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Biomaterials 34 (2013) 4945e4955 Contents lists available at SciVerse ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials Review Using extracellular matrix for regenerative medicine in the spinal cord Fabio Zomer Volpato a, b,1, Tobias Führmann a,1, Claudio Migliaresi b, Dietmar W. Hutmacher a, Paul D. Dalton a, * a b Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Ave, Kelvin Grove 4059, Australia BIOtech Research Centre, University of Trento, 101 Via delle Regole, Trento 38123, Italy a r t i c l e i n f o a b s t r a c t Article history: Received 22 January 2013 Accepted 20 March 2013 Available online 15 April 2013 Regeneration within the mammalian central nervous system (CNS) is limited, and traumatic injury often leads to permanent functional motor and sensory loss. The lack of regeneration following spinal cord injury (SCI) is mainly caused by the presence of glial scarring, cystic cavitation and a hostile environment to axonal growth at the lesion site. The more prominent experimental treatment strategies focus mainly on drug and cell therapies, however recent interest in biomaterial-based strategies are increasing in number and breadth. Outside the spinal cord, approaches that utilize the extracellular matrix (ECM) to promote tissue repair show tremendous potential for various application including vascular, skin, bone, cartilage, liver, lung, heart and peripheral nerve tissue engineering (TE). Experimentally, it is unknown if these approaches can be successfully translated to the CNS, either alone or in combination with synthetic biomaterial scaffolds. In this review we outline the first attempts to apply the potential of ECM-based biomaterials and combining cell-derived ECM with synthetic scaffolds. Ó 2013 Elsevier Ltd. All rights reserved. Keywords: Spinal cord injury Tissue engineering Extracellular matrix Decellularization 1. Introduction Unlike most other organs/tissues, the central nervous system (CNS) has a limited capacity to regenerate and traumatic injuries to the CNS are associated with a poor prognosis. Injuries to the spinal cord lead to functional motor and sensory loss by disrupting long distance projecting axons in white matter tracts, so far no widely accepted treatment strategies are available [1]. The only commonly used interventions are surgical stabilization of damaged vertebrae and intensive rehabilitation [1]. Different experimental treatment strategies are under investigation to promote recovery after spinal cord injury (SCI), with cell and/or pharmaceutical delivery as the most common approaches [2,3]. Biomedical materials are used in regenerative medicine approaches to replace or restore the anatomic structure and function of damaged or missing tissues following any injury or disease by combining the topographical cues of biomaterials with cells or bioactive molecules [1]. Tissue engineered scaffolds intended for CNS repair are often based on particular extracellular matrix (ECM) molecules (e.g. fibrin, collagen, fibronectin [4e6]), other natural polymers (alginate, agarose, chitosan [7e9]) or synthetic polymers (e.g. poly(-hydroxy acids), poly(2-hydroxyethyl methacrylate), * Corresponding author. Fax: þ61 7 3138 6030. E-mail address: [email protected] (P.D. Dalton). 1 These authors contributed equally to this work and are first authors. 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.03.057 polyethylene glycol [10e12]). While natural/synthetic polymer blends are widely accepted as potential materials [13,14] the use of ECM grafts is uncommon [15e19]. However decellularized tissue grafts used for regeneration in other systems (e.g. peripheral nerves [20,21], heart [22], and skin [23]) have shown great promise. Here we discuss the possibility of combining orientated scaffolds of synthetic biomaterials with cell-derived ECM to promote CNS repair. 2. Pathophysiology of SCI The severity of SCI depends on the level, type and intensity of injury but includes permanent locomotor and sensory deficits, and may lead to neuropathic pain, spasticity, urinary and respiratory dysfunction, metabolic problems as well as psychological problems. The primary, mechanical injury leads to damage of numerous nerve fibre pathways in the white matter and possibly also cells in the grey matter. The mechanical disruption of the highly organized cytoarchitecture of the spinal cord has devastating effects on both the primary injury zone and the injury parenchyma. The primary injury is followed by secondary degenerative events including apoptosis, bleeding, excitotoxicity, free-radical production, inflammation, ischemia, oedema, scarring and cystic cavitation, all of which contribute to an increase in tissue loss [24,25]. Inflammatory processes are a major contributor to secondary degeneration, which take place over a time scale of hours, days, weeks and even months after the injury [26]. Microglia and also 4946 F.Z. Volpato et al. / Biomaterials 34 (2013) 4945e4955 astrocytes represent intrinsic immunocompetent cells. If the bloodspinal cord barrier is disrupted, extrinsic inflammatory cells