Hydrogel Mediated Delivery of Trophic Factors for Neural Repair Joshua S

Advanced Review Hydrogel mediated delivery of trophic factors for neural repair Joshua S. Katz1 and Jason A. Burdick∗

Neurotrophins have been implicated in a variety of diseases and their delivery to sites of disease and injury has therapeutic potential in applications including spinal cord injury, Alzheimer’s disease, and Parkinson’s disease. Biodegradable polymers, and specifically, biodegradable water-swollen hydrogels, may be advantageous as delivery vehicles for neurotrophins because of tissue-like properties, tailorability with respect to degradation and release behavior, and a history of biocompatibility. These materials may be designed to degrade via hydrolytic or enzymatic mechanisms and can be used for the sustained delivery of trophic factors in vivo. Hydrogels investigated to date include purely synthetic to purely natural, depending on the application and intended release profiles. Also, flexibility in material processing has allowed for the investigation of injectable materials, the development of scaffolding and porous conduits, and the use of composites for tailored molecule delivery profiles. It is the objective of this review to describe what has been accomplished in this area thus far and to remark on potential future directions in this field. Ultimately, the goal is to engineer optimal biomaterials to deliver molecules in a controlled and dictated manner that can promote regeneration and healing for numerous neural applications.  2008 John Wiley & Sons, Inc. Wiley Interdiscipl. Rev. Nanomed. Nanobiotechnol. 2009 1 128–139

isruption of central nervous system (CNS) or Neurotrophins have been widely investigated for Dperipheral nervous system (PNS) tissues such their influence on cell mortality, differentiation, and as the spinal cord, optic nerve, and motor neurons function in both the CNS and the PNS.6 These neu- can severely affect a patient’s motor, sensory, rotrophins include factors such as nerve growth factor and autonomic functions, and depending on the (NGF), brain-derived neurotrophic factor (BDNF), severity of the injury, the patient’s quality of life neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5), can decline dramatically.1–3 Unfortunately, current and glial derived neurotrophic factor (GDNF). Neu- clinical treatment options are severely limited for rotrophins bind to tropomyosin-related kinase (Trk) many of these injuries and diseases and are unable receptors (TrkA for NGF, TrkB for BDNF and NT- to restore complete function to these patients. For 4/5, and TrkC for NT-3) and the pan-neurotrophin instance, in the spinal cord, one significant barrier receptor p75.7 The functions of neurotrophins in to regeneration is the extremely complex cascade of vivo are many and include controlling neural cell events (e.g., inflammation, glial scarring, release of growth and survival, influencing glial development, inhibitory molecules) that occurs after injury that and functions in non-neural tissues such as in the must be addressed to restore functional recovery to cardiovascular and immune systems.8–12 Addition- the patient.4,5 However, one promising therapy is the ally, neurotrophins can mediate axon signals or act delivery of neurotrophins that can influence the local on myelinating glia to influence the remyelination function of cells within and surrounding the injury site. of axons.13,14 For example, neurotrophins can promote axonal growth, neuronal survival, and plasticity ∗ Correspondence to: Jason A. Burdick, Department of Bioengineer- after injury to the spinal cord.15 Lu and coworkers16 ing, University of Pennsylvania, 240 Skirkanich Hall, 210 S. 33rd Street, Philadelphia, PA 19104, USA. recently illustrated the ability of NT-3 in combination E-mail: [email protected] with cyclic adenosine monophosphate to induce regen- 1Department of Bioengineering, University of Pennsylvania, 240 eration of sensory axons past a spinal cord lesion. Skirkanich Hall, 210 S. 33rd Street, Philadelphia, PA 19104, USA Additionally, the overexpression of neurotrophins DOI: 10.1002/wnan.010 after injury induced sprouting of corticospinal tract

