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Objectives: Evaluate the remineralisation ability of experimental resin-based materials containing Fluoride-Doped Calcium Phosphate (FDCP), applied in mineral-deficient dentine in combination with a biomimetic dual-analogue primer., Methods: Artificial dentine carious lesions were created in occlusal cavities of human molars. An experimental resin-based adhesive and flowable composite, containing FDCP (10wt% and 20wt%, respectively), were applied (±) with a biomimetic primer containing polyacrylic acid and sodium tripolyphosphate. A glass-ionomer cement and a conventional adhesive/composite were used as comparative materials. All specimens were submitted to chewing simulation in artificial saliva (AS). Subsequently, these were cut into slabs and submitted to Knoop microhardness testing and Raman spectroscopy (AS: 0, 15, 30 and 60 days). Further specimens were analysed through confocal scanning microscopy (AS: 0, 60 days). Total demineralised dentine and disc-shape materials were analysed through FTIR, SEM and EDX (Ca/P ratio) after prolonged contact in AS. Data were statistically analysed (two-way ANOVA and multiple comparison Bonferroni; α=0.05)., Results: Only the experimental FDCP materials induced a significant increase in microhardness after storage in AS, especially in those specimens pre-treated with the biomimetic primer, except at 50 µm underneath the interface. Raman and CLSM analysis corroborated such results. The ability of the experimental FDCP resin-based materials to induce apatite-like deposition was demonstrated through SEM-EDX and FTIR., Conclusions: Resin-based materials containing FDCP can mineralise the mineral-deficient dentine, especially when combined with a biomimetic dual-analogue primer., Clinical Significance: Such Innovative ion-releasing restorative systems applied after selective caries removal might promote remineralisation of fully and partially demineralised dentine and increase the durability of dental restorations., Competing Interests: Declaration of competing interest We wish to confirm that there are no known conflicts of interest associated with this publication, and there has been no significant financial support for this work that could have influenced its outcome., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

A particular challenge to the field of neuroscience involves translating findings from 2D in vitro systems to 3D in vivo environments. Standardized cell culture environments that adequately reflect the properties of the central nervous system (CNS) such as the stiffness, protein composition, and microarchitecture in which to study 3D cell-cell and cell-matrix interactions are generally lacking for in vitro culture systems. In particular, there remains an unmet need for reproducible, low-cost, high-throughput, and physiologically relevant environments comprised of tissue-native matrix proteins for the study of CNS microenvironments in 3D. Advances in the field of biofabrication over the past number of years have facilitated the production and characterization of biomaterial-based scaffolds. Typically developed for tissue engineering applications, they also provide sophisticated environments in which to study cell-cell and cell-matrix interactions and have been used for 3D modeling for a range of tissues. Here, we describe a simple and scalable protocol for the production of biomimetic, highly porous freeze-dried hyaluronic acid scaffolds with tunable microarchitecture, stiffness, and protein composition. Furthermore, we describe several different approaches that can be used to characterize a range of physicochemical properties and how to employ the scaffolds for the 3D culture of sensitive CNS cells in vitro. Finally, we detail several approaches for the study of key cell responses within the 3D scaffold environments. Overall, this protocol describes the manufacture and testing of a biomimetic and tunable macroporous scaffold system for neuronal cell culture applications. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Scaffold manufacture Basic Protocol 2: Scaffold characterization Basic Protocol 3: Cell culture and analysis of neurons in scaffolds., (© 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.)

Nerve guidance conduits (NGCs) are sub-optimal for long-distance injuries with inflammation and poor vascularization related to poor axonal repair. This study used a multi-factorial approach to create an optimized biomaterial NGC to address each of these issues. Through stepwise optimization, a collagen-chondroitin-6-sulfate (Coll-CS) biomaterial was functionalized with extracellular matrix (ECM) components; fibronectin, laminin 1 and laminin 2 (FibL1L2) in specific ratios. A snap-cooled freeze-drying process was then developed with optimal pore architecture and alignment to guide axonal bridging. Culture of adult rat dorsal root ganglia on NGCs demonstrated significant improvements in inflammation, neurogenesis and angiogenesis in the specific Fib:L1:L2 ratio of 1:4:1. In clinically relevant, large 15 mm rat sciatic nerve defects, FibL1L2-NGCs demonstrated significant improvements in axonal density and angiogenesis compared to unmodified NGCs with functional equivalence to autografts. Therefore, a multiparameter ECM-driven strategy can significantly improve axonal repair across large defects, without exogenous cells or growth factors., Competing Interests: Declaration of competing interests At the time of writing, Simon Archibald is an employee of Integra Life Sciences., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

