Peptide-Tethered Hydrogel Scaffold Promotes Recovery from Spinal

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Peptide-tethered Hydrogel Scaffold Promotes Recovery from Spinal Cord Transection via Synergism with Mesenchymal Stem Cells Li-Ming Li, Min Han, Xinchi Jiang, Xianzhen Yin, Fu Chen, Tian-Yuan Zhang, Hao Ren, Jiwen Zhang, Zhong Chen, Tingjun Hou, HongWei Ouyang, Yasuhiko Tabata, Youqing Shen, and Jian-Qing Gao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b12829 • Publication Date (Web): 06 Jan 2017 Downloaded from http://pubs.acs.org on January 8, 2017

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ACS Applied Materials & Interfaces

Peptide-tethered Recovery

Hydrogel

from Spinal

Scaffold

Cord

Promotes

Transection

via

Synergism with Mesenchymal Stem Cells Liming Li #,a, Min Han #,a, Xinchi Jiang a, Xianzhen Yin b, Fu Chen c, Tianyuan Zhang a, Hao Ren d

, Jiwen Zhang b, Tingjun Hou c, Zhong Chen e, Hongwei Ouyang

d, f

, Yasuhiko Tabata g,

Youqing Shen h, Jianqing Gao*, a, f a

Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou

310058, Zhejiang, China. b

Center for Drug Delivery System, Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, Shanghai 201210, China c

d

College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China Center for Stem Cell and Tissue Engineering, School of Medicine, Zhejiang University,

Hangzhou 310058, China. e

Department of Pharmacology, Key Laboratory of Medical Neurobiology of The Ministry of

Health of China, Zhejiang Province Key Laboratory of Neurobiology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.

ACS Paragon Plus Environment

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f

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Dr. Li Dak Sum & Yip Yio Chin Center for Stem Cell and Regenerative Medicine, Zhejiang

University, Hangzhou 310058, China g

Department of Biomaterials, Field of Tissue Engineering, Institute for Frontier Medical

Sciences, Kyoto University, Kyoto, Japan. h

Center for Bionanoengineering and Key Laboratory of Biomass Chemical Engineering of

Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China

KEYWORDS. hyaluronic acid, hydrogel scaffold, mesenchymal stem cells, adhesive peptide, spinal cord injury, tissue engineering

ABSTRACT. Spinal cord injury (SCI) is one of the most devastating injuries. Treatment strategies for SCI are required to overcome comprehensive issues. Implantation of biomaterial scaffolds and stem cells has been demonstrated to be promising strategies. However, a comprehensive recovery effect is difficult to achieve. In the comprehensive treatment process, the specific roles of the implanted scaffolds and of stem cells in combined strategy are usually neglected. In this study, a peptide-modified scaffold is developed based on hyaluronic acid and an adhesive peptide PPFLMLLKGSTR. Synchrotron radiation micro computed tomography measurement provides insights to the three-dimentional inner topographical property and perspective porous structure of the scaffold. The modified scaffold significantly improves cellular survival and adhesive growth of mesenchymal stem cells during 3D culture in vitro. After implantation in transected spinal cord, the modified scaffold and mesenchymal stems are


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found to function in synergy to restore injured spinal cord tissue, with respective strengths. Hindlimb motor function scores exhibit the most significant impact of the composite implant at two weeks post injury, which is the time secondary injury factors begin to take hold. Investigation on the secondary injury factors including inflammatory response and astrocyte overactivity at 10 days post injury reveals the possible underlying reason. Implants of the scaffold, cells and especially the combination of both elicit inhibitory effects on these adverse factors. The study develops a promising implant for spinal cord tissue engineering and reveals the roles of the scaffold and stem cells. More importantly, the results provide the first understanding of the bioactive peptide PPFLMLLKGSTR concerning its functions on mesenchymal stem cells and spinal cord tissue restoration.