128  2008JohnWiley&Sons,Inc. Volume1,January/February2009 WIREs Nanomedicine and Nanobiotechnology Hydrogel mediated delivery of trophic factors axons past the injury site.17 Techniques such as gene INJECTABLE HYDROGELS therapy, delivery via stem cells, and polymeric delivery Hydrogels are made injectable through numerous vehicles are being investigated for the supplementation means including free-radical polymerizations (i.e., 6 of neurotrophins to injured neural tissues. thermal, photo, or redox initiation), self-assembly There are several methods which have been of materials, or ionic crosslinking.27 One of the explored for the delivery of neurotrophins and drugs biggest advantages to using injectable materials for to the nervous system, including mini-pumps, genet- these applications is the non-invasiveness of hydrogel ically modified cells, and polymer formulations.18–20 delivery, which can limit further tissue damage. For Hydrogels are water-swollen insoluble polymer net- instance, disruption of the dura cover to many tissues works that have a wide range of chemical compo- (including the brain and spinal cord) results in the sitions and properties.21–23 Hydrogels can be formed loss of many potentially stimulatory molecules, which through a variety of mechanisms, including both phys- could be avoided with injection through the dura. ical (e.g., ionic or hydrogen bonding) and chemical Additionally, many imaging techniques could be used in combination with the injection procedures to gelation (e.g., covalent bonding). Through alterations potentially deliver these hydrogels in a closed surgery. in the chemical structure, important properties such as Several clinically used biomaterials are already swelling, degradation (e.g., hydrolytic or enzymatic), injected in vivo, such as poly(methyl methacrylate) and mechanics can be controlled. The delivery of bone cements28,29 and photocurable resins for filling large molecules, such as neurotrophins, is typically dental caries,30 and neural applications could benefit accomplished by encapsulating the molecules during from similar procedures. This section focuses on the gelation, which are subsequently released via diffu- various injectable hydrogels that have been explored sion and degradation mechanisms. This process is for delivery of growth factors for neurological relatively complex and dynamic as the hydrogel mesh applications. size changes as the material degrades and swells. With Agarose is a polysaccharide derived from recent advances in polymer synthesis and our under- seaweed and comprised of repeating galactopyranose standing of biological polymers, our ability to control units. It has been used for a variety of biomedical hydrogels, and consequently, molecule delivery is con- applications and can be thermally induced to form a 24 hydrogel through intermolecular hydrogen bonding stantly improving. 31,32 There are numerous factors that make hydrogels interactions. At elevated temperatures when hydrogen bonds cannot form, agarose solutions do ideal delivery vehicles for neurotrophic factors and not gel. However, as the solution is cooled, hydrogen repair of neural tissue. First, this approach does not bonds begin to form, leading to gelation. Jain and introduce either live tissue (e.g., grafts) or viral vectors, coworkers33 used cooled nitrogen gas to gel solutions eliminating potential issues with graft rejection and of agarose in situ. Following injury to the spinal adverse responses. Next, hydrogel delivery eliminates cord, a solution of agarose containing BDNF-loaded the need for devices like pumps and catheters that can microtubules was pipetted into the injury site. The malfunction. Finally, hydrogels can provide constant solution was then cooled by nitrogen gas which and tailorable delivery of either one or numerous was passed over a bath of dry ice to produce a molecules to a desired in vivo location. Because of gel. A schematic of this cooling system is shown in the short in vivo half life of neurotrophins, sustained Figure 1(a). The presence of BDNF greatly enhanced delivery to the injury site results in significantly better the regeneration of axons and their ability to penetrate recovery compared to a single injection.25,26 Because into and through the scaffold. of the complexity of injuries, the appropriate delivery Another natural polymer, collagen, crosslinks profile depends on the injured tissue and timing of at physiological conditions through ionic interactions with salts present in solution. Hamann and therapies. Several hydrogels have been investigated coworkers35,36 injected aqueous solutions of collagen for the controlled delivery of neurotrophins and containing growth factors (epidermal growth factor it is the objective of this review to outline past (EGF) and/or FGF-1) into the intrathecal space sur- work in this area and look forward to future rounding the spinal cord as a drug delivery system. directions. The hydrogels investigated have ranged in Rather than acting as a mechanical support for axonal composition from purely synthetic (e.g., poly (ethylene regeneration as seen in many other systems, this sys- glycol) (PEG)) to purely natural (e.g., collagen), and tem chemically supports the regenerative response to in physical structure from uniform gels to porous spinal cord injury (SCI) by the delivery of therapeu- scaffolds and composite materials. tic agents directly and locally to the site of injury.

