While advances in technology and medicine have improved both longevity and quality of life in patients living with a spinal cord injury, restoration of full motor function is not often achieved. This is due to the failure of repair and regeneration of neuronal connections in the spinal cord after injury. In this review, the complicated nature of spinal cord injury is described, noting the numerous cellular and molecular events that occur in the central nervous system following a traumatic lesion. In short, postinjury tissue changes create a complex and dynamic environment that is highly inhibitory to the process of neural regeneration. Strategies for repair are outlined with a particular focus on the important role of biomaterials in designing a therapeutic treatment that can overcome this inhibitory environment. The importance of considering the inherent biological response of the central nervous system to both injury and subsequent therapeutic interventions is highlighted as a key consideration for all attempts at improving functional recovery.
“One having a crushed vertebra in his neck; he is unconscious of his two arms (and) his two legs, (and) he is speechless. An ailment not to be treated” [
Current strategies to treat spinal cord injury have focused on restoring function via enhancement of neuronal survival after injury, regeneration of damaged axons, and neuroplasticity of spared axons. Ideally, a single treatment paradigm would be used to accomplish all of these tasks simultaneously. Unfortunately, however, research efforts have thus far demonstrated no single therapy or treatment that will reverse the damage after SCI. Such findings center on the fact that the spinal cord is a unique and complex environment, posing many challenges to the restoration of function. Given that combinations of pharmacologic and rehabilitative therapies may be necessary to address all of these challenges, researchers in this field need to consider the biological implications of each type of therapy in conjunction with the inherent response to spinal cord injury. Therefore, this paper is aimed at providing a comprehensive discussion of the challenges posed by the postinjury response of spinal cord, current strategies aimed at enhancing functional repair, and the potential use of biomaterials in aiding the recovery process.
On average, there are approximately 12,500 newly reported cases of SCI in the United States each year, with a prevalence estimated to be approximately 276,000 persons [
The human spinal cord, on average, is approximately 45 cm long in males and 42-43 cm long in females [
While the anatomic level of the injury determines what regions and appendages of the body are affected, the completeness of the SCI determines the severity of loss in function and sensation. There are two categories into which SCIs can be classified: complete and incomplete. In a complete injury, the spinal cord is severed into two distinct stumps, axotomizing all of the ascending and descending axonal tracts. Incomplete injuries, on the other hand, axotomize some motor and sensory axonal tracts without separating the spinal cord into two distinct sections. The more complete the injury, the more severe the resulting impairment. In a complete lesion, the lack of tissue connectivity between the two cut ends results in physical retraction of the stumps, essentially negating any chance of translesion recovery. The spared rim of white matter in an incomplete lesion, however, holds the spinal cord together and provides a potential bridge for axonal regrowth, thus allowing for the possibility that some limited translesion recovery may occur. Unfortunately, even though a majority of SCI cases are the result of incomplete lesions [
The basic anatomic organization of the spinal cord places the white matter tracts on the outer periphery, making them susceptible to physical trauma [
The postinjury changes that occur in neurons are thought to be a result of a disruption in sustained neurotrophic support. As neurons rely on neurotrophins for survival, any alterations in the availability of such molecules could result in irreparable damage. In general, neurotrophic support can be provided through autocrine or paracrine sources or from axonal connections with a neuronal target [
It is well known that supraspinal neurons lack a strong intrinsic regenerative response following an axotomy in the spinal cord. This class of neurons, which includes the corticospinal tract neurons (CST), vestibulospinal tract (VST) neurons, and rubrospinal tract (RuST) neurons, has, therefore, become the most frequent targets of neural regeneration research. Although the observed lack of a regenerative response fostered the belief that CNS neurons were incapable of undergoing any type of regeneration, some studies have demonstrated the contrary. It has been noted that CNS neurons, particularly those that were axotomized near the neuronal cell body, are able to grow axons within a peripheral nerve graft [
