Metamorph image analysis program

L5 was fixed in 4% buffered formalin for 1 week and then stored for a few weeks in 70% ethanol. Thereafter, L5 was embedded in Technovit 9100 New® (Heraeus Kulzer GmbH, Wehrheim, Germany) and cut longitudinally using a Leica microtome (RM 2165, Leica Instruments GmbH) to a thickness of 5 µm. The sections were deacrylated, stained with Toluidine Blue O (Merck, Darmstadt, Germany), and mounted with Eukitt (O. Kindler GmbH, Freiburg, Germany) [30 (link)]. The sections were digitalized using a digital camera (Leica DFC490) and a zoom stereo microscope (Leica DMRXE) and analyzed with the aid of the MetaMorph image analysis program (Leica, Bensheim, Germany). Three randomly chosen fields of 0.1 mm² within the histological section were taken for the analyses. The following parameters were measured according to ASBMR nomenclature: osteoblast number per bone perimeter (N.Ob/B.Pm), osteoclast number per B.Pm (N.Oc/B.Pm), and osteocyte number per bone area (Ot/B.Ar) [24 (link), 31 (link)]. The criteria for the morphological identification of osteoblasts and osteoclasts were as follows. Cuboid-shaped cells that covered trabecular bone were counted as osteoblasts, whereas multinucleated cells that were resorbing bone were counted as active osteoclasts [32 (link)].

The tibia samples were exposed to sequential ascending concentrations of ethanol and embedded in Technovit^® 9100 medium (Heraeus Kulzer GmbH, Wehrheim, Germany). Sections 150 µm in thickness were cut transversally 2 cm distal to the knee surface using a diamond saw microtome (Leica SP 1600, Leica Instruments GmbH, Nussloch, Germany). Three sections of the tibia were mounted with Eukitt medium (O. Kindler GmbH, Freiburg, Germany), digitalized using a digital camera (Leica DFC490) and a zoom stereo microscope (Leica DMRXE), and analyzed with the aid of the MetaMorph image analysis program (Leica, Bensheim, Germany) (Figure 3J). The following parameters were measured: the thickness of CG-stained new bone at the endosteal and periosteal sites of the cortical bone, marrow diameter (Ma.Dm), bone diameter (B.Dm), and the bone-to-marrow diameter ratio (B.Dm/Ma.Dm) [40 (link)].