Multichanneled Nerve Guidance Conduit with Spatial Gradients of

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Multi-channeled Nerve Guidance Conduit with Spatial Gradients of Neurotrophic Factors and Oriented Nanotopography for Repairing the Peripheral Nervous System Yo-Cheng Chang, Ming-Hong Chen, Shih-Yung Liao, HsiChin Wu, Chen-Hsiang Kuan, Jui-Sheng Sun, and Tzu-Wei Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b12567 • Publication Date (Web): 09 Oct 2017 Downloaded from http://pubs.acs.org on October 11, 2017

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ACS Applied Materials & Interfaces

Multi-channeled Nerve Guidance Conduit with Spatial Gradients of Neurotrophic Factors and Oriented Nanotopography for Repairing the Peripheral Nervous System Yo-Cheng Chang1, Ming-Hong Chen2, Shih-Yung Liao3, Hsi-Chin Wu4, Chen-Hsiang Kuan5, JuiSheng Sun6, Tzu-Wei Wang1* 1

Department of Materials Science and Engineering, National Tsing Hua University, Hsinchu,

Taiwan 2

Department of Neurosurgery, Cathay General Hospital, Taipei, Taiwan

3

Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua

University, Hsinchu, Taiwan 4

Department of Materials Engineering, Tatung University, Taipei, Taiwan

5

Division of Plastic Surgery, Department of Surgery, National Taiwan University Hospital, Taipei,

Taiwan 6

Department of Orthopedic Surgery, National Taiwan University Hospital, Taipei, Taiwan

*Corresponding Author: [email protected]

Keywords: multi-channeled biomimetic structure; neurotrophic concentration gradient; nanotopography; sequential controlled release; tissue engineering

1 ACS Paragon Plus Environment


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ABSTRACT

Peripheral nerve injuries, causing sensory and motor impairment, affect a great number of patients annually. It is therefore important to incorporate different strategies to promote nerve healing. Among the treatment options, however, the efficacy of nerve conduits is often compromised by their lack of living cells, insufficient growth factors and absence of the extracellular matrix (ECM)-like structure. To improve the functional recovery, we aimed to develop a natural biodegradable multi-channeled scaffold characterized with aligned electrospun nanofibers and neurotrophic gradient (MC/AN/NG) to guide axon outgrowth. The gelatin-based conduits mimicked the fascicular architecture of natural nerve ECM. The multi-channeled (MC) scaffolds, crosslinked with microbial transglutaminase, possessed sustainable mechanical stability. Meanwhile, the release profile of dual neurotrophic factors: nerve growth factor (NGF) and brainderived neurotrophic factor (BDNF) exhibited a temporal-controlled manner. In vitro, the differentiated neural stem cells effectively extended their neurites along the aligned nanofibers. Besides, in the treated group, the cell density increased in high NGF concentration regions of the gradient membrane, and the BDNF significantly promoted myelination. In a rabbit sciatic nerve transection in vivo model, the MC/AN/NG scaffold showed superior nerve recovery and less muscle atrophy comparable to autograft. By integrating multiple strategies to promote peripheral nerve regeneration, the MC/AN/NG scaffolds as nerve guidance conduits showed promising results and efficacious treatment alternatives for autologous nerve grafts.


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INTRODUCTION Peripheral nerve injuries drastically affect patients’ life quality and result in major socioeconomic loss. When injuries are extensive or the nerve defects are large, spontaneous functional recovery is less likely and sensor/motor impairments are often permanent. To bridge such a gap, especially larger than 10mm, autologous nerve grafts or artificial nerve conduits are often required.1 Autologous nerve grafts have superior clinical outcomes but drawbacks of limited source, donor site morbidities, and possible size mismatch.2-4 To overcome the limitations associated with nerve autografts, artificial nerve conduits have been developed as an alternative treatment for peripheral nerve regeneration. Commercially available nerve conduits are mostly single lumen conduits which yield inferior outcomes compared to Autografts.5 By increasing the surface area of the conduit for cell growth, nerve regeneration can be improved. Forms of multichannel lumens are reported to optimally mimicking the natural spatial arrangement of the extracellular matrix (ECM) and axons in neural tissue.6 In addition, ECM-mimicking fibrous structures can be prepared by electrospinning technique, yielding aligned fibrous matrices that act as internal fillers in artificial nerve conduit.7 Such alignment is beneficial to guide the cell growth in a certain direction.8 A neurotrophic factor-rich environment is essential for nerve regeneration. Ideally, the nerve guidance conduits should promote unidirectional growth of axons, and allow diffusion of growth factors and nutrients.9 Neurotrophic growth factor (NGF) plays a vital role in nerve cells growth, differentiation, regeneration, and neurotransmitter homeostasis.10 NGF prevents neuronal degeneration in animal models with encouraging results and has led to several clinical trials in humans.11 Also, brain-derived neurotrophic factor (BDNF) is important in neural stem



