Tissue engineering with glial cells and a novel biodegradable matrix to promote functional repair following experimental spinal cord injuries

It has been well known for centuries that injuries to the spinal cord usually result in dramatic consequences for the individual concerned, without the prospect for healing or cure. The ancient Egyptians declared that spinal cord injury (SCI) was “a condition not to be treated”. In the late 1800’s to early 1900’s, the Spanish neuroscientist Santiago Ramon y Cajal demonstrated that the consequences of SCI were due to the failure of the central nervous system (CNS) to regenerate. Until the late 1940’s, the outcome of any individual suffering from SCI, and in particular those victims of both world wars, led to the judgement, that “injuries to the spinal cord are essentially a death sentence. If the injury itself didn’t prove fatal, then the complications … became fatal” (C.T. Liverman et. al., 2005). Over the last 30 years, numerous research groups have focused on numerous aspects of acute or chronic spinal cord injury, and a wide variety of intervention strategies have been developed, some of which are currently subject to clinical trials. Within the last years, more and more has been gained showing the potential for axonal regeneration within the nervous system of both experimental animals and humans. While most of the regenerative capacity has been identified in the peripheral nervous system (PNS), it could also be demonstrated that axonal regeneration and compensatory sprouting takes place in the injured CNS. Such findings have led to the development of a number of approaches to support or enhance the regenerative capacity of the PNS and CNS. Within the CNS, the main thrust of these approaches focused either on new surgical methods, novel medications, cell based intervention strategies or, most recently, the application of tissue engineering strategies using artificial matrices. The aim of the present study was based on the two latter approaches, combining growth promoting glial cells and a newly developed, highly orientated growth promoting three dimensional matrix in a tissue engineering strategy to bridge acute spinal cord lesions in adult rats. The matrix used in this study was a prototype in the early stage of development. The investigation therefore focussed on three main aspects: The first part of the thesis addresses the issue of cytocompatibility. Multiple qualities of porcine collagen matrices were tested with a range of neural cell types in order to choose the best quality matrix for subsequent experiments. Inferior quality matrices could be identified and removed from further investigation by the instability of the substrate or by the poor growth of cells. The best matrix supported orientated growth, migration and proliferation of PNS and CNS glia. Furthermore, highly orientated axonal growth will be shown in an in vitro assay using adult rat dorsal root ganglion explants. The second part of the thesis addresses issue of the biocompatibility with adult rat CNS tissues. The matrix was found to be biocompatible for up to 6 months following implantation into the acutely lesioned adult rat spinal cord. The matrix was not rejected in any way. On the contrary, there was a moderate cell infiltration into the matrix, with early and steady vascularization as well as long term axonal regeneration. Differences in the extent of graft-host integration, depending on prior glial seeding of the matrix, were also investigated. The third and final part of the thesis addresses the functional consequences of implanting the matrix into acutely spinal cord injured rats. A clear and statistically significant improvement of food pellet retrieval was demonstrated by the objective “staircase test”. The data are discussed in the context of the latest developments in experimental SCI intervention strategies, in particular, those which employ tissue engineering approaches to attempt to bridge the lesion site

Tissue engineering with glial cells and a novel biodegradable matrix to promote functional repair following experimental spinal cord injuries

