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Tissue Engineering and Regenerative Medicine

Sericin Nerve Guidance Conduit Delivering Therapeutically Repurposed Clobetasol for Functional and Structural Regeneration of Transected Peripheral Nerves Lei Zhang, Wen Yang, Hongjian Xie, Hui Wang, Jian Wang, Qiangfei Su, Xiaolin Li, Yu Song, Guobin Wang, Lin Wang, and Zheng Wang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01297 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on January 29, 2019

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ACS Biomaterials Science & Engineering

Sericin Nerve Guidance Conduit Delivering Therapeutically Repurposed Clobetasol for Functional and Structural Regeneration of Transected Peripheral Nerves

Lei Zhang,#, † Wen Yang,#, † Hongjian Xie,#, † Hui Wang,#, ‡ Jian Wang,† Qiangfei Su,† Xiaolin Li,† Yu Song,† Guobin Wang,*, § Lin Wang,*, †, || and Zheng Wang*, †, § † Research

Center for Tissue Engineering and Regenerative Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China ‡ Department

of Medical Genetics, Basic School of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China §

Department of Gastrointestinal Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China || Department

of Clinical Laboratory, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China Address correspondence to: Zheng Wang, Phone: 86-27-85726612. E-mail: [email protected] Or to: Lin Wang, Phone: 86-27-85726612. E-mail: [email protected] Or to: Guobin Wang, Phone: 86-27-85726612. E-mail: [email protected]

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ABSTRACT: Peripheral nerve injury often causes significant function loss. Autologous nerve grafting as a gold-standard repair strategy for treating such an injury is limited by donor nerve supply. Tissue-engineered nerve guidance conduits (TENGCs) as a promising alternative for autografting are challenged by large nerve gaps. Herein, we fabricate a glutaraldehyde-crosslinked sericin nerve guidance conduit (GSC) incorporated with clobetasol, a glucocorticoid receptor agonist, for repairing a 10-mm long sciatic nerve gap in a rat model. The GSC exhibits biocompatibility and regeneration-favorable physicochemical properties. GSC’s degradation products promote the secretion of neurotrophic factors in Schwann cells. By repurposing clobetasol for peripheral nerve regeneration, our work uncovers clobetasol’s previously unknown functions in promoting Schwann cell proliferation and upregulating the expression of myelin-related genes. Importantly, the implantation of this clobetasol-loaded GSC in vivo leads to successful regeneration of the transected sciatic nerve. Strikingly, the regeneration outcome is functionally comparable to that of autologous nerve grafting (evidenced by three parameters). Specifically, the static sciatic index (SSI), relative reaction time (RRT) and nerve conduction velocity (NCV) in Clobetasol/GSC group are -74.55, 1.30 and 46.4 mm/s at Week 12, respectively, while these parameters are -64.53, 1.23 and 49.8 mm/s in Autograft group. Thus, this work represents the first report unveiling clobetasol’s potential in peripheral nerve regeneration, reveals the feasibility of applying a sericin conduit for repairing a large nerve defect, and demonstrates the effectiveness of the clobetasol-loaded-GSC based strategy in transected nerves’ regeneration.

KEYWORDS: sericin, nerve guidance conduit, clobetasol, peripheral nerve regeneration, large nerve gap 2


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INTRODUCTION Peripheral nerve injuries result in disabilities, significantly affecting patients’ quality of life.1-2 Over 200,000 new cases of peripheral nerve injuries occur in the United States annually, consuming approximately 1.5-billion-dollar worth of healthcare.3-4 Among these injuries, nerve transection is the most severe, causing a loss of nerve continuity. The distal stump of a transected nerve eventually undergoes Wallerian degeneration that involves complex cellular and molecular events, such as disintegration of axonal membrane, activation of immune cells, and recruitment of macrophages.5 Currently, the widely-accepted, gold-standard therapy for repairing large nerve defect (> 5 mm) is autologous nerve grafting.6 However, it is limited by a lack of donor nerve supply, multiple surgeries required for completing the grafting, local tissue damage at donor sites, and possible mismatching between defective nerves and grafted segments.1 To overcome these limitations, alternative approaches have been investigated for bridging transected nerves, including allografts and biomaterials-based tissue-engineered nerve guidance conduits (TENGCs),1 which is thought to be a primary alternative.7 Development of TENGCs for bridging nerve gap using natural biomaterials, including gelatin, alginate, chitosan, collagen, silk fibroin and zein, has been extensively explored.8-11 To functionally enhance TENGCs’ regeneration-promoting capability, some components, such as neurotrophic factors, cell-derived extracellular matrix, and stem cells as well as Schwann cells, were encapsulated into TENGCs.2, 12-14 Although these TENGCs promote nerve gap bridging, their repair efficacy remains suboptimal to that of autologous nerve grafting.15 Thus, TENGCs with higher regenerative efficacy are highly desired. Sericin, a major component of silk fibers from silkworm cocoons, is now increasingly utilized in regenerative medicine due to the recent revelation of its pro-regeneration 3



bioactivities.16-17 These properties include cell adhesiveness, cell-proliferation-promoting effect under serum-free condition,17-20 and notably, sericin’s neurotrophic, neuroprotective, myelination-promoting activities that are particularly valuable for repair of injured nerves.21-23 Despite sericin’s neuron-favorable properties, whether sericin-based TENGCs are able to facilitate nerve regeneration over a large gap in vivo, such as a 10-mm defect in rats (a critically sized defects model where the defect would not self-heal),24-25 remains unknown. Clobetasol, a glucocorticoid receptor agonist commonly used for treating inflammatory skin diseases,26 is a potential remyelinating agent for central nervous system as it increases the number of mature oligodendrocytes through activating glucocorticoid receptor signaling.27 However, it is unclear whether clobetasol can benefit peripheral nerve regeneration after transection injury. We hypothesize that the incorporation of clobetasol into sericin-based TENGCs might improve regenerative outcomes. To achieve this, we have prepared a glutaraldehyde-crosslinked sericin conduit (GSC) loaded with clobetasol in hope of bridging a large gap (10 mm) caused by nerve transection injury. The conduit’s mechanical properties, biocompatibility and cytotoxicity were comprehensively characterized. Clobetasol’s effects on peripheral nerve regeneration were investigated. We found that (1) the degradation products of the sericin conduit promoted Schwann cells to secrete neurotropic factors (NT-3 and NT-4); (2) clobetasol activated PI3K/Akt signaling to enhance Schwann cell proliferation, and promote Schwann cells to produce positive myelination regulators (NRG1 and EGR2). Then the clobetasol-loaded GSC was utilized to treat a 10-mm long sciatic nerve defect in a preclinical animal model. Histological analysis suggested that axonal regrowth and the restoration of the diameter of myelinated nerve fibers were close to that of autologous nerve grafting. Furthermore, such a clobetasol-loaded GSCs repairing strategy led to a regenerative 4


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outcome functionally comparable to that of autologous nerve grafting, which is evidenced by the determination of static sciatic index (SSI), relative reaction time (RRT) and nerve conduction velocity (NCV). Together, our study not only demonstrates the effectiveness of the sericin-based conduit in the repair of large nerve gaps, but also reveals the functional roles of clobetasol in promoting peripheral nerve regeneration.