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cells (NSCs) proliferation, differentiation and directional migration.12-14 Adding exogenous BDNF enhances myelination, whereas the removal of endogenous BDNF inhibits the formation of mature myelin internodes.15 Due to the haptotactic and chemotactic behavior of nerve cells, establishing the environment with neurotrophic gradients is an effective strategy for guiding the nerve growth, migration, and cell differentiation.16 To optimize peripheral nerve regeneration, four different strategies were integrated to develop a gelatin-based artificial nerve conduit in this study. First of all, a gelatin-based multichanneled structure is designed. Compared to single lumen structure, the multi-channeled structure can improve mechanical properties, decrease nerve dispersion, and mimic the natural nerve fascicular structure. Second, we created aligned nanofibers by the electrospinning technique to induce axonal growth unidirectionally.17-18 Then, the neurotrophic factors concentration gradient is established to promote neural stem cell differentiation and axonal outgrowth. Finally, we generated a dual growth factor enriched environment to achieve a longterm synergistic effect. Dual growth factors are loaded either in the gelatin scaffold or embedded in nanoparticles for controlled release. In the early stage, NGF is expected to protect injured axons and enhance axonal regeneration, while in the later stage, BDNF is expected to increase myelin proteins expression. The suitability of this scaffold as a promising nerve replacement for peripheral nerve injury was studied in vitro and in vivo (Scheme 1). To the best of our knowledge, few studies have incorporated these important strategies and successfully integrated them into one system.


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Scheme 1. Schematic illustration of the fabrication process of the nerve conduit with aligned fibers for axon outgrowth direction guidance and neurotrophic concentration gradient to attract the regenerated axon.



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EXPERIMENTAL SECTION Electrospinning of aligned nanofibrous (AN) membrane. A 19wt% gelatin (Mn = 50-100 kDa, Sigma, St. Louis, USA) and 1wt% polyethylene oxide (PEO) (Mn = 900kDa) solution was prepared by dissolving in 40% acetic acid. The polymer solution was ejected at 0.9 mL/hr using a syringe pump onto a rotating collector at a distance of 7 cm. The rotating speed of the electrospinning apparatus was varied from 500 to 2000 rpm. The fibers were crosslinked by dehydrothermal treatment (DHT) at 140 °C for 48 hrs in a vacuum environment. In the subsequent step, the fibers were soaked in 30 U microbial transglutaminase (mTG) solution for 2hrs. To quantify fiber size and observe fiber morphology, the fibers were imaged using scanning electron microscopy (SEM). Synthesis of gelatin nanoparticles (GNs) and GNs loaded with BDNF (BDNF--GN). Gelatin nanoparticles (GNs) were synthesized by desolvation as demonstrated in our previous published work.19 The nanoparticles were then crosslinked by 100 μL of 15% EDC/NHS (4:1) for 2 hrs. The fabricated GNs were purified and resuspended in deionized water. The BDNF-GNs were created to encapsulate growth factors on the surface of or into the GNs. 5μL of BDNF solution with a concentration of 0.1 μg/ μL was adsorbed in 10 mg GN at 4 °C for 12 hrs. The BDNF-GNs were then washed in deionized water to remove residual solution. Fabrication of the MC, MC/AN, and MC/AN/NG scaffolds. A 10wt% gelatin solution was prepared with distilled de-ionized water (DDI-water) at room temperature. The multi-channeled (MC) scaffold with controlled, parallel-channel architecture were fabricated using a stainless-steel rod fixed on the injecting mold. Cylindrical molds with a diameter of 3.0 mm and 4-channels were constructed. Before pouring the gelatin solution into the mold, the AN membranes were fabricated by electrospinning and were then rolled around the stainless-steel rod. For the multi-


