Smx 90ct

Animals were sacrificed using a pentobarbital I.V. overdose (Narkorens, Meral GmbH, Hallbergmoos, Germany). The area of the augmented soft tissue was removed from the animal’s calvarium bone en-bloc along with the surrounding tissues then fixed in 10% neutralized formalin for 1 week. Random micro-computed tomography (micro CT) scanning of two specimens was done (SMX-90CT, Shimadzu, Kyoto,Japan); one specimen from each group. These two specimens were plastic-embedded using Technovit 7200 Heraeus Kulzer GmbH,Wehrheim, Germany) with the distractor device (TPDD) in place.
In the rest of the specimens (n = 9 for each time point), (TPDD) was carefully dissected from the surrounding soft tissue using a sharp blade. The generated soft tissue was then peeled from the underlying calvarial bone using a sharp periosteal elevator. The samples were then embedded in paraffin, stained with AZAN stain and observed under an optical microscope (BZ-8000, Keyence, Osaka, Japan)

The porosity of the 3D printed dosage forms was assessed by X-ray microtomography (XμCT) (SMX-90 CT; Shimadzu Corp., Kyoto, Japan). Images were taken using 40 kV and 60 mA, with 360° rotation and 1024 × 1024 resolution. Images were reconstructed using the InspeXio SMX-90CT software program (Shimadzu Corp., Kyoto, Japan) and the porosity measurements were carried out with the VGSTUDIOMAX 3.5 software (Volume Graphics GmbH, Heidelberg, Germany) after applying a Gaussian filter and surface determination using a standardised threshold. After segmentation, the percentage of volume occupied by the 3D printed structure and the total volume, including the empty spaces, was calculated. The porosity (%) was calculated based on the percentage of empty volume in the 3D printed formulation.

Body composition (lean mass, fat mass, bone mineral content, and BMD) of the mice was measured using dual X-ray absorptiometry (DXA) (QDR-4500A, Hologic, Waltham, MA, USA). The instrument was calibrated before scanning sessions using a phantom with known BMD, according to the manufacturer's guidelines. Body composition excluding the skull was assessed every four weeks from the fourth week of experimental period. All scans were performed with the animals positioned prone and spread, with tape attached to each limb on the platform. The lean or fat mass percentage was calculated as lean or fat mass divided by total body weight. At the end of the study, the microarchitectures of the femora and the fifth lumbar vertebrae from sacrificed mice were analyzed using micro-computed tomography (micro-CT) scanning (SMX-90CT, Shimadzu, Kyoto, Japan). The cortical bone and trabecular bone were separated manually on each slice by a cursor line. The three-dimensional structure was analyzed using the TRI 3D-BON (RACTOC System Engineering Co., Tokyo, Japan) program. In this study, the morphometric parameters calculated for both skeletal sites included bone volume fraction (BV/TV, %), trabecular thickness (µm), trabecular number (1/mm), and trabecular separation (µm). Femoral images were also evaluated for cortical thickness (µm) and cortical cross-sectional area (mm²).