The complex orchestration of molecular changes in the spinal cord following any trauma creates an environment that is well documented as hostile to the regenerative processes [
Immune response following SCI. Trauma to the spinal cord elicits an immune response, which begins almost immediately after injury. Neutrophils are the first immune cells to respond to the lesion site, arriving within the first few hours after injury, and remaining for up to 3 days after injury. Vascular macrophages are the second class of immune cell to arrive at the lesion, arriving after the initial infiltration of neutrophils. Activation and infiltration of vascular macrophages subsequently activate and recruit microglial cells, which can persist in the lesion site for months after injury [
The glial scar has been widely accepted as a primary reason for the lack of a maintained regenerative response following SCI. However recent evidence is beginning to cast a new light on the glial scar as an important protective barrier preventing further secondary tissue damage [
Reactive astrogliosis produces an upregulation of CSPGs in the tissue surrounding the lesion site (reviewed by [
Upregulation and expression of CSPGs. Almost immediately following an SCI, astrocytes located within the area of trauma begin to undergo hypertrophy, synthesizing and secreting CSPGs, including neurocan, phosphacan, and brevican. Additionally, the infiltration of vascular macrophages, activated microglial cells, and OPCs results in the increase in the proteoglycans NG2 and versican. The temporal expression of these proteoglycans is important to factor into any treatment, as they have differential effects on the regenerative process. Neurocan and versican are upregulated quickly following injury, with maximal expression observed 2 weeks after injury. Their expression begins to wane at longer times, approaching base levels by 8 weeks after injury. Brevican is also upregulated after injury, reaching maximal expression 2 weeks after injury. However unlike neurocan and versican, brevican expression remains elevated over time. Phosphacan is initially downregulated following SCI, with significantly reduced levels 1 week after injury. The expression begins to increase at longer times after injury and peaks around 8 weeks after injury. NG2 expression can be generally correlated to the infiltration of vascular macrophages, activated microglia, and OPCs, with maximal expression being found 1 week after injury. This differential expression pattern of CSPGs plays a large role in governing the regenerative response as many CSPGs are inhibitory to both process of atonal regeneration and remyelination (adapted from [
While CSPGs, overall, exert a largely inhibitory influence to the regenerative process, the specific inhibitory nature varies among the different proteoglycans.
Neurocan and phosphacan are also both highly inhibitory to OPC process outgrowth and differentiation [
Overall, the inhibitory influence of CSPGs on the postinjury environment is a major barrier to the regenerative process. Further complicating this matter is the temporal expression of these molecules. While the induction of the CSPG synthesis begins immediately after injury, the upregulation of specific CSPGs happens at different intervals. Brevican, neurocan, and versican expression is found to be maximal at two weeks after injury, while NG2 achieves peak expression one-week after injury (Figure
The glial scar appears to be a paradoxical structure, identified as highly inhibitory to axonal regeneration, while also protecting and isolating the damaged tissue. The dual nature of the glial scar suggests that while it will need to be modified in order to create a permissive environment, the scar is necessary to prevent additional tissue damage. Additionally, the fact that reactive astrogliosis is a response that is graded to the nature of the CNS insult [
Both direct physical destruction and indirect damage due to inflammatory activity result in the death of oligodendrocytes, the myelin producing cell of the CNS. Oligodendrocytes are particularly sensitive to SCI [
Loss of oligodendrocytes creates an excess of myelin breakdown products in the lesion. This myelin debris contains variety of myelin proteins, including Myelin Associated Glycoprotein (MAG), Myelin Oligodendrocyte Glycoprotein (MOG), Nogo-66, and Nogo-A, all of which have been demonstrated to be highly inhibitory to regenerating neurons [
As the macrophages and microglia clear cellular debris, the lesion cavity will eventually become nothing more than a fluid filled cyst called a syrinx [
Given the substantial role that cellular and molecular responses play in the regenerative ability of the spinal cord, it is critical that these aspects are considered when attempting to successfully approach the development and use of methods aimed at neurorestoration following SCI. Therefore, the remainder of review will be devoted to discussion of common research strategies for enhancing repair as well as the potential role of biomaterials in promoting more substantial neurorestorative effects.
