Mimicking Extracellular Matrix via Engineered Nanostructured Biomaterials for Neural Repair.

Extracellular matrix (ECM) consists of proteins, proteoglycans, and different soluble molecules. ECM provides structural support to mammalian cells. ECM is responsible for important cell functions, as well as assembling cells into various tissues and organs, regulating growth and cell-cell interaction. Recent studies have shown the potential of nanostructured biomaterials to mimic native ECM. Developing tailor-made biomaterials that mimic the complex nanoscale mesh of local ECM is not a trivial endeavor: bio-inspired biomaterials are designed to supply a healthy ECMlike structure, capable of filling the lesion cavity, favoring transplanted cell engraftment, providing physical support to endogenous neurogenesis and also tuning the inflammatory response to protect spared neurons. The strategies used to manufacture biomimetic hydrogel scaffold represent particularly important prospects of novel therapies for CNS regeneration. During this review, we describe with details the most promising regulatory pathways from ECM involved in the CNS injury and regeneration and we draw a line to the biomimetic potential of engineered nanostructured biomaterials aimed at mimicking extracellular matrix constructs and favoring the release of pro-regenerative agents. Lastly, a brief overview of their application in clinical trials is provided. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.

INTRODUCTION

Spinal cord injury (SCI), traumatic brain injury (TBI), ischemic stroke, and malignant gliomas are the leading cause of morbidity and mortality in the United States (US) [1-3]. Unfortunately, their damages and subsequent complications have enormous physical and sociological consequences on the affected patients.

About 18.000 new SCI cases occur each year, and approximately 291.000 people are living with chronic SCI in the US [4]. Instead, 155 people under 45 years of age expire every day because of TBI, becoming the principal cause of death and disability [5]. Cerebrovascular diseases kill 140.000 American people each year, while incidence and mortality are projected to continue to increase in low- and middle- income countries [6]. On the other hand, 700.000 people in the US are living with brain cancers, and one-third of malignant brain tumors are diagnosed each year [7, 8].

After trauma, CNS is unable to revert axonal regrowth as an excessive deposition of extracellular matrix (ECM), such as an upregulation of chondroitin sulfate proteoglycans

(CSPGs) takes place, and acts as a physic-chemical barrier against axonal sprouting [9-12].

During development, CSPGs help with establishing the formation of synapses, while in adulthood they are largely responsible for the formation of gliotic scar barriers in the injured brain and spinal cord, thus hampering any spontaneous nervous repair [13, 14].

Removal of CSPGs has been shown to increase neural regeneration in injuries at the CNS: therefore, treatments focused on the demolition of the inhibitory glial scar at the site of injury are becoming of growing interest [15-17].

To this purpose, novel biomaterials have been increasingly used as scaffolds to support the development of controlled depot systems for pro-plastic agents [18], but also for providing physical support and biochemical cues fostering axon regrowth [19].

Nanostructured scaffolds mimic the native ECM in terms of both nanostructure and biomimetic functionalities so as to foster CNS regeneration after injury or disease [20, 21]. They can be employed for: 1)the investigation of still unknown mechanisms acting in healthy and diseased individuals (e.g. in organoids mimicking living tissues in vitro and for drug screenings); 2) as substrates triggering nervous fiber migration and extension; as carriers for 3) cell transplants, 4) pro-plastic agents or cytokines to be released in situ.

In this review, we focus on recent progresses in the understanding of the several ECM-related pathways involved in nervous regeneration, and we present a series of biomimetic nanostructured scaffolds acting as biomimicry of ECM organization and functions: precious tools to improve the understanding of pathological mechanisms and to develop new treatments. In summary, we discuss the role of ECM composition and topographical signals in neural regeneration and we correlate it to the study and development of new optimized nanomaterials for CNS regeneration.

ECM, a Key Regulator of CNS Plasticity

The neural ECM account approximately 20% of the brain and spinal cord [22]. Synthesized by resident cells, the ECM is a dynamic three-dimensional “scaffold” composed of proteins and polysaccharides that modulate receptor binding on the cellular surface, neural plasticity, and axon regeneration in terms of injury repair, moderate damage from mechanical stressors when strained [23, 24].

The interstitial matrix stands for the ECM that surrounds the cells in a tissue, while the pericellular matrix refers to the ECM that is in intimate contact with cells. In both development and disease states, the ECM components, acting as ligands, can be seen as pivotal regulators for the retention and controlled release of growth factors, cytokines and chemokines [25-27].

