title: Bone marrow cell differentiation induced by mechanically damaged osteocytes in 3D gel-embedded culture.
abstract: Osteocytes are suggested to have a crucial role in the initial resorptive phase of bone turnover after microdamage. To study the role of osteocytes in targeted remodeling, we developed an in vitro model, in which osteocytes can be locally damaged and their interactions with bone marrow cells studied. Our results show that the damaged osteocytes activate the osteoclast precursors by soluble factors and thus can control the initial phase of targeted remodeling.
  
  
  BACKGROUND
  Microdamage in bone contributes to fractures and acts as a stimulus for bone remodeling. Besides the targeted remodeling, some remodeling may also be random to serve metabolic purposes. Osteocytes have been considered to provide a crucial role in the activation of osteoclastic bone resorption adjacent to the damaged site. This study was aimed to develop a relevant in vitro model of the targeted remodeling and to show that damaged osteocytes can induce the initial bone resorptive stage.
  
  
  METHODS
  We developed a new device, in which osteocyte-like cell line MLO-Y4 cells were 3D cultured, subjected to local scratching, and assayed for cell viability. NIH3T3-3 cells were used as a control. Bone marrow cells were cultured on the top of the mechanically damaged MLO-Y4 cells, and the formation of TRACP+ cells was assayed. Additionally, the conditioned medium from scratched cultures was added to bone marrow cultures, and the TRACP activity in cell lysates was quantified. The macrophage-colony stimulating factor (M-CSF) and RANKL secretion in the conditioned medium was assayed by ELISA.
  
  
  RESULTS
  Scratching induced the death of MLO-Y4 cells. When bone marrow cells were cultured over the gel-embedded MLO-Y4 cells, the application of mechanical scratching induced TRACP+ cell differentiation on gel surface. The cells with TRACP+ could be observed in the very restricted region along the scratching path. Additionally, mechanically damaged osteocytes secreted M-CSF and RANKL, and the conditioned medium showed the potential to induce TRACP+ cells in bone marrow culture.
  
  
  CONCLUSIONS
  These findings indicate that soluble factors secreted from damaged osteocytes can locally induce and activate the initial phase of osteoclastic cell formation. This study directly shows the association between the damaged osteocytes and the initiation of resorptive stage in bone remodeling.
authors.affiliation: Department of Biorobotics, Faculty of Engineering, Kyushu Sangyo University, Fukuoka, Japan. kurata@ip.kyusan-u.ac.jp
authors.forename: Kosaku
authors.initials: K
authors.lastname: Kurata
authors.forename: Terhi J
authors.initials: TJ
authors.lastname: Heino
authors.forename: Hidehiko
authors.initials: H
authors.lastname: Higaki
authors.forename: H Kalervo
authors.initials: HK
authors.lastname: Väänänen
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D002352:Carrier Proteins / Q000032:analysis; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002478:Cells, Cultured; D005782:Gels; D016173:Macrophage Colony-Stimulating Factor / Q000032:analysis; D008562:Membrane Glycoproteins / Q000032:analysis; D051379:Mice; D010011:Osteocytes / Q000473:pathology* / Q000502:physiology*; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D013314:Stress, Mechanical
chemical_list: D002352:Carrier Proteins; D005782:Gels; D008562:Membrane Glycoproteins; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; D016173:Macrophage Colony-Stimulating Factor