Preparation of MPBN
MPBN was synthesized as previously reported [22, 34]. First, K3[Fe(CN)6] (395 mg, P7380, Solarbio) and PVP (5 g, Mw = 40 000, P8290, Solarbio) were added to HCl solution (40mL, 1 mol L− 1) to obtain the clear solution. The solution was stirred for 30 min at room temperature and then heated at 80 °C for 24 h without stirring. The color of the solution changed to deep blue from yellow. After that, the PBN were obtained by centrifugation (12000 rpm, 20 min) and washed with deionized (DI) water three times. Then, PBN (40 mg) and PVP (200 mg) were dissolved in 40 mL of HCl solution (1 mol L− 1) under magnetic stirring. After stirring for 4 h at room temperature, the solution was transferred into an autoclave and heated at 120–130°C for 3 h. The MPBN were collected by centrifugation (12000 rpm, 20 min) and washed with DI water three times.
Preparation of IOF
Vertical deposition method was used to prepare the opal crystal templates [32]. Briefly, a 2 mm diameter glass column was vertically immersed in a flask containing a solution of monodisperse silica nanoparticles (2% w/v ethanol solution, M120353, aladdin) at constant temperature (37°C) and humidity incubator until the solution volatilized and silica particles were self-assembled on glass, forming a colloid crystal array junction. Then PLGA solution (15% w/v in ethyl acetate, DG-50DLGH065, Jinan Dagang Biological Engineering, China) was infiltrated into the colloidal crystal arrays and the film solidified after the ethyl acetate volatilized. The IOF neuroconduit, with a diameter of 2 mm, was fabricated through the etching process of silicon dioxide nanoparticles using 4% hydrofluoric acid.
MPBN scavenged free radical assay
The activities of MPBNs scavenging radicals were determined using commercial DPPH kits (A153-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Briefly, Add different concentrations of MBPN to 1,1-diphenyl-2-picrylhydrazyl free radical for reaction, and measure the absorbance at the wavelength of 517 nm with a spectrophotometer.
Characterization of MPBN
The microstructure and component energy spectra of MPBN were observed by transmission electron microscopy (Tecnai G2 F30, FEI, USA). Dynamic light scattering (DLS) measurement was conducted on ZetasizerNanosize (Nano ZS90). X-ray photoelectron spectroscopy (XPS, Thermo Scientifific Escalab 250Xi), UV − vis − NIR spectrophotometry (TU-1901, China), X-ray diffraction XRD (D8 ADVANCE, BRUKER, Germany), Fourier transform infrared spectroscopy (FTIR) (Thermo Scientifific Nicolet iS5), and an automatic specifific surface and porosity analyzer (ASAP 2460, micromeritics, USA) were used.
SOD-like activity of MPBN
The O2•− scavenging ability of MBPNs was demonstrated using the superoxide anion radical assay kit (A052-1-1) of Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Briefly, using the xanthine/xanthine oxidase method based on the production of O2•−. Add different concentrations of MPBN to the reaction, and measure the absorbance at the wavelength of 550 nm with a spectrophotometer.
POD-like activity of MPBN
In the PBS solution (pH 6.5, 10 mM, 2 mL), MB solution (1 mg mL− 1, 20 µL), H2O2 (1 M, 20 µL), and MPBN solution (the final concentration was 100 µg mL-1) were mixed together. After incubating at 37°C for 2 h, the solutions were centrifuged and the absorbance was measured by UV-Vis spectroscopy. Hydroxyl radical test kit (A018-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) also prove the production of •OH. Add different concentrations of MBPN to the reaction, add Griess reagent, and measure the absorbance at the wavelength of 550 nm on a spectrophotometer.
CAT-like activity of MPBN
Oxygen generation in catalase-like activity assays of the MPBN was recorded by D09100 portable Dissolved Oxygen Meters (Senyuan Electric Co. LTD). In detail, MPBN (the final concentration was 100 µg mL− 1) was suspended in 3% H2O2 solution.
