A small-molecule PI3Kα activator for cardioprotection and neuroregeneration.

Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development1-5. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia-reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.

Advanced Formulation Approaches for Emerging Therapeutic Technologies.

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.

Serum neurofilament light chain measurements following nerve trauma.

BACKGROUND: Optimal functional recovery following peripheral nerve injuries (PNIs) is dependent upon early recognition and prompt referral to specialist centres for appropriate surgical intervention. Technologies which facilitate the early detection of PNI would allow faster referral rates and encourage improvements in patient outcomes. Serum Neurofilament light chain (NfL) measurements are cheaper to perform, easier to access and interpret than many conventional methods used for nerve injury diagnosis, such as electromyography and/or magnetic resonance imaging assessments, but changes in serum NfL levels following traumatic PNI have not been investigated. This pre-clinical study aimed to determine whether serum NfL levels can: (1) detect the presence of a nerve trauma and (2) delineate between different severities of nerve trauma. METHODS: A rat sciatic nerve crush and common peroneal nerve crush were implemented as controlled animal models of nerve injury. At 1-, 3-, 7- and 21-days post-injury, serum samples were retrieved for analysis using the SIMOA® NfL analyser kit. Nerve samples were also retrieved for histological analysis. Static sciatic index (SSI) was measured at regular time intervals following injury. RESULTS: Significant 45-fold and 20-fold increases in NfL serum levels were seen 1-day post-injury following sciatic and common peroneal nerve injury, respectively. This corresponded with an eightfold higher volume of axons injured in the sciatic compared to the common peroneal nerve (p < .001). SSI measurements post-injury revealed greater reduction in function in the sciatic crush group compared with the common peroneal crush group. CONCLUSIONS: NfL serum measurements represent a promising method for detecting traumatic PNI and stratifying their severity. Clinical translation of these findings could provide a powerful tool to improve the surgical management of nerve-injured patients.

Local Administration of Minocycline Improves Nerve Regeneration in Two Rat Nerve Injury Models.

Peripheral nerve injuries are quite common and often require a surgical intervention. However, even after surgery, patients do not often regain satisfactory sensory and motor functions. This, in turn, results in a heavy socioeconomic burden. To some extent, neurons can regenerate from the proximal nerve stump and try to reconnect to the distal stump. However, this regenerating capacity is limited, and depending on the type and size of peripheral nerve injury, this process may not lead to a positive outcome. To date, no pharmacological approach has been used to improve nerve regeneration following repair surgery. We elected to investigate the effects of local delivery of minocycline on nerve regeneration. This molecule has been studied in the central nervous system and was shown to improve the outcome in many disease models. In this study, we first tested the effects of minocycline on SCL 4.1/F7 Schwann cells in vitro and on sciatic nerve explants. We specifically focused on the Schwann cell repair phenotype, as these cells play a central role in orchestrating nerve regeneration. Finally, we delivered minocycline locally in two different rat models of nerve injury, a sciatic nerve transection and a sciatic nerve autograft, demonstrating the capacity of local minocycline treatment to improve nerve regeneration.

Culture Conditions for Human Induced Pluripotent Stem Cell-Derived Schwann Cells: A Two-Centre Study.

Adult human Schwann cells represent a relevant tool for studying peripheral neuropathies and developing regenerative therapies to treat nerve damage. Primary adult human Schwann cells are, however, difficult to obtain and challenging to propagate in culture. One potential solution is to generate Schwann cells from human induced pluripotent stem cells (hiPSCs). Previously published protocols, however, in our hands did not deliver sufficient viable cell numbers of hiPSC-derived Schwann cells (hiPSC-SCs). We present here, two modified protocols from two collaborating laboratories that overcome these challenges. With this, we also identified the relevant parameters to be specifically considered in any proposed differentiation protocol. Furthermore, we are, to our knowledge, the first to directly compare hiPSC-SCs to primary adult human Schwann cells using immunocytochemistry and RT-qPCR. We conclude the type of coating to be important during the differentiation process from Schwann cell precursor cells or immature Schwann cells to definitive Schwann cells, as well as the amounts of glucose in the specific differentiation medium to be crucial for increasing its efficiency and the final yield of viable hiPSC-SCs. Our hiPSC-SCs further displayed high similarity to primary adult human Schwann cells.

