Signalling by interfacial motifs is a fundamental biological mechanism that underpins numerous aspects of morphogenesis and wound healing . Tools that enable further insight into how immobilized ligands regulate cell behaviour will not only provide mechanistic understanding but also the basis for therapeutic approaches such as tissue engineering . Our results show that a generic immobilisation system with a variable motif sandwiched between expression enhancing and self assembly domains (Tol-motif-ST) can present the motifs in a functional state at the surface of a SAM. The fusion protein is easily produced in large amounts in E coli and is easy to convert from a highly water soluble state to a SAM inserted form on gold . Analysis by SPR of the Tol-ST proteins on a gold surface interestingly provided evidence that Tol-OPN-ST and Tol-BMP-ST pack at higher surface densities than Tol-ST alone. This may arise from flexibility introduced into the protein by the inclusion of the short OP and BMP motifs. The highest flexibility is expected from the OP domain as there are three linker sequences found in this sequence whilst the BMP motif is shorter. In addition analysis of Tol-GFP (data not shown) assembly by SPR shows this rigid construct to be similar to Tol alone, supporting the possibility that protein flexibility plays a role in its packing on the surface. Thus retention of the N terminal Tol domain does not inhibit self assembly and likely serves to solubilise and protect the flexible peptides from degradation, presenting the biologically active motifs in a more constrained structure.
The biological activity of the Tol-ST surfaces was first determined in simple cell adhesion assays performed in the absence of serum. The abundant cell adhesion on Tol-OPN-ST, in contrast to the other surfaces, is most likely mediated by α9β1 integrin as described for endothelial cells [11–14]. Calvarial derived osteoblasts have not been previously shown to express α9β1 integrins but they can be found on periodontal ligament cells  and cells of the osteoclast lineage . The adhesion and spreading of cells described here is a possible demonstration of α9β1 integrins activity in these cells but an alternative mechanism cannot be discounted.
Surface assembly techniques provide the ability to control surface density of cell interaction motifs. Reducing the apparent density of Tol-OP-ST had significant effects on cell shape, morphology and numbers of vinculin-positive focal contacts. This revealed a threshold level of Tol-OP-ST below which cytoskeletal tensioning and remodelling cannot be supported (2.7 × 109 molecules/mm2), and above which the cells can interact with the surface initiating morphological change and regulation of cell behaviour. The estimated density measured here compares with other approaches that have attempted to address this issue where cells of fibroblastic origin exhibited increased cell adhesion and spreading on integrin ligands of the order 109 molecules/mm2 [23–25]
The way in which cells adhere and interact with the surrounding matrix is known to not only influence cell shape but also to control cell response and gene expression . Indeed this has been shown to be a mechanism that is important in the regulation of osteoblast differentiation [26, 27] and response to BMPs [28–30]. To demonstrate that Tol-ST surfaces could be used to probe this phenomenon, different densities of Tol-OPN-ST were characterised for their ability to support BMP dependent signalling. A SMAD-responsive reporter construct showed significantly greater activation in response to srBMP-2 when cells were cultured on Tol-OPN-ST at 2.7 × 109-3.3 × 1010 molecule/mm2 compared to 2.3 × 108 molecules/mm2 and lower. It is particularly noteworthy that the apparent threshold for formation of abundant cell adhesion plaques (1.6 × 1010 molecules/mm2) is higher than that for BMP receptor activation and signal transduction (2.7 × 1010 molecules/mm2). This suggests that the mechanisms regulating BMP receptor signalling may well be independent of cytoskeletal organisation controlled by integrin adhesion to Tol-OPN-ST. On other substrates, αvβ integrins  and Focal Adhesion Kinase (FAK)  have both been implicated in the osteoblastic response to BMP. Our results support general matrix-dependent BMP signalling in terms of FAK activity since weakly adherent cells demonstrated poor BMP responses whilst well adhered and spread cells exhibited elevated BMP effects. Nevertheless this does not exclude a role for integrin subunit specific responses with BMPRI and BMPRII directly associating with αv and β1 integrins , on appropriate external ECM ligands, to give altered responses.
Tol-BMP-ST maintained BMP-like activity and like srBMP-2 was able to activate the Smad signalling cascade. This is in agreement with the work of Saito et al (2003) who showed that a synthetic BMP2 knuckle peptide was able to induce alkaline phosphatase activity in C2C12 cells but less effectively than srBMP2 . Previous work [32, 33] has shown that immobilised BMP2 more effectively activates differentiation of osteoblasts than soluble BMP2. Tol-BMP-ST was immobilised on gold surfaces, back-filled with TEG-thiol and then compared with soluble recombinant BMP-2 for the ability to regulate osteoblast activity and bone formation. Immobilized Tol-BMP-ST showed activation of SMAD responsive elements driving luciferase expression in transfected cells. Interestingly the level of activation of signalling by the immobilised ligand was comparable to soluble recombinant BMP2, demonstrating its enhanced activity compared to soluble Tol-BMP-ST. The activity of this BMP2 knuckle peptide is postulated to be mediated through its interaction with BMP receptors I and II (BMPRI and BMPRII) . Activation of preformed or induced BMPR complexes by soluble BMP results in initiation of intracellular signalling cascades that include the Smad pathway. In addition, BMPR complexes are believed to be internalised by the cell in processes which both alter the signalling dynamic and lead to loss of or attenuation of activation [34–38]. It is therefore likely that Tol-BMP-ST surfaces facilitate prolonged activation of BMP signalling since the receptor complex would be retained at the cell surface by the immobilised ligand and prevented from trafficking into the cell for example via lipid rafts. This is supported by the work of others which shows that immobilised recombinant BMP2 causes prolonged activation of osteogenic gene expression and enhanced osteoblast differentiation [39, 40].
When cells were plated on Tol-BMP-ST under routine culture conditions, cell differentiation occurred during long term experiments with evidence of the formation of abundant bone-like nodules. In contrast, few mineralised matrix deposits were observed on Tol-OPN-ST and bare gold. Immobilised BMP has previously been shown to support osteogenic differentiation in vitro [32, 40, 41] using MC3T3-E1 cells and BMP2 that was immobilized by a heat treatment process. Data presented here use primary cells that are a heterogeneous population that retain the potential to differentiate to several different lineages but this is the first time that a tethered ligand alone and in the complete absence of other agents like dexamethasone not only induces ostegenic differentiation but also extensive deposition of a mineralized bone-like matrix. This illustrates the enhanced and sustained signalling provided by the Tol-BMP-ST surface reinforced cell regulator cues that drive the temporal control of cell phenotype. Furthermore Tol-BMP-ST on gold reproduces the asymmetric directional control of bone formation observed in vivo, a process that is believed to be guided by immobilised ligands. The formation of bone-like nodules in vitro requires extensive cell motility with the formation of three-dimensional cell aggregates and therefore the immobilised signal derived from Tol-BMP-ST is likely to be relayed to cells away from the surface by cell:cell interactions and paracrine signalling.
Spatial control of osteoblast differentiation was demonstrated by using microcontact printing of Tol-BMP-ST. This approach has been used widely in cell culture studies  with predominantly ECM motifs but this is the first time that cell differentiation motifs have been presented in this way. This showed that it is immobilised Tol-BMP-SP and not ligand lost from the surface that is directing cell behaviour. Work using an inkjet approach has recently reported similar findings with recombinant BMP2 spotted on a fibrin layer  illustrating the potential for patterns of immobilised biological ligands to control cell behaviour in a way that is spatially restricted.