In vitro model of neurotrauma using the chick embryo to test regenerative bioimplantation.

Effective repair of spinal cord injury sites remains a major clinical challenge. One promising strategy is the implantation of multifunctional bioscaffolds to enhance nerve fibre growth, guide regenerating tissue and modulate scarring/inflammation processes. Given their multifunctional nature, such implants require testing in models which replicate the complex neuropathological responses of spinal injury sites. This is often achieved using live, adult animal models of spinal injury. However, these have substantial drawbacks for developmental testing, including the requirement for large numbers of animals, costly infrastructure, high levels of expertise and complex ethical processes. As an alternative, we show that organotypic spinal cord slices can be derived from the E14 chick embryo and cultured with high viability for at least 24 days, with major neural cell types detected. A transecting injury could be reproducibly introduced into the slices and characteristic neuropathological responses similar to those in adult spinal cord injury observed at the lesion margin. This included aligned astrocyte morphologies and upregulation of glial fibrillary acidic protein in astrocytes, microglial infiltration into the injury cavity and limited nerve fibre outgrowth. Bioimplantation of a clinical grade scaffold biomaterial was able to modulate these responses, disrupting the astrocyte barrier, enhancing nerve fibre growth and supporting immune cell invasion. Chick embryos are inexpensive and simple, requiring facile methods to generate the neurotrauma model. Our data show the chick embryo spinal cord slice system could be a replacement spinal injury model for laboratories developing new tissue engineering solutions.

A benchtop brain injury model using resected donor tissue from patients with Chiari malformation.

The use of live animal models for testing new therapies for brain and spinal cord repair is a controversial area. Live animal models have associated ethical issues and scientific concerns regarding the predictability of human responses. Alternative models that replicate the 3D architecture of the central nervous system have prompted the development of organotypic neural injury models. However, the lack of reliable means to access normal human neural tissue has driven reliance on pathological or post-mortem tissue which limits their biological utility. We have established a protocol to use donor cerebellar tonsillar tissue surgically resected from patients with Chiari malformation (cerebellar herniation towards the foramen magnum, with ectopic rather than diseased tissue) to develop an in vitro organotypic model of traumatic brain injury. Viable tissue was maintained for approximately 2 weeks with all the major neural cell types detected. Traumatic injuries could be introduced into the slices with some cardinal features of post-injury pathology evident. Biomaterial placement was also feasible within the in vitro lesions. Accordingly, this 'proof-of-concept' study demonstrates that the model offers potential as an alternative to the use of animal tissue for preclinical testing in neural tissue engineering. To our knowledge, this is the first demonstration that donor tissue from patients with Chiari malformation can be used to develop a benchtop model of traumatic brain injury. However, significant challenges in relation to the clinical availability of tissue were encountered, and we discuss logistical issues that must be considered for model scale-up.

Systematic Alignment Analysis of Neural Transplant Cells in Electrospun Nanofibre Scaffolds.

Spinal cord injury is debilitating with functional loss often permanent due to a lack of neuro-regenerative or neuro-therapeutic strategies. A promising approach to enhance biological function is through implantation of tissue engineered constructs, to offer neural cell replacement and reconstruction of the functional neuro-architecture. A key goal is to achieve spatially targeted guidance of regenerating tissue across the lesion site to achieve an aligned tissue structure lost as a consequence of injury. Electrospun nanofibres mimic the nanoscale architecture of the spinal cord, can be readily aligned, functionalised with pro-regenerative molecules and incorporated into implantable matrices to provide topographical cues. Crucially, electrospun nanofibers are routinely manufactured at a scale required for clinical use. Although promising, few studies have tested whether electrospun nanofibres can guide targeted spatial growth of clinically relevant neural stem/precursor populations. The alignment fate of daughter cells (derived from the pre-aligned parent cells) has also received limited attention. Further, a standardised quantification methodology to correlate neural cell alignment with topographical cues is not available. We have adapted an image analysis technique to quantify nanofibre-induced alignment of neural cells. Using this method, we show that two key neural stem/precursor populations of clinical relevance (namely, neural stem cells (NSCs) and oligodendrocyte precursor cells), reproducibly orientate their growth to aligned, high-density electrospun nanofiber meshes, but not randomly distributed ones. Daughter populations derived from aligned NSCs (neurons and astrocytes) maintained their alignment following differentiation, but oligodendrocytes did not. Our data show that pre-aligned transplant populations can be used to generate complex, multicellular aligned-fibre constructs for neural implantation.