MATERIALS AND METHODS Fabrication of Glutaraldehyde Crosslinked Sericin Conduit (GSC) and Glutaraldehyde Crosslinked Sericin Film (GSF). Sericin solution (10%, w/v) was obtained from Bombyx mori, 140 Nd-s cocoons using a modified LiBr extraction process.20 Briefly, 0.5 g cocoons were cut up and dissolved in 27.5 mL LiBr solution (6 M). The mixture (in a 50 mL centrifuge tube) was placed in a water bath at 35 °C for 24 hours and then subjected to centrifugation (3500 rpm, 5 minutes) for separating the solid residue. The supernatant was mixed with 6.88 mL Tris-HCl solution (1 M, pH 9.0) in dialysis membranes (MWCO 3500 Da) for dialysis in double distilled water for 24 hours at room temperature. The solution was then concentrated in PEG-6000 solution. Next, the concentrated sericin solution (500 μL) was crosslinked with glutaraldehyde (25%, w/v) (5 μL) to generate a sericin hydrogel in a tailored conduit mold or on a platform at 37 °C. The conduit mold consisted of a hollow plastic tube with a 3.20-mm inner diameter and a cylindrical iron bar with a 1.40-mm diameter. The hydrogel was sequentially subjected to freeze (-80 °C for 8 hours) and lyophilization for obtaining the GSC or GSF.

Measurement of Gelation Time. The gelation time was determined as illustrated 5



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previously.28 The time from the beginning of adding glutaraldehyde into the sericin solution to the moment when the mixture became sticky and no more downflow could be observed along the tube wall was considered as the gelation time.

Determination of Degree of Crosslinking. The percentage of free-amino group content of crosslinked sericin hydrogels was detected using ninhydrin (NHN) assay.29 The ninhydrin solution was prepared as previously described.20 Detection was carried out as follows: sericin solution (10%, w/v) (100 μL) was firstly mixed with glutaraldehyde (25%, w/v) (1 μL) in a 1.5-mL centrifuge tube. After different periods of time, the mixture sample was heated to 100 °C with 100 μL ninhydrin solution for 15 minutes. When the solution cooled to room temperature, the optical absorbance of the solution was quantified (570 nm) with a microplate reader (TECAN). The uncrosslinked sericin solutions served as the controls. The degree of crosslinking was calculated as the following formula: Degree of crosslinking (%) = (O.D. valueu - O.D. valuec)/O.D. valueu × 100%

(1)

where O.D. valueu represents the optical absorbance values of the uncrosslinked sericin solution, and O.D. valuec represents the the optical absorbance values of the crosslinked sample, respectively.

GSC Incorporated with Clobetasol. Clobetasol was added into sericin solution (10%, w/v) to achieve the concentration of 5 μM before gelling. Clobetasol-loaded GSCs were fabricated as mentioned above. All clobetasol-loaded GSCs were stored in a -80 °C freezer prior to use.

Determination of the Inner Diameter and Wall Thickness of GSC. The conduit was 6


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firstly immersed in PBS at 37 °C for 72 hours. Subsequently, the cross-section of the GSC was photographed. To determine the inner diameter and the wall thickness of a GSC, the representative images were assessed by Image-Pro Plus software (version 6.0.0.260, Media Cybernetics, Inc., MD, USA). Each assessment contains three samples of GSCs.

Scanning Electron Microscopy. The porous microarchitecture of a GSC was observed and analyzed under a scanning electron microscope (SEM) (Zeiss, ULTRA PLUS-43-13, Germany) with a given operating voltage of 3 kV. In order to improve surface conductivity, samples were pre-coated with gold before investigation. To determine the average pore size of a GSC, the representative SEM images were assessed by Image-Pro Plus software (version 6.0.0.260, Media Cybernetics, Inc., MD, USA). Ninety random pores were selected from the representative SEM images for calculating the average pore size.

Swelling Ratio Analysis. The test for water absorption of a GSC was carried out as follows:30 the initial weight of a GSC (noted as M1) was measured; then the GSC was immersed in PBS (pH 7.4) at room temperature; at Day 1, 2, 3, 4 and 5, the swollen mass of the GSC (noted as M2) was determined. The swelling ratio was determined according to the following equation: Swelling (%) = (M2 - M1)/ M1 × 100%

(2)

where M1 represents the initial mass of a GSC, and M2 represents the swollen mass of the GSC.

Porosity Analysis. The porosity of the GSC was characterized by employing the ethanol 7



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displacement method.2 Briefly, the dried weight (noted as Wd) and total volume (noted as V) of a GSC were determined. Next, the GSC was immersed in ethanol for 24 hours. After immersion, the weight of the GSC (noted as Wi) was measured. The porosity was calculated by using the following formula: Porosity (%) = (Wi - Wd)/(V × ρ) × 100%

(3)

where Wd (g) represents the dried weight of a GSC, and Wi (g) is the weight of the GSC after immersion; V (cm3) represents the total volume of a GSC; ρ (0.789 g/cm3) is the density of ethanol.

Assessment of Mechanical Properties. The Young’s modulus and the bending stiffness of a GSC were measured by a universal testing machine (Instron 5848 MicroTester, MA, USA) as previously described.11 Samples were tailored to be 1-cm long and immersed in PBS at 37 °C for 72 hours before testing at the compression speed of 1 mm/min.

Fourier Transform Infrared Spectroscopy. The secondary structures of GSCs, clobetasol-loaded GSCs, and sericin protein were examined by a Fourier transform infrared (FTIR) spectroscope (Nicolet 6700, Thermo Electron Co., USA). The freeze-dried samples were smashed into powders before investigation. The absorption peak was analyzed with a ZnSe ATR cell within a range of 4000-400 cm-1 using the OMNIC software.

Assessment of RSC96 Schwann Cells’ Growth on GSFs. GSFs were tailored to an appropriate shape and then attached to 96-well plates. RSC96 Schwann cells (5,000 cells per well) were seeded on GFSs or on the glass sides of 96-well plates (serve as control). Cells 8


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were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, HyClone, UT, USA) containing fetal bovine serum (10%, FBS; Gibco, CA, USA) in an incubator (37 °C, 5% CO2). Cell viability of RSC96 Schwann cells was determined by a Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) at Day 0, 1, 3, 5 and 7 after seeding according to the user manual.

