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.

Miniaturized Biosensors to Preserve and Monitor Cultural Heritage: from Medical to Conservation Diagnosis.

The point-of-care testing concept has been exploited to design and develop portable and cheap bioanalytical systems that can be used on-site by conservators. These systems employ lateral flow immunoassays to simultaneously detect two proteins (ovalbumin and collagen) in artworks. For an in-depth study on the application of these portable biosensors, both chemiluminescent and colorimetric detections were developed and compared in terms of sensitivity and feasibility. The chemiluminescent system displayed the best analytical performance (that is, two orders of magnitude lower limits of detection than the colorimetric system). To simplify its use, a disposable cartridge was designed ad hoc for this specific application. These results highlight the enormous potential of these inexpensive, easy-to-use, and minimally invasive diagnostic tools for conservators in the cultural heritage field.

Innovative perspective for the cleaning of historical iron heritage: novel bio-organogel for the combined removal of undesired organic coatings and corrosion.

An innovative green organogel was designed to simultaneously tackle inorganic compounds (i.e., iron corrosion) and organic substances (i.e., acrylic coatings) as undesired materials possibly present on the surface of altered indoor metal artworks. Poly-3-hydroxybutyrate (PHB), ethyl lactate (EL), and deferoxamine B (DFO) were employed in the formulation as thickening agent, organic solvent, and complexing agent, respectively, aiming to propose a sustainable and less harmful chemical cleaning method for metal care. The components were selected because they are bio-sourced, renewable, biodegradable, and non- or low-toxic materials. A multi-modal protocol of analysis was carried out to characterise the newly designed PHB-EL-DFO organogel. The cleaning performance of the novel formulation was assessed on mild steel mock-ups presenting both corrosion and organic coating to be removed. The conducted multi-analytical approach verified that the PHB-EL-DFO gel was able to tackle the two undesired materials simultaneously in an adjustable and easy-to-use way thanks to a modular application. Supplementary Information: The online version contains supplementary material available at 10.1186/s40494-024-01288-0.

Microporous electrospun nonwovens combined with green solvents for the selective peel-off of thin coatings from painting surfaces.

Over the last few decades, significant research efforts have been devoted to developing new cleaning systems aimed at preserving cultural heritage. One of the main objectives is to selectively remove aged or undesirable coatings from painted surfaces while preventing the cleaning solvent from permeating and engaging with the pictorial layers. In this work, we propose the use of electrospun polyamide 6,6 nonwovens in conjunction with a green solvent (dimethyl carbonate). By adjusting the electrospinning parameters, we produced three distinct nonwovens with varying average fiber diameters, ranging from 0.4 µm to 2 µm. These samples were characterized and tested for their efficacy in removing dammar varnish from painted surfaces. In particular, the cleaning process was monitored using macroscale PL (photoluminescence) imaging in real-time, while post-application examination of the mats was performed through scanning electron microscopy. The solvent evaporation rate from the different nonwovens was evaluated using gravimetric analysis and Proton Transfer Reaction- Time-of-Flight. It was observed that the application of the nonwovens with small or intermediate pore sizes for the removal of the terpenic varnish resulted in the swollen resin being absorbed into the mats, showcasing a peel-off effect. Thus, this protocol eliminates the need for further potentially detrimental removal procedures involving cotton swabs. The experimental data suggests that the peel-off effect relates to the microporosity of the mats, which enhances the capillary rise of the swollen varnish. Furthermore, the application of these systems to historical paintings underwent preliminary validation using a real painting from the 20th century.

Alternative SERRS probes for the immunochemical localization of ovalbumin in paintings: an advanced mapping detection approach.

