Osteomeasurexp software

Bone histomorphometry was carried out in Ctsk^Cre/WT;Notch2^tm1.1Ecan;Hes1^Δ/Δ, Ctsk^Cre/WT;Hes1^Δ/Δ, and Ctsk^Cre/WT;Rosa^Hes1 mice, and sex-matched controls were injected with calcein 20 mg/kg and demeclocycline 50 mg/kg at a 5 or 7 days of interval and sacrificed 2 days after demeclocycline administration. For static cancellous bone histomorphometry and to assess for the presence of TRAP-positive multinucleated cells, bones were decalcified in 14% EDTA for 14 days and embedded in paraffin, and 7 μm sections were stained for the presence of TRAP and counterstained with hematoxylin and analyzed at a 100× magnification using OsteoMeasureXP software (Osteometrics). Stained sections were used to draw bone tissue and measure trabecular separation, number and thickness, and eroded surface, as well as to count osteoblast and osteoclast number. To assess dynamic parameters of bone histomorphometry, undecalcified femurs were embedded in methyl methacrylate, and 5 μm sections were cut using Microm microtome (Richards-Allan Scientific). Mineralizing surface per bone surface and mineral apposition rate were measured on unstained sections visualized under UV light and a triple diamidino-2-phenylindole/fluorescein/Texas red set long-pass filter, and bone formation rate was calculated (85 (link)).

We processed the imaging samples by using an Olympus DP71 microscope (Olympus Scientific Solutions Americas Inc., Waltham, MA, USA). We analyzed the human specimens according to five sequential sections per stain and used anatomical landmarks to ensure comparability, including the presence of bone marrow and bone matrix. Moreover, serial sagittal sections of HO lesions were obtained. We determined the number of positively stained cells in each of five random visual fields in five sequential sections per specimen for each group and normalized the values to the number per millimeter of the adjacent bone surface (for TRAP staining quantification) or per square millimeter in the HO area. We conducted a quantitative analysis using the OsteoMeasureXP Software (OsteoMetrics, Inc., Decatur, GA, USA). For CD31⁺ and EMCN⁺ vessel quantification, we calculated the areas in red (CD31⁺) and green (EMCN⁺) at the whole HO site of each slide in three sequential sections per specimen for each group and normalized the values to those of the control specimens (set to 1). For chondrocyte quantification, we quantified all cells in the brown (Col II⁺) area and considered them as col II⁺ chondrocytes. Quantifications were performed using the software ImageJ 1.53c (National Institutes of Health, Bethesda, MD, USA).