Given the complexity of the biological response to SCI, a number of different therapeutic approaches have been developed to target one or more of the issues preventing functional recovery. In general, spinal cord injury research focuses on a few broad topics: neutralization of inhibitory elements within the postinjury environment; promotion of neuronal survival (neuroprotection); stimulation of axonal regeneration and/or plasticity (neuroregeneration); and remyelination of denuded axons. Research in each one of these areas has yielded important insight into the ability for neurorestoration of the functional spinal cord as a result of postinjury environment manipulation.
Neutralization of inhibitory factors in the postinjury environment is one promising approach for enhancing the regenerative response following an SCI. While there are many different inhibitory elements that can be targeted within the postinjury environment, the most progress has been made on developing agents to neutralize the inhibitory influence of either CSPGs or myelin debris.
CSPGs expressed in and around the glial scar are widely accepted as a primary reason for the lack of axonal regeneration and/or remyelination following an SCI. However, the inhibitory nature of the CSPGs can actually be neutralized using the enzyme chondroitinase ABC (cABC). Chondroitinase is an enzyme produced by the bacteria
One of the major limitations of cABC as a treatment for SCI is the mode of administration. Chondroitinase is a very labile enzyme that when reconstituted does not retain its biological activity for very long, due to its thermal instability. When incubated at 37°C, the enzymatic activity of cABC, in solution, is gone by 7–10 days [
While the neutralization of CSPGs has demonstrated promise in reversing their inhibitory effects, a similar result has also been noted via modulation of the activity of the PTP
The myelin debris released into the lesion environment presents additional inhibition to the regenerative ability of the injured axons, which becomes further compounded by the slow phagocytic clearance of the debris [
Significant increases in both the number of axons regenerating and the overall length of the regenerating axons have been found following infusion or other systemic deliveries of the Nogo-A antibody [
Similar effects on axonal regeneration have also been noted following administration of an antibody that is specific to the potent inhibitory domain of Nogo-A, IN-1 [
Taken together, these studies collectively demonstrate that the use of cABC, Nogo-A, or IN-1 to neutralize the inhibitory elements found within the postinjury environment has potential to aid the neuroregenerative response following SCI.
CSPGs interfere with axonal regeneration by inducing collapse of axonal growth cones, producing premature abortion of the normal regenerative response. Axonal collapse is thought to be a result of molecular signaling events activated within the axon itself. Exposure of the damaged axonal tip to the CSPGs and myelin debris found within the lesion results in the activation of inhibitory signaling pathways, such as RhoA/Rock. This then triggers the breakdown of actin filaments and results in the cessation of axon growth [
Microtubule stabilizing anticancer drugs, which achieve their anticancer properties by interfering with cellular division, have recently shown promise in the field of axonal regeneration. Two such drugs are paclitaxel (Taxol) and Epothilone B [
Axonal regeneration may be stimulated after injury through direct modulation of signaling pathways within the axons. While the molecular signaling events that occur within the axon are complex and numerous, there are a few that warrant discussion due to their ability to facilitate axonal regrowth. One such molecular signaling target is Phosphate and Tensin homologue (PTEN), which is a negative regulator of the mammalian target of rapamycin (mTOR). Recent studies have demonstrated that silencing this molecule results in significant axonal growth [
In addition to PTEN, suppressor of cytokine signaling 3 (SOCS3), which is a negative regulator of Janus kinase/signal transducers and activators of transcription (JAK/STAT), has been described as inhibitory to axonal regeneration. For example, conditionally knocking out SOCS3 in mice results in a significant increase in the number of axons that cross a crush injury to the optic nerve [
Finally, another target for axonal regeneration therapies is the Krüppel-like factors (KLF) family of transcription factors. This family of transcription factors plays a large and important role in the regulation of neural growth and regeneration by either suppressing or enhancing axonal growth abilities. Interestingly, KLF family members known to be inhibitory to axonal growth (KLF 4 and 9) have been found to be upregulated postnatally, while those that are growth promoting (KLF 6 and 7) are downregulated at this time [
These studies, when considered collectively, indicate that both strategies that target growth inhibitory signaling elements and therapies that stabilize the growth cone may be necessary in order to achieve the long distance growth needed for functional recovery following SCI.