The composition of the ECM is tissue- and organ-specific: it can largely vary from one body site to another, and from one physiological state to a pathological one [28]. These differences in composition are the result of continuousundergoing controlled remodeling, essential to provide the tissue with the necessary biomechanical and chemical properties [29].

For example, after injury, an upregulation of neural ECM production is considered a protective mechanism as it acts as a physical barrier decreasing the diffusion of membrane-associated molecules from the injured area to other part of CNS [30-32].

Neural ECM is composed by fibrous proteins (collagen, elastin), glycoproteins (laminin, fibronectin) and polysaccharides (glycosaminoglycans, CSPGs). The CNS is wealthy in numerous subtypes of CSPGs. The main component of CSPGs are sulfated glycosaminoglycans (GAGs) attached to a variable core protein: GAG side-chains are characterized by negative charge conferred by sulphation of acidic sugar residues [33]. As mentioned before, the expression of many ECM-related molecules, such as CSPGs, is substantially upregulated following injury, resulting in a dense isolation of the injured site, inhibiting neural regeneration and causing permanent deficits.

For several months after injury, most cellular components close to the lesion epicenter contribute to CSPG enrichment [34, 35]. For example, reactive astrocytes, mainly present in the white matter, synthesize brevican, neurocan, and phosphacan; while vascular macrophages, activated microglia and endogenous oligodendroglial precursor cells (OPCs) account for the increased expression of NG2 and versican (Table ) [36, 37].

A summary scheme of the Lectican family members in the nervous tissue is illustrated in Fig. (.

Fig. (1)

Schematic classification of the Lectican family members. Aggrecan, versicans, neurocan and brevican share a similar homology of globular domains. These modules are at the N-terminus (G1 domain), and a C-terminus (G3 domain). The GAG side chain is attached to the core protein and varies in number among the different lectican family members. Chondroitin sulfate consists of repeating disaccharide unit composed of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc). Each monosaccharide may be sulfated on different residues. CS-A: carbon (C) 4 of the GalNAc. CS-C: C6 of the GalNAc. CS-D: C2 of the GlcA and C6 of the GalNAc. CS-E: C4 and C6 of GalNAc. (A higher resolution/colour version of this figure is available in the electronic copy of the article).

Biosynthesis, Function and Structure of CSPGs

The ECM is a dynamic microenvironment, constantly subject to enzymatic degradation and molecules deposition from the cells [39]. Soluble hyaluronan-binding CSPGs, the main components of neural ECM [40, 41], are characterized by interaction with laminin, fibronectin, tenascin, HA and collagen [42]. Besides playing an important role in the process of neurite outgrowth, CSPGs are essential for their ability to bind extracellular signals and cellular receptors [43]. Indeed, at the N-terminal of the core protein, there is a globular domain (G1), responsible for mediating CSPGs linkage to hyaluronic acid (HA). Instead, the C-terminal comprises an epidermal growth factor (EGF) domain and a complement regulatory-protein-like domain, complemented with a c-type lectin binding domain (G3) that allows the linking of simple sugars, other GAGs, and glycoproteins (Fig. ) [43]. Most importantly, many of the functional properties of CSPGs are attributed to the interaction between attached GAGs side-chains and cell surface receptors [29].

The length of polymer GAGs may vary, based on the repetition of a disaccharide unit, from a sugar chain of just 10kDa to 40 kDa, while the core protein (ranging from 300 to 1700 amino acids) can have a variable number (from 1 to over 100) of members of GAG-chains attached, resulting in a broad range of functional complexities (e.g. aggrecan (120 binding sites), versican (20 binding sites), neurocan (7 binding sites) and brevican (3 binding sites). In addition, different types of GAGs may be crafted as a result of sulphation and epimerization modifications [44, 45]. The sulfated CS moieties [46] are a class of GAGs made of linear polysaccharides, including repeating disaccharide units composed of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) [47]. Polyanions-rich GAGs (GlcA and GalNAc) bind polycations and cations like Na+ and K+, attracting water molecules by osmotic pressure into ECM and forming a hydrogel-like meshwork with remarkable high-water content. However, in the literature, the stiffness role of GAGs remains discussed [48]. Indeed, the stiffness of nervous tissue can alter neural development. For example, axons preferentially grow towards a less stiff environment. By contrast, in a more challenging environment, the axons avoided this area [49]. Lastly, by limiting the passage of massive macromolecules into ECM, and permitting relatively fast diffusion of tiny molecules, the GAGs also maintain tissue boundaries, direct differentiation, and drive cell migration [50, 51].

