Page 155 - Physico-Chemical Niche Conditions for Bone Cells

stiffness affects the relation between ligand density and cell area [35]. Since the SLBs used in the current study have a lower elastic modulus than glass [23] and present RGD-peptides in a mobile manner as consequence of the SLB fluidity [17,21], the relationship between ligand density and cell spreading is possibly different on SLBs with mobile RGD-peptides compared to substrates with immobilized RGD-peptides. SLBs provide the opportunity to investigate in detail the relationship between pre-osteoblast spreading and substrate stiffness, ligand variety, mobility, and density. Nevertheless, this is only relevant if a relationship between pre- osteoblast spreading and osteogenic phenotype exists, but such a relationship has not been established. Interestingly, MSCs with a large surface area are more osteogenic than MSCs with a small surface area [16,36–38]. Therefore, for studies investigating the effect of substrate stiffness and ligand presentation on pre-osteoblast morphology, it should be established whether there is a relation between pre-osteoblast morphology and the osteogenic state.
Reduced focal adhesion formation in pre-osteoblasts cultured on RGD-functionalized SLBs compared to PLL indicating lower adhesion strength Cells on SLBs without and with RGD showed less and smaller phospho-paxillin clusters than cells on PLL-coated glass. These differences likely resulted from differences in phosphorylation of paxillin and not from differences in protein content, since there were no differences in paxillin mRNA. The size of phospho-paxillin clusters is indicative of the strength of the adhesions and the forces applied to the adhesions, either by contraction of the actin cytoskeleton or as a result of external mechanical perturbations [6]. Therefore, the decreased number and smaller phospho-paxillin clusters on SLBs compared to PLL-coated glass suggest that cells did adhere less firm to SLBs, which substantiates the reduced cell adhesion shown in the current study.
Cells on SLBs with 0.5 μM RGD showed more phospho-paxillin clusters than on SLBs with 0.2 or 1.0 μM RGD. This indicates that ligand density on the fluid SLBs modulated focal adhesion formation, as did the density of immobile ligands [39].
Phosphorylation of paxillin not only implies adhesion but also activation of focal adhesion kinase and other signaling molecules in the adhesion complex [36]. These signaling molecules activate signaling pathways such as mitogen-activated protein kinase, which play a critical role in osteogenic differentiation of MSCs by stimulating RUNX2 gene expression [6,36]. MSCs with large focal adhesions are more osteogenic than cells with small focal adhesions [16,36–38]. MC3T3-E1 pre-osteoblasts show increased focal adhesion formation accompanied by decreased osteocalcin expression and matrix mineralization [40]. Therefore, it is unclear whether the less abundant and smaller focal adhesions in pre-osteoblasts cultured on RGD-functionalized SLBs in comparison to PLL-coated glass indicated that cells were more or less osteogenic on SLBs than on PLL. Thus, we also investigated the effect of RGD- functionalized SLBs on osteogenic gene expression.
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