It has been well known for centuries that injuries to the spinal cord usually result in dramatic consequences for the individual concerned, without the prospect for healing or cure. The ancient Egyptians declared that spinal cord injury (SCI) was “a condition not to be treated”. In the late 1800’s to early 1900’s, the Spanish neuroscientist Santiago Ramon y Cajal demonstrated that the consequences of SCI were due to the failure of the central nervous system (CNS) to regenerate. Until the late 1940’s, the outcome of any individual suffering from SCI, and in particular those victims of both world wars, led to the judgement, that “injuries to the spinal cord are essentially a death sentence. If the injury itself didn’t prove fatal, then the complications … became fatal” (C.T. Liverman et. al., 2005). Over the last 30 years, numerous research groups have focused on numerous aspects of acute or chronic spinal cord injury, and a wide variety of intervention strategies have been developed, some of which are currently subject to clinical trials. Within the last years, more and more has been gained showing the potential for axonal regeneration within the nervous system of both experimental animals and humans. While most of the regenerative capacity has been identified in the peripheral nervous system (PNS), it could also be demonstrated that axonal regeneration and compensatory sprouting takes place in the injured CNS. Such findings have led to the development of a number of approaches to support or enhance the regenerative capacity of the PNS and CNS. Within the CNS, the main thrust of these approaches focused either on new surgical methods, novel medications, cell based intervention strategies or, most recently, the application of tissue engineering strategies using artificial matrices. The aim of the present study was based on the two latter approaches, combining growth promoting glial cells and a newly developed, highly orientated growth promoting three dimensional matrix in a tissue engineering strategy to bridge acute spinal cord lesions in adult rats. The matrix used in this study was a prototype in the early stage of development. The investigation therefore focussed on three main aspects: The first part of the thesis addresses the issue of cytocompatibility. Multiple qualities of porcine collagen matrices were tested with a range of neural cell types in order to choose the best quality matrix for subsequent experiments. Inferior quality matrices could be identified and removed from further investigation by the instability of the substrate or by the poor growth of cells. The best matrix supported orientated growth, migration and proliferation of PNS and CNS glia. Furthermore, highly orientated axonal growth will be shown in an in vitro assay using adult rat dorsal root ganglion explants. The second part of the thesis addresses issue of the biocompatibility with adult rat CNS tissues. The matrix was found to be biocompatible for up to 6 months following implantation into the acutely lesioned adult rat spinal cord. The matrix was not rejected in any way. On the contrary, there was a moderate cell infiltration into the matrix, with early and steady vascularization as well as long term axonal regeneration. Differences in the extent of graft-host integration, depending on prior glial seeding of the matrix, were also investigated. The third and final part of the thesis addresses the functional consequences of implanting the matrix into acutely spinal cord injured rats. A clear and statistically significant improvement of food pellet retrieval was demonstrated by the objective “staircase test”. The data are discussed in the context of the latest developments in experimental SCI intervention strategies, in particular, those which employ tissue engineering approaches to attempt to bridge the lesion site

Neurite outgrowth promoting effects of enriched and mixed OEC/ONF cultures.

Olfactory ensheathing cell (OEC) transplants stimulate axon regeneration and partial functional recovery after spinal cord injury. However, it remains unclear whether enriched OEC or mixed transplants of OEC and olfactory nerve fibroblasts (ONF) are optimal for stimulating axon regrowth. The neurite outgrowth stimulating effects of enriched OEC, ONF, and mixed OEC/ONF cultures on neonatal cerebral cortical neurons were compared using co-cultures. We show that (1) OEC are more neurite outgrowth promoting than ONF, and (2) ONF do not enhance the neurite outgrowth stimulating effects of OEC in mixed OEC/ONF cultures. Hence, our data indicate that there is no preference for the use of enriched OEC or mixed OEC/ONF cultures with respect to stimulation of neurite growth in vitro

Functional improvement following implantation of a microstructured, type-I collagen scaffold into experimental injuries of the adult rat spinal cord

The formation of cystic cavitation following severe spinal cord injury (SCI) constitutes one of the major barriers to successful axonal regeneration and tissue repair. The development of bioengineered scaffolds that assist in the bridging of such lesion-induced gaps may contribute to the formulation of combination strategies aimed at promoting functional tissue repair. Our previous in vitro investigations have demonstrated the directed axon regeneration and glial migration supporting properties of microstructured collagen scaffold that had been engineered to possess mechanical properties similar to those of spinal cord tissues. Here, the effect of implanting the longitudinally orientated scaffold into unilateral resection injuries (2 mm long) of the mid-cervical lateral funiculus of adult rats has been investigated using behavioural and correlative morphological techniques. The resection injuries caused an immediate and long lasting (up to 12 weeks post injury) deficit of food pellet retrieval by the ipsilateral forepaw. Implantation of the orientated collagen scaffold promoted a significant improvement in pellet retrieval by the ipsilateral forepaw at 6 weeks which continued to improve up to 12 weeks post injury. In contrast, implantation of a non-orientated gelatine scaffold did not result in significant functional improvement. Surprisingly, the improved motor performance was not correlated with the regeneration of lesioned axons through the implanted scaffold. This observation supports the notion that biomaterials may support functional recovery by mechanisms other than simple bridging of the lesion site, such as the local sprouting of injured, or even non-injured fibres