COMPATIBILITY OF FLUOROCHROME LABELING PROTOCOL WITH RAMAN SPECTROSCOPY TO STUDY BONE FORMATION

Oral Communication presented at the ";Forum des Jeunes Chercheurs";, Brest (France) 2011

Dexamethasone in osteogenic medium strongly induces adipocyte differentiation of mouse bone marrow stromal cells and increases osteoblast differentiation

BACKGROUND: Osteoblasts and adipocytes share a common mesenchymal stem cell origin. Therefore, it has been suggested that the accumulation of marrow adipocytes observed in bone loss is caused by a shift in the commitment of mesenchymal stem cells from the osteogenic pathway to the adipogenic pathway. Supporting this hypothesis the competition between adipogenic and osteogenic lineages was widely demonstrated on partially homogeneous cell populations. However, some data from mouse models showed the existence of an independent relationship between bone mineral content and bone marrow adiposity. Therefore, the combination of adipogenesis and osteogenesis in primary culture would be helpful to determine if this competition would be observed on a whole bone marrow stromal cell population in a culture medium allowing both lineages. In this aim, mouse bone marrow stromal cells were cultured in a standard osteogenic medium added with different concentrations of Dexamethasone, known to be an important regulator of mesenchymal progenitor cell differentiation.RESULTS: Gene expression of osteoblast and adipocyte markers, biochemical and physical analyses demonstrated the presence of both cell types when Dexamethasone was used at 100 nM. Overall, our data showed that in this co-differentiation medium both differentiation lineages were enhanced compared to classical adipogenic or osteogenic culture medium. This suggests that in this model, adipocyte phenotype does not seem to increase at the expense of the osteoblast lineage.CONCLUSION: This model appears to be a promising tool to study osteoblast and adipocyte differentiation capabilities and the interactions between these two processes

Comparability of Raman Spectroscopic Configurations: A Large Scale Cross-Laboratory Study

This is the final version. Available on open access from the American Chemical Society via the DOI in this recordThe variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a 'primary' setup and the test data are generated on 'replicate' setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable. However, Raman spectra can be highly sensitive to the measurement conditions, and they change from setup to setup even if the same samples are measured. Although increasingly recognized as an issue, the dependence of the Raman spectra on the instrumental configuration is far from being fully understood and great effort is needed to address the resulting spectral variations and to correct for them. To make the severity of the situation clear, we present a round robin experiment investigating the comparability of 35 Raman spectroscopic devices with different configurations in 15 institutes within seven European countries from the COST (European Cooperation in Science and Technology) action Raman4clinics. The experiment was developed in a fashion that allows various instrumental configurations ranging from highly confocal setups to fibre-optic based systems with different excitation wavelengths. We illustrate the spectral variations caused by the instrumental configurations from the perspectives of peak shifts, intensity variations, peak widths, and noise levels. We conclude this contribution with recommendations that may help to improve the inter-laboratory studies.COST (European Cooperation in Science and Technology)Portuguese Foundation for Science and TechnologyNational Research Fund of Luxembourg (FNR)China Scholarship Council (CSC)BOKU Core Facilities Multiscale ImagingDeutsche Forschungsgemeinschaft (DFG, German Research Foundation

Bone molecular modifications induced by diagenesis followed-up for 12 months

After death, diagenesis takes place. Numerous processes occur concomitantly, which makes it dif-ficult to identify the diagenetic processes. The diagenetic processes refer to all processes (chemical or physical) that modify the skeletal remains. These processes are highly variable depending on the environmental factors (weather, temperature, age, sex, etc.), especially in the early stages. Numerous studies have evaluated bone diagenetic processes over long timescales (~millions of years), but fewer have been done over short timescales (between days and thousands of years). The objective of the study is to assess the early stages of diagenetic processes by Raman micro-spectroscopy over 12 months. The mineral and organic matrix modifications are monitored through physico-chemical parameters. Ribs from six humans were buried in soil. The modifica-tions of bone composition were followed by Raman spectroscopy each month. The decrease in the mineral/organic ratio and carbonate type-B content and the increase in crystallinity reveal that minerals undergo dissolution-recrystallization. The decrease in collagen cross-linking indicates that collagen hydrolysis induces the fragmentation of collagen fibres over 12 months.THIS DATASET IS ARCHIVED AT DANS/EASY, BUT NOT ACCESSIBLE HERE. TO VIEW A LIST OF FILES AND ACCESS THE FILES IN THIS DATASET CLICK ON THE DOI-LINK ABOV