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(a) to shear stresses (Figure 1(b)). A volume of 10 µL of HAMC was injected into the intrathecal space following a clip compression injury to rats. While

N2 Tank functional recovery of the animals was better with the injection of the HAMC than with an injection of Dry ice box artificial cerebrospinal fluid (aCSF) into the intrathecal Aluminum rod space, the difference was not significant after 1 week. surrounded by dry ice However, no growth factors were injected with the HAMC in this study, and the presence of such factors could potentially cause significant improvement in the animal’s recovery. (b) PEG has been widely investigated as a biomaterial for many years, primarily because it is a 40,41 Liquid relatively inert material. As protein adsorption to PEG hydrogels is minimal, non-specific protein binding and cellular interactions can be avoided. Hubbell Gel and coworkers42,43 modified PEG hydrogels with both Dura mater degradable units and then reactive groups to form intrathecal space biodegradable hydrogels based on PEG. The chemi- Spinal cord cal structure of this material is shown in Figure 2(a). These synthetic macromers form a hydrogel via a rad- FIGURE 1| Examples of techniques for in situ gelation of hydrogel ical polymerization, which typically is initiated using solutions. (a) For agarose, nitrogen is passed from the tank through a a photoinitiation process. For the encapsulation and bath of dry ice and acetone (box) through an aluminum rod surrounded release of a growth factor (e.g., neurotrophin), the by dry ice to cool the solution and induce gelation. (b) Shear forces, PEG macromer is dissolved in a buffer solution con- induced through syringe injection, allow methylcellulose and taining the photoinitiator and the growth factor. This acetate-modified hyaluronan (HAMC) gels to flow and then re-gel at solution is exposed to light to form a hydrogel and physiological temperatures in vivo, potentially in the intrathecal space. the growth factor is released through a combination of diffusion and degradation.43–45 This hydrogel Significantly higher levels of cavitation were observed has been explored extensively for both tissue engi- in animals that did not receive the growth factors at neering and drug delivery applications,46,47 primarily the site of injury. However, in a functional recovery because of the control that is afforded over the tempo- test with this specific delivery system, there was no ral material properties. For instance, degradation can significant difference between the treated animals and be altered through parameters such as the molecular the controls. weight of the PEG, the type (e.g., lactic vs caproic To address some of the issues associated acid) of degradable groups, the number of degradable with the injectable collagen system, such as slow groups, and the concentration of the macromer in gelation and cell infiltration within the dura, Gupta solution. and coworkers34 created mechanically-reversible gels For the delivery of neurotrophins, Burdick consisting of a blend of methylcellulose and acetate- and coworkers48 monitored the release kinetics of modified hyaluronan (HAMC). Methylcellulose (MC) several factors from PEG hydrogels. Example release is a modified natural material that gels with increasing profiles are shown in Figure 2(b) for a range of temperature. As hydrogen bonds break, hydrophobic neurotrophins. They found that the release kinetics interactions force the MC to gel. Hyaluronan of these factors were controlled by changes in (HA) is a naturally occurring polysaccharide that the network crosslinking density, which influences may spontaneously gel in water though hydrophilic neurotrophin diffusion and subsequent release from associations with the water and has been explored the gels, with total release times ranging from widely for biological applications.37–39 However, weeks to several months. The release and activity of when sheared, the gel breaks as the molecules align one neurotrophic factor, ciliary-neurotrophic factor with the direction of the shear. One limitation (CNTF), was assessed with a cell based proliferation of using unmodified HA alone is that it quickly assay and an assay for neurite outgrowth from retinal disperses when injected in vivo because of its high explants. CNTF released from a degradable hydrogel water solubility. HAMC blends gel at room and above an explanted retina was able to stimulate physiologic temperatures, but flow when subjected outgrowth of a significantly higher number of