The process of CS synthesis is enzymatically controlled: it begins in the endoplasmic reticulum (ER), and finishes in the Golgi apparatus, where Chondroitin synthase enzyme mediates the CS addition to the protein [52-55] (Fig. ).

In addition to such heterogeneity coming from the different chain lengths, CS chains may present significant structural differences due to sulfotransferases (SULTs). Based on the sulfation pattern, several CS disaccharide units have been identified: CS-A (C4S of the GalNAc); CS-C unit (C6S of the GalNAc); CS-E (C4S and C6S of the GalNAc); CS-D (C2S of the GlcA and C6S of the GalNAc) [50].

In the healthy CNS, the ECM “self-organize” into perineuronal nets (PNNs), specialized mesh-like structures around points of synaptic contact surrounding inhibitory interneurons, and peri-capillary matrix (PCM), surrounding mature neurons and capillaries [56]. In the mature CNS, PNNs act as inhibitors to the formation of new connections in the neural circuitry, and at the same time, they preserve established connections [57]. CSPGs and Hyaluronan (HA) are the major components of PNNs, while tenascin-C (Tn-C), tenascin-R (Tn-R), members of the hyaluronan and proteoglycan link proteins (HAPLNs) family, fibronectin, collagen and laminin are the minor components [58].

PNN content in the juvenile brain comprises an elevated amount of permissive CS-C, decreasing over time and reaching its minimum in the adult brain. CS-A has an opposite trend and is mostly expressed in the PNN of the adult organism.

Pathophysiology after CNS Injury

Various damages, from traumatic to degenerative lesions can compromise the human brain and spinal cord, resulting in relevant complications and reducing life expectations [3, 14, 59].

Numerous axon-inhibitory molecules, classified in myelin-associated molecules and ECM constituents, are upregulated in the injured CNS environment [13, 60].

Within the first 24 hours after trauma, in order to contain damage spreading, the upregulation of CSPGs is quickly stimulated by cytokines and chemokines from activated microglial cells, which play an important role in inflammatory process regulation and CNS homeostasis preservation [61, 62].

Specifically, the synthesis of CSPG molecules have different temporal patterns, and may last up to six months after lesion. Immediately, as early as one day after trauma in the spinal cord of rats, neurocan is firstly produced, followed by brevican and versican. NG2 is usually expressed at one week after injury [31], while after approximately eight weeks, phosphacans reach the peak levels [63].

In addition, following a traumatic insult, the regionalization of CSPGs expression close to lesion area is also variable [64]. Neurocan is expressed at 100 to 500 µm from the lesion area, [62], while brevican is deposited within 300 µm. On the other hand, phosphacan and versican are expressed [64, 65] in a larger area, i.e. up to 600 um from the impact site (Fig. ).

Moreover, role of GAGs may vary depending on their degrees of sulfation: indeed, CS chains can inhibit or promote axon growth depending on sulphation patterns: e.g. the CS-A sulfated GAG chains, are more inhibitory than CS-C sulfated GAG chains to axon regeneration [66, 67]. After SCI and TBI, CS-A unit content material expands in the lesion region and in peri-contusion tissue.

In particular, in contusive SCI and TBI results in a massive death of neurons and glia at the site of injury, cut of ascending and descending axons, and damage to the vasculature. This harm results in vascular disruption at the lesion site that, in turn, results in the release of factors that contribute to the microglial response. Indeed, microglial cells react within hours after injury by accumulating around the lesion epicenter and secreting pro-inflammatory cytokines and chemokines that contribute to the inflammatory response. Astrocytes proliferate, enter a hypertrophic state, upregulate expression of glial fibrillary acid protein (GFAP), and secrete cytokines, chemokines, growth factors and CS-A sulfated GAG chains [68, 69].

Increased inflammation results in secondary damage to neurons and oligodendrocytes, as well as axonal dieback characterized by dystrophic endings. Myelin debris and CSPGs, each repressive to axon regeneration, accumulate within the lesion core and also the glial scar. Hematogenous macrophages begin to infiltrate the lesion and attract perivascular fibroblast that becomes independent from blood vessels and contribute to the fibrotic scar formation.

