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A Thermosensitive heparin-poloxamer hydrogel bridge aFGF to treat spinal cord injury Qingqing Wang, Yan He, Yingzheng Zhao, Huixu Xie, Qian Lin, Zili He, Xiaoyan Wang, Jiawei Li, Hongyu Zhang, Chenggui Wang, Fanghua Gong, Xiaokun Li, Huazi Xu, Qingsong Ye, and Jian Xiao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b13155 • Publication Date (Web): 09 Feb 2017 Downloaded from http://pubs.acs.org on February 9, 2017

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ACS Applied Materials & Interfaces

A Thermosensitive heparin-poloxamer hydrogel bridge aFGF to treat spinal cord injury

Qingqing Wang, †,‡# Yan He, ‡, §# Yingzheng Zhao, ‡# Huixu Xie, Qian Lin, ‡ Zili He, †,‡ Xiaoyan Wang, † Jiawei Li, †,‡ Hongyu Zhang, ‡ Chenggui Wang, †,‡ Fanghua Gong, ∥

‡

Xiaokun

Li, ‡ Huazi Xu, *,†,‡ Qingsong Ye, *,†,‡,§ Jian Xiao *,†,‡

†

Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, 325035 China

‡

WMU-JCU Joint Research Group for Stem Cell and Tissue Engineering, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, 325035, China § UQ-WMU Joint Research Group for Regenerative Medicine, Oral Health Centre, University of Queensland, Brisbane 4006, Australia ∥

State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China Corresponding Authors *E-mail: [email protected] (J.X.). *E-mail: [email protected] (Q.S.Y). *E-mail: [email protected] (H.Z.X). Author Contributions # Q.Q.W. , Y.H. and Y.Z.Z. contributed equally to this work. Notes The authors declare no competing financial interest.

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ABSTRACT: Acidic fibroblast growth factor (aFGF) exerts a protective effect on spinal cord injury (SCI) but is limited by the lack of physicochemical stability andthe ability to cross the blood spinal cord barrier (BSCB). As promising biomaterials, hydrogels contain substantial amounts of water and a 3D porous structureand are commonly used to load and deliver growth factors. Heparin can not only enhance growth factor loading onto hydrogels but also can stabilize the structure and control the release behavior. Herein, a novel aFGF-loaded thermosensitive heparin-poloxamer (aFGF-HP) hydrogel was developed and applied to provide protection and regeneration after SCI. To assess the effects of the aFGF-HP hydrogel, BSCB restoration, neuron and axonal rehabilitation, glial scar inhibition, inflammatory response suppression and motor recovery were studied both in vivo and in vitro. The aFGF-HP hydrogels exhibited sustained release of aFGF and protected the bioactivity of aFGF in vitro. Compared to groups i.v. administered either drug-free HP hydrogel or aFGF alone, the aFGF-HP hydrogel group revealed prominent and attenuated disruption of the BSCB, reduced neuronal apoptosis, reactive astrogliosis and increased neuron and axonal rehabilitation both in vivo and in vitro. This work provides an effective approach to enhance recovery after SCI and provide a successful strategy for SCI protection.

KEYWORDS:

spinal cord injury, acidic fibroblast growth factor, control release,

thermosensitive hydrogel, neuroprotection


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1. INTRODUCTION Spinal cord injury (SCI) is a crippling and severely disabling disease with almost 12,000 new cases occurring annually. SCI causes a significant loss of sensory andmotor functions as well as a wide range of disabilities via two primary phases.

1

The initial mechanical injury causes a

structural disturbance; this is followed by long-term secondary damage comprising inflammation, apoptosis, oxidative stress and the formation of glial scars. 2 Currently, injury stabilization by decompression surgery, secondary complication prevention and rehabilitation via drug administration are considered essential for functional recovery after SCI. The molecules involved with neurological recovery after SCI are designed to protect surviving tissue against degeneration, 3

inhibit inflammation, 4 promote regenerative growth of lesioned axons 5 and reduce glial scars, 6-7

all of which are barriers of neuron axon regeneration after SCI. Acidic fibroblast growth factor (aFGF) is one of powerful factors involved in the protection and regeneration of the nervous system clinical trial.

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8-11

and has been demonstrated as safe and feasible in a

A proteomics study indicated that aFGF can reduce the number of apoptotic

neurons and the inflammatory reaction after SCI; 13-15 however, as a macromolecular protein, aFGF has poor penetrability of the blood spinal cord barrier (BSCB). Thus, aFGF delivery via either subcutaneous or intravenous administration is ineffective in SCI. In situ administration can help aFGF bypass the BSCB, but the effects of aFGF are limited due to its limited shelf life and susceptibility to biochemical variations in the body. Therefore, identifying a more effective route of aFGF administration and maintaining aFGF sustained release are urgently needed. Several recent reviews summarized the development of the cells and drugs delivery strategies into the spine cord and reviewed the role of hydrogel in SCI treatments trategies. 16-18 Many new materials



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such as three-dimensional biomimetic hydrogel

19-20

, nanoparticles

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, novel scaffolds

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were

used to carry substances (drugs, antibodies, peptides, or other proteins) and/or cells for spinal cord injury treatment. Besides, with the deep understanding of the mechanisms of spinal cord injury, many selective delivery tools which could selectively treat/target the aim cells or tissue have been developed.

24-25

Hydrogels are non-toxic, biodegradable, 3D porous structures that can act as

promising protective agents and vehicles to load and deliver biological macromolecules.

