title: Osteoblastic and osteoclastic differentiation of human mesenchymal stem cells and monocytes in a miniaturized three-dimensional culture with mineral granules.
abstract: The pathologies of the skeleton have a significant socioeconomic impact on our population. Although therapies have improved the treatment of osteosarcoma and osteoporosis, their efficacy still remains limited. In this context, we developed a miniaturized 3-D culture model of bone cells on calcium phosphate ceramics. Human bone marrow mesenchymal stem cells (MSCs) were three-dimensionally cultured on particles of biphasic calcium phosphate (BCP, 125-200μm) in osteogenic media. The MSCs seeded on the BCP particles adhered and proliferated, producing abundant collagenous extracellular matrix (ECM). Light and confocal laser scanning microscopy showed that the MSCs created bridges between the BCP particles and formed a 3-D structure. Energy dispersive X-ray analysis in a scanning electron microscope confirmed the mineralization of the collagen matrix. The 96-well sized bone constructs were tested by immunohistology and transcription analysis, proving cell differentiation. Both techniques corroborated the osteoblastic differentiation with high production of bone sialoprotein and osteocalcin. Peripheral blood CD14-positive monocytes (MOs) were pre-differentiated into osteoclasts prior to seeding on the 3-D constructs. Multinucleated and tartrate-resistant acid phosphatase-positive cells were also identified at the surface of the 3-D constructs after 90days of culture. In addition, cell viability within these constructs was measured by flow cytometry. In summary, we have developed a miniaturized 3-D culture of bone cell precursors with osteoblasts and osteoclasts. This 3-D culture may make it possible to test the effects of new drugs for bone healing, osteoporosis and osteosarcomas, in more appropriate cell-cell and cell-matrix interactions than conventional 2-D cultures.
authors.affiliation: INSERM, UMR957, Nantes, France.
authors.forename: Anne Laure
authors.initials: AL
authors.lastname: Gamblin
authors.affiliation: INSERM, UMR957, Nantes, France.
authors.forename: Audrey
authors.initials: A
authors.lastname: Renaud
authors.affiliation: INSERM, UMR957, Nantes, France.
authors.forename: Céline
authors.initials: C
authors.lastname: Charrier
authors.affiliation: Université de Nantes, Nantes Atlantique Universités, Laboratoire de Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives, Nantes, France; Plate forme MicroPICell, SFR Santé François Bonamy, Inserm UMS 016, CNRS 3556, Nantes, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Hulin
authors.affiliation: Laboratoire de Physique des Matériaux et Nanostructures, UMR 6502, Institut des Matériaux Jean Rouxel, 2 Rue de la Houssinière, BP 32229, 44322 Nantes Cedex 03, France.
authors.forename: Guy
authors.initials: G
authors.lastname: Louarn
authors.affiliation: INSERM, UMR957, Nantes, France; Université de Nantes, Nantes Atlantique Universités, Laboratoire de Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives, Nantes, France; CHU, Hôtel Dieu, Nantes, France.
authors.forename: Dominique
authors.initials: D
authors.lastname: Heymann
authors.affiliation: INSERM, UMR957, Nantes, France; Université de Nantes, Nantes Atlantique Universités, Laboratoire de Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives, Nantes, France.
authors.forename: Valérie
authors.initials: V
authors.lastname: Trichet
authors.affiliation: INSERM, UMR957, Nantes, France; Université de Nantes, Nantes Atlantique Universités, Laboratoire de Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives, Nantes, France; CHU, Hôtel Dieu, Nantes, France. Electronic address: pierre.layrolle@inserm.fr.
authors.forename: Pierre
authors.initials: P
authors.lastname: Layrolle
mesh_terms: D061249:Batch Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000295:instrumentation / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D008904:Miniaturization; D009000:Monocytes / Q000166:cytology* / Q000502:physiology; D010006:Osteoblasts / Q000166:cytology*; D010010:Osteoclasts / Q000166:cytology*; D023822:Tissue Engineering / Q000295:instrumentation* / Q000379:methods; D054457:Tissue Scaffolds
keywords: 3-D model; Bone; Human mesenchymal stromal/stem cells; Human monocytes; Osteoblasts