Introduction
The Spinal Cord Injury (SCI) Subgroup's primary goal is to understand the changes that occur in the spinal cord, adjacent tissue and across the human body in response to spinal cord injury, with a focus on developing a therapy that can overcome factors inhibiting recovery. In our Human practices page, we have detailed how we have contacted and learned from Researchers, Doctors and Experts in multiple fields related to regenerative medicine and SCI. This was to understand the short-term and long-term impacts of spinal cord injuries in a patient’s motor and sensory functions, mental health, and quality of life. Through the development of this understanding we have been able to transform initial ideas into concrete therapy plans, which function in unison to create a novel SCI therapy.
Our goals are to ensure that our therapy is efficient, accessible and most importantly safe for patients, end users and the environment.
As described in our Project Design, our therapy is composed of three main parts:
The ideas for our project were chosen and designed after having gained a clear understanding of what is happening following a SCI and how it affects the human body. It is therefore important to understand the mechanisms underlying a SCI, in order to understand the impact of our project and its applications.
Structure and Function of the Spinal Cord
The spinal cord is made up of four different regions, rostral to caudal: the cervical, thoracic, lumbar and sacral regions. These different regions can be visually distinguished from one another and are related to different motor functions. When considering spinal cord injuries, the location at which they occur have different consequences for those affected.
Figure 1: The structure and functions of the spinal cord. From rostral to caudal, the spinal cord can be divided into 4 sections: cervical, thoracic, lumbar and sacral. Each section with its innervating nerves control different bodily functions. The cervical region controls the diaphragm (breathing), head and arm movements. The thoracic region controls the trunk and abdominals. The lumbar region is responsible for hip and leg movements. The sacral region is responsible for sexual and bowel functions.
In Phase I of our project, we largely focused on developing therapeutic strategies to aid recovery from cervical spinal cord lesions as these patients typically constitute the largest demographic of SCI patients around the world, presenting severe motor deficits. While the distribution differs slightly from country to country, a 2019 paper reported that, of the 12,500 new cases of SCI in North America each year, 50% affect the cervical region (more specifically, C5), 35% afflict the thoracic region, and 11% in the lumbar region. The remaining 4% fall under the sacral vertebrae (Alizadeh, Dyck and Karimi-Abdolrezaee, 2019).
Furthermore, studies report that cervical injury patients are willing to spend 2–3 months being less independent, while recovering from surgery to improve motor function (Anderson, Fridén and Lieber, 2008). This year, after heeding the advice from experts and stakeholders we have modified our therapy to be implemented in more specific regions of the spinal cord.
Type of Injuries Considerations
Independently of where injuries happen these can be complete, affecting all motor function below the injury site, or incomplete, allowing for some amount of motor function and sensation. Assessing the extent of the injury is extremely important as it acts as a frame of reference for improvement in motor and sensory functions and helps to set realistic recovery goals. The American Spinal Injury Association (ASIA) impairment scale or AIS is used for the assessment of the functional impairments as a result of SCI and is important when considering surgical and rehabilitation protocols and the success of regenerative therapies. Further information about how the AIS scale is structured can be found in the Project Design page.
Complete lesions are blanketed under AIS A whereas incomplete lesions consist of AIS B, AIS C, and AIS D. The final category of the AIS scale is AIS E, where patients retain normal motor and sensory function. At this stage of development in our treatment, we would like to focus on AIS A patients who have no motor or sensory function (Roberts, Leonard and Cepela, 2017). The classification of spinal cord injuries in regards to the presence of residual function was given additional attention this year leading to our decision to only extend our treatment to AIS A patients as our preliminary target population concurring with the advice given by Dr. Jerry Silver in regards to possible surgical damages to patients with remaining motor functions.
In order to guarantee the safety of our therapy we looked at which motor functions are affected as a consequence of injuries at different levels of the spinal cord and how resecting the glial scar or operating at these levels might affect the patient. The cervical spinal cord is home to the motor phrenic nerve pool (C5 and above) which forms an essential constituent of respiratory function (Alilain et al., 2011) as well as input into vocal and hand function from C5 - C7 (Waxenbaum et al., 2021). Further potential damage - secondary to surgery - to structures at higher vertebrae could lead to grave consequences, the most severe of which is death. Neurosurgeon Mr. Gordan Grahovac also advised us of the difficulties with operating on higher cervical regions of the spinal cord and suggested shifting our focus to target injuries at lower locations to reduce possible complications during surgery. Alternatively, thoracic spinal cord injuries, occurring lower down in the cord, have less severe potential outcomes (Alizadeh, Dyck and Karimi-Abdolrezaee, 2019) and more optimistic outlooks when injured (Nas et al., 2015). However, this does limit our population-to-treat pool and would hinder our capabilities in proving the extent to which our therapeutic approach can enhance recovery.
In consideration of the aforementioned issues raised by Dr Jerry Silver and Mr. Gordan Grahovac, we have decided to implant our scaffold in injuries at the thoracic and lower cervical (C6 - C7) levels. We have also decided to implement our therapy for AIS A patients exclusively following Mr Gordon Grahovac’s explanation of possible consequences during surgery in patients with incomplete lesions, so as to not negatively affect any residual respiratory and motor function that the patient might possess. Nevertheless, we hope to expand the reach of our therapeutic strategy to lumbar and sacral spinal cord injuries, alongside those in other AIS classes further down the line, once the the potential of our therapy has been assessed in vivo.
Phases of SCI:
Physical damage to the spinal cord initiates a cascade of physiological events both at the injury site and surrounding spinal cord tissue. Initial damage to the spinal cord - the primary injury, results in a plethora of events categorised as a secondary injury which can be temporally divided into acute, sub-acute, and chronic phases.
The acute phase begins immediately following SCI and is characterised by internal bleeding, cytokine and chemokine release, ionic imbalance, neurotransmitter accumulation (excitotoxicity), free radical formation, calcium influx, edema formation, inflammation, and cell necrosis (Alizadeh, Dyck and Karimi-Abdolrezaee, 2019).
