Measuring Bone Volume at Multiple Densities by Micro-computed Tomography

Abstract

Bone strength is controlled by both bone mass, and the organization and quality of the bone material. The current standard method for measuring bone mass in mouse and rat studies is micro-computed tomography. This method typically uses a single threshold to identify bone material in the cortical and trabecular regions. However, this single threshold method obscures information about the mineral content of the bone material and depends on normal morphology to separately analyze cortical and trabecular structures. To extend this method to identify bone mass at multiple density levels, we have established a protocol for unbiased selection and application of multiple thresholds using a standard laboratory-based micro-computed tomography instrument. This non-invasive method can be applied to longitudinal studies and archived samples and provides additional information about bone structure and strength.

Bone Geometry Is Altered by Follistatin‐Induced Muscle Growth in Young Adult Male Mice

“…Multilevel Otsu thresholding was used to segregate bone into low‐, high‐, and mid‐density components as described recently. ( 61,62 ) Briefly, four‐level Otsu thresholding was used across the proximal shaft of the tibia (2.1–6.2 mm, excluding trabecular bone) to categorize the gray pixels into three bone densities: low density (calibrated to 0.438–0.963 g/cm 3 calcium hydroxylapatite [CaHA]), mid‐density (calibrated to 0.963–1.517 g/cm 3 CaHA), and high density (calibrated to >1.517 g/cm 3 CaHA). The lowest density quartile (0–0.438 g/cm 3 CaHA) was excluded as background or noise.…”

STAT3 Hyperactivation Due to SOCS3 Deletion in Murine Osteocytes Accentuates Responses to Exercise- and Load-Induced Bone Formation

“…This was achieved by measuring proportions of low-, mid-, and highdensity bone, as previously. (22,30) We measured the tibial metaphysis, since this has been reported, at least in male mice, to be most responsive to treadmill running. (24) In control mice, we observed no effect of treadmill exercise on total bone volume or on the proportions of low-, mid-, or high-density bone (Fig.…”

“…Proximal tibial bone volume was determined at three bone density thresholds using an un-biased Otsu thresholding algorithm as previously described (22,30) ; the three thresholds were determined for each study based on the cortical bone of the control (Socs3 f/f ) sedentary or non-loaded groups. In the exercise study, these parameters were measured at the following densities: low density from 0.342 to 0.744 g/cm 3 , mid-density from 0.744 to 1.059 g/cm 3 , and high density was all bone of tissue density greater than 1.059 g/cm 3 .…”

Gut microbiome depletion and repetitive mild traumatic brain injury differentially modify bone development in male and female adolescent rats

“…Due to the nature of the interventions and our a-priori hypotheses that these would result in significant changes in bone morphology, the morphology could not be considered “normal”. We therefore used multilevel Otsu thresholding to establish thresholds for measuring bone volume fraction at low, mid, and high density to control for significant heterogeneity between sexes, as well as treatment and injury groups ( Walker et al, 2020 ; Walker et al, 2021 ). This iterative algorithm separates pixels from raw images based on mean grey level intensity into different classes ( Otsu, 1979 ).…”