are recruited (neutrophils, macrophages, monocytes, lymphocytes, and natural killer cells). These inflammatory cells express proteolytic enzymes which degrade ECM proteins and contribute further to bloodebrain barrier degradation and oedema. Inflammatory cells and (to a lesser degree) astrocytes express a wide range of proinflammatory cytokines and chemokines (e.g. interleukin (IL)-a, interferon (INF)-g, tumour necrosis factor (TNF)-a) as well as reactive oxygen species, oxidative enzymes, and metalloproteinases, which exacerbate secondary tissue damage and the formation of an inhibitory glial scar [27,28]. However, the immune response may also contribute to tissue repair by clearance of hemorrhagic and necrotic tissue, reducing the lesion size and the release of trophic factors and cytokines which can be neuroprotective and promote axonal regeneration (e.g. interleukin (IL)10, transforming growth factor (TGF-b)) [27,29]. The final, chronic stage of SCI is characterized by Wallerian degeneration, death of oligodendrocytes, fluid-filled cyst formation within grey and white matter and the development of the glial scar [3,25]. The glial scar represents a major mechanical and chemical barrier (e.g. chondroitin sulphate proteoglycans (CSPGs)) to axonal regeneration [30]. In lesions that spare the dura mater, the scar is composed primarily of astrocytes, but in more severe lesions (which open or rupture the meninges) invading connective tissue elements (e.g. fibroblasts, endothelial cells, ependymal cells) intermingle with the astrocytes [31,32]. Furthermore, depending on lesion type and severity, Schwann cells migrate from the adjacent nerve roots into the lesion side and come into close contact with the reactive astrocytes [31,32]. In the end, this glial scar encapsulates a cavity, which can be many times the size of the initial wound [33]. Fig. 1 shows such a fluid-filled cavity six days after a unilateral compression injury to the rodent spinal cord. Surrounding the edge of the cavity, an intense staining of GFAP can be seen, which identifies the intermediate filaments within astrocytes. It is important to note that GFAP, a common marker for astrocytes, identifies internal filaments of this cell. The cell membrane of astrocytes, however, is markedly larger and the density of such reactive astrocytes at the edge of the cyst is particularly intense. Internally, such a cyst is almost exclusively filled with activated macrophages at this time point, emphasizing the role that inflammation plays in the cyst formation. There is also recent evidence that intermediate filaments of glial cells significantly contribute to the local stiffness of the tissue. Kas and colleagues correlated the GFAP intensity with the viscoelastic properties of glia-rich retinal slices that underwent ischemia [34]. It is an important finding since increasing the stiffness of glial scar tissue would further impede any neurite growth into this region. Conversely, the formation of a glial scar has a beneficial role during the acute phase (1e2 weeks) after SCI. When reactive astrocytes were eliminated or prevented from contributing to scar formation after CNS injury, the result was a failure in bloodebrain barrier repair, accompanied by massive inflammatory cell infiltration, increased loss of neurons and oligodendrocytes, and the eventual worsening of the functional outcome [35,36]. The lack of any functionally significant axon regeneration is mainly due to an imbalance of local axon growth-promoting and growth-inhibitory mechanisms. Neurotrophic factors and guidance Fig. 1. AeC: Immunohistological sections of the rodent spinal cord six days after injury showing a debris-filled cyst, surrounded by a dense glial scar, which is impermeable to regenerating axons. B: Higher magnification of the glial scar with GFAP staining. C: Glial scar stained for CD68þ cells, which are activated macrophages/microglia. The insetin C is luxol fast blue that stained myelin and visualizes the white and grey matter, with the red line showing the plane of sectioning for AeC. D: injection of a single astrocyte with a florescent dye demonstrates the volume of an astrocyte. E: Glial scar at 4 weeks stained for neurocan, an ECM known for inhibiting neurite outgrowth. AeC: Unpublished Images. D: Republished with permission of the Society for Neuroscience, from Wilhelmsson et al. [210]; permission conveyed through Copyright Clearance Center, Inc. and E: Reprinted from Huang et al. [211] with permission from Elsevier. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) F.Z. Volpato et al. / Biomaterials 34 (2013) 4945e4955 cues (e.g. from the ECM) are only poorly expressed at the lesion site, while the presence of potent molecular (myelin inhibitors: NOGOA, myelin-associated glycoprotein (MAG), oligodendrocyte-myelin glycoprotein (OMgp)) and physical barriers (glial scar) prevent axonal regeneration [31,37]. The CNS contrast with injuries of the peripheral nervous system, which has the capacity to regenerate significant distances; nerve gaps of approximately 4 cm in humans can be bridged with nerve guidance channels [38]. Such a “critical gap” equivalent for the spinal cord has been described as overcoming scar and myelin mediated inhibition[31]. 