Abstract
Autologous nerve transplantation (ANT) is currently considered the gold
standard for treating long-distance peripheral nerve defects. However,
several challenges associated with ANT, such as limited availability of
donors, donor site injury, mismatched nerve diameters, and local
neuroma formation, remain unresolved. To address these issues
comprehensively, we have developed porous poly(lactic-co-glycolic acid)
(PLGA) electrospinning fiber nerve guide conduits (NGCs) that are
optimized in terms of alignment and conductive coating to facilitate
peripheral nerve regeneration (PNR) under electrical stimulation (ES).
The physicochemical and biological properties of aligned porous PLGA
fibers and poly(3,4-ethylenedioxythiophene):polystyrene sodium
sulfonate (PEDOT:PSS) coatings were characterized through assessments
of electrical conductivity, surface morphology, mechanical properties,
hydrophilicity, and cell proliferation. Material degradation
experiments demonstrated the biocompatibility in vivo of
electrospinning fiber films with conductive coatings. The conductive
NGCs combined with ES effectively facilitated nerve regeneration. The
designed porous aligned NGCs with conductive coatings exhibited
suitable physicochemical properties and excellent biocompatibility,
thereby significantly enhancing PNR when combined with ES. This
combination of porous aligned NGCs with conductive coatings and ES
holds great promise for applications in the field of PNR.
Keywords: Peripheral nerve defect, Nerve guide conduit, Electrospinning
fibers, Conductive coating, Electrical stimulation
Graphical abstract
This study focuses on the use of porous poly(lactic-co-glycolic acid)
(PLGA) fiber conduits with optimized alignment and conductive coating
for guiding peripheral nerve regeneration (PNR) under electrical
stimulation (ES). PLGA was chosen to prepare porous electrospun fibers
and to explore the optimal alignment and porogenic conditions.
Poly(3,4-ethylenedioxythiophene):polystyrene sodium sulfonate
(PEDOT:PSS) was chosen as the conductive coating and its optimal
coating concentration was explored. The physicochemical properties of
the coated PLGA electrospun fibers and their effects on nerve cell
behavior were fully characterized. The electrospun NGCs were then
implanted into rats and ES was administered to investigate the effect
of promoting PNR and to explore the mechanism of nerve regeneration.
[35]Image 1
[36]Open in a new tab
List of abbreviations
3D
Three-dimensional
AChE
Acetyl cholinesterase
ANT
Autologous nerve transplantation
ATP
Adenosine triphosphate
CCK-8
Cell counting kit-8
CLSM
Confocal laser scanning microscope
CMAPs
Compound muscle action potentials
CNS
Central nervous system
CPs
Conducting polymers
DAPI
4′,6-diamidino-2-phenylindole
DCM
Dichloromethane
DEG
Differentially expressed genes
DMEM
Dulbecco's modified eagle medium
DMF
Dimethylformamide
DMSO
Dimethyl sulfoxide
ECM
Extracellular matrix
EDX
Energy dispersive X-Ray
ELISA
Enzyme linked immunosorbent assay
ES
Electrical stimulation
FDA
Food and drug administration
FTIR
Fourier transform infrared
GFAP
Glial fibrillary acidic protein
GM
Gastrocnemius muscle
GO
Gene ontology
GSEA
Gene set enrichment analysis
H&E
Hematoxylin-eosin
IL-10
Interleukin-10
IT
Intermediary toe
KEGG
Kyoto encyclopedia of genes and genomes
MAP-2
Microtubule association protein-2
MAPK
Mitogen-activated protein kinase
MBP
Myelin basic protein
M-CSF
Macrophage stimulating factor
MEP
Motor endplate
NF200
Neurofilament-200
NGC
Nerve guide conduit
PANI
Polyaniline
PBS
Phosphate buffer saline
PC-12
Pheochromocytoma-12
PCL
Polycaprolactone
PEDOT
Poly(3,4-ethylenedioxythiophene)
PFA
Paraformaldehyde
PI
Propidium iodide
PL
Paw length
PLGA
Poly(lactic-co-glycolic acid)
PLLA
Poly(l-lactic acid)
PNI
Peripheral nerve injury
PNR
Peripheral nerve regeneration
PNS
Peripheral nervous system
PPy
Polypyrrole
PSS
Polystyrene sulfonate
ROS
Reactive oxygen species
RPMI-1640
Roswell park memorial institute-1640
SC
Schwann cell
SD
Sprague-Dawley
SEM
Scanning electron microscopy
SFI
Sciatic functional index
TCP
Tissue culture polystyrene
TEM
Transmission electron microscope
TNF-α
Tumor necrosis factor-α
TS
Toe spread
Tuj-1
Beta3-Tubulin
UVO
Ultraviolet ozone
XPS
X-ray photoelectron spectroscopy
1. Introduction
Peripheral nerve injury (PNI) refers to damage occurring in the
peripheral nerve plexus, nerve trunk, or its branches, typically
resulting from traumatic factors such as lacerations, motor vehicle
accidents, compression injuries, and excessive stretching [[37]1]. The
management of long-distance (over 5 cm) nerve defects poses a
significant surgical challenge due to the inability to directly suture
nerves [[38]2]. The current treatment for long-distance nerve defects
is autologous nerve transplantation (ANT) [[39]3]. However, ANT has
several inherent disadvantages, including limited availability of
donors, functional loss in the donor region, formation of neuromas, and
mismatched nerve diameters [[40]4]. To overcome these limitations, a
tissue-engineered nerve guide conduit (NGC) was developed [[41]5]. The
NGC effectively isolates the regenerated nerve axon from surrounding
scar tissue, therefore preventing compression of the nerve by adjacent
tissues [[42]6]. Additionally, the NGC plays a crucial role in
accurately directing nascent neural tissue towards its intended target
organ [[43]7].
Various techniques were employed for the fabrication of NGCs, including
freeze-drying, solvent casting, phase separation, gas foaming,
three-dimensional (3D) printing, and electrospinning [[44]8].
Electrospinning is an electrostatically driven process utilized to
produce random or aligned fibers with diameters ranging from nanometers
to microns [[45]9]. The desired morphology of electrospinning fiber was
achieved by adjusting process parameters, environmental conditions, and
solution properties [[46]5]. Many previous studies have demonstrated
the indispensability of electrospinning fibers with aligned porous
morphology for nerve regeneration, as they effectively enhance neuronal
cell adhesion, proliferation, and guided growth while also modulating
cytokine expression to a moderate extent [[47]10,[48]11]. Zamani et al.
found that electrospinning poly(lactic-co-glycolic acid) (PLGA) porous
cylindrical fibers significantly enhanced the attachment, growth, and
proliferation of human A-172 nerve cells [[49]12]. Filimona et al.
validated that the surface porous topology of electrospinning film
prevented microbial colonization and reduced the risk of postoperative
infections, which is crucial in neural tissue engineering [[50]13].
Although numerous previous studies have been conducted on the
application of aligned electrospinning with surface porous morphology
in neural tissue engineering, a majority of these studies solely
focused on cellular experiments rather than validating their effects on
neural regeneration in animal models. Zhang et al. demonstrated that
the combination of aligned electrospinning NGCs and electrical
stimulation (ES) effectively enhanced peripheral nerve regeneration
(PNR), thereby suggesting that employing multiple treatment modalities
may yield a favorable synergistic effect [[51]14]. Due to its poor
electrical conductivity, PLGA requires modification with a conductive
coating to enhance neurostimulation by ES. Among the various options
available, poly(3,4-ethylenedioxythiophene) (PEDOT) stands out as the
most extensively studied polythiophene derivative due to its superior
electrochemical stability, enhanced conductivity, and improved thermal
stability compared to polypyrrole (PPy) and polyaniline (PANI)
[[52]15]. Unlike other conductive polymers, PEDOT doped with
polystyrene sulfonate (PSS) can be easily dispersed in aqueous solution
while maintaining excellent conductivity [[53]16]. Therefore, the
utilization of PEDOT:PSS represents a promising approach for developing
conductive scaffolds that facilitate cell adhesion and promote cellular
growth and differentiation [[54]17].
