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\begin{document}

\title{Mechanical-cellular electrical conversion model reshapes the immune
microenvironment of peripheral nerve by modulating neutrophil
extracellular traps}



\author[1]{Yaowei Lv}%
\author[2]{Xiangyun Yao}%
\author[3]{lei Zhan}%
\author[2]{Jinye Shi}%
\author[4]{Xiangyang Wang}%
\author[4]{Hede Yan}%
\author[2]{Xu Wang}%
\author[3]{Chen Huang}%
\author[1]{Yun Qian}%
\author[2]{Yuanming Ouyang}%
\affil[1]{Shanghai Sixth People's Hospital}%
\affil[2]{Shanghai 6th Peoples Hospital Affiliated to Shanghai Jiaotong University School of Medicine Department of Orthopaedic Surgery}%
\affil[3]{Donghua University}%
\affil[4]{The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University}%


\vspace{-1em}



  
  \date{June 14, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Perturbations of the immune homeostasis following peripheral nerve
injury (PNI) disturbs growth microenvironment that delays nerve repair.
Although extensive efforts have been made to stimulate nerve
regeneration, their efficacy is limited by energy deficiency and
persistent and overactive inflammation. It is not yet clear how
exogenous implantable neural electrical stimulation system regulates
immune homeostasis and promotes peripheral nerve regeneration. Here
reports a self-powered immunoactive scaffold based on piezoelectric and
electroconductive materials. Such in situ electrical stimulation
technique regulates lasting and high-level inflammatory cytokines
infiltrated in injured nerve tissue, modulates aberrant neutrophil
activities and promotes fast revascularization. By benefiting immune
balance and angiogenesis, this electroactive scaffold averts
growth-suppression following PNI and robustly facilitates neural
regeneration. Therefore, this piezoelectric model represents an
effective tool for PNI immunotherapy.%
\end{abstract}%



\sloppy


Mechanical-cellular electrical conversion model reshapes the immune
microenvironment of peripheral nerve by modulating neutrophil
extracellular traps

Yaowei Lv\textsuperscript{a,b,1}, Xiangyun Yao\textsuperscript{a,1}, Lei
Zhan\textsuperscript{c,1}, Jinye Shi\textsuperscript{a,b,d,1}, XiangYang
Wang\textsuperscript{e}, Hede Yan\textsuperscript{e}, Xu
Wang\textsuperscript{a,b}, Chen Huang\textsuperscript{c,***},Yun
Qian\textsuperscript{a,b,**}, Yuanming Ouyang\textsuperscript{a,b,*}

a National Center for Orthopedics, Department of Orthopedics, Sixth
People's Hospital Affiliated to Shanghai Jiao Tong University School of
Medicine, Shanghai, 200233, China;

b Shanghai Engineering Research Center for Orthopaedic Material
Innovation and Tissue Regeneration, Shanghai, 200233, China;

c State Key Laboratory for Modification of Chemical Fibers and Polymer
Materials, College of Textiles, Donghua University, Shanghai, 201620,
China

d College of Fisheries and Life Science, Shanghai Ocean University,
Shanghai, 201306, China

e Department of Orthopaedics, The Second Affiliated Hospital of Wenzhou
Medical University, Wenzhou, 325003, China.

1 These authors contributed equally to this work.

\section*{Keywords}

{\label{keywords}}

piezoelectricity, piezoelectric nanogenerator, angiogenesis, peripheral
nerve regeneration, tissue engineering

\section*{Abstract}

{\label{abstract}}

Perturbations of the immune homeostasis following peripheral nerve
injury (PNI) disturbs growth microenvironment that delays nerve repair.
Although extensive efforts have been made to stimulate nerve
regeneration, their efficacy is limited by energy deficiency and
persistent and overactive inflammation. It is not yet clear how
exogenous implantable neural electrical stimulation system regulates
immune homeostasis and promotes peripheral nerve regeneration. Here
reports a self-powered immunoactive scaffold based on piezoelectric and
electroconductive materials. Such \emph{in situ} electrical stimulation
technique regulates lasting and high-level inflammatory cytokines
infiltrated in injured nerve tissue, modulates aberrant neutrophil
activities and promotes fast revascularization. By benefiting immune
balance and angiogenesis, this electroactive scaffold averts
growth-suppression following PNI and robustly facilitates neural
regeneration. Therefore, this piezoelectric model represents an
effective tool for PNI immunotherapy.

\section*{Highlights}

{\label{highlights}}

\begin{itemize}
\tightlist
\item
  The optimal electrical stimulation parameters for neural regeneration
  were identified.
\item
  The electrical stimulation model can enhance rapid vascularization of
  endothelial cells and provide them with energy.
\item
  Electrical stimulation may reshape the immune microenvironment by
  regulating the formation of neutrophil extracellular traps (NETs)
\end{itemize}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Graphical abstract}

A summary diagram of the mechanisms by which peripheral nerve conduits
promote nerve repair. Self-generated piezoelectric nanofilms produce
micro-currents. The neutrophil senses electrical stimulation and breaks
down the extracellular trap, which promotes nerve reconstruction.

\section*{1. Introduction}

{\label{introduction}}

The repair of nerve injuries remains a challenging issue in clinical
treatment{[}1{]}. Electrical stimulation proved effective in promoting
nerve regeneration in clinical practice{[}2, 3{]}. However, current
applications face several challenges: 1) The frequency and amplitude of
exogenous electrical stimulation, which are crucial for peripheral nerve
regeneration, are difficult to control. In clinical settings, high
electric currents often lead to complications such as pain, electrical
injuries, and psychological trauma{[}4{]}. 2) The intrusive power supply
equipment is bulky, difficult to replace, has a short lifespan, and may
trigger immune rejections{[}5{]}. 3) Invasive electrodes produce
electrical stimulation to cells at the injury site, which is
uncontrollable{[}6, 7{]}. Ideally, an implantable neural electrical
stimulation system should be controllable and compatible with neural
preferences.

Piezoelectric materials offer an in-built power source for peripheral
nerve repair{[}8, 9{]}. The advancement of pliable materials, which
convert passively generated mechanical energy into electrical energy, is
crucial for sustainable energy production. This kind of mechanical
energy can be generated by muscle contractions, produced either by the
autonomic nervous system, or the body's self-regulating cells {[}10{]}.
However, harnessing this energy is often challenging as the placement of
implanted electrodes in areas such as the heart, aorta, and mediastinum
typically necessitates open-heart surgery. This often leads to
significant trauma, postoperative infections, and inevitable immune
rejection{[}11{]}. Skeletal muscle contracture usually results in
limited mechanical deformation, which restricts the generation of
adequate nerve electrical stimulation signals from the implanted
piezoelectric material and impedes nerve regeneration{[}12{]}. Following
PNI, various cells within the nerve tissue attempt to migrate across the
nerve stump{[}13{]}. Intriguingly, these minute cell-level deformations,
when acted upon by piezoelectric materials, can generate electrical
signals that are sufficiently reactive to excitable cells{[}14{]}. To
generate ample electrical stimulation \emph{in vivo} , bioactive
materials must leverage small deformations to produce electrical signals
that can sufficiently stimulate nerve electrical excitation{[}15{]}.
Indeed, enhancing the piezoelectric, ferroelectric, and dielectric
properties of biomaterials has been a focal point in nerve repair tissue
engineering{[}16{]}.

