Recent studies on peptide hydrogels have shown that ultrashort peptides (<8 amino acids) can self-assemble into hydrogels. Ultrashort peptides can be designed to incorporate antimicrobial motifs, such as positively charged lysine residues, so that the peptides have inherent antimicrobial characteristics. Antimicrobial hydrogels represent a step change in tissue engineering and merit further investigation, particularly in applications where microbial infection could compromise healing. Herein, we studied the biocompatibility of dental pulp stem/stromal cells (DPSCs) with an ultrashort peptide hydrogel, (naphthalene-2-ly)-acetyl-diphenylalanine-dilysine-OH (NapFFεKεK-OH), where the epsilon (ε) amino group forms part of the peptide bond rather than the standard amino grouping. We tested the antimicrobial properties of NapFFεKεK-OH in both solution and hydrogel form against Staphylococcus aureus, Enterococcus faecalis and Fusobacterium nucleatum and investigated the DPSC secretome in hydrogel culture. Our results showed NapFFεKεK-OH hydrogels were biocompatible with DPSCs. Peptides in solution form were efficacious against biofilms of S. aureus and E. faecalis, whereas hydrogels demonstrated antimicrobial activity against E. faecalis and F. nucleatum. Using an angiogenic array we showed that DPSCs encapsulated within NapFFεKεK-OH hydrogels produced an angiogenic secretome. These results suggest that NapFFεKεK-OH hydrogels have potential to serve as novel hydrogels in tissue engineering for cell-based pulp regeneration.

In light of the increasing levels of antibiotic resistance, nanomaterials and novel biologics are urgently required to manage bacterial infections. To date, commercially available self-assembling peptide hydrogels have not been studied extensively for their ability to inhibit micro-organisms relevant to tissue engineering sites such as dental root canals. In this work, we assess the biocompatibility of dental pulp stem/stromal cells with commercially available multicomponent peptide hydrogels. We also determine the effects of dental pulp stem/stromal cell (DPSC) culture in hydrogels on growth factor/cytokine expression. Furthermore, to investigate novel aspects of self-assembling peptide hydrogels, we determine their antimicrobial activity against the oral pathogens Staphylococcus aureus, Enterococcus faecalis, and Fusobacterium nucleatum. We show that self-assembling peptide hydrogels and hydrogels functionalized with the adhesion motif Arg-Gly-Asp (RGD) are biocompatible with DPSCs, and that cells grown in 3D hydrogel cultures produce a discrete secretome compared with 2D-cultured cells. Furthermore, we show that soluble peptides and assembled hydrogels have antimicrobial effects against oral pathogens. Given their antibacterial activity against oral pathogens, biocompatibility with dental pulp stem/stromal cells and enhancement of an angiogenic secretome, multicomponent peptide hydrogels hold promise for translational use.

Alport syndrome (AS) is a rare disease characterized by defective glomerular basement membranes, caused by mutations in COL4A3, COL4A4, and COL4A5, which synthesize collagen type IV. Patients present with progressive proteinuria, hematuria and podocyte loss. There is currently no cure for Alport syndrome, and this is mainly due to its complex and variable pathogenesis, as well as the lack of models that can faithfully mimic the human phenotype. Here we have developed a novel human culture model of Alport syndrome and used it to study the effects of different mutations on podocyte development and biology. First, we established a differentiation protocol that allowed us to generate podocyte spheroids from patient-derived human induced pluripotent stem cells (hiPSCs). We have then carried out discovery proteomics and demonstrated that a total of 178 proteins were differentially expressed between Alport (AS1 and AS3) and control (LT) podocytes. GO analysis indicated alterations in several metabolic pathways, such as oxidative phosphorylation, RNA maturation, chromatin condensation, and proliferation. Although functional assays showed no changes in lactate production and mitochondrial potential compared to healthy controls, immunofluorescence and electron microscopy analysis showed key morphological changes related to the phenotypical maturation of Alport podocytes. Moreover, the studied mutations led to persistent proliferation, increased reactive oxygen species (ROS) production and the concomitant expression of peroxisome proliferator-activated receptors α and γ (PPARα and PPARγ) in podocytes. These data on patient-derived podocytes provide evidence that collagen mutations, in addition to playing a central role in the defective development of the glomerular filtration barrier, cause significant alterations in podocyte development and metabolism very early in development, even before the formation of the filtering apparatus. In conclusion, our study provides a new methodological platform for the differentiation of podocytes and to study human podocytopathies in a personalized manner, and reveals new insights into the etiopathogenesis and pathobiology of Alport syndrome.

The synthesis of hydrogel scaffolds with inherent antimicrobial activity has advantages for their use in tissue engineering. An ultra-short naphthalene lysine conjugated peptide, NapFFK’K’, containing naphthalene (Nap) as a molecule of high aromaticity for gel strength, phenylalanine (F) and epsilon variant lysine (K’) has previously been shown by us to self-assemble forming hydrogels with inherent antimicrobial properties against a limited number of pathogens tested. The aim of this work was to extend the antimicrobial activity studies on NapFFK’K’ including pathogenic bacteria associated with dental infections. NapFFK’K’ was synthesised using the 9-fluorenylmethoxucarbonyl Solid Phase Peptide Synthesis. Peptide purity was analysed by mass spectrometry. Hydrogel formulation was achieved by suspending the peptide in sterile deionized water followed by addition of NaOH and HCl. Hydrogels were tested at peptide concentrations of 1 %, 1.5 % and 2 % w/v against the Gram-positive bacteria Enterococcus faecalis and Staphylococcus aureus, and the Gram-negative bacterium Fusobacterium nucleatum. Bacteria inoculumns were exposed on hydrogel surface for 24 h. Bacterial susceptibility assay, employing the Miles and Misra method, was used to determine antimicrobial activity of hydrogels after 24 h incubation. Our results show that peptide hydrogels exhibit antimicrobial properties against both groups of bacteria but at different peptide concentrations. The 1 % peptide hydrogels was most effective against Gram-positive bacteria whereas the 2 % peptide hydrogel was effective against Fusobacterium nucleatum. Given the efficacy of the self-assembling NapFFK’K’ peptide hydrogels against oral pathogens, they may have potential use in tissue engineering approaches for regenerative endodontic treatments.