A combined experimental and computational framework to evaluate the behavior of therapeutic cells for peripheral nerve regeneration.

Recent studies have explored the potential of tissue-mimetic scaffolds in encouraging nerve regeneration. One of the major determinants of the regenerative success of cellular nerve repair constructs (NRCs) is the local microenvironment, particularly native low oxygen conditions which can affect implanted cell survival and functional performance. In vivo, cells reside in a range of environmental conditions due to the spatial gradients of nutrient concentrations that are established. Here we evaluate in vitro the differences in cellular behavior that such conditions induce, including key biological features such as oxygen metabolism, glucose consumption, cell death, and vascular endothelial growth factor secretion. Experimental measurements are used to devise and parameterize a mathematical model that describes the behavior of the cells. The proposed model effectively describes the interactions between cells and their microenvironment and could in the future be extended, allowing researchers to compare the behavior of different therapeutic cells. Such a combinatorial approach could be used to accelerate the clinical translation of NRCs by identifying which critical design features should be optimized when fabricating engineered nerve repair conduits.

Modelling-informed cell-seeded nerve repair construct designs for treating peripheral nerve injuries.

Millions of people worldwide are affected by peripheral nerve injuries (PNI), involving billions of dollars in healthcare costs. Common outcomes for patients include paralysis and loss of sensation, often leading to lifelong pain and disability. Engineered Neural Tissue (EngNT) is being developed as an alternative to the current treatments for large-gap PNIs that show underwhelming functional recovery in many cases. EngNT repair constructs are composed of a stabilised hydrogel cylinder, surrounded by a sheath of material, to mimic the properties of nerve tissue. The technology also enables the spatial seeding of therapeutic cells in the hydrogel to promote nerve regeneration. The identification of mechanisms leading to maximal nerve regeneration and to functional recovery is a central challenge in the design of EngNT repair constructs. Using in vivo experiments in isolation is costly and time-consuming, offering a limited insight on the mechanisms underlying the performance of a given repair construct. To bridge this gap, we derive a cell-solute model and apply it to the case of EngNT repair constructs seeded with therapeutic cells which produce vascular endothelial growth factor (VEGF) under low oxygen conditions to promote vascularisation in the construct. The model comprises a set of coupled non-linear diffusion-reaction equations describing the evolving cell population along with its interactions with oxygen and VEGF fields during the first 24h after transplant into the nerve injury site. This model allows us to evaluate a wide range of repair construct designs (e.g. cell-seeding strategy, sheath material, culture conditions), the idea being that designs performing well over a short timescale could be shortlisted for in vivo trials. In particular, our results suggest that seeding cells beyond a certain density threshold is detrimental regardless of the situation considered, opening new avenues for future nerve tissue engineering.

Low frequency oscillating gradient spin-echo sequences improve sensitivity to axon diameter: An experimental study in viable nerve tissue.

Mapping axon diameters within the central and peripheral nervous system could play an important role in our understanding of nerve pathways, and help diagnose and monitor an array of neurological disorders. Numerous diffusion MRI methods have been proposed for imaging axon diameters, most of which use conventional single diffusion encoding (SDE) spin echo sequences. However, a growing number of studies show that oscillating gradient spin echo (OGSE) sequences can provide additional advantages over conventional SDE sequences. Recent theoretical results suggest that this is especially the case in realistic scenarios, such as when fibres have unknown or dispersed orientation. In the present study, we adopt the ActiveAx approach to experimentally investigate the extent of these advantages by comparing the performances of SDE and trapezoidal OGSE in viable nerve tissue. We optimise SDE and OGSE ActiveAx protocols for a rat peripheral nerve tissue and test their performance using Monte Carlo simulations and a 800 mT/m gradient strength pre-clinical imaging experiment. The imaging experiment uses excised sciatic nerve from a rat's leg placed in a MRI compatible viable isolated tissue (VIT) maintenance chamber, which keeps the tissue in a viable physiological state that preserves the structural complexity of the nerve and enables lengthy scan times. We compare model estimates to histology, which we perform on the nerve post scanning. Optimisation produces a three-shell SDE and OGSE ActiveAx protocol, with the OGSE protocol consisting of one SDE sequence and two low-frequency oscillating gradient waveform sequences. Both simulation and imaging results show that the OGSE ActiveAx estimates of the axon diameter index have a higher accuracy and a higher precision compared to those from SDE. Histology estimates of the axon diameter index in our nerve tissue samples are 4-5.8 µm and these are excellently matched with the OGSE estimates 4.2-6.5 µm, while SDE overestimates at 5.2-8 µm for the same sample. We found OGSE estimates to be more precise with on average a 0.5 µm standard deviation compared to the SDE estimates which have a 2 µm standard deviation. When testing the robustness of the estimates when the number of the diffusion gradient directions reduces, we found that both OGSE and SDE estimates are affected, however OGSE is more robust to these changes than the SDE. Overall, these results suggest, quantitatively and in in vivo conditions, that low-frequency OGSE sequences may provide improved accuracy of axon diameter mapping compared to standard SDE sequences.

