RGD-functionalized supported lipid bilayers modulate pre-osteoblast adherence and promote osteogenic differentiation

Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam Movement Sciences, Amsterdam, The Netherlands Laboratory of Biointerface Chemistry, TechMed Centre and MESA+ Institute for Nanotechnology, University of Twente, Enschede, The Netherlands Laboratory for Myology, Faculty of Behavioural and Movement Sciences, Vrije Universiteit Amsterdam, Amsterdam Movement Sciences, Amsterdam, The Netherlands


| INTRODUCTION
Biomaterials are used to replace bone in pathological conditions such as tooth loss, osteoarthritis, or large bone defects as a result of trauma or tumor removal (Farré-Guasch et al., 2013). In the case of tooth loss or osteoarthritis, the tooth or joint is replaced by a prosthesis, which has to integrate into the native bone. When large bone defects are concerned, resorbable grafts are inserted which in time are replaced by native bone. The integration of both implants and bone grafts starts with cell recruitment, adhesion, proliferation, and differentiation (Farré-Guasch et al., 2013;Gittens, Olivares-Navarrete, Schwartz, & Boyan, 2014;Shah, Thomson, & Palmquist, 2019). Implant integration requires that osteoprogenitors and mesenchymal stem cells are recruited, attach to the implant, proliferate, and differentiate into bone forming osteoblasts (Gittens et al., 2014;Shah et al., 2019). To allow bone graft integration, osteoclast precursors that differentiate and fuse to become bone graft resorbing osteoclasts are also needed (Farré-Guasch et al., 2013). The most important challenge in bone tissue engineering is the development of biomaterials that promote adhesion, proliferation, and differentiation of osteoprogenitors and osteoclast precursors, while repelling adhesion of bacteria, that may cause infection, and cells that produce a membranous structure between the biomaterial and the bone leading to implant failure (Gittens et al., 2014;Shah et al., 2019). For improved bone regeneration and seamless biomaterial integration into the bone, innovative biomimetic coatings for biomaterials are still needed.
Adhesion, proliferation, and differentiation of osteoprogenitors are affected by integrin binding and focal adhesion formation. Cells adhere to extracellular matrix (ECM) primarily by the binding of integrin receptors to proteins within the ECM (Sun, Guo, & Fässler, 2016). Integrin binding induces the formation of adhesion complexes where integrins cluster together, and where scaffolding and signaling proteins are recruited and attach to the actin cytoskeleton (Geiger, Spatz, & Bershadsky, 2009;Marie, Hay, & Saidak, 2014;Sun et al., 2016). Focal adhesions strengthen osteogenic cell attachment to the ECM and induce cell spreading and morphology changes by remodeling the actin cytoskeleton (Porté-Durrieu et al., 2004;Takai, Landesberg, Katz, Hung, & Guo, 2006). The signaling resulting from focal adhesion formation regulates the activity of transcription factors that direct cell growth, proliferation, survival, and differentiation toward osteoblasts (Marie et al., 2014;Sun et al., 2016;Takai et al., 2006). Several approaches have been employed to improve the adherence and differentiation of cells on biomaterials. Improved osseointegration is observed when using implants with a rough surface compared to a smooth surface (Gittens et al., 2014;Lim et al., 2007). Surface chemistry also influences cellular adhesion (Keselowsky, Collard, & García, 2004).
Osteoprogenitor adhesion to RGD-functionalized biomaterials is not yet optimal possibly due to the immobile ligand presentation preventing the cells from rearranging the ECM to optimize cell-ECM interaction as occurs in vivo (Koçer & Jonkheijm, 2017). Ligand immobilization likely inhibits integrin clustering (Glazier & Salaita, 2017) and thereby decreases cell adhesion strength and signaling resulting in differentiation (Marie et al., 2014). Supported lipid bilayers (SLBs) provide a platform for functionalizing biomaterials with mobile ligands, including RGD (Glazier & Salaita, 2017;Koçer & Jonkheijm, 2017;van Weerd, Karperien, & Jonkheijm, 2015). SLBs are made of phospholipids and comparable to natural cell membranes. They are nonfouling in nature (van Weerd et al., 2015). SLBs are extensively applied into modern clinical use owing to their biophysical and chemical versatility (Ashley et al., 2011;Glazier & Salaita, 2017;Soler et al., 2018). They are applied with micro-and nano-array format, which has opened new avenues to create biochip strategies, for example, sensing strategy for diagnostics, carrier role for vaccines, theranostics, and labeling capability for imaging (Ashley et al., 2011), immunoassays (Soler et al., 2018), and tissue engineering approaches for multiple cellular processes (Glazier & Salaita, 2017).
