An injectable hydrogel enhances tissue repair after spinal cord injury by promoting extracellular matrix remodeling methods
Aim. Evidence-backed execution summary for An injectable hydrogel enhances tissue repair after spinal cord injury by promoting extracellular matrix remodeling methods from An injectable hydrogel enhances tissue repair after spinal cord injury by promoting extracellular matrix remodeling.
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rat
Subject model for the experiment.
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Results
reagent used in the protocol.
- Use
- I-5 was synthesized through conjugation of imidazole to the polymer II (CP-2) by carbodiimide cross-linking between the amine group of 1-3 aminopropylimidazole and the carboxyl group of the polymer II (Fig. ). The existence of the imidazole group in I-5 was confirmed by the presence of imidazole peaks in...
I-5 hydrogel eliminates cystic cavities after contusive SCI
reagent used in the protocol.
- Use
- A previous study showed that cystic cavities start to form as early as 1 week after contusion injury and progressively enlarge up to the 4-week time point. At 1 week after injury, we also observed quite large cystic cavities already formed at the epicenter (Supplementary Fig. ). At this time point, non-cystic...
Methods
reagent used in the protocol.
- Use
- IleOEt·HCl (23.81 g, 121.67 mmol) in dry tetrahydrofuran (THF) containing trimethylamine (TEA) was added slowly to poly(dichlorophosphazene) (10.00 g, 86.29 mmol) dissolved in dry THF. The reaction mixture was incubated at dry ice bath for 12 h and room temperature for 36 h....
Methods
reagent used in the protocol.
- Use
- Step 3: [NP(IleOEt) 1.45 (Succinic acid) 0.13 (Imi) 0.01 (AMPEG750) 0.41 ] n (I-5). The carboxylic acid terminated polymer II (10 g, 16.55 mmol) was dissolved in dried THF. After cooling to 0 °C, diisopropylcarbodiimide (DIC; 3.58 ml, 23.17 mmol) and N -hydroxysuccinimide (NHS; 2.6...
Animals and surgical procedures
reagent used in the protocol.
- Use
- All animal protocols were approved by the Institutional Animal Care and Use Committee of Ajou University School of Medicine. Adult female Sprague Dawley rats (250-300 g) were used in this study. Spinal contusion injury was inflicted using the Infinite Horizon impactor (Precision Systems and Instrumentati...
I-5 hydrogel eliminates cystic cavities after contusive SCI
A previous study showed that cystic cavities start to form as early as 1 week after contusion injury and progressively enlarge up to the 4-week time point. At 1 week after injury, we also observed quite large cystic cavities already formed at the epicenter (Supplementary Fig. ). At this time point, non-cystic...
- Use
- A previous study showed that cystic cavities start to form as early as 1 week after contusion injury and progressively enlarge up to the 4-week time point. At 1 week after injury, we also observed quite large cystic cavities already formed at the epicenter (Supplementary Fig. ). At this time point, non-cystic...
Methods
IleOEt·HCl (23.81 g, 121.67 mmol) in dry tetrahydrofuran (THF) containing trimethylamine (TEA) was added slowly to poly(dichlorophosphazene) (10.00 g, 86.29 mmol) dissolved in dry THF. The reaction mixture was incubated at dry ice bath for 12 h and room temperature for 36 h....
- Use
- IleOEt·HCl (23.81 g, 121.67 mmol) in dry tetrahydrofuran (THF) containing trimethylamine (TEA) was added slowly to poly(dichlorophosphazene) (10.00 g, 86.29 mmol) dissolved in dry THF. The reaction mixture was incubated at dry ice bath for 12 h and room temperature for 36 h....
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Results
I-5 was synthesized through conjugation of imidazole to the polymer II (CP-2) by carbodiimide cross-linking between the amine group of 1-3 aminopropylimidazole and the carboxyl group of the polymer II (Fig. ). The existence of the imidazole group in I-5 was confirmed by the presence of imidazole peaks in the 1 H-NMR spectrum (Supplementary Fig. ) and by an increase of the C=O peak, corresponding to amide bond stretching in Fourier Transform InfraRed (FT-IR) spectroscopy data (Supplementary Fig. ). The molecular weight of I-5 ranged from approximately 14 kDa to 18 kDa The viscosity of I-5 hydrogel was measured at a wide range of temperatures (Fig. ). The viscosity rose abruptly when the temperature reached 30 °C, causing a phase transition from sol to gel state. The viscosity at body temperature was ∼600 Pa·s, which indicates th...
