Christopher Parmenter

Extracellular matrix (ECM) derived biomaterials are becoming more prevalent for regenerative medicine applications as they can provide bio‐instructive cues to damaged tissues, facilitating constructive remodeling of the injured site. Existing clinical products based on ECM can be found in sheet or powder formats, however recent clinical trials have utilized ECM hydrogels. Further exploration of ECM hydrogels and modulation of their mechanics, degradation and swelling behaviors could provide increased utility of these materials. Mechanical modulation through crosslinking can be achieved through application of chemical reagents such as glutaraldehyde (GA). However long‐term effects of GA warrant increased research of alternatives such as the plant‐based derivatives genipin (GP) and proanthocyanidins (PA). In this work, crosslinking was confirmed through alterations to the mechanical moduli of bECM hydrogels with both GP and PA conferring significantly increased moduli compared to non‐crosslinked controls. In vitro examination determined that cytotoxic effects were observed for PA to SH‐SY5Y cells at the concentration used, however no cytotoxicity was observed from PA when tested on L929 cells and primary tenocytes. There was no cytotoxicity observed from GP treatment. Therefore, use of these reagents could provide a further toolkit to manipulate physicochemical properties and increase utility of ECM hydrogels.

Polyurea greases play an important role in the operation of electric vehicles, and it is understood that their performance is principally determined by their microstructure. Consequently, developing a complete understanding of the structural characteristics of polyurea greases is crucial for the future engineering of new greases. One of the most widely used techniques for urea grease analysis is scanning electron microscopy (SEM). However, this requires the removal of the base oil component prior to analysis and hence does not give a true picture of the grease microstructure. Alternative techniques, including polarized optical microscopy (POM) and cryogenic SEM (cryo-SEM), do not require base oil removal, but fail to provide the chemical information required to definitively discriminate between the base oil and thickener components. In this study, we pioneered the use of a novel combination of native- and near-native-state chemical imaging techniques, namely, confocal Raman microscopy (CRM) and cryogenic secondary ion mass spectrometry (cryo-SIMS), to characterize a commercial polyurea grease sample. It was possible to detect peaks diagnostic of polyurea in CRM, and the urea species known to be present in the grease in cryo-SIMS. In the chemical imaging analyses, distinct particles of polyurea (∼250–280 μm2) were observed, broadly consistent with observations from POM of the same material. Such findings increase confidence that POM, the most straightforward native-state technique applicable for polyurea grease analysis, can be reliably used to understand the key structure-performance relationships that will ultimately guide the development of future polyurea greases.

Solvated supramolecular hydrogels present unique challenges in nanoscale morphological characterization because of their fragile fibrous nature and low concentration of the solid component. In this study, imidazolium-based hydrogels containing either diketopyrrolopyrrole (DPP) or zinc(II) phthalocyanine (ZnPc) fluorophores were imaged using confocal laser scanning microscopy (CLSM) of fully solvated gels and cryogenic scanning electron microscopy (cryo-SEM) was used to observe the corresponding xerogels. The DPP@Gel systems exhibit strong fluorescence and are effectively imaged using CLSM, with fibre morphologies that closely correlate with those seen with cryo-SEM. In contrast, the analogous imidazolium gel system containing a sulfonated zinc phthalocyanine (ZnPc@Gel) yields poor CLSM images because of the relatively weak emission and sample disruption during compression, whereas cryo-SEM enables clear visualization of the native fibrous network. These results demonstrate the complementary nature of CLSM and cryo-SEM and highlight the value of cryo-SEM as a very useful tool for imaging soft nanomaterials with low fluorescence or limited optical contrast.

Tooth enamel is characterised by an intricate hierarchical organization of apatite nanocrystals that bestows high stiffness, hardness, and fracture toughness. However, enamel does not possess the ability to regenerate, and achieving the artificial restoration of its microstructure and mechanical properties in clinical settings has proven challenging. To tackle this issue, we engineer a tuneable and resilient supramolecular matrix based on elastin-like recombinamers (ELRs) that imitates the structure and function of the enamel-developing matrix. When applied as a coating on the surface of teeth exhibiting different levels of erosion, the matrix is stable and can trigger epitaxial growth of apatite nanocrystals, recreating the microarchitecture of the different anatomical regions of enamel and restoring the mechanical properties. The study demonstrates the translational potential of our mineralising technology for treating loss of enamel in clinical settings such as the treatment of enamel erosion and dental hypersensitivity.

Extracellular vesicles (EVs) may contain a variety of molecular cargo, including proteins and nucleic acids. Vault particle components have been repeatedly reported in the literature as EV cargo. Here, we demonstrated that vault RNA (vtRNA) and major vault protein (MVP) were highly abundant in EV pellets enriched by differential centrifugation (DC) by qPCR and western blotting, respectively. RNase and proteinase treatment of DC‐derived pellets demonstrated that most vtRNA and MVP were not enclosed and protected within an EV membrane. Vault‐like particles were visualised in 100k DC pellets by cryo‐transmission electron microscopy. EVs were enriched by size exclusion chromatography, and western blotting of individual fractions showed co‐elution of EV markers and vault particle proteins. Immunocapture of EVs post‐ultracentrifugation (100k DC pellet) showed co‐purification of MVP, whereas EVs isolated by direct immunocapture from conditioned medium were MVP‐negative. The current study highlights the importance of determining the topology of putative EV‐associated components to determine if they are EV cargo or contaminants that have been co‐purified.

