Micro engineered 3D constructs for CNS repair

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

Abstract

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.

Technical Summary

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.
 
Description Our aim was to develop scaffolds that filled with appropriate cells and transplanted into a spinal cord lesion will guide and support functional repair as part of an integrated advanced therapy.

Our ultimate aim was to develop scaffolds that filled with appropriate cells and transplanted into a spinal cord lesion will guide and support functional repair as part of an integrated advanced therapy. Our objective for this grant was to develop a prototype scaffold for future transplantation into rat models of spinal cord injury. To test the supporting structure and material properties of a prototype 3D PCL scaffolds we used mixed neural cells (we term myelinating cultures) that comprise the many cells that make up the CNS and can differentiate as seen in vivo to form internodes of myelin and nodes of Raniver.



During the course of the grant we have:

• Continued to develop the 2D and 3D scaffold development with specific focus on how pores and pillars which contribute to the 3D design influence glial-axonal interactions.

• Optimised surface topography and structure in 2D and 3D, testing other substrates and materials. PCL was optimal coated in PLL.

• Found astrocytes on PCL secreted factors that delayed myelination but did not inhibit it.

• Tubular design: was complex to load with cells but a bioreactor was designed to maintain the cultures. The cell biology of a range of cells was studied on the constructs.

• Examined viability of a range of cells on layered scaffold mimicking the 3D structure of the scaffold and found this can be affected in the centre of the scaffold.. However the inclusion of pores within the construct alleviated the problem.
Exploitation Route The long term aim is to use scaffold for the repair of spinal cord injury in the clinic. The data suggest transplantation of such a scaffold should not affect the biology of regenerating CNS cells and therefore the next stage would be to assess the prototype scaffold in rat models of CNS injury
Sectors Pharmaceuticals and Medical Biotechnology

 
Description To share information about our constructs and ability to support cell survival, growth and differentiation.
First Year Of Impact 2007
Sector Pharmaceuticals and Medical Biotechnology
 
Description Development of polymer scaffolds for use in the repair of spinal cord injury, MRS
Amount £110,000 (GBP)
Organisation Medical Research Scotland 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2014 
End 09/2018
 
Description NC3Rs PhD studentship
Amount £90,000 (GBP)
Organisation National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2012 
End 09/2015
 
Description 16th International Neuroscience Winter Conference, Symposium "New strategies targeting diverse neural cells for regenerative neurobiology" Solden, Austria, 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact sharing information and sparked quesitons

potential collaboration
Year(s) Of Engagement Activity 2014
 
Description American Society for Neurochemistry, Symposium " New model to investigate myelination-remyelination", Long Beach, California, 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact conference workshop which instigated discussion

none
Year(s) Of Engagement Activity 2014
 
Description FENS Featured Regional Meeting 2013, Prague, 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact stimlated questions and discussion

possible new grant writing/collaboration
Year(s) Of Engagement Activity 2013
 
Description ISN-ASN main meeting Stem Cells and Biomaterials for the Treatment of Spinal Cord Injury Cancun. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact discussion and questions

was asked to present at another meeting
Year(s) Of Engagement Activity 2013
 
Description In Vitro Toxicology Society (IVTS). November 2016. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact present a talk on work using cultures
Year(s) Of Engagement Activity 2016
URL http://www.ivts.org.uk/site/
 
Description Invited Speaker for the ISN-ESN Myelin Biology: unraveling functions, mechanisms and therapies satellite meeting in Paris, August 2017. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presented work
Year(s) Of Engagement Activity 2017
URL https://www.neurochemistry.org/biennial-meeting/satellite-meetings.html
 
Description Invited speaker for the annual meeting of the In Vitro Toxicology Society (IVTS). 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact presented various methods to study disease in vitro
Year(s) Of Engagement Activity 2016
 
Description Invited talk Biomatcell international seminar, 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Presented data about bioreactors and scaffolds at Biomatcell international seinar. knowledge exchange

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description pint of science 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Pint of science is organised to allow communications of work to the public
Year(s) Of Engagement Activity 2017
URL https://pintofscience.com/