Exosomes from Human Gingiva-Derived Mesenchymal Stem Cells Combined with Biodegradable Chitin Conduits Promote Rat Sciatic Nerve Regeneration methods
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2. Materials and Methods
With the approval of the ethical committee of the Peking University Hospital of Stomatology, gingivae were collected from four healthy patients without a history of periodontal disease, such as upon wisdom tooth extraction. Human GMSCs were isolated as described in published protocols [ ]. The gingival tissues were treated aseptically, washed several times with PBS, and cultured in a medium containing 2 mg/mL protease (Gibco) at 4°C. After terminating the digestion, the epithelial layer and the lamina propria were separated, and the lamina propria was collected and shredded. Tissues were cut into pieces and digested with collagenase IV at 37°C for 1 hour and centrifuged at 1000 rpm for 10 minutes, and the supernatant was discarded. The cells were suspended in a 10 cm cell culture dish and cultured in a humidified cell culture incubator containing 5% CO 2 at...
2.3. Multipotent Differentiation of GMSCs
GMSCs were plated at 5 × 10 5 cells/well in 6-well plates in MSC growth medium; upon reaching 60% fusion, medium was replaced with osteogenic induction medium supplemented with dexamethasone, L-glutamine, ascorbic acid, and β -glycerophosphate. The culture medium was changed every three days. After 4 weeks, in vitro mineralization was assayed by Alizarin Red S staining.
2.3.2. Adipogenic Differentiation
When GMSCs reached 60% fusion, the medium was replaced with adipogenic induction culture medium consisting of MEM medium with 10 µ M human insulin, 1 µ M dexamethasone, 200 µ M indomethacin, and 0.5 mM 3-isobutyl-1-methylxanthine. The medium was replaced every three days for 14 days. Oil Red O staining was performed to detect intracellular lipid vacuole characteristic of adipocytes.
2.8. Animals and Surgical Procedures
Chitin conduits (diameter: 1.2 mm) were patented by the Peking University and China Spinning and Weaving Institute, and these can retain structure for at least 6 weeks in vivo and undergo complete biodegradation (please refer to Chinese patent ZL01136314 for technical details). Eight-week-old SD rats were provided by Beijing Weitong Lihua Co. Ltd. We followed the guideline for ethical review of animal welfare of China (GB/T 35892-2018), and all experiments complied with the relevant regulations of the Medical Ethics Committee of Peking University People's Hospital (approval no. 2102000024). Twenty-four SD rats weighing 200-220 g were randomly divided into three groups, with eight rats in each, as follows: the hollow chitin conduit group, chitin conduit plus GMSC-derived exosome group, and autograft group. All rats were anesthetized with a sodium pentobarbital solutio...
2.4. Culture and Proliferation Assay of the Schwann Cell
Three-day-old Sprague-Dawley (SD) mice were used. The bilateral sciatic nerves were cut, and its epicardium was removed. Sciatic nerve was collected and digested with 0.2% NB4 collagenase for 10 min at 37°C. Schwann cell culture medium (10% fetal bovine serum, 1% penicillin/streptomycin, and 10 -2 M/mL forskolin) was added to resuspend the cells. Schwann cell was seeded in a cell culture flask. After 48 h, Schwann cell was digested and seeded in 96-well plates and 6-well plates cultured with exosomes (100 µ g/mL). After culture for 1, 3, and 5 days, 10 µ L of CCK solution was added to 96-well plates. The absorbance at 450 nm was measured with a microplate reader. Five days after culture, Schwann cell in 6-well plates was identified by immunofluorescence staining using rabbit anti-S100 antibody.
2.5. Isolation and Identification of Exosomes
GMSCs were cultured at 3 × 10 6 cells/75 cm 2 density with 15 mL medium containing exosomes-free fetal bovine serum for 48-72 h; then, the medium was collected in a 15 mL centrifugal tube and centrifuged at 3000 × g for 10 min; the supernatant was then carefully collected and transferred to a new tube and placed on ice. The supernatant from 10 mL of cells was extracted, and 2.5 mL of the exosome extraction reagent (HieffTM Quick exosome isolation kit, 41201ES25) was added. The supernatant was mixed via vortex oscillation for 1 minute, and then placed at 4°C for 2 h. The tube containing the mixture was removed and centrifuged for 60 minutes at 4°C and 10000 × g; then, the supernatant was discarded, and the pellet was collected. The centrifugal sediment was beaten uniformly with 100 µ L of PBS for...
