Surface staining is widely used for histological studies involving undecalcified thick bone sections. Individual osteons, particularly newly formed ones stained with toluidine blue (TB), show various color intensities. We studied the correlations between TB color intensity and the differences in stiffness and degree of mineralization of individual osteons in undecalcified histological sections of goat tibial diaphysis, measured by scanning acoustic microscopy (SAM) and contact microradiography (CMR), respectively. Results showed that all three measurements correlated significantly with each other (r = 0.567 - 0.786; all P < or = 0.01). The TB surface staining intensity of individual osteons correlated better with the reflection coefficient (stiffness index) measured by SAM (r = 0.713) than with the aluminum step-wedge equivalent thickness measured on CMR micrographs (r = 0.567). The aluminum step-wedge equivalent thickness of individual osteons on CMR correlated slightly better with the SAM reflection coefficient (r = 0.786) than with the TB surface staining intensity (r = 0.713). The results of this study suggest that TB surface staining may be used as a simple method for indicating differences in stiffness and degree of mineralization in individual osteons in comparative histological studies.

Single human osteons were isolated by propagating fractures along their natural boundaries. Furthermore, decalcified osteons were mechanically manipulated to expose their interlamellar interfaces, making possible the study of collagen fibers by means of scanning electron microscopy (SEM). Isolation of single osteons of a wide range of lengths, as described in this paper, makes possible for the first time the study of their mechanical properties in all possible modes. SEM studies of exposed lamellar interfaces reveal that collagen fiber orientations are more complex than previously suggested and suggest further studies in an effort to solve past controversies on collagen fiber orientations in human bone.

Scanning acoustic microscopy (SAM) of Haversian bone at high frequency clearly shows differences in the brightness (gray) levels of individual osteons, which were observed to correspond to the differences in reflection coefficients measured with the SAM technique: the darker the bony regions on the SAM micrographs, the lower the SAM reflection coefficient values, and vice versa. We studied the correlation between differences in brightness and the related reflection coefficients of osteons on undecalcified transverse sections of goat tibial diaphysis using a 400-MHz burst mode lens on the Olympus UH3 SAM. Results showed that there was a strong linear correlation between brightness intensities and the corresponding reflection coefficients, with a correlation coefficient r = 0.99 ( P << 0.001) using gray level 210 as cutoff. This result suggested that the image brightness intensity of osteonic bone could be measured retrospectively to provide information on the stiffness of the corresponding bone matrix.

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