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(a) O POROUS SCAFFOLDS AND GUIDANCE CHANNELS R R O O While the primary goal of injectable hydrogels is n to chemically support axonal regeneration through O neurotrophin delivery locally at the site of injury, it may be advantageous to also physically support O O cellular function and growing axons. To date, the R = or 5 m m majority of this support has come through the O O development of nerve guidance channels, which aim to template regenerating axons through the injury 50 (b) 100 site. While originally designed from non-degradable, non-hydrogel materials, more recent scaffolds have 80 begun to incorporate many features common among tissue engineering scaffolds, such as porosity and 60 swelling. 40 Poly(hydroxyethyl methacrylate) (poly(HEMA)) hydrogels have been widely explored as a biocompat- 20 ible, non-degradable material for drug delivery and Cumulative % release tissue engineering applications.51,52 Gels form through 0 the free-radical polymerization of the methacrylate 0 5 10 15 20 25 Time (days) units in the presence of a crosslinker (i.e., dimethacry- late). These materials have been used recently for the FIGURE 2| (a) Structure of PEG modified with degradable groups delivery of neurotrophins to cells for axonal regen- and reactive acrylates to allow for degradation and eration. Piotrowicz and Shoichet53 synthesized nerve photopolymerization, respectively. (b) Cumulative release of guidance channels from copolymers of HEMA and • neurotrophins ciliary-neurotrophic factor (CNTF) ( ), BDNF ( ), and methyl methacrylate (MMA). They incorporated NGF NT-3 ( ) from 10 wt% PEG hydrogels. (Reprinted, with permission, into the channels either by adding poly (lactic-co- from Ref. 48. Copyright 2006 Elsevier). glycolic acid) (PLGA) microspheres to the formulation or by adding a second layer of HEMA containing NGF to the interior wall of the channel. Sustained neurites than controls without CNTF. Finally, unique release of NGF was observed for both systems over microsphere/hydrogel composites were developed to a 30-day period, but the release was much higher simultaneously deliver multiple neurotrophins with for the HEMA/NGF coated nerve guidance channels. 48 individual release rates. Shoichet and coworkers54,55 also induced concentra- This system was also exploited for application to tion gradients of NGF and NT-3 into macroporous 49 the injured spinal cord. The failure of injured axons HEMA nerve guidance channels using a gradient to regenerate in the mature CNS can have significant mixer. Neurite outgrowth within the channels was implications in spinal cord injury. However, the observed to follow the gradient of NGF. Additionally, delivery of neurotrophins can promote axon growth they found that NGF and NT-3 work synergistically, in injured CNS and thus, the delivery of NT-3 was with less NGF required to successfully guide neurite used in an attempt to alter anatomical and behavioral growth when NT-3 was present. outcomes. NT-3 was delivered to a dorsal hemisection Belkas and coworkers56 filled HEMA-co-MMA lesion in adult rats using PEG hydrogels by injecting nerve tubes with collagen and implanted them into the macromer and initiator solution directly on the rats that had 10 mm sections of the sciatic nerve lesion and exposing to visible light (using a dental removed. A bimodal response was observed in curing lamp) for 60 s for gelation. The treated animals which approximately 60% of the rats improved showed improved recovery in both an open-field BBB in comparison to rats which received autografts in test and in a horizontal ladder walk test compared place of the nerve tube, while approximately 40% to untreated rats. Also, the treated animals showed saw no significant healing, potentially as a result of much greater axon growth in both the corticospinal channel collapse. In an expansion of this work, Tsai and raphespinal tracts over control animals. Thus, this and coworkers57 filled HEMA-co-MMA hydrogel provides great scope to the use of injectable hydrogels guidance channels with MatrigelTM, MC, fibrin, or for trophic factor delivery to influence outcomes in type I collagen. Some channels also contained either injured patients. FGF-1 or NT-3. Channels were implanted into rats

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(a) (b)

