Aligned chitosan nanofiber hydrogel grafted with peptides mimicking bioactive brain-derived neurotrophic factor and vascular endothelial growth factor repair long-distance sciatic nerve defects in rats methods
Aim. Evidence-backed execution summary for Aligned chitosan nanofiber hydrogel grafted with peptides mimicking bioactive brain-derived neurotrophic factor and vascular endothelial growth factor repair long-distance sciatic nerve defects in rats methods from Aligned chitosan nanofiber hydrogel grafted with peptides mimicking bioactive brain-derived neurotrophic factor and vascular endothelial growth factor repair long-distance sciatic nerve defects in rats.
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rat
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Methods
reagent used in the protocol.
- Use
- Chitosan, poly (ethylene glycol) (average MW ca. 4,000 kDa) and sodium tripolyphosphate (STPP) were purchased from Sigma-Aldrich. The aligned chitosan nanofiber hydrogel (ACG) was fabricated by electrospinning as reported previously,,. Briefly, 2% electrospinning chitosan solution was prepared by dissolving 0.2 g...
Methods
reagent used in the protocol.
- Use
- The functional polypeptides RGI and KLT were synthesized by solid-phase technology by Shanghai Qiangyao Biotechnology Co., Ltd. High-performance liquid chromatography (HPLC) was performed to verify that the polypeptides had > 95% purity. Samples of 2.5 mg RGI and 2.5 mg KLT polypeptide powders were dissolved in 25 m...
Isolation and culture of Schwann cells
reagent used in the protocol.
- Use
- Five Sprague-Dawley rats were used to obtain cultures of primary Schwann cells within 3 days after birth, as reported previously. Briefly, the bilateral sciatic nerves of the rats were collected after sterilization. The sciatic nerve epicardial membrane was then peeled off using microforceps, and the sciatic nerve...
RT-PCR and quantitative PCR
reagent used in the protocol.
- Use
- RNA was extracted from Schwann cells in all groups using TRIzol reagent (Invitrogen). cDNA was synthesized using a Synthesis Kit (Roche). Real-time PCR was performed using SYBR Green PCR master mix (Roche) with the forward and reverse primers listed in Table. To analyze changes in gene expression, the results of th...
Western blotting (WB)
reagent used in the protocol.
- Use
- Schwann cells and sciatic nerves were harvested from the experimental and control groups for WB. Briefly, Schwann cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors (Applygen) for protein extraction. Then, aliquots of 20 µg of protein were separated by sodium dodecyl...
Surgical process
reagent used in the protocol.
- Use
- Sprague-Dawley rats weighing 200-220 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Animals were treated in accordance with the Laboratory Animal Guideline for Ethical Review of Animal Welfare of China (GB/T 35892-2018). All experiments were performed in compliance with the relevant...
Immunofluorescence
reagent used in the protocol.
- Use
- After culture for 5 days, Schwann cells were fixed with paraformaldehyde, washed with PBS, and incubated for 2 h at 37°C with rabbit anti-S100 antibody (S2644; Sigma-Aldrich). After rinsing with PBS, samples were incubated with goat anti-rabbit IgG H+L (Alexa Fluor 594) (ab150084; Abcam) in the dark for 1 h at...
Cutting of ultrathin sections and transmission electron microscopy (TEM)
reagent used in the protocol.
- Use
- Regenerative nerves in all groups were removed after 12 weeks, fixed in 2.5% glutaraldehyde overnight, post-fixed by immersion in 1% osmium tetroxide solution for 3 h, and dehydrated through a graded acetone series (30%, 50%, 70%, 90%, and 100%). The samples were then embedded in Epon 812 epoxy resin and cut into 80...
Polarized light microscopic observation
The chitosan hydrogel obtained by electrospinning was cut into small segments (~5 cm in length) for observation under a polarizing microscope (UPT203i; Leica). First, the polarizer was adjusted in the orthogonal state to achieve complete extinction of the dark field of vision. Then, bunches of chitosan hydrogels wer...
