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Sustained Local Release of NGF from a Chitosan-Sericin Composite Scaffold for Treating Chronic Nerve Compression Lei Zhang, Wen Yang, Kaixiong Tao, Yu Song, Hongjian Xie, Jian Wang, Xiaolin Li, Xiaoming Shuai, Jinbo Gao, Panpan Chang, Guobin Wang, Zheng Wang, and Lin Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14691 • Publication Date (Web): 29 Dec 2016 Downloaded from http://pubs.acs.org on December 30, 2016

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Sustained Local Release of NGF from a Chitosan-Sericin Composite Scaffold for Treating Chronic Nerve Compression Lei Zhang1,2,#, Wen Yang1, #, Kaixiong Tao3, #, Yu Song1, Hongjian Xie1, Jian Wang1, Xiaolin Li1, Xiaoming Shuai3, Jinbo Gao3, Panpan Chang1, Guobin Wang3,*, Zheng Wang1,3,*, Lin Wang1,2,*

1

Research Center for Tissue Engineering and Regenerative Medicine, Union Hospital, Tongji

Medical College, Huazhong University of Science and Technology, Wuhan, China 430022

2

Department of Clinical Laboratory, Union Hospital, Tongji Medical College, Huazhong

University of Science and Technology, Wuhan, China 430022

3

Department of Gastrointestinal Surgery, Union Hospital, Tongji Medical College, Huazhong

University of Science and Technology, Wuhan, China 430022

#These authors contribute equally to this work *Correspondence to: Guobin Wang, [email protected] Zheng Wang, [email protected] Lin Wang, [email protected]

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Abstract Chronic nerve compression (CNC), a common form of peripheral nerve injury, always leads to chronic peripheral nerve pain and dysfunction. Current available treatments for CNC are ineffective as they usually aim to alleviate symptoms at the acute phase with limited capability towards restoring injured nerve function. New approaches for effective recovery of CNC injury are highly desired. Here we report for the first time a tissue-engineered approach for the repair of CNC. A genipin cross-linked chitosan-sericin 3D scaffold for delivering nerve growth factor (NGF) was designed and fabricated. This scaffold combines the advantages of both chitosan and sericin, such as high porosity, adjustable mechanical properties and swelling ratios, the ability of supporting Schwann cells growth and improving nerve regeneration. The degradation products of the composite scaffold upregulate the mRNA levels of the genes important for facilitating nerve function recovery, including glial-derived neurotrophic factor (GDNF), early growth response 2 (EGR2) and neural cell adhesion molecule (NCAM) in Schwann cells, while downregulate two inflammatory genes’ mRNA levels in macrophages, tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β). Importantly, our tissue-engineered strategy achieves significant nerve functional recovery in a preclinical CNC animal model by decreasing neuralgia, improving nerve conduction

velocity

(NCV),

accelerating

microstructure

restoration

and

attenuating

gastrocnemius muscles dystrophy. Together, this work suggests a promising clinical alternative for treating chronic peripheral nerve compression injury.

Key Words: chronic nerve compression; tissue engineering approach; chitosan-sericin scaffold; NGF; functional restoration

2


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Introduction Chronic nerve compression (CNC) often results in peripheral nerve damage to individuals. 90% of all diagnosed compression neuropathies is carpal tunnel syndrome (CTS), a clinical form of CNC, affecting 4.9% to 7.1% of the worldwide population each year.1-2 CNC impairs the structure and microvasculature of peripheral nerves and causes myelin loss of axons at the nerve fascicle of periphery nerves.3

Currently, there is no effective clinical intervention for the treatment of CNC injury.4 Nonsurgical interventions, such as corticosteroid injection and mobilization interventions, are often applied for mild-to-moderate carpal tunnel syndrome5-6 focusing on alleviating symptoms at the acute phase, but are not capable of restoring nerve microstructure, which is however fundamentally important for nerve function recovery. Similarly, the surgical treatment where an operation is used to release the tunnel that causes nerve pressure,7 which does not yield satisfactory long-term results likely due to scar-tissue formation around or within the nerve,8-10 leading to nerve atrophy.11

Aiming to improve the restoration of nerve function and microstructure of compressed nerves, we hypothesize a well-designed, tissue-engineered scaffold delivering an appropriate cytokine to injured sites might achieve this goal. To this end, we proposed to generate a chitosan (CS) and sericin (SS) composite scaffold with the capability of delivering a neurotrophic factor. This design was based on two reasons: (1) chitosan and sericin, two natural biomaterials,12-13 have been demonstrated to possess unique advantages in peripheral nerve repair;14 (2) their limitations can be mutually compensated by their individually unique properties. Chitosan, a polysaccharide, can be readily acquired from chitin in shrimp shells, crabs, and lobsters by deacetylation.15-16 Owing to its good biodegradability, biocompatibility, antimicrobial activity and anti-adhesion ability, chitosan has been widely utilized in diverse biomedical fields, such as wound healing, drug delivery, biosensors and nerve regeneration.17-19 Recent studies reveal that degradation products of chitosan are able to stimulate proliferation of Schwann cells,20 accelerate 3



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differentiation of PC12 cells,21 and promote peripheral nerve regeneration.20 Sericin, one of the main components of silkworm silk, has been increasingly studied for tissue regeneration and regenerative medicine.22-23 Previous studies indicate that sericin has excellent biocompatibility, biodegradation and various bioactivities, including anticoagulation, oxidation resistance, enhancing cell adhesion and promoting cell proliferation.24 Our previous work demonstrated that sericin hydrogels effectively promote axon extension and prevent the death of neurons under hypoxia conditions.25 When sericin was fabricated into a genipin cross-linked sericin nerve guidance conduit, it promoted the functional regeneration of transected peripheral nerves.26

Despite chitosan’s neural favorable properties, pure chitosan has its limitations. Chitosan is brittle with limited mechanical endurance; it reportedly causes inflammatory responses.27-28 Interestingly, in these two aspects, sericin naturally possesses the advantages. Sericin has been used to improve mechanical properties of various other materials.24, 29 Although sericin was suggested to induce inflammation previously, later research revealed that this only occurs when sericin is associated with fibroin.30 Pure sericin itself does not elicit inflammation responses.30-31 Thus, given that both of chitosan and sericin have neurotrophic effects and their properties appear to be mutually compensatory, we hypothesize that a composite scaffold fabricated using these two types of biomaterials may combine the advantages of both components and enhanced nerve-regeneration.

