Page 140 - Physico-Chemical Niche Conditions for Bone Cells

RGD-functionalized supported lipid bilayers modulate pre-osteoblasts
MATERIALS AND METHODS
SLB formation
SLBs were formed by vesicle fusion on a glass support as described [16]. Briefly, large unilamellar vesicles consisting of DOPC (melting transition temperature −20°C; Avanti Polar Lipids, Alabaster, AL) were formed by extrusion of a lipid suspension of multilamellar vesicles in MilliQ water through 100 nm membranes (Whatman Nucleopore Track-Etched polycarbonate membrane filter; Whatman, Zwijndrecht, The Netherlands). Vesicle formation was verified using dynamic light scattering (DLS; typical size 102 ± 34 nm with polydispersity index of 0.52; Microtrac Inc., Montgomeryville, PA). Vesicle suspension was sterilized by filtering through 0.2 μm membranes (Nalgene Syringe Filter; Thermo Scientific, Waltham, MA).
Glass bottom wells of 96-well plates (Sensoplate, F-bottom; Greiner Bio-One, Amsterdam, The Netherlands) were incubated with 1 M NaOH for 1 hr to make the surface hydrophilic (Figure 1). After rinsing with MilliQ water, large unilamellar vesicles (0.2 mg/mL in 0.5´ PBS; 100 μL/well) were put onto the glass surface and incubated for 1 hr. During incubation, the vesicles adsorbed to the glass, ruptured, and fused to form SLBs (Figure 1). After incubation, SLBs were first rinsed with PBS to remove excess vesicles, followed by rinsing with serum-free α-Modified Eagle's Medium (α-MEM; Gibco, Paisly, UK) containing 300 μg/mL penicillin (Sigma-Aldrich, St. Louis, MO) and 250 μg/mL streptomycin (Sigma- Aldrich).
Scratch assay and fluorescent recovery after photobleaching
Homogeneous SLB formation and fluidity were confirmed by confocal microscopy. To this end, Texas Red conjugated 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TR-DHPE, Molecular Probes, Thermo Fisher Scientific, Eugene, OR) lipid was introduced in large unilamellar vesicles at 0.2 mol %. SLBs of these vesicles were visualized using confocal microscopy (Nikon A1 confocal microscope, Nikon Instruments Europe B.V., Tokyo, Japan) to confirm homogeneous SLB formation. Scratch assays were performed by scratching the SLBs with a pipet tip and visualizing the recovery using confocal microscopy. Images were taken every 2 min. Fluorescent recovery after photobleaching was performed as described before [16]. Briefly, a 10 μm spot was bleached and recovery was visualized using confocal microscopy. The mobile fraction and diffusion coefficient were derived from the FRAP data using ImageJ (National Institutes of Health, Bethesda, MD) and FRAPAnalyser (University of Luxembourg, Luxembourg).
138