Polypyrrole/polylactic acid nanofibrous scaffold cotransplanted with bone marrow stromal cells promotes the functional recovery of spinal cord injury in rats

Summary Aims The objective of this study was to analyze the efficacy of polypyrrole/polylactic acid (PPy/PLA) nanofibrous scaffold cotransplanted with bone marrow stromal cells (BMSCs) in promoting the functional recovery in a rat spinal cord injury (SCI). Methods Female Sprague‐Dawley rats were randomly divided into three groups (n = 18/group): control group, PPy/PLA group, and PPy/PLA/BMSCs group. The SCI was induced in all rats. Consequently, rats in PPy/PLA/BMSCs group were transplanted with 1 × 105 BMSCs after implantation of PPy/PLA, while those in the PPy/PLA group were implanted with PPy/PLA only; no implantation was performed in the control group. Six weeks after surgery, immunofluorescence microscopy, electron microscope, and polymerase chain reaction (PCR) techniques were performed to assess the changes in the injured spinal cord tissues. Results Electrophysiology and locomotor function testing suggested that PPy/PLA nanofibrous scaffold cotransplanted with BMSCs could promote the functional recovery of the spinal cord. Six weeks after the operation, lower amount of scar tissue was found in the PPy/PLA group compared with the control group. Abundant neurofilament (NF) and neuron‐specific marker (NeuN) positive staining, and myelin formations were detected in the injured area. In addition, the transplantation of BMSCs not only improved the efficacy of PPy/PLA but also managed to survive well and was differentiated into neural and neuroglial cells. Conclusions The implantation of PPy/PLA nanofibrous scaffold and BMSCs has a great potential to restore the electrical conduction and to promote functional recovery by inhibiting the scar tissue formation, promoting axon regeneration, and bridging the gap lesion.


| INTRODUC TI ON
Spinal cord injury (SCI) is characterized by the loss of sensory and motor function caudal to the level of injury. Although many research studies have addressed the management of SCI, thus far no effective treatment has been developed. The main treatments for SCI include surgery, while the use of drugs and rehabilitation have shown to improve the neurological function to some extent. However, there are still many limitations for these treatment modalities. SCI causes a series of pathophysiological events, such as massive inflammation, edema, demyelination, cell death, vascular destruction, and glial scar, which affect the axons regeneration. 1,2 So far, various biomaterial scaffolds in the form of nerve guidance conduits have been widely developed and tested in vivo. These materials have the ability to improve functional recovery in nervous system injury by promoting new axon formation that span across the lesion gap. [3][4][5][6][7][8] Yet, the nerves conduction velocity (NCV) of regenerated nerves has shown to be significantly lower compared with the healthy nerves. Recent studies on biomaterials engineering have focused on obtaining the optimal functional recovery, and thus on examining scaffold materials that possess the ability to conduct electricity, and in turn promote nerve regeneration. 9,10 As a result, electro conducting polymers and their effects in promoting nerve regeneration have been widely investigated.
Polypyrrole (PPy) is a well-known conducting polymer used in biomedical applications to enhance the nerve regeneration by electrical stimulation. 11 PPy can easily be synthetized and offer good cytocompatibility and conductivity. [12][13][14] In vitro studies have suggested that PPy can be used as a promising scaffold material for cell growth. Recently, an in vivo study was carried out to confirm the viability of PPy/polymer composite material as a scaffold for promoting peripheral nerve regeneration. Signs of PPy degradation were observed after 3 months after implantation, while a more significant reduction was seen after 6 months. 17 However, to our knowledge, there is a scarcity of hitherto reports on the study of the biocompatibility of PPy/polymer composite nerve conduits in central nervous system (CNS) injuries.
Polypyrrole/polylactic acid (PPy/PLA) is a potential stem cell seeding biomaterial used for nerve tissue engineering. 18,19 Bone marrow stromal cells (BMSCs) are regarded as an ideal candidate type of cell for transplantation due to low immunorejection, rapid propagation, and easy accessibility. 20,21 Furthermore, BMSCs can release a series of factors that may provide trophic support and can differentiate into different types of cell, such as neurons, oligodendrocytes, and astrocytes, which can replace the lost tissue. 22,23 It has been shown that BMSCs are very beneficial in SCI. 24 In this study, the efficacy of a PPy/PLA nanofibrous scaffold was examined for spinal cord injury treatment, and BMSCs were applied to optimize the functional recovery. The polymerization continued for 6 hours at 18°C. PPy nanoparticles were collected through centrifuge and were washed with deionized water and alcohol several times before they were vacuum-dried at 60°C for 24 hours and ground to fine powders for further use.

