Controlled Release of Growth Factors from Multilayered Fibrous

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Article Cite This: ACS Biomater. Sci. Eng. 2018, 4, 576−586

Controlled Release of Growth Factors from Multilayered Fibrous Scaffold for Functional Recoveries in Crushed Sciatic Nerve Min-Ho Hong,†,□ Hye Jin Hong,‡,□ Haejeong Pang,‡ Hyo-Jung Lee,† Seong Yi,*,† and Won-Gun Koh*,‡ †

ACS Biomater. Sci. Eng. 2018.4:576-586. Downloaded from pubs.acs.org by KAOHSIUNG MEDICAL UNIV on 09/27/18. For personal use only.

Department of Neurosurgery, Spine and Spinal Cord Institute, College of Medicine, Yonsei University, Seoul 03722, Republic of Korea ‡ Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul 03722, Republic of Korea S Supporting Information *

ABSTRACT: In this study, we designed and fabricated a multilayered fibrous scaffold capable of the controlled release of multiple growth factors for sciatic nerve regeneration in rats. The scaffold consists of three layers prepared by sequential electrospinning, where the first layer is fabricated using polycaprolactone (PCL)-aligned electrospun nanofibers for the attachment and differentiation of cells toward the direction of the sciatic nerve. The second and third layers are fabricated using poly(lactic-co-glycolic acid) 6535 (PLGA 6535) and 8515 (PLGA 8515), respectively. The resultant three nanofiber layers were stacked and fixed by incorporating hydrogel micropatterns at both ends of nanofiber scaffold, which also facilitated the surgical handling of the multilayered scaffolds. The PLGA layers acted as reservoirs to release growth factors neurotrophin (NT-3), brain-derived neurotrophic factor (BDNF), and platelet-derived growth factor (PDGF). The different biodegradation rate of each PLGA layer enabled the controlled release of multiple growth factors such as NT-3, BDNF, and PDGF with different patterns. In a rat model, the injured nerve was rolled up with the multilayered scaffold loading growth factors, and behavior tests were performed five weeks after surgery. Sciatic functional index (SFI) and mechanical allodynia analysis revealed that the fast release of NT-3 and BDNF from PLGA 6535 and subsequent slow release of PDGF from PLGA 8515 proved to be the greatest aid to neural tissue regeneration. In addition to the biochemical cues from growth factors, the aligned PCL layer that directly contacts the injured nerve could provide topographical stimulation, offering practical assistance to new tissue and cells for directional growth parallel to the sciatic nerve. This study demonstrated that our multilayered scaffold performs a function that can be used to promote locomotor activity and enhance nerve regeneration in combination with align-patterned topography and the controlled release of growth factors. KEYWORDS: multilayered fibrous scaffold, controlled release of growth factor, sciatic nerve regeneration, electrospinning, hydrogel micropattern

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INTRODUCTION

pain and donor site morbidity, mismatch of donor nervous tissue size, fascicular inconsistency between the autograft and the proximal and the distal stumps of the injured site, the need for additional surgery, and a limited supply of suitable nerves.6 For these reasons, the development of artificial nerve grafts is thought to be a promising alternative to autologous nerve grafts. In the last few decades, a variety of polymer-based nerve grafts, mostly in the form of nerve guidance conduits, has been developed and tested in animal and/or clinical practice for neural tissue regeneration.7 In developing a polymer-based nerve conduit, topographical and biochemical cues have been incorporated because they are known to play important roles

The nervous system is broadly divided into the central nervous system (CNS, two major structures: brain and spinal cord) and the peripheral nervous system (PNS, nerves located throughout the rest of the body). In terms of axonal regeneration after injury, the PNS is more capable than the CNS. However, the spontaneous repair of peripheral nerves is still deficient in poor functional recovery.1,2 PNS injury causes the atrophy of effector muscles and the impairment of behaviors (such as gait) because there is axon demyelination and degradation induced by distal stump.3 Generally, the most widely used technique for regenerating an injured nerve is using an autograft in the clinical field.4 However, the functional results of using an autograft are unstable, and the lack of a donor nerve also retards the application of the autograft in a clinical setting.5 Moreover, there are many intrinsic drawbacks to autograft implantation, including © 2018 American Chemical Society

Received: October 24, 2017 Accepted: January 3, 2018 Published: January 3, 2018 576

DOI: 10.1021/acsbiomaterials.7b00801 ACS Biomater. Sci. Eng. 2018, 4, 576−586

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ACS Biomaterials Science & Engineering

Figure 1. Schematic representation for fabrication of growth factor-loaded multilayered nanofiber scaffold.

behavior and biological tests were performed to demonstrate the successful regeneration of neural tissue at the site of the injured sciatic nerve.

in the regeneration of the nervous system. According to previous studies, the aligned topography that mimics the aligned architecture of native nerve anatomy is a critical parameter in the design of nerve conduits, showing the ability to promote the directional growth of nerve tissues.8,9 Compared with topographical cues, biochemical cues (for example, growth factors loaded into the scaffold) could offer a more progressive microenvironment that allows cell migration and neuronal differentiation.10−12 Among diverse types of nerve conduits, electrospun nanofiberbased scaffolds have received great attention for nerve regeneration.13 Electrospinning produced porous scaffolds consisting of fibers with nano- or micrometer scale, which recapitulate aspects of the native extracellular matrix (ECM). Especially, the aligned electrospun fibers are well-suited for neural tissue engineering because they can provide the spatial guidance for neurite outgrowth and axonal elongation.14 On the other hand, various strategies have been studied to incorporate biochemical cues such as growth factors into the electrospun fibers.15,16 Despite the successful results of developing fiberbased nerve conduits with either topographical or biochemical cues, little effort has been made to create scaffolds combining drug-releasing and aligned features, which would provide not only biochemical cues but also topographical guidance for peripheral nerve regeneration.17,18 In particular, to the best of our knowledge, there has been no report on the development of an aligned fibrous nerve conduit from which multiple drugs are released in a controlled manner. In the present study, we designed and fabricated multilayered fibrous scaffolds for sciatic nerve regeneration in rats. The multilayered scaffolds consisted of three layers that were combined together by a hydrogel patterning process. The first layer was prepared as aligned PCL fibers to provide a topographical cue, while the second and third layers were prepared from randomly oriented PLGA fibers with different lactide/glycolide ratios. The different biodegradation rates of each PLGA layer enabled the controlled release of multiple growth factors such as neurotrophin (NT-3), brain-derived neurotrophic factor (BDNF), and platelet-derived growth factor (PDGF) with different patterns, providing spatiotemporal biochemical cues.19,20 After confirming the successful fabrication of the multilayered scaffolds and their capability to control the release behavior of multiple growth factors, in vivo studies such as

