Page 141 - Physico-Chemical Niche Conditions for Bone Cells

RGD functionalization
To allow cell attachment and growth, SLBs were functionalized by inserting cholesterol- conjugated RGD-peptides (chol-RGD) (Figure 1). These peptides consisting of the amino acids KGSGRGDSG were synthesized, purified, and conjugated to cholesterol using established methods available in our group (Figure S1: Mass spectrum of chol- RGD). Different concentrations of chol-RGD in PBS (0.2, 0.5, and 1.0 μM) were added to the SLBs and incubated for at least 2 hr.
Figure 1 Glass bottom well plates were treated by 1 M NaOH for 1 hr to obtain a hydrophilic surface, followed by incubation with large unilamellar vesicles for 1 hr to form SLB coatings. Different concentrations of cholesterol-conjugated RGD-peptides in PBS were added to SLB-coated glass bottom surfaces and incubated for ≥2 hr. Density, morphology, and gene expression of pre-osteoblasts cultured for 17 hr or 1 week on functionalized SLB-coated glass bottom surfaces were analyzed using confocal microscopy and RT-PCR. LUV, large unilamellar vesicles; SLB, supported lipid bilayer.
Cell culture
MC3T3-E1 pre-osteoblasts were maintained in a-MEM (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco, Paisly, UK), 300 μg/mL penicillin (Sigma-Aldrich), 250 μg/mL streptomycin (Sigma- Aldrich), and 1.25 μg/mL fungizone (Gibco, Paisly, UK) [25,26]. At 80% confluency, the cells were harvested using 0.25% trypsin and 0.1% EDTA in PBS [25]. For experiments, cells of passage 24–34 were used. Cells were seeded at 2 ´ 103 cells/cm2 on different substrates and cultured for 17 hr or 1 week in a-MEM with 10% FBS, 300 μg/mL penicillin, and 250 μg/mL streptomycin. Cells were cultured for 17 hr on different substrates,
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