Self-assembling peptide nanofiber hydrogels for central nervous ...

Front. Mater. Sci. 2015, 9(1): 1–13 DOI 10.1007/s11706-015-0274-z

REVIEW ARTICLE

Self-assembling peptide nanofiber hydrogels for central nervous system regeneration Xi LIU1,2, Bin PI3, Hui WANG1, and Xiu-Mei WANG (✉)2 1 National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, Suzhou 215123, China 2 Institute for Regenerative Medicine and Biomimetic Materials, Key Laboratory of Advanced Materials, School of Materials Science and Engineering, Tsinghua University, Beijing 100084, China 3 Department of Orthopedics, The First Affiliated Hospital of Soochow University, Suzhou 215006, China

© Higher Education Press and Springer-Verlag Berlin Heidelberg 2015

ABSTRACT: Central nervous system (CNS) presents a complex regeneration problem due to the inability of central neurons to regenerate correct axonal and dendritic connections. However, recent advances in developmental neurobiology, cell signaling, cell–matrix interaction, and biomaterials technologies have forced a reconsideration of CNS regeneration potentials from the viewpoint of tissue engineering and regenerative medicine. The applications of a novel tissue regeneration-inducing biomaterial and stem cells are thought to be critical for the mission. The use of peptide nanofiber hydrogels in cell therapy and tissue engineering offers promising perspectives for CNS regeneration. Self-assembling peptide undergo a rapid transformation from liquid to gel upon addition of counterions or pH adjustment, directly integrating with the host tissue. The peptide nanofiber hydrogels have mechanical properties that closely match the native central nervous extracellular matrix, which could enhance axonal growth. Such materials can provide an optimal three dimensional microenvironment for encapsulated cells. These materials can also be tailored with bioactive motifs to modulate the wound environment and enhance regeneration. This review intends to detail the recent status of selfassembling peptide nanofiber hydrogels for CNS regeneration. KEYWORDS: regeneration

self-assembling peptide; hydrogel; central nervous system (CNS); nerve

Contents 1 Introduction 2 Nanofiber biomaterials for CNS regeneration 2.1 Traditional nanofiber biomaterials for CNS 2.2 Self-assembling peptide nanofiber hydrogel scaffolds 3 Self-assembling peptide nanofiber hydrogels for CNS Received October 15, 2014; accepted November 24, 2014 E-mail: [email protected]

regeneration 3.1 RADA self-assembling peptide 3.2 RADA self-assembling peptide hydrogel in CNS regeneration 3.2.1 Pure RADA for CNS treatment 3.2.2 RADA/cells for CNS treatment 3.2.3 Functionalized RADA for CNS treatment 3.3 Peptide amphiphile 3.4 PA peptide hydrogel in CNS regeneration 3.5 Multidomain peptide and its application in CNS regeneration

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4 Conclusion and future perspectives Abbreviations Acknowledgements References

1

Introduction

Injury to the central nervous system (CNS) including the brain and spinal cord is a leading cause of permanent severe neurological disabilities and death. Most people suffering from CNS injuries would endure irreversible disabilities as a result of these insults. It is estimated that more than 2 million people suffer from traumatic brain injuries (TBIs) annually [1], over 500,000 people per year suffer from stroke, and approximately 12,000 new cases of spinal cord injuries (SCIs) occur each year in US [https:// www.nscisc.uab.edu/, 2013]. The associated social-economic burden of caring for patients with CNS injuries is very high. Taking SCIs for example, the annual costs are estimated to be $9.7 billion in United States [2]. Unfortunately, there are no effective therapies to treat CNS injuries. Recent therapies that can dissolve blood clots show promise for ischemic stroke [3–4]. However, less than 2% of stroke patients are typically treated with this approach. Steroid treatment for SCIs can produce some benefits [5–7], but less than 1% of persons experienced complete neurologic recovery by hospital discharge. The major causes of CNS injury result from pathological biochemical events that still remain poorly understood. In fact, the inherent challenge of developing therapies aimed at treating CNS injury is rooted in the complexity of its pathobiology. For the restoration of function following CNS trauma, damaged axons must regenerate cross and beyond the site of injury [8]. Even a small amount of neuronal, dendritic, or axonal regeneration would therefore result in life-changing improvements. Therefore, a considerable amount of work has been performed in the advances of therapeutic approaches to CNS diseases. Molecular and cell therapy are the main methods in clinical CNS treatment. The application of growth factors, including nerve growth factor (NGF), brain-derived neurotrophic factors (BDNFs), and neurotrophin 3 (NT-3), has been shown to augment morphological and sometimes functional recovery [9–14]. Moreover, nerve growth inhibitor blockers have been tested to improve neural regeneration after CNS injury [15–18]. Neural stem cells (NSCs), neural progenitor cells (NPCs), olfactory ensheathing cells (OECs), and Schwann