Neurotrophic factors can effectively promote nerve tissue regeneration when utilized with tissue-engineered scaffolds. Nerve growth factor (NGF), a widely used neurotrophic factor, is capable of stimulating nerve fiber growth,32 promoting axonal branching and elongation,33 and providing neuroprotection.34-35 More importantly, NGF reportedly promotes the myelination and the restoration of nerve’s microstructure in peripheral nerve system.36 Thus, NGF may be an effective therapeutic factor for treating impaired nerves caused by CNC.

In this study, we have prepared a 3D crosslinked chitosan-sericin (CS-SS) composite scaffold as 4


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a NGF delivery vehicle for repairing CNC with a specific aim to promote functional and nerve microstructural recovery. Through the experimental screen and property comparisons, the ratio (v/v) of CS to SS, 1:1, was chosen as the most optimal formulation for scaffold fabrication and NGF delivery. Our designed scaffold was highly porous and biodegradable with good mechanical strength. The degradation products of this composite scaffold demonstrated good biocompatibility, neurotrophic function and anti-inflammation ability. More importantly, our in vivo data from a sciatic nerve compression rat model showed that the sustained local release of NGF from a CS-SS composite scaffold led to a functional recovery and the structural regeneration evidenced by the comprehensive analysis including mechanical and thermal algesia tests, electrophysiological and histological analysis. Thus, our data collectively demonstrate that this tissue-engineering based therapeutic strategy may have great potential of being translated into clinical treatment of CNC injury.

Materials and Methods

Chitosan-sericin Composite Scaffold Fabrication Procedure. Sericin was isolated from Bombyx mori, 140 Nd-s cocoons by LiBr extraction method as described in our previous work.26 Before dissolution, cocoon pieces were sterilized by 75% ethanol for 10 minutes and then washed with PBS. The final concentration of the sericin solution was 10% (w/v). Chitosan solution (1%, w/v) was prepared as follow: first, dissolved 0.5 g chitosan powder in 1% (v/v) acetic acid in a 50 ml plastic centrifuge tube and then placed the tube onto a shaker at 37°C to achieve complete dissolution. Secondly, adjusted pH value of chitosan solution to 5.90~6.00 using 4% sodium hydroxide. Finally, filtered the solution through a 0.22-µm filter. Then the chitosan and sericin solutions were mixed at five predetermined volume ratios (CS/SS), which were 5:0 (CS100/SS0), 4:1 (CS80/SS20), 1:1 (CS50/SS50), 1:4 (CS20/SS80), and 0:5 (CS0/SS100). For crosslinking, the volume ratio of each mixed CS/SS solutions to genipin solution (1%, w/v) was 3:1. After gelling in a mold, hydrogels were placed at -80°C and then put into a lyophilizer to obtain scaffolds. To fabricate the NGF-loaded CS-SS scaffolds, 200 ng NGF 5



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(10 µl) was added into 267 µl of the CS/SS/genipin mixed solution and the lyophilized scaffolds were obtained as described above. The NGF-loaded scaffolds were stored at -20°C.

Analysis of the Microstructures, Mechanical Properties and Secondary Structures of the CS-SS Scaffolds. To investigate the microstructures of the CS-SS scaffolds, specimens were prepared and analyzed by a scanning electron microscope (Zeiss, ULTRA PLUS-43-13, Germany) as previously described.26 The mean pore size was calculated from the representative images (90 random pores in total, 30 pores per sample, 3 samples per group) using Image-Pro Plus (version 6.0.0.260, Media Cybernetics, Inc., MD, USA). The compressive modulus of the CS-SS scaffolds was examined by a universal testing machine (Instron 5848 MicroTester, MA, USA) as described in our previous work (3 samples per group).26 The secondary structure of the CS-SS scaffolds was assessed by an Fourier transform infrared (FTIR) spectroscope (Nexus, Thermal Nicolet, USA) as previously described.26 The Dynamic Swelling and Porosity of the Scaffolds. The swelling behavior of the composite scaffolds was analyzed as previously reported.37 The dry weights of CS-SS scaffolds were measured as W1 (initial weight). The weights of the swollen scaffolds were measured as W2 (swollen weight). The swelling ratio of the CS-SS scaffold was calculated as below: Swelling ratio (%) =

W2 - W1 × 100% W1

The porosity of the composite scaffolds was measured using ethanol replacement method as previously described.38

Cell Lines and the Source of Different Cells. RSC96 Schwann cells, RAW264.7 cells and PC12 cells were all obtained from the Cell Bank, Type Culture Collection, Chinese Academy of Sciences, Shanghai, China.

Analysis of Cell Proliferation on Composite Scaffolds. The medium used for culturing 6


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RSC96 Schwann cells contained Dulbecco’s Modified Eagle’s Medium (DMEM, HyClone, UT, USA), 10% fetal bovine serum (FBS; Gibco, CA, USA) and 1% penicillin-streptomycin (HyClone, UT, USA). The scaffolds were coated on a 96-well plate. Before being used for cell culture, the 96-well plate was embedded in 75% ethanol for 30 minutes and washed with sterilized PBS for three times. RSC96 Schwann cells were seeded in the 96-well plate (2,000 cells/well) and then incubated in a humidified incubator (37°C, 5% CO2). The proliferation of RSC96 Schwann cells was analyzed at indicated time points (Day 0, 1, 3, 5, 7 and 9) by CCK-8 assay as previously described.39

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). Total cellular RNA was extracted from indicated cells and complementary DNA was obtained by RNA reverse transcription. The qRT-PCR was performed and the relative levels of gene expression were obtained as previously reported.31 Detailed information of the primers was shown in Table S1.