In other different pathological conditions, such as in brain tumors, matrix proteolysis is favored instead of matrix production. In this case, the abnormal growth of the tumor cells is linked to increased degradation of the ECM and the progressive destruction of the normal architecture of the brain. These mechanisms facilitate tumor spreading and metastasis [70].

CSPGs Restrict Axon Growth And Regeneration: Roles of PTPσ, LAR1, and Ng1/3 Receptors

CSPGs negatively regulate the targeting of mitochondria and ER to inhibit the lamellipodial extension and axon regeneration of neuronal cell spreading [71, 72].

High-affinity of CSPGs acts by binding several receptor proteins, including two transmembrane proteins of the Leukocyte antigen related (LAR) phosphatase subfamily, Protein Tyrosine Phosphatase σ (PTPσ), Nogo Receptor 1 (NgR1) and NgR3 [16].

LAR and PTPσ belong to the subfamily of PTP receptors, commonly found on growth cones and also involved in axonal guidance during embryogenesis as well as after injury [73]. PTPσ interacts with the negative charges of GAG chains through a positive charge region of Ig-like domain, inducing dephosphorylation of tropomyosin-related kinase B (TrkB), and are responsible for transducing CSPG repulsion which leads to a down-regulation of dendritic spine formation [74-76].

LAR interacts with CSPGs similarly to PTPσ, leading to the inactivation of Akt and RhoA, thus inhibiting axonal growth directly (Fig. ) [77].

NgR1 and NgR3 induce neurite outgrowth inhibition through the activation of intracellular RhoA – RhoKinase, while Akt-GSK 3beta and Ras-Raf-MEK-ERK pathways are inactivated (Fig. ) [78]. Together with PTPσ, they bind to monosulfated CS-B, the disulfated CS-D and CS-E but not to CS-A or CS-C [74].

Plasticity Following CSPGs Modification in CNS Injury: the Function of Cell Adhesion Molecules in Neurite OUTGROWTH

Glial Scar formation is a pathological hallmark of CNS injuries that defines severe tissue damage [79].

The ECM is a rich source of signaling molecules and has been increasingly recognized as an important regulator of proliferation, differentiation, survival, migration of neurons, and plasticity in the CNS [80].

Recent studies have shown that CSPGs can actively modulate neural plasticity through specific interaction of CS chains with its binding partners [81]. Diversity in structure of CS chains is correlated to the diverse functions of CSPGs [82]. As mentioned above, CS chains can either inhibit or promote axon regeneration via binding to inhibitory receptors such as PTPσ or facilitatory receptor such as specific cell adhesion molecule (CAM).

CAMs are essential during development for cell migration: they are transmembrane proteins located on the cell surface, mediating cell-cell and cell-ECM interactions. The majority of the CAMs mediate homophilic interactions in which a CAM on the cell surface interacts with the same type of CAM on another cell surface, some CAMs are also capable of heterophilic binding with other CAMs, cell surface receptors or ECM molecules [83].

In particular, Contactin-1, a glycosylphosphatidyl inositol (GPI) – linked membrane glycoprotein is implicated in neurogenesis binding with high affinity for CS-E [65]. Binding of CS-E to contactin-1 induces intracellular downstream signaling and leads to axonal growth [84]. In addition, CS-E acts as a co-receptor and/or reservoir for neurogenic factors, such as midkine and BDNF, and stimulates neurite outgrowth [84].

Instead, phosphacan and neurocan feature high binding affinity for adhesion molecules specific to the CNS, Neural-CAM (NCAM) and neuron-glia CAM (Ng-CAM), thus interfering with their interactions and indirectly inhibiting motor neuron sprouting [43, 85].

STRATEGIES TO MIMIC THE EXTRACELLULAR MATRIX

Biomimetics in Nanostructured Scaffolds

Taking inspiration from the role of resident ECM, advances in regenerative medicine and tissue engineering have played an increasingly prominent role in providing promising “healing effects” on injured nervous tissue [86]. In particular, tissue-engineering strategies were produced to favor transplanted cell engraftment and cytokine release, along with neurotrophic factors, for nervous regeneration [87].

