Raman spectroscopy of osteosarcoma cells

Osteosarcoma is the most common primary malignant bone tumor. In the last years, several studies have demonstrated that the increase of Hydroxyapatite (HA) and Interleukin-6 (IL-6) syntheses compared to those expressed by normal osteoblasts could be used to detect the degree of malignancy of osteosarcoma cells. Conventional biochemical methods widely employed to evaluate bone cell differentiation, including normal and cancerous phenotypes, are time consuming and may require a large amount of cells. HA is a mineral form of calcium phosphate whose presence increases with maturation of osteosarcoma cells. Analogously, IL-6 is a fundamental cytokine whose production is highly increased in osteosarcoma cells. In this study, we employ Raman spectroscopy to the identification and discrimination of osteosarcoma cells from osteo-differentiated mesenchymal stromal cells (MSCs) by detecting the presence of HA and IL-6. However, while the identification of HA is facilitated by the characteristic peak at 960 cm-1, corresponding to symmetric stretching (P-O) mode, the quantification of IL-6 it is much more elusive, being its Raman signal characterized by cysteine, but also by phenylalanine, amide I II and III whose signals are common to other proteins. Supported by an accurate multivariate analysis, the results show that Raman spectroscopy is a high sensitivity technique dealing out a direct and quantitative measurement of specific mineralization levels of osteosarcoma cells. In turn, by exploiting the Surface-Enhanced Raman Scattering stimulated by internalized Gold Nanoshells (AuNSs) and combined with scanning probe microscopies, we were able to employ Raman spectroscopy to study subcellular components locally

Constitutive Expression of Pluripotency-Associated Genes in Mesodermal Progenitor Cells (MPCs)

Background: We recently characterized a progenitor of mesodermal lineage (MPCs) from the human bone marrow of adults or umbilical cord blood. These cells are progenitors able to differentiate toward mesenchymal, endothelial and cardiomyogenic lineages. Here we present an extensive molecular characterization of MPCs, from bone marrow samples, including 39 genes involved in stem cell machinery, differentiation and cell cycle regulation. Methodology/Principal Findings: MPCs are cytofluorimetrically characterized and quantitative RT-PCR was performed to evaluate the gene expression profile, comparing it with MSCs and hESCs lines. Immunofluorescence and dot-blot analysis confirm qRT-PCR data. MPCs exhibit an increased expression of OCT4, NANOG, SALL4, FBX15, SPP1 and to a lesser extent c-MYC and KLF4, but lack LIN28 and SOX2. MPCs highly express SOX15. Conclusions/Significance: MPCs express many pluripotency-associated genes and show a peculiar Oct-4 molecular circuit. Understanding this unique molecular mechanism could lead to identifying MPCs as feasible, long telomeres, target cells for reprogramming with no up-regulation of the p53 pathway. Furthermore MPCs are easily and inexpensively harvested fro

Polysaccharide hydrogels for multiscale 3D printing of pullulan scaffolds

Structurally and mechanically similar to the extracellular matrix (ECM), biomimetic hydrogels offer a number of opportunities in medical applications. However, the generation of synthetic microenvironments that simulate the effects of natural tissue niches on cell growth and differentiation requires new methods to control hydrogel feature resolution, biofunctionalization and mechanical properties. Here we show how these goals can be achieved by using a pullulan-based hydrogel, engineered in composition and server as cell-adhesive hydrogel, 3D photo-printable in dimension, ranging from the macro- to the micro-scale dimensions, and of tunable mechanical properties. For this, we used absorbers that limit light penetration, achieving 3D patterning through stereolithography with feature vertical resolution of 200 μm and with overall dimension up to several millimeters. Furthermore, we report the fabrication of 3D pullulan-modified hydrogels by two-photon lithography, with sub-millimetric dimensions and minimum feature sizes down to some microns. These materials open the possibility to produce multiscale printed scaffolds that here we demonstrate to be inert for cell adhesion, but biologically compatible and easily functionalizable with cell adhesive proteins. Under these conditions, successful cell cultures were established in 2D and 3D. Keywords: Hydrogel, Biomaterials, Polysaccharide, Pullulan, 3D printing, Two photon laser lithography, Mesenchymal stromal cell

