1,721,031 research outputs found
Magnetic assistive and hydrogel technology for enhanced survival and function of neurons
Get PDFNeurons are the targets of injury and disease in many neurological conditions, and achieving neuronal survival/repair is a key goal for regenerative medicine. In this context, genetic engineering of neurons offers a platform for (i) basic research to enhance our understanding of neuronal biology in normal, disease/and injury conditions; and (ii) for regenerative medicine to enhance the functionality of neurons. Although, a wide range of attempts have been made to promote gene delivery to primary neurons, these cells are still difficult to genetically engineer, and current methods rely heavily on viral vectors which pose safety considerations. Magnetic nanoparticles (IONPs) are currently of great interest in regenerative medicine including for non-viral gene delivery by the 'magnetofection' strategy, i.e when used with applied magnetic fields. This project aimed to examine (i) the influence of two novel uniaxial and biaxial oscillating magnetic field devices on primary neuronal transfection efficiency, and (ii) examine the safety of magnetofection using histological and electrophysiological studies. In order to do this, a robust protocol to derive primary cortical neurons was first established.A second issue is that surgical delivery of Neurons results in low survival. Additionally, most basic research has relied on neurons grown on 'hard‘ substrates such as plastic, which do not mimic the mechanical properties of the in vivo microenvironment. To address these limitations, primary cortical neurons were grown in a 3-dimensional 'soft' collagen hydrogel construct which can serve both as a protective cell delivery system and a 'neuromimetic' substrate. The safety of the established protocol was evaluated by electrophysiological analyses on neurons. The findings demonstrate that the safety of magnetofection is magnetic field dependent, and at optimal conditions, electrophysiological properties of the nano-engineered neurons were normal. Secondly, I have shown that collagen hydrogels can support the 3D growth of neurons and electrophysiological studies can be carried out on the construct neurons; small differences were found between neurons grown on hard and soft materials. Finally, the amenability of genetic engineering of neurons within hydrogels using IONPs has been shown
Developing neural transplant cell sprays for neurological injuries
Get PDFRegeneration of the central nervous system following penetrating traumatic brain injury (pTBI) represents a significant clinical challenge. Neural cell therapies have the potential to promote regeneration in central neural injury. However, such therapy is currently clinically unavailable. Neural cell transplantation methods have significant drawbacks, notably high cell loss during surgical delivery, so have limitations for pTBI treatment. Spray delivery may have benefits including rapid cell administration in a homogenous distribution whilst being minimally invasive and has not yet been tested for neural cell delivery. The hypothesis is that cell spray technology could be utilised for neural transplantation and that this may offer significant advantages over current administration routes. Additionally, for rapid cell transplantation, no transportation system exists to facilitate remote delivery. Transport media and novel polymer solutions exist for the transport of medical grade tissue however these have not yet been directly tested for neural cell transplant populations. A further hypothesis is that a novel tissue transport or polymer solution could facilitate remote neural cell delivery for transplantation applications. To test these hypotheses, primary rodent mixed glial cultures were used to generate oligodendrocyte pre-cursor cell (OPC) and astrocyte populations which were spray delivered. Additionally, Hibernate-A a CO2 independent nutrient media was assessed for the storage and transportation potential of OPCs and astrocytes at 4oC and room temperature. Cells were assessed for viability and properties which underpin their therapeutic potential. Both cell types could survive, proliferate, differentiate (OPCs), retain cellular markers and retain normal features following spray delivery and on return to standard culture conditions following low temperature storage. AtelerixTM polymer gel transportation system was also evaluated for encapsulation of neural cells demonstrating the potential for mechanical protection during transit for these therapies.These findings suggest neural cell transplant populations could be transported without detrimental effects to remote settings (such as a military hospital) and then rapidly spray delivered to patients with pTBI
Developing tools for inter-site transport of brain tissue models in tissue engineering research
