Controlled spatial and conformational display of immobilised bone morphogenetic protein-2 and osteopontin signalling motifs regulates osteoblast adhesion and differentiation in vitro

Abstract

BackgroundThe interfacial molecular mechanisms that regulate mammalian cell growth and differentiation have important implications for biotechnology (production of cells and cell products) and medicine (tissue engineering, prosthetic implants, cancer and developmental biology). We demonstrate here that engineered protein motifs can be robustly displayed to mammalian cells in vitro in a highly controlled manner using a soluble protein scaffold designed to self assemble on a gold surface.ResultsA protein was engineered to contain a C-terminal cysteine that would allow chemisorption to gold, followed by 12 amino acids that form a water soluble coil that could switch to a hydrophobic helix in the presence of alkane thiols. Bioactive motifs from either bone morphogenetic protein-2 or osteopontin were added to this scaffold protein and when assembled on a gold surface assessed for their ability to influence cell function. Data demonstrate that osteoblast adhesion and short-term responsiveness to bone morphogenetic protein-2 is dependent on the surface density of a cell adhesive motif derived from osteopontin. Furthermore an immobilised cell interaction motif from bone morphogenetic protein supported bone formation in vitro over 28 days (in the complete absence of other osteogenic supplements). In addition, two-dimensional patterning of this ligand using a soft lithography approach resulted in the spatial control of osteogenesis.ConclusionThese data describe an approach that allows the influence of immobilised protein ligands on cell behaviour to be dissected at the molecular level. This approach presents a durable surface that allows both short (hours or days) and long term (weeks) effects on cell activity to be assessed. This widely applicable approach can provide mechanistic insight into the contribution of immobilised ligands in the control of cell activity.

BMP growth factor signaling in a biomechanical context

“…Studies in 2D culture systems investigated biochemical and mechanical effectors without active force application by immobilizing BMP ligands and peptide fragments thereof on different biomaterial surfaces, such as gold surfaces or biopolymeric films . Furthermore, the influence between ligand density and surface topography has been assessed in 2D TiO 2 nanotube‐based culture systems .…”

Osteogenic differentiation of 3D cultured mesenchymal stem cells induced by bioactive peptides

“…Plenty of reports which look into the effect of these peptides on osteogenesis have been published . However, culture conditions of cells used in this work differ significantly.…”

“…Other reports looked only at the synergistic effect of peptides together with added osteogenic supplements . There are also plenty of studies that are interested in the enhanced effect of peptides together with hydroxyapatite matrices, with further studies dealing with the effect of peptides on the differentiation of osteoblasts which are already determined for osteogenic line . In contrary, we selected pMSCs as a cell type, and PCL nanofibres as a scaffold for MSC cultivation with no addition of osteogenic supplements.…”

Matricellular proteins and biomaterials

“…OPN segments have also been immobilized onto gold in various patterns to influence cell adhesion and shape as well as response to growth factors (Mieszawska and Kaplan, 2010; Mitchell et al, 2010). Functionalizing biomaterials with osteopontin has proven to be a useful technique for influencing cell behavior, limiting the FBR to implants, and encouraging tissue regeneration, particularly for orthopedic implants.…”