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EXPERIMENTAL SECTION

Materials. Polycaprolactone (PCL; MW 80 000), polyethylene glycol diacrylate (PEG-DA; MW 575), poly(D,L-lactide-co-glycolide) 6535 (PLGA 6535; MW 40 000−75 000), PLGA 8515 (MW 50 000− 75 000), 2,2,2-trifluoroethanol (TFE), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), Tween 20, Triton X-100, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolum bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, United States). NT-3, BDNF, PDGF, and all the biotinylated detection antibodies for ELISA were purchased from PeproTech (Rocky Hill, NJ, United States). Horseradish peroxidase (HRP) for ELISA and 3,3′,5,5′tetramethylbenzidine (TMB) were purchased from Thermo Fisher Scientific Inc. (Rockford, IL, United States). The mouse neural stem cells (mNSC) were purchased from CRL-2925TM, American Type Culture Collection (Manassas, VA, United States). Dulbecco’s modified Eagle’s medium (DMEM/F12), fetal bovine serum (FBS), trypsinEDTA, and penicillin were purchased from Gibco (Grand Island, NY, United States). Phosphate buffered saline (PBS) was purchased from HyCloneTM Laboratories Inc. (South Logan, UT, United States). Ketamine hydrochloride was purchased from Yuhan (Seoul, Korea), and xylazine hydrochloride was purchased from Bayer Korea (Ansan, Korea). A protein extraction solution for Bradford was purchased from PRO-PREPTM, Intron Biotechnology (Sungnam, Korea). Polyvinylidene difluoride membrane was purchased from Millipore (Schwalbach, Germany). Class III β-tubulin antibody (Tuj1; ab18207), anti-NT3 antibody (ab53685), anti-PDGF antibody (ab125268), β-actin antibody (ab8226), and microtubule associated protein-2 (MAP2; ab32454) were purchased from Abcam (Cambridge, UK). HRP-conjugated secondary antibodies and anti-BDNF antibody (sc-65514) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). All the secondary antibodies used in immunohistochemistry were purchased from Jackson ImmunoResearch (West Grove, PA, United States). Fabrication of Multilayered Scaffold Loaded with Multiple Growth Factors. The growth factor-loaded, multilayered fibrous scaffolds were fabricated by combining sequential electrospinning and photolithographic hydrogel patterning as shown in Figure 1. Each nanofiber matrix was electrospun separately and combined into one whole scaffold by patterning with a PEG-DA hydrogel. The incorporation of hydrogel micropatterns into nanofibers made handling convenient and firmly fixed the different layers of nanofibers together. The resultant nanofiber scaffolds were vertically divided into three different regions (triple-layered nanofiber scaffolds), which consist of aligned PCL fibers, PLGA 6535 fibers, and PLGA 8515 fibers as the first, 577

DOI: 10.1021/acsbiomaterials.7b00801 ACS Biomater. Sci. Eng. 2018, 4, 576−586

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ACS Biomaterials Science & Engineering second, and third layers, respectively. For the first layer, electrospinning of 20 wt % of PCL in TFE was conducted around a rotating drum at a speed of 850 rpm for 2 h. The flow rate of PCL solution injection was 0.5 mL/h with a voltage of 8.0 kV, and the distance between the 23G needle and the collecting plate was approximately 80 mm. For the second PLGA 6535 nanofiber layer, 40 wt % PLGA 6535 in TFE was electrospun with the following condition: 0.5 mL/h, 11 kV, 10 cm distance, and 23G needle for 1 h. The third layer was fabricated through electrospinning of 50 wt % PLGA 8515 in HFIP under the same condition as the one for PLGA 6535. When the growth factor-loaded nanofibers were prepared, 1 μg each of NT-3, BDNF, and PDGF was added separately or together to different PGLA solutions. After all the nanofiber sheets were obtained, they were stacked in a proper order and ready for the PEG hydrogel patterning. The multilayered scaffold sheet was put through O2 plasma treatment (FemtoScience, Seoul, Korea) to facilitate the infiltration of hydrogel precursor solution. After precursor solution permeated through the nanofiber scaffold, the photomask was placed onto the resultant nanofibers and exposed to 365 nm, 300 mW/cm2 UV light (EFOS Ultracure 100ss Plus, UV spot lamp, Mississauga, Ontario, Canada) for 0.5 s. The resulting scaffold was washed with distilled water to remove the unreacted precursor solution. The resultant triple-layered nanofiber scaffolds were divided into four groups depending on the composition of growth factors within the different PLGA nanofiber layers (Table 1).

Scanning electron microscopy (SEM; JSM-7001F, JEOL, Tokyo, Japan) was performed to examine the thickness and structure of layers (alignedPCL, PLGA 6535, PLGA 8515, and PEG-DA hydrogel). Porosity of PCL layer and average pore size was obtained using Porosimeter (Quantachrome Inst. PM33GT, Boynton Beach, FL, United States). Growth Factors Release Test. The release behavior of growth factors from multilayered scaffold with (6535, 8515, and experimental groups) or without growth factors (control group) were monitored in vitro for 10 weeks. The scaffold was immersed in a well plate containing 2 mL of PBS and stored at 37 °C in an incubator (n = 15). The amount of the released growth factors was determined through ELISA at predetermined time points for 10 weeks. A 96-well plate was coated with capture antibodies of either NT-3, BDNF, or PDGF. The sample solution, which had been incubated with experimental fiber sheets, was loaded in a volume of 100 μL/well, and then 100 μL/well of biotinylated detection antibodies was conjugated after 2 h of incubation. After additional incubation for 2 h, the further conjugation of HRP and reaction with TMB in the dark would finally result in a colorimetric change of the solutions. The final result was measured with spectrophotometric plate reader at 450 nm (VersaMaxTM ELISA microplate reader, Molecular Devices, Sunnyvale, CA). In Vitro Cytotoxicity Assay. The mNSC was used for the in vitro assay. The cells were cultured in DMEM/F12 supplemented with 10% FBS and 5% penicillin under 5% CO2 at 37 °C. Cells were subcultured every 2 days before reaching confluence using PBS and trypsin/EDTA, and used for the experiment between passage 5 and 10. To culture the cells on the scaffolds, the scaffolds were placed in 6-well plates and sterilized with 70% ethanol. The in vitro biocompatibility of the scaffolds was tested with an MTT assay, which is a colorimetric assay that measures the activity of enzymes that reduce MTT to formazan dyes, giving purple colors. The MTT assay was performed according to the manufacturer’s protocol. In detail, 7 × 104 cells/scaffold was seeded on the scaffold and cultured in 6-well culture plate under 5% CO2 at 37 °C. On the first, third, fifth, and seventh day of culture, the culture medium was removed, and MTT reagent was added to the wells and then incubated for 1 h. After removing the MTT reagent, DMSO was added to each well, and the plates were shaken for 15 min. The optical density (OD) at 570 nm was measured using the spectrophotometric plate reader.