Get PDFPenetrating traumatic brain injury (pTBI) remains one of the leading causes of death in the younger population in the UK, as no clinical regenerative therapies are available. Experimental therapies currently being screened for efficacy are wide ranging and complex (such as biomaterial implantation, nanotherapies and electrostimulation) which require effective multi-disciplinary collaboration between biological and physical scientists, such as engineers, chemists and physicists. Generating complex and pathomimetic models of pTBI requires costly equipment, expert training and access to animal facilities, making them logistically inaccessible to the majority of physical scientists, representing a bottleneck in neurotherapy development.Developing the capacity for inter-site transport of complex brain injury models would significantly ease this logistical barrier. Specifically, complex primary models of pTBI can be transported to sites remote from the site of biological model production for testing, promoting multi-disciplinary collaboration and efficiency in cross-disciplinary research.Our laboratory recently developed two multicellular and in vitro primary cortical models of pTBI (a glial and neuronal model) which offer significant advantages as facile but complex, injury simulating and pathomimetic models of pTBI. The objective of this study was to establish if HibernateTM, a commercially available neural tissue storage medium, could be used safely for storage and transport of these model at room temperature (RT) (removing the need for cold chain transport), without detriment to neural cell viability, maturation or reactivity.Findings indicate there is no effect of HibernateTM storage at RT for four hours on neural cell culture confluency, overall cell viability or proportions of each cell type. Moreover, neurons and oligodendrocytes show no significant decrease in maturation after storage, nor do astrocytes and microglia show any significant increase in reactivity. This indicates transportation of primary neural models is feasible and could facilitate multi-site transport of complex brain tissue models for neuroregenerative research
Delivering nanoengineered neural stem cells within neurosurgical grade biomaterials
Get PDFAchieving neural regeneration after spinal cord injury (SCI) represents a significant challenge. Neural stem cell (NSC) therapy offers replacement of damaged cells and delivery of pro-regenerative factors, but >95% of cells die when transplanted to sites of neural injury. Biomaterial scaffolds provide cellular protective encapsulation to improve cell survival. However, current available scaffolds are overwhelmingly not approved for human use, presenting a major barrier to clinical translation. Surgical biomaterials offer the unique benefit of being FDA-approved for human implantation. Specifically, a neurosurgical grade material, DuraGenTM, used predominantly for human duraplasty has many attractive features of an ideal biomaterial scaffold. Here, we have investigated the use of DuraGenTM as a three-dimensional (3D) cell encapsulation device for potential use in combinatorial, regenerative therapies. To show the feasibility of enhancing the therapeutic potential of this construct, we genetically engineered the NSCs prior to DuraGenTM encapsulation, which could offer the opportunity to increase expression of therapeutic biomolecules at the site of injury. A combination of magnetofection and minicircle technologies were used for genetic engineering of the NSCs.I show that DuraGenTM can support the survival (ca 95% viability at 12 days) and 3D growth of NSCs. Key parameters including maintenance of NSC phenotype, proliferative capacity and differentiation into astroglial lineage cells, neurons and oligodendrocytes were unaffected by DuraGenTM. Furthermore, proof of concept of the capacity of DuraGenTM to maintain a viable genetically engineered NSC transplant population is demonstrated using reporter protein expression which could be detected for up to eight days (latest time point examined) within the construct. The findings support the concept that a ‘combinatorial therapy’, consisting of NSCs engineered to produce therapeutic biomolecules and protected within the DuraGenTM construct, is a promising clinically translatable neuro-regenerative therapy
New in vitro model of traumatic brain injury to assess biomaterial based regenerative strategies
Get PDFPenetrating traumatic brain injury (pTBI) causes significant neural damage and debilitation. The management of pTBI is largely supportive currently, with no clinically established regenerative therapies. Researchers have previously evaluated the regenerative potential of biomaterial constructs called hydrogels in pTBI. To screen biomaterials for regenerative application, clinically predictive models of pTBI are required. However, there is a lack of facile, high throughput, pathomimetic in vitro pTBI models capable of evaluating biomaterial implantation. This thesis aimed to develop methods to i) establish a high throughput and facile culture system containing the major glial cell types, which play an important role in biomaterial handling in the central nervous system ii) introduce reliable and characterizable penetrating lesions into the cultures iii) implant DuraGen PlusTM – an Food and Drug Administration (FDA) approved neurosurgical grade biomaterial into the lesion iv) visualize cell-biomaterial interactions using simple light microscopy v) refine the model to establish a high throughput neuronal model containing all of the neural cell