Preview

PDF, English Download (48MB) | Terms of use

Abstract

Injury to the adult spinal cord damages ascending and descending spinal fiber tracts thereby disrupting proper information transmission between the brain, spinal cord and periphery of the body. Restoring neural connectivity beyond the site of injury is the essential prerequisite for functional recovery to occur. Without intervention, central nervous system (CNS) axons fail to regenerate, resulting in tremendous impairment of sensorimotor function as well as autonomic dysfunction and, consequently, a significant reduction of the patients’ quality of life. Hence, a massive effort has been spent to develop effective repair strategies for spinal cord injury (SCI) including cell transplantation and biomaterial implantation. However, functional axonal growth past the lesion site remains insufficient due to inappropriate implant integration, detri-mental fibroglial scarring and failure of spinal axons to grow beyond the site of injury. Recently, astrocytes were identified as essential key players for neuroregeneration due to their neuro-protective and supportive functions after CNS injury. Further, immature astrocytes not only fulfil scaffolding functions during development, but might also adapt to the harsh lesion envi-ronment without adopting detrimental phenotypes. Thus, astrocyte are prime candidates to provide structural as well as trophic support for growing axons in combination with biomaterial implants at SCI lesion sites. In the present study, novel alginate-based hydrogel implants with a defined channel micro-structure were combined with cellular grafts of immature astrocytes derived either from the cortex or the spinal cord of neonatal Fischer-344 rats to: (1) provide a physical guidance structure for regrowing axons at the site of injury; and (2) establish a permissive cellular growth substrate within and beyond the hydrogel implant supporting axonal crossing of the lesion cavity of a cervical unilateral hemisection of the spinal cord in adult rats. First, alginate-based hydrogel implants were modified with polypetides to improve their bio-compatibility and cell viability in vitro and in vivo. Afterwards, immature astrocytes from neona-tal rat cortex were cultivated and enriched in vitro. Seeding of alginate-based hydrogel im-plants with immature cortex-derived astrocytes improved axonal regrowth compared to non-seeded hydrogel implants following SCI. The grafted astrocytes interacted with the host as-trocytic network and aligned into tissue bridges structurally guiding axons across the host-graft interface. To elucidate whether astrocytes with a spinal cord identity would elicit superior pro-regenerative effects after SCI, immature astrocytes were isolated from the spinal cord of neonatal rats and compared with cortex-derived astrocytes. Phenotypic characterization re-vealed minor molecular and morphological differences between both astrocyte populations in vitro and in vivo. Particularly, cortex-derived astrocytes were found to have a more mature phenotype compared to spinal cord-derived astrocytes in vitro, however, both cell populations adopted a differentiated morphology and expressed functional molecular astrocytic markers in vivo after transplantation into the intact spinal cord. After SCI, seeded hydrogel implants to-gether with additional caudal grafts of either immature astrocyte population further enhanced axonal growth through the implantation site and promoted revascularization. The grafted cells connected with the host spinal parenchyma facilitating tissue bridging between implant and host. Finally, seeded hydrogel implants in combination with rostral and caudal immature astro-cyte grafts were shown to additionally increase axonal growth through the hydrogel implants after SCI by 70% compared to the previous transplantation paradigms. Thus, the combination of biomaterial implantation with cell transplantation superiorly promotes axonal growth through sites of acute SCI compared to treatment paradigms based only upon biomaterial implants. Moreover, additional grafts of immature astrocytes into the surrounding host tissue improve host-graft interactions by formation of a continuous cellular substrate spanning the SCI lesion site. Nonetheless, axonal re-entry into the distal host spinal cord may require additional trophic attraction.