Biomaterials, mimicking biochemical signals withinside the CNS, corresponding to small peptides, glycans, and even proteins or polysaccharides, permits the controlled healing and organization of neural cells to hold out appropriate diagnostic or therapeutic activities [88]. In the CNS, degeneration, injuries to nervous fibers or aberrant extracellular environments can cause a number of diseases [89]. Mechanical properties of the tissues are altered in tissue development and in pathologies: thereby highlighting the important role played by ECM stiffness. Indeed, fibrous tissues become typically stiffer than the initial tissues. While, after skin injury, a rise of ECM production is important for wound-healing processes, the process of gliotic scarring in the CNS results is more complex than in soft tissues. In this regard, neural tissue engineering has been addressing the following aims: 1)functional regeneration of lacking nervous tissue; 2) rescue of broken neural tissue; 3)neural protection of spared nervous tissue from secondary damage [90]. For example, polymeric structures developed for slow-drug release trap the molecule of interest in a natural or artificial polymer matrix to let it release slowly over time: possible strategies for this are physical, ionic entrapments or covalent linking. The control of shape, size and composition of the polymer matrix may also improve the stabilization of the bioactive molecule and facilitate a steady, sustained release over a period of time [91]. Moreover, in terms of host-tissue response, cell encapsulation in nature-inspired biomaterials allows the diffusion of nutrients for the grafted allogeneic or xenogeneic cells, also keeping good biocompatibility and anti-inflammatory effect of the cell-construct [20].

Foreign Body Reaction of Biomaterials in CNS Regeneration

The CNS has restricted regenerative capacity, however, appears tolerant regarding biomaterial implants [92].

One of the most important strategies for the regeneration of CNS is to create an artificial scaffold that mimic the physiological ECM and spatially guides nervous regeneration. The foreign body reaction (FBR), composed of macrophages and immune cells, describes the host’s inflammatory response to implanted material [93].

After biomaterial implantation, immune response could initiate a notable tissue reaction capable of causing additional neuronal loss death [88]. In case of long-term approaches, such response may turn into chronic response, lasting throughout the lifespan of the bioabsorbable implant and further worsening the initial injury. Therefore, the biocompatibility of neural implants has to be carefully designed, weighted and verified [94].

Generally, in neural tissue engineering, efforts are being made thru numerous artificial as well as natural materials, alone or mixed with cells, growth factors or drugs. Natural-based materials used for CNS regeneration encompass purified ECM-molecules, like collagen, fibronectin, hyaluronic acid, and other naturally occurring polysaccharides (alginate, agarose, chitosan) or their combinations. However, they may contain residual molecules or impurities: consequently, a strict control of their composition is difficult and represents a hurdle to clinic application [95, 96]. On the other hand, chemically synthesized materials fail to imitate the complicated morphological form and chemistry of the local ECM and require various artifices to enhance their bioactivity and cellular adhesiveness.

Indeed, to look at host tissue reaction to the implantation of biomaterials in CNS, it’s crucial to recognize that macrophages are responsible for producing numerous biologically active agents to implanted biomaterials. Macrophage interactions with foreign and implanted materials were appreciably studied in the literature [97, 98]. GFAP positive reactive astrocytes are also present around the implants. The post-surgery response of astrocytes is characterized by hyperplasia and hypertrophy of astrocytic cell bodies. Some studies demonstrated that astrocytes positively influence the neural repair process [99, 100, 101]: they facilitate neuronal sprouting and exert a cytotrophic effect on neurons through the secretion of growth factors and guidance molecules such as neural ECM.

Nanomaterials for Management of CNS Injuries and Drug Delivery

The regenerative techniques for the regeneration of injured nervous system are limited and specifically allow partial functional recovery [102], thus, it is vital to broad new and powerful techniques for nervous tissue regenerative therapies.

Indeed, any damage to the CNS is excessive ample to produce neuronal cell death, followed by a cascade of biochemical events that ultimately result in liquefactive necrosis. The necrotic core goes to be basined and isolated by a glial scar.

As discussed above, thanks to their versatile capability and tunable biological behavior, biocompatible nanomaterials (natural or synthetic) are ideal candidates for neural regeneration [102].

From a material point-of-view, the brain can be considered an elastic solid. Measurements of the Young’s modulus of the brain showed its heterogeneousness, varying between 0.1 and 2kPa, and with differences in white and grey matters [103].

Indeed, mechanical properties play a vital role in the evolution of nanomaterials. For example, biomaterial stiffness affects cell spreading, motility and differentiation [104, 105]. The foremost representative example is actually the tailoring ability of the self-assembling peptides (SAPs), which can drastically influence cell fate and dictate 3D cell behavior (Fig. ) [106, 107].

