Materials
PLGA (80:20, Mw: 50,000- 75,000) and chitosan (low molecular weight with a deacetylation degree of >75%), Collagenase type I, 4,6-diamidino-2-phenylindole (DAPI), laminin, trypsin–ethylene diamine tetra acetic acid (EDTA) and anti-neurofilament 160/200 were supplied by Sigma-Aldrich (USA). N, N-dimethyl formamide (DMF) and chloroform were purchased from Merck (Germany). Dulbecco’s Modified Eagles Medium (DMEM) /F12, phosphate buffered saline (PBS), fetal bovine serum (FBS) and penicillin–streptomycin was purchased from Invitrogen. BDNF and AuNPs were obtained from R&D (USA) and US NANO (USA), respectively. Anti-S100, anti-myelin basic protein (anti-MBP), rabbit anti-mouse FITC, and goat anti-mouse alexa flour were purchased from Abcam, fresh frozen plasma (FFP) was used to prepare thrombin, and cryoprecipitate was used to prepare fibrinogen (FFP and cryoprecipitate were obtained from the Blood Bank of Isfahan Province (Isfahan, Iran).
Electrospinning and characterization of PLGA nanofibers
All of materials which used in this study were prepared from Sigma-Aldrich, St. Louis, MO, USA except it was mentioned in the text. Electrospun scaffolds with mild modification were fabricated as described in our previous study [35]. PLGA (80:20, Mw: 50,000- 75,000) was dissolved in chloroform: DMF (Merck, Darmstadt, Germany) mixture with a volume ratio of 80: 20 to obtain a concentration of 20% (w/v). High voltage electric field of 21 kV was used to draw the polymer solution fed at a rate of 250 µL/h into nanofibers over a distance of 15 cm from the needle tip to an aluminum-wrapped rotating drum. The drum rotation speed was gently decreased from 2500 to 300 rpm. Therefore, a highly aligned nanofibrous inner surface was formed, and as the drum rotation speed was decreased, fiber orientation gradually diminished. Electrospinning process continued for 9 h, and randomly oriented fibers of outer surface endowed the scaffold with isotropic mechanical properties, while the gradual alteration in fiber orientation could avoid sheet delamination. Scanning electron microscopy (SEM) (SEM, Seron Technology AIS 2500, India) was used to assessed the morphology of PLGA. Therefore, electrospun PLGA samples were coated with a thin layer of gold prior to taking SEM images.
PLGA sheets coating with laminin containing BDNF and AuNPs encapsulated chitosan nanoparticles
AuNP or BDNF encapsulated chitosan nanoparticles were produced by the ionotropic gelation method . After emulsification of 50 ppm AuNPs (US Research Nanomaterials, Inc, Houston, TX, USA) [18] or 5 µg/ ml BDNF (R&D systems, Minneapolis, MN, USA) with 0.1 % chitosan solution, the solution was mixed on a magnetic stirrer for 15 min. Then, 0.03% TPP of an aqueous solution as a cross linker was added dropwise into the previous solution.The 20 µg/ ml of laminin was used for supply suspention of chitosan nanospheres containing BDNF or AuNPs, and finally was coated on the PLGA scaffold at 4°C temperature for 24 h [24] .
Design and fabrication of nerveconduit
PLGA sheet was cut to the size (14×20 mm) and exposed to UV light for sterilization. The membrane was coated with chitosan nanospheres encapsulated BDNF or AuNPs mixed by 20 µg/ ml laminin. Next, the electrospun mat rolling 2.5 rounds around a mandrel to create a tubular structure. The edge of conduit was sealed with cyanoacrylate glue. All these procedures were performed under sterile conditions.
Rat-ADSCs isolation, expansion
All of the experimental procedures involving animals were conducted in accordance with the guidelines given by National Institute of Health Guide for the Care and Use of Laboratory Animals, and approved by the Animal Ethics Committee of Isfahan University of Medical Sciences (No: IR.MUI.REC.1396.3.207). Under sterile conditions, the isolated tissue was placed on ice to the stem cell laboratory. Briefly, the adipose tissue surrounding the inguinal region was cut into tiny segments and treated with 0.075% collagenase type I and shaken at 37°C for 35 to 40 min. The resultant to neutralizing enzyme activity was added to the DMEM (Gibco Grand Island, NY, USA) containing 10% fetal bovine serum (Gibco Grand Island, NY, USA) into each tube. After centrifugation (1200 rpm for 5 min), the upper fat tissue layer and supernatant were discarded. The cell pellet was transferred into culture flasks containing DMEM + 10% FBS and transferred into an incubator at 5% CO2 and 37°C and saturated humidity. After 3-4 days, the medium was refreshed, and the cells were sub-cultured until passage 4. In this study, h-ADSCs obtained from passage 3-4 were used for the experiments.
