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. 2017 Aug 19;38(3):703–713. doi: 10.1007/s10571-017-0535-8

Polyurethane/Gelatin Nanofibrils Neural Guidance Conduit Containing Platelet-Rich Plasma and Melatonin for Transplantation of Schwann Cells

Majid Salehi 1, Mahdi Naseri-Nosar 1, Somayeh Ebrahimi-Barough 1, Mohammdreza Nourani 2, Arash Khojasteh 3, Saeed Farzamfar 4, Korosh Mansouri 5, Jafar Ai 1,✉
PMCID: PMC11481952  PMID: 28823058

Abstract

The current study aimed to enhance the efficacy of peripheral nerve regeneration using a biodegradable porous neural guidance conduit as a carrier to transplant allogeneic Schwann cells (SCs). The conduit was prepared from polyurethane (PU) and gelatin nanofibrils (GNFs) using thermally induced phase separation technique and filled with melatonin (MLT) and platelet-rich plasma (PRP). The prepared conduit had the porosity of 87.17 ± 1.89%, the contact angle of 78.17 ± 5.30° and the ultimate tensile strength and Young’s modulus of 5.40 ± 0.98 MPa and 3.13 ± 0.65 GPa, respectively. The conduit lost about 14% of its weight after 60 days in distilled water. The produced conduit enhanced the proliferation of SCs demonstrated by a tetrazolium salt-based assay. For functional analysis, the conduit was seeded with 1.50 × 104 SCs (PU/GNFs/PRP/MLT/SCs) and implanted into a 10-mm sciatic nerve defect of Wistar rat. Three control groups were used: (1) PU/GNFs/SCs, (2) PU/GNFs/PRP/SCs, and (3) Autograft. The results of sciatic functional index, hot plate latency, compound muscle action potential amplitude and latency, weight-loss percentage of wet gastrocnemius muscle and histopathological examination using hematoxylin–eosin and Luxol fast blue staining, demonstrated that using the PU/GNFs/PRP/MLT conduit to transplant SCs to the sciatic nerve defect resulted in a higher regenerative outcome than the PU/GNFs and PU/GNFs/PRP conduits.

Keywords: Gelatin, Melatonin, Neural guidance conduit, Platelet-rich plasma, Polyurethane, Schwann cells

Introduction

Neural repair following peripheral nerve damages is limited, often requires sophisticated surgical procedures to bridge the injury gaps (Dai et al. 2013). The gold standard to bridge the peripheral nerve gaps >5 mm is the autologous nerve grafts. However, this treatment is associated with a variety of clinical complications, such as donor site morbidity, limited length of the nerve that can be harvested, and the formation of neuroma (Nectow et al. 2012). To cope with these limitations, artificial neural guidance conduits (NGCs) have been introduced by researchers as a substitute for their ease of mass production (Salehi et al. 2017). Despite of their advantages, current designs are not as efficacious as autografts and are limited by a narrow range of options (De Ruiter et al. 2009). Thus, development of the NGCs which can match the effectiveness of an autologous nerve graft would be beneficial to the field of peripheral nerve surgery.

To produce NGCs with ultimately good neural regeneration, material selection and optimization is an important step. From natural polymers to the synthetic polymers and blends, there are numerous options to capitalize on different properties of each material, such as mechanical strength, biocompatibility and degradability (De Ruiter et al. 2009). Natural polymers are characterized by their poor mechanical properties, causing the NGCs to kink or collapse, and undergo fast degradation in vivo (De Luca et al. 2014). Polyurethanes are synthetic polymers used in a wide variety of applications because of their extensive structure/property diversity such as excellent mechanical properties, elasticity and flexibility, high elongation, and good biocompatibility (Chen et al. 2015). However, cell affinity towards synthetic polymers is generally poor as a consequence of their low hydrophilicity and lack of surface cell-recognition sites. Improving the hydrophilic property and incorporation of cell-recognition sites such as RGD (Arg-Gly-Asp) and extracellular matrix (ECM) bioactive proteins have been carried out to enhance the cell-synthetic polymer interactions (Ghasemi-Mobarakeh et al. 2008). Gelatin is a natural polymer derived from collagen and has an almost identical composition as that of collagen (Liu et al. 2009). The selection of gelatin as a conduit material can decrease the concerns of immunogenicity and pathogen transmission associated with collagen (Parenteau-Bareil et al. 2010). To introduce gelatin into NGCs, one potential strategy is to incorporate it in the form of nanofibrils into NGCs. Such small dimension of fibrils can physically mimic the structural properties of native ECM (Salehi et al. 2017; Lu et al. 2015).