Enhancing the regenerative potential of stem cell-laden, clinical-grade implants through laminin engineering.

Protected delivery of neural stem cells (NSCs; a major transplant population) within bioscaffolds has the potential to improve regenerative outcomes in sites of spinal cord injury. Emergent research has indicated clinical grade bioscaffolds (e.g. those used as surgical sealants) may be repurposed for this strategy, bypassing the long approval processes and difficulties in scale-up faced by laboratory grade materials. While promising, clinical scaffolds are often not inherently regenerative. Extracellular molecule biofunctionalisation of scaffolds can enhance regenerative features such as encapsulated cell survival/distribution, cell differentiation into desired cell types and nerve fibre growth. However, this strategy is yet to be tested for clinical grade scaffolds. Here, we show for the first time that Hemopatch™, a widely used, clinically approved surgical matrix, supports NSC growth. Further, functionalisation of Hemopatch™ with laminin promoted homogenous distribution of NSCs and their daughter cells within the matrix, a key regenerative criterion for transplant cells.

Delivery of chondroitinase by canine mucosal olfactory ensheathing cells alongside rehabilitation enhances recovery after spinal cord injury.

Spinal cord injury (SCI) can cause chronic paralysis and incontinence and remains a major worldwide healthcare burden, with no regenerative treatment clinically available. Intraspinal transplantation of olfactory ensheathing cells (OECs) and injection of chondroitinase ABC (chABC) are both promising therapies but limited and unpredictable responses are seen, particularly in canine clinical trials. Sustained delivery of chABC presents a challenge due to its thermal instability; we hypothesised that transplantation of canine olfactory mucosal OECs genetically modified ex vivo by lentiviral transduction to express chABC (cOEC-chABC) would provide novel delivery of chABC and synergistic therapy. Rats were randomly divided into cOEC-chABC, cOEC, or vehicle transplanted groups and received transplant immediately after dorsal column crush corticospinal tract (CST) injury. Rehabilitation for forepaw reaching and blinded behavioural testing was conducted for 8 weeks. We show that cOEC-chABC transplanted animals recover greater forepaw reaching accuracy on Whishaw testing and more normal gait than cOEC transplanted or vehicle control rats. Increased CST axon sprouting cranial to the injury and serotonergic fibres caudal to the injury suggest a mechanism for recovery. We therefore demonstrate that cOECs can deliver sufficient chABC to drive modest functional improvement, and that this genetically engineered cellular and molecular approach is a feasible combination therapy for SCI.

Stiffness-matched biomaterial implants for cell delivery: clinical, intraoperative ultrasound elastography provides a 'target' stiffness for hydrogel synthesis in spinal cord injury.

Safe hydrogel delivery requires stiffness-matching with host tissues to avoid iatrogenic damage and reduce inflammatory reactions. Hydrogel-encapsulated cell delivery is a promising combinatorial approach to spinal cord injury therapy, but a lack of in vivo clinical spinal cord injury stiffness measurements is a barrier to their use in clinics. We demonstrate that ultrasound elastography - a non-invasive, clinically established tool - can be used to measure spinal cord stiffness intraoperatively in canines with spontaneous spinal cord injury. In line with recent experimental reports, our data show that injured spinal cord has lower stiffness than uninjured cord. We show that the stiffness of hydrogels encapsulating a clinically relevant transplant population (olfactory ensheathing cells) can also be measured by ultrasound elastography, enabling synthesis of hydrogels with comparable stiffness to canine spinal cord injury. We therefore demonstrate proof-of-principle of a novel approach to stiffness-matching hydrogel-olfactory ensheathing cell implants to 'real-life' spinal cord injury values; an approach applicable to multiple biomaterial implants for regenerative therapies.

Safe nanoengineering and incorporation of transplant populations in a neurosurgical grade biomaterial, DuraGen PlusTM, for protected cell therapy applications.

