Full length articleSelf-assembling peptides optimize the post-traumatic milieu and synergistically enhance the effects of neural stem cell therapy after cervical spinal cord injury
Graphical abstract
Introduction
Cervical spinal cord injury (SCI) is a disastrous event, which can lead to lifelong disability and loss of independence. Cervical lesions can cause paresis or paralysis of the upper and lower extremities and can additionally affect bladder and bowel function, respiration, and trunk stability. More than 50% of spine injuries involve the cervical region [1], [2], [3]; which is distinct from the thoracolumbar region in both anatomy and pathophysiology. In particular, differences exist in vascular organization, volume of gray and white matter, and neural segmentation. This makes cervical trauma models highly clinically-relevant, particularly when moving towards translation.
Following severe SCI, a secondary injury cascade is initiated which results in loss of neurons, release of cytotoxic factors, demyelination, and development of intramedullary cystic cavitation. Although surviving axons persist in a subpial rim of white matter, possessing the potential to support regeneration, the hostile microenvironment limits endogenous repair [7]. Furthermore, cystic cavities and deposited astroglial/proteoglycan scar form a physical barrier to axonal outgrowth and cell migration [4], [5], [6], [8], [9], [10].
Exogenous cell transplantation thus emerges as a promising treatment strategy to overcome structural tissue-damage and generate functional recovery. Stem cells and their progeny may promote neuroprotection by modifying the toxic microenvironment, inhibiting inflammatory signaling, and releasing growth factors [11]. Furthermore, in the sub-acute stage transplanted cells can support host axon regrowth by reducing neurite growth-inhibitory substances, releasing trophic factors, enhancing remyelination of axons and supplying an extracellular matrix [12]. Of particular interest are neural precursor cells (NPCs) which can differentiate into both neurons and glia [13]. NPCs have been shown to promote remyelination [14], [15] and support functional recovery [16], making them very promising for the treatment of spinal cord injuries. Unfortunately, transplanted cells have poor survival rates. Efforts are currently underway to improve engraftment and differentiation through combinatorial approaches with growth factors, sonic hedgehog proteins and chondroitinase [17], [18].
Scaffolds, such as collagens, fibrin or plasma, represent an exciting bioengineered strategy to bridge the lesion cavity and shape the inhibitory post-traumatic microenvironment [19]. Agarose hydrogels or alginates reduce astroglial and fibrotic scarring and serve as a matrix to deliver drugs or growth factors, both supporting outgrowth and regeneration of axons [20], [21], [22], [23]. NeuroGel (poly-(N-[2-hydroxypropyl]methacrylamid)-hydrogel) additionally supports angiogenesis, whereas fibronectin shows convincing effects of orientated growth of axons within the damaged spinal cord [24], [25], [26], [27]. Some studies also used multicomponent polymers or collagens in order to both provide a scaffold and to transport or deliver factors (e.g., neurotrophin-3) or cells (e.g., olfactory nerve ensheathing cells or neural stem cells) within the damaged spinal cord [28], [29], [30].