With the advancement of production techniques during the previous decades, structures of NGCs have been greatly improved to satisfy different kinds of requirements including porous and fibrillated structures with good permeability and degradability, along with proper mechanical properties to resist collapse when they are used in vivo (Cui et al. 2008). Electrospinning and thermally induced phase separation (TIPS) are two production techniques which are able to successfully produce such structures (Liu et al. 2013; Salehi et al. 2016a, b). Electrospinning is one of the most established processes for producing nanofibrillated structures that mimic the ECM (Wang et al. 2013), whereas the TIPS can produce porous structures with high permeability to nutrients (Salehi et al. 2015). Recently, enrichment of NGCs’ structures with agents that can enhance the outgrowth of axons has been suggested as a strategy to improve the peripheral nerve repair (Pfister et al. 2007). A number of neuroscientists demonstrated that pineal neurohormone melatonin (MLT) has an effect on the morphologic features of the neural tissue, suggesting its neuroprotective, free radical scavenging, antioxidative, and analgesic effects in degenerative diseases of peripheral nervous system (Odaci and Kaplan 2009; Stavisky et al. 2005; Kong et al. 2008). At present, it is widely accepted that MLT possesses useful effects on axonal sprouting, myelination of developing peripheral nerves, inhibition of collagen accumulation and neuroma formation following traumatic injuries of peripheral nervous system (Turgut and Kaplan 2011; Atik et al. 2011). Another agent which its positive effects on neural repair have been demonstrated is platelet-rich plasma (PRP) (Farrag et al. 2007). The enhancing effect of PRP is based on the premise that a large number of platelets in the PRP release a significant amount of growth factors that aid the healing process. These growth factors act locally to recruit undifferentiated cells to the site of injury, trigger mitosis in these cells, and induce angiogenesis (Abbasipour-Dalivand et al. 2015). Another strategy being developed to improve the peripheral nerve repair is cell therapy which represents a powerful approach to repair the neural damages (Salehi et al. 2017; Frattini et al. 2012). Schwann cells (SCs) are a promising candidate which play an important role in peripheral nerve regeneration by providing bioactive substrates on which axons migrate and release molecules that regulate axonal proliferation (Webber et al. 2011; Sun et al. 2009).

In this study, an NGC was produced from polyurethane and gelatin nanofibrils through the combination of electrospinning and TIPS techniques for transplantation of SCs. Prior to implantation, NGCs were seeded with SCs and filled with PRP gel containing MLT. This conduit simultaneously provided a structural guidance for axonal regeneration, as well as a carrier for therapeutic agents and SC transplantation. This construct was tested in a rat sciatic nerve defect, which simulates the clinical long-gap peripheral nerve injury, in order to assess its ability to support the neural repair in clinical settings.

Materials and Methods

Chemicals

The materials and solvents were purchased from Sigma-Aldrich (St. Louis, USA) and Merck (Darmstadt, Germany) respectively unless otherwise noted.

Preparation of NGC

Production of Gelatin Nanofibrils via Electrospinning

Gelatin solution [40% (w/v)] was prepared by dissolving the gelatin powder (bovine skin, type B) in acetic acid aqueous solution [75% (v/v)] on a magnetic stirrer at room temperature. The solution was loaded into a 10-mL disposable syringe ended to an 18-gauge stainless steel needle connected to a high voltage source (Fanavaran Nano-Meghyas, Tehran, Iran) which was set to 20 kV. A syringe pump (Fanavaran Nano-Meghyas, Tehran, Iran) was used to feed the solution at the constant flow rate of 0.50 mL/h. The extruded solution was collected at room temperature on an aluminum-foil 15 cm away from the needle tip. The electrospun mat was transferred to a nitrogen tank and after 24 h crushed to small pieces (GNFs). The morphology of the GNFs was observed by scanning electron microscope (SEM; KYKY Technology Development, Beijing, China) at an accelerating voltage of 26 kV, after sputter coating with gold for 180 s using a sputter coater (KYKY Technology Development, Beijing, China).

