*4.6. Micro-Computed Tomography Analysis*

The left ankle joints of the rats were fixed in 70% ethanol and scanned with a μ-CT system (TOSCANER-32300μFD, Toshiba, Tokyo, Japan). The reconstructed data sets were examined with three-dimensional data analysis software (TRI/3-D-BON, Ratoc System Engineering Co., Tokyo, Japan; *n* = 4 for each group) [35]. The regions of interest were defined in the whole talus bones. To analyze the talus, the following trabecular bone parameters in the whole talus were evaluated: bone volume fraction, Tb.Th, and bone mineral content per tissue volume (BMC/TV). We also assessed indirect parameters, including MSV, which is the mean volume of all the parts of an object that can be unobscured in all the directions from a point inside the object [36]. The degree of bone erosion and bone formation were also analyzed using 3D-μ-CT. Eroded bone surface per repaired bone surface (Es/Rps) on whole talus was determined with the software automatically according to the software program. We set the concave surface search range up to 0.5 mm, and the absorption surface extraction radius of curvature was 240 μm or less.