3. Natural ECM of the CNS The ECM contributes only approximately 20% to the total tissue volume within the CNS [39]. While structural proteins such as elastin, collagen and fibronectin are the major component of most tissues’ ECM, the uninjured adult CNS only sparingly contains these elements. The ECM of the spinal cord is composed mostly of glycosaminoglycans (hyaluronic acid) and proteoglycans (agrin, aggrecan, brevican, neurocan, phosphacan, versican) [40]. Other components include laminin, nidogen, reelin, netrin-1, tenascin-C and tenascin-R, Slit-1, -2, as well as attached growth factors like fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF). A schematic drawing of the major ECM proteins of the CNS is shown in Fig. 2. Additionally, a specialized extracellular matrix called perineuronal nets (PNN) can be found in the maturing CNS [41,42]. PNN are layers of condensed pericellular matrix surrounding the cell body and dendrites of many neurons. They control and stabilize the formation of synapses and connections [43,44]. The main components of PNN are hyaluronan (HA), link proteins, CSPGs and tenascin-R [41,43]. It is thought, that this structure plays an important role in CNS regeneration and its formation is directly correlated to decrease in neuron plasticity. Since the PNN are considered a specialized form of the ECM, they will be combined and described as ECM in the context of this review. For a detailed overview on ECM and PNN see Refs. [45] and [44], respectively. In addition to the storage of cytokines and growth factors, the ECM can also regulate their activity [46]. In the neural stem cell (NSC) niche, FGF-2 binds to N-sulphated heparan sulphate proteoglycans (HSPGs) to help maintain the NSC phenotype [47]. Moreover, intracellular signalling activated by integrins can modulate pathways activated by cytokines [48,49]. Another mechanism to regulate cellular function by the ECM is transduction of mechanical signals. CNS ECM molecules have also been recognized for being multivalent, which means that they can bind to either identical or multiple other ECM ligands showing opposite 4947 effects. An example is tenascin-C that has both adhesive and antiadhesive domains, each recognized by different receptors, and specific binding interactions may engender contrary effects (i.e. tenascin and tenascineglycoprotein conjugates may either promote or inhibit axonal growth) [50]. It is important to appreciate that the ECM of embryonic, postnatal and adult spinal cord tissue is different. For example, PNN appear only late in development and are related to the increase in neural activity [44]. Furthermore, laminin (e.g. laminin 111 or a1b1g1 e previously known as laminin-1 [51]) a widely used ECM molecule for promoting neurite growth in vitro, is widely expressed during early embryogenesis, but not usually in contact with neurons during adulthood [52,53]. In total, there are at least 15 different isoforms of laminin in vivo; this diversity and distribution (both location and in time) of laminin demonstrates the complex and specific function of this extracellular constituent. 4. ECM of the glial scar The injury-induced glial scar is both a physical and chemical barrier to axonal regeneration. The chemical nature of the glial scar in particular has been the subject of considerable research, yet our understanding is not complete [54]. The glial scar in vivo differs in distribution and type; and its major constituents (e.g. tenascin, HSPGs, CSPGs and keratan sulphate proteoglycans (KSPGs)) are mainly inhibitory for neurite outgrowth [55e57]. These ECM molecules are already abundant in the normal CNS but are highly upregulated during gliosis, e.g. by astrocytes and fibroblasts [55]. Structurally, CSPG contains a single core protein and one or more polysaccharide glycosaminoglycan (GAG) chains, which are the main inhibitory part. Examples of CSPG implicated in hindering regeneration after injury include aggrecan, brevican, neurocan, neuronglial antigen 2 (NG2), phosphacan and versican [55,58]. CSPGs suppress axonal regeneration via negatively charged sulphate [59] or via binding and blocking growth-promoting effects of some ECM molecules, such as laminin and tenascin [60,61]. Most axonal growth inhibitors in the CNS limit axonal regeneration via binding their interacting receptor proteins on the membrane [62,63]. Two studies indicate that receptor protein tyrosine phosphatase s (RPTP s) acts as a receptor for CSPGs and partially mediates the ability of CSPGs to restrict regenerative neurite outgrowth [64,65]. Most recently, the leukocyte common antigenrelated phosphatase (LAR) was recognized as a functional receptor for CSPG axon growth inhibitors [66]. Nevertheless, axon “growth-promoting” molecules are also expressed (e.g. by reactive astrocytes), for example laminin, fibronectin, decorin, and poly-sialylated neural cell adhesion Fig. 2. Schematic drawing of the major ECM proteins of the CNS. 4948 F.Z. Volpato et al. / Biomaterials 34 (2013) 4945e4955 