Functional recovery not correlated with axon regeneration through olfactory ensheathing cell-seeded scaffolds in a model of acute spinal cord injury.

The implantation of bioengineered scaffolds into lesion-induced gaps of the spinal cord is a promising strategy for promoting functional tissue repair because it can be combined with other intervention strategies. Our previous investigations showed that functional improvement following the implantation of a longitudinally microstructured collagen scaffold into unilateral mid-cervical spinal cord resection injuries of adult Lewis rats was associated with only poor axon regeneration within the scaffold. In an attempt to improve graft-host integration as well as functional recovery, scaffolds were seeded with highly enriched populations of syngeneic, olfactory bulb-derived ensheathing cells (OECs) prior to implantation into the same lesion model. Regenerating neurofilament-positive axons closely followed the trajectory of the donor OECs, as well as that of the migrating host cells within the scaffold. However, there was only a trend for increased numbers of regenerating axons above that supported by non-seeded scaffolds or in the untreated lesions. Nonetheless, significant functional recovery in skilled forelimb motor function was observed following the implantation of both seeded and non-seeded scaffolds which could not be correlated to the extent of axon regeneration within the scaffold. Mechanisms other than simple bridging of axon regeneration across the lesion must be responsible for the improved motor function.

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 (2mm 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.

Transplantation of human fetal tissue for neurodegenerative diseases: validation of a new protocol for microbiological analysis and bacterial decontamination.

Restorative cell therapy concepts in neurodegenerative diseases are aimed at replacing lost neurons. Despite advances in research on pluripotent stem cells, fetal tissue from routine elective abortions is still regarded as the only safe cell source. Progenitor cells isolated from distinct first-trimester fetal CNS regions have already been used in clinical trials and will be used again in a new multicenter trial funded by the European Union (TRANSEURO). Bacterial contamination of human fetal tissue poses a potential risk of causing infections in the brain of the recipient. Thus, effective methods of microbial decontamination and validation of these methods are required prior to approval of a neurorestorative cell therapy trial. We have developed a protocol consisting of subsequent washing steps at different stages of tissue processing. Efficacy of microbial decontamination was assessed on rat embryonic tissue incubated with high concentrations of defined microbe solutions including representative bacterial and fungal species. Experimental microbial contamination was reduced by several log ranks. Subsequently, we have analyzed the spectrum of microbial contamination and the effect of subsequent washing steps on aborted human fetal tissue; 47.7% of the samples taken during human fetal tissue processing were positive for a microbial contamination, but after washing, no sample exhibited bacterial growth. Our data suggest that human fetal tissue for neural repair can carry microbes of various species, highlighting the need for decontamination procedures. The decontamination protocol described in this report has been shown to be effective as no microbes could be detected at the end of the procedure.

Cytocompatibility of a novel, longitudinally microstructured collagen scaffold intended for nerve tissue repair.

Traumatic injury to the nervous system induces functional deficits as a result of axonal destruction and the formation of scar tissue, cystic cavitation, and physical gaps. Bioengineering bridging materials should ideally act as cell carriers for the implantation of axon growth-promoting glia, as well as supporting integration with host cell types. Here, we describe the cytocompatibility of a novel, micro-structured porcine collagen scaffold containing densely packed and highly orientated channels that, in three-dimensional (3D) tissue culture, supports attachment, proliferation, aligned process extension, and directed migration by populations of glial cells (olfactory nerve ensheathing cells and astrocytes) and orientated axonal growth by neurons (differentiated human SH-SY5Y neuroblastoma cell line). The seeded glia required several weeks to penetrate deeply into the highly porous scaffold, where they adopted an orientated morphology similar to that displayed in simple 2D cultures. The direct interaction between SH-SY5Y-derived nerve fibers and the collagen scaffold also resulted in highly orientated axonal growth. It is likely that biocompatible scaffolds that are capable of promoting glial cell attachment, migration, and highly orientated process outgrowth will be important for future repair strategies for traumatically injured nervous tissues.

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.

The blood supply of the cat's visual cortex and its postnatal development.

We examined the blood supply of the cat's visual cortex using alkaline phosphatase histochemistry to demonstrate the capillary endothelial cells. In the adult, layer 4 is marked by a band that is of obviously greater density, extends throughout areas 17 and 18, and ends abruptly at the 18/19 border. We quantified blood vessel density in area 17, observing a 23% greater density in layer 4 than in supragranular and infragranular layers. This difference reflects a laminar difference in metabolic rate. In three animals studied using the metabolic marker 2-deoxyglucose, layer 4 was 25% denser than the other layers. The band of greater density in layer 4 is not present in newborn kittens, but becomes apparent at about 5 weeks of age. Early in development, the endothelial cells form filopodia as the capillaries grow and branch. The density of blood vessels decreases slightly during the first week of postnatal life, but increases between 1 and 6 weeks of age, so that by 6 weeks, the blood supply of the visual cortex resembles that seen in the adult. This pattern resembles that of cortical metabolism seen with 2-deoxyglucose [J. Cereb. Blood Flow Metab. 11 (1991) 35], but the increase in vascular density precedes that in glucose metabolism.