In the field of analytical chemistry, many scientific efforts have been devoted to develop experimental procedures for the characterization of organic substances present in heterogeneous artwork samples, due to their challenging identification. In particular, performances of immunochemical techniques have been recently investigated, optimizing ad hoc systems for the identification of proteins. Among all the different immunochemical approaches, the use of metal nanoparticles - for surface enhanced Raman scattering (SERS) detection - remains one of the most powerful methods that has still not been explored enough for the analysis of artistic artefacts. For this reason, the present research work was aimed at proposing a new optimized and highly efficient indirect immunoassay for the detection of ovalbumin. In particular, the study proposed a new SERRS probe composed of gold nanoparticles (AuNPs) functionalised with Nile Blue A and produced with an excellent green and cheap alternative approach to the traditional chemical nanoparticles synthesis: the laser ablation synthesis in solution (LASiS). This procedure allows us to obtain stable nanoparticles which can be easily functionalized without any ligand exchange reaction or extensive purification procedures. Moreover, the present research work also focused on the development of a comprehensive analytical approach, based on the combination of potentialities of immunochemical methods and Raman analysis, for the simultaneous identification of the target protein and the different organic and inorganic substances present in the paint matrix. An advanced mapping detection system was proposed to achieve the exact spatial location of all the components through the creation of false colour chemical maps.

Analysis of paint cross-sections: a combined multivariate approach for the interpretation of µATR-FTIR hyperspectral data arrays.

The present research is aimed at introducing a suitable approach for the exploitation of the hyperspectral data obtained by µATR-FTIR analyses of paint cross-sections. The application of principal component analysis for chemical mapping is well-established, even if a very limited number of applications to µFTIR data have been reported so far in the field of analytical chemistry for cultural heritage. Moreover, in many cases, chemometric tools are under-utilized and the outcomes under-interpreted. As a consequence, results and conclusions may be considerably compromised. In an attempt to overcome such drawbacks, the present work is proposing a comprehensive and efficient procedure based on an interactive brushing approach, which combines the structural information of the score scatter plots and the spatial information of the principal component (PC) score maps. In particular, the study demonstrates not only how the multivariate approach may provide more information than the univariate one, but also how the integration of different chemometric tools may allow a more comprehensive interpretation of the results with respect to the studies up to now reported in the literature. The examination of the average spectral profile of each score cluster, jointly with the loading analysis, is functional to characterize each area investigated on the basis of its spectral features. A multivariate comparison with spectra of standard compounds, projected in the PC score space, helps in supporting the chemical identification. The approach was validated on two real case studies.

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.

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.

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.

An effective strategy for the monitoring of microplastics in complex aquatic matrices: Exploiting the potential of near infrared hyperspectral imaging (NIR-HSI).

Contamination by microplastics (MP) represents a critical environmental challenge with potential consequences at ecosystem, economic and societal levels. As the marine system is the final sink for MP, there is an urgent need to develop methods for the monitoring of synthetic particles in different marine compartments and sample matrices. Extensive evaluations are hindered by time and costs associated with to conventional MP spectroscopic analyses. The potential of near infrared hyperspectral imaging (NIR-HSI) has been recently evaluated. However, NIR-HSI has been poorly studied so far, limitedly to the detection of large particles (>300 µm), and its capability for direct characterization of MP in real marine matrices has not been considered yet. In the present study, a rapid near infrared hyperspectral imaging (NIR-HSI) method, coupled with a customised normalised difference image (NDI) strategy for data processing, is presented and used to detect MP down to 50 µm in environmental matrices. The proposed method is largely automated, without the need for extensive data processing, and enabled a successful identification of different polymers, both in surface water and mussel soft tissue samples, as well as on real field samples with environmentally occurring MP. NIR-HSI is applied directly on filters, without the need for particles pre-sorting or multiple sample purifications, avoiding time consuming procedures, airborne contaminations, particle degradation and loss. Thanks to the time and cost effectiveness, a large-scale implementation of this method would enable to extensively monitor the MP presence in natural environments for assessing the ecological risk related to MP contamination.

Quantifying spatial variation in the uptake of microplastic by mussels using biodeposit traps: A field-based study.

Spatial uptake patterns of microplastics (MP) by marine species are largely unexplored under field conditions. A novel "biodeposit trap" that measure uptake and egestion of MP by suspension-feeders through the analysis of their biodeposits, was designed and used to estimate the spatial variation of these processes by mussels in field conditions. Traps containing wild or farmed mussels or control empty shells were deployed at three sites characterised by different MP concentrations and water flow conditions. A different MP dimensional composition was observed between MP pools present in biodeposit and control traps, with the latter shifted towards higher dimensional range (0.05-5 mm). Conversely, mussels accumulated small MP (0.02-0.05 mm) into their biodeposits without any significant difference between wild and farmed specimens. MP uptake rates were on average 4-5 times higher at the site where MP contamination was expected to be highest and where water flow conditions were considered moderate.