Moreover, they can act as a depot system to mediate the controlled release of active molecules like GFs, neurotrophic and neuroprotective factors [108].

For SCI treatment, biomaterial scaffolds, as a substitute for lost neural ECM, appear considerably attention-grabbing. Ideally, any such material should provide physical support and establish a “biochemically attractive” milieu in order to facilitate cell migration, attachment, axon growth, improve revascularization and be integrated resembling the host tissue-scaffold interface. In preclinical tests, various hydrogels have shown necessary therapeutic potential for SCI repair [109, 20].

Among the synthetic polymers, the methacrylate-derived pHEMA (poly-(2-hydroxyethyl) methacrylate) is a hydrogel exhibiting mechanical properties similar to those of nerve tissue, and has been used primarily in SCI. It provides sensible cell adhesion and neural development once combined with ECM molecules, resembling laminin or collagen [110]. Neurogel, a cross-linked pHEMA hydrogel, has led to implant revascularization and a “bridge” promoting neural structure restoration and reducing glial scarring [111].

Polyethylene glycol (PEG) is a hydrophilic, non-toxic, perishable and bioabsorbable synthetic hydrogel. Injectable and liquid, PEG can be crosslinked by photopolymerization: in the presence of a light source, the photons absorbed by the photo-initiators, such as thiol or norbornene, trigger the formation of free radicals. These reactive molecules react with the vinyl bonds in the pre-polymer PEG, leading to the formation of chemical crosslinks between the polymer chains [112]. Moreover, PEG-based hydrogels can be used to generate implantable microspheres for drug release (e.g. of neurotrophic factors) into the spinal lesion site [113].

On the other hand, since neural cell reacts to electrical stimulation, presumably, promising neural scaffolds should feature relevant electrical conduction to help neurite outgrowth and thereby elevate nerve regeneration. In this regard, conductive smart biomaterials were utilized in nervous tissue engineering. Wu et al. synthesized the conductive biodegradable polyurethane (PU) supported by aniline oligomer that improved the Schwann cells (SCs) myelin gene expression and neurotrophin secretion for peripheral nerve regeneration [114].

In another study, Wu’s group used a conductive PU micropatterned film based on polyglycerol sebacate-co-aniline pentamer: they obtained significant increments of NGF gene expression from Schwann cells and promoted the alignment, elongation, and neurite extension of PC12 cells in in vitro at 7DIV [115].

Moreover, a core-shell composite biomimetic scaffold based on aligned conductive nanofibrous yarns of PCL, silk fibroin, and carbon nanotubes has been prepared to mimic the aligned nerve fiber structure, resulting in a promising biomimetic scaffold for nerve tissue construction. Indeed, after culturing PC12 and dorsal root ganglia (DRG) cells for 3DIV they detected aligned neuronal outgrowth and cell migration along the direction of nanofibers [116].

Instead, self-assembling peptides (SAP) have shown distinctive properties, making them promising candidates for a number of applications [117, 118]. SAPs are capable of forming nano-structured fibers once expose to an external stimulus, such as shifts in ionic strength or pH [119].

The mostly used SAPs are recognized by a brief and repetitive sequence of amino acids. Their sequence contains alternated hydrophilic and hydrophobic residues that permit the peptides to self-assemble into ordered supramolecular nano-architectures, like nanofibers, nanotubes or nanovesicles. The straightforward modulation permits ad hoc aspect for the specific tissue to be regenerated.

Most SAPs are soluble in water, where they form stable β-sheet structures due to their charged aminoacids. Self-assembly, ruling the sol-gel transition, is triggered by a change in ionic strength, pH or temperature. SAPs are able to form a hydrogel scaffold with 99% of water. Hydrophobic interactions amongst the uncharged sidechains of the peptides, as well as the ionic attraction among the positive and negative charges of peptide molecules are essential for self-assembling.

Synthetic SAPs mimic the 3D microenvironments of natural biological tissues and are made of amino acids biodegraded via canonical cellular pathways: consequently, they have a promising biocompatibility.

