The objective of this study was to design a novel method of sustained delivery of potentially therapeutic agents to the transected spinal cord. This was accomplished by incorporating dbcAMP-encapsulating PLGA microspheres in an OPF hydrogel, which was then molded into the cylindrical shape of the spinal cord. Subsequent transplantation of this microsphere-embedded scaffold facilitated study of the biological effects of dbcAMP in the presence of no implanted cells, SCs, or MSCs within the injured spinal cord. We report biological effects as a result of this dbcAMP delivery in vitro and in vivo and propose that this delivery platform may be suitable for testing the effects of other potentially therapeutic agents on SCI.
This method of embedding microspheres within the scaffold architecture represents an attractive approach to microsphere delivery within the transected spinal cord. An alternative approach would have been to deliver microspheres within the channels.56
However, this approach introduces a number of complications such as (1) potential obstruction of regeneration and (2) inability to utilize the channels for cell delivery. Another limiting factor is channel size. Smaller diameter channels have been deemed optimal for spinal regeneration,59
which limits the maximum size of microspheres that could potentially be loaded within the channel itself. Our delivery method enables release of the therapeutic agent from the microsphere-embedded scaffold, without occupying the channel spaces designed for regenerating axons and cell transplantation. Further, we demonstrate that microspheres embedded within the OPF hydrogel degrade within the pores they originally occupied, confirming that they do not degrade or swell into the channels themselves.
The in vitro
studies served as a platform to establish release and biological activity of dbcAMP. We confirm that the ability to control the release of compounds using microsphere technology remains unaffected by incorporation into a hydrogel substrate. Encapsulation of dbcAMP within 50:50 PLGA microspheres lead to a more prolonged release of the compound compared with direct injection into OPF in vitro
. Increases in intracellular cAMP concentrations have been shown to enhance neurite outgrowth in PC12 cells.61–64
We demonstrate that exposure of PC12 cells to the dbcAMP-encapsulating microspheres resulted in neurite outgrowth, thereby confirming that functionality was unaffected by the encapsulation process.
Generation of dbcAMP-releasing scaffolds was achieved by injecting the PLGA microsphere–OPF hydrogel solution into the glass cylindrical mold and inserting seven parallel wires. We were initially concerned that the size of the scaffold needed for implantation into the spinal cord would limit the volume of dbcAMP microspheres, which could be incorporated, thereby reducing any potential effects. However, we showed that low concentrations of dbcAMP (0.5
nmol) could promote the same extent of neurite outgrowth as higher concentrations (128
nmol). This data coupled with the scaffold release data instilled confidence that our delivery system would provide enough dbcAMP to produce biological effects in an in vivo
Re-establishing motor functionality below the site of an SCI requires that axons regenerate through the site of injury and form functional synapses with their targets. Assessment of this regeneration through the implanted scaffold was performed by staining for the neuron-specific neurofilament. Transversely sectioned axon profiles could then be counted using previously validated methods.58,59
The rationale for elevating cAMP at the site of injury stemmed from previous research demonstrating its effectiveness in promoting extension of axons to their peripheral targets.51–55,65
Despite these previous findings, our results pointed toward a dbcAMP-induced inhibition of axonal regeneration. This was significant when one compared the regenerative potential of SCs versus SCs + dbcAMP. SCs have consistently been shown to enhance axonal regeneration within the peripheral and central nervous system.14,66,67
However, when coupled with sustained delivery of dbcAMP, this regenerative potential was lost. Axonal counts for animals receiving SCs + dbcAMP were comparable with those observed in the control group (which showed a base-line level of endogenous axonal regeneration) and were significantly lower than those animals receiving SCs alone.
Contrary to these observations, in the presence of MSCs, dbcAMP was shown to rescue MSC-induced inhibition of axonal regeneration. The reason for these conflicting results is unclear. dbcAMP may be exerting varying effects on the SCs and MSCs, thereby altering their regenerative potential. Previous studies indicated that exposure of SCs to cAMP elevators induced expression of galactocerebroside and other molecules characteristic of mature SCs68–72
and resulted in differential expression of extracellular matrix genes.73
We speculate that the regenerative potential of SCs in the injured CNS requires maintenance of the cells in a dedifferentiated state. In the case of MSCs, cAMP elevators have been shown to stimulate neuron-like morphology.74,75
However, these morphological changes were not consistent with a real process of transdifferentiation into mature functional neurons.76,77
Indeed, specific application of dbcAMP to MSCs has been shown to have cytotoxic effects.76
These effects could potentially be involved in negating inhibition of axonal regeneration in the presence of dbcAMP.