Polyvinylidene fluoride (PVDF) is favored for its easy preparation,
excellent mechanical flexibility, and good biological safety. It has
become a prominent component of piezoelectric materials and a popular
type of biomaterial in the field of tissue engineering{[}17{]}. The
molecular chain of PVDF contains a strongly electronegative fluorine
atom, thus providing a remarkable dipole moment. Among the five
crystalline polymorphic forms of PVDF, the \selectlanguage{greek}β \selectlanguage{english}phase has exceptional
piezoelectric properties{[}18{]}. However, the stiffness and
piezoelectric coefficient (D33) of conventional PVDF are less than those
of crystalline or ceramic piezoelectric materials. Various methods have
been developed to increase the piezoelectric properties of PVDF, such as
co-polymerization with other polymers, incorporation of nanoparticles,
mechanical drawing, annealing, or polarization. All these methods
promote the formation of \selectlanguage{greek}β \selectlanguage{english}phase in PVDF{[}19{]}. PVDF is frequently
co-polymerized with trifluoroethylene (TrFE). The piezoelectric
efficiency of the P (VDF-TrFE) copolymer is not constant and depends on
the factors including the copolymer content, the loading mechanical
force and the loading frequency{[}20{]}. The addition of fluorine atoms
expands the chain spacing and reduces the phase transition activation
energy, encouraging the transition from \selectlanguage{greek}α \selectlanguage{english}phase to \selectlanguage{greek}β \selectlanguage{english}phase without
mechanical stretching{[}21{]}. Nanoparticles such as zinc oxide{[}22{]},
graphene oxide{[}23{]} and barium titanate{[}24{]} can effectively
induce \selectlanguage{greek}β-\selectlanguage{english}phase formation. The addition of these nanoparticles as
nucleating agent can effectively promote the formation of \selectlanguage{greek}β \selectlanguage{english}phase in
PVDF. Lastly, the synthesis process, such as the synthesis temperature
and the electrospinning conditions, may also affect the formation of \selectlanguage{greek}β
\selectlanguage{english}phase.

The disruption of the immune microenvironment following PNI often serves
as a significant barrier to nerve regeneration{[}25{]}. In clinical
contexts, our understanding of the cellular and molecular mechanisms
that underpin immune cell responses to these disruptions remain limited.
Most of the existing focus has been on the immune reprogramming of
macrophages{[}26-28{]}. Neutrophils, the most abundant immune cells in
human peripheral blood, are the first to arrive at sites of tissue
damage{[}29{]}. These cells combat infection and inflammation through a
variety of processes, including intracellular degradation, extracellular
digestion, and extracellular entrapment{[}30{]}. However, the specific
role of neutrophils in the context of PNI remains largely undefined.

In this study, we designed a piezoelectric conductive composite nerve
scaffold. The complex activity of cell migration generates minute
deformations on the scaffold surface, which in turn leads to the
creation of electrical signals. These signals play a crucial role in
regulating cellular metabolism and biological behavior. The generated
microcurrents have been observed to limit the excessive inflammatory
response of neutrophils following PNI, thereby promoting the
regeneration of the damaged peripheral nerve tissue (Fig 1). Moreover,
nerve conduits fabricated from piezoelectric materials exhibit excellent
mechanical and electrical properties, as well as dependable biological
safety. Following three months of \emph{in vivo} animal experimentation,
the nerve conduits demonstrated significant efficacy in facilitating
nerve repair. Given the premise of biological safety, the
electrophysiological evaluation outcomes of the nerve conduits, coupled
with their ability to promote angiogenesis, were found to be comparable
with those achieved through autologous nerve transplantation.

\section*{2. Results}

{\label{results}}

\textbf{2.1. Excessive neutrophil after PNI inhibits nerve
regeneration.}

After peripheral nerve damage, the bioelectrical signal is immediately
interrupted. The immune system responds vigorously in real time. To
investigate the effect of early immune response following acute nerve
injury, we established a model of peripheral nerve transection and
conducted a preliminary experiment without any intervention. According
to prior research, neutrophils in the rats were the initial immune cells
to arrive at the injury site within a span of 6-8 hours, reaching a peak
approximately at the 12-hour mark. After a period of 1-2 days,
neutrophils completed their homing reaction, following which, their
count steadily decreased to a low level{[}31{]}. Consequently, we
decided to analyze the degree of inflammation and cellular status in
both injured and normal nerves, at 12 hours post-injury.

To measure the inflammation level subsequent to nerve transection
without any treatment, we employed histopathology and immunofluorescence
assays (Figure 2A-B). Upon examining the longitudinal section of the
proximal part of the sciatic nerve injury, we observed an aggregation of
neutrophils in the early phase of injury (Figure 2C). These neutrophils
were primarily located in the epineurium, exhibiting an increased
aggregation closer to the injury site (Figure S1), which foreboded that
immune system managing to redeem the disturbed immune microenvironment
and start regenerate programs. Similar outcomes were drawn from MPO
immunofluorescence experiment (Figure 2D).

