The mammalian vestibular system has two types of sensory receptors (hair cells), type I and type II. Understanding the roles of type I and II hair cells in the vestibular system's control of motor behaviors is difficult because most primary vestibular neurons receive inputs from both hair cell types. To test if type I hair cells are required for motor behaviors, we ablated them from peripheral zones of vestibular epithelia in young adult (3-6 months) Fbxo2CreERT2::Rosa26DTA (experimental) mice of both sexes, then examined motor behaviors and brainstem neuronal responses. Over 90% of peripheral type I hair cells were ablated from vestibular organs by one week post-tamoxifen, while central type I and all type II hair cell numbers did not change significantly out to 8 weeks post-tamoxifen. Right after ablation, mice displayed no obvious locomotor abnormalities. However, they could only balance on a rotarod or beam for a few seconds, and gains of the horizontal vestibulo-ocular reflex were reduced by 50%. Deficits persisted to 8 weeks post-tamoxifen, with one exception: 3-6 month old mice showed partial recovery of rotarod performance after 4 weeks post-tamoxifen, likely due to adaptive motor strategies. Remarkably, 16 month-old mice with type I hair cell ablation failed to recover rotarod function. Motion-induced CFOS expression in vestibular brainstem neurons was nearly eliminated after hair cell ablation, suggesting inputs from vestibular organs had changed significantly. This study demonstrates that peripheral type I hair cells are essential for a vestibular reflex and for some tasks requiring balance and motor coordination.Significance Statement This study demonstrates that a specific subpopulation of vestibular sensory receptor cells (peripherally located type I hair cells) is required in adult mice to maintain normal vestibular function, including the vestibulo-ocular reflex, balance and motor coordination, and neuronal responses to a motion stimulus. These findings have implications for the underlying pathology in some types of balance disorders and may inform new strategies to restore vestibular function after hair cell damage or degeneration.

Tissue engineering has the potential to overcome the limitations of using autografts in nerve gap repair, using cellular biomaterials to bridge the gap and support neuronal regeneration. Various types of therapeutic cells could be considered for use in aligned collagen-based engineered neural tissue (EngNT), including Schwann cells and their precursors, which can be derived from human induced pluripotent stem cells (hiPSCs). Using Schwann cell precursors may have practical advantages over mature Schwann cells as they expand readily in vitro and involve a shorter differentiation period. However, the performance of each cell type needs to be tested in EngNT. By adapting established protocols, hiPSCs were differentiated into Schwann cell precursors and Schwann cells, with distinctive molecular profiles confirmed using immunocytochemistry and RT-qPCR. For the first time, both cell types were incorporated into EngNT using gel aspiration-ejection, a technique used to align and simultaneously stabilise the cellular hydrogels. Both types of cellular constructs supported and guided aligned neurite outgrowth from adult rat dorsal root ganglion neurons in vitro. Initial experiments in a rat model of nerve gap injury demonstrated the extent to which the engrafted cells survived after 2 weeks and indicated that both types of hiPSC-derived cells supported the infiltration of host neurons, Schwann cells and endothelial cells. In summary, we show that human Schwann cell precursors promote infiltrating endogenous axons in a model of peripheral nerve injury to a greater degree than their terminally differentiated Schwann cell counterparts.

Aligning the material properties of organic semiconducting polymers to effectively interface with biological matter is critical for their use in bioelectronic devices. Synthetic modification and advanced processing techniques have typically been employed to promote cell adhesion and growth. In this study we apply UV-Ozone (UVO) treatment as a simple and accessible alternative for modifying pDPP3T films. Exposure to UVO increases polarity of the semiconductor surface, as confirmed by contact angle and XPS analysis. Surface treatment at and above the optimized time (t ≥ 30 s) consequently led to enhanced Schwann cell growth, with comparable behaviour to standard tissue culture plastic (TCP). Simultaneously, prolonged exposure begins to cause significant changes to the polymer's optical properties, with gradual photobleaching leading to the reduction in semiconducting behavior above 30 s. Leveraging the optimal biointerfacing properties of the UVO-treated pDPP3T, the validity of the technique in supporting cell viability and proliferation upon a semiconducting polymer was tested using electrical impedance spectroscopy. This work demonstrates the potential to more easily integrate conjugated polymers with biological environments, widening the opportunity to explore the interplay between ion diffusion and semiconductor electroactivity in the presence of biological cells.