The inhibitory nature of the postinjury environment is well described, and the physiologic and metabolic stresses experienced by the neuron are extensive. While it is clear that the neutralization of inhibitory elements found within the post-SCI environment has beneficial effects on axonal sprouting/growth, the overall health of neurons following injury still needs to be maintained. If the neuron dies, then any hope of a regenerative response is lost. Therefore, another active research area in SCI regeneration has focused specifically on the neuron, identifying ways to promote neuronal survival, axonal regeneration, and axonal plasticity through the use of neurotrophic (NT) agents and other growth promoting molecules.
Neurotrophic molecules consist of a family of proteins which are structurally similar and bind to one of three tyrosine kinase (Trk) surface receptors or the p75 neurotrophic receptor (
Another family of growth promoting molecules is the glial derived neurotrophic factors, which require two surface receptor components. The GDNF family of molecules directly binds to one of four GDNF family receptor alphas (GFR
The NT and GDNF family of molecules represents only a small sample of the plethora of neurotrophic substances and growth factors that may contribute to the regenerative quality of the CNS. Additional neurotrophic agents, which have also been shown to be potent in enhancing neuronal survival or axonal regeneration, include leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) [
Studies have demonstrated that classes of efferent neurons such as CST, RuST, and coerulospinal and reticulospinal neurons are receptive to the NT agents, BDNF, GDNF, NT-3, and NT-4/5, with varying responses of neuronal survival, axonal sprouting, and even axonal growth [
Overall,
Timing of NT delivery is another critical aspect when it comes to the use of NT agents to promote repair in the lesioned spinal cord. The lesion site and surrounding tissue present a very dynamic environment, with a multitude of events occurring concurrently (Figure
Chronology of postinjury events. The lesion site in an injured spinal cord is also a very dynamic environment that undergoes many different changes, as the lesion changes from an acute injury to a chronic injury. In addition to the inhibitory environment established after SCI, the ever-changing nature of these postinjury events needs to be factored into the design of any therapeutic treatment [
Microarray studies examining the postinjury response of specific classes of CNS neurons have also demonstrated how critical the issue of timing is in regard to the regenerative response. In a study examining the response of short thoracic propriospinal (TPS) neurons to axotomy, a strong upregulation in the genes for the receptors of GDNF and LIF was observed 3 days after injury [
Given the sheer number NT and growth factor receptors that are expressed on neurons and the differential expression of these receptors in efferent and afferent neuron populations, it is likely that different combinations of these NT molecules will be needed in order to elicit full regenerative potential after injury. To this end, studies have shown enhanced regenerative responses in retinal ganglion cells following application of a combination of BDNF, CNTF, fibroblast growth factor (FGF2), and NT-3, as compared with use of each factor independently [
Administering neurotrophic agents after SCI results in an increase in the percentage of neurons spared from atrophy and apoptosis, as well as an enhancement of axonal sprouting or regeneration, when compared to control groups [
An additional avenue that can enhance functional recovery after SCI is the process of remyelination. As previously discussed, the survival of myelin producing oligodendrocytes can be limited by both direct and indirect factors following SCI. While the exact axonal cues that mediate oligodendrocyte survival have not been fully elucidated, both
Remyelination of axons depends upon the health and availability of OPCs. Upon the completion of initial axon myelination, populations of adult OPCs remain throughout the brain and spinal cord. In order for OPCs to be successful in remyelinating axons, they must be able to proliferate, migrate towards the site of demyelination, make contact with an axon, and then mature into myelin forming oligodendrocytes [
The hypothesis that adult axons are no longer capable of myelination has been addressed in a series of different studies. In the adult retina, the nerve fiber layer contains naturally unmyelinated axons, as OPCs are unable to migrate out of the optic nerve and myelinate these axons. However, when OPCs are transplanted into the nerve fiber layer and then the layer is examined 4 weeks after OPC transplantation, axons have been found to undergo myelination [
Interestingly, experimental therapies commonly used to stimulate axonal sprouting and regeneration after SCI have also demonstrated effects on the biology of OPCs. Supplementing the lesion site with various neurotrophic factors such as BDNF, NT-3 [
