Shedding Light on Osteosarcoma Cell Differentiation: Impact on Biomineralization and Mitochondria Morphology.

Osteosarcoma (OS) is the most common primary malignant bone tumor and its etiology has recently been associated with osteogenic differentiation dysfunctions. OS cells keep a capacity for uncontrolled proliferation showing a phenotype similar to undifferentiated osteoprogenitors with abnormal biomineralization. Within this context, both conventional and X-ray synchrotron-based techniques have been exploited to deeply characterize the genesis and evolution of mineral depositions in a human OS cell line (SaOS-2) exposed to an osteogenic cocktail for 4 and 10 days. A partial restoration of the physiological biomineralization, culminating with the formation of hydroxyapatite, was observed at 10 days after treatment together with a mitochondria-driven mechanism for calcium transportation within the cell. Interestingly, during differentiation, mitochondria showed a change in morphology from elongated to rounded, indicating a metabolic reprogramming of OS cells possibly linked to an increase in glycolysis contribution to energy metabolism. These findings add a dowel to the genesis of OS giving new insights on the development of therapeutic strategies able to restore the physiological mineralization in OS cells.

Near-infrared hyperspectral imaging to map collagen content in prehistoric bones for radiocarbon dating.

Many of the rarest prehistoric bones found by archaeologists are enormously precious and are considered to be part of our cultural and historical patrimony. Radiocarbon dating is a well-established technique that estimates the ages of bones by analysing the collagen still present. However, this method is destructive, and its use must be limited. In this study, we used imaging technology to quantify the presence of collagen in bone samples in a non-destructive way to select the most suitable samples (or sample regions) to be submitted to radiocarbon dating analysis. Near-infrared spectroscopy (NIR) that was connected to a camera with hyperspectral imaging (HSI) was used along with a chemometric model to create chemical images of the distribution of collagen in ancient bones. This model quantifies the collagen at every pixel and thus provides a chemical mapping of collagen content. Our results will offer significant advances for the study of human evolution as we will be able to minimise the destruction of valuable bone material, which is under the protection and enhancement of European cultural heritage and thus allow us to contextualise the valuable object by providing an accurate calendar age.

Towards the non-destructive analysis of multilayered samples: A novel XRF-VNIR-SWIR hyperspectral imaging system combined with multiblock data processing.

The new challenge in the investigation of cultural heritage is the possibility to obtain stratigraphical information about the distribution of the different organic and inorganic components without sampling. In this paper recently commercialized analytical set-up, which is able to co-register VNIR, SWIR, and XRF spectral data simultaneously, is exploited in combination with an innovative multivariate and multiblock high-throughput data processing for the analysis of multilayered paintings. The instrument allows to obtain elemental and molecular information from superficial to subsurface layers across the investigated area. The chemometric strategy proved to be highly efficient in data reduction and for the extraction and integration of the most useful information coming from the three different spectroscopies, also filling the gap between data acquisition and data understanding through the combination of principal component analysis (PCA), brushing, correlation diagrams and maps (within and between spectral blocks) on the low-level fused. In particular, correlation diagrams and maps provide useful information for the reconstruction of a stratigraphic structure without the need to take any sample, thanks to the effective account for inter-correlation among data (variables), which is able to effectively characterize the possible combinations of components located in the same depth level. The highly innovative technology and the data processing strategy are applied for the multi-level characterization of a complex painting reproduction as an illustrative pilot study.

A non-invasive diagnostic tool for cellulose acetate films using a portable miniaturized near infrared spectrometer.

This article tests the suitability of a new method to monitor the degree of substitution of cellulose acetate films, by employing a compact and inexpensive near-infrared miniaturized spectrometer (908.1-1676.2 nm) that can be easily applied in situ. The present study compares the analytical performance of the proposed method against conventional diagnostic strategies based on benchtop micro-attenuated total reflectance Fourier transform Infrared (µATR -FTIR) measurements in the mid-infrared spectral range. The novel calibration function exploits the shifts in the first overtone of the hydroxyl stretching 2νOH band of probe materials and was created using a set of analytical standards with different degrees of substitution. The robustness of the method was assessed by application on a group of sixteen historical cinematographic films. The accurate condition assessment of these films was performed in situ, in a non-invasive manner. The proposed analytical procedure is quick and easy-to-implement, and therefore it constitutes a rapid method to guide conservation strategies regarding film storage and digitalization in cultural institutions, including museums and cinematheques. Potential applications on three-dimensional objects and industrial processes are possible.