BACKGROUND:The static sciatic index is commonly used in rat models of nerve crush injury to quantify functional recovery from new therapies under evaluation. However, it is challenging to standardize these measurements across different investigations, and the process is labor intensive. MATERIAL/METHODS:A new machine learning method was previously developed that performs these measurements automatically and consistently. Here, the approach is tested using two data sets that use different experimental setups, and end-user requirements are evaluated. RESULTS:The model's outputs presented a nerve regeneration profile comparable to the manual measurements and outperformed the latter by having a much tighter standard deviation (± 5- ± 10 compared to ± 10 - ± 50). CONCLUSION:An inexpensive automatic tool that can perform functional analysis for nerve repair research was developed and tested. The software is available open source to facilitate its dissemination and use in quantifying recovery from peripheral nerve crush injury.

Parkinson's disease is a neurodegenerative condition associated with the progressive loss of dopaminergic neurons. This leads to neurological impairments with heightening severity and is globally increasing in prevalence due to population ageing. Cell transplantation has demonstrated significant promise in altering the disease course in the clinic, and stem cell-derived grafts are being investigated. Current clinical protocols involve systemic immunosuppression to prevent graft rejection, which could potentially be avoided by encapsulating the therapeutic cells in a locally immunosuppressive biomaterial matrix before delivery. Here we report the progression of an immunomodulatory encapsulation system employing ultrapure alginate hydrogel beads alongside tacrolimus-loaded microparticles in the encapsulation of dopaminergic neuron progenitors derived from human induced pluripotent stem cells (hiPSCs). The hiPSC-derived progenitors were characterised and displayed robust viability after encapsulation within alginate beads, producing dopamine as they matured in vitro. The encapsulation system effectively reduced T cell activation (3-fold) and protected progenitors from cytotoxicity in vitro. The alginate bead diameter was optimised using microfluidics to yield spherical and monodisperse hydrogels with a median size of 215.6 +/- 0.5 mu m, suitable for delivery to the brain through a surgical cannula. This technology has the potential to advance cell transplantation by locally protecting grafts from the host immune system.

Mesenchymal stem cells have shown promise in many areas of regenerative medicine due to the anti-inflammatory and pro-regenerative effects of the secreted factors. However, successful delivery remains problematic, particularly for delivery to areas such as the brain. Spray delivery is a method investigated in wound care and lung injury, which may be applicable for brain delivery to patients already requiring surgery. To retain therapeutic mesenchymal stem cells at the delivery site, biomaterials can be employed; pectin is a biocompatible, sprayable, and mucoadhesive material, which could prove suitable for spray delivery of cells for therapeutic uses. The biocompatibility of four grades of low-methyl pectin gelled by addition of calcium was assessed using SH-SY5Y cells. After, mesenchymal stem cells were suspended within the four different grades of low-methyl pectin solutions and sprayed using a syringe-driven spray device. The suitability was then assessed by cell viability testing, flow cytometry to test for surface markers, and differential gene expression studies to understand the effects of both the pectin and the spraying process on the gene expression of the cells. All four grades of low-methyl pectin were biocompatible with SH-SY5Y cells. The syringe-driven spray device delivered human mesenchymal stem cells to well plates with high viability, and suspending these cells in pectin solutions for spraying did not negatively affect the viability. The grade of pectin named CU-701 was the best grade based on results of the flow cytometry, whereby the surface marker expression was not altered from the control cells. The RNA sequencing showing the differential expression showed that the process of spraying the cells did not alter gene expression compared to the control, however the pectin, and the presence of calcium used to induce gelation of the pectin, did lead to altered gene expression in cells. Spraying is a suitable delivery method for the mesenchymal stem cells, showing no detrimental effect on the cells. Pectin shows little effect on the viability of the cells, however the use of calcium to gel the pectin appears to affect the expression of several genes.