2.6. Isolation and Culture of Dorsal Root Ganglion (DRG) Cells with Exosomes
SD mice born within 24 hours were sacrificed. Then, 75% alcohol was used to sterilize the backs of mice; vertebrae were exposed and removed and then placed in DMEM-F12 medium containing 10% FBS. The vertebral body was cut centrally along the sagittal plane, and the DRG was extracted from the intervertebral foramen and placed in a new culture dish. The DRG epineurium was then peeled off and plated onto poly-lysine-coated glass coverslips in 6-well plates. The DRGs were cultured with exosomes (100 µ g/mL) in MEM medium (Gibco) supplemented with B-27 (Gibco) and GlutaMAX (Gibco). The control had no exosomes.
2.7. Immunofluorescence
After culture for 5 days, immunofluorescence staining was performed to evaluate the effect of exosomes on DRGs. First, DRG samples were fixed with paraformaldehyde for 30 minutes and then blocked with 10% goat serum for 1 hour. Next, mouse anti-neurofilament 200 (N0142; Sigma) and rabbit anti-S100 (S2644; Sigma) were applied as the primary antibodies for overnight incubation at 4°C. Goat anti-mouse IgG (Alexa Fluor® 488; Abcam) and goat anti-rabbit (Alexa Fluor® 594; Abcam) secondary antibodies were then incubated with the samples in the dark for 1 hour. Three randomly selected DRG samples from each group were used for statistical analysis. The three longest axons per quadrant were measured and used to calculate the mean length of each DRG axon using the Image-Pro Plus 6.0 image analysis software.
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Evidence quotes (8)
With the approval of the ethical committee of the Peking University Hospital of Stomatology, gingivae were collected from four healthy patients without a history of periodontal disease, such as upon wisdom tooth extraction. Human GMSCs were isolated as described in published protocols [ ]. The gingival tissues were treated aseptically, washed several times with PBS, and cultured in a medium containing 2 mg/mL protease (Gibco) at 4°C. After terminating the digestion, the epithelial layer and the lamina propria were separated, and the lamina propria was collected and shredded. Tissues were cut into pieces and digested with collagenase IV at 37°C for 1 hour and centrifuged at 1000 rpm for 10 minutes, and the supernatant was discarded. The cells were suspended in a 10 cm cell culture dish and cultured in a humidified cell culture incubator containing 5% CO 2 at 37°C for 24 hours in alpha-MEM medium containing 10% FBS and 100 U/mL penicillin/100 µ g/mL streptomycin. The original culture medium was discarded, and the floating cells were removed. Fresh medium was added. The medium was then replaced every two days. When cell fusion reac...
GMSCs were plated at 5 × 10 5 cells/well in 6-well plates in MSC growth medium; upon reaching 60% fusion, medium was replaced with osteogenic induction medium supplemented with dexamethasone, L-glutamine, ascorbic acid, and β -glycerophosphate. The culture medium was changed every three days. After 4 weeks, in vitro mineralization was assayed by Alizarin Red S staining.
When GMSCs reached 60% fusion, the medium was replaced with adipogenic induction culture medium consisting of MEM medium with 10 µ M human insulin, 1 µ M dexamethasone, 200 µ M indomethacin, and 0.5 mM 3-isobutyl-1-methylxanthine. The medium was replaced every three days for 14 days. Oil Red O staining was performed to detect intracellular lipid vacuole characteristic of adipocytes.
