Page 92 - Physico-Chemical Niche Conditions for Bone Cells

PFF affects pre-osteoblast behavior
stress fiber was stained using Alexa Fluor 488 (Invitrogen, 1:100) for 40 min at room temperature in the dark. Afterwards, nuclei were stained by using 4’,6-diamidino-2- phenylindole (DAPI; Merck, Whitehouse Station, NJ, USA) in PBS (1:1000) for 10 min in the dark at room temperature. Cells were washed gently 3 times for 15 min with PBS, and mounted in Vecta-shield (Vector Laboratories, Burlingame, CA, USA) for visualization by LSCM. To measure cell ratio (length/width), Cell images before and after 1 h PFF treatment were taken by normal light microscopy (Leica, Solms, Gemany). Cell F-actin fluorescence intensity, cell orientation (angle), and cell ratio (length/width) were analyzed using Image-Pro Plus software (Media Cybernetics, Houston, TX, USA). Cell F-actin fluorescence intensity along the cell long axis was quantified by using the tool “Line profile” in Image-Pro Plus software. Quantification of cell orientation (angle) and ratio (length/width) were done as follows (1) The tool of “Irregular AOI” was chosen to outline the cell. The parameters of “trace” were set, including thresh=3, smooth=0, speed=5, noise=5. (2) The tools of “Multiple AOI” “NEW AOI” were used to outline more cells. (3) The parameters of “angle” (between the long axis of the cell and the vertical line), length (major axis of the cell) and width (minor axis of the cell) were selected in the “select measurements” of “Measure”. (4) “Convert AOI(s) To Object(s)” was selected in “Edit” of “Count and measure objects”. (5) “Count” was chosen in the diagram of “Count and size” in “Count and measure objects”. (6) “Measurement data” was chosen in “View” of “Count and measure objects”. (7) The data was saved and used to prepare the figures. To quantify the cell orientation, 169 (control) and 133 (PFF) cells from 4 glass slides from 4 independent experiments were analyzed. To quantify the cell ratio, 76 (control) and 80 (PFF) cells from 3 glass slides from 3 independent experiments were analyzed.
Scanning electron microscopy (SEM; XL20, Fei Company, Eindhoven, The Netherlands) was used to visualize static control and PFF-treated MC3T3-E1 pre-osteoblasts at 6 h post-incubation. Cells were washed with phosphate-buffered saline (PBS), and fixed with 4% paraformaldehyde (Merck, Whitehouse Station, NJ, USA), 1% glutaraldehyde (Merck), and 0.1 M natriumcacodylate at 4°C overnight. Then the samples were dehydrated in a graded ethanol series (35, 50, 70, 80, 90, and 100%), and air-dried overnight with hexamethyldisilazane (HMDS; Sigma-Aldrich, St. Louis, MO, USA). To evaluate cell morphology, the specimens were sputter-coated with gold and examined using SEM at an accelerating voltage of 15 kV. The cells and regions of cells were selected randomly for SEM analysis of pseudopodia. The magnification was ´10000. Quantification of cell pseudopodia length was performed using Image-Pro Plus software. The tool of “line” in “Features” of “Manual measurements” was chosen to draw and measure the length of pseudopodia. The data was saved from “Export data” (data to “Features”, output data to “File”) in “Input/Output” of “Manual measurements”. Six cells from 6 glass slides from 3 independent experiments (n=3;
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