High transplant cell loss is a major barrier to translation of stem cell therapy for pathologies of the brain and spinal cord. Encapsulated delivery of stem cells in biomaterials for cell therapy is gaining popularity but experimental research has overwhelmingly used laboratory grade materials unsuitable for human clinical use - representing a further barrier to clinical translation. A potential solution is to use neurosurgical grade materials routinely used in clinical protocols which have an established human safety profile. Here, we tested the ability of Duragen Plus™ - a clinical biomaterial used widely in neurosurgical duraplasty procedures, to support the growth and differentiation of neural stem cells- a major transplant population being tested in clinical trials for neurological pathology. Genetic engineering of stem cells yields augmented therapeutic cells, so we further tested the ability of the Duragen Plus™ matrix to support stem cells engineered using magnetofection technology and minicircle DNA vectors- a promising cell engineering approach we previously reported (Journal of Controlled Release, 2016 a &b). The safety of the nano-engineering approach was analysed for the first time using sophisticated data-independent analysis by mass spectrometry-based proteomics. We prove that the Duragen Plus™ matrix is a promising biomaterial for delivery of stem cell transplant populations, with no adverse effects on key regenerative parameters. This advanced cellular construct based on a combinatorial nano-engineering and biomaterial encapsulation approach, could therefore offer key advantages for clinical translation.

Less is more: Investigating the influence of cellular nanoparticle load on transfection outcomes in neural cells.

Genetic engineering of cell transplant populations offers potential for delivery of neurotherapeutic factors to modify the regenerative microenvironment of the injured spinal cord. The use of magnetic nanoparticle (MNP)-based vectors has reduced the traditional reliance on viral methods and their associated obstacles in terms of scale up and safety. Studies utilizing magnetic assistive platforms for MNP-mediated gene delivery have found transfection efficiency in astrocytes (a major transplant and homeostatic neural cell type) to be both frequency- and amplitude-dependent. It is widely assumed that increased intracellular particle load will enhance transfection efficiency in a cell population. Therefore, we tested repeat delivery of MNP:plasmid complexes in conjunction with oscillating magnetic field parameters-a process termed "magneto-multifection"-in astrocytes of primary origin in an attempt to enhance transfection levels. We show (a) levels of transfection using magneto-multifection equal that seen with viral methods; (b) reporter protein expression using two reporter plasmids shows a diverse profile of single/dual transfected cells with implications for delivery of a "cocktail" of neurotherapeutic proteins; and (c) contrary to expectation, an inverse relationship exists between particle load and reporter protein expression.

Electrophysiological properties of neurons grown on soft polymer scaffolds reveal the potential to develop neuromimetic culture environments.

Tissue engineering methodologies for various physiological systems are seeing a significant trend towards 3D cell culture in or on 'soft' polymeric hydrogel materials, widely considered to provide a more biomimetic environment for cell growth versus 'hard' materials such as glass or plastic. Progress has been slower with 3D neural cell culture with current studies overwhelmingly reliant on hard substrates. Accordingly, our knowledge of the alterations in electrochemical properties of neurons propagated in soft materials is relatively limited. In this study, primary cortical neurons and glial cells were seeded onto the surface of collagen hydrogels and grown in vitro for 7-8 days. At this time, neurons had formed a complex neurite web interspersed with astrocytes. Neuronal patch clamp recordings revealed voltage-gated Na+ and K+ currents in voltage clamp and action potentials in current clamp. When measured at voltages close to maximum activation, both currents were >1 nA in mean amplitude. When compared to primary cortical neurons cultured on glass coverslips, but otherwise under similar conditions (Evans et al., 2017), the Na+ current from hydrogel neurons was found to be significantly larger although there were no differences in the K+ current amplitude, membrane potential, input resistance or cell capacitance. We speculate that the larger size of the neuronal voltage-dependent Na+ current in the hydrogels is related to the better biomimetic properties of the soft material, being close to values reported for neurons recorded in brain slices. The results highlight the potential benefits offered by neuronal culture on soft and biomimetic polymeric materials for neural tissue engineering studies.