Novel branched copolymer surfactants (BCS) allow the formation of oil-in-water emulsions that exhibit a temperature-induced liquid-to-gel transition. If the temperature of this transition is between room and body temperature (ca 25 and 37 °C, respectively), then the emulsions form a gel in situ upon contact with the body. A major advantage of this in situ gelation is the potential to manipulate the materials at room temperature in the low viscosity liquid state, then administer them to the body to initiate a switch to a retentive gel state, which could be used to deliver drugs to challenging sites such as the nasal mucosa. There are, however, several important factors which have not been explored for thermoresponsive BCS-stabilised emulsions to progress their use towards this application. Neither the delivery of drugs from the materials, the retention on tissue, nor the impact of co-formulated drugs on the thermoresponsive behaviours, are known. Furthermore, it has not been demonstrated that the materials are compatible with devices to generate sprays of the correct profiles for nasal administration. In this study we investigate the potential of thermoresponsive BCS-stabilised emulsions for the nasal delivery of licensed molecular therapeutics to examine the potential of BCS emulsion systems as a carrier for medicines. It was found that thermoresponsive behaviours can be maintained in the presence of drug substances, and that the liberation of the incorporated drugs occurs in a sustained manner. The BCS appear to have comparable cytotoxicity to common excipients and significantly enhanced retention on nasal tissue compared to even well-established mucoadhesives. The emulsions were incorporated into a spray device to demonstrate that the materials can be atomised with a plume appropriate for nasal administration prior to in situ gelation.

IntroductionDamage to peripheral nerves is common in major trauma cases, and current options for surgical repair are often not sufficient to promote satisfactory recovery of sensory and motor function. In this study we describe the development of a biomaterial scaffold with aligned nanofibrous topography and encapsulated neurotrophic factor, designed to direct and enhance axonal regeneration and so effectuate faster return of function.MethodsGlial cell line-derived neurotrophic factor (GDNF) was loaded into aligned polycaprolactone (PCL) nanofibres using emulsion electrospinning, and the biomaterial was characterised alongside random and aligned PCL scaffolds without growth factor.Results and discussionThis fabrication route produced fine and uniform nanofibres with sustained release of GDNF over at least four weeks, and the aligned topography was able to orientate the growth of Schwann cells. Finally, the GDNF-loaded aligned nanofibrous scaffold significantly enhanced and directed the outgrowth of primary rat neurons cultured on its surface, demonstrating its promise as a pro-regenerative biomaterial for the surgical repair of nerve injury.

In addition to proteins, discussed in the Chapter "Advances in Vaccine Adjuvants: Nanomaterials and Small Molecules", there are a wide range of alternatives to small molecule active ingredients. Cells, extracellular vesicles, and nucleic acids in particular have attracted increasing research attention in recent years. There are now a number of products on the market based on these emerging technologies, the most famous of which are the mRNA-based vaccines against SARS-COV-2. These advanced therapeutic moieties are challenging to formulate however, and there remain significant challenges for their more widespread use. In this chapter, we consider the potential and bottlenecks for developing further medical products based on these systems. Cells, extracellular vesicles, and nucleic acids will be discussed in terms of their mechanism of action, the key requirements for translation, and how advanced formulation approaches can aid their future development. These points will be presented with selected examples from the literature, and with a focus on the formulations which have made the transition to clinical trials and clinical products.

Electrical stimulation has shown promise in clinical studies to treat nerve injuries. This work aimed to create an aligned bioelectronic construct that can be used to bridge a nerve gap, directly interfacing with the damaged nerve tissue to support regeneration. The conductive three-dimensional bioelectronic scaffolds described herein are composite materials, comprised of conductive polypyrrole (PPy) nanoparticles embedded in an aligned collagen hydrogel. The bioelectronic constructs were seeded with dorsal root ganglion (DRG) derived primary rat neurons and electrically stimulated in vitro. The PPy loaded constructs supported a 1.7-fold increase in neurite length in comparison to control collagen constructs. Furthermore, upon electrical stimulation of the PPy-collagen construct, a 1.8-fold increase in neurite length was shown. This work illustrates the potential of bioelectronic constructs in neural tissue engineering and lays the groundwork for the development of novel bioelectronic materials for neural interfacing applications.

The mechanical behaviour of peripheral nerves is known to vary between different nerves and nerve regions. As the field of nerve tissue engineering advances, it is vital that we understand the range of mechanical regimes future nerve implants must match to prevent failure. Data on the mechanical behaviour of human peripheral nerves are difficult to obtain due to the need to conduct mechanical testing shortly after removal from the body. In this work, we adapt a 3D multiscale biomechanical model, developed using asymptotic homogenisation, to mimic the micro- and macroscale structure of a peripheral nerve. This model is then parameterised using experimental data from rat peripheral nerves and used to investigate the effect of varying the collagen content, the fibril radius and number density, and the macroscale cross-sectional geometry of the peripheral nerve on the effective axial Young's moduli of the whole nerve. Our results indicate that the total amount of collagen within a cross section has a greater effect on the axial Young's moduli compared to other measures of structure. This suggests that the amount of collagen in a cross section of a peripheral nerve, which can be measured through histological and imaging techniques, is one of the key metrics that should be recorded in the future experimental studies on the biomechanical properties of peripheral nerves.