Preparation of the Degradation Products of Composite Scaffolds. Different composite scaffolds were immersed in 10 ml PBS in 15 ml centrifuge tubes. Then the tubes were placed on a shaker (200 rpm) at 37°C until the scaffolds were completely degraded. The solutions were collected and lyophilized to obtain the powder of degradation products. The powder was weighed and dissolved in PBS making the concentration of 10 g/l. The degradation products solutions were sterilized by filtering through a 0.22-µm filter. All the sterilized solutions were stored at room temperature before use.

RSC96 Schwann Cells, RAW264.7 Cells and PC12 Cells Culture with the Degradation Products of Composite Scaffolds. For qRT-PCR test, cells were seeded in 6-well plates (100,000 cells/well for RSC96 and 50,000 cells/well for RAW264.7) in DMEM containing 0.1% FBS. 24 hours later, the original medium was discarded and 1.5 ml DMEM containing 10% FBS was added. Then 500 µl sterilized solution of degradation products or PBS (as the negative control) was added, making the final concentration of degradation products 2.5 g/l. 24 hours later, 7



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the cells were collected. The relative mRNA levels of GDNF, EGR2, NCAM, BDNF, NGF, NT-4, NRG1, TNF-α and IL-1β in these cells were analyzed using qRT-PCR. To analyze proliferation of RSC96 Schwann cells, cells were seeded in 48-well plates (5,000 cells/well) in DMEM containing 0.1% FBS. 24 hours later, the original medium was discarded and 200 µl DMEM containing 1% FBS was added. Then 100 µl sterilized solution of degradation products or PBS (as the negative control) was added, making the final concentration of degradation products 3.33 g/l. The proliferation of Schwann cells was measured at different time points (Day 0, 1, 3, 5 and 7) using MTT assay as previously described.40 The culture medium was renewed every other day with fresh medium supplemented with the solution of degradation products or PBS. For cell differentiation assessment, PC12 cells were cultured in 24-well plates (6,000 cells/well) in F12 Ham Kaighn’s Modification Medium (F12K, HyClone, UT, USA) containing 10% horse serum (HS; Gibco, CA, USA) and 5% FBS. 24 hours later, the original medium was discarded and then 300 µl differentiation medium (F12K containing 5% FBS and 50 ng/ml NGF) was added. Next, 200 µl sterilized solution of degradation products or PBS (as the negative control) was added. The final concentration of degradation products was 4.0 g/l. 24 hours later, cells were examined and photographed in an inverted phase contrast microscope (Olympus IX71, Japan).

In Vitro NGF Release. The composite scaffold (cylinder shape, with the diameter of 1.0 cm and the height of 0.4 cm) delivering 200 ng NGF was incubated in 1.0 ml PBS at 37°C. PBS was collected and replaced daily from Day 1 to Day 40. The collected PBS was stored at -20°C for later tests. The daily release amount of NGF from the composite scaffold was analyzed using enzyme-linked immunoabsorbent assay (ELISA) with an ELISA kit (Wuhan Boster Biological Technology, Ltd., Wuhan, China). The bioactivity of NGF released from the composite scaffolds was assessed by examining its effect on neurite outgrowth of PC12 cells. Undifferentiated cells were cultured in the 24-well plates (6,000 cells/well) in regular culture medium (F12K, 5% FBS, 10% HS). The solution 8


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collected at Day 1 was lyophilized and made up into a solution of 10 ng/ml using regular culture medium. Prior to being used for cell culture, the sterile medium was prepared by filtration sterilization. The cells were observed and photographed at Day 0 and Day 7 after treatment. The rate of neurite-bearing cells was calculated as previously described.19

Surgical Technique. The animal model of chronic sciatic nerve compression was established as previously described.41 A silastic tube was used to compress the right sciatic nerve. The left sciatic nerve (Sham group) received the same operation as did the right side but without compression or treatment. After 4 weeks, operations were performed to remove the silastic tube. The four experimental groups were: Blank control group (only removing the silastic tube), Scaffold group (after the removal of the silastic tube, a CS50/SS50 scaffold was placed around the nerve), NGF group (after the removal of the silastic tube, a solution containing 200 ng NGF (100 µl) was injected to the nerve directly), NGF/scaffold group (after the removal of the silastic tube, a CS50/SS50 scaffold delivering 200 ng NGF was placed around the nerve). There were 16 rats in each group. All the rats were randomized into the indicated experimental groups according to the result of nerve conduction studies (see below) at Week 4 after compression. The outliers of the nerve conduction studies were excluded. All in vivo experiments performed in this study were approved by the Institutional Animal Care and Use Committee, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.

Thermal Withdrawal Latency Test. To test thermal withdrawal latency, an infrared heat stimulus device (UGO BASILE Plantar Test 37370, Comerio, VA, Italy) was utilized as previously described.42 The test was performed at Week 2 and Week 4 after treatment. The time (seconds) from the beginning of stimulation to the moment that rats lifted their feet was recorded. The mean value of thermal withdrawal latency per limb was calculated by averaging the results from 5 tests.

Mechanical Withdrawal Threshold Test. The mechanical withdrawal threshold test was 9



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done using Von Frey hairs (North Coast Medical, Inc., CA, USA), and the threshold was measured by the up-down method as previously described.43 The test was performed at Week 2 and Week 4 after treatment. The mean value of pressure threshold per limb was calculated by averaging the results of 3 tests.

Nerve Conduction Study. The nerve conduction velocity (NCV) was recorded using an electromyography recorder (CareFusion, CA, USA) as previously reported.26 The assessment of nerve conduction was performed 3 times: Week 4 after compression, Week 2 and Week 4 after treatment.

