Downloaded from http://rsfs.royalsocietypublishing.org/ on February 22, 2018
Interface Focus (2012) 2, 278–291 doi:10.1098/rsfs.2012.0016 Published online 21 March 2012
REVIEW
Hyaluronic acid-based scaffold for central neural tissue engineering Xiumei Wang*, Jin He, Ying Wang and Fu-Zhai Cui Institute for Regenerative Medicine and Biomimetic Materials, State Key Laboratory of New Ceramics and Fine Processing, Department of Materials Science and Engineering, Tsinghua University, Beijing 100084, People’s Republic of China Central nervous system (CNS) regeneration with central neuronal connections and restoration of synaptic connections has been a long-standing worldwide problem and, to date, no effective clinical therapies are widely accepted for CNS injuries. The limited regenerative capacity of the CNS results from the growth-inhibitory environment that impedes the regrowth of axons. Central neural tissue engineering has attracted extensive attention from multi-disciplinary scientists in recent years, and many studies have been carried out to develop cell- and regenerationactivating biomaterial scaffolds that create an artificial micro-environment suitable for axonal regeneration. Among all the biomaterials, hyaluronic acid (HA) is a promising candidate for central neural tissue engineering because of its unique physico-chemical and biological properties. This review attempts to outline current biomaterials-based strategies for CNS regeneration from a tissue engineering point of view and discusses the main progresses in research of HA-based scaffolds for central neural tissue engineering in detail. Keywords: central neural tissue engineering; hyaluronic acid; central nervous system; regeneration; scaffold
1. INTRODUCTION
divided into two phases: primary injury and secondary injury. Primary injury is an acute phase of injury that is caused by contusion, laceration, compression or severe rotation of the tissue, transection of axons and local blood vessel damage, and finally it leads to cell necrosis at the injury epicentre [1,2]. Except for direct cell death and haemorrhage, there is no more damage to the CNS caused by the primary injury [3]. However, the advent of secondary injury slowly deteriorates the condition, resulting in complete and permanent trauma to cells and axons, and functional disabilities in the CNS. Secondary injury is the chronic phase injury, which lasts several months or years. After injury, owing to death of neural cells, breakdown of blood–brain barrier and influx of inflammatory cells, reactive gliosis is initiated and glial scars are formed [4]. Although, at the beginning, glial scars seclude the injury site from the healthy tissue and play an important role in restoration of the blood– brain barrier, they finally develop into a tenacious and rubbery membrane acting as a physical barrier that blocks the outgrowth, penetration and reconnection of axons [5]. Besides this physical barrier, in the glial scar, there are several kinds of molecules that act as a biochemical barrier, including Nogo, myelin-associated glycoprotein (Mag), oligodendrocyte myelin glycoprotein (Omgp) and chondroitin sulphate proteoglycan (CSPG), all of which have been demonstrated to be potent inhibitors of neurite outgrowth [6–13].
Damage to the central nervous system (CNS) including the brain and the spinal cord, which is mostly caused by trauma, tumour or disease, usually results in severe neurological impairments with irretrievable nervous system functional devastations or limitations such as paralysis, limited mobility and sensory loss. In China, it is roughly estimated that more than 1 500 000 people live with spinal cord injury (SCI), and there are approximately 10 000 new cases every year. Therefore, functional recovery of injured CNS has been extremely significant in clinical cases for improving the qualities of patients’ lives. Unlike peripheral nervous system (PNS) injury, completely functional restoration in damaged CNS is not possible in most clinical cases. Consequently, CNS regeneration with central neuronal connections and restoration of synaptic connections has been a longstanding worldwide problem, which has attracted extensive attention from multi-disciplinary scientists. The CNS has limited capacity for regeneration mainly because of the growth-inhibitory environment that impedes the regrowth of axons. Owing to injuries, a series of complex cellular and biochemical reactions are triggered within the CNS and they are usually *Author for correspondence (
[email protected]). One contribution of 11 to a Theme Issue ‘Biomaterials research in China’. Received 6 January 2012 Accepted 20 February 2012
278
This journal is q 2012 The Royal Society
Downloaded from http://rsfs.royalsocietypublishing.org/ on February 22, 2018
Review. HA-based scaffold for CNS regeneration Based on the knowledge of physiology of the CNS and pathology of CNS injuries, the main factors that deprive the CNS of the ability to regenerate are multi-faceted and can be summarized as: inability of adult neurons to proliferate; absence of neurotrophic factors; formation of glial scars; existence of inhibitory molecules. To date, no effective clinical therapies are widely accepted for CNS injuries, although some are commonly applied, including methylprednisolone treatment and cell therapy, both of which fail to achieve complete recovery because they overcome only some but not all of the inhibitory factors. Hence, a multifaceted approach is needed to design and produce a system that integrates several factors to conquer all the inhibitory factors. Tissue engineering uses scaffolds, living cells and regulators to develop ideal biological substitutes for restoring or regenerating damaged body tissues. Consequently, by integrating the use of biomaterials, cells and regulators, such tissue engineering strategy sheds new light on CNS regeneration. Since it is biomaterials (scaffolds) that play a critical role in tissue engineering by acting as biodegradable engineered extracellular matrix (ECM) for in vitro or in vivo cell attachment, proliferation, migration, differentiation and tissue formation, the key to successful CNS regeneration with tissue engineering approaches lies in developing materials-based strategies to overcome the inhibitory environment. Recently, higher requirements are raised in bioactivity of materials [14,15]. Instead of merely being able to promote cell adhesion, migration and proliferation, the truly bioactive materials are expected to be capable of inducing certain cellular responses and activating certain gene expressions in the patient’s tissue, so as to make use of their self-healing potential. These ideal properties are underlaid by a special design of the biomaterial system and it is up to the materials scientists to achieve this goal with multi-disciplinary knowledge. Here in this review, we first summarize current biomaterials-based strategies for central neural tissue engineering, and then we focus on recent progress in designing and fabricating hyaluronic acid (HA)-based scaffolds.
2. CURRENT BIOMATERIALS-BASED STRATEGIES FOR CENTRAL NERVOUS SYSTEM REGENERATION FROM A TISSUE ENGINEERING POINT OF VIEW As we all know, tissue engineering triad consists of scaffolds, cells and regulators (biomolecules), all of which are quite critical for tissue repair or regeneration. However, cells and/or biomolecules are thought to be not definitely necessary to be loaded into scaffolds and cultured for a while in vitro before transplantation. There is no doubt that if a biomaterial scaffold itself has sufficient bioactivities to recruit endogenous cells and growth factors to help tissue regeneration, exogenous cells and molecules could be omitted. Because in vitro applications of cells and/or biomolecules have encountered a number of translational, manipulation, safety and regulatory problems, currently biomaterials-based strategies for in situ tissue engineering have been an Interface Focus (2012)
