Improved Axonal Regeneration of Transected Spinal Cord Mediated by Multichannel Collagen Conduits Functionalized with Neurotrophin-3 Gene

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ORIGINAL ARTICLE Improved axonal regeneration of transected spinal cord mediated by multichannel collagen conduits functionalized with neurotrophin-3 gene

LYao1,2, W Daly1, B Newland1,SYao3, W Wang1, BKK Chen4, N Madigan4, A Windebank4 and A Pandit1

Functionalized biomaterial scaffolds targeted at improving axonal regeneration by enhancing guided axonal growth provide a promising approach for the repair of spinal cord injury. Collagen neural conduits provide structural guidance for neural tissue regeneration, and in this study it is shown that these conduits can also act as a reservoir for sustained gene delivery. Either a G-luciferase marker gene or a neurotrophin-3-encoding gene, complexed to a non-viral, cyclized, PEGylated transfection vector, was loaded within a multichannel collagen conduit. The complexed genes were then released in a controlled fashion using a dual release system both in vitro and in vivo. For evaluation of their biological performance, the loaded conduits were implanted into the completely transected rat thoracic spinal cord (T8–T10). Aligned axon regeneration through the channels of conduits was observed one month post-surgery. The conduits delivering neurotrophin-3 polyplexes resulted in signiﬁcantly increased neurotrophin-3 levels in the surrounding tissue and a statistically higher number of regenerated axons versus the control conduits (Po0.05). This study suggests that collagen neural conduits delivering a highly effective non-viral therapeutic gene may hold promise for repair of the injured spinal cord.

Gene Therapy (2013) 20, 1149–1157; doi:10.1038/gt.2013.42; published online 25 July 2013 Keywords: non-viral gene delivery; neural conduits; 2-(Dimethylamino)ethyl methacrylate; spinal cord injury

INTRODUCTION scaffolds serve to bypass or prevent the formation of the glial Spinal cord injury (SCI) is a devastating condition, which often scar and allow axons to regenerate in a more growth-permissive results in life-long disability and a broad range of secondary environment. However, when the regenerated axon number complications.1 The application of cell therapy, molecular therapy reaches the limitation of their re-growth capacity in such and biomaterial scaffolds in SCI has increased the understanding scaffolds, functional restoration becomes limited. This limited of the factors contributing to, and the requirements for, spinal regenerative capacity may be primarily due to a poor regenerative cord repair.2–12 However, significant improvements are still environment with an insubstantial supply of growth-promoting required for the advancement of therapeutic strategy to clinical factors. practice. One method of extending the regenerative capacity is the use A number of natural and synthetic biomaterial scaffolds have of cell or molecular therapies. Cell therapies such as the use of been implanted in models of SCI. These include compression, peripheral Schwann cells or stem cells enclosed within the hemisection and complete transection SCI models.11–14 biomaterial scaffold have proven efficacy in enhancing spinal Compression allows study in a more clinically relevant model for cord repair.13,17–20 However, a lot remains to be understood about spinal cord repair. However, analysis of regeneration in this setting the interaction and behavior of the transplanted cells in such a can be confusing, as origins of regenerating axons or processes hostile microenvironment. In particular, studies have shown that make it difficult to isolate (for example, regenerating axons may implanted neural stem cells result in increased allodynia over the be sprouting from remaining uninjured axons).13 Complete course of the treatment.13,21 Alternatively, molecular therapies transection allows analysis of repair in a more isolated that enhance axonal regeneration by delivering growth- environment, and regenerative processes can be more readily promoting and survival factors (for example, nerve growth traced to their site of origin. For regeneration of the injured cord, factor, brain-derived neurotrophic factor and neurotrophin-3 biomaterial scaffolds can provide the structural support necessary (NT-3) and/or reduce inhibitory components of the glial scar for repair and provide appropriate architecture for topographical (chondroitinase ABC) have shown promise for repair.17–19,22–26 guidance to the regenerating axons. Biomaterials-based scaffolds However, one major limitation restricts their applicability for have been applied to the injured spinal cord in a number of repair. The use of such therapies shows limited improvement configurations and designs (for example, single- and multichannel when used in single dose administration, as they cannot maintain conduits and hydrogels) and have shown the ability to promote a constant biological effect in vivo and multiple doses are required regeneration within the injured spinal cord.11,13,15,16 These for maintenance.27,28 Ideally, this biological effect would be