cells have been applied to replace lost neurons and create a beneficial environment for neural regeneration [19–25]. Although cell and molecular therapies produced some positive benefits, injecting the cells or pharmacological agents into the host can be problematic because of the large cavities that form [21,26–29]. A number of studies have shown that the effects of cell or molecular treatment are enhanced when utilized in combination with biomaterial scaffolds, necessitating the development of a delivery matrix for CNS regeneration to overcome the main limitations of traditional methods [30–31]. Here, we will discuss the use of self-assembling peptide nanofiber hydrogel scaffolds for brain and spinal cord injury treatment in detail.

2 Nanofiber biomaterials for CNS regeneration Acting as a substitute of extracellular matrix (ECM), biomaterial scaffold is a crucial structure component for supporting cells and provides a permissive microenvironment that influences the function of cells [32]. It can also be used to deliver drugs at a rate designed to match the physiological need of the tissue [33–34]. Tissue engineered scaffolds can be used to fill the cavities, guide cell proliferation, direct cell differentiation, promote tissue growth, bridge structure gaps for the reconnection of neuronal process, influence the survival of transplanted cells, serve as a bridge for endogenous cell migration and axonal elongation. A promising innovation in tissue engineering is the development of biologically compatible nanofiber scaffolds that can serve as a permissive bridge for axonal regeneration or as cell/drug carriers [35–37]. The porous structure of the nanofiber matrix could recapitulate the key characteristics of natural ECM with the associated topographical cues, cellular adhesion sites, biochemical and physiological signals [38–39]. 2.1

Traditional nanofiber biomaterials for CNS

A variety of biodegradable materials have been processed into nanofiber scaffolds using electrospinning or solution phase separation for CNS regeneration [39–42]. There are primarily two kinds of biomaterials: naturally derived materials and synthetic macromolecular polymers. Natural scaffolds, such as collagen, fibrin, hyaluronic acid, alginate, and fibronectin, are mainly derived directly

Xi LIU et al. Self-assembling peptide nanofiber hydrogels for CNS regeneration

from ECM and have inherent merits of biological recognition including the presentation of biological receptor-binding ligands for cell adhesion, proteolytic degradation, and remodeling in vivo [43–49]. They also impart intrinsic signals within the structure that can enhance tissue formation [50]. Some, but not all, natural materials also allow for cell infiltration [51]. However, since these materials are obtained from natural sources, major concerns include homogeneity of the product, immunogenicity, pathogen transmission, and weak mechanical in vivo strength are associated with the use of natural materials [52]. In addition, most natural materials are soluble in aqueous media unless crosslinking is introduced. Synthetic materials, such as poly(lactic acid) (PLA), polyethylene glycol (PEG) and poly(lactic-co-glycolic acid) (PLGA), have know compositions and can be custom designed with specific mechanical or degradation properties or to minimize the immune response [53–57]. Unfortunately, the degradation byproducts of synthetic materials may cause pH changes around the implantation site, leading to necrosis, delayed apoptosis, and pain [58– 59]. In addition, the occasional rigidity of the structures with small pores of these natural or synthetic materials restricts both the migration of cells in and out of the material as well as the growth of axons [49,60]. Most of them are showed limited ability to integrate with surrounding tissue and hold the injected cells in the lesion cavities [28,61]. There are also some inherent limitations associated with the traditional scaffold fabrication methods. Though electrospinning is the most widely used method to fabricate nanofiber scaffolds, there are some inherent limitations associated with it, such as small pore size and limited scaffold thickness. These pose a problem for tissue engineers because we often aim to form fully cellularized three dimensional tissue constructs to fill the nerve injury cavities. Moreover, cells cannot penetrate the scaffold due the fact that all mammalian cells range in size from several micrometers to several hundred micrometers in size [62].