Table 1. Representative Groups in This Study control group aligned PCL layer PLGA 6535 layer PLGA 8515 layer

6535 group

8515 group

NT-3, BDNF, and PDGF NT-3, BDNF, and PDGF

experimental group

NT-3 and BDNF PDGF

The formation of multiple layer structure was visualized with a confocal laser scanning microscope (LSM700, Carl Zeiss, Oberkochen, Germany), where each layer was incorporated with rhodamine B (red), toluidine blue (blue), and FITC (green) fluorophores, respectively.

Figure 2. Photos showing the surgical procedure for animal studies. 578

DOI: 10.1021/acsbiomaterials.7b00801 ACS Biomater. Sci. Eng. 2018, 4, 576−586

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ACS Biomaterials Science & Engineering Surgical Procedures and Animal Care. This research was carried out in strict accordance with the recommendations contained in the Guideline of Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International) and approved by the Institutional Animal Research Ethics Committee of the Yonsei Medical Center (IACUC Approval no. 2015-0375). In this study, male adult Sprague−Dawley rats (300 ± 30 g, Orient, Korea) were used in accordance with the approved guidelines. The animal room was artificially illuminated from 8:00 am to 8:00 pm and conditioned at 24 ± 3 °C with 40−60% humidity. The rats were anesthetized with ketamine hydrochloride (1.15 μL/g) and xylazine hydrochloride (0.5 μL/g). After exposing the left sciatic nerves of rats, they were clamped by a 100g force microclamp (jaw width = 2 mm) for 10 s. The injured sciatic nerves were rolled up by the multilayered scaffold (10 × 15 mm2). We wrapped the crushed nerve three times with the scaffold. Following the injury and scaffold implantation, the muscle and skin were closed with a running suture of polyglactin 3/0 (Vicryl, Johnson and Johnson, Markham, ON). The above-mentioned surgical procedure is shown in Figure 2. Behavior Studies. All rats were subjected to a series of behavior analyses after injury to their sciatic nerves. Recovery of the left hindlimb locomotor activity was considered as a proof of adequate muscle reinnervation and functional recovery of the sciatic nerve lesion and was monitored by analyzing the free-walking pattern, which was the method referenced from a previous study.21 For this test, rats were placed on a track (length of 500 mm and width of 100 mm, covered with a white sheet of paper at the bottom and ending in a dark box) and were allowed to walk after painting a dark dye on the plantar surface of the hind paws (Supplementary Video 1). The rats’ footprints were used to calculate the sciatic functional index (SFI). Three values were measured from the footprints: a distance from the heel to the third toe, which is indicated as the print length (PL), a distance from the first to the fifth toe (TS), and a distance from the second to the fourth toe, which is named as the intermediary toe spread (ITS) (Figure 3). All three measurements were

and MAP2 exhibited by proteins. In detail, animals were terminally sacrificed in a CO2 chamber and perfused through the ascending aorta with saline solution followed by 4% paraformaldehyde (pH 7.4) after defined survival times (2 and 5 weeks). After the perfusion, the sciatic nerve rolled with the scaffold was removed and postfixed in the same fixative for 6 h, then dehydrated with 30% sucrose overnight. The cryostat samples were cut at 10−20 μm both perpendicularly and longitudinally to the main nerve axis. The cryostat sections were rinsed with 0.3% Tween 20. Nonspecific primary antibody binding was blocked using 10% normal donkey serum diluted in 0.3% Triton X-100 (Tuj1; 1:2000 and MAP2; 1:2000). The samples were incubated with primary antibodies for 2 h at room temperature. After being rinsed with 0.3% Tween 20, the primary antibodies were bound by secondary antibodies for an hour. After rinsing with 0.3% Tween 20, the sections were examined under a confocal laser scanning microscope. Statistical Analysis. The data of the behavioral experiments and a quantitative analysis of immunohistochemical evaluation were statistically analyzed by one-way ANOVA using SPSS 23 statistical software (IBM, Armonk, NY), and a post hoc analysis was performed according to Tukey’s method. The significant difference between each group was determined at p < 0.05, whereas p < 0.001 was considered a highly significant difference.

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RESULTS Characterization of Multilayered Scaffold. The structure and configuration of the scaffolds were observed with SEM and confocal microscopy (Figure 4). As shown in Figure 4A, hydrogels were successfully incorporated at both ends of a triple-layered nanofiber scaffold. Because all the nanofiber layers were prepared with hydrophobic polymers, the multilayered scaffold without hydrogel formed an aggregated clump after contact with water as shown in Figure 4B. However, the incorporation of a PEG hydrogel micropattern facilitated the handling of nanofibers as shown in Figure 4B. The upper PCL layer that directly contacted the injured nerve was electrospun in an aligned form to support the elongated guidance of neuronal cells when inserted within the in vivo environment, as demonstrated in previous studies,23−25 while the other two layers releasing growth factors had randomly oriented nanofibers. The aligned and random orientations of each fibrous sheet were clearly distinguished and observed through the SEM results as shown in Figure 4C. The porosity was approximately 52.48%, and average pore size was 34.06 μm, respectively. These results indicate that porosity of PCL layer was large enough to allow the diffusion of growth factors through the fibrous scaffold. The formation of triple-layered nanofibers was verified by incorporating different fluorescent dyes into each layer (first PCL layer with rhodamine B, second PLGA 6535 layer with toluidine blue, and third PLGA 8515 layer with FITC). A cross-sectional fluorescence image of the scaffold demonstrated that the nanofiber scaffold was clearly divided into three different vertical regimes (Figure 4D). The SEM image shown in Figure 4D reveals that the overall thickness of the scaffold was approximately 140 μm, where the aligned PCL layer was approximately 30 μm thick and the PLGA 6535 and PLGA 8515 layers represented similar thicknesses (55−60 μm). Hydrogel micropatterning plays the role of a stapler, firmly fixing different layers of nanofibers together. The in vitro biocompatibility of the multilayered scaffolds was evaluated using an MTT assay as shown in Figure 4E, where absorbance values are proportional to the number of viable cells seeded to the scaffolds. It is apparent that the mNSCs remained viable and could proliferate within the all scaffold groups. In Vitro Release Profile of Growth Factors. Three different growth factors (NT-3, BDNF, and PDGF) were loaded