C-caspase 3 acts as an early signifier of nerve damage, whereas KI67
serves as an indicator of initial nerve tissue repair. We assessed the
metabolic variations in the injured nerve through measuring the
expression levels of both C-caspase 3 and KI67 in the damaged and the
normal nerves (Figure2 E-F). According to \emph{in
vivo}immunofluorescent images, the elevated C-caspase 3 signifies injury
and apoptosis in a greater number of nerve-related cells, while the rise
in KI67 suggests that nerve repair commences at the injury's initial
phase (Figure 2G-H). The immunofluorescence results of IL-1, IL-6, and
TNF-\selectlanguage{greek}α \selectlanguage{english}also reported the immune disorder of the nerve after injury
(Figure 2I-O). To investigate the impact of neutrophil NET on neuronal
metabolism, we conducted \emph{in vitro} experiments, excluding the
interference of other cells. The activated neutrophil by Phorbol
12-myristate 13-acetate (PMA) released an abundant amount of NET,
leading to reduced proliferation and increased apoptosis in Schwann
cells (Figure 2P, Figure S2). Prior research has demonstrated that
excessive activation of NET considerably hampers tissue
repair{[}32{]},and that diminishing its expression aids in inflammation
control{[}29{]}. Similarly, the regulation of skeletal proteins through
electrical stimulation can influence the migratory ability of
cells{[}6{]}. we aim to apply this stimulation to the regulation of
immune cells. In conclusion, the aforementioned experiments confirmed
the conversion of the immune state in nerve tissue during the early
injury phase, and this disruption exhibited a regulable function.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 1.} Increased neutrophil infiltration of the damaged
nerve. (A) HE staining of Sciatic nerve in the longitudinal section with
or not with injury. Scale bars=1000 \selectlanguage{greek}µ\selectlanguage{english}m; 50\selectlanguage{greek}μ\selectlanguage{english}m; 20\selectlanguage{greek}μ\selectlanguage{english}m (B) Myeloperoxidase
(MPO) immunofluorescent staining image of the nerve. Scale bars=500 \selectlanguage{greek}µ\selectlanguage{english}m;
20\selectlanguage{greek}μ\selectlanguage{english}m (C) Quantitative analysis of the number of neutrophil cells in the
epineurium. (D) Mean fluorescent intensity (MFI) of MPO positive cell.
(E) Immunofluorescence staining for C-casepase-3 in Sciatic nerve. (F)
Immunofluorescence staining for Ki67 in Sciatic nerve. (G) MFI of
C-caspase 3 positive cell. (H) MFI of Ki67 positive cell. (I)
Immunofluorescence staining for IL-1 in Sciatic nerve. (J)
Immunofluorescence staining for IL-6 in Sciatic nerve. (K)
Immunofluorescence staining for TNF-\selectlanguage{greek}α \selectlanguage{english}in Sciatic nerve. (L) Merged
images of inflammatory markers from I to K. (M) MFI of IL-1 positive
cell. (N) MFI of IL-6 positive cell. (O) MFI of TNF-\selectlanguage{greek}α \selectlanguage{english}positive cell. (P)
Quantitative analysis of live dead cells analysis.

\textbf{2.2. The P(VDF-TrFE)/rGO/PCL nerve conduit converted mechanical
electrical signals into cellular electrical signals.}

Can tissue engineering scaffolds be used as regulater for interrupted
nerve current signals? It has been documented that piezoelectric
materials can influence cell behavior and viability via microcurrents
created by mechanical deformation{[}6, 14{]}. In an attempt to restore
the microenvironment of nerve regeneration disturbed in the early injury
phase, we developed a piezoelectric conductive composite scaffold to
ascertain whether it can modulate the aforementioned inflammatory
process. We have fabricated fiber films of varying concentrations using
electrospinning.

We employed electrospinning technology to fabricate three types of films
(rGO/PCL (rP), 10\%P (VDF-TrFE)/rGO/PCL (10\% PrP) and 20\%P
(VDF-TrFE)/rGO/PCL (20\% PrP)), which were subsequently folded into
tubular scaffolds for peripheral nerve regeneration (Figure 2A). The
electrospun fibers exhibited a relatively uniform morphology, and the
incorporation of rGO did not alter the fiber morphology (Figure 2B).
Moreover, rGO was uniformly loaded onto the fiber surface. The initial
modulus of all three scaffolds were comparable. Notably, when P
(VDF-TrFE) content reached 10\%, the resultant film displayed the
highest initial modulus (Figure 2C). Interestingly, the addition of P
(VDF-TrFE) enhanced crystallinity of electrospun fibers, indicating that
PrP possessed superior piezoelectric properties (Figure 2D). It is worth
mentioning that due to the low concentration of rGO used in this study,
its characteristic peaks could not be detected by Fourier-transform
infrared spectroscopy (FTIR, Figure 2E), which was consistent with
previous research findings\textsuperscript{{[}33{]}}. Due to the
hydrophobic nature of rGO and P (VDF-TrFE), remarkable hydrophilicity
was observed among rP, 10\% PrP and 20\% PrP films, and the blended
films remained hydrophobic as well (Figure 2F). Intriguingly, when
identical small loads were applied, both 10\% and 20\% PrP films
generated similar voltages that were significantly higher than those
produced by rGO/PCL film, and thereby confirming excellent
piezoelectricity conferred by P (VDF-TrFE) (Figure 2G-I). To further
evaluate the electrical characteristics of P (VDF-TRFE)/rGO/PCL
conduits, we tested the dielectric spectrum and dielectric loss of fiber
(Figure 2J-K). In order to ascertain the impact of the current produced
by these materials on neutrophils, we evaluated the intracellular
calcium levels in neutrophils exposed to different materials. Flow
cytometry results demonstrated that the 10\% PrP membrane significantly
augmented the calcium influx in cells compared to other groups (Figure
2L).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 2.} Fabrication and performance analyses of materials.
(A) Schematic diagram of electrospinning material preparation. (B)
Scanning electron microscopy (SEM) of fibrous membranes. Scale bars=5 \selectlanguage{greek}µ\selectlanguage{english}m
(C) Stress-strain curves of nerve conduits. (D) X-ray diffraction
patterns. (E) Fourier transform infrared spectroscopy. (F) Water contact
angle. Electro-mechanical conversion analysis of (G) rGO/PCL, (H) 10\%P
(VDF-TrFE) /rGO/PCL and (I) 20\%P (VDF-TrFE)/rGO/PCL. (J) Dielectric
spectrum of nerve conduits. (K) Dielectric loss of nerve conduits. (L)
Intracellular calcium transients induced by different conduits.

\textbf{2.3. The cellular electrical signals generated by piezoelectric
materials promote the rapid vascularization of endothelial cells.}

What are the electrical stimulation parameters suitable for
neurovascularization? In our previous experiments, we determined that
the optimal concentration of reduced graphene oxide (rGO) is
2\%{[}34{]}. To explore the ideal concentration of Copolymer P
(VDF-TrFE) in composite piezoelectric scaffolds, we integrated 0, 10\%
and 20\% P (VDF-TrFE) into 2\% rGO/PCL scaffolds and seeded cells onto
the surface. Schwann cells and endothelium, integral to peripheral nerve
repair, were thus chosen for the experiment with RSC96 and human
umbilical vein endothelial cells (HUVEC) to examine the biocompatibility
and functionality of PrP. Flow cytometry results indicated no
significant difference in the number of apoptotic Schwann cells cultured
across varying concentrations of the copolymer (Figure 3A). Cell
Counting Kit-8 (CCK8), a widely used method for evaluating cytotoxicity
and proliferation, showed an increase in OD value as cell proliferation
increases, darkening the solution. After intervals of 6H, 12H, 24h, and
48h, CCK8 results indicated that scaffolds with 10\% copolymer were less
toxic than those in the other two groups (Figure 3B).

Insufficient intraneuronal vascularization can affect peripheral nerve
regeneration {[}25{]}. To assess the angiogenic activity associated with
different concentrations of PrP, we conducted a Transwell test, a tube
formation test, and evaluated mitochondrial membrane potential in HUVEC
cells. The Transwell results indicated that the migratory ability of
cells in the 10\% PrP group surpassed that of the other two groups
(Figure 3C-E). Interestingly, despite the 10\% PrP group having a lower
migratory ability than the control group, its angiogenic capacity
exceeded that of the normal group in the tube formation experiment
(Figure 3F-J). This implies that 10\% PrP may enhance angiogenesis by
coordinating endothelial cell functions, rather than simply boosting the
migration ability (Figure 3K). The mitochondrial membrane potential
experiment exhibited a similar pattern (Figure 3L-N).