1Network of Excellence for Functional Biomaterials, National University of Ireland, Galway, Ireland; 2Department of Biological Sciences, Wichita State University, Wichita, KS, USA; 3Section of Cellular Pathology and Molecular Genetics, Department of Molecular and Clinical Cancer Medicine, Institute of Translational Medicine, University of Liverpool, Liverpool, UK and 4Department of Neurobiology, Mayo Clinic, Rochester, MN, USA. Correspondence: Dr A Pandit, Network of Excellence for Functional Biomaterials (NFB), National University of Ireland, Galway, Ireland. E-mail: [email protected] Received 27 January 2013; revised 16 April 2013; accepted 17 June 2013; published online 25 July 2013 Conduits carrying NT-3 for spinal cord repair L Yao et al 1150 maintained throughout the regenerative process, without the material properties such as the rate of biodegradation and its need for multiple injections or further invasive therapy. mechanical properties can be directly controlled by crosslinking of Gene therapy offers an alternative strategy, which would allow the core solution.42 The flexibility of the fabrication process and the maintenance of such biological effects. NT-3, an attractive properties of the collagen molecule allow the incorporation/ candidate growth factor for neuronal repair, is involved in axon loading of factors within or on the surface of conduit material; path-finding by mediating its chemoattractive property and such that they are released in a controlled and regulated manner. promoting growth of the spinal cord axons.29,30 Viral vectors are In this instance non-viral polyplexes are incorporated for gene a well-established system for gene delivery to the central nervous therapy. It was hypothesized that controlled delivery of a NT-3 system because of their long-term, stable transduction.31,32 A growth-promoting gene from the multichannel collagen conduit previous study demonstrated that neural conduits delivering viral would result in greater axonal regeneration than simply relying on vectors encoding NT-3 can improve axonal growth within the structural guidance alone. injured spinal cord.33 Although recombinant viral vectors efficiently transduce cells, their clinical therapeutic efficacy may be compromised by the potential risk of insertional mutagenesis, immunogenicity and difficulty of scaling-up.34–36 Alternatively, RESULTS non-viral gene vectors avoid these perceived safety concerns by Release profile of polyplexes from multichannel collagen conduits generating functional proteins while remaining episomal in the and in vitro cell transfection host cell. To employ a non-viral strategy a highly efficient gene Multichannel collagen conduits were fabricated with the tube vector with a low-toxicity profile and a vector that protects and outer diameter, length and channel diameter of 2.6, 2.0, and delivers the plasmid of interest is required. The high transfection 0.66 mm respectively (Figure 1). The fabrication method of the capability of 2-(dimethylamino) ethyl methacrylate-based multichannel collagen conduits was modified from the previously polymers has recently been shown through in vitro studies.37,38 reported method.42,43 To create additional guidance cues for In this study a cyclized 2-(dimethylamino) ethyl methacrylate- spinal axon growth, a layer of aligned collagen fibers was based polymer that consists of single polymer chain looped with fabricated into the inner wall of the channels (Figures 1d–f). itself in a knot structure (distinctly different from hyperbranched The knot-complexed pGluc showed a rapid release from the or dendrimer polymers) was used to deliver a NT-3-encoding gene collagen film and from the 7-channel neural conduits during the in vivo. first 24 h in phosphate-buffered saline (PBS) solution at 37 1C. In this investigation, upregulation of growth-promoting cues About 50% of the loaded polyplexes were released within 24 h through sustained delivery of a non-viral gene vector, was used in and an additional 30% of the loaded polyplexes were released in combination with a multichannel collagen conduit to promote the following 13 days (Figure 2a). The conduits surface-loaded repair of the injured spinal cord. Previous studies in our group with polyplexes also showed significantly higher transfection have demonstrated that multichannel collagen conduits that capability than did the collagenase-digested conduits for both 3T3 resemble the structure of the multiple nerve basal lamina tubes fibroblast cells and adipose-derived stem cells (Figures 2b and c). can control axonal dispersion in peripheral nerve regeneration.39 By combining the two methods, the biomaterial conduit ideally Similar devices using synthetic 7-channel conduits made from a obviated the trade-off between fast release (high transfection) for variety of polymers have been successfully used for spinal cord the initial transection and sustained delivery (lower transfection repair.2,13,16,40 However, the use of a more natural material, such as capability) for maintenance of NT-3 expression. In addition, collagen, has a number of major advantages over synthetic polyplexes released from collagenase-digested conduits showed polymers and may hold additional benefits for repair. It possesses significantly higher transfection than that seen from a comparison multiple cell-adhesive and signaling domains that have been group comprised of polyplexes formed with a commercially shown to improve axonal growth and repair.41 The conduit available polyethylenimine (PEI).

Figure 1. Fabrication of multichannel collagen conduits with an inner layer of collagen fibers in the channels of the conduit. Photographs (a–c) show the multichannel collagen conduits with an internal layer of collagen fibers. Panels (a and b) show the conduits in their dry non- hydrated form. Panel (c) shows conduits hydrated post overnight incubation in PBS at room temperature. (d) Photograph of the fabricated parallel collagen fibers. (e) Diagram shows the inner layer of fibers in the conduit channels and (f) shows the structure and dimension of the conduits. Scale bar, 100 mm.

Gene Therapy (2013) 1149 – 1157 & 2013 Macmillan Publishers Limited Conduits carrying NT-3 for spinal cord repair L Yao et al 1151

Figure 2. In vitro cell transfection with released pGluc polyplexes. (a) Release proﬁle of polyplexes from surface-loaded multichannel collagen conduits and collagen ﬁlm. (b) Transfection of 3T3 cells with pGluc polyplexes released from surface-loaded neural conduits (* compared with control group Po0.01) or digested neural conduits fabricated by the in situ loading method. (^ compared with PEI group and control group Po0.01). (c) Transfection of adipose-derived stem cells with pGluc polyplexes released from surface-loaded neural conduits (* compared with PEI group and control group Po0.01), or digested neural conduits fabricated by the in situ loading method (^ compared with PEI group and control group Po0.01).