have been utilizing self-assembly to understand and mimic biology for years [71–72]. Among them, self-assembling peptide nanofiber scaffold has shown great versatilities in creating self-assembled nanostructures with biological functionalities [73–82]. By engineering the amino acid sequence, the secondary structure of peptides can be manipulated to optimize the interactions between adjacent peptides. Typically, these peptides are composed of alternating positive and negative amino acids that assemble into nanofibrous networks capable of entrapping water. Self-assembling peptides have been considered for application in CNS because they are able to create a permissive three dimensional (3D) environment for cell migration, glial scar prevention, and axonal extension [83– 84]. The benefits of self-assembling peptides over some natural and/or synthetic materials are that: (i) selfassembling peptide scaffold is a molecular designed bioactive matrix without exogenous proteins, posing a minimized risk of carrying biological pathogens or contaminants, thus appears to be immunologically inert; (ii) liquid self assembling peptide scaffold can be injected directly into the lesion site and fill the cavities, regardless of their size and shape; (iii) self-assembling peptides are amendable to functionalization to mimic the naturally occurring proteins; (iv) self-assembling peptides allow for high cell implantation densities and show highly potential for controlled drug release [32,85–87]. The use of selfassembling peptide nanofiber scaffolds for CNS is a novel approach by means of a synthetic biological nanofiber hydrogel that fills the cavities and links the damaged nerve segments of the lesion. This connection allows the migration of cells, promotes rapid and selective differentiation of neural stem/progenitor cells into neurons, alleviate glial scarring, and regenerate blood vessels and axons, leading to functional return of behaviors.

3 Self-assembling peptide nanofiber hydrogels for CNS regeneration 3.1

2.2

3

RADA self-assembling peptide

Self-assembling peptide nanofiber hydrogel scaffolds

Peptide nanofiber scaffold prepared via self-assembling process is an area of growing interest for CNS regenerative medicine [32,63]. Self-assembly is one of the most powerful ways to prepare nanostructure materials and offers great opportunities for the creation of novel biomaterials [64–70]. In the field of biomaterials, scientists

The motif of RADA self-assembling peptide is characterized by periodic repeats of ionic hydrophilic and hydrophobic amino acids, leading to the peptide to fold into βsheet secondary structure with distinct hydrophobic and hydrophilic surfaces [88–89]. During assembly in aqueous conditions, the hydrophobic alanines form overlapping hydrophobic interactions, while on the hydrophilic aspect,

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alternate positive and negative charged peptides pack together through intermolecular ionic interactions in a checkerboard-like manner. The β-sheets stack to form nanofibers of approximately 10 nm in diameter. Upon addition of phosphate buffered saline (PBS) or physiological medium, the nanofibers aggregated into scaffolds with about 99% water content [86,90–94]. In particular, the self-assembling process will not be disrupted with the attachment of short peptide ligands, allowing the formation of scaffold with cell-specific peptides that facilitate cell adhesion, proliferation, and differentiation. Bioactive short motifs can be directly conjugated to the C-terminus of RADA16-I during solid-phase synthesis (Fig. 1). Usually a spacer comprising two glycine residues is added to guarantee a flexible and correct exposure of the motifs to cell surface receptors. Different functional motifs in various ratios can be incorporated in the same scaffold. Usually 1% functionalized peptide solution was mixed with 1% pure RADA16-I solution with volume ratio of 1:1 to get 1% functionalized peptide mixture. Upon exposure to solution with neutral pH, the functionalized sequences self-assemble leaving the functional peptide motifs extending out from the RADA16-I β-sheet double-tape. Nanofibers take part to the overall scaffold thus giving microenvironments functionalized with specific biological stimuli [95–101].