1. Introduction Spinal cord injury (SCI) is among the most devastating traumatic injuries and can result in permanent neurological damage and locomotor deficit.1 Primary injury results in an irreversible loss of neurons and a disruption of axons in spinal cord tissue. After primary injury, serious inflammatory response and glial scar formation can cause further damage to the normal nerve tissues near the lesion site. Complications of secondary injury and the extremely limited selfrecovery capacity of central nervous system (CNS) tissue make hamper efforts to restore neurons and functions after SCI.2, 3, 4 Stem cell and biomaterial implantation are promising strategies to facilitate nerve tissue regeneration after SCI.5 Stem cells are broadly considered to be effective candidates to replace lost neurons.6 However, implanted cells struggle to survive and function in the injured tissue


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because of the absence of extracellular matrix (ECM) and restricted inflammatory microenvironment after spinal cord trauma. Therefore, scaffolds that are based upon biomaterials have been extensively studied to bridge the injured tissues and provide cellular niches for stem cells. Scaffolds applied alone or in combination with stem cells have been demonstrated to be vitally important in spinal cord regeneration.7,

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Constructing a permissive three-dimensional

(3D) scaffold niche for stem cells and neural tissues has become a key project for nerve tissue regeneration. The soft natural neural tissue and complicated microenvironment, which includes cytotoxic neurotransmitters, lead to strict demands for scaffold construction. To mimic the natural neural tissue ECM, scaffolds are required to be hospitable in materials, mechanically soft and adhesive for stem cells and host neurons. In this way, the implantation of scaffolds and cells is being used to overcome the issues of initial injury and secondary injury, including neuron loss, axonal disruption, glial scar barriers, and inflammatory responses. Hyaluronic acid (HA) is a polysaccharide that broadly exists in natural ECM. Favorable functions of HA in CNS tissue repair have been revealed through several studies.9, 10, 11 These functions include an anti-inflammatory and inhibitory effect on glial scar formation. Scaffolds constructed based upon HA have been demonstrated to be safe and enhancing of spinal cord regeneration.12,

13, 14

HA can react with cell surface receptors, such as CD44. However, this

impact is not enough for cellular adhesive growth in 3D HA scaffolds. In the native matrix, cells adhere to the ECM through the reaction between integrin and laminin. PPFLMLLKGSTR, a motif sequence derived from laminin-5 α3 chain, has been confirmed to be the major binding site for α3β1 integrin.15 Tethering of this peptide on chitin16, 17 and collagen materials18, 19 have been reported to promote cell adhesion and survival. The promoting effects of this peptide on neurite adhesion have also been studied.20 However, to the best of our knowledge, there is no report


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regarding the function of PPFLMLLKGSTR on stem cells. In particular, the incorporation of HA scaffolds during CNS tissue regeneration and its impacts of bioactive peptide are poorly studied. Among stem cells, bone marrow mesenchymal stem cells (MSCs) are easily isolated and expanded in vitro. MSCs are a population of multipotent stem cells that can differentiate into neuronal cells under certain conditions. Animal studies have shown a positive effect of MSCs on spinal tissue recovery, which may be attributed to the function of MSCs in integration into injured tissues,21, 22 the secretion of neurotrophic factors23 and the immunoregulatory functions of MSCs.24,

25, 26

Although nerve tissue regenerative effects of MSCs and hydrogel scaffolds

implantation have been reported,27, 28 scant attention has been devoted to the synergetic and independent roles of both cells and cell niches in the developed implant. In this study, a hydrogel scaffold was constructed using HA and modified by the peptide PPFLMLLKGSTR to promote the adhesive growth of MSCs. Design of the study is illustrated in Scheme 1. We evaluated the cellular adhesion and viability in the scaffold and observed significant improvement in cellular behaviors after peptide modification. Internal topography and porous structures of the modified hydrogel scaffold were further presented through synchrotron radiation micro computed tomography (SR-µCT) measurement. Next, the modified scaffold was investigated for in vivo spinal cord repair. The scaffold supported MSCs to survive in the lesion site and to be integrated into host tissue. Implantation of MSC-encapsulated scaffold significantly facilitated nerve tissue integration and neuronal regeneration and also showed an inhibitory effect on glial scar formation and inflammatory cell infiltration. To make clear the single roles of both the modified scaffolds and MSCs in the combinatory implant, scaffolds and MSCs were also implanted and investigated independently, thereby indicating that the recovery


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of the transected spinal cord was attributed to the synergistic effect of the PPFLMLLKGSTR peptide-modified scaffold and the inner sustained MSCs. Scheme 1. Illustration of the experimental scheme.