To establish if differing effects of dbcAMP in the presence of SCs versus MSCs were due to histopathological variations, we assessed the volume of scar and cyst formation in the interface rostral and caudal to the implanted scaffold. Interestingly, we found that in the presence of SCs and MSCs, dbcAMP showed a trend toward reduced cyst and scar formation (when compared with those groups receiving SCs or MSCs alone). This is contradictory to the findings of Fouad et al.
, who reported deleterious effects of continuous cAMP delivery in the injured CNS.78
These researchers delivered cAMP analogs via osmotic minipumps for up to 4 weeks in two models of incomplete SCI. They reported an absence of progressive locomotor recovery, sporadic micro-hemorrhage formation and cavitation, enhanced macrophage infiltration, and tissue damage at regions beyond the immediate application site. These conflicting findings may be attributable to application of lower levels of dbcAMP, or to the use of a transection SCI model compared with an incomplete SCI model.
Disruption of the vasculature can exacerbate damage following SCI, thereby limiting restoration of function.79–81
Restoring oxygen and glucose supply to cells in the vicinity of the injury requires neovascularization of the lesioned area. To assess if increased axonal regeneration is associated with increased capillary formation and with differences in the effects of dbcAMP in the presence of SCs versus MSCs, von Willebrand factor staining of endothelial cells was performed on the transversely sectioned scaffold. SCs and MSCs were shown to significantly increase the number of capillaries formed compared with the control group. The greatest level of capillary formation occurred in the group receiving MSCs. This is not surprising given previous findings demonstrating the angiogenic differentiation potential of MSCs in vitro
and in other injury models.82–85
In this study, we suggest that the lack of co-localization of the transplanted GFP-MSCs with the von Willebrand factor–stained endothelial cells points toward a promotion of angiogenesis by MSCs rather than the differentiation of MSCs themselves into endothelial cells. Indeed, we have previously demonstrated that MSCs remain in an undifferentiated state following delivery to the injured spinal cord.57
It is also worth noting that a perivascular origin for MSCs has been hypothesized.86
The rationale for this hypothesis comes from a very elegant study by Crisan et al.
Cell sorting for pericytes and subsequent expansion showed that these cells were multipotent for osteogenic, chondrogenic, adipogenic, and myogenic lineages, all hallmarks of MSCs.87
In our study, the presence of dbcAMP significantly decreased capillary number in the group receiving MSCs, suggesting an alteration of MSC angiogenic potential resulting from sustained dbcAMP delivery. This result was surprising to us. One would have expected to see increased angiogenesis in the animals that received both MSCs + dbcAMP, given that MSC inhibition of axonal regeneration is negated in the presence of dbcAMP. This, however, was not the case. We speculate that the presence of dbcAMP results in decreased functionality of MSCs (i.e., reduced inhibition of axonal regeneration and reduced angiogenesis).
The MSCs + dbcAMP group also showed a significant improvement in motor function when compared with all other groups at 4 weeks. However, SCs were shown to promote the greatest level of axonal regeneration, which was not coupled with such functional improvements. This difference in functionality between the groups may be attributable to (1) the generation of more functionally mature neurons; (2) successful synapsing of regenerating axons with their targets; or (3) reduced tissue destruction (as was observed in the presence of dbcAMP).
Our findings raise a number of questions with regard to the mechanisms involved in (1) dbcAMP-mediated inhibition of axonal regeneration in the presence of SCs; (2) dbcAMP-mediated rescue of MSC-induced inhibition of axonal regeneration; (3) dbcAMP-mediated reduction of capillary formation in the presence of MSCs; and (4) functional improvements brought about by MSCs + dbcAMP delivery. We aim to address these questions in future studies.