Dxm1200f ccd

To investigate coating degradation, titanium disks with CS-(HA-LDc)n were immersed in phosphate-buffered saline (PBS) buffer (pH = 7.4) at 37 °C. Every 2 days, titanium substrates were taken out and rinsed with deionized water for visualization with a Nikon Eclipse 80i fluorescence microscope (Nikon Corp., Melville, NY, USA) with DXM1200F CCD. The average fluorescence intensity was measured as described. Each group contained three samples.

For transient expression, subconfluent HEK293 cells and HN4 cells were seeded at a density of 2 × 10⁴ cells/well onto the CS-(HA-LDc)5 coating surface in 24-well culture plates. Then, 1 day, 4 days, 7 days, and 9 days later, the medium was gently removed and samples were fixed with 4% paraformaldehyde/PBS at room temperature for 20 min. After washing with PBS, the cells were incubated with rhodamine phalloidin for 30 min, followed by counterstaining with Hoechst 33258 DNA dye for 5 min in the dark. Transfection efficiency was detected using a Nikon Eclipse 80i fluorescence microscope (Nikon Corp., Melville, NY, USA) with DXM1200F CCD. Triplicate samples were used in all cases. The transfection efficiency was calculated using the following equation:

Green fluorescent protein (GFP) expression efficiency was expressed as mean ± standard deviation for three different substrates and was replicated in a separate second run.

Plasmid DNA (pDNA) was labeled with rhodamine using a Label IT labeling kit according to the manufacturer’s protocol. The procedure for the layer-by-layer assembly of loading rhodamine-labeled cDNA onto the titanium surface was performed as described but in the dark. Then cDNA was visualized with a Nikon Eclipse 80i fluorescence microscope (Nikon Corp., Melville, NY, USA) with DXM1200F CCD. The fluorescence of the rhodamine-labeled cDNA on the surface of the plate was analyzed over a 1.2 mm² area using Image-Pro version 6.0 software (Media Cybernetics Corp., USA). Results were obtained using a calibration curve, which was prepared by depositing known amounts of plasmid DNA onto the CS and HA-adsorbed titanium substrate surface. CS-(HA-Lip)9 without plasmid was established as a blank control. Three samples from each group were analyzed.