X. Wang et al.
279
attractive area. Biomaterials scientists have been trying to design and fabricate ideal biomaterial scaffolds, which are capable of delivering chemical, physical and biological cues to regulate cell attachment, proliferation, migration, differentiation and neotissue formation by acting as biodegradable engineered ECM. Therefore, biomaterials-based strategies have great promise for tissue engineering and regenerative medicine. Biomaterial scaffolds are not only simply ‘tissue-engineered scaffolds’ for cell delivery or cell migration, but also ‘cell- and regeneration-activating systems’ for in situ tissue engineering. Numerous studies have focused on design and fabrication of biomaterials. Here, we classify current progress according to the three factors in central neural tissue engineering. 2.1. Scaffolds For use in central neural tissue engineering, biomaterials should meet the following criteria. Biomaterials should: integrate well with host tissue without inducing inflammatory reaction and glial scar formation; have similar physical properties to the brain or the spinal cord; allow infiltration of cells and axons, and transportation of nutrients and metabolites; exhibit a suitable rate of degradation with no inflammation caused by the degradation products. On the basis of these requirements, it is found that among all the biomaterials, hydrogels, electrospun nanofibres and self-assembling peptides are ideal candidates and have been applied in many studies. Hydrogels are three-dimensional networks of hydrophilic polymer held together by chemical or physical cross-linking. Hydrogels are glassy in the dry state but they swell in water and form elastic gels, retaining a large quantity of water in their mesh-like structures. Many kinds of hydrogels have been used and they can be classified into naturally derived hydrogels, namely HA, chitosan, alginate, agarose, fibrin, and methylcellulose, and synthetic hydrogels, namely poly(2-hydroxyethyl methacrylate), poly[N-(2-hydroxypropyl) methacrylamide] and polyethylene glycol. Made of polysaccharides, glycosaminoglycans or ECM constituents, naturally derived hydrogels are inherently bioactive and allow for cell attachment. Besides, under physiological conditions, naturally derived hydrogels are degradable via enzymatic action, which facilitates infiltration of cells and axons into the hydrogels [16]. However, batch variation and the risk of disease transmission may limit the application of naturally derived hydrogels. For synthetic hydrogels, they are biologically inert and cells hardly adhere to them, which makes it necessary to adopt modification such as tethering of natural polymers or adhesive motifs. Electrospinning is effective in producing fine fibres of nanoscale diameters that range from several nanometres to 1 mm [17]. Made of nanofibrous meshes, electrospun scaffolds exhibit high surface-to-volume ratio and high porosity, and thus mimic the hierarchical structures of laminin and collagen of the ECM, which facilitates cell and axon penetration, offers guidance cues to neurite extension and enhances scaffold–tissue integration. A lot of polymers have been employed to produce electrospun fibres, including chitosan [18], poly-L-lactic acid
Downloaded from http://rsfs.royalsocietypublishing.org/ on February 22, 2018
280
Review. HA-based scaffold for CNS regeneration
(PLLA) [19,20], polycaprolactone (PCL) [21], polylactic-glycolic acid (PLGA) [22], polyether sulphone [23], poly(L-lactic acid)-co-poly(3-caprolactone) [24] and polyurethane [25]. Self-assembly is another method for producing nanofibre networks. Via hydrophobic interaction, oligopeptides or amphiphilic peptides assemble into self-assembling peptide nanofibre scaffolds (SAPNSs), with hydrophobic backbones forming the core, and the hydrophilic head groups forming the sheath [26]. Compared with electrospun fibres, SAPNSs more closely mimic the hierarchical structure of ECM owing to smaller fibre diameters and high-density presentation of peptide sequences. In order to enhance neural regeneration, many studies have been conducted to optimize the design and fabrication of scaffolds. The effect of modulus of elasticity, architectural structures and modification with ECM components has been extensively investigated. 2.1.1. Modulus of elasticity of scaffolds Modulus of elasticity of scaffolds has recently been shown to be a key factor that influences cell behaviours. Different adhesion and morphologies of astrocytes and neurons in response to changes in modulus were observed [27]. Soft gels tended to suppress the adhesion and proliferation of astrocytes, while modulus did not significantly alter the actin formation and neurite extension. In cultures of dissociated embryonic cortices, it was further demonstrated that the control of modulus was effective in screening the cells that attached onto the surface. Moreover, modulus was reported to be able to direct stem cells into different lineages. Mesenchymal stem cells (MSCs) were directed into neurogenic lineages when they were cultured on soft gels that had modulus similar to that of brain tissue [28]. Neural stem cells (NSCs) favoured neuronal lineages on soft gels with modulus less than 1 kPa [29,30]. Thus, modulus should be paid attention to when scaffolds are designed and prepared. Hydrogels with modulus similar to that of the brain or spinal cord tissue are ideal for use in CNS regeneration. 2.1.2. Architecture There have been many studies on repair of the PNS by tubes or nerve guides, with some promising results observed [31,32]. These anisotropic structures offer physical guidance to the migration of cells and the penetration of axons. Thus, in CNS studies, uniaxial structures are prepared and the effects of guidance cues are investigated. There are several ways to create uniaxial structures with hydrogels. Templating, which is straightforward and reproducible, is highly effective in fabricating scaffolds with longitudinal channels [33]. After removal of PCL fibres by acetone, uniaxial channels are formed where PCL fibres had been. Thus, the sizes of the channels can be directly controlled by the diameter of the fibres. It is reported that the implantation of templated agarose scaffolds resulted in linear and organized penetration of axons and blood vessels, in distinction to the random orientation without scaffolds [34]. However, Interface Focus (2012)
X. Wang et al. an in vivo study surprisingly showed that channels with different diameters did not differ in their ability to support axon migration [35]. Moreover, the tubular structure is far from perfect. Wong et al. [36] highlighted the importance of microstructures. In this study, the effect of scaffolds with different complex structure was studied, demonstrating that open-path designs better promoted axonal regeneration, while closed designs resulted in the encapsulation of fibrous tissues and the enlargement of defects. Freeze-drying is another way to fabricate uniaxial hydrogel scaffolds [37,38]. With a gradient in temperature, scaffolds with honeycomb structure were created [38]. Along with other factors, linear growth of axons within channels was observed and the axons successfully bridged the lesion. Many studies have shown that the orientations of electrospun nanofibres are vital in controlling cell behaviours. Aligned electrospun fibres are mainly fabricated by collecting the polymer stream at a metal grounded drum or plate that rotates at a suitable speed [19, 39– 41]. It was reported that the orientation of neurite extension was highly dependent on the fibres [20]. Further analysis measuring the length of neurites showed that the aligned fibres increased neurite extension by 20 per cent in length compared with random controls. This effect was more vividly shown by samples combining both random controls and aligned fibres [42]. At the border of aligned and random nanofibres, the neurites from the same dorsal root ganglion (DRG) cells grew without any preference in direction on the side of random fibres, while on the other side where electrospun fibres were aligned, they grew along the fibre alignment; apparently, the neurites were much longer on aligned fibres. If pluripotent cells were incorporated, the alignment was shown to influence cell differentiation, with more cells being induced towards neuronal lineages on aligned fibres for mouse embryonic stem cells (ESCs) [21] and MSCs [43].
2.1.3. Modification of engineered extracellular matrix components The ECM molecules are important components of the nervous system. Composed of a heterogeneous lattice of proteoglycans and glycoproteins, ECM provides structural support and anchorage for cells and regulates cell adhesion, migration and proliferation [44]. Among all the ECM molecules, laminin and fibronectin, together with their peptide motifs, have been recognized to facilitate nerve development and offer a neuroprotective function in the nervous system after injury [45– 47]. Thus, laminin, fibronectin, collagen and their peptide motifs have been used to modify scaffolds, so as to improve cell adhesion and axon sprouting, and enhance neural regeneration. Incorporation of whole ECM molecules, either by coating, blending, covalently immobilizing, or as fillers in tubes, makes scaffolds more biocompatible, especially for synthetic scaffolds that lack bioactive domains to interact with cells. Modification with laminin, either by covalent binding, physical adsorption or blending, was observed to significantly enhance neurite extension
Downloaded from http://rsfs.royalsocietypublishing.org/ on February 22, 2018
Review. HA-based scaffold for CNS regeneration compared with negative controls [48]. In this study, blending was shown to be more effective than covalent binding and physical adsorption, which might result from the higher laminin quantity coupled to PLLA nanofibres by blending. The function of collagen in promoting spinal cord regeneration was investigated in vivo [49]. One year after implantation, regeneration of axon and functional recovery were achieved in groups with chitosan tubes filled by semifluid type I collagen. This modification stimulated regenerative nerve fibres to penetrate the lesion site and grow into the distal end, while this effect was not observed in negative controls. The incorporation of ECM components may influence differentiation of pluripotent cells, but it is still debatable and extensive studies are needed [50,51]. Peptides of laminin, collagen and fibronectin are alternative ECM molecules in modification of scaffolds. Arg – Gly – Asp (RGD), Ile – Lys – Val– Ala –Val (IKVAV) and Tyr– Ile – Gly – Srg– Arg (YIGSR) are the most widely used. Modification of YIGSR better enhanced neurite extension compared with control groups without YIGSR both in vitro and in vivo [52]. Similar results were reported in studies on modification of RGD and IKVAV [53,54]. Schense et al. [55] systematically investigated the effect of peptide concentration and combination on neurite extension. Not all peptides had linear relationships between neurite outgrowth and incorporated peptide concentration. Different co-crosslinked peptides elicited various effects on the neurite extension, depending on the combination. 2.2. Regulators Delivery of antibodies, neurotrophic factors or therapeutic drugs to lesion sites in the CNS can alleviate inflammation, protect spared tissue around the lesion and promote neural regeneration. Direct injections of these bioactive molecules have a relatively short half-life [56]; thus, scaffolds are used to achieve sustained release of bioactive molecules. Here, we limit our discussion to the scaffold-based delivery of neurotrophic factors and therapeutic drugs, while delivery of antibodies will be discussed in §3.4. 2.2.1. Delivery of neurotrophic factors Some of the most commonly used growth factors to promote neural regeneration are neurotrophic factors, including nerve growth factor (NGF) [57], neurotrophin-3 (NT-3) [58], brain-derived neurotrophin factor (BDNF) [59], glial cell line-derived neurotrophic factor (GDNF) [60] and ciliary neurotrophic factor (CNTF) [61]. Different methods have been developed to deliver these growth factors, namely physical embedding, heparin-binding and covalent-binding. Physical embedding is a simple method to deliver neurotrophic factors. Factors are blended with hydrogel solution and they evenly distribute within the hydrogel scaffolds after gelation [62,63]. Delivery of GDNF in this way resulted in promotion of axonal outgrowth and suppression of cystic cavitation [62]. To prolong the in vivo availability and reduce running away, carriers are introduced to deliver growth factors. It was shown that the dosage of NT-3 to induce NSCs to commit Interface Focus (2012)