A variety of functional motifs have been successfully conjugated to RADA16-I without disruption of selfassembling properties and nanofiber formations. Although their nanofiber structures appear to be indistinguishable from the RADA16-I scaffold, the appended functional motifs significantly influenced cell behaviors: two-unit RGD binding sequence PRG (PRGDSGYRGDS) enhances cell adhesion and proliferation [97–99], vascular endothelial growth factor (VEGF) mimicking peptide KLT (KLTWQELYQLKYKGI) promotes migration and lumen formation of endothelial cells [98,101], bone marrow homing peptide SKPPGTSS significantly enhanced neural cell survival [96], osteopontin cell adhesion motif DGR (DGRGDSVAYG) enhanced mouse pre-osteoblast cell proliferation and differentiation [97], and heparin binding domain FHRRIKA improve the growth factor secretion ability of human adipose stem cells [102]. 3.2 RADA self-assembling peptide hydrogel in CNS regeneration 3.2.1

Pure RADA for CNS treatment

RADA self-assembling peptide scaffolds support not only the growth of PC12 cells and neuron differentiation of NSCs but also the formation of functional synapses in vivo

Fig. 1 Molecular models of designer peptides and schematic illustrations of self-assembling peptide RADA16-I nanofiber scaffolds. RADA16-I contains 16 amino acids with alternating polar and nonpolar pattern and forms stable β-sheet double-tape structure. The side chains are distributed into two sides, polar side and non-polar side, to undergo self-assembly. Functional peptide motifs are extending out from the RADA16-I β-sheet double-tape. The typical atomic force microscopy (AFM) morphology of the self-assembling functionalized peptide solutions and scanning electron microscopy (SEM) morphology of the functionalized peptide nanofiber scaffold gel are also presented. (Reproduced from Ref. [101] with permission from the Royal Society of Chemistry)


[96,103]. The initial study of RADA for CNS injury repair was conducted by Ellis-Behnke et al. [84]. Using a mammalian visual system as a model in which a tissue gap in the hamster midbrain was caused by deep transection of the optic tract, 10 μL of 1% RADA solution was used to bridge the gap. The gap was reduced in all peptide-treated animals within the first 24 h. Compared with saline, peptide scaffold appeared to create a seamless junction to knit the tissue together across the lesion site which led to axonal growth, partially functional restoring the optic tract and the return of functional vision. Guo et al. further applied RADA peptide to reconstruct cerebral parenchyma in a rat TBI model [104]. Histological, immunohistochemical and apoptosis studies were performed at 2 d, 2 weeks, and 6 weeks after surgery. It was found that RADA peptide scaffold integrated very well with the host tissue with no obvious gaps. Peptide scaffold could support host cells migration into the scaffold due to its similar nanoscale structures with the natural ECM and the formed appropriate host-scaffold interface. Moreover, peptide scaffold significantly reduced the apoptosis around the lesion site and effectively mitigated reactive gliosis and inflammation. When using noninvasive manganese enhanced magnetic resonance imaging (MEMRI) for real-time in vivo monitoring, Liang et al. could observe that RADA was helpful to heal a chronic optic tract lesion and regenerate axons in CNS [105]. 3.2.2

RADA/cells for CNS treatment

Recently, quite a few integrative strategies of peptide scaffold incorporated with cells have been conducted with aim to initiate axonal extension or block axon regeneration

