In police investigations and crime novels, in order to discover the culprit, it is often necessary to find and analyze fingerprints first. We used this same approach to solve an enigma of a pathology of the ear, cholesteatomas (CHO), a condition where an epithelial mass forms in the middle ear, causing inflammation, pain and hearing loss. The mystery of this pathology: which cell erodes the middle ear incus bone?
Incus bone erosion is considered a typical characteristic of advanced CHO; however, it is still a matter of debate if resorption lacunae on the surface of incus bones are caused by osteoclast1 action. To address this issue, we observed resorption bays in the neck of an osteoporotic human femur (indubitably of osteoclastic origin) and those present in incus bone affected by CHO.
We used BSE images obtained by our scanning electron microscope Hitachi VP-SEM SU 3500, equipped with a four-quadrant BSE detector that allows the obtention of four images simultaneously in only one scan. The four pictures obtained were reconstructed into a 3D image using HitachiMap 3D software based on Mountains® and processed to extract quantitative information.
Above: (a) FE SEM 700X, CHO incus bone resorption bay formed by several lacunae, osteoclast snake trail pathway is visible (arrows). At the centre of the resorption bay a small promontory rises, which is relatively resistant to resorption. (b) FE SEM,600X, osteoclastic resorption bay formed by several lacunae on osteoporotic human femur neck (arrows), they are unequivocally of osteoclast origin and are undistinguishable from those in fig a.
Obtaining this type of data was extremely useful in implementing the morphological classification parameters generally used to characterize the formation and resorption of bone surfaces. In fact, acquirement quantitative data of resorption lacunae, such as area, mean depth and volume allowed us to compare lacunae derived from different sources (femur and incus) and finally, assess if they had the same origin. Resorption lacunae area, mean depth and volume were calculated with Hitachi Map 3D. For further detail, we performed a single lacuna selection on the 3D image reconstruction, followed by automatic measurements of the area, the mean depth, and the volume.
Above: (a) Extracted area of a resorption bay, 3D reconstructed image. (b) A delimited single lacuna from which HitachiMap 3D software calculated morphological parameter values.
The data, collected and statistically analyzed, showed that no significant differences exist between the two groups. Therefore, if the fingerprints are the same, then the culprit is the same: in this case the osteoclast. This quantitative approach implemented morphological characterization, allowing us to state that surface erosion of the incus was due to osteoclast action.
The novel scanning characterization approaches used in this study allowed for the first time the 3D imaging of incus bone erosion and its quantitative measurement, opening up a new era of quantitative SEM morphology on biological samples.
1 A type of bone cell that breaks down bone tissue
Above: Electron microscopy unit of the Human Anatomy division, SAIMLAL Department, Sapienza University of Rome, Rome, Italy. From left to right: Mr Ezio Battaglione, Prof. Selenia Miglietta, Prof. Michela Relucenti, Prof. Giuseppe Familiari, Prof. Stefania A.Nottola, Dr. Orlando Donfrancesco. On the background, the portrait of Prof. Pietro Motta, founder of the laboratory in 1963.
Instrument & software used
Scanning Electron Microscope Hitachi VP-SEM SU 3500 + Hitachi Map 3D, software based on Mountains® technology