Figure 3. Formula to calculate the SFI, which is derived by multiple linear regression (PL; print length, ITS; intermediary toe spread, TS; toe spread). obtained from the experimental (E) and the normal (N) sides. The measured values were inserted in the equations, and the SFI values were calculated (n = 12).21 As an additional behavior study, a basal pain sensitivity was measured before and after the implantation of the scaffolds.22 The rats were placed on an elevated wire grid. For mechanical allodynia, the plantar surface on the hind paw was stimulated with a series of von Frey hairs. In this study, mechanical paw withdrawal threshold (PWT) measurement was assessed by a dynamic plantar esthesiometer (Ugo Basile, Varese, Italy). The threshold was taken as the lowest force that evokes a brisk withdrawal response (1−50g in 50 s). When the rat withdrew its hind paw, the mechanical stimulus was automatically withdrawn (n = 12) (Supplementary Video 2. Immunohistochemistry. The immunohistochemistry (IHC) staining was performed to detect specific expression patterns of Tuj1 579

DOI: 10.1021/acsbiomaterials.7b00801 ACS Biomater. Sci. Eng. 2018, 4, 576−586

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Figure 4. Triple-layered nanofibrous scaffolds incorporated into hydrogel micropattern. (A) Photographic image and magnified SEM image of the scaffold (scale bar; 200 μm). (B) Photographic image of the scaffold with PEG hydrogel pattern (left) and without PEG hydrogel pattern (right) after wetting with water. (C) SEM images of aligned PCL fibrous layer and randomly oriented PLGA fibrous layers (scale bar; 10 μm). (D) Cross-sectional images taken by confocal microscopy and SEM (scale bar; 100 μm). (E) MTT assay result of all groups (n = 3). There is no significant difference.

Figure 5. Release profiles of growth factors loaded into multilayered scaffold from (A) 6535 group, (B) 8515 group, and (C) experimental group for 10 weeks (n = 15).

into the second and third nanofiber layers in the multilayered scaffold, which were made with PLGA with a lactide/glycolide ratio 65:35 (PLGA 6535) and 85:15 (PLGA 8515), respectively, as grouped in Table 1. It is expected that growth factors in PLGA 6535 released faster than those in PLGA 8515 because the PLGA with the lower lactide/glycolide ratio degraded faster than the

PLGA with the higher lactide/glycolide ratio. Therefore, our multilayered scaffold can realize the independent release of multiple growth factors in a controlled manner. Figure 5 shows the controlled release of different growth factors, which has three different scenarios. First, all the growth factors were released quickly by loading all of them in PLGA 6535 layer as shown in 580

DOI: 10.1021/acsbiomaterials.7b00801 ACS Biomater. Sci. Eng. 2018, 4, 576−586

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ACS Biomaterials Science & Engineering Figure 5A (6535 group). Second, all the growth factors were loaded into PLGA 8515 (8515 group) and released more slowly than in the first case (Figure 5B). Finally, Figure 5C shows the release behaviors from the experimental group, where NT-3 and BDNF were loaded into the PLGA 6535 layer and PDGF was loaded into the PLGA 8515 layer. In this experimental group, NT-3 and BDNF were released faster than PDGF, which showed more sustained release behavior. For all three graphs, the profiles showed sustained release. Moreover, the release profile from PLGA 6535 reached a plateau after six weeks, while the one from PLGA 8515 began to exhibit a plateau after eight weeks. Sciatic Functional Index and Mechanical Allodynia. To determine whether the local delivery of growth factors from the multilayered scaffold at the injury site was effective in peripheral nerve regeneration, we first assessed motor function in rat with a biceps femoris injury of the sciatic nerve. Motor recovery after sciatic nerve injury was evaluated using the SFI, which is a reliable method to estimate the recovery of innervation of foot muscles. Figure 6 shows the SFI values of all four groups at 0, 1, 2, 3, 4, and

Figure 7. Mechanical allodynia in rats 0, 1, 3, 7, 14, 21, 28, and 35 days after injury of the sciatic nerve and implantation of scaffold. Mechanical allodynia as mechanical withdrawal thresholds in g (n = 12; statistically significant difference with respect to experimental group. p < 0.05; * control group, † 6535 group, ‡ 8515 group. p < 0.001, ** control group, †† 6535 group).

these two groups for all testing times. According to these two behavior tests, the fast release of three different growth factors simultaneously did not cause any improvement compared with the control group that did not have any growth factors. At the same time, the slow, more sustained, and simultaneous release of three growth factors brought much better results than the control group did. However, the best results were obtained from the sequential release of different growth factors, that is, the fast release of NT-3 and BDNF, and subsequent slow release of PDGF, as we hypothesized. Immunohistochemical Evaluation of Regenerated Nerves. Immunohistochemical evaluation was carried out as shown in Figure 8. First, we assessed neural tissue regeneration by looking at the expression of MAP2 and Tuj1 at 5 weeks postinjury. The center of the injured sciatic nerves was imaged with confocal fluorescence microscopy. As shown in Figure 8A, differentiated neurons were clearly labeled with MAP2 (red) and Tuj1 (green). Figure 8A indicates that a similar level of MAP2 expression was observed for all the groups. In the case of Tuj1, however, the 8515 and experimental groups exhibited higher Tuj1 expression than the other groups, which is consistent with the previous results of SFI and mechanical allodynia. Figure 8B (left graph) indicates that the experimental group showed even higher Tuj1 expression than the 8515 group, representing the highest expression among the four groups; the expression intensity of all groups showed a significant difference compared to the experimental group (p < 0.05). On the other hand, there was no significant difference for MAP2 expression between each group (Figure 8B, right graph). Tuj1 and MAP2 revealed the early and late stages of neuronal differentiation, representing newly differentiated neurons and undifferentiated pre-existing neurons, respectively.26 Therefore, these results indicated that the optimally controlled release of multiple growth factors induced the migration of neural stem cells and their subsequent differentiation into new neurons. In Figure 8C, the experimental group had alignment at the upper layer, and this topographical stimulation offered practical assistance to new tissue and cells for the directional growth parallel to the sciatic nerve, as demonstrated in previous studies.23,27

Figure 6. SFI for the control, 6535, 8515, and experimental groups performed weekly through 5weeks. Experimental group had the highest SFI after 2 weeks (indicating improved reinnervation compared to other groups by 5 weeks) (n = 12; statistically significant difference with respect to experimental group. p < 0.05; * control group, † 6535 group, ‡ 8515 group. p < 0.001, ** control group, †† 6535 group).