These findings suggest that 10\% PrP demonstrates relatively low
cytotoxicity and a robust capacity for promoting angiogenesis and
preserving endothelial mitochondrial energy. Consequently, we selected
10\% PrP as the material for further \emph{in vivo} biosafety and
efficacy evaluations. Unless otherwise specified in subsequent content,
all references to PrP refer to 10\% p (VDF-TRFE)/rGO/PCL nerve conduits.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 3.} Evaluation of P (VDF-TrFE)/ rGO/ PCL nerve
scaffolds\emph{in vitro} . (A) Apoptosis of Schwann cells measured by
flow cytometry (B) Cell Counting Kit-8 (CCK-8) experiment. (C)
Assessment of cell migration ability. Scale bars=50 \selectlanguage{greek}µ\selectlanguage{english}m (D) Quantitative
analysis of cell number passing through Transwell pores (E) Quantitative
analysis of the crossing index of the Migration Experiment (F)
Angiogenic experiments under bright field and (G) fluorescent
microscope. Scale bars=500 \selectlanguage{greek}µ\selectlanguage{english}m (H) Quantitative analysis of the nodes
number of the angiogenic experiment. (I) Quantitative analysis of the
junction number in angiogenic experiment. (J) Quantitative analysis of
the MFI of calcein. (K) The illustration of angiogenesis experiment (L)
Mitochondrial membrane potential assay kit with JC-1. Scale bars=50 \selectlanguage{greek}µ\selectlanguage{english}m
(M) Quantitative analysis of the mean fluorescence intensity of
activated and (N) inactivated mitochondria measured by mitochondrial
membrane potential.

\textbf{2.4. 10\% p (VDF-TRFE)/rGO/PCL nerve conduits promote the
recovery of damaged Sciatic nerve in a biologically safe manner \emph{in
vivo}.}

Ensuring the biosafety of neural implants in tissue engineering is
crucial. We verified the organ and metabolic toxicity of nerve conduits
by implanting them into rats for a period of three months and monitoring
organ and blood homeostasis. Pathological changes were absent in tissues
of the heart, liver, spleen, kidneys, and lungs (Figure S3A).
Confirmation of organ and hematology biosafety was achieved through
tests of liver and kidney function, as well as electrolyte levels.
Additionally, a blood test was conducted (Figure S3B). The results
demonstrated that the PrP conduit exhibited robust biosafety, aligning
with previous \emph{in vitro} safety tests, thus supporting further
validation.

To evaluate whether electrical stimulation via our custom-designed nerve
conduits can promote peripheral nerve regeneration, we conducted a
series of experiments \emph{in vivo} . We initiated our study by
assessing neuromuscular recovery in rats through a series of behavioral
tests. Specifically, we trained the rats to navigate from one end of the
track to the other. The shape and pressure of their footprints were
recorded using pressure-sensitive acrylic plates (Figure 4A), a
well-established method for analyzing motion recovery {[}35{]}. We
observed that the poorer the nerve recovery, the more indistinct and
elongated the footprint appeared, and the less pressure was exerted by
the foot (Figure 4B-C). Our results revealed that the nerve recovery in
rats treated with 10\% PrP paralleled that of autologous nerve
transplantation (Figure 4D), with a significant increase in footprint
pressure (Figure 4E).

Following this, we proceeded to evaluate nerve conduction. Under
anesthesia, the rats were incised along the initial cut to reveal the
nerve. One electrode was introduced into the gastrocnemius muscle, while
the other was used to stimulate both ends of the nerve defect, allowing
us to evaluate the axonal and myelin functionality of the nerve. Our
data indicated that the neural function in rats treated with 10\% PrP
had recuperated to a level comparable to autologous nerve
transplantation (Figure 4F-H). We subsequently harvested the sciatic
nerves and performed histological staining, immunofluorescence, and
transmission electron microscopy experiments to quantitatively observe
the nerve recovery (Figure 4I-K). The results demonstrated that this
specially designed piezoelectric material effectively promoted the
regeneration of the myelin sheath (Figure 4L-N). Histological sections
of gastrocnemius muscle tissues further bolstered our findings (Figure
4O). In summary, this piezoelectric material can promote regeneration
after PNI.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 4.} The neuromuscular recovery was observed after 3
months. (A) A schematic of behavioral analysis. (B) Rats walking
footprints. (C) The pressure of each hind foot on the board. (D)
Quantitative analysis of Sciatic nerve index. (E) Quantitative analysis
of pressure recovery index. (F) Quantitative analysis of nerve
conduction velocity (NCV) and (G) distal compound motor action potential
(DCMAP) (H) Action potential waveform of the Sciatic nerve (I)
Immunofluorescence staining for S100 and NF200 in sciatic nerve. Scale
bars=200 \selectlanguage{greek}µ\selectlanguage{english}m (J) HE staining of the Sciatic nerve. Scale bars=100 \selectlanguage{greek}µ\selectlanguage{english}m (K)
Representative electron micrographs of sciatic nerve cross sections.
Scale bars=10 \selectlanguage{greek}µ\selectlanguage{english}m (L) MFI of S100 positive cell. (M) MFI of NF200
positive cell. (N) Analysis of myelin sheath thickness by calculation of
the g-ratio. (O) Masson and HE staining of gastrocnemius. Scale bars=200
\selectlanguage{greek}µ\selectlanguage{english}m

\textbf{2.5. Piezoelectric stimulation works to repair damaged nerves by
inhibiting NET and promoting rapid vascularization.}