In the sub-acute phase of injury, the blood-brain barrier (BBB) is disrupted and microglia are activated. This is followed by an up regulation of proliferated vascular fibroblasts and inhibitory extracellular matrix (ECM) components such as transforming growth factor β and interleukins. Finally, inflammatory cytokines increase proteoglycan production. This sequence of events results in the induction of scarring, forming the glial scar (Silver and Miller, 2004).
In the chronic phase, astrocytes and OPCs of the lesion penumbra mediate ECM remodeling via the upregulation of CSPGs by interacting with CSPG receptors on approaching axons, causes growth cone dystrophy of approaching axons and chronic regeneration failure (Tran, Warren and Silver, 2021).
We’ve gone over the various characteristics of the acute, sub-acute, and chronic SCI phases accessible and now need to put these phases in the context of our therapeutic strategy. We’ve the following general figures for the beginning and end of each phase: the acute phase generally lasts up to 2 days post-injury, with the sub-acute phase typically continuing from the 2-day point up until 4 weeks post-injury when the chronic phase begins (Fehlings and Hawryluk, 2010).
Introducing the Glial Scar
The structure of the mature glial scar has multiple constituents, with fibroblasts at the center of the lesion secreting collagens, fibronectin and laminin. Surrounding this center is a thick boundary of astrocytes and microglia. At the lesion border, astrocytes and NG2 oligodendrocyte progenitor cells (OPCs) that produce chondroitin sulfate proteoglycans (CSPGs) are responsible for inhibiting axonal regrowth (Tran, Warren and Silver, 2021). Macrophages, namely the pro-inflammatory M1 subtype, are entrapped in the glial scar lesion core by the astrocytes and microglia: this greatly up-regulates inflammation, but prevents the inflammatory response from spreading away from the lesion (Tran, Warren and Silver, 2021).
This glial scar has a dual role, both protecting tissue and inhibiting axonal regeneration. It stabilises the fragile CNS tissue post-injury by repairing the BBB: reactive astrocytes divide instantaneously and surround the lesion core, limiting the inflammatory response and cellular degeneration. However, the cellular constituents in the glial scar alongside the remodelled extracellular matrix (ECM) strongly limit axonal regrowth. In the acute phase after injury, upon physical contact with the injured axonal tip, infiltrating macrophages mediate axonal retraction by releasing proteases (Tran, Warren and Silver, 2021).
Figure 2: The inhibitory environment of an SCI lesion posed by the glial scar. The glial scar and its cellular constituents contribute to the inhibition of axonal regeneration. In the acute to subacute phase, upon disruption of the BBB, infiltrating macrophages and the ones trapped in the glial scar come into physical contact with approaching axons and mediate axonal die-back. The scar core consists of collagen secreting fibroblasts which contribute to the hardness of the scar. Surrounding the core, astrocytes secrete CSPGs which interact with their receptors PTP sigma (PTPσ) on axonal end bulbs and the ECM to mediate axonal retraction, as well as sequester growth factors. On the scar border, OPCs synapse with approaching axons to cause axonal entrapment. Figure inspired from Tran, Warren and Silver, 2021
Resecting the glial scar
We are resecting the glial scar during the scaffold implementation surgery to limit the inhibitory environment in the SCI since our therapy aims to provide stability in the damaged environment and increase neuroplasticity and regeneration. Surgical Protocols explaining this removal process can be found in our proposed implementation and they discuss how this process would be done to ensure it is the most efficient and as safe as possible. The decision to remove the glial scar was mostly based on the fact that CSPGs are highly upregulated in this scar and we have found several other publications which corroborate our decision.
A 2018 study reported that chronic glial scars have less of a balance between neuroprotective and inhibitory effects, leaning more towards inhibition. They mention research carried out by Mr. Dai Jianwu and colleagues who looked at proteomic analyses of subacute and chronic scar tissues which showed a larger volume of inhibitory factors at 8 weeks post-SCI versus 2 weeks post-SCI. Removal of the glial scar at ~5 weeks also displayed encouraging nerve fibre regeneration (Li et al., 2018) which links to an additional study where it was found that recovery plateaus and inhibitory mechanisms are enhanced at 4 weeks after an SCI (Hu et al., 2010). Hence, this acts as a major obstacle for the axonal regeneration that we are attempting to achieve through our therapy (Li et al., 2018).
In terms of potential negative effects, there were 5 surgeries from 3 hospitals including Affiliated Hospital of Logistics University of CAPF, The First Affiliated Hospital of Soochow University, and First Affiliated Hospital of PLA General Hospital. These surgeries were carried out on complete injury (AIS A) patients, where in their glial scars were resected and NeuroRegen scaffold implanted; these surgeries were followed up in 2016 (Xiao et al., 2016). It was reported that glial scar resection did not result in any deterioration on the ASIA Impairment Scale grade nor sensory or motor function. There was also no neurological decline among the 5 patients, indicating that glial scar resection has little to no adverse effects following correct surgical and post-surgical protocols (Xiao et al., 2016).
Regeneration Mechanisms After Injury
Post-SCI, injured axons retract and fail to regenerate, thus not forming functional connections with target neurons beyond the injury site, preventing functional recovery (Rodemer, Gallo and Selzer, 2020). As aforementioned, CSPGs in the ECM of a SCI site inhibit axonal regrowth through sequestering growth factors and entrapping axons.
The growth cone at the tip of the axon is a sensory-motility structure with a specialized cytoskeleton and protein distributions that contribute to axonal outgrowth. These cytoskeletons consist of actin, microtubules (MT), and neurofilaments. Actin and MT are polar and highly dynamic, playing important roles in elongating and guiding axonal and neurite outgrowth. They are composed of monomers and polymerize at their positive ends (Miller and Suter, 2018).