Bacterial adaptability to diverse environments drives infection, persistence, and antibiotic resistance. Although hydrogels are increasingly used to model such conditions, the factors governing hydrogel-dependent bacterial growth is complex. Here, we focus on agarose hydrogels and investigate how their material properties influence bacterial proliferation. Using two agarose types – hydroxyethyl substituted and unsubstituted – at varying concentrations, we tested four bacterial species ( E. coli, P. fluorescens, S. aureus, B. subtilis ) across five nutrient media yielding 120 conditions. Growth consistently decreased with increasing hydrogel stiffness and water loss in unsubstituted and substituted agarose hydrogels, regardless of species. Media effects were largely due to their impact on hydrogel properties rather than nutrient content. Furthermore, electrostatic repulsion between Gram positive bacteria and anionic unsubstituted agarose suppressed growth in high concentration gels. These findings demonstrate that bacterial growth in agarose systems is primarily shaped by gel mechanics and surface interactions, informing the design of infection models and antibacterial materials.

Mechanically resilient hydrogels with ion‐electron mixed transport properties effectively bridge biology with electronics. An ideal bioelectronic interface can be realized through introducing electronically conductive polymers into supramolecular hydrogels. However, inhomogeneous morphologies of conducting polymers, such as poly(3,4‐ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS), have limited mechanical properties and ion‐electron interactions. Here, supramolecular conductive hydrogels that possess homogeneous ionic and electronic transport are achieved. The materials demonstrate high toughness (620 kJ m ⁻³ ), stretchability (>1000%), softness (10.5 kPa), and conductivity (5.8 S cm ⁻¹ ), which surpasses commonly used inhomogeneous PEDOT:PSS‐based hydrogels. The homogeneous network leads to higher charge injection capacitance and lower skin impedance compared to commercial electrodes or commonly used inhomogeneous PEDOT:PSS conducting networks. This significant advance arises from the homogeneous incorporation of the hydrophilic self‐doped conducting polymer S‐PEDOT, which has polymerized within a supramolecular polymer network template mediated by high‐binding affinity host‐guest crosslinks. Furthermore, the compatibility of S‐PEDOT with hydrophilic secondary networks enables the realization of fully dryable and reswellable electronic devices, facilitating reusability and improving their ease of handling. It is anticipated that achieving such material architectures will offer a promising new direction in future synthesis and implementation of conductive hydrogels in the field of bioelectronics.

Ventilator-associated pneumonia is defined as pneumonia that develops in a patient who has been on mechanical ventilation for more than 48 hours through an endotracheal tube. It is caused by biofilm formation on the indwelling tube, which introduces pathogenic microbes such as Pseudomonas aeruginosa, Klebsiella pneumoniae and Candida albicans into the patient’s lower airways. Currently, there is a lack of accurate in vitro models of ventilator-associated pneumonia development. This greatly limits our understanding of how the in-host environment alters pathogen physiology and the efficacy of ventilator-associated pneumonia prevention or treatment strategies. Here, we showcase a reproducible model that simulates the biofilm formation of these pathogens in a host-mimicking environment and demonstrate that the biofilm matrix produced differs from that observed in standard laboratory growth medium. In our model, pathogens are grown on endotracheal tube segments in the presence of a novel synthetic ventilated airway mucus medium that simulates the in-host environment. Matrix-degrading enzymes and cryo-scanning electron microscopy were employed to characterize the system in terms of biofilm matrix composition and structure, as compared to standard laboratory growth medium. As seen in patients, the biofilms of ventilator-associated pneumonia pathogens in our model either required very high concentrations of antimicrobials for eradication or could not be eradicated. However, combining matrix-degrading enzymes with antimicrobials greatly improved the biofilm eradication of all pathogens. Our in vitro endotracheal tube model informs on fundamental microbiology in the ventilator-associated pneumonia context and has broad applicability as a screening platform for antibiofilm measures including the use of matrix-degrading enzymes as antimicrobial adjuvants.

Extracellular vesicles (EVs) may contain a variety of molecular cargo including proteins and nucleic acids. Vault particle components have been repeatedly reported in the literature as EV cargo. Here, we demonstrated by small RNA sequencing that vault RNA (vtRNA) were highly abundant in EV pellets enriched by differential centrifugation. EVs were prepared by commonly used enrichment methods and biochemical assays used to determine whether vault particle components were bona fide EV cargo. EVs were isolated by differential centrifugation, size exclusion chromatography (SEC) and Dynabead immunocapture. RNase and proteinase treatment of EV preparations demonstrated that most vtRNA and major vault protein (MVP) were not enclosed and protected within the EV membrane. Vault-like particles were visualised in differential centrifugation pellets by cryo-transmission electron microscopy. EVs enriched by size exclusion chromatography and those isolated by immunocapture post-ultracentrifugation showed co-purification of MVP, whereas EVs isolated by direct immunocapture from conditioned medium were MVP-negative. Taken together, commonly used isolation techniques, such as differential centrifugation and SEC, can lead to contamination of EVs with vault particles. The current study highlights the importance of determining the topology of putative EV-associated components to determine if they are EV cargo or contaminants that have been co-purified.