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channeled scaffold combined with aligned nanofibers and neurotrophic gradient (MC/AN/NG scaffold), the aligned nanofiber membranes were fabricated by electrospinning and rolled around a 500-μm-thick stainless-steel rod. The stainless-steel with aligned nanofibers membranes were then fixed in the mold. Next, neurotrophic gradient (NG) was established using gradient maker (CBS Scientific, Temecula, CA). The 500 ng/mL NGF solution and BDNF-GN solution in chamber A was mixed with an increasingly lower volume of 0 ng/mL NGF and BDNF-GN-free solution in chamber B, and delivered into the mold. The overall procedure is briefly illustrated in Scheme 1. Analysis of the physiochemical properties of the fabricated scaffolds. Determination of the degree of crosslinking was carried out using a modified TNBS (2,4,6-trinitrobenzene sulphonic acid) method described by Nagai et al.20 In brief, 5 mg specimens from various concentrations of mTG-crosslinked gelatin scaffolds were weighed into individual test tubes. 1 mL of 0.01% w/v TNBS solution and 1 mL of 0.1 M sodium hydrogen carbonate (NaHCO3, pH 8.5) were added. The solution was further treated with 2 mL of 6 N HCl at 60 °C for 1.5 hrs. The absorbance of the solutions was determined at 355 nm by microplate reader (Molecular Devices, SpectraMax Plus 384). The degree of crosslinking was then calculated using the following equation: Crosslinking degree (%) = [1-(absorbance of crosslinked gelatin solution/absorbance of non-crosslinked gelatin solution)] × 100. Mechanical properties of various concentrations mTG-crosslinked gelatin scaffolds were determined using Instron 8848 microtester. Three samples were taken from each group for mechanical test. The Young’s modulus and ultimate tensile strength was obtained by the stress-strain curve. The biodegradation of gelatin scaffolds was investigated in vitro by using lysozyme. Lysozyme is known as the major enzyme in human serum responsible for enzymatic degradation of biodegradable materials.21 Briefly, gelatin scaffolds were treated with 10 and 100



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U/g mTG (weight = 0.1-0.2 g) then incubated with 1.5 ug/mL lysozyme in PBS. All the specimens were incubated at 37 °C for 10 weeks. After degradation, the gels of remaining gelatin were dried in an oven at 37 °C. The dried gels were weighed and weight loss was calculated using the following equation: Weight loss (%) = (Wi-Wr/Wi)×100, where Wi denotes the initial weight of the scaffold and Wr the remaining weight of the scaffold at time (t). The values were expressed as the mean ± standard deviation. Quantification of neurotrophic gradient and release profile of neurotrophic concentrations by ELISA kit. MC/AN/NG scaffolds were cut into five parts and diluted ten times (10x) with DDI-water. Each specimen was measured for absorbance at 450 nm by microplate reader (Molecular Devices, SpectraMax Plus 384). To evaluate the growth factor release profile of MC/AN/NG scaffolds, the scaffold was immersed in PBS buffer solution and incubated at 37 °C. At specific time intervals, the supernatant was taken to determine the absorption spectra with a microplate reader. Individual release profiles of neurotrophic factors including NGF and BDNF were examined with incubation time. To analyze the release kinetics of growth factors, an NGF ELISA kit (Abnova, KA0400) and a BDNF ELISA kit (USCNK, SEA011Hu) were utilized. In vitro cell culture study. To investigate the effects of the neurotrophic gradient on dNSC cell behavior, dNSCs (HCN-A94-2, kindly provided by Professor Fred H. Gage of the Salk Institute, UCSD) were seeded on neurotrophic gradient gelatin scaffolds. All dNSCs were cultured for 7 days in Schwann cell medium at 37 °C in a humidified atmosphere with 5% CO2. The cell culture medium was changed every 3 days. An optical microscope was used to observe dNSCs on the neurotrophic gradient gelatin scaffolds. Neurite length and cell density was measured by Image J. Immunocytochemistry (ICC) was carried out to observe the cell morphology of dNSCs on


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aligned nanofibrous scaffolds. In brief, the cells were fixed with 4% formaldehyde in PBS of pH 7.4 for 20 mins at room temperature and washed twice with ice cold PBS. dNSCs were then permeabilized with PBS containing 0.25% Triton X-100 for 10 mins and washed three times. Next, the cells were treated with blocking solution containing 1% BSA, 0.2% Tween 20 and 0.3 M glycine for 30 mins. Primary and secondary antibodies of each marker were applied for 1 hr at room temperature. dNSCs were stained with DAPI for 1 min and mounted with mounting medium for long-term preservation. The list of primary and secondary antibodies was shown in Supplementary Table S1. To investigate the effect of the neurotrophic gradient on SCs associated with myelin-specific proteins, dNSCs-SCs were co-cultured on neurotrophic gradient gelatin scaffolds. SCs were harvested from 6 week old Spargue Dawley rats. NSCs (1×104 cells/cm2) cocultured with SCs (1×104 cells/cm2) were seeded on neurotrophic gradient gelatin scaffolds. Cultures were fixed at 3, 7, and 14 days after using antibodies to detect specific myelin basic protein (MBP). An optical microscope was used to observe the MBP-labeled area. The MBP area ratio was calculated from these micrographs using Image J software. In vivo rabbit animal study. New Zealand white rabbits weighing between 3 and 3.5 kg were used. The animals were allocated to seven groups. A 15-mm sciatic nerve defect was created and bridged under seven different conditions. For the surgical procedure, rabbits were anesthetized using 2.5-3% isoflurane delivered with a mask. Dissection was performed with the EyeMag Pro (Carl Zeiss, Inc., Oberkochen, Germany). A 15-mm segment of the sciatic nerve was resected before the bifurcation of the nerve into the tibial and peroneal nerve branches. The proximal and distal nerve ends were inserted into the 15-mm-long scaffolds and sewed with 6-0 monofilament polyamide sutures. The wound was subsequently closed. The same procedure was performed for autologous nerve grafts. At 8, 16, and 24 weeks, the sciatic nerve was harvested, fixed in 4%