RSC96 Schwann Cells Culture with GSC Extraction Solution. The GSC extraction solution was prepared using a previously described method.31 Briefly, a 10-mm GSC was completely immersed into 1 mL DMEM supplemented with 2% FBS. After a 24-hour incubation (37 °C, 5% CO2), the medium (GSC extraction solution) was collected and sterilized with 0.22 μm filters before use. Another 1 mL DMEM supplemented with 2% FBS was added for preparing the new GSC extraction solution. RSC96 Schwann cells (3,000 cells per well) were seeded in 48-well plates and cultured in a cell culture incubator (37 °C, 5% CO2). After 24 hours, the original medium was replaced with the GSC extraction solution or the regular medium (DMEM supplemented with 2% FBS) (control group). Half of the GSC extraction solution and the regular medium were changed every day. Cells were viewed and photographed via a phase contrast microscope (Olympus IX71, Japan) at Day 0, 7 and 14 after seeding. Cell viability of RSC96 Schwann cells at Day 0, 1, 3, 5, 7, 11, 15 and 21 after seeding was quantified by CCK-8 assay.32 The relative cell viability at each time point was calculated by dividing the O.D. value of the GSC extraction solution group by that of the control group.

Clobetasol Treatment on RSC96 Schwann Cells. For evaluating whether clobetasol has 9



effects on cell proliferation, RSC96 Schwann cells (5,000 cells per well) were seeded in 48-well plates and maintained in DMEM supplemented with 2% FBS. After 24 hours, cells were treated with 0.09% DMSO (vehicle control), 5 μM clobetasol, 5 μM clobetasol together with 10 μM RU486 (a glucocorticoid receptor antagonist), and 10 μM RU486, respectively. Cells were viewed and photographed via a phase contrast microscope (Olympus IX71, Japan) at Day 1, 3 and 5. Six random fields were selected from different treatment groups for calculating the relative cell density. Cell viability at Day 0, 1, 3 and 5 after different treatments was measured by CCK-8 assay.32 To further investigate whether clobetasol affects cell proliferation through PI3K/Akt signaling pathway, RSC96 Schwann cells (3,000 cells per well) were seeded in 48-well plates and maintained in DMEM supplemented with 2% FBS. After 24 hours, cells were pretreated with 10 μM MK2206 (an Akt inhibitor) or 0.09% DMSO (vehicle control) for 12 hours. Thereafter, cells were stimulated with 5 μM clobetasol or 0.09% DMSO (vehicle control). The assessment of the relative cell density and the cell viability was performed as above-mentioned.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Analysis. For evaluating the influence of a GSC on Schwann cells, RSC96 Schwann cells (200,000 cells per well) were cultured on a GSF in a well of a 6-well plate with DMEM containing 10% FBS. Twenty-four hours later, cells were collected for total cellular RNA extraction. For assessing clobetasol’s function upon peripheral nerve regeneration, RSC96 Schwann cells (200,000 cells per well) were seeded in 6-well plates and maintained in DMEM containing 10% FBS. Twenty-four hours later, the medium was replaced and then the cells were treated with 5 μM clobetasol or 0.09% DMSO (vehicle control). After a 24-hour stimulation, cells were 10


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collected for total cellular RNA extraction. After RNA reverse transcription, qPCR experiments were performed as previously mentioned.33 The relative mRNA levels of NT-3, NT-4, BDNF, NGF, CNTF, GDNF, NRG1 and EGR2 were detected. The primer sequences were listed in Table S1 of the Supplementary data.

Enzyme-Linked Immunosorbent Assay (ELISA). RSC96 Schwann cells (200,000 cells per well) were cultured on a GSF or on the glass side in a well of a 6-well plate with DMEM containing 10% FBS. Twenty-four hours later, the culture supernatant was collected. Neurotrophin-3 and Neurotrophin-4 secreted by RSC96 Schwann cells in the culture supernatant were detected using ELISA kits (Cusabio Biotech Co., Ltd., Wuhan, China).

Western Blotting. RSC96 Schwann cells (200,000 cells per well) were seeded in 6-well plates and allowed to grow adhering for 24 hours. To investigate the proliferation-associated signaling pathways in RSC96 Schwann cells influenced by clobetasol, cells were treated with 0.09% DMSO (vehicle control), 5 μM clobetasol, 5 μM clobetasol together with 10 μM RU486 or 10 μM RU486, respectively. To examine clobetasol’s functional roles on myelination, cells were treated with 5 μM clobetasol or 0.09% DMSO (vehicle control). After 24 hours, cells were collected and protein samples were prepared. Proteins were separated by electrophoresis and then transferred onto nitro cellulose (NC) filter membranes. After blocking, primary antibody incubation and secondary antibody incubation, specific bands were visualized by chemiluminescence assay and photographed using a BioSpectrum®600 Imaging System (Upland, CA, USA). The detailed information on primary antibodies was 11



listed below: EGR2 (dilution 1:500; Proteintech Group, IL, USA), NRG1 (dilution 1:500; Proteintech Group, IL, USA), β-actin (dilution 1:5,000; Proteintech Group, IL, USA), GR (dilution 1:500; Proteintech Group, IL, USA), p-GR (dilution 1:500; Proteintech Group, IL, USA), Akt (dilution 1:1,000; Cell Signaling Technology, MA, USA), p-Akt (Ser 473) (dilution 1:1,000; Cell Signaling Technology, MA, USA), and GAPDH (dilution 1:5,000; Proteintech Group, IL, USA). The original western blotting was shown in the Supporting Information.

In Vitro Clobetasol Release. A 10-mm GSC loaded with 20 μg clobetasol was soaked in 1.0 mL acetic acid-ammonium acetate buffer solution (pH 7.4) at 37 °C. From Day 1 to Day 40, the buffer solution was collected daily and supplemented with 1.0 mL new buffer solution. The collected buffer solution was frozen at -80 °C and then lyophilized to obtain the powders. The powders were dissolved with 500 μL acetonitrile and filtered with 0.22 μm filters for later tests. To determine the daily release amount of clobetasol, 15 μL solution sample was injected into a high-performance liquid chromatography-tandem mass spectrometer (HPLC-MS) (Thermo, MA, USA). Data were analyzed and processed using the Waters Quanlynx 4.0 software.

Sciatic Nerve Surgery. All the Sprague-Dawley (SD) rats (250-300 g) for in vivo experiments were purchased from the Experimental Animals Center of Tongji Medical College, Huazhong University of Science and Technology. Sixty-four rats in total were evenly divided into 4 groups randomly: the non-intervention group, the autologous nerve graft 12


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group (the nerve tissue was connected with a resected nerve segment, recorded as Autograft group), the GSC group (the nerve tissue was connected with a GSC, recorded as GSC group) and the GSC delivering clobetasol group (the nerve tissue was connected with a clobetasol-loaded GSC, recorded as Clobetasol/GSC group). The animals were firstly anesthetized with pentobarbital sodium (3%, 1 mL/kg) and then placed on an operating table. The right hind limbs were subjected to shaving and sterilization. An incision was made for exposing sciatic nerve, followed by cutting a 10-mm long nerve segment off from the middle portion of the nerve. For non-intervention group, the nerve defect was left untreated. For Autograft group, the resected nerve segment was re-sutured to both the proximal and distal stumps. For GSC and Clobetasol/GSC group, each nerve stump was placed into the lumen with a depth of 1 mm and then anchored to the conduit (12 mm) with 8/0 nylon sutures. The permission of animal care and surgery was obtained from the Institutional Animal Care and Use Committee, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.

