Micro-PIXE and histochemical studies of Zn and Ca distribution in normal bone

To better understand the role of zinc in bone mineralization we studied the distribution of Ca and Zn by μPIXE profiling and of Zn by histochemical mapping in cortical bone from metacarpal bovine bone (1 year old). For μPIXE, rectangular samples 1-2 mm thick were cut, ethanol fixed, dried at 55 deg. C, and polished. The measurements were carried out at the Rossendorf Nuclear Microprobe with a 3.1 MeV proton beam focused to a spot of ∼ 3 μm, at ∼ 4 μm effective resolution. Unique scans ∼ 500 x 12 μm (128 x 3 pixels) and side-to-side sequences of scans were done for Ca and Zn, taking the average of the three lines for the profiles of the elements. The noise in Zn profiles was filtered by Fast Fourier Transform (FFT). For histochemistry, samples were embedded in polymethyl-methacrylate and Zn was stained in thin sections by the sulphide-silver reaction (Timm's method). In the histochemical maps, Zn is stained at the bone surfaces. The periosteal surface layer is about 50 μm thick. The endosteal layer is more uneven, but generally up to 200 μm thick. The osteonic bone in the central zone is stained in single osteons at the 5-20 μm thick internal layer surrounding Havers' canal and a very thin layer representing the intracanalicular spaces. A comparison of the μPIXE side-to-side Ca profiles and FFT-filtered Zn profiles showed significant Zn changes associated with every Ca variation, whether small or large. As the line randomly crossed both zones of compact mineralized tissue and zones of void-porous non-mineralized tissue (chiefly osteonic canals), bone surfaces were identified as the regions where the Ca profile abruptly decayed, and the Zn profile was higher there than in the compact regions. Thus both μPIXE and histochemistry showed that Zn mainly localized at the surfaces of various structures in bone tissue. (authors)