Development and characterization of genetically engineered M13 bacteriophage as tissue engineering materials

In vivo cell polarization is mitigated by topographical and chemical cues supplied by the extracellular nanofibrous protein matrix and the neighboring cells. To replace or repair tissues functionally dependent on cell alignment, engineers must design materials to simulate these environments. Here we investigated the use of M13 bacteriophage (phage), filamentous bacterial viruses as aligned nanofibrous tissue engineering matrices.;The phage shell is made of 2700 copies of protein pVIII that can be engineered to create a dense and uniform display of biofunctional peptides available for cell interaction. However, any genetic modifications to pVIII must be able to accommodate its biological roles in phage replication process. To express an inherently unfavorable positively charged RGD motif within the negative N-terminus of pVIII we devised a novel method to circumvent bacterial biological censorship. By constraining our peptide, among the degenerate flanking residues, we were able to obtain a library of phage containing the RGD sequence surrounded by variable naturally selected residues.;Concentrated phage solutions can self-assemble into directionally defined liquid crystalline structures due to their long-rod shape and monodispersity. Therefore after expressing RGD- and IKVAV cell-signaling peptides on the phage pVIII proteins we used them as building blocks for the construction of aligned nanofiber scaffolds. Through viability and proliferation assays we showed the biocompatibility of phage materials with neural progenitor cells (NPCs) and fibroblasts. We demonstrated the specificity of cell interaction with phage presented ligands by differences in cell adhesion to RGD and IKVAV phage substrates as compared to and RGE- and wildtype phage controls. We constructed two dimensional aligned phage films by shearing, and three-dimensional aligned nanofibrous phage scaffolds by micropipette spin injection. Finally we showed that the aligned structure of phage scaffolds was able to control the cell polarization and elongation in both two and three dimensions.;As phage have several proteins that can be engineered to present functional peptides or linker molecules (i.e. HPQ biotin-like motifs), they offer promising opportunities to construct highly functionalized aligned matrices for challenging medical problems, such as the regeneration of nerve tissue, or for controlled in vitro model systems to study complicated cell signaling environments.