Bone erosion is considered a typical characteristic of advanced or complicated cholesteatoma (CHO), although it is still a matter of debate if bone erosion is due to osteoclast action, being the specific literature controversial. The purpose of this study was to apply a novel scanning characterization approach, the BSE 3D image analysis, to study the pathological erosion on the surface of human incus bone involved by CHO, in order to definitely assess the eventual osteoclastic resorptive action. To do this, a comparison of BSE 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 software Hitachi 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 the morphological characterization, allowing us to state that surface erosion of the incus is due to osteoclast action. Moreover, our observation and processing image workflow are the first in the literature showing the presence not only of bone erosion but also of matrix vesicles releasing their content on collagen bundles and self-immuring osteocytes, all markers of new bone formation on incus bone surface. On the basis of recent literature, it has been hypothesized that inflammatory environment induced by CHO may trigger the osteoclast activity, eliciting bone erosion. The observed new bone formation probably takes place at a slower rate in respect to the normal bone turnover, and the process is uncoupled (as recently demonstrated for several inflammatory diseases that promote bone loss) thus resulting in an overall bone loss. 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 a new era of quantitative SEM morphology.
Consensus-based recommendations for the definition of advanced or complicated cholesteatoma (CHO) [
We observed eighteen incus bones recovered during surgical procedures of CHO removal obtained with patients’ informed consent and 1 unaffected incus bone (the control) from cadaver.
We studied eighteen femoral neck biopsies from postmenopausal women with hip arthrosis and osteoporosis who underwent surgical hip substitution, 1 femoral neck biopsy from woman without osteoporosis. BMD and T-score to assess bone osteoporosis condition were evaluated by DEXA (Hologic Delphi) before the surgical operation. Samples were obtained with patients’ informed consent.
The study was approved by the Institutional Ethics Board and adhered to the tenets of the Declaration of Helsinki.
Samples were fixed immediately upon recovery in 2.5% glutaraldehyde in PBS at 4°C for 48 h, then immersed in a 3% hydrogen peroxide solution for 48 h at room temperature (for bone marrow removal), and then rinsed with distilled water. Samples were then sonicated in a sonic device [
Samples were fixed immediately upon recovery in 2.5% glutaraldehyde in PBS at 4°C for 48 h; then, they were gently sonicated in an ultrasonic device (to remove excess of keratinizing squamous epithelium that would have prevented surface observation). Fifteen samples were prepared for SEM (as previously described for femur neck) and sputter coated with platinum using an Emitech K 550 sputter coater (Emitech, Corato, Italy). Observations were conducted by a Hitachi FE SEM S 4000 operating at 7 kV and by a Hitachi SU 3500 (Hitachi High-Technologies Europe GmbH, Mannheim, Germany), at 10 kV in SE mode.
Three samples, after fixation in 2.5% glutaraldehyde in PBS at 4°C for 48 h, were only gently sonicated in a sonic device [
Hitachi SU 3500 is equipped with a four-quadrant BSE detector that allows to acquire four images simultaneously with only one scan. The four pictures are then integrated into 3D images and processed to extract quantitative information (all those steps were performed by the software Hitachi Map 3D 7.4 Digital surf, Besançon, France). To obtain this kind of data is extremely useful to implement the morphological classification parameters usually used to characterize resorbing and forming bone surfaces. In fact, acquisition of quantitative resorption pit information such as area, mean depth, and volume allows to compare pits from different sources (femur and incus) and finally assess if they have the same origin. Regions containing resorption bay were analyzed in both incus bone and femur neck samples. BSE 3D images of well delimited resorption pits were acquired, 4 images were combined by the software, and 3D reconstruction was obtained. Resorption pit area, mean depth, and volume were extracted by MountainsMap software after 3D image reconstruction. In more detail, we performed single pit selection on the 3D image reconstruction, followed by automatic measurement of area, mean depth, and volume. Data were collected and statistically analyzed by SPSS statistical software. The following test was performed: summary statistic to assess the normality of distribution of pit area, mean depth, and volume values; independent sample
The variable pressure scanning electron microscopy used in this study (VP-SEM, Hitachi SU3500) is equipped with dual energy dispersive X-ray spectroscopy (dEDS, Bruker XFlash® 6|60) detectors. This instrument has the ability to perform simultaneously multimodal imaging and spatial distribution chemical mapping, a truly powerful analytical approach to study biological surfaces in their native state. The XFlash® 6|60 is particularly suitable for applications with relatively low X-ray yield, as common in the area of nanoanalysis.
Incus bone areas were classified as resorptive and forming bone surfaces, according to widely accepted morphological criteria described in literature [
Each CHO incus sample was observed by SEM at low magnification following a precise scanning pathway, in order to assess the general bone morphology and define areas suitable for high magnification observations. This method allowed counting of nutrient foramina opening onto the surface (49 foramina on 18 bones) and identification of areas with marked bone erosion and, interestingly, areas with new bone formation. It is still a matter of debate if bone erosion is due to osteoclast action; moreover, new bone formation was never been described in the incus bone affected by CHO. To get an insight on these findings, we performed observations at magnifications ranging from 400x to 600x, 3D image reconstruction, and EDS analysis.
Before showing images of samples with resorption areas, two images of normal surfaces are presented (Figure
(a) SE mode, 400x. Incus bone surface from cadaver, normal surface. (b) SE mode, 400x. Trabecular bone from patient without osteoporosis, normal surface.
