title: Osteoclast differentiation from human blood precursors on biomimetic calcium-phosphate substrates.
abstract: The design of synthetic bone grafts to foster bone formation is a challenge in regenerative medicine. Understanding the interaction of bone substitutes with osteoclasts is essential, since osteoclasts not only drive a timely resorption of the biomaterial, but also trigger osteoblast activity. In this study, the adhesion and differentiation of human blood-derived osteoclast precursors (OCP) on two different micro-nanostructured biomimetic hydroxyapatite materials consisting in coarse (HA-C) and fine HA (HA-F) crystals, in comparison with sintered stoichiometric HA (sin-HA, reference material), were investigated. Osteoclasts were induced to differentiate by RANKL-containing supernatant using cell/substrate direct and indirect contact systems, and calcium (Ca++) and phosphorus (P5+) in culture medium were measured. We observed that OCP adhered to the experimental surfaces, and that osteoclast-like cells formed at a rate influenced by the micro- and nano-structure of HA, which also modulate extracellular Ca++. Qualitative differences were found between OCP on biomimetic HA-C and HA-F and their counterparts on plastic and sin-HA. On HA-C and HA-F cells shared typical features of mature osteoclasts, i.e. podosomes, multinuclearity, tartrate acid phosphatase (TRAP)-positive staining, and TRAP5b-enzyme release. However, cells were less in number compared to those on plastic or on sin-HA, and they did not express some specific osteoclast markers. In conclusion, blood-derived OCP are able to attach to biomimetic and sintered HA substrates, but their subsequent fusion and resorptive activity are hampered by surface micro-nano-structure. Indirect cultures suggest that fusion of OCP is sensitive to topography and to extracellular calcium.
  
  
  
  The novelty of the paper is the differentiation of human blood-derived osteoclast precursors, instead of mouse-derived macrophages as used in most studies, directly on biomimetic micro-nano structured HA-based surfaces, as triggered by osteoblast-produced factors (RANKL/OPG), and influenced by chemistry and topography of the substrate(s). Biomimetic HA-surfaces, like those obtained in calcium phosphate cements, are very different from the conventional calcium phosphate ceramics, both in terms of topography and ion exchange. The role of these factors in modulating precursors' differentiation and activity is analysed. The system is closely reproducing the physiological process of attachment of host cells and further maturation to osteoclasts toward resorption of the substrate, which occurs in vivo after filling bone defects with the calcium phosphate grafts.
authors.affiliation: Orthopaedic Pathophysiology and Regenerative Medicine Unit, Istituto Ortopedico Rizzoli, Bologna, Italy. Electronic address: gabriela.ciapetti@ior.it.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Ciapetti
authors.affiliation: Orthopaedic Pathophysiology and Regenerative Medicine Unit, Istituto Ortopedico Rizzoli, Bologna, Italy; Department of Biomedical and Neuromotor Sciences, University of Bologna, Italy. Electronic address: gemma.dipompo@ior.it.
authors.forename: Gemma
authors.initials: G
authors.lastname: Di Pompo
authors.affiliation: Orthopaedic Pathophysiology and Regenerative Medicine Unit, Istituto Ortopedico Rizzoli, Bologna, Italy. Electronic address: sofia.avnet@ior.it.
authors.forename: Sofia
authors.initials: S
authors.lastname: Avnet
authors.affiliation: Department of Biomedical and Neuromotor Sciences, University of Bologna, Italy. Electronic address: desiree.martini@unibo.it.
authors.forename: Desirée
authors.initials: D
authors.lastname: Martini
authors.affiliation: Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia, BarcelonaTech (UPC), Barcelona, Spain. Electronic address: anna.diez-escudero@upc.edu.
authors.forename: Anna
authors.initials: A
authors.lastname: Diez-Escudero
authors.affiliation: Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia, BarcelonaTech (UPC), Barcelona, Spain. Electronic address: edgar.montufar@upc.edu.
authors.forename: Edgar B
authors.initials: EB
authors.lastname: Montufar
authors.affiliation: Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia, BarcelonaTech (UPC), Barcelona, Spain; Institute for Bioengineering of Catalonia, Barcelona, Spain. Electronic address: maria.pau.ginebra@upc.edu.
authors.forename: Maria-Pau
authors.initials: MP
authors.lastname: Ginebra
authors.affiliation: Orthopaedic Pathophysiology and Regenerative Medicine Unit, Istituto Ortopedico Rizzoli, Bologna, Italy; Department of Biomedical and Neuromotor Sciences, University of Bologna, Italy. Electronic address: nicola.baldini@ior.it.
authors.forename: Nicola
authors.initials: N
authors.lastname: Baldini
mesh_terms: D040761:Biomimetic Materials / Q000737:chemistry / Q000494:pharmacology; D018786:Bone Substitutes / Q000737:chemistry / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects*; D017886:Durapatite / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D023461:Myeloid Progenitor Cells / Q000378:metabolism*; D049329:Nanostructures / Q000737:chemistry*; D010010:Osteoclasts / Q000378:metabolism*; D053245:RANK Ligand / Q000494:pharmacology
chemical_list: D018786:Bone Substitutes; D053245:RANK Ligand; C505688:TNFSF11 protein, human; D017886:Durapatite
keywords: Bone resorption; Differentiation; Hydroxyapatite; Ionic exchange; Osteoclasts; Topography