Histological Examination. The sciatic nerve was isolated at Week 2 and Week 4 after treatment. Then the nerve samples were fixed in 4% paraformaldehyde. To visualize myelin sheath, cross-sections of the harvested sciatic nerves were stained with Luxol fast blue (LFB) as previously reported.44 The immunofluorescence staining was done as previously described.26 The detailed information of antibodies was shown as follow: the mouse anti-MBP antibody (1: 400 diluted; R&D systems, Inc., MN, USA) was used to identify myelin sheath; the rabbit anti-β3-tubulin antibody (1: 400 diluted; R&D systems, Inc., MN, USA) was used to identify axons. The secondary antibodies were Cy3-conjugated Affinipure donkey anti-mouse IgG (1: 500 diluted; Jackson ImmunoResearch, Inc., PA, USA) and FITC-labeled goat anti-rabbit IgG (1: 500 diluted; Wuhan Boster Biological Technology, Ltd., Wuhan, China). Cell nuclei were stained with DAPI (Wuhan Boster Biological Technology, Ltd., Wuhan, China). The image capturing of stained cross-sections and the quantification of β3-tubulin positive areas were done as previously reported.26 The relative density of myelinated nerve fibers was calculated by dividing the counts of myelin sheaths by the indicated area (104 µm2) (6 randomly selected images per animal, 6-8 animals per group).

Transmission Electron Microscopy. A transmission electron microscope (H7650, Hitachi, Tokyo, Japan) was utilized to view myelin sheath. The preparation of ultrathin cross-sections of 10


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sciatic nerves, image capturing procedure and the quantification of myelin sheath thickness were done as previously described.26

Weighing Gastrocnemius Muscles and Masson Trichrome Staining. The operational and contralateral side gastrocnemius muscles were isolated at Week 2 and Week 4 after treatment. The quantification of relative wet weight of gastrocnemius muscles, Masson trichrome staining procedure, image capturing procedure and quantification of diameters of the muscle fibers were all performed as previously described.26 (6 randomly selected images for each animal, 6 animals per group)

Statistical Analysis. Data were analyzed by the SPSS software (SPSS Inc.) using one-way ANOVA analysis with Turkey post-test. ANOVA p < 0.05 indicates statistical significance. Data were presented as means ± SD or means ± SEM.

Results

Characterizaitons of the CS-SS Crosslinked Scaffolds CS-SS hydrogels were fabricated (Fig. 1a) through crosslinking the mixed chitosan and sericin via genipin. The dark blue appearance of the hydrogels was due to genipin reacting with primary amino groups of chitosan and sericin.25, 45 Five different mixing ratios (v/v) of CS to SS, 5:0 (CS100/SS0), 4:1 (CS80/SS20), 1:1 (CS50/SS50), 1:4 (CS20/SS80) and 0:5 (CS0/SS100), were tested. Lyophilized scaffolds (Fig. 1b) were highly porous with the porosity reaching 80% (Fig. 1c) (Table 1). The pore sizes of the pure CS scaffolds and the pure SS ones were approximately 150 µm, slightly larger than the other composite scaffolds with different CS-to-SS ratios (Table 1) ranging from 130 to 146 µm. Such a pore size scale was suitable for the growth of nerve cells, as the size of 50-160 µm could effectively provide neighboring nerve cells with oxygen and nutrients.46

11



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The mechanical assessment revealed that the amount of sericin in the composite scaffold was positively correlated with the compressive modulus ranging from 25.4 kPa (CS100/SS0) to 47.8 kPa (CS0/SS100) (Table 1), suggesting that a convenient way of adjusting mechanical strength by altering CS-to-SS ratios. The secondary structural transformation of different CS-SS scaffolds was examined by FTIR spectroscopy (Fig. 1d). The sericin powder showed its three characteristic absorption peaks at 1600-1690 cm−1, 1480-1575 cm−1 and 1229-1301 cm−1, which were assigned to amide I, amide II and amide III groups, respectively.47 Chitosan powder displayed a characteristic structure of saccharine at the peak of roughly 1400 cm−1 and an amino characteristic peak at 1591 cm−1.48 As sericin content decreased concomitantly with chitosan content increasing in the composite scaffolds, the characteristic absorption peaks of sericin gradually disappeared with the characteristic absorption peaks of chitosan progressively appearing (Fig. 1d), indicating a successful fabrication of CS-SS composite scaffolds. Swelling property reflects the water absorbing ability of a scaffold, which might affect the diffusion of encapsulated bioactive agents.24 We next examined the swelling properties of the CS-SS composite scaffolds. The equilibrium swelling percentage decreased with the content of sericin increasing (Fig. 1e), indicating a content-based, adjustable swelling capability.

12


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Figure 1. Characterizations of the chitosan-sericin hydrogels and scaffolds. a and b) Photograph of the chitosan-sericin hydrogels (a) and the corresponding scaffolds (b) crosslinked by genipin at different chitosan-to-sericin ratios (v/v). Scale bar, 1 cm. c) SEM images of the chitosan-sericin scaffolds with porous microstructure. Scale bar, 500 µm. d) Fourier transform infrared spectra of the chitosan-sericin scaffolds. FTIR analysis show the absorption peaks of the pure chitosan scaffold (CS100/SS0, black line), the three chitosan-to-sericin scaffolds (CS80/SS20, red line; CS50/SS50, green line; CS20/SS80 blue line), and the pure sericin scaffold (CS0/SS100, cyan line). e) Swelling kinetics of the five chitosan-sericin scaffolds in PBS with pH 7.4 at 37oC (3 samples for each analysis, data presented as mean ± SD). 13



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Table 1. Pore size, porosity and compressive modulus of different CS-SS scaffolds CS-SS

CS100/SS0

CS80/SS20

CS50/SS50

CS20/SS80

CS0/SS100

Pore sizea(µm)

157.7±43.5

130.6±43.2

146.3±30.8

136.4±40.2

152.0±37.1

Porosityb(%)

83.3±1.0

84.1±1.1

85.3±1.1

82.5±1.4

90.7±1.0

25.4±1.9

31.0±1.1

34.7±2.2

36.0±0.8

47.8±2.3

scaffolds

Compressive modulusc(kPa) a

The pore size was averaged from 30 random pores per sample, 3 samples in total.

b

The ethanol replacement method was used for calculating the porosity.

c

The uniaxial compression test was used for examining the compressive modulus.

