Bioactive Self-Assembling Peptide Hydrogel ... - ACS Publications

Subscriber access provided by Kaohsiung Medical University

Tissue Engineering and Regenerative Medicine

Bioactive Self-Assembling Peptide Hydrogel Functionalized with BDNF- and NGF- mimicking Peptides Synergistically Promote Peripheral Nerve Regeneration Changfeng Lu, Yu Wang, Shuhui Yang, Chong Wang, Xun Sun, Jiaju Lu, Heyong Yin, Wenli Jiang, Haoye Meng, Feng Rao, Xiu-Mei Wang, and Jiang Peng ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00536 • Publication Date (Web): 18 Jun 2018 Downloaded from http://pubs.acs.org on June 28, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering Bioactive Self-Assembling Peptide Hydrogel Functionalized with BDNF- and NGF- mimicking Peptides Synergistically Promote Peripheral Nerve Regeneration Changfeng Lu†,‖,1, Yu Wang†,‖,1, Shuhui Yang‡,1, Chong Wang†,‖, Xun Sun†,§, Jiaju Lu‡, Heyong Yin⊥, Wenli Jiang†, Haoye Meng†,‖, Feng Rao†, Xiumei Wang‡,*, Jiang Peng†,‖,* †

Institute of Orthopedics, Chinese PLA General Hospital, Fuxing Road 28#, Beijing 100853, PR China; Beijing Key Lab of

Regenerative Medicine in Orthopedics, Fuxing Road 28#, Beijing 100853, PR China; Key Lab of Musculoskeletal Trauma & War Injuries, PLA, Fuxing Road 28#, Beijing 100853, PR China ‡

State Key Laboratory of New Ceramics and Fine Processing, School of Materials Science and Engineering, Tsinghua University,

Beijing 100084, China §

‖

School of Medicine, Nankai University, Weijin Road 94#, Tianjin 300071, PR China

Co-innovation Center of Neuroregeneration, Nantong University, Nantong, Jiangsu Province 226007, PR China

⊥

Experimental Surgery and Regenerative Medicine, Department of Surgery, Ludwig-Maximilians-University (LMU),

Nussbaumstr. 20, Munich 80336, Germany. 1 These authors contributed equally to this paper. Corresponding Authors *E-mail: [email protected] *E-mail: [email protected]

ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 35

Abstract Various artificial materials have been fabricated as alternatives to autologous nerve grafts in peripheral nerve regeneration, and these afford positive recovery effects without the disadvantages of the gold standard. In this study, we prepared a three-dimensional

functionalized

self-assembling

peptide

nanofiber

hydrogel

containing

two

neurotropic

peptides

(CTDIKGKCTGACDGKQC and RGIDKRHWNSQ derived from NGF and BDNF, respectively), which reflected the structure and properties of the neural extracellular matrix. The material was used to promote axonal regrowth and functional recovery. Scanning electron microscopy revealed a three-dimensional porous matrix within the hydrogel. Circular dichroism spectroscopy and atomic force microscopy confirmed that the peptides displayed β-sheet structure and self-assembled into long nanofibers. Rheology measurements and atomic force microscopy indicated that the elasticity of the peptide hydrogels was close to that of the nerve tissue matrix. In vitro work with Schwann cells and dorsal root ganglia showed the hydrogels exhibited good cell compatibility. Furthermore, the hydrogel containing CTDIKGKCTGACDGKQC and RGIDKRHWNSQ promoted the neurite outgrowth of PC12 cells significantly compared with non-functionalized peptide. In vivo, the hydrogels were placed into chitosan tubes and used to bridge 10-mm-long sciatic nerve defects in rats. We found that the combination of CTDIKGKCTGACDGKQC and RGIDKRHWNSQ accelerated axonal regeneration and afforded good functional recovery, suggesting that they synergistically facilitate peripheral nerve regeneration. Keywords Peripheral nerve regeneration; Self-assembling peptide; Neurotropic peptides; Nanofiber; Hydrogel 1. Introduction Peripheral nerve injury caused by trauma or neuropathic disease remains a major clinical problem worldwide, significantly affecting quality of life and creating enormous financial burdens.1 Therefore, peripheral nerve regeneration has attracted a great deal of attention. It is known that peripheral nerves can regenerate; but regeneration must be rapid to allow functional recovery of both nerves and muscles. Currently, the gold standard therapy for peripheral nerve regeneration is autologous nerve graft implantation into large nerve defects, with the grafts reflecting the natural, anatomical internal topography.2, 3 However, associated problems include microstructural mismatch, a requirement for donor-site surgery, few donor sources, and excessive graft length.4, 5