Animal survival post-surgery Complete spinal cord transection surgery was performed on 32 rats and the transected spinal cord was implanted with one of the two experimental groups: (a) non-functionalized control multichannel conduits (without polyplexes, n ¼ 16) or (b) multichannel conduits carrying plasmid DNA encoding NT-3 (pNT-3) polyplexes (n ¼ 16). All the animals survived throughout the intended time period. No severe self-mutilation was observed in any animals post-surgery.

pNT-3 polyplexes improved axonal regeneration through the multichannel collagen conduits Four weeks post-implantation of the neural conduits into the 2 mm spinal transection, the spinal cords were re-exposed and isolated for histological study and for protein analysis (Figures 3a and b). All samples showed neural tissue ingrowth within the conduit (Figures 3c and d). When the connective tissue and the remaining conduit outer wall were removed, neural tissue bundles were visible (Figure 3e). Longitudinal sections (Figures 4a–f) and cross-sections (Figures 4g and i) of the implanted conduits were processed for immunohistochemical analysis, and anti-neurofilament antibodies were applied to assess axonal growth within the channels of conduit. Both cross-sections and longitudinal sections showed axonal regeneration in the channels of the conduits. The longitudinal sections showed regenerated axons growing into the channels from both rostral and caudal sides of the conduit entry zone. Although axons were randomly orientated at the rostral and caudal sides of spinal cord tissue close to conduits (Figures 4a and f), axons in the conduit channels grew parallel to the longitudinal axis of the conduits (Figures 4b–e). To assess the ability of the delivered gene vector to promote repair, the number of regenerated axons from cross-sections at both one-quarter and three-quarter positions of the isolated conduit was counted. At both one-quarter and three-quarter Figure 3. Morphology of isolated spinal cord and conduits 4 weeks positions, the NT-3 group showed a significantly greater number after surgery. (a) Dorsal side of the spinal cord. (b) Ventral side of the of axons for the entire scaffold than did the control group spinal cord. (c) Neural tissue bundles are visible in multichannel collagen conduits. (Figure 5). The axon number of the NT-3 group at one-quarter and three-quarter positions of the isolated conduit (1711.3±687.1 and 1662.4±558.5, respectively) was significantly higher than that was significantly higher than that of the control group seen in the control group (P 0.05) (Figure 6b). o (63.3 pg mg À 1±21.2 pg mg À 1, P 0.01). To confirm enhanced expression of human NT-3 within the o injured spinal cord, NT-3 protein level was quantified using an enzyme-linked immunosorbent assay (ELISA) assay (Figure 6a). Measurement of microglia in the regenerated neural tissue The NT-3 protein content (expressed as weight ratio of NT-3 (pg) The cross-sections of the tissue samples were labeled with to tissue (mg)) in the NT-3 group (122.5 pg mg À 1±24.7 pg mg À 1) anti-ED-1 antibody to identify and quantify the presence of

& 2013 Macmillan Publishers Limited Gene Therapy (2013) 1149 – 1157 Conduits carrying NT-3 for spinal cord repair L Yao et al 1152

Figure 4. Longitudinal sections and cross-sections show axonal growth in the channels of multichannel collagen conduits. Spinal cord axons grow into the channels from the rostral side (a and d) or caudal side (c and f) of the conduits. The red dash lines show the borders of the conduits. (b and e) The aligned axons in the channels of conduits. Axons on the cross-sections at one-quarter (g) and three-quarter (h) positions of conduits from rostral side to caudal side. (i) Diagram shows the positions of the taken images (a–h). Scale bar, 50 mm.

Figure 5. Comparison of axons in the cross-sections of control conduits and conduits delivering pNT-3 polyplexes. (a–g) shows axons in the channels of the seven-channel control conduit taken at the one-quarter position of the conduit; (a0–g0) shows axons in the matching channels of control conduit at three-quarter position of the conduit; (h–n) shows axons in the seven-channels at one-quarter position of the treated conduit carrying NT-3 polyplexes; (h0–n0) shows axons in the matching channels of NT-3 polyplex conduit at the three-quarter position of the conduit. Scale bar, 200 mm.

phagocytic cells (that is, microglia). The average number of Assessment of functional recovery microglia in the observed ﬁelds for the control group and NT-3 The Basso, Beattie and Bresnahan (BBB) locomoter rating scale was group was 4.6±1.6 and 3.8±1.5 respectively (Figure 6c). assessed to evaluate the functional recovery of the animals at 2 No signiﬁcant difference was found between groups. weeks and 4 weeks post-surgery. At 4 weeks, the BBB score of the