FIGURE 3| Insertion of collagen-filled HEMA-co-MMA nerve tubes into a complete transection spinal cord injury. (a) Injury pre-insertion. (b) Filling of tubes with collagen. (c) Half-implanted tube. (d) Fully-inserted nerve tube at site of injury. (Reprinted, with permission, from Ref. 57. Copyright 2006 Elsevier). that had undergone a complete spinal cord transection channels within poly(HEMA) hydrogels. The gels (Figure 3). The presence of the growth factors altered were created by pouring a HEMA solution contain- the density and improved the orientation of the ing a biodegradable crosslinker and photoinitiator growing axons. Two channel formulations (fibrin over templated poly(methyl methacrylate) (PMMA) and multiple channels within a larger channel) led spheres, which is a technique to create highly-ordered to consistent improvement in recovery as measured by porous hydrogels.61 Selective blocking of the UV light BBB score. and exploitation of reaction behavior was used to To better mimic the mechanical properties of create open channels of several hundred microns in the spinal cord, Bakshi and coworkers58 designed diameter, while the rest of the gel contained open, HEMA microporous gels containing 85% water. highly interconnected pores of approximately fifty These gels had compressive moduli of 3–4 kPa, microns in diameter. While the authors did not address similar to that of the spinal cord. The flexibility the potential application of this system to neural of the gels allows them to easily fit into a defect regeneration, it is easy to postulate that within the caused by spinal cord injury, and the presence of system, the large pores could facilitate axonal growth the micropores allows for cells to migrate and grow and guidance, while the smaller surrounding pores through the gels. Gels were implanted into injured enabled the rapid transport of nutrients to and from rats and after 1, 2, and 4 weeks, the rats were the growing cells. sacrificed and examined for response to the gels. All Stokols and Tuszynski62,63 created agarose gels prompted a moderate inflammatory response, scaffolds with linearly-oriented channels by growing though there was little scarring. In gels soaked ice crystals along a temperature gradient through a in BDNF prior to implantation, axon regeneration solution of agarose followed by freeze drying. A was observed, though only transiently (2 weeks). In representative image of the scaffolds is shown in order to make the nerve conduits bioactive, Yu and Figure 4, showing longitudinal pores with a honey- Shoichet59 copolymerized HEMA with 2-aminoethyl comb structure cross-section. BDNF was incorporated methacrylate, which can be easily modified with into the gels by swelling the freeze-dried gels in peptides. In this work, two peptides derived from the presence of the protein. Prior to implantation laminin (i.e., YIGSR and IKVAV) were used. Peptide- in rats, the channels were also filled with collagen. modified conduits templated on polycaprolactone The presence of a growth factor allowed for 2–3 times (PCL) fibers (which were subsequently removed, as many axons to penetrate through the channels yielding hollow channels) were much more conducive compared to negative controls (no BDNF), and the to cell growth and neurite extension compared to immune response was minimal. However, the authors unmodified channels. do not report on the functional recovery of the In a recent study, Bryant and coworkers60 used animals. Stokols, Tuszynski, and coworkers64 also a photomask and ultraviolet light to selectively gel templated agarose channels on polystyrene fibers, certain regions of a precursor solution, creating yielding a scaffold with uniform channels following

132  2008JohnWiley&Sons,Inc. Volume1,January/February2009 WIREs Nanomedicine and Nanobiotechnology Hydrogel mediated delivery of trophic factors

(a) (b)

(c)

FIGURE 4| Aligned agarose nerve tubes synthesized by ice crystallization. (a, b) SEM images of freeze-dried tubes a| longitudinally and (b) cross-sectionally. (c) Axonal penetration in vivo through the agarose tubes. Scale bars = 100 µm. (Reprinted, with permission, from Ref. 63. Copyright 2006 Elsevier). polystyrene dissolution. Prior to implantation, the peptide, as patterned via the light activation. This authors filled the channels with bone marrow stromal work reports the spatial control of only two dimen- cells (MSCs) or MSCs engineered to produce BDNF. sions or at best a gradient in the z direction. However, BDNF production by the MSCs in vivo greatly it is easy to see how such a system could easily enhanced the ability of regenerating axons to penetrate be expanded to three dimensional patterning using into the scaffold and span the length of the scaffold. advanced microscopy and laser techniques and could However, the functional recovery of the animals was incorporate the delivery of neurotrophic factors. not reported. To study how cells move along a molecular gradient, Dodla and Bellamkonda70 immobilized laminin-1 (LN-1) in an agarose gel. Gradients of PATTERNED AND COMPOSITE different degrees were created by diffusion of LN- HYDROGELS 1 through the gel followed by photoimmobilization. To provide spatial chemical cues and to enhance Interestingly, the lower concentration gradients of material properties, numerous techniques such as LN-1 did a better job of promoting directionality patterning and the use of composite materials have in neurite extension and growth. These results are been investigated. As demonstrated by Nomura and potentially useful for the development of regenerative coworkers,65 both the mechanical and chemical prop- materials that could be modified with gradients of erties of implants are important for the development neurotrophins and used in vivo for axonal growth of a successful implant. Loading one material within and guidance. another material allows for further tuning of both Fibrin is another natural polymer that has 71,72 types of properties in tandem with each other. been widely used in the biomaterials field. 73–77 The 2-nitrobenzyl moiety is well-known as a Sakiyama-Elbert and coworkers have shown photo-protecting group, and recently has found a progress in the controlled release of neurotrophins niche in the development of functional biomaterials from fibrin gels through interactions with heparin which can be patterned in multiple dimensions.66,67 In or peptides. Heparin interacts non-covalently with a pioneering work, Luo and Shoichet68,69 conjugated various neurotrophins, such as NGF, BDNF and NT- agarose polymers with 2-nitrobenzyl cysteine. Follow- 3. Heparin was attached to fibrin gels that contained ing gelation, selected portions of the gels were exposed immobilized heparin binding peptides within the to ultraviolet light, liberating the thiol sidechain of the matrix to influence the release of neurotrophins from blocked cysteine. Further conjugation of the liberated the fibrin gels. A schematic of this process is illustrated cysteine residues allowed for biofunctionalization of in Figure 5. Decreased release rates from the gels were the gels with small peptides or proteins. Rat dorsal observed and reported for both NGF74 and NT-3,75 root ganglia were then seeded on the gels and seen and were observed to significantly improve neurite to grow through the columns of gel containing the extension in vitro.74 In in vivo studies, regeneration of

