Page 142 - Physico-Chemical Niche Conditions for Bone Cells

RGD-functionalized supported lipid bilayers modulate pre-osteoblasts
since we have shown previously stable cell spreading on RGD-functionalized SLBs within this time period [16]. Osteogenic gene expression was measured after 1 week [27,28].
Immunocytochemistry
To assess cell morphology and focal adhesion formation, cells were cultured for 17 hr on different substrates. Then cells were fixated using 4% paraformaldehyde in PBS and permeabilized using 0.2% Triton X-100 (Serva Electrophoresis GmbH, Heidelberg, Germany) in PBS. Nonspecific binding of antibodies was prevented by blocking with 5% normal goat serum (NGS; Life Technologies, ThermoFisher, Carlsbad, CA). Cells were incubated with primary antibodies against integrin a5 (rat monoclonal antibody, Abcam AB25251, dilution 1:100; Abcam, Cambridgeshire, UK) and phosphorylated paxillin (phospho-Tyr31, rabbit polyclonal antibody, Invitrogen 44-720G, dilution 1:100; Fisher Scientific, Carlsbad, CA) in 5% NGS for 1 hr at room temperature. After washing five times for 10 min, cells were incubated for 1 h at room temperature with Alexa Fluor 488 goat-anti-rat (Invitrogen A11006; dilution 1:500; Fisher Scientific) and Alexa Fluor 555 goat anti- rabbit (Invitrogen A21428, dilution 1:500; Fisher Scientific) in 5% NGS. After washing three times for 10 min with PBS, 40,6- diamidine- 20-phenylindole dihydrochloride (DAPI) was added for 30 min at room temperature to stain the nuclei. Cells were mounted using Vectashield (Vector Laboratories Inc, Burlingame, CA).
Confocal microscopy
Samples were imaged using a Nikon A1+ confocal laser scanning microscope (Nikon Instruments Europe B.V.). To obtain an overview of one well with cells, 36 z-stack images (slice thickness 3.0 μm) obtained with a 20 ´ objective (numerical aperture 0.8) were stitched together in a 6 ´ 6 configuration. To visualize single cells, a 60 ´ objective was used to obtain z-stack images with a slice thickness of 0.175 μm. From every well, z-stacks of 10–30 cells were taken and analyzed as described below.
Image analysis
To investigate the cell density, a maximal z-projection was made from the stitched z-stacks using ImageJ (National Institutes of Health). An area measuring 1000 ´ 1000 pixels (1253 ´ 1253 μm) from the center of this image was selected, and the number of cells in this image was counted using a cell counter plugin for ImageJ (De Vos, 2019). The cell density per cm2 was calculated from the cell number acquired in the selected area.
For two-dimensional (2D) morphology measures, maximal z projections were made from single cell z-stack images. Morphological parameters were measured from the resulting
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