Metaphyseal trabecular bone separation is bimodal: a methodological update

Micro-computed tomography with morphometric analysis is the gold standard methodology for skeletal phenotyping of small animal models of bone disease. Metaphyseal trabecular bone is the most common site of assessment. The 2010 guidelines for the assessment of bone microstructure recommends a minimal set of parameters to be reported; bone volume fraction (BV/TV), and trabecular number (Tb.N), thickness and separation (Tb.Sp). Utilising three different models of osteoporosis (rat spinal cord injury, mouse ovariectomy and mouse ageing), we demonstrate that metaphyseal Tb.Sp is bimodal (Figure 1A) both in the femur of tibia. We propose that metaphyseal Tb.Sp should be reported as two distinct values - preliminarily named Tb.Sp1 (representing trabecular thickening/thinning) and Tb.Sp2 (representing marrow cavity expansion). 2 BV/TV, Tb.N and Tb.Sp analyses were performed on metaphyseal trabecular bone VOIs. The outputs from Tb.Sp analysis are the volume-weighted average Tb.Sp, Tb.Sp histogram, and Tb.Sp dataset (Figure 1). Metaphyseal Tb,Sp histograms appeared bimodal (or multimodal), which is particularly clear in osteoporotic datasets (Figure 1A). A global threshold (350µm) was applied, separating the two peaks in the distribution, enabling acquisition of Tb.Sp1 and Tb.Sp2 VOIs. The Tb.Sp1 (or Tb.Sp2) VOI was then applied to the original dataset and the Tb.Sp analysis was rerun, generating results for Tb.Sp1 (or Tb.Sp) (Figure 1C). For the age-induced osteoporosis datasets (14-, 36- and 62-weeks-old), a monotonic decrease in BV/TV and Tb.N, and increase in Tb.Sp was observed (p < 0.01) confirming age-induced osteoporosis. Tb.Sp histograms were multimodal (Figure 1A). Segmenting the Tb.Sp dataset based on sphere diameter enabled the distinction of effects due to intra-trabecular thinning (first peak) captured by Tb.Sp1, from those due to complete resorption of centrally-located trabeculae, which widens the metaphyseal marrow cavity, captured by Tb.Sp2 (Figure 1C). All 3 types of Tb.Sp were different (p<0.01), indicating that both trabecular thinning/thickening and marrow cavity expansion contribute to Tb.Sp. In the 62weeks-old dataset Tb.Sp2 became dominant (Figure 1B). Similar results were observed for ovariectomy and spinal cord injury models. We envisage that this methodological tweak should enable a more sensitive distinction of skeletal phenotypes.