A three-dimensional collagen construct to model lipopolysaccharide-induced activation of BV2 microglia.

BACKGROUND: We report a novel method of culturing microglia in three dimension (3D) using collagen as a substrate. By culturing microglia within a matrix, we aim to emulate the physical state of microglia embedded within parenchyma. METHODS: BV2 microglia cell suspensions were prepared with type I collagen and cast into culture plates. To characterise the BV2 microglia cultured in 3D, the cultures were evaluated for their viability, cell morphology and response to lipopolysaccharide (LPS) activation. Conventional monolayer cultures (grown on uncoated and collagen-coated polystyrene) were set up concurrently for comparison. RESULTS: BV2 microglia in 3D collagen matrices were viable at 48 hrs of culture and exhibit a ramified morphology with multiplanar cytoplasmic projections. Following stimulation with 1 µg/ml LPS, microglia cultured in 3D collagen gels increase their expression of nitric oxide (NO) and CD40, indicating their capacity to become activated within the matrix. Up to 97.8% of BV2 microglia grown in 3D cultures gained CD40 positivity in response to LPS, compared to approximately 60% of cells grown in a monolayer (P<.05). BV2 microglia in 3D collagen gels also showed increased mRNA and protein expression of inflammatory cytokines IL-6, TNF-α and the chemoattractant MCP-1 following LPS stimulation. CONCLUSIONS: In summary, BV2 microglia cultured in 3D collagen hydrogels exhibit multiplanar cytoplasmic projections and undergo a characteristic and robust activation response to LPS. This culture system is accessible to a wide range of analyses and provides a useful new in vitro tool for research into microglial activation.

Formation of vascular-like structures using a chemotaxis-driven multiphase model.

We propose a continuum model for pattern formation, based on the multiphase model framework, to explore in vitro cell patterning within an extracellular matrix (ECM). We demonstrate that, within this framework, chemotaxis-driven cell migration can lead to the 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 found to reduce the spatial scale of patterning to within a biologically plausible range for capillary structures. 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.

Electrospun aligned tacrolimus-loaded polycaprolactone biomaterials for peripheral nerve repair.

Background: Efficacious repair of peripheral nerve injury is an unmet clinical need. The implantation of biomaterials containing neurotrophic drugs at the injury site could promote nerve regeneration and improve outcomes for patients. Materials & methods: Random and aligned electrospun poly-ε-caprolactone scaffolds containing encapsulated tacrolimus were fabricated, and the gene expression profile of Schwann cells (SCs) cultured on the surface was elucidated. On aligned fibers, the morphology of SCs and primary rat neurons was investigated. Results: Both scaffold types exhibited sustained release of drug, and the gene expression of SCs was modulated by both nanofibrous topography and the presence of tacrolimus. Aligned fibers promoted the alignment of SCs and orientated outgrowth from neurons. Conclusion: Electrospun PCL scaffolds with tacrolimus hold promise for the repair of peripheral nerve injury.