X. Wang et al.
281
neuronal lineages was much lower in the groups employing chitosan carriers, compared with those of direct addition [64]. Sakiyama-Elbert et al. developed a heparin-based delivery system for sustained release of growth factors. This system has been successfully applied to deliver b-fibroblast growth factor [65], NGF [66], plateletderived growth factor [67] and NT-3 [68]. The potential of this heparin-based delivery system for use in repairing CNS injuries was investigated in vivo [58]. Despite some promising results in the short term, functional recovery study between a group with heparin-NT-3 and a negative control did not show a significant difference after 12 weeks, indicating that from a clinical point of view, the time period for growth factor delivery should be further prolonged. Recently, many studies tried to covalently immobilize neurotrophic factors onto scaffolds. Compared with physical adsorption, a higher concentration of growth factors was obtained via immobilization [69]. Instead of compromising the bioactivity, immobilization in fact increased the efficacy of growth factors [57]. It was reported that immobilization of vascular endothelial growth factor (VEGF) outperformed in cell adhesion and viability even at a much lower concentration compared with soluble VEGF [70]. Thus, immobilization of neurotrophic factors increases the efficiency, reduces the required dosage and makes it possible to provide a long-term delivery. 2.2.2. Delivery of therapeutic drugs Methylprednisolone, which is capable of improving neurologic recovery, is widely used for acute SCI. However, high doses of methylprednisolone increase the risk of unwanted side effects. So it is imperative to find an effective way to deliver methylprednisolone to lesion sites in doses as small as possible. A delivery system was developed to provide a sustained release of methylprednisolone [71]. Methylprednisolone was encapsulated in PLGA nanoparticles embedded in agarose hydrogels. Results showed that this system successfully delivered methylprednisolone to injured spinal cord tissue without compromising its bioactivity. The inflammation and cystic cavitation were significantly reduced owing to the presence of methylprednisolone. 2.3. Cells Several cell types have been used for CNS injury studies, including Schwann cells (SCs) [72], hippocampal neurons [73], DRGs [74], olfactory ensheathing cells (OECs) [75], NSCs [76,77], neural progenitor cells (NPCs) [67], ESCs [51] and MSCs [78,79]. In many studies, cell – scaffold complexes are fabricated and implanted into CNS lesion sites to evaluate their abilities in inducing neural regeneration. It was observed that implantation of such cell – scaffold complexes resulted in suppression of inflammation and astrocytic scarring around the lesion [80– 82], reduction of lesion volume [78], infiltration of regenerating axons into the implant [80,82,83], ingrowth of new blood vessels [82,83], formation of synapses [84] and even functional recovery [78,83,85].
Downloaded from http://rsfs.royalsocietypublishing.org/ on February 22, 2018
282
Review. HA-based scaffold for CNS regeneration (a)
X. Wang et al. (b)
Figure 1. The structure of HA hydrogels observed using scanning electron microscopy at (a) lower and (b) higher magnification. Reproduced with permission from Hou et al. [94], q 2006 Elsevier. Scale bars, (a) 50 mm; (b) 20 mm.
To further enhance regeneration, cell co-culture system is introduced. Co-culture of endothelial cells and NPCs led to enhanced formation of tubular structures [86]. After implantation into lesions in spinal cord, the coculture implant was found to be highly effective in promoting angiogenesis, with a twofold increase in functional vessels over the implant with endothelial cells alone [87]. Since OECs and SCs are important cells that ensheath axons, co-culture of SCs with NSCs/NPCs may further enhance axonal formation and infiltration compared with encapsulation of either cells alone. 3. RESEARCH OF HYALURONIC ACIDBASED SCAFFOLDS FOR CENTRAL NERVOUS SYSTEM REGENERATION As discussed earlier, different kinds of biomaterials have been investigated for their potential in promoting axonal regeneration. Among them, HA is one of the best candidates for the following reasons: as a major component of soft connective tissue, HA is widely found in most organs and tissues [88,89], especially in the CNS [90]; owing to its high biocompatibility, HA plays a beneficial role in wound healing [91,92]; in recent studies, it has been shown that implantation of HA scaffolds reduces glial scar formation [93,94]. To further enhance axonal regeneration with HA scaffolds, a series of strategies are implemented, and promising results are observed [95,96]. Here, we briefly discuss recent progress on the researches of HA-based scaffolds for CNS regeneration. 3.1. Pure hyaluronic acid hydrogels as scaffolds for central nervous system regeneration HA hydrogels have an interconnected porous structure (figure 1) [94] that allows transportation of nutrition and penetration of cells, nerve fibres and blood vessels. Thus, HA hydrogels are used as implants to enhance neural regeneration. HA was demonstrated to be effective in reducing scar formation and enhancing neural regeneration in both the PNS [97] and CNS [98]. An in vivo study showed that treatment with HA hydrogels significantly inhibited glial scarring, with much smaller gliosis thickness and fewer glial fibrillary acidic protein (GFAP)-positive cells around the scarring area [98]. However, according to a recent study, only HA hydrogels with high molecular weight had such an effect to Interface Focus (2012)
inhibit astrocytic activation, macrophage/microglia infiltration and CSPG deposition [99]. It was hypothesized that this effect resulted from interaction between HA and certain receptors via different states of aggregation [100]. However, further experiments are essential to confirm the results because in the original study, a control group treated with low molecular weight HA was absent, where the influence of low molecular weight cannot directly be shown. Besides molecular weight, the modulus of HA hydrogels was reported to be capable of influencing differentiation of NPCs [101]. The majority of NPCs cultured in hydrogels with similar modulus to that of neonatal brain tissue differentiated into neurons with extended long processes, while those cultured in hydrogels with similar modulus to that of adult brain tissue mostly differentiated into astrocytes. 3.2. Hyaluronic acid hydrogels blended with other materials as scaffolds for CNS regeneration A dominant disadvantage of HA is that cells do not adhere to its surface. Hence, HA hydrogels were blended with other materials to promote cell adhesion and thus enhance neural regeneration. Spector and his colleague reported that blending HA with collagen enabled the fabrication of scaffolds with suitable mechanical properties for CNS regeneration [102]. In vitro experiments showed that the NSCs cultured in HA – collagen scaffolds favoured neuronal differentiation, which was in accordance with the results reported by Forsberg-Nilsson and co-workers [103]. 3.3. Modification of hyaluronic acid hydrogels Another way to promote cell adhesion is modifying HA hydrogels with ECM components, namely laminin, RGD, IKVAV, poly-D-lysine (PDL) and poly-L-lysine (PLL). Our group reported that the modification of HA with laminin better improved tissue reconstruction than that of negative controls [104]. Figure 2a,b, shows the reactive astrocytes around the boundary of the host tissues and the HA hydrogel scaffold by immunostaining after 12 weeks. Fewer GFAP-positive astrocytes were found in the groups with laminin modification. Statistical analysis showed that after 12 weeks, modification of laminin reduced one-third of astrocytes around the boundary. Silver staining revealed the presence of argyrophilic processes that grew into the HA
Downloaded from http://rsfs.royalsocietypublishing.org/ on February 22, 2018
Review. HA-based scaffold for CNS regeneration (a)
X. Wang et al.
283
(b)
*
*
(d)
(c)
* *
Figure 2. Reactive astrocytes around the boundary of normal tissue and HA hydrogel scaffold by immunostaining for GFAP after 12 weeks: (a) HA hydrogels with laminin modification, (b) HA hydrogels without laminin modification. Silver staining showing argyrophilic processes that grew into the lesion area: (c) HA hydrogels with laminin modification, (d ) HA hydrogels without laminin modification. Black asterisks indicate normal tissue. Reproduced with permission from Hou et al. [104], q 2006 Elsevier. Scale bars, (a,b) 60 mm; (c,d) 20 mm.
(a)
(b)
T
G G T
Figure 3. Immunostaining photomicrographs of HA hydrogels implanted to rat brains. Cells were immunostained as GFAPpositive in (a) HA –RGD hydrogels and (b) HA hydrogels. G is the hydrogel implant. T is host tissue. Reproduced with permission from Cui et al. [53], q 2006 Springer. Scale bars, 100 mm.
scaffolds in the modified groups (figure 2c), not only around the boundary of the lesion site, but also into the epicentre of the lesion, while no such processes were found in the unmodified counterpart (figure 2d ). However, laminin modification did not affect the new blood formation. Hence, HA hydrogels modified with laminin improved neural regeneration mainly by reducing reactive astrocytes gathering around the lesion boundary and promoting new fibre formation within the scaffolds. Besides laminin, special domains of laminin such as RGD and IKVAV were immobilized to HA hydrogels to promote cell adhesion. RGD modification was demonstrated to significantly enhance cell migration into implants (figure 3), finally resulting in the Interface Focus (2012)
formation of collagen-like bundles and neurofibrils in the hydrogel implants [53]. IKVAV modification was demonstrated to have similar effects [105]. Modification with ECM proteins or peptides makes it possible for cells to adhere to HA hydrogels, which facilitates HA scaffolds to serve as a promising candidates for cell delivery in stem cell therapies for neural regeneration. PDL is frequently used as a coating material before cultures of neuronal cells [106]. In order to make HA an effective substrate for axonal extension, lysine was covalently bound to HA and it was found that PDL modification was effective in promoting cell adhesion and migration [93]. Rat cortical cells failed to attach to untreated HA hydrogels even after 5 days of culture in serum-containing medium, while in PDL-treated
Downloaded from http://rsfs.royalsocietypublishing.org/ on February 22, 2018
284
Review. HA-based scaffold for CNS regeneration (a)
X. Wang et al. (b)
5 µm
1 µm
Figure 4. Scanning electron microscopy images of neural cells that adhered to HA–PDL hydrogel. Boxed section in (a) is enlarged in (b) to show the connection (white arrow) between neurons [93].