Combining elemental and immunochemical analyses to characterize diagenetic alteration patterns in ancient skeletal remains.

Bones and teeth are biological archives, but their structure and composition are subjected to alteration overtime due to biological and chemical degradation postmortem, influenced by burial environment and conditions. Nevertheless, organic fraction preservation is mandatory for several archeometric analyses and applications. The mutual protection between biomineral and organic fractions in bones and teeth may lead to a limited diagenetic alteration, promoting a better conservation of the organic fraction. However, the correlation between elemental variations and the presence of organic materials (e.g., collagen) in the same specimen is still unclear. To fill this gap, chemiluminescent (CL) immunochemical imaging analysis has been applied for the first time for collagen localization. Then, Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry (LA-ICP-MS) and CL imaging were combined to investigate the correlation between elemental (i.e., REE, U, Sr, Ba) and collagen distribution. Teeth and bones from various archeological contexts, chronological periods, and characterized by different collagen content were analyzed. Immunochemical analysis revealed a heterogeneous distribution of collagen, especially in highly degraded samples. Subsequently, LA-ICP-MS showed a correlation between the presence of uranium and rare earth elements and areas with low amount of collagen. The innovative integration between the two methods permitted to clarify the mutual relation between elemental variation and collagen preservation overtime, thus contributing to unravel the effects of diagenetic alteration in bones and teeth.

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.

Effects of imidazolium ionic liquids on growth, photosynthetic efficiency, and cellular components of the diatoms Skeletonema marinoi and Phaeodactylum tricornutum.

This article describes the toxic effects of imidazolium ionic liquids bearing alkyl (BMIM), monoethoxy (MOEMIM), and diethoxy (M(OE)(2)MIM) side chains toward two marine diatoms, Skeletonema marinoi and Phaeodactylum tricornutum. MOEMIM and M(OE)(2)MIM cations showed a lower inhibition of growth and photosynthetic efficiency with respect to their alkyl counterpart, with both algal species. However, a large difference in sensitivity was found between S. marinoi and P. tricornutum, the first being much more sensitive to the action of ionic liquids than the second one. The effects of salinity on BMIM Cl toxicity toward S. marinoi revealed that a decrease from salinity 35 to salinity 15 does not influence the biological effects toward the alga. Finally, Fourier transform infrared (FT-IR) microscopy of algal cells after ionic liquids exposure allowed us to detect an alteration of the organic cellular components related to silica uptake and organization. On the basis of these results, the different behavior of the two diatom species can be tentatively ascribed to different silica uptake and organization in outer cell walls.

Application of ATR-far-infrared spectroscopy to the analysis of natural resins.

This study proposes FTIR spectroscopy in the far-infrared region (FarIR) as an alternative method for the characterisation of natural resins. To this purpose, standards of natural resins belonging to four different categories (sesquiterpenic, i.e. elemi, shellac; diterpenic, i.e. colophony, Venice turpentine; diterpenic with polymerised components, i.e. copal, sandarac; triterpenic, i.e. mastic and dammar) used as paint varnishes have been analysed by FarIR spectroscopy in ATR mode. Discrimination between spectral data and repeatability of measurements have been magnified and verified using principal component analysis, in order to verify the effectiveness of the method in distinguishing the four resin categories. The same samples were analysed in the MidIR range, but the spectral differences between the different categories were not evident. Moreover, the method has been tested on historical samples from the painting "La Battaglia di Cialdiran" (sixteenth century) and from a gilded leather (seventeenth century). In the first case, FarIR spectroscopy allowed confirmation of the results obtained by analytical pyrolysis. In the latter, FarIR spectroscopy proved successfully, effective in the identification of the superficial resin layer that could not be detected with the bulk chromatographic analyses.

Development of a multiplexed chemiluminescent immunochemical imaging technique for the simultaneous localization of different proteins in painting micro cross-sections.