| MATERIAL AND ME
Polypyrrole nanoparticles were homogeneously dispersed in DMF by ultrasonication at a concentration of 5.6% (w/v) and then were stirred continuously overnight at room temperature to form the PPy suspension. PLA was dissolved in DCM at a concentration of 18.75% (w/v). The PPy suspension or pure DMF was mixed with the PLA solution while stirring to the point where DCM:DMF reached 2:1, in order to achieve the PPy/PLA suspension or the PLA solution, respectively.
The electro-spinning device (Ucalery Co., Ltd.) was set up with a homemade high-speed collecting drum. The prepared PPy/PLA suspension or PLA solution was added into a plastic syringe with a stainless-steel needle. The outer diameter of the needle was 0.6 mm.
The flow rate of the suspension was 1 mL/h. The applied voltage was 15 kV. The aligned films were achieved at the speed of 2500 r/ min. The electrospun films were dried in a vacuum drier at 40°C for 3 days.

| Animals and BMSCs
Female Sprague-Dawley rats, 200-250 g, were obtained from Vital River Laboratories, China. All the animals were housed in an environment with temperature of 22 ± 1°C, relative humidity of 50% ± 1%, and a light/dark cycle of 12/12 hours All animal studies (including the rats euthanasia procedure) were done in compliance with the regulations and guidelines of Beijing Neurosurgical Institute institutional animal care (Permit Number: 201301020).
Efforts were made to minimize animal suffering during the procedures.
Rats were anesthetized through intraperitoneal injection of chloral hydrate solution (0.4 mL/100 g). BMSCs were collected from femurs and tibias out of adult Sprague-Dawley rats. The cells were plated at a density of 1 × 10 5 per cm 2 in Dulbecco's modified Eagle's medium/ nutrient mixture F-12 (GIBCO, USA) supplemented with 15% fetal bovine serum (GIBCO, USA), in a humidified atmosphere containing 5%CO 2 /95% air at 37°C. The medium was replaced with a new one every third day. The cells were digested with trypsin (GIBCO, USA) and passaged upon reaching 90% confluence. After five passages, cells were suspended with normal saline to a concentration of 1 × 10 5 cell/mL. The percentage of cell viability was determined by the average counts from five different randomly chosen microscopic fields. Flow cytometry was performed to detect the cell purity. If the percentage of CD105, CD90, and CD73 were all higher than 95%, and the percentage of CD45 and CD34 were lower than 5%, the cells were used for transplantation.

| Establishment of rat model with complete transected spinal cord and the transplantation of BMSCs
The rats were randomly divided into three groups (n = 18/group: control group, PPy/PLA group, and PPy/PLA/BMSCs group). Briefly, all animals were subjected to intraperitoneal anesthesia using 10% chloral hydrate solution (0.4 mL/100 g). A 2.5-cm midline skin incision was then performed along the vertebrae T7-T10. The thoracolumbar fascia and paraspinal musculature were incised along the spinous processes implanted with PPy/PLA only; no implantation was performed in a control group. Then, the incision was closed by suturing the muscles and the skin in order. Antibiotic (penicillin 10 000 units) was given after the surgery, and the rats were observed until they were all conscious. Manual bladder and peritoneal exercise were performed twice a day until the recovery of the bladder and intestinal reflex.

