This study presents a methodology for measuring the thickness of trabecular rods directly from anaglyphs. Macerated sagittal slices of T12 vertebral bodies from 15 subjects were examined by scanning electron microscopy (SEM). Two digital images (the second image tilted 5 degrees ) were recorded, and a 3D anaglyph was created. The thickness of the trabecular rods (Tb.Th((rods))), and the anatomical orientation of the trabecular rods were measured using an image analyser. Conventional 2D histomorphometry was performed on adjacent bone slices. A total of 1559 rod measurements were made from the 15 vertebral bone slices, with a mean Tb.Th((rods)) of 123 +/- 36 microm. The rod thickness in males (128 +/- 34 microm) was significantly greater than that in females (119 +/- 37 microm, P < 0.001). Tb.Th((rods)) changed significantly with age in the males: the thicker rods in the younger men reduced with age to a thickness similar to that in women. 3D measurements were significantly larger than the 2D estimates, and there was no correlation between the two methods of measurement. An inverse correlation was found between the number of rods and the bone volume fraction (BV/TV), indicating that decreased BV/TV is associated with an increased number of rods. The vertical rods (132 +/- 39 microm) were significantly thicker than the horizontal rods (116 +/- 33 microm, P < 0.001). The determination of rod numbers, and their orientation and individual thicknesses enables a greater understanding of cancellous bone architecture in both individuals and populations, and will allow more reliable finite element modelling. Direct measurements from 3D anaglyphs of intact specimens provide new data that show previously unrecognised age- and sex-related changes.
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