Different strategies are being investigated for treatment of spinal cord injuries, one of the most promising being application of neurotrophic factors, which have been shown to prevent neuronal death and stimulate regeneration of injured axons. Ex vivo gene therapy has emerged as the leading delivery method at the site of the injury, and we have shown previously that encapsulating genetically engineered fibroblasts in an immunoprotective alginate capsule can permit implantation of the factor-secreting cells without need for immunosuppression. This strategy could be greatly enhanced by providing the sprouting neurons with a permissive substrate upon which to attach and grow. We report here studies on the modification of an alginate gel surface by either coating it with laminin or by covalent attachment of YIGSR peptide. Using NB2a neuroblastoma cells, we found that native alginate elicited minimal cell attachment ( approximately 1.5%); however, YIGSR-alginate conjugate elicited a fivefold increase in numbers of cells attached using peptide ratios of 0.5 and 1 mg/g alginate, ranging from 9.5% of the cells at the lower ratio, to about 44% at the higher. Only a further 19% increase was obtained at an increased peptide density of 2 mg/g alginate ( approximately 63% over control). Laminin-coated gels showed approximately 60% cell attachment. However, laminin coating did not stimulate differentiation and neurite growth, whereas both numbers and lengths of outgrowths increased with increasing peptide density on peptide-modified alginate. We demonstrate here the ability of the peptide-modified alginate gels to allow adhesion of NB2a neuroblastoma cells and to promote neurite outgrowth from these cells when attached to the peptide-modified alginate surface. Also, we show that the adhesion of NB2a neuroblastoma cells and neurite outgrowth from the attached cells is a function of the peptide density on the gel surface.

Encapsulation of cells has the potential to provide a protective barrier against host immune cell interactions after grafting. Previously we have shown that alginate encapsulated BDNF-producing fibroblasts (Fb/BDNF) survived for one month in culture, made bioactive neurotrophins, survived transplantation into the injured spinal cord in the absence of immune suppression, and provided a permissive environment for host axon growth. We extend these studies by examining the effects of grafting encapsulated Fb/BDNF into a subtotal cervical hemisection on recovery of forelimb and hindlimb function and axonal growth in the absence of immune suppression. Grafting of encapsulated Fb/BDNF resulted in partial recovery of forelimb usage in a test of vertical exploration and of hindlimb function while crossing a horizontal rope. Recovery was significantly greater compared to animals that received unencapsulated Fb/BDNF without immune suppression, but similar to that of immune suppressed animals receiving unencapsulated Fb/BDNF. Immunocytochemical examination revealed neurofilament (RT-97), 5-HT, CGRP and GAP-43 containing axons surrounding encapsulated Fb/BDNF within the injury site, indicating axonal growth. BDA labeling however showed no evidence of regeneration of rubrospinal axons in recipients of encapsulated Fb/BDNF, presumably because the amounts of BDNF available from the encapsulated grafts are substantially less than those provided by the much larger numbers of Fb/BDNF grafted in a gelfoam matrix in the presence of immune suppression. These results suggest that plasticity elicited by the BDNF released from the encapsulated cells contributed to reorganization that led to behavioral recovery in these animals and that the behavioral recovery could proceed in the absence of rubrospinal tract regeneration. Alginate encapsulation is therefore a feasible strategy for delivery of therapeutic products produced by non-autologous engineered fibroblasts and provides an environment suitable for recovery of lost function in the injured spinal cord.

Grafting of genetically modified cells that express therapeutic products is a promising strategy in spinal cord repair. We have previously grafted BDNF-producing fibroblasts (FB/BDNF) into injured spinal cord of adult rats, but survival of these cells requires a strict protocol of immune suppression with cyclosporin A (CsA). To develop a transplantation strategy without the detrimental effects of CsA, we studied the properties of FB/BDNF that were encapsulated in alginate-poly-L-ornithine, which possesses a semipermeable membrane that allows production and diffusion of a therapeutic product while protecting the cells from the host immune system. Our results show that encapsulated FB/BDNF, placed in culture, can survive, secrete bioactive BDNF and continue to grow for at least one month. Furthermore, encapsulated cells that have been stored in liquid nitrogen retain the ability to grow and express the transgene. Encapsulated FB/BDNF survive for at least one month after grafting into an adult rat cervical spinal cord injury site in the absence of immune suppression. Transgene expression decreased within two weeks after grafting but resumed when the cells were harvested and re-cultured, suggesting that soluble factors originating from the host immune response may contribute to the downregulation. In the presence of capsules that contained FB/BDNF, but not cell-free control capsules, there were many axons and dendrites at the grafting site. We conclude that alginate encapsulation of genetically modified cells may be an effective strategy for delivery of therapeutic products to the injured spinal cord and may provide a permissive environment for host axon growth in the absence of immune suppression.

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