Calculation of Static Sciatic Index (SSI). Sciatic nerves’ motor function is often assessed using static sciatic index (SSI): the value 0 indicates normal function, while the value -100 reflects a total function loss.34-35 The plantar views of the rats’ paws were photographed and assessed by the Image-Pro Plus software for quantification of the following parameters: the length of toe spread (TS) and the length of intermediate toe spread (ITS). SSI measurements were performed according to the following equation:34 SSI index = 108.44 × (TSo - TSc)/TSc + 31.85 × (ITSo - ITSc)/ITSc - 5.49

(4)

where TSo is the length of toe spread of the operated side, TSc is the length of toe spread of 13



the contralateral side, ITSo is the length of intermediate toe spread of the operated side, ITSc is the length of intermediate toe spread of the contralateral side, respectively.

Nerve Sensory Function Analysis. The thermal withdraw latency (TWL) was analyzed by an infrared heat stimulus device (UGO BASILE Plantar Test 37370, Comerio, VA, Italy) at indicated time points. The rats’ paws were stimulated by an infrared beam. The animals would lift their paws if they could no longer bear the pain. The time from the beginning of the stimulation to the moment rats moved their paws off was considered as the TWL. Five independent tests were performed to determine the average TWL. The relative reaction time (RRT) was calculated by dividing the TWL of the injured side by that of the contralateral side. The closer the numerical values of RRT are to "1", the more significant recovery of sensory function is obtained.

Nerve Conduction Analysis. To determine the nerve conduction velocity (NCV) of the regenerated nerve, rats in indicated groups were anesthetized and then the sciatic nerve of the injured side was re-exposed at Week 8 and 12 after implantation. NCV was detected and then recorded with an electromyography recorder (CareFusion, CA, USA) at a given pulse mode.21 A higher value of NCV is reportedly indicates a better restoration of nerve conduction function.

Morphological Observation and Histological Analysis of Regenerated Nerves. The rats in indicated groups were sacrificed at Week 8 and 12 after implantation, respectively. Next, the regenerated sciatic nerves and the contralateral sciatic nerves were isolated carefully. The 14


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macroscopic appearance of the regenerated nerve was captured. All harvested nerve tissue samples were immersed in 4% paraformaldehyde and then included in paraffin for later histological examination. To assess the microstructural restoration of regenerated sciatic nerves, the immunofluorescence staining was performed on 8-μm thick paraffin sections. Nerve sample slices were firstly incubated with appropriate primary antibodies: the mouse anti-S100 antibody (1:400 diluted; Abcam, MA, USA) for identifying Schwann cells, the mouse anti-myelin basic protein (MBP) antibody (1:500 diluted; Abcam, MA, USA) for identifying myelin sheaths, and the rabbit anti-β-3 tubulin antibody (1:1000 diluted; Abcam, MA, USA) for identifying axons and the rabbit anti-CD34 antibody (1:250 diluted; Abcam, MA, USA) for identifying micro-vessels, respectively. The corresponding 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). DAPI (Wuhan Boster Biological Technology, Ltd., Wuhan, China) was used to identify cell nuclei. To calculate the diameters of the myelinated fibers, Luxol fast blue (LFB) staining was employed to visualize myelin sheath as reported earlier.36 In brief, paraffin sections with the thickness of 3 μm were firstly dehydrated and then incubated in 0.1% LFB solution at 37 °C overnight. After that, the sections sequentially underwent cooling, incubation in 0.05% lithium carbonate solution, and differentiation in 70% ethanol. The stained slices were photographed with an inverted fluorescence microscope (Olympus IX71, Japan). The quantitative analyses on S100 and β-3 tubulin positive regions were performed by calculating the total positive area relative to 100 μm2 area (8 animals per group, 3 random fields per animal). The myelin sheath thickness was calculated by averaging 90 axons per group. The percentage of myelinated nerve fibers was 15



quantified by the counts of myelinated nerve fibers relative to the total number of axons in a given area (8 animals per group, 3 random fields per animal). The above analyses were carried out using the Image-Pro Plus software.

Target Muscles Analysis. The rats in indicated groups were sacrificed at Week 8 and 12 after implantation, respectively. The gastrocnemius muscles of both limbs were removed carefully. The weight of gastrocnemius muscles was determined and recorded. Then all harvested muscle samples were immersed in 4% paraformaldehyde and then included in paraffin for later histological examination. Gastrocnemius muscle fibers (dark red-stained areas) were visualized by Masson trichrome staining. The relative wet weight of gastrocnemius muscle was the ratio of the wet weight of the injured side relative to the wet weight of the contralateral side (8 animals per group). Quantifications of the average percentage of collagen fiber area (blue-stained areas) and the average diameter of muscle fibers were carried out using the Image-Pro Plus software (8 animals per group, 3 random fields per animal). The diameter of gastrocnemius muscle fibers was calculated by averaging 90 muscle fibers per group.

Statistics. Data were presented as mean ± SD. Statistical comparisons were done using Student’s t-test or one-way analysis of variance (ANOVA) with the SPSS software (SPSS Inc., IL, USA). p < 0.05 indicated statistically significant.

RESULTS Characterizations of Glutaraldehyde Crosslinked Sericin Hydrogels and Sericin 16


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Conduits (GSCs). Sericin was extracted from the cocoons of Bombyx mori (140 Nd-s) silkworm strain via the LiBr extraction method as previously described.20 The addition of glutaraldehyde into the sericin solution (10%, w/v) led to the generation of sericin hydrogels (Figure S1a). We further examined the gelation dynamics of the sericin hydrogels. The assessment of crosslinking rate showed that 10% sericin took nearly 294 seconds to form a hydrogel (Figure S1b). The degree of crosslinking increased gradually after adding glutaraldehyde and the sericin hydrogels took approximately 2 hours to reach saturated crosslinking (Figure S1c). These results demonstrate that the glutaraldehyde crosslinking is successful. By using abovementioned sericin, a GSC was fabricated in a pre-designed mold. The GSC was a 10-mm long, yellowish, hollow tube (Figure 1a) with a 1.5-mm inner diameter, a 0.91-mm wall thickness, and 0.18 MPa of Young’s modulus (Table 1) in water-saturated state. The conduit was flexible with the bending stiffness of 0.35 Nmm2, which allowed it to be bendable while returned to its original position after external force was removed (Table 1; Figure 1b), reflecting the conduit’s resilience. Such a property would allow the conduit to withstand crush that might be caused by body movement during regeneration.37 Futher, this conduit was highly porous (porosity, 82.56%) with the average pore size of 36.73 μm (Table 1; Figure 1c) and pores were interconnected (Figure 1c). These features would facilitate nutrient influx from neighbouring tissue, benefiting nerve regeneration.38-39 The water absorbing ability of a conduit was important for loading therapeutic agents.19 The conduit achieved the water absorbing equilibrium after a 24-hour immersion in PBS (Figure 1d), suggesting a rapid water-absorbing capacity. The secondary structures of sericin, the GSC, and the clobetasol-loaded GSC were 17



