Page 153 - Physico-Chemical Niche Conditions for Bone Cells

DISCUSSION
This study aimed to investigate whether differences exist in adhesion, morphology, focal adhesion formation, proliferation, and osteogenic potential of pre-osteoblasts on RGD- functionalized SLBs compared to unfunctionalized SLBs and PLL-coated glass substrate. We showed for the first time the feasibility of culturing MC3T3-E1 pre-osteoblasts, as a model for osteoprogenitors, on RGD-functionalized supported lipid bilayers. It was shown that (a) pre- osteoblasts did adhere to SLBs with and without RGD, albeit 30–45% less than to PLL-coated glass; (b) cells cultured on SLBs with and without RGD were ~35% smaller and 10% more elongated with more protrusions than cells cultured on PLL-coated glass; (c) cells cultured on PLL-coated glass showed 70–85% more and ~50% larger phospho-paxillin clusters than cells cultured on SLBs with and without RGD; (d) osteopontin mRNA expression levels were 2.5- fold higher in cells cultured for 17 hr on SLBs with 0.2 μM RGD than in those cultured on PLL; (e) after 1 week of culture there was a trend towards increased COL1a1 expression in cells cultured on SLBs compared to PLL. These results suggest that pre-osteoblasts cultured on SLBs with RGD were more osteogenic than cells on PLL-coated glass, despite their smaller size and lower phospho-paxillin content, indicating that application of RGD-functionalized SLBs with variable fluidities to study the mechanisms underlying cell fate and function in relation to physical and chemical substrate properties is promising, as well as clinical application of RGD-functionalized SLBs as coating on biomaterials for enhanced bone regeneration and osteointegration.
Reduced adhesion of pre-osteoblasts on RGD-functionalized SLBs compared to PLL- coated Glass
After 17 hr of culture, cell density on SLBs without or with RGD was lower than on PLL-coated glass, which might be explained by the larger variety of ligands for cell attachment on PLL- coated substrates than on SLBs. PLL is a positively charged molecule that interacts with negatively charged sites on adhesion complexes at cell surfaces [30]. The positive charge of PLL molecules likely allows serum proteins to adsorb to the glass, which provides a multitude of ligands for integrin receptors. SLBs are nonfouling, that is, serum proteins do not or barely adsorb, preventing cells from attaching to the surface [18]. Therefore, negligible numbers of ligands for integrins are presented on SLBs, unless they are functionalized with integrin- binding peptides, such as the RGD peptide used in this study [18]. RGD-functionalized SLBs present only one type of ligand for integrins, while serum proteins adsorbed to PLL probably contain additional peptide sequences that serve as ligands for other integrins, such as sequences in collagen to which a different class of integrins attaches. Therefore, the variety
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