Rm 2155 biocut microtome

Rat femurs or human femoral head regions of interest were collected. The overlying tissues were removed prior to the fixation in 4% paraformaldehyde (PFA) for 48 hours. Bone tissues were decalcified in EDTA (14%, PH=7.4) (Sigma-Aldrich) at 37 °C for 14 days, in which the EDTA solution was changed every day till the bone became soft for sectioning. Next, the bone samples were placed in the automatic tissue processor for dehydration, followed by paraffin-embedding. Sections of 5-μm thickness were cut using a Leica RM 2155 Biocut Microtome (Leica Microsystems) and collected onto glass slides. Haematoxylin and eosin (HE) staining and tartrate-resistant acid phosphatase (TRAP) staining were implemented to visualise the bone microstructures and osteoclasts. Stained and mounted bone sections were scanned with Uscope MXII-20 (ProSciTech). Osteoclast parameters including osteoclast number per bone surface (N.Oc/BS) and osteoclast surface per bone surface (Oc.S/BS) were analysed using Bioquant Osteo software (Bioquant Image Analysis Corporation).

Rat femoral head samples were collected at 6 weeks after the establishment of the model. After 48 h of fixation in 4% paraformaldehyde (PFA) and 14 days of decalcification in EDTA (14%, PH = 7.4) (Sigma-Aldrich) at 37°C, the femoral head samples were embedded in paraffin and sectioned. Sections of 5-μm thickness were cut using a Leica RM 2155 Biocut Microtome (Leica Microsystems) and collected onto glass slides. Haematoxylin and eosin (HE) staining and TRAP staining were implemented to visualize the bone microstructures and osteoclasts.
Femurs were collected, soft tissues were dissected, and femoral heads were scanned using a Skyscan Micro-CT instrument (Bruker) with the following parameters: source current of 385 μA, source voltage of 65 kV, pixel size of 9 μm, AI filter of 1.0 mm, and rotation step of 0.4°. The images were reconstructed using NRecon software (Bruker), and the data was analyzed using the CTAn program (Bruker). A refined volume of interest, positioned 0.5 mm above the growth plate and with a height of 0.25 mm, was generated. The subchondral region of interest (ROI) in the trabecular bone was manually determined using a constant threshold range of 50–255. Parameters such as bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular separation (Tb.Sp), and trabecular thickness (Tb.Th) were compared among the three groups.