Gene Therapy (2013) 1149 – 1157 & 2013 Macmillan Publishers Limited Conduits carrying NT-3 for spinal cord repair L Yao et al 1153 with either cells or exogenous growth factors, and been implanted within the injured spinal cord.13,16 Oriented neural tissue was observed to grow from both rostral and caudal sides within the channels of the biomaterial scaffolds and, in some cases, regenerated axons extended up to 14 mm distal from the conduit into the cord evident from fast blue tracing of the regenerated tracts.9,11,14,16,47 Based on these studies a natural seven-channel conduit was fabricated from collagen type I with similar dimensional properties to that of the previous studies (that is, an internal channel diameter of 660 mm). It was shown that aligned collagen fibers within a biomaterial conduit resulted in significantly increased axonal regeneration and more accurate nerve regeneration.43,48–51 In this study, we fabricated aligned collagen fibers into the inner wall of the conduit channels. It was assumed that the addition of structure to the inner wall of the conduit would provide additional topographical guidance to the regenerating axons. Resulting from this combination, randomly oriented axons were observed at the boundaries of the conduit entry zone (Figures 4a and f). Directly at the conduit entry zone, axons were seen to become oriented by the narrow conduit channels both rostrally and caudally (Figures 4d and c, respectively). Moving further into the conduit, clear, aligned (Figures 4b and e) and parallel axonal growth was seen to grow within the channels of the conduits. This data suggests that the combination of collagen fibers and channels can effectively direct axonal growth. Although many of the previously used multichannel conduits Figure 6. Measurement of NT-3 level in the tissue extraction were fabricated using synthetic polymers, we have used bovine- homogenate and quantification of regenerated axons, microglia, derived collagen type I as a biomaterial to fabricate these conduits and functional assessment. (a) ELISA assay shows increased NT-3 for the intrinsic presence of cell-adhesive and signaling domains, level in the NT-3 group (* compared with control group Po0.05). which offer advantages over synthetic polymers for axonal (b) Regenerated axons in the conduits of NT-3 group are regeneration.9,11,13,14,16 Furthermore, the mechanical and significantly higher than control groups (* compared with control degradable properties of collagen conduits have been group Po0.05). (c) Measurement of ED-1 microglia did not thoroughly characterized and can be readily tailored using a show significant difference between the experimental groups. suitable crosslinking regime.42,52 In particular, the degradation rate (d) BBB scores were measured and quantified at 2-week and 4-week post-surgery. of such collagen neural conduits can be modified by adjusting the crosslinking strength according to the duration of the in vivo experiments offering advantages over synthetic polymers, which degrade at a slower rate. NT-3 group (5.5±1.9) is higher than that of the control group To promote axonal regeneration, neurotrophins are major (3.6±2.4, P ¼ 0.07) (Figure 6d). therapeutic factors that have been delivered to the injured central nervous system. However, because of their relatively short half-life, neurotrophins can function only transiently.27,28 Non-viral gene DISCUSSION delivery into the spinal cord offers a promising approach for the Primary injury of spinal cord causes direct neurological damage to treatment of spinal cord traumatic injury. In a study by Takahashi the spinal cord leading to the necrosis of tissue at the site of injury et al.53, adult rats with spinal cord hemisection were injected with due to trauma and local ischemia from damage to the lipoplexed DNA plasmid encoding human Bcl-2 gene into the surrounding vasculature. The injury provokes a cascade-like normal side the spinal cord (or caudal to the hemisection). secondary reaction and further injury to the neural tissue. Tissue Administration of the Bcl-2 gene in the spinal cord prevented destruction causes the death of neurons and glial cells, retrograde cell death and also minimized atrophy. In another demyelination of intact axons, increased ischemia, inflammation study, a lipoplexed plasmid encoding glial cell-derived and results in swelling in the segments immediately above and neurotrophic factor was injected after SCI. Real-time polymerase below the site of injury.15 A number of cells migrate to the site of chain reaction showed the increased expression of glial cell- injury (for example, macrophages, microglia, neuroglia such as derived neurotrophic factor mRNA in the injected areas at 7 days astrocytes and eventually Schwann cells from the periphery) and after injection. Four weeks after glial cell-derived neurotrophic participate in both the inflammatory response and scar tissue factor gene transfer, regeneration down the corticospinal tracts formation.44,45 The cyst/glial scar formed from reactive astrocytes was confirmed using anterograde tract tracing. This study and the infiltrated cells forms both a chemical and physical barrier demonstrated that in vivo transfer of glial cell-derived to axonal repair. The implantation of biomaterial nerve conduits neurotrophic factor cDNA can promote axonal regeneration and have been previously shown to prevent/reduce scar formation demonstrate some locomotion functional recovery.54 The use of and simultaneously creating a suitable microenvironment for polymeric vectors has also been reported recently, where plasmid repair.11,13,14,16,46 The use of multichannel neural conduits has DNA complexed with polyethylene glycol-grafted PEI showed demonstrated the ability to provide structural support and enhanced gene expression after repeated intrathecal injections.55 guidance to regenerating nerve tissue while retaining their Plasmid gene delivery by direct injection locally into the spinal structural integrity. Seven-channel conduits have been made cord, however, may not be efficient as the plasmids are quickly from a number of synthetic materials including poly(lactide-co- eliminated by metabolism and the flow of cerebrospinal fluid. glycolide), poly(e-caprolactone fumarate), oligo(polyethyelene Furthermore, repeated injection or continuous infusion may cause glycol) fumarate and have been used alone or in combination extra trauma to the spinal cord and introduce infection. Plasmid

