Osteomeasure bone histomorphometry software

Left tibias were dissected and fixed in 10% buffered formaldehyde, decalcified in 14.3% EDTA, and then embedded in paraffin wax. Sections were cut longitudinally at 3 μm thickness and stained using a tartrate-resistant acid phosphatase (TRAP) staining kit (Sigma, 386A), according to the manufacturer’s protocol. The number of osteoblasts (N.Ob/B.Pm) and the number of osteoclasts (N.Oc/B.Pm) were determined on a 1.5 mm length of lateral and medial endocortical surfaces respectively, using a Leitz DMRB microscope (Leica Microsystems, Wetzlar, Germany). All histomorphometric nomenclature were based on the published guidelines³⁹ (link) and were obtained using the Osteomeasure bone histomorphometry software (OsteoMetrics, Inc. Decatur, GA, USA).

Bone samples were divided for histologic and histomorphometric evaluation. Samples were cut and fixed in 10% (w/v) formalin (Bio-Optica) for 3 days. For histomorphometric analysis, undecalcified samples were processed for plastic embedding, using a previously described protocol. Briefly, samples were dehydrated in a graded series of ethanol [50, 70 and 100% (v/v)], followed by immersion in xylol for 24 h. The undecalcified bone samples were infiltrated with a plastic embedding mixture using a three-step protocol. In each step, the samples were infiltrated for three consecutive days with daily freshly made solutions, containing 75% (v/v) of methylmethacrylate (MMA, Sigma-Aldrich) and 25% (v/v) of dibutyl phthalate (Prolabo) with increase in concentrations (0 g/mL; 0.01 g/mL and 0.025 g/mL) of benzoyl peroxide (Sigma-Aldrich). Polymerization was carried out at 37 °C for a week. The plastic blocks, containing the processed bone samples, were cut into 7 µm sections using a tungsten knife (Leica). After deplasticization, the sections were stained with toluidine blue staining. Trabecular separation (Tb.Sp) was determined using the Osteomeasure bone histomorphometry software (OsteoMetrics, OsteoMetrics, Inc.). The percentage of adipose tissue was calculated using a 15 to 11 points grid [38 (link), 39 (link)].