After spinal cord injury (SCI), tissue engineering scaffolds offer a potential bridge for regeneration across the lesion and support repair through proregenerative signaling. Ideal biomaterial scaffolds that mimic the physicochemical properties of native tissue have the potential to provide innate trophic signaling while also minimizing damaging inflammation. To address this challenge, taking cues from the spinal cord's structure, the proregenerative signaling capabilities of native cord components are compared in vitro. A synergistic mix of collagen-IV and fibronectin (Coll-IV/Fn) is found to optimally enhance axonal extension from neuronal cell lines (SHSY-5Y and NSC-34) and induce morphological features typical of quiescent astrocytes. This optimal composition is incorporated into hyaluronic acid scaffolds with aligned pore architectures but varying stiffnesses (0.8-3 kPa). Scaffolds with biomimetic mechanical properties (<1 kPa), functionalized with Coll-IV/Fn, not only modulate primary astrocyte behavior but also stimulate the production of anti-inflammatory cytokine IL-10 in a stiffness-dependent manner. Seeded SHSY-5Y neurons generate distributed neuronal networks, while softer biomimetic scaffolds promote axonal outgrowth in an ex vivo model of axonal regrowth. These results indicate that the interaction of stiffness and biomaterial composition plays an essential role in vitro in generating repair-critical cellular responses and demonstrates the potential of biomimetic scaffold design., (© 2021 Wiley-VCH GmbH.)

Injury to the peripheral or central nervous systems often results in extensive loss of motor and sensory function that can greatly diminish quality of life. In both cases, macrophage infiltration into the injury site plays an integral role in the host tissue inflammatory response. In particular, the temporally related transition of macrophage phenotype between the M1/M2 inflammatory/repair states is critical for successful tissue repair. In recent years, biomaterial implants have emerged as a novel approach to bridge lesion sites and provide a growth-inductive environment for regenerating axons. This has more recently seen these two areas of research increasingly intersecting in the creation of 'immune-modulatory' biomaterials. These synthetic or naturally derived materials are fabricated to drive macrophages towards a pro-repair phenotype. This review considers the macrophage-mediated inflammatory events that occur following nervous tissue injury and outlines the latest developments in biomaterial-based strategies to influence macrophage phenotype and enhance repair.

Inhalation offers a means of rapid, local delivery of siRNA to treat a range of autoimmune or inflammatory respiratory conditions. This work investigated the potential of a linear 10 kDa Poly(ethylene glycol) (PEG)-modified 25 kDa branched polyethyleneimine (PEI) (PEI-LPEG) to effectively deliver siRNA to airway epithelial cells. Following optimization with anti- glyceraldehyde 3-phosphate dehydrogenase (GAPDH) siRNA, PEI and PEI-LPEG anti-IL8 siRNA nanoparticles were assessed for efficacy using polarised Calu-3 human airway epithelial cells and a twin stage impinger (TSI) in vitro lung model. Studies were then advanced to an in vivo lipopolysaccharide (LPS)-stimulated rodent model of inflammation. In parallel, the suitability of the siRNA-loaded nanoparticles for nebulization using a vibrating mesh nebuliser was assessed. The siRNA nanoparticles were nebulised using an Aerogen ® Pro vibrating mesh nebuliser and characterised for aerosol output, droplet size and fine particle fraction. Only PEI anti-IL8 siRNA nanoparticles were capable of significant levels of IL-8 knockdown in vitro in non-nebulised samples. However, on nebulization through a TSI, only PEI-PEG siRNA nanoparticles demonstrated significant decreases in gene and protein expression in polarised Calu-3 cells. In vivo, both anti-CXCL-1 (rat IL-8 homologue) nanoparticles demonstrated a decreased CXCL-1 gene expression in lung tissue, but this was non-significant. However, PEI anti-CXCL-1 siRNA-treated rats were found to have significantly less infiltrating macrophages in their bronchoalveolar lavage (BAL) fluid. Overall, the in vivo gene and protein inhibition findings indicated a result more reminiscent of the in vitro bolus delivery rather than the in vitro nebulization data. This work demonstrates the potential of nebulised PEI-PEG siRNA nanoparticles in modulating pulmonary inflammation and highlights the need to move towards more relevant in vitro and in vivo models for respiratory drug development.

