Novel Peptide-Based Platform for the Dual Presentation of Biologically Active Peptide Motifs on Biomaterials Carlos Mas-Moruno,* ,†,‡,§ Roberta Fraioli, †,‡,§ Fernando Albericio, ‡,⊥,#,|| Jose ́ María Manero, †,‡ and F. Javier Gil †,‡ † Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgical Engineering, Technical University of Catalonia (UPC), ETSEIB, Avenida Diagonal 647, 08028 Barcelona, Spain ‡ Biomedical Research Networking Centre in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Campus Río Ebro, Edificio I+D Bloque 5, 1a planta, C/Poeta Mariano Esquillor s/n, 50018 Zaragoza, Spain § Centre for Research in NanoEngineering (CRNE), Universitat Polite ́ cnica de Catalunya, C/Pascual i Vila 15, 08028 Barcelona, Spain ⊥ Institute for Research in Biomedicine (IRB-Barcelona), Barcelona Science Park, C/Baldiri Reixac 10, 08028 Barcelona, Spain # Department of Organic Chemistry, University of Barcelona, C/Martí i Franque ̀ s 1-11, 08028 Barcelona, Spain || School of Chemistry & Physics, University of KwaZulu-Natal, 4001 Durban, South Africa *S Supporting Information ABSTRACT: Biofunctionalization of metallic materials with cell adhesive molecules derived from the extracellular matrix is a feasible approach to improve cell-material interactions and enhance the biointegration of implant materials (e.g., osseointegration of bone implants). However, classical biomimetic strategies may prove insufficient to elicit complex and multiple biological signals required in the processes of tissue regeneration. Thus, newer strategies are focusing on installing multifunctionality on biomaterials. In this work, we introduce a novel peptide-based divalent platform with the capacity to simultaneously present distinct bioactive peptide motifs in a chemically controlled fashion. As a proof of concept, the integrin-binding sequences RGD and PHSRN were selected and introduced in the platform. The biofunctionalization of titanium with this platform showed a positive trend towards increased numbers of cell attachment, and statistically higher values of spreading and proliferation of osteoblast-like cells compared to control noncoated samples. Moreover, it displayed statistically comparable or improved cell responses compared to samples coated with the single peptides or with an equi- molar mixture of the two motifs. Osteoblast-like cells produced higher levels of alkaline phosphatase on surfaces functionalized with the platform than on control titanium; however, these values were not statistically significant. This study demonstrates that these peptidic structures are versatile tools to convey multiple biofunctionality to biomaterials in a chemically defined manner. KEYWORDS: biofunctionalization, cell adhesive peptide, cell adhesion, peptide platform, titanium, RGD peptide, PHSRN peptide ■ INTRODUCTION It is nowadays well-established that the biofunctionalization of metallic materials with cell adhesive molecules derived from the extracellular matrix (ECM) is a powerful approach to stimulate and direct cell behavior (i.e., cell adhesion, proliferation and dif- ferentiation), and thus improve and accelerate the biointegration of an implant material within the organism. 1-3 Such biomimetic strategy relies on the interaction of short cell binding peptide sequences of the ECM with cell-expressed receptors such as integrins, which trigger intracellular biochemical signals and mediate specific cellular functions. 4 In regard to this, the functionalization of Ti surfaces with proteins or peptides with affinity for integrins expressed by osteoblasts has shown to enhance osteoblast adhesion to these surfaces in vitro and improve implant osseointegration in vivo. 1,5,6 Nonetheless, there is a long-standing debate concerning which is the best biomimetic approach to convey functionality to biomaterials. 7 The use of native proteins (e.g., fibronectin, 8,9 type I collagen 10-12 ) or recombinant protein fragments (e.g. FNIII- (7-10), 13-15 FNIII(9-10) 16 ) from the ECM provides multiple and dynamic bioactive epitopes that efficiently engage and activate complex cellular responses. Nonetheless, this approach presents some limitations related to the low stability of proteins towards changes of pH and temperature, the risks of infection and immunogenicity associated with their use, the difficulty in obtaining chemically defined structures, and their high costs of production. 17,18 Received: January 7, 2014 Accepted: March 27, 2014 Published: March 27, 2014 Research Article www.acsami.org © 2014 American Chemical Society 6525 dx.doi.org/10.1021/am5001213 | ACS Appl. Mater. Interfaces 2014, 6, 6525-6536