Production of NGC via TIPS

Polyurethane (PU; Tecoflex® SG-80A, Thermedics, Woburn, USA) with a total concentration of 6% (w/v) was dissolved in 1,4-dioxane on a magnetic stirrer at room temperature for 24 h. The GNFs were dispersed in PU solution [GNFs to PU: 30% (w/w)] and stirred for 1 h. The mixture was transferred to a −80 °C freezer for 1 h and then freeze-dried (Telstar, Terrassa, Spain) for 48 h. The produced structure was cut into 14-mm tubes (1 mm inner diameter, 3 mm outer diameter) using a water jet cutter. In order to crosslink the gelatin, the tubes were immersed in 1% glutaraldehyde solution for 24 h and then extensively washed in order to remove the residual glutaraldehyde.

Characterization of NGC

The water contact angle was measured using a static contact angle measuring device (KRUSS, Hamburg, Germany) and the contact angles of three points on each sample were averaged. The mechanical properties of three samples of each NGC were measured using a universal testing machine (Santam, Karaj, Iran).

The in vitro degradation was investigated by immersing each NGC in 5 mL of distilled water at 37 °C. After 30 and 60 days, the sample was removed from the water, dried at room temperature and its weight was measured. The degradation was quantified by calculating the weight-loss percentage using the following equation (Naseri-Nosar et al. 2016); where W 0 is the initial weight of the sample and W 1 is its dry weight after removing from the water. Three weight-loss percentages of each NGC were averaged.

Weight-loss%=W0-W1W0×100.

A liquid displacement method was used to determine the porosity of each NGC using the following equation (Salehi et al. 2016a); where V 1 is the initial volume of 96% ethanol, V 2 is its volume after soaking the NGC (and ethanol filled the pores), and V 3 is the volume of the ethanol after removing the NGC. Three porosity percentages for each NGC were averaged.

Porosity%=V1-V3V2-V3×100.

Cell Culture Studies

Primary rat Schwann cells (SCs) isolated from the sciatic nerves of adult male Wistar rats were provided by Dr. S. Ebrahimi-Barough (Tehran University of Medical Sciences, Tehran, Iran). The cells were cultured in Dulbecco’s modified Eagle’s medium: nutrient mixture F-12 (DMEM/F12; Gibco, Grand Island, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco, Grand Island, USA), 100 unit/mL of penicillin, and 100 mg/mL of streptomycin in a humidified incubator at 37 °C with 5% CO2. The proliferation of SCs on NGCs was assessed by 3-(4,5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium Bromide (MTT; Sigma-Aldrich, St. Louis, USA) assay kit according to the manufacturer’s instruction. The NGCs were cut in a size of a well in a 96-well plate, sterilized by ultraviolet light (254 nm) irradiation in a laminar flow hood for 60 min and then were seeded with 5 × 103 SCs. The cells in the wells of the plate without NGCs were treated as positive control. All experiments were performed in triplicate and the absorbance values were read by an Awareness Technology microplate-reader (Palm City, USA) at 570 nm.

In Vivo Studies

Animal experiments were approved by the ethical committee of Tehran University of Medical Sciences and were carried out in accordance with the university’s guidelines.

PRP Isolation

The PRP was extracted according to Kajikawa et al. (2008). Three donor rats were euthanized and decapitated in order to collect their complete body blood. An average of 7–8 mL blood was retrieved per rat and mixed with 1 mL sodium citrate aqueous solution [3.80% (w/v)]. The whole blood was centrifuged at 1500 rpm for 10 min to separate the platelet-rich layer. The layer then was centrifuged at 3000 rpm for 10 min to separate a cell pellet within the supernatant. Most of the supernatant was removed and the pellet then was resuspended in the residual supernatant. The extracted PRP was stored at 4 °C.