The process of PNR is intricate, and achieving satisfactory therapeutic
outcomes with a single material, scaffold, or treatment proves
challenging. Henceforth, the future research direction should focus on
composite treatments involving multiple materials or approaches
[[55]18]. Prabhakaran et al. suggested that the combination of ES with
topographical cues synergistically promoted axonal growth, surpassing
the effects of monotherapy [[56]19]. In another study, agarose NGCs
were combined with a PEDOT conductive coating to enhance their
mechanical properties and significantly improve conductivity. Although
ES was not applied in the experiment, the desired nerve repair effect
was achieved, leading to the successful restoration of motor function
in the lower limbs of rats [[57]18]. Other studies have primarily
focused on utilizing topographic cues or ES to influence schwann cell
(SC) migration [[58]20,[59]21]. However, there is a scarcity of reports
regarding the combination of ES, topographic cues, surface topography,
and conductive coatings for PNR.
Here, the electrospinning fibers were integrated with optimized porous
alignment and highly conductive materials, in conjunction with ES, to
regulate nerve cell behavior and facilitate neural repair in vivo, as
illustrated in [60]Scheme 1. We fabricated aligned PLGA fibers and
investigated the optimal preparation conditions for their surface
porous morphology by varying solvent ratios. Considering the
cytotoxicity of high concentrations of PEDOT:PSS solutions, we
systematically explored gradient dilution to achieve an optimal
concentration of conductive coatings that would provide good
conductivity without compromising biocompatibility for the
electrospinning fibers. The resulting composite NGCs exhibited
significantly enhanced nerve regeneration in rats under ES, offering a
facile approach to obtain a well-suited NGC for nerve tissue
engineering applications in PNI.
Scheme 1.
[61]Scheme 1
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Schematic illustration of porous aligned PEDOT:PSS-coated PLGA
electrospinning NGC for promoting nerve regeneration under ES.
2. Materials and methods
2.1. Materials
PLGA ((LA): (GA) = 75:25; the molecular weight was 8 × 10^4 g mol^−1)
was provided by Changchun SinoBiomaterials Co., Ltd (Changchun, P. R.
China). Dimethylformamide (DMF, 0.948 g cm^−³) was purchased from
Energy Chemical Co., Ltd (Shanghai, P. R. China). Dichloromethane (DCM,
1.325 g cm^−³) was purchased from XiLong Scientific Co., Ltd (Shenzhen,
P. R. China). PEDOT:PSS, neurofilament-200 (NF200) antibody, glial
fibrillary acidic protein (GFAP) antibody, and beta3-Tubulin (Tuj-1)
antibody were purchased from Sigma-Aldrich (Shanghai, P. R. China). The
rat hematoxylin-eosin (H&E) kit, the masson kit, the Roswell park
memorial institute-1640 (RPMI-1640) medium, and the Dulbecco's modified
eagle medium (DMEM) were purchased from Servicebio Co., Ltd (Wuhan, P.
R. China). Cell counting kit-8 (CCK-8) was purchased from Beyotime
Biotechnology Co., Ltd (Shanghai, P. R. China).
4′,6-diamidino-2-phenylindole (DAPI) was purchased from Solarbio Co.,
Ltd (Beijing, P. R. China). Phalloidin-FITC conjugate kit was purchased
from Thermo Fisher Scientific Co., Ltd (Shanghai, P. R. China). Clear
tissue culture polystyrene (TCP) plates were purchased from Corning
Costar Co., Ltd (Cambridge, MA, USA). The living/dead cell double
staining kit and acetylcholinesterase staining kit were purchased from
Bestbio Co., Ltd (Shanghai, P. R. China). Myelin basic protein (MBP)
antibody, rat pheochromocytoma-12 (PC-12) cells, and rat SCs were
purchased from Bihe Biochemical Technology Co., Ltd (Shanghai, P. R.
China). Sprague-Dawley (SD) rats were purchased from the animal
experiment center of Jilin University (Changchun, P. R. China).
Collagenase IV and DNase I were purchased from Sigma-Aldrich (Shanghai,
P. R. China). Antibodies of CD11b, CD86, and CD206 were purchased from
eBioscience Co., Ltd (Santiago, USA). Intracellular staining buffer was
purchased from BIOCREATIVE Co., Ltd (Beijing, P. R. China). Enzyme
linked immunosorbent assay (ELISA) kits were purchased from Servicebio
Co., Ltd (Wuhan, P. R. China). RNA nano 6000 assay kit of the
bioanalyzer 2100 system was purchased from Agilent Technologies Co.,
Ltd (CA, USA). Acetyl cholinesterase (AChE) kits were purchased from
Bestbio Co., Ltd (Nanjing, P. R. China).
2.2. Preparation and characterization of PLGA electrospinning fibers under
varying roller receiver speeds
2.52 g PLGA was dissolved in a mixture of 10 mL DMF and 10 mL DCM
(10 wt%). The mixture was stirred for over 12 h until no undissolved
PLGA particles were visible. Subsequently, the PLGA solution was
aspirated into a 1 mL syringe under a device voltage of 14 kV and with
a distance of 15 cm between the needle tip and the roller collector
encapsulated in copper foil. The resulting electrospinning fibers
obtained at different roller receiver speeds (0 rpm, 500 rpm, 1000 rpm,
1500 rpm, 2000 rpm, and 2500 rpm) were named as follows: PLGA-0,
PLGA-500, PLGA-1000, PLGA-1500, PLGA-2000, and PLGA-2500, respectively.
Scanning electron microscopy (SEM, Inspect-F50, FEI, Eindhoven,
Finland) was employed to observe the alignment and surface morphology
characteristics of the electrospinning fibers. Additionally,
hydrophilicity characterization of the PLGA fibers was conducted by
measuring their water contact angle using a contact angle meter (KRUSS,
Hamburg, Germany).
2.3. Preparation and characterization of porous PLGA fibers with varying
ratios of DCM and DMF blends
The PLGA was dissolved using varying ratios of DCM mixed with DMF, as
indicated in [63]Table 1, all for a 10 wt% PLGA solution.
Table 1.
Various ratios of DCM and DMF were utilized to dissolve PLGA for the
preparation of electrospinning fibers.
The solvent's volume ratio DCM DMF PLGA
12:1 3 mL 0.25 mL 0.47 g
10:1 3 mL 0.3 mL 0.47 g
8:1 3 mL 0.38 mL 0.48 g
6:1 3 mL 0.5 mL 0.49 g
4:1 2.6 mL 0.65 mL 0.45 g
3:1 2.4 mL 0.8 mL 0.44 g
2:1 2.2 mL 1.1 mL 0.44 g
[64]Open in a new tab
We employed SEM to conduct morphological characterization of PLGA
fibers. The densities and porosities were determined using the liquid
phase displacement technique with ethanol as the displacing agent. To
enhance hydrophilicity, the PLGA fiber films underwent a 50s treatment
with an ultraviolet ozone (UVO) cleaner (42–220, Jelight, USA). The
quality of the fiber film was M, followed by immersion in ethanol
(volume V1) for 5 min, resulting in a total volume of ethanol and film
denoted as V2. After removing the fiber film from ethanol, the
remaining volume of ethanol was measured as V3. Subsequently, equation
[65](1) was utilized to calculate the density (ρ) of the fiber film.