A number of natural polymer biomaterial-based nerve guidance conduits (NGCs) are developed to facilitate repair of peripheral nerve injuries. Cross-linking ensures mechanical integrity and desired degradation properties of the NGCs; however, common methods such as formaldehyde are associated with cellular toxicity. Hence, there is an unmet clinical need for alternative nontoxic cross-linking agents. In this study, collagen-based NGCs with a collagen/chondroitin sulfate luminal filler are used to study the effect of cross-linking on mechanical and structural properties, degradation, biocompatibility, and immunological response. A simplified manufacturing method of genipin cross-linking is developed, by incorporating genipin into solution prior to freeze-drying the NGCs. This leads to successful cross-linking as demonstrated by higher cross-linking degree and similar tensile strength of genipin cross-linked conduits compared to formaldehyde cross-linked conduits. Genipin cross-linking also preserves NGC macro and microstructure as observed through scanning electron microscopy and spectral analysis. Most importantly, in vitro cell studies show that genipin, unlike the formaldehyde cross-linked conduits, supports the viability of Schwann cells. Moreover, genipin cross-linked conduits direct macrophages away from a pro-inflammatory and toward a pro-repair state. Overall, genipin is demonstrated to be an effective, safe, biocompatible, and anti-inflammatory alternative to formaldehyde for cross-linking clinical grade NGCs., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Bacterial resistance to antibiotics is widely regarded as a major public health concern with last resort MRSA treatments like vancomycin now encountering resistant strains. TFDs (Transcription Factor Decoys) are oligonucleotide copies of the DNA-binding sites for transcription factors. They bind to and sequester the targeted transcription factor, thus inhibiting transcription of many genes. By developing TFDs with sequences aimed at inhibiting transcription factors controlling the expression of highly conserved bacterial cell wall proteins, TFDs present as a potential method for inhibiting microbial growth without encountering typical resistance mechanisms. However, the efficient protection and delivery of the TFDs inside the bacterial cells is a critical step for the success of this technology. Therefore, in our study, specific TFDs against S. aureus were complexed with two different types of nanocarriers: cationic nanostructured lipid carriers (cNLCs) and chitosan-based nanoparticles (CS-NCs). These TFD-carrier nanocomplexes were characterized for size, zeta potential and TFD complexation or loading efficiency in a variety of buffers. In vitro activity of the nanocomplexes was examined alone and in combination with vancomycin, first in methicillin susceptible strains of S. aureus with the lead candidate advancing to tests against MRSA cultures. Results found that both cNLCs and chitosan-based carriers were adept at complexing and protecting TFDs in a range of physiological and microbiological buffers up to 72 hours. From initial testing, chitosan-TFD particles demonstrated no visible improvements in effect when co-administered with vancomycin. However, co-delivery of cNLC-TFD with vancomycin reduced the MIC of vancomycin by over 50% in MSSA and resulted in significant decreases in viability compared with vancomycin alone in MRSA cultures. Furthermore, these TFD-loaded particles demonstrated very low levels of cytotoxicity and haemolysis in vitro. To our knowledge, this is the first attempt at a combined antibiotic/oligonucleotide-TFD approach to combatting MRSA and, as such, highlights a new avenue of MRSA treatment combining traditional small molecules drugs and bacterial gene inhibition., Competing Interests: Prof. Michael McArthur is CSO and Co-Founder of Procarta Biosystems which specialises TFD technology. No funding was received from Procarta Biosystems. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Neurotrophic factor delivery via biodegradable nerve guidance conduits may serve as a promising treatment for the repair of large peripheral nerve defects. However, a platform for controlled delivery is required because of their short in vivo half-life and their potential to impede axonal regeneration when used in supraphysiological doses. In this study, we investigated the dose-dependent, synergistic and temporal effects of NGF and GDNF on neurite outgrowth, adult dorsal root ganglia axonal outgrowth, Schwann cell migration and cytokine production in vitro. Using the optimal dose and combination of NGF and GDNF, we developed a PLGA microparticle-based delivery platform to control their delivery. The dose-dependent effects of both NGF and GDNF individually were found to be non-linear with a saturation point. However, the synergistic effect between NGF and GDNF was found to outweigh their dose-dependent effects in terms of enhancing Schwann cell migration and axonal outgrowth while allowing a 100-fold reduction in dose. Moreover, a temporal profile that mimics the physiological flux of NGF and GDNF in response to injury, compared to one that resembles an early burst release delivery profile, was found to enhance their bioactivity. The optimized NGF- and GDNF-loaded microparticles were then incorporated into a guidance conduit, and their capacity to enhance nerve regeneration across a 15 mm sciatic nerve defect in rats was demonstrated. Enhanced nerve regeneration was seen in comparison to non-treated defects and very encouragingly, to a similar level compared to the clinical gold standard of autograft. Taken together, we suggest that this delivery platform might have significant potential in the field of peripheral nerve repair; allowing spatial and temporal control over the delivery of potent neurotrophic factors to enhance the regenerative capacity of biomaterials-based nerve guidance conduits., (Copyright © 2019 Elsevier B.V. All rights reserved.)