& 2013 Macmillan Publishers Limited Gene Therapy (2013) 1149 – 1157 Conduits carrying NT-3 for spinal cord repair L Yao et al 1154 DNA delivered in a controlled manner by a combinatorial MATERIALS AND METHODS biomaterials approach, may overcome these problems. Fabrication of multichannel collagen conduit with inner layer of In addition to the structural guidance for axonal re-growth, the parallel collagen fibers constant release of the plasmid DNA, from the scaffolds, Collagen fibers were fabricated by extruding an atellocollagen solution could continuously transfect cells and maintain expression of (5 mg ml À 1) at a rate of (0.3 ml min À 1) using a syringe pump (KD-Scientific therapeutic molecules. An example of such a study used surface- 200, KD-Scientific, Holliston, MA, USA) into a fiber formation buffer (118 mM mediated DNA delivery from multichannel poly(lactide-co-glycolide) phosphate buffer and 20% polyethylene glycol (molecular weight 8000), bridges coated with fibronectin to deliver lipoplexes to the spinal pH 7.55, 37 1C) for 5 min and then transferring the fibers into an incubation 1 cord hemisection model.11 The lipoplex-mediated expression in buffer (6.0 mM phosphate buffer and 75 mM sodium chloride, pH 7.1, 37 C) for another 5 min. The collagen fibers were crosslinked with 0.5 mM 1-ethyl- the neural tissue persisted for at least 3 weeks. 3-(3-dimethylaminopropyl) carbodiimide and 0.5 mM N-hydroxysuccinimide In this study, a highly effective transfection agent was required, 56 in 50 mM 2-(N-morpholino)ethansulfonate, pH 5.5 for 30 min, as per which transfects neuronal cell types such as astrocytes. To this previous work.43 Thereafter, the collagen fibers were thoroughly washed end the recently developed cyclic knot polymer was used to with distilled water for 1 h to remove excess crosslinker and subsequently complex and protect the plasmid encoding NT-3. By subsequent air-dried (Figure 1d). loading to collagen conduits, a combined approach was utilized The aligned, extruded collagen fibers were attached onto the surface of coupling conduit implantation with sustained gene delivery in a seven stainless steel wires to be incorporated into the multichannel single surgical procedure. The higher number of regenerated conduit fabrication process. The conduit was fabricated by sequentially axons in the NT-3 group ,therefore, corresponds to the increased inserting stainless steel wires into the molds and air-drying the collagen on stainless steel wires. The air-dried collagen conduit on the wires was then NT-3 level (as confirmed by ELISA) in the neural tissue implanted treated with a crosslinking solution (30 mM carbodiimide and 10 mM with conduits. This study suggests that the collagen neural N-hydroxysuccinimide in 50 mM 2-(N-morpholino)ethansulfonate solution), conduit delivering a functional gene is an effective approach to pH 5.5 for 5 h. After washing with 0.1 M NaH2PO4 and distilled water, the improve axonal regeneration. The functional recovery of the rats multichannel collagen conduit was freeze-dried on the wire. Molds and after injury was studied using BBB scoring. The BBB score of NT-3 wires were removed from the collagen conduits after freeze-drying. group is higher than control group at both 2 weeks and 4 weeks after the surgery. The result suggested that pNT-3 in the conduits Cyclized knot polymer synthesis improved axonal regeneration, which may result in better 38 functional recovery. Although the difference was not statistically The transfection agent was synthesized as previously reported. Briefly, a highly controlled synthesis method, known as deactivation-enhanced significant, this study indicated that an increased animal number atom transfer radical polymerization, was employed.59 This allows the in the functional study may yield significance as there was a high polymerization of single or multiple di-vinyl monomers without the variation within the experimental groups. For future studies it will formation of an insoluble gel.59,60 The three monomers ethylene glycol be interesting to see if these regenerated axons extend distally dimethacrylate, used as the knotting agent; 2-(dimethylamino) ethyl into the conduit, and how that regeneration distance would methacrylate, the complexing agent; and poly(ethylene glycol) methyl compare to previous studies. This may be achieved through the ether methacrylate, for PEGylation were polymerized in a ‘one-pot’ reaction use of appropriate anterograde and retrograde tracers.57 at 50 1C in tetrahydrofluran at a ratio of 10:82:8, respectively using