Anne Bernhardt1, Matthias Schumacher, Michael Gelinsky. 1. Centre for Translational Bone, Joint and Soft Tissue Research, University Hospital, Medical Faculty Carl Gustav Carus, Technische Universität Dresden , Dresden, Germany .
Abstract
OBJECTIVES: 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. MATERIALS AND 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. CONCLUSION: 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.
OBJECTIVES: 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. MATERIALS AND 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. CONCLUSION: 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: David Kilian; Max von Witzleben; Matthew Lanaro; Cynthia S Wong; Corina Vater; Anja Lode; Mark C Allenby; Maria A Woodruff; Michael Gelinsky Journal: J Funct Biomater Date: 2022-06-08
Authors: Tilman Ahlfeld; Anja Lode; Richard Frank Richter; Winnie Pradel; Adrian Franke; Martina Rauner; Bernd Stadlinger; Günter Lauer; Michael Gelinsky; Paula Korn Journal: Int J Mol Sci Date: 2021-01-26 Impact factor: 5.923
Authors: Paula Korn; Tilman Ahlfeld; Franziska Lahmeyer; David Kilian; Philipp Sembdner; Ralph Stelzer; Winnie Pradel; Adrian Franke; Martina Rauner; Ursula Range; Bernd Stadlinger; Anja Lode; Günter Lauer; Michael Gelinsky Journal: Front Bioeng Biotechnol Date: 2020-03-25