Characterization of a Novel Aspect of Tissue Scarring Following Experimental Spinal Cord Injury and the Implantation of Bioengineered Type-I Collagen Scaffolds in the Adult Rat: Involvement of Perineurial-like Cells?

Numerous intervention strategies have been developed to promote functional tissue repair following experimental spinal cord injury (SCI), including the bridging of lesion-induced cystic cavities with bioengineered scaffolds. Integration between such implanted scaffolds and the lesioned host spinal cord is critical for supporting regenerative growth, but only moderate-to-low degrees of success have been reported. Light and electron microscopy were employed to better characterise the fibroadhesive scarring process taking place after implantation of a longitudinally microstructured type-I collagen scaffold into unilateral mid-cervical resection injuries of the adult rat spinal cord. At long survival times (10 weeks post-surgery), sheets of tightly packed cells (of uniform morphology) could be seen lining the inner surface of the repaired dura mater of lesion-only control animals, as well as forming a barrier along the implant-host interface of the scaffold-implanted animals. The highly uniform ultrastructural features of these scarring cells and their anatomical continuity with the local, reactive spinal nerve roots strongly suggest their identity to be perineurial-like cells. This novel aspect of the cellular composition of reactive spinal cord tissue highlights the increasingly complex nature of fibroadhesive scarring involved in traumatic injury, and particularly in response to the implantation of bioengineered collagen scaffolds.

Fibroadhesive scarring of grafted collagen scaffolds interferes with implant-host neural tissue integration and bridging in experimental spinal cord injury.

Severe traumatic spinal cord injury (SCI) results in a devastating and permanent loss of function, and is currently an incurable condition. It is generally accepted that future intervention strategies will require combinational approaches, including bioengineered scaffolds, to support axon growth across tissue scarring and cystic cavitation. Previously, we demonstrated that implantation of a microporous type-I collagen scaffold into an experimental model of SCI was capable of supporting functional recovery in the absence of extensive implant-host neural tissue integration. Here, we demonstrate the reactive host cellular responses that may be detrimental to neural tissue integration after implantation of collagen scaffolds into unilateral resection injuries of the adult rat spinal cord. Immunohistochemistry demonstrated scattered fibroblast-like cell infiltration throughout the scaffolds as well as the presence of variable layers of densely packed cells, the fine processes of which extended along the graft-host interface. Few reactive astroglial or regenerating axonal profiles could be seen traversing this layer. Such encapsulation-type behaviour around bioengineered scaffolds impedes the integration of host neural tissues and reduces the intended bridging role of the implant. Characterization of the cellular and molecular mechanisms underpinning this behaviour will be pivotal in the future design of collagen-based bridging scaffolds intended for regenerative medicine.

Cell-enrichment with olfactory ensheathing cells has limited local extra beneficial effects on nerve regeneration supported by the nerve guide Perimaix.

The reconstruction of peripheral nerve injuries is clinically challenging, and today, the autologous nerve transplantation is still considered as the only gold standard remedy for nerve lesions where a direct nerve coaptation is not possible. Nevertheless, the functional merits of many biomaterials have been tested as potential substitutes for the autologous nerve transplant. One of the strategies that have been pursued is the combination of bioengineered nerve guides with cellular enrichment. In this present study, we combined the previously evaluated collagen-based and microstructured nerve guide Perimaix with olfactory ensheathing cell enrichment. Rat sciatic nerve defects of 20 mm were either bridged by a cell-seeded or nonseeded nerve guide or an autologous nerve transplant. Animals were monitored for 12 weeks for structural and functional parameters. Seeded cells survived on Perimaix, and following implantation aligned along the microstructured Perimaix framework. Axonal densities within the cell-seeded nerve guides were higher than in the nonseeded nerve guides and were comparable to the autograft. Additionally, cell-seeding had local beneficial effects on myelination within the nerve guide, as myelin sheath thickness was enhanced when compared with the empty scaffold. Nevertheless, for bridging the nerve gap of 20 mm, both the cell-seeded as well as nonseeded scaffolds were equally efficient regarding the functional outcome, which did not differ between the autograft, seeded or nonseeded groups. Our data demonstrate that olfactory ensheathing cell enrichment has local effects on nerve regeneration in combination with the Perimaix nerve guide. Surprisingly, for traversing the lesion gap, additional cell-seeding is not crucial.

Pre-differentiation of mesenchymal stromal cells in combination with a microstructured nerve guide supports peripheral nerve regeneration in the rat sciatic nerve model.

Many bioartificial nerve guides have been investigated pre-clinically for their nerve regeneration-supporting function, often in comparison to autologous nerve transplantation, which is still regarded as the current clinical gold standard. Enrichment of these scaffolds with cells intended to support axonal regeneration has been explored as a strategy to boost axonal regeneration across these nerve guides Ansselin et al. (1998). In the present study, 20 mm rat sciatic nerve defects were implanted with a cell-seeded microstructured collagen nerve guide (Perimaix) or an autologous nerve graft. Under the influence of seeded, pre-differentiated mesenchymal stromal cells, axons regenerated well into the Perimaix nerve guide. Myelination-related parameters, like myelin sheath thickness, benefitted from an additional seeding with pre-differentiated mesenchymal stromal cells. Furthermore, both the number of retrogradely labelled sensory neurons and the axon density within the implant were elevated in the cell-seeded scaffold group with pre-differentiated mesenchymal stromal cells. However, a pre-differentiation had no influence on functional recovery. An additional cell seeding of the Perimaix nerve guide with mesenchymal stromal cells led to an extent of functional recovery, independent of the differentiation status, similar to autologous nerve transplantation. These findings encourage further investigations on pre-differentiated mesenchymal stromal cells as a cellular support for peripheral nerve regeneration.

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.

Assessing motor outcome and functional recovery following nerve injury.

Behavioral tests significantly contribute to our understanding of nerve function after experimental lesions and/or therapeutic intervention. In particular, the rat sciatic nerve has proven to be a valuable animal model to study nerve injury and repair. Here, we describe how to optimally use the commercially available CatWalk system to obtain a detailed and objective analysis of dynamic and static gait parameters.