SLBs can be functionalized with peptides derived from natural proteins, for example, ECM proteins, growth factors, cytokines, antibacterial agents, which influence cellular function. One of the intrinsic properties of SLBs is that they are fluid, that is, the phospholipids laterally diffuse through the layers (Glazier & Salaita, 2017;van Weerd et al., 2015). Since the ligands are anchored to the phospholipids, they diffuse through the lipid layers as well, facilitating the clustering of integrins and their ligands (Glazier & Salaita, 2017). The fluidity of SLBs and thereby the mobility of the ligands can be adjusted by changing the fatty acid composition and ligand density (Glazier & Salaita, 2017;Koçer & Jonkheijm, 2017;van Weerd et al., 2015). in vitro studies with mesenchymal stem cells (MSCs) (Koçer & Jonkheijm, 2017) and C2C12 myoblasts (Bennett et al., 2018) on RGD-functionalized SLBs with variable fluidity have shown contradictory results. Increased numbers of MSCs are attached to more fluid SLBs consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) than to more solid SLBs consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), with cells on DOPC exhibiting a larger cell area than on DPPC (Koçer & Jonkheijm, 2017). Osteogenic differentiation is enhanced in MSCs cultured on more fluid SLBs (Koçer & Jonkheijm, 2017). On the other hand, C2C12 myoblasts show a larger cell area and higher expression of myogenic markers on the more solid DPPC compared to the more fluid DOPC (Bennett et al., 2018). These studies indicate that the degree of ligand mobility modulates progenitor adhesion as well as osteogenic and myogenic differentiation.
Whether ligand mobility also modulates osteoprogenitor adhesion and differentiation is unknown.
The combination of viscosity and stiffness (i.e., viscoelasticity) changes the cell response (Bennett et al., 2018). The viscoelasticity of SLBs might resemble the natural environment of osteoprogenitors (osteoid), indicating that SLBs may be a suitable substrate to stimulate osteogenic differentiation of osteoprogenitors.
Application of RGD-functionalized supported lipid bilayers as coating for biomaterials requires that osteoprogenitors, like MSCs, adhere, proliferate, and differentiate on these substrates. Therefore, the aim of this study was to investigate whether differences exist in adhesion, focal adhesion formation, morphology, proliferation, and osteogenic potential of pre-osteoblasts cultured on RGDfunctionalized SLBs compared to unfunctionalized SLBs and serumattracting poly-L-lysine (PLL), which is a commonly used preosteoblast culture substrate (Bakker et al., 2016;Takai et al., 2006). This study realized for the first time pre-osteoblast adhesion and enhanced differentiation on RGD-functionalized SLBs, which could point to a new horizon in the management of bone regeneration using biomaterials. These results, together with the possibility to adjust SLB fluidity and to incorporate additional proteins that can optimize cellular function, for example, growth factors, cytokines, and/or antibacterial agents, as well as SLBs nonfouling nature make SLBs highly promising as substrate to develop innovative biomimetic coatings for biomaterials in bone regeneration. To the best of our knowledge, this is a novel approach to enhance osteoblast differentiation on biomaterials for improved bone regeneration and seamless biomaterial integration into the bone.
Scratch assays were performed by scratching the SLBs with a pipet tip and visualizing the recovery using confocal microscopy. Images were taken every 2 min. Fluorescent recovery after photobleaching was performed as described before (Koçer & Jonkheijm, 2017). Briefly, a 10 μm spot was bleached and recovery was visualized using confocal microscopy. The mobile fraction and diffusion coefficient were derived from the FRAP data using ImageJ (National Institutes of Health, Bethesda, MD) and FRAPAnalyser (University of Luxembourg, Luxembourg).

