Quantitative bone histology: a new method.

A method is described for quantitative histology of undecalcified bone using an embedding medium suitable for histochemistry. Six normal subjects were given tetracycline markers 24 and 3 d prior to the biopsy which was taken from the posterior iliac spine using an 8 gauge Jamshidi needle. The tissue was embedded in a mixture of equal parts methylmethacrylate and hydroxyethylmethacrylate. Three consecutive sections were cut and (1) stained histochemically for acid phosphatase activity to identify osteoclast cytoplasm, (2) stained with pyronin to identify osteoblast cytoplasm, (3) mounted unstained for tetracycline fluorescence. Tracings of the 3 sections were superimposed using a camera lucida attachment to the microscope, and the tracings quantitated with an electronic digitizer. The major parameters were bone area (17.1 +/- 4.4%) (mean +/- SD), osteoblast or forming surface (5.9 +/- 2.7%) and osteoclast or resorbing surface (1.0 +/- 0.3%). Bone apposition rate was 1.0 +/- 0.2 micrometers/day and resorption velocity (calculated by assuming bone formation and resorption to be equal) 8.4 +/- 4.1 micrometers/day. Mineralization was assessed by osteoid area (3.8 +/- 2.5%), osteoid surface (13.9 +/- 6.6%), tetracycline uptake at the mineralizing front (62.3 +/- 13.5%), and mineralization lag time (18.1 +/- 2.5 days).