Functional rewiring across spinal injuries via biomimetic nanofiber scaffolds

The regrowth of severed axons is fundamental to reestablish motor control after spinal-cord injury (SCI). Ongoing efforts to promote axonal regeneration after SCI have involved multiple strategies that have been only partially successful. Our study introduces an artificial carbon-nanotube based scaffold that, once implanted in SCI rats, improves motor function recovery. Confocal microscopy analysis plus fiber tracking by magnetic resonance imaging and neurotracer labeling of long-distance corticospinal axons suggest that recovery might be partly attributable to successful crossing of the lesion site by regenerating fibers. Since manipulating SCI microenvironment properties, such as mechanical and electrical ones, may promote biological responses, we propose this artificial scaffold as a prototype to exploit the physics governing spinal regenerative plasticity

Biological response to structured and functionalized substrates for nerve tissue regeneration

After a lesion in the CNS, glial cells play a fundamental role, being the mediators of both the inhibitory and the beneficial response for neural regeneration. The tissue engineering approach consists in the use of biomaterials to help the regeneration and guide the regenerative capable cells to create a permissive environment. The main working hypothesis of this thesis is that we can promote a favourable environment for CNS regeneration identifying material properties which can modulate neuronal cells behaviour. In a first place we analyzed glial and neuronal response to two very different biopolymers, PMMA and chitosan. Wettability, surface and mechanical properties were characterized for both materials. Then line pattern of different dimensions in the micrometrical range were introduced. The response of glial cell and neurons were analyzed in terms of cell adhesion, morphology and differentiation state. Finally, we studied the behaviour of glial cells on glass model surfaces functionalized by self assembling monolayers with different wettability (OH, COOH, NH2, CH3), in order to identify the specific role that wettability plays in determining cell response. The dates suggest that the adhesion, the morphology and the differentiation state of neuron and glial cells can be controlled by choosing the proper combination of material properties and physical patterns. Overall, line patterns resulted to be a suitable tool to use in biomaterial design for nerve regeneration. However, the performance of each material must be analyzed with attention, since the combination of material properties, which most of the time is not predictable, play important roles in the biological activity

The expression and functional analysis of neurite outgrowth inhibitors in the nervous system of Xenopus laevis

Includes bibliographical references (leaves 115-128).Generally, the factors contributing to success or failure of axon regeneration lie in the intrinsic properties of the injured neurons, as well as the surrounding microenvironment of the transected axon. Mammalian neurons may lack the intrinsic ability to survive after trauma, or to re-express genes required for axonal regrowth. Moreover, several proteins inhibitory to neurite growth, such as Tenascin-R (TN-R) and Nogo-A, have been identified in mammals. These proteins are associated with oligodendrocytes and myelin and are considered major inhibitory components of the CNS environment

BIOENGINEERED SCAFFOLDS TO INDUCE ALIGNMENT AND PROMOTE AXON REGENERATION FOLLOWING SPINAL CORD INJURY

Scaffolds delivered to injured spinal cords to stimulate axon connectivity often act as a bridge to stimulate regeneration at the injured area, but current approaches lack the permissiveness, topology and mechanics to mimic host tissue properties. This dissertation focuses on bioengineering scaffolds through the means of altering topology in injectables and tuning mechanics in 3D-printed constructs as potential therapies for spinal cord injury repair. A self-assembling peptide scaffold, RADA-16I, is used due to its established permissiveness to axon growth and ability to support vascularization. Immunohistochemistry assays verify that vascularized peptide scaffolds promote axon infiltration, attenuate inflammation and reduce astrogliosis. Furthermore, magnetically-responsive (MR) RADA-16I injections are patterned along the rostral-caudal direction in both in-vitro and in-vivo conditions. ELISA and histochemical assays validate the efficacy of MR hydrogels to promote and align axon infiltration at the site of injury. In addition to injectable scaffolds, this thesis uses digital light processing (DLP) to mimic the mechanical heterogeneity of the spinal cord caused by white and gray matter, and demonstrate that doing so improves axon infiltration into the scaffold compared to controls exhibiting homogeneous mechanical properties. Taken together, this work contributes to advancing the field of tissue engineering and regenerative medicine by demonstrating the potential of bioengineered scaffolds to repair the damaged spinal cord

Neurogenesis and apoptosis in the developmentally regulated loss of spinal cord regeneration.

