Chapter 5: Summary and Future prospects
B) The ways in which lipoproteins may be modified to act as contrast agents. (Adapted with permission from Cormode et al, 2010)
2.7 Nanotechnology in Neuroregeneration
Although axonal regeneration in the PNS occurs much more efficiently than in the CNS, reinnervation and functional recovery are often poor. This is particularly the case for lesions in which large spaces between nerve stumps are created. This gap, lacking the Schwann cells necessary for axonal regrowth, poses a major obstacle for regenerating axons. In these cases, the golden standard for treatment of PNS damage has been the use of autologous nerve grafts for bridging the two ends.
However, as discussed above, these grafts pose several disadvantages. Instead, nanotechnology may hold the answer for the development of alternatives to these autologous grafts, in the form of tissue engineered nerve grafts (TENGs). Although in the most classical sense a TENG consists essentially of a hollow tube connecting the two nerve stumps, more recently developed TENGs rely on a combination of physical and biochemical cues to actively facilitate the growth of axons through it.
These cues are often introduced in the form of nanofibers.Nanofibers possess a number of advantages for their use as scaffolds for neural regeneration, since (1) their physical properties can be tailored to specifically meet the requirements of a particular type of lesion; (2) their large surface area – to – volume ratio enhances the presentation of biochemical cues; and (3) they can be made from a number of biocompatible and degradable materials.
Electrospinning is one of the most used methods for the fabrication of nanofibers.
This technique allows for the production of fibers with a wide range of dimensions, which can be easily regulated through variations in the polymer solution, electric field strength, and field pattern used in the fabrication process. Synthetic materials, including poly(ε-caprolactone) (PCL) (Panseri et al., 2008), poly(ε-caprolactone-co- ethyl ethylene phosphate) (PCLEEP) (Chew et al., 2007), and poly(L-lactic acid) (PLLA) (Yang et al., 2005); are the most common component used for the fabrication of electrospun nanofibers for nerve regeneration. Fig 2.18 describes a study with GDNF incorporated aligned PCLEEP electrospun nanofibrous conduits. Kim et al.
(2008), for example, used poly(acrylonitrile-co-methylacrylate) (PAN-MA) fibers (with a diameter of 400–600 nm) to bridge a 17 mm rat tibial nerve lesion gap. They observed that, following a 16 week recovery period, these rats exhibited a sensory and motor recovery on par with that of rats with autologous grafts. Notably, this effect was only seen in animals with PAN-MA fibers aligned with the axis of axonal regeneration, suggesting that the topographical cues provided by the nanofibers are
a crucial component of their regenerative potential. Similarly, in a recent study Jiang et al. (2012) implanted either PCL microfibers (981±83 nm diameter) or nanofibers (251 ±32 nm diameter) into a 15 mm rat sciatic nerve lesion. Regeneration through the graft, as seen by retrograde labelling, was much greater in nanofiber than in microfiber grafts; a finding which serves to highlight the importance of nanofiber scale in their properties as regenerative scaffolds. Naturally occurring materials, such as chitosan (Wang et al., 2008), have also been employed as nanofibers scaffolds.
Table 2.2: Nanomaterials used in Neuroregeneration
Although these materials more closely resemble the tissue they are intended to replace, potentially exhibiting greater biocompatibility, their weak mechanical properties limit their usefulness as nerve graft scaffolds.
The other major alternative to electrospinning for the fabrication of nanofibers is self- assembly. This technique consists in the design of molecules, particularly peptides, capable of assembling by noncovalent bonding into polymeric structures directly in their target areas (Zhang et al., 2003). Self-assembled peptide nanofibers(SAPNs)
are characterised by a very small diameter (5-10 nm) in contrast to electrospun fibres, which often are orders of magnitude larger. These dimensions closely resemble those of extracellular matrix (ECM) proteins, making SAPNs a close imitation of the natural cellular environment. Additionally, these fibersbreak down intoL-amino acids which, being common in all organisms, are non-toxic. Several self-assembling peptide (sapeptide) sequences have been used for the formation of SAPN scaffolds. Arginine-alanine-aspartate sequences (RAD16), for example, have been shown to support adhesion and facilitate neurite outgrowth of primary neurons and neuronal cell lines (PC12) in vitro(Holmes et al, 2000).Sapeptides can also be modified so as to include certain motifs, introducing functional properties in the final SAPNs which may be desirable in the final scaffold. For example, Gelain et al.