Proliferation of Schwann Cells on CS-SS Scaffolds Schwann cells are critical in the functional recovery of chronically compressed nerves. For instance, Schwann cells promote the formation of myelin sheaths in peripheral system.49-50 We determined whether the scaffolds affected Schwann cells’ behavior by seeding Schwann cells on the CS-SS scaffolds. RSC96 Schwann cells were capable of proliferating on the five scaffolds for over 9 days (Fig. 2a), suggesting scaffolds’ low cytotoxicity. Among the composite scaffolds, CS50/SS50 showed the most significant cell-growth promoting effect (p < 0.05) (Fig. 2a) likely due to the combined efficacy derived from chitosan’s proliferation stimulating effect20 and sericin’s known cell adhesion capability.26, 51

Effects of Scaffolds’ Degradation Products on Schwann Cells, Macrophages and Pheochromocytoma Cells

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The degradation products of CS-SS scaffolds are a primary source of foreign molecules at implantation sites, which might affect biological behaviors of cells within that microenvironment. Considering that chitosan can promote Schwann cell proliferation,20 accelerate differentiation of PC12 cells48 but cause inflammatory responses,28 we set to determine the effects of the degradation products of these composite scaffolds on the cells types involved in CNC, including Schwann cells (RSC96), macrophages (Raw264.7) and pheochromocytoma cells (PC12). We first analyzed the influence of CS-SS scaffolds’ degradation products on the repair related genes. With sericin content increasing within the scaffolds, the treatment using the degradation products of the five scaffolds gradually upregulated the mRNA levels of glial cell line-derived neurotrophic factor (GDNF), early growth response 2 (EGR2) and neural cell adhesion molecule (NCAM) (Fig. 2b-2d), which are crucial for promoting peripheral nerve regeneration,52 helping Schwann cells to enter into the myelinating stage,53 and participating in early initiation of myelination by mediating interactions between axons and glial cells.54 Further, the mRNA levels of other neuronal factors, including brain-derived neurotrophic factor (BDNF), NGF, neurotrophin-4 (NT-4), and neuregulin-1 (NRG1), remained largely unchanged (Fig. S1). Of note, the upregulation that the degradation products of the CS50/SS50 could lead to for the three genes (GDNF, EGR2 and NCAM) was less than 50% (ranging from 30% to 40%) of the levels that the degradation products of CS0/SS100 could reach, suggesting that CS’s degradation products might slightly suppress the gene-upregulating effect of SS’s degradation products. These results indicate that the addition of sericin confers the scaffolds with the ability of promoting the transcription of neurotrophic factors and myelination positive regulators, consistent with the previous study showing that sericin protein and pure sericin hydrogel’s degradation products had neurotrophic and neuroprotective effects on primary cortical neurons.25 In response to peripheral nerve compression, infiltrating macrophages contribute significantly to inflammatory responses.55 Cytokines secreted from macrophages, such as tumor necrosis factor alpha (TNF-α) and interleukine-1 beta (IL-1β), play key roles in developing and maintaining pain in peripheral nerve compression.56 We next examined whether degradation products of the scaffolds might influence the expression of TNF-α and IL-1β in RAW264.7 cells. Compared to the pure CS scaffold, the degradation products of the sericin-containing scaffolds downregulated TNF-α and IL-1β mRNA levels with the pure SS scaffold exhibiting the most significant suppressive effects (p < 0.05) (Fig. 2e, 2f), suggesting that the addition of sericin provides the 15



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ability of reducing the transcription of inflammatory cytokines, which was similarly observed in the previous study where sericin protein suppressed the production of pro-inflammatory cytokines (TNF-a and IL-18) in infarcted mouse hearts.31 We then evaluated whether scaffolds’ degradation products inherited the proliferation-promoting ability of CS-SS scaffolds in Schwann cells. The degradation products of the five scaffolds supported Schwann cell proliferation during 7-day culture (Fig. S2a). The cells treated with the degradation products of the scaffolds containing the increasing amount of chitosan (CS50/SS50, CS80/SS20, CS100/SS0) exhibited progressively enhanced proliferative activity (Fig. 2g), suggesting that chitosan’s proliferation promoting activity appears to be more potent than sericin. Notably, the growth-promoting effect of the degradation products of the CS50/SS50 scaffold was approximately 50% of that of the CS100/SS0 scaffold, suggesting that SS’s degradation products do not interfere the capability of CS’s degradation products in promoting cell proliferation. Next, we studied the effect of the degradation products of the five scaffolds on rat PC12 cells (pheochromocytoma cells) differentiation induced by NGF. The images showing the morphology of PC12 cells were obtained from phase-contrast microscopy (Fig. S2b). The cells with the membrane processes whose length was equal or longer than the cell body were considered differentiated. After treating PC12 cells with degradation products, the differentiation rate in each treatment group was all over 90%, which was higher than that in the negative control group (Fig. 2h). These data suggest that all the degradation products effectively promote PC12 cell differentiation.