Sciatic Nerve Defect

Twelve healthy adult male Wistar rats (3 months old, weighing 250–270 g) were purchased from Pasteur Institute (Tehran, Iran). The rats were randomly divided into four groups: (1) Autograft, (2) PU/GNFs/SCs, (3) PU/GNFs/PRP/SCs, and (4) PU/GNFs/PRP/MLT/SCs. The SC loading was carried out 24 h before implantation by seeding 1.50 × 104 allogeneic cells on each NGC. For surgical procedure, the animals were anesthetized by intraperitoneal injection of Ketamine 5%/Xylazine 2% (25% (v/v), 0.10 mL/100 g body weight). A skin incision was made at the left lower limb of the animals and a 10–mm-long segment of their sciatic nerve was resected. For rats in the NGC groups, the nerve defect was bridged by inserting both proximal and distal nerve stumps into the NGC for 2 mm at each junction. The PRP gel was prepared by mixing 100 μL of PRP with 16.65 μL of 10% (w/v) CaCl2 aqueous solution and 16.65 μL of 300 IU thrombin. After the proximal nerve ending was sutured, 150 μL of the prepared gel was injected into the cell-seeded NGC using an insulin syringe, and later, the distal nerve ending was sutured to the other end of the NGC. The MLT/PRP gel was prepared by blending 3 mg of MLT with 100 μL of PRP and then 150 μL of the MLT/PRP gel was injected into the cell-seeded NGC. For the autograft group, the nerve defect was bridged with the resected nerve segment, which was reversed and anastomosed to the proximal and distal nerve stumps. The NGC and autograft groups were attached using No. 6-0 polyglycolic acid suture (SUPA medical devices, Tehran, Iran) to the proximal and distal stumps.

Walking-Foot-Print Analysis

Four, 8, and 12 weeks post surgery, the rats’ footprints were recorded for the analysis of sciatic functional index (SFI). The rats’ hind paws were soaked in ink and they were placed inside an acrylic corridor (43 cm length, 8.70 cm width, and 5.50 cm height) lined with a millimeter paper and ended with a darkened goal box. SFI was calculated using the following equation (Dijkstra et al. 2000); where PL is the distance from the heel to the top of the third toe, TS is the distance between the first and the fifth toe, and IT is the distance from the second to the fourth toe. NPL, NTS, and NIT were the measures from the non-operated foot and OPL, OTS, and OIT were from operated one. An index of 0 represented the normal function while −100 represented the complete loss of function. The SFI for three rats in each group were averaged.

SFI=-38.30×(OPL-NPL) / NPL)+109.50×(OTS-NTS) / NTS+13.30×(OIT-NIT) / NIT-8.80

Hot Plate Latency Test

Twelve weeks post surgery, the rats were evaluated for hot plate latency (HPL), by placing their injured limb on a hot plate (56 °C) and recording the time until they jumped or licked their paws. Following the response, the rats were removed from the plate. The cut-off time for their reaction was set at 12 s. Latencies from three rats in each group were averaged.

Nerve Conduction Test

Twelve weeks post surgery, the compound muscle action potential (CMAP) amplitude and latency of the sciatic nerve were measured. The animals were anesthetized by intraperitoneal injection of Ketamine 5%/Xylazine 2% (25% (v/v), 0.10 mL/100 g body weight). The sciatic nerve proximal to the site of the injury was stimulated with an electric stimulus (3–5 mA) using needle electrodes. The CMAP amplitude and latency were recorded from the needle and cap electrodes placed on the gastrocnemius muscle (filtering frequency of 10 Hz to 10 kHz, the sensitivity of 2 mV/division and sweep speed of 1 ms/division), using an electromyographic recorder (Negarandishegan, Tehran, Iran). The measurements for three rats in each group were averaged.

Gastrocnemius Muscle Wet Weight-Loss

At the end of 12th week post-surgery, the animals were sacrificed under anesthesia and the posterior gastrocnemius muscles on the injured and uninjured hind limbs were harvested and immediately weighed to determine the wet weight-loss of muscles using the following equation (Naseri-Nosar et al. 2017). The percentages for three rats in each group were averaged.

Gastrocnemius muscle wet weight-loss%=1-Wet weight of the muscle on the injured sideWet weight of the muscle on the uninjured side×100.

Histopathological Examination

The rats’ sciatic nerves at the end of 12th week post-surgery were fixed in a 10% buffered formalin, and after processing and embedding in paraffin, were cross-sectioned and stained with hematoxylin–eosin (H&E) and Luxol fast blue (LFB). The prepared samples were examined under a light microscope (Carl Zeiss, Thornwood, USA) with a digital camera (Olympus, Tokyo, Japan).