molecule (PSA-NCAM) have also been reported [31,67,68]. Neurotrophins (e.g. nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF)), embedded within the ECM, support neuronal survival and differentiation, guide axonal growth, and regulate synaptic plasticity in a variety of neuronal populations in vitro and in vivo[69,70]. The effect of ECM on axonal growth is complex and understanding interactions depends on its context. In general, the growth cone extension within the ECM environment is affected by the 1) isoform of the ECM molecule, 2) combination of ECM molecules, 3) age of the neuron, 4) type of the neuron and 5) recent history of the growth cone environment. While the ECM of the CNS can be broadly considered as “inhibitory” or “growth promoting”, the reality is different since ECM molecules interact with each other to control this effect. For example, an “inhibitory” ECM molecule (such as phosphacan, neurocan) can inhibit neurite growth, yet promote outgrowth when combined with another ECM molecule (such as fibronectin) in vitro. “Growth-promoting” ECM can also inhibit neurite growth through strong adhesion, diminishing cell-membrane release and this can be regulated during development. For example embryonic neurons on laminin in vitro can regulate their integrin receptors significantly, controlling the extent of adhesion, however adult neurons are less capable of this, and growth can actually be slowed on laminin through excessive adhesion [71,72]. Fig. 3 schematically shows how intermediate concentrations of ECM provide the most neurite outgrowth. Although the expression of both growth-inhibitory and growth-promoting ECM molecules increases after injury, overall the environment is inhibitory to regeneration [73]. Therefore to induce regeneration the final composition of growth-promoting and growth-inhibitory molecules has to be balanced towards promoting tissue regeneration. One way to achieve this could be the transplantation of scaffolds made primarily from growthpromoting ECM. 5. Regenerative medicine strategies to promote recovery after SCI Since the pathophysiology of SCI is both complex and resistant to therapeutics, many conceptually different ways to promote recovery have been attempted, including cell-based therapies, gene therapy, drug, antibody and growth factor delivery [3,74]. Cellbased therapies include the transplantation of Schwann cells [75,76], olfactory ensheathing cells (OECs) [77,78], astrocytes [79,80], neural progenitor cells [81,82], mesenchymal stromal cells [83,84], or induced pluripotent stem cells (iPS) [85,86] either alone or in combination with a growth-promoting scaffold, into the lesion site to promote axon regeneration or tissue sparing. Such cells are capable to produce neurotrophic factors such as NGF, BDNF, neurotrophin-3 (NT-3), ciliary neurotrophic factor (CNTF), glial cellderived neurotrophic factor (GDNF) and leukemia inhibitory factor (LIF), in addition to proteins such as laminins, fibronectin, collagen I/III and IV [87e90], all of which are able to promote recovery after CNS injury. Schwann cells have been the most investigated in cell transplantation studies to promote neural repair after CNS injury, due to their crucial role in PNS repair [76,91e93]. These cell-based strategies have been reported to promote functional improvement in experimental models of CNS, but the mechanism(s) of action remain poorly defined (e.g. Refs. [94,95]). Cell-based therapies are reviewed in depth elsewhere [96,97]. Drug delivery therapies include inhibition of the inflammatory response (e.g. inhibition of leukocyte, macrophage/monocyte, and neutrophil infiltration [98e100], inhibition of inflammatory angiogenesis [101], administration of classical immunosuppressives [102]), stimulation of the inflammatory response (e.g. administration of T-cells, transplantation of stimulated homologous macrophages, vaccination with myelin self-antigens [103e107]), suppression of myelin-associated inhibitor molecules (e.g. NOGO-A, MAG, OMgp) and its pathways [108e111], and CSPGs digestion (e.g. administration of chondroitinase ABC (ChABC) or hyaluronidase) [112e114]. Reports Fig. 3. Both ‘growth-promoting’ and ‘growth-inhibiting’ molecules may prevent migration of growth cones in regions of injury. (a) For non-neuronal cells, adhesion to the substratum increases linearly with amount of bound matrix protein, yet peak motility is only supported by a narrow range of adhesion states. Low levels of attachment/matrix inhibit motility by reducing traction (grip) while high levels of attachment/matrix inhibit motility by reducing the ability of cells to release from the substratum. (b) In regions of CNS injury, growth cone motility may be reduced by more than a single mechanism. Factors providing mechanical barriers to migration may physically block growth cone advance (red). Factors that antagonize the function of cell-matrix receptors or directly induce growth cone collapse will prevent migration by reducing traction (blue). ECM molecules and receptors that increase growth cone adhesion will prevent migration by preventing release from the substratum (orange). Finally, high levels of neurotrophic molecules (i.e. biglycan) or molecules that recruit growth factors (in italics) may retain growth cones in regions injury due to high trophic support (yellow). Reprinted from Condic et al. [71] with permission. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) F.Z. Volpato et al. / Biomaterials 34 (2013) 4945e4955 suggest that inhibition of inflammation promotes tissue sparing and improves functional recovery in animal models of SCI [100,101,115]. Axonal growth-inhibitory molecules such as Nogo, MAG, CSPGs, activate the Rho pathway [108e111]. Therefore inhibition of the Rho/ ROCK signalling pathway demonstrated beneficial effects through modulation of the inflammatory response after injury [116,117]. Nevertheless, there are negative effects associated to the inhibition of such signalling pathways, i.e. increased spinal cord tissue atrophy [118], decreased axonal sprouting/regeneration, impaired functional recovery [119,120], and increase astroglial activation and CSPGs deposition [119]. The administration of ChABC has shown to effectively degrade CSPGs present in the ECM [112,113], allowing significant axon regeneration both in vitro and in vivo. The complexity of the CNS and its response to injury dictates that the latest advances in bioengineering and macromolecular chemistry have to be implemented in the design of scaffolds intended to promote CNS tissue repair. Researchers have developed multi-therapeutic strategies, combining various elements to promote repair [76,121e123]. Biomaterials are well-positioned to deliver such combinatorial approaches for SCI repair, matrices or scaffolds have been used in many of the strategies outlined above. Biodegradability, mechanical strength, channels/fibres, porosity, capability of cell adhesion and electrical activity are some of the parameters that affect the regenerative capacity of a given design [1,124]. A number of general issues may need to be considered for the choice of biomaterial and the design of scaffolds, including: 1. Biocompatibility, minimizing adverse tissue reactions in vivo; 2. Topographic cues for orientated tissue integration and cellular stimulation; 3. Cytocompatibility, promoting optimal cell adhesion, migration and axon outgrowth; 4. Physical properties (e.g. elasticity, strength, tenacity) that approach those of the host tissue; 5. Biodegradability with the production of non-toxic by-products. Any tissue engineered scaffold implantation would require major surgery and might not be suitable for many types of spinal cord injuries. Injuries where the cord is lacerated (e.g. gun or knife injuries) are the most likely candidates for the application of such an approach. However, there is a significant opportunity to implant and understand fundamental tissue engineering (TE) scaffold integration within the CNS. Therefore, the immediate need for degradability of these scaffolds/materials remains open for discussion. While few researchers will argue for a non-degradable clinical product, the majority of current SCI in vivo models extend out to only 4e6 weeks. With such short time periods, rapid degradation may induce localized responses which may the lead to poor outcomes [125]. Since many significant challenges remain with SCI (i.e. robust regeneration/repair, migration of fibres out of scaffolds), degradability remains an optional requirement for biomedical materials in the spinal cord. One important factor when designing biomaterials is that their mechanical properties are similar to the spinal cord. Soft, high water content hydrogels are the more widely used materials and significant in vivo data supports their use. As one example, the Shoichet laboratory developed hydrogel guidance channels that were mechanically similar to the spinal cord [11,126] to contain and protect a particular therapy. These “guidance channels” wrap around the proximal and distal stumps of transected spinal cord and can be filled with a gel-like matrix or glial cells [76,92,127]. Surprisingly, these guidance channels supported significant axonal ingrowth, which mainly occurred on the inner surface on the tubes. However, since these channels were soft, they collapsed slightly during implantation, and efforts were made to strengthen them. 4949 However, making the channels more resistant to collapse resulted in syringomyelia of the spinal cord [128]. Therefore scaffolds and biomedical materials used within the spinal cord should be soft and compliant, with suitable mechanical properties otherwise other complications could arise. The elastic modulus of the spinal cord (including pia/dura) is approximately 230 kPa [11], while that of the grey/white matter is 2e5 kPa [129]. 