| Confocal microscopy
Samples were imaged using a Nikon A1+ confocal laser scanning microscope (Nikon Instruments Europe B.V.). To obtain an overview of one well with cells, 36 z-stack images (slice thickness 3.0 μm) obtained with a 20× objective (numerical aperture 0.8) were stitched together in a 6 × 6 configuration. To visualize single cells, a 60× objective was used to obtain z-stack images with a slice thickness of 0.175 μm. From every well, z-stacks of 10-30 cells were taken and analyzed as described below.

| Image analysis
To investigate the cell density, a maximal z-projection was made from the stitched z-stacks using ImageJ (National Institutes of Health). An F I G U R E 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 area measuring 1000 × 1000 pixels (1253 × 1253 μm) from the center of this image was selected, and the number of cells in this image was counted using a cell counter plugin for ImageJ (De Vos, 2019).
The cell density per cm 2 was calculated from the cell number acquired in the selected area. To analyze focal adhesions, summated z-projections of the lower 15 slices of single cell images were made using ImageJ. From the resulting 2D images, integrin α 5 staining intensity and phosphopaxillin cluster number and size were measured using ImageJ as described (Horzum, Ozdil, & Pesen-Okvur, 2014).

| Gene expression analysis
After 17 hr or 1 week of culture, cells were lysed using TRIreagent (Invitrogen; Fisher Scientific). Total RNA was extracted using  Figure 4) were not normally distributed and tested per category using mixed model ANOVA with category as within-subjects factor, substrate as between-subjects factor, and percentage of cells as the dependent variable. A significant interaction effect (category*substrate) was considered T A B L E 1 Primer sequences used for real-time PCR Target  gene Primer sequence Annealing

| Cell density
To investigate the adherence of MC3T3-E1 pre-osteoblasts on SLBs with and without RGD, the number of cells per cm 2 was quantified ( Figure 3). Cells did adhere to SLBs with and without RGD. Cell density on SLBs with RGD (1353 ± 255 cells/cm 2 , mean ± SEM of 3 RGD-concentrations used) and on SLBs without RGD (1621 ± 266 cells/cm 2 ) was 30-45% lower than on PLL-coated glass (2299 ± 426 cells/cm 2 ; Figure 3; p < .001). There were no significant differences in cell density between SLBs with and without RGD.

| Cell morphology
To investigate the effect of RGD-functionalized supported lipid bilayers on cellular morphology, parameters for cell size (cell surface area, cell volume) and shape of the surface area (elongation, circularity, and On all substrates more than 75% of the cells had a form factor less than 0.2, and no cell had a form factor higher than 0.7, confirming that the cells were generally elongated (Figure 4f). No significant differences were observed between cells on SLBs with and without RGD, although the averages for eccentricity, form factor, and extent seemed to indicate that cells on SLBs with 0.5 μM RGD were somewhat more elongated, less circular, and exhibited more protrusions than cells on SLBs without RGD (Table 2).

| Proliferation
To investigate whether RGD-functionalized SLBs influence proliferation of MC3T3-E1 pre-osteoblasts we investigated gene expression of Ki67 and Cyclin D1 (CCND1) ( Figure 6). After 17 hr, the expression levels of both Ki67 and CCND1 in cells cultured on SLBs without RGD or on SLBs with 0.5 or 1.0 μM RGD were similar to those in cells cultured on PLL-coated glass (Figure 6a). There was a trend toward upregulation of Ki67 and CCND1 expression on SLBs with 0.2 μM RGD compared to SLBs without RGD (not significant). After 1 week, Ki67 and CCND1 expression did not differ between substrates, and were lower than after 17 hr of culture (Figure 6b).

| Osteogenic differentiation state after 17 hr of culture
To investigate the initial osteogenic gene expression response to RGD-  The morphological parameter values for surface area, volume, eccentricity, form factor, and extent are mean ± SEM. SLB ± RGD values are mean ± SEM of pooled data of all SLB experimental groups. Cells on SLBs without or with RGD had a smaller surface area, smaller volume, larger eccentricity, smaller form factor, and lower extent than cells on PLL. Cells on SLBs with 0.5 μM RGD exhibited a larger surface area than cells on SLBs without RGD or with 1.0 μM RGD. n > 300 cells. Significantly different from PLL, *p < .05, **p < .001. # Significant effect of RGD, p < .05. Abbreviations: PLL, poly-L-lysine; SLB, supported lipid bilayer.
trend toward 1.5-fold higher mRNA expression levels of the early osteogenic marker RUNX2 in cells cultured on SLBs with 0.2 μM RGD compared to those cultured on PLL-coated glass (Figure 6a; n.s.). There were no differences regarding the expression of the late osteogenic marker COL1a1 between the different substrates ( Figure 6a). ALP expression, also a late osteogenic marker, was too low to be determined (Figure 6a).