groups cells adhered to HA hydrogels and formed interconnected networks, as shown in figure 4. In a traumatic brain injury rat model, groups implanted with PDL-treated HA hydrogels showed improved regeneration of blood vessels and reconstruction of new ECM. These results further demonstrated that PDLmodification was effective in making HA hydrogels more biocompatible and promoting angiogenesis. 3.4. Delivery of bioactive agents using hyaluronic acid-based scaffolds 3.4.1. Delivery of Nogo receptor antibodies using hyaluronic acid-based scaffolds Studies have shown that the three axonal growth inhibitors Nogo, Mag and Omgp require interactions with Nogo receptor (NgR) to exert their inhibitory influence [107– 109], and blocking of NgR by using polyclonal Nogo receptor antibodies (anti-NgRs) successfully blocked the inhibition of neurite outgrowth by Mag in a dose-dependent manner [109]. Hence, sustained delivery of anti-NgRs in the CNS after injury is effective in overcoming the growth inhibitory environment and promoting axonal regeneration. Our group first studied the controlled release of anti-NgRs via HA hydrogels [110]. The antibodies were conjugated to the HA backbone by a condensation reaction, with the amount of conjugated antibodies being 135 mg antibody per milligram hydrogel. The release was pH-dependent, with nearly 80 per cent of antibodies being released for up to 400 h in pH 7.4 buffer solution. Further in vitro experiments demonstrated that the condensation reaction did not compromise the bioactivity of anti-NgRs. Based on the HA-based delivery system, the effect of controlled release of anti-NgRs via HA hydrogels was evaluated both in vitro and in vivo. In an in vitro study, release of anti-NgRs enhanced adhesion, viability and neurite extension of DRG on HA hydrogels [94]. As shown in figure 5a,b, many more neurites were observed in groups with anti-NgRs than those without. This effect was more vividly shown by releasing anti-NgRs from a certain point and thus creating a concentration gradient of the antibodies. DRG neurites extended specifically towards where anti-NgRs were released, as shown in figure 5c. Interface Focus (2012)
Our group then investigated the effect of controlled release of anti-NgRs in a rat stroke model by using HA hydrogels [95]. In behavioural tests, 20 weeks after implantation, the groups with HA–anti-NgRs obtained all pellets in less attempts than the control with HA did, which indicated that the release of anti-NgRs improved functional recovery. The results well matched those of histochemical investigations. There are more nerve fibres and b-tubulin-III positive neurons in the HA–anti-NgR hydrogels than in negative controls, as shown in figure 6. In an SCI model, similar results were observed, with few NF-positive fibres discovered in the implants without anti-NgRs, while several NF-positive axons were observed entering the HA–anti-NgR–PLL implants [96]. These in vivo results demonstrated that controlled delivery of antiNgRs supported axonal regeneration, which was of great potential for clinical applications. 3.4.2. Delivery of neurotrophic factors using hyaluronic acid-based scaffolds Park et al. [111] reported the effect of controlled release of BDNF both in vitro and in vivo by using HA hydrogels. BDNF was incorporated into HA hydrogels by electrostatic interaction. The release of BDNF significantly changed the morphology and gene expression of MSCs in culture. In a rat SCI model, HA implants with BDNF better promoted functional recovery as measured by Basso, Beattie and Bresnahan (BBB) scores. Hence, HA hydrogels had the potential to be ideal carriers of BDNF. Recently, our group reported the effect of using PLGA microspheres as carriers for growth factors [112]. VEGF and BDNF were incorporated into PLGA microspheres by a water-in-oil-in-water emulsion technique, and PLGA microspheres were distributed in HA solution before gelation. The release curves of this HA hydrogel/ PLGA microsphere composite began with an initial burst, followed by a stable release phase, with about 12 per cent of total loading released after 100 h. 3.5. Delivery of neural cells using hyaluronic acid-based scaffolds On the basis of studies on the modifications and delivery of bioactive agents, HA hydrogels are investigated for the purpose of delivering neural cells to the lesion sites of the CNS, so as to facilitate
Downloaded from http://rsfs.royalsocietypublishing.org/ on February 22, 2018
Review. HA-based scaffold for CNS regeneration (a)
(b)
(c)
(d)
X. Wang et al.
285
Figure 5. Effect of controlled release of anti-NgRs on DRG cells. After 72 h, DRG cells hardly grew on the surface of (a) HA hydrogels, while many neurites were observed on the surface of (b) HA hydrogels with anti-NgRs released. Neurites of DRG cells were observed to grow towards (c) where anti-NgRs were released (black arrow indicates the gradient of anti-NgRs), while such directional preference was absent in (d) the groups without anti-NgRs. Reproduced with permission from Hou et al. [94], q 2006 Elsevier. Scale bars, 50 mm.
(a)
(b)
(c)
(d) T
G
T
G
Figure 6. Histochemical results eight weeks after implantation. More nerve fibres were found in (a) HA– anti-NgR hydrogels than in (b) HA hydrogels; more b-tubulin-III positive neurons in (c) the HA– anti-NgR hydrogels than in (d ) HA hydrogels. G, hydrogel implant; T, host tissue. Reproduced with permission from Ma et al. [95], q 2006 IOP Publishing Ltd. Scale bars, 100 mm.
axonal regeneration and functional recovery. The biocompatibility of HA hydrogels with different neural cells, namely was NPCs, hippocampal neurons and NSCs, was investigated. Interface Focus (2012)
After being cultured for 7 days, hippocampal neurons exhibited different morphologies on different HA hydrogels, as shown in figure 7 [113]. Neurons on hydrogels without modification remained rounded, while those on
Downloaded from http://rsfs.royalsocietypublishing.org/ on February 22, 2018
286
Review. HA-based scaffold for CNS regeneration
X. Wang et al.
(a)
(b)
(c)
(d)
(e)
( f)
(g)
(h)
Figure 7. Morphology of hippocampal neurons cultured on different HA hydrogels for 7 days. (a,e) HA hydrogels without modification. (b, f ) HA –anti-NgR hydrogels. (c,g) HA– PLL hydrogels. (d ,h) HA–PLL– anti-NgR hydrogels [113]. Scale bars, 100 mm.
(a)
(b)
(c)
(d)
Figure 8. Adhesion of NSCs on HA-based hydrogels after 5 days of culture. (a) HA; (b) HA–anti-NgR– PLL; (c) HA–PLL; (d) HA– anti-NgR. Scale bars, (a) 5 mm, (b–d) 10 mm.
hydrogels modified with PLL or hydrogels cross-linked with anti-NgRs exhibited multi-polar morphology. In addition, neurons were more evenly distributed on hydrogels with both PLL and NgR. Interestingly, NPCs were observed to behave similarly on these hydrogels [114]. Our recent work focuses on the delivery of NSCs via HA-based scaffolds. The adhesion of NSCs to HA hydrogels is shown in figure 8. The modifications with PLL and the delivery of anti-NgRs helped neurospheres Interface Focus (2012)
to attach to and spread on HA hydrogels. Incorporation of PLGA microspheres encapsulating VEGF and BDNF further promoted NSC adhesion and proliferation [112]. After 5 days of culture, neurites extended along the wall of the scaffold and formed an extensive network (figure 9). The proliferation assay showed that NSCs on the HA hydrogel with growth-factor-containing PLGA microspheres grew faster than negative controls. In our latest work, the
Downloaded from http://rsfs.royalsocietypublishing.org/ on February 22, 2018
Review. HA-based scaffold for CNS regeneration (a)
(b)
X. Wang et al.
287
(c)
nestin Hoechst
0
µm
250
0
µm
75
10 µm
Figure 9. The immunostaining and morphology of NSCs on HA hydrogel composite after 5 days of culture. (a,b) Immunostaining of nestin (green) showed widely distributed NSCs. 40 ,6-Diamidino-2-phenylindole (DAPI) was counterstained in blue. (c) NSCs adhered to hydrogels were observed by scanning electron microscopy. Reproduced with permission from Wang et al. [112], q 2011 Springer.
NSC–HA–PLL–anti-NgR complex was implanted into infarction lesions of rat brain and its capability to induce neural regeneration was evaluated. Our results showed that NSCs within the HA scaffolds were able to stay alive for up to 24 weeks after implantation. The most inspiring result was that 10 weeks after implantation, both synapse formation and expression of synaptophysin were observed. Since synaptophysin is the marker of synapse regeneration, its presence suggested that neurons derived from grafted NSCs formed synapses and began to reconstruct nervous circuits, which was an important step towards functional recovery.
4. CONCLUDING REMARKS By developing a materials-based system containing biomaterials, regulators and cells, great progress has been made in promoting CNS regeneration in recent years. The integration of different factors overcomes the multi-faceted inhibitory environment in the CNS after injury and successfully facilitates infiltration of cells, formation of nerve fibres and blood vessels, and finally functional recovery. In recent years, HA hydrogels have been extensively studied as a candidate for repairing CNS injuries. Owing to its unique properties, HA significantly reduced glial scar formation after implantation to lesion sites, which makes it attractive in designing and fabricating scaffolds for CNS regeneration. Different ECM components have been applied to modify HA hydrogels, aiming at enhancing axonal regrowth. The controlled release of anti-NgRs further enhances the regeneration of nerve fibres into the scaffolds. Combining with ECM modification and release of antibodies, HA-based scaffolds are investigated in neural cell culture, showing that HA-based scaffolds are highly biocompatible. Further in vivo studies find the existence of synaptophysin and formation of synapses. Thus, HAbased scaffolds have great potential for use in central neural tissue engineering. To date, there is still no widely accepted therapy that is capable of achieving complete functional recovery. Interface Focus (2012)
Despite some inspiring results, such as axonal growth into the distal ends, myelination of new nerve fibres and formation of synapses, only partial functional recovery is observed, and in the long term, scaffolds in fact do not make a difference in functional recovery as observed by BBB scores. This is confirmed by the finding that 1 year after implantation the number of regenerative axon fibres is much less than that of axons in spared tissues [49]. Hence, there is still a long way to go. Future design and fabrication of scaffolds for CNS regeneration will certainly focus on development of a materials-based system integrating different strategies of design and fabrication of scaffolds, delivery of bioactive agents and cells to trigger intrinsic selfregenerative ability. Scaffolds of hybrids or composites, which are made from blending of different kinds of electrospun nanofibres, or made of hydrogel –electrospun fibre composites, may have some unexpected properties in influencing cell behaviour and directing cell migration. In co-culture systems, different combinations of supporting cells and pluripotent cells may result in specific control over lineage of pluripotent cells, especially when combinations of growth factors are present. Scaffolds with special designer gradients of growth factors or ECM components may outperform in directing axonal outgrowth and promoting functional recovery. Although regeneration of the injured CNS is challenging, it is promising that integration of scaffolds, regulators and cells will provide auspicious therapies to achieve a complete recovery of CNS injuries. This work was supported by 973 programme (grant no. 2011CB606205) and the National Natural Science Foundation of China (grant nos. 50973052 and 50803031).