20, 26

Several GF hydrogel systems have been reported for the treatment of SCI, including a hyaluronan, methylcelluloseand hydrogel or nanoparticle composite with PDGF, 27-28 a beta hairpin peptide hydrogel containing NGF, 29 a hyaluronic acid hydrogel with BDNF, 30 a HEMA-MOETACL hydrogel containing bFGF

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and a VEGF-loaded alginate hydrogel. 32 The most ideal hydrogels

for SCI recovery should be thermosensitive, have a high loading capacity and offer maximal protection of the growth factors. However, none of these hydrogels are optimal. Identifying the most appropriate biocompatible material for loading growth factors that could act as a scaffold and maximize their potential in promoting a comprehensive recovery of SCI is still a big challenge. In this work, we designed anovel thermosensitive aFGF-infused heparin-poloxamer hydrogel that could load and transferaFGF to the injured spinal cord in a localized and sustained manner. We infused modified heparin, a sulfated polysaccharide and a representative antithrombotic drug, into a poloxamer hydrogel to bind aFGF and implement controlled release behaviors as well as maximize the effectiveness of the molecule while minimizing the biochemical modifications that are common of biologically active proteins such ascytokines and growth factors, especially aFGF. 33-35

Heparin-containing hydrogels can immobilize and protect high-affinity heparin-bound aFGF

from degradation36, release growth factor in a sustained manner, 36-38 enhance the aFGF binding


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affinity to receptors on the cell surface to activate more intracellular signaling pathways and increase both the stability and activity of the growth factor. 33, 39 Furthermore, the HP hydrogel has a controlled sol-to-gel transition temperature and is suitable for orthotopic injection. Our results indicate that the aFGF-HP hydrogel has the potential for not only the localized and sustained transmission of aFGF to the injured spinal cord but also comprehensive tissue regeneration and recovery of SCI. Here, we provide extensive evidence that this method may expedite the applications of aFGF in the clinical treatment of SCI.



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2. MATERIALS AND METHODS 2.1. Preparation of P and HP hydrogels. Poloxamer 407 was purchased from Badische Anilin Soda Fabrik Ga (Shanghai, China). The synthesis of HP according to EDC/NHS method as previously described. 35 First, poloxamer 407 (1 mM) reacted with diaminoethylene (3 mM) to form a mono amine-terminated Poloxamer (MATP). Next, 0.5mM MATP was reacted with 0.5 mM heparin salt by 0.5 mM EDC and 0.25 mM NHS in 0.5 M MES buffer for 1 day. The amine groups of poloxamer 407 was coupled with carboxyl ones of heparin specifically and resulted in amide bond formation. After that, the mixture was dialyzed 72 h and lyophilized. Finally, the heparin-Poloxamer (HP) was obtained. Lyophilized powder of P or HP was mixed in aFGF solution with modest stirring, then the mixture was stored at 4°C overnight to form the aFGF loaded hydrogel method. 40 2.2. Characterization of aFGF-HP hydrogels. The gelation temperature measurements of the aFGF-HP hydrogel were measured as previously reported.

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The micromorphology of the

prepared aFGF-HP hydrogels was observed by scanning electron microscopy (SEM). The aFGF-HP hydrogels and drug-free HP hydrogels were freeze dried and sputter coated with gold followed by scanning observation. Rheological measurements of the aFGF-P and aFGF-HP were carried out using the discovery hybrid rheometer. The transition temperature and amplitude sweep were measured using the stainless steel parallel plate flat plates (25 mm). The shear frequency was set to 10 rad/s; shear strain was set to 1%. 2.3. Release profiles of aFGF from aFGF-HP hydrogels. The release profiles ofactivated aFGF from the aFGF-HP hydrogels were analyzed by adding 100 µl of aFGF-HP hydrogel (containing 100 ng aFGF) into 500 µl of 0.9% saline and incubating at 37°C. At specific time


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points (12 h and 1, 3, 5, 7, 14, 21, and 28 d), the solution was centrifuged for 10 min at 12000 g, and the supernatant was collected and replaced with the same volume of fresh saline. The aFGF concentration in the supernatant was measured by using an aFGF enzyme-linked immunosorbent assay kit (ELISA, Westang System, Shanghai, China). 2.4. Spinal cord injury model and drug treatment. Adult female Sprague-Dawley rats (220-250 g, n = 108) were obtained from the Animal Center of the Chinese Academy of Science (Shanghai, China). The care and use of all animals conformed to guidelines set forth by the Chinese National Institutes of Health. All the rats were housed under controlled environmental conditions. To induce a spinal cord injury (SCI), all the animals were anesthetized by 8% (w/v) chloral hydrate (3.5 mL/kg, i.p.). The ninth ribs and T9 vertebrae were located using a locating pin and confirmed by an animal digital X-ray machine (Kubtec Model XPERT.8; KUB Technologies Inc.). Afterwards, a laminectomy was performed at the T9 vertebrae after the vertebral column was exposed. The spinal cord was fully exposed, and a moderate crushing injury was performed using a vascular clip for 1 min (30 g forces, Oscar, China). 41 After SCI, 10 µl of HP hydrogel, an aFGF solution or aFGF-HP hydrogel was orthotopically injected (OI) at a dose of 2 µg/µl using a microsyringe. The aFGF solution, which was dissolved in saline (500 µl, 0.04 µg/µl) and intravenously (IV) injected through the tail vein was set as a control (aFGF IV). Postoperative monitoring included manual bladder emptying three times a day. Subsequently, the rats were sacrificed at 1, 3, 7, 14, 28 or 56 d after treatment. 2.5. Measurement of blood-spinal cord barrier disruption. The integrity of the BSCB was investigated with Evan’s Blue dye extravasation assay according to our previous report. 42 4 mL/kg of 2% Evan’s Blue dye solution was injected through the tail vein at 1 d after inducement of SCI.