How does overactivated NET in the early stage of PNI affect regeneration
in the long term? We undertook preliminary animal studies to evaluate
the influence of early neutrophil activity on neurological recovery.
Generally, we implanted various materials into the cut nerve. After a
span of 12 hours post-injury, we extracted the nerves to check their
impact on the neutrophil. HE staining hinted that the nerve repair
effect could potentially be obtained by manipulating neutrophil behavior
(Figure 5A). Following this, we detected Histone H3 (HH3) and
myeloperoxidase (MPO) (Figure 5B), specific markers indicating the
presence of neutrophil extracellular traps (NETs), and CD31, specific
markers indicating the occurrence of angiogenesis (Figure 5C). Our
findings revealed a substantial expression of extracellular NETs in the
rP group, but the NETs` expression in the PrP and autologous
transplantation groups was over 1.5 times lower (Figure 5D-E). The
nerves subjected to electrical stimulation have more granulation
tissues, indicating a higher degree of vascularization (Figure
5F).\emph{In vitro} results mirrored these findings. In essence, we
isolated rat peripheral blood neutrophils, cultured them on different
materials, and then detected NET expression using Western blot (Figure
5G-H). Further, we co-cultured endothelial cells with neutrophils
treated with varied materials and conducted a scratch assay (Figure 5I).
The data showed a superior outcome in the PrP group compared to the rP
group, and strikingly, the effect of the PrP group was nearly identical
to the non-activated neutrophil group on endothelial cells (Figure 5J).
Interestingly, when we explored the correlation between this early
immune regulation and long-term tissue repair, macrophages showed an
interesting adjustment. (Figure 5K.) The M1 polarization of macrophages
decreased in the nerve conduits with early electrical treatment. (Figure
5L-M) These results suggest that electrical stimulation could regulate
the immune response in the early stage of injury, enhancing angiogenesis
and nerve regeneration, and the early inflammatory regulation may
indicate an increase in the benefits of long-term immune
microenvironment-mediated nerve regeneration.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 5.} Nerve conduits regulated neutrophil behavior. (A) The
neutrophil in the epineurium. Scale bars=25 \selectlanguage{greek}µ\selectlanguage{english}m (B) Histone H3 and
myeloperoxidase immunofluorescent staining of regenerative nerves. Scale
bars=100 \selectlanguage{greek}µ\selectlanguage{english}m (C) Immunofluorescence staining for CD31 in sciatic nerve.
Scale bars= 20 \selectlanguage{greek}µ\selectlanguage{english}m (D) Quantitative analysis of the mean fluorescence
intensity of Histone H3. (E) Quantitative analysis of the mean
fluorescence intensity of MPO. (F) Quantitative analysis of the mean
fluorescence intensity of CD31. (G) Western blot analysis of Histone H3
expression in neutrophil. (H) Relative expression levels of Histone H3.
(I) Wound-Healing Assay after co-culture of neutrophil and endothelial
cells. Scale bars=100 \selectlanguage{greek}µ\selectlanguage{english}m (J) Quantitative analysis of the repair index
of endothelial cells. (K) Immunofluorescence staining for CD68 and iNOS
in sciatic nerve. Scale bars= 50 \selectlanguage{greek}µ\selectlanguage{english}m (L) MFI of CD68. (M) MFI of iNOS.

\section*{3. Discussion}

{\label{discussion}}

Increasing attention has been paying to the impact of implant
bioelectricity on tissue repair, particularly regarding the generation
and maintenance of bioelectricity in peripheral nerve tissue
engineering{[}36{]}. It has been observed that reconstructing the normal
electrophysiological microenvironment can significantly facilitate the
repair of injured sites or wound tissue{[}37{]} Consequently, numerous
methods of energy substitution are being explored. Ultrasound
stimulation, often utilized as a source of mechanical stress \emph{in
vitro} , is one such method{[}21{]}. Biomaterials have even been found
to stimulate nerve repair by generating currents in response to changes
in an external magnetic field{[}38{]}. However, these stimuli can be
cumbersome and challenging to convert for tissue repair purposes. For
instance, nerve damage patients are often required to be exposed
frequently to an electric field for brief periods, ranging from seconds
to minutes{[}39{]}. Piezoelectric materials offer a promising solution.
They have been demonstrated to promote tissue regeneration via in situ
electrical stimulation, independent of exogenous mechanical friction
electrical stimulation as a power source{[}2, 10, 37{]}. Endothelial and
immune cells can cause minute deformations on these materials, which can
generate sufficient electrical signals to modify cellular
metabolism{[}14, 40{]}. Research has shown that the electrical
stimulation from piezoelectric materials may serve as an internal
generator for peripheral nerve regeneration{[}41{]}. For example,
electrospinning scaffolds composed of P
(VDF-TrFE)/BaTiO\textsubscript{3} nanoparticles, as established by
NUNES-PEREIRA J {[}42{]}, can generate voltages up to 5.02 V and 25 mW
of power under mechanical stimulation. This provides robust electrical
support for the recovery of injured nerves.

The preparation of ZnO/PCL piezoelectric composite scaffolds was
successful, demonstrating remarkable properties for neural
recovery{[}41{]}. These endogenous piezoelectric materials were found to
prompt the rapid regeneration of a damaged sciatic nerve within a span
of 4 weeks. The data suggests that the optimal voltage range to
encourage nerve regeneration lies between 0.2 mV and 10 V{[}43{]}.
Interestingly, the introduction of nanoscale currents in the vicinity of
peripheral nerves appears adequate to modify the metabolic
microenvironment, thereby facilitating nerve regeneration{[}44{]}.
However, it is crucial to note that excessive electrical stimulation
could prove counterproductive, posing a threat to the cytoskeleton and
the cells' migration capacity, which could be harmful to tissue
repair{[}6{]}.

NETs significantly contribute to extracellular DNA that impairs wound
healing{[}32{]}. They are also known to enhance the migratory
capabilities of tumor cells{[}45{]},and can foster chemotherapy
resistance{[}46{]}. Neutrophil over-activation can lead to the
transformation of macrophages into M1-type macrophages, thereby
intensifying the inflammatory response{[}47{]}. Neutrophils secrete
chemokines that boost the macrophages' capacity to clear myelin
sheaths{[}48{]},and simultaneously serve as a backup for Wallerian
degeneration, as no notable delay in myelin denaturation is observed
post suppressed macrophage activity{[}49{]}. This ''relay mode'' of
neutrophil macrophages suggests a potential role of neutrophil signal
crosstalk with macrophages in inflammatory responses post nerve
injury{[}50{]}. Prior research indicates that inhibiting NET formation
can encourage the prompt arrival of macrophages at PNI sites, fostering
regeneration{[}31{]}. Neutrophils are inherently flexible in adapting to
environmental signals, not even limited by their initial stage of
maturation.{[}51{]} This means that neutrophils in the neural
regenerative microenvironment may follow the same process, adjusting
their own immune function status under the guidance of the nerve guide
conduits, and thus transforming into a niche that promotes peripheral
nerve repair. Our aim is to regulate NETs through biophysical methods to
expedite nerve tissue repair. However, specific mechanism by which
piezoelectric materials modulate the behavior of immune cells during the
initial phase of inflammatory injury remains unclear. Excess DNA, a
pivotal source of extracellular DNA at injury sites, is often implicated
in the cyclic GMP-AMP (cGAS)-Stimulator of Interferon Genes (STING)
pathway {[}52{]}. Indeed, the activation of this pathway is intricately
linked with vascular, neuronal, and interstitial cell injuries {[}53,
54{]}. This indicates the feasibility of biomaterials inhibiting the
formation of neutrophil extracellular traps to improve cells'
regenerative capacity in PNI.

In our research, P (VDF-TrFE) /rGO/PCL emerges as a promising material
for nerve regeneration{[}55{]}. Prior studies have underscored the
advantages of biomimetic materials in facilitating bioelectrical signal
transmission during peripheral nerve regeneration{[}56-58{]}. An
appropriate local voltage potential has the capacity to instigate a
conformational shift in the voltage-gated channel, leading to
intracellular calcium transients{[}23{]}. The in-situ electrical
properties of piezoelectric materials are influenced by both the
material's form and the magnitude of the cell adhesion force{[}40{]}.
Reduced graphene oxide, a commendable conductor within the graphene
family, can proficiently convey electron flow, thereby enhancing the
piezoelectric attributes of the materials{[}59{]}. Superior cell
adhesion properties when applied to bio-conductive materials can foster
more effective mechano-bioelectric signal interactions between the
materials and the regenerating cells {[}40, 60{]}.