REFERENCES 1 LaPlaca, M. C., Simon, C. M., Prado, G. R. & Cullen, D. K. 2007 CNS injury biomechanics and experimental models. In Neurotrauma: new insights into pathology and treatment (eds J. T. Weber & A. I. R. Maas), pp. 13 –26. Amsterdam, The Netherlands: Elsevier Science.
Downloaded from http://rsfs.royalsocietypublishing.org/ on February 22, 2018
288
Review. HA-based scaffold for CNS regeneration
2 Hulsebosch, C. E. 2002 Recent advances in pathophysiology and treatment of spinal cord injury. Adv. Physiol. Educ. 26, 238– 255. (doi:10.1152/advan.00039.2002) 3 Beattie, M. S., Hermann, G. E., Rogers, R. C. & Bresnahan, J. C. 2002 Cell death in models of spinal cord injury. In Spinal cord trauma: regeneration, neural repair and functional recovery (eds L. McKerracher, G. Doucet & S. Rossignol), pp. 37– 47. Amsterdam, The Netherlands: Elsevier Science. 4 Yiu, G. & He, Z. 2006 Glial inhibition of CNS axon regeneration. Nat. Rev. Neurosci. 7, 617 –627. (doi:10.1038/ nrn1956) 5 Silver, J. & Miller, J. H. 2004 Regeneration beyond the glial scar. Nat. Rev. Neurosci. 5, 146 –156. (doi:10. 1038/nrn1326) 6 Chen, M. S., Huber, A. B., van der Haar, M. E., Frank, M., Schnell, L., Spillmann, A. A., Christ, F. & Schwab, M. E. 2000 Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature 403, 434 –439. (doi:10.1038/ 35000219) 7 GrandPre, T., Nakamura, F., Vartanian, T. & Strittmatter, S. M. 2000 Identification of the Nogo inhibitor of axon regeneration as a reticulon protein. Nature 403, 439 –444. (doi:10.1038/35000226) 8 Prinjha, R., Moore, S. E., Vinson, M., Blake, S., Morrow, R., Christie, G., Michlovich, D., Simmons, D. L. & Walsh, F. S. 2000 Neurobiology: inhibitor of neurite outgrowth in humans. Nature 403, 383– 384. (doi:10.1038/ 35000287) 9 Huber, A. B., Weinmann, O., Brosamle, C., Oertle, T. & Schwab, M. E. 2002 Patterns of Nogo mRNA and protein expression in the developing and adult rat and after CNS lesions. J. Neurosci. 22, 3553–3567. 10 Wang, X. X., Chun, S. J., Treloar, H., Vartanian, T., Greer, C. A. & Strittmatter, S. M. 2002 Localization of Nogo-A and Nogo-66 receptor proteins at sites of axon-myelin and synaptic contact. J. Neurosci. 22, 5505–5515. 11 McKerracher, L., David, S., Jackson, D. L., Kottis, V., Dunn, R. J. & Braun, P. E. 1994 Identification of myelin-associated glycoprotein as a major myelin-derived inhibitor of neurite growth. Neuron 13, 805 –811. (doi:10. 1016/0896-6273(94)90247-X) 12 Davies, S. J. A., Goucher, D. R., Doller, C. & Silver, J. 1999 Robust regeneration of adult sensory axons in degenerating white matter of the adult rat spinal cord. J. Neurosci. 19, 5810–5822. 13 Vinson, M., Strijbos, P. J. L. M., Rowles, A., Facci, L., Moore, S. E., Simmons, D. L. & Walsh, F. S. 2001 Myelin-associated glycoprotein interacts with ganglioside GT1b: a mechanism for neurite outgrowth inhibition. J. Biol. Chem. 276, 20 280 –20 285. (doi:10.1074/jbc. M100345200) 14 Gumera, C., Rauck, B. & Wang, Y. 2011 Materials for central nervous system regeneration: bioactive cues. J. Mater. Chem. 21, 7033–7051. (doi:10.1039/c0jm04335d) 15 Hench, L. L. & Polak, J. M. 2002 Third-generation biomedical materials. Science 295, 1014–1017. (doi:10. 1126/science.1067404) 16 Namba, R. M., Cole, A. A., Bjugstad, K. B. & Mahoney, M. J. 2009 Development of porous PEG hydrogels that enable efficient, uniform cell-seeding and permit early neural process extension. Acta Biomater. 5, 1884–1897. (doi:10.1016/j.actbio.2009.01.036) 17 Smith, L. A. & Ma, P. X. 2004 Nano-fibrous scaffolds for tissue engineering. Colloids Surf. B 39, 125– 131. (doi:10. 1016/j.colsurfb.2003.12.004)
Interface Focus (2012)
X. Wang et al. 18 Liu, T., Teng, W. K., Chan, B. P. & Chew, S. Y. 2010 Photochemical crosslinked electrospun collagen nanofibers: synthesis, characterization and neural stem cell interactions. J. Biomed. Mater. Res. A 95A, 276–282. (doi:10.1002/jbm.a.32831) 19 Hurtado, A., Cregg, J. M., Wang, H. B., Wendell, D. F., Oudega, M., Gilbert, R. J. & McDonald, J. W. 2011 Robust CNS regeneration after complete spinal cord transection using aligned poly-L-lactic acid microfibers. Biomaterials 32, 6068–6079. (doi:10.1016/j.biomaterials. 2011.05.006) 20 Corey, J. M., Lin, D. Y., Mycek, K. B., Chen, Q., Samuel, S., Feldman, E. L. & Martin, D. C. 2007 Aligned electrospun nanofibers specify the direction of dorsal root ganglia neurite growth. J. Biomed. Mater. Res. A 83A, 636–645. (doi:10.1002/jbm.a.31285) 21 Xie, J., Willerth, S. M., Li, X., Macewan, M. R., Rader, A., Sakiyama-Elbert, S. E. & Xia, Y. 2009 The differentiation of embryonic stem cells seeded on electrospun nanofibers into neural lineages. Biomaterials 30, 354–362. (doi:10.1016/j.biomaterials.2008.09.046) 22 Lee, J. Y., Bashur, C. A., Goldstein, A. S. & Schmidt, C. E. 2009 Polypyrrole-coated electrospun PLGA nanofibers for neural tissue applications. Biomaterials 30, 4325–4335. (doi:10.1016/j.biomaterials.2009.04.042) 23 Christopherson, G. T., Song, H. & Mao, H. Q. 2009 The influence of fiber diameter of electrospun substrates on neural stem cell differentiation and proliferation. Biomaterials 30, 556– 564. (doi:10.1016/j.biomaterials. 2008.10.004) 24 Jin, G. Z., Kim, M., Shin, U. S. & Kim, H. W. 2011 Neurite outgrowth of dorsal root ganglia neurons is enhanced on aligned nanofibrous biopolymer scaffold with carbon nanotube coating. Neurosci. Lett. 501, 10 –14. (doi:10. 1016/j.neulet.2011.06.023) 25 Carlberg, B., Axell, M. Z., Nannmark, U., Liu, J. & Kuhn, H. G. 2009 Electrospun polyurethane scaffolds for proliferation and neuronal differentiation of human embryonic stem cells. Biomed. Mater. 4, 045004. (doi:10.1088/1748-6041/4/4/045004) 26 Zhao, X., Pan, F., Xu, H., Yaseen, M., Shan, H., Hauser, C. A. E., Zhang, S. & Lu, J. R. 2010 Molecular self-assembly and applications of designer peptide amphiphiles. Chem. Soc. Rev. 39, 3480–3498. (doi:10.1039/b915923c) 27 Georges, P. C., Miller, W. J., Meaney, D. F., Sawyer, E. S. & Janmey, P. A. 2006 Matrices with compliance comparable to that of brain tissue select neuronal over glial growth in mixed cortical cultures. Biophys. J. 90, 3012–3018. (doi:10.1529/biophysj.105.073114) 28 Engler, A. J., Sen, S., Sweeney, H. L. & Discher, D. E. 2006 Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689. (doi:10.1016/j.cell.2006.06.044) 29 Leipzig, N. D. & Shoichet, M. S. 2009 The effect of substrate stiffness on adult neural stem cell behavior. Biomaterials 30, 6867–6878. (doi:10.1016/j.biomaterials. 2009.09.002) 30 Hynes, S. R., Rauch, M. F., Bertram, J. P. & Lavik, E. B. 2009 A library of tunable poly(ethylene glycol)/poly(Llysine) hydrogels to investigate the material cues that influence neural stem cell differentiation. J. Biomed. Mater. Res. A 89A, 499–509. (doi:10.1002/jbm.a.31987) 31 Meek, M. F., Den Dunnen, W. F. A., Schakenraad, J. M. & Robinson, P. H. 1999 Long-term evaluation of functional nerve recovery after reconstruction with a thin-walled biodegradable poly(DL-lactide-1-caprolactone) nerve guide, using walking track analysis and electrostimulation tests. Microsurgery 19, 247–253. (doi:10.1002/(SICI)10982752(1999)19:5,247::AID-MICR7.3.0.CO;2-E)