F I G U R E 4
Legend on next page.

| Osteogenic differentiation state after 1 week of culture
To further elucidate the effect of RGD-functionalized SLBs on osteogenic differentiation of pre-osteoblasts, gene expression of RUNX2, OPN, COL1a1, and ALP was also analyzed after 1 week of culture (Figure 6b).
There was a 1.8-fold higher expression of COL1a1 in cells cultured on SLBs with 0.5 μM RGD than on PLL-coated glass (Figure 6b; p < .02). In cells cultured on SLBs without RGD and with 0.2 μM and 1.0 μM RGD, the mean expression of COL1a1 mRNA was 1.4-fold higher than in cells cultured on PLL (Figure 6b; n.s.). Compared to 17 hr, gene expression levels of OPN, COL1a1, and ALP were higher after 1 week, while RUNX2 expression was lower ( Figure 6). This shows that after 1 week the expression of osteogenic genes in cells cultured on RGD-functionalized SLBs was comparable to the expression in cells cultured on PLL, if not higher.

| DISCUSSION
This study aimed to investigate whether differences exist in adhesion, Therefore, the variety of ligands for integrins was likely larger on PLLcoated 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 Percentage of cells adhered to PLL, SLBs without or with RGD with a small (<1 × 10 3 μm 2 ), medium (1-2 × 10 3 μm 2 ), or large (>2 × 10 3 μm 2 ) surface area. There was a lower percentage of cells with a small (p < .001) or a medium surface area (p < .05) and a higher percentage of cells with a large surface area (p < .001) on PLL than on SLBs with or without RGD. (c) Percentage of cells adhered to PLL, and to SLBs without or with RGD with small (<4 × 10 3 μm 3 ), medium (4-8 × 10 3 μm 3 ), or large (>8 × 10 3 μm 3 ) cell volume. There was a lower percentage of cells with a small volume on PLL than on SLBs with or without RGD (p < .05). (d) Eccentricity, a measure for cell elongation, equals 0 for a circle and 1 for a line segment. There was a higher percentage of cells with an eccentricity value larger than 0.9 and a lower percentage of cells with an eccentricity value smaller than 0.8 on SLBs with or without RGD than on PLL (p < .05). (e) Extent, a measure for the number of extensions, has a value between 0 and 1. With an increasing number of extensions, the extent value decreases. There was a lower percentage of cells with an extent value lower than 0.25 and a higher percentage of cells with an extent value between 0.25 and 0.5 on PLL then on SLBs with or without RGD (p < 0.05). (f) FormFactor, a measure for cell shape (4π × area/perimeter; Shah et al., 2019, equals 1 for a perfect circle). On all substrates ±80% of the cells had a FormFactor <0.2 indicating that cells were elongated. There were no differences between substrates. n = 4 separate experiments, 20-140 cells/substrate/experiment (in total 80-560 cells/experiment) **p < .001, *p < .05. PLL, poly-L-lysine coated glass; SLBs, supported lipid bilayers Ki67 and CCND1, as well as RUNX2 was similar in cells cultured on the different substrates. OPN expression was higher in cells cultured on SLBs with 0.2 μM RGD than on PLL (p < .05). COL1a1 expression was not different between cells cultured on the different substrates. ALP expression was too low to be determined. (b) Gene expression in MC3T3-E1 pre-osteoblasts after 1 week of culture. Relative mRNA expression of proliferation markers Ki67 and CCND1, as well as osteogenic markers RUNX2, OPN, and ALP were similar in cells cultured on the different substrates. COL1a1 expression was increased in cells cultured on SLBs compared to PLL. n = 5. *p < .05; **p < .02; CCND1, Cyclin D1; OPN, osteopontin; SLBs, supported lipid bilayers; PLL, poly-L-lysine coated glass; COL1a1, collagen type I α1 chain; ALP, alkaline phosphatase; n.d., not detectable 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 (1975)

