# Imaging Bone Multi-Scale Structure: Towards Improved
Implants

Corinne Mahfouz, Thomas Reiss, Patrick Aimedieu, Benjamin Smaniotto,
Elsa Vennat, Sophie Lecann

► To cite this version:

Corinne Mahfouz, Thomas Reiss, Patrick Aimedieu, Benjamin Smaniotto, Elsa Vennat, et al. Imaging Bone Multi-Scale Structure: Towards Improved Implants. Multidisciplinary Biomechanics Journal, 2025, 50th congress of the.., 10.46298/mbj.16240. hal-05191795v2

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Submitted on 23 Oct 2025

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# Imaging Bone Multi-Scale Structure:
Towards Improved Implants

Corinne Mahfouz<sup>a, c*</sup>, Thomas Reiss<sup>a</sup>, Patrick Aimedieu<sup>b</sup>,
Benjamin Smaniotto<sup>a</sup>, Elsa Vennat<sup>a</sup>, Sophie Le Cann<sup>c</sup>

<sup>a</sup> LMPS, Université Paris-Saclay, CentraleSupélec, CNRS, Gif-sur-Yvette, France

<sup>b</sup> Navier, CNRS, ENPC, UGE, Champs-sur-Marne, France

<sup>c</sup> MSME, CNRS, UPEC, Créteil, France

*Corresponding author: corinne.mahfouz@centralesupelec.fr

Received date: 06/04/2025

Accepted date: 27/06/2025

Publication date: 27/10/2025

**Keywords:** bone, microstructure, SEM, micro-CT, image analysis

© 2025 The Authors
Licence CC-BY 4.0
Published by Société de Biomécanique

## 1. Introduction

Bone is a biological composite with a heterogeneous multi-scale structure (Buss et al. 2022) that adapts to the presence of an implant. This osseointegration process around implants is a multiscale phenomenon (Shah et al. 2019, Deering et al. 2023) and is characterized by the formation of new bone at the bone-implant interface. The dynamics of osseointegration involve structural and compositional changes in the bone around implants and its studies often require animal experimentation (in our case on a rabbit model). In order to characterize newly formed bone at the interface, a comparative approach with healthy rabbit bone will be adopted. The aim of this work is to gain a deeper understanding of the bone microstructure of the healthy rabbit model, for which data is limited, particularly at small scales.

## 2. Methods

### 2.1 X-ray micro-computed tomography (micro-CT)

First, three half healthy rabbit tibiae (distal halves, 6 cm in length) were scanned with a micro-CT scanner (North Star Imaging, Tomograph X50, and RX Solutions, Ultratom, voxel size 13 µm).

Then, three smaller samples of 4 x 1 x 4 mm³ were sectioned from the middle shaft of the tibia and scanned with a higher resolution (RX Solutions, Ultratom, voxel size 2 µm).

### 2.2 Scanning electron microscopy (SEM)

The same samples were processed (dehydration in graded ethanol, embedding, polishing, slicing) and

visualized with a Scanning Electron Microscope (SEM) (Zeiss Leo Gemini 1530) in backscattered electron (BSE) mode.

### 2.3 Image segmentation and analysis

Segmentation, 2D and 3D morphology measurements were done using Dragonfly software (Comet Technologies Canada Inc., Montreal, Canada.).

On micro-CT images, intra-cortical porosities were segmented from bone by grey-level automatic thresholding (Otsu). A volume criterion of [80-2400 µm³] was used to classify osteocyte lacunae. Larger porosities were counted as canals.

To analyze SEM images, a deep-learning model was created in Dragonfly to segment bone, porosities and cracks to exclude the latter from quantifications. A surface criterion of [15-240 µm²] was used to classify osteocyte lacunae, larger porosities were counted as canals.

## 3. Results and discussion

Key structural parameters in healthy rabbit bone were analyzed and quantified at different scales. At the macroscale, trabecular bone was distinguished from cortical bone using global micro-CT scans and both morphological set of parameters such as trabecular or cortical thickness were assessed. Their ranges were in accordance with the literature. Large scale porosities in cortical bone, resorption spaces or “cutting cones” (~ 100 µm maximum in diameter), were also resolved in the global micro-CT scan. They attest of the ongoing remodeling
process (bone resorption and subsequent bone formation). At the microscale, two types of porosities were identified: osteonal canals (~ 20 µm) and osteocyte lacunae (~ 10 µm) and were quantified in 3D on local higher resolution micro-CT scans (Table 1), consistently with literature (Pazzaglia et al. 2010). Osteonal canals (in blue) form a highly inter-connected network and appear to have a preferred longitudinal direction, in reference to the long axis of the bone (Fig.1 (a)). Osteocyte lacunae (in purple) were of ellipsoidal shape and seemed to also follow this direction, with their long axis oriented longitudinally.