6. ECM as scaffolds/matrices for regenerative medicine Biomaterials science includes the implantation of artificial, 3D, highly orientated and biodegradable scaffolds [121,130] into the injured spinal cord. Such oriented, cell-invasive scaffolds are often derived from natural polymers, including ECM-molecules [6,8], synthetic polymers [11] or combinations of both [13,14]. Treatment strategies to promote regeneration of other tissues and organs focus also on decellularised tissues and cells intended to promote regeneration, known as ECM-derived scaffolds (ECM-scaffolds). In the context of this review, these scaffolds do not include scaffolds derived from purified ECM molecules (e.g. collagen, fibrin alone). Such ECM-scaffolds demonstrated tremendous potential in both experimental animal studies and clinical trials in non-CNS organs [21,131e133]. Applications of ECM-scaffolds include blood vessels [134,135], skin [23], bone [136], cartilage [137,138], trachea [132], lung [139,140], heart [22,141,142] and peripheral nerves [20,21]. However, only few preliminary studies tried to harvest this potential for CNS repair [17,18,143,144]. In their normal environment cells are surrounded by ECM [145], therefore three-dimensional scaffolds derived of or functionalized with native ECM are thought to play an important role in regenerative medicine to promote regeneration of new tissues and organs [146]. The ECM functions not only as a supporting material but also as a regulator of cellular functions such as cell survival, proliferation, morphogenesis and differentiation [145,147]. Moreover, the ECM can modulate signal transduction activated by various bioactive molecules such as growth factors and cytokines [48,49]. The ECM molecules and networks change their compositions, structures and biomechanical properties according to each specific tissue and organ. Their compositions and properties also dynamically change and remodel during development [148]. ECM from developing tissue is thought to have greater beneficially effects compared to adult tissue: i.e. immature astrocytes demonstrated a greater ability to promote neurite outgrowth and scaffold integration compared to more mature astrocytes [149,150]. Ideally, the scaffolds and substrates used for regenerative medicine can provide the same or similar macro- and micro-environment to cells as that of an ECM existing in vivo and assemble the infiltrating cells and secreted matrices into functional tissues and organs [151]. If degradable, the scaffolds will be replaced by the cells own matrices during regeneration. An alternative to using the full ECM-derived matrix is the use of purified ECM proteins (e.g. fibrin, laminin, collagen, fibronectin). However, these approaches lack in replicating the morphological structure of native ECM. Furthermore, usually one single protein is applied to the scaffold production, whilst the ECM is a combination of several proteins, growth factors and other cytokines. Various scaffolds or hydrogels based on purified ECM proteins have been developed [1,80,152,153] and used for treatment strategies to promote recovery of the injured spinal cord and are reviewed in more detail elsewhere [1]. Another, even more reductive approach, is the use of short bioactive peptide sequences, such as arginine-glycine-aspartic acid (RGD), tyrosine-isoleucine-glycine-serine-arginine (YIGSR) and isoleucine-lysine-valine-alanine-valine (IKVAV). Such bioactive peptides can be used for surface modification or as part of a 4950 F.Z. Volpato et al. / Biomaterials 34 (2013) 4945e4955 self-assembling peptide and are reviewed for neural applications elsewhere [154]. This approach uses synthetic peptide fragments from the active protein to specifically change cell behaviour [155e 158]. For example, neurite extension in vitro is greatest with an optimized concentration of RGD, with low or excessive amounts shown to reduce the extent of neurite outgrowth [159]. Peptide combinations can induce negative, additive and synergistic effects on neurite extension in vitro [159]. For example combinations of RGD- and IKVAV-modified fibrin matrices reduced neurite extension compared to either peptide alone. On the other hand, other peptide combinations can synergistically promote neurite extension e the combination of YIGSR and IKVAV promotes robust adhesion and neurite outgrowth on fluorinated polymer surfaces [160]. In situ gelling hydrogels have attracted significant recent interest for neural tissue engineering [161,162]. This includes selfassembling peptides, which aggregate into nanoscale structures under certain conditions and can be combined with bioactive peptides such as IKVAV to impart a biological effect [163e166]. Such in situ gelling materials have a high water content and have been injected directly into [165,166] or next to the spinal cord [162,167]. The intrathecal injection of in situ gelling hydrogels based on hyaluronic acid/methyl cellulose has been combined with drug delivery for enhanced release [167e170]. In situ gelling hydrogels are reviewed extensively elsewhere [171,172]. However, tissue-ECM is derived from multiple cell types and composed of many kinds of proteins, including growth factors and cytokines, and has a complex structure. Although the complexity is advantageous for promoting repair, current decellularization protocols lack the specificity to remove growth-inhibitory molecules while retaining growth-promoting molecules. Furthermore, it would be difficult to reconstruct a scaffold or substrate that has the same composition as that of an in vivo ECM using purified ECM proteins, short peptides or conventional physicochemical methods. In addition to the difficulty of mimicking the ECM composition, the complex microstructure of extracellular space makes accurately reproducing this environment extremely challenging. Researchers have therefore been focussing on natural tissues to develop novel biomaterial scaffolds based on cell-derived ECM [151,158]. Moreover, synthetic peptides might be initially useful for the functionalization of scaffolds to promote cell adhesion however the final product would consist of a biomaterial scaffold with cell-derived ECM. In contrast to cell transplantation, ECM alone has the advantage of not requiring immunosuppression treatment [143,174]. 