title: Focus Ion Beam/Scanning Electron Microscopy Characterization of Osteoclastic Resorption of Calcium Phosphate Substrates.
abstract: This article presents the application of dual focused ion beam/scanning electron microscopy (FIB-SEM) imaging for preclinical testing of calcium phosphates with osteoclast precursor cells and how this high-resolution imaging technique is able to reveal microstructural changes at a level of detail previously not possible. Calcium phosphate substrates, having similar compositions but different microstructures, were produced using low- and high-temperature processes (biomimetic calcium-deficient hydroxyapatite [CDHA] and stoichiometric sintered hydroxyapatite, respectively). Human osteoclast precursor cells were cultured for 21 days before evaluating their resorptive potential on varying microstructural features. Alternative to classical morphological evaluation of osteoclasts (OC), FIB-SEM was used to observe the subjacent microstructure by transversally sectioning cells and observing both the cells and the substrates. Resorption pits, indicating OC activity, were visible on the smoother surface of high-temperature sintered hydroxyapatite. FIB-SEM analysis revealed signs of acidic degradation on the grain surface under the cells, as well as intergranular dissolution. No resorption pits were evident on the surface of the rough CDHA substrates. However, whereas no degradation was detected by FIB sections in the material underlying some of the cells, early stages of OC-mediated acidic degradation were observed under cells with more spread morphology. Collectively, these results highlight the potential of FIB to evaluate the resorptive activity of OC, even in rough, irregular, or coarse surfaces where degradation pits are otherwise difficult to visualize.
authors.affiliation: 1 Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgical Engineering, Technical University of Catalonia (UPC) , Barcelona, Spain .|2 Center for Research in NanoEngineering (CRnE) , UPC, Barcelona, Spain .
authors.forename: Anna
authors.initials: A
authors.lastname: Diez-Escudero
authors.affiliation: 1 Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgical Engineering, Technical University of Catalonia (UPC) , Barcelona, Spain .|2 Center for Research in NanoEngineering (CRnE) , UPC, Barcelona, Spain .
authors.forename: Montserrat
authors.initials: M
authors.lastname: Espanol
authors.affiliation: 1 Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgical Engineering, Technical University of Catalonia (UPC) , Barcelona, Spain .|2 Center for Research in NanoEngineering (CRnE) , UPC, Barcelona, Spain .
authors.forename: Edgar B
authors.initials: EB
authors.lastname: Montufar
authors.affiliation: 3 Orthopaedic Pathophysiology and Regenerative Medicine Unit , IstitutoOrtopedico Rizzoli, Bologna, Italy .|4 Department of Biomedical and Neuromotor Sciences, University of Bologna , Bologna, Italy .
authors.forename: Gemma
authors.initials: G
authors.lastname: Di Pompo
authors.affiliation: 3 Orthopaedic Pathophysiology and Regenerative Medicine Unit , IstitutoOrtopedico Rizzoli, Bologna, Italy .
authors.forename: Gabriela
authors.initials: G
authors.lastname: Ciapetti
authors.affiliation: 3 Orthopaedic Pathophysiology and Regenerative Medicine Unit , IstitutoOrtopedico Rizzoli, Bologna, Italy .|4 Department of Biomedical and Neuromotor Sciences, University of Bologna , Bologna, Italy .
authors.forename: Nicola
authors.initials: N
authors.lastname: Baldini
authors.affiliation: 1 Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgical Engineering, Technical University of Catalonia (UPC) , Barcelona, Spain .|2 Center for Research in NanoEngineering (CRnE) , UPC, Barcelona, Spain .|5 Institute for Bioengineering of Catalonia , Barcelona, Spain .
authors.forename: Maria-Pau
authors.initials: MP
authors.lastname: Ginebra
mesh_terms: D001862:Bone Resorption / Q000000981:diagnostic imaging / Q000378:metabolism*; D002130:Calcium Phosphates / Q000378:metabolism*; D002478:Cells, Cultured; D017886:Durapatite / Q000378:metabolism; D006801:Humans; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D010010:Osteoclasts / Q000378:metabolism* / Q000648:ultrastructure; D013379:Substrate Specificity
chemical_list: D002130:Calcium Phosphates; D017886:Durapatite
keywords: bone regeneration; calcium phosphate; focus ion beam; osteoclast; resorption; scanning electron microscopy