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microspheres. The released EGF from the nerve guidance channels was able to successfully promote the G G G G formation of neurospheres in culture for the first 14 days of release. However, by day 21, the released EGF was no longer biologically active. The lack of avail- ability of bioactive EGF is a very serious potential G G G limitation that must be addressed for the devel- G G opment of sustained release implants. Others have entrapped numerous neurotrophins in microspheres G 79,80 81 G for delivery in vivo. Yu and Bellamkonda loaded Heparin poly(sulfone) guidance channels with LN-1-modified Fibrin agarose and lipid microtubules loaded with NGF for Peptide G Growth factor slow release. The channels were implanted in a 10- mm defect in the sciatic nerve of rats. Recovery and FIGURE 5| Schematic of growth factor immobilization to a fibrin regeneration were statistically the same as autografts, matrix through interactions with heparin. (Reprinted, with permission, though neither group returned to ‘normal’ function. from Ref. 76. Copyright 2006 Elsevier). As an additional example, Yang and coworkers82 formed porous nerve conduits of PLGA 75/25 contain- axons was significantly improved by the inclusion of ing NGF (illustrated in Figure 6). Successful release of heparin into the fibrin matrices to control the release NGF was observed and release could be sustained up of NGF73 or NT-3.76 As another method to slow the to a 40-day period by changes in porosity, mechanism release of NGF from fibrin matrices, phage display of NGF incorporation, and polymer molecular weight. was used to positively select an NGF binding peptide, Others have used non-porous scaffolding for neural 83,84 which was then immobilized in the fibrin matrix.77 An regeneration applications, but this is beyond the improved response in neurite extension was observed scope of this review. relative to free NGF in a fibrin matrix and the response was both dose and pH dependent. Towards another composite structure, Nomura FUTURE DIRECTIONS and coworkers65 carried out a study similar to that carried out by Belkas and coworkers56 (discussed As reviewed above, a substantial amount of work above) with HEMA or HEMA-co-MMA channels has been performed in the area of hydrogel delivery loaded with fibrin, acidic fibroblast growth factor of trophic factors. This work has led to significant (aFGF), and heparin and reinforced with a PCL advances in the development of potential therapeutics coil. They employed the coil to alleviate the for individuals with damage to their neural system. problems of tube collapse that was observed in However, recent advances in the engineering of the previous study.56 The coil was successful in materials, polymer synthesis, and neural biology open preventing collapse of the nerve tubes; however, no up further avenues for research in this area. Several axonal regeneration was observed as a result of the important and developing technologies are outlined development of syringomyelia (a cyst which causes below. nerve degeneration) and migration of the rostral Stimuli responsive hydrogels are being developed stump. This work emphasizes the importance of for numerous applications, where some external stim- mechanical properties of the hydrogel towards the uli (pH, temperature, light) are used to alter hydro- successful regeneration of the tissues. gel properties, and consequently, molecule release. Combining synthetic polymers as drug releasing For instance, poly(N-isopropylacrylamide) hydrogels devices within a natural polymer matrix, Goraltchouk respond and deswell with increased temperatures,85 and coworkers78 incorporated PLGA 50/50 micro- which can lead to the release of growth factors. Das spheres containing bovine serum albumin (BSA) or and coworkers86 recently incorporated light-sensitive epidermal growth factor (EGF) within chitosan/chitin gold nanoshells into these hydrogels, where the gold nerve guidance channels. PLGA is an example of a nanoshells absorb light at a predefined wavelength, poly(α-hydroxy ester) that has been widely explored heat up, and initiate the hydrogel thermal response. as a degradable material for tissue engineering and In this system, light can be transmitted transdermally drug delivery. They observed controlled release of to trigger release, leading to a well defined release protein from the microspheres into solution over an profile. Stayton and coworkers87,88 have developed 84-day period compared to only 70 days for the free several novel copolymer systems that are responsive