Design, fabrication and characterization of composite piezoelectric ultrafine fibers for cochlear stimulation

Sensorineural hearing loss, primed by dysfunction or death of hair cells in the cochlea, is the main cause of severe or profound deafness. Piezoelectric materials work similarly to hair cells, namely, as mechano-electrical transducers. Polyvinylidene fluoride (PVDF) films have demonstrated potential to replace the hair cell function, but the obtained piezoresponse was insufficient to stimulate effectively the auditory neurons. In this study, we reported on piezoelectric nanocomposites based on ultrafine PVDF fibers and barium titanate nanoparticles (BTNPs), as a strategy to improve the PVDF performance for this application. BTNP/PVDF fiber meshes were produced via rotating-disk electrospinning, up to 20/80 weight composition. The BTNP/PVDF fibers showed diameters ranging in 0.160-1.325 μm. Increasing collector velocity to 3000 rpm improved fiber alignment. The piezoelectric β phase of PVDF was well expressed following fabrication and the piezoelectric coefficients increased according to the BTNP weight ratio. The BTNP/PVDF fibers were not cytotoxic towards cochlear epithelial cells. Neural-like cells adhered to the composite fibers and, upon mechanical stimulation, showed enhanced viability. Using BTNP filler for PVDF matrices, in the form of aligned ultrafine fibers, increased the piezoresponse of PVDF transducers and favored neural cell contact. Piezoelectric nanostructured composites might find application in next generation cochlear implants

Development of tissue-engineered constructs for ossicular chain replacement

Ossicular chain (OC) is the bony part of middle ear dedicated to sound transmission. Chronic inflammations, infections and traumas occurring in the lifespan result in a set of severe diseases known as “conductive hearing loss”. To recover an acceptable hearing threshold, the damaged OC have to be surgically replaced with artificial prostheses. However, despite many efforts aimed at fabricating optimal replacements, all the synthetic prostheses are subject to extrusion, i.e., a type of rejection due to lack of biointegration. For these reasons, it is necessary to envision novel strategies for the OC substitution. In recent years, we have proposed an approach for OC reconstruction based on tissue engineering (TE), in which mesenchymal stromal cells (MSCs) are cultured under osteogenic differentiation regimen on bioresorbable 3D scaffolds up to obtain new bone substitutes with appropriate shape and dimensions (Danti et al., 2009; Danti et al., 2010). In this study, human MSCs were osteo-differentiated on different types of OC scaffolds fabricated in our laboratories. TE constructs were analyzed via biochemical assays, molecular biology and histo-morphological methods. An extensive analysis on native ossicles was performed to compare the results obtained in the constructs with the mature tissues. The results showed that the cells were viable, colonized the scaffolds and produced extracellular matrix molecules at intra- and extra-cellular level. MSC differentiation towards the osteogenic lineage was demonstrated by the production of mineralized matrix and specific osteogenic markers. Moreover, we assessed that all the investigated molecules were also expressed in the native tissues, even if at different expression levels, indicating that it was obtained a preliminary step for the creation of TE constructs to be employed, in perspective, as OC substitutes in the otologic surgery

Assessing climate change impacts on crops by adopting a set of crop performance indicators