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Two hours later, rats were anaesthetized and sacrificed and the spinal cords were sectioned with a cryostat 30 µm coronal slices. The fluorescence of Evan’s Blue was observed with a confocal fluorescence microscope. 2.6. Transmission electron microscopy (TEM). Spinal cord tissues were fixed in 2.5% (w/v) glutaraldehyde solution overnight, post-fixed in 2% (v/v) osmium tetroxide and blocked with 2% (v/v) uranyl acetate. Tissues were embedded in Araldite after dehydration in a series of acetone washes. Semi-thin section and toluidine blue staining were performed for observation of location. Finally, ultra-thin sections of at least six blocks per sample were cut and observed using a TEM. 2.7. Tissue preparation. Animals were anesthetized with 8% (w/v) chloralic hydras (3.5 mL/kg, i.p.) at specific time points after SCI. For Nissl staining, immunohistochemistry, etc., 0.5 cm section of the spinal cord was dissected out, post-fixed by 4% paraformaldehyde for 6 h and then embedded in paraffin. Longitudinal or transverse sections (5µm thick) were mounted on slides for following staining. For western blot test, a spinal cord segment (0.5 cm length) at the contusion epicenter was dissected and stored at -80℃ immediately. 2.8. Western blot. The supernatant of tissue or cells was collected for protein assay. The extracts were first quantified with BCA reagents. 80 µg proteins were separated on 10% gels and transferred onto PVDF membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% (w/v) milk (Bio-Rad) in TBST for 120 min and incubated with the primary antibody solutions overnight at 4℃ followed by treated with horseradish peroxidase-conjugated secondary antibodies for 60 min. Signals were visualized by Chemi DocXRS + Imaging System (Bio-Rad). All experiments were repeated three times. 2.9. Histology, immunofluorescence and immunohistochemistry. Longitudinal or transverse


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sections mounted on slides were prepared as described above. Transverse sections for histopathological examination were treated by H&E staining, Nissl staining and LFB staining following the manufacturer’s instructions. Brightfield images were acquired using light microscopy. For immunofluorescence, longitudinal sections were treated with primary antibodies targeting the following proteins: NF-200 (1:2000, Abcam), GFAP (1:2000, Abcam), microtubule-associated protein 2 (MAP-2, 1:300, Santa Cruz) and CD68 (1:400,Abcam). The transverse sections were treated with primary antibodies targeting NeuN (1:400,Abcam), Claudin5 (1:200,Santa Cruz), and cleaved caspase-3 (1:300，Cell Signaling Technologies). The sections were washed four times with PBST and incubated with AlexaFluor 568, AlexaFluor 488 or AlexaFluor 647 donkey anti-rabbit/mouse secondary antibodies for 1 h at 37°C. Afterwards, the sections were washed with PBS, incubated with DAPI for 7 min, rinsed with PBS and finally sealed with a coverslip. For immunohistochemistry, the longitudinal sections were treated with primary antibodies targeting the following proteins: myelin basic protein 2 (MBP-2, 1:400，Santa Cruz), Nestin (1:200，Abcam), and GAP-43 (1:200，Cell Signaling Technologies) and followed by incubated with horseradish peroxidase-conjugated secondary antibodies overnight at 4°C. Next, the sections were developed with DAB and counterstained with hematoxylin. All the images were captured using a confocal fluorescence microscope (Nikon, Japan). 2.10. Locomotion recovery evaluation. Locomotion recovery analyses, including the Basso-Beattie-Bresnahan (BBB) locomotion scale, inclined plane test, and footprint test

43

were

performed at 0, 3, 7, 14, 21 and 28 d. In brief, the BBB scores range from 0 points to 21 points. The inclined plane test 44 was also performed to assess functional improvement at each time point and the footprint analysis was performed by dipping the animal’s hindpaws with red dye as



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previously described. 45 Outcome measures were obtained by 5 independent examiners who were blinded to the experimental conditions. 2.11. Statistical analysis. All data was presented as the mean ± standard error of the mean (SEM). Differences between groups in BBB scores and inclined plane test were analyzed with use of generalized linear mixed models. Dynamic interaction between aFGF and HP hydrogel were measured by a 2 × 2 factorial trial. Statistical analysis of the other data was performed using one-way analysis of variance (ANOVA). P values < 0.05 were considered statistically significant.


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3. RESULTS 3.1. Characterization of the aFGF-HP hydrogel. The relationship between the HP concentration and the gelation temperature is shown in Figure 1B. Considering the body temperature of animals and humans (37°C), the aFGF-HP hydrogel with an HP concentration of 17% was observed to have a suitable gelation temperature. To test the effects of the HP and P hydrogels on aFGF release, the in vitro release profile of aFGF from aFGF-HP was recorded. Less than 10% of the aFGF was detected in the supernatant after synthesis of the aFGF-HP and aFGF-P hydrogels, indicating that the loading efficiency of these two hydrogels was greater than 90%. An initial burst release (~18%) of aFGF from aFGF-P was observed on day 1, and another ~7% was released over the following 4 d. However, a negligible amount of aFGF was released from the aFGF-P hydrogel after day 5, with approximately 25% of loaded aFGF released by day 28 (Figure 1C). In contrast, a sustained release behavior of aFGF was observed in the aFGF-HP hydrogel. Approximately 55% of the loaded aFGF was released from the aFGF-HP hydrogel by day 28, which was two times greater than that in the aFGF-P hydrogel (Figure 1C). As shown in Figure 1 D and E, Storage modulus (G’) and loss modulus (G”) respectively representing elastic and viscous behavior of these two gels. Varying temperatures were performed to reveal the gelation process of these two systems (Figure 1D). Amplitude sweep was conducted to analyze the physical nature of the hydrogels (Figure 1E). The two systems showed a similar sol-to-gel transition at between 21-26 °C. The sol-to-gel transition was moderate and the transition temperature was similar to room temperature, making the gels suitable for manipulation in vitro and applications in vivo. The amplitude sweep displayed the similar intersection at moderate strain amplitude of about 6.5%. The results indicated that these two hydrogels were both relatively soft