Page 3 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering Hence, good alternative treatments, including a microenvironment that supports neurogenesis, are in great demand.6 Bioengineered tissue scaffolds often seek to mimic not only natural nanofibrous structures that are 50 to 500 nm in size but also the biochemical composition of the extracellular matrix (ECM) in efforts to improve nerve regeneration.6-8 Almost all tissue is composed of nanofibrous structures (those of collagen, bone, cartilage, nerves, and skin).9-11 In vivo, the nerve ECM is a three-dimensional nanofibrous structure featuring pores at the nanometer scale allowing communication among fibers. Both the surface receptors and the functional cell domains are also nano-scale in nature, facilitating cell-to-cell interactions. Accordingly, scaffolds with three-dimensional nanostructures may maximally mimic the structure of the natural ECM and thus perform biological functions.8, 12, 13 Natural bottom-up self-assembly is a new method by which to fabricate nano-scale biomaterials. It features spontaneous molecular changes from a state of disorder to a state of order that depend on the formation of non-covalent bonds (e.g., charge-based and hydrogen bonds, and ionic bonds).14 Self-assembling peptides (SAPs) undergo ordered self-assembly into stable secondary structures, forming membranes or hydrogels, which may be used for in vivo regeneration of brain tissue, peripheral nerves,

and

heart

and

bone

tissue

and

as

three-dimensional

scaffolds

for

cell

culture.15-22

RADA16-I

(Ac-RADARADARADARADA-CONH2) is a typical (and widely used) self-assembling peptide exhibiting high-level biocompatibility, low cytotoxicity, a 3D structure facilitating cell growth, and good integration into variously shaped wounds. The material is composed of repeated segments containing positively charged arginine (R) and hydrophobic alanine (A) and negatively charged aspartic acid (D).6, 23 RADA16-I forms very stable β-sheet structures self-assembling into nanofibers about 10 nm in diameter when exposed to a salt solution or alkaline pH. A 3D hydrogel containing > 99% water and pores 5–200 nm in diameter, resembling the structure of the natural ECM, is thus created. RADA16-I facilitated peripheral nerve growth.6, 16, 17, 24 Furthermore, various bioactive short-peptide motifs (analogs of growth factors and ECM macromolecules) are easily conjugated to the C-terminus of RADA16-I to balance the acidity of the solution and to promote specific cellular responses.17, 22, 25 Short functional motifs of IKVAV (Ile-Lys-Val-Ala-Val) and RGD (Arg-Gly-Asp) have been conjugated to RADA16-I and then used to trigger peripheral and central nerve regeneration.26-28 The specific functions of the SAPs depend principally on the motifs chosen. Growth factors and neurotrophic factors, including nerve growth factor (NGF) and brain-derived neurotrophic factor



(BDNF), stimulate morphological differentiation and regulate neuronal gene expression and are required by mature neurons to maintain neuronal function and phenotype.29 NGF, a homodimeric protein, plays crucial roles in neuronal survival, differentiation, and growth.30 Exogenous NGF can prevent or delay peripheral neuropathies in various tissues.31 Most importantly, both in vitro and in vivo studies have shown that NGF promotes survival, differentiation, and the function of peripheral sensory and sympathetic cells.32 BDNF affords neuroprotection and promotes the rescue and regeneration of injured neurons.33 BDNF promoted axonal regrowth after sciatic nerve injury and motor recovery after peripheral nerve injury.34-37 However, the use of growth factors and neurotrophic factors is sometimes limited by high costs, short half-lives, controversial sources, and patient vulnerabilities.38 The peptide CTDIKGKCTGACDGKQC mimics the biological activities of NGF, exhibiting better pharmacokinetics and fewer side-effects than NGF itself, both in vitro and in vivo.39 The short peptide RGIDKRHWNSQ derived from BDNF was linked to the C-terminus of RADA16-I to fabricate a scaffold that filled the injured brain cavity, reduced the surrounding reactive gliosis, and provided neurotrophic factors to the milieu.40 CTDIKGKCTGACDGKQC and RGIDKRHWNSQ are promising short motifs for attachment to RADA16-I when it is sought to enhance peripheral nerve repair. Furthermore, considering that NGF mediates sensory recovery and BDNF motor recovery, a combination of NGF and BDNF may further accelerate the regrowth of regenerating axons and allow better recovery of important muscles. Our principal aim was to construct novel scaffolds using functionalized self-assembling peptides based on RADA16-I to mimic the functions of NGF and BDNF, and investigated the synergetic effect of CTDIKGKCTGACDGKQC and RGIDKRHWNSQ on promoting peripheral nerve regeneration. We first used both the BDNF-derived peptide RGIDKRHWNSQ and the NGF-derived peptide CTDIKGKCTGACDGKQC to repair peripheral nerves. The two peptides CTD and RGI were synthesized by direct extension from the C-terminus of RADA16-I (solid phase synthesis), and to improve function further, two amino acids (GG) were placed between RADA16-I and both CTD and RGI. These functionalized self-assembling peptides were then mixed with pure RADA16-I to obtain stable hydrogels. Four kinds of nanofiber hydrogels RADA16-I (RAD), RADA16-I with CTD (RAD/CTD), RADA16-I with RGI (RAD/RGI), and RADA16-I with both CTD and RGI (RAD/CTD+RGI) were used in this study. Circular dichroism, atomic force microscopy, scanning electron microscopy and rheology measurements were used to characterize the secondary structures, the morphologies and mechanical properties of the self-assembling nanofiber hydrogels.