Statistical Analysis

The results were statistically analyzed by Minitab 17 software using Student’s t test and the data were expressed as the mean ± standard deviation (SD). In all evaluations, P < 0.05 was considered as the statistically significant.

Results

Incorporation of GNFs Enhanced the Hydrophilicity and Degradability of PU but Decreased its Tensile Strength and Modulus

The SEM image of GNFs (Fig. 1a) revealed that the fibrils were randomly distributed and had a uniform and smooth morphology. The fibrils’ average diameter and distribution were statistically calculated using ImageJ (National Institutes of Health, Bethesda, USA) and OriginPro 2015 (Origin Lab, Northampton, USA) by analyzing a total of 20 random points per image. The fibrils had the average diameter of 304 ± 55.38 nm. In order to increase the cell-loading capacity of NGCs, they were produced highly porous using TIPS technique. The porosity also facilitates the oxygen and nutrient diffusion into the NGCs following implantation in vivo. Figure 1b illustrates the porous structure of PU/GNFs conduit. As Table 1 shows, incorporation of GNFs had almost no effect on the average porosity percentage of PU conduit (87.17 ± 3.01% vs 87.17 ± 1.89% for PU and PU/GNFs conduits, respectively). The GNF incorporation significantly (P < 0.05) decreased the average contact angle of PU conduit from 99.33 ± 4.16° to 78.17 ± 5.30° for the PU/GNFs conduit.

Fig. 1.

Fig. 1

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Characterization of the neural guidance conduits. a Representative SEM micrograph of the electrospun gelatin nanofibrils. The histogram is the diameter distribution from the corresponding SEM image, b representative SEM micrograph of the PU/GNFs conduit, c histogram comparing the weight-loss percentages of the conduits in distilled water after 30 and 60 days, and d the effect of conduits on the proliferation of SCs evaluated by MTT assay. Values represent the mean ± SD, n = 3, *P < 0.05, **P < 0.01 (obtained by Student’s t test)

Table 1.

Characterization of the neural guidance conduits

graphic file with name 10571_2017_535_Taba_HTML.gif
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Values represent the mean ± SD, n = 3, *P < 0.05 (obtained by Student’s t test).

Figure 1c displays the average weight-loss percentages of the NGCs in distilled water after 30 and 60 days. The weigh-loss percentages for PU conduit after 30 and 60 days (0.67 ± 0.15% and 2.37 ± 0.31%, respectively) revealed that its weight remained relatively constant during the test. Incorporation of GNFs significantly (P < 0.01) enhanced the degradation of PU/GNFs conduit which is evident from its weight-loss percentages after 30 days (7.43 ± 1.02%) and 60 days (14 ± 1.60%).

Since the tensile properties have a direct influence on suturing quality of NGCs during the grafting surgery, they were measured and reported in Table 1 (Wang et al. 2009). Incorporation of GNFs decreased the ultimate tensile strength of PU conduit from 6.80 ± 1.14 MPa to 5.40 ± 0.98 MPa and Young’s Modulus from 3.54 ± 1.04 GPa to 3.13 ± 0.65 GPa. All observed differences in the tensile properties results were not statistically significant.

Regenerative Outcome Achieved by PU/GNFs/PRP/MLT/SCs Conduit was Higher Than the PU/GNFs and PU/GNFs/PRP Conduits

The positive effect of NGCs on the physiology of SCs is desirable (Salehi et al. 2017). Thus, the effect of prepared NGCs on the proliferation of SCs was investigated using MTT assay. Incorporation of PRP and MLT with the PU/GNFs conduit not only did not induce any toxic effects towards SCs, it enhanced their proliferation as evidenced by increasing their MTT absorbance values after 48 h and 72 h (Fig. 1d). However, no statistically significant differences were observed between the absorbance values of NGCs. The positive control had significantly higher absorbance values in both time intervals. Figure 2a shows an attached SC to the PU/GNFs/PRP/MLT conduit. The cell body exhibits elongation and establishment of connections to the conduit through its outgrowths which means the conduit favored the growth and normal morphology of SCs.

Fig. 2.