5 weeks after the scaffold implantation, which improved with time for all groups. The experimental group and 8515 group showed superior progressive improvements compared to the 6535 and the control groups after 2 weeks. The improvements in the outcome of the experiment group were found to be statistically significant compared to the control and 6535 groups at all times after implantation. A highly significant effect of the groups was observed for the SFI values (p < 0.001) with the experimental group being different from the control and the 6535 groups at 4 and 5 weeks. For the result at 5 weeks, there was a significant difference between the experimental and the 8515 groups (p < 0.01). Next, the sensory function was measured by the pain reaction elicited after a touch stimulus using von Frey hairs. As shown in Figure 7, the ipsilateral mechanical allodynia following the compression injury increased after the implantation of multilayered scaffolds in all groups, and similar trends were observed with SFI results. The experimental group had significantly higher threshold values than the 6535 and control groups after two weeks. Although the experimental group also had higher values than the 8515 group, there was no significant difference between 581

DOI: 10.1021/acsbiomaterials.7b00801 ACS Biomater. Sci. Eng. 2018, 4, 576−586

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ACS Biomaterials Science & Engineering

Figure 8. Histochemical analysis of in vivo results. (A) Transverse sections of centered location of the injured sciatic nerves. Double immunofluorescence for MAP2 (red) and Tuj1 (green). Cell nuclei were counterstained with DAPI (N; sciatic nerve, S; scaffold). The dotted area was observed with higher magnification (20×). Scale bar: 200 μm (5×) and 50 μm (20×). (B) Quantitative analysis of Tuj1 and MAP2 expression resulted from immunofluorescent assay (n = 16; statistically significant difference with respect to experimental group. p < 0.05). (C) DAPI-stained cells in the aligned PCL layer in experimental group (the arrows selectively indicate elongated nucleus of cells located at the aligned PCL fiber layer).

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with aligned PCL fibers for directional guidance during the sciatic nerve regeneration. The second and third layers, where the growth factors were loaded, were prepared with PLGA 6535 and PLGA 8515, respectively, expecting that the different biodegradation rates between PLGA 6535 and PLGA 8515 would allow us to control the release behavior of different growth factors. A multilayered nanofibrous scaffold was formed by

DISCUSSION In this work, we investigated the efficacy of hydrogelincorporated, multilayered nanofiber scaffolds, which were capable of the local delivery of multiple growth factors in a controlled manner, on the sciatic nerve regeneration. The multilayered nanofiber scaffolds consisted of three layers. The first layer, which contacted the injured nerve tissue, was prepared 582

DOI: 10.1021/acsbiomaterials.7b00801 ACS Biomater. Sci. Eng. 2018, 4, 576−586

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ACS Biomaterials Science & Engineering

growth factors is the degradation of PLGA, the degradation rate of PLGA is a critical factor to determine the release kinetics. The biodegradation rate of the PLGA copolymers is dependent on the various factors such as molar ratio of the lactic and glycolic acids in the polymer chain, molecular weight of the polymer, the degree of crystallinity, and the Tg of the polymer. In this study, different release patterns of multiple growth factors were achieved by using PLGA with different molar ratios of the lactic and glycolic acids. As shown in Figure 5, growth factors loaded in PLGA with the lower lactide/glycolide ratio (PLGA 6535) were released faster than the PLGA with the higher lactide/glycolide ratio (PLGA 8515). The release kinetics were mainly dependent on the types of PLGA, not the types of growth factors, confirming that degradation rate is the major factor that determines the release patterns of different growth factors. The behavioral tests are crucial for the quantitative analysis of the motor recovery during the neural tissue regeneration.38 In this study, we conducted SFI and mechanical allodynia analysis for five weeks using the rat as a model animal. For the tissue regeneration in the sciatic nerve of the adult rat model, a fiveweek recovery period was chosen in accordance with the injured short gap (2 mm) model to probe the effect of controlled release growth factors to the injured site.39,40 SFI has been used widely for evaluating the recovery of motor function as a result of regeneration of the injured sciatic nerve. Some studies do not recommend the use of SFI for the evaluation of motor function recovery because the animals show a proclivity to attack their denervated hind limbs after sciatic nerve transection.41,42 To minimize this problem, we did not transect the sciatic nerve but injured the tissue with a microclamp. As mentioned in the in vivo results above, the release rate of growth factors had a significant effect on the regeneration of neural tissue. According to the behavior studies, the 6535 group did not enhance the regeneration of the injured sciatic nerve, showing similar recovery with the control group that did not contain any growth factors. On the other hand, the 8515 and experimental groups, where PDGF was released slowly, resulted in significantly better recovery in terms of both the SFI value and the mechanical allodynia force than the 6535 and control groups. In particular, the result of SFI at week five shows that the experimental group was significantly higher than the 8515 group. The results of mechanical allodynia followed the same trend with those of SFI, but it was not significantly different between the experimental and 8515 groups. From these animal studies, it can be determined that the control and the 6535 groups are evidently less effective than the 8515 and the experimental groups. In addition, even though the 6535 groups released all three growth factors, its resulting effect was almost the same as the control group that lacks growth factors. Lastly, the result of the experimental group, where two groups of growth factors were released sequentially, was better than the 8515 group, where all growth factors were released slowly. Taken together, these results indicate that the most crucial factor in nerve regeneration is the slow and sustained release of PDGF. As previously mentioned, NT-3 and BDNF are categorized as neurotrophic factors that function in cellular proliferation at the early stage of neural recovery. Moreover, unlike PDGF, BDNF has been shown to promote and enhance locomotor activity when it is applied with NT-3.43−45 As soon as a neural system experiences an injury, neurotrophic factors, such as NT-3 and BDNF, are secreted in a burst from neighboring cells to stimulate cellular proliferation to replace injured cells. As the proliferation stage has sufficiently processed, neural differentiation will