Unlike the adult mammal, the chick can successfully regenerate its spinal cord until embryonic day (E) 13. Multiple factors may contribute to the subsequent loss of regenerative capacity, although most research has concentrated on axonal re-growth inhibition as a key issue. The number of viable cells remaining in the spinal cord could also be important and may be affected by cell survival and cell replacement. In this thesis the early response of the chick spinal cord to injury has been investigated, focusing on cell death and the potential to replace lost cells by neurogenesis. Pharmacological reduction of haemorrhage after injury at El5 resulted in reduced apoptosis and cavitation, suggesting that blood-borne factors, such as the serine protease thrombin, may cause apoptosis. Endogenous thrombin expression and activity after injury was investigated. Thrombin was not up-regulated after injury at El5 however, evidence suggests that the activity of other serine proteases was increased. In parallel, in organotypic slice cultures, exogenous thrombin treatment did not increase apoptosis. These results provide new information about the contribution of serine proteases to apoptosis in the chick, suggesting that, although thrombin is not of primary importance, other serine proteases could play a greater role. Next, the contribution of neurogenesis to regeneration at Ell was examined. Changes in the expression and phosphorylation of the early neuronal marker, doublecortin, in response to injury were observed. Although increased proliferation in the grey matter was observed, no increase in neurogenesis after injury was detected. Surprisingly, ongoing neurogenesis was discovered in the normal spinal cord at Ell. These results challenge established views about the timing of neurogenesis in the chick spinal cord and suggest that ongoing proliferation and neurogenesis may contribute to the regenerative capacity at this stage. This thesis presents insights into factors involved in the early response of the chick spinal cord to injury, providing new information about the contribution of neurogenesis and cell survival to regenerative capacity

Investigating the role of ephrin signalling in spinal cord injury.

Spinal cord injury in adult mammals commonly leads to the permanent loss of motor and sensory function in regions of the body below the level of injury. The inability of the central nervous system to regenerate is, in part, due to the presence of growth-inhibitory agents surrounding the lesion site. This thesis presents a previously unreported, inhibitory interaction between ephrinB2 expressed on reactive astrocytes and the EphA4 receptor present on lesioned corticospinal tract axons. This interaction appears to mediate the unusually large retraction of the corticospinal tract away from spinal cord injury sites. An attempt to interfere with this interaction by implanting a cell line secreting the ephrinA5 receptor binding domain is reported. While this approach induced improvements in regenerative sprouting from the corticospinal tract, complications with immune rejection and cell proliferation stopped further investigation. A second intervention using a small peptide with high affinity and specificity for the EphA4 receptor is also reported. Intrathecal infusion of this peptide for 14 or 28 days after injury reversed the retraction of the corticospinal tract and induced improvements in regenerative sprouting from corticospinal and rubrospinal tracts following dorsal or lateral white matter transection injuries. Sprouts were seen to migrate long distances, often to the astrocyte margin of the lesion cavity. Astrocyte behaviour following injury was also altered with the formation of astrocytic 'bridges' into the lesion cavity along which regenerating axons grew. Functional recovery was also enhanced with improvements in the paw reaching assay within 10 days of a unilateral dorsal column lesion with a 30% recovery of function at 28 days post-operation. The simplicity of this intervention and direct translation to human application make it a promising candidate for use in combinatorial approaches to human spinal cord injury treatment

Growth control mechanisms in neuronal regeneration

AbstractNeurons grow during development and extend long axons to make contact with their targets with the help of an intrinsic program of axonal growth as well as a range of extrinsic cues and a permissive milieu. Injury events in adulthood induce some neuron types to revert to a regenerative state in the peripheral nervous system (PNS). Neurons from the central nervous system (CNS), however, reveal a much lower capacity for regenerative growth. A number of intrinsic regeneration-promoting mechanisms have been described, including priming by calcium waves, epigenetic modifications, local mRNA translation, and dynein-driven retrograde transport of transcription factors (TFs) or signaling complexes that lead to TF activation and nuclear translocation. Differences in the availability or recruitment of these mechanisms may partially explain the limited response of CNS neurons to injury