[MATH: ρ=MV2‐V3 :MATH]
(1)
where M was in “g”, and V2 and V3 were in “mL”.
The porosity (ε) of the fiber film was determined using equation
[66](2):
[MATH: ε=V1‐V3V2‐V3 :MATH]
(2)
where V1, V2, and V3 were in “mL”.
We determined the water absorption of the fiber film with an initial
mass of M0. The fiber films were immersed in deionized water for 24 h.
After being removed from the water, excess moisture on the surface of
the fiber films was absorbed using filter paper, and its quality was
measured as M1. PLGA fiber films were subjected to vacuum drying at
40 °C for 24 h and then weighed as M2. The water absorption (δ) of the
fiber film was calculated using equation [67](3):
[MATH: δ=M1‐M2M0 :MATH]
(3)
M0, M1, and M2 were in “g”.
2.4. The impact of PEDOT:PSS-coated porous PLGA fibers on cellular
proliferation
To coat the porous PLGA fibers, we employed the PEDOT:PSS solution.
Previous research has indicated that incorporating propanol or dimethyl
sulfoxide (DMSO) into the PEDOT:PSS solution enhances its solubility
and conductivity [[68]22]. However, the use of organic solvents may
potentially harm the mechanical properties and surface morphology of
PLGA fiber films. Therefore, we chose to mix an equal volume of
deionized water with PEDOT:PSS to avoid any potential damage. The
UVO-treated fiber films were immersed in aqueous PEDOT:PSS solution for
1 h, dried under vacuum at 30 °C for 3 h, and subsequently rinsed with
deionized water to eliminate any residual PEDOT:PSS from the surface
[[69]22].
With the PLGA fiber films, both coated and uncoated, placed in 96-well
plates, we added 5 × 10^3 PC-12 cells suspended in 200 μL of RPMI-1640
medium to each well. Following a 24 h incubation period, the cell
culture medium was aspirated and replaced with a mixture of CCK-8
reagent (dissolved at a concentration of 5 mg mL^−1) in serum-free
medium at a ratio of 1:10. Subsequently, 100 μL of the mixture was
added to each well and further incubated until an orange color
developed. Proliferation rates were assessed using the Bio-Rad
microplate detector (Bio-Rad 550, Hercules, California, USA). Cell
experiments were conducted at both the 48 h and 72 h time points
following previously described methods.
2.5. The exploration of the optimal coating concentration for PEDOT:PSS
We employed gradient dilution to explore the optimal coating
concentration of PEDOT:PSS using rat PC-12 cells and SC cells for
experiments. A total of 50 wells arranged in 5 rows and 10 columns were
selected on a 96-well plate. Subsequently, each well was supplemented
with 5 × 10^3 PC-12 cells and 200 μL of culture medium, followed by
incubation for 24 h. The cell culture medium was then aspirated from
each well, after which the leftmost five wells received an addition of
100 μL of PEDOT:PSS solution. Next, these wells were mixed thoroughly
with an additional supplementation of 100 μL of cell culture medium
before transferring a volume of 100 μL from this mixture into the
second column's 5 wells. This process was repeated by adding another
100 μL cell culture medium and thorough mixing before pipetting the
resulting mixture into the third column's wells for further dilution.
This sequential procedure continued until reaching the tenth column
where a final volume of well-mixed liquid measuring at 200 μL was
obtained, discarding the 100 μL mixture.
After incubation for 24 h, the mixture was removed, followed by the
addition of a mixture containing CCK-8 reagent solution and cell
culture medium. The resulting mixture was further incubated until an
orange color developed and subsequently analyzed using a microplate
reader.
2.6. Preparation and characterization of porous PLGA fiber films coated with
the optimal concentration of PEDOT:PSS
The porous PLGA fiber films were coated with PEDOT:PSS solution at the
optimal concentration for promoting cell proliferation. We employed a
digital multimeter (DLX890C+, Delixi Group Co., Ltd, Zhejiang, China)
to determine the electrical conductivity of the PLGA fiber films. The
cross-sectional area of the fiber film was determined by measuring its
width and thickness using vernier calipers, and the conductivity (σ)
was calculated based on equation [70](4):
[MATH: σ=LAR :MATH]
(4)
R was the resistance of the fiber film in “MΩ”; L was the distance
between the two electrodes in “cm”; A was the cross-sectional area of
the fiber film in “cm^2”.
We examined the surface morphology of PLGA fiber films after PEDOT:PSS
coating using SEM. The presence of the coating on the fiber films was
studied through mapping and energy dispersive X-Ray (EDX) spectroscopy.
X-ray photoelectron spectroscopy (XPS) was employed to analyze the
elemental sulfur present on the surface of PLGA fiber films. The
mechanical properties of PLGA fiber films were evaluated using a
universal testing machine (Shimadzu, Kyoto, Japan). We conducted
mechanical property tests on the fiber films in both parallel and
perpendicular orientations to the fiber alignment, with the
stress-strain curves providing us with the maximum tensile strength
data. Fourier transform infrared (FTIR) spectroscopy (Bio-Red Win-IR,
Bruker, Karlsruhe, Germany) was employed to analyze uncoated PLGA fiber
films, PEDOT:PSS conductive coating, and coated PLGA fiber films. The
hydrophilicity of the coated PLGA fiber films was evaluated by
measuring their water contact angle.
2.7. The in vitro and in vivo degradation of porous PLGA fiber films coated
with PEDOT:PSS
We prepared non-porous, porous, and coated porous PLGA fiber films for
in vitro degradation experiments. The weight of each fiber film was
accurately measured, followed by immersion in 1 % elastase at 37 °C.
The fiber films were removed every 10 days for vacuum drying and
subsequent weighing to determine the remaining weight as a percentage
of the initial weight at each time point.
The rats were anesthetized with a 2 % solution of pentobarbital sodium
through intraperitoneal injection, and subsequently, an “L” shaped
incision was made on the dorsal region of each rat. Following the
separation of the subcutaneous tissue using a mosquito hemostat, the
fiber films were implanted into the subcutaneous fascial layer and
secured in place with sutures. Local tissues were excised at 1, 2, and,
3 months post-implantation of the fiber films, and paraffin sections
were prepared after fixation of the tissues using a 4 %
paraformaldehyde (PFA) solution. The rats' weights were recorded every
10 days following implantation.
2.8. The impact of PLGA fiber films with a coated porous surface on cellular
behavior
We conducted cellular experiments using SC and PC-12 cells and employed
SEM to characterize the cell morphology on the surface of the fiber
films. Additionally, we performed cytoskeleton staining to assess the
impact of electrospinning fiber alignment on cell growth and
morphology. Furthermore, we evaluated the toxicity of PEDOT:PSS on
nerve cells through living/dead cell double staining.
The porous PLGA fiber films were coated with the optimal concentration
of PEDOT:PSS solution. Subsequently, the coated porous PLGA fiber films
were placed in 24-well plates, and a small well containing
2 × 10^4 cells was prepared with the fiber film for subsequent
incubation. Following aspiration of the medium, the PLGA fiber films
were washed twice using phosphate buffer saline (PBS). The
electrospinning fiber films were fixed using 4 % PFA for 30 min.
Sequentially, ethanol at concentrations of 30 %, 50 %, 70 %, 80 %,
90 %, 95 %, and pure ethanol was added to dehydrate the cells adhering
to the PLGA fiber films for 30 min before their morphology was
characterized using SEM.
The coated porous PLGA fiber films were placed into a 24-well plate,
and 1 × 10^4 cells were seeded into each well of the plate and
incubated. After removing the medium and rinsing the fiber films with
PBS, the films were fixed with PFA. Following removal of PFA and
rinsing of the fiber film with PBS, acetone at −20 °C was added for
5 min. The acetone was aspirated and the film was rinsed with PBS.