Fig. 2

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a SEM-micrograph (pseudo-colored) of a SC on the PU/GNFs/PRP/MLT conduit exhibiting elongation and establishment of connections to the conduit and b surgical implantation of the prepared conduit in a 10-mm sciatic nerve defect in rat

The prepared NGCs were surgically implanted into a 10-mm sciatic nerve defect of rats (Fig. 2b). As shown in Fig. 3a, the SFI values had the same trend in all time intervals and autograft > PU/GNFs/PRP/MLT/SCs > PU/GNFs/PRP/SCs > PU/GNFs/SCs. The autograft as the gold standard of nerve bridging showed improved SFI value from −66.85 ± 0.77 at the end of 4th week to −51 ± 3.95 and −17.44 ± 0.99 at the end of 8th and 12th weeks, respectively. The PU/GNFs/PRP/MLT/SCs group had the SFI value of −70.17 ± 3.20 after 4 weeks. At the end of 8th and 12th weeks, its SFI reached to the values of −56.90 ± 2.01 and −22.18 ± 1.44, respectively. The SFI values for the PU/GNFs/PRP/SCs group were −75.63 ± 2.32, −60.30 ± 7.06 and −24.77 ± 1.59 after 4, 8 and 12 weeks, respectively. The PU/GNFs/SCs group had the lowest SFI in all time intervals with the values of −77.67 ± 1.02, −66.83 ± 0.85, and −31.10 ± 4.88 at the end of 4th, 8th, and 12th weeks, respectively.

Fig. 3.

Fig. 3

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Functional analysis results 12 weeks post surgery. a Histogram comparing the sciatic functional index (SFI), b histogram comparing the hot plate latency results, c electromyographic results (compound muscle action potential (CMAP) amplitudes and latencies), and d histogram comparing the gastrocnemius muscle wet weight-loss percentages. Values represent the mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.005 (obtained by Student’s t test)

The HPL test was performed to evaluate the nociceptive function of injured limbs, and the results were presented in Fig. 3b. The PU/GNFs/PRP/MLT/SCs group had the smallest HPL time (7.33 ± 1.15 s) among the NGC groups. The HPL time for the PU/GNFs/PRP/SCs and PU/GNFs/SCs were 8 ± 1 and 8.67 ± 1.15 s, respectively. The autograft group had the smallest HPL time (6.33 ± 0.58 s) among all study groups. The differences observed between all HPL times were not statistically significant.

The CMAP amplitude and latency indirectly show the number of regenerated motor neurons and the maturation of nerve fibers, respectively (Yu et al. 2011). Figure 3C shows that the autograft group possessed the highest CMAP amplitude (19.88 ± 1.79 mV). The PU/GNFs/PRP/MLT/SCs group had the closest amplitude (12.58 ± 6.02 mV) to the autograft group. However, their difference was not statistically significant. Significant differences only were observed between the autograft group and PU/GNFs/PRP/SCs (10.05 ± 3.09 mV, P < 0.05) and PU/GNFs/SCs (3.59 ± 0.25 mV, P < 0.005). The CMAP latency results were concomitant with the CMAP amplitudes. The autograft group had the lowest latency (1.43 ± 0.04 ms) but its difference was only statistically significant compared with the PU/GNFs/SCs group (2.83 ± 0.11 ms, P < 0.005). The PU/GNFs/PRP/MLT/SCs group had smaller latency (1.60 ± 0.28 ms) than the PU/GNFs/SCs (P < 0.05) and PU/GNFs/PRP/SCs (2.05 ± 0.42 ms) groups.

The gastrocnemius muscle atrophy can indirectly show the motor neuron defect (Evans et al. 1995). Thus, the wet weight-loss percentage of the gastrocnemius muscle was measured to represent the sciatic nerve regeneration efficacy. Figure 3d shows that the PU/GNFs/PRP/MLT/SCs group had smaller weight-loss percentage (8.20 ± 2.91%) than the PU/GNFs/PRP/SCs (12.81 ± 7.45%) and PU/GNFs/SCs (16.63 ± 8.47%) groups. The autograft group had the smallest weight-loss percentage (7.15 ± 0.99%) among all study groups. The differences observed between all weight-loss percentages were not statistically significant.

