Axonal regeneration is fundamental for spinal cord repair and functional recovery after spinal cord injury [13
]. The aim of this study was to use a model system to quantitatively compare the regenerative capacity of four polymer types as implants within a spinal cord transection model. Schwann cells have been consistently shown to be one of the most effective therapeutic cell types in transplantation and regeneration after experimental spinal cord injury [23
]. These cells reduce the size of spinal cysts, remyelinate axons [13
] and improve functional recovery in spinal cord injury [51
]. They have also been shown to produce a number of growth factors that initiate and support the growth of axons, and that these cells express cell adhesion molecules on their surface for axon guidance [54
]. The polymers used here included PLGA [10
], PCLF [34
], OPF and OPF+ [12
]. We have shown improved regeneration capacity with schwann cell-seeded PLGA scaffolds over scaffolds seeded with neuronal stem cells [14
]. With regards to OPF and schwann cell viability, tissue culture data also showed that the positively charged hydrogel polymer supported both neuronal outgrowth from dorsal root ganglia explants, improved schwann cell attachments and migration and contributed to neurite myelination [12
]. In the present study, significantly higher axonal counts were seen in PCLF and OPF+ groups compared to the PLGA groups as a control. PLGA scaffolds have been extensively studied by our research group [11
] and were thus used as the benchmark to compare novel polymers such as fumarate composites. The present study is the first to demonstrate the enhanced capacity of OPF+ to support axonal regeneration in vivo
when combined with schwann cell delivery.
A variety of biomaterials have been used for implantation into the injured cord in an attempt to recover neurologic function [57
]. However, few studies have shown a high degree of polymer integration with the surrounding tissue [61
]. The major difficulties were the filling and holding of the injected cells in the lesion cavities [65
]. These cavities or cysts became an important physical obstacle for axonal regeneration, structural rebuilding and functional recovery. Compared to PLGA, OPF, OPF+ and PCLF conduits showed better interaction with the surrounding host tissue, filling the gap and building a bridge across which axons were able to grow into the implants. They also exhibited smoother integration into the host tissue, although we were not able to demonstrate a significant difference in scar volumes compared to the PLGA group. To improve the integration of the implants, we have been developing and modifying our surgical techniques to minimize the injury/trauma induced by the transection/implant on the rat transection model. Such modifications have included making smaller laminectomies, minimizing the incision through dura, implementing quick and complete transection of the cord, rigid fixation and ensuring there is a gap between spinal cord ends that accurately accommodates the scaffold size. Other techniques have included minimizing bleeding, suturing the muscle tightly in order to hold and fix the implanted scaffold in good alignment with the rostral and caudal stumps [43
Rapid progress in study of hydrogel chemistry has produced the materials more suitable for the spinal cord in respect to their mechanical properties. Highly hydrated and soft polymers have similar properties to spinal cord tissue [10
]. Our results show that PLGA and PCLF have higher flexural modulus compared to OPF and OPF+, indicating that they are more resistant to bending. However, at the same loading condition, PCLF has a compression modulus of 8.4 MPa, which is about 20 times higher than PLGA and 44 times higher than fresh spinal cord. OPF and OPF+ contain a large amount of water that decreases both flexural and compression modulus of these materials similar to fresh spinal cord tissue with high porosity and large water content. In our experience, OPF polymers, charged and non-charged, possess the best texture or physical properties compatible with normal spinal cord tissues. PLGA is relatively rigid compared to the soft OPF and OPF+ hydrogels, while PCLF has more flexibility in bending as compared to PLGA. Among the four polymers in the present study, PCLF showed significantly higher compression modulus than other three polymers (PLGA, OPF and OPF+) as well as the fresh spinal cord. This may explain why the volume of cyst in the PCLF polymer group was significantly higher than the volume of cyst in PLGA scaffolds. PCLF scaffolds may lead to more compressive damage of the blood vessels and leave bigger volumes of cyst in the injured area of the transected cord.
