Surface guidance of stem cell behavior: chemically tailored co-presentation of integrin-binding peptides stimulates osteogenic differentiation in vitroand bone formationin vivo

Surface modification stands out as a versatile technique to create instructive biomaterials that are able to actively direct stem cell fate. Chemical functionalization of titanium has been used in this work to stimulate the differentiation of human mesenchymal stem cells (hMSCs) into the osteoblastic lineage, by covalently anchoring a synthetic double-branched molecule (PTF) to the metal that allows a finely controlled presentation of peptidic motifs. In detail, the effect of the RGD adhesive peptide and its synergy motif PHSRN is studied, comparing a random distribution of the two peptides with the chemically-tailored disposition within the custom made synthetic platform, which mimics the interspacing between the motifs observed in fibronectin. Contact angle measurement and XPS analysis are used to prove the efficiency of functionalization. We demonstrate that, by rationally designing ligands, stem cell response can be efficiently guided towards the osteogenic phenotype: In vitro, PTF-functionalized surfaces support hMSCs adhesion, with higher cell area and formation of focal contacts, expression of the integrin receptor α5β1 and the osteogenic marker Runx2, and deposition a highly mineralized matrix, reaching values of mineralization comparable to fibronectin. Our strategy is also demonstrated to be efficient in promoting new bone growth in vivo in a rat calvarial defect. These results highlight the efficacy of chemical control over the presentation of bioactive peptides; such systems may be used to engineer bioactive surfaces with improved osseointegrative properties, or can be easily tuned to generate multi-functional coatings requiring a tailored disposition of the peptidic motifs. Statement of significance Organic coatings have been proposed as a solution to foster osseointegration of orthopedic implants. Among them, extracellular matrix-derived peptide motifs are an interesting biomimetic strategy to harness cell-surface interactions. Nonetheless, the combination of multiple peptide motifs in a controlled manner is essential to achieve receptor specificity and fully exploit the potentiality of synthetic peptides. Herein, we covalently graft to titanium a double branched molecule to guide stem cell fate in vitro and generate an osseoinductive titanium surface in vivo. Such synthetic ligand allows for the simultaneous presentation of two bioactive motifs, thus is ideal to test the effect of synergic sequences, such as RGD and PHSRN, and is a clear example of the versatility and feasibility of rationally designed biomolecules.