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and suitable for the biological application on the spinal cord. The morphology of the HP and aFGF-HP hydrogels was also observed under SEM. Compared to the smooth surface of the HP hydrogel, the aFGF-HP hydrogel had a porous structure resembling a cribriform plate (Figure 1F) where the aFGF proteins can be adsorbed. Furthermore, the HP hydrogel itself showed no toxicity in PC12 cells (Figure 1G and 1H). Based on the characteristics observed, the aFGF-HP hydrogel sustains thermosensitivity feature as well as a 3D porous structure, both of which are favorable for the localized and sustained delivery of aFGF. However, heparin-containing hydrogels can immobilize and protect aFGF from degradation, which allows the release of aFGF in a sustained manner. 3.2. aFGF-HP attenuated BSCB disruption by preventing the loss of tight junction and adherens junction proteins after SCI. BSCB disruption is one of the most serious types of damage from SCI and is maximally disrupted at 24 h post-SCI. 46 To observe the effects of aFGF-HP on BSCB integrity, we examined the permeability of the BSCB at day 1 after SCI injury by using the Evans Blue assay (n = 5). As shown in Figure 2B-D, compared with the sham group, the SCI rats indicated a significant increase in the amount of EB extravasation after SCI. aFGF administration reduced the levels of extravasation to varying degrees, and the levels of EB were lowest in the aFGF-HP group, which were similar to those in the sham group. In addition, the fluorescence intensity of EB was ordered as the aFGF-HP/OI group > aFGF/OI group > aFGF/IV group> HP/OI group (Figure 3E-H, P < 0.01). It is well accepted that tight junctions (TJs) and adherens junctions (AJs) are involved in maintaining the integrity of the BSCB.

47

Next we

examined the effect of aFGF-HP on the alterations of SCI-induced TJ and AJ protein expression by Western blotting and immunofluorescence. Our results showed that the levels of AJs


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(P120-catenin) as well as the TJs (Occludin, Claudin5) were decreased after SCI and that decreases were significantly ameliorated in the aFGF-treated groups, especially in the aFGF-HP/OI group (Figure 4A-D). To further confirm the effects of aFGF-HP on protecting the BSCB, we applied H2O2 to HBMVECs and measured the paracellular permeability of FITC-dextran to evaluate the permeability of the BSCB. As shown in Figure S2A, compared with the H2O2 group, the penetrability to FITC-dextran was decreased most frequently in the aFGF-HP group. Furthermore, the expression levels of proteins associated with AJs (β-catenin) and TJs (Claudin5) were enhanced with aFGF-HP treatment to a greater degree than the levels observed in the other groups (SFigure 2B-D). The results showed that aFGF deters BSCB disruption by inhibiting the degradation of AJ and TJ proteins during the initial stages after SCI. In addition, in situ administration of aFGF shows exerts a more pronounced protective effect of the BSCB than i.v. administration, and HP enhances this effect of aFGF. 3.3. aFGF-HP reduces the apoptosis of neurons in vivo and in vitro. Cell apoptosis rapidly occurs after the initial trauma, after which a broad and prolonged apoptotic event arises around the epicenter of the contusion. 48-49 To evaluate the role of the aFGF-HP hydrogel in modulating cellular apoptosis after SCI, immunohistochemistry staining for NeuN (red) and cleaved caspase-3 (green) as well as TUNEL staining were performed on neurons at 7 d post injury (dpi). As shown in Figure 5A, 5B and Figure 6A, the percentage of the cleaved caspase-3-positive neurons and the number of TUNEL-positive cells in the SCI group were noticeably increased. The aFGF treatments reduced the number of apoptotic cells to varying degrees, but the aFGF-HP hydrogel resulted in a more remarkable reduction of neuronal apoptosis. The expression of cleaved caspase-3 was further tested by Western blotting in each group at either 7 dpi or 6 h after H2O2



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treatment. As shown in Figure 6B, 6C, and Figure S3C-E, aFGF-HP substantially inhibited the apoptosis of neurons either caused by SCI in the rats or induced by H2O2 in PC-12 cells. The microstructures of these neurons were also examined by transmission electron microscopy (Figure 6D). The neurons in the sham group had normal mitochondria and an ER with many ribosomes. However, the organelles in neurons from the SCI group were almost completely disintegrated. The organelles were protected by the aFGF treatments, with few organelles disintegrated in the aFGF-HP/OI group, suggesting that HP protects aFGF from modifications. Taken together, these data revealed the hierarchy of the effects on reducing neuronal apoptosis to benefit of the functional recovery of SCIas follows: aFGF-HP/OI group > aFGF/OI group > aFGF/IV group> HP/OI group. 3.4. aFGF-HP promotes the rehabilitation of the neurons in vivo and in vitro. To further test the effects of aFGF-HP on the promoting neuronal rehabilitation, the expression levels of GAP-43 and Nestin, which are two classic indicators of neural regeneration, were tested in each group by western blotting and immunohistochemistry at 7 dpi. As shown in Figure 7A, 7B and 7C, minimal GAP-43 and Nestin were expressed in the sham group. In contrast, the expression levels of GAP-43 and Nestin in the presence of aFGF treatment were significantly increased. Among all the groups, the aFGF-HP/OI group showed the highest GAP-43 and Nestin levels. These results in the aFGF-HP/OI group were confirmed by immunohistochemistry, providing further evidence of the effect of aFGF-HP on neuronal rehabilitation (Figure 7D, 7E and 7F). Finally, the histological morphology in each group was evaluated using HE and Nissl stain at 5 mm rostral and 5 mm caudal of the spinal cord. As shown in Figure 8A, 8B and 8C, in contrast to the sham group, severe damage to the central gray matter and dorsal white matter was obvious in the SCI rats. The


