Introduction: In cell-material interaction, knowledge of the dependence of cell behavior on topographical features is relevant for designing implant surfaces. Cells are sensitive to their underlying topography and especially micro-topography offers cues that evoke large ranges of cell responses. Human MG-63 osteoblastic cells growing on top of micro-pillared structures altered their actin organization and the changed cell architecture resulted in a decreased synthesis of extracellular matrix proteins collagen-I, bone sialo protein-2[1] and fibronectin as well as impaired ATP induced mobilization of intracellular calcium[2]. But the complex interplay between the topography-induced cell morphology and functional changes is not completely understood.
Material and Methods: Regular geometric micro-pillared structures (diameters: width 5 µm; length 5 µm; height 5 µm) were used as artificial surfaces. The micro-pillared topography was produced by deep-reactive ion etching of silicon wafers and coated with 100 nm titanium. Human MG-63 osteoblastic cells (ATCC, CRL-1427) were grown for 24 and 96 h on the surfaces. Cellular morphology was analyzed by scanning electron microscopy (SEM), gene expression of alkaline phosphatase, caveolin-1 (Cav-1), collagen type 1 (Col1), fibronectin (FN) and osteocalcin by real time PCR, protein expression of Cav-1, phosphorylated Cav-1, Col1, FN and bone sialo protein 2 by western blot and catalase expression by luminex assay. Protein localization of Cav-1 and cholesterol was determined by confocal microscopy. Via fluorescence quantification by FACS, micro-plate reader of microscopy reactive oxygen species (ROS) generation, intercellular ATP/ADP amount and mitochondrial activity were determined.
Results: Cav-1 and cholesterol were found to be clustered on top of the micro-pillars after 24 h and the SEM showed MG-63 osteoblasts engulfing the micro-pillars after 96 h. Gene expression of osteoblast marker genes was impaired after 24 h as well as the protein expression after 96 h of cells grown on the micro-pillars. The energy metabolism was disturbed by the micro-pillars. This was indicated by a higher mitochondrial activity and reduced intercellular ATP amounts; accompanied with higher ROS levels and protein expression of ROS composition enzymes.
Discussion: Our recent experiments revealed an attempted caveolae-mediated phagocytosis of the surface-fixed micro-pillars by osteoblasts attended by increased energy demands resulting in higher cell stress, e.g. ROS generation. Phagocytosis is known to reorganize the actin cytoskeleton[3] as observed on the micro-pillared topography and is also characterized to be an energy-dependent process[4] and therefore the topography-triggered phagocytosis may lead to the observed impaired osteoblastic cell signaling.
German Research Foundation (DFG) graduate school welisa (1505/2)
References:
[1] Matschegewski C, Staehlke S, Loeffler R, Lange R, Chai F, Kern DP, Beck U, Nebe BJ. Cell architecture – cell function dependencies on titanium arrays with regular geometry. Biomaterials 2010;31:5729-5740. doi:10.1016/j.biomaterials.2010.03.073
[2] Staehlke S, Koertge A, Nebe B. Intracellular calcium dynamics dependent on defined microtopographical features of titanium. Biomaterials 2015,46:48-57. doi:10.1016/j.biomaterials.2014.12.016
[3] May RC, Machesky LM. Phagocytosis and the actin cytoskeleton. J Cell Sci 2001;114:1061-1077.
[4] Borregaard N, Herlin T. Energy metabolism of human neutrophil during phagocytosis. J Clin Invest 1982;70:550-557.