After SCI, the CNS transitions from being capable of regenerating axons to failing to regrow them due to the impact of intrinsic and extrinsic factors (Rodemer, Gallo and Selzer, 2020). Pre-SCI, actin filaments polymerize to guide axonal outgrowth, elaborating filopodia and lamellipodial veils in the peripheral region of the growth cones (Miller and Suter, 2018). Post-SCI, the imbalance of factors in the microenvironment of the lesion, including the upregulation of cofilin and the inhibition of myosin II, drive actin-based structures within axons, causing axons to extend on CSPGs which leads to axonal dystrophy and/or collapse (Rodemer, Gallo and Selzer, 2020).
MT polymerization from tubulin monomers at the plus end is the primary contributor to axonal elongation, while microtubule motor protein transportation regulates the rate of axonal elongation. For instance, inhibition of the motor protein kinesin 5 encourages axons to extend onto CSPGs, inhibiting axonal regeneration (Rodemer, Gallo and Selzer, 2020).
Understanding Axonal Inhibition
As mentioned before, CSPGs are one of the major inhibitory molecules in the microenvironment of spinal cord injury lesions. They have a core protein with unbranched carbohydrate chains known as glycosaminoglycan (GAG) chains. Varying numbers of these chains are attached to the core protein by O-linkage to serine residues. Chondroitin sulfate is not the only type of sulfated GAG chain: there is also dermatan sulfate (DS), heparan sulfate (HS) and keratan sulfate (KS). All of these types have slightly different functions, but broadly inhibit axonal regrowth following SCI, except HS (Crespo et al., 2007).
Highly sulfated chains trap more growth factors and are therefore more inhibitory to axonal regrowth. Hyalectans are a major family of CSPGs in the CNS and can bind to hyaluronic acid (HA) in the ECM by their N-terminal domain. Their carboxyl terminal domain can bind to the GAG chains of other CSPGs, creating complexes of CSPG-HA meshes in the ECM, which are all linked together by tenascins. This contributes to the dense inhibitory environment at the lesion site (Crespo et al., 2007).
Hyaluronan is a GAG chain expressed on cell surfaces. It remains at cell surfaces either bound to specific cell surface receptors such as CD44 or still associated with hyaluronan synthase as it is being synthesized (Crespo et al., 2007). Hyaluronan can be bound by the N terminal domain of CSPG core proteins and tenascin to form perineuronal nets (PNNs) that surround neuronal cell bodies and dendrites. Hyaluronan is the most important component of PNNs: when they are removed the PNNs are absent (Galtrey and Fawcett, 2007).
PNNs have multiple putative roles in the healthy adult CNS after their formation following the critical period in the development of the CNS which ends in humans at approximately 8 years old. Their main role is encasing synapses to increase their stability and prevent new synapses from disrupting the existing connections. In this way, PNNs act to reduce plasticity following the closure of the critical period (Fawcett, Oohashi and Pizzorusso, 2019). This reduced plasticity prevents functional recovery, so targeting PNNs with ChABC will encourage the formation of new synaptic connections.
The sulfated GAG chains of CSPGs are present in PNNs as well and, in general, CSPGs function by this mechanism of binding growth factors but also by interacting with PTP sigma (PTPσ) receptors on axonal end bulbs. Releasing these factors by digesting the GAG chains increases the plasticity of the CNS and creates a more permissive environment for axonal regrowth in spinal cord injury (Fawcett, Oohashi and Pizzorusso, 2019). More information about how CSPGs act to create a growth inhibitory environment after SCI can be found in the CSPG guide created for further understanding of the actions of this molecule.
PTP Sigma Interactions
Chronically, CSPGs cause growth cone dystrophy of approaching axons, as aforementioned. The interacting behaviours of such CSPGs with PTPσ (CSPGs/PTPσ) is determined by the sulfation pattern of their chains, wherein having a wide range of different proteoglycans with different sulfation patterns ensures complex interactions within the ECM. When PTPσ binds with heparan sulfate proteoglycans (HSPGs), PTPσ-mediated protease secretion may degrade surrounding ECM including CSPGs, permitting axonal outgrowth. When PTPσ is bound by CSPGs, the complex disrupts autophagy, a critical homeostatic process in which unwanted proteins are engulfed by autolysosomes and degraded by lysosomal enzymes (Tran, Warren and Silver, 2020). You can find more information about the mechanism by which this disruption happens by clicking on the cross above Figure 3 below.
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Figure 3: Axonal growth cone interaction with CSPGs. On the left side, the binding of PTPσ of the growth cone with HSPGs in the ECM causes axonal outgrowth.
Our therapy focuses on degrading CSPGs in the SCI injury to create a more permissive environment to guide axonal elongation and promote functional recovery. On the right side, when PTPσ binds CS-E which is highly up-regulated post-SCI, axonal retraction occurs. CS-E/PTPσ monomerizes PTPσ, disrupting autophagosome-lysosome fusion, and traps approaching axons by causing them to synapse with OPCs in the ECM. Figure adapted from Tran, Warren and Silver, 2021.
It is also worth mentioning that MFP adheres to GAG side chains in the ECM, however, these are the chains that ChABC cleaves. Upon discussion with Professor James Fawcett and further literature research, we found that ChABC digestion of the GAG side chains of CSPGs leaves behind a residual ‘stub’ (Crespo et al., 2007). Professor Fawcett advised us that these stubs are identical in terms of components and sulfation pattern to the original intact chain, however, they are just one heterodimer. Stubs are a substitute for the intact chains in terms of MFP adherence and they also have minimal contribution to the inhibitory microenvironment, therefore the implementation of ChABC in our therapy will not affect the adhesive properties of our MFP and will still reduce growth inhibition.
Last year our team researched the parts necessary to allow for axonal conduction and regeneration after a spinal cord injury. They understood the effects of the glial scar and CSPGs in impeding neural regeneration/axonal regrowth and set up to find the last piece of our therapy that would create a more promoting environment to increase the success of our therapy. They, therefore, focused on protease ChABC as a possible part of our holistic therapy to target the cellular and molecular growth inhibition present after a SCI.