a Spinal cord injury results in the influx of a large number of copper/ligand complex activated by the addition of ascorbic acid. By the different cell types to the site of injury (for example, macrophages, suppression of gelation, several grams of single copolymer polymer chains cyclized upon themselves at a molecular weight of 32 kDa were obtained microglia, oligdendrocyte precursors, meningeal cells and astro- and were then purified by hexane precipitation. This was followed by cytes). Although these cells can be potentially transfected, it was extensive dialysis in distilled water and subsequent freeze-drying leaving critical to analyze whether the amount of cyclic knot polymer used the polymer ready for use as a dry powder. for complexing the NT-3 plasmid resulted in a greater infiltration of a phagocytic cells, which may be inhibitory for repair.58 As previously reported, seven-channel conduits have been shown to In vitro polyplex release profile from the collagen conduits and create an isolated microenvironment for repair.13 It was critical to subsequent functionality analysis analyze whether the introduction of such polyplexes into the To study the release profile of polyplexes from the conduit, 1 mg of Gaussia channels of the conduit resulted in an increased influx of princeps luciferase plasmid (pGluc) labeled with Cy5 using a Label ITCy5 Labeling Kit (Mirus Bio, Madison, WI, USA) was complexed to the knot phagocytic cells, that is, microglia. However, no significant polymer by addition at a 4:1 polymer-to-plasmid w/w ratio in dH2O for differences in ED-1 positive cells was found between groups 15 min.61 Collagen films were fabricated by spreading the collagen solution indicating that the 4 mgofin situ-loaded complexed plasmid, plus on a flat Teflon surface (weigh boat) and air-dried. The collagen films were the 3 mg loaded on the surface, cause no rise in the number of then crosslinked with 1 mM carbodiimide and 1 mM N-hydroxysuccinimide microglia recruited to the site of implantation. Further study of the for 1 h. The labeled polyplexes were loaded onto the crosslinked collagen interaction of the surrounding tissue may be warranted to ensure film or multichannel collagen conduits post-assembly, as outlined in there are no adverse effects in that area, future studies will Figure 7b. The polyplexes-loaded scaffolds were placed into a 96-well plate address this. with 200 ml PBS solution and incubated at 37 1C. At 24-hour, 48-hour, In conclusion, multichannel collagen conduits can be 1-week and 2-week time points, the conduits were transferred to another row of wells containing fresh PBS solution. At the end of the 2-week time functionalized with polyplexes by two methods: in situ loading period, the conduits were removed from the final set of wells and the PBS or surface loading, each displaying a different transfection solution from each time point was analyzed using a Varioskan Flash profile in vitro. By combining the two loading methods, NT-3 Multimode Reader (Thermo Scientific, Essex, UK). levels were shown to be statistically increased during nerve To study the functionality of polyplexes delivered by means of the regeneration and maintained up to 4 weeks in vivo.Thiscyclic collagen conduits, two methods of loading and subsequent release were knot polymer was used for enhanced and sustained NT-3 performed, with subsequent transfection analysis as outlined in Figures 7a delivery within the injured spinal cord and shown to maintain and b. For this study pGluc complexed either with the knot polymer or PEI expression levels up to one month in vivo. Sustained expression (at a 2:1 polymer to plasmid w/w ratio) (Sigma, St Louis, MO, USA) were resulted in significantly increased aligned axonal regeneration used. The scaffolds were then loaded with these polyplexes in one of two ways: (1) the polyplexes were mixed with the initial atellocollagen solution within the channels of the multichannel collagen conduit used to fabricate the conduit followed by crosslinking with 1 mM compared with the control group. This study suggests that the carbodiimide and 1 mM N-hydroxysuccinimide for 1 h to form an in situ- combination of biomaterial scaffolds and sustained non-viral gene loaded conduit directly (Figure 7a); (2) the polyplexes were added to the delivery provides synergistic effects of structural guidance and surface/inner channels (that is, surface loaded) of the conduit fabricated effective molecular therapy for spinal cord regeneration. using collagen solution alone without polyplexes (Figure 7b). To study