Downloaded from http://rsfs.royalsocietypublishing.org/ on February 22, 2018
Review. HA-based scaffold for CNS regeneration 32 Mackinnon, S. E. & Dellon, A. L. 1990 Clinical nerve reconstruction with a bioabsorbable polyglycolic acid tube. Plast. Reconstr. Surg. 85, 419 –424. 33 Flynn, L., Dalton, P. D. & Shoichet, M. S. 2003 Fiber templating of poly(2-hydroxyethyl methacrylate) for neural tissue engineering. Biomaterials 24, 4265–4272. (doi:10.1016/s0142-9612(03)00334-x) 34 Stokols, S., Sakamoto, J., Breckon, C., Holt, T., Weiss, J. & Tuszynski, M. H. 2006 Templated agarose scaffolds support linear axonal regeneration. Tissue Eng. 12, 2777– 2787. (doi:10.1089/ten.2006.12.2777) 35 Scott, J. B., Afshari, M., Kotek, R. & Saul, J. M. 2011 The promotion of axon extension in vitro using polymer-templated fibrin scaffolds. Biomaterials 32, 4830– 4839. (doi:10.1016/j.biomaterials.2011.03.037) 36 Wong, D. Y., Leveque, J. C., Brumblay, H., Krebsbach, P. H., Hollister, S. J. & LaMarca, F. 2008 Macroarchitectures in spinal cord scaffold implants influence regeneration. J. Neurotraum. 25, 1027 –1037. (doi:10. 1089/neu.2007.0473) 37 Stokols, S. & Tuszynski, M. H. 2004 The fabrication and characterization of linearly oriented nerve guidance scaffolds for spinal cord injury. Biomaterials 25, 5839–5846. (doi:10.1016/j.biomaterials.2004.01.041) 38 Stokols, S. & Tuszynski, M. H. 2006 Freeze-dried agarose scaffolds with uniaxial channels stimulate and guide linear axonal growth following spinal cord injury. Biomaterials 27, 443–451. (doi:10.1016/j.biomaterials.2005.06.039) 39 Mukhatyar, V. J., Salmeron-Sanchez, M., Rudra, S., Mukhopadaya, S., Barker, T. H., Garcia, A. J. & Bellamkonda, R. V. 2011 Role of fibronectin in topographical guidance of neurite extension on electrospun fibers. Biomaterials 32, 3958–3968. (doi:10.1016/j.bio materials.2011.02.015) 40 Cooper, A., Bhattarai, N. & Zhang, M. Q. 2011 Fabrication and cellular compatibility of aligned chitosan-PCL fibers for nerve tissue regeneration. Carbohydr. Polym. 85, 149 –156. (doi:10.1016/j.carbpol.2011.02.008) 41 Yang, F., Murugan, R., Wang, S. & Ramakrishna, S. 2005 Electrospinning of nano/micro scale poly(L-lactic acid) aligned fibers and their potential in neural tissue engineering. Biomaterials 26, 2603 –2610. (doi:10.1016/ j.biomaterials.2004.06.051) 42 Xie, J., MacEwan, M. R., Li, X., Sakiyama-Elbert, S. E. & Xia, Y. 2009 Neurite outgrowth on nanofiber scaffolds with different orders, structures, and surface properties. ACS Nano 3, 1151 –1159. (doi:10.1021/ nn900070z) 43 Cho, Y. I., Choi, J. S., Jeong, S. Y. & Yoo, H. S. 2010 Nerve growth factor (NGF)-conjugated electrospun nanostructures with topographical cues for neuronal differentiation of mesenchymal stem cells. Acta Biomater. 6, 4725–4733. (doi:10.1016/j.actbio.2010.06.019) 44 Platt, C. I., Krekoski, C. A., Ward, R. V., Edwards, D. R. & Gavrilovic, J. 2003 Extracellular matrix and matrix metalloproteinases in sciatic nerve. J. Neurosci. Res. 74, 417 –429. (doi:10.1002/jnr.10783) 45 Duan, W. M., Zhao, L. R., Westerman, M., Lovick, D., Furcht, L. T., McCarthy, J. B. & Low, W. C. 2000 Enhancement of nigral graft survival in rat brain with the systemic administration of synthetic fibronectin peptide V. Neuroscience 100, 521 –530. (doi:10.1016/S03064522(00)00299-2) 46 Zhao, L. R., Spellman, S., Kim, J., Duan, W. M., McCarthy, J. B. & Low, W. C. 2005 Synthetic fibronectin peptide exerts neuroprotective effects on transient focal brain ischemia in rats. Brain Res. 1054, 1–8. (doi:10. 1016/j.brainres.2005.04.056)
Interface Focus (2012)
X. Wang et al.
289
47 King, V. R., Hewazy, D., Alovskaya, A., Phillips, J. B., Brown, R. A. & Priestley, J. V. 2010 The neuroprotective effects of fibronectin mats and fibronectin peptides following spinal cord injury in the rat. Neuroscience 168, 523–530. (doi:10.1016/j.neuroscience.2010.03.040) 48 Koh, H. S., Yong, T., Chan, C. K. & Ramakrishna, S. 2008 Enhancement of neurite outgrowth using nanostructured scaffolds coupled with laminin. Biomaterials 29, 3574–3582. (doi:10.1016/j.biomaterials.2008.05.014) 49 Li, X., Yang, Z., Zhang, A., Wang, T. & Chen, W. 2009 Repair of thoracic spinal cord injury by chitosan tube implantation in adult rats. Biomaterials 30, 1121– 1132. (doi:10.1016/j.biomaterials.2008.10.063) 50 Prabhakaran, M. P., Venugopal, J. R. & Ramakrishna, S. 2009 Mesenchymal stem cell differentiation to neuronal cells on electrospun nanofibrous substrates for nerve tissue engineering. Biomaterials 30, 4996–5003. (doi:10.1016/j.biomaterials.2009.05.057) 51 Hashemi, S. M., Soudi, S., Shabani, I., Naderi, M. & Soleimani, M. 2011 The promotion of stemness and pluripotency following feeder-free culture of embryonic stem cells on collagen-grafted 3-dimensional nanofibrous scaffold. Biomaterials 32, 7363–7374. (doi:10.1016/j. biomaterials.2011.06.048) 52 Borkenhagen, M., Clemence, J. F., Sigrist, H. & Aebischer, P. 1998 Three-dimensional extracellular matrix engineering in the nervous system. J. Biomed. Mater. Res. 40, 392– 400. (doi:10.1002/(SICI)10974636(19980603)40:3,392::AID-JBM8.3.0.CO;2-C) 53 Cui, F. Z., Tian, W. M., Hou, S. P., Xu, Q. Y. & Lee, I. S. 2006 Hyaluronic acid hydrogel immobilized with RGD peptides for brain tissue engineering. J. Mater. Sci. Mater. Med. 17, 1393–1401. (doi:10.1007/s10856-006-0615-7) 54 Suzuki, M., Itoh, S., Yamaguchi, I., Takakuda, K., Kobayashi, H., Shinomiya, K. & Tanaka, J. 2003 Tendon chitosan tubes covalently coupled with synthesized laminin peptides facilitate nerve regeneration in vivo. J. Neurosci. Res. 72, 646–659. (doi:10.1002/ jnr.10589) 55 Schense, J. C., Bloch, J., Aebischer, P. & Hubbell, J. A. 2000 Enzymatic incorporation of bioactive peptides into fibrin matrices enhances neurite extension. Nat. Biotechnol. 18, 415–419. (doi:10.1038/74473) 56 Krewson, C. E., Klarman, M. L. & Saltzman, W. M. 1995 Distribution of nerve growth-factor following direct delivery to brain interstitium. Brain Res. 680, 196–206. (doi:10.1016/0006-8993(95)00261-N) 57 Yu, L. M. Y., Wosnick, J. H. & Shoichet, M. S. 2008 Miniaturized system of neurotrophin patterning for guided regeneration. J. Neurosci. Methods 171, 253–263. (doi:10.1016/j.jneumeth.2008.03.023) 58 Mo, L. H., Yang, Z. Y., Zhang, A. F. & Li, X. G. 2010 The repair of the injured adult rat hippocampus with NT-3-chitosan carriers. Biomaterials 31, 2184–2192. (doi:10.1016/j.biomaterials.2009.11.078) 59 Jain, A., Kim, Y. T., McKeon, R. J. & Bellamkonda, R. V. 2006 In situ gelling hydrogels for conformal repair of spinal cord defects, and local delivery of BDNF after spinal cord injury. Biomaterials 27, 497– 504. (doi:10. 1016/j.biomaterials.2005.07.008) 60 Wang, Y. C., Wu, Y. T., Huang, H. Y., Lin, H. I., Lo, L. W., Tzeng, S. F. & Yang, C. S. 2008 Sustained intraspinal delivery of neurotrophic factor encapsulated in biodegradable nanoparticles following contusive spinal cord injury. Biomaterials 29, 4546– 4553. (doi:10.1016/ j.biomaterials.2008.07.050) 61 Burdick, J. A., Ward, M., Liang, E., Young, M. J. & Langer, R. 2006 Stimulation of neurite outgrowth by