Subsequently, the fiber film was stained using a phalloidin solution
for 90 min, followed by 3 times rinses with PBS. Next, the fiber films
were stained with a DAPI solution for 10 min and again rinsed 3 times
with PBS. Finally, the stained cells were captured using a confocal
laser scanning microscope (CLSM, T-PMT, Zeiss, Japan).
A sterile coverslip was placed on the bottom of a 6-well plate, and
2 × 10^5 cells along with 3 mL of cell culture medium mixed with the
PEDOT:PSS solution were added into each well. The plate was then
incubated for 48 h. Live cells were stained using calcein AM, while
dead cells were stained using propidium iodide (PI). The coverslip was
carefully lifted from the bottom of the 6-well plate and placed upside
down onto a slide, which was subsequently captured using a fluorescence
microscope (ECLIPSE C1, Nikon, Japan).
To assess the impact of electrospinning fibers on macrophages at the
PNR sites, we employed ELISA to study cytokine secretion. Rat
macrophages were cultured on electrospinning fibers, and cell
supernatants were collected after 24, 48, and 72 h. The concentrations
of tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) in the
supernatants were quantified using ELISA kits.
2.9. The procedures of animal experimentation
The female SD rats, weighing 220–250 g and aged 4–6 weeks, were
provided with adequate water and food. All animals underwent a two-week
acclimatization period before the animal experiments. A 10 mm sciatic
nerve defect model was created to study the efficacy of coated aligned
porous NGC combined with ES in promoting sciatic nerve regeneration in
rats. The rats were randomly divided into 6 groups, each consisting of
10 individuals: nerve defect group, PLGA-1500 group, porous PLGA-1500
group, coated porous PLGA-1500 group, coated porous PLGA-1500 + ES
group, and autograft group. After administering ether inhalation
anesthesia to rats, a 2 % solution of pentobarbital sodium was
intraperitoneally administered at a dosage of 2 mL kg^−1 body weight.
Once the anesthesia took effect, a longitudinal incision was made along
the posterior aspect of the left femur to expose the sciatic nerve by
gently separating the muscle tissue. The sciatic nerve was surgically
resected to create a 10 mm nerve defect, and the area of the nerve
defect was implanted with the NGC made of PLGA fiber film. In the
autograft group, the resected nerve segments were utilized to bridge
the nerve gaps after inversion, and each rat received an intramuscular
injection of 80,000 units of penicillin post-surgery for infection
prevention.
A Rigol DG1022 signal generator (Puyuan Jingdian Technology Co. LTD,
Beijing, China) was utilized to administer ES treatment on rats in the
postoperative ES group every other day for a total of 5 sessions.
Following previous research findings, we configured the stimulation
parameters to include a frequency of 20 Hz, a duty cycle of 50 %, and
an operating voltage of 100 mV. Platinum wire electrodes were
positioned within the proximal and distal tissues of the nerves, with
each stimulation session lasting for 2 h [[71]14]. The recovery
progress of lower extremity nerves in rats was evaluated at both the
2-month and 3-month time points after treatment.
2.10. The analysis of walking tracks
To evaluate the recovery of the lower extremity function of rats, a
walking track analysis was conducted on 5 rats from each group at 2 and
3 months post-treatment. A white paper was placed on the bottom of the
plexiglass runway, while the hind feet and toes of the rats were
blackened with the dye. A light source positioned at the end of the
runway was utilized to stimulate forward movement in the rats, thereby
leaving their footprints imprinted on the paper. The following
parameters were obtained from the footprints of the rats: paw length
(PL), which refers to the distance from hindfoot to distal middle toe;
intermediary toe spread (IT), which represents the distance between the
second and fourth toes; Toe spread (TS), indicating the distance
between the first and fifth toes. The sciatic functional index (SFI)
was calculated using [72]formula (5):
[MATH: SFI=‐38.3×(EPL‐NPL)NPL+109.5×(ETS‐NTS)NTS+13.3×(EIT‐NIT)NIT‐8.8 :MATH]
(5)
where E denoted the left footprints, while N represented the right
footprints.
2.11. Electrophysiological analysis of the sciatic nerve and characterization
of implanted aligned electrospinning NGCs
The bilateral compound muscle action potentials (CMAPs) of the lower
extremities in rats were recorded at 2 and 3 months post-treatment
using a 9033A07 EMG/evoked potentiometer (Bendi Medical Equipment Co.,
Ltd, Shanghai, China). Following anesthesia induction, longitudinal
incisions were made posterior to the femur to expose the nerve. The
gastrocnemius muscle (GM) was penetrated by one recording electrode in
a vertical manner, while another recording electrode was inserted
vertically into the rat at the Achilles tendon. The grounding electrode
was placed in the rat's tail, with the stimulation electrode positioned
at the proximal region of the regenerated nerve. Pulses at 50 Hz were
used to stimulate the sciatic nerves, and for each group, the
amplitudes and latencies of CMAP were recorded. The electrospinning
NGCs within the sciatic nerve defect area were extracted and subjected
to vacuum drying, followed by SEM observation to assess fiber
alignment.
2.12. Histological and immunofluorescence examination of the GM and
regenerated sciatic nerve
We consulted the previous study for the current experimental section
[[73]14]. We assessed the histopathology of regenerated nerves and GM
specimens from rats at 2 and 3 months post-treatment. The bilateral GMs
of rats were measured in terms of weight, and the percentage of
bilateral muscle weight was calculated using equation [74](6):
[MATH: Weight(%)=Weight(E)Weight(N) :MATH]
(6)
Weight (E) and Weight (N) denoted the muscle weight of the GM on the
experimental and normal sides, respectively.
The GM and regenerated nerves were prepared, stained, and subsequently
observed under a microscope (ECLIPSE C1, Nikon, Japan). The diameter of
the regenerated nerve fibers and the thickness of myelin sheaths were
characterized using a transmission electron microscope (TEM,
HT7800/HT7700, hitachi, Japan). Immunofluorescence staining for NF200,
MBP, GFAP, and Tuj-1 was performed to detect neural axon regeneration,
followed by image acquisition using CLSM. The diameters of GM fibers,
regenerated nerve fibers, and myelin sheath thickness were quantified
using nano measurer software ([75]http://www.downxia.com) based on the
image results. Additionally, the immunofluorescence results of the
regenerated nerves were analyzed using ImageJ software
([76]http://rsb.info.nih.gov/ij/).
2.13. Immunocytometric analysis and transcriptomic assay were conducted on
the regenerated sciatic nerve, while motor endplate (MEP) assay was performed
on GM
After the administration of anesthesia, the rats were subjected to
cardiac perfusion using a saline solution. The regenerated nerves were
then extracted and diced into small fragments, which were subsequently
placed in a digestion buffer containing RPMI-1640 medium, collagenase
IV, and DNase I for 1 h. Following this step, the samples underwent
filtration through a 70 μm nylon filter to eliminate any undigested
debris, while cells were collected by centrifugation. Finally, the
cells were stained with 1 μL of CD11b and CD86 antibodies for 30 min at
4 °C under light protection. After the completion of staining, the
buffer was added to suspend the staining, and subsequent washing steps
were performed. Following fixation with PFA and additional buffer
washes, permeabilization was achieved using intracellular staining
buffer. After further washing with buffer, cells were stained with 1 μL
CD206 antibody for 40 min under light-protected conditions.
Subsequently, after discontinuing the staining and performing a final
round of cell washing using buffer, the samples were analyzed by flow
cytometry (Becton, Dickinson and Company, USA).