7. Decellularized tissue intended for CNS regeneration Treatment strategies intended to promote CNS regeneration based on decellularized tissue use primarily peripheral nerves [15,16,19,175], however there are examples of decellularized nonneuronal [18] tissue used in the CNS. All of these strategies are mostly early attempts and only give a hint of the potential of ECMbased strategies. Approaches used include acellular peripheral nerve grafts implanted after optic nerve injury [15,16,19,144] and into the diencephalon [175] (in experimental rat and hamster models, respectively). Interestingly, the conclusions were that the seeding of Schwann cells into the decellularized nerves was important for axon regeneration, with insignificant regeneration with acellular nerves alone. Despite the lack of axonal regeneration, acellular nerves assisted the survival of retinal ganglion cells, possibly due to a release of neurotrophic factors [16]. However, none of protocols to decellularize the peripheral nerves included a step to remove myelin, which could have affected its growth promoting properties. A recent study investigated the potential of acellular muscle, which contains laminin, fibronectin and collagen, to promote recovery after SCI in rats [18]. The chemically extracted acellular muscle was implanted into spinal cord after lateral hemi-section and was able to promote neuronal survival, neovascularisation and axonal ingrowth into the lesion site, which followed the orientation of the ECM-scaffold. Furthermore, ED1 and glial fibrillary acidic protein (GFAP) immunoreactivity was comparable to the injured controls, indicating that ECM-scaffolds do not exacerbate inflammation or gliosis [18]. Finally, CNS-derived acellular scaffolds have been investigated on their potential to promote repair. Brain, spinal cord or optic nerve tissue was decellularized either through chemical treatments [174] or a combination of freeze-drying and chemical treatments [17,143]. The acellular scaffolds contained laminin, fibronectin, myelin and growth factors (vascular endothelial growth factor (VEGF), FGF-2) [17,174]. Decellularized CNS scaffolds promoted angiogenesis of chick embryo chorioallantoic membranes [143], proliferation, migration and differentiation of neuronal cells in vitro [17,174] and elicited no immune response after subcutaneous transplantation in vivo [174]. Similar to the acellular peripheral nerve grafts, myelin was not completely removed, which would hamper the benefits of CNS-derived ECM-scaffolds after transplantation into the CNS [17]. 8. 3D hybrid-scaffolds: combining synthetic biomaterials with natural ECM Outside to the CNS, several studies demonstrated the capacity of scaffolds with in vitro deposited ECM to promote tissue repair (e.g. in cartilage, skin and bone [177e180]). This type of hybrid scaffold, schematically shown in Fig. 4, is initially produced though conventional or additive manufacturing principles then temporarily seeded with cells to produce ECM that partially remains after decellularization. Following a similar logic, the same concept could be applied to the CNS. The shape and morphology of the 3D scaffold could then be controlled to provide mechanical stability and a guidance structure for the seeded cells, as orientation guides axonal growth [121,130,154]. Studies that focus on both synthetic and natural polymers for SCI regeneration, such as polycaprolactone [13], poly(lactic-co-glycolic acid) [83,181], fibrin [152,182,183], collagen [13,184,185] and hydrogels [186,187] could be used as underlying substrates for natural ECM deposition. A schematic for the combinations of such hybrid-scaffolds is shown in Fig. 4. The potential source of ECM proteins and growth factors for CNS applications is diverse, with many cells secreting ECM proteins and molecules that are able to enhance axonal regeneration and neuroprotection [31,68,154,173]. Since the ECM-derived from cells might contain inhibitory elements it is important to chose a cell type and developmental stage where the ECM is overall more growth promoting than inhibitory. For example, only mature oligodendrocytes produce myelin, together with its inhibitory factors [188], while oligodendrocyte progenitor cells produce neurotrophic factors like BDNF [189]. Other cell types, which have overall growth-promoting properties, include immature astrocytes, Schwann