| Neurobehavioral testing and electrophysiology
The locomotor recovery of the animals was assessed by two independent observers in an open field (diameter of plastic pool: 90 cm) using the 21-point Basso, Beattie, and Bresnahan (BBB) open-field locomotor score from 1 to 6 weeks after SCI. The BBB scale was used to assess hind limb locomotor recovery including joint movements, stepping ability, coordination, and trunk stability. The function of both hind limbs was detected. Rats were tested once a week using a blind approach, and the duration of each session was 2 minutes per rat.
Motor evoked potential (MEP) was measured using the Neurosoft electrophysiology monitoring system (neuron spectrum 5, Russia).
MEP was obtained using stainless-steel needle electrode, Neurosoft software. Briefly, rats were anesthetized with 10% chloral hydrate solution (0.4 mL/100 g). The stimulating electrode was then inserted into the scalp, and the recording electrode was introduced into the tibial muscle. The reference electrodes were inserted subcutaneously above the nostril, the peritoneum, and the tail. MEP was obtained by electrical stimulation at 0.1 ms duration, 10 mA intensity, and at a frequency of 1 Hz, and the latent period of MEP was analyzed.

| Tissue processing for light and fluorescent microscope
After 6 weeks, the rats were anesthetized using intraperitoneal injection of 10% chloral hydrate solution (0.4 mL/100 g). The T6-T10 spinal cords were then collected (six specimens/group), fixed in 4% paraformaldehyde containing 30% sucrose at 4°C for 48 hours, and embedded in OCT (Sakura Finetechnical, Japan). Samples were then divided into 20-µm sections, which were stained with hematoxylin and eosin for histological examination. Immunofluorescent staining was performed to assess nerve regeneration, spared neurons, survival, and differentiation of transplanted BMSCs.

| Histological analysis
For histological analysis, the spinal cord specimens were managed the same as above reported. Immunofluorescent staining was performed on 20-μm sections, which were stained with the following antibodies:

| Ultra-structure analysis by both transmission and scanning electron microscope
Six weeks postimplantation, the spinal cords (six specimens each group) were examined by transmission electron microscopy (Hitachi TEM, Japan). The rats were anesthetized as mentioned above. Then, the heart was exposed. Next, 300 mL of 1× PBS followed by 300 mL 4% paraformaldehyde was perfused through the vascular system.
The spinal cord tissue was fixed with 2.5% glutaraldehyde and 2% paraformaldehyde. Ultra-thin sections were achieved with an ultramicrotome (Leica EM UC6, Germany) and stained with uranyl acetate and lead citrate. Observation was performed by TEM (Hitachi).

| Quantitative real-time RT-PCR
Total RNA was extracted from the spinal cord tissue (six specimens each group) including the graft sites using the Trizol reagent (Invitrogen).  Table 1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was used as a control. The relative expression between a given sample and a reference sample was calculated using the 2 −ΔΔCt method.

| Quantitative analysis
Immunofluorescence imaging was used to analyze the morphological aspects of each experimental group. All quantitative cell analyses

| Statistical analysis
All the data were expressed as the mean ± standard deviation (SD).
Statistics were calculated with the software SPSS 17.0. Differences between groups were compared with one-way ANOVA, Fisher's least significant difference (LSD) method was used for the post hoc test. The differences in the repeated measurements were analyzed with multivariate ANOVA. A significant difference was indicated by P < 0.01 and P < 0.05.

| In vitro culture and characterization of BMSCs
Bone marrow stromal cell were collected from femurs and tibias of adult Sprague-Dawley rats. Cells were collected after adherence and were then cultured ( Figure 1A) and passaged for five times. The cell purity was detected by flow cytometry (FCM). In vitro culture showed a rapid proliferation of BMSCs ( Figure 1B). The phenotypes of BMSCs used in our study were positive for CD105 (90.74%), CD90 (99.91%), and CD73 (98.49%) and negative for CD45 (1.62%) and CD34 (0.69%); Figure 1C).

| Structure of the PPy/PLA nanofibrous scaffold and implantation of the scaffold
We first tested the morphology of the electrospun nanofibers con- F I G U R E 3 A, B, The electrophysiological recovery postoperation. The latent period of the PPy/PLA/BMSCs group showed significant improvement compared with the PPy/PLA and control groups (the length of the latent period was measured from green dot to red dot). C, The behavioral assessment using the BBB locomotor rating scale. The BBB analysis was performed continually starting from the first week after operation. BBB analysis indicated that the PPy/ PLA/BMSCs-treated group showed the greatest functional recovery compared with the control group or those treated with PPy/PLA alone. **P < 0.01, *P < 0.05