I-5 hydrogel eliminates cystic cavities after contusive SCI
A previous study showed that cystic cavities start to form as early as 1 week after contusion injury and progressively enlarge up to the 4-week time point. At 1 week after injury, we also observed quite large cystic cavities already formed at the epicenter (Supplementary Fig. ). At this time point, non-cystic lesions were filled with ED-1 positive macrophages surrounded by glial fibrillary acidic protein (GFAP) positive astrocytes (Supplementary Fig. ). We chose to examine the presence of cystic cavities 4 weeks after injection because a previous study reported that there is no further expansion of the cystic cavities after the 4-week time point. At 5 weeks after injury (4 weeks after injection), cystic cavities became larger extending rostrocaudally more than 2.0 mm away from the epicenter in animals injected with control PBS (Fig. ). Surprisingly, I-5 injec...
Methods
IleOEt·HCl (23.81 g, 121.67 mmol) in dry tetrahydrofuran (THF) containing trimethylamine (TEA) was added slowly to poly(dichlorophosphazene) (10.00 g, 86.29 mmol) dissolved in dry THF. The reaction mixture was incubated at dry ice bath for 12 h and room temperature for 36 h. AEtOH (1.56 g, 25.50 mmol) and AMPEG750 (37.53 g, 50.04 mmol) were dissolved in dry THF including TEA and added to the mixture. The reaction mixture was stirred at room temperature for 24 h and then at 40-50 °C for 24 h. AMPEG750 (18.76 g, 25.02 mmol) in dried THF was added to the reaction mixture and stirred at room temperature for 24 h and then at 40-50 °C for 24 h. The reaction mixture was filtered; the filtrate was concentrated and poured into n-hexane to obtain a precipitate,...
Discussion
Several structural changes may explain how neural function for locomotion was recovered by I-5 injection. We found that filling cavity spaces with fibrotic matrix resulted in preservation of motor neurons and myelination. In animals with control PBS injection, myelin immunoreactivity was severely reduced especially in the spared white matter surrounding cavity spaces. We speculate that fluid within the cystic spaces may contain elevated levels of toxic substances that can contribute to secondary degeneration of motor neurons and/or demyelination of the white matter. Filling cystic spaces with fibrotic matrix may minimize exposure to toxic materials in the cystic fluid, leading to preservation of myelin and motor neurons. Another possibility is that the formation of fibrotic matrix in the lesion center may increase the stability of post-injury spinal cord. Spinal cord tissue with a lar...
Methods
Succinic anhydride (2.76 g, 27.58 mmol) and 4-(dimethylamino)pyridine (DMAP) (3.37 g, 27.56 mmol) dissolved in dried THF, were added separately into EP (27.12 g, 45.94 mmol) in dried THF. The reaction mixture was stirred at 40 °C for 24 h. The products were dialyzed with a dialysis membrane (Spectra/Por, MWCO: 10-12 kDa) against methanol for 4 days at room temperature and against distilled water for 4 days at 4 °C. Freeze-drying was carried out after dialysis to obtain polymer II. Yield: 91%. 1 H NMR (CDCl 3 ), δ (ppm): 0.8-1.0 (s, 6H), 1.1-1.3 (b, 3H), 1.3-1.6 (b, 2H), 1.6-1.9 (b, 1H), 2.5-2.8 (b, 2H), 2.8-3.3 (b, 2H), 3.4-3.8 (b, 62H), 3.9 (s, 1H), 4.0-4.3 (b, 3H).
Methods
Step 3: [NP(IleOEt) 1.45 (Succinic acid) 0.13 (Imi) 0.01 (AMPEG750) 0.41 ] n (I-5). The carboxylic acid terminated polymer II (10 g, 16.55 mmol) was dissolved in dried THF. After cooling to 0 °C, diisopropylcarbodiimide (DIC; 3.58 ml, 23.17 mmol) and N -hydroxysuccinimide (NHS; 2.67 g, 23.16 mmol) were added and the mixture was stirred for 30 min in order to activate the carboxyl groups of the polymer. The mixture was moved to 1-3-aminopropylimidazole (1.45 g, 11.54 mmol) dissolved in distilled THF. The reaction mixture was stirred at 0 °C for 6 h and then at room temperature for 18 h. Afterwards, the solution was concentrated and purified by precipitation with 1 M potassium fluoride solution. The precipitate was dialyzed with a dialysis membrane (Spectra/Por, Spectrum Laboratories, mol...