Raman diagnostic of the reactivity between ZnSO₄ and CaCO₃ particles in humid air relevant to heterogeneous zinc chemistry in atmosphere

Laboratory experiments using Raman imaging demonstrated the behaviour of ZnSO4⋅7H2O (goslarite) microparticles in contact with a 1014 CaCO3 (calcite) surface under three different experimental conditions representative of remote atmosphere. Contact between the ZnSO4⋅7H2O particles and the CaCO3 surface in humid air (RH ~40-80%) did not induce any deliquescence and chemical phenomena. In contrast, condensation of a water drop at the ZnSO4⋅7H2O-CaCO3 interface caused free dissolution of the ZnSO4⋅7H2O particle and rapid precipitation of Zn4SO4(OH)6 onto the CaCO3 surface. This coating inhibited the surface reaction and subsequent drying resulted in the deposition of residual ZnSO4⋅7H2O, then ZnSO4⋅H2O (gunningite) and CaSO4⋅2H2O (gypsum) superimposed onto the Zn4SO4(OH)6 layer. The deposition of ZnSO4⋅7H2O particles in a water drop, previously in contact with a CaCO3 particle for a long time, resulted in the coprecipitation of Zn4SO4(OH)6 and Zn5(CO3)2(OH)6 (hydrozincite). Subsequent drying caused the deposition of residual ZnSO4⋅7H2O, ZnSO4⋅H2O and CaSO4⋅2H2O as small particles. These results indicated the possible fates of ZnSO4 particles in a humid atmosphere, when externally mixed with CaCO3 mineral dust after atmospheric events such as aggregation, water condensation and evaporation. This study indicated the fundamental role of water that typically existed on the surface of aerosol particles in the troposphere. These heterogeneous chemical processes have substantial consequences on particle size and solubility, and thus on bioavailability and toxicity of metal-rich particles

Dual-energy computed-tomography-based discrimination between basic calcium phosphate and calcium pyrophosphate crystal deposition in vivo.

Dual-energy computed tomography (DECT) is being considered as a non-invasive diagnostic and characterization tool in calcium crystal-associated arthropathies. Our objective was to assess the potential of DECT in distinguishing between basic calcium phosphate (BCP) and calcium pyrophosphate (CPP) crystal deposition in and around joints in vivo. A total of 13 patients with calcific periarthritis and 11 patients with crystal-proven CPPD were recruited prospectively to undergo DECT scans. Samples harvested from BCP and CPP calcification types were analyzed using Raman spectroscopy and validated against synthetic crystals. Regions of interest were placed in BCP and CPP calcifications, and the following DECT attenuation parameters were obtained: CT numbers (HU) at 80 and 140 kV, dual-energy index (DEI), electron density (Rho), and effective atomic number (Z <sub>eff</sub> ). These DECT attenuation parameters were compared and validated against crystal calibration phantoms at two known equal concentrations. Receiver operating characteristic (ROC) curves were plotted to determine the highest accuracy thresholds for DEI and Z <sub>eff</sub> . Raman spectroscopy enabled chemical fingerprinting of BCP and CPP crystals both in vitro and in vivo. DECT was able to distinguish between HA and CPP in crystal calibration phantoms at two known equal concentrations, most notably by DEI (200 mg/cm <sup>3</sup> : 0.037 ± 0 versus 0.034 ± 0, p = 0.008) and Z <sub>eff</sub> (200 mg /cm <sup>3</sup> : 9.4 ± 0 versus 9.3 ± 0, p = 0.01) analysis. Likewise, BCP calcifications had significantly higher DEI (0.041 ± 0.005 versus 0.034 ± 0.005, p = 0.008) and Z <sub>eff</sub> (9.5 ± 0.2 versus 9.3 ± 0.2, p = 0.03) than CPP crystal deposits with comparable CT numbers in patients. With an area under the ROC curve of 0.83 [best threshold value = 0.0 39, sensitivity = 90. 9% (81.8, 97. 7%), specificity = 64.6% (50.0, 64. 6%)], DEI was the best parameter in distinguishing between BCP and CPP crystal depositions. DECT can help distinguish between crystal-proven BCP and CPP calcification types in vivo and, thus, aid in the diagnosis of challenging clinical cases, and in the characterization of CPP and BCP crystal deposition occurring in osteoarthritis

