Fatigue failure of osteocyte cellular processes: implications for the repair of bone.

The physical effects of fatigue failure caused by cyclic strain are important and for most materials well understood. However, nothing is known about this mode of failure in living cells. We developed a novel method that allowed us to apply controlled levels of cyclic displacement to networks of osteocytes in bone. We showed that under cyclic loading, fatigue failure takes place in the dendritic processes of osteocytes at cyclic strain levels as low as one tenth of the strain needed for instantaneous rupture. The number of cycles to failure was inversely correlated with the strain level. Further experiments demonstrated that these failures were not artefacts of our methods of sample preparation and testing, and that fatigue failure of cell processes also occurs in vivo. This work is significant as it is the first time it has been possible to conduct fatigue testing on cellular material of any kind. Many types of cells experience repetitive loading which may cause failure or damage requiring repair. It is clinically important to determine how cyclic strain affects cells and how they respond in order to gain a deeper understanding of the physiological processes stimulated in this manner. The more we understand about the natural repair process in bone the more targeted the intervention methods may become if disruption of the repair process occurred. Our results will help to understand how the osteocyte cell network is disrupted in the vicinity of matrix damage, a crucial step in bone remodelling.</p

FLA3 is an essential protein in the bloodstream form o<i>f T. brucei.</i>

<p>Panel A. Northern blot analysis of 15 µg of total RNA from a cloned FLA3 bloodstream RNAi cell line grown under induced or noninduced conditions for 24 h. The probe used for hybridisation was the same fragment employed for RNAi. The lower part of the panel presents the ethidium bromide staining of the RNA as a control for loading. The arrow indicates the presence of the dsRNA (∼ 600 bp) in the induced cells. Panel B. An immunoblot analysis confirmed loss of the FLA3 protein in the induced cells after 30 h induction. Each lane contained 5×10⁶ cells and a loading control was performed with antibodies against trypanosome actin. Panel C. The effect of knock down of FLA3 on the growth of a representative cloned FLA3 RNAi cell line cultured in the presence (○) or absence of tetracycline (•). Panel D. Analysis of the number of nuclei and kinetoplasts per cell in a FLA3 RNAi cell line cultured in the presence of tetracycline. For each time point individual cells were assessed for the presence of kinetoplast and nucleus and scored as 1N1K, 1N2K, 2N2K and cells with clearly more than 2N. The results were expressed as the mean ± SD of four separate surveys of 50 cells.</p

Knockdown of FLA3 leads to flagellar detachment in the bloodstream form of <i>T. brucei.</i>

<p>Panel A. FLA3 RNAi cells were grown in the presence of tetracycline. Samples were removed, fixed and processed for phase contrast and fluorescence microscopy to assess flagellar attachment (Phase) and DNA content (DAPI) of the cells. Bar = 10 µm. Panel B. At each time point the number of cells with detached flagella was assessed as a % of the total number of cells in the population. The results were expressed as the mean ± SD of four separate surveys of 50 cells.</p

Localization of FLA3 in the bloodstream form of <i>T. brucei</i>.

<p>Trypanosomes were purified from infected blood, fixed and processed for immunofluorescence using the affinity purified α-FLA3 antibodies. Upper panels present a merge of the fluorescence and phase images and reveal the location of FLA3 (green) and the nucleus/kinetoplast (blue); lower panels present the fluorescence image alone. Bar = 10 µm. Panel A. FLA3 was detected as a punctate pattern of staining along the FAZ. The black arrows in the upper panel (α-FLA3) indicate the absence of FLA3 along the free flagellum. Non specific binding was investigated by including the peptide antigen (10 µg.ml⁻¹) (α-FLA3+ pep). Panel B. FLA3 is associated with existing and new (arrows) FAZ. Panel C. FLA3 remains associated with the cell body at regions where the flagellum has become detached (black arrows in the phase/fluorescence merge). The white arrows indicate more pronounced staining of FLA3 frequently observed at the posterior and anterior end of the attachment zone (lower panels).</p

The effect of the loss of FLA3 on the flagellar attachment zone.

<p>Panel A. Detail of the flagellar attachment zone in noninduced FLA3 RNAi cells showing the regularly spaced FAZ filament structure (arrow). Panel B. Detail of the flagellar attachment zone in an induced FLA3 RNAi cells at the point where the flagellum became detached reveals the loss of the regularly spaced FAZ filament structure (arrows). The panel on the left presents a close up of the region where detachment occurred. Bar = 500 nm.</p

Co-localization of FLA3 with markers for the FAZ and flagellum.

<p>Cells were probed using affinity purified α-FLA3 rabbit antibodies and a mouse monoclonal antibody against FAZ1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052846#pone.0052846-Vaughan2" target="_blank">[19]</a> or PFR2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052846#pone.0052846-Kohl4" target="_blank">[18]</a>. The cells were examined using an Olympus Fluoview 1000 confocal microscope. The panels are presented as merges of the phase and the individual immunofluorescence images plus a combined antibody fluorescence merged image. Bar = 10 µm. Panel A. The location of FLA3 (green), FAZ1 (magenta) and the nucleus/kinetoplast stained with DAPI (blue). Panel B. The location of FLA3 (green), PFR2 (magenta) and the nucleus/kinetoplast (blue). PFR2 but not FLA3 is associated with the flagellum and the free flagellum at the anterior end of the cell (black arrow).</p