- Use
- The chitosan hydrogel obtained by electrospinning was cut into small segments (~5 cm in length) for observation under a polarizing microscope (UPT203i; Leica). First, the polarizer was adjusted in the orthogonal state to achieve complete extinction of the dark field of vision. Then, bunches of chitosan hydrogels wer...
Examination of mechanical properties
The mechanical properties of chitosan hydrogel were measured using a material testing machine (ZwickRoell). Briefly, the two ends of the chitosan hydrogel were fixed on the clamp, and then stretched at a rate of 1 mm/min. The Young's elastic modulus of the chitosan hydrogel was determined from the linear slope of th...
- Use
- The mechanical properties of chitosan hydrogel were measured using a material testing machine (ZwickRoell). Briefly, the two ends of the chitosan hydrogel were fixed on the clamp, and then stretched at a rate of 1 mm/min. The Young's elastic modulus of the chitosan hydrogel was determined from the linear slope of th...
Surgical process
Sprague-Dawley rats weighing 200-220 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Animals were treated in accordance with the Laboratory Animal Guideline for Ethical Review of Animal Welfare of China (GB/T 35892-2018). All experiments were performed in compliance with the relevant...
- Use
- Sprague-Dawley rats weighing 200-220 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Animals were treated in accordance with the Laboratory Animal Guideline for Ethical Review of Animal Welfare of China (GB/T 35892-2018). All experiments were performed in compliance with the relevant...
Cutting of ultrathin sections and transmission electron microscopy (TEM)
Regenerative nerves in all groups were removed after 12 weeks, fixed in 2.5% glutaraldehyde overnight, post-fixed by immersion in 1% osmium tetroxide solution for 3 h, and dehydrated through a graded acetone series (30%, 50%, 70%, 90%, and 100%). The samples were then embedded in Epon 812 epoxy resin and cut into 80...
- Use
- Regenerative nerves in all groups were removed after 12 weeks, fixed in 2.5% glutaraldehyde overnight, post-fixed by immersion in 1% osmium tetroxide solution for 3 h, and dehydrated through a graded acetone series (30%, 50%, 70%, 90%, and 100%). The samples were then embedded in Epon 812 epoxy resin and cut into 80...
Sciatic nerve index analysis
A CatWalk XT gait analysis system (Noldus), which automatically records movement, was used to analyze the motor function of the rats. First, the camera was adjusted to the appropriate position. Before recording, rats were familiarized with the test environment. The sciatic function index (SFI) was measured by the Br...
- Use
- A CatWalk XT gait analysis system (Noldus), which automatically records movement, was used to analyze the motor function of the rats. First, the camera was adjusted to the appropriate position. Before recording, rats were familiarized with the test environment. The sciatic function index (SFI) was measured by the Br...
Evaluation of the electrical conduction of regenerative nerves
The gastrocnemius muscle and sciatic nerve from the operated side of the rats were subjected to electrophysiological evaluation. A Medelec Synergy electrophysiological system (Oxford Instrument Inc.) was used to measure the electrical activity. The stimulating electrodes were placed at the proximal and distal ends o...
- Use
- The gastrocnemius muscle and sciatic nerve from the operated side of the rats were subjected to electrophysiological evaluation. A Medelec Synergy electrophysiological system (Oxford Instrument Inc.) was used to measure the electrical activity. The stimulating electrodes were placed at the proximal and distal ends o...
Evaluation of the electrical conduction of regenerative nerves
Statistical analysis. The data were analyzed using SPSS software (ver. 17.0; SPSS Inc.) with one-way ANOVA followed by Tukey's post hoc multiple comparison test. The results were shown with mean ±SEM. In all analyses, p <0.05 was taken to indicate statistical significance.
- Use
- Statistical analysis. The data were analyzed using SPSS software (ver. 17.0; SPSS Inc.) with one-way ANOVA followed by Tukey's post hoc multiple comparison test. The results were shown with mean ±SEM. In all analyses, p <0.05 was taken to indicate statistical significance.