(2006) studied the effect of functionalisation of RADA16(arginine-alanine-aspartate- arginine) sapeptides with bone marrow homing motifs. Functionalised SAPN scaffolds were found to support neural stem cell proliferation to a greater degree than pure RADA16 SAPN scaffolds.
Axonal regeneration in the CNS often fails, due to a combination of inhibitory environment and lack of growth supporting factors. Although these limiting factors have often been addressed individually, no therapy currently exists to treat CNS lesions in humans. It is likely that, given the multimodal nature of the problem, the issues limiting regeneration in the CNS will have to be addressed simultaneously to achieve successful regeneration. Given the wide range of unique properties that they can exhibit, nanotechnologies are thus an attractive platform for the development of these therapies.
As in the PNS, nanofiber scaffolds have been used to promote the regeneration of axons through lesions in the CNS. Although electrospunfibers have sometimes been used for this purpose (Meiners et al., 2007), SAPNs are more commonly employed due to their ability to polymerise in situ, minimising the need for surgical intervention.
Tysseling‑Mattiace et al. (2008) injected laminin motif-derived IKVAV (isolucine- lysine-valine-alanine-valine) sapeptidesinto mouse thoracic (T10) spinal cord compression injuries. These mice not only had reduced glial scar formation, but also exhibited enhanced functional recovery 9 weeks after injury. RADA16 sapeptide scaffolds have also been used in in vivo CNS regeneration studies, in both spinal
cord (Guo et al., 2007) and optic tract (Ellis-Behnke et al., 2006) lesion models (Fig 2.19).
Fig 2.19: Self-Assembling Peptide Nanofiber Scaffold (SAPNS) mediated repair for the animal brain. (i) (a) Molecular model of the RADA16-I molecular building block. (b) Molecular model of numerous RADA16-I molecules undergo self assembly to form well ordered nanofibers with the hydrophobic alanine sandwich inside and hydrophilic residues on the outside. (c) The SAPNS is examined by using scanning electron microscopy. (Scale bar,500 nm.) (ii) SAPNS allows axons to regenerate through the lesion site in brain. The dark-field composite photos are parasagittal sections from animals 30 days after lesion and treatment. (a) Section from brain of 30-day-old hamster with 10μl of saline injected in the lesion at P2. The cavity shows the failure of the tissue healing. The retinal projections, in light green at the top left edge of the cavity, have stopped and did not cross the lesion.
Arrows indicate path and extent of knife cut. (b) A similar section from a 30-day-old hamster with a P2 lesion injected with 10μl of 1%SAPNS. The site of the lesion has healed, and axons have grown through the treated area and reached the caudal part of the superior colliculus (SC). Axons from the retina are indicated by light-green fluorescence. The boxed area is an area of dense termination of axons that have crossed the lesion. Arrows indicate path and extent of knife cut. (c) Enlarged view of boxed area in b. The regrown axons, shown in white, were traced with cholera-toxin fragment B labeling by using immunohistochemistry for amplification of the tracer. (iii) Optic tract regeneration and functional return of vision. This SAPNStreated adult animal turns toward the stimulus in the affected right visual field in small steps, prolonged here by movements of the stimulus away from the animal. Each frame is taken from a single turning movement, at times 0.00 (a), 0.27 (b), 0.53 (c), and 0.80 (d) sec from movement initiation. The animal reached the stimulus in the last frame. This is 29% slower than most turns by a normal animal. The recording was made 6 weeks after surgery and treatment when the animal started to show a response.(
Adapted with permission from Ellis-Behnke et al, 2006)
More complex approaches to CNS regeneration have also been taken. One such example is the combination of nanofiber scaffolds with stem cell therapy. Neural stem cells (NSCs) can aid regenerating axons through the formation of a growth- supportive microenvironment, and are considered to hold great therapeutic potential
for the treatment of CNS injury and disease(Martino and Pluchino, 2006). Poly(lactic- co-glycolic acid) (PLGA) scaffolds containing pores for axonal guidance, and an underlying layer seeded with NSCs, were implanted in rats with spinal cord hemisection lesions (Teng et al., 2002). Implantation of this scaffold led to axonal regeneration and a functional recovery which was superior to that seen in rats implanted with the scaffold or NSCs alone.