ChABC
ChABC is a bacterial enzyme that digests the GAG chains of CSPGs to encourage plasticity and axonal regeneration in the CNS (Bradbury and Carter, 2011). With the aim of providing a novel and holistic treatment for SCI, based on last year and this year’s literature review, we have decided to implement microinjections of ChABC during our scaffold implantation surgery, and a second round of injections after scaffold implantation, the timing of which will be determined by the axonal regrowth scores from imaging data. ChABC microinjections will increase the permissiveness of the SCI environment for axonal regeneration, regenerated axons would grow across the scaffold and make significant connections with target host neurons on the other side. This will encourage functional recovery in the sensory, motor and autonomic facets of nervous system functions, including sensation detection, locomotion, sexual and bowel functional improvement.
Mechanism of Action of ChABC
As described in our Project Design, ChABC is composed of two enzymes that work together to degrade chondroitin sulfate: an endolyase and an exolyase. The endolyase depolymerizes the chondroitin sulfate GAG chains and the exolyase degrades the resulting tetra- and hexasaccharides into disaccharides. There are other types of chondroitinases, namely AC-I, AC-II and B, that target different GAG chains or sulfation patterns. ChABC is the best choice as it cleaves all types and has been highly characterized in contrast to the other types (Crespo et al., 2007).
There is evidence for 4 different mechanisms by which ChABC affects the CNS. ChABC promotes neurite outgrowth in the glial scar seemingly because it allows access to growth-promoting sites on the laminin (Crespo et al., 2007). It also promotes regrowth by releasing the factors bound to the GAG chains of CSPGs. Growth-promoting and neurotrophic factors can then provide their positive effect and bound inhibitory factors can diffuse away from the lesion and decrease inhibition (Galtrey and Fawcett, 2007). ChABC degradation of CSPGs produces chondroitin-6-sulfate as a product which has been found to promote neurite outgrowth of some neurons and induce microglial cells to take on a neuroprotective role. Finally, the digestion of hyaluronan by ChABC produces oligosaccharides and smaller chains of hyaluronan that induce the expression of cytokines and macrophages that are pro-inflammatory. So this digestion starts a reparatory process that allows axonal sprouting and functional recovery (Crespo et al., 2007).
Figure 4: The therapeutic effect of ChABC treatment. Left: M1 macrophages creates an inflammatory microenvironment, while inhibitory constituents of the glial scar trap growth promoting neurotrophic factors, both cause approaching axons to retract. Right: upon digestion of CSPGs by ChABC, M1 macrophages transform into pro-inflammatory M2 macrophages, neurotrophic factors are released. Together, creating a permissive environment for axonal sprouting. Figure adapted from Bradbury and Carter, 2011.
PTPσ as mentioned before is a CSPG receptor on axons. The mRNA expression of PTPσ elevates dramatically in dystrophic stabilized growth cones, inhibiting axonal growth via CSPGs/PTPσ interaction (Hu et al., 2021). Alongside digesting CSPGs, ChABC was found to reduce the expression of PTPσ mRNA. This sustained effect, together with the decrease of caspase (a family of cysteine proteases involved in apoptosis) activity in the axons, ChABC reduces retrograde neuronal apoptosis signaling, preventing axonal dieback and promoting axonal regeneration (Hu et al., 2021).
Thermostabilising ChABC
Following a literature review of the advantages of ChABC and its effectiveness in increasing plasticity and promoting axonal sprouting in the SCI microenvironment (Lee, McKeon and Bellamkonda, 2009), we contacted researchers working with ChABC to discuss its enzymatic activity in the human body and establish a delivery method that would be compatible with our scaffold. Based on our discussions with Dr Bradbury and Dr Hettiaratchi it was clear that a long-term delivery of ChABC was of primary interest; in vivo studies in rat models indicated greater functional improvements after prolonged release of this enzyme, with significantly improved grasping capacities after 8 weeks of delivery (Burnside et al., 2018). However, ChABC is not stable for prolonged periods at body temperatures and is thus unsuitable for long-term delivery within the human body as shown by the decline in its enzymatic activity at temperatures between 37 and 39 degrees (Tester, Plaas and Howland, 2007). In an attempt to circumvent this issue, Dr Bradbury developed a method of viral ChABC delivery, accompanied by a kill-switch, allowing constant release within the SCI environment (Burnside et al., 2018). Dr Hettiaratchi on the other hand focused on mutating the ChABC enzyme in order to make it more thermostable for a prolonged enzymatic activity at the lesion site and thus a wider window of plasticity after SCI (Hettiaratchi et al., 2019).
There are many different species of bacteria that contain ChABC, with varying amino acid sequences. In order to find the best sequence to use for our therapy in terms of stability and efficacy, a Multiple Sequence Alignment (MSA) was carried out on 250 different sequences containing the ChABC lyase active site. From this MSA, a platonic sequence of the active site was determined, i.e. the most common residues at conserved sites were written out to create an ‘ideal’ sequence to compare to individual sequences. Proteins that most closely match this platonic sequence will be the most naturally stable protein sequences. The PDB codes of the two sequences with the closest match to the platonic sequence are 1HN0 and 2Q1F. Therefore, these two sequences became the best candidates for use in our therapy.
Only 4 of the 250 sequences have been experimentally reviewed, according to Uniprot, including 1HN0 and 2Q1F. These 4 were therefore put through another MSA to exclude the non-reviewed sequences, and their functional domains identified using Uniprot. It was discovered that 1HN0 has one additional functional domain than 2Q1F: the Pfam code for which is PF02884. Upon review, PF02884 was found to be a lyase C-terminal domain. A structural comparison of the C-terminal ends of the protein sequences for 1HN0 and 2Q1F showed the additional domain clearly. An additional lyase domain means that 1HN0 has a higher efficacy than 2Q1F.
