Shandon tbd 1 decalcifier

Samples from radio-sterilized DMB and DCC scaffolds and control native bovine cancellous bone blocks were fixed in 10% neutral buffered formalin (Thermo Fischer Scientific, USA), and decalcified using ready-made decalcification solution (Shandon^™ TBD-1^™ Decalcifier, Thermo Fischer Scientific, USA) with periodic examination for softening of the samples. Decalcification was performed in both DMB and DCC samples to prepare good-quality paraffin sections. The decalcification period for DMB samples, however, was shorter than for DCC samples. Upon completing the decalcification process, the bone samples were rinsed in running tap water thoroughly for 2 h followed by paraffin embedding and sectioning into 7μm thick sections. Bone sections were stained with nuclear dye Hematoxylin and the counterstain Eosin (Sigma-Aldrich). Images of tissue sections were captured using an inverted microscope (Olympus IX73, Japan).

Paraffin embedding was used to perform the histological processing of all samples (VNOs and OBs). In one of the individuals, the complete NC was pre-decalcified; it was immersed in a decalcifying solution (Shandon TBD-1 Decalcifier, Thermo, Pittsburgh, PA, USA) and continuously stirred for thirty hours. The samples were then washed under running water for two hours, and were cut into several blocks which were serially cut from the incisor papilla to the caudal end of the vomeronasal cartilage in order to obtain information on the changes in the VNO throughout its length. Following this process, all blocks were paraffin embedded.
Cutting The samples were cut with a Leica Reichert Jung microtome with a thickness of 4–8 μm, depending on the tissue to be processed. We opted for thinner cuts in the study of the VNO and thicker cuts in the study of the AOB, as these allow a better visualisation of the nerve and glial processes.

Following the micro-CT analysis, the right maxillary samples, fixed in 10% neutral buffered formalin, were decalcified using Shandon TBD-1 decalcifier (Thermo Scientific, USA). The decalcified samples were embedded into paraffin blocks and sectioned using a microtome into 5 µm sections. Samples were subjected to hematoxylin and eosin (H&E) stain. The slides were observed blindly using an inverted microscope with ocular’s magnification of 10X (Nikon Eclipse Ts2R, Nikon Instruments Inc., Melville, NY, USA). Slides were initially inspected at 4X objective magnification. Then, area of interests at the pressure and tension sites were inspected at 20X objective magnification. The histological images were taken in TIFF format and analyzed qualitatively for the bone resorption and bone formation. Six samples per group (three sections per sample) were considered for the histological assessment. Intraclass correlation coefficient (ICC) was used to confirm intra-rater reliability (ICC score 0.98).