While our study demonstrates that piezoelectric fibrous materials
modulate neutrophil inflammatory responses during the early stages of
nerve injury, potentially aiding nerve regeneration, we need to
acknowledge that neutrophil extracellular traps (NETs) are not entirely
detrimental to nerve repair. In fact, an optimal concentration of NETs
may promote nerve repair. Lower NET concentrations may stimulate cell
proliferation via the NF-KB pathway{[}61{]}. Recent research has
revealed that neutrophil populations with immature leukocyte phenotypes
possess neural repair-promoting functions {[}62{]}. This has spurred our
interest in deciphering the specific microenvironmental changes within
neural tissue that enable the functional reprogramming of neutrophils,
shifting from patrolling behavior to specialized, tissue-specific
tasks{[}63{]}. Our study, however, did not definitively determine the
ideal NET concentration for nerve regeneration, marking a substantial
limitation. Furthermore, the precise electrical stimulation intensity
required to yield the optimal NET concentration remains unclear. These
questions necessitate further experimental exploration and hold
significant implications for our comprehensive understanding of the role
piezoelectric materials play in nerve repair.

\section*{4. Conclusion}

{\label{conclusion}}

Our research demonstrated that the production of neutrophil NETs could
impede early nerve repair post-injury. Consequently, we engineered a
piezoelectric conductive composite nerve conduit, specifically a PrP
nerve conduit, which targets early-stage neutrophils at the injury site
by deploying an electric current generated through physical deformation.
This electrical stimulation disrupts NET formation, thereby eliminating
barriers to micro-vessel formation and axonal regeneration. PrP has
exhibited promising biosafety and efficacy as an intracellular plant
following nerve injury. In conclusion, the effectiveness of
piezoelectric materials in peripheral nerve repair has been established,
and the PrP nerve conduit may present a competitive edge in future
translational applications.

\section*{5. Materials and method}

{\label{materials-and-method}}

\textbf{5.1. Materials}

P (VDF-TrFE) Solvene\selectlanguage{ngerman}®~250 /P400 (900904, Sigma-Aldrich, USA) was
provided by Shanghai Chongjing Biotechnology Co., Ltd., while the
reduced graphene oxide from tea polyphenols (XF247, XFNANO, China) and
polycaprolactone (P871874, Sigma-Aldrich, USA) were supplied by Shanghai
Yasheng Biotechnology. Aceton and N, N-dimethylformamide (DMF) were
purchased from Macklin (Shanghai, China).

\textbf{5.2. Fabrication and characterization of electroactive P
(VDF-TrFE)/rGO/PCL scaffolds}

P (VDF-TrFE)/rGO/PCL scaffolds were fabricated using the electrospinning
method. The copolymer P (VDF-TrFE) (7525), 2\%rGO, and 16\% PCL were
dissolved uniformly in a mixed solvent comprised of DMF and acetone
(6:4). This was done in accordance with the optimal spinning conditions
as identified in previous reports{[}21{]}. The copolymer concentration
within the solution was adjusted to 0\%, 10\%, and 20\%. The injection
speed was set at 1 ml/h and a DC voltage of 15 kV was applied between
the injector and the mould. The collection distance was maintained at 10
cm. Prior to each experiment, the film was cut into a circular piece of
the corresponding aperture size using a perforator and then detached
from the foil. The membrane was subsequently immersed in 75\% ethanol
and exposed to ultraviolet light for 1 hour. The material was then
air-dried and set aside for future use.

The surface morphology of electrospun films was examined using a
Scanning Electron Microscope (SEM). The microstructure of the
electrospun fibers was observed at 5kv following a gold spraying
treatment of 30 seconds. A tensile apparatus was employed to determine
the mechanical properties of the piezoelectric materials, with the
stress-strain curves being drawn based on the elastic modulus and
elongation at break. The X-ray Diffractometer (XRD) was set with
experimental parameters adjusted to 1.542 Å of copper radiation, to
characterize the crystal structures of different materials between the
angles of 5 to 80 degrees. The Fourier Transform Infrared (FTIR)
spectroscopy (NEXUS-670) was used to evaluate the Fourier transform
transmittance in the range of 500 cm to 4000 cm. The contact angle of
different materials when interacting with water was measured using a
Contact Angle Goniometer (OCA15EC, Germany).

\textbf{5.3. Cell culture}

The RSC96 cell line, procured from Zhongqiao Xinzhou (ZQ0154), was
cultured in Dulbecco's Modified Eagle's Medium (DMEM, GIBCO, USA),
supplemented with 10\% Fetal Bovine Serum (FBS, GIBCO) and 1\%
penicillin/streptomycin solution (GIBCO, USA). Primary Human Umbilical
Vein Endothelial Cells were obtained from Seiye Biotechnology Co., Ltd.
(HUVEC-20001, Cyagen), and these endothelial cells were cultured in
specialized media (HUVEC-90011, Cyagen). The culturing was conducted at
37°C in a 5\% CO2 incubator.

\textbf{5.4. Cytotoxicity assay}

We employed a cell counting kit-8 (CCK8) (C6005, NCM Biotech, China) to
identify the piezoelectric biomaterials with minimal toxicity to Schwann
cell growth. Sterilized films were positioned into 24-well plates, and
cells were seeded at a density of 2*10\textsuperscript{\^{}}5 into these
plates with different materials. Following a 2-hour period, the cells
fully adhered to the membrane surface, marking the beginning of the
treatment process. Treatment endpoints were established at 6, 12, 24,
and 48 hours post the commencement of the treatment. At the conclusion
of the treatment, the complete medium was discarded and replaced with
basal medium containing 10\% CCK8 reagent. After a further 3 hours, the
supernatant was gently removed from each well and the absorbance at 450
nm was recorded.

\textbf{5.5. Apoptosis experiment}

We utilized an Annexin-V-FITC/PI apoptosis assay kit
(abs50001\selectlanguage{english},absin\selectlanguage{english},China) to assess cell apoptosis following material
treatment. Once the Schwann cells on the membrane achieved confluence of
over 85\%, they were digested with trypsin (sans EDTA), centrifuged, and
subsequently resuspended in 250\selectlanguage{greek}µ\selectlanguage{english}l of 2\% Annexin-V-FITC solution. This
was followed by a 15-minute incubation period at room temperature,
shielded from light. Prior to the flow cytometry analysis, which was
carried out 5 minutes before loading the sample into the machine, a
250\selectlanguage{greek}µ\selectlanguage{english}l aliquot of 2\% PI solution was added. The flow cytometry analysis
was conducted using a FACS celesta (BD, USA).

