title: Formation of osteoclasts on calcium phosphate bone cements and polystyrene depends on monocyte isolation conditions.
abstract: OBJECTIVE
  Peripheral blood mononuclear cells (PBMC) are an attractive source for the generation of osteoclasts in vitro, which is an important prerequisite for the examination of resorption and remodeling of biomaterials. In this study, different preparation methods are used to obtain cell populations with a rising content of CD14(+) monocytes. We wanted to address the question whether there is a correlation between content of CD14(+) cells in the preparation and functionality of formed osteoclasts.
  
  
  METHODS
  PBMC obtained by density gradient centrifugation with and without further purification by plastic adherence or immunomagnetic separation of CD14(+) cells were seeded on both cell culture polystyrene and a calcium phosphate bone cement (CPC) and cultivated under stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappa B ligand (RANKL). Cell cultures were characterized by histological and fluorescent staining of multinucleated cells that were positive for tartrate-resistant acid phosphatase (TRAP) activity and the presence of actin rings, respectively. Furthermore, activities of osteoclast marker enzymes TRAP and carbonic anhydrase II (CA II) were quantified. For osteoclasts cultured on CPC, resorption pits were visualized using scanning electron microscopy (SEM).
  
  
  RESULTS
  Monocytes of all preparations were successfully differentiated into multinucleated osteoclasts showing TRAP and CA II activity on both cell culture plastic and CPC. Preparations involving an additional plastic adherence step exhibited only a minor increase of TRAP and CA II activity in the second week of cultivation. Furthermore, the number of resorption pits on CPC was reduced in these cultures compared with immunomagnetically enriched monocytes and preparations without additional plastic adherence steps. Optimal results with regard to yield, number of multinucleated osteoclasts, activity of TRAP and CA II, and resorption of CPC were obtained by simple density gradient centrifugation.
  
  
  CONCLUSIONS
  All examined monocyte preparation protocols were suitable for the generation of osteoclasts on both polystyrene and CPC. Highly purified monocytes are not mandatory to obtain functional osteoclasts for investigation of biomaterial resorption.
authors.affiliation: Centre for Translational Bone, Joint and Soft Tissue Research, University Hospital, Medical Faculty Carl Gustav Carus, Technische Universität Dresden , Dresden, Germany .
authors.forename: Anne
authors.initials: A
authors.lastname: Bernhardt
authors.forename: Matthias
authors.initials: M
authors.lastname: Schumacher
authors.forename: Michael
authors.initials: M
authors.lastname: Gelinsky
mesh_terms: D000135:Acid Phosphatase / Q000378:metabolism; D000328:Adult; D001843:Bone Cements / Q000494:pharmacology*; D002130:Calcium Phosphates / Q000494:pharmacology*; D002256:Carbonic Anhydrases / Q000378:metabolism; D002469:Cell Separation / Q000379:methods*; D048430:Cell Shape / Q000187:drug effects; D002478:Cells, Cultured; D005434:Flow Cytometry; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D009000:Monocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects; D011137:Polystyrenes / Q000494:pharmacology*; D053245:RANK Ligand / Q000494:pharmacology; D000071681:Tartrate-Resistant Acid Phosphatase
chemical_list: D001843:Bone Cements; D002130:Calcium Phosphates; D007527:Isoenzymes; D018950:Lipopolysaccharide Receptors; D011137:Polystyrenes; D053245:RANK Ligand; D016173:Macrophage Colony-Stimulating Factor; C020243:calcium phosphate; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase; D002256:Carbonic Anhydrases