2. Experimental Section Materials and animals. HA (MW 2.3 MDa) was provided by Novozymes (Beijing, China). Adipic dihydrazide (ADH) was purchased from Aladdin (Shanghai, China). Ethyl N, Ndimethylaminopropyl carbodiimide (EDC) and 1, 10-carbonyldiimidazole (CDI) was purchased from Sigma-Aldrich (St Louis, MO). Male Sprague Dawley (SD) rats at three weeks of age for MSCs insulation and female SD rats (220-250 g) for spinal cord injury study were purchased from Slac Laboratory Animal Co. Ltd. (Shanghai, China). All animal procedures and experiments complied with the guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Zhejiang University.


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Preparation of hydrogel scaffold and peptide modification. The hydrogel scaffold was prepared through crosslinking of HA and ADH. Sodium salt of HA (0.05 g) was dissolved in distilled water (7 ml) and was fully stirred for complete dissolution. The pH of the HA solution was adjusted to 4.5 with HCl (0.1 M) before ADH (0.1 g) was added. After fully mixing, EDC (0.35 g) was added and vigorously stirred for rapid mixing. Next, the mixture was placed into 12-well plates for gel formation at room temperature (25 °C). The formed hydrogel was lyophilized after washed in PBS five times. Lyophilized scaffolds were cut into slices measuring 1 mm in thickness. For peptide modification, the hydrogel scaffold was dehydrated with a series of graded acetone before a 15-min activating reaction by CDI (500 mg) in dry acetone (5 ml).29 The peptide solution of 10 ml was prepared with 2 mg of the peptide PPLFMLLKGSTR dissolved by DMSO (200 µl) and NaHCO3 (100 mM, PH= 8.4) added to the final volume. The activated scaffold was washed by NaHCO3 for five times to remove the remaining CDI and the residual acetone before the peptide tethering reaction. The reaction system was gently stirred for 36 h at room temperature (25 °C), and then the scaffold was thoroughly washed by PBS for five times before lyophilization. Samples of the peptide solution before and after the reaction were collected and examined for peptide concentration using a BCA protein assay kit (Beyotime, Shanghai, China). The reactants and products in scaffold preparation and modification were detected by nuclear magnetic resonance (NMR). Hydrogels were measured for the storage and loss modulus with a plate-to-plate rheometer (Physica MCR, Anton Paar, Ashland, VA) using a 25 mm plate at 37 °C under a constant strain of 0.1% and frequency ranging from 0.1 rad/s to 10 rad/s.


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Before in vitro cell culture and in vivo use, the scaffolds were immersed in 75% ethanol for sterilization and then washed by PBS and cell culture medium. SR-µCT measurement. The internal structure of the peptide-modified scaffold was characterized using SR-µCT tomography with the BL13W1 beam line (Shanghai Synchrotron Radiation Facility, SSRF). After consideration of the X-ray spectral flux profile, sample properties include composition and density, synchrotron radiation X-ray at 13.0 keV was used for the scans to obtain higher spectral flux and reduction in the imaging times required. To enhance the contrast, the sample-to-detector distance was set at 12 cm after a series of preliminary experimental studies. After penetrating the sample, the X-ray was converted into visible light by a Lu2SiO5: Ce scintillator (10 µm thickness). The projections were magnified using diffraction-limited microscope optics (×10 magnification) and digitized using a highresolution 2048 pixel × 2048 pixel sCMOS camera (ORCA Flash 4.0 Scientific CMOS, Hamamatsu K.K, Shizuoka Pref., Japan). The pixel size was 0.65 µm and the exposure time was 1 s. For each acquisition, 1080 projections over 180° were collected. Light field images (i.e. Xray illumination on the beam path without the sample) and dark field images (i.e. X-ray illumination off) were also collected during each acquisition to account for the electronic noise and variations in the brightness of the X-ray source. The CT scan was a 3D map of the X-ray phase contrast. Further analysis of the data was required for a quantitative description of the microstructure. The projected images for the samples were reconstructed using the X-TRACT SSRF CWSx64 (Commonwealth Scientific and Industrial Research Organization, Australia, http://www.ts-imaging.net/Default.aspx) to perform a direct filtered back projection algorithm. To enhance the quality of reconstructed slices, propagationbased phase contrast extraction was carried out. After phase retrieval and reconstruction, a