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formaldehyde, and washed with PBS. After fixation, neural tissue in each group was embedded in FSC 22 frozen section media (Leica). Sections (6-10 μm) were cut on a cryostat microtome (Leica CM1900, Wetzlar, Germany) and stained with 1% osmium tetroxide.22-23 Image J software was used to analyze the photographs for determination of the axon diameter, thickness of the myelin sheath and diameter of the myelinated fibers. For all animals of the experimental groups, the electrophysiological tests were examined before tissue harvesting at 8, 16, and 24 weeks after implantation. A bipolar stimulating electrode was placed under the sciatic nerve at a location 20 mm proximal to the graft site, and a recording electrode was placed in the gastrocnemius muscle. Then the compound muscle action potential (CMAP, an indicator reflecting the muscle atrophy and muscle reinnervation) was recorded with a Cadwell’s Cascade® intraoperative neuromonitoring (IONM) system (Cascade 3.0 Classic software). The peak amplitude of CMAP and the nerve conduction velocity (NCV, an important indicator reflecting the functional recovery of injured nerves) values were calculated.24 The sciatic nerve innervates gastrocnemius muscle in rabbits and starts to atrophy after nerve injury.25 In order to evaluate the nerve reinnervation, the relative gastrocnemius muscle weight (RGMW), the diameter of muscle fiber and the average percentage of collagen fiber area were measured. The gastrocnemius muscle from both limbs was harvested after the rabbits were sacrificed. Immediately, the muscle weight was recorded. The transverse sections (4-6 μm) were cut on a microtome (Thermo Fisher Scientific HM 315, Waltham, USA) and stained with a Masson trichrome stain kit (Sigma HT15, St. Louis, USA). The diameter of gastrocnemius muscle fiber and the average percentage of collagen fiber area were calculated from random fields per animal (n = 3-4 animals per group) with the Image J software. Statistical analysis. All data are expressed as the mean ± standard error of the mean unless otherwise indicated. The significance of the effect of selected parameters on the outcome variables


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was analyzed by multifactor analysis of variance (ANOVA). Group comparisons were made by Fisher’s protected least significant difference (PLSD). Statistical significance was accepted at a level of p < 0.05.



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RESULTS Characterization of aligned nanofibrous membrane. Gelatin/PEO nanofibrous membranes were prepared by electrospinning. Electrospinning fibers were collected on a 20-cm diameter cylindrical collector at rotation speeds of 500, 1000, 1500, and 2000 rpm. The effect of the rotation speed on fiber diameter and orientation is shown in Figures 1(A)-(D). The results showed that the alignment of nanofibers became more directionally oriented when the rotation speed increased. This effect was clearly observed when the speed over 1500 rpm. The voltage was maintained at 18 kV and the flow rate at 0.9 mL/hr, using a 25-gauge needle and a traveled distance of 7 cm. When rotation speeds were set at 1500 and 2000 rpm, the fiber diameters were 256 ± 12.3 and 235.7 ± 10.2 nm, respectively, and the fiber orientations were highly aligned with the rotational direction. As the rotation speed increased, the fibers were structured more compactly with smaller pores. To address this issue, PEO was added into the system as a sacrificial fiber because it can be hydrolyzed by immersion in an aqueous environment. When the stacking of PEO fibers were dissolved in water, the pore size were then significantly increased from few micrometers to hundreds of micrometers. The seeded neural stem cells were then able to migrate between the interconnected porous structure. In addition, the gelatin/PEO aligned nanofibers with interstacked nanofibrous structure were analogous to the extracellular matrix in scale. To maintain fiber morphology, gelatin/PEO nanofibrous membrane was crosslinked using a two-step process beginning with DHT followed by soaking the membrane in microbial transglutaminase (mTG) solution. The SEM result of gelatin/PEO nanofibers crosslinked by DHT treatment at 140 °C for 48 hrs was shown in Figure 1(E). The gelatin/PEO nanofibers treated with DHT and then further crosslinked by immersion into 30 U mTG solution at 37 °C for 2 hrs was shown in Figure 1(F). The fiber diameter of the nanofibers after DHT and DHT/mTG treatments were shown in Figure