SAPs sequences can be tuned to reproduce the biomechanical characteristics of the CNS tissue and can be decorated with biologically active functional motifs capable of interacting with cells. Functional motifs can be selected from the literature (e.g. RGD, IKVAV, YIGDSR, etc.) or obtained by running assays with random libraries of peptides [19]. If SAPs are made of alternated residues hydrophilic/hydrophobic sequences, they are usually prone to ß-sheet formation [120]. As such, they can be extended with biological moieties one or both C- and N-termini [121]. Differently functionalized SAPs can self-integrate and give a multi-functionalized scaffold as long as they share the same self-assembling backbone sequence [20]. Functional motifs, interspaced by the self-assembling backbone sequences with 1-5 Gly residues (providing enough flexibility and motif exposure), flank the self-assembled nanofibers and are available for cell binding: thus, they can enhance cellular viability and coax cellular differentiation in vitro. Thanks to phage display technology, functional motifs were selected specifically for nervous tissue regeneration [121]. This technology makes use of libraries of bacteriophage displaying random combinations of short peptides (from 7 to 11 residues) [122-125]. After incubation with target cells, phage expressing sequences with the highest avidity for target cell surface molecules are captured and amplified. Consequently, a phage display library of 7-residue peptides was panned against the progeny of differentiating murine NSCs. Then, SAPs were functionalized with the discovered sequences at N-terminal of the sequence [106].

Numerous SAPs have been differently used to enhance neural regeneration after traumatic CNS.

One of first attempts to explicitly tackle on the regeneration of brain injury were according by Ellis-Bhenke et al. [126], using (RADA)4, describing a good restoring part of impaired vision in hamsters.

Wu’s research lab demonstrated that SAP-treatment diminished inflammatory response and astrogliosis reducing tissue loss after peripheral nerve injury [127]. Others also tested the (RADA)4 as an efficient vehicle for the delivery and graft of hIPSCs within the brain [128].

Shi functionalized (RADA)4 with RGIDKRHWNSQ motif derived from BDNF to favor MSC differentiation and promoting neuron outgrowth and synapse formation [129].

When used as a carrier for stem cells, (RADA)4 diminished neuroinflammation, injury size and reactive gliosis: suggesting the positive impact given by SAP decoration for the treatment of traumatic brain injury [130].

Sharing, an analogous self-assembly mechanism, LDLK12 has also been widely used for tissue engineering [131-133, 20].

Recently LDLK12 has been functionalized with the addition of assorted functionalizing sequences. In 2012 it had been shown that the SSLSVND amide sequence promotes the variation and differentiation of neural stem cells by influencing cellular activity without making use of additional soluble factors [97]. In 2013, the KLPGWSG functional motif sequence was demonstrated to bind to murine adult NSC and to be involved in stem cell fate determination [134].

In another work, SAPs functionalized with an epitope from Tenascin-C were injected into the rostral migratory stream in rodent adult brains, showing a with successful migration of resident neural progenitor cells to the injured cortex [135].

Intriguingly, in the ischemic brain, Nisbet et al. [136], used SAP functionalized with a laminin-derived motif, amalgamated with BDNF and embryonic stem cells, to improve nervous regeneration and blood vessel infiltration into the implants.

The ability to synthesize nanofibrous scaffolds in the shape of conduits or as aligned microfibers using the electrospinning technique has been widely used in neural tissue engineering [98]. In this regard, biodegradable electrospun scaffolds based on ali- phatic polyesters, such as poly(D,L-lactide-co-glycolide) (PLGA) and poly(ε-caprolactone) (PCL), are widely studied for varied biomedical applications due to their biocompatibility, biodegradability, excellent fiber-forming properties and their approval by Food and Drug Administration (FDA) for clinical usage [137-140].

PCL electrospun nanofibers show remarkable mechanical strength, but also a prolonged biodegradation profile, making them potentially unsuited for neural regenerative therapies where persistent scaffold leftovers might hamper tissue regeneration. Moreover, its inert and hydrophobic nature can affect protein adsorption eventually yielding unfavorable cell adhesion sites. Because of these characteristics, new approaches improving its hydrophilicity, biodegradability and controllable mechanical properties are needed.

A previous study showed that electrospun polymeric guidance channels loaded with functionalized SAPs, once transplanted into the cavity caused by chronic SCI, provided a major growth of freshly formed nerve tissue among and within the guidance channels over a six-month timeframe, fostering functional regeneration and behavioral recovery [98].

Recently, chemically cross-linked SAPs, due to their improved biomechanics, were electrospun into resilient self-standing microchannels entirely made of SAPs with tunable functionalization, flexibility and bioabsorption times to suit the specific need of various neural regenerative applications [141].

CNS Regeneration Via Biomaterials in Clinical Trials

In nanomedicine, bio-inspired nanomaterials are used to not only to produce novel methodologies for targeted drug delivery [142], but also to produce novel scaffolds potentially acting as medical devices.

