Imagej software

The relative density of the sintered samples was calculated as the ratio between the experimental density determined by the Archimedes’ method and the composite theoretical density (7.618 g cm⁻³) calculated as the weighted average of PZT (8.006 g cm⁻³) and CFO (5.304 g cm⁻³) crystallographic densities.
The crystalline phases were identified by X-ray powder diffraction using a Bruker D8 Advance X-ray diffractometer (θ-θ equipped with a LINXEYE detector (Bruker, Karlsruhe, Germany), using Cu K_α radiation. Patterns were recorded in the 15° ≤ 2θ ≤ 80° range with 2.4°/min scanning rate.
The microstructure of the sintered samples was investigated by scanning electron microscopy (SEM-FEG, Carl Zeiss Sigma NTS GmbH, Oberkochen, Germany), embedding the cross sections in epoxy resin and then polishing them down to 0.25 µm finish. The grain size distributions of the sintered samples were calculated via image analysis of the SEM micrographs using ImageJ software (Java, ORACLE, Redwood City, CA, USA).

The positive blue staining of β-galactosidase was used to estimate cellular senescence. For senescence determination, cells were seeded in 24-well plates at a concentration of 10,000 cells per well. β-galactosidase staining was analyzed at 96 h after senescence induction by 0.5 mM of H₂O₂, using a Senescence Cells Histochemical Staining Kit (Sigma–Aldrich CS0030, St. Louis, (MO), USA)). Briefly, the culture medium was aspirated from the wells, and the cells were washed twice with 1 mL of PBS. Then, 1.5 mL of fixing buffer was added per well and incubated for 7 min at room temperature. After washing twice with PBS, 1 mL of freshly prepared dying solution, prepared following kit instructions, was added to the wells, and incubated overnight at 37 °C without CO₂. The plates were sealed with parafilm to avoid drying. Finally, the wells were washed with PBS, and the cells were scored for β-galactosidase-positive staining using light microscopy. Five representative images from each well were captured, total cell number and β-galactosidase-positive cells were obtained by using Image-J software (Oracle Corporation, Santa Clara, (CA), USA), and the percentage of positive cells with respect to total cells in each imaged was determined.

For histochemical examination, muscles samples were fixed in 10% neutral buffered formalin and embedded in paraffin. Four µm-thick sections were stained with Hematoxylin and Eosin (HE) and Masson Trichrome with aniline blue (MT) (Bio-Optica, Milan, Italy). Histological images were evaluated using a photomicroscope (Axiophot; Carl-Zeiss, Oberkochen, Germany) with an attached digital camera controller (DS-L1 camera control unit, Nikon).
From HE and MT stained sections, five histological images per slide were randomly taken at 200x magnification and were analyzed with ImageJ software (Java-based image processing program, Oracle corporation, Austin, Texas, USA). HE stained histological images were used to study the inflammatory cells density (cells/mm²), blood vessels density (blood vessels/mm²), blood vessels area (µm²) and percentage of centrally nucleated fibers with respect to total muscle fibers. MT stained histological images were used to assess areas of fibrillar collagen to estimate the percentage of fibrotic areas (FA) development.