In addition to NT treatment, another method for promoting remyelination of spared axons is through the use of antibodies to block the protein Leucine Rich Repeat and Ig Domain Containing 1 (LINGO-1). LINGO-1 is highly inhibitory to the myelination process and is selectively expressed in both oligodendrocytes and neurons. The expression of this protein is developmentally controlled, is known to be upregulated following CNS disease or injury, and inhibits the differentiation and maturation of OPCs via the activation of RhoA pathway [
Promotion of remyelination has also been attempted via cellular transplantation. Transplanting cells, such as OPCs [
Remyelination of spared axons is an enticing avenue of research, given that it may explain an apparent disconnect within the reported findings of many axonal regeneration studies, which have demonstrated paradoxical functional recovery without full anatomical regeneration. While most studies can show evidence of increased axonal sprouting into the spinal cord lesion site, very few studies show that these axons grow beyond the lesion [
The complex nature of the spinal cord injury dictates that multiple agents will be needed to maximize repair (reviewed by [
One major limitation of many promising treatment strategies for spinal cord injuries is the method of delivery. Most of the therapeutic agents described above have to be delivered via an injection, series of injections, implantation of a pump or intrathecal catheter, use of a viral vector, or implantation of fibroblasts or other cells genetically engineered to produce a given NT or cABC [
At the present time, there is no agreement on the optimal characteristics for biomaterials used to repair of the damaged spinal cord [
The use of biomaterials in the spinal cord generally falls into one of three classes: guidance channels and scaffolds, hydrogels, and nanoparticles. Each one of these can be produced with different chemical compositions, uses, and biological compatibilities. The bioengineering and design of these materials is a very active research field and have been extensively described in the literature [
Guidance channels have been proposed as far back as the late 1800s, with the thought that demineralized bone tubes could be used to fix nerve gaps [
In the case of peripheral nerve injury, there are several nerve conduits that are FDA approved for the repair of peripheral nerve gaps that are 30 mm or less (reviewed by [
Various forms of scaffolds have been designed to be placed into the spinal cord lesion in order to provide a bridge through the cavitations formed following injury (reviewed by [
Scaffolds and guidance channels can incorporate both growth promoting molecules and a variety of cells that may assist in speeding axon growth through the lesion. The growth factors can be incorporated or attached to the scaffold itself [
One important feature of any scaffold is that it should not elicit a host reaction to the implant. It was noted early that any implant into the spinal cord that was not biodegradable would activate a tissue response that ultimately resulted in the implant being encapsulated in reactive cells and separated from the host tissue [
The advantage of scaffolds is that they bridge an area of the lesion that is inhospitable with axon regeneration. There are numerous studies that can demonstrate enhanced axonal growth and even some motor improvement, when such techniques are utilized in an experimental model of SCI (reviewed by [
Hydrogels are water saturated polymers that can be developed to mimic the three-dimensional physical properties of the host environment (reviewed by [
Hydrogels can be classified into two general categories: natural and synthetic, referring to the origin of the molecules being used. Mammalian ECM-based natural polymers such as collagen, fibronectin, hyaluronic acid, or combinations are often used in hydrogel creations because of their biocompatibility and the fact that they are part of the naturally occurring ECM. Such substances can be used as a cell-delivery vehicle to promote neurite outgrowth while also providing structural support to the regenerating tissues [
Synthetic polymers are being developed which can be optimized for maximal protein, cell binding, and rates of degradation. Some of the most common synthetics used for CNS repair have been developed from poly(hydroxyethyl methacrylate) (pHEMA) and derivatives, poly-ethylene-glycol (PEG)/poly-ethylene oxide (PEO), poly(vinyl alcohol) (PVA), and poly(alpha-hydroxyacids). Being synthetic, these substances have some inherent advantages over the natural molecules. They can be manufactured easily, and the properties of the polymers can be customized, to maximize the desired capabilities. For example, the surface of the polymer gel can be optimized for cell attachment. Control of degradation rate can protect or release cells that are transplanted in the hydrogel, depending on the need and the role of these cells in the repair process. Since they are not derived from animals, the potential for allergic reactions to the hydrogel is also minimal [