\textbf{5.6. Mitochondrial membrane potential experiment}

The assessment of mitochondrial depolarization in human umbilical vein
endothelial cells was conducted in accordance with the manufacturer's
instructions (C2003s, Beyotime, China). Specifically, JC-1 staining
solution (5 \selectlanguage{greek}μ\selectlanguage{english}g/ml) was introduced into the cell culture dishes, followed
by a 20-minute incubation period in the cell incubator. Subsequently,
the dishes were washed twice with a buffer solution, then observed under
a fluorescence microscope. The fluorescence intensities were measured at
490 nm and 530 nm excitation wavelengths. It is noteworthy that a higher
mitochondrial membrane potential correlates with an increase in red
light intensity, whereas a stronger green light intensity indicates the
opposite.

\textbf{5.7. Angiogenesis experiment}

HUVECs naturally develop into reticular tubules on the Matrigel surface
at elevated concentrations, thereby simulating angiogenesis in living
organisms{[}64{]}. A volume of 10 \selectlanguage{greek}μ\selectlanguage{english}l of Matrigel\selectlanguage{ngerman}® (354234, Corning, USA)
at a concentration of 10 mg/ml was evenly spread in the lower
compartment of a \selectlanguage{greek}μ-\selectlanguage{english}slide plate (81506,ibidi,German). The upper
compartment was then inoculated with a 50 \selectlanguage{greek}μ\selectlanguage{english}l cell suspension at a
density of 2*10\^{}5 cells/ml. The setup was incubated and observed
after 24 hours. Following the final observation in the bright field,
Calcein-AM/PI (C542, Dojindo, Japan) staining was performed for
additional observation. Image J software was utilized to count the
vascular nodes and branches.

\textbf{5.8. Transwell cell migration assay}

The Matrigel was combined with the basal medium at a ratio of 1:8 and
subsequently added to the upper compartment of the Transwell Plate.
Endothelial cells were then seeded onto various membranes and incubated
for 2-3 days. Following digestion, cells were centrifuged, re-suspended
in basic medium, and then inoculated into the upper compartment of the
Transwell Plate. Next, complete medium was added to the lower
compartment. After a period of 12 hours, the chamber was removed and
gently swabbed with a cotton bud to clean the inner side of the upper
compartment. The cells were then stained with crystal violet. The Image
J software was used to quantify the number and average area of cells
that migrated through the chamber.

\textbf{5.9. Neutrophil extraction}

Initially, we collected peripheral blood samples from Rats. Utilizing a
peripheral blood neutrophil separation kit (P9200, Solarbio, China), we
carefully combined 16 ml of fresh blood samples with an equal volume of
AC mixture (liquid A: liquid C at a ratio of 2:1), followed by
centrifugation at 700g for 25 minutes. Post-centrifugation, the
neutrophils congregated in a layer described as a white mist at the
bottom. This cell layer was gently aspirated, rinsed, and subjected to a
second centrifugation (250g for 10 minutes). Subsequently, the cells
were re-suspended in 1 ml of Lysate and incubated at room temperature
for a 5-minute lysis period. After lysis, the supernatant was removed by
a further round of centrifugation (250g for 10 minutes). Finally, the
cells were re-suspended in a 1640 basal medium before being seeded onto
the material.

\textbf{5.10. Neutrophil purity test}

Following each neutrophil extraction, we conducted purity assessments
and cell count measurements. Utilizing a water-blocking pen (BC004,
Biosharp, China), we placed droplets of the neutrophil suspension onto a
glass slide pre-coated with poly-lysine (ZLI-9802, ZSGB-bio, China),
after which we dried the slide in a CO2-free incubator for a period of
two hours. The characteristic lobulated nuclei of the neutrophils became
identifiable after staining with diff staining solution (G1540,
Solarbio, China) or a DAPI-containing tablet (P0131, Beyotime, China).

\textbf{5.11. Extraction of neutrophil extracellular traps}

Using a previously detailed method, neutrophils were extracted from the
peripheral blood and neutrophil extracellular traps (NETs) were
subsequently isolated{[}45{]}. The neutrophil NETs expression inducer,
Phorbol 12-myristate 13-acetate (PMA), was procured from MedChemExpress
(MCE, America). In brief, we subjected the neutrophils to a 4-hour
stimulation using 500 nm PMA, ensuring their complete activation. Upon
reaching the designated time point, we discarded the supernatant and
gently rinsed the NETs at the base of the dish with chilled PBS,
followed by a 10-minute centrifugation at 1000 g, at a temperature of 4
\selectlanguage{ngerman}°C. We then collected the cell-free supernatants containing the NETs
(DNA-protein complexes) for subsequent experimentation.

\textbf{5.12. Live-Dead Cell test}

We propagated the RSC96 cell line in 6-well plates, maintaining a growth
density of 500,000 cells per well. The cells were then treated for 6
hours with either a purified NET solution or PBS. Following this
treatment, the immunofluorescence intensity of live and dead cells was
measured using Calcein-AM/PI (Solarbio, China).

\textbf{5.13. Calcium ion flow experiment}

We utilized the Fluo-4 Calcium Assay Kit (S1061s, Beyotime, China) to
evaluate the calcium influx in neutrophils post-treatment. The harvested
neutrophils were positioned in orifice plates either with or without
materials. Following a 6-hour treatment period, the cells were
transferred into a centrifuge tube and subjected to fluo-4 staining
solution. The levels of calcium influx within the cells were
subsequently measured using flow cytometry.

\textbf{5.14. Wound-Healing Assay}

Neutrophils treated with various piezoelectric films were centrifuged,
and the supernatant was meticulously collected. A 200\selectlanguage{greek}µ\selectlanguage{english}l Spear tip was
utilized to create a grid of horizontal and vertical scratches on the
endothelial cell culture dish. The intersection points of the scratches
served as coordinates to conveniently record each scratch's location.
Loose cells were gently rinsed off with PBS and the medium was replaced
with basic medium. Subsequently, the supernatant from the treated
neutrophils was added to the endothelial cell culture medium. Scratch
repair at the same location was documented at 0 and 24 hours
post-treatment using an inverted microscope. The width of the scratches
was analyzed using Image J software to assess the migratory capacity of
the endothelial cells.

\textbf{5.15. Western blot test}

To evaluate the influence of various piezoelectric films on neutrophil
traps, neutrophils treated with these films were centrifuged to yield
their total protein content. Following quantitative electrophoresis, the
proteins were transferred onto a PVDF membrane. This membrane was then
blocked using 5\% skim milk and incubated with specific antibodies.
Protein expression was subsequently determined via a chemiluminescent
technique. ImageJ was utilized for the processing of grayscale value
data, and the expression level of the target protein was normalized
accordingly. The primary antibodies employed included Histone H3
(1:1000, AB281584, abcam, UK) and \selectlanguage{greek}β-\selectlanguage{english}actin (1:5000, LF201, Epizyme,
China). The secondary antibodies used were HRP-labeled Goat Anti-Mouse
IgG (1:10000, LF 101, Epizyme, China) and HRP-labeled Goat Anti-Rabbit
IgG (1:10000, LF 102, Epizyme, China).