While a wide range of experimental and commercial transfection reagents are currently available, persistent problems remain regarding their suitability for continued development. These include the transfection efficiency for difficult-to-transfect cell types and the risks of decreased cell viability that may arise from any transfection that does occur. Therefore, research is now turning toward alternative molecules that improve the toxicity profile of the gene delivery vector (GDV), while maintaining the transfection efficiency. Among them, cell-penetrating peptides, such as octa-arginine, have shown significant potential as GDVs. Their pharmacokinetic and pharmacodynamic properties can be enhanced through peptidomimetic conversion, whereby a peptide is modified into a synthetic analogue that mimics its structure and/or function, but whose backbone is not solely based on α-amino acids. Using this technology, novel peptidomimetics were developed by co- and postpolymerization functionalization of substituted ethylene oxides, producing poly(ethylene glycol) (PEG)-based peptidomimetics termed "PEGtides". Specifically, a PEGtide of the poly(α-amino acid) oligo-arginine [poly(glycidylguanidine)] was assessed for its ability to complex and deliver a small interfering ribonucleic acid (siRNA) using a range of cell assays and high-content analysis. PEGtide-siRNA demonstrated significantly increased internalization and gene inhibition over 24 h in Calu-3 pulmonary epithelial cells compared to commercial controls and octa-arginine-treated samples, with no evidence of toxicity. Furthermore, PEGtide-siRNA nanocomplexes can provide significant levels of gene inhibition in "difficult-to-transfect" mouse embryonic hypothalamic (mHypo N41) cells. Overall, the usefulness of this novel PEGtide for gene delivery was clearly demonstrated, establishing it as a promising candidate for continued translational research., Competing Interests: The authors declare no competing financial interest.

In a recent report, the World Health Organisation (WHO) classified antibiotic resistance as one of the greatest threats to global health, food security, and development. Methicillin-resistant Staphylococcus aureus (MRSA) remains at the core of this threat, with persistent and resilient strains detectable in up to 90% of S. aureus infections. Unfortunately, there is a lack of novel antibiotics reaching the clinic to address the significant morbidity and mortality that MRSA is responsible for. Recently, nanomedicine strategies have emerged as a promising therapy to combat the rise of MRSA. However, these approaches have been wide-ranging in design, with few attempts to compare studies across scientific and clinical disciplines. This review seeks to reconcile this discrepancy in the literature, with specific focus on the mechanisms of MRSA infection and how they can be exploited by bioactive molecules that are delivered by nanomedicines, in addition to utilisation of the nanomaterials themselves as antibacterial agents. Finally, we discuss targeting MRSA biofilms using nano-patterning technologies and comment on future opportunities and challenges for MRSA treatment using nanomedicine., Competing Interests: The authors declare no conflict of interest.