The H&E staining image of the PU/GNFs/SCs group (Fig. 4a) presented axonal swelling with a large number of activated SCs and blood vessels. Both PU/GNFs/SCs and PU/GNFs/PRP/SCs (Fig. 4b) groups presented loose and disorganized myelinated nerve fibers. Activated SCs were also observed in less number in the PU/GNFs/PRP/MLT/SCs (Fig. 4c) and autograft (Fig. 4d) groups. Dense, round, uniform, and ordered myelin lamellar structures were only observed in the PU/GNFs/PRP/MLT/SCs and autograft groups. The lamellar spaces in the autograft group were expanded and separated from each other more than the ones for the PU/GNFs/PRP/MLT/SCs group (Chen et al. 2016; Shi et al. 2013).

Fig. 4.

Fig. 4

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Histological analysis of the hematoxylin–eosin stained sciatic nerve cross sections at the end of 12th week post-surgery. a PU/GNFs/SCs, b PU/GNFs/PRP/SCs, c PU/GNFs/PRP/MLT/SCs and d Autograft. B blood vessel, V vacuolar degeneration, L lamellar space, S Schwann cell. Scale bar 500 µm and ×100 magnification

The LFB staining was carried out to observe the changes of myelin sheath (Fig. 5). The LFB stained myelin fibers appears to be blue in color with a gray background. The myelin swelling was evident in the PU/GNFs/SCs (Fig. 5a) and PU/GNFs/PRP/SCs (Fig. 5b) groups. Visible signs of vacuolization and shrinkage of myelin were observed for the PU/GNFs/PRP/MLT/SCs (Fig. 5c) and autograft (Fig. 5d) groups (Wu et al. 2014).

Fig. 5.

Fig. 5

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Histological analysis of Luxol fast blue stained sciatic nerve cross sections at the end of 12th week post-surgery. a PU/GNFs/SCs, b PU/GNFs/PRP/SCs, c PU/GNFs/PRP/MLT/SCs and d Autograft. Scale bar 500 µm and ×100 magnification

Discussion

A degradable NGC can obviate the need for a second surgery for its removal after regeneration (Gristina et al. 1990). Incorporation of GNFs enhanced the in vitro hydrolytic degradation of PU by increasing its hydrophilicity and consequently further interaction with water. Chan–Chan et al. (Chan–Chan et al. 2010) suggested that the very PU used in this study can be degraded to a higher extent in vivo during the inflammatory process both by low pH and the activity of cholesterol esterase, a hydrolytic enzyme produced by monocyte-derived macrophages. Incorporation of GNFs also reduced the tensile properties of the PU conduit. The fibril-matrix interfacial bonding is a determinant factor of tensile properties in composites (El-Shekeil et al. 2012). The low interfacial adhesion between the hydrophobic PU and hydrophilic GNFs caused they debond at lower stress during the stretching.

SCs have been used alone or in combination with NGCs to support the repair and regeneration of peripheral nerve injuries (Luo et al. 2015). Activating the proliferation of SCs can effectively promote the nerve regeneration following a peripheral nerve injury (Chang et al. 2014). The effects of PRP are based on the release of growth factors such as platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factor II (FGF-II), and transforming growth factor-β (TGF-β), which their proliferative effects on SCs have been demonstrated by several studies (Davis and Stroobant 1990; Sondell et al. 1999; Danielsen et al. 1988; Ridley et al. 1989). Furthermore, Chang et al. (2014) demonstrated the role of MLT on neural regeneration by promoting the proliferation of SCs. The proliferative effect of MLT on SCs is via binding to its MT1 receptor and activating the intracellular ERK1/2 pathway (Chang et al. 2014; Ogata et al. 2006). Thus, the exogenous treatment of MLT can promote the proliferation of SCs and consequently enhance the reinnervation (Corfas et al. 2004). The results of cell proliferation (Fig. 1d) and functional analysis (Fig. 3a–d) demonstrated that the simultaneous administration of PRP and MLT could increase the cell proliferation in vitro and enhance the recovery rate of injured animals in vivo.

Conclusion

In this study, we successfully produced and examined a MLT/PRP-containing PU/GNFs conduit as a carrier to transplant SCs to the sciatic nerve defect of rats. Our results revealed that the prepared conduit enhanced the regeneration of the created defect. However, it was unsuccessful to restore the animal’s function as well as the autograft, as the gold standard of nerve gap bridging, indicating the need for further optimization of the produced conduit.

Acknowledgement

This study was funded by Tehran University of Medical Sciences (Grant No. 95-01-87-31294).

Compliance with Ethical Standards

Conflicts of interest

The authors declare that they have no conflicts of interest.

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