The rat sciatic nerve model is commonly used to test novel therapies for nerve injury repair. The static sciatic index (SSI) is a useful metric for quantifying functional recovery, and involves comparing an operated paw versus a control paw using a weighted ratio between the toe spread and the internal toe spread. To calculate it, rats are placed in a transparent box, photos are taken from underneath and the toe distances measured manually. This is labour intensive and subject to human error due to the challenge of consistently taking photos, identifying digits and making manual measurements. Although several commercial kits have been developed to address this challenge, they have seen little dissemination due to cost. Here we develop a novel algorithm for automatic measurement of SSI metrics based on video data using casted U-Nets. The algorithm consists of three U-Nets, one to segment the hind paws and two for the two pairs of digits which input into the SSI calculation. A training intersection over union error of 60 % and 80 % was achieved for the back paws and for both digit segmentation U-Nets, respectfully. The algorithm was tested against video data from three separate experiments. Compared to manual measurements, the algorithm provides the same profile of recovery for every experiment but with a tighter standard deviation in the SSI measure. Through the open-source release of this algorithm, we aim to provide an inexpensive tool to more reliably quantify functional recovery metrics to the nerve repair research community.

As we navigate the transition from the Fourth to the Fifth Industrial Revolution, the emerging fields of biomanufacturing and biofabrication are transforming life sciences and healthcare. These sectors are benefiting from a synergy of synthetic and engineering biology, sustainable manufacturing, and integrated design principles. Advanced techniques such as 3D bioprinting, tissue engineering, directed assembly, and self-assembly are instrumental in creating biomimetic scaffolds, tissues, organoids, medical devices, and biohybrid systems. The field of biofabrication in the United Kingdom and Ireland is emerging as a pivotal force in bioscience and healthcare, propelled by cutting-edge research and development. Concentrating on the production of biologically functional products for use in drug delivery, in vitro models, and tissue engineering, research institutions across these regions are dedicated to innovating healthcare solutions that adhere to ethical standards while prioritising sustainability, affordability, and healthcare system benefits.

The study aimed to develop a potential biomaterial for neuroregeneration by the electrospinning of the biocompatible polymer polyvinylidene fluoride (PVDF) with innovative functionalized boron nitride quantum dots (F-BNQDs) synthesized via green chemistry using the amino acid taurine as a functionalization. The F-BNQDs were characterized and were found to be 16-21 nm in diameter, showed good photoluminescent characteristics, and their elemental composition was confirmed by x-ray photoelectron spectroscopy, Fourier-transform infrared (FTIR), and ultraviolet spectroscopy. The PVDF with F-BNQDs was prepared at three different concentrations, and the resulting electrospun fibers were characterized by scanning electron microscopy, FTIR, thermogravimetric analysis, contact angle, biodegradation, water uptake, Schwann cell cytotoxicity, and cell behavior studies. The results of the study showed that the addition of F-BNQDs to PVDF resulted in enhanced mechanical properties, decreased contact angle, increased degradability, and water uptake. The lactate dehydrogenase assay revealed that 5% BN-PVDF had the lowest cytotoxicity. Fluroscent 4',6-diamidino-2-phenylindole (DAPI) staining showed that the increase in F-BNQD concentration up to 5% BN-PVDF enhanced cell behavior on the electrospun fibers. Therefore, the study concluded that 5% BN-PVDF would be suitable for further testing as a potential biomaterial for neuroregeneration in vivo.HighlightsFunctionalized boron nitride quantum dots (F-BNQDs) were prepared using amino acid taurine.The F-BNQDs were characterized and electrospun with polyvinylidene fluoride (PVDF).The incorporation of F-BNQDs to PVDF has enhanced its physicochemical properties.All samples showed a single beta phase PVDF.Schwann cells showed excellent compatibility and cell adhesion on F-BNQDs/PVDF. Functionalized boron nitride quantum dots preparation and incorporation with PVDF.image