The GM specimens were prepared as frozen sections, which were then
immersed in pre-cooled 10 % calcium formaldehyde for 10 min.
Subsequently, the sections were thoroughly rinsed with distilled water.
Following this, the sections were incubated in an AChE incubation
solution at 37 °C for 2 h, ensuring they were kept away from light
until they attained a light brown coloration. The sections underwent
further rinsing under running water and subsequently underwent staining
with hematoxylin stain for 5 min. This was followed by another round of
rinsing under running water lasting for 10 min. Finally, after routine
sealing procedures had been carried out, photographs were captured
using the microscope (ECLIPSE C1, Nikon, Japan).
The animal tissue specimens were cryopreserved at −80 °C immediately
after isolation for optimal preservation. RNA integrity was evaluated
using the Bioanalyzer 2100 system with the RNA nano 6000 assay kit.
Total RNA was utilized as input material for the preparation of RNA
samples. The index-coded samples were clustered on a cBot cluster
generation system using TruSeq PE cluster kit v3-cBot-HS (Illumia),
following the manufacturer's instructions. After cluster generation,
the library preparations were sequenced using an Illumina Novaseq
platform, resulting in the generation of 150 bp paired-end reads. The
raw data (raw reads) in fastq format underwent initial processing
through fastp software. Reference genome and gene model annotation
files were directly downloaded from the genome website. The mapped
reads for each sample were assembled using StringTie (v1.3.3b) in a
reference-based approach [[77]23]. Featurecounts v1.5.0-p3 was employed
to quantify the number of reads mapped to each gene.
The clusterProfiler package was utilized to perform Gene ontology (GO)
enrichment analysis on differentially expressed genes (DEG), with gene
length bias correction applied. GO terms exhibiting corrected P-values
less than 0.05 were deemed significantly enriched by DEG. The kyoto
encyclopedia of genes and genomes (KEGG) serves as a database resource
for understanding high-level functions and utilities of biological
systems, including cells, organisms, and ecosystems, based on
molecular-level information derived from large-scale molecular datasets
generated through genome sequencing and other high-throughput
experimental technologies ([78]http://www.genome.jp/kegg/). We utilized
the clusterProfiler package to assess the statistical enrichment of
differential expression genes in KEGG pathways. Gene set enrichment
analysis (GSEA) is a computational approach employed to determine if a
predefined gene set exhibits significant and consistent differences
between two biological states. The GSEA analysis tool
([79]http://www.broadinstitute.org/gsea/index.jsp), along with GO and
KEGG datasets, were independently employed for conducting GSEA.
2.14. The assessment of nutritional status and the testing of organ toxicity
in rats
The nutritional status of the rats was evaluated based on changes in
body weight over 3 months. The heart, liver, spleen, lung, and kidney
specimens were fixed in PFA solution, followed by dehydration and
preparation of paraffin sections. These sections were then
deparaffinized and rehydrated by using xylene immersion. Subsequently,
all slices were stained with H&E dye, rinsed with water, and dehydrated
using graded ethanol. Finally, the sections underwent two rounds of
xylene soaking before capturing photomicrographs using the microscope
(ECLIPSE C1, Nikon, Japan).
2.15. Statistical analysis
Data were presented as the mean ± standard deviation (SD) and were
analyzed with the GraphPad Prism 7.04 software (Graphpad Inc., San
Diego, CA, USA). The Student's t-test was used for statistical
analysis. Statistical significance was set at *P < 0.05 and high
statistical significance was set as **P < 0.01 and ***P < 0.001.
3. Results and discussion
3.1. The PLGA electrospinning fibers were prepared using various roller
receiver speeds
In the study of electrospinning fiber alignment, a solution was
prepared using equal volume ratios of DCM and DMF. Due to its high
volatility, an excessive amount of DCM can lead to rapid viscosity
increase in the solution, resulting in clogging of the jet needle and
failure in the preparation process. Conversely, an excessive proportion
of DMF may generate numerous beaded fibers that adversely affect the
surface morphologies of the fibers. Therefore, for our study on
electrospinning fiber alignment, we opted to mix these two solutions in
equal volumes and successfully obtained PLGA fibers with uniform
morphology and excellent alignment ([80]Fig. 1A).
Fig. 1.
[81]Fig. 1
[82]Open in a new tab
Preparation of aligned porous electrospinning fibers and their physical
properties. (A) SEM images depict PLGA fibers at various roller speeds.
(B) The alignment of PLGA fibers is quantified at different rotational
speeds (n = 100, n represents the number of electrospinning fibers for
each speed). (C) SEM images display PLGA electrospinning fibers
prepared using different volume ratios of DCM/DMF polymer solutions.
(D) and (E) The hydrophilicity of PLGA fiber films with different
alignments is evaluated (n = 7, n indicates the number of samples
tested in each group). (F) Fiber diameter measurements are conducted on
PLGA fiber films at different roller speeds to assess their size
distribution and uniformity (n = 100, n represents the number of
electrospinning fibers for each rotational speed; * indicates P < 0.05
compared with PLGA-0 group). (G) External phase, (H) density, (I)
porosity, and (J) water absorption of different PLGA fiber films
(n = 3, n indicates the number of samples tested in each group). All
statistical data are represented as mean ± SD.
The alignment of the electrospinning fibers is influenced by the type
of receiver employed. Isotropic electrospinning fibers are obtained
when a plane receiver is utilized, whereas anisotropic fibers are
achieved with a roller collector. The impact of rotational speed on
fiber alignment was analyzed through SEM images at a magnification of
5 × 10^3. The fibers exhibited an isotropic morphology when a plane
collector (0 rpm) was utilized, as depicted in [83]Fig. 1A. However,
the alignment of the fibers was limited when a roller collector
operated at a lower speed (500 rpm). As the roller speed increased, so
did the alignment of fibers. Nevertheless, once the speed surpassed a
critical value, further increments resulted in diminished fiber
alignment. The continuous centrifugal and shear force generated by the
high-speed roller collector resulted in the fibers being easily pulled
in different directions, thereby diminishing their overall alignment
[[84]24]. Utilizing a methodology employed in previous studies, we
determined that the alignment of PLGA fibers at 500 rpm, 1000 rpm,
1500 rpm, 2000 rpm, and 2500 rpm was calculated to be 45 %, 66 %, 77 %,
71 %, and 74 % respectively [[85]25] ([86]Fig. 1B).
The water contact angles of the PLGA fiber films prepared at different
roller receiver speeds were shown in [87]Fig. 1D, exhibiting values of
123.51 ± 0.98°, 123.58 ± 1.61°, 122.21 ± 2.32°, 118.63 ± 2.49°,
120.62 ± 2.14°, and 117.71 ± 1.62° ([88]Fig. 1E). These results
consistently demonstrated the hydrophobic nature of all PLGA fiber
films fabricated in our study. The diameters of the isotropic PLGA
fibers measured 0.67 ± 0.02 μm ([89]Fig. 1F). The electrospinning
fibers prepared using the roller receiver exhibited significantly
smaller diameters compared to the isotropic fibers, which can be
attributed to fiber stretching induced by the centrifugal force
generated through rotation of the roller receiver [[90]26].