stacking each layer, where the three layers of different nanofiber matrix were bound together by hydrogel micropatterns. The incorporation of hydrogel micropatterns not only has fixed different layers of nanofibers together but also facilitated the handling of multilayered scaffolds (Figures 4A−D). When nanofiber scaffolds come in contact with water without hydrogel micropatterns, they become clumped and difficult to handle due to the hydrophobic nature of each layer. Combining the hydrogel micropatterns at both ends of the multilayered scaffold can solve these problems and allow a surgeon to easily roll up the scaffold around the damaged nerve tissue in animal studies. The nanofibrous scaffold materials used in this study (PCL, PLGA, and PEG) are FDA-approved to be implanted into the human body for specific applications such as drug delivery and tissue engineering scaffolds. The fabricated multilayered fibrous scaffold was also biocompatible as expected, showing proper cell proliferations during the initial seven days of culture (Figure 4E). The cells were seeded on and grown within the fibrous structure because it is well-known that the electrospun fibrous structure closely mimics the extracellular matrix and helps the cells to attach, migrate, and proliferate more efficiently.13,28 To distinguish from previous nerve conduit studies that have used cell-seeded aligned scaffolds, we examined only the effects of multiple growth factor delivery on the neural tissue regeneration for this study. Therefore, cells were not seeded on the scaffolds, and the first layer was fixed with aligned nanofibers, where the only variable was the pattern of growth factor delivery. Three different growth factors, NT-3, BDNF, and PDGF, were chosen to generate different delivery patterns of growth factors. NT-3 is known to proliferate fibroblasts to help the nerve regeneration in a rat model.29 BDNF also brought about similar effects with NT-3, causing the increase of the axon growth in the spinal cord.30 However, NT-3 and BDNF are not typically used as a singular effecter; instead, both growth factors act together to promote axonal regeneration and behavioral outcomes.31,32 In addition, the combined secretion of NT-3 and BDNF usually occurs in vivo for cellular proliferation before the differentiation stage.33 Unlike these growth factors, PDGF has not been extensively studied with respect to its neurogenic potential, but it may still be a good assistant because it is known to play an important role in nerve regeneration by acting as a mitogen and protecting against neuronal degeneration.34−36 PDGF is also known to induce the neurogenesis in the postmitotic stage, where the differentiation stage would start to dominate the proliferative states. Therefore, it can be hypothesized that later appearance of PDGF would further extend the neurogenesis process.37 On the basis of these previous studies, we chose a combination of NT-3 plus BDNF as one group and PDGF as another group. We also expected that a sequential release of these two groups of three growth factors would further improve the functional recovery of the injured nerves, which would finally lead to neuronal regeneration. Therefore, we chose the scaffold capable of fast release of NT-3 plus BDNF and slow release of PDGF as an experimental group. The three different scaffolds, which were absent of any growth factor release and capable of fast and of slow release of all the growth factors simultaneously, were chosen as the control group, 6535 group, and 8515 group, respectively. Two groups of growth factors were loaded into PLGA 6535 and/ or PLGA 8515. Both in vitro and in vivo, the PLGA copolymer undergoes degradation in an aqueous environment (hydrolytic degradation or biodegradation) through cleavage of its backbone ester linkages. Because the major driving force for the release of 583

DOI: 10.1021/acsbiomaterials.7b00801 ACS Biomater. Sci. Eng. 2018, 4, 576−586

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ACS Biomaterials Science & Engineering ORCID

commence to further promote the regeneration of neural tissue.32,46 Because PDGF functions at the postmitotic stage and helps to protect the neurons during the regeneration process, it is important to sustain the PDGF release toward the poststage of the process. This is the reason why the experimental groups, in which PDGF is released at the later stage, show better functionality than the other two groups, including the control and 6535 groups. For the 6535 group, where PDGF is quickly released at an earlier stage, the PDGF could not actively support the differentiation process and thus, the neural regeneration did not occur. Moreover, the differentiation stage normally begins after the proliferation stage completes.47−49 Therefore, it is advisable to induce the differentiation process after the proliferation process. In summary, it is definitely evident from the results that slow and sustained release of PDGF is more critical in neuronal regeneration than the initial fast release of NT-3 and BDNF. However, as widely known nerve growth factors, the NT-3 and BDNF are surely helpful in the process of nerve regeneration. For this reason, the experimental group showed the best result among the four groups, showing synergistic effects with initial release of NT-3 and BDNF together and the later release of PDGF in combination. Therefore, it is true that NT-3 and BDNF have minor roles compared to that of PDGF, but they are still very important growth factors in the nervous system.

Won-Gun Koh: 0000-0002-5191-2531 Author Contributions □

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIP) (Grants 2015R1D1A1A01060444, 2017M3D1A1039289, 20090093823, 2014M3A7B4051596, 2016R1A6A3A11932752, and 2017R1A2B3011586) and the Technology Innovation Program funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea) (Grant 10062712, Development of spinal fusion implant and its manufacturing system; the functionality optimized, patient-customized in terms of bioactive materials to meet the clinical needs).

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ABBREVIATIONS CNS, central nervous system; PNS, peripheral nervous system; PCL, polycaprolactone; PLGA, poly(lactic-co-glycolic acid); PEG, poly(ethylene glycol); NT-3, neurotrophic factor 3; BDNF, brain-derived neurotrophic factor; PDGF, plateletderived growth factor; TFE, 2,2,2-trifluoroethanol; HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolum bromide; DMSO, dimethyl sulfoxide; HRP, horseradish peroxidase; TMB, 3,3′,5,5′tetramethylbenzidine; mNSC, mouse neural stem cell; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; PBS, phosphate buffered saline; Tuj1, class III β-tubulin; MAP2, microtubule associated protein-2; SEM, scanning electron microscopy; SFI, sciatic functional index; PL, print length; ITS, intermediary toe spread; PWT, paw withdrawal threshold; IHC, immunohistochemistry

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CONCLUSION The combination of sequential electrospinning and hydrogel patterning generated multilayered fibrous scaffolds which were used as a growth factor-releasing nerve conduit for sciatic nerve regeneration. Multilayered scaffolds consisted of three layers, where the first, second, and third layers were prepared from aligned PCL fibers and randomly oriented PLGA 6535 and 8515 fibers, respectively. Two different PLGA layers with different lactide/glycolide ratios were used to achieve the controlled release of three different growth factors such as NT-3, BDNF, and PDGF, while the first PCL layer was used to provide guided nerve growth. Among the various combinations of growth factor release, the fast release of NT-3 and BDNF as well as the slow release of PDGF gave the best results in terms of nerve regeneration, which was confirmed by behavioral tests such as SFI and mechanical allodynia analysis. Furthermore, the morphology analysis of regenerated tissue revealed that the growth direction of tissue is similar to the direction of the real spinal cord due to an aligned fibrous PCL layer. The developed multilayered fibrous scaffold tailored the chemical and morphological properties of the materials and biological factors to give important cues for neural tissue engineering.