Vestibular hair cells (HCs) are mechanoreceptors that sense head motions by modulating the firing rate of vestibular ganglion neurons (VGNs), whose central processes project to vestibular nucleus neurons (VNNs) and cerebellar neurons. We explored vestibular function after HC destruction in adult Pou4f3+/DTR (DTR) mice, in which injections of high-dose (50 ng/g) diphtheria toxin (DT) destroyed most vestibular HCs within 2 weeks. At that time, DTR mice had lost the horizontal vestibulo-ocular reflex (aVORH), and their VNNs failed to upregulate nuclear cFos expression in response to a vestibular stimulus (centrifugation). Five months later, 21 and 14% of HCs were regenerated in utricles and horizontal ampullae, respectively. The vast majority of HCs present were type II. This degree of HC regeneration did not restore the aVORH or centrifugation-evoked cFos expression in VNNs. The failure to regain vestibular pathway function was not due to degeneration of VGNs or VNNs because normal neuron numbers were maintained after HC destruction. Furthermore, sinusoidal galvanic stimulation at the mastoid process evoked cFos protein expression in VNNs, indicating that VGNs were able to regulate VNN activity after HC loss. aVORH and cFos responses in VNNs were robust after low-dose (25 ng/g) DT, which compared to high-dose DT resulted in a similar degree of type II HC death and regeneration but spared more type I HCs in both organs. These findings demonstrate that having more type I HCs is correlated with stronger responses to vestibular stimulation and suggest that regenerating type I HCs may improve vestibular function after HC loss.

Engineered neural tissue (EngNT) is a stabilised aligned cellular hydrogel that offers a potential alternative to the nerve autograft for the treatment of severe peripheral nerve injury. This work aimed to automate the production of EngNT, to improve the feasibility of scalable manufacture for clinical translation. Endothelial cells were used as the cellular component of the EngNT, with the formation of endothelial cell tube-like structures mimicking the polarised vascular structures formed early on in the natural regenerative process. Gel aspiration-ejection for the production of EngNT was automated by integrating a syringe pump with a robotic positioning system, using software coded in Python to control both devices. Having established the production method and tested mechanical properties, the EngNT containing human umbilical vein endothelial cells (EngNT-HUVEC) was characterised in terms of viability and alignment, compatibility with neurite outgrowth from rat dorsal root ganglion neurons and formation of endothelial cell networks in vitro. EngNT-HUVEC manufactured using the automated system contained viable and aligned endothelial cells, which developed into a network of multinucleated endothelial cell tube-like structures inside the constructs and an outer layer of endothelialisation. The EngNT-HUVEC constructs were made in various sizes within minutes. Constructs provided support and guidance to regenerating neurites in vitro. This work automated the formation of EngNT, facilitating high throughput manufacture at scale. The formation of endothelial cell tube-like structures within stabilised hydrogels provides an engineered tissue with potential for use in nerve repair.

We propose a continuum model for pattern formation, based on the multiphase model framework, to explore in vitro cell patterning within an extracellular matrix. We demonstrate that, within this framework, chemotaxis-driven cell migration can lead to formation of cell clusters and vascular-like structures in 1D and 2D respectively. The influence on pattern formation of additional mechanisms commonly included in multiphase tissue models, including cell-matrix traction, contact inhibition, and cell-cell aggregation, are also investigated. Using sensitivity analysis, the relative impact of each model parameter on the simulation outcomes is assessed to identify the key parameters involved. Chemoattractant-matrix binding is further included, motivated by previous experimental studies, and to augment the spatial scale of patterning to within a biologically plausible range. Key findings from the in-depth parameter analysis of the 1D models, both with and without chemoattractant-matrix binding, are demonstrated to translate well to the 2D model, obtaining vascular-like cell patterning for multiple parameter regimes. Overall, we demonstrate a biologically-motivated multiphase model capable of generating long-term pattern formation on a biologically plausible spatial scale both in 1D and 2D, with applications for modelling in vitro vascular network formation.

All-atom molecular dynamics (MD) simulations are an essential structural biology technique with increasing application to multimillion-atom systems, including viruses and cellular machinery. Classical MD simulations rely on parameter sets, such as the AMBER family of force fields (AMBERff), to accurately describe molecular motion. Here, we present an implementation of AMBERff for use in NAMD that overcomes previous limitations to enable high-performance, massively-parallel simulations encompassing up to two billion atoms. Single-point potential energy comparisons and case studies on model systems demonstrate that the implementation produces results that are as accurate as running AMBERff in its native engine.