A classification of in vivo bone labels after double labeling in canine bones

Labeling patterns were classified after double bone labeling of four male beagles, 10 months of age. Calcein and oxytetracycline were given on the 18th and the 7th day prior to simultaneous iliac and 11th rib biopsies. Undecalcified sections stained with the Villanueva bone stain were studied by epifluorescence microscopy. Five structures were identified and classified: the first or green label, the interlabel layer of mineralized bone, the second or yellow label, the post double-labeled mineralized bone layer, and osteoid seams. Doubly plus singly labeled surface equalled 40.8 +/- 8.4% of the total trabecular surface of the ilium. Doubly labeled surface as a percent of the total labeled surfaces equaled 55.5% in trabeculae and 68.1% in osteons, whereas green first-singly labeled surface equaled 24.2% and 11.9%, respectively, and yellow second-singly labeled surface equaled 20.3% and 20.0%, respectively. Unequivocal examples appeared in both biopsy sites of all four dogs of bone-forming systems that lacked one or the other label, or both, and also of systems in which cessation of mineralization or of new matrix formation occurred between the two labels and between the second label and the day of biopsy. The findings prove that the On-Off states in active bone-forming sites that have been postulated by other investigators do exist. Since widely different labeling patterns appeared in different bone-forming centers in the same bone and the same animal, a local factor rather than a systemic one should control those differences at the level of the BMU.