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
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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 ).
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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 ).
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