ACG-RGI/KLT Promotes motor function and electrical conduction recovery
In rats, the denervated muscles begin to atrophy after sciatic nerve injury, which leads to limb paralysis. By recording rat footprints, the degree of reinnervation and functional recovery could be quantified. Twelve weeks after the operation, the gait of the rats in each group was examined by CatWalk XT 10.6 gait a...
- Use
- In rats, the denervated muscles begin to atrophy after sciatic nerve injury, which leads to limb paralysis. By recording rat footprints, the degree of reinnervation and functional recovery could be quantified. Twelve weeks after the operation, the gait of the rats in each group was examined by CatWalk XT 10.6 gait a...
Evaluation of the electrical conduction of regenerative nerves
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Statistical analysis. The data were analyzed using SPSS software (ver. 17.0; SPSS Inc.) with one-way ANOVA followed by Tukey's post hoc multiple comparison test. The results were shown with mean ±SEM. In all analyses, p <0.05 was taken to indicate statistical significance.
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Methods
Chitosan, poly (ethylene glycol) (average MW ca. 4,000 kDa) and sodium tripolyphosphate (STPP) were purchased from Sigma-Aldrich. The aligned chitosan nanofiber hydrogel (ACG) was fabricated by electrospinning as reported previously,,. Briefly, 2% electrospinning chitosan solution was prepared by dissolving 0.2 g of chitosan powder with 10 mL of 2% acetic acid. Then, 0.2% polyethylene oxide (PEO) was added to the electrospinning solution to improve its viscosity. The polymer solution was mixed for 30 min on a rotary mixer until the liquid was evenly mixed. During the electrospinning process, polymer solution was ejected through a 2.5 mL syringe needle at a rate of 3 mL/h, and charged with 4 kV positive potential to form a solution jet. A circular collector was used to collect the aligned chitosan nanofibers at a speed of 60 rpm with 1% STPP aqueous solution (Figure A). The chitosan...
Methods
The functional polypeptides RGI and KLT were synthesized by solid-phase technology by Shanghai Qiangyao Biotechnology Co., Ltd. High-performance liquid chromatography (HPLC) was performed to verify that the polypeptides had > 95% purity. Samples of 2.5 mg RGI and 2.5 mg KLT polypeptide powders were dissolved in 25 mL of ultrapure water. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N -hydroxysuccinimide (NHS) were added to COOH:EDC:NHS in a molar ratio of 10:10:1. The chitosan nanofibers hydrogel grafted RGI and KLT (ACG-RGI/KLT) was constructed by immerse chitosan nanofibers hydrogel into the above solution containing RGI and KLT on shaking bed for 24 h.
Polarized light microscopic observation
The chitosan hydrogel obtained by electrospinning was cut into small segments (~5 cm in length) for observation under a polarizing microscope (UPT203i; Leica). First, the polarizer was adjusted in the orthogonal state to achieve complete extinction of the dark field of vision. Then, bunches of chitosan hydrogels were placed in the center of the object stage for imaging. Two bunches of chitosan hydrogels were superimposed at 90° and placed in the center of the object stage for imaging.
Scanning electron microscopy (SEM) analysis
Chitosan hydrogel was fixed with glutaraldehyde for 30 min and then dehydrated through a graded acetone series (30%, 50%, 70%, 90%, and 100%); this was followed by critical point drying. Samples were imaged by SEM (6700F; JEOL).
Examination of mechanical properties
The mechanical properties of chitosan hydrogel were measured using a material testing machine (ZwickRoell). Briefly, the two ends of the chitosan hydrogel were fixed on the clamp, and then stretched at a rate of 1 mm/min. The Young's elastic modulus of the chitosan hydrogel was determined from the linear slope of the strain as < 5%, and the stress at gel fracture was taken as the tensile strength. The corresponding strain was defined as the fracture strain.