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aFGF-treated groups presented decreased damage to varying degrees-the quantitative results indicated that the aFGF-HP/OI group showed the highest percentage of preserved tissuethan the other aFGF treatment groups. The number of ventral motor neurons (VMNs) was counted. As shown in Figure 8A, 8D and 8E, a great loss of VMNs after SCI; however, aFGF (especially the aFGF-HP hydrogel) significantly mitigated the loss of VMNs. The results demonstrated that the aFGF-HP hydrogel can reduce neuron loss and ameliorate the pathological morphology of the injured tissues. Collectively, the aFGF-HP hydrogel combined with an in situ injection revealed the best protective effects to neurons by reducing neuron apoptosis and promoting neuron rehabilitation. 3.5. aFGF-HP promotes remyelination and axonal rehabilitation in vivo and in vitro. Remyelination and axonal regeneration is a critical aspect of sensory and motor function recovery after SCI. 50-53 To determine the effect of the aFGF-HP hydrogel on remyelination, the degree of myelin sheath destruction was assessed using LFB staining at 28 dpi. As shown in Figure 9A and 9B, the hierarchy of the amount of LFB-positive myelin was as follows: aFGF-HP/OI group > aFGF/OI group > aFGF/IV group> HP/OI group. The microstructure of the myelin was also examined by TEM (Figure 9A). In the sham group, the structure of the myelin sheath was clear, and the layers were closely packed with a large number of microtubules in the axon. However, the myelin sheath in the SCI group presented vacuolar changes, acantholysis and a loss of microtubules. The degree of myelin sheath destruction was reduced after the aFGF treatments, with the aFGF-HP/OI group showing an obvious and more pronounced effect on myelin sheath restoration than the other treatment groups. Furthermore, we examined the expression of myelin basic proteins (MBPs), which are structural proteins and constituents of the myelin sheath, in each



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group in vivo and in vitro by Western blotting and immunohistochemistry (Figure 9C-F and Figure S4C, S4E). The results revealed that MBP protein expression was significantly decreased after SCI but that aFGF treatment increased the expression with the following hierarchy: aFGF-HP/OI group > aFGF/OI group > HP/OI group. Additionally, to determine the effects of aFGF-HP on axonal rehabilitation, we examined the expression of MAP-2, which is a structural protein and a constituent of axon microtubules, in each group by immunofluorescence and western blotting (Figure 10A and 10B). The results revealed that the number of MAP-2-positive axons in the SCI group was significantly decreased and disorganized at 28 dpi; however, compared to the SCI group, the aFGF-HP/OI group presented tighter and more continuous MAP-2-positive axons, which was similar to the observed morphology in the sham group. The results were further confirmed by Western blotting to detect MAP-2 both in vivo and in vitro (Figure 10C-D and Figure S4C-D). In addition, Figure S4A and S4B show the neurite length of the PC12 cells. Compared to the control group, the neurite length was reduced in the H2O2 group but was increased in the aFGF-HP group. The hierarchy of the neurite length was as follows: aFGF-HP/OI group > aFGF/OI group > aFGF/IV group> HP/OI group. Taken together, aFGF exerted an effect on promoting axonal growth and remyelination, and the aFGF-HP hydrogel can maximize this effect. 3.6. aFGF-HP attenuates the reactive astrogliosis after SCI and prevents scattering of inflammatory cells. Although the relationship between glial scar formation (mainly formed by activated astrocytes) and axonal regeneration is still debated, 54-55 it is widely accepted that glial scars provide a vital role in the recovery after SCI. 56-57 To this end, the effects of the aFGF-HP hydrogel on glial scars were tested. We first measured the expression of GFAP (a marker of


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astrocyte activation) at 1, 3, 7, 14, 28, and 56 dpi by using immunofluorescence. As shown in Figure 11B and 11C, the astrogliosis presented hypertrophy and hyperplasia at 7 dpi and appeared as broad bundles interlacing and blending with the neighboring neurons at 14 dpi. A glial scar was formed at 28 dpi and did not significantly change after this. Based on these results, 28 dpi was selected for evaluating the degree of astrogliosis. We examined the expression of GFAP and vimentin (a marker of reactive astrocytes) in vivo and in vitro by western blotting (Figure 11D-E, Figure S5A- C). A marked increase in the levels of these two proteins was observed in the SCI group than in the sham group; however, the aFGF-HP hydrogel treatment reduced the protein levels of GFAP and vimentin significantly. The chondroitin sulfate proteoglycans (CSPGs) secreted from astrocytes is the primary inhibitors of axonal regeneration. Neurocan, a glial scar-related CSPG, was markedly increased after injury but significantly inhibited after aFGF-HP hydrogel treatment both in vivo and in vitro (Figure 11D, 11G and Figure S5A, S5D). These results were further confirmed by immunofluorescence staining. As shown in Figure 12A, 12B and 12C, the crush injury around the lesion site led to highly reactive astrogliosis and the formation of a glial scar. Immunofluorescence revealed that aFGF-HP significantly reduced GFAP expression, downregulated the number of reactive astrocytes, and reversed their morphological alterations while concomitantly reducing the thickness of scar. Furthermore, it has been reported that glial scars can limit inflammation and inhibit the proliferation of inflammatory cells. Therefore, double staining for GFAP (red) and CD68 (green) was performed to observe the glial scar and inflammatory cells. As shown in Figure 12A, inflammatory cells were present and scattered after SCI. The aFGF-HP hydrogel treatment markedly reduced the number of CD68+ cells and prevented inflammatory cell scattering. These results showed that aFGF-HP can weaken