\textbf{5.16. Animal surgeries and procedures}

The male Sprague-Dawley rats utilized in this study were sourced from
the Laboratory Animal Center of Shanghai Sixth People's Hospital. All
animal and experimental protocols were scrutinized and sanctioned by the
Animal Experimental Ethics Committee of Shanghai Sixth People's
Hospital. Five animals per group were procured from Xipulbikai
Experimental Animal, Shanghai.

Rats were weighed to the nearest 0.1 grams and anesthetized via an
intraperitoneal injection of sodium pentobarbital, dosed at 30 mg/kg of
body weight. After a brief period of 5 minutes, the rats' muscles
relaxed and both pain and corneal reflexes ceased. A surgical incision
of 5cm was made on the right hind limb of each rat, which was in a prone
position. The gluteus maximus muscle and deep fascia were delicately
separated until the sciatic nerve was revealed. A portion of this nerve,
measuring 10mm, was then excised. For the experimental groups, a nerve
conduit, 12 mm in length, was used to bridge the severed nerve ends. The
transected nerve was gingerly inserted 1mm into the nerve conduit and
meticulously sutured with a 6-0 suture under microscopic observation.
For the autogenous nerve transplantation group, the severed nerve was
rotated 180\selectlanguage{ngerman}° before suturing. The muscle and epidermis were then closed
with a 4-0 suture. Post-operatively, the rats were warmed under a light
until they regained consciousness.

\textbf{5.17. Behavioral analysis}

Following a recovery period of three months, we utilized a gait analyzer
(VisuGait, XR-XFP101, China) to record the relative data pertaining to
the rats' ambulatory trajectories. The rats were permitted to traverse a
secure, dimly lit tunnel with a pressure-sensitive, transparent acrylic
sheet situated beneath. The unique characteristics of these plates
ensured comprehensive recording of the rats' movements via an
under-tunnel camera, while also transmitting data from their footprints
to a connected computer. Quantitative evaluation of the Sciatic nerve
recovery was conducted via the interactive paw print measurement module
for the injured right hind limb. The footprint analysis machine
automatically measures relevant parameters of the footprint and
calculates the sciatic nerve function index (SFI), which range from 0
(indicating normal function) to 100 (denoting complete loss of
function), based on the preset formula.

\textbf{5.18. Electrophysiological Assessment}

In alignment with the model, rats were anesthetized using sodium
pentobarbital. Following anesthesia, the right hind limb of the rat was
shaved. Subsequently, we reopened the nerve defect site, mirroring the
original incision, thereby exposing the two severed nerve ends. Needle
electrodes were then strategically implanted into the right
gastrocnemius muscle of the rats, maintaining a consistent stimulation
intensity and frequency of 20 ma and 1.5 Hz, respectively. Finally, the
resultant electromyography (EMG) was meticulously recorded and analyzed
utilizing Image J.

\textbf{5.19. Hematologic evaluation}

The rats were carefully positioned supine and secured. Following this, a
dissection was performed along the anterior abdominal midline, involving
the skin, fascia, and muscle. The abdominal cavity was subsequently
exposed by delicately opening the intestines and passively separating
the retroperitoneum, thereby revealing the abdominal aorta. Whole blood
was then procured using a disposable blood collection needle and an
anticoagulant blood collection vessel. Arterial blood was collected
separately using an EP tube. After a waiting period of 30 minutes,
centrifugation was applied to collect the serum. Both the whole blood
and serum were then utilized to assess various physiological indicators.

\textbf{5.20. Histological experiments}

Following blood collection, the animals are euthanized via an overdose
of anaesthetic. The sciatic nerve and muscles are harvested, alongside
the heart, lungs, liver, spleen, and kidneys. It is crucial to mention
that for paired organs, only the left side is procured, and in case of
lobes, the largest lobe is chosen. The collected tissues are
subsequently fixed at 4°C in 4\% paraformaldehyde solution for 24 hours,
following which paraffin-embedded sections are prepared on the next day.
For Hematoxylin-Eosin (HE) staining, the sections are initially stained
with hematoxylin, subsequently with eosin, and finally dehydrated and
sealed. Myelin sheath thickness was determined by g-ratio analysis by
dividing the axonal diameter by the fiber diameter (diameter of axon
including the myelin sheath). For Masson staining, the sections undergo
staining with potassium dichromate, Ponceau red, phosphomolybdic acid
and aniline blue, followed by differentiation with glacial acetic acid.
For immunofluorescence experiments, the chosen primary antibodies are
Histone H3 (1:2000, AB281584, abcam) and Myeloperoxidase (1:50,
AB300650, abcam), and the secondary antibodies are Abflo 488-conjugated
Goat Anti-Rabbit IgG (1:200, AS053, ABclonal, China) and CY3 conjugated
Goat Anti-Rat IgG (1:500, A0507, Beyotime, China).

\textbf{5.21. Statistical analysis}

The presented data are expressed as the mean ± standard deviation,
derived from at least three independent experiments. The determination
of statistical significance was carried out using Student's t test or
ANOVA, with Bonferroni correction applied to the P values.

\section*{Supporting Information}

{\label{supporting-information}}

Supporting Information is available from the Wiley Online Library or
from the author.

\section*{Acknowledgments}

{\label{acknowledgments}}

This work was supported by the National Key R\&D Program of China (No.
2021YFC2400800), the Projects of the National Natural Science Foundation
of China (Nos. 82072452, 82272475); the Science and Technology
Commission of Shanghai Municipality (No. 20DZ2254900) ; the Sino-German
Mobility Programme (No. M-0699); the Excellent Youth Cultivation Program
of Shanghai Sixth People's Hospital, No. ynyq202201; the Medical
Engineering Co-Project of University of Shanghai for Science and
Technology, 10-22-310-520; and a grant from Shanghai Municipal Health
Commission, No. 202040399.

\section*{Conflicts of interest
declaration}

{\label{conflicts-of-interest-declaration}}

The authors declare no conflict of interest.

\section*{Data Availability Statement}

{\label{data-availability-statement}}

All data that support the findings of this study are included in the
paper and the Supporting Information. Additional data related to this
paper are available from the corresponding author upon reasonable
request.

\section*{Author Contribution}

{\label{author-contribution}}

Y.L., X.Y., L.Z. and J.S. contributed equally to this work. Y.L. and
L.Z. performed the experience and Y.L. drafted the manuscript. X.Y. and
J.S. participated in the design of the experiment and reviewed the
manuscript. Y.Q. reviewed the literature and commented on the
manuscript. Y.O., Y.Q., and C.H. revised the manuscript. All authors
read and approved the final version.

Received: ((will be filled in by the editorial staff))

Revised: ((will be filled in by the editorial staff))

Published online: ((will be filled in by the editorial staff))

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