Biology subjects

Boscaro, D.

Publications and source records attributed to Boscaro, D..

3 recordsLinked to original sources

bioRxiv2026

Ultra-structural analysis of mineralized extracellular matrix in osteogenic monolayers and spheroids: comparison of sample preparation methods

Accurate evaluation of extracellular matrix (ECM) mineralization at the nano-scale is essential for establishing relevant in vitro bone models. This is particularly important with the development and increased application of three-dimensional (3D) cell models for biological research. Transmission electron microscopy (TEM) allows to perform ultra-structural analysis of cells and ECM organization, but its application in in vitro bone models remains limited, due to the potential alteration or loss of the mineral phase during sample preparation. In this study, we compared two TEM sample preparation methods the conventional chemical fixation and the anhydrous methods to evaluate their ability to preserve the mineralized ECM in MC3T3-E1 cells cultured as monolayers and as alginate-encapsulated bone spheroids. Chemical fixation preserved cellular ultra-structure and collagen organization, allowing for detailed assessment of cells and ECM organization. Although mineral deposits were detected and their needle-like morphology assessed, characterization of more immature deposits was partially limited by the effects of uranyl acetate and the overall sample preparation process, which could lead to alteration or loss of less stable mineral phases. The anhydrous preparation method resulted in limited preservation of cellular and ECM morphology and did not allow reliable identification of mineral deposits. When applied to spheroids, the chemical fixation method preserved the 3D architecture, collagen-rich ECM and inner mineral deposits, confirming spheroids as a relevant model for bone studies. Overall, these results highlight the need for optimized sample preparation strategies that preserve both ultra-structure and mineral components for accurate nano-scale characterization of bone mineralization.

biophysics↗

bioRxiv2026

Label-free imaging of matrix mineralization in alginate-encapsulated bone spheroids using Coherent Raman Scattering microscopy

Three-dimensional (3D) cell cultures, such as spheroids, are increasingly used to perform advanced studies on bone matrix mineralization. However, their full characterization remains challenging. Traditional colorimetric and fluorescent assays using dyes, such as Alizarin Red S (ARS) and calcein, are effective in monolayer cell cultures, but fail to provide reliable information when used in complex 3D cell constructs. In this study, we investigated the application of Coherent Raman Scattering microscopy for label-free, comprehensive characterization of extracellular matrix (ECM) mineralization in alginate-encapsulated bone spheroids. After confirming that traditional staining techniques are unreliable for mineral detection in spheroids, Stimulated Raman Scattering (SRS) microscopy was used to detect phosphate-rich mineral deposits at a Raman shift of 960 cm-1, while Second Harmonic Generation (SHG) microscopy was used in association with SRS to provide complementary information on the deposition and organization of the collagenous matrix. SRS was used to detect lipid-rich regions at a Raman shift of 2857 cm-1 to perform cell localization. SRS imaging revealed the presence of phosphate-rich regions in the spheroids, including the core regions, usually challenging to characterize in intact 3D constructs. Raman spectral scans on SRS-positive regions confirmed the specificity of the phosphate signal. In addition, comparison of SRS and Coherent Anti-Stokes Raman Scattering (CARS) demonstrated the advantage of SRS in terms of reduced background compared to CARS for lipid imaging. Taken together, our results demonstrated that SRS, in combination with SHG, provides a promising and powerful approach to perform label-free, chemically specific characterization of intact 3D bone models.

biophysics↗

bioRxiv2025

Characterization of ECM produced by MC3T3-E1 cellular spheroids encapsulated in alginate hydrogel

The application of cellular spheroids in bone tissue engineering research has gained significant interest in the last decade. Compared to monolayer cell cultures, the 3D architecture allows for more physiological cell-cell and cell-extracellular matrix (ECM) interactions that make cellular spheroids a suitable model system to investigate bone ECM in vitro. The use of 3D model systems requires fine-tuning of experimental methods used to study cell morphology, ECM deposition and mineralization, and cell-ECM interactions. In this study, we use MC3T3-E1 cellular spheroids encapsulated in an alginate hydrogel to study and characterize the deposited ECM. Spheroid shape and structure were evaluated using confocal microscopy. The deposited collagenous ECM was characterized using quantitative assay and microscopy, in particular Second Harmonic Imaging Microscopy (SHIM), hydroxyproline (HYP) assay and Transmission Electron Microscopy (TEM). The use of hydrogel constructs allows easy handling and imaging of the samples and helps to preserve the spheroids stability by preventing cells from adhering to the culture dish surface. We use a non-modified alginate hydrogel that does not facilitate cell attachment and therefore functions as an inert encapsulating scaffold. Constructs were cultured for up to 4 weeks. SHIM, HYP assay and TEM confirmed the deposition of collagenous matrix by the spheroid, with most of it taking place between week 2 and 4. We demonstrated that alginate encapsulated bone spheroids are a promising model for studying bone ECM in vitro.

bioengineering↗