The identification and localization of organic components in the complex stratigraphy of paintings play a crucial role in studies of painting techniques and authentication, restoration, and conservation of artworks. Much scientific effort has been expended for the development of analytical approaches suitable for the investigation and characterization of organic substances, allowing high sensitivity, specificity, and spatial resolution. Proteins (e.g., ovalbumin, casein, and collagen from different animal sources) are one of the classes of organic substances most widely used as painting materials. The analytical techniques commonly used for their analysis (micro Fourier transform infrared spectroscopy, chromatographic techniques, and proteomic approaches) have limits related to the lack of specificity or to the absence of information concerning the stratigraphic localization of the detected proteins. Immunological techniques are a promising alternative approach for the characterization of proteins in artworks. Thanks to the high specificity of antigen-antibody reactions, these techniques are widely used for the analysis of proteins in bioanalytical and clinical chemistry and recently they have been successfully applied in the field of science for conservation of cultural heritage. The present research aimed to develop an ultrasensitive chemiluminescent immunochemical procedure for the simultaneous localization of ovalbumin and bovine casein (two common proteins found in binding media or varnishes of artistic and archaeological samples) in resin-embedded painting micro cross-sections. The possibility of performing the simultaneous identification of different proteins in painting cross-sections is of particular relevance in the field of cultural heritage because samples are often small and available in a limited number; therefore, the maximum amount of information must be obtained from each of them.

Performance evaluation of mapping and linear imaging FTIR microspectroscopy for the characterisation of paint cross sections.

Different Fourier transform infrared microspectroscopic techniques, using attenuated total reflection (ATR) mode and single-element mercury-cadmium-telluride (MCT) detector (mapping) or multielement MCT detector (raster scanning), are compared with each other for the characterisation of inorganic compounds and organic substances in paint cross sections. All measurements have been performed on paint cross sections embedded in potassium bromide, a transparent salt in the mid-infrared region, in order to better identify the organic materials without the interference of the usual embedding resin. The limitations and advantages of the different techniques are presented in terms of spatial resolution, data quality and chemical information achieved. For all techniques, the chemical information obtained is found to be nearly identical. However, ATR mapping performed with a recently developed instrumentation shows the best results in terms of spectral quality and spatial resolution. In fact, thin organic layers (approximately 10 microm) have been not only identified but also accurately located. This paper also highlights the recent introduction of multielement detectors, which may represent a good compromise between mapping and imaging systems.

Rapid and direct detection of small microplastics in aquatic samples by a new near infrared hyperspectral imaging (NIR-HSI) method.

Microplastic (MP) contamination is a critical environmental challenge with a strong impact on the ecosystems, economy and potentially for human health. The smaller the MP size, the greater is the environmental risks as well as the analytical difficulties in detecting and characterising the particles. .We propose a rapid near infrared hyperspectral imaging (NIR-HSI) method that enables the chemical identification and characterisation of small MP (down to 80 µm) in aquatic samples, directly on filters, with no pre-sorting step needed. By considerably reducing the procedural steps, the time of analysis and costs our method addresses the urgent need of cost-effective and robust tools for extensive monitoring of MP in natural systems.

Organogel Coupled with Microstructured Electrospun Polymeric Nonwovens for the Effective Cleaning of Sensitive Surfaces.

Hydrogels and organogels are widely used as cleaning materials, especially when a controlled solvent release is necessary to prevent substrate damage. This situation is often encountered in the personal care and electronic components fields and represents a challenge in restoration, where the removal of a thin layer of aged varnish from a painting may compromise the integrity of the painting itself. There is an urgent need for new and effective cleaning materials capable of controlling and limiting the use of solvents, achieving at the same time high cleaning efficacy. In this paper, new sandwich-like composites that fully address these requirements are developed by using an organogel (poly(3-hydroxybutyrate) + γ-valerolactone) in the core and two external layers of electrospun nonwovens made of continuous submicrometric fibers produced by electrospinning (either poly(vinyl alcohol) or polyamide 6,6). The new composite materials exhibit an extremely efficient cleaning action that results in the complete elimination of the varnish layer with a minimal amount of solvent adsorbed by the painting layer after the treatment. This demonstrates that the combined materials exert a superficial action that is of utmost importance to safeguard the painting. Moreover, we found that the electrospun nonwoven layers act as mechanically reinforcement components, greatly improving the bending resistance of organogels and their handling. The characterization of these innovative cleaning materials allowed us to propose a mechanism to explain their action: electrospun fibers play the leading role by slowing down the diffusion of the solvent and by conferring to the entire composite a microstructured rough superficial morphology, enabling to achieve outstanding cleaning performance.

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.