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Figure 4. Fluorescence microscope images of adherent MC3T3-E1 (A) and L929 (B) cells on the specimens. The cells were fixed with 3.7% formaldehyde in PBS and permeabilized by treatment with 0.1% Triton X-100 in PBS for 1 min. The cells were then incubated for 3 hrs with a rhodamine-conjugated phalloidin solution. After the cells were washed with water, stress fiber formation and cell morphology were visualized with the use of a fluorescence microscope. The left panel displays adherent cells on a control specimen. No stress fiber formation is observed in cells on the control specimens, and the cells adhere loosely to the surface compared with those on Ti-Cl. The right panel displays adherent cells on Ti-Cl. These adherent cells have already begun to form stress fibers and have widely extended cytoplasm. (C) The adsorbed fibronectin on each specimen was visualized by means of FITC labeling. The specimens were soaked in 
minimal essential medium containing 10% fetal bovine serum (37°C, 30 min). The specimens were washed with PBS 3 times, blocked with 5% BSA for 1 hr, and immersed in a 2000-fold dilution of monoclonal anti-human fibronectin for 2 hrs. After another 3 washes with PBS, they were immersed in a 32-fold dilution of FITC-conjugated anti-mouse IgG for 1 hr. Following another 3 washes with PBS, the adsorbed fibronectin on each specimen was visualized with the use of a fluorescence microscope. The left panel shows an FITC image of adsorbed fibronectin on a control specimen. The right panel shows an FITC image of adsorbed fibronectin on Ti-Cl. Quantitative analysis of the images (C) was performed on a Windows <model> computer equipped with the public domain program NIH Image (developed at the US National Institutes of Health and available from the Internet by anonymous FTP from zippy.nimh.nih.gov from the National Technical Information Service, Springfield, VA; part number PB95-500195GEI). The results are expressed as the mean ± SD of 6 specimens (n = 6). The findings were analyzed statistically by Student’s t tests. Significant differences were considered to exist when p < 0.01. Bars = 10 µm (A,B) and 40 µm (C).