1HN0 is thus the sequence that we chose for our therapy. The thermostability of wild-type 1HN0 at physiological temperature is still very low, and so we decided to adapt Dr Hettiaratchi’s method to thermostabilize ChABC (Hettiaratchi et al., 2020). 7 of the mutations that led to an improved thermostability were chosen, namely: K194E, A228K, S274P, N288D, S343N, K654D, R670T, Q781E.
We have compared this solution to Bradbury's viral vector below.
ChABC is one of many compounds that stimulate plasticity in the injured spinal cord. In most experiments these compounds tend to be applied as closely as possible to the time of injury to increase the natural window of plasticity after an injury. For human patients, however, it is not always possible to apply a regenerative treatment soon after injury as many patients are either too ill, or it is not possible to fit another intervention into the clinical plan. Therefore, we are hoping to administer ChABC to the lesion site through two microinjections at different times throughout our therapy, one during the surgery to implant the scaffold in the chronic phase, and another once axonal regrowth has been confirmed to increase synaptic formations, the timeframe of these injections will be further discussed below.
Our Solution
Delivering ChABC
The use of microinjections is a technique of delivering foreign chemical or biological components into living cells. They have been used as drug delivery systems since they allow for highly localised drug delivery and produce little tissue damage and inflammatory responses due to their small glass-tip size with diameters ranging from 0.1 to 10 µm.
Furthermore, microinjections have proven beneficial when delivering ChABC to the SCI microenvironment, allowing for extensive CSPG digestion, as indicated by Tom and Houlé (2008) where microinjections of ChABC were delivered rostrally and caudally to the lesion. This is the same approach that we are planning to implement within the present therapy. More importantly, the ChABC, delivered through microinjections, lead to functionally active fibers being found in the spinal cord tissue surrounding the lesion site. This same study demonstrated that microinjections themselves do not induce much trauma, nor a noticeable inflammatory response . When compared to intrathecal delivery systems where it is hard to elucidate the actual concentration of ChABC reaching the spinal cord tissues, and where the risks associated with this delivery method are higher, microinjections are a far better alternative (Tom et Houlé, 2008).
Although Tom and Houlé (2008) did not report changes in functional recovery, other researchers have made efforts to more explicitly investigate such outcomes. In this regard, Wiersma, Fouad and Winship (2017) administered injections of ChABC into the cervical region of chronic stroke rat models at approximately 28 days post-stroke and assessed the efficacy of this treatment.The ChABC injections alone depicted axonal outgrowth but only modest sensorimotor recovery in the murine models when compared to controls; Collectively, their results suggested that a combination of ChABC injections and intense physical rehabilitation yielded the best functional recovery, the reach training and plasticity results were much more promising when ChABC was combined with rehabilitative training Overall, combining ChABC injections with intense rehabilitative training - as early as possible post-implementation of ChABC - provided the most optimum outcome in the context of functional recovery (Wiersma, Fouad and Winship 2017).
We are microinjecting our thermostabilized ChABC during the scaffold implantation procedure to decrease the number of procedures the patient needs, thus reducing the risk of possible complications. ChABC will also prevent the reformation of another glial scar after resection (Cheng et al., 2015) allowing for the regrowth of the axons within our scaffold. Furthermore, an injection prior to the surgery can create further inflammation, delaying our therapy’s time frame since a clear measurement of the lesion is not possible when inflammation occurs (Seif et al., 2019). MRI can be used in the acute phase to determine the extent of haemorrhage (hypointensity) and oedema (hyperintensity). This oedema increases in the first 48 h and decreases gradually in the 3 weeks post-injury. At approx. 1 month post injury (sub-acute phase) the lesion is remodelled and MRI (T2 weighted) can be used to identify the cyst size and the preserved tissue bridges (Lammertse et al., 2007; Seif et al., 2019). It is after the remodelling of the lesion that we are equipped to segment the lesion from MRI scans to print our scaffold with the correct dimensions suited for each patient’s lesion characteristics.
Throughout our therapy, axonal regrowth can be measured using Diffusion Tensor Imaging (DTI-MRI modality) which uses water movement to detect physical barriers such as axons and myelin. Two important metrics are fractional anisotropy (FA) and apparent diffusion coefficient (ADC). Low FA values and high ADC values suggest disorganised axon fibres. We would be imaging the lesion site with our scaffold implanted to detect how the neurons are progressing along its microarchitecture and synapsing. We would expect high FA and low ADC values for a successful therapy (Aung, Mar and Benzinger, 2013).
Once axonal regrowth has been confirmed following surgery, further injections of ChABC should be administered at rostral and caudal ends of the lesion site when neurites grow to the other end of the scaffold to ensure they can exit the structure and form new synaptic connections. The first set of ChABC injections would be to degrade the CSPGs and allow neurite outgrowth into the implemented scaffold. The second set would degrade any CSPGs that have been synthesized since the first injections when the neurites reach the ends of the scaffold to enable their exit as well as digesting the PNNs on target neurons to allow the formation of new synapses and functional recovery. Without these further injections, the regenerated axons would remain stuck in the scaffold and there would be no functional recovery. The ChABC implementation timeframe was guided and confirmed by Dr. Jerry Silver, who suggested the imaging technique to visualise when to administer the second dose of ChABC and confirmed the potential of multiple, time staggered injections.
ChABC in our therapy
Potential treatments for SCI are currently proposed to be implemented during the acute phase, <96 hours post-injury. Acute SCI patients are often polytraumatic, however, and have many complications that make a complex surgery unfeasible (Bonner and Smith, 2013). It is also unrealistic to expect individuals to be able to give informed consent on the implementation of a novel therapy while in a polytraumatic state so close to the time of injury. Once the lesion stabilises in the chronic phase at approximately 4 weeks post-injury (Adams and Gallo, 2017), it will be possible to use MRI to visualise the lesion which is necessary to design and print the personalised scaffold and the patient can give informed consent.