analyzed using Fourier transform infrared (FTIR) spectroscopy. Pure sericin had three feature peaks, which were amide I (C=O stretching) ranging from 1600 cm-1 to 1690 cm-1, amide II (N-H bending) ranging from 1480 cm-1 to 1575 cm-1, and amide III (C-N stretching) ranging from 1229 cm-1 to 1301 cm-1, respectively (Figure 1e).40-41 Among these, the amide I band is most commonly used for detecting the secondary structures of sericin protein. After crosslinking, the amide I peaks of the GSC (1658 cm-1) and the clobetasol-loaded GSC (1658 cm-1) slightly changed when compared with the uncrosslinked sericin powder (1654 cm-1) (Figure 1e). Meanwhile, the amide II and amide III peaks were similarly observed for the uncrosslinked sericin powder, the GSC and the clobetasol-loaded GSC (Figure 1e). These results indicate that neither glutaraldehyde crosslinking or clobetasol loading leads to significant transformation of the secondary structures of sericin. Together, the high porosity, appropriate pore size, and the bendability collectively confer GSC’s structural advantages towards promoting peripheral nerve regeneration.

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Figure 1. Characterization of glutaraldehyde-crosslinked sericin conduits (GSC). a) The mold used for the fabrication of the conduit (left panel) and the cross-sectional (middle panel) and lateral (right panel) appearance of a GSC. Scale bar, 5 mm. b) Before- and after-bent photographs of a GSC. Scale bar, 1 cm. c) SEM micrographs of the cross-section of a GSC. The white dotted regions in I, II and III were enlarged in II, III and IV, respectively. Scale bar, 200 μm (upper panel, left); 100 μm (upper panel, right); 20 μm (lower panel, left); 10 μm (lower panel, right). d) The swelling properties of a GSC in PBS with pH 7.4 at 37 °C. e) FTIR spectra of sericin protein powder, GSC powder, and clobetasol-loaded GSC powder. Data were expressed as mean ± SD (n = 3).

Table 1. Characterizations of Glutaraldehyde-Crosslinked Sericin Conduits (GSCs) Pore sizea)

Porosity a)

Inner diameter a)

Wall thickness a)

(μm)

(%)

(mm)

(mm)

36.73 ± 4.37b)

82.56 ± 2.12

1.50 ± 0.12

0.91 ± 0.05

a)

Young’s

Bending

modulus a)

stiffness a)

(MPa)

(Nmm2)

0.18 ± 0.05

0.35 ± 0.07

Each assessment contains three samples of GSCs. Data were presented as mean ± SD; b)

Ninety random pores were selected for calculating the average pore size.

Biocompatibility of the GSF and GSC. Schwann cells are indispensable to peripheral 19



nerve regeneration.42 We next determined whether the sericin-based scaffold influenced Schwann cells’ survival and proliferation. RSC96 Schwann cells were cultured onto the glutaraldehyde crosslinked sericin films (GSFs) that were pre-cast on the bottom of the wells in 96-well plates. RSC96 Schwann cells were able to grow on the GSF for 7 days (Figure 2a). Given that neurotrophins secreted by Schwann cells are critical for accelerating peripheral nerve regeneration,43 we examined the impact of the GSF on expression of several key neurotrophins in RSC96 Schwann cells. Twenty-four hours after being cultured onto the GSF, RSC96 Schwann cells significantly up-regulated the mRNA levels of neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) (Figure 2b), but not the other neurotrophins (for instance, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF) and glial cell derived neurotrophic factor (GDNF)) (Figure S2). In addition, GSF group exhibited higher protein levels of NT-3 and NT-4 over 7 days (Figure 2c). These observations suggest that the GSF promotes RSC96 Schwann cells to produce selected neurotrophic factors. We next assessed the cytotoxicity of the GSC. As previously described,31 the GSC extraction solution was collected and applied to RSC96 Schwann cells for a 21-day culture. Throughout this time period, the relative cell viability stayed nearly 100% (Figure 2d; Figure S3), suggesting GSC’s good cytocompatibility.

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Figure 2. In vitro biocompatibility testing of glutaraldehyde crosslinked sericin films (GSFs) and cytotoxicity testing of GSCs. a) The RSC96 Schwann cells were cultured on a GSF and a cell culture plate (control), and the cell viability was analyzed by CCK-8 assay within 7 days. b) The RSC96 Schwann cells were cultured on a GSF and a cell culture plate (control). Twenty-four hours later the relative mRNA levels of neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) were detected. c) The RSC96 Schwann cells were cultured on a GSF and a cell culture plate (control). ELISA for detecting NT-3 and NT-4 proteins in the culture supernatant was performed after 2, 4 and 7 days. d) The relative cell viability determined by the O.D. value in the GSC group (RSC96 Schwann cells cultured with the GSC extraction solution) relative to the O.D. value in the control group (RSC96 Schwann cells cultured with regular medium) at given time points. Data were expressed as mean ± SD (n = 3). **, p < 0.01; ***, p < 0.001; Student’s t-tests.

Clobetasol’s Effects on RSC96 Schwann Cells. While clobetasol reportedly promotes myelination in central nervous system,27 little is known about its effects on peripheral nerves. The cell counting and CCK-8 assay showed that clobetasol significantly enhanced RSC96 Schwann cells’ proliferation, which was abolished by RU486, a glucocorticoid receptor antagonist (Figure 3a; Figure S4a, b),27 indicating that clobetasol acts through glucocorticoid receptors. Consistently, the phosphoproteomic analyses on clobetasol-treated RSC96 21



Schwann cells show significantly increased phosphorylation of glucocorticoid receptors, which could be also blocked by RU486 (Figure 3b). Meanwhile, PI3K/Akt signaling, a pathway known to regulate cell proliferation, was activated in clobetasol-treated RSC96 Schwann cells (Figure 3b). The blockade of PI3K/Akt signaling using MK2206, an Akt inhibitor, abrogated clobetasol’s proliferation-promoting effect on RSC96 Schwann cells (Figure 3c; Figure S4c, d). These results suggest that clobetasol acts through glucocorticoid receptors to activate PI3K/Akt signaling to promote proliferation. Of note, the clobetasol treatment on RSC96 Schwann cells significantly increased the mRNA and protein levels of two myelin-critical genes, neuregulin 1 (NRG1) and early growth response 2 (EGR2) (Figure 3d, e), indicating that clobetasol may promote myelination in peripheral nervous system.