cells, stem cells and olfactory ensheathing cells (OECs). ECM produced by such cells contains neurotrophic factors such as NGF, BDNF, neurotrophin-3 (NT-3), ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF) and leukemia inhibitory factor (LIF), in addition to proteins such as laminins, fibronectin, collagen I/III and IV [87e90]. Schwann cells have been investigated in cell transplantation studies to promote neural repair after CNS injury, due to their crucial role in PNS repair [76,91e93]. Schwann cells have been shown to express the surface adhesion molecules N-CAM, Ng-CAM/L1 and J1 F.Z. Volpato et al. / Biomaterials 34 (2013) 4945e4955 4951 Fig. 4. Schematic diagram of a hybrid-scaffold containing synthetic fibers deposited by additive manufacture technologies and cell-derived ECM. The hybrid-scaffold is decellularized to minimize immunological response, whilst maintaining the growth-promoting molecules (e.g. laminin, collagen IV, NT-3, BDNF). [190,191]; neurotrophic factors such as NGF, BDNF, GDNF and insulin-like growth factor (IGF) [192,193]; and the ECM molecules laminin and fibronectin [154,194], all of which are able to promote recovery after CNS injury. Furthermore certain cytokines (e.g. interleukin (IL)-1 alpha, beta, IL-6, TNF-alpha), which can be preserved during decellularisation [195] are known to influence the inflammatory response and are able to modulate the secondary injury [196]. Therefore Schwann cells would make a good starting point for future investigations. One current challenge with transplantation of live Schwann cells is that they form boundaries with astrocytes in vitro and in vivo, so the bridges will not integrate with the spinal cord [176,197]. These sharp boundaries that form between Schwann cells and host tissue are a barrier to regenerating axons, particularly axons attempting to leave the growth-promoting Schwann cell environment and reenter the host spinal cord [176]. One possibility is that Schwann cell-derived ECM is highly growthpromoting, preventing axons to reenter the host spinal cord, especially if the matrix is slowly degraded. Another challenge of cell transplantation into chronic lesions is poor cell survival [208]. Since scaffolds or matrices do not depend on cell survival, this is an alternative approach to promote repair in a chronic lesion [209]. Therefore, it is envisioned that ECM-scaffolds will be beneficial for acute, sub-acute and chronic lesions. Different time points of implantation could be fine-tuned to target different aspects of the pathophysiology of spinal cord injury, e.g. while neuroprotection is a good target for scaffolds used in the acute phase, chronic lesions may benefit more from factors promoting axonal regeneration. The efficiency of such hybrid-scaffolds depends largely on the availability of growth-promoting ECM molecules. Several groups have reported that ECM-scaffolds rapidly degrade after implantation by enzymatic (collagenase and matrix metalloproteinases (MMP’s)) and cellular mechanisms [198e201]. Leukocytes, such as neutrophils, macrophages, monocytes, lymphocytes and microglia, as well as astrocytes all express such proteolytic enzymes. In tendon replacement, approximately 60% of the ECM-scaffold is degraded within 4 weeks, and completely in 3 months [200]. The mechanical properties of the matrix rapidly decreased due to the nature of its components. However, the kinetics behind ECM degradation are still largely unknown, and further studies of the inflammatory response to transplanted ECM and how it will affect its composition 4952 F.Z. Volpato et al. / Biomaterials 34 (2013) 4945e4955 and bio-availability are necessary. Decellularization and sterilization protocols can also affect the biomechanical [202e204] and biological (e.g. strength and functionality of growth factors) [205,206] properties of the scaffolds. However, different crosslinking methods are able to reduce the degradation kinetics and prolong scaffold stability [198,207]. Furthermore, after implantation, the scaffold is in a dynamic environment which is not only characterized by the degradation of the ECM-scaffold but also by the migration of host cells onto the scaffold, which can deposit new ECM [199,200]. Further complementary therapies might be combined with the hybrid-scaffold, i.e. to reduce the effect of the inflammatory response and associated ECM degradation. 9. Conclusions The lack of functionally significant axon regeneration after SCI is mainly due to an imbalance of local axon growth-promoting and growth-inhibitory mechanisms. Over the past decade, biomaterials and regenerative medicine research has diversified and developed rapidly. While the use of decellularized tissue has been widely embraced in non-CNS tissues, its efficacy in the spinal cord is still not fully understood and has been partially restricted by the inhibitory ECM molecules that remain after processing. The native ECM is a complex combination of proteins and polysaccharides that play an important role in cellular behaviour such as attachment, differentiation, and proliferation. 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