Isolation and culture of Schwann cells
Five Sprague-Dawley rats were used to obtain cultures of primary Schwann cells within 3 days after birth, as reported previously. Briefly, the bilateral sciatic nerves of the rats were collected after sterilization. The sciatic nerve epicardial membrane was then peeled off using microforceps, and the sciatic nerve was cut into 1-mm segments, enzymatically dissociated in 0.2% collagenase NB4 (17454; SERVA) for 20 min at 37 °C, centrifuged, and resuspended in Dulbecco's modified Eagle's medium (DMEM)/F-12 (Gibco) containing 10% fetal bovine serum (FBS) (10099141; Gibco). The Schwann cells were then seeded in culture dishes and cultured with DMEM/F-12 containing 10% FBS, 2 mM forskolin, and 2 ng/mL heregulin.
RT-PCR and quantitative PCR
RNA was extracted from Schwann cells in all groups using TRIzol reagent (Invitrogen). cDNA was synthesized using a Synthesis Kit (Roche). Real-time PCR was performed using SYBR Green PCR master mix (Roche) with the forward and reverse primers listed in Table. To analyze changes in gene expression, the results of three replicates in three independent experiments were averaged. Levels of gene expression were normalized relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
Surgical process
Sprague-Dawley rats weighing 200-220 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Animals were treated in accordance with the Laboratory Animal Guideline for Ethical Review of Animal Welfare of China (GB/T 35892-2018). All experiments were performed in compliance with the relevant regulations laid out by the Medical Ethics Committee of Peking University People's Hospital. Rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution (30 mg/kg body weight) and randomly divided into four groups: hollow NGC group, NGC containing ACG group, NGC containing ACG-KLT/NGI group, and autograft group. The sciatic nerves of the right hind limbs of rats were cut to leave a 15 mm gap defect. The nerve stumps were bridged with nerve grafts as described above, under a microscope using 10-0 sutures (Figure ).
Measurement outputs
What raw and processed outputs should exist?
Chitosan hydrogel was fixed with glutaraldehyde for 30 min and then dehydrated through a graded acetone series (30%, 50%, 70%, 90%, and 100%); this was followed by critical poin...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
RNA was extracted from Schwann cells in all groups using TRIzol reagent (Invitrogen). cDNA was synthesized using a Synthesis Kit (Roche). Real-time PCR was performed using SYBR...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Regenerative nerves in all groups were removed after 12 weeks, fixed in 2.5% glutaraldehyde overnight, post-fixed by immersion in 1% osmium tetroxide solution for 3 h, and dehyd...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Gastrocnemius muscles were removed and their wet weight ratios were measured, followed by fixation in 4% paraformaldehyde overnight. Muscle samples were embedded in paraffin, cu...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect the raw assay or blot output and retain identifiers for each sample and experimental group.
inferred from protocolPreprocessing / cleaning
Chitosan hydrogel was fixed with glutaraldehyde for 30 min and then dehydrated through a graded acetone series (30%, 50%, 70%, 90%, and 100%); this was followed by critical point drying.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Chitosan hydrogel was fixed with glutaraldehyde for 30 min and then dehydrated through a graded acetone series (30%, 50%, 70%, 90%, and 100%); this was followed by critical poin...; RNA was extracted from Schwann cells in all groups using TRIzol reagent (Invitrogen). cDNA was synthesized using a Synthesis Kit (Roche). Real-time PCR was performed using SYBR...; Regenerative nerves in all groups were removed after 12 weeks, fixed in 2.5% glutaraldehyde overnight, post-fixed by immersion in 1% osmium tetroxide solution for 3 h, and dehyd...; Gastrocnemius muscles were removed and their wet weight ratios were measured, followed by fixation in 4% paraformaldehyde overnight. Muscle samples were embedded in paraffin, cu....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
Chitosan hydrogel was fixed with glutaraldehyde for 30 min and then dehydrated through a graded acetone series (30%, 50%, 70%, 90%, and 100%); this was followed by critical poin...; Statistical analysis. The data were analyzed using SPSS software (ver. 17.0; SPSS Inc.) with one-way ANOVA followed by Tukey's post hoc multiple comparison test. The results wer...; At 12 weeks after the operation, the regenerated nerves in each group were removed after cardiac perfusion and subjected to osmium acid staining. The results of HE and toluidine...; The diameter of regenerated myelinated nerve fibers and the myelin sheath thickness were evaluated by TEM (Figure A). The results showed that the regenerated myelinated nerve fi...