The intention of research is to line up a regulatory technological know-how basis to support FDA’s regulatory and steering roles in nanotechnology and its effect on the human body.

Five nanomedicine-related clinical trials are approved (Table ). In one of the studies, autologous transplantation of neural stem cells (NSC) with the biocompatible RMx Biomatrix, for traumatic SCI is presently within the recruitment phase (NCT0232666). In another study, a company, InVIvo Therapeutics, is testing PLGA poly-L-lysine-based scaffolds seeded with NSC for the treatment of complete thoracic traumatic acute SCI. Another functional collagen scaffold is additionally being tested for transplantation in acute SCI patients by the Chinese Academy of Sciences (NCT02510365). An analogous clinical trial based on collagen-scaffold-containing MSCs for transplantation in SCI has also been supported by the identical institute (NCT02352077).

The Neuro-Spinal Scaffold is the first safe human implanted porous bioresorbable polymer scaffold in a 25 years old man with T11-T12 fracture dislocation resulted in a motocross accident. The bioresorbable scaffold was implanted into the traumatic cavity of SCI, and after 3 months neurological functions had improved to an L1 AIS C incomplete SCI (NCT03762655).

Another study is intended to assess the role of functional neural regeneration collagen scaffold transplantation mixed with electrical stimulation in SCI patients (NCT03966794).

Biomaterials for the treatment of ischemic stroke and brain tumor are undergoing clinical trials. RGTA are synthetic PGs scaffold mimicking ECM for the treatment of acute ischemic stroke (NCT04083001). The nanomaterial NPt-Ca is used for glioma brain, in a pilot study against malignant brain tumors not showing any improvement to conventional therapies.

CONCLUSION

ECM is related to a range of basic, physiological and pathological processes. Following CNS injury, akin to SCI or TBI, abnormal ECM dynamics lead to tissue fibrosis and congenital defects, where even minor changes in ECM synthesis or degradation can promote pathogenesis. Nanostructured biomaterials with multiple advantages are exploited as tissue engineering tools for several different pathologies in order to boost the efficacy of regenerative medicine strategies.

In addition, nanostructured biomaterials are employed in the controlled release of drugs, growth factors, antibody and implants. Moreover, the functionalization and spatial orientation of ECM mimicking nanofibers can affect cells functions, as supported by altered cellular behavior, as well as stem cell differentiation [143].

In the near future, additional research will help the development of novel scaffolds with tailor-made properties customized for each specific CNS pathology and with high translational potential in clinical settings.

Table 1

Cellular sources of the lecticans. These members are produced by neurons and glial cells.

Cell	Proteoglycan	CNS specific	Location	Refs.
Activated microglial cells	NG2	No	TM & ECM	[36, 37]
Oligodendrocyte progenitor cells	NG2VersicanPhosphacan (in vivo)	NoYesYes	TM & ECMECMECM	[36, 37][36, 37]
Neurons	AggrecanBrevican (in vivo)NeurocanPhosphacan (in vivo)Versican (in vitro)	NoYesYesYesYes	ECMECMECMECMECM	[38, 40][40, 43][35, 43][43]
Astrocytes	BrevicanNeurocan (in vitro)Phosphacan (in vivo)Versican (in vitro)	YesYesYesYes	ECMECMECMECM	[43][43][43]

Table 2

List of ongoing clinical trials with biomaterials used for CNS regeneration.

Study	Clinical Study Identifier	Conditions	Description
Neuro-Spinal Scaffold	NCT03762655	Traumatic spinal cord injury	Porous bioresorbable polymer scaffold composed of (PLGA_PLL)
NeuroRegen scaffolds	NCT02510365	Acute spinal cord injury	Collagen scaffolds with MSCs were transplanted into the injury site
Collagen scaffolds	NCT03966794	Complete spinal cord injury	Collagen scaffold transplantation combined with epidural electrical stimulation for spinal cord injury repair
RGTA (ReGeneraTingAgent)	NCT04083001	Acute, ischemic stroke (AIS)	Injectable medical device of synthetic polysaccharides mimicking HSPGs in anterior circulation AIS
Nanomaterial NPt-Ca	NCT03250520	Glioma	Administration of Platinum acetylacetonate (1% wt) supported by sol-gel technology functionalized titania in High grade recurrent brain tumor