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simple linear rescaling is utilized to transform the reconstructed CT images to a grey value of 0 to 255 (8 bit grey level). The 3D rendered data were analyzed with the commercially available Amira (Version 6.0, FEI, USA) and Image Pro Analyzer 3D software (Version 7.0, Media Cybernetics, Inc., USA). After the 3D reconstruction, the resolved images have a high quality phase contrast. The 3D microstructure of matrix can be visualized at a high resolved level. Isolation and culture of rat MSCs. MSCs for in vitro experiments were isolated from the bone marrow of 3-week old male SD rats according to the method described previously.30 Briefly, the femurs and tibias were harvested after the rats were sacrificed. The ends of the bones were removed and the marrow was flushed out using DMEM (Gibco BRL, Gaithersberg, USA) supplemented with 10% FBS, L-glutamine, penicillin (100 IU/mL), and streptomycin penicillin (100 µg/mL). The marrow was filtered through a 100-µm mesh and centrifuged for 5 min at 1200 rpm to collect the cells. Next, the cells were suspended with 10 mL culture medium in a 100-mm dish and cultured at 37 °C and 5% CO2 atmosphere. The medium was changed on the next day and every two days thereafter. Cells of passage two or three were used for experiments. For in vivo study, to track the exogenous implanted cells, green fluorescent protein (GFP) labelled MSCs (Xingming, Shanghai, China) were used. GFP-MSCs were cultured according to the manual protocol. Before 3D cell culture, the scaffolds were balanced using cell culture medium. Sub-confluent MSCs or GFP-MSCs were detached from the flask. A total of 5 × 104 cells in 20 µL culture medium were injected from the scaffold surface into interior using a pipette. The overflow were repeatedly injected for several times to ensure that enough cells were seeded into the scaffold.


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In vitro cell viability and morphology in scaffolds. MSCs were seeded into unmodified hydrogel scaffolds and peptide modified scaffolds respectively to investigate cellular viability and morphology in the different scaffolds. Detection for cell viability. After 1 day, 7 days, 14 days and 21 days of culture, MSCs encapsulated in scaffolds were stained using Live/Dead cell-staining reagent (Invitrogen Corp., Carlsbad, California, USA). The samples were observed using laser scanning confocal microscopy (BX61W1-FV1000, Olympus, Japan). In randomly selected fields, scaffolds were scanned according to the z-axis between the boundaries of cell growth. Thickness between the boundaries was measured and differently coloured cells were numbered to characterize the function of the scaffolds to support MSC growth. Cytotoxicity of scaffolds-soaked culture medium was evaluated by MTS assay with the use of flow chambers. Culture medium in chambers without scaffolds was used as the control system. Immunocytofluorescense staining for cell adhesion and phenotype. To investigate whether the scaffolds can support adhesive growth and neuronal differentiation of MSCs, cells in scaffolds were stained for actin, focal adhesion proteins and neural markers. For actin and focal adhesion proteins staining, MSCs were cultured in scaffolds for 3 days. For neural markers staining, MSCs were cultured in the scaffold for 7 days forβ3-tubulin staining, 14 days for microtubule associated protein 2 (MAP2) staining and 21 days for neurofilament (NF) staining. MSCsencapsulated scaffolds were rinsed thoroughly by PBS and fixed with 4% paraformaldehyde (Boster, Wuhan, China) for 15 min. Then, after being washed by PBS, the samples were permeabilized in 0.1% Triton-X100 (Amresco, Ohio, America) for 10 min and blocked with goat serum (Boster) for 20 min. Primary antibodies (β3-tubulin, MAP2, NF, Vinculin, Paxillin) were incubated with the samples for 1 h at 37 °C. After being washed by PBS three times, samples