In recent years, self-assembling peptides (SAPs) have been developed for tissue engineering and factor delivery. As a novel treatment approach, SAPs can be injected directly into the epicenter of the lesion to self-assemble into 3D nanofibers which scaffold the intramedullary cavity, modify the microenvironment, and serve as a structural framework for the integration and axonal outgrowth of transplanted cells [31], [32]. In SCI treatment, SAPs containing IKVAV (IKVAV PA) have been shown to reduce astrogliosis and cell death in addition to promoting regeneration [33]. Another SAP, RADA 16-1, has been shown to bridge the injured spinal cord and elicit axon regeneration [34]. Since RADA 16-1 is acidic, with a pH of 3–4, it cannot be applied directly to nervous tissue as it causes inflammation and cysts making pre-buffering mandatory [34]. K2(QL)6K2 (QL6) is a novel SAP introduced by Dong et al. and provided by Covidien (Medtronic Inc.) [35], [36]. It is a multidomain peptide (MDP) with an ABA structure. The central B block consists of a variable number of glutamine and leucine (QL). The alternating hydrophilic and hydrophobic pattern allow all glutamine side chains to lie on one side creating a driving force for the hydrophobic faces to pack against one another forming a hydrophobic sandwich. To mitigate the hydrophobic character and thus, poor solubility, the A block containing a variable number of charged amino acids (lysine) was added to both termini [35]. The K2(QL)6K2 type is soluble in water at a pH of 7.4 and showed a very strong β-sheet conformation and a population of nanofibers with uniform diameter (6 ± 1 nm), controlled length (120 ± 30 nm), and no amorphous aggregates [35]. On the other hand, adding a fourth lysine on the terminal endings totally changes the secondary structure from a β-sheet to an unstable α-helix conformation [35]. The K2(QL)6K2 (QL6) β-sheet conformation is stable and may find use in understanding and treatment of various diseases caused by protein aggregation or as nanostructured scaffold for bioengineering [35]. When applied alone, QL6 reduces post-traumatic apoptosis, inflammation and astrogliosis leading to electrophysiological and behavioral improvements after spinal cord injury [36]. Furthermore, QL6 has been shown to support co-transplanted NPCs and to biodegrade over several weeks making it an ideal candidate in SCI [36], [41].
With this background, we hypothesized that QL6 supports NPC survival and differentiation by modifying the microenvironment and that this effect can be further enhanced by creating a lead time between QL6 injection and NPC transplantation. In the current study, we examined whether pre-treatment with QL6 SAPs injected into the injured spinal cord tissue immediately after SCI positively shapes the hostile microenvironment to support subsequent NPC therapy. By using this novel strategy, we expect enhanced NPC survival, integration and differentiation with QL6 pretreatment. Furthermore, we anticipate reduced inflammation and tissue scarring and attenuation of neurological deficits.
Section snippets
Animals
A total of 90 female Wistar rats (250 g; Charles River Laboratories, Wilmington, MA) were used. All experimental protocols were approved by the animal care committee of the University Health Network in accordance with the policies established in the Guide to the Care and Use of Experimental Animals prepared by the Canadian Council of Animal Care.
Experimental groups
This study contained two major study-arms with different survival times following SCI (28 days and 9 weeks). Each study arm was composed of 5 groups:
Group
NPC survival and differentiation
To quantify the number of surviving NPCs, YFP+/DAPI+ cells were counted 28 days after SCI. Animals which received QL6 pretreatment one day after trauma showed a significantly larger number of surviving NPCs (18,088 ± 1208 vs. 11,493 ± 1228; p < 0.05; n = 8) (Fig. 1A, B).
Furthermore, the number and percentage of NPCs that differentiated into neurons (YFP+/DAPI+/NeuN+) or oligodendrocytes (YFP+/DAPI+/APC+) were increased by QL6 pretreatment compared with NPCs + vehicle. 1570 ± 163 vs. 672 ± 98 of surviving NPCs
Discussion
SAPs are a promising treatment option for SCI. After injection into the injured spinal cord, SAPs self-assemble into engineered nanofiber scaffolds which can bridge the intramedullary cavity and modulate the microenvironment. Gliosis and cell death can be reduced and cystic areas diminished [54], [55]. The QL6 form of SAPs is characterized by a periodic repetition of alternating ionic hydrophilic and hydrophobic amino acids (glutamine and leucine) [41]. In a previous study, we showed that QL6
Conclusion
In this study, we demonstrate that QL6 SAPs injected into the injured cervical spinal cord 24 h after trauma shape the hostile post-traumatic microenvironment to improve conditions for NPC transplantation. The number of surviving NPCs and the profile of differentiation was significantly improved, astrogliosis and tissue-scarring was reduced, and the degree of preserved perilesional tissue was increased. Together, these findings provide key evidence that QL6 can mitigate components of the
Disclosures
The authors have no conflicts of interest to disclose.
Acknowledgements
We would like to acknowledge the funding support for this work from the Canadian Institutes of Health Research (CIHR) and the Krembil Family Foundation. Klaus Zweckberger was funded by a grant from the German Research Society (DFG).
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