Page 154 - Physico-Chemical Niche Conditions for Bone Cells

RGD-functionalized supported lipid bilayers modulate pre-osteoblasts
of ligands for integrins was likely larger on PLL-coated substrates than on RGD-functionalized SLBs, providing cells with more opportunities to adhere.
In this study, cell adhesion on SLBs with and without RGD was similar. This is unexpected and might reveal a lack of interaction between Chol-RGD and SLBs. However, this is highly unlikely, since quartz crystal microbalance with dissipation monitoring (QCM-D) revealed successful interaction between Chol-RGD and SLB (data not shown). Therefore, it is unlikely that the similar cell densities on SLBs with and without RGD can be explained by a lack of interaction between Chol-RGD and SLB. It also seems unlikely that the similar cell densities on SLBs with and without RGD result from a charged surface on SLBs. Mazia, Schatten, and Sale [31] have performed electrokinetic measurements showing that the zeta potential of DOPCSLBs approaches zero with increasing salt concentrations up to 10 mM KCl at pH 7.4. In our study, we used a-MEM with 5.3 mM KCl and 117 mM NaCl at pH 7.4. With this high ionic strength, the zeta potential is expected to be nearly zero. Therefore, it seems unlikely that the effect of SLBs without RGD on pre-osteoblasts is the result of surface charge.
Ligand mobility and density on RGD-functionalized SLBs affect pre-osteoblast morphology
Osteoblasts cultured on SLBs with and without RGD were generally smaller, more elongated, and showed more protrusions than cells cultured on PLL-coated glass. Our findings that the cell surface area was smaller and more elongated, are similar to those observed for MSCs on RGD-functionalized SLBs [16]. C2C12 myoblasts cultured on RGD-functionalized SLBs are also smaller than myoblasts cultured on glass [21]. This indicates that cells spread less on RGD-functionalized SLBs than they do on serum-coated glass.
The smaller cells on RGD-functionalized SLBs in comparison to serum-coated glass may be explained by possible variation in types of integrin ligands present on these substrates. It is also possible that cell morphology is affected by the mobile ligand presentation on the viscoelastic SLBs. Cells sense their environment by pulling on their attachments and sensing the resistance of the matrix to this pulling [32]. Since RGDs in the SLBs can diffuse through the bilayer, pulling of the cells on their attachments may displace these attachments towards the center of the cell, causing a smaller cell area on SLBs. Therefore, SLB fluidity affects cell morphology and behavior.
Not only variety and lateral mobility of the ligands but also variation in RGD density may determine cell spreading. Our data show that cells cultured on SLBs with 0.5 μM RGD were larger than cells cultured on SLBs with 1.0 μM RGD, suggesting that there is an optimum ligand density for cell spreading on RGD-functionalized SLBs. The density of immobilized RGD-peptides has been shown to be positively related to MC3T3-E1 cell area [33,34]. However, in these studies, cells were cultured on very stiff (glass) substrates, while substrate
152