Operation procedure and experimental groups
In this study, 40 mature (12-week-old) Wistar male rats (weighing ~200–250 g) purchased from Pasteur Institute, Tehran, Iran and were housed under standard conditions (at 18-24°C, 12 h light/dark) with free access to laboratory pellet chow and water. Acclimatization of animals began 10 days before the experiment date. Each rat was kept in one cage during experimental period.
All of the experimental procedures involving animals were conducted in accordance with the guidelines given by International Council for Laboratory Animal Science (ICLAS) for the Care and Use of Laboratory Animals, and approved by the Animal Ethics Committee of Isfahan University of Medical Sciences (No: IR.MUI.REC.1396.3.207). The rats were randomly divided into five groups (each, n = 8), including autograft group (Auto ) as positive control, PLGA conduit coated by laminin and filled with DMEM/F12 (PL), PLGA conduit coated by laminin and filled with 2×106 rADSCs according to previous study [36] (PLC), PLGA conduit coated by laminin containing BDNF-CNPs, AuNPs-CNPs and filled with 2×106 rADSCs (PLGBC) and PLGA conduit coated by laminin containing BDNF-CNPs, AuNPs-CNPs and filled with 2×106 rADSCs suspended in fibrin matrix (PLGBCF) transplanted in 10 mm sciatic gap.
Each rat was anesthetized with intraperitoneal injection of Xylazine 10mg/kg and Ketamine 100mg/kg. The thigh areas on left sides were shaved and disinfected with povidone-iodine.
Then, the sciatic nerve was exposed by skin and muscle-splitting incision.
The 10 mm of sciatic nerve segment was removed and PLGA conduit coated by laminin and filled with DMEM/F12 (PL), PLGA conduit coated by laminin and filled with 2×106 rADSCs for each animal (PLC) and PLGA conduit coated by laminin containing BDNF-CNPs, AuNPs-CNPs and filled with 2×106 rADSCs for each animal (PLGBC) implanted in different groups.
Also, the 10 mm segment of sciatic nerve was excised, reversed, and without rotation reattached using 10-0 nylon as an autograft group. Finally, in the fibrin matrix group include PLGA conduit coated by laminin containing BDNF-CNPs, AuNPs-CNPs and filled with 2×106 rADSCs suspended in fibrin matrix (PLGBCF) for each animal, 60 μl human fibrinogen containing 2×106 cells with 40 μl thrombin was injected into the nerve conduit.
The proximal and distal cut ends of the sciatic nerve were inserted 2 mm into the ends of the nerve conduit and fixed with a 10-0 nylon epineural suture.
Then, the muscle and skin were closed with 7-0 nylon sutures. Finally, the rats had free access to water and food, and housed in controlled animal rooms under artificial 12-hour light/dark cycles.
Sciatic function index
Functional recovery of the sciatic nerve was evaluated by the sciatic functional index (SFI) obtained from rat footprints on the white paper covered on the box floor. Three obvious footprints were selected from each rat, and finally three different parameters were measured: (i) heel to the third toe (print length, PL), (ii) the first toe to the fifth toe (toe spread, TS), and (iii) the second toe to the fourth toe (intermediate toe spread, ITS). The SFI was calculated by the following formula:
SFI = −38.3 (EPL − NPL)/NPL + 109.5(ETS − NTS)/NTS + 13.3(EIT − NIT)/NIT − 8.8
E is the experimental sides, and N is the normal sides. Generally, a value of close to 0 indicates the normal function and a value of close to -100 indicates the higher impairment [37].
Pinprick test
To determine of sensory recovery, the pinprick test was performed. The experimental limb of rats was pinched with standardized forceps from the toe to the heel. In the pinprick test limb withdraw response to painful stimulus was graded from 0 to 3. Pinprick test was evaluated from heel to toes and measured values include: 0 = no response, 1 = heel, 2 = dorsum of foot (mid-foot), and 3 = toes [38].
Electrophysiology
For each animal, electrophysiological testing was performed in a 12-week post-surgery period. After anesthesia , the electrophysiologic method was performed by placing stimulation electrode in the proximal end of the regenerated nerve and recording electrodes in the mid of gastrocnemius muscle. The compound muscle action potential (CMAP) has been used to estimate the numbers of repaired motor nerve fibers. The CMAP parameters were analyzed for each rat; for instance, the CMAP latency parameter was assessed from the stimulation site to the start of the response and measured in milliseconds.
