Page 145 - Physico-Chemical Niche Conditions for Bone Cells

RESULTS
Functionalized glass slides with SLB
Homogeneous coatings of SLBs conjugated with Texas Red-DHPE (1,2-dihexadecanoyl-sn- glycero-3-phosphoethanol-amine) were formed on glass surfaces as shown by confocal microscopy (Figure 2a). The fluidity of the SLBs was demonstrated by showing that a scratch in the layer gradually disappeared resulting in full SLB recovery (Figure 2b). Fluorescent recovery after photobleaching revealed recovery of fluorescence over time, also demonstrating the fluidity of the SLBs (Figure 2c). In an earlier study we determined the diffusion coefficient (0.91 ± 0.21 μm2) and the mobile fraction (90.3 ± 3.5%) [16].
Figure 2 Supported lipid bilayer characterization. (a) Typical example of DOPC-SLB conjugated with Texas Red-DHPE (0.2 mol %). Bar: 100 μm. (b) Typical example of a scratch assay. t0, immediately after a scratch was made in the bilayer; t1, 15 min after t0; t2, 30 min after t0. The images show that SLBs recover from scratching. (c) Typical example of fluorescent recovery after photobleaching. t0, immediately after bleaching; t1, 10 s after t0 showing recovery of fluorescence after bleaching. Bar: 10 μm. SLB, supported lipid bilayer; DOPC, 1,2-dioleoyl-sn-glycero- 3-phosphocholine; SLB, supported lipid bilayer; DHPE, 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine
Cell density
To investigate the adherence of MC3T3-E1 pre-osteoblasts on SLBs with and without RGD, the number of cells per cm2 was quantified (Figure 3). Cells did adhere to SLBs with and without RGD. Cell density on SLBs with RGD (1353 ± 255 cells/cm2, mean ± SEM of 3 RGD- concentrations used) and on SLBs without RGD (1621 ± 266 cells/cm2) was 30-45% lower than on PLL-coated glass (2299 ± 426 cells/cm2; Figure 3; p < 0.001). There were no significant differences in cell density between SLBs with and without RGD.
Chapter 6
143
6