Multiscale Femoral Neck Imaging and Multimodal Trabeculae Quality Characterization in an Osteoporotic Bone Sample.

Although multiple structural, mechanical, and molecular factors are definitely involved in osteoporosis, the assessment of subregional bone mineral density remains the most commonly used diagnostic index. In this study, we characterized bone quality in the femoral neck of one osteoporotic patients as compared to an age-matched control subject, and so used a multiscale and multimodal approach including X-ray computed microtomography at different spatial resolutions (pixel size: 51.0, 4.95 and 0.9 µm), microindentation and Fourier transform infrared spectroscopy. Our results showed abnormalities in the osteocytes lacunae volume (358.08 ± 165.00 for the osteoporotic sample vs. 287.10 ± 160.00 for the control), whereas a statistical difference was found neither for shape nor for density. The osteoporotic femoral head and great trochanter reported reduced elastic modulus (Es) and hardness (H) compared to the control reference (−48% (p < 0.0001) and −34% (p < 0.0001), respectively for Es and H in the femoral head and −29% (p < 0.01) and −22% (p < 0.05), respectively for Es and H in the great trochanter), whereas the corresponding values in the femoral neck were in the same range. The spectral analysis could distinguish neither subregional differences in the osteoporotic sample nor between the osteoporotic and healthy samples. Although, infrared spectroscopic measurements were comparable among subregions, and so regardless of the bone osteoporotic status, the trabecular mechanical properties were comparable only in the femoral neck. These results illustrate that bone remodeling in osteoporosis is a non-uniform process with different rates in different bone anatomical regions, hence showing the interest of a clear analysis of the bone microarchitecture in the case of patients' osteoporotic evaluation.

A new miniaturised short-wave infrared (SWIR) spectrometer for on-site cultural heritage investigations.

The paper reports on the development of an analytical method based on the use of a new miniaturised short-wave infrared (SWIR) spectrometer for the analysis of cultural heritage samples. The spectrometer is a prototype characterised by small dimension (45.0 mm in diameter x 47.5 mm in height x 60 g weight), easily handled and transferable out of research laboratories. The prototype enables the acquisition of spectra in the SWIR range of 1200-2200 nm, which is a unique feature for miniaturised spectrometers. The exploitation of this spectral range allows the detection of a high number of combination and overtone bands, which guarantees significant diagnostic power to the instrument. The present study lays a significant foundation to the development of analytical strategies based on miniaturised NIR spectrometers working in the SWIR spectral range for the characterization of complex samples such as cultural heritage specimens. Analytical performances of the new spectrometer were assessed on archaeological bones, cinematographic films and bronze patinas. The selected cases of study present challenging conservation issues not properly addressed, and their analyses usually require to be performed on-site, in places not easily accessible by restorers, archaeologists and/or scientists. The data acquired with the prototype, combined with a multivariate data analysis approach, show the possibility to i) differentiate between the materials used as a support for cinematographic film namely cellulose nitrate (CN), cellulose acetates (CA) and polyethylene terephthalate (PET); ii) sort out archaeological bone fragments according to their collagen content as an initial screening test for bones characterization; iii) differentiate between corrosion products on outdoor bronze sculpture, which is important for assessing the state of conservation of the artwork. The prototype enabled rapid information acquisition to guide restoration strategies, which need to be supported in real time by quick and easy analytical procedures.

Macroscopic mid-FTIR mapping and clustering-based automated data-reduction: An advanced diagnostic tool for in situ investigations of artworks.