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inhibitors. Guo and his coworkers incorporated NPCs and Schwann cells in 1% RADA, and transplanted them into dorsal column lesion of spinal cord of rats [106]. The grafted cells survived very well in the implants with some of them migrating into the host tissue. Moreover, it was found that large numbers of host cells migrated into the implants. Many blood vessels and extensive axonal regrowth were also observed in the implants with hematoxylin-eosin (HE) staining and anti-neurofilament (anti-NF) immunohistochemistry staining respectively (Fig. 2). It was reported that it is important and necessary to pre-treat peptide in culture medium before transplanting it into the injured spinal cord, so as to buffer the pH. The untreated peptide scaffold elicited inflammation in the host spinal cord tissue, creating distinct gaps and cysts surrounding the implants. However, RADA hydrogel could be administered directly to the injured brain with good results [104]. RADA also showed spinal cord regeneration efficiency despite the delay in treatment [107]. Moradi et al. transplanted RADA nanofiber scaffold with Schwann cells to spinal cord 7 d after injury. Immunohistochemical analysis of grafted lumber segments at 8 weeks after grafting revealed reduced astrogliosis and considerably increased infiltration of endogenous S100+ cells into the injury site. Notably, the peptide treated group and peptide with Schwann cells treated group displayed significant behavioral improvement compared with Schwann cells alone treated group according to Basso, Beattie and Bresnahan (BBB) result, indicating that the nanostructure of the RADA peptide is necessary for the beneficial effects on functional recovery after SCI. Hou et al. also confirmed that the nanofiber matrix was essential for functional

Fig. 2 Integration of implants within the injured spinal cord. (a) RADA implants integrated very well with host tissue, with no obvious cavities or gaps and only slight inflammation. Many host cells migrated into the implants, shown by 4′,6-diamidino-2-phenylindole hydrochloride (DAPI) staining. (b) Implantation of pre-cultured RADA with green fluorescent protein (GFP) NPCs, transplanted NPCs were found to migrate into the host tissue. (c) A lkaline phosphatase (AP) histochemistry staining showed (arrows) that blood vessels grew into the implants. (Reproduced with permission from Ref. [106], Copyright 2007 Elsevier)

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recovery of CNS when using RADA self-assembling peptide with motor neurons for SCI in rats [108]. 3.2.3

Functionalized RADA for CNS treatment

In addition to deliver cells, RADA self-assembling peptide scaffold was used to deliver bioactive factors in CNS therapy. For example, CT04, a cell permeable RhoA inhibitor, was successfully incorporated into RADA and implanted in a complete transection lesion at T9 level of the spinal cord [109]. The combination of RADA peptide and CT04 not only reconstructed the gap in the injured spinal cord and provided regrowth-promoting scaffold reducing the physical obstacles after injury, but also exerted inactivation of RhoA to ameliorate the hostile microenvironment of perilesional site to elicit significant axonal regeneration and motor functional recovery. The nanofiber peptide scaffold-based delivery of cells or bioactive factors is a potentially effective therapeutic strategy by reknitting lesion gap, attenuating secondary injury and improving axonal regeneration. Another important advantage of self-assembling peptide scaffold is its ability to be tailor made to conjugate biologically active peptide motifs to provide specific biological stimuli spatially and temporally. A class of bone marrow homing peptides stimulated adult mouse NSCs adhesion and differentiation in vitro and enhanced neural regeneration in vivo [110–112]. Gelain et al. injected 1% BMHP1 (DAGGGGPFSSTKT) functionalized RADA (RADABMHP1) peptide immediately after contusion in the rat spinal to bridge the gap. Semi-quantitative reverse transcription and polymerase chain reaction (SqRT-PCR) revealed that RADA-BMHP1 peptide scaffold up-regulated GAP-43, trophic factors, and ECM remodeling proteins 7 d after surgery. Histological analysis showed that RADA-BMHP1 peptide scaffold increased cellular infiltration, basement membrane deposition and axon regeneration within the cyst. BBB test indicated that RADA-BMHP1 peptide scaffold significantly improved both hindlimbs’ motor performance and forlimbshindlimbs coordination. Cheng et al. further encapsulated NSCs with IKVAV functionalized RADA (RADA-IKVAV) peptide scaffold to repair cerebral neocortex loss in brain tissue engineering [113]. The injected RADA-IKVAV peptide solution immediately in situ formed the 3D hydrogel filling up the cavity and bridging the gaps. Histology staining showed that the self-assembling peptide hydrogel not only enhanced survival of encapsulated NSCs but also reduced the formation of glial astrocytes.