Animals and surgical procedures
All animal protocols were approved by the Institutional Animal Care and Use Committee of Ajou University School of Medicine. Adult female Sprague Dawley rats (250-300 g) were used in this study. Spinal contusion injury was inflicted using the Infinite Horizon impactor (Precision Systems and Instrumentation). Animals were anesthetized with 4% chloral hydrate (10 ml/kg, injected intraperitoneally), followed by a dorsal laminectomy at the 10th thoracic vertebral level (T10-11) to expose the dorsal surface of the spinal cord. Then, standardized contusion at a force of 200 kdyn was performed with the Infinite Horizon impactor. After the injury, muscles were sutured in layer and the skin was stapled. Bladder care was provided twice daily until spontaneous voiding resumed. I-5 injection was performed 1 week after contusion injury to avoid potential tissue damage relat...
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I-5 was synthesized through conjugation of imidazole to the polymer II (CP-2) by carbodiimide cross-linking between the amine group of 1-3 aminopropylimidazole and the car...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
A previous study showed that cystic cavities start to form as early as 1 week after contusion injury and progressively enlarge up to the 4-week time point. At 1 week after inju...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
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Acquisition
Collect the raw assay or blot output and retain identifiers for each sample and experimental group.
inferred from protocolPreprocessing / cleaning
Biomaterial-based treatment has been proposed as a strategy to promote tissue repair by bridging cavity spaces -.
from paperScoring or quantification
Quantify the primary readouts for this experiment: I-5 was synthesized through conjugation of imidazole to the polymer II (CP-2) by carbodiimide cross-linking between the amine group of 1-3 aminopropylimidazole and the car...; A previous study showed that cystic cavities start to form as early as 1 week after contusion injury and progressively enlarge up to the 4-week time point. At 1 week after inju....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
Biomaterial-based treatment has been proposed as a strategy to promote tissue repair by bridging cavity spaces -. Implanting various tissue-engineered scaffolds or matric...; A previous study showed that cystic cavities start to form as early as 1 week after contusion injury and progressively enlarge up to the 4-week time point. At 1 week after inju...
from paperReporting output
Report representative outputs alongside summary comparisons for I-5 was synthesized through conjugation of imidazole to the polymer II (CP-2) by carbodiimide cross-linking between the amine group of 1-3 aminopropylimidazole and the car..., A previous study showed that cystic cavities start to form as early as 1 week after contusion injury and progressively enlarge up to the 4-week time point. At 1 week after inju....
inferred from protocolStructured statistical methods
Biomaterial-based treatment has been proposed as a strategy to promote tissue repair by bridging cavity spaces -. Implanting various tissue-engineered scaffolds or matric...; A previous study showed that cystic cavities start to form as early as 1 week after contusion injury and progressively enlarge up to the 4-week time point. At 1 week after inju...
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Evidence quotes (7)
I-5 was synthesized through conjugation of imidazole to the polymer II (CP-2) by carbodiimide cross-linking between the amine group of 1-3 aminopropylimidazole and the carboxyl group of the polymer II (Fig. ). The existence of the imidazole group in I-5 was confirmed by the presence of imidazole peaks in the 1 H-NMR spectrum (Supplementary Fig. ) and by an increase of the C=O peak, corresponding to amide bond stretching in Fourier Transform InfraRed (FT-IR) spectroscopy data (Supplementary Fig. ). The molecular weight of I-5 ranged from approximately 14 kDa to 18 kDa The viscosity of I-5 hydrogel was measured at a wide range of temperatures (Fig. ). The viscosity rose abruptly when the temperature reached 30 °C, causing a phase transition from sol to gel state. The viscosity at body temperature was ∼600 Pa·s, which indicates that the material injected in vivo has physical strength sufficient to support and maintain the shape of the hydrogel. The rapidity of the gelation process was visualized by injecting polymer solution into distilled water at either 4 or 37 °C (Fig. ). Immediately after injection of po...
