Full length articleSurface guidance of stem cell behavior: Chemically tailored co-presentation of integrin-binding peptides stimulates osteogenic differentiation in vitro and bone formation in vivo
Graphical abstract
Introduction
Several therapies for the treatment of injured bone tissue are currently established, yet recent findings on the natural healing processes of the organism suggest new routes for improvement. The role of stem cells in the restoration of damaged tissue has been fully recognized [1], [2], and much effort is nowadays dedicated to the understanding of the underlying mechanisms [3], with a view to establishing novel clinical treatments [4], [5]. Mesenchymal stem cells (MSCs) are non-haematopoietic, heterogeneous pluripotent cells that are capable of differentiating into several mesodermal and non-mesodermal cell lineages [6]. As traumatic injury occurs, these progenitor cells are mobilized from their niche and recruited to the damaged tissue, in order to contribute to the reparative process [7]. Their contribution to the natural healing response of the body has been described to be via in situ differentiation into cells that directly replace the damaged tissue and paracrine action (or trophic activity [8]), which controls the injury-related inflammatory response [9].
Particular attention has been given to the engineering of biomaterials that control the commitment of MSCs to specific lineages, such as neuronal [10], chondrogenic [11], cardiac [12], and osteoblastic [13]. However, harnessing MSC fate remains a major challenge. Addressing such challenge would be of great significance in the orthopaedic and maxillofacial field, where the capacity to stimulate the osteogenic differentiation of MSC on the surface of clinically-relevant materials (e.g. on titanium and its alloys) [14] would translate into higher rates of implant osteointegration and improved long-term functionality. Surface modification strategies can be used for this purpose. As a matter of fact, variations of the surface stiffness [15], chemical composition [16], [17], topography [18], [19], and hydrophilicity [20] have been proven to influence MSC response.
Among chemistry-based strategies of surface functionalization, the anchoring of integrin-binding ligands is a particularly interesting solution to guide osteodifferentiation [21], [22]. Integrins are a family of heterodimeric transmembrane receptors, constituted by two non-covalently bound α and β subunits, that are responsible for the communication of signals from the extracellular matrix (ECM) to the nucleus, and vice versa [23]. Although, a complete view on how integrin ligands influence the response of progenitor cells has not been achieved yet, peptides derived from the ECM have been used to modulate cell fate. This is the case of the two integrin-binding motifs present in the cell attachment site of fibronectin (FN): the Arg-Gly-Asp (RGD) peptide [24], which interacts with several integrin subtypes [25], and the Pro-His-Ser-Arg-Asn (PHSRN) motif, whose synergic effect increases the affinity of RGD for the integrin receptor α5β1 [26]. This integrin receptor has been proved to be important in osteogenesis and thus its specific engagement opens promising prospects in bone regeneration [22], [27], [28], [29]. Presentation of RGD and PHSRN sequences at the proper distance is crucial to preserve their synergic behavior [30], [31]. The effect of these two ECM-derived motifs on cell adhesion has been studied in the literature [30], [32], [33], [34], [35], but their capacity to induce osteogenic differentiation of MSCs on metallic substrates has been only achieved by using engineered recombinant fragment of FN encompassing the whole cell attachment site of the protein [36], [37], [38]. Nonetheless, the limitations associated to the use of proteins urge the development of alternative synthetic approaches that mimic and retain their integrin-binding specificity but offer higher chemical control, safety and stability [39]. To mimic the 30–40 Å distance that separates the motifs in FN [40], [41] within a synthetic ligand, several linkers have been proposed, including polyglycine chains, (Gly)6 [42], [43] or (Gly)13 [35], as well as (Ser-Gly)5 units [32]. In our study we use a novel design based on a double-branched molecule (Fig. 1), which uses 4 aminohexanoic acid (Ahx) molecules linked via a lysine (Lys) residue as spacer. When the branches adopt an open conformation, such spacer covers approximately the same distance as 12 Gly [44]. Hence, the aim of this study is to characterize the response of human MSCs (hMSCs) on Ti surfaces functionalized with a novel synthetic platform (PTF) [44], in which the RGD and PHSRN motifs are chemically inserted in a controlled fashion, and compare it with the presentation of the motifs alone or in a geometrically random distribution (Fig. 1). To the best of our knowledge, the effect on hMSCs and the in vivo response due to such synthetic system of ligand presentation, which mimics the biological orientation and spacing of RGD and PHSRN sequences present in FN, has never been addressed before. To this end, we realized a straightforward three-step procedure to anchor the integrin-binding ligands to Ti, and studied the effect on hMSCs behavior in terms of cell number, spreading, focal contacts, proliferation, gene expression and mineralization. The capacity of the PTF to promote bone formation in vivo was evaluated in a calvarial defect model in rats.
Section snippets
Synthesis of the bioactive molecules
The platform (Ac-Arg-Gly-Asp-Ser-Ahx-Ahx) (Ac-Pro-His-Ser-Arg-Asn-Ahx-Ahx)-Lys-βAla-Cys-NH2 (PTF) and the linear peptides MPA-Ahx-Ahx-Ahx-Arg-Gly-Asp-Ser-OH (RGD) and MPA-Ahx-Ahx-Ahx-Pro-His-Ser-Arg-Asn-NH2 (PHSRN) (Ahx: aminohexanoic acid; MPA: 3-mercaptopropionic acid) were manually synthesized by solid-phase peptide synthesis methods following the Fmoc/tBu strategy and using 2-chlorotrityl chloride (CTC, 200 mg, 1.0 mmol/g) or Fmoc-Rink amide MBHA resin (200 mg, 0.45 mmol/g) as solid supports,
Functionalization of Ti: evolution of physicochemical properties of the surface
The sessile drop method was used to measure the contact angle of three 1 μL drops of either water (Fig. 2) or diiodomethane. The nitric acid treatment (HNO3) resulted in a significant decrease of water contact angle, compared to polished samples (P). In contrast, silanization with APTES increased this value. Anchoring of the peptidic ligands in all cases resulted in a significant increase in wettability. Diiodomethane contact angle was not significantly affected by the functionalization process
Discussion
The aim of this study is to develop a bioactive surface that guides the osteogenic differentiation of hMSCs by means of a chemically controlled presentation of integrin-binding ligands. In detail, the RGD and PHSRN sequences synergically bind integrin α5β1, which is known to be involved in several important events of bone biology [22], [53], [54], [54], [55], provided that the distance between the motifs mimics the one found in the protein [31], [32], [35]. For the synergic effect to take
Conclusions
This work explores the possibility of guiding cell fate in vitro and fostering in vivo osteointegration of a clinically-relevant material such as Ti, by generating a controlled disposition of two synergic peptidic sequences, namely RGD and PHSRN. Our custom synthesized molecule (PTF) is designed so that the distance between the sequences mimics the one found in the integrin-binding site of FN. This straightforward control of motifs disposition is demonstrated to positively influence the
Acknowledgements
The authors thank the Spanish Government for financial support through Project No. MAT2015-67183-R (MINECO-FEDER), co-funded by the European Union through European Regional Development Funds. R.F. and C.M.-M thank the Government of Catalonia for financial support through a pre-doctoral and post-doctoral fellowship, respectively. C.M.-M also thanks the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7-PEOPLE-2012-CIG, REA Grant Agreement No. 321985)
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