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the glial scar and prevent inflammatory cell scattering. In addition, the effect of aFGF-HP on attenuating reactive astrogliosis and preventing inflammatory cell scattering was better than that of the other treatment groups (P < 0.05). 3.7. aFGF-HP promotes axonal generation across the glial scar. Whether the axons above the injured site can cross the scar in the injured area has become the focus of SCI repair in recent years. 54, 58 Double staining for GFAP (red) and NF-200 (green) was performed to observe whether neurofilaments can across the glial scar surrounding the lesion site (Figure 12A and 12B). In the SCI group, the neurofilaments were completely lost in the injury epicenter. The HP- and aFGF/IV-treated animals showed only a mild increase in the number of NF-200-positive axons that passed through the scar. By contrast, the aFGF-HP treatment group presented a pronounced number of NF-200-labeled fibers around the lesion site that had grown beyond the glial scar to a greater degree than the aFGF/OI group. Biotinylated dextran amine is an effective anterograde tracing neural tracer that is an important tool for detection axonal growth. 59 To provide further evidence that the aFGF-HP hydrogel can promote axonal growth across the scar, frozen sections of the T12-L1 plane (under the injured site) were tested using BDA staining. As shown in Figure 13C and 13D, the number of BDA-positive axons was abundant in the normal sham rats but sharply decreased in the SCI group. The aFGF-HP hydrogel treatment increased the number of BDA-positive fibers approximately 8- to 10-fold over the number in the SCI group, which was an additional 2- to 3-fold over the levels observed in aFGF/OI group. The hierarchy of therapeutic activity in the regeneration process of SCI was as follows: aFGF-HP/OI group > aFGF/OI group > aFGF/IV group> HP/OI group. These data indicated that the aFGF-HP hydrogel can promote axonal growth across the scar, which is of great benefit for the functional recovery of SCI.


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3.8. aFGF-HP promotes the motor function after SCI. aFGF-HP exerted a positive effect on BSCB protection, neuroprotection, remyelination and axonal rehabilitation as well as attenuated reactive astrogliosis and promoted axonal growth across the scar. Whether these effects can be translated into motor function recovery requires further examination. We use three behavioral tests, the inclined plane test, the BBB locomotion scale and footprint test, to measure functional recovery. The hind legs of all the rats lost function immediately after SCI and showed restoration in a time-dependent manner. The results showed significant differences in the recovery of motor function among the experimental groups. By estimating the BBB score and the angle of incline, the aFGF-HP/OI group showed the most significant effect followed by the aFGF/OI group (Figure 14A and 14B). The footprint test can intuitively show the restoration of hind leg movement at 28 dpi. The rats treated with the aFGF-HP hydrogel exhibited coordinated crawling with the tail raised up and almost achieved the functional levels of the control group, whereas rats in the SCI group were still dragging their hind legs (Figure 14C). All these data indicate that the aFGF-HP/OI group showed maximum improvements regarding functional recovery to a higher degree than the aFGF/OI group, aFGF/IV group, HP/OI group and SCI group. Furthermore, the dynamic interaction between aFGF and the HP hydrogel as measured by a 2 × 2 factorial trial can enhance the motor function recovery (P < 0.01). By comparison, the effect of the aFGF/OI group was shown to be better than that of the aFGF/IV group, indicating that an in situ injection is the preferred administration method for these drugs (P < 0.01). Taken together, the aFGF-HP hydrogel in association with an in situ injection could effectively improve motor function recovery after SCI. 3.9. aFGF-HP inhibits the ER stress signaling pathway. Our previous reports have indicated



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that the ER stress signal contributes to the induction of apoptosis in neuronal injury diseases. 60-61 To determine the relationship between aFGF-HP and the regulation of ER stress in vivo, we detected proteins involved in the ER stress-induced apoptosis signaling pathway by western blotting and immunofluorescence staining. As shown in Figure 15A, immunofluorescence staining revealed that the number of cells positive for CHOP, the most significant ER stress-induced apoptosis protein, was increased in spinal cord lesions in the SCI group. However, the number of positive cells was reduced in the aFGF-treated groups, particularly in the aFGF-HP/OI group. In addition, the levels of ER stress-related proteins (GRP78, ATF-6, PDI and CHOP) were significantly increased at 7 dpi but decreased after aFGF treatment; furthermore, the protein levels in the aFGF-HP-treated rats were much lower than those in the aFGF group, which was in accordance with the immunofluorescence staining results (Figure 15B-F). All these data revealed the critical protective role of aFGF-HP in mitigating sustained ER stress.


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4. DISCUSSION aFGF is highly expressed in motor neurons and is released in response to sublethal cell injury. 62-63 It is well accepted that aFGF has direct neurotrophic activity and exerts beneficial effects to combat the negative consequences of SCI. 9, 64 Exogenous aFGF was also been used to repair human non-acute SCI. 12 Despite these properties, the practical therapeutic use of aFGF to treat acute SCI is limited due to its short half-life, destabilization in vivo and undesirable effects at high systemic levels. In our study, several novel biomaterials were developed, including an HP thermosensitive hydrogel, 35, 65 a H2S-releasing nano-fibrous coating

66

and an acellular matrix

scaffold. 67 Among these, HP hydrogel was found to exert the best properties for loading and releasing aFGF versus than any other FGFs, including bFGF.

65

In our HP-Hydrogel, the

poloxamer was used as a primary material, which is an FDA-approved and highly safe pharmaceutical adjuvant for venous injection with heparin to load aFGF. On the one hand, unlike other positively charged FGFs, HP and aFGF are negatively charged, which allows for relatively easy release of aFGF from HP hydrogels. 65 On the other hand, compared with poloxamer alone, HP can load a much higher amount of aFGF and release it in a slow and sustained manner, which can be beneficial for SCI repair. This phenomenon could be largely attributed to heparin, which can conjugate with the hydrogel though its -COOH and -OH groups and bind to the growth factor with its -SH group. 33 In addition, heparin can protect aFGF from acid or thermal inactivation, and protease degradation as well as stabilize the conformation, thereby effectively protecting aFGF activity. 36, 38, 68 With these advantages, HP is a promising candidate for delivering aFGF for SCI therapy. In addition, the lack of a comprehensive method to evaluate the effects of therapeutics on



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human SCI hinders the development of new therapies. Previous studies found that the BSCB disruption

69

, neurons loss 3, demyelination

70

, axonal impairment

55

and astrocytic gliosis

57

are

common indicators that reflect the neurological damage of SCI. In these years, a lot of biological materials were developed for spinal cord injury treatment. Some of them were especial for providing the best environment for adhesion and proliferation of cells.