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Figure 2. All of the five scaffolds support Schwann cells proliferation and the effects of scaffold’s degradation products on RSC96 Schwann cells, RAW264.7 macrophages or PC12 pheochromocytoma cells. a) RSC96 Schwann cells were cultured on the surfaces of the five chitosan-sericin scaffolds. The cell proliferation was quantified by CCK-8 assay during the 17



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period of 9-day culture. b, c, d) The relative mRNA levels of glial cell derived neurotrophic factor (GDNF) (b), early growth response 2 (EGR2) (c) and neural cell adhesion molecule (NCAM) (d) of RSC96 Schwann cells were examined 24 hours after the cells seeded on 6-well plates were treated with PBS or degradation products of chitosan-sericin scaffolds. e, f) The relative mRNA levels of tumor necrosis factor alpha (TNF-α) (e) and interleukine-1 beta (IL-1β) (f) of RAW264.7 cells were examined 24 hours after the cell seeded on 6-well plates were treated with PBS, degradation products of chitosan-sericin scaffolds or 10 g/l LPS. g) The proliferation of RSC96 Schwann cells after being treating with PBS or degradation products of chitosan-sericin scaffolds was assessed by MTT assay. h) Differentiation rate of PC12 cells 24 hours after being treating with PBS or degradation products of chitosan-sericin scaffolds. The cells treated with PBS were negative control (N.C.). Data were presented as mean ± SD, 3 independent experiments for each analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001; One-way ANOVA analysis.

Evaluation of NGF Release Profile from CS-SS Scaffolds The amount of NGF released from the scaffolds with the five different chitosan-to-sericin ratios was analyzed. No significant differences in release kinetics of NGF were detected. The 90% of NGF was rapidly released during the first 8 days and completely released within 40 days (Fig. 3a and 3b). These results indicate that NGF delivered by the scaffolds has stable, sustained release kinetics. Rat PC12 cells were used for testing the bioactivity of NGF released from the scaffolds. The medium containing released NGF promoted the neurite outgrowth from PC12 cells (Fig. 3c) much more significantly than the control group (p < 0.01) during a 7-day culture (Fig. 3d), indicating that NGF released from CS-SS scaffolds remains functionally active.

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Figure 3. Characterizations of NGF release and assessment of NGF’s bioactivity in vitro. a) The daily release rates of NGF from the chitosan-sericin scaffolds in PBS (pH=7.4) at 37oC over 40 days. b) The cumulative release of NGF from the chitosan-sericin scaffolds. (Each scaffold was loaded with 200 ng NGF). c) Phase contrast micrographs of PC12 cells that were cultured in 48-well platesand treated with PBS or NGF released from chitosan-sericin scaffolds at Day 0 and Day 7. I) The cells were maintained in the medium without NGF. II-IV) The cells were maintained in the medium containing NGF (10 ng/ml) released from five scaffolds: II) CS100/SS0, III ) CS80/SS20, IV ) CS50/SS50, V ) CS20/SS80, VI ) CS0/SS100. Scale bar, 50 µm. (See “Materials and Methods” for details). d) Quantification of the percentage of neurite-bearing PC12 cells in c) at Day 7. The percentage is calculated by counting 100 cells per group in randomly captured photographs. The cells treated with PBS were the negative control (N.C.). **, p < 0.01; Data were presented as mean ± SD, 3 samples for each analysis. One-way ANOVA analysis.

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The Composite Scaffold with 1:1 Chitosan-to-sericin Possesses Optimal Neurotropic Properties The above results together revealed that CS50/SS50 composite scaffolds’ degradation products effectively retained the two biomaterials’ advantages and exhibited the most potent regenerative capability among all the tested scaffolds (Table 2). Given the better performance of the CS50/SS50 scaffold than the other scaffolds in the aforementioned in vitro studies, we chose the CS50/SS50 scaffold as the NGF delivery vehicle for the rest of study in a preclinical chronic sciatic nerve compression rat model. Table 2. The assessment of neuronal-favorable molecular and cell biological impacts across the degradation products of the five scaffolds Group Up-regulate GDNF in RSC96 cellsa

CS100/SS0

CS80/SS20

CS50/SS50

CS20/SS80

CS0/SS100

×

√

√

√

√

Up-regulate EGR2 in RSC96 cellsa

√

√

√

√

√

Up-regulate NCAM in RSC96 cellsa

√

√

√

√

√

×

×

√

×

√

×

√

√

√

√

√

√

√

×

×

Downregulate TNF-α in RAW264.7 cellsb Downregulate IL-1β in RAW264.7 cellsb Promote proliferation of RSC96 cellsa

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Promote √ √ √ √ √ differentiation a of PC12 cells √ indicates a significant difference, × indicates no significant difference. a Each group was compared to N.C. group (The cells treated with PBS were N.C. (negative control)). b Each group was compared to LPS group (The cells treated with LPS were the positive control).

Functional Restoration of the Chronically Compressed Sciatic Nerve To evaluate the composite scaffold’s therapeutic efficacy in vivo, we used the CS50/SS50 scaffold to deliver NGF (NGF/scaffold group) to treat chronically compressed nerves in a rat model (Fig. 4a). The round flake-like scaffolds were folded and adhered to the injured nerve after decompression. The degradation of the scaffold could be visually detected 2 weeks after transplantation, became obvious 2 more weeks later and was almost completed 8 weeks after transplantation (Fig. 4b). As chronic nerve compression causes constant mechanical and thermal algesia, we used mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) as two indicators to evaluate the functional recovery in rats. A high MWT and TWL indicates low mechanical algesia and thermal algesia, respectively, reflecting a good functional recovery.43, 57 No differences were detected across all the four treatment groups at Week 2. However, at Week 4, the MWT and TWL in the NGF/scaffold group were comparable to those in the sham group (p > 0.05) and significantly higher than the scaffold only and NGF only groups (p < 0.001) (Fig. 4c, 4d), indicating that the sustained local release of NGF from the CS50/SS50 scaffold effectively restores mechanical and heat sensitivity, indicating a nerve functional recovery. Chronic nerve compression often results in a gradual decrease in nerve conduction velocity (NCV).58 We used NCV as another important indicator for the function recovery of injured nerves.41 There were no differences across all the treatment groups at Week 2. In contrast, at Week 4 the NCV of the NGF/scaffold group was comparable to that of the sham group (p > 0.05) and significantly faster than that of the other three treatment groups (p < 0.05) (Fig. 4e). These results indicate that CS50/SS50 scaffold releasing NGF restores nerve conductivity. 21