Our scaffold will therefore increase axonal regrowth due to its topography and serve as a solid base through which axons can migrate and grow into. Our Mussel Foot Protein (MFP) will keep the scaffold in place and serve as a matrix to promote axonal regeneration and reduce Foreign Body Reaction (FBR) by reducing the movement of the scaffold in the spinal cord. Finally, our ChABC is the final piece to the puzzle: reducing the amount of CSPGs in the SCI microenvironment and increasing plasticity, therefore making this environment more permissive for axonal growth and synaptic formation, making our therapy functionally restorative.
The integration of this enzyme into our project was a well investigated decision made after having contacted experts in SCI, most of which were familiar with ChABC. This type of communication was important to understand how our projects and constituents are perceived as well as to get advice on how to better implement these parts in our therapy, for example how to maximise ChABC’s enzymatic activity in the human body, or which injury levels we should focus on to prevent damage to the patient during surgery. The timeframe for its implementation was also rethought multiple times while heeding the advice given to us by multiple researchers. The choice of its delivery method was also not an easy one and there were several instances in which we had to prioritize safety over efficiency. But this is a choice we would make every time. These calls are also important to further understand the spinal cord microenvironment and learn from the incredible amount of experience these professionals have, so that we can become more confident with the future decisions we make in our therapy.
We were as thorough as possible in our research and identified certain further considerations regarding our products that we believed were worth further research to ensure safety of our therapy. These considerations are listed below and how we went about further understanding them and adapting our project according to the advice given by experts.
Further Research
Foreign Body Reaction
As we are implementing a foreign solid material into the spinal cord environment, it is important to consider reactivity of the immune system to said object. Foreign objects, such as medical devices inserted into the body, can initiate a response known as the foreign body reaction (FBR). The mechanism and progression of FBR is illustrated in Figure 5. Any foreign material, even if biocompatible to the SC environment, can provoke this response. Detrimental effects of the FBR can include harmful inflammation in the patient, or even fibrosis that encapsulates the medical device and hinders its functionality (Carnicer-Lombarte et al., 2021). Our scaffold is made of polycaprolactone (PCL), a foreign biomaterial that has the potential to induce FBR. Therefore, we conducted extensive research into the topic, in an attempt to ensure its lessened severity.
Figure 5: The progression of the foreign body reaction following surgical implantation of the scaffold. The immune reaction to the provisional matrix leads to the formation of a fibrotic capsule which is stabilised by new blood vessels.
As this fibrosis could affect the function of our scaffold, it was important to take these factors into consideration. Multiple properties of the biomaterial used can affect the intensity of the Vroman effect, including hydrophobicity, surface topography, shear stress/strain, etc. We have contacted researchers familiar with FBR such as Dr Alejandro Carnicer, an expert in the field of inflammatory responses caused by foreign materials in the CNS, to ensure that our scaffold would not produce a massive FBR. Firstly, we ensured the biocompatibility of our therapy in the SCI to reduce FBR initiated due to chemical and biological problems. We have also modelled our scaffold to be porous, thus decreasing its Young's modulus to match that of the spinal cord microenvironment in an attempt to reduce inflammation caused by mismatch between tissue types. Furthermore, our scaffold will have round edges, as sharp ones cause more damage. Our MFP will also increase adhesion to the microenvironment, restricting movement of our scaffold within the body and therefore reducing inflammation-causing capillary and tissue damage.
The high number of pathways involved in the FBR allow numerous targets for pharmacological manipulation of the immune response. Hence, methods to downregulate the FBR using pharmacological treatments (e.g. targeting TGF-beta to prevent fibrotic encapsulation of the biomaterial) have been suggested by other researchers, which we have discussed in our proposed implementation.
Rehabilitation
Alongside our therapy and scaffold implantation it is extremely important to define clear patient-specific rehabilitation protocols to be applied alongside and following surgical intervention, subsequently aiding the recovery process. Rehabilitation protocols following SCI are generally provided by a multidisciplinary team including the patients’ family, physiotherapists, and psychologists, amongst others (Nas et al., 2015). Referrals may also be needed to pain clinics for those who experience neuropathic pain. Rehabilitation protocols focus not only on restoring patient’s functional capacities but most importantly on improving patients quality of life with holistic treatments aimed towards mental health and adaptability of both the patient and the world surrounding them, many times with the help of assistive devices (Harvey, 2016). We have put together a comprehensive guide regarding rehabilitation protocols and their importance which can be found here.
Combinatorial approaches
Even though we have set out to create a holistic design for our therapy there is always room for combinatorial approaches in further stages. We have put together a comprehensive table consisting of different agents such as Growth factors, OPcs and more that can potentially be combined with our therapy and improve its efficacy. This table also serves as a guide for entrepreneurial applications and collaborations for our therapy. You can find more information about how these combinations can lead to new entrepreneurship avenues for our therapy in our Proposed Implementation.
Table 1: The effects of combining ChABC with other agents. A comparison between using ChABC alone as well as the benefits and limitations with each agent is illustrated in separate columns.