Preparation of IOF-MBPN
The IOF conduit and MPBN water mixture (0.5 mL, 100 µg mL-1) were placed in a centrifuge tube (1.5 mL) and stored at -20 °C overnight for preparation. Subsequently, the centrifuge tube was transferred to a vacuum freeze dryer without the lid to facilitate the production of the final MPBN-loaded IOF conduit using vacuum freeze-drying technology. The microporous structure of IOF-MPBN was observed by scanning electron microscopy. Fourier transform infrared spectroscopy also confirmed that IOF was attaching MPBN. The mechanical properties of IOF were measured on a tensile testing machine (3343, instron, USA). The 2 cm length of the IOF and IOF-MPBN was clamped with a 1.5 cm inter-clamp distance and pulled longitudinally at a rate of 1 mm s-1 until rupture. The slope of the stress-strain curve in elastic region represented the Young’s modulus. The maximum stress and strain at rupture were measured.
Cell culture
Schwann cell line (RSC96) and macrophage cell line (RAW264.7) were obtained from the National Collection of Authenticated Cell Cultures (Chinese Academy of Science). The cells were cultured with Dulbecco’s modified eagle medium high glucose supplemented by 10% fetal bovine serum and 1% penicillinstreptomycin solution.
Cell viability assay
To evaluate the influence of MPBN on cell vitality, a standard CCK-8 (Solarbio, China) was determined. Briefly, RSC96 cells were pre-seeded into 96-well plates for 24 h, and then incubated with different concentrations (0, 5, 10, 25, 50, 100, 200, 400 µg mL− 1) of MPBN for another 24 h. Subsequently, the cells with appropriate MPBN concentration were selected and incubated with or without the H2O2 (100 µM) for 4 h. CCK-8 solution (10 µL) was added to each well and incubated for another 2 h, respectively. To evaluate the influence of IOF and IOF-MPBN on cell vitality, RSC96 cells suspension was co-cultured with IOF and IOF-MPBN in the 96-well plates. Following 1, 2, and 4 days of culturing, the culture medium was substituted with a working medium containing a 10% solution of CCK-8. The absorbance was measured at 450 nm using a microplate Reader in all the above experiments. Three independent experiments were performed.
Live/dead cell staining assay
The RSC96 cells were pre-incubated in 6-well plates for 24 h. Afterward, the cells were washed three times with a PBS solution (10 mM, pH 7.4). Subsequently, both IOF and MPBN-IOF were added to the aforementioned medium and incubated for an additional 24 h. Furthermore, the cells were exposed to a combination of H2O2 (100 µM) and the aforementioned substances for a duration of 4 h. Then, the cells were stained with Calcein-AM (4 × 10− 6 M) and PI solutions (4 × 10− 6 M) in PBS buffer solution and incubated for 30 min. Finally, the cells were washed several times with PBS buffer solution and observed by confocal laser scanning microscopy (CLSM) to examine their live/dead status. The Calcein-AM and PI were excited with lasers at 488 and 543 nm, respectively.
Repercussions of MPBN intervention on SC cells
The RSC96 cells were pre-inoculated onto the slides of a 12-well plate and incubated for 24 h. After washing the cells with PBS (10mM, pH 7.4) solution three times, MPBN was added to the aforementioned medium for further culture for an additional 24 h. The cells were subsequently fixed using 4% paraformaldehyde. Subsequently, after washed with PBS three times, cells were permeabilized using 0.1% TritoX-100 in PBS for approximately 5 min at room temperature, and washed with PBS three times. The cells were then blocked with 5% bovine serum albumin (BSA) in PBS at room temperature for 1 h, and incubated with the recommended diluted primary antibody for 1.5 h at room temperature. After washed with PBS, the cells were incubated with appropriate diluted fluorescent dye-linked secondary antibody (abcam) for additional 1 h, washed with PBS three times, and stained with DAPI for 3 min. Finally, the cells were washed with PBS twice, and fluorescence images of each slide were obtained via a fluorescence microscope. The primary antibody employed was pAMPK antibody (1:200,381164, ZENBIO, USA), SIRT1 antibody (1:200, 13161-1-AP, proteintech, USA), PGC-1 antibody (1:200, 66369-1-Ig, proteintech, USA), SOD2 antibody (1:500, 24127-1-AP, Proteintech, USA).