In addition to the rotational speed of the receiver, the diameter of
the electrospinning fibers is closely correlated with other preparation
parameters. Ramacciotti et al. demonstrated that an increase in polymer
solution concentration resulted in a corresponding enlargement of fiber
diameter, potentially attributed to the higher viscosity of
concentrated solutions and slower formation of Taylor's cone,
necessitating a stronger electric field force for electrospinning fiber
preparation [[91]27]. The electric field force was enhanced by
increasing the applied voltage, resulting in a decrease in the diameter
of the prepared fibers. It was observed that higher voltages led to
smaller fiber diameters. Other preparation parameters, such as
temperature, humidity, and the distance between the jetting needle and
receiver, had minimal impact on electrospinning fiber diameter
[[92]27]. The previous study demonstrated a direct correlation between
the velocity of polymer solution spraying and the resulting fiber
diameter, potentially attributed to reduced stretching time under the
influence of an electric field. The inadequate stretching was
identified as a contributing factor for larger fiber diameters
[[93]28].
3.2. The porous PLGA fibers were fabricated by blending varying ratios of DCM
and DMF
We fabricated electrospinning fibers with a rough, porous surface
structure using a phase separation method ([94]Fig. 1C). PLGA particles
were dissolved in a mixture of DCM and DMF at varying volume ratios
(10:1, 8:1, 6:1, 4:1, 3:1, 2:1). The presence of porous structures was
attributed to the solvent evaporation rate during fiber preparation.
The formation of porous structures in PLGA fibers was facilitated by a
higher percentage of volatile DCM in the solvent. However, if the DCM
ratio exceeded 8:1, severe clogging occurred at the injection port and
only a few PLGA fibers could reach the roller receiver. Increasing the
DMF ratio beyond a certain level resulted in the disappearance of the
porous structure and the development of a banded groove structure on
the fiber surface (at ratios of 4:1 and 3:1), which may be attributed
to insufficient volatility of the solvent to form the porous structure
by liquid phase separation. As the DMF ratio continued to increase, the
banded grooves on the fiber surface gradually disappeared and were
replaced by a smooth surface (2:1). In order to achieve the optimal
structure of PLGA fibers, we ultimately selected a 6:1 ratio for fiber
preparation.
Porous fibers are of interest for various applications, such as
filtration or tissue engineering repair [[95]29]. For instance,
specific surface topologies play a crucial role in influencing cell
behavior and facilitating specific adsorption processes. Bognitzki et
al. have concluded that the selection of appropriate parameters and
solvents during electrospinning can directly yield porous fibers
[[96]30]. The porous morphology of the fibers is achieved through phase
separation during the electrospinning process, resulting in spinodal or
binodal types of phase morphologies within the fibers. This also leads
to a rapid increase in the jet surface within a few milliseconds.
Solvent evaporation occurs on time scales significantly below the
second-range, allowing for the crossing of phase boundaries and the
formation of structures through phase separation [[97]30].
The previous study revealed that the utilization of volatile solvents,
such as DCM, resulted in the formation of polymer fibers with a regular
porous structure. Their interpretation was that rapid phase separation
during the electrospinning process led to the creation of a consistent
phase morphology [[98]30]. It appeared that solvent-rich regions
transformed pores. Substituting DCM with a less volatile solvent
notably diminished the propensity for pore formation, which aligned
with our experimental findings.
The porous structure did not disrupt the structural guidance for
neurons in the aligned fibers, as evidenced by numerous previous
studies. For instance, Kim et al. demonstrated that fibrous scaffolds
composed of porous and aligned polycaprolactone (PCL)/silk/quercetin
exhibited superior nerve repair capabilities compared to aligned nerve
scaffolds [[99]31]. Additionally, Zhou et al. showed that elliptical
nano-pore surfaces on aligned electrospinning poly(l-lactic acid)
(PLLA) fibers enhanced the cellular response of vascular smooth muscle
cells [[100]32].
The fiber diameter tended to decrease as the percentage of DCM
decreased, as depicted in [101]Fig. 1C, aligning with previously
reported findings [[102]28]. We posit that the variation in the DCM
ratio primarily influenced the surface morphology of the fibers rather
than their diameter. Furthermore, the impact of fibers with different
diameters on neuronal cells remained a subject of debate. Daud et al.
concluded that thicker fibers exhibited promotion of nerve axon growth
[[103]28]. However, Yao et al. demonstrated no significant variance in
the promotion of axon growth by fibers with different diameters
[[104]33]. It is noteworthy that the morphology of electrospinning
fibers is influenced by various factors, including diverse polymers and
solvent ratios. In our preparation process, when the ratio of DCM to
DMF was 12:1, the highly volatile DCM rapidly evaporated and led to a
rapid increase in solution viscosity. Consequently, the formation of
Taylor's cone was delayed due to the increased viscosity, resulting in
solidification and blockage of the injection needle. This phenomenon
was also observed at a ratio of 10:1.
As shown in [105]Fig. 1G, the porous PLGA fiber films were prepared
using a mixed solution of DCM/DMF (V:V = 6:1), and their appearance
resembled that of the nonporous PLGA fiber films. The densities of the
nonporous and porous fiber films were calculated to be
0.10 ± 0.01 g cm^−3 and 0.09 ± 0.01 g cm^−3, respectively. There was no
significant difference in densities observed between the two types of
fiber films, possibly due to the minimal impact of the nanoscale porous
structure on their densities ([106]Fig. 1H).
The porosity of nonporous and porous PLGA fiber films was determined
using the ethanol displacement technique, yielding values of
87.91 ± 3.11 % and 86.93 ± 2.43 %, respectively. The inability of
ethanol to penetrate nanoscale pores can be attributed to its surface
tension. Given that both fiber films were prepared under identical
rotational speed, their alignments were similar, resulting in
comparable porosities ([107]Fig. 1I). The water absorption capacities
of the nonporous and porous PLGA films were 214.11 ± 2.32 % and
214.08 ± 0.96 %, respectively. However, due to their similar
porosities, there was no significant difference observed in their water
absorption rates ([108]Fig. 1J).
3.3. The impact of PEDOT:PSS-coated porous PLGA fibers on cellular
proliferation
Conducting polymers (CPs) are polymers with delocalized electrons in
the backbone and whose backbone atoms are connected to π-bonds. The
conjugated backbone provides a pathway for electron migration,
resulting in enhanced electrical conductivity [[109]22]. CPs are
generally considered non-toxic and have no impact on cell growth
[[110]34]. Numerous CPs have been utilized in the field of tissue
engineering, including PPy, PANI, PEDOT, and poly(3-hexylthiophene)
[[111]22]. The conducting polymer PEDOT is commonly doped with PSS to
form a stable aqueous suspension of particles [[112]35]. Due to its
exceptional chemical stability and conductivity, PEDOT finds
applications in diverse fields including energy reserves, sensors,
conductor electrode materials, biotechnology, and medicine [[113]36].
Ghasemi-Mobarakeh et al. reported that the incorporation of CPs in
tissue engineering has been shown to enhance cell adhesion and
proliferation [[114]37]. Similarly, Shahini et al. achieved
satisfactory outcomes by utilizing PEDOT:PSS in bone tissue engineering
[[115]38]. In this study, we employed the dip-coating technique to
uniformly coat PLGA fiber films with PEDOT:PSS solution and
investigated its impact on nerve cell growth ([116]Fig. S1A).
We assessed the proliferation of PC-12 cells at various time points
using the CCK-8 reagent. As seen in [117]Fig. S1B, uncoated PLGA fiber
films exhibited significant promotion of cell proliferation at 24h,
48h, and 72h, whereas coated fiber films demonstrated inhibition of
cell proliferation (P < 0.001). The pre-treatment of both sets of PLGA
fiber films with UVO enhanced their hydrophilicity, potentially
facilitating cell adhesion and proliferation [[118]39]. A previous
study indicated that high concentrations of PEDOT:PSS solution may
exhibit cytotoxicity [[119]40]. Babaie et al. reported that lower
concentrations of PEDOT:PSS solution can enhance cellular activity
[[120]41]. The conductivity of PEDOT:PSS facilitates cell signaling and
promotes the adsorption of cell surface proteins [[121]42]. Therefore,
we planned to perform a gradient dilution of the PEDOT:PSS solution to
study its optimal coating concentration.