from paperReporting output
Report representative outputs alongside summary comparisons for Chitosan hydrogel was fixed with glutaraldehyde for 30 min and then dehydrated through a graded acetone series (30%, 50%, 70%, 90%, and 100%); this was followed by critical poin..., RNA was extracted from Schwann cells in all groups using TRIzol reagent (Invitrogen). cDNA was synthesized using a Synthesis Kit (Roche). Real-time PCR was performed using SYBR..., Regenerative nerves in all groups were removed after 12 weeks, fixed in 2.5% glutaraldehyde overnight, post-fixed by immersion in 1% osmium tetroxide solution for 3 h, and dehyd..., Gastrocnemius muscles were removed and their wet weight ratios were measured, followed by fixation in 4% paraformaldehyde overnight. Muscle samples were embedded in paraffin, cu....
inferred from protocolStructured statistical methods
Chitosan hydrogel was fixed with glutaraldehyde for 30 min and then dehydrated through a graded acetone series (30%, 50%, 70%, 90%, and 100%); this was followed by critical poin...; Statistical analysis. The data were analyzed using SPSS software (ver. 17.0; SPSS Inc.) with one-way ANOVA followed by Tukey's post hoc multiple comparison test. The results wer...; At 12 weeks after the operation, the regenerated nerves in each group were removed after cardiac perfusion and subjected to osmium acid staining. The results of HE and toluidine...; The diameter of regenerated myelinated nerve fibers and the myelin sheath thickness were evaluated by TEM (Figure A). The results showed that the regenerated myelinated nerve fi...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
Chitosan, poly (ethylene glycol) (average MW ca. 4,000 kDa) and sodium tripolyphosphate (STPP) were purchased from Sigma-Aldrich. The aligned chitosan nanofiber hydrogel (ACG) was fabricated by electrospinning as reported previously,,. Briefly, 2% electrospinning chitosan solution was prepared by dissolving 0.2 g of chitosan powder with 10 mL of 2% acetic acid. Then, 0.2% polyethylene oxide (PEO) was added to the electrospinning solution to improve its viscosity. The polymer solution was mixed for 30 min on a rotary mixer until the liquid was evenly mixed. During the electrospinning process, polymer solution was ejected through a 2.5 mL syringe needle at a rate of 3 mL/h, and charged with 4 kV positive potential to form a solution jet. A circular collector was used to collect the aligned chitosan nanofibers at a speed of 60 rpm with 1% STPP aqueous solution (Figure A). The chitosan nanofibers were collected and washed in sterile phosphate-buffered saline (PBS) for 6 h to remove the PEO and STPP, followed by immersion in 75% ethanol for 24 h.
The functional polypeptides RGI and KLT were synthesized by solid-phase technology by Shanghai Qiangyao Biotechnology Co., Ltd. High-performance liquid chromatography (HPLC) was performed to verify that the polypeptides had > 95% purity. Samples of 2.5 mg RGI and 2.5 mg KLT polypeptide powders were dissolved in 25 mL of ultrapure water. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N -hydroxysuccinimide (NHS) were added to COOH:EDC:NHS in a molar ratio of 10:10:1. The chitosan nanofibers hydrogel grafted RGI and KLT (ACG-RGI/KLT) was constructed by immerse chitosan nanofibers hydrogel into the above solution containing RGI and KLT on shaking bed for 24 h.
