Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Femur preparation and µCT evaluation
• Data

Workflow and sampling

Step	Procedure	Equipment	Data collected
1	Mice are euthanized	Isoflurane inhalation	-
2	Left femurs are harvested and fixed	-	-
3	Femurs are analyzed via µCT	µCT system	Parameters determined in the metaphyseal trabecular bone

Equipment and supplies

• µCT System (µCT 50, Scanco Medical AG, Switzerland)

Reagents and solutions

• 5% isoflurane
• 4% paraformaldehyde
• 70% ethanol

Procedure: Femur preparation and µCT evaluation

1. Mice are intraperitoneally euthanized with cervical dislocation performed approximately one min after breathing stops owing to 5% isoflurane inhalation.
2. Left femurs are harvested and fixed for 24h in 4% paraformaldehyde solution, and then stored in 70% ethanol.
3. Whole femurs are examined by a µCT system as previously described (Hiram-Bab et al., 2015).
4. Scans are performed at a 10 µm resolution in all three spatial dimensions.
5. Mineralized tissues are differentially segmented by a global thresholding procedure as described by Ruegsegger et al, 1996.
6. All morphometric parameters are determined by a direct 3D approach as described by Hildebrand et al., 1999.
7. Parameters analyzed are determined in the metaphyseal trabecular bone.

References

Hildebrand T, Laib A, Muller R, Dequeker J, Ruegsegger P. Direct three-dimensional morphometric analysis of human cancellous bone: microstructural data from spine, femur, iliac crest, and calcaneus. J Bone Miner Res. 1999. 14:1167-74.

Hiram-Bab S, Liron T, Deshet-Unger N, Mittleman M, Gassmann M, Rauner M, et al., Erythropoietin directly stimulates osteoclast precursors and induces bone loss. FASEB J. 2015. 29:1890-900.

Ruegsegger P, Koller B, Muller R. A microtomographic system for the nondestructive evaluation of bone architecture. Calcif Tissue Int. 1996. 58:24-9.

Data collected by investigator

• Femur length
• Trabecular bone volume fraction
• Trabecular thickness
• Trabecular number
• Trabecular connectivity density