Particle–Particle Chemistry between Micrometer-Sized PbSO₄ and CaCO₃ Particles in Turbulent Flow Initiated by Liquid Water

A mixture of natural and anthropogenic particles is ubiquitous in the troposphere and exerts an important influence on air quality. This work reports the study of mixing and heterogeneous chemistry of particles of natural-like mineral dust (CaCO₃) and anthropogenic-like microparticle (PbSO₄) in turbulent air flow under varying relative humidity. Sparse monolayers of laboratory-generated particles were collected on substrates using impaction. The grain size distribution and chemistry of micrometer-sized particles were determined as CaCO₃–PbSO₄ internal and external mixtures by Raman imaging, scanning electron microscopy, and time-of-flight static secondary ionization mass spectrometry. The condensation of a thin water layer on mixed aggregates initiates the formation of complex internal mixtures of Pb₃(CO₃)₂(OH)₂, PbCO₃, CaSO₄·2H₂O, CaCO₃, and PbSO₄ fine particles. These heterogeneous chemistry processes which may occur in ambient air can increase dramatically the amounts of hazardous breathable particles

Resolving the internal structure of individual atmospheric aerosol particle by the combination of Atomic Force Microscopy, ESEM-EDX, Raman and ToF-SIMS imaging

In this study, internal structures of individual aerosol particles were resolved by using micro-analytical techniques in combination. We demonstrated the practical applicability of the combined use of Atomic Force Microscopy (AFM), Environmental Scanning Electron Microscopy coupled with Energy-Dispersive X-ray analysis (ESEM-EDX), Raman Microspectrometry (RMS) and Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) to provide morphological, elemental, molecular and outer surface imaging of the same individual airborne particles for the first time. The characterization of single particles collected in the industrial atmosphere influenced by marine air masses demonstrated the physicochemical evolution of the particles in a short time period. The marine-derived particles were mainly encountered as genuine sea salts internally mixed with reacted sea salts such as NaNO3 and liquid NO3- which are covered by an organic thin layer. The particles collected downwind the industrial area were solid particles composed of an internal mixture of iron oxides and of marine-derived particles coated with an organic layer. The formation of these particles is a result of coalescence, agglomeration and drying processes occurring in the atmosphere during the transport of particles in a short time period (~15min). It is demonstrated that the combined use of the different types of spectral and imaging data from the same individual particles in atmospheric aerosol sample provides richer information on their physicochemical characteristics than when those techniques were used alone or when two techniques in combination

Dexamethasone in osteogenic medium strongly induces adipocyte differentiation of mouse bone marrow stromal cells and increases osteoblast differentiation

Background: Osteoblasts and adipocytes share a common mesenchymal stem cell origin. Therefore, it has been suggested that the accumulation of marrow adipocytes observed in bone loss is caused by a shift in the commitment of mesenchymal stem cells from the osteogenic pathway to the adipogenic pathway. Supporting this hypothesis the competition between adipogenic and osteogenic lineages was widely demonstrated on partially homogeneous cell populations. However, some data from mouse models showed the existence of an independent relationship between bone mineral content and bone marrow adiposity. Therefore, the combination of adipogenesis and osteogenesis in primary culture would be helpful to determine if this competition would be observed on a whole bone marrow stromal cell population in a culture medium allowing both lineages. In this aim, mouse bone marrow stromal cells were cultured in a standard osteogenic medium added with different concentrations of Dexamethasone, known to be an important regulator of mesenchymal progenitor cell differentiation. Results: Gene expression of osteoblast and adipocyte markers, biochemical and physical analyses demonstrated the presence of both cell types when Dexamethasone was used at 100 nM. Overall, our data showed that in this co-differentiation medium both differentiation lineages were enhanced compared to classical adipogenic or osteogenic culture medium. This suggests that in this model, adipocyte phenotype does not seem to increase at the expense of the osteoblast lineage. Conclusion: This model appears to be a promising tool to study osteoblast and adipocyte differentiation capabilities and the interactions between these two processes