Figure 3. Cellular and molecular effects of clobetasol on RSC96 Schwann cells. a-c) The 22


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RSC96 Schwann cells were treated with DMSO (vehicle control), clobetasol, clobetasol combined with RU486 (a glucocorticoid antagonist), or RU486 alone. a) The cell viability of RSC96 Schwann cells at Day 0, 1, 3 and 5 in indicated groups were determined by CCK-8 assay. b) Western blot of glucocorticoid receptors (GR), phosphorylated GR (p-GR), total Akt, and phosphorylated Akt (p-Akt) in RSC96 Schwann cells after indicated treatments for 24 hours. Clo represented clobetasol treatment. d, e) The RSC96 Schwann cells were treated with DMSO (vehicle control), clobetasol, clobetasol together with MK2206 (an Akt inhibitor), or MK2206 alone. c) The cell viability of RSC96 Schwann cells at Day 0, 1, 3 and 5 in indicated groups were determined by CCK-8 assay. d, e) RSC96 Schwann cells were treated with 5 μM clobetasol or DMSO (vehicle control) for 24 hours. Then the relative mRNA levels d) and proteins e) of early growth response protein 2 (EGR2) and neuregulin 1 (NRG1) of RSC96 Schwann cells were detected. Clo represented clobetasol treatment. Data were expressed as mean ± SD (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001; N.S., not significant; Student’s t-tests (d) or One-way ANOVA analysis (a, c).

Analysis on Clobetasol Release Kinetics from the GSC. Next, we examined the release kinetics of clobetasal from a GSC. Clobetasol was continuously released from the GSC over 40 days (Figure 4a), during which three release phases were observed: a fast release from Day 1 to Day 12 (Phase I), a moderate release from Day 12 to Day 20 (Phase II), and then a slow-downed release from Day 20 to Day 40 (Phase III) (Figure 4a). The cumulative release gave rise to a sustained release profile (Figure 4b).

Figure 4. Clobetasol in vitro release profile. Daily release a) and cumulative release b) of clobetasol from a GSC at 37 °C in acetic acid-ammonium acetate buffer solution. Data were expressed as mean ± SD (n = 3).

23



Functional Assessment of Regenerated Sciatic Nerves. Next, the GSC and the clobetasol-loaded GSC (Clobetasol/GSC) as well as autologous nerves (Autograft) were implanted to bridge a 10-mm gap in a transected sciatic nerve in a rat model (See the Materials and methods for details). The SSI of the Clobetasol/GSC group (-74.55 at Week 12) was significantly higher than that of the GSC group (-95.91 at Week 12) (p < 0.05) (Figure 5a, b), and importantly, comparable to the Autograft group (-64.53 at Week 12) (p > 0.05) (Figure 5b), indicating that the Clobetasol/GSC implantation leads to motor function recovery as effective as autografting. Meanwhile, sciatic nerve sensory function was assessed by thermal withdrawal latency.44 The relative reaction time of the Clobetasol/GSC group (1.30 at Week 12) was close to that of the Autograft group (1.23 at Week 12), significantly less than that of the GSC group (1.57 at Week 12) (p < 0.05) (Figure 5c), revealing that the Clobetasol/GSC treatment results in autografting-comparable sensory function recovery. Similarly, nerve conduction velocity (NCV), a key indicator of nerve function,45 in the Clobetasol/GSC group (46.4 mm/s at Week 12) matched that in the Autograft group (49.8 mm/s at Week 12) (p > 0.05), while higher than GSC group (35.6 mm/s at Week 12) (p < 0.05) (Figure 5d).

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Figure 5. Functional assessment of regenerated sciatic nerves. a) The hind paws of the rats from the GSC group (GSC), the clobetasol-loaded GSC group (Clobetasol/GSC), and autologous nerve grafts group (Autograft) were photographed from the plantar side at 8 and 12 weeks postoperatively. The blue arrowheads indicated the experimental hind limbs. The lengths of black lines (the linear distance from the first to the fifth toe, noted as “TS”) and red lines (the linear distance from the second to the fourth toe, noted as “ITS”) were measured. b) Quantitative analysis of the static sciatic index (SSI) from hind paws (see the Materials and methods for details) at 8 and 12 weeks postoperatively (n = 8 per group). c) Quantification of the relative thermal withdrawal latency of hind paws (noted as the relative reaction time; see the Materials and Methods for details) at 8 and 12 weeks postoperatively (n = 8 per group). d) Quantification of nerve conduction velocity (NCV) on the experimental side at 8 and 12 weeks postoperatively (n = 8 per group). Data were expressed as mean ± SD. *, p < 0.05; **, p < 0.01; N.S., not significant; One-way ANOVA analysis.

GSC Degradation and Regrowth of Sciatic Nerve Fibers. The degradation of the conduits became visually apparent by Week 4, and was later enhanced over time, and completed by Week 12 (Figure 6a). Such degradability would spare retrieval surgery, avoiding secondary surgical injury. While the conduit was degrading, the nerve fragment 25



connecting the two stump ends were regenerated in all of the operated animals with no obvious infection or neuroma observed (Figure 6b). However, we observed that the two transected nerve stumps failed to bridge and re-connect at Week 8 and Week 12 in the non-intervention group (Figure S5), indicating no spontaneous regeneration, demonstrating that a sericin nerve guidance conduit is essential for nerve regeneration in vivo. The number of Schwann cells (visualized by anti-S100 staining) within the regenerated nerves in the Clobetasol/GSC group was slightly lower (8.03% at Week 8; 10.29% at Week 12) than that in the Autograft group (11.63% at Week 8; 13.95% at Week 12), but significantly higher than the GSC group (4.90% at Week 8; 6.20% at Week 12) (Figure 6c, d). This was consistent with the in vitro observation that clobetasol was capable of promoting the proliferation of Schwann cells. Moreover, anti-β-3 tubulin staining for visualizing neuronal axons’ growth revealed that the number of regenerated axons in the Clobetasol/GSC group was higher than the GSC group and, importantly, comparable to the Autograft group (Figure 6c, e).

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Figure 6. Photographs and histological assessments of regenerated sciatic nerves in the Autograft group, the GSC group and the Clobetasol/GSC group. a) In vivo degradation of the GSC. The white dotted lines outline the shape of a GSC. Scale bar, 5 mm. b) Photographs of regenerated sciatic nerves at 8 and 12 weeks post-implantation. Scale bar, 5 mm. c) Triple immunofluorescence staining was used to visualize the cross-sections of the central portions of the regenerated nerves at 8 and 12 weeks post-implantation: S100 (Schwann cells, red), β3-tubulin (axons, green) and DAPI (nuclei, blue). The enlarged images were for the white dotted regions from the upper “Merged” panel. The myelinated nerve fibers were indicated by white arrowheads. Scale bar, 50 μm. d, e) Quantification of S100 d) and β3-tubulin e) positive 27



area per 100 µm2 at 8 and 12 weeks post-implantation (n = 8 animals per group, 3 random fields per animal). Data were expressed as mean ± SD. *, p < 0.05; **, p < 0.01; N.S., not significant; One-way ANOVA analysis.