In vitro anti-oxidation assay
Intracellular ROS production levels were detected by DCFH-DA fluorescent probe staining. Briefly, RSC96 cells were pre-seeded into 6-well plates for 24 h, and then incubated with MPBN (100 µg mL-1) and H2O2 (100 µM). After 4 h, the medium was removed and DCFH was added (final concentration 1 × 10− 6 M) and incubated for 30 min. Finally, all the cells were viewed by inverted microscope with laser at 488 nm. To study the differences in expression of oxidative stress-related enzymes. RSC96 cells were treated as before and labeled with immunofluorescence, and observed by inverted microscope.The primary antibodies were SOD2 antibody (1:500, 24127-1-AP, Proteintech, USA).
In vitro anti-inflammatory assay
The expression of inflammation-related indicators was assessed through immunofluorescence staining. Briefly, Raw 264.7 cells were seeded onto 12-well plates at a density of 5 × 104 cells for 24 h, followed by treatment with lipopolysaccharide (LPS) (1 µg mL− 1) and MPBN(100 µg mL− 1) for another 24 h. Cells were washed for three times in PBS (5 min) and fixed by 4% paraformaldehyde at room temperature for about 10 min, and subsequently subjected to immunofluorescence staining imaging. The primary antibodies were CD206 antibody (1:500, 60143-1-Ig, Proteintech, USA), CD86 antibody (1:500, 13395-1-AP, Proteintech, USA), and IL-1 antibody (1:500, 16765- 1-AP, Proteintech, USA), IL-6 antibody (1:500, 21865-1-AP, Proteintech, USA), TNF-α antibody (1:500, 17590-1-AP, Proteintech, USA), COX-2 (1:500,66351-1-Ig, proteintech, USA).
Sciatic nerve defect model
The animal surgery and post-surgery experiments were performed according to the guidelines approved by the Animal Ethics Committee for Wenzhou Medical University. Forty Sprague Dawley male rats (180–200 g) were purchased from Wenzhou Gaofei Biotechnology Co., LTD. Rats were acclimated at least 1 week prior to the start of the experiment, and maintained in an air-conditioned room (21 ± 2°C) under a 12 h day-night cycle with food and water provided enough. All animal housing, care, feeding, and experimental procedures were in compliance with the National Guidelines for Animal Protection. The experimental animals were randomly divided into 3 groups: (i) autograft group (n = 10); (ii) IOF group (n = 15); (iii) IOF-MPBN group (n = 15). All rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution (40 mg kg-1). The right sciatic nerve was carefully exposed, a section of the sciatic nerve was excised, and the nerve stump was retracted leaving a 10 mm long defect. Then the gap was bridged with a 10 mm scaffold. In the autograft group, a 10 mm nerve segment was flipped and re-sutured. The animals were housed under normal light conditions with free access to food and water supply. The digital photographs of the scaffolds and the implantation surgery were provided.
Behavioral tests
To study hindlimb recovery, walking track test was performed every two weeks. For walking track analysis, the paws on both sides were dyed with red pigment, and the animals were placed on white paper to collect footprints. The length between the first and fifth toes was measured as “TS”, the length between the third toe and the heel was measured as “PL”, and the length between the second and fourth toes was measured as “IT”. The SFI was calculated with the following formula and “E” was used as the abbreviation for the “experimental side” and “N” as the abbreviation for the “non-experimental side”.
Electrophysiological assessment
After appropriate anesthesia, the animals received electromyography tests. In brief, a recording electrode was inserted into the gastrocnemius muscle while a stimulating electrode was placed upon the injured nerve segment. Electrical stimulation was applied at the proximal end of the injured nerve. The amplitude and latency of nerve conduction were recorded after the stimulation current passed through the nerve conduit.