Clinically available hollow nerve guidance conduits (NGCs) have had limited success in treating large peripheral nerve injuries. This study aims to develop a biphasic NGC combining a physicochemically optimized collagen outer conduit to bridge the transected nerve, and a neuroconductive hyaluronic acid-based luminal filler to support regeneration. The outer conduit is mechanically optimized by manipulating crosslinking and collagen density, allowing the engineering of a high wall permeability to mitigate the risk of neuroma formation, while also maintaining physiologically relevant stiffness and enzymatic degradation tuned to coincide with regeneration rates. Freeze-drying is used to seamlessly integrate the luminal filler into the conduit, creating a longitudinally aligned pore microarchitecture. The luminal stiffness is modulated to support Schwann cells, with laminin incorporation further enhancing bioactivity by improving cell attachment and metabolic activity. Additionally, this biphasic NGC is shown to support neurogenesis and gliogenesis of neural progenitor cells and axonal outgrowth from dorsal root ganglia. These findings highlight the paradigm that a successful NGC requires the concerted optimization of both a mechanical support phase capable of bridging a nerve defect and a neuroconductive phase with an architecture capable of supporting both Schwann cells and neurons in order to achieve functional regenerative outcome., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A major limitation with current tissue-engineering approaches is creating functionally vascularized constructs that can successfully integrate with the host; this often leads to implant failure, due to avascular necrosis. In order to overcome this, the objective of the present work was to develop a method to incorporate growth factor-eluting alginate microparticles (MPs) into freeze-dried, collagen-based scaffolds. A collagen-hydroxyapatite (CHA) scaffold, previously optimized for bone regeneration, was functionalized for the sustained delivery of an angiogenic growth factor, vascular endothelial growth factor (VEGF), with the aim of facilitating angiogenesis and enhancing bone regeneration. VEGF was initially encapsulated in alginate MPs by spray-drying, producing particles of < 10 µm in diameter. This process was found to effectively encapsulate and control VEGF release while maintaining its stability and bioactivity post-processing. These VEGF-MPs were then incorporated into CHA scaffolds, leading to homogeneous distribution throughout the interconnected scaffold pore structure. The scaffolds were capable of sustained release of bioactive VEGF for up to 35 days, which was proficient at increasing tubule formation by endothelial cells in vitro. When implanted in vivo in a rat calvarial defect model, this scaffold enhanced vessel formation, resulting in increased bone regeneration compared to empty-defect and VEGF-free scaffolds. This biologically functionalized scaffold, composed entirely of natural-based materials, may offer an ideal platform to promote angiogenesis and tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

