SEM BSE 3D image analysis of human incus bone affected by cholesteatoma ascribes to osteoclasts the bone rrosion and VpSEM dEDX analysis reveals new bone formation

Bone erosion is considered a typical characteristic of advanced or complicated cholesteatoma (CHO), although it is still a matter ofdebate if bone erosion is due to osteoclast action, being the specific literature controversial. The purpose of this study was to apply anovel scanning characterization approach, the BSE 3D image analysis, to study the pathological erosion on the surface of humanincus bone involved by CHO, in order to definitely assess the eventual osteoclastic resorptive action. To do this, a comparison ofBSE 3D image of resorption lacunae (resorption pits) from osteoporotic human femur neck (indubitably of osteoclastic origin)with that of the incus was performed. Surface parameters (area, mean depth, and volume) were calculated by the softwareHitachi MountainsMap© from BSE 3D-reconstructed images; results were then statistically analyzed by SPSS statistical software.Our findings showed that no significant differences exist between the two groups. This quantitative approach implements themorphological characterization, allowing us to state that surface erosion of the incus is due to osteoclast action. Moreover, ourobservation and processing image workflow are the first in the literature showing the presence not only of bone erosion but alsoof matrix vesicles releasing their content on collagen bundles and self-immuring osteocytes, all markers of new bone formationon incus bone surface. On the basis of recent literature, it has been hypothesized that inflammatory environment induced byCHO may trigger the osteoclast activity, eliciting bone erosion. The observed new bone formation probably takes place at aslower rate in respect to the normal bone turnover, and the process is uncoupled (as recently demonstrated for severalinflammatory diseases that promote bone loss) thus resulting in an overall bone loss. Novel scanning characterizationapproaches used in this study allowed for the first time the 3D imaging of incus bone erosion and its quantitative measurement,opening a new era of quantitative SEM morphology.