Nanoparticle-Based Imaging of Clinical Transplant Populations Encapsulated in Protective Polymer Matrices.

A recent clinical trial proves that autologous olfactory mucosal cell (OMC) transplantation improves locomotion in dogs with naturally occurring spinal injuries comparable to human lesions. However, not all dogs respond to the treatment, likely due to the transplantation procedures involving injections of cell suspensions that are associated with cell death, uneven cell distribution, and cell washout. Encapsulating cells in protective hydrogel matrices offers a tissue engineering solution to safely achieve 3D growth of viable transplant cells for implantation into injury sites, to improve regenerative outcomes. It is shown for the first time that canine OMCs (cOMCs) can be propagated with high viability in 3D collagen matrices. Further, a method to incorporate cOMCs pre-labeled with clinical-grade iron oxide nanoparticles into the constructs is described. Intraconstruct labeled cells are visualized using magnetic resonance imaging, offering substantial promise for in vivo tracking of cOMCs delivered in protective matrices.

Noninvasive imaging of nanoparticle-labeled transplant populations within polymer matrices for neural cell therapy.

AIM: To develop a 3D neural cell construct for encapsulated delivery of transplant cells; develop hydrogels seeded with magnetic nanoparticle (MNP)-labeled cells suitable for cell tracking by MRI. MATERIALS & METHODS: Astrocytes were exogenously labeled with MRI-compatible iron-oxide MNPs prior to intra-construct incorporation within a 3D collagen hydrogel. RESULTS: A connective, complex cellular network was clearly observable within the 3D constructs, with high cellular viability. MNP accumulation in astrocytes provided a hypointense MRI signal at 24 h & 14 days. CONCLUSION: Our findings support the concept of developing a 3D construct possessing the dual advantages of (i) support of long-term cell survival of neural populations with (ii) the potential for noninvasive MRI-tracking of intra-construct cells for neuroregenerative applications.

A proteomic investigation into mechanisms underpinning corticosteroid effects on neural stem cells.

Corticosteroids (CSs) are widely used clinically, for example in pediatric respiratory distress syndrome, and immunosuppression to prevent rejection of stem cell transplant populations in neural cell therapy. However, such treatment can be associated with adverse effects such as impaired neurogenesis and myelination, and increased risk of cerebral palsy. There is increasing evidence that CSs can adversely influence key biological properties of neural stem cells (NSCs) but the molecular mechanisms underpinning such effects are largely unknown. This is an important issue to address given the key roles NSCs play during brain development and as transplant cells for regenerative neurology. Here, we describe the use of label-free quantitative proteomics in conjunction with histological analyses to study CS effects on NSCs at the cellular and molecular levels, following treatment with methylprednisolone (MPRED). Immunocytochemical staining showed that both parent NSCs and newly generated daughter cells expressed the glucocorticoid receptor, with nuclear localisation of the receptor induced by MPRED treatment. MPRED markedly decreased NSC proliferation and neuronal differentiation while accelerating the maturation of oligodendrocytes, without concomitant effects on cell viability and apoptosis. Parallel proteomic analysis revealed that MPRED induced downregulation of growth associated protein 43 and matrix metallopeptidase 16 with upregulation of the cytochrome P450 family 51 subfamily A member 1. Our findings support the hypothesis that some neurological deficits associated with CS use may be mediated via effects on NSCs, and highlight putative target mechanisms underpinning such effects.

A fusion of minicircle DNA and nanoparticle delivery technologies facilitates therapeutic genetic engineering of autologous canine olfactory mucosal cells.

Olfactory ensheathing cells (OECs) promote axonal regeneration and improve locomotor function when transplanted into the injured spinal cord. A recent clinical trial demonstrated improved motor function in domestic dogs with spinal injury following autologous OEC transplantation. Their utility in canines offers promise for human translation, as dogs are comparable to humans in terms of clinical management and genetic/environmental variation. Moreover, the autologous, minimally invasive derivation of OECs makes them viable for human spinal injury investigation. Genetic engineering of transplant populations may augment their therapeutic potential, but relies heavily on viral methods which have several drawbacks for clinical translation. We present here the first proof that magnetic particles deployed with applied magnetic fields and advanced DNA minicircle vectors can safely bioengineer OECs to secrete a key neurotrophic factor, with an efficiency approaching that of viral vectors. We suggest that our alternative approach offers high translational potential for the delivery of augmented clinical cell therapies.