A previous study showed that cystic cavities start to form as early as 1 week after contusion injury and progressively enlarge up to the 4-week time point. At 1 week after injury, we also observed quite large cystic cavities already formed at the epicenter (Supplementary Fig. ). At this time point, non-cystic lesions were filled with ED-1 positive macrophages surrounded by glial fibrillary acidic protein (GFAP) positive astrocytes (Supplementary Fig. ). We chose to examine the presence of cystic cavities 4 weeks after injection because a previous study reported that there is no further expansion of the cystic cavities after the 4-week time point. At 5 weeks after injury (4 weeks after injection), cystic cavities became larger extending rostrocaudally more than 2.0 mm away from the epicenter in animals injected with control PBS (Fig. ). Surprisingly, I-5 injection resulted in an almost complete disappearance of cystic cavities in all the animals that received I-5 injection (Fig. ). Instead of cystic cavities, the epicenter region was filled with eosin-stained ECM-like tissue (Fig. ). GFAP immunostaining showed that the ECM-like tissue was lar...
IleOEt·HCl (23.81 g, 121.67 mmol) in dry tetrahydrofuran (THF) containing trimethylamine (TEA) was added slowly to poly(dichlorophosphazene) (10.00 g, 86.29 mmol) dissolved in dry THF. The reaction mixture was incubated at dry ice bath for 12 h and room temperature for 36 h. AEtOH (1.56 g, 25.50 mmol) and AMPEG750 (37.53 g, 50.04 mmol) were dissolved in dry THF including TEA and added to the mixture. The reaction mixture was stirred at room temperature for 24 h and then at 40-50 °C for 24 h. AMPEG750 (18.76 g, 25.02 mmol) in dried THF was added to the reaction mixture and stirred at room temperature for 24 h and then at 40-50 °C for 24 h. The reaction mixture was filtered; the filtrate was concentrated and poured into n-hexane to obtain a precipitate, which was re-precipitated twice in the same solvent system. The polymer product was further purified by dialysis with a dialysis membrane (Spectra/Por, MWCO: 10-12 kDa) against methanol for 4 days at room temperature and against distilled water for 4 days at 4 °C. The dialyzed...
Several structural changes may explain how neural function for locomotion was recovered by I-5 injection. We found that filling cavity spaces with fibrotic matrix resulted in preservation of motor neurons and myelination. In animals with control PBS injection, myelin immunoreactivity was severely reduced especially in the spared white matter surrounding cavity spaces. We speculate that fluid within the cystic spaces may contain elevated levels of toxic substances that can contribute to secondary degeneration of motor neurons and/or demyelination of the white matter. Filling cystic spaces with fibrotic matrix may minimize exposure to toxic materials in the cystic fluid, leading to preservation of myelin and motor neurons. Another possibility is that the formation of fibrotic matrix in the lesion center may increase the stability of post-injury spinal cord. Spinal cord tissue with a large fluid-filled cavity would be more vulnerable to physical alterations such as tissue collapse or adhesion to bones and tendons, which could result in functional exacerbation. Presence of NF positive and 5-HT axons within the FN-rich matrix implies that the newly remodeled fibrotic matrix is permis...
Succinic anhydride (2.76 g, 27.58 mmol) and 4-(dimethylamino)pyridine (DMAP) (3.37 g, 27.56 mmol) dissolved in dried THF, were added separately into EP (27.12 g, 45.94 mmol) in dried THF. The reaction mixture was stirred at 40 °C for 24 h. The products were dialyzed with a dialysis membrane (Spectra/Por, MWCO: 10-12 kDa) against methanol for 4 days at room temperature and against distilled water for 4 days at 4 °C. Freeze-drying was carried out after dialysis to obtain polymer II. Yield: 91%. 1 H NMR (CDCl 3 ), δ (ppm): 0.8-1.0 (s, 6H), 1.1-1.3 (b, 3H), 1.3-1.6 (b, 2H), 1.6-1.9 (b, 1H), 2.5-2.8 (b, 2H), 2.8-3.3 (b, 2H), 3.4-3.8 (b, 62H), 3.9 (s, 1H), 4.0-4.3 (b, 3H).