3.3

Peptide amphiphile

Another well-known self-assembling peptide nanofiber scaffold is peptide amphiphile (PA) from Samuel I. Stupp group [114–117]. This family of molecules consists of a hydrophobic alkyl tail connected to a short peptide sequence. Upon addition of counterions or pH adjustment, these molecules can spontaneously self-assemble into networks of well-defined cylindrical nanofibers in aqueous environments. The cylinders have a diameter in the range of 6–8 nm and consist of β-sheet assemblies that tend to be parallel to the nanofibers. This process is thought to be triggered by peptide charge screening using counterions and facilitated by the collapse of the fatty acid as well as hydrogen bond formation among adjacent peptide segments [80,118]. Typical structures of PA design are including four key structural features. Block 1 is the long alkyl tail that conveys hydrophobic character to the molecule and makes the molecule amphiphilic; block 2 is the beta-sheet forming segment; block 3 is the charged peptide segment; block 4 is the bioactive segment (epitope). From the biological point of view, the most important feature of this material is the epitope, which imparts versatility by simple alteration of the sequence and presenting density. When PA molecules self-assemble to cylindrical nanofibers from aqueous solution, the epitopes display on the surfaces. Because each molecule contains an epitope sequence at its hydrophilic terminus, the nano-structures assembled in an aqueous medium are able to display bioactive sequence perpendicular to their long axis at nearly van der Walls density. Due to the intensified the epitope density, PA nanofiber scaffold could offer effective biological stimuli for specific tissue need. Many modifications have been made to endow varied biological properties for a variety of tissue engineering applications [119–120]. 3.4

PA peptide hydrogel in CNS regeneration

PA peptide hydrogel was studied extensively in neural tissue engineering [121–123]. PA molecules containing IKVAV sequences were found to induce a very rapid differentiation of neurons and to suppress the development of astrocytes when NPCs were encapsulated in vitro within the 3D nanofiber network [83]. In vivo injection of PAIKVAV nanofibers into a spinal cord compression lesion reduced astrogliosis and cell death, increased the number of oligodendroglia at the site of injury [124]. Furthermore, the self-assembling nanofiber scaffold promoted the regenera-


tion of both descending motor and ascending sensory fibers through the lesion site and resulted in significant behavioral improvement. Tysseling et al. further found that PAIKVAV improved functional recovery after SCIs in two different species (rat and mouse) and in two different injury models (contusion and compression) [125]. However, no significant functional recovery was observed with the injection of PA alone, indicating the IKVAV epitope was essential for the effects on sensory axons after SCIs. Serotonergic fibers were distributed significantly higher caudal to the injury site in the PA-IKVAV group. The PAIKVAV treated group also trended higher both in the total number neurons adjacent to the lesion and in the number of long propriospinal tract connections from the thoracic to the lumbar cord. PA-IKVAV also showed promising in intracranial nerve regeneration. Injection of PA-IKVAV into the hippocampus of transgenic mice model of Alzheimer’s disease significantly improved cognitive impairment and enhanced neurogenesis in the hippocampus [126]. Enzyme-linked immunosorbent assay (ELISA) demonstrated that PAIKVAV also significantly reduced the levels of soluble Aβ40, Aβ1-42. Aβ plaques in both the frontal cortex and the hippocampus were also reduced by the treatment of PAIKVAV according to Thioflavin staining. Interestingly, it is possible to achieve long range alignment of PA selfassembling nanofiber peptide scaffold through a thermal induction process [127]. The anisotropic peptide scaffolds enlarge the application of peptide materials and could also provide directional cues to neurons that can promote and guide neurite growth [128–129]. 3.5 Multidomain peptide and its application in CNS regeneration