In addition, the amplitude (AMP) of CMAPs was calculated by the potential difference between maximum negative and positive peaks of the CMAP signal in millivolts [39]. Electrophysiological analysis was calculated using the eProbe software. Finally, the CMAP parameters were compared among different groups.
Muscle mass
After sacrificing of animals (with 100 mg/kg sodium pentobarbital i.p.), gastrocnemius muscles were removed from normal and injured sides and weighed while still wet by an electronic balance (A&D Weighing EK-3000I Portable Balance, 3000g Capa
city). The recovery rate and muscle atrophy were calculated by the wet weight of gastrocnemius muscle on the experimental side/the wet weight of gastrocnemius muscle on the normal contralateral side × 100% [40].
Histological study
Operated nerve and gastrocnemius muscle of both limbs were dissected from the surrounding tissues 12 weeks after conduit implantation. The middle segment of muscles and nerve were fixed in 10% neutral buffered formalin for 48 hours, and after tissue processing, paraffin blocks were cut into 5 μm thick sections. The transvers sections of gastrocnemius muscle were stained with Masson’s trichrome and examined under a light microscope. The mean diameter of the muscle fibers was calculated using the Digimizer software by an image analysis system. Nerve samples were stained with toluidine blue to label the myelin sheath. Briefly, the fixed nerves were dehydrated in a graded ethanol series and embedded in paraffin. The specimens were cut into 5 μm thick cross sections with a microtome, stained with 0.125% toluidine blue solution, observed and evaluated by under a light microscope [41].
The G-ratio (the axon-to-fiber diameter ratio) is an important parameter to estimate degree of myelination [42].To calculate the mean number of myelinated axons, nine images were randomly captured at 400× magnification for each group, and 5 fields of all images were counted to obtain the mean numbers of myelinated nerve fibers. The average diameter of the sciatic regenerated nerve fibers, axon, myelin and the mean number of myelinated axons were calculated using the Digimizer software by an image analysis system.
Immunohistochemistry staining
In this study, S100 β as a specific marker for Schwann cells, myelin basic protein (MBP) as a myelin sheath marker, and neurofilaments-200 (NF200) as a growing axon marker were used. Nerve specimens were fixed with 4% paraformaldehyde for 6 h and embedded in paraffin and then cut into 3-μm thick sections. For double immunostaining analysis of NF200 and MBP or NF200 and S100 in the sections, antigen unmasking was performed by 10 mM sodium citrate buffer, pH= 6.0, at 90 °C for 10 min. Fallowing, were incubated for 30 min in blocking serum (0.5% normal goat serum to cover unspecific binding) and then incubated for overnight at 4°C with the primary antibodies diluted involve: anti- S100 β (1:500), anti-major basic protein anti-MBP (1:500), and NF200 (1:100) )(Abcam, Cambridge, MA). They were washed three times with PBS and incubated in secondary antibodies: rabbit anti mouse FITC (1:500) and goat anti mouse Alexa Flour (1:1000) (Abcam).
Finally, all sections were incubated with DAPI for cell nuclei staining, and immunohistochemistry results were examined under a fluorescent microscope (Olympus BX51, Japan). Then, the total area of the images was measured for the intensity of NF200, S100 and MBP staining using the Image-j software [43].
Real-time reverse transcription polymerase chain reaction
The level of S100, MBP, GFAP and Nestin expression in the rat sciatic nerve tissue in were assessed using real time RT-PCR. Total RNA was isolated using the Total RNA Prep Kit (BIOFACT). RNA was reverse transcribed using the BioFact™ 5X RT Pre-Mix cDNA Synthesis Kit (BIOFACT) according to the manufacturer’s protocol. The real-time PCR was performed using BioFact™ 2X Real-Time PCR Master Mix Kit (BIOFACT) and the StepOne Plus™ quantitative Real Time PCR Detection System (Applied Biosystems). The level of β-actin was used as the control housekeeping gene. Table 1 lists the sequence of the used primers (metabion, Germany) [23].
Statistical analysis
Statistical differences between the different groups were tested by the one-way ANOVA followed by post-hoc, tukey’s test using SPSS software version 22.0 (SPSS Inc., Chicago, IL, USA). Data were presented as mean ± standard error and P-values less than 0.05 were considered statistically significant.