Building a bridge towards guided neuronal regeneration in spinal cord repair
Design: Integrating current knowledge into building the bridge
1. Bridge Design
As mentioned, the bridge that will allow axonal growth from proximal to distal region will require an "on-ramp" (where the regenerating axons can interface with the bridge), the bridge (which will direct the axonal regeneration), and the "off-ramp" (where the axons will exit, reattach with the distal stump, and reinnervate). Also as mentioned, many difficulties emerged in the development of the "off-ramp", due to incongruities between the Schwann cell and/or PNS glial cell type bridge construction, and the CNS environment of the region of the distal stump. Hence, the design of the "off-ramp" is of critical importance. An overall schematic of the bridge is shown below.
2. Axon Growth in the bridge
a. Axonal growth can be related both to surface adhesion and cellular signaling; thus the bridge must cater to both components to stimulate directional axonal growth. Several studies have discerned a variety of unfavorable conditions for axonal growth including level of adhesion (too high or too low) and type of matrix or cellular material. [10] Further studies have more critically characterized the connection between signaling and growth, because of the variation in control of growth via adhesion. [11]
b. Because of the precedence of the growth cone in axonal growth, a major function of cell adhesion molecules to promote axonal growth is to promote growth-cone signaling. [12] Thus, the bridge should incorporate such molecules, including laminin-derived IKVAV peptide and tenascin-C derived VFDNFVLK peptide, or the desired carbohydrates and peptides can be synthetically attached to desired matrix or cell molecules and incorporated within the bridge.
c. Other molecules have the property of limiting the growth of oligodendrocyte progenitor and meningeal cells which would intefere with axonal growth. N-CAM adhesion molecules mediate interactions between astrocytes (glial cells of the CNS) and neurons [13]. L1 and L1-Fc adhesion molecules promote neuronal growth and do not promote astrocyte/fibroblast growth. [14] The bridge should consider such a distribution of such molecules as well.
d. Because axonal growth is also associated with the expression of various genes, the bridge should include molecules (i.e. timed release) which promote associated gene expression. There are a variety of neurotrophins, chemokines, trophic factors, and soluble factors involved in transcriptional and/or posttranscriptional modifications towards gene expression, which should be incorporated. [15]
e. Similar to axonal regeneration in the PNS, directed axonal regeneration should consider directed growth along a fiber or inside a tube, with promoting growth factors that direct the growth cone. In the PNS, axons regenerate along the neural tube with contact areas both on the inner surface of the neural tube and the Schwann cells inside the tube. [12] Axonal growth is quite ordered. An ordered system either of multiple parallel fibers and/or multiple channel tubes should restrict the pathway and provide the best direction. Initial trial studies have considered some optimal conditions (10μm on a planar surface or 20-50μm in a small diameter fiber) [16] ; however, more characteristic studies will have to be conducted.
Extensive research has been conducted in the axonal responses to various neurotrophic factors—some of this is summarized below. The bridge design will incorporate a variety of these neurotrophic factors in an intelligent design.
3. Material of the Bridge
Polymeric materials incite glial scar formation, which directly inhibits axonal growth. [17] Polymeric materials may also induce irritation, toxicity, and/or an immune response. Various hydrophobic structures have been noted to prevent attachment of competing meningeal cells. [18] Clearly the ideal material would have both favorable structural, immuno, and flex properties; furthermore, the best material should also be biodegradable, in conjunction with the time for nerve regeneration and possibly reinnervation.
Various biodegradable polymers have been utilized in cell attachment and neuronal growth applications, including poly(l-lactic acid) and poly(glycolic acid). [19] Coating these polymers with matrix molecules such as laminin further increases cellular attachment. [20] These polymers can be chemically cross-linked with desired molecules that will interact with cell-surface receptors or will release into the growth area and influence the growth process. 
Natural polymers such as collagen and fibronectin should be considered for their structural characteristics. Polypeptide polymers should also be considered and can furthermore be engineered to promote self-assembly. These polymers are immunogenic and may require further chemical derivatization. Poly(l-lactic acid) has a favorable, physiological-like degradation as compared to poly(glycolic acid) as shown beside.
4. On-Ramp
The bridge must contain factors and must facilitate axonal growth towards it. Therefore, post-injury, the axons must grow past some of the gliar scar tissue before entering the bridge material. Schwann cells, used in PNS neural growth post-injury applications, would work well facilitating axonal growth. A number of secretions including netrin-1, laminin, tenascin, and a variety of proteoglycans, have demonstrated chemoattractant properties in axonal growth.[21] A number of these factors should be provided that will allow the axons to grow beyond the glial scar. Timing post injury is also important, because scar tissue will remain in the reforming process, inhibiting axonal growth.
5.Off-Ramp
As previously mentioned, one of major problems in axonal growth in the CNS is the uninviting astrocyte region for growth. Olfactory sheathing cells have been demonstrated to permit migration of axons, and should be incorporated in the construction of the off-ramp. The sharp boundary from the permissive bridge graft to the glial scar inhibits further axonal growth; hence the off-ramp should either promote a gradual transition to the uninviting region or somehow turn off the mechanism in growth cones as they cross this boundary. This should be addressed with factors that can reduce the glial scar formation, such as suramin [22], chondroitin sulfate proteoglycans [23], and/or long charged glycosaminoglycans [GAG] chains [24]. Also, eliminating the growth of competing cells, such as oligodendrocytes as previously mentioned, can also stimulate axonal growth in the inhibitory area.
6. Bridge Position
It may be necessary to modify the bridge to provide for individual pathways for motor and sensory nerve growth and these individual growth pathways can be controlled by the respective soluble factors The general fitting of the bridge will require accurate fitting near the proximal and distal stumps and the area surrounding the bridge should be clear of scarring so new scarring can form around the bridge. The bridge should be made of porous material, such as the hydrogel examples [25], for good vascularization.
7. Functional Recovery
Stimulation is an important key in reinnervation. For example, locomotor training on humans with various degrees of SCI found restoration of some motor behavior. [26] In addition, as previous mentioned, electrical stimulation has found success in reinnervation. In all cases, successful axonal regeneration will require complete myelination, and treatments to promote myelination such as oligodendrocyte progenitor cells [27] should be considered.