Magnetic Nanoparticle-Mediated Gene Delivery to Two- and Three-Dimensional Neural Stem Cell Cultures: Magnet-Assisted Transfection and Multifection Approaches to Enhance Outcomes.

Neural stem cells (NSCs) have high translational potential in transplantation therapies for neural repair. Enhancement of their therapeutic capacity by genetic engineering is an important goal for regenerative neurology. Magnetic nanoparticles (MNPs) are major non-viral vectors for safe bioengineering of NSCs, offering critical translational benefits over viral vectors, including safety, scalability, and ease of use. This unit describes protocols for the production of suspension (neurosphere) and adherent (monolayer) murine NSC cultures. Genetic engineering of NSCs with MNPs and the application of 'magnetofection' (magnetic fields) or 'multifection' (repeat transfection) approaches to enhance gene delivery are described. Magnetofection of monolayer cultures achieves optimal transfection, but neurospheres offer key advantages for neural graft survival post-transplantation. A protocol is presented which allows the advantageous features of each approach to be combined into a single procedure for transplantation. The adaptation of these protocols for other MNP preparations is considered, with emphasis on the evaluation of procedural safety. © 2017 by John Wiley & Sons, Inc.

Nanoengineering neural stem cells on biomimetic substrates using magnetofection technology.

Tissue engineering studies are witnessing a major paradigm shift to cell culture on biomimetic materials that replicate native tissue features from which the cells are derived. Few studies have been performed in this regard for neural cells, particularly in nanomedicine. For example, platforms such as magnetic nanoparticles (MNPs) have proven efficient as multifunctional tools for cell tracking and genetic engineering of neural transplant populations. However, as far as we are aware, all current studies have been conducted using neural cells propagated on non-neuromimetic substrates that fail to represent the mechano-elastic properties of brain and spinal cord microenvironments. Accordingly, it can be predicted that such data is of less translational and physiological relevance than that derived from cells grown in neuromimetic environments. Therefore, we have performed the first test of magnetofection technology (enhancing MNP delivery using applied magnetic fields with significant potential for therapeutic application) and its utility in genetically engineering neural stem cells (NSCs; a population of high clinical relevance) propagated in biomimetic hydrogels. We demonstrate magnetic field application safely enhances MNP mediated transfection of NSCs grown as 3D spheroid structures in collagen which more closely replicates the intrinsic mechanical and structural properties of neural tissue than routinely used hard substrates. Further, as it is well known that MNP uptake is mediated by endocytosis we also investigated NSC membrane activity grown on both soft and hard substrates. Using high resolution scanning electron microscopy we were able to prove that NSCs display lower levels of membrane activity on soft substrates compared to hard, a finding which could have particular impact on MNP mediated engineering strategies of cells propagated in physiologically relevant systems.

Part II: Functional delivery of a neurotherapeutic gene to neural stem cells using minicircle DNA and nanoparticles: Translational advantages for regenerative neurology.

Both neurotrophin-based therapy and neural stem cell (NSC)-based strategies have progressed to clinical trials for treatment of neurological diseases and injuries. Brain-derived neurotrophic factor (BDNF) in particular can confer neuroprotective and neuro-regenerative effects in preclinical studies, complementing the cell replacement benefits of NSCs. Therefore, combining both approaches by genetically-engineering NSCs to express BDNF is an attractive approach to achieve combinatorial therapy for complex neural injuries. Current genetic engineering approaches almost exclusively employ viral vectors for gene delivery to NSCs though safety and scalability pose major concerns for clinical translation and applicability. Magnetofection, a non-viral gene transfer approach deploying magnetic nanoparticles and DNA with magnetic fields offers a safe alternative but significant improvements are required to enhance its clinical application for delivery of large sized therapeutic plasmids. Here, we demonstrate for the first time the feasibility of using minicircles with magnetofection technology to safely engineer NSCs to overexpress BDNF. Primary mouse NSCs overexpressing BDNF generated increased daughter neuronal cell numbers post-differentiation, with accelerated maturation over a four-week period. Based on our findings we highlight the clinical potential of minicircle/magnetofection technology for therapeutic delivery of key neurotrophic agents.