| Therapy |
Therapeutic effect of ChABC alone |
Therapeutic effect when combined |
Limitations |
| Viral vector (Lentiviral and adeno-associated virus) (Zhao et al., 2011) |
- Shorter enzymatic activity time leading to less functional recovery
| - Sustained ChABC activity for more than 4 weeks
- Easy administration
- Local and long-distance axon projections
- Regeneration of the corticospinal tract (CST) and functional recovery (forelimb and grasp functions) in SCI rat models
|
- Possible integration in the host’s genome ( specifically lentiviral)
- Decline in expression over time due to episomal loss by degradation
- Small packaging capacity(specially AAV)
- Strong cell-mediated immune response -possible increase of FBR in our therapy
|
|
Cells (Schwann cells/olfactory ensheathing cells OECs) (Fouad et al., 2009) |
- Effects enhanced in combinatorial therapy
|
- Improvement in graft integration, axon regeneration across the lesion, locomotive recovery, and bladder function improvement in rat models with complete T8 contusion
|
- Insufficient cell survival and differentiation
- Hard to control the directional exit of axons from the caudal end of the transplant and re-enter into host tissue
|
| Neurotrophins + neural progenitor cells (ChABC+polymer scaffold+ lentiviral neurotrophin-3) (Massey et al., 2008)
| - Effects enhanced in combinatorial therapy
|
- Successful differentiation and migration of neural precursor cells
- Increased CST and serotonergic axon plasticity
- Significantly improved ladder-walking
|
- Limitations associated with lentiviral vectors, mentioned before
|
| Neuroprotective/conditioning agents (clenbuterol+ChABC) (Bai et al., 2010)
| - Effects were only observed in combined groups
|
- Decrease in CSPGs and collagen deposition
- Regenerated axons through the lesion were observed
- Recovery of locomotive function
|
- Failure of CST regeneration (critical for locomotive functional recovery)
|
| NDMA receptor type (NR2D subunit of the NMDA receptor increased NT3) (Garcia-Alias et al., 2011)
| - Electrical conductivity across the lesion is only re-established in the combined group
|
- Increased axonal sprouting
- Strengthening of neuronal circuits in the spinal cord
- Improved BBB locomotor test
|
- Weak NT3-medidated effect and NR2D expression in adult rats with chronic left lateral hemisection
- Failed to promote connections to motoneurons in the second postnatal week, leading to formation of perineuronal nets
|
| Blocking myelin inhibitors (anti-Nogo-A antibody) (Zhao et al., 2013)
| - Electrical conductivity across the lesion is only re-established in the combined group
|
- Significantly improved axon sprouting and regeneration
- Recovery in CST-dependent tasks compared to single treatments
- Stimulated the growth of axons with diameters >3 µm alongside finer ones
|
- Anti-Nogo-A antibodies used in the study don’t address CSPGs upregulation in the ECM
- Possibility of binding to the amino-Nogo-A region of myelin-associated molecules, thus inhibiting axon regeneration, and destabilizing synaptic transmission
|
| Rehabilitation (Wang et al., 2011) |
- CSPGs were digested throughout the cord 2–3 mm rostral and 3–4 mm caudal to the injury
|
- Animals that received both ChABC and task-specific rehabilitation showed the greatest recovery in skilled paw reaching
|
- Possible limitations might be lack of rehabilitation devices to support the patient's weight
|
| Mesenchymal stromal /stem cells (Lee et al., 2015)
| - Sustained partial decrease in CSPG levels for 8-12 weeks
|
- Significantly better functional recovery 8 weeks after transplantation
- Increased expression of digested CSPGs (2B6), b3 tubulin, and NF-M
|
- The levels of COX2 (P < 0.05), and tumor necrosis factor-a were higher in the treatment groups
- Possibly degraded CSPGs, stimulate macrophages to migrate into injured sites, leading to further inflammation
|
| Low Level Laser (LLL)- chronic pain management and functional recovery (Janzadeh et al., 2020) |
- Removal of CSPGs from the glial scar without effect on cytotoxic edema
|
- LLL, applied 30 minutes post-surgery, limited inflammatory response
- LLL+ChABC caused a reduction in the levels of glycogen synthase kinase-3β, implicated in demyelination and Wallerian degeneration leading to an increase in axon number and degree of myelination
- Cavity size was reduced
- Functional recovery (BBB score) improved
- Decreased neuropathic pain
|
- LLL alone induced a fibrotic scar, which is suboptimal for axon outgrowth
- Inflammation considerations needed
|
Different approaches to administering ChABC
Prior to our decision in administering thermostabilised ChABC using microinjections, we were particularly interested in a hydrogel model due to their low mechanical properties and prolonged drug delivery capacities. Furthermore, hydrogels have prior literature evidence of successfully releasing thermostabilised ChABC for long periods of time as well as localising it to the area of injury.
Displayed in the table below you can find research completed in an effort to compare and contrast the efficacy and effectiveness of various hydrogels. This was done in order to assess their suitability within our project. Ultimately, we have decided not to implement hydrogels due to limited studies on the ways in which each material would interact with both MFP and PCL alongside our inability to carry out these experiments in the lab given the project’s timeframe. Nevertheless, the incorporation of hydrogels would prove an exciting outlet towards further developing our product in the future and can act as a possible avenue of collaboration with biosynthetic companies.
Table 2: The different types of hydrogel that can be used to deliver ChABC. The methods, results, and discussion of each hydrogel are outlined in separate columns.
| Hydrogel | Incorporation of ChABC | Results | Discussion |
| Agarose (Lee, McKeon and Bellamkonda, 2009) |
- SeaPlaque agarose (Cambrex) was combined with ChABC/trehalose/microtubes
- Trehalose enabled thermostabilization of ChABC
- Microtubes and agarose gel allowed for a sustained release of the trehalose-stabilised ChABC
|
- In-vivo experiments on animals measured successful digestion of CSPGs using trehalose-stabilised ChABC at the lesion site
- ChABC actively digested CSPGs for 2 weeks, resulting in low levels of CSPG for 6 weeks
|
- Potential to cause detrimental sprouting which can lead to neuropathic pain and autonomic dysreflexia. However, no evidence of hyperalgesia or allodynia was indicated within the test subjects
- Combination therapy with neurotrophic factors (NT-3) potentiates the effects of ChABC and promotes locomotor-centric functional recovery
|
|
Methylcellulose(1) (Hettiaratchi et al., 2020) |
- Mutagenesis was carried out on ChABC to produce ChABC-37 (37 mutations) which increased activity from 16.8 hours to 4.4 days.