71-72

Some of them were

designed to achieve the drug target and controlled release delivery. 29, 31 Each material had its own characteristics. To the best of our knowledge, we provided the first multifaceted evidence that a novel aFGF-loaded HP hydrogel administered via in situ injection (which can bypass the BSCB more efficiently than intravenous injection) could effectively promote the neuroprotective effect and recovery after SCI. BSCB integrity plays a crucial role in sustaining the function of the spinal cord. Sarah A et al. showed that the BSCB was disrupted within 1 hafter injury and remained open for 5 d, with maximum permeability observed at 24 h post injury. 73 In the current study, we first discovered that aFGF treatment decreased the extravasation of EB dye at 1 dpi as well as reduced the penetrability of an H2O2-treated cell monolayer to FITC-dextran. Another interesting finding was that the reduction of TJ and AJ proteins such as β-catenin and claudin-5 was inhibited, which is closely related to the penetrability of the BSCB during SCI. Those results suggested that aFGF could prevent BSCB disruption. Regarding the effects of the different administration routes, our results showed that the aFGF/OI group had a better effect on BSCB protection than the aFGF/IV group, which suggested that in situ administration of aFGF can bypass the BSCB and more effectively concentrate the drug at the lesion site than intravenous administration. In addition, the aFGF-HP/OI group had a more pronounced effect than the aFGF/OI group, which exhibited less


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release of aFGF. This result can be explained by the protective effect of HP on aFGF, which creates a synergistic effect. In conclusion, we discovered that aFGF-HP exerted a better protective effect in preserving the BSCB during the early stage of SCI at 1 dpi. Previous studies have reported that aFGF exerts direct and potent neurotrophic activity and promotes axonal growth. 10-11 In this study, our results showed that aFGF can reduce the level of neural apoptosis and increase the expression of GAP43 and Nestin, all of which are associated with neural restoration. In addition, we showed that aFGF reduced white matter injury in the spinal cord by inhibiting acantholysis of the myelin sheath and the reduced MBP expression. Lastname et al found that microtubule stabilization can promote axon growth;

74

our results

expanded on this notion by showing that aFGF facilitated the axon regeneration in vivo and in vitro and increased the expression of MAP-2, indicating that the positive effect of aFGF on axonal growth was associated with microtubule stabilization. In addition, the aFGF-HP/OI group exhibited a better effect than either the aFGF/OI or aFGF/IV groups, suggesting that the slow and sustained release of aFGF from the aFGF-HP hydrogel can significantly promote the neuroprotective effect. In conclusion, the potential of aFGF on preventing neuron cell death as well as promoting neuronal axon regeneration after SCI can be achieved upon in situ administration of the aFGF-HP hydrogel. It is widely regarded that excessive scar tissue formationis a major factor for inhibiting axonal regeneration, 75 Furthermore, it has been verified that preventing the proliferation of scar-forming astrocytes or removing the inhibitory CSPGs secreted from reactive astrocytes could effectively improve functional deficiency after SCI. 57, 76 However, Mark A. Anderson et al. recently found that preventing astrocyte scar formation failed to produce spontaneous regrowth of the axons



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through the lesions. 56 In contrast, our results showed that both the physical and chemical barriers, including astroglial proliferation, glial scar formation, and Neurocan secretion from reactive astrocytes, were markedly reduced after aFGF treatment both in vivo and in vitro. Moreover, more NF-200-positive fibers were detected at the lesion site after SCI. Current studies have revealed that reactive astrogliosis limits the inflammatory response, and reducing astrocyte scar formation induced the widespread infiltration of inflammatory cells. 77 However, our data indicated that aFGF treatment did not induce infiltration of CD68+ (a marker of macrophages) cells but rather reduced the number of CD68+ cells. In summary, our results suggest that administration of aFGF ameliorated the barriers induced by reactive astrocytes and confined the inflammatory region,which can also support axonal regrowth beyond the glial scars. In addition, sustained local delivery of aFGF-HP via in situ injection locally and continuouslyreleased aFGF, which exerted better effects than the other tested administration routes of aFGF, suggesting that aFGF treatment via a slow-release carrier might be a more validmethod for providing nerve regeneration during the later stages of SCI. Previous studies have shown that aFGF plays vital neuroprotective and neuroregenerative roles through the PI3K/Akt and MAPK/ERK pathways; 13, 78-79 however, the detailed mechanism is still unclear. Our previous reports have suggested that the ER stress signaling pathway may play a direct role in inducing apoptosis in neuronal injury diseases.

80-81

Our study is the first to

demonstrate that ER stress-associated proteins, including GRP78, ATF-6, caspase 12, PDI, and CHOP, were obviously increased after SCI and were decreased after treatment with the aFGF-HP hydrogel in vivo. Our results showed that the aFGF-HP hydrogel treatment exerted neuroprotective and neuroregenerative effects and improved functional recovery after SCI in part


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by inhibiting ER stress-induced apoptosis.



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5. CONCLUSIONS A novel aFGF heparin-poloxamer thermosensitive hydrogel was shown to be the optimal formulation for loading and releasing aFGF versus other FGFs based on data in clinical trials. The aFGF-HP hydrogel, when administered via in situ injection, effectively enhanced the recovery of SCI. aFGF-HP exerted neuroprotective effects and created advantageous conditions for BSCB protection, neuroprotection, axonal regeneration, reactive astrogliosis suppression and functional restoration by inhibiting ER stress. In addition, the positive effects of aFGF-HP were better achieved by in situ administration to bypass the BSCB. The aFGF-HP hydrogel could facilitate and prolong aFGF delivery to the damaged spinal cord and maximize the effects of aFGF while minimizing the adverse effects (such as carcinogenicity or tumorigenicity). aFGF-HP, as a slow-release carrier, represents a more effective approach for neuroprotection and could be a clinically feasible therapeutic approach for patients suffering from SCI.