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(a) Side view (b) Top view Side view

Top view Center rod Fixers Screens Fixers

PINS Center rod PINS

Screens

FIGURE 6| PLGA porous nerve conduits. (a, b) Schematics for fabrication of single and multiple lumen conduits, respectively. (c, d) Images of single and multiple lumen porous conduits. Scale bars: (c) 700 µm, (d) 500 µm. (Reprinted, with permission, from Ref. 82. Copyright 2005 Elsevier). to pH. These polymers are used as gene delivery vehi- Finally, recent trends are towards combinatorial cles which swell at low pH and facilitate release from approaches for the delivery of multiple stimulatory the endosome, allowing more specific, local release of factors that together can have an additive effect on the gene. Such a polymer could be potentially use- regeneration. One example by Lu and coworkers16 ful for intracellular delivery or delivery around the illustrated that the delivery of both cAMP and injury where the pH has been shown to be slightly NT-3 provided elevated recovery in animals with lower than surrounding tissues.89 Thus, advances in spinal cord injury over the delivery of only one polymer synthesis and processing are leading to novel of the molecules. Similar results were also seen approaches for growth factor, and potentially, neu- in the work done by Moore and coworkers with rotrophin delivery. NT-3 and NGF.55 Biomaterials, and specifically, In a different area, Mi and coworkers identified hydrogels, are ideal candidates for the delivery pleiotrophin (PTN, also known as heparin binding of multiple factors, each with individual release neurotrophic factor) as a neurotrophic factor that profiles, as a result of our excellent control over aids in the recovery of damaged motor neurons in the polymer behavior. Additionally, various stem cells and spinal cord.90 Following nerve injury, a significant novel scaffolding may be combined in combination upregulation of PTN was observed in recovering with neurotrophin delivery to accelerate healing and nerves near the site of injury for up to 1 month. recovery. In vitro studies showed that PTN could induce axonal growth in a culture of spinal cord explants in the direction of the PTN source. Following a sciatic CONCLUSION nerve transection, HEK−293PTN cells were delivered in a silicone tube to the site of injury. After 8 The application of synthetic and natural materials for weeks, there was a ten-fold increase in axon density neurotrophin delivery has only recently been used and compared to control, and some functional recovery is finding widespread success. While no single system was observed. Additionally, when HEK−293PTN cells explored has yet to provide what many would consider were transplanted in gelfoams to the site of facial to be complete recovery, the results from many of nerve injury in mouse pups, 63% of the facial motor these studies are promising and for many of these neurons recovered compared to 12% of the controls. applications, incremental improvements correlate to The emergence of PTN as another neurotrophic significant enhancement in quality of life. Continued factor that plays a role in the recovery of injured research in this field will shed new light on the role neurons should further aid in the search for the each neurotrophin plays in the healing of neural ideal delivery system/delivery agent(s) combination. tissues, as well as on the role that the physical material

Volume 1, January/February 2009  2008 John Wiley & Sons, Inc. 135 Advanced Review www.wiley.com/wires/nanomed can play in the delivery of the neurotrophin and novel trophic factors, and combinatorial approaches support of growing axons at the injury site. Recent that will allow for promising treatment methods for trends and expected directions in this ﬁeld promise patients with debilitating neurological conditions. even more advanced systems of responsive materials,

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