Gene Therapy (2013) 1149 – 1157 & 2013 Macmillan Publishers Limited Conduits carrying NT-3 for spinal cord repair L Yao et al 1155

Figure 7. Two methods used for loading the multichannel neural with polyplexes with subsequent analyses. Conduits for in vitro analysis were loaded by mixing polyplexes and the collagen solution before fabrication (a) or loaded to the surface post-fabrication (b). A dual loading method of in situ loading followed by surface loading was used for the preparation of scaffolds for in vivo analysis (c). functionality of the in situ-loaded polyplexes, conduits were digested with to two subgroups: control collagen conduits (without polyplexes) and bacterial collagenase type II (Sigma C6885), which was dissolved in 35 ml conduits containing pNT-3 polyplexes (NT-3 group). Animals were PBS (3 U of collagenase per mg of collagen sample). The solution was anesthetized with intraperitoneal injection of ketamine and xylazine. The then collected for transfection analysis. To study the functionality of the back was shaved and aseptically prepared using povidone-iodine. Vet surface-loaded polyplexes, the treated conduits were placed directly into ointment was applied to protect the eyes from dehydration during the 48-well plate wells with cultured cells. relatively long procedure. To maintain body temperature during surgery, To establish the in vitro efficacy of the released polyplexes, both the animals were kept on a heating pad at 37 1C. A microsurgical microscope standard cell line 3T3 fibroblasts and adipose-derived stem cells were used was used throughout the surgical procedure. After skin incision and in the study. The cells were seeded at a density of 8000 cells per well in laminectomy at the T8–T10 vertebral levels, the spinal cord was completely 48-well plates for the gene transfection study. Twenty-four hours after cell transected with a no. 11 blade. After transection, the spinal cord was seeding, the conduits loaded with polyplexes or the solution containing retracted from the lesion site. A cotton sponge was inserted into the gap of the digested conduits was added into the cell culture wells. After another the transected spinal cord to staunch excess bleeding and create a 2 mm 72 h, the cell culture medium was collected for analysis using BioLux gap. A 2-mm-long multichannel collagen conduit was implanted in the gap Gaussia Luciferase Assay Kit (E3300) (New England Biolabs, Ipswich, MA, and aligned with the rostral and caudal spinal cord stumps. Muscles and USA). skin were then sutured separately. Post-operative care was included twice daily monitoring for wound sepsis and bladder expression. Antibiotics (Baytril, Bayer HealthCare, LLC, KS, USA) and analgesics (Buprenorphine, Fabrication of multichannel collagen conduits functionalized with Fort Dodge Animal Health, Southampton, UK) were administered for the NT-3 polyplexes first week post-surgery, and saline was administered 3 days post-surgery. pNT-3 (pcDNA3.3-TOPO (Invitrogen, Grand Island, NY, USA) human NT-3) was complexed with the knot polymer at a polymer-to-plasmid weight ratio of 4:1. The polyplexes were incorporated into the conduits in a two- Tissue processing step procedure outlined in Figure 7c. First, pNT-3 (4 mg) polyplexes were One month post-surgery, the animals were killed by an overdose of added into the collagen solution (12 mg ml À 1) and mixed gently to ensure sodium pentobarbitone (intraperitoneally) (for immunohistochemical uniform distribution of the polyplexes within the solution (in situ loading). studies) or CO2 asphyxiation (for protein analysis). For the immunohisto- Then the solution was used to fabricate the multichannel conduits with chemical study, 11 rats in each group were transcardially perfused with 4% inner layer aligned collagen fibers. Second, after the conduits were paraformaldehyde in PBS. The spinal cord was then removed and post fabricated, pNT-3 (3 mg) polyplexes were loaded onto the surface and fixed in 4% paraformaldehyde. The samples were cryoprotected in 30% within the channels of the multichannel collagen conduits before being sucrose and the region containing the scaffold was embedded in Cryogel air-dried (surface loaded). This created a conduit that combined surface (Instrumedics, Richmond, IL, USA) and frozen for sectioning. The samples loading and in situ loading based on the results of previously described were then continuously sectioned transversely or longitudinally. For release studies. protein analysis, the spinal cords of five rats in each group were isolated without performing transcardial perfusion. The isolated tissue was frozen using liquid nitrogen and stored in a À 80 1C freezer for further analysis. Surgical procedure In this study a total of 32 adult female Sprague Dawley rats weighing between 220 and 280 g were used. The multichannel conduits with or Immunohistochemistry without polyplexes were implanted into the completely transected rat The sample sections for immunohistochemical analysis were permeabilized spinal cord. All experimental procedures were conducted in accordance with 0.5% Triton X-100. Then the sections were exposed to blocking with national and institutional guidelines, and all procedures were solution (2% goat serum, 2% bovine serum albumin). All antibodies were approved by the institutional animal ethics committee and by the National diluted in PBS with 1% bovine serum albumin. The sections were Board under the Cruelty to Animals Act. The rats were randomly assigned incubated with primary antibody for two hours at room temperature.