| The recovery of electrophysiology and the assessment of functional recovery in SCI
Motor evoked potentials (MEPs) assay was performed before surgery, and at 3 and 6 weeks after operation to assess the functional recovery of the injured spinal cord. The short latent period in the MEP assay is considered a normal nerve conduction. Immediately after injury, the rats treated with PPy/PLA/BMSCs (5.36 ± 0.15) and PPy/ PLA (5.53 ± 0.10) showed a shorter latent period of the lower extremity compared with the control group (6.06 ± 0.12, ** P < 0.01). At the 3-week postoperation, a slight yet significant improvement was observed in each group. In addition, 6 weeks after the operation, the PPy/PLA/BMSCs group showed a shorter latent period (5.08 ± 0.09) compared with PPy/PLA (5.47 ± 0.09) and compared with control groups (5.71 ± 0.08) ( Figure 3A,B). These data verified the role of PPy/ PLA and BMSCs in the electrophysiological improvement of SCI.

| PPy/PLA nanofibrous scaffold combined with BMSC transplantation reduces the scar tissue formation
Six

| Axon regeneration and remyelination in the injured area
Axon regeneration is essential for the nerve functional recovery.
The effect of treatment on axonal sprouting following SCI was PPy/PLA groups (1.66 ± 0.16) ( Figure 5E). Based on these data, we believe that treatment with PPy/PLA/BMSCs has the ability to enhance axonal regeneration within the lesion site, following SCI.
Myelin formation is another important process for the nerve function recovery. It has been reported that improving myelination is beneficial for functional recovery in spinal cord injury. Therefore, in order to investigate the efficacy of nanofibrous scaffold to promote myelin formation, TEM was employed and at week 6th post-SCI. Briefly, rats treated with PPy/PLA/BMSCs had more myelinated axons (22.4 ± 1.1; ** P < 0.01) than those treated with PPy/PLA (14.6 ± 1.1) or the control group (8.6 ± 1.1) (Figure 5B,D).

| PPy/PLA/BMSCs reduce neuron apoptosis and vascular formation in rat SCI
The long-term neurological deficits after spinal cord injury may be due

| In vivo survival and differentiation of transplanted BMSCs in SCI
Six weeks after the operation, immunofluorescence assay was performed

| D ISCUSS I ON
Given that spontaneous axonal regeneration after spinal cord injury is limited, 25  After injury, a sequence of progressive pathological changes occurs. The lesion cavity is gradually filled by scar tissue, which becomes a permanent barrier for nerve regeneration. 27  activate the voltage-gated ion channels on cell membranes and induce a modulation in the intracellular signaling pathways, which in turn may impact the cell behavior, such as cell apoptosis. 34 Last but not the least, angiogenic factors, such as VEGF, also have neuroprotective effects. 35 Increased expression of VEGF and its receptor during hypoxic/ischemic injury to the brain and spinal cord suggests that VEGF could have a neuroprotective role in these pathophysiological processes. 36 A study by Rong et al 37 has shown that improving the expression of VEGF may reduce the expression of caspase-3 protein, thus promoting nerve repair following SCI. Our results are consistent with the previous study, which suggest that this treatment strategy may provide neuroprotection.
In connection and restore the local neuronal connectivity 39 by connecting the host tissue and the implanted nanofibrous scaffold as described in Figure 6D. Nonetheless, further investigation is needed to verify the interaction between host tissues, transplanted cells, and the scaffold. However, the significant improvement in functional recovery in the PPy/PLA/BMSCs group supports this hypothesis.

| CON CLUS IONS
An aligned PPy/PLA nanofibrous scaffold was fabricated and grafted into a complete transected spinal cord to promote nerve regeneration and to recover nerve conduction. The results showed that the PPy/PLA nanofibrous scaffold can inhibit scar tissue formation and induce the axonal regeneration and myelination in the lesion area. New vascular formation was also promoted after implantation, creating a better microenvironment, which reduced neuronal apoptosis. Furthermore, the transplanted BMSCs survived and were differentiated into neural and neuroglial cells. This work demonstrated the remarkable potential of the implantation of the PPy/ PLA nanofibrous scaffold combined with BMSCs transplantation for nerve regeneration and for motor functional recovery following SCI.

CO N FLI C T O F I NTE R E S T
The authors have no commercial, proprietary, or financial interest in the products or companies described in this article.