The present study describes a multivariate strategy that can be used for automatic on-site processing of reflection mode macro FTIR mapping (MA-rFTIR) data obtained during investigation of artworks. The chemometric strategy is based on the integration of principal component analysis (PCA) with a clustering approach in the space subtended by the three lowest-order principal components and allows to automatically identify the regions of interest (ROIs) of the area scanned and to extract the average FTIR spectra related to each ROI. Thanks to the automatic data management, in-field HSI (hyperspectral imaging)-based analyses may be performed even by staff lacking specific advanced chemometric expertise, as it is sometimes the case for conservation scientists or conservators with a scientific background. MA-rFTIR was only recently introduced in the conservation field and, in this work the technique was employed to characterize the surface of metallic artefacts. The analytical protocol was employed as part of a rapid procedure to evaluate the conservation state and the performance of cleaning methods on bronze objects. Both activities are commonly part of restoration campaigns of bronzes and require an on-site analytical procedure for efficient and effective diagnosis. The performance of the method was first evaluated on aged standard samples (bronzes with a layer of green basic copper hydroxysulphate, treated with different organic coatings) and then scrutinized in situ on areas of the 16th century Neptune fountain statue (Piazza del Nettuno, Bologna, Italy) by Gianbologna.

Characterization of outdoor bronze monument patinas: the potentialities of near-infrared spectroscopic analysis.

The corrosion products usually found on outdoor bronzes are generated by the interaction between the metal alloy and the atmospheric pollutants. To protect the external surface of bronzes, different organic materials (natural or synthetic) can be applied, creating over time a patina consisting of a complex mixture of inorganic and organic degraded components. The correct chemical characterization of patina constituents is fundamental to define the state of conservation of a metal artwork and address proper restoration actions. In this paper, we evaluated the potentialities of near-infrared (NIR) reflectance microscopy (4000-7500 cm-1) as complementary method to mid-infrared (MIR) analyses for the characterization of bronze patinas. Although NIR spectroscopy has been already used in the field of heritage science, its application for the characterization of bronze patinas is almost unexplored. In this paper, several corrosion products usually found on the surface of outdoor bronze sculptures were synthesized, characterized, and submitted to the NIR-MIR total reflection analysis to build up a reference spectral database. We devoted particular attention to the NIR features of copper hydroxychlorides, such as atacamite and paratacamite, which have not been studied in detail up to now. A selection of organic-based formulations, commonly used by restorers to protect the bronze surface against the outdoor aggressive environment, were also considered as references. Successively, NIR-MIR reflectance microscopy was successfully employed for the analysis of patina micro-samples collected from the bronze statues of the Neptune Fountain (sixteenth century) located in Bologna. The obtained results demonstrate the ability of NIR spectroscopy to identify organic and inorganic patina constituents, even in mixtures. In addition, the study can be considered as a proof of concept for the possible future application of the technique for in situ diagnostic campaigns on bronze sculptures.

An explorative chemometric approach applied to hyperspectral images for the study of illuminated manuscripts.

Hyperspectral imaging (HSI) is a fast non-invasive imaging technology recently applied in the field of art conservation. With the help of chemometrics, important information about the spectral properties and spatial distribution of pigments can be extracted from HSI data. With the intent of expanding the applications of chemometrics to the interpretation of hyperspectral images of historical documents, and, at the same time, to study the colorants and their spatial distribution on ancient illuminated manuscripts, an explorative chemometric approach is here presented. The method makes use of chemometric tools for spectral de-noising (minimum noise fraction (MNF)) and image analysis (multivariate image analysis (MIA) and iterative key set factor analysis (IKSFA)/spectral angle mapper (SAM)) which have given an efficient separation, classification and mapping of colorants from visible-near-infrared (VNIR) hyperspectral images of an ancient illuminated fragment. The identification of colorants was achieved by extracting and interpreting the VNIR spectra as well as by using a portable X-ray fluorescence (XRF) spectrometer.

A Multivariate Methodological Workflow for the Analysis of FTIR Chemical Mapping Applied on Historic Paint Stratigraphies.

In the field of applied researches in heritage science, the use of multivariate approach is still quite limited and often chemometric results obtained are often underinterpreted. Within this scenario, the present paper is aimed at disseminating the use of suitable multivariate methodologies and proposes a procedural workflow applied on a representative group of case studies, of considerable importance for conservation purposes, as a sort of guideline on the processing and on the interpretation of this FTIR data. Initially, principal component analysis (PCA) is performed and the score values are converted into chemical maps. Successively, the brushing approach is applied, demonstrating its usefulness for a deep understanding of the relationships between the multivariate map and PC score space, as well as for the identification of the spectral bands mainly involved in the definition of each area localised within the score maps.