This article reports the production and testing of fibrous materials loaded with tacrolimus, a drug known to improve nerve regeneration, for the surgical repair of peripheral nerve injury. Materials were created with either a randomly orientated structure or an aligned structure that mimics the anatomy of native nerve, and both displayed long-term release of the loaded drug. Schwann cells, which are a critical cell type in nerve regeneration, were grown on the materials and their behaviour was positively influenced by the fibrous surfaces and/or the presence of tacrolimus. Neurons grown on the aligned materials demonstrated directional outgrowth, which may be also beneficial for increasing the rate of regeneration. These materials have the potential to improve outcomes of nerve repair for patients.

In silico model for automated calculation of functional metrics in animal models of peripheral nerve injury repair.

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.

Aligned Bioelectronic Polypyrrole/Collagen Constructs for Peripheral Nerve Interfacing.

Electrical stimulation has shown promise in clinical studies to treat nerve injuries. This work is aimed to create an aligned bioelectronic construct that can be used to bridge a nerve gap, directly interfacing with the damaged nerve tissue to provide growth support. 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 are seeded with dorsal root ganglion derived primary rat neurons and electrically stimulated in vitro. The PPy loaded constructs support 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 is 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.

Perspectives on optimizing local delivery of drugs to peripheral nerves using mathematical models.

Drug therapies for treating peripheral nerve injury repair have shown significant promise in preclinical studies. Despite this, drug treatments are not used routinely clinically to treat patients with peripheral nerve injuries. Drugs delivered systemically are often associated with adverse effects to other tissues and organs; it remains challenging to predict the effective concentration needed at an injured nerve and the appropriate delivery strategy. Local drug delivery approaches are being developed to mitigate this, for example via injections or biomaterial-mediated release. We propose the integration of mathematical modeling into the development of local drug delivery protocols for peripheral nerve injury repair. Mathematical models have the potential to inform understanding of the different transport mechanisms at play, as well as quantitative predictions around the efficacy of individual local delivery protocols. We discuss existing approaches in the literature, including drawing from other research fields, and present a process for taking forward an integrated mathematical-experimental approach to accelerate local drug delivery approaches for peripheral nerve injury repair. This article is categorized under: Neurological Diseases > Molecular and Cellular Physiology Neurological Diseases > Computational Models Neurological Diseases > Biomedical Engineering.

Mathematical modelling with Bayesian inference to quantitatively characterize therapeutic cell behaviour in nerve tissue engineering.

Cellular engineered neural tissues have significant potential to improve peripheral nerve repair strategies. Traditional approaches depend on quantifying tissue behaviours using experiments in isolation, presenting a challenge for an overarching framework for tissue design. By comparison, mathematical cell-solute models benchmarked against experimental data enable computational experiments to be performed to test the role of biological/biophysical mechanisms, as well as to explore the impact of different design scenarios and thus accelerate the development of new treatment strategies. Such models generally consist of a set of continuous, coupled, partial differential equations relying on a number of parameters and functional forms. They necessitate dedicated in vitro experiments to be informed, which are seldom available and often involve small datasets with limited spatio-temporal resolution, generating uncertainties. We address this issue and propose a pipeline based on Bayesian inference enabling the derivation of experimentally informed cell-solute models describing therapeutic cell behaviour in nerve tissue engineering. We apply our pipeline to three relevant cell types and obtain models that can readily be used to simulate nerve repair scenarios and quantitatively compare therapeutic cells. Beyond parameter estimation, the proposed pipeline enables model selection as well as experiment utility quantification, aimed at improving both model formulation and experimental design.

Engineered neural tissue made using hydrogels derived from decellularised tissues for the regeneration of peripheral nerves.

