Probing the microporosity and 3D spatial distribution of calcium phosphate cement/hydrogel biomaterials using FIB/SEM at cryogenic temperatures

May 05, 2023

Mouad Essani (1), Baptiste Charbonnier (2), Nicolas Stephant (1), Hilel Moussi (1) (2), Pierre Weiss (3), Jean Le Bideau (1) and Patricia Abellan (1)
Materials Advances. Issue 11 (May 5 2023). DOI:


Considerable clinical success has been achieved ever since calcium phosphate cements (CPCs) were introduced in bone regenerative therapies. The development of novel CPC/hydrogel composites with increased mechanical and biological properties is critical for the next generation of bone substitutes. Focused ion beam/scanning electron microscopy (3D FIB/SEM) tomography combined with energy dispersive spectrometry (EDS) provide the ability to elucidate the spatial distribution of the different phases (organic/inorganic) with tens of nanometer resolution, which is crucial information for the development of biomaterials with optimized biodegradation. Characterization under cryogenic conditions by using cryo-FIB/SEM in combination with cryo-EDS allows us to resolve the sub-micrometer structural features of such composites. In this study, we present a multidimensional, low dose structural and chemical analysis of calcium deficient hydroxyapatite (CDHA) mixed with two different hydrogels: gelatin and silanized hyaluronic acid (Si-HA). We show that, depending on the chemical nature of the hydrogel, CDHA/hydrogel compounds can exhibit different properties, particularly microporosity and phase distribution at the (sub) microscale. The incorporation of Si-HA and gelatin both resulted in an increase of the amount of microporosity within the composites. While Si-HA formed a homogeneous mixture with CDHA, a separate phase of gelatin was observed inside the CDHA/gelatin compound. Our results also indicate that structural modification occurs when applying sample preparation protocols or analytical methods that are subject to altering the hydration level within these biomaterials and thus, cryogenic temperatures are necessary for their characterization using electron microscopy.

How Our Software Was Used

3D volume reconstruction of FIB-SEM data, segmentation of structural features and quantification of microporosity (defined in biomaterial science as a pore size of less than 10 μm) were performed using Dragonfly. In addition, thickness meshes were used to determine the mean pore size for each compound.

Author Affiliation

(1) Nantes Université, CNRS, Institut des Matériaux de Nantes Jean Rouxel (IMN), F-44000 Nantes, France
(2) Nantes Université, INSERM, UMR 1229 RMeS/ONIRIS, Regenerative Medicine and Skelton Laboratory, F-44042 Nantes, France
(3) Nantes Université, CHU Nantes, INSERM, UMR 1229 RMeS/ONIRIS, Regenerative Medicine and Skeleton Laboratory, F-44042 Nantes, France