AbstractThe impact of climate change on the agricultural systems of three major islands in the Mediterranean basin, namely Sicily, Crete and Cyprus, was evaluated using a suite of specifically calibrated crop models and the outputs of a regional circulation model for Representative Concentration Pathway (RCP) 4.5 and 8.5 downscaled to 12 km of resolution and tested for its effectiveness in reproducing the local meteorological data. The most important annual (wheat, barley, tomato and potato) and perennial (grapevine and olive tree) crops were selected to represent the agricultural systems of the islands. The same modelling framework was used to test the effectiveness of autonomous adaptation options, such as shifting sowing date and the use of varieties with different growing season length. The results highlighted that, on average, warmer temperatures advanced both anthesis and maturity of the selected crops, but at different magnitudes depending on the crop and the island. Winter crops (barley, wheat and potato) experienced the lowest impact in terms of yield loss with respect to the baseline, with even some positive effects, especially in Sicily where both wheat and barley showed a general increase of 9% as compared to the baseline, while potato increased up to + 17%. Amongst perennial crops, olive tree showed low variation under RCP 4.5, but on average increased by 7% under RCP 8.5 on the three islands. Climate change had a detrimental effect specifically on tomato (− 2% on average in RCP 8.5 and 4.5 on the three islands) and grapevine (− 7%). The use of different sowing dates, or different varieties, revealed that for winter crops early autumn sowing is still the best option for producing wheat and barley in future periods on the three islands under both future scenarios. For tomato and potato, advancing sowing date to early winter is a winning strategy that may even increase final yield (+ 9% for tomato and + 17% for potato, on average). For grapevine, the use of late varieties, while suffering the most from increasing temperatures and reduced rainfall (− 15%, on average), is still a valuable option to keep high yield levels with respect to earlier varieties, which even if showing some increases with respect to the baseline have a generally much lower production level. The same may be applied to olive tree although the production differences between late and early varieties are less evident and climate change exerts a favourable influence (+ 4 and + 3% for early and late varieties, respectively)

In vitro study on the generation of tympanic membrane substitutes via tissue engineering

The tympanic membrane (TM) is an anatomical structure with unique histological and physiological features playing a fundamental role in sound transmission. In particular, the middle layer of the pars tensa, which represents the widest and thickest surface portion of the TM, consists of connective tissue mainly composed of collagen types II and III fibers, while collagen type I is present at a lesser extent [1]. Several pathologies affect the TM, including otitis media, tympanosclerosis, cholesteatoma and perforation that require reconstructive surgery depending on the lesion extent [2]. To this purpose, the temporalis fascia is currently considered as the gold standard material. However, due to limited graft availability, fully synthetic substitutes are also applied, with poorly satisfactory outcomes. For these reasons new strategies for TM replacement are still needed. In this study, we employed a tissue engineering (TE) approach for the regeneration of TM substitutes selecting some biocompatible and bioresorbable polymeric matrices to be cultured with human bone marrow-derived mesenchymal stem cells (MSCs). We set up a cell differentiation protocol using an appropriate mix of growing factors to obtain the in vitro differentiation of MSCs into TM fibroblasts. Furthermore, because of the role played by mechanical forces in TM motion, these engineered substitutes underwent mechanical stress during the culture. The obtained biohybrid constructs were characterized about cellular viability assays, gene expression quantification as well as histochemical and immunohistochemical analyses. Moreover, native TMs from cadavers were investigated for assessment and optimization of the engineered constructs. Our results showed that MSCs were able to grow and differentiate properly on the selected biomaterials and to synthesize appropriate extracellular matrix molecules. Moreover, the applied mechanical forces seem to promote TM-fibroblastic differentiation, increasing the production of collagen type II, that is a peculiarity of TM structure

Mesodermal Progenitor Cells (MPCs) Differentiate into Mesenchymal Stromal Cells (MSCs) by Activation of Wnt5/Calmodulin Signalling Pathway