3.4. Exploring the optimal coating concentration of PEDOT:PSS solution for
coating
Our study investigated the impact of varying concentrations of
PEDOT:PSS solution on cellular proliferation. The presence of a
high-concentration solution significantly impeded cell growth; however,
upon dilution, cells exhibited improved growth potential. We conducted
a gradient dilution of the PEDOT:PSS solution using PBS ([122]Fig. 2A).
At a concentration of 0.55 wt%, no significant change in color was
observed for the diluted PEDOT:PSS solution. As the concentration
decreased to 0.017 wt%, the solution gradually lightened in color and
approached transparency. Different concentrations of PEDOT:PSS solution
were employed to assess their effects on PC-12 cell and SC
proliferation.
Fig. 2.
[123]Fig. 2
[124]Open in a new tab
The impact of gradient dilution of the PEDOT:PSS solution on cell
proliferation. (A) The external phase of the gradient dilution of the
PEDOT:PSS solution. (B) The impact of gradient dilution of the
PEDOT:PSS solution on the proliferation of PC-12 and SC cells (n = 5, n
represents the number of experimental replicates at each coating
concentration). All statistical data are represented as mean ± SD (*
indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001).
The proliferation of both cells at different time points was assessed
using CCK-8 reagent ([125]Fig. 2B). Even when the PEDOT:PSS solution
was diluted to a concentration of 0.138 wt%, the growth of PC-12 cells
remained significantly inhibited after 24 h of incubation. However,
when a solution with a concentration of 0.069 wt% was used in cell
culture, the proliferation of the cells showed significant improvement
while still exhibiting some degree of inhibition.
The cell proliferation in the 0.034 wt% solution exhibited further
enhancement upon dilution of the coating solution. Subsequent dilutions
did not yield significant differences in cell proliferation between
neighboring concentrations, yet overall trends indicated a gradual
increase in cell proliferation with solution dilution, followed by a
decline after reaching a certain concentration. The proliferation of
PC-12 cells at the time points of 48 and 72 h, as well as SCs,
exhibited a similar pattern. This could be attributed to the impact of
highly concentrated CP solution on cell growth and proliferation due to
its permeability and toxicity, while the hydrophilic and conductive
properties of PEDOT:PSS facilitated cell adhesion when the solution was
appropriately diluted [[126]43]. The CP can induce an electric field in
the cell membrane, and this alteration of ion channels and
bioelectricity within the membrane may further enhance cell
proliferation [[127]43]. Based on the experimental results from 6 time
points, a solution concentration of 0.017 wt% was determined as the
optimal concentration for promoting cell proliferation. Therefore, we
selected this specific concentration to prepare the coated conduit.
3.5. The optimal concentration of PEDOT:PSS was used to coat porous PLGA
fiber films
The majority of nerve tissue engineering research on PEDOT has focused
on its application as an electrode material. In contrast to PANI, PEDOT
is soluble and can be chemically modified in various organic solvents,
making it suitable for a wide range of implantable nerve scaffolds
[[128]44]. Additionally, PEDOT:PSS exhibits both ionic and electronic
conductivity due to its porous nature, enabling the exchange of ions
between the material and the biological medium. Although previous
reports have suggested that PEDOT may degrade during ES, other studies
have reported that PEDOT can be stabilized over approximately 100
million pulses using parameters matched to peripheral nervous system
(PNS) [[129]45]. Therefore, it is crucial to select an appropriate
concentration of PEDOT:PSS coating for effective ES treatment in PNR.
The external phase of the PLGA fiber film, after being coated with the
optimal concentration of PEDOT:PSS solution, is shown in [130]Fig. 3A.
The electrical conductivity of NGC also plays a crucial role in
effectively promoting PNR [[131]14]. As illustrated in [132]Fig. 3B,
the aligned fiber films exhibited anisotropic conductivity values of
0.07 ± 0.01 S cm^−1 parallel to the fibers and 0.02 ± 0.01 S cm^−1
perpendicular to the fibers (p < 0.001). This disparity can be
attributed to the preferential movement of electrons along the fiber
direction while hindering their movement perpendicular to it [[133]46].
Zhang et al. demonstrated that the disparity in electrical conductivity
between parallel and perpendicular orientations of electrospinning
fibers was more than tenfold [[134]14]. In contrast, our findings
revealed that the discrepancy in conductivity between these two
directions was less than fivefold, which can be attributed to the
interconnection of adjacent fibers through the PEDOT:PSS coating,
thereby reducing the variance in conductivity.
Fig. 3.
[135]Fig. 3
[136]Open in a new tab
External phase and characterization of porous PLGA electrospinning
fibrous films coated with the optimal concentration of PEDOT:PSS. (A)
The external phase of the coated porous PLGA electrospinning fiber
film. (B) The electrical conductivity of coated porous PLGA
electrospinning fiber films (n = 9, n represents the number of samples
tested in each group). The surface morphology of (C) uncoated and (D)
coated porous PLGA fiber films. (E) The mapping and EDX results of
porous PLGA fiber films coated with optimal concentrations of PEDOT:PSS
(n = 3, n indicates the number of samples tested in each group). (F)
The mechanical properties of PLGA fiber films were tested.
Stress-strain curves were obtained for uncoated PLGA fiber films in
both parallel (G) and perpendicular (H) directions to the fiber
alignment (n = 3, n indicates the number of samples tested in each
group). Stress-strain curves were obtained for coated PLGA fiber films
in both parallel (I) and perpendicular (J) directions to the fiber
alignment (n = 3, n indicates the number of samples tested in each
group). (K) The in vitro hydrophilicity of coated and uncoated PLGA
fiber films (n = 10, n indicates the number of samples tested in each
group). All statistical data are represented as mean ± SD (***
indicates P < 0.001).
The SEM images of the porous PLGA fibers before and after coating with
appropriate concentrations of PEDOT:PSS are presented in [137]Fig. 3C
and D, respectively. Before coating, the PLGA fibers exhibited a smooth
surface except for the presence of porous structures. In contrast, the
PEDOT:PSS coating did not cover the pores but was uniformly distributed
on the fiber surface, providing an ideal foundation for nerve cell
adhesion and proliferation. The coated PLGA fiber films were analyzed
using mapping testing and EDX spectroscopy to detect the presence of
sulfur elements ([138]Fig. 3E). Elemental sulfur was exclusively found
in the PEDOT:PSS coating, while no traces were observed in the PLGA
film. Mapping testing results demonstrated a uniform distribution of
conductive coatings on the surface of PLGA films, as evidenced by the
presence of sulfur elements throughout. Additionally, EDX analysis
confirmed the successful coating of the PLGA film surface with
PEDOT:PSS.
To further investigate the conductive coating on the film, XPS testing
was conducted on the coated film to analyze elemental sulfur. As shown
in [139]Fig. S2, the 2P binding energies of elemental sulfur in PSS and
PEDOT were approximately 169 eV and 165 eV, respectively, which aligned
with the previous research [[140]47]. These findings additionally
corroborated the presence of PEDOT:PSS conductive coatings on PLGA
films.
The mechanical properties of the coated and uncoated fiber films were
evaluated using an electronic universal testing machine ([141]Fig. 3F).
The stress-strain curve demonstrated that the uncoated PLGA fiber film
exhibited favorable tensile properties, with its elastic response
attributed to the excellent flexibility within the range of elastic
deformation ([142]Fig. 3G) [[143]48]. Conversely, when subjected to
stress-strain tests perpendicular to the fiber alignment, the samples
easily detached due to a lack of opposing forces in the vertical
direction caused by anisotropic electrospinning fibers ([144]Fig. 3H).