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1(E) and Figure 1(F). The values were 222.7 ± 9.9 nm and 851.8 ± 63.9nm, respectively. We found that the nanostructure could still be preserved after DHT and mTG two-step crosslinking process although some fusion of fibers were noticed. From the results, fibers with nano- and submicro- scale were both existed.

Figure 1. SEM images of electrospun gelatin/PEO nanofibers. At a rotation speed of (A) 500 rpm, (B) 1000 rpm, (C) 1500 rpm and (D) 2000 rpm. (E) gelatin/PEO nanofibers crosslinked by DHT treatment at 140 °C for 48 hrs. (F) gelatin/PEO nanofibers treated with DHT and then further crosslinked by immersion into 30 U mTG solution at 37 °C for 2 hrs (DHT/mTG).

Characterization of multi-channeled scaffold with aligned nanofibers (MC/AN scaffold). An important indicator in the prognosis of nerve injuries is the preservation of parallel microchannels of the nerve guidance conduit.26 Therefore, one of the major goals of our design is to incorporate a multi-channeled scaffold with aligned nanofibers as a nerve guide. MC/AN scaffold was molded into a cylindrical structure 3 mm in diameter and 15 mm in length as shown in Figures 2(A), (B). The MC/AN scaffold contained four channels, each approximately 500 um in diameter, a size



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chosen to mimic the neural fascicle structure. The components for the assembly of nerve conduit fabrication was shown in Figure 2(C). The cross section and the inner surface of the MC/AN scaffold with longitudinally aligned electrospun fibers were shown in Figures 2(D), (E). The porous structure of the scaffold and the aligned fibrous topography could be clearly observed.

Figure 2. Photographs of the MC/AN scaffold. (A) Cross-section view and (B) side view of MC/AN scaffold. (C) The assembly of molds for nerve conduit fabrication. (D, E) SEM images of cross-section of MC/AN scaffold with longitudinally aligned fibers inside the channel (shown at 50 and 2000 × magnification).

Physiochemical properties of fabricated scaffolds. The degree of crosslinking can be evaluated by the residual number of ε-amino groups. Picrylsulfonic acid (2,4,6-Trinitrobenzenesulfonic acid solution, TNBS) methods are usually used to monitor chemical modifications of ε-amino groups in the side chains of gelatin. The degree of crosslinking of 10 wt% gelatin scaffolds with different


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mTG concentrations (10, 30, 50 100 U/g-gelatin) was shown in Figure 3(A). The result showed that the degree of crosslinking was significantly affected by mTG concentration. When mTG concentration increased from 10 U/g gelatin to 100 U/g gelatin, the degree of cross-linking of the gelatin scaffolds increased from 10.16 ± 3.5 % to 22.49 ± 2.52 % at 24hrs. The result was similar to the group at 12 hrs. The enzyme lysozyme, found in certain human body fluids, has been reported to be synthesized during active phagocytosis after nerve injury.27 To evaluate the degradation kinetics of the scaffolds, gelatin scaffolds were incubated in PBS solution with and without lysozyme (Figure 3(B)). After crosslinking, the gelatin scaffolds showed a progressive weight loss with a stable degradation rate. During a 10-week period, scaffolds incubated in PBS solution containing lysozyme with mTG 10 U/g-gelatin and 100 U/g-gelatin showed losses in mass of up to 80% and 40%, respectively. As the concentration of the cross-linking agent increased, the degradation rate decreased. The rate of degradation was also increased with the presence of lysozyme.

Figure 3. (A) Degree of crosslinking of 10 wt% gelatin scaffold with different mTG concentration (U/g-gelatin) for 12 hrs and 24 hrs at 37 °C. (B) Percentage of weight loss of mTG-crosslinked gelatin scaffolds. Scaffolds were treated with mTG 10 and 100 U/g-gelatin and degraded in PBS



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solution with and without lysozyme (1.5 ug/mL) at 37 °C for various time periods. Error bars indicate SD (n = 4), *p

Multichanneled Nerve Guidance Conduit with Spatial Gradients of

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