Mesenchymal Stromal Cells (MSCs) remain poorly characterized because of the absence of manifest physical, phenotypic, and functional properties in cultured cell populations. Despite considerable research on MSCs and their clinical application, the biology of these cells is not fully clarified and data on signalling activation during mesenchymal differentiation and proliferation are controversial. The role of Wnt pathways is still debated, partly due to culture heterogeneity and methodological inconsistencies. Recently, we described a new bone marrow cell population isolated from MSC cultures that we named Mesodermal Progenitor Cells (MPCs) for their mesenchymal and endothelial differentiation potential. An optimized culture method allowed the isolation from human adult bone marrow of a highly pure population of MPCs (more than 97%), that showed the distinctive SSEA-4+CD105+CD90(neg) phenotype and not expressing MSCA-1 antigen. Under these selective culture conditions the percentage of MSCs (SSEA-4(neg)CD105+CD90(bright) and MSCA-1+), in the primary cultures, resulted lower than 2%.We demonstrate that MPCs differentiate to MSCs through an SSEA-4+CD105+CD90(bright) early intermediate precursor. Differentiation paralleled the activation of Wnt5/Calmodulin signalling by autocrine/paracrine intense secretion of Wnt5a and Wnt5b (p<0.05 vs uncondictioned media), which was later silenced in late MSCs (SSEA-4(neg)). We found the inhibition of this pathway by calmidazolium chloride specifically blocked mesenchymal induction (ID₅₀ =  0.5 µM, p<0.01), while endothelial differentiation was unaffected.The present study describes two different putative progenitors (early and late MSCs) that, together with already described MPCs, could be co-isolated and expanded in different percentages depending on the culture conditions. These results suggest that some modifications to the widely accepted MSC nomenclature are required

Comparison of site sensitivity of crop models using spatially variable field data from Precision Agriculture

Site conditions and soil properties have a strong influence on impacts of climate change on crop production. Vulnerability of crop production to changing climate conditions is highly determined by the ability of the site to buffer periods of adverse climatic situations like water scarcity or excessive rainfall.  Therefore, the capability of models to reflect crop responses and water and nutrient dynamics under different site conditions is essential to assess climate impact even on a regional scale. To test and improve sensitivity of models to various site properties such as soil variability and hydrological boundary conditions, spatial variable data sets from precision farming of two fields in Germany and Italy were provided to modellers. For the German 20 ha field soil and management data for 60 grid points for 3 years (2 years wheat, 1 year triticale) were provided. For the Italian field (12 ha) information for 100 grid points were available for three growing seasons of durum wheat. Modellers were asked to run their models using a) the model specific procedure to estimate soil hydraulic properties from texture using their standard procedure and use in step b) fixed values for field capacity and wilting point derived from soil taxonomy. Only the phenology and crop yield of one grid point provided for a basic calibration. In step c) information for all grid points of the first year (yield, soil water and mineral N content for Germany, yield, biomass and LAI for Italy) were provided. First results of five out of twelve participating models are compared against measured state variables analysing their site specific response and consistency across crop and soil variables.(Main text to be published in a peer-reviewed journal

Comparison of site sensitivity of crop models using spatially variable field data from Precision Agriculture

Site conditions and soil properties have a strong influence on impacts of climate change on crop production. Vulnerability of crop production to changing climate conditions is highly determined by the ability of the site to buffer periods of adverse climatic situations like water scarcity or excessive rainfall.  Therefore, the capability of models to reflect crop responses and water and nutrient dynamics under different site conditions is essential to assess climate impact even on a regional scale. To test and improve sensitivity of models to various site properties such as soil variability and hydrological boundary conditions, spatial variable data sets from precision farming of two fields in Germany and Italy were provided to modellers. For the German 20 ha field soil and management data for 60 grid points for 3 years (2 years wheat, 1 year triticale) were provided. For the Italian field (12 ha) information for 100 grid points were available for three growing seasons of durum wheat. Modellers were asked to run their models using a) the model specific procedure to estimate soil hydraulic properties from texture using their standard procedure and use in step b) fixed values for field capacity and wilting point derived from soil taxonomy. Only the phenology and crop yield of one grid point provided for a basic calibration. In step c) information for all grid points of the first year (yield, soil water and mineral N content for Germany, yield, biomass and LAI for Italy) were provided. First results of five out of twelve participating models are compared against measured state variables analysing their site specific response and consistency across crop and soil variables.(Main text to be published in a peer-reviewed journal