Chitin conduits (diameter: 1.2 mm) were patented by the Peking University and China Spinning and Weaving Institute, and these can retain structure for at least 6 weeks in vivo and undergo complete biodegradation (please refer to Chinese patent ZL01136314 for technical details). Eight-week-old SD rats were provided by Beijing Weitong Lihua Co. Ltd. We followed the guideline for ethical review of animal welfare of China (GB/T 35892-2018), and all experiments complied with the relevant regulations of the Medical Ethics Committee of Peking University People's Hospital (approval no. 2102000024). Twenty-four SD rats weighing 200-220 g were randomly divided into three groups, with eight rats in each, as follows: the hollow chitin conduit group, chitin conduit plus GMSC-derived exosome group, and autograft group. All rats were anesthetized with a sodium pentobarbital solution (30 mg/kg body weight), and then, the right hind limb was prepared. The sciatic nerve was cut and exposed, and 10 µ L PBS was administered to the hollow chitin conduit group, retaining a 10 mm gap. The exosome group received 10 µ L PBS containing 10 µ...
Three-day-old Sprague-Dawley (SD) mice were used. The bilateral sciatic nerves were cut, and its epicardium was removed. Sciatic nerve was collected and digested with 0.2% NB4 collagenase for 10 min at 37°C. Schwann cell culture medium (10% fetal bovine serum, 1% penicillin/streptomycin, and 10 -2 M/mL forskolin) was added to resuspend the cells. Schwann cell was seeded in a cell culture flask. After 48 h, Schwann cell was digested and seeded in 96-well plates and 6-well plates cultured with exosomes (100 µ g/mL). After culture for 1, 3, and 5 days, 10 µ L of CCK solution was added to 96-well plates. The absorbance at 450 nm was measured with a microplate reader. Five days after culture, Schwann cell in 6-well plates was identified by immunofluorescence staining using rabbit anti-S100 antibody.
GMSCs were cultured at 3 × 10 6 cells/75 cm 2 density with 15 mL medium containing exosomes-free fetal bovine serum for 48-72 h; then, the medium was collected in a 15 mL centrifugal tube and centrifuged at 3000 × g for 10 min; the supernatant was then carefully collected and transferred to a new tube and placed on ice. The supernatant from 10 mL of cells was extracted, and 2.5 mL of the exosome extraction reagent (HieffTM Quick exosome isolation kit, 41201ES25) was added. The supernatant was mixed via vortex oscillation for 1 minute, and then placed at 4°C for 2 h. The tube containing the mixture was removed and centrifuged for 60 minutes at 4°C and 10000 × g; then, the supernatant was discarded, and the pellet was collected. The centrifugal sediment was beaten uniformly with 100 µ L of PBS for mixing and transferred to a new 1.5 mL centrifugal tube. The 1.5 mL centrifugal tube containing exosome was centrifuged at 4°C for 2 min at 12000 × g. The precipitation was discarded, and the supernatant was retained.
SD mice born within 24 hours were sacrificed. Then, 75% alcohol was used to sterilize the backs of mice; vertebrae were exposed and removed and then placed in DMEM-F12 medium containing 10% FBS. The vertebral body was cut centrally along the sagittal plane, and the DRG was extracted from the intervertebral foramen and placed in a new culture dish. The DRG epineurium was then peeled off and plated onto poly-lysine-coated glass coverslips in 6-well plates. The DRGs were cultured with exosomes (100 µ g/mL) in MEM medium (Gibco) supplemented with B-27 (Gibco) and GlutaMAX (Gibco). The control had no exosomes.
After culture for 5 days, immunofluorescence staining was performed to evaluate the effect of exosomes on DRGs. First, DRG samples were fixed with paraformaldehyde for 30 minutes and then blocked with 10% goat serum for 1 hour. Next, mouse anti-neurofilament 200 (N0142; Sigma) and rabbit anti-S100 (S2644; Sigma) were applied as the primary antibodies for overnight incubation at 4°C. Goat anti-mouse IgG (Alexa Fluor® 488; Abcam) and goat anti-rabbit (Alexa Fluor® 594; Abcam) secondary antibodies were then incubated with the samples in the dark for 1 hour. Three randomly selected DRG samples from each group were used for statistical analysis. The three longest axons per quadrant were measured and used to calculate the mean length of each DRG axon using the Image-Pro Plus 6.0 image analysis software.
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