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Figure 4. Surgery images of the different groups, degradation study of the chitosan-sericin scaffolds in vivo, and functional assessment of the sciatic nerve. a) The upper panel is the graphical representation of the CNC model generation and the relevant treatments. The lower panel shows the corresponding surgical photographs of the upper ones. A 1-cm length of silastic tube was used to wrap the right sciatic nerve to provide chronic compression (Tube on compressed nerve). After the tube was removed, the compressed nerves received no treatment (Blank control), the scaffold (CS50/SS50) only treatment (Scaffold), the NGF only treatment (NGF), and the NGF-loaded scaffold treatment (NGF/scaffold), respectively. Scale bar, 1cm. b) The degradable chitosan-sericin scaffolds in vivo were observed at Week 2, Week 4 and Week 8 after the implantation. Scale bar, 1cm. c) Hindpaw thermal withdrawal latency was determined 22


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using plantar test at Week 2 and 4 after treatment. d) Hind limb mechanical withdrawal thresholds were determined using the von Frey behavioral test at Week 2 and 4 after treatment. e) Quantification of nerve conduction velocity (NCV) in the corresponding groups at Week 2 and 4 after treatment. (Sham, left sciatic nerve receiving sham operation; n = 6-8 per group.) *, p < 0.05; **, p < 0.01; ***, p < 0.001; N.S., not significant; Data were presented as mean ± SEM. One-way ANOVA analysis. Histological Restoration of Nerve Fibers We next evaluated the histological recovery of the compressed nerve. The middle portions of the sciatic nerves of the animals receiving treatment were cross-sectioned and stained for myelin basic protein (MBP) and β3-tubulin to visualize myelin sheath and axons, respectively (Fig. 5a). In the study of immunofluorescence staining, the density of myelinated nerve fibers and axons in NGF/scaffold group was significantly higher in comparison to the other treatment groups at Week 2 and Week 4 (p < 0.001) (Fig. 5b and 5c). These results indicate that the CS50/SS50 scaffold releasing NGF effectively improves the morphological and microstructural restoration of the compressed nerve. Luxol fast blue (LFB) staining was also used for visualizing myelin sheaths (Fig. S3a). The density of myelinated nerve fibers determined by LFB staining across the different treatments was consistent with that determined by MBP staining (Fig. S3b). To further assess the regeneration of injured nerves, TEM was used to determine the myelin sheath thickness of axons. The thickness of myelin sheath in NGF/scaffold group was similar to that of the sham group (p > 0.05) (Fig. 5d, 5e), significantly higher than the other treatment groups at Week 4 (p < 0.01) (Fig. 5d, 5e). These results demonstrate that the scaffold releasing NGF effectively restores the key microstructure of the compressed sciatic nerve, which may account for the observed nerve functional recovery, such as improved NCV.

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Figure 5. Histological and microstructure evaluation of the sciatic nerves. a) The representative images show anti-MBP staining (red), anti-β3-tubulin staining (green) and DAPI staining (blue) for the cross-sections of the middle regions of the sciatic nerves from the indicated treatment 24


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groups at Week 2 and Week 4 after treatment. Scale bar, 50 µm. b) Quantification of the number of the myelinated nerve fibers per 104 µm2 in the indicated groups at Week 2 and 4 after treatment (6 randomly selected images in each animal, n = 6 animals per group). c) Quantification of β3-tubulin-positive area per 100 µm2 in the five groups as indicated at Week 2 and 4 after treatment (6 randomly selected images in each animal, n = 6 animals per group). d) TEM images of the cross-sections of the middle regions of the sciatic nerves from the sham group and the other four treatment groups. The images in the lower panel are enlarged areas boxed (white dotted-line) in the upper panel. The myelin sheaths in corresponding groups are shown by yellow arrowheads. Scale bar, 5 µm. e) Quantification of the myelin sheath thickness in the five groups as indicated (100 axons randomly selected in each group). *, p < 0.05; **, p < 0.01; ***, p < 0.001; N.S., not significant; Data were presented as mean ± SEM. One-way ANOVA analysis.

Analyses on Gastrocnemius Muscle Chronic sciatic nerve compression induces the atrophy of gastrocnemius muscle, one of the target organs of sciatic nerves. This atrophy was often manifested as a reduction in muscle weight and the size of muscle fibers.59 NGF/scaffold group had a higher wet weight of gastrocnemius muscle (Fig. 6a) and a larger diameter of the muscle fibers (Fig. 6b) in comparison to the other treatment groups at Week 2 and Week 4 (p < 0.05), demonstrating that our tissue-engineered approach suppresses the atrophy of gastrocnemius muscles, suggesting a functional recovery of the sciatic nerve.

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Figure 6. Analyses on morphology and microstructure of gastrocnemius muscles. a) The representative images show the morphology of gastrocnemius muscles in the corresponding groups at Week 2 and 4 after the four different treatments. Scale bar, 1 cm. The relative wet weight of gastrocnemius muscle in the experimental side is presented as percentage of the contralateral side (n = 6-8 per group). b) The representative Masson trichrome staining shows the microstructure of gastrocnemius muscles in the five groups as indicated. Scale bar, 20 µm. The diameters of muscle fibers were quantified (6 randomly selected images in each animal, n = 6 animals per group). *, p < 0.05; **, p < 0.01; ***, p < 0.001; N.S., not significant; Data were presented as mean ± SEM. One-way ANOVA analysis. 26


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Discussion The traditional treatments for CNC, such as surgical decompression2, 7 and local corticosteroid injection,5-6 were not tailored towards nerve microstructural restoration, which may partially account for the poor functional recovery and unsatisfying longer-term outcome of CNC.11 Aiming at improving the function of compressed nerves, we proposed a strategy based on a chitosan-sericin (CS-SS) composite scaffold designed to release NGF for treating CNC. The CS-SS composite scaffold combined the advantages of both biomaterials with their disadvantages mutually compensated. This is the first time that these two biomaterials are used to fabricate a 3D composite scaffold with genipin as the cross-linker and utilized in vivo for chronic nerve compression treatment.

