Page 156 - Physico-Chemical Niche Conditions for Bone Cells

RGD-functionalized supported lipid bilayers modulate pre-osteoblasts
Effect of RGD-functionalized SLBs on osteogenic gene expression
To further assess the initial response of pre-osteoblasts to RGD-functionalized SLBs, we examined their osteogenic state by analyzing osteogenic gene expression. Cells cultured on SLBs with and without RGD for 17 hr did express the osteogenic genes RUNX2, COL1a1, and OPN. Cells on SLBs with 0.2 μM RGD showed increased OPN expression and a trend toward elevated RUNX2 expression compared to PLL.
The ECM protein osteopontin is expressed in bone, as well as in other organs, for example, kidney, heart, and inner ear [41,42]. It is a signaling molecule that is upregulated in osteoblasts in response to mechanical loading [43]. Osteopontin is an essential protein in bone involved in matrix remodeling and tissue calcification [41]. Thus, the higher OPN expression in pre-osteoblasts cultured on SLBs suggest that these cells were more osteogenic than on PLL-coated glass.
The 17 hr culture period in our study was rather short to investigate the osteogenic phenotype of pre-osteoblasts. Changes in gene expression of RUNX2, COL1a1, and ALP are usually only observed after several days of culture [27,28]. MSCs cultured for 10 days on RGD-functionalized SLBs are more osteogenic, that is, they show increased ALP activity when more fluid SLBs are used compared to less fluid SLBs [16], indicating that initial culturing of MSCs on more fluid RGD-functionalized SLBs guides osteogenic differentiation of MSCs. Therefore, the osteogenic state of MC3T3-E1 pre-osteoblasts after 1 week of culture on RGD- functionalized SLBs was investigated. It was observed that the cells were still well attached to the surface and nicely spread. Compared to the 17 hr time point, the cell layer was more confluent, indicating that cells had proliferated on SLBs.
Gene expression levels of RUNX2 and OPN on all substrates were lower after 1 week than after 17 hr of culture. In contrast, the expression levels of COL1a1 and ALP were higher after 1 week than after 17 hr. These results are in line with the normal osteogenic differentiation pattern [27], showing that pre-osteoblasts grow and differentiate well on RGD- functionalized SLBs.
Pre-osteoblasts cultured on SLBs showed increased expression of COL1a1 compared to cells cultured on PLL, suggesting that pre-osteoblasts cultured on SLBs may be more osteogenic than on PLL. The lack of effect on expression of other osteogenic genes may be the result of the possible degradation of the SLBs over time during culture. This degradation of SLBs has been shown for charged SLBs consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC) and 1,2-dioleoyl-3-trimethyl-ammonium-propane (DOTAP) during culture of neuronal cells [44]. Degradation of SLBs may allow serum proteins to adsorb to the hydrophilic glass creating an environment comparable to that on PLL-coated glass. If so, the slightly higher expression of COL1a1 in cells cultured on SLBs suggests that the initial
154