The conductive properties of certain nanomaterials also make them well suited to combine neural scaffolding with electrical stimulation. Although studies have mainly limited to in vitro testing, neuronal cultures have been reported to exhibit increased neurite outgrowth in response to electrical stimulation (Kimura et al., 1998).
Electrospun nanofibers coated with electrically conductive polymers have been studied by several groups as potential platforms for the fabrication of regenerative scaffolds. Lee et al. (2009) cultured cells of the PC12 neuronal cell line ona mesh of poly(lactic-co-glycolic acid) (PLGA) nanofibers coated with a nano-thick layer of electro-conducting polypyrrole (PPy).Not only did the nanofibers support the extension of neurites from the cells, but neurite length and number was greatly increased when cells were electrically stimulated. Carbon nanotubes also hold great potential for the development of multifunctional scaffolds. Although they are non- degradable; their size (~1 nm diameter),electrical conductivity, and excellent mechanical properties make them good candidates for their use as scaffolds for neural regeneration. Moreover, in vivo studies in rat SCI lesion models have shown that carbon nanotube scaffolds, functionalised with polyethylene glycol, are capable of supporting axonal regeneration through the lesion and promote functional recovery (Roman et al., 2011).
Finally, more traditional treatments like the direct delivery of growth-supportive compounds to lesions have also benefitted from nanotechnology. Although agents such as Epidermal growth factor receptor (EGFR) inhibitors have been shown to reduce the inhibitory effect of CSPGs and myelin on axon regeneration (Koprivica et al., 2005), sustained delivery is necessary for its therapeutic effects to be seen.
Since systemic administration is not a desirable delivery route due to associated side-effects, consequence of the role played by EGFR in other tissues (Fakih and Vincent, 2010), a nanotechnology platform was developed for the sustained, local delivery of EGFR inhibitors (Robinson et al., 2011). PLGA microspheres and
nanospheres loaded with the EGFR inhibitor Tyrosine Kinase Inhibitor (TKI) AG1478 were administered to the eye of rats following injury of the optic nerve. Although both microspheres and nanospheres caused axons to regenerate through the site of injury 2 weeks later, survival of these axons at 4 weeks post-lesion was only observed in nanosphere-injected animals. This observation was attributed to the ease of administration and more stable drug release profile of nanospheres compared to their larger counterparts, and serves as an example of the advantages that nanotechnology can have over seemingly similar approaches to axonal regeneration (Fig 2.20).
Fig 2.20 Promoting Optic Nerve Regeneration through sustained delivery of EGFR TKI AG1478 by nanospheres (i) Optic nerve crush injury model. (a) Diagram of the rat eye and optic nerve illustrating the surgical approach for the animal studies testing the effectiveness of the microspheres and nanospheres. (b) Diagram illustrating the area of detail for the immunohistological images examining nerve regeneration. (ii) Poly(lactic-co-glycolic acid) (PLGA) microspheres and nanospheres containing the EGFR TKI 4-(3-chloroanilino)-6,7- dimethoxyquinazoline (AG1478) were fabricated for intravitreal administration in a rat optic nerve crush injury model. Two weeks after intravitreal delivery, microspheres and nanospheres could be detected in the vitreous using coumarin-6 fluorescence, but fewer microspheres were observed compared to the nanospheres. At four weeks only nanospheres could be detected. AG1478 microspheres and nanospheres promoted optic nerve regeneration at two weeks, and at four weeks evidence of regeneration was found only in the nanosphere-injected animals. This observation could be attributed to the ease of administration of the nanospheres versus the microspheres, which in turn led to an increased amount of spheres delivered to the vitreous in the nanosphere group compared to the microsphere group.(Adapted with permission from Robinson et al, 2011).