Downloaded from http://rsfs.royalsocietypublishing.org/ on February 22, 2018
290
62
63
64
65
66
67
68
69
70
71
72
73
74
Review. HA-based scaffold for CNS regeneration neurotrophins delivered from degradable hydrogels. Biomaterials 27, 452– 459. (doi:10.1016/j.biomaterials. 2005.06.034) Iannotti, C., Li, H. Y., Yan, P., Lu, X. B., Wirthlin, L. & Xu, X. M. 2003 Glial cell line-derived neurotrophic factor-enriched bridging transplants promote propriospinal axonal regeneration and enhance myelination after spinal cord injury. Exp. Neurol. 183, 379 –393. (doi:10. 1016/S0014-4886(03)00188-2) Piantino, J., Burdick, J. A., Goldberg, D., Langer, R. & Benowitz, L. I. 2006 An injectable, biodegradable hydrogel for trophic factor delivery enhances axonal rewiring and improves performance after spinal cord injury. Exp. Neurol. 201, 359–367. (doi:10.1016/j.expneurol.2006.04.020) Li, X., Yang, Z. & Zhang, A. 2009 The effect of neurotrophin-3/chitosan carriers on the proliferation and differentiation of neural stem cells. Biomaterials 30, 4978–4985. (doi:10.1016/j.biomaterials.2009.05.047) Sakiyama-Elbert, S. E. & Hubbell, J. A. 2000 Development of fibrin derivatives for controlled release of heparin-binding growth factors. J. Control. Release 65, 389 –402. (doi:10.1016/S0168-3659(99)00221-7) Willerth, S. M., Johnson, P. J., Maxwell, D. J., Parsons, S. R., Doukas, M. E. & Sakiyama-Elbert, S. E. 2007 Rationally designed peptides for controlled release of nerve growth factor from fibrin matrices. J. Biomed. Mater. Res. A 80A, 13 –23. (doi:10.1002/jbm.a.30844) Johnson, P. J., Tatara, A., Shiu, A. & Sakiyama-Elbert, S. E. 2010 Controlled release of neurotrophin-3 and platelet-derived growth factor from fibrin scaffolds containing neural progenitor cells enhances survival and differentiation into neurons in a subacute model of SCI. Cell Transplant. 19, 89–101. (doi:10.3727/ 096368909X477273) Taylor, S. J., McDonald, J. W. & Sakiyama-Elbert, S. E. 2004 Controlled release of neurotrophin-3 from fibrin gels for spinal cord injury. J. Control. Release 98, 281–294. (doi:10.1016/j.jconrel.2004.05.003) Rahman, N., Purpura, K. A., Wylie, R. G., Zandstra, P. W. & Shoichet, M. S. 2010 The use of vascular endothelial growth factor functionalized agarose to guide pluripotent stem cell aggregates toward blood progenitor cells. Biomaterials 31, 8262– 8270. (doi:10.1016/j.biomaterials.2010.07.040) Shen, Y. H., Shoichet, M. S. & Radisic, M. 2008 Vascular endothelial growth factor immobilized in collagen scaffold promotes penetration and proliferation of endothelial cells. Acta Biomater. 4, 477 –489. (doi:10.1016/j.actbio. 2007.12.011) Chvatal, S. A., Kim, Y.-T., Bratt-Leal, A. M., Lee, H. & Bellamkonda, R. V. 2008 Spatial distribution and acute anti-inflammatory effects of methylprednisolone after sustained local delivery to the contused spinal cord. Biomaterials 29, 1967–1975. (doi:10.1016/j.biomaterials. 2008.01.002) Schnell, E., Klinkhammer, K., Balzer, S., Brook, G., Klee, D., Dalton, P. & Mey, J. 2007 Guidance of glial cell migration and axonal growth on electrospun nanofibers of poly-1caprolactone and a collagen/poly-1-caprolactone blend. Biomaterials 28, 3012–3025. (doi:10.1016/j.bio materials. 2007.03.009) Xu, T., Molnar, P., Gregory, C., Das, M., Boland, T. & Hickman, J. J. 2009 Electrophysiological characterization of embryonic hippocampal neurons cultured in a 3D collagen hydrogel. Biomaterials 30, 4377–4383. (doi:10. 1016/j.biomaterials.2009.04.047) Novikova, L. N., Mosahebi, A., Wiberg, M., Terenghi, G., Kellerth, J. O. & Novikov, L. N. 2006 Alginate hydrogel
Interface Focus (2012)
X. Wang et al.
75
76
77
78
79
80
81
82
83
84
85
86
87
and matrigel as potential cell carriers for neurotransplantation. J. Biomed. Mater. Res. A 77A, 242–252. (doi:10. 1002/jbm.a.30603) Shen, Y. X., Qian, Y. Q., Zhang, H. X., Zuo, B. Q., Lu, Z. F., Fan, Z. H., Zhang, P., Zhang, F. & Zhou, C. L. 2010 Guidance of olfactory ensheathing cell growth and migration on electrospun silk fibroin scaffolds. Cell Transplant. 19, 147–157. (doi:10.3727/096368910x492616) Frampton, J. P., Hynd, M. R., Shuler, M. L. & Shain, W. 2011 Fabrication and optimization of alginate hydrogel constructs for use in 3D neural cell culture. Biomed. Mater. 6, 015002. (doi:10.1088/1748-6041/6/1/015002) Freudenberg, U. et al. 2009 A star-PEG-heparin hydrogel platform to aid cell replacement therapies for neurodegenerative diseases. Biomaterials 30, 5049– 5060. (doi:10.1016/j.biomaterials.2009.06.002) Lu, D., Mahmood, A., Qu, C., Hong, X., Kaplan, D. & Chopp, M. 2007 Collagen scaffolds populated with human marrow stromal cells reduce lesion volume and improve functional outcome after traumatic brain injury. Neurosurgery 61, 596– 602. (doi:10.1227/01.neu. 000028004/.85651.4f ) Barralet, J. E., Wang, L., Lawson, M., Triffitt, J. T., Cooper, P. R. & Shelton, R. M. 2005 Comparison of bone marrow cell growth on 2D and 3D alginate hydrogels. J. Mater. Sci. Mater. Med. 16, 515–519. (doi:10. 1007/s10856-005-0526-z) Hejcl, A. et al. 2010 HPMA–RGD hydrogels seeded with mesenchymal stem cells improve functional outcome in chronic spinal cord injury. Stem Cells Dev. 19, 1535– 1546. (doi:10.1089/scd.2009.0378) Kadoya, K. 2009 Combined intrinsic and extrinsic neuronal mechanisms facilitate bridging axonal regeneration one year after spinal cord injury. Neuron 64, 165–172. (doi:10.1016/j.neuron.2009.09.016) Guo, J., Su, H., Zeng, Y., Liang, Y.-X., Wong, W. M., Ellis-Behnke, R. G., So, K.-F. & Wu, W. 2007 Reknitting the injured spinal cord by self-assembling peptide nanofiber scaffold. Nanomed. Nanotechnol. Biol. Med. 3, 311– 321. (doi:10.1016/j.nano.2007.09.003) Xiong, Y., Qu, C. S., Mahmood, A., Liu, Z. W., Ning, R. Z., Li, Y., Kaplan, D. L., Schallert, T. & Chopp, M. 2009 Delayed transplantation of human marrow stromal cell-seeded scaffolds increases transcallosal neural fiber length, angiogenesis, and hippocampal neuronal survival and improves functional outcome after traumatic brain injury in rats. Brain Res. 1263, 183–191. (doi:10.1016/ j.brainres.2009.01.032) Yu, H. W., Cao, B., Feng, M. Y., Zhou, Q., Sun, X. D., Wu, S. L., Jin, S. Z., Liu, H. W. & Jin, L. H. 2010 Combinated transplantation of neural stem cells and collagen type I promote functional recovery after cerebral ischemia in rats. Anat. Rec. 293, 911–917. (doi:10.1002/ar.20941) Mahmood, A., Qu, C. S., Ning, R. Z., Wu, H. T., Goussev, A., Xiong, Y., Irtenkauf, S., Li, Y. & Chopp, M. 2011 Treatment of TBI with collagen scaffolds and human marrow stromal cells increases the expression of tissue plasminogen activator. J. Neurotraum. 28, 1199– 1207. (doi:10.1089/neu.2010.1694) Ford, M. C., Bertram, J. P., Hynes, S. R., Michaud, M., Li, Q., Young, M., Segal, S. S., Madri, J. A. & Lavik, E. B. 2006 A macroporous hydrogel for the coculture of neural progenitor and endothelial cells to form functional vascular networks in vivo. Proc. Natl Acad. Sci. USA 103, 2512– 2517. (doi:10.1073/pnas.0506020102) Rauch, M. F., Hynes, S. R., Bertram, J., Redmond, A., Robinson, R., Williams, C., Xu, H., Madri, J. A. & Lavik, E. B. 2009 Engineering angiogenesis following