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REFERENCES

(1) Schmidt, C. E.; Leach, J. B. Neural tissue engineering: strategies for repair and regeneration. Annu. Rev. Biomed. Eng. 2003, 5 (1), 293−347. (2) Sulaiman, W.; Gordon, T. Neurobiology of peripheral nerve injury, regeneration, and functional recovery: from bench top research to bedside application. Ochsner J. 2013, 13 (1), 100−108. (3) Allodi, I.; Udina, E.; Navarro, X. Specificity of peripheral nerve regeneration: interactions at the axon level. Prog. Neurobiol. 2012, 98 (1), 16−37. (4) Millesi, H.; Meissl, G.; Berger, A. The interfascicular nerve-grafting of the median and ulnar nerves. J. Bone Jt. Surg. 1972, 54 (4), 727−750. (5) Dedkov, E. I.; Kostrominova, T. Y.; Borisov, A. B.; Carlson, B. M. Survival of Schwann cells in chronically denervated skeletal muscles. Acta Neuropathol. 2002, 103 (6), 565−574. (6) Arslantunali, D.; Dursun, T.; Yucel, D.; Hasirci, N.; Hasirci, V. Peripheral nerve conduits: technology update. Med. Devices: Evidence Res. 2014, 7, 405. (7) Zorlutuna, P.; Vrana, N. E.; Khademhosseini, A. The expanding world of tissue engineering: the building blocks and new applications of tissue engineered constructs. IEEE Rev. Biomed. Eng. 2013, 6, 47−62. (8) Yang, F.; Murugan, R.; Wang, S.; Ramakrishna, S. Electrospinning of nano/micro scale poly (L-lactic acid) aligned fibers and their potential in neural tissue engineering. Biomaterials 2005, 26 (15), 2603−2610. (9) Xie, J.; Willerth, S. M.; Li, X.; Macewan, M. R.; Rader, A.; SakiyamaElbert, S. E.; Xia, Y. The differentiation of embryonic stem cells seeded on electrospun nanofibers into neural lineages. Biomaterials 2009, 30 (3), 354−362.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.7b00801. Supplementary videos 1 and 2 showing the experiments to investigate the free-walking pattern and basal pain sensitivity of injured rats, respectively (ZIP)

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M.-H.H. and H.J.H. contributed equally to this work.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. 584

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ACS Biomaterials Science & Engineering (10) Xie, J.; MacEwan, M. R.; Schwartz, A. G.; Xia, Y. Electrospun nanofibers for neural tissue engineering. Nanoscale 2010, 2 (1), 35−44. (11) Ghasemi-Mobarakeh, L.; Prabhakaran, M. P.; Morshed, M.; NasrEsfahani, M.-H.; Ramakrishna, S. Electrospun poly (ε-caprolactone)/ gelatin nanofibrous scaffolds for nerve tissue engineering. Biomaterials 2008, 29 (34), 4532−4539. (12) Su, F.-C.; Wu, C.-C.; Chien, S. Roles of microenvironment and mechanical forces in cell and tissue remodeling. J. Med. Biol. Eng. 2011, 31, 233−244. (13) Nisbet, D.; Forsythe, J. S.; Shen, W.; Finkelstein, D.; Horne, M. K. A review of the cellular response on electrospun nanofibers for tissue engineering. J. Biomater. Appl. 2009, 24 (1), 7−29. (14) Lim, S. H.; Liu, X. Y.; Song, H.; Yarema, K. J.; Mao, H.-Q. The effect of nanofiber-guided cell alignment on the preferential differentiation of neural stem cells. Biomaterials 2010, 31 (34), 9031−9039. (15) Willerth, S. M.; Rader, A.; Sakiyama-Elbert, S. E. The effect of controlled growth factor delivery on embryonic stem cell differentiation inside fibrin scaffolds. Stem Cell Res. 2008, 1 (3), 205−218. (16) Johnson, P. J.; Tatara, A.; Shiu, A.; Sakiyama-Elbert, S. E. Controlled release of neurotrophin-3 and platelet-derived growth factor from fibrin scaffolds containing neural progenitor cells enhances survival and differentiation into neurons in a subacute model of SCI. Cell Transplant. 2010, 19 (1), 89−101. (17) Li, X.; Li, M.; Sun, J.; Zhuang, Y.; Shi, J.; Guan, D.; Chen, Y.; Dai, J. Radially Aligned Electrospun Fibers with Continuous Gradient of SDF1α for the Guidance of Neural Stem Cells. Small 2016, 12 (36), 5009−5018. (18) Madduri, S.; Papaloizos, M.; Gander, B. Trophically and topographically functionalized silk fibroin nerve conduits for guided peripheral nerve regeneration. Biomaterials 2010, 31 (8), 2323−2334. (19) Park, T. G. Degradation of poly (lactic-co-glycolic acid) microspheres: effect of copolymer composition. Biomaterials 1995, 16 (15), 1123−1130. (20) Makadia, H. K.; Siegel, S. J. Poly lactic-co-glycolic acid (PLGA) as biodegradable controlled drug delivery carrier. Polymers 2011, 3 (3), 1377−1397. (21) Bain, J.; Mackinnon, S.; Hunter, D. Functional evaluation of complete sciatic, peroneal, and posterior tibial nerve lesions in the rat. Plast. Reconstr. Surg. 1989, 83 (1), 129−136. (22) Ji, R.-R.; Befort, K.; Brenner, G. J.; Woolf, C. J. ERK MAP kinase activation in superficial spinal cord neurons induces prodynorphin and NK-1 upregulation and contributes to persistent inflammatory pain hypersensitivity. J. Neurosci. 2002, 22 (2), 478−485. (23) Jiang, X.; Cao, H. Q.; Shi, L. Y.; Ng, S. Y.; Stanton, L. W.; Chew, S. Y. Nanofiber topography and sustained biochemical signaling enhance human mesenchymal stem cell neural commitment. Acta Biomater. 2012, 8 (3), 1290−1302. (24) Wang, Y.; Yao, M.; Zhou, J.; Zheng, W.; Zhou, C.; Dong, D.; Liu, Y.; Teng, Z.; Jiang, Y.; Wei, G. The promotion of neural progenitor cells proliferation by aligned and randomly oriented collagen nanofibers through β1 integrin/MAPK signaling pathway. Biomaterials 2011, 32 (28), 6737−6744. (25) Lee, M. R.; Kwon, K. W.; Jung, H.; Kim, H. N.; Suh, K. Y.; Kim, K.; Kim, K.-S. Direct differentiation of human embryonic stem cells into selective neurons on nanoscale ridge/groove pattern arrays. Biomaterials 2010, 31 (15), 4360−4366. (26) Callahan, L. A. S.; Xie, S.; Barker, I. A.; Zheng, J.; Reneker, D. H.; Dove, A. P.; Becker, M. L. Directed differentiation and neurite extension of mouse embryonic stem cell on aligned poly (lactide) nanofibers functionalized with YIGSR peptide. Biomaterials 2013, 34 (36), 9089− 9095. (27) Jha, B. S.; Colello, R. J.; Bowman, J. R.; Sell, S. A.; Lee, K. D.; Bigbee, J. W.; Bowlin, G. L.; Chow, W. N.; Mathern, B. E.; Simpson, D. G. Two pole air gap electrospinning: Fabrication of highly aligned, three-dimensional scaffolds for nerve reconstruction. Acta Biomater. 2011, 7 (1), 203−215. (28) Furth, M. E.; Atala, A.; Van Dyke, M. E. Smart biomaterials design for tissue engineering and regenerative medicine. Biomaterials 2007, 28 (34), 5068−5073.