Microstructural Restoration within Regenerated Sciatic Nerves. Remyelination of regenerated axons is critical for functional recovery during nerve regeneration.42 The staining for MBP (myelin marker) and β3-tubulin (axon marker) was used to assess myelination of nerve fibers (Figure 7a). The Clobetasol/GSC group (74.63% at Week 8; 80.70% at Week 12) had the slightly lower number of myelinated nerve fibers than that in autografting (90.30% at Week 8; 91.33% at Week 12), but much higher than that in the GSC group (58.93% at Week 8; 64.62% at Week 12) (Figure 7a, b), indicating that clobetasol enhances myelination. Moreover, the average diameter of myelinated nerve fibers (visualized by luxol fast blue (LFB) that stained myelin sheath) in the Clobetasol/GSC group was close to that of the Autograft group (Figure 7c, d). Immunofluorescence staining for CD34 was performed to assess vascularization in the process of nerve regeneration. The micro-vessel density (CD34 positive area) in Clobetasol/GSC group was significantly higher than that in GSC group, but slightly lower than that in Autograft group (Figure S6), indicating that clobetasol facilitates vascularization. Taken together, these observations suggest that the Clobetasol/GSC treatment effectively restores the microstructure and the morphology of nerve fibers.

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Figure 7. Histological analysis of the myelination in the Autograft group, the GSC group and the Clobetasol/GSC group. a) Triple immunofluorescence staining for visualizing the cross-sections of the central portions of the regenerated nerves at 8 and 12 weeks post-implantation: MBP (myelin sheath, red), β3-tubulin (axons, green) and DAPI (nuclei, blue). The enlarged images were for the white dotted regions from the upper “Merged” panel. The myelinated nerve fibers were indicated by white arrowheads. Scale bar, 50 μm. b) Quantitative analysis of the average percent of myelinated nerve fibers at 8 and 12 weeks postoperatively. (n = 8 animals per group, 3 random fields per animal). c) Luxol fast blue (LFB) staining for detecting the myelin sheath in the cross-sections of the central portions of the regenerated nerves at 8 and 12 weeks postoperatively. Scale bar, 10 μm. d) Quantification 29



of the mean diameters of the myelinated fibers at 8 and 12 weeks postoperatively. (n = 8 animals per group, 3 random fields per animal). Data were expressed as mean ± SD. *, p < 0.05; **, p < 0.01; N.S., not significant; One-way ANOVA analysis.

Evaluation of Gastrocnemius Muscle. Nerve gap defects lead to functional deterioration and atrophy of targeting muscles, which may be partly prevented by regenerated nerves re-innervating these muscles.2 The relative wet weight of gastrocnemius muscle and the diameter of the muscle fibers in the Clobetasol/GSC group were higher than that of the GSC group (p < 0.05) (Figure 8a-d), but moderately lower than that of the autografting group, suggesting that the inclusion of clobetasol into the GSC partially alleviates skeletal muscle atrophy after a loss of innervation. Further, the percentage of fibrotic tissue (determined using Masson trichrome staining) in the Clobetasol/GSC group was much less than the GSC group, while close to the autografting group (Figure 8e), indicating that clobetasol’s integration into the GSC effectively suppresses formation of fibrotic tissue.

Figure 8. Recovery of atrophied gastrocnemius muscles. a) The gross observation of the 30


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gastrocnemius muscles of both hind limbs at 8 and 12 weeks postoperatively. The gastrocnemius muscles of experimental hind limbs were indicated by blue arrowheads. Scale bar, 10 mm. b) Quantitative analysis of the relative wet weight of gastrocnemius muscle (the wet weight of injured sides relative to the wet weight of contralateral sides) at 8 and 12 weeks postoperatively. (n = 8 per group). c) Masson trichrome staining images of the transverse sections of the gastrocnemius muscles (injured side) in the corresponding groups at 8 and 12 weeks postoperatively. Scale bar, 50 μm. d, e) Statistical results of the mean diameters of gastrocnemius muscle fibers d) and the average percentage of collagen fiber area e) at 8 and 12 weeks postoperatively. (n = 8 animals per group, 3 random fields per animal). Data were expressed as mean ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001; One-way ANOVA analysis.

Table 2. Assessment of in vivo regeneration outcomes (Week 12) Parameter

Autograft

SSI relative reaction time NCV (mm/s) S-100 positive area (%) β-3 tubulin positive area (%) percent of myelinated nerve fibers (%) diameter of myelinated nerve fibers (μm) relative wet weight of gastrocnemius muscle (%) diameters of gastrocnemius muscle fibers (μm) percentage of collagen fiber area (%)

-64.53 ± 16.16 1.23 ± 0.11 49.8 ± 6.3 13.95 ± 1.18 17.19 ± 1.57 90.30 ± 2.74 4.86 ± 0.24 70.68 ± 6.26 57.94 ± 3.29 2.32 ± 0.35

Group (Mean ± SD) Clobetasol/GSC GSC -74.55 ± 9.75* 1.30 ± 0.11* 46.4 ± 6.1* 10.29 ± 1.14#, * 15.65 ± 1.16* 80.70 ± 3.38#, * 4.59 ± 0.15* 42.75 ± 7.07#, * 50.90 ± 1.95#, * 3.36 ± 0.28#, *

-95.91 ± 7.09 1.57 ± 0.20 35.6 ± 6.5 6.20 ± 1.33 11.76 ± 2.18 64.63 ± 3.59 3.44 ± 0.37 23.33 ± 5.89 39.62 ± 2.08 5.39 ± 0.48

* p < 0.05 compared with GSC group; # p < 0.05 compared with Autograft group.

DISCUSSION Effective repair of a large gap defect in a peripheral nerve is challenging.15 Here, we develop a glutaraldehyde-crosslinked sericin nerve guidance conduit embedded with clobetasol, an agonist for glucocorticoid receptors, to treat a large nerve gap (10 mm) caused by nerve transection injury in rats. Our results demonstrate that this tissue-engineered approach effectively promotes functional and structural regeneration in sciatic nerve. As a promising alternative for repair of transected peripheral nerves, an engineered nerve guidance conduit is required to possess physicochemical properties favoring nerve 31



