We showed that tissue engineering with a biodegradable polymer scaffold loaded with SCs supports regeneration that can be reliably quantified. This scaffold provides a platform in which the contributions of extracellular architecture, donor cells, and microenvironment may be separately manipulated to assess their contributions to successful regeneration. The donor cells re-created a peripheral nerve microenvironment within the channels of scaffold, with typical extracellular basal lamina and collagen fibrils. In addition, we showed that retrograde labeling with FB is a reproducible and quantitative method for measuring regeneration after spinal cord injury.
FB is a diamidino dye that is taken up by cells, is transported over long distances, and remains in the cytoplasm. Axons, dendrites, and neuron cell bodies are rendered visible when the dye is sufficiently concentrated. After the dye is injected, it diffuses away from the injection site through the extracellular space. Varying volumes of dye (0.1–1.0
μL) have been injected in spinal cord injury studies (Xu et al., 1995
; Russo and Conte, 1996
; Asada et al., 1998
; Puigdellivol-Sanchez et al., 2002
; Takami et al., 2002a
). To our knowledge, this is the first study to examine the range of distribution of dye label at different times after injection and with different volumes of injection. This information is important when interpreting the results of dye tracing as a marker of regeneration in spinal cord injury. A critical question is the distance traveled by axons that have regenerated through an environment that is permissive for axonal growth and then entered a nonpermissive environment.
The observed relationship between time and volume of dye injection decreases the concern that dye might have diffused long distances before being taken up by axons. The label within the halo appeared to be in all cells, confirming that the dye is taken up by cells if it is present in the extracellular space. With these findings, and previous observations that 7 days is required to allow retrograde transport to the most distant cell bodies, we concluded that injection of 0.6
μL and tissue harvest after 7 days would provide reliable data and allow us to account for volume of diffusion in the interpretation of our findings.
Under positive control conditions (nontransected animals), neurons were labeled around the injection site (P1), in the cord (P2-P4), and in the pons, midbrain, deep subcortical nuclei, and frontal cortex (). Under negative control conditions (insertion of scaffold containing BMP but no cells), no labeling was seen rostral to the scaffold. This showed that BMP did not support regeneration and that dye did not leak out of the cord and falsely label rostral cells. The uniform number of cells labeled in P1 (~6,000 cells) indicated uniform injection in all of the groups.
Longitudinal sections showed FB labeling of axons growing throughout the scaffold in transected spinal cords, but labeled axons were seen only within segments close to the injection site (P1, P2, or within the scaffold). In these adjacent areas, punctate extracellular labeling was seen in close proximity to the cell bodies (). This was never observed in more distant segments (P3, P4, and brain), probably because label was concentrated enough in cells to visualize axon terminals on dendritic spines. Cells visualized in controls had the typical morphology of the motor system: long projecting neurons in the rostral cord (P3, P4), pons, deep subcortical nuclei, basal ganglia, and frontal cortex (). In future studies, it will be interesting to more specifically identify labeled neurons above the craniocervical junction using more detailed anatomical and immunohistochemical techniques. In this study, no neurons were labeled above this level in the transected animals.
After spinal cord transection and scaffold implantation, neurons were labeled in the rostral cord. When dye was injected close to the caudal interface of the scaffold and cord (0-mm group, and ), 736 neurons (P2
P4) were labeled after 1 month compared with 46,418 labeled neurons (P2
P4) in positive controls (). This indicates that 1.6% of neurons projected axons into and through the scaffold. The axons may originate from several different types of neurons. They might be coming from motor system interneurons that projected axons to the caudal cord before transection. This would represent true regeneration. They might originate as collaterals from neurons that were close to the transection site but did not previously project to the caudal cord. Finally, the axons might originate from ventral horn cells that previously projected into roots. After injury, and under the influence of SC in the scaffold, they projected into the scaffold. Each of the latter possibilities, although aberrant, might result in return of function below the lesion. Although the findings demonstrate that a small number of axons grew through the scaffold into the distal cord, we do not have evidence that they reached an appropriate target and established functional connections.
