagen-containing ECM formation over time in the chondrocyte pellets. This culture system can be implemented as a medium-throughput platform for Raman applications and screening microtissue formation, since with these agarose-microwell arrays relatively large numbers of cell pellets could be screened in a short time in situ, without having to transfer the pellets onto microscopic slides. Moreover, in this manner the culture system is suitable for long-term, real-time live-cell measurements

Experimental investigation of the late phase of spent fuel pool accidents

Experimental programmes have been carried out in order to investigate the behaviour of nuclear fuel components in high-temperature air atmosphere, which characterises the main conditions of the late phase of spent fuel pool accidents. The tests provided new data on the oxidation of zirconium cladding in different atmospheres, on the oxidation and release of ruthenium from fuel pellets and on the integral behaviour of fuel bundles. The integral test confirmed that water injection into the spent fuel storage pool is the right measure to terminate a severe accident

A zinc oxide-modified hydroxyapatite-based cement facilitated new crystalline-stoichiometric and amorphous apatite precipitation on dentine.

Aim: To evaluate the remineralization ability of two dentin canal sealer cements. Methodology: Dentin surfaces were subjected to: i) 37% phosphoric acid (PA) or ii) 0.5 M ethylenediaminetetraacetic acid (EDTA) conditioning prior to the application of two experimental hydroxyapatite-based cements, containing sodium hydroxide (calcypatite) or zinc oxide (oxipatite), respectively. Samples were stored in simulated body fluid during 24 h or 21 d. Remineralization of the dentin surfaces were studied by Raman spectroscopy, mapping with K-means cluster and hierarchical cluster analysis were done. Nano-roughness and collagen fibrils width measurements were performed by means of an atomic force microscopy. Results: PA+oxipatite promoted both the highest dentin mineralization and crystallographic maturity at the dentin surface. Non-crystalline amorphous-like apatites were also formed. Dentin treated with PA+calcypatite attained the roughest surface with minimal fibril width. Crosslinking of collagen only raised in the group PA+oxipatite, after 21 d. The maximum relative mineral concentration and structure of collagen referred to amide I and ratio amide III/AGEs was achieved after using PA+calcypatite at 21 d time point. EDTA produced a lower stoichiometric hydroxyapatite with decreased maturity, at the expense of the carbonate band widening, though it favored the nucleation of carbonated calcium phosphate. Conclusions: Surfaces treated with PA+oxipatite attained the highest dentin remineralization with both crystalline-stoichiometric and amorphous apatites, at long term. EDTA conditioning facilitated amorphous-bulk mineral precipitation. This amorphization, more intense after using oxipatite, provided an ion-rich environment favoring in situ dentin remineralization.This work was supported by the Ministry of Economy and Competitiveness (MINECO) [Project MAT2014-52036-P] and European Regional Development Fund (FEDER)

Ions-modified nanoparticles affect functional remineralization and energy dissipation through the resin-dentin interface

The aim of this study was to evaluate changes in the mechanical and chemical behavior, and bonding ability at dentin interfaces infiltrated with polymeric nanoparticles (NPs) prior to resin application. Dentin surfaces were treated with 37% phosphoric acid followed by application of an ethanol suspension of NPs, Zn-NPs or Ca-NPs followed by the application of an adhesive, Single Bond (SB). Bonded interfaces were stored for 24 h, submitted to microtensile bond strength test, and evaluated by scanning electron microscopy. After 24 h and 21 d of storage, the whole resin-dentin interface adhesive was evaluated using a Nano-DMA. Complex modulus, storage modulus and tan delta (δ) were assessed. AFM imaging and Raman analysis were performed. Bond strength was not affected by NPs infiltration. After 21 d of storage, tan δ generally decreased at Zn-NPs/resin-dentin interface, and augmented when Ca-NPs or non-doped NPs were used. When both Zn-NPs and Ca-NPs were employed, the storage modulus and complex modulus decreased, though both moduli increased at the adhesive and at peritubular dentin after Zn-NPs infiltration. The phosphate and the carbonate peaks, and carbonate substitution, augmented more at interfaces promoted with Ca-NPs than with Zn-NPs after 21 d of storage, but crystallinity did not differ at created interfaces with both ions-doped NPs. Crosslinking of collagen and the secondary structure of collagen improved with Zn-NPs resin-dentin infiltration. Ca-NPs-resin dentin infiltration produced a favorable dissipation of energy with minimal stress concentration trough the crystalline remineralized resin-dentin interface, causing minor damage at this structure.This work was supported by the Ministry of Economy and Competitiveness (MINECO) [Project MAT2014-52036-P]

Raman Spectroscopy and Regenerative Medicine: A Review

The field of regenerative medicine spans a wide area of the biomedical landscape—from single cell culture in laboratories to human whole-organ transplantation. To ensure that research is transferrable from bench to bedside, it is critical that we are able to assess regenerative processes in cells, tissues, organs and patients at a biochemical level. Regeneration relies on a large number of biological factors, which can be perturbed using conventional bioanalytical techniques. A versatile, non-invasive, non-destructive technique for biochemical analysis would be invaluable for the study of regeneration; and Raman spectroscopy is a potential solution. Raman spectroscopy is an analytical method by which chemical data are obtained through the inelastic scattering of light. Since its discovery in the 1920s, physicists and chemists have used Raman scattering to investigate the chemical composition of a vast range of both liquid and solid materials. However, only in the last two decades has this form of spectroscopy been employed in biomedical research. Particularly relevant to regenerative medicine are recent studies illustrating its ability to characterise and discriminate between healthy and disease states in cells, tissue biopsies and in patients. This review will briefly outline the principles behind Raman spectroscopy and its variants, describe key examples of its applications to biomedicine, and consider areas of regenerative medicine that would benefit from this non-invasive bioanalytical tool

First aid competition on secondary medical school in Havlíčkův Brod

Bakalářská práce se zabývá tématem "Soutěž první pomoci na Střední zdravotnické škole v Havlíčkově Brodě". Práce se skládá z teoretické a praktické části. V teoretické části jsou sepsány některé metody výuky, základní informace o First Aid Rescue Group-záchranné, definice první pomoci a některé postupy. Praktická část se zabývá soutěží v první pomoci a rozborem hodnotících tabulek.Bachelor thesis is dedicated to topic " First aid competition on secondary medical school in Havlíčkův Brod". The thesis consists of theoretical and practical parts. In the theoretical part are written some teaching methods, basic information about First Aid Rescue Group-rescue, definition and first aid guidelines. Practical part deals with first aid competition and by analyzing the scoreboards.Fakulta zdravotnických studiíHodnocení vedoucího: C Hodnocení oponenta: D Doplňující otázky k obhajobě: 1. Proč jste do souhrnu výsledků uvedl pouze deset družstev a ne dvacet? 2. Jak budete dál pracovat s výsledky, kterých jste dosáhl? Obhajoba bakalářské práce s prezentací velmi dobrá.Dokončená práce s úspěšnou obhajobo