Micro engineered 3D constructs for CNS repair

Spinal cord injury often leads to a paralysis of parts of the body (i.e. the late actor Chris Reeves 'Superman') - which can be in most severe cases affect all four limbs, and in others just the bladder, the legs and lower parts of the body etc. This is due to the direct or indirect damage to the nerve fibres (known as axons) running from the brain along the spinal cord, and those carrying sensory information from the periphery back to the brain. After the injury the area damaged will fill with non-functional scar tissue. Unfortunately there is very little recovery after injury and no restoration of function - even over many years. The biological aspects of spinal cord repair after injury is a complex problem that is not completely understood and is actively being investigated. Leading neurosurgeons agree that future treatment envisaged for the repair of spinal cord injury will be a combination of a cellular transplant with pharmacological intervention. Popular candidates for transplantation are support cells or glia (astroglia, olfactory ensheathing cells) that normally envelope and guide regenerating nerve processes. It is hoped that these cells will aid axonal regeneration across the graft through the site of the scar into normal CNS tissue. As the scar environment is inhibitory to axonal outgrowth due to the presence of many inhibitory molecules (a molecule involved in this is even called nogo!) intervention with drugs is necessary to either overcome inhibitory signals or to remove the inhibitory molecules Our recent data on cellular transplantation using olfactory glia has shown that they can support the ingrowth of many axons, although very few if any appear to exit the graft. Anatomical examination of the grafts gives an impression that the axons are wrapped by olfactory glia but there is no alignment of these axons or order within the graft. It is clear that CNS repair is a complex process and a single treatment like glial cell transplantation is not sufficient for restoring spinal cord function. Within this grant we intend to develop a scaffold based guidance system for axonal outgrowth. The envisaged scaffold will be a polymer with internal tiny (ca. 1/2 hair diameter wide) guidance tubes filled with transplanted glia. On the inside of these tubes we place even smaller local guidance structures. This scaffold will guide the axons and at the same time protect them from the regeneration limiting scar environment. The principles of fabrication developed for the computer industry allow us to design flat sheet of hard material (silicon) with very fine detail. In order to create 3-dimensional scaffolds we are creating polymer replicates of these structures and then roll the structured sheet up using a small device akin to a cigarette-roller. We are therefore able to make such scaffolds with very high accuracy and repeatability. By using biodegradable polymers for the scaffold the device can be left within the body slowly dissolving and being replaced with the bodies own material, all the while instructing the nerve extensions. As it is not beforehand obvious how the nerve helper cells and the extending axons interact within such an artificial environment we will investigate the cellular response in molecular detail and use the information gained to inform the construct design in a constant dialog. One example of the structural features modified, which are expected to have a significant effect on cellular survival and orientation, is the size number and distribution of perforations / which are needed to allow nutrients to enter the tube. We will also investigate which cellular transplant is best used in combination with our microstructured implant. We hope to have at the end a device and selected a cell type that together could enter in vivo testing of spinal cord injury.

Spinal cord injuries results in paralysis and a loss of sensation as communication between the brain and neurons below the injury is interrupted. Repair and regeneration of axons in the mammalian CNS does not occur mainly due to the formation of an inhibitory scar around the site of injury. However, a growing number of reports suggest that this situation is not inevitable and that axotomised fibres in the adult spinal cord can be encouraged to regenerate by appropriate interventions. It is envisaged that this will include a combination of cellular transplantation with pharmacological treatment. Recent data has shown that olfactory ensheathing cells can promote the ingrowth of many axons although very few if any appear to exit the graft. It is clear that CNS repair is a complex process and a single treatment like glial cell transplantation is not sufficient for restoring spinal cord function. Within this grant we intend to develop a scaffold based guidance system for axonal outgrowth and present the axons to the interface of graft and normal tissue. Our previous data have demonstrated that microtopography can act through an overlaid astrocyte layer and lead to aligned neurites generated from embryonic spinal cord cultures in long-term culture and that these can be myelinated by endogenous oligodendrocytes. The polymer scaffolds will be microengineered to promote porosity, cell survival, and axonal guidance and filled with transplanted glia. This scaffold will guide the axons and at the same time protect them from the regeneration limiting scar environment. Semiconductor fabrication technology allows us to prepare masters with high detail and repeatability. In order to create 3D scaffolds, polymer replicates of these structures are made and rolled into tubes. Biodegradable polymers will be used for the scaffold which slowly degrades during the repair process. At the end of the grant we hope to have a device that could enter in vivo testing of spinal cord injury.