Part I: Minicircle vector technology limits DNA size restrictions on ex vivo gene delivery using nanoparticle vectors: Overcoming a translational barrier in neural stem cell therapy.

Genetically engineered neural stem cell (NSC) transplant populations offer key benefits in regenerative neurology, for release of therapeutic biomolecules in ex vivo gene therapy. NSCs are 'hard-to-transfect' but amenable to 'magnetofection'. Despite the high clinical potential of this approach, the low and transient transfection associated with the large size of therapeutic DNA constructs is a critical barrier to translation. We demonstrate for the first time that DNA minicircles (small DNA vectors encoding essential gene expression components but devoid of a bacterial backbone, thereby reducing construct size versus conventional plasmids) deployed with magnetofection achieve the highest, safe non-viral DNA transfection levels (up to 54%) reported so far for primary NSCs. Minicircle-functionalized magnetic nanoparticle (MNP)-mediated gene delivery also resulted in sustained gene expression for up to four weeks. All daughter cell types of engineered NSCs (neurons, astrocytes and oligodendrocytes) were transfected (in contrast to conventional plasmids which usually yield transfected astrocytes only), offering advantages for targeted cell engineering. In addition to enhancing MNP functionality as gene delivery vectors, minicircle technology provides key benefits from safety/scale up perspectives. Therefore, we consider the proof-of-concept of fusion of technologies used here offers high potential as a clinically translatable genetic modification strategy for cell therapy.

'Stealth' nanoparticles evade neural immune cells but also evade major brain cell populations: Implications for PEG-based neurotherapeutics.

Surface engineering to control cell behavior is of high interest across the chemical engineering, drug delivery and biomaterial communities. Defined chemical strategies are necessary to tailor nanoscale protein interactions/adsorption, enabling control of cell behaviors for development of novel therapeutic strategies. Nanoparticle-based therapies benefit from such strategies but particle targeting to sites of neurological injury remains challenging due to circulatory immune clearance. As a strategy to overcome this barrier, the use of stealth coatings can reduce immune clearance and prolong circulatory times, thereby enhancing therapeutic capacity. Polyethylene glycol (PEG) is the most widely-used stealth coating and facilitates particle accumulation in the brain. However, once within the brain, the mode of handling of PEGylated particles by the resident immune cells of the brain itself (the 'microglia') is unknown. This is a critical question as it is well established that microglia avidly sequester nanoparticles, limiting their bioavailability and posing a major translational barrier. If PEGylation can be proved to promote evasion of microglia, then this information will be of high value in developing tailored nanoparticle-based therapies for neurological applications. Here, we have conducted the first comparative study of uptake of PEGylated particles by all the major (immune and non-immune) brain cell types. We prove for the first time that PEGylated nanoparticles evade major brain cell populations - a phenomenon which will enhance extracellular bioavailability. We demonstrate changes in protein coronas around these particles within biological media, and discuss how surface chemistry presentation may affect this process and subsequent cellular interactions.

Endocytotic potential governs magnetic particle loading in dividing neural cells: studying modes of particle inheritance.

AIM: To achieve high and sustained magnetic particle loading in a proliferative and endocytotically active neural transplant population (astrocytes) through tailored magnetite content in polymeric iron oxide particles. MATERIALS & METHODS: MPs of varying magnetite content were applied to primary-derived rat cortical astrocytes ± static/oscillating magnetic fields to assess labeling efficiency and safety. RESULTS: Higher magnetite content particles display high but safe accumulation in astrocytes, with longer-term label retention versus lower/no magnetite content particles. Magnetic fields enhanced loading extent. Dynamic live cell imaging of dividing labeled astrocytes demonstrated that particle distribution into daughter cells is predominantly 'asymmetric'. CONCLUSION: These findings could inform protocols to achieve efficient MP loading into neural transplant cells, with significant implications for post-transplantation tracking/localization.