Another type of well-studied self-assembling peptide is the multidomain peptides (MDPs) that utilize molecular frustration to control the organization and extent of assembly [130–134]. MDPs consist of an ABA block motif in which the central B contains alternative hydrophobic and hydrophilic amino acids. The alternation pattern allows the hydrophobic amino acids side chains lie on one face of the peptide and the hydrophilic amino acids side chains on the other when in an extended β-sheet conformation creating a facial amphiphile (Fig. 3). In aqueous environment, two of these hydrophobic faces will pack against one another forming a “hydrophobic sandwich” which consequently stabilizes and reinforces the fully extended β-sheet conformation. Once in this con-

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Fig. 3 Proposed model of MDPs nanofiber self-assembly. The nanofiber forms through the assembly of peptides stabilized by βsheet hydrogen bonding down the long axis of the structure. This tape-like structure has a hydrophilic face and hydrophobic face. Two such tapes assemble to bury the hydrophobic interface and present hydrophilic and charged amino acids to the aqueous environment. Alteration hydrophobic amino acid and charged A amino acids result in different degree of self-assembly, morphology and mechanical properties. (Reproduced with permission from Ref. [130], Copyright 2007 American Chemical Society)

formation intermolecular backbone hydrogen bonding can readily take place between additional peptides eventually growing into high aspect ratio fibers. The hydrophobic amino acids in central B domain are the primary driving force for self-assembly. The flanked A block of the MDPs is composed of a variable number of charged amino acids whose electrostatic repulsion at pH 7 and is used to mediate the assembly of central B block. This region makes the peptide soluble in water. The relative sizes of blocks A and B dictate the peptide secondary structure which in turn controls the resulting nanostructure. When forces favoring assembly (block B) are properly balanced with forces favoring disassembly (block A) discrete individually dispersed and fully soluble nanofibers with controlled length. At the same time, these charges provide a handle to control the second step of selfassembly into a cross-linked hydrogel through the use of oppositely charges ions. Self-assembling MDPs can tolerate a wide variety of amino acids in the B region as long as the alternating preference for water is preserved. The amino acids in the hydrophobic core can either be aliphatic amino acids or the aromatic amino acids [135]. The nanofiber morphology, as well as the mechanical properties of peptide hydrogel, can be controlled by the selection of amino acids. In addition, like RADA and PA peptides, the strong assembly forces of MDPs permit the incorporation of bioactive motifs such as cell adhesion sequences for tissue engineering applications [136].

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K2(QL)6K2, a typical representative of MDPs, affords optimal self-assembly properties among the family. K2(QL)6K2 self-assembles into β-sheet at neutral pH and forms nanofibers approximately 120 nm long, 6 nm wide, and 2 nm high. K2(QL)6K2 peptide nanofiber could be triggered to form hydrogel in the presence of multivalent anions such as phosphate, commonly present in PBS or cell culture media, which makes it possible to trap cells in three dimension. K2(QL)6K2 peptide scaffold alone, without any bioactive adjunct, could provide favorable support for neuronal growth and functional repair post-SCI [137]. In vitro, it was found that K2(QL)6K2 peptide scaffold enhanced neuronal differentiation and suppressed astrocytic development of NPCs. In vivo, K2(QL)6K2 peptide scaffold led to a significant reduction in post-traumatic apoptosis, inflammation and astrogliosis. Zhao et al. further combined K2(QL)6K2 peptide scaffold and NPCs, and injected them into SCI site [138]. NPCs and peptide scaffold were distributed both caudal and rostral to the injury site, and behavioral analysis showed that the combinatorial transplantation significantly improved locomotor score. It is important to note that K2(QL)6K2 self-assembled into βsheet at neutral pH, very close to physiologic pH; when apply directly onto nerve tissue, there is no need to prebuffering to counteract the acidity of the peptides.