The stress-strain curve obtained from tensile testing conducted
parallel to the fiber direction on the coated fiber film is shown in
[145]Fig. 3I. Following the coating process, enhancements were observed
in the tensile elastic limit, elastic modulus, and strength limit of
the fiber film; however, a reduction was noted in its breaking
elongation. The accumulation of conductive coating at the joints of
different fibers may contribute to this phenomenon, as the adhesive
nature of the coating impedes fiber elongation and sliding between
them. Consequently, tensile strength increases while deformation
capacity [[146]49]. The change in mechanical properties of the coated
film was observed not only parallel to the fiber alignment but also
perpendicular ([147]Fig. 3J). The increased viscosity of the conductive
coating enhanced adhesion between fibers, increasing the tensile
strength. However, it also heightened material brittleness and
consequently decreased breaking elongation.
Although the porous structure has some impact on the mechanical
properties of electrospinning fibers, the presence of a conductive
coating enhances their mechanical strength. The elastic modulus of
uncoated fiber film parallel to the fibers was 2.29 ± 0.15 MPa
([148]Fig. 3G), whereas it increased to 5.33 ± 0.19 MPa after coating
([149]Fig. 3I). These improved mechanical properties provide sufficient
support for nerve regeneration [[150]50].
The FTIR spectra of uncoated PLGA fiber film, PEDOT:PSS conductive
coating and coated PLGA fiber film were presented in [151]Fig. S3. The
absorption peak (-OH) at both ends of PLGA was observed at 3509 cm^−1.
Meanwhile, the stretching vibration peak (-C Created by potrace 1.16,
written by Peter Selinger 2001-2019 O) of PLGA appeared at 1759 cm^−1,
exclusively in the spectrum of PLGA and not in PEDOT:PSS. A distinctive
peak (-SO[3]^-) was detected at 1224 cm^−1 solely in the PEDOT:PSS
conductive coating but absent in the PLGA. The stretching vibration
peak (–COO–) at 1174 cm^−1 was observed exclusively in PLGA and not in
PEDOT:PSS. Additionally, the characteristic peak (-C-O-C-) at
1094 cm^−1 was identified solely in PEDOT:PSS. These findings from the
FTIR spectra indicate the successful coating of PLGA fiber film with a
conductive PEDOT:PSS coating.
The water contact angle serves as an indicator of the hydrophilicity of
the electrospinning fiber film. A water contact angle exceeding 90°
indicates its hydrophobic nature, whereas a value below 90° suggests
acceptable hydrophilicity [[152]22]. While hydrophilicity plays a
crucial role in cell adhesion to the surface of the fiber film, it also
significantly influences cell proliferation [[153]22]. The water
contact angles of the uncoated and coated PLGA films were
123.07 ± 2.01° and 54.03 ± 3.94°, respectively (p < 0.001) ([154]Fig.
3K). Consequently, the hydrophilicity of the coated film was
significantly enhanced, thereby promoting cell adhesion and
proliferation.
3.6. The in vitro and in vivo degradation of porous PLGA fiber films coated
with PEDOT:PSS
The polymer PLGA is widely recognized for its exceptional
biocompatibility, excellent biodegradability, and ease of fabrication,
making it highly suitable for various applications in tissue
engineering. A notable advantage of PLGA NGCs lies in their
biodegradable nature, eliminating the need for a secondary surgical
procedure to remove them. To ensure optimal performance, the
degradation rate of NGCs must align with the pace of nerve
regeneration. The optimal degradation time of PLGA allows for
sufficient mechanical support for PNR without hindering its progress.
After 100 days of degradation, the nonporous PLGA fiber film, porous
PLGA fiber film, and coated porous PLGA fiber film degraded to
51.26 ± 0.93 %, 52.15 ± 0.76 %, and 51.24 ± 0.21 % of their original
qualities, respectively ([155]Fig. S4). Due to the UVO treatment
applied to the fiber films, elastase could effectively penetrate the
films. The appropriate degradation rate of PLGA fiber films creates
favorable conditions for PNR.
Additionally, PLGA is among the limited number of biomaterials that
have been approved by the food and drug administration (FDA) for both
experimental and clinical applications [[156]51]. Numerous previous
studies have demonstrated the suitability of PLGA as a material for PNR
[[157][52], [158][53], [159][54], [160][55]]. Faroni et al. revealed
that cylindrical NGCs composed of PLGA exhibited excellent flexibility,
biodegradability, permeability, and facilitated easy suturing of
transected nerve stumps. When the NGC was surgically implanted into a
12-mm gap in the rat sciatic nerve, resulting in the successful PNR
[[161]56]. Furthermore, PLGA has been extensively investigated for its
ability to provide adequate mechanical support for nerve regeneration
in numerous studies [[162]25,[163]57,[164]58]. These findings
demonstrate the reliability of using PLGA for the preparation of NGCs.
In addition to its applications in PNR, PLGA has also been utilized in
various other medical fields without limitations imposed by its
degradation products, including skin grafting, wound closure, and
micro- and nanoparticles. Various applications of PLGA drug microsphere
preparation have also been reported, including the utilization of PLGA
microspheres as carriers for protein and enzyme drugs, which is a
prominent area of research [[165]59]. Additionally, PLGA is employed as
a drug carrier in Lupron Depot, an effective treatment for advanced
prostate cancer.
We implanted uncoated and coated PLGA fiber films into the subcutaneous
fascia layer of the rat dorsum for in vivo degradation testing
([166]Fig. S5A). The weight changes of the rats were monitored over 3
months post-implantation. Body weight variations can reflect both rat
growth and potential implant toxicity. All groups exhibited an increase
in body weight following fiber film implantation, indicating no
significant impact on their growth ([167]Fig. S5B).
[168]Fig. 4 displays the uncoated and coated PLGA fiber films at 1, 2,
and, 3 months post-implantation, along with the corresponding H&E
staining results. At the mark point, the uncoated PLGA fiber film was
observed to be encapsulated by soft tissue, accompanied by an
inflammatory infiltrate surrounding it 1 month after surgery. This
phenomenon primarily resulted from the in vivo degradation of PLGA,
which is a complex process involving various cell types such as
eosinophils and macrophages [[169]60]. After 2 months post-surgery,
only a minimal amount of residual PLGA was observable, and the cellular
infiltration had essentially subsided. 3 months following implantation,
complete disappearance of PLGA occurred, rendering the implanted area
indistinguishable from normal tissue. Regarding the coated fiber film,
no conspicuous aggregation of inflammatory cells surrounding the
coating was observed, thus confirming its excellent biocompatibility.
The presence of undegraded coated PLGA films at the 3-month
postoperative mark suggests that the conductive coating effectively
impedes cell-PLGA interaction and retards in vivo degradation of the
film. The conductive coating exhibited excellent biocompatibility as
evidenced by the absence of significant cellular infiltration in its
vicinity.
Fig. 4.
[170]Fig. 4
[171]Open in a new tab
The external phase of in vivo degradation and H&E staining were
performed on PLGA fiber films at various time points. No evident edema,
oozing, or inflammation was observed in the implanted area of the fiber
film during the external phase evaluation. Additionally, H&E staining
revealed no significant aggregation of inflammatory cells within both
the PLGA fiber film and its coating area, indicating excellent
biocompatibility of the implanted material. The magnified area is
indicated by a black square, while PLGA fiber films are denoted by
black arrows and PEDOT:PSS coating by red arrows (n = 3, n indicates
the number of samples tested in each group). (For interpretation of the
references to colour in this figure legend, the reader is referred to