The close linear relationship between number of axons labeled and actual injection distance at 1 and 2 months () suggests a stochastic process in which a few axons are able to penetrate into the distal cord. The flattening of the linear relationship between number of axons labeled and actual injection distance and the fewer labeled neurons after 2 months suggest that regeneration in this model occurs mostly within a narrow time window after transection injury or that axons regenerating into the distal cord may not have made successful connections and may have retracted or degenerated. This would be similar to the phenomenon of “pruning” in the regenerating peripheral nerve (Brushart et al., 1998
When dye was injected rostral to the scaffold, the numbers of labeled neurons in P3 and P4 () were very close to those in control P3 and P4 segments after 1 month (), which confirms the consistency of injection. The number of trans-scaffold-labeled neurons 1 month after rostral injection (P2
P1, ) was close to the number of neurons labeled in the reverse direction (P2
P4, ) at the same time point.
The use of cell-loaded, biodegradable polymer scaffolds is one approach to bridging the gap after injury of the spinal cord (Xu et al., 1999
; Oudega et al., 2001
; Friedman et al., 2002
; Teng et al., 2002
). Microengineered polymer structures potentially can provide guidance, organized cell architecture, and soluble growth or guidance factors impregnated into the polymer matrix. Biologically active molecules may be covalently attached to polymer surfaces, and computer-guided stereolithography can construct intricate architecture (Friedman et al., 2002
). We used PLGA because it is approved by the U.S. Food and Drug Administration, is readily molded into a simple 7-channel scaffold, and supports regeneration in the nervous system (de Ruiter et al., 2007
). Novel polymers derived from synthetic and biological sources are emerging rapidly (Friedman et al., 2002
). This is the first detailed study of the use of PLGA in a spinal cord scaffold. Our results confirm that the material is compatible with regeneration and differentiation in the spinal cord.
SCs express axonal growth supporting cell adhesion molecules on their surface (Daniloff et al., 1986
; Bixby et al., 1988
; Kleitman et al., 1988
; Martini and Schachner, 1988
) and produce axonal growth–promoting substrates such as laminin (Cornbrooks et al., 1983
; Bozyczko and Horwitz, 1986
; Ard et al., 1987
) and fibronectin (Cornbrooks et al., 1983
; Bozyczko and Horwitz, 1986
). Almost three decades ago, SCs were shown to support CNS regeneration (Richardson et al., 1980
). This observation has been confirmed by many others (Baron-Van Evercooren, 1994
; Raisman, 1997
; Bunge and Pearse, 2003
; Oudega et al., 2005
; Oudega and Xu, 2006
). In the present study, SCs were introduced in the scaffold, but we cannot exclude the possibility that endogenous SCs from proximal spinal roots migrated into the injured spinal cord (Bresnahan, 1978
; Bunge et al., 1994
; Takami et al., 2002a
Although SCs can support CNS regeneration, they may not be practical for use with a clinical approach. A major disadvantage is that they create peripheral nerve architecture within the CNS. Electron microscopy showed that individual SCs ensheathed axons and generated typical peripheral nerve myelin surrounded by a basal lamina () (Windebank et al., 1985
). In the more compact CNS architecture, 1 oligodendrocyte provides myelin for multiple axons and there is no basal lamina. In addition, SCs generated an extensive extracellular collagen matrix (). The basal lamina and extracellular collagen matrix provide flexible mechanical strength that protect the peripheral nervous system (Podratz et al., 2001
). For the spinal cord, which has very limited mechanical strength, the spinal column bone provides protection and allows much closer packing of neural cell elements. We estimate that if the spinal cord had the architecture of the sciatic nerve it would have a diameter of 8.8
mm. This reiterates the importance of finding other cells such as stem cells or olfactory ensheathing glia to support regeneration in the spinal cord.
The construct studied here promoted limited regeneration. We showed that long-distance regeneration can occur from CNS neurons that project through a scaffold construct into distal tissue. We have no evidence of functional reconnection, and the number of successfully regenerating axons was small. However, this study serves as an initial step toward reliably quantifying the effects of architecture, cell type, and growth factors on regeneration, from which future studies can be built.