Page 4 of 35

Page 5 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering Next, the hydrogels served as 3D or 2D cell culture matrices for growth of Schwann cells or dorsal root ganglia (DRG) to evaluate cell attachment and compatibility. And PC12 cells were cultured on different hydrogels for investigating the neurite growth induced by the hydrogels. Finally, the hydrogels were injected into hollow chitosan tubes and used to bridge 10-mm-long sciatic nerve defects in rats. The axonal regeneration and functional recovery were assessed. 2. Materials and Methods All experimental procedures involving animals were performed in accordance with the Guides for the Care and Use of Laboratory Animals of the Chinese Ministry of Public Health and the US National Institutes of Health. 2.1 Peptide synthesis and fabrication of self-assembling peptide hydrogels The self-assembling peptide RADA16-I (Ac-RADARADARADARADA-CONH2) and the functionalized peptides CTD (Ac-RADARADARADARADA-GG-CTDIKGKCTGACDGKQC-NH2)

and

RGI

(Ac-RADARADARADARADA-GG-RGIDKPHWNSQ-NH2) (purity > 90%) were custom-synthesized and purified by China Peptides Co. Ltd. (Shanghai, China). Peptide purities and identities were confirmed by analytical high-performance liquid chromatography (HPLC). Peptide powders were dissolved in distilled water to 1% (w/v), filter-sterilized with Acrodisc Syringe Filters (0.2-µm-pore-sized HT Tuffrun membranes; Pall Crop., Ann Arbor, MI, USA), and sonicated for 30 min (VCX 130PB, Sonics, CT, USA). According to previous study on different ratios of functional motifs (1%, 10%, 40%, 70% and 100%) for peptide functions, a content of 40%-70% was optimal.41 So 50% of functionalized peptide was used as standard in this study. Fewer functionalized motifs may cause inadequate activity while too many motifs can weaken the mechanical strength of hydrogels, which further influences the regeneration. In addition, the main purpose of this study is to evaluate the synergistic effects of RGI and CTD to simulate neuro-related growth factors. Pure RGI and CTD were used as the control groups and a ratio of 1:1 with RADA16-I was adopted. To be comparable, RGI and CTD composite group had a total functional peptide of 50%, of which RGI and CTD had a ratio of 1:1. Under the above consideration, functionalized peptide solutions were mixed with a 1% (w/v) solution of pure RADA16-I at a volume ratio of 1:1 to obtain the functionalized peptide mixtures RAD/CTD and RAD/RGI or at a volume ratio of 2:1:1 to obtain the RAD/CTD+RGI mixture. Self-assembling peptide hydrogels were fabricated within cell culture transwell inserts (10 mm in diameter, Millipore,