(29) Grill, R.; Murai, K.; Blesch, A.; Gage, F.; Tuszynski, M. Cellular delivery of neurotrophin-3 promotes corticospinal axonal growth and partial functional recovery after spinal cord injury. J. Neurosci. 1997, 17 (14), 5560−5572. (30) Liu, Y.; Kim, D.; Himes, B. T.; Chow, S. Y.; Schallert, T.; Murray, M.; Tessler, A.; Fischer, I. Transplants of fibroblasts genetically modified to express BDNF promote regeneration of adult rat rubrospinal axons and recovery of forelimb function. J. Neurosci. 1999, 19 (11), 4370− 4387. (31) Cheng, H.; Cao, Y.; Olson, L. Spinal cord repair in adult paraplegic rats: partial restoration of hind limb function. Science 1996, 273, 510−512. (32) Horner, P. J.; Gage, F. H. Regenerating the damaged central nervous system. Nature 2000, 407 (6807), 963. (33) Namiki, J.; Kojima, A.; Tator, C. H. Effect of brain-derived neurotrophic factor, nerve growth factor, and neurotrophin-3 on functional recovery and regeneration after spinal cord injury in adult rats. J. Neurotrauma 2000, 17 (12), 1219−1231. (34) Pencea, V.; Bingaman, K. D.; Wiegand, S. J.; Luskin, M. B. Infusion of brain-derived neurotrophic factor into the lateral ventricle of the adult rat leads to new neurons in the parenchyma of the striatum, septum, thalamus, and hypothalamus. J. Neurosci. 2001, 21 (17), 6706− 6717. (35) Erlandsson, A.; Enarsson, M.; Forsberg-Nilsson, K. Immature neurons from CNS stem cells proliferate in response to platelet-derived growth factor. J. Neurosci. 2001, 21 (10), 3483−3491. (36) Kawabe, T.; Wen, T.-C.; Matsuda, S.; Ishihara, K.; Otsuka, H.; Sakanaka, M. Platelet-derived growth factor prevents ischemia-induced neuronal injuries in vivo. Neurosci. Res. 1997, 29 (4), 335−343. (37) Mohapel, P.; Frielingsdorf, H.; Häggblad, J.; Zachrisson, O.; Brundin, P. Platelet-derived growth factor (PDGF-BB) and brainderived neurotrophic factor (BDNF) induce striatal neurogenesis in adult rats with 6-hydroxydopamine lesions. Neuroscience 2005, 132 (3), 767−776. (38) Griffin, J. W.; Pan, B.; Polley, M. A.; Hoffman, P. N.; Farah, M. H. Measuring nerve regeneration in the mouse. Exp. Neurol. 2010, 223 (1), 60−71. (39) Angius, D.; Wang, H.; Spinner, R. J.; Gutierrez-Cotto, Y.; Yaszemski, M. J.; Windebank, A. J. A systematic review of animal models used to study nerve regeneration in tissue-engineered scaffolds. Biomaterials 2012, 33 (32), 8034−8039. (40) Georgiou, M.; Bunting, S. C.; Davies, H. A.; Loughlin, A. J.; Golding, J. P.; Phillips, J. B. Engineered neural tissue for peripheral nerve repair. Biomaterials 2013, 34 (30), 7335−7343. (41) Ao, Q.; Fung, C.-K.; Tsui, A. Y.-P.; Cai, S.; Zuo, H.-C.; Chan, Y.S.; Shum, D. K.-Y. The regeneration of transected sciatic nerves of adult rats using chitosan nerve conduits seeded with bone marrow stromal cell-derived Schwann cells. Biomaterials 2011, 32 (3), 787−796. (42) Vleggeert-Lankamp, C. L. The role of evaluation methods in the assessment of peripheral nerve regeneration through synthetic conduits: a systematic review. J. Neurosurg. 2007, 107 (6), 1168−1189. (43) Boyce, V. S.; Tumolo, M.; Fischer, I.; Murray, M.; Lemay, M. A. Neurotrophic factors promote and enhance locomotor recovery in untrained spinalized cats. J. Neurophysiol. 2007, 98 (4), 1988−1996. (44) Boyce, V. S.; Park, J.; Gage, F. H.; Mendell, L. M. Differential effects of brain-derived neurotrophic factor and neurotrophin-3 on hindlimb function in paraplegic rats. Eur. J. Neurosci. 2012, 35 (2), 221− 232. (45) Jakeman, L. B.; Wei, P.; Guan, Z.; Stokes, B. T. Brain-derived neurotrophic factor stimulates hindlimb stepping and sprouting of cholinergic fibers after spinal cord injury. Exp. Neurol. 1998, 154 (1), 170−184. (46) Huang, E. J.; Reichardt, L. F. Neurotrophins: roles in neuronal development and function. Annu. Rev. Neurosci. 2001, 24 (1), 677−736. (47) Lichtenfels, M.; Colomé, L.; Sebben, A. D.; Braga-Silva, J. Effect of platelet rich plasma and platelet rich fibrin on sciatic nerve regeneration in a rat model. Microsurgery 2013, 33 (5), 383−390. (48) Caldwell, M. A.; He, X.; Wilkie, N.; Pollack, S.; Marshall, G.; Wafford, K. A.; Svendsen, C. N. Growth factors regulate the survival and 585

DOI: 10.1021/acsbiomaterials.7b00801 ACS Biomater. Sci. Eng. 2018, 4, 576−586

Article

ACS Biomaterials Science & Engineering fate of cells derived from human neurospheres. Nat. Biotechnol. 2001, 19 (5), 475. (49) Zhang, Y.; Huang, J.; Huang, L.; Liu, Q.; Shao, H.; Hu, X.; Song, L. Silk Fibroin-Based Scaffolds with Controlled Delivery Order of VEGF and BDNF for Cavernous Nerve Regeneration. ACS Biomater. Sci. Eng. 2016, 2 (11), 2018−2025.

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