Self-assembling peptide-based hydrogels have The formation of hydrogels by self-assembling peptides 44 involves two distinct processes; the self-assembly of the 45 peptides themselves to form thin fibrillar structures and the 46 entanglement and association of these fibrils into a threef1 47 dimensional percolated network (Figure 1). Developing a 48 fundamental understanding of these two processes at all length 49 scales is crucial as the properties of the final materials will not 50 only depend on the intrinsic properties of the fibers, but also on In this work we were interested in understanding how 77 network topology affects the mechanical properties of the 78 hydrogel and how it can be used to design materials with 79 tailored properties. For this purpose we decided to use a family 80 of octapeptides based on the same design that are known to 81 self-assemble into β-sheet rich fibrils. This approach allowed us 82 to keep the fiber structure identical across all the systems and 83 focus on the effect of network topology on the mechanical 84 properties of the hydrogels formed. As mentioned above due to 85 the design chosen interfiber interactions are controlled mainly 86 by the hydrophilic residues. Of particular interest to us was 87 arginine, which is a residue that has a guanidine side group. 88 were assessed in an oscillatory mode, using a stress-controlled ) < 2.4, with q = 171 (4π/λ) sin(θ/2), where θ is the scattering angle. The collected data 172 was corrected for the detector efficiency and dark current background. 173 Counter normalization was achieved by using the incoherent scattering 174 of an amorphous hydrogenous poly(methyl methacrylate) secondary 175 standard. After ensuring the scattering was isotropic, the data were 176 radially averaged to obtain a one-dimensional scattering curve. Under 177 these conditions, the normalized intensity scattered by a sample is

β-Sheet forming peptides have attracted significant interest for the design of hydrogels for biomedical applications. One of the main challenges is the control and understanding of the correlations between peptide molecular structure, the morphology, and topology of the fiber and network formed as well as the macroscopic properties of the hydrogel obtained. In this work, we have investigated the effect that functionalizing these peptides through their hydrophobic face has on their self-assembly and gelation. Our results show that the modification of the hydrophobic face results in a partial loss of the extended β-sheet conformation of the peptide and a significant change in fiber morphology from straight to kinked. As a consequence, the ability of these fibers to associate along their length and form large bundles is reduced. These structural changes (fiber structure and network topology) significantly affect the mechanical properties of the hydrogels (shear modulus and elasticity).

An attractive strategy for the regeneration of tissues has been the use of extracellular matrix analogous biomaterials. Peptide-based fibrillar hydrogels have been shown to mimic the structure of extracellular matrix offering cells a niche to undertake their physiological functions. In this study, the capability of an ionic-complementary peptide FEFEFKFK (F, E, and K are phenylalanine, glutamic acid, and lysine, respectively) hydrogel to host human mesenchymal stem cells in three dimensions and induce their osteogenic differentiation is demonstrated. Assays showed sustained cell viability and proliferation throughout the hydrogel over 12 days of culture and these human mesenchymal stem cells differentiated into osteoblasts simply upon addition of osteogenic stimulation. Differentiated osteoblasts synthesized key bone proteins, including collagen-1 (Col-1), osteocalcin, and alkaline phosphatase. Moreover, mineralization occurred within the hydrogel. The peptide hydrogel is a naturally biodegradable material as shown by oscillatory rheology and reversed-phase high-performance liquid chromatography, where both viscoelastic properties and the degradation of the hydrogel were monitored over time, respectively. These findings demonstrate that a biodegradable octapeptide hydrogel can host and induce the differentiation of stem cells and has the potential for the regeneration of hard tissues such as alveolar bone.

Understanding peptide self-assembly mechanisms and stability of the formed assemblies is crucial for the development of functional nanomaterials. Herein, we have adopted a rational design approach to demonstrate how a minimal structural modification to a nonassembling ultrashort ionic selfcomplementary tetrapeptide FEFK (Phe4) remarkably enhanced the stability of self-assembly into β-sheet nanofibers and induced hydrogelation. This was achieved by replacing flexible phenylalanine residue (F) by the rigid phenylglycine (Phg), resulting in a constrained analogue PhgEPhgK (Phg4), which positioned aromatic rings in an orientation favorable for aromatic stacking. Phg4 self-assembly into stable β-sheet ladders was facilitated by π-staking of aromatic side chains alongside hydrogen bonding between backbone amides along the nanofiber axis. The contribution of these noncovalent interactions in stabilizing self-assembly was predicted by in silico modeling using molecular dynamics simulations and semiempirical quantum mechanics calculations. In aqueous medium, Phg4 β-sheet nanofibers entangled at a critical gelation concentration ≥20 mg/mL forming a network of nanofibrous hydrogels. Phg4 also demonstrated a unique surface activity in the presence of immiscible oils and was superior to commercial emulsifiers in stabilizing oil-in-water (O/W) emulsions. This was attributed to interfacial adsorption of amphiphilic nanofibrils forming nanofibrilized microspheres. To our knowledge, Phg4 is the shortest ionic self-complementary peptide rationally designed to self-assemble into stable β-sheet nanofibers capable of gelation and emulsification. Our results suggest that ultrashort ionic-complementary constrained peptides or UICPs have significant potential for the development of cost-effective, sustainable, and multifunctional soft bionanomaterials.