| Ligand mobility and density on RGDfunctionalized 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 (Koçer & Jonkheijm, 2017). C2C12 myoblasts cultured on RGD-functionalized SLBs are also smaller than myoblasts cultured on glass (Bennett et al., 2018). This indicates that cells spread less on RGD-functionalized SLBs than they do on serumcoated 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 (Discher, Janmey, & Wang, 2005). 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 (Arnold et al., 2004;Arnold et al., 2008;Huang et al., 2009). However, in these studies, cells were cultured on very stiff (glass) substrates, while substrate stiffness affects the relation between ligand density and cell area (Oria et al., 2017).
Since the SLBs used in the current study have a lower elastic modulus than glass (Picas et al., 2012) and present RGD-peptides in a mobile manner as consequence of the SLB fluidity (Bennett et al., 2018;Glazier & Salaita, 2017), 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 (Frith, Mills, & Cooper-White, 2012;Guo, Lu, Merkel, Sterling, & Guelcher, 2016;Koçer & Jonkheijm, 2017;Wang et al., 2013). Therefore, for studies investigating the 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 (Burridge, Turner, & Romer, 1992).
Phosphorylation of paxillin not only implies adhesion but also activation of focal adhesion kinase and other signaling molecules in the adhesion complex (Khatiwala, Kim, Peyton, & Putnam, 2009).
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 (Khatiwala et al., 2009;Marie et al., 2014). MSCs with large focal adhesions are more osteogenic than cells with small focal adhesions (Frith et al., 2012;Guo et al., 2016;Koçer & Jonkheijm, 2017;Wang et al., 2013). MC3T3-E1 pre-osteoblasts show increased focal adhesion formation accompanied by decreased osteocalcin expression and matrix mineralization (Kong, Polte, Alsberg, & Mooney, 2005). 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. 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 (van Esterik et al., 2016), 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 preosteoblasts 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 (Afanasenkau & Offenhäusser, 2012).
Degradation of SLBs may allow serum proteins to adsorb to the hydrophilic glass creating an environment comparable to that on PLLcoated glass. If so, the slightly higher expression of COL1a1 in cells cultured on SLBs suggests that the initial culturing on SLBs induces signaling that stimulates COL1a1 expression even after the SLBs have started to degrade. To further elucidate the long-term effect of SLBs on cells, SLBs exhibiting higher stability are required. This can be achieved by for example, polymerization of diacetylene-containing lipids introduced within the SLB (Morigaki et al., 2013). An important consideration while targeting SLB stability is preserving SLB fluidity, that is, its biomimicry as desired for certain biomedical applications (Deng et al., 2008;Morigaki et al., 2013).

| Future perspectives for SLBs as coating for biomaterials in bone
Taken together, the results of this study are a first indication that SLBs may be promising as coating for biomaterials in bone, although SLBs have to be further developed to optimize adhesion and differentiation of osteoprogenitors. Changing fatty acid composition and thereby the lateral mobility of SLBs and attached ligands likely affects osteoprogenitor adhesion and differentiation. Osteoprogenitors adhere and differentiate better on substrates with higher rigidity (Wang et al., 2013). Preparing SLBs of lipids with a higher melting transition temperature and thereby lower lateral mobility (e.g., 1-myristol-2-palmityol-sn-glycero-3-phosphocholine [MPPC], melting transition temperature 35 C) may provide an environment where cells experience slightly more resistance when pulling on their attachments, increasing focal adhesion formation and thereby adhesion strength and probably also osteogenic differentiation.
Another way to optimize SLBs for osteoprogenitor adhesion and differentiation may be SLB functionalization with more than one peptide. Immobilization of the short peptide GFOGER, the major binding locus for integrins on collagen type I, to nonfouling substrates induces adhesion of osteoblasts to a level comparable to adhesion on full collagen type I-coated substrates (Reyes & García, 2003). Furthermore, immobilization of RGD together with its synergy sequence PHSRN to poly(ethylene glycol) hydrogels improves osteoblast adhesion compared to RGD alone (Benoit & Anseth, 2005). Osteoprogenitors also adhere with a higher affinity to RGD-peptides with a cyclic conformation than to RGD-peptides with a linear conformation (Porté-Durrieu et al., 2004). The current study used a peptide with a linear conformation and therefore adhesion can likely be improved by functionalizing SLBs with a cyclic RGD peptide. Furthermore, peptides derived from growth factors or cytokines can be incorporated into the SLBs to optimize cellular function, for example, vascular endothelial growth factor (VEGF) to stimulate vascularization.
An advantage of SLBs is their nonfouling nature. This intrinsic resistance of SLBs to the adsorption of proteins and cellular adhesion likely prevents bacteria from attaching to the surface, lowering the risk of infection when SLBs are used as a coating for biomaterials. To further reduce the infection risk, antimicrobial proteins/ peptides can be incorporated into the SLBs as well (Chen & Chen, 2006). Future research will have to elucidate how changing fatty acid composition of the SLBs and functionalization with other peptides can be used to develop innovative coatings for biomaterials in bone regeneration.

| CONCLUSIONS
This study realized for the first time pre-osteoblast adhesion and enhanced differentiation on RGD-functionalized SLBs, which could point to a new horizon in the management of bone regeneration using biomaterials. These results, together with the possibility to adjust SLB fluidity and to incorporate additional proteins that can optimize cellular function, for example, growth factors, cytokines and/or antibacterial agents, as well as SLBs non-fouling nature make SLBs highly promising as substrate to develop innovative biomimetic coatings for biomaterials in bone regeneration.