Billerica, MA, USA) as described by Wang et al.16, 18 Briefly, sterilized inserts were placed in 24-well culture plates, and 400 µL of culture medium was added to each well. After adequate insert madefaction, 100-µL amounts of peptide solutions were gently added, followed by incubation at 37°C for 15 min to allow gelation. Another 400 µL of culture medium was then carefully added to each hydrogel, followed by incubation at 37°C for 15 min. The entire medium in the well was changed at least four times to equilibrate the hydrogel to the physiological pH followed by incubation at 37°C overnight. 2.2 Characterization 2.2.1 Circular dichroism (CD) We obtained CD spectra of peptide secondary structures by using Chirascan plus (Applied Photophysis, UK). Peptide solutions (1% [w/v] in water) were diluted to working concentrations of 0.01% (w/v), and 200-µL amounts of diluted samples were analyzed at room temperature with a 1-mm path length. CD spectra were recorded over the range 190–260 nm, with 1-nm steps and a 3-nm bandwidth. All samples were evaluated three times and the data averaged. To further investigate the secondary structures of peptides, the software CDPro was applied to estimate secondary structure contents. The peptides’ secondary structure fractions were measured by CONTINLL algorithm compared to a set of selected reference proteins (Ibasis10 [SMP56], λ = 240 – 190 nm). 2.2.2 Atomic force microscopy (AFM) Pure RADA16-I and functionalized peptide solutions (1% w/v) were diluted to 0.01% (w/v), dropped onto freshly cleaved mica surfaces for 30 s, and then rinsed with 300 µL distilled water. After air-drying, the samples were immediately subjected to AFM (Bruker Dimension ICON, Billerica, MA, USA) using a silicon scanning probe (ScanAsyst-Air\SNL-A) operating in the contact mode; the scan area was 1 × 1 µm, and the scan rate was 1.01 Hz. In addition, the mechanical properties of the self-assembling peptide hydrogels were measured by AFM.42 The measurements were performed by using V-shaped silicon SNL-D probes (nominal spring constant 0.06 N/m, Bruker Nano, Inc., Malvern, PA, USA). Briefly, the cantilever tips were modified by attaching 20-µm-diameter microspheres. Then, 500 measurements were made randomly on the surface of each hydrogel. Force-indentation plots were fitted with a Hertz model to determine Young’s moduli for all samples.


Page 6 of 35

Page 7 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering 2.2.3 Scanning electron microscopy (SEM) After gelation, the hydrogels were fixed in 2.5% (w/v) glutaraldehyde for 30 min and dehydrated through a graded series of ethanol baths (30, 50, 70, 80, 90, 95, and 100%; all v/v) for 30 min in each bath; they were then subjected to CO2 critical-point drying (Samdri-PVT-3D instrument, Tousimis, USA). Fresh fracture surfaces were sputter-coated with a layer of Pt prior to SEM (Zeiss, Germany). 2.2.4 Rheology Mechanical properties of the hydrogels were measured by MCR rheometer (Aaton Paar Physica MCR301/302, Austria) equipped with an 8-mm diameter parallel plate and a 0.75-mm truncation gap. After gelation, the hydrogel was positioned on the sample loading stage. Dynamic frequency sweep test (0.1 – 10 rad/s at 0.5% strain) was performed for each sample at room temperature (25 ℃). The storage modulus (G’) and loss modulus (G’’) of RAD, RAD/CTD, RAD/RGI and RAD/CTD+RGI hydrogels were determined. 2.3 Cell culture assay 2.3.1 Isolation and 3D culture of Schwann cells (SCs) Schwann cells were harvested and purified as described previously.8, 12 Briefly, the sciatic nerves were completely dissected and removed from 3-day-old Sprague–Dawley rats; they were enzymatically dissociated with 1 mL of 0.2% (w/v) collagenase NB4 (Sigma-Aldrich) after trituration for 5 min. The mixtures were stirred at 37°C for 10 min, centrifuged, and resuspended in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F12) supplemented with 10% (v/v) fetal bovine serum. Cell culture transwell inserts were prepared as described above. Purified SCs were suspended in 10% (w/v) sucrose immediately before seeding, and peptide solutions were mixed with 20% (w/v) sucrose at a volume ratio of 2:1. Twenty-microliter amounts of cell suspension (500,000 cells) were quickly mixed with 100-µL amounts of the sucrose-containing peptide solutions. The mixtures were gently added to the inserts, and 400-µL amounts of Schwann cell culture medium were carefully and slowly layered onto the hydrogels to allow for gelation. The plates were incubated at 37°C for 15 min, and the medium was changed; this was followed by a further 30 min of incubation and at least two further medium changes to ensure that the 3D cell cultures were at physiological pH. After growth for 3 days, the SCs were fixed with 4% (v/v) paraformaldehyde for 30 min, permeabilized with