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ASSOCIATED CONTENT Supporting Information Cell culture and cell viability assay; Schematic of aFGF-HP hydrogel on different cells; detection for aFGF-HP hydrogel on TJ and AJ proteins in endothelial cells; survival rate and apoptosis detection for aFGF-HP hydrogelin vitro; detection for aFGF-HP hydrogel onaxonal length of PC12 cells; the effect of aFGF-HP hydrogel on attenuating the reactive astrogliosis in vitro.

ACKNOWLEDGMENTS This study was partly supported by a research grant from the National Natural Science Funding of China (81572237, 81572227, 81372112), Zhejiang Provincial Natural Science Foundation of China (LY14H170002), Zhejiang Science and Technology Project (2016C33107), Zhejiang Provincial Program for the Cultivation of High-level Innovative Health talents (to J. X.).



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FIGURE LEGENDS Figure 1. Characterization of aFGF-HP hydrogel. (A) Schematic of the preparation of aFGF-HP. (B) The gelation temperature of aFGF-HP with different concentration of HP. (C) The release profile of active aFGF fromaFGF-HP and aFGF-P hydrogel. (D) Storage (G’) and loss (G”) moduli of aFGF-Pand aFGF-HPhydrogelsas a function of temperature from 10 to 40°C. (E) Amplitude sweep of aFGF-Pand aFGF-HP hydrogels displaying storage modulus (G’) and loss modulus (G”). (F) SEM images of the lyophilized HP hydrogel and aFGF-HP hydrogel. (G The survival rate of PC12 cells with or without treatment of HP using PI/annexin V-FITC staining (H) Quantification results of the survival rate of PC12 cells from E. All experiments were performed in triplicate and data are presented as Mean ± SEM.

Figure 2. Schematic of aFGF-HP thermosensitive hydrogels enhance the recovery of spinal cord injury. The protection of aFGF-HP containing BSCB protection, neuroprotection, remyelination, attenuation of astrogliosis, axon elongation in three different stage after SCI, which are the main obstacles to recovery of spinal cord injury.

Figure 3. aFGF-HP attenuates BSCB disruptionat 24 h post-SCI. (A) Schematic of the structure of BSCB and the BSCB protection of aFGF-HP. (B) Representative images of whole spinal cords with Evan’s Blue dye staining at 24 h post-SCI. (C) Quantification intensity results of Evans Blue from A by software Image J. (D) Quantification data of EB content of spinal cord (µg/g). (E) Representative confocal images showing the fluorescence of Evans Blue Dye extravasation from the transverse area of the spinal cord of each group. Scale bar = 1000 µm. (F-H) Quantification of


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the fluorescence intensity of Evan’s Blue in each group at rostral 5 mm, caudal 5 mm, and lesion site. All data represent Mean values ± SEM, n = 4. #P < 0.05, ##P < 0.01 versus the SCI group, *P < 0.05, **P < 0.01 versus the aFGF-HP group.

Figure 4. aFGF-HP attenuates BSCB disruption by preventing loss of tight junction and adheren junction proteins at 24 h after SCI. (A) Immunofluorescence staining of tight junction protein Claudin5 (red) in each group. Scale bar = 10 µm. (B) Representative western blots result of adheren junction protein P120, tight junction protein tight junction protein. (C-D) Quantification of western blot data from B. All data represent Mean values ± SEM, n = 5. #P < 0.05, ##P < 0.01 versus the SCI group, *P < 0.05, **P < 0.01 versus the aFGF-HP group.

Figure 5. aFGF-HP reduces cell apoptosis at lesion site at 7 days after injury. (A) Immunofluorescence staining for TUNEL (green) of sections from the injured spinal cord in each group. Scale bar = 50µm. (B) Quantitative estimation of apoptotic and TUNEL cells from five independent sections between 5 mm from the injury epicenter. All data represent Mean values ± SEM, n = 5. #P < 0.05, ##P < 0.01 versus the SCI group, *P < 0.05, **P < 0.01 versus the aFGF-HP group.

Figure 6. aFGF-HP reduces neuron apoptosis at 7 days after SCI. (A) Immunofluorescence images show that C-caspase3 (green) co-localize in neuron (NeuN, red) in each group. Scale bar = 20 µm. (B) Protein expressions of C-caspase3 in the spinal cord segment at the contusion epicenter. GAPDH was used as the control and band density normalization. (C) The optical density analysis



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of C-caspase3 proteins. (D, a-f) Transmission electron microscopy images show the microstructure of the neurons in each group. Values were expressed as the mean ± SEM, n = 5 per group. #P < 0.05,

##

P < 0.01 versus the SCI group, *P < 0.05, **P < 0.01 versus the aFGF-HP

group.

Figure 7. aFGF-HP enhances neuron restoration at 7 days after SCI. (A) Protein expressions of GAP-43 and Nestin in each group. (B-C) Quantification of western blot data from B. (D-F) Immunohistochemisty staining and quantification data of GAP-43 and Nestin in each group. Scale bar = 50 µm. Values were expressed as the mean ± SEM, n= 5 per group. #P < 0.05, ##P < 0.01 versus the SCI group, *P < 0.05, **P < 0.01 versus the aFGF-HP group.

Figure 8. aFGF-HP decreases the damage of tissue structure and the loss of neurons at 28 days after SCI. (A) Representative images from HE, Nissl staining at 28 day post injury (dpi). (B-C) Quantification of the percent of preserved tissue at rostral 5 mm, caudal 5 mm of the spinal cord. (D-E) Counting analysis of VMN at rostral 5 mm, caudal 5 mm of the spinal cord. Values were expressed as the mean ± SEM, n = 5 per group. #P

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