Hydrogel scaffolds also have advantages in spinal cord regeneration with a three-dimensional porous structure, their chemical and physical properties, and their diffusion parameters. Biocompatibility also extends to the degree of inflammation the material may cause in vivo
. We have previously investigated the biocompatibility and biodegradation of both OPF and OPF+ in a rat cage model [9
] and did not observe any increased inflammatory response compared to empty cage and PLGA control groups (unpublished data). These materials can therefore provide a scaffold for ingrowth of neural tissue that will subsequently degrade after regeneration. Gradients and surface charge modification for cell adhesion can also be constructed on functionalized synthetic polymers [10
]. The influence of surface charge on cell growth has been investigated and it is now well known that many types of cells adhere better to positively charged surface [66
]. OPF+ polymers may have a better attachment attribute in vivo
so the implanted growth-promoting cells attach to the channel walls. This may facilitate the newly regenerated axons infiltrating into the central part of the channels by providing a contact guidance for the axonal regeneration and allow nerve fibers to extend across the entire channels of the scaffold. In the present study, highest percentage of the centralized infiltrating axons in the channels happened in the OPF+ polymer groups. Moreover, polymers with the centralized pattern showed significant higher number of regenerated axons compared to the dispersed pattern. Another recent study using an in vivo
hemisection model of spinal cord injury assessed the ingrowth of neuronal tissue elements in hydrogels with different charges. Neurofilaments were similarly demonstrated to infiltrate the central part of the hydrogels most plentifully in the copolymers with positive charges [67
In a previous study we developed a synthetic, positively charged hydrogel as a substrate for nerve cell attachment and neurite outgrowth in cultures. Positively charged OPF improved primary sensory rat neuron attachment and differentiation dose-dependently. Positively charged hydrogels also helped to support attachment of dorsal root ganglion (DRG) explants that contain sensory neurons and SCs. Taken together all these findings indicate that charged OPF hydrogels are able to sustain both primary nerve cells and neural supporting cells that are important for neural regeneration [12
]. Others studied the injury and repair of transected sciatic nerves in adult mice by charged guidance channels [68
]. Significantly more myelinated axons has been found in the nerves that regenerated in positively charged channels compared to those in uncharged channels. Although the mechanism for the biological effects of the positive electrical charge is not clear, a defined calcium concentration may be important for optimal neurite outgrowth [69
]. These data indicate that the existence of positive charge on a cell attachment surface is a critical factor in the subsequent behaviors of the cells that anchor on the surfaces. The positively charged OPF hydrogels used in the present study represent a group of promising candidate scaffolds for neural tissue engineering applications.
Spinal cord injury results in the formation of scars and cysts. In the acute phase of spinal cord injury, hemorrhage occurs around the injured sites, leading to a disruption in oxygen and nutrient supply to all cells and tissues in the injured area. The fluid-filled cysts caused by the inflammatory response together with the vascular damage owing to the spinal cord injury can thereafter form and expand progressively at the injured site, and subsequently contribute to cell death and inhibition of axonal regeneration [70
]. Glial scar formation occurs usually in the second phase of spinal cord injury. This scar has been well demonstrated as another major inhibitory barrier to axonal regeneration after spinal cord injury [70
]. Biomaterial scaffolds with salt-leached porous poly (epsilon-caprolactone) have been designed to compare different channel architecture and the influence that the overall design may have in spinal cord transection. Wong and colleagues [74
] demonstrated that open-path designs permitted larger penetration of GFAP-labeled neural tissues from both stumps and resulted in less scarring than the conventional implant designs (no open-path designs). The open-path design, which is very similar to our scaffolds, provided contact guidance and allowed nerve axons to extend across the entire length of the implanted scaffolds [74
]. Scaffolds with optimal designs may impede scarring and subsequent cyst formation. PLGA scaffolds have been demonstrated to prevent scarring and cyst formation in the animal models of SCI [63
]. In this study we also found a smaller volume of cyst in PLGA polymer group than the PCLF polymer group. This would be compatible with our previous observation that minimizing vascular damage reduces cyst formation [76
]. Thus scaffolds with mechanical properties most closely resembling the host tissue produce better outcomes.
The scaffold could serve as a bridge to facilitate to guide the regenerating axons across the injury site and to recover connections with the target of innervation to support functional recovery [54
]. Functional recovery was not seen in any of the animal groups at both 2 and 4 weeks, as assessed by the BBB locomotor scale. We hypothesized that a certain threshold number of regenerated axon numbers would be required to exert a significant and detectable functional recovery after the injury in this time frame. Clearly, not all the counted axons reach and connect to target neurons correctly and functionally, and indeed we observed variablility in the direction of axonal growth that was dependent upon the polymer type. It is also probable that a four week time frame is insuffient to observe functional recovery following complete transection. It is unlikely that regenerated axons have been able to arrive at the correct destination and estalish functional connections. We recently demonstrated by axon tracing studies that regenerated axons through schwann cell loaded PLGA scaffold extended for up to 15 mm beyond the scaffold into the distal cord. However, even after 2 months functional improvement was not observed [13
]. Nevertheless, the negative results in functional assssment are also consistent with our previous studies [14
]. Future studies will be designed to explore approaches to enhance connectivity using optimized scaffolds for time-released delivery of therapeutic agents.