Page 8 of 35

0.3% (v/v) Triton X-100 for 5 min, and incubated in 10% (v/v) Normal Goat Serum (Solarbio) for 10 min at room temperature. The samples were then stained with a primary antibody against S100 (1:200; mouse polyclonal antibody, Abcam) overnight at 4°C. The secondary antibody Alexa 488 (1:200; goat anti-mouse polyclonal antibody, Abcam) was then added, and incubation continued for 1 h. After washing three times with PBS, the samples were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min, washed three times with PBS, and imaged via confocal laser scanning microscopy (LSM710, Leica, Germany). 2.3.2 Isolation and 2D culture of rat dorsal root ganglia (DRG) DRG were isolated from 12-h-old Sprague–Dawley rats using a previously described method.8 In brief, DRG were extracted through the lateral intervertebral foramen, and their outer membranes were removed under a microscope using microforceps. Cell culture transwell inserts were prepared as described above. The medium in the wells was removed, and the purified DRG were cultured on self-assembling peptide nanofiber hydrogels that had been fabricated in the inserts and grew for over 6 days. The culture medium [DMEM/F12 (Gibco, USA) with 2% [w/v] B-27 (Gibco, USA) and 1% [w/v] penicillin-streptomycin] was changed every 2 days. Then, DRG on the hydrogels were fixed in 4% (v/v) paraformaldehyde for 30 min, permeabilized with 0.3% (v/v) Triton X-100 for 5 min, and incubated with 10% (w/v) Normal Goat Serum (Solarbio) for 10 min at room temperature. Next, the DRG were incubated with a mouse anti-tubulin primary antibody (1:1000; Abcam) at 4°C overnight and then with the secondary antibody for 1 h. Finally, the nuclei were stained with DAPI, followed by confocal laser scanning microscopy. 2.3.3 Analysis of PC12 cell neurite outgrowth on hydrogels PC12 cell line was obtained from Tiandz, Inc. (China) and cultured in RPMI Medium 1640 (Invitrogen, 11875-093) containing 10% horse serum (Gibco) and 5% fetal bovine serum (Gibco). The PC12 cells were seeded on the hydrogels RAD, RAD/RGI, RAD/CTD and RAD/CTD+RGI at a density of 2.5 × 104 cells/ hydrogel, and then incubated in an atmosphere containing 5% CO2 at 37℃ for 48 h to allow cell attachment. Then the medium was changed with PC12 differentiation media (RPMI Medium 1640, 1% horse serum, 50 ng/mL Nerve Growth Factor) for an additional 3 days for neurite differentiation. After that, PC12 cells were stained with Rhodamine Phalloidin (Cytoskeleton, Inc., US) and imaged by confocal laser scanning microscopy. The length of neurite outgrowth in different groups was measured by the software Image Pro Plus 6.0. 2.4 The rat sciatic nerve defect model of peripheral nerve injury


Page 9 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering 2.4.1 Surgical procedure Fifty healthy male Sprague–Dawley rats aged 8 weeks and weighing 200–250 g were kept in the Experimental Animal Center of the Chinese PLA General Hospital. The rats were randomly divided into five groups (10 rats/group): the hollow-chitosan-nerve-conduit (Hollow) group; the chitosan-nerve-conduit-filled-with-RAD/CTD-nanofiber-hydrogel (RAD/CTD) group; the RAD/RGI-nanofiber-hydrogel (RAD/RGI) group, the RAD/CTD+RGI-nanofiber-hydrogel (RAD/CTD+RGI) group; and the autologous-nerve-graft (Autograft) group. During surgery, all rats were anesthetized via injection of 3% (w/v) sodium pentobarbital solution (2.5 mg/100 g body weight), and the hair of the right femur was removed. Next, the sciatic nerve of the right hind leg was exposed by making a skin incision and splitting the muscles, and a segment of the nerve was removed, creating a 10-mm gap after retraction of the nerve ends. These gaps were bridged using the various conduits or autologous nerves, and the muscle and skin were sutured. All rats were then housed and fed as usual, and all changes were monitored. 2.4.2 Immunofluorescence and immunohistochemical staining Two weeks after surgery, two rats in each group were killed, and the tissues in the original nerve gap were harvested and cut into 10-µm-thick longitudinal sections with the aid of a freezing microtome (Leica, Germany). The sections were stained with NF200 (1:800, Abcam) to reveal regenerated axons and P0 (1:200, Abcam) to show myelin protein, following a published method.8 Briefly, the sections were incubated with 10% (v/v) Normal Goat Serum (Solarbio) for 10 min at room temperature; they were then stained with a mixture of two primary antibodies against NF200 and P0 (binding to axons and myelin protein, respectively), followed by the addition of the secondary antibodies Alexa 488 and Alexa 594 for 1 h. After washed three times with PBS, the sections were stained with DAPI for 5 min. The frozen sections were stained with hematoxylin-eosin (HE). All stained slides were observed using an Axio-Scan instrument (Zeiss, Germany) 2.4.3 Immunohistochemical analysis and transmission electron microscopy (TEM) At 12 weeks after surgery, five of the remaining rats in each group were killed, and the tissue-containing conduits were harvested. Next, the distal ends of regenerated nerves were removed and fixed in pre-cooled 2.5% (w/v) glutaraldehyde for 3 h. They were then post-fixed in a 1% (w/v) osmium tetraoxide solution for 1 h, washed, dehydrated, embedded in Epon 812 epoxy resin, and cut into 700-nm-thick semi-thin sections and 70-nm-thick ultrathin sections. The semi-thin sections were stained with