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were incubated with secondary antibodies for 1 h at 37 °C. Details for the primary and secondary antibodies are listed in Table S1. Rhodamine-phalloidine (Invitrogen) was used to label actin filaments with a 30 min incubation at room temperature. Cell nuclei were stained with diamidino-phenyl-indole (DAPI) (Invitrogen) by incubating for 8 min at room temperature. The samples were observed using laser scanning confocal microscopy (BX61W1-FV1000, Olympus, Japan, and LSM710NLO, Zeiss, Germany) and Z-stack images were taken. Scanning electron microscopy. Scaffolds cultured with MSCs were examined by scanning electron microscopy (SEM) after 5 days of culture. The samples were washed by PBS and fixed in glutaraldehyde for 2 h and in osmium tetroxide for 1 h. Then, fixative was washed off and the samples were dehydrated with graded ethanol. Finally, a fracture was made on the samples and examined by SEM (S3000N, Hitachi, Japan) after drying and coating with gold. Fracture of lyophilized scaffold was also coated with gold and observed under SEM to investigate the inner porous structure of the scaffold. Surgery for spinal cord transection and implant embedding. Spinal cord transection surgery were conducted on female SD rats weighting 220-250 g. For the transplantation treatment, rats were randomly separated into four groups: spinal cord injury group (SCI) without implant, peptide-modified scaffold group (Pep-Sca) with an implant of the modified scaffold, MSC group (MSC) with implantation of MSCs, and Pep-Sca-MSC group with an implant of MSC-embedded modified scaffold (full implant). Scaffolds were spinal cord-shaped, 1.5 mm in thickness. MSCembedded scaffolds were seeded with 5 × 104 MSCs. All scaffolds were prepared and incubated in cell culture medium 1 day before washed by PBS for transplantation.


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For surgical procedures, rats were anaesthetized with 10% chloralic hydras. After a laminectomy, the dorsal surface of the T9-10 segment was exposed. The spinal cord was transected to make a 1.5 mm gap using microscissors. After exact hemostasis，the prepared implants were embedded into the gap using microforceps and were ensured to fit the lesion cavity. In MSC group, 5 × 104 MSCs in 20µL PBS were implanted into the lesion cavity. Finally, the muscle and skin were sutured. For postsurgical care, animals received manual bladder expression twice daily until reflexive bladder control returned. Behavioural assessment. Over the duration of 10 weeks post-implantation, hindlimb locomotor function was assessed weekly using the Basso, Beattie and Bresnahan (BBB) open-field locomotor test. Rats were placed on a piece of non-slip floor mat in an open-field. Each animal was observed for 4 min by two examiners blinded to the treatment. At least six rats were tested in each treatment group each time. Immunohistochemistry. To make multiaspect evaluation on the effect of the implants in nerve tissue recovery, spinal cords were harvested at 10 days, 4 weeks and 8 weeks post-surgery. Animals were perfused with isotonic physiological saline followed by 4% paraformaldehyde (Boster) under deep anaesthesia. Spinal cords were dissected for a 1.5-cm longitudinal segment spanning the injury sites and cryosectioned at 20 µm or 8 µm. For fluorescence labelling, primary antibodies of GFP, β3-tubulin, NF, glial fibrillary acidic protein (GFAP) and CD31, CD68 were incubated with the sections at 20 µm overnight at 4 °C. Alexa Fluor 488- and 594-conjugated secondary antibodies were incubated with the sections at 37 °C for 1 h. Nuclei were labelled using DAPI. For biotin labelling of GFAP and CD68 on sections at 8 µm, Horse Radish Peroxidase (HRP)-conjugated corresponding secondary


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antibodies were used. Haematoxylin and eosin (H&E) staining was conducted on sections at 20 µm or 8 µm to investigate the joint of the transected tissue and the cellular morphology in the lesion site. Morphological quantification. For investigation of cell viability in scaffolds, living cells (labelled green) and dying cells (co-localized by green and red to be yellow) in Live/Dead assay were counted. Dead cells were not analysed because they can be lost from the scaffolds during the culture and staining procedure. For each sample, 10-15 randomized visual fields (20× lens) were analysed by ImageJ 1.48v software. For standardization of in vivo results, every fifth section in at least ten sections were stained and two visual fields (20× lens) in each of the caudal, central, rostral parts were analysed in one section. For quantitation of GFP-positive MSCs, visual fields in lesion sites and in tissue near to the implants were respectively processed. Pixel areas of GFP labelling and NF labelling were measured and converted to mm2 by ImageJ and the results were averaged over all sections in one group. For CD68 labelling with biotin, percentage of CD68-positive cells were calculated by counting the positively labelled cell number, while nuclei labelling indicated total cell number by ImageJ and dividing the values. All results were analysed by OriginPro software. Statistical analysis. The quantitative data were reported as the mean ± standard deviations (SD). Statistical analysis was performed using Student’s t-test. Differences were determined to be significant at P

Peptide-Tethered Hydrogel Scaffold Promotes Recovery from Spinal

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