types. The findings of this study show that the key pathological features of injury seen in pTBI can be reliably replicated, in this novel, facile, high throughput, multi-glial model. Specifically, peri-lesional astrocytes have markedly different responses to injury versus distal astrocytes showing hypertrophic palisading astrocytes and glial fibrillary acidic protein (GFAP) upregulation analogous to reactive astrogliosis in vivo. In addition, microglia and oligodendrocyte precursor cells (OPCs) were observed to infiltrate the lesion core similar to processes seen in pTBI models in vivo. Furthermore, DuraGen PlusTM could be implanted into the lesions to visualize cell-biomaterial interactions. Finally, early pilot data shows that use of an alternative chemical medium can further support the growth of neurons, resulting in a model containing all neural cell types in a technically simple and high throughput experimental system
Cell models to evaluate oligodendrocyte lineage cell growth on biomaterials
Get PDFOligodendrocyte lineage and neuronal cells are essential neural populations required for axonal remyelination after spinal cord injury (SCI). Injection of an exogenous cell population into the site of neural injury has posed translational barriers, such as greater than 95% cell death without the use of neural cell protective delivery systems. Additionally, there is a lack of biomaterials approved for human implantation which could act as a neural cell delivery system. Furthermore, majority of scaffolds often have an isotropic structure, and may lack the ability to direct and guide axonal growth and remyelination through the damaged regions in SCI.This thesis demonstrates the growth and maturation of oligodendrocyte lineage cells in the FDA approved, neurosurgical grade, three-dimensional (3D) biomaterial DuraGen PlusTM when co-seeded with astrocytes, which act as a supportive cell population for oligodendrocyte lineage survival. This cellular construct showed potential for use as an implantable scaffold in neural injuries which may result in demyelination. Additionally, encapsulating neuronal enriched cells within DuraGen PlusTM demonstrated that the scaffold enables the maturation of neurons. However, future work is required to improve viability of the cells within the construct. There is scope to produce a pre differentiated neural circuit in the DuraGen PlusTM matrix that can be integrated into lesioned sites in the CNS to promote regeneration. Moreover, oligodendrocyte lineage cells were seeded onto a poly-L-caprolactone (PCL), 3D aligned Cellevate nanomatrix to demonstrate the cell alignment capabilities of the matrix. The findings show that Cellevate was able to sustain the survival and maturation of oligodendrocyte precursors (OPCs) and promoted the alignment of OPCs within the scaffold. It thereby mimicked the in vivo neuro-cytoarchitecture that would aid in guiding axonal remyelination. Oligodendrocyte cell alignment, however, was not as prominent in the matured constructs and hence, future work could focus on further scaffold modifications which may be required to enhance cellular alignment
Developing in vitro models of traumatic injury for neural tissue engineering applications
Get PDFPenetrating traumatic brain injury (pTBI) and spinal cord injury (SCI) cause significant neurological damage and debilitation. The management of these central nervous system (CNS) injuries is largely supportive, with no clinically available regenerative therapies. Regeneration is difficult to achieve because of the inhibitory microenvironment in CNS injury sites. Researchers are constantly developing and testing new therapies to aid regeneration of neural tissue, however, these are heavily reliant on live animal experimentation. There is an urgent need for clinically predictive, in vitro models of neurological injury which satisfy requirements such as: mimicry of complex neural architecture, patho-mimicry, high throughput and facile technical procedures, and being in line with the Reduction, Replacement and Refinement of animal experimentation. Two important approaches for regeneration of damaged CNS tissue are biomaterial application and electrical stimulation (ES) therapy. This thesis aims to develop and evaluate novel in vitro models of CNS injury for evaluation of biomaterial and ES therapy. The thesis goals were to: i) compare and contrast an ex vivo (organotypic) model of SCI and TBI with implantation of a neurosurgical grade scaffold (DuraGenTM); ii) develop a technical method for a 2D and 3D culture model of cortical injury, and iii) establish a platform for electrophysiological studies of the injury environment using multi-electrode arrays (MEAs). Pathological responses such as glial scarring (astrogliosis), microglial activation and neuronal outgrowth were assessed. I provide evidence that these newly developed models replicate key pathological features of injury, and can be reliably used for assessment of regenerative therapies, including neural cell responses to biomaterial implantation, and for interfacing with bioelectronic recording/stimulation systems
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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