While hydrogels show promise as a potential strategy to maximize repair of the spinal cord, there is no obvious polymer or combination of polymers that are optimal for this application. Therefore the identification of such agents is an active field of research. There are concerns that the mechanical strength of the hydrogel is not sufficient to sustain the lesion and that hydrogels have a shorter durability than fabricated scaffolds because they degrade quickly. More importantly, there is no directionality of the microchannels formed after polymerization
An alternative approach to treatment of the damaged spinal cord is the use of nanoparticles, which can be used to administer growth factors, NT, and antagonists to inhibitory substances in the lesion. Nanoparticles and microspheres are polymer derived particles that can degrade over time to release any encapsulated agents. These are being widely tested in a myriad of drug delivery applications in multiple tissues, from alleviating tissue rejections from allografts, to targeting cancer cells [
Nanosphere delivery of growth factors and other agents can be successfully used to treat the spinal cord lesion. Drug delivery after an SCI is difficult because of the loss of vascularization and the instability of some of the more promising agents. Chondroitinase ABC is one such agent: it can degrade the glycan residues attached to CSPGs that inhibit axonal growth, neutralizing the effects of these CSPGs. However, it is highly unstable in solution, losing most of its activity within days [
Nanoparticles and nanospheres that deliver GDNF, BDNF, and NT3 are being developed for several applications in the CNS, including SCI (reviewed by [
Nanosphere delivery of therapeutic molecules is attractive for treatment of SCI for many reasons: they are minimally invasive and provide sustained local drug release, which results in a higher dose locally without systemic side effects. Moreover, administration of multiple agents of growth factors could be accomplished by an injection of a mix of nanospheres containing growth factors, cABC, and Nogo antagonists. However, there are many questions that need to be answered prior to use of nanoparticles in a clinical setting. Some of the more critical questions concern the release rates and dose of the agents released from nanoparticles. For example, how much of a particular agent will be required before therapeutic effects occur? Moreover, how long will such agents need to be released into the post-lesion site? cABC and methylprednisolone may be needed acutely, but NT and other molecules may be needed at later stages following injury. A formulation that is released for months has not yet been manufactured in any experimental condition. Nanoparticles could be included in hydrogels to extend release times at the lesion site, if needed [
Spinal cord injuries are complex and difficult to repair. Research efforts thus far have characterized many molecular events that occur at the injury site, allowing for the identification of several avenues for therapeutic intervention. In short, interventions aimed at promoting functional recovery following spinal cord injury may be targeted towards the neutralization of inhibitory proteoglycans, support of neuronal survival, and stimulation of axonal regeneration and remyelination. The clinical consensus is that there is no single therapeutic agent that can effectively address these issues and that maximal restoration of motor function will most likely be achieved with a mix of agents, each optimized to target a specific aspect of the postinjury response. The other significant problem that persists in developing treatments for SCI relates to the delivery of therapeutic agents to the spinal cord at the appropriate time to facilitate repair. Axonal regeneration is a slow process which, depending on the size of the lesion, could take many months or years in humans. Unfortunately, at present, little is known about how long various therapeutic agents will be needed, their biological half-life and bioactivity
The use of biomaterials provides a promising avenue for addressing the above-mentioned concerns regarding spinal cord repair. Such materials can be utilized not only to deliver therapeutic agents but also to provide physical support for the damaged tissue. Additionally, both natural and synthetic polymers can be used to fabricate several types of structures that can release therapeutic agents with customizable release kinetics. While extensive literature on scaffolds, hydrogels, and nanoparticles exists, there is currently no consensus as to which material is most optimal in repairing the lesioned spinal cord. Ultimately, as new biological requirements of damaged spinal tissue are discovered, the development of biomaterials specialized for the treatment of SCI will need to consider how each of these requirements plays a role in the natural injury response process and potential for functional recovery.
All contributing authors have no conflict of interests.