Page 10 of 35

1% (w/v) toluidine blue/1% (w/v) borax solutions and observed under a light microscope to evaluate the density of myelinated axons. After stained with lead citrate and uranyl acetate, the 70-nm-thick ultrathin sections were subjected to TEM (Philips, Holland), and images of 10 random fields in each section were taken to determine the thickness of the myelin sheaths and the diameter of the myelinated nerve fibers. Image Pro Plus 6.0 software was used to analyze the pictures. 2.4.4 Electrophysiological assessment At 12 weeks after surgery, the sciatic nerves on the injured side were re-exposed under anesthesia with sodium phenobarbital, and electrical stimulation (3 mA) was applied between the proximal and distal nerve stumps. The compound muscle action potentials (CMAPs) were recorded at the target gastrocnemius muscles using a recording electrode. The CMAP latencies and peak amplitudes were calculated and compared between the groups. 2.4.5 Motor functional assessment using the CatWalk gait analysis system The extent of the functional motor recovery was evaluated using the CatWalk XT 9.0 gait analysis system (Noldus, Wageningen, the Netherlands) at 2, 4, 6, 8, 10, and 12 weeks after surgery. The rats (n=3/group/time point) were placed on the right side of the runway (which had a glass surface and black plastic walls), and each run was captured by a high-speed camera located under the runway. The stand time, contact area, and impact intensities of the right injured hind paw (RH) and the normal left hind paw (LH) were recorded. The Sciatic Function Index (SFI) was calculated using the following formula:

(1), where ETS reflected the experimental toe spread; NTS reflected the normal toe spread; EPL reflected the experimental print length; NPL reflected the normal print length; EIT reflected the experimental inter-toe spread; and NIT reflected the normal inter-toe spread. 2.4.6 Ultrasonography of the gastrocnemius muscle At 6 weeks after surgery, color ultrasonography was used to assess the morphology and elasticity of the gastrocnemius muscles of the injured and contralateral sides. A TOSHIBA Apolio 500 instrument fitted with a linear array probe operating at a frequency of 5–14 MHz was used for this purpose, and real-time shear-wave elastography (SWE) was performed. Briefly, the


Page 11 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering

surface projection of the gastrocnemius muscle was fully exposed, and 2D ultrasound and color Doppler flow imaging were performed to reveal the size, shape, and echo of the muscle and the nature of the blood supply. The system was then switched to the continuous excitation mode and the probe pressure released. After the images were stabilized by adjusting the sampling frame size, the images were frozen and the mode changed to the speed propagation graph mode. For images exhibiting good timelines, regions of interest were selected using the default circle of the instrument and the Young’s modulus value (the EI) was calculated. The standard deviations were automatically recorded. 2.4.7 Determination of muscle wet weight ratios and remodeling of muscle fibers At 12 weeks after surgery, gastrocnemius muscles harvested from the injured and contralateral hindlimbs (n=5/group/time point) were immediately weighed to derive wet weight ratios and then fixed in 4% (v/v) paraformaldehyde at 4°C. Muscle samples were transversely cut into 7-µm-thick paraffin sections and subjected to Masson’s trichrome staining. For each sample, images were taken of 10 randomly chosen fields under a light microscope, and the data were quantitatively analyzed with the aid of Image Pro Plus 6.0 software. 2.5 Statistical analysis All numerical data are presented as means ± standard deviations. Comparisons were performed using analysis of variance (ANOVA) and the acceptable level of statistical significance was set at p

Bioactive Self-Assembling Peptide Hydrogel ... - ACS Publications

Recommend Documents