A tissue-engineered scaffold suitable for peripheral nerve injury should have proper porous structure, compressive modulus, swelling ratios and biodegradability and facilitate functional regeneration.60 We selected the CS50/SS50 scaffold for in vivo experiments because CS50/SS50 were tested to be the optimal formulation for the repair of CNS injury (Table 2). CS50/SS50 scaffold possessed excellent physical and mechanical properties that benefits nerve repair. Its porosity was over 80%, which was reportedly to promote the exchange of nutrition and metabolism during nerve function recovery.61 Secondly, CS50/SS50 scaffold’s mechanical strength, 34.7 kPa, was lower than the native peripheral nerve tissue, 11.7 MPa,60 which would not cause physical compression on nerves. Similarly, CS50/SS50 scaffold’s rapid swelling property could help avoid the secondary nerve compression resulting from persistent swelling. Moreover, the scaffold’s biodegradability spared the need of surgical retrieval of the implants, thus avoiding secondary surgical injury that may cause additional pain to the patients.

In addition, the biocompatibility of the scaffolds and their degradation products had significant impact on nerve function recovery. Our in vitro experiments showed that Schwann cells adhered and proliferated on the CS-SS scaffolds. Given that Schwann cells promote myelination in chronic nerve compression and participate in development and functional recovery of peripheral 27



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nerves,41,

49-50

this interaction between the scaffolds and Schwann cells would benefit the

recovery of compressed nerves. Since cell-based peripheral nerve system regeneration has become a recent research focus,62 the CS-SS scaffolds bearing a biocompatible, functional interface with Schwann cells might make them a promising cell delivery vehicle for Schwann-cell based CNC repair. Inflammatory responses during nerve injury caused by CNC56 can inhibit the repair and regeneration of peripheral nerves.63 Interestingly, our data indicate that the degradation products of the CS-SS composite scaffolds downregulated the mRNA levels of inflammatory factors (TNF-α and IL-1β) in macrophages, which might reduce inflammatory and pain responses, providing a microenvironment favorable for in vivo nerve repair. Moreover, the severe infection or tissue-adhesion around the nerve was not observed during repair, which might be associated with chitosan’s antimicrobial and anti-postoperative-adhesion effects.17-18

The traditional therapeutic drugs used in the management of CNC were corticosteroids or nonsteroidal anti-inflammatory drug (NSAID).5 These drugs can relieve symptoms, but their long-term benefits remain questionable.64-65 Surgical treatment largely aims to reduce nerve compression with no additional intervention for preventing subsequent nerve atrophy. Thus, in our regimen, NGF, a cytokine with neurotrophic effect,32-35 was included to suppress nerve atrophy through NGF activating Schwann cells to form myelin sheath.36 Supporting this, our data on the functional indicators, MWT, TWL and NCV, collectively provide strong evidence showing that NGF inclusion in the treatment strategy significantly improved functional recovery. The atrophy and function impairment of gastrocnemius muscle, a sciatic nerve-controlled muscle, was suppressed. Consistently, the nerve fiber microstructure recovery, a basis for functional recovery, was also significant in NGF/scaffold group. The number and thickness of myelinated nerve fibers as well as axons increased drastically. This improvement was in part due to a sustained, local release of NGF from the composite scaffold (NGF’s short half-life precludes the option of directly injecting NGF to injury sites).66-67 Taken together, the functional, morphological and microstructural improvements in the animals receiving the NGF-loaded scaffold treatment demonstrate the potential of this tissue-engineering strategy in treating CNC 28


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injury.

Conclusion We for the first time empolyed chitosan and sericin to fabricate a 3D composite scaffold using genipin as a crosslinker. This composite scaffold possesses the advantages of both chitosan and sericin, including high porosity, adjustable mechanical properties and swelling ratios, and the ability of supporting the growth of Schwann cells. The degradation products of the composite scaffold also have various excellent properties, such as promoting the proliferation of Schwann cells and inducing PC12 cells differentiation, up-regulating the mRNA levels of GDNF, EGR2, NCAM, down-regulating the mRNA levels of some inflammatory genes. In addition, we applied this tissue-engineered scaffold with NGF encapsulated for the treatment of CNC. This novel therapeutic strategy achieved significant functional and histological recovery when compared to that of traditional surgical decompression, suggesting a promising alternative for clinically treating peripheral nerve compression.

ASSOCIATED CONTENT Supporting Information: quantitative real-time PCR analyses on RSC96 Schwann cells; phase contrast photographs of RSC96 Schwann cells and PC12 cells treated with degradation products of scaffolds; luxol fast blue (LFB) staining of the compressed sciatic nerve; qRT-PCR oligonucleotide primers.

AUTHOR INFORMATION Corresponding Authors E-mail: [email protected] Email: [email protected] Email: [email protected] 29



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Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS The work is supported by the National Natural Science Foundation of China Programs 81272559, 81402875 and 81671904, the International Science and Technology Corporation Program of Chinese Ministry of Science and Technology S2014ZR0340, the Science and Technology Program of Chinese Ministry of Education 113044A, the Frontier Exploration Program of Huazhong University of Science and Technology 2015TS153, the Natural Science Foundation Program of Hubei Province 2015CFA049, and the Hubei Province “Hundreds of Talents” Program.

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Graphic Abstract

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Sustained Local Release of NGF from a Chitosan ... - ACS Publications

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