Engineered neural tissue (EngNT) promotes in vivo axonal regeneration. Decellularised materials (dECM) are complex biologic scaffolds that can improve the cellular environment and also encourage positive tissue remodelling in vivo. We hypothesised that we could incorporate a hydrogel derived from a decellularised tissue (dECMh) into EngNT, thereby providing an alternative to the currently used purified collagen I hydrogel for the first time. Decellularisation was carried out on bone (B-ECM), liver (LIV-ECM), and small intestinal (SIS-ECM) tissues and the resultant dECM was biochemically and mechanically characterised. dECMh differed in mechanical and biochemical properties that likely had an effect on Schwann cell behaviour observed in metabolic activity and contraction profiles. Cellular alignment was observed in tethered moulds within the B-ECM and SIS-ECM derived hydrogels only. No difference was observed in dorsal root ganglia (DRG) neurite extension between the dECMh groups and collagen I groups when applied as a coverslip coating, however, when DRG were seeded atop EngNT constructs, only the B-ECM derived EngNT performed similarly to collagen I derived EngNT. B-ECM EngNT further exhibited similar axonal regeneration to collagen I EngNT in a 10 mm gap rat sciatic nerve injury model after 4 weeks. Our results have shown that various dECMh can be utilised to produce EngNT that can promote neurite extension in vitro and axonal regeneration in vivo. STATEMENT OF SIGNIFICANCE: Nerve autografts are undesirable due to the sacrifice of a patient's own nerve tissue to repair injuries. Engineered neural tissue (EngNT) is a type of living artificial tissue that has been developed to overcome this. To date, only a collagen hydrogel has been shown to be effective in the production and utilisation of EngNT in animal models. Hydrogels may be made from decellularised extracellular matrix derived from many tissues. In this study we showed that hydrogels from various tissues may be used to create EngNT and one was shown to comparable to the currently used collagen based EngNT in a rat sciatic nerve injry model.

Improving the biological interfacing capability of diketopyrrolopyrrole polymers via p-type doping.

Polydiketopyrrolopyrrole terthiophene (DPP3T) is an organic semiconducting polymer that has been widely investigated as the active layer within organic electronic devices, such as photovoltaics and bioelectronic sensors. To facilitate interfacing between biological systems and organic semiconductors it is crucial to tune the material properties to support not only cell adhesion, but also proliferation and growth. Herein, we highlight the potential of molecular doping to judiciously modulate the surface properties of DPP3T and investigate the effects on Schwann cell behaviour on the surface. By using p-type dopants FeCl3 and Magic Blue, we successfully alter the topography of DPP3T thin films, which in turn alters cell behaviour of a Schwann cell line on the surfaces of the films over the course of 48 hours. Cell numbers are significantly increased within both DPP3T doped films, as well as cells possessing larger, more spread out morphology indicated by cell size and shape analysis. Furthermore, the viability of the Schwann cells seeded on the surfaces of the films was not significantly lowered. The use of dopants for influencing cell behaviour on semiconducting polymers holds great promise for improving the cell-device interface, potentially allowing better integration of cells and devices at the initial time of introduction to a biological environment.

Development of ibuprofen-loaded electrospun materials suitable for surgical implantation in peripheral nerve injury.

The development of nerve wraps for use in the repair of peripheral nerves has shown promise over recent years. A pharmacological effect to improve regeneration may be achieved by loading such materials with therapeutic agents, for example ibuprofen, a non-steroidal anti-inflammatory drug with neuroregenerative properties. In this study, four commercially available polymers (polylactic acid (PLA), polycaprolactone (PCL) and two co-polymers containing different ratios of PLA to PCL) were used to fabricate ibuprofen-loaded nerve wraps using blend electrospinning. In vitro surgical handling experiments identified a formulation containing a PLA/PCL 70/30 molar ratio co-polymer as the most suitable for in vivo implantation. In a rat model, ibuprofen released from electrospun materials significantly improved the rate of axonal growth and sensory recovery over a 21-day recovery period following a sciatic nerve crush. Furthermore, RT-qPCR analysis of nerve segments revealed that the anti-inflammatory and neurotrophic effects of ibuprofen may still be observed 21 days after implantation. This suggests that the formulation developed in this work could have potential to improve nerve regeneration in vivo.

Considerations for the use of biomaterials to support cell therapy in neurodegenerative disease.

Using biomaterials to complement cell therapies for a range of neurodegenerative disorders provides a potential opportunity to improve cell survival, integration and regeneration. Materials can be developed to serve as cell or drug delivery systems, temporary scaffolds or longer-term encapsulation structures. However, as yet clinical translation has been limited, with much work still required to achieve this. This chapter discusses a number of considerations for using biomaterials to support cell therapies, which are likely to be essential to achieve successful translation.