regeneration.10 Given that sericin exhibits neurotrophic, neuroprotective and myelination-promoting activities,21-23 it could be a raw biomaterial for fabricating nerve guidance conduits. By crosslinking sericin with glutaraldehyde, the resultant GSC exhibited regeneration-favorable mechanical and structural properties. An appropriate mechanical property should provide not only sufficient mechanical strength resisting compression from the surrounding, but also appropriate flexibility to offset recipients’ body motions.46 Sericin at the concentration of 10% was able to provide sufficient mechanical strength. Meanwhile, it remained bioactive and rarely denatured at room temperature. The Young’s modulus of the GSC, 0.18 MPa, higher than that of a commercial NeuraGen® conduit (80 kPa),47 would help maintain the conduit shape during axon regeneration. One the other hand, the resilience (bending stiffness of 0.35 Nmm2) could not only weaken the adhesion to the surrounding tissues but also confer the conduit with the ability to prevent possible rupture caused by body movement during nerve reconstruction.37, 48 The inner diameter of GSC reaching water absorbing equilibrium (1.50 mm) was larger than the cross-sectional diameter of rat sciatic nerve (approximately 1.1 mm),49 which could permit axonal growth through the tube.50 The successful re-connection of nerve stumps in GSC and Clobetasol/GSC group suggested that the wall thickness was able to hold a suture connecting the nerve stumps and the conduit during regeneration process.51 Moreover, the conduit’s rapid swelling behavior would allow adjacent tissues and regenerating nerves to avoid continuous compression after implantation. Further, GSC’s 82.56% porosity and pore size of 37 μm should favor nerve regeneration given that 80% or higher of porosity in a conduit wall was thought to be optimal,38, 52 and the pore size between 20-50 μm was found to effectively support cell migration, facilitate nutrition uptake and promote metabolic exchange.53-54 32


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It would be ideal that TENGCs bear biological properties functionally benefiting Schwann cells, a key type of cells during nerve repair, towards effective regeneration.10 Notably, our GSC was not only biologically compatible with Schwann cells, but also appears to possess a bioactivity of promoting Schwann cells’ secretion of regeneration-favorable cytokines, NT-3 and NT-4 (members of neurotrophin gene family), two axonal guidance molecules playing important functional roles in stimulating nerve regeneration,43, 55-56 and preventing denervation-induced muscle atrophy.57 Given that the immature Schwann cells secrete many neurotrophic factors (including BDNF, NGF and NT-4) but not NT-3,5 the up-regulated NT-3 in Schwann cells activated by the GSC would help complete the cytokine profile required for effective nerve regeneration. A common way to improve regenerative effects is to incorporate neurotropic factors into TENGs that subsequently release these factors once implanted in vivo.58 Since most of neurotrophins are proteins with poor stability, short half-lives, and high cost,15, 59-60 we explored the possibility of repurposing clobetasol, a FDA-approved drug known for its therapeutic effects on inflammatory skin diseases,26 to serve as a potential neurotrophin alternative. This notion was well justified by our cell biological and biochemical data that uncovered clobetasol’s two regeneration-favorable activities: (1) it activated PI3K/Akt signaling to promote the proliferation of RSC96 Schwann cells that are fundamentally critical to nerve regenerations;61 (2) clobetasol was capable of stimulating the production of two myelin-related genes in RSC96 Schwann cells, NRG1 and EGR2. Given that NRG1 is a potent inducer of myelination and EGR2 activates Schwann cells to enter the myelinating stage,62-63 the enhanced production of these two factors would contribute to the improved remyelination observed for the regenerated peripheral nerves. These newly-identified features 33



in conjunction with clobetasol’s known anti-inflammatory function, good stability and easy storage,64-65 make clobetasol a promising neurotropic agent for peripheral nerve regeneration. Indeed, our in vivo experiment in a rat sciatic transection injury model (10-mm gap) indicated that (1) the GSC alone leads to a moderate recovery in motor and sensory functions as well as nerve conductivity, which is further improved to be comparable to autografting by the inclusion of clobetasol into the GSC; (2) the GSC allows successful regeneration of the nerve fragment re-bridging the large defect with moderate quantitative recovery of nerve fibers and the incorporation of clobetasol further elevates the quantitative recovery of nerve fibers to a level close to autografting (Table 2). Such favorable outcome was evidenced by the three restored key functional parameters (SSI for motor function, relative reaction time for sensory function, and NCV for nerve conductivity), and the recovered morphology and microstructure of nerve fibers (myelination, diameter, and axons density). This clobetasol-boosted regeneration was likely in part because of clobetasol’s aforementioned two new activities. Nevertheless, although the clobetasol-loaded GSC effectively suppressed fibrosis formation to a level similar to the autografting, it seemed not equally effective in preventing the atrophy of gastrocnemius muscle, revealing a need of further improving the clobetasol-loaded GSC towards maintaining the function and structure of target muscles. Further, this conduit could to be functionalized with other bioactive groups for effectively delivering Schwann cells or be incorporated with topological cues in the inner surface for repairing even larger nerve gaps.

CONCLUSION The application of clobetasol-loaded GSC to the rats with transected sciatic nerves resulted in 34


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an effective regeneration of injured nerves. Such high effectiveness was attributable to three main mechanisms: (1) the nerve guidance conduit performed regeneration-favorable mechanical and structural properties; (2) the degradation products of the sericin conduit promoted Schwann cells to secrete neurotropic factors (NT-3 and NT-4); (3) clobetasol activated PI3K/Akt signaling to enhance Schwann cell proliferation, and promoted Schwann cells to produce positive myelination regulators (NRG1 and EGR2). Of note, the inability of the GSC on its own to achieve this effectiveness reflected the important role of clobetasol in peripheral nerve regeneration, which was never reported before. This sericin conduit possesses the ability to be further functionalized for repairing even larger nerve gaps, suggesting its clinical translational potential.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications websites: Formation and gelation dynamics of the sericin hydrogels, quantitative real-time PCR analysis on RSC96 Schwann cells, phase contrast micrographs of RSC96 Schwann cells, RSC96 Schwann cells were treated with glucocorticoid antagonist (RU486) or Akt inhibitor (MK2206), a sciatic nerve with a 10-mm defect cannot be spontaneously reconnected and self-regenerated, assessment of vascularization in sciatic nerve regeneration at Week 12 postoperatively, oligonucleotide primers for qRT-PCR, the inner diameter and wall thickness of the sericin conduit in different state and original data of Western blotting (PDF)

AUTHOR INFORMATION Corresponding Authors 35



* Phone: 86-27-85726612. E-mail: [email protected]. * Phone: 86-27-85726612. E-mail: [email protected]. * Phone: 86-27-85726612. E-mail: [email protected]. Author Contributions # These

authors contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors appreciate Miss Yan Zhang and Mr. Bo Cai (Huazhong University of Science and Technology, Wuhan, China) for their kind assistance and suggestions. This work was supported by the National Natural Science Foundation of China Programs (81572866 and 81671904), the International Science and Technology Corporation Program of Chinese Ministry of Science and Technology (2014DFA32920), and 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).

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For Table of Contents Use Only Sericin Nerve Guidance Conduit Delivering Therapeutically Repurposed Clobetasol for Functional and Structural Regeneration of Transected Peripheral Nerves Lei Zhang,#, † Wen Yang, #, † Hongjian Xie, #, † Hui Wang, #, ‡ Jian Wang,† Qiangfei Su,† Xiaolin Li,† Yu Song,† Guobin Wang,*, § Lin Wang,*, †, || and Zheng Wang*, †, §

4.76 cm high x 8.38 cm wide

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Sericin Nerve Guidance Conduit Delivering ... - ACS Publications

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