A series of well-defined star-shaped polypeptides were successfully synthesised by the ring opening polymerisation (ROP) of the N-carboxyanhydride (NCA) of ε-carbobenzyloxy-l-lysine (ZLL) using a range of generations of polypropylene imine (PPI) dendrimers as multifunctional initiators. The monomer feed ratio and dendrimer generation were varied to afford a series of polypeptide dendrimer hybrids with superior structural versatility and functionality. Subsequent protecting group removal yielded star-shaped poly(lysine) of controlled variation in polypeptide chain length and arm multiplicity. Star-shaped PLL polymers were used to prepare pDNA and siRNA to form "polyplexes" to determine their ability to complex different nucleic acid cargoes and were compared with linear PLL polyplex controls. Significant differences in size and surface charge were seen between star-shaped PLL polyplexes and linear PLL polyplexes for both cargoes. The star-shaped polypeptides were capable of more effective complexation of both nucleic acids at low N/P ratios compared to linear PLL as evidenced by zeta potential and electrophoretic data. This was particularly evident in siRNA polyplexes as linear PLL failed to completely complex siRNA into nanocomplexes of appropriate size for cell transfection i.e. <200 nm in size, while star poly(lysine) formed siRNA polyplexes <100 nm at certain N/P ratios, albeit strongly dependent on the particular molecular weight and architecture, as analysed by dynamic light scattering (DLS). Atomic force microscopy (AFM) identified discrete spherically shaped polyplexes for all star-shaped polypeptide-based polyplexes while linear PLL formed elongated irregular shaped complexes. This difference in morphology may go some way towards explaining the 300-fold increase in luciferase expression seen for star-shaped PLL polyplexes G5(64)-PLL 40 compared to linear PLL pGLuc polyplexes in epithelial cells. Each of the PPI-PLL polymers appeared to be capable of protecting the nucleic acid cargoes from degradation by the relevant nuclease enzyme as effectively as the positive control polyethyleneimine (PEI) polyplexes. Overall the promising nucleic acid complexation, sizing, morphology and protection capacity of two different genetic "cargoes" highlight the potential of polypeptide dendrimer hybrids as gene delivery vectors.

The emergence of RNAi offers a potentially exciting new therapeutic paradigm for respiratory diseases. However, effective delivery remains a key requirement for their translation into the clinic and has been a major factor in the limited clinical success seen to date. Inhalation offers tissue-specific targeting of the RNAi to treat respiratory diseases and a diminished risk of off-target effects. In order to deliver RNAi directly to the respiratory tract via inhalation, 'smart' non-viral carriers are required to protect the RNAi during delivery/aerosolization and enhance cell-specific uptake to target cells. Here, we review the state-of-the-art in therapeutic aerosol bioengineering, and specifically non-viral siRNA delivery platforms, for delivery via inhalation. This includes developments in inhaler device engineering and particle engineering, including manufacturing methods and excipients used in therapeutic aerosol bioengineering that underpin the development of smart, cell type-specific delivery systems to target siRNA to respiratory epithelial cells and/or alveolar macrophages.

The healing potential of scaffolds for tissue engineering can be enhanced by combining them with genes to produce gene-activated matrices (GAMs) for tissue regeneration. We examined the potential of using polyethyleneimine (PEI) as a vector for transfection of mesenchymal stem cells (MSCs) in monolayer culture and in 3D collagen-based GAMs. PEI-pDNA polyplexes were fabricated at a range of N/P ratios and their optimal transfection parameters (N/P 7 ratio, 2μg dose) and transfection efficiencies (30±8%) determined in monolayer culture. The polyplexes were then loaded onto collagen, collagen-glycosaminoglycan and collagen-nanohydroxyapatite scaffolds where gene expression was observed up to 21 days with a polyplex dose as low as 2μg. Transient expression profiles indicated that the GAMs act as a polyplex depot system whereby infiltrating cells become transfected over time as they migrate throughout the scaffold. The collagen-nHa GAM exhibited the most prolonged and elevated levels of transgene expression. This research has thus demonstrated that PEI is a highly efficient pDNA transfection agent for both MSC monolayer cultures and in the 3D GAM environment. By combining therapeutic gene therapy with highly engineered scaffolds, it is proposed that these GAMs might have immense capability to promote tissue regeneration., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Aims: Delivery of siRNA to the lungs via inhalation offers a unique opportunity to develop a new treatment paradigm for a range of respiratory conditions. However, progress has been greatly hindered by safety and delivery issues. This study developed a high-throughput method for screening novel nanotechnologies for pulmonary siRNA delivery., Methodology: Following physicochemical analysis, the ability of PEI-PEG-siRNA nanoparticles to facilitate siRNA delivery was determined using high-content analysis (HCA) in Calu-3 cells. Results obtained from HCA were validated using confocal microscopy. Finally, cytotoxicity of the PEI-PEG-siRNA particles was analyzed by HCA using the Cellomics multiparameter cytotoxicity assay., Conclusion: PEI-PEG-siRNA nanoparticles facilitated increased siRNA uptake and luciferase knockdown in Calu-3 cells compared with PEI-siRNA.