Using local micro-CT scans in these conditions, it was not possible to resolve noteworthy variations in the surrounding bone matrix. However, local variations of grey-values intensities, often indicating mineralized areas in bone were seen in SEM images. Zones of relatively more mineralized bone tissue were identified in the interstitial space between osteons. At high magnifications (x10 000), “lamellae” of roughly 2 µm in thickness were formed around osteonal canals. This can be seen as concentric rings of alternating grey-level intensities around the canal (Fig.1 (b)). Smaller porosities, canaliculi (~ 250 nm) connected to lacunae appearing as low-grey level features in Fig.1 (b) could also be identified at this level.

## 4. Conclusions

Healthy rabbit bone structure was analyzed through a multi-scale and multimodal correlative approach. 3D micro-CT high resolution scans allowed visualization and quantification of the intra-cortical canal network and osteocyte lacunae. This analysis was completed by a 2D SEM analysis of porosities of the same samples. SEM imaging revealed the local heterogeneity in the bone matrix and smaller scale canaliculi (~ 250 nm). This work will serve as a baseline for the rabbit model in bone multi-scale research and will be used as reference for future studies of newly formed bone at the bone-implant interface. Further studies will explore local mechanical properties, through nanoindentation tests, of both healthy and newly formed (at the bone-implant interface) tissue.

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<figure><img src="image_4.png" /><figcaption>**Figure 1.** (a). 3D visualization of segmented canal network and osteocyte lacunae on local micro-CT scan of cortical bone. (b). cross-sectional SEM image</figcaption></figure>

<table><caption>**Table 1.** Morphology analysis of cortical bone porosities in 3D (micro-CT)</caption><thead><tr><th>Cortical Porosity</th><th>Parameter</th><th>Mean Value ± SD</th></tr></thead><tbody><tr><td rowspan="3">Osteonal Canals</td><td>Volume Fraction (%)</td><td>2.29 ± 0.91</td></tr><tr><td>Thickness (µm)</td><td>18.20 ± 3.88</td></tr><tr><td>Volume Fraction (%)</td><td>1.41 ± 0.82</td></tr><tr><td rowspan="3">Osteocyte Lacunae</td><td>Thickness (µm)</td><td>10.64 ± 1.51</td></tr><tr><td>Individual Volume (µm³)</td><td>307.90 ± 169.47</td></tr><tr><td>Sphericity</td><td>0.84 ± 0.064</td></tr></tbody></table>
**Conflict of Interest Statement**

None

**Funding**

This work was funded by the ANR MuBIA (23-CE51-0018), Paris Ile-de-France Region (DIM “Respore”) and the CNRS (MITI interdisciplinary program).

**References**

Buss, D. J., Kröger, R., McKee, M. D., & Reznikov, N. (2021). Hierarchical organization of bone in three dimensions: A twist of twists. *Journal of Structural Biology*, *X*, **6**, 100057. https://doi.org/10.1016/j.yjsbx.2021.100057

Shah, F. A., Thomsen, P., & Palmquist, A. (2019). Osseointegration and current interpretations of the bone-implant interface. *Acta Biomaterialia*, **84**, 1-15. https://doi.org/10.1016/j.actbio.2018.11.018

Deering, J., Chen, J., Mahmoud, D., Tang, T., Lin, Y., Fang, Q., Wohl, G. R., Elbestawi, M., & Grandfield, K. (2023). Characterizing mineral ellipsoids in new bone formation at the interface of Ti6Al4V porous implants. *Advanced Materials Interfaces*, *10*(24), 2300333. https://doi.org/10.1002/admi.202300333

Pazzaglia, U. E., Zarattini, G., Giacomini, D., Rodella, L., Menti, A. M., & Feltrin, G. (2010). Morphometric analysis of the canal system of cortical bone: An experimental study in the rabbit femur carried out with standard histology and micro-CT. *Anatomia, histologia, embryologia*, *39*(1), 17-26. https://doi-org/10.1111/j.1439-0264.2009.00973.x