The chitosan hydrogel obtained by electrospinning was cut into small segments (~5 cm in length) for observation under a polarizing microscope (UPT203i; Leica). First, the polarizer was adjusted in the orthogonal state to achieve complete extinction of the dark field of vision. Then, bunches of chitosan hydrogels were placed in the center of the object stage for imaging. Two bunches of chitosan hydrogels were superimposed at 90° and placed in the center of the object stage for imaging.
Chitosan hydrogel was fixed with glutaraldehyde for 30 min and then dehydrated through a graded acetone series (30%, 50%, 70%, 90%, and 100%); this was followed by critical point drying. Samples were imaged by SEM (6700F; JEOL).
The mechanical properties of chitosan hydrogel were measured using a material testing machine (ZwickRoell). Briefly, the two ends of the chitosan hydrogel were fixed on the clamp, and then stretched at a rate of 1 mm/min. The Young's elastic modulus of the chitosan hydrogel was determined from the linear slope of the strain as < 5%, and the stress at gel fracture was taken as the tensile strength. The corresponding strain was defined as the fracture strain.
Five Sprague-Dawley rats were used to obtain cultures of primary Schwann cells within 3 days after birth, as reported previously. Briefly, the bilateral sciatic nerves of the rats were collected after sterilization. The sciatic nerve epicardial membrane was then peeled off using microforceps, and the sciatic nerve was cut into 1-mm segments, enzymatically dissociated in 0.2% collagenase NB4 (17454; SERVA) for 20 min at 37 °C, centrifuged, and resuspended in Dulbecco's modified Eagle's medium (DMEM)/F-12 (Gibco) containing 10% fetal bovine serum (FBS) (10099141; Gibco). The Schwann cells were then seeded in culture dishes and cultured with DMEM/F-12 containing 10% FBS, 2 mM forskolin, and 2 ng/mL heregulin.
RNA was extracted from Schwann cells in all groups using TRIzol reagent (Invitrogen). cDNA was synthesized using a Synthesis Kit (Roche). Real-time PCR was performed using SYBR Green PCR master mix (Roche) with the forward and reverse primers listed in Table. To analyze changes in gene expression, the results of three replicates in three independent experiments were averaged. Levels of gene expression were normalized relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
Sprague-Dawley rats weighing 200-220 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Animals were treated in accordance with the Laboratory Animal Guideline for Ethical Review of Animal Welfare of China (GB/T 35892-2018). All experiments were performed in compliance with the relevant regulations laid out by the Medical Ethics Committee of Peking University People's Hospital. Rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution (30 mg/kg body weight) and randomly divided into four groups: hollow NGC group, NGC containing ACG group, NGC containing ACG-KLT/NGI group, and autograft group. The sciatic nerves of the right hind limbs of rats were cut to leave a 15 mm gap defect. The nerve stumps were bridged with nerve grafts as described above, under a microscope using 10-0 sutures (Figure ).
Machine-readable layer
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"text": "Chitosan, poly (ethylene glycol) (average MW ca. 4,000 kDa) and sodium tripolyphosphate (STPP) were purchased from Sigma-Aldrich. The aligned chitosan nanofiber hydrogel (ACG) was fabricated by electrospinning as reported previously,,. Briefly, 2% electrospinning chitosan solution was prepared by dissolving 0.2 g of chitosan powder with 10 mL of 2% acetic acid. Then, 0.2% polyethylene oxide (PEO) was added to the electrospinning solution to improve its viscosity. The polymer solution was mixed for 30 min on a rotary mixer until the liquid was evenly mixed. During the electrospinning process, polymer solution was ejected through a 2.5 mL syringe needle at a rate of 3 mL/h, and charged with 4 kV positive potential to form a solution jet. A circular collector was used to collect the aligned chitosan nanofibers at a speed of 60 rpm with 1% STPP aqueous solution (Figure A). The chitosan..."
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"datePublished": "2020",
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