Histochemical Detection of Zinc in Mineralizing Rat Bone: Autometallographic Tracing of Zinc Ions in the Mineralization Front, Osteocytes, and Osteoblasts

Abstract

Zinc (Zn) in biological tissue is found in 2 pools. One pool is firmly bound to proteins; ie, Zn is involved in maintaining the 3-dimensional form of molecules or is part of the active site of enzymes. The other pool comprises a pool of free or loosely-bound Zn ions, often stored in vesicles, where they have been found to make peptides and proteins clump.It has been known for some time that bone contains high levels of Zn, whereas, to our knowledge, Zn ions have not previously been histochemically recognized in bone. The aim of the study was, therefore, to detect and localize Zn ions in bone cells and mineralizing bone tissues by the autometallographic (AMG) sulfide silver method.Rats were transcardially perfused with a sodium sulfide solution, whereby nanometer sized Zn sulfide crystals were created at sites containing free or loosely-bound Zn ions. The femora and tibiae were removed and cut on a diamond saw. The 150-μm sections, which included undecalcified cortical bone, were subjected to AMG development, whereby Zn sulfide crystallites were silver-amplified.We found AMG silver grains localized intracellularly in osteoblasts and osteocytes and extracellularly in mineralizing osteoid. Ultrastructurally, the intracellular Zn ions were located in secretory vesicles, and, the extracellular Zn ions were found in matrix vesicles.It has been hypothesized that Zn ions act as an important regulator of calcium influx in matrix vesicles, and their detection inside these vesicles is therefore interesting. During mineralization, Zn ions may participate in the nuclear ization and growth of hydroxyapatite crystals. (The J Histotechnol 22:85, 1999)