Images of CHO incus bone surface showed 67% of nutritive foramina surrounded by large resorption bays that seem to radiate from nutritive foramen opening (Figures
(a) SE mode, 270x. Nutritive foramen from CHO incus bone. On the right side of the image, large resorption bays, extending since into the foramen, are visible. On the left corner of the picture, osteocytic lacunae are visible. (b) BSE-COMP mode, 270x of same sample. Darker (demineralized) areas correspond to deeper resorption bays. This field shows both bone resorption and bone formation phenomena.
Observed at higher magnification CHO incus bone resorption bays and pits (Figure
(a) FE SEM 700x, CHO incus bone resorption bay at higher magnification, osteoclast snake trail pathway is visible (arrows). At the center of the resorption bay, a small promontory rises being relatively resistant to resorption. (b) FE SEM, 600x, osteoclastic resorption bay on osteoporotic human femur neck (arrows), they are unequivocally of osteoclast origin and are undistinguishable from those in (a).
To definitely assess if incus bone resorption bay is a product of osteoclasts action, we used Hitachi MountainsMap© software to perform a 3D reconstruction from 4 BSE mode images (Figure
3D reconstruction from 4 images in BSE mode. Each resorption bay contains several pits.
A small area was extracted from a 3D-reconstructed image, and each single pit in the small area was analyzed by the software to calculate: area, mean depth, and volume (Figures
(a) The extracted area of a resorption bay from a larger 3D-reconstructed image. (b) A delimited single pit from which software calculated parameter values.
We analyzed 79 pits, for each considered parameter values which were recorded and statistically evaluated by SPSS statistical software. Firstly, a summary statistic was performed on data collected for each parameter, to assess normality of distribution (Table
Summary statistic of area, mean depth, and volume values.
Pit | Distribution | Area |
Mean depth |
Volume |
---|---|---|---|---|
Incus | Normal | |||
Femur neck | Normal |
Independent sample
Area | Mean depth | Volume | ||||
---|---|---|---|---|---|---|
Incus | Femur | Incus | Femur | Incus | Femur | |
Sample size | 79 | 79 | 79 | 79 | 79 | 79 |
Arithmetic mean | 120.48 | 121.34 | 0.799 | 0.784 | 96.48 | 94.99 |
95% CI for the mean | 118.57 to 122.39 | 116.15 to 126.54 | 0.77 to 0.82 | 0.74 to 0.82 | 93.51 to 99.45 | 89.69 to 100.28 |
Variance | 72.95 | 538.45 | 0.011 | 0.025 | 173.31 | 559.32 |
St deviation | 8.54 | 23.20 | 0.10 | 0.16 | 13.16 | 23.65 |
St error mean | 0.96 | 2.61 | 0.011 | 0.018 | 1.49 | 2.66 |
Levene |
Graphs represent distribution of pit measurement data (from the left to the right): incus area vs. femur area; incus mean depth vs. femur mean depth; incus volume vs. femur volume.
The detailed incus surface observation allowed another interesting finding, the observation of new bone-forming areas on incus surface. Our SEM images are the first to show this process on incus. Mineralizing vesicles releasing their content on collagen bundles are shown in Figures
SE, BSE comp 5000x, new bone formation on CHO incus bone surface. (a) SE mineralizing matrix vesicles releasing their content on collagen bundles (arrows). (b) BSE comp mineralizing matrix vesicles (arrows) appear as bright and rough spheres. Collagen fibres and bundles with variable mineralization degree are visible. Mineralized areas appear lighter at BSE mode.
These areas were also analyzed in uncoated samples by variable pressure SEM dEDS analysis. Variable pressure SEM allows the observation of uncoated samples, avoiding metal coating disturbance during elemental analysis. Areas containing calcified matrix vesicles (Figure
BSE Comp, 3000x, dEDS analysis, confirmation of new bone formation on CHO incus bone. (a) VP SEM BSE image shows matrix vesicles (arrows). (b) Elemental distribution (dEDS analysis) allows identification of chemical species, calcium in matrix vesicles (yellow) and sulphur in extracellular matrix (red).
A later stage in new bone formation is represented by osteocyte self-immuring in forming bone areas. In Figure
New bone formation on CHO incus bone surface (a), FE SEM, 250x, osteocytic lacunae (arrows) formed by self-immuring osteocytes. (b) SE, 5000x, high magnification of an osteocytic lacuna, the floor appears less mineralized and spotted by deep holes to accommodate osteocyte cellular processes.
Prominent theories on bone resorption in CHO are osteoclast activation; pressure necrosis; and acid lysis, enzyme mediation, and inflammatory mediation [
In some studies [
Our results showed that no difference exists between area, mean depth, and volume values between incus and femur resorption pit, allowing us to state that surface erosion on the incus is due to osteoclast action.
Osteoclasts are multinucleated cells, they differentiate from monocyte-lineage hematopoietic precursor cells [
Inflammatory cells and an osteoclast on incus affected by CHO surface, FE SEM 3000x. Active macrophage (blue), lymphocyte (red), and osteoclast (yellow).
Bone homeostasis is maintained balancing bone-resorbing osteoclast and bone-forming osteoblast activity, alteration of this balance causes bone loss, that is not recovered by new bone formation. In fact, in inflammation, disease-like RA bone erosion results from excessive bone resorption and markedly limited bone formation [
The innovative quantitative approach used in this paper implements the classical surface morphological characterization, allowing us to state that surface erosion of the incus is due to osteoclast action. Moreover, our observation and processing image workflow are the first in the literature showing the presence not only of bone erosion but also of matrix vesicles releasing their content on collagen bundles and self-immuring osteocytes, all markers of new bone formation on incus bone surface. On the basis of recent literature [
Data are stored in computer of our institution and are available upon request.
The authors declare that they have no conflicts of interest.
This study was funded by the Sapienza University of Rome Research projects funding.