Formation of Ultracracks in Methacrylate-Embedded Undecalcified Bone Samples by Exposure to Aqueous Solutions

Back-scattered electron (BSE) imaging allows the visualization and evaluation of mineralized bone structures down to the micrometer range. To produce undecalcified bone sections with adequate structural and surface integrity, bone specimens are usually resin-embedded, followed by cutting, grinding , and polishing procedures. In samples prepared this way, so-called ultracracks were detected as black clefts in the lamellar bone matrix by BSE-imaging at magnifications ranging from 1000x to 3000x. By charging phenomena in the secondary electron (SE) mode of the scanning electron microscope (SEM), these clefts can be proven to be open cracks in the sample surface, and thus, as being created after embedding. These ultracracks seem to be a swelling effect of the bone matrix when it is exposed to water on the sample surface, followed by shrinking during drying . They did not occur, when water-free preparation techniques, like micromilling, were used and all water contact with the sample surface was avoided. This observation using the BSE-technique in SEM, and the simple method of discrimination between cracks existing before embedding and cracks newly generated during or after embedding, seem important for ultrastructural investigations of mineralized bone tissue, particularly for the evaluation of microcracks after loading or for the study of bone-implant interfaces

A New Scanning Electron Microscopy Approach to the Quantification of Bone Mineral Distribution: Backscattered Electron Image Grey-Levels Correlated to Calcium Kα-Line Intensities

The introduction of backscattered electron (BSE) imaging in scanning electron microscopy (SEM) has led to new possibilities for the evaluation of mineral distributions in bone on a microscopic level. The different grey-levels seen in the BSE-images can be used as a measure for the local mineral content of bone. In order to calibrate these BSE-grey-levels (BSE-GL) and correlate them to mineral contents, various attempts, using reference samples with known weighted mean atomic number and/or using simulated bone tissues with known hydroxyapatite concentrations, have been made. In contrast, a new approach is presented here based on measurements of the X-ray intensities of the calcium Kα-line on selected areas of real bone samples; the measured intensities are then related to the corresponding BSE-GL. A linear positive correlation between weight percent (wt%) calcium and BSE-GL was found. When the BSE-mode is standardized using carbon and aluminum as references, the different mineral contents in bone samples can be recorded as BSE-GL, calibrated to wt% of calcium or hydroxyapatite (HA), respectively. The resulting mineral concentration histograms have a dynamic range from O to 89 wt% HA and have a binwidth resolution of 0.45 wt% HA. The presented modifications of the BSE method strongly enhance its feasibility in the field of bone research and its application as a special diagnostic tool for bone diseases