- ChABC-37 underwent a fusion with the Src homology 3 (SH3) domain
- ChABC-37-SH3 was released - via an affinity-based method - from methylcellulose hydrogels modified with SH3-binding peptides
|
- In-vitro experiments indicated sustained activity for 7 days when ChABC-37-SH3 was released from the methylcellulose hydrogels
- Resistance to trypsin
|
- Study lasted only for 7 days
- ~62% of ChABC-37-SH3 was released but this is expected to increase for in-vivo experiments since the hydrogel will dissolve
- To ensure that all protein is released, they have developed a new methylcellulose hydrogel which should speed up the degradation rate whilst increasing resorption.
|
| Methylcellulose(2) (Hettiaratchi et al., 2019) |
- ChABC was thermostabilised using site-directed mutagenesis which doubled its half life
- Covalent modification was then applied through poly(ethylene glycol) chains i.e. PEGylation which resulted in a 10-fold increase in activity
- PEG-N1000G-ChABC underwent a fusion with SH3 and was consequently released from methylcellulose hydrogels modified with SH3 binding peptides
|
- In-vivo experiments on murine models had lower CSPG levels in the peri-lesion area at 14 and 28 days post cortical stroke injury (49% and 40%, respectively) when compared to controls
|
- PEGylation increases half-life of ChABC and does not disrupt binding with the SH3 binding peptide-modified methylcellulose hydrogel but slows down the release profile in-vitro
- Slowed release profile is counteracted by the better penetration of tissues and well-sustained delivery of PEG-N1000G-ChABC
- Encourages the delivery of ChABC with other neurotrophic factors or cell lines to further enhance functional recovery
|
| Self-assembling peptides (SAPs) nanofiber hydrogel (Raspa et al., 2021) |
- 2 types of self-assembling peptides were used: FAQ and CK
- Both mixed with ChABC at 0.1 unit/ml through 2 separate methods (blend and via injection)
|
- Control experiments in-vitro showed that most of the active ChABC was released on day 0 and decreased significantly in the following days
- Release profiles of active ChABC from the SAP hydrogels were measured for 42 days during in-vitro experiments. The injection method lasted 14 days and the blend method lasted 28 days
- SAP hydrogels enabled sustained delivery of ChABC which encouraged neurite outgrowth and enhanced locomotor function recovery
|
- Beneficial effects in-vivo are likely to be attributed to the enhanced ChABC stability alongside the delivery system used: SAP hydrogels
- Has potential to be utilised for peripheral nervous system (PNS) injuries e.g. stroke which would also benefit from ChABC
|
| Fibrin
(Hyatt et al., 2010)
|
- Genipin was utilised for further crosslinking to increase the density of the gel (and ensure a sustained release of active ChABC)
- These implants contained 1.25 microliters of ChABC (200U/ml)
- They were placed directly above the spinal cord lesion in in-vivo models
|
- Fibrin gel delivery system postulated to offer thermostability and reduce loss of activity
- 24.4% of the initial dose of bioactive ChABC was detected around the lesion site 3 weeks post-implantation compared to 4.4% with intraspinal injections
- Levels of GAG chains were 37% lower in lesions treated using the fibrin versus intraspinal injections
|
- This study did not assess locomotor recovery in the rat models meaning that we cannot be sure that this therapeutic strategy would result in the desired functional improvement across motor, sensory, and autonomic impediments of spinal cord injury
|
As explained in the information above, we researched extensively into hydrogels as a potential method of administration. In the end we found that microinjections are the better fit with our therapy as a whole. Hydrogels would block the pores of our scaffold as would ChABC given its relatively large protein size. Microninjections on the other hand, would not overload the scaffold with ChABC and do not contribute any additional material into the pores of the scaffold. By using a thermostable ChABC, the issue of ChABC degrading too quickly from microinjections is overcome.
Spinal Cord Atrophy
In the chronic phase, long term effects such as spinal cord atrophy are also common and can have lifelong consequences. Spinal cord atrophy refers to the degradation and eventual reduction in volume of the spinal cord, this is mediated by the cellular processes apoptosis and necrosis and is exacerbated by the resulting inflammatory environment. The process itself occurs on the sagittal plane of the spinal cord, i.e., at the vertebrae of the site of injury. Early stage atrophy after injury has been reported to contribute to poor prognosis and a greater loss of myelin at the surrounding axons (Bang and Kim, 2009).
Multiple studies have shown that atrophy of the spinal cord (decreased myelin content and increased iron concentration levels) following a trauma to the region can be assessed using MRI modalities such as T1-weighted or T2*-weighted assessments (Chandra et al., 2011; Talbott et al., 2015). These measurements give an idea of the amount of neurodegeneration happening in the spinal cord following injury and can be used as a prognosis for patient recovery and to assess the amount of myelination and structural strength of the spinal cord after our therapy. Research suggests that atrophy in the spinal cord after a SCI is a continuous process with decreasing rates with time, common values reported have suggested atrophy of up to 7% (5mm2) in the first years which reaches 14% at 2 years post injury and up to 30 % at 14 years (Seif et al., 2019).
These measurements alongside those regarding muscle and bone wasting can be compared to motor and sensory scores to provide a more broad view of what is happening at the level of patient recovery using non-invasive methods. If our therapy is successful we would be expecting reduced cord atrophy by 6 months (less than the approximate 7%) leading to predicted recovery in lower extremity motor score at 2 years post-injury.
Future Investigations
While our therapy has enormous potential, there are some improvements that can be made. Our mutagenesis increased the thermostability of ChABC, but it is only preliminary as ChABC can be further stabilised to increase its efficacy in removing inhibition in the SCI microenvironment. Another improvement to increase the efficacy of ChABC as part of our therapy would be to incorporate a delivery mechanism similar to hydrogels that will keep ChABC localised for a longer period of time with a prolonged release, but that does not interfere with the other components of the therapy. Adding other factors to the scaffold such as OPCs or growth factors would further encourage axonal regrowth alongside ChABC to improve reconnection of neurons. Finally, expanding our patient pool further to incorporate spinal cord lesions at all sites in the cord would allow all SCI patients to benefit from our therapy.
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