& 2013 Macmillan Publishers Limited Gene Therapy (2013) 1149 – 1157 Conduits carrying NT-3 for spinal cord repair L Yao et al 1156 The cross-sections (taken at one-quarter length and three-quarter length 7 Ogawa Y, Sawamoto K, Miyata T, Miyao S, Watanabe M, Nakamura M et al. intervals) and the longitudinal sections of the isolated neural conduit Transplantation of in vitro-expanded fetal neural progenitor cells results in neu- samples were labeled with anti-neurofilament monoclonal antibody (1:200, rogenesis and functional recovery after spinal cord contusion injury in adult rats. J N2912, Sigma) to identify and quantify regenerating axons. Regenerated Neurosci Res 2002; 69: 925–933. axons from cross-sections at both one-quarter and three-quarter positions 8 Teng YD, Lavik EB, Qu X, Park KI, Ourednik J, Zurakowski D et al. Functional of the isolated conduit (rostral to the caudal) were labeled with anti- recovery following traumatic spinal cord injury mediated by a unique polymer neurofilament antibodies and the axon number was counted. scaffold seeded with neural stem cells. Proc Natl Acad Sci USA 2002; The cross-sections at the mid-point of the scaffold-cord interface were 99: 3024–3029. labeled with an anti-ED-1 antibody (1:250, MAB1435, Millipore, Billerica, 9 Moore MJ, Friedman JA, Lewellyn EB, Mantila SM, Krych AJ, Ameenuddin S et al. MA, USA). Following exposure to primary antibodies, the sections were Multiple-channel scaffolds to promote spinal cord axon regeneration. Biomaterials incubated with DyLight 488-conjugated donkey anti-mouse (1:250, 2006; 27: 419–429. Jackson ImmunoResearch Europe, Suffolk, UK) for 1 h. Nuclei were stained 10 Lee H, McKeon RJ, Bellamkonda RV. Sustained delivery of thermostabilized chABC with 4,6-diamidino-2-phenylindole. Fluorescent images of the sections enhances axonal sprouting and functional recovery after spinal cord injury. Proc were viewed under a microscope (Olympus dp70, Olympus, Tokyo, Japan). Natl Acad Sci USA 2010; 107: 3340–3345. Six fields of view were randomly selected and captured at a magnification 11 De Laporte L, Yan AL, Shea LD. Local gene delivery from ECM-coated poly(lactide- of 400 Â for each section using image analysis software (Image Pro, Media co-glycolide) multiple channel bridges after spinal cord injury. Biomaterials 2009; Cybernetics, Buckinghamshire, UK). The number of ED-1-labeled cells was 30: 2361–2368. measured using a 192-point grid. A 192-point grid was overlaid on each 12 Afshari FT, Kwok JC, White L, Fawcett JW. Schwann cell migration is integrin- field of view, and the number of ED-1 positive cells that intersected with a dependent and inhibited by astrocyte-produced aggrecan. Glia 2010; 58: grid point was counted. 857–869. 13 Olson HE, Rooney GE, Gross L, Nesbitt JJ, Galvin KE, Knight A et al. Neural stem ELISA assay cell- and schwann cell-loaded biodegradable polymer scaffolds support axonal The frozen tissue samples containing the scaffolds were suspended in regeneration in the transected spinal cord. Tissue Eng Pt A 2009; 15: 1797–1805. tissue extraction reagent (Sigma) and then homogenized using a 14 De Laporte L, Yang Y, Zelivyanskaya ML, Cummings BJ, Anderson AJ, Shea LD. TissueRuptor (Qiagen, Valencia, CA, USA). Homogenate was centrifuged Plasmid releasing multiple channel bridges for transgene expression after spinal at 10 000 g for 10 min to remove particulates. The amount of NT-3 in the cord injury. Mol Ther 2009; 17: 318–326. tissue homogenate was quantified using an ELISA kit (DY267, R&D Systems, 15 Straley KS, Foo CW, Heilshorn SC. Biomaterial design strategies for the treatment Minneapolis, MN, USA). The absorbance of the samples was read at 450 nm of spinal cord injuries. J Neurotrauma 2010; 27: 1–19. using a Varioskan Flash Multimode Reader (Thermo Scientific, Essex, UK). 16 Chen BK, Knight AM, Madigan NN, Gross L, Dadsetan M, Nesbitt JJ et al. The NT-3 content in the homogenate was then analyzed and normalized to Comparison of polymer scaffolds in rat spinal cord: A step toward quantitative the total protein content, as analyzed using the Bio-Rad Protein assay assessment of combinatorial approaches to spinal cord repair. Biomaterials 2011; (Bio-Rad, Hercules, CA, USA). 32: 8077–8086. 17 Schnell L, Schneider R, Kolbeck R, Barde YA, Schwab ME. Neurotrophin-3 enhances sprouting of corticospinal tract during development and after adult Assessment of locomotor recovery spinal cord lesion. Nature 1994; 367: 170–173. BBB locomotor score was used to evaluate motor functional recovery of 18 Huang WC, Kuo WC, Hsu SH, Cheng CH, Liu JC, Cheng H. Gait analysis of spinal experimental and control animals 2 and 4 weeks after surgery. Each rat was cord injured rats after delivery of chondroitinase ABC and adult olfactory mucosa observed in a clean and open plastic box for 5 min to record hind-limb progenitor cell transplantation. Neurosci Lett 2010; 472: 79–84. joint movements, weight support, toe clearance, tail position and 19 Jefferson SC, Tester NJ, Howland DR, Chondroitinase ABC. promotes recovery of coordination of gait and paw position. adaptive limb movements and enhances axonal growth caudal to a spinal hemisection. J Neurosci 2011; 31: 5710–5720. 20 Wang JM, Zeng YS, Wu JL, Li Y, Teng YD. Cograft of neural stem cells and schwann Statistical analysis cells overexpressing TrkC and neurotrophin-3 respectively after rat spinal cord The data were expressed as means±s.d. and analyzed by using a one-way transection. Biomaterials 2011; 32: 7454–7468. ANOVA (post-hoc Bonferroni) with SPSS version 17.0 software package 21 Hofstetter CP, Holmstrom NA, Lilja JA, Schweinhardt P, Hao J, Spenger C et al. (SPSS, Chicago, IL, USA). P o0.05 was considered statistically significant. Allodynia limits the usefulness of intraspinal neural stem cell grafts; directed differentiation improves outcome. Nat Neurosci 2005; 8: 346–353. 22 Xu XM, Gueńard V, Kleitman N, Aebischer P, Bunge MB. A combination of BDNF CONFLICT OF INTEREST and NT-3 promotes supraspinal axonal regeneration into Schwann cell grafts in The authors declare no conflict of interest. adult rat thoracic spinal cord. Exp Neurol 1995; 134: 261–272. 23 Sterne GD, Brown RA, Green CJ, Terenghi G. Neurotrophin-3 delivered locally via fibronectin mats enhances peripheral nerve regeneration. Eur J Neurosci 1997; 9: ACKNOWLEDGEMENTS 1388–1396. 24 Patist CM, Mulder MB, Gautier SE, Maquet V, Jerome R, Oudega M. 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