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Conclusion and future perspectives

Based on the understanding of rules and interactions governing self-assembly, novel self-assembling amino acids as building blocks have been designed for biomaterials. The modular intrinsic property of β-sheet selfassembling peptides and the strong forces that govern self-assembly makes them amendable to incorporate functional motifs. A various number of bioactive motifs have been added for cell adhesion, differentiation, and migration. With the combinatorial properties of nanofiber structure, high water content, bioactive motifs, and injectability, selfassembling peptide hydrogels achieved desirable therapeutic effects in central nervous system. They are capable of filling the heterogeneous injury defects, and can facilitate cell-scaffold interaction, support cell proliferation, migration, and neural differentiation. Numerous in vivo models have demonstrated that significant neuroprotection and axonal regeneration have been achieved in central nervous injury models after treatment with self-

assembling peptide hydrogel. More importantly, injured sensory and locomotor function was shown to recover in a number of central nervous injury models using different animal species. Collectively, extensively studies evidenced the great therapeutic potential of self-assembling peptide hydrogel in CNS clinical application. Based on the current advances of material fabrication techniques and cell–material interaction knowledge, future directions would consider the introduction of updated topographical cues and more bioactive motifs or growth factors into the peptide materials design. By designing and fabricating scaffolds with combination of several benefits, it is promising that the neural growth-inhibitory environment may be overcome in the lesion sit in the future, and injured central nervous tissue will be successfully repaired with robust and organized nerve regeneration, achieving more significant functional recovery.

Abbreviations 3D AFM AP BBB BDNF CNS DAPI ECM ELISA GFP HE MDP MEMRI

three dimensional atomic force microscopy alkaline phosphatase Basso, Beattie and Bresnahan brain-derived neurotrophic factor central nervous system 4′,6-diamidino-2-phenylindole hydrochloride extracellular matrix enzyme-linked immunosorbent assay green fluorescent protein hematoxylin-eosin multidomain peptide manganese enhanced magnetic resonance imaging NF neurofilament NGF nerve growth factor NPC neural progenitor cell NSC neural stem cell NT-3 neurotrophin 3 OEC olfactory ensheathing cell PA peptide amphiphile PBS phosphate buffered saline PEG polyethylene glycol PLA poly(lactic acid) PLGA poly(lactic-co-glycolic acid) SCI spinal cord injury SEM scanning electron microscopy SqRT-PCR semi-quantitative reverse transcription and polymerase chain reaction


TBI VEGF

traumatic brain injury vascular endothelial growth factor

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[12] Ramer M S, Priestley J V, McMahon S B. Functional regeneration of sensory axons into the adult spinal cord. Nature, 2000, 403(6767): 312–316

Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant Nos. 51303119 and 51203108), Natural Science Foundation of Jiangsu Province (BK20130309, BK201341421, BK2011355), Natural Science Foundation of the Jiangsu Higher Education Institutions (13KJB430019), National Science Foundation for Post-doctoral Scientists of China (2013M541724 and 2014T70545), Tsinghua University Initiative Scientific Research Program (20121087982), and 973 Program (2011CB606205).

[13] Philips M F, Mattiasson G, Wieloch T, et al. Neuroprotective and behavioral efficacy of nerve growth factor-transfected hippocampal progenitor cell transplants after experimental traumatic brain injury. Journal of Neurosurgery, 2001, 94(5): 765–774 [14] Kaplan G B, Vasterling J J, Vedak P C. Brain-derived neurotrophic factor in traumatic brain injury, post-traumatic stress disorder, and their comorbid conditions: role in pathogenesis and treatment. Behavioural Pharmacology, 2010, 21(5–6):

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