Nerve structure assessment
Semi-thin section and toluidine blue staining were performed to visualize peripheral nerve myelin structure. Take a sample of the nerve at the end of the nerve conduct and immerse it in 2.5% glutaraldehyde. The samples were then post-fixed by 1% OsO4 and dehydrated by ethanol. Next, the samples were embedded in resin, and semi-section was performed. Finally, the samples were immersed in preheated TB dye solution. Images were acquired by an optical microscope.
High-magnification images of nerve structures were acquired by TEM
The nerve samples were treated with glutaraldehyde and OsO4. Then ethanol dehydration and resin penetration were performed. An ultramicrotome was used to produce 80 nm ultra-thin sections and the sections were placed on cuprum grids. The sections were stained by uranium acetate-saturated alcohol and lead citrate. The sections were observed with a TEM under 80k voltage. Quantitative analysis of nerve ultrastructure was performed following previous work [57].
Histological assessment
The animals were sacrificed by overdose anesthesia 3 months after surgery. The bilateral gastrocnemius muscles and target nerve tissue were harvested and the samples were preserved in 4% paraformaldehyde. Then the samples underwent a stand Paraffin-embedded sections or frozen slice and were prepared into 5 μm slides. For H&E staining, the slides were first immersed in Hematoxylin solution, then rinsed by alcohol, and finally dipped in the Eosin dye for 5 min. For immunofluorescence staining of neural tissue sections, the section was permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature, followed by three washes with PBS. After blocking with 5% BSA, section was then incubated with primary antibodies targeting S100 (1:200, 11250-1-AP, Proteintech, USA), NF-H (1:200, ab4680, Abcam, USA), MBP (1:200, ab7349, Proteintech, USA), CD206 (1:500, 60143-1-Ig, Proteintech, USA), CD86 (1:500, 13395-1-AP, Proteintech, USA), Gap-43 (1:20, ab75810, Abcam, USA), SIRT1 (1:200,13161-1-AP, proteintech, USA), PGC-1(1:200,66369-1-Ig, proteintech, USA), SOD2(1:500, 24127-1-AP, Proteintech, USA) followed by incubation with Alexa Fluor 594 or Alexa Fluor 488-labled secondary antibodies at room temperature. DAPI (Solarbio, China) counterstaining was used to visualize the nuclei. The immunofluorescent images were acquired via a fluorescence microscope. Image J software were used to analyze acquired images.
Transcriptome analysis
The total RNA extracted from the regenerated tissue in the IOF-MPBN neuroconduit was designated as the experimental group, while the RNA extracted from the IOF neuroconduit served as the control group. Then, the samples were dispatched for standard RNA-seq at GENE DENOVO Co. Ltd (Guangzhou, China). The global transcriptome profiles were compared between the two groups, and DEGs were identified using DESeq2 (version 1.24.0) analysis. The threshold was set as p < 0.05 and fold change cutoff of 2. Then KEGG enrichment, Gene Ontology terms, and Reactome analysis were conducted with a p-value cutoff < 0.05. The online analysis platform for the RNA-seq was Omicsmart (https://www.omicsmart.com/). The genome version used in this study was “Ensembl_release109”. The sequencing data is provided in Table S1 (Supporting Information).
In vivo biosafety assay
The potential toxicity of the IOF-MPBN in vivo was studied by examining the major organ morphology [58]. Four months after surgery, the major organs (heart, liver, spleen, lung and kidney) were harvested and prepared into paraffin-embedded slides. Then the samples received standard H&E staining and were characterized by a light microscope.
Statistical analysis
Data presentation was mean ± standard deviation. The statistical analysis method was one-way analysis of variance analysis (ANOVA) followed by Tukey’s multiple comparison test. For comparison between two groups, the statistical analysis method was two-tailed Student’s t-test. The data normalization method was mentioned in the corresponding figure legend. The software for statistical analysis was GraphPad Prism (version 9.0) and Origin Pro (version 2022). P > 0.05 was considered as no significant difference (ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001).