Downloaded from http://rsfs.royalsocietypublishing.org/ on February 22, 2018
Review. HA-based scaffold for CNS regeneration
88
89
90
91
92
93
94
95
96
97
98
99
100
101
spinal cord injury: a coculture of neural progenitor and endothelial cells in a degradable polymer implant leads to an increase in vessel density and formation of the blood –spinal cord barrier. Eur. J. Neurosci. 29, 132 –145. (doi:10.1111/j.1460-9568.2008.06567.x) Mori, M., Yamaguchi, M., Sumitomo, S. & Takai, Y. 2004 Hyaluronan-based biomaterials in tissue engineering. Acta Histochem. Cytochem. 37, 1–5. (doi:10.1267/ahc.37.1) Laurent, T. C., Laurent, U. B. G. & Fraser, J. R. E. 1996 The structure and function of hyaluronan: a overview. Immunol. Cell Biol. 74, A1–A7. (doi:10.1038/icb.1996.32) Costa, C., Tortosa, R., Domenech, A., Vidal, E., Pumarola, M. & Bassols, A. 2007 Mapping of aggrecan, hyaluronic acid, heparan sulphate proteoglycans and aquaporin 4 in the central nervous system of the mouse. J. Chem. Neuroanatomy 33, 111 –123. (doi:10.1016/j. jchemneu.2007.01.006) Manuskiatti, W. & Maibach, H. I. 1996 Hyaluronic acid and skin: wound healing and aging. Int. J. Dermatol. 35, 539 –544. (doi:10.1111/j.1365-4362.1996.tb03650.x) Chen, W. Y. J. & Abatangelo, G. 1999 Functions of hyaluronan in wound repair. Wound Repair Regen. 7, 79 –89. (doi:10.1046/j.1524-475X.1999.00079.x) Tian, W. M., Hou, S. P., Ma, J., Zhang, C. L., Xu, Q. Y., Lee, I. S., Li, H. D., Spector, M. & Cui, F. Z. 2005 Hyathree-dimensional luronic acid –poly-D-lysine-based hydrogel for traumatic brain injury. Tissue Eng. 11, 513 –525. (doi:10.1089/ten.2005.11.513) Hou, S., Tian, W., Xu, Q., Cui, F., Zhang, J., Lu, Q. & Zhao, C. 2006 The enhancement of cell adherence and inducement of neurite outgrowth of dorsal root ganglia co-cultured with hyaluronic acid hydrogels modified with Nogo-66 receptor antagonist in vitro. Neuroscience 137, 519 –529. (doi:10.1016/j.neuroscience.2005.09.029) Ma, J., Tian, W.-M., Hou, S.-P., Xu, Q.-Y., Spector, M. & Cui, F.-Z. 2007 An experimental test of stroke recovery by implanting a hyaluronic acid hydrogel carrying a Nogo receptor antibody in a rat model. Biomed. Mater. 2, 233 –240. (doi:10.1088/1748-6041/2/4/005) Wei, Y. T., He, Y., Xu, C. L., Wang, Y., Liu, B. F., Wang, X. M., Sun, X. D., Cui, F. Z. & Xu, Q. Y. 2010 Hyaluronic acid hydrogel modified with nogo-66 receptor antibody and poly-(L)-lysine to promote axon regrowth after spinal cord injury. J. Biomed. Mater. Res. B. 95B, 110–117. (doi:10. 1002/jbm.b.31689) Ozgenel, G. Y. 2003 Effects of hyaluronic acid on peripheral nerve scarring and regeneration in rats. Microsurgery 23, 575 –581. (doi:10.1002/micr.10209) Lin, C.-M. et al. 2009 Hyaluronic acid inhibits the glial scar formation after brain damage with tissue loss in rats. Surg. Neurol. 72(Suppl. 2), S50– S54. (doi:10. 1016/j.wneu.2009.09.004) Khaing, Z. Z., Milman, B. D., Vanscoy, J. E., Seidlits, S. K., Grill, R. J. & Schmidt, C. E. 2011 High molecular weight hyaluronic acid limits astrocyte activation and scar formation after spinal cord injury. J. Neural Eng. 8, 046033. (doi:10.1088/1741-2560/8/4/046033) Campo, G. M., Avenoso, A., Campo, S., D’Ascola, A., Nastasi, G. & Calatroni, A. 2010 Molecular size hyaluronan differently modulates toll-like receptor-4 in LPSinduced inflammation in mouse chondrocytes. Biochimie 92, 204 –215. (doi:10.1016/j.biochi.2009.10.006) Seidlits, S. K., Khaing, Z. Z., Petersen, R. R., Nickels, J. D., Vanscoy, J. E., Shear, J. B. & Schmidt, C. E. 2010 The effects of hyaluronic acid hydrogels with
Interface Focus (2012)
102
103
104
105
106
107
108
109
110
111
112
113
114
X. Wang et al.
291
tunable mechanical properties on neural progenitor cell differentiation. Biomaterials 31, 3930–3940. (doi:10. 1016/j.biomaterials.2010.01.125) Wang, T.-W. & Spector, M. 2009 Development of hyaluronic acid-based scaffolds for brain tissue engineering. Acta Biomater. 5, 2371–2384. (doi:10.1016/j.actbio.2009.03.033) Brannvall, K., Bergman, K., Wallenquist, U., Svahn, S., Bowden, T., Hilborn, J. & Forsberg-Nilsson, K. 2007 Enhanced neuronal differentiation in a three-dimensional collagen-hyaluronan matrix. J. Neurosci. Res. 85, 2138–2146. (doi:10.1002/jnr.21358) Hou, S. P., Xu, Q. Y., Tian, W. M., Cui, F. Z., Cai, Q., Ma, J. & Lee, I. S. 2005 The repair of brain lesion by implantation of hyaluronic acid hydrogels modified with laminin. J. Neurosci. Methods 148, 60–70. (doi:10. 1016/j.jneumeth.2005.04.016) Wei, Y. T., Tian, W. M., Yu, X., Cui, F. Z., Hou, S. P., Xu, Q. Y. & Lee, I.-S. 2007 Hyaluronic acid hydrogels with IKVAV peptides for tissue repair and axonal regeneration in an injured rat brain. Biomed. Mater. 2, S142 –S146. (doi:10.1088/1748-6041/2/3/s11) Yavin, E. & Yavin, Z. 1974 Attachment and culture of dissociated cells from rat embryo cerebral hemispheres on polylysine-coated surface. J. Cell Biol. 62, 540–546. (doi:10.1083/jcb.62.2.540) Fournier, A. E., GrandPre, T. & Strittmatter, S. M. 2001 Identification of a receptor mediating Nogo-66 inhibition of axonal regeneration. Nature 409, 341–346. (doi:10. 1038/35053072) Wang, K. C., Koprivica, V., Kim, J. A., Sivasankaran, R., Guo, Y., Neve, R. L. & He, Z. G. 2002 Oligodendrocyte-myelin glycoprotein is a Nogo receptor ligand that inhibits neurite outgrowth. Nature 417, 941–944. (doi:10.1038/nature00867) Domeniconi, M. et al. 2002 Myelin-associated glycoprotein interacts with the Nogo66 receptor to inhibit neurite outgrowth. Neuron 35, 283–290. (doi:10.1016/ S0896-6273(02)00770-5) Tian, W. M., Zhang, C. L., Hou, S. P., Yu, X., Cui, F. Z., Xu, Q. Y., Sheng, S. L., Cui, H. & Li, H. D. 2005 Hyaluronic acid hydrogel as Nogo-66 receptor antibody delivery system for the repairing of injured rat brain: in vitro. J. Control. Release 102, 13–22. (doi:10.1016/j.jconrel. 2004.09.025) Park, J., Lim, E., Back, S., Na, H., Park, Y. & Sun, K. 2010 Nerve regeneration following spinal cord injury using matrix metalloproteinase-sensitive, hyaluronic acid-based biomimetic hydrogel scaffold containing brain-derived neurotrophic factor. J. Biomed. Mater. Res. A 93A, 1091–1099. (doi:10.1002/jbm.a.32519) Wang, Y., Wei, Y. T., Zu, Z. H., Ju, R. K., Guo, M. Y., Wang, X. M., Xu, Q. Y. & Cui, F. Z. 2011 Combination of hyaluronic acid hydrogel scaffold and PLGA microspheres for supporting survival of neural stem cells. Pharm. Res. 28, 1406–1414. (doi:10.1007/s11095-011-0452-3) Wei, Y. T., Sun, X. D., Xia, X., Cui, F. Z., He, Y., Liu, B. F. & Xu, Q. Y. 2009 Hyaluronic acid hydrogel modified with Nogo-66 receptor antibody and poly(L-lysine) enhancement of adherence and survival of primary hippocampal neurons. J. Bioact. Compat. Polym. 24, 205–219. (doi:10.1177/0883911509102266) Pan, L. J., Ren, Y. J., Cui, F. Z. & Xu, Q. Y. 2009 Viability and differentiation of neural precursors on hyaluronic acid hydrogel scaffold. J. Neurosci. Res. 87, 3207–3220. (doi:10.1002/jnr.22142)