Step 3: [NP(IleOEt) 1.45 (Succinic acid) 0.13 (Imi) 0.01 (AMPEG750) 0.41 ] n (I-5). The carboxylic acid terminated polymer II (10 g, 16.55 mmol) was dissolved in dried THF. After cooling to 0 °C, diisopropylcarbodiimide (DIC; 3.58 ml, 23.17 mmol) and N -hydroxysuccinimide (NHS; 2.67 g, 23.16 mmol) were added and the mixture was stirred for 30 min in order to activate the carboxyl groups of the polymer. The mixture was moved to 1-3-aminopropylimidazole (1.45 g, 11.54 mmol) dissolved in distilled THF. The reaction mixture was stirred at 0 °C for 6 h and then at room temperature for 18 h. Afterwards, the solution was concentrated and purified by precipitation with 1 M potassium fluoride solution. The precipitate was dialyzed with a dialysis membrane (Spectra/Por, Spectrum Laboratories, molecular weight cut-off: 12-14 kDa) against distilled water for 3 days at 4 °C and the dialyzed solution was freeze-dried to obtain the final product (I-5). Yield: 79%. 1 H NMR (CDCl3), δ (ppm): 0.8-1.0 (s, 6H), 1.1-1.3 (b, 3H), 1.3-1.6 (b, 2H), 1.6-...
All animal protocols were approved by the Institutional Animal Care and Use Committee of Ajou University School of Medicine. Adult female Sprague Dawley rats (250-300 g) were used in this study. Spinal contusion injury was inflicted using the Infinite Horizon impactor (Precision Systems and Instrumentation). Animals were anesthetized with 4% chloral hydrate (10 ml/kg, injected intraperitoneally), followed by a dorsal laminectomy at the 10th thoracic vertebral level (T10-11) to expose the dorsal surface of the spinal cord. Then, standardized contusion at a force of 200 kdyn was performed with the Infinite Horizon impactor. After the injury, muscles were sutured in layer and the skin was stapled. Bladder care was provided twice daily until spontaneous voiding resumed. I-5 injection was performed 1 week after contusion injury to avoid potential tissue damage related to the injection itself. I-5 hydrogel solution (10%) was maintained on ice until loaded into a 26 G Hamilton syringe (model 701RN) to prevent gelation. Animals were anesthetized and the dorsal surface of the spinal cord with the previous contusion injury was re-exposed. After the contused a...
Machine-readable layer
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"text": "I-5 was synthesized through conjugation of imidazole to the polymer II (CP-2) by carbodiimide cross-linking between the amine group of 1-3 aminopropylimidazole and the carboxyl group of the polymer II (Fig. ). The existence of the imidazole group in I-5 was confirmed by the presence of imidazole peaks in the 1 H-NMR spectrum (Supplementary Fig. ) and by an increase of the C=O peak, corresponding to amide bond stretching in Fourier Transform InfraRed (FT-IR) spectroscopy data (Supplementary Fig. ). The molecular weight of I-5 ranged from approximately 14 kDa to 18 kDa The viscosity of I-5 hydrogel was measured at a wide range of temperatures (Fig. ). The viscosity rose abruptly when the temperature reached 30 °C, causing a phase transition from sol to gel state. The viscosity at body temperature was ∼600 Pa·s, which indicates th..."
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"name": "I-5 hydrogel eliminates cystic cavities after contusive SCI",
"text": "A previous study showed that cystic cavities start to form as early as 1 week after contusion injury and progressively enlarge up to the 4-week time point. At 1 week after injury, we also observed quite large cystic cavities already formed at the epicenter (Supplementary Fig. ). At this time point, non-cystic lesions were filled with ED-1 positive macrophages surrounded by glial fibrillary acidic protein (GFAP) positive astrocytes (Supplementary Fig. ). We chose to examine the presence of cystic cavities 4 weeks after injection because a previous study reported that there is no further expansion of the cystic cavities after the 4-week time point. At 5 weeks after injury (4 weeks after injection), cystic cavities became larger extending rostrocaudally more than 2.0 mm away from the epicenter in animals injected with control PBS (Fig. ). Surprisingly, I-5 injec..."
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"text": "IleOEt·HCl (23.81 g, 121.67 mmol) in dry tetrahydrofuran (THF) containing trimethylamine (TEA) was added slowly to poly(dichlorophosphazene) (10.00 g, 86.29 mmol) dissolved in dry THF. The reaction mixture was incubated at dry ice bath for 12 h and room temperature for 36 h. AEtOH (1.56 g, 25.50 mmol) and AMPEG750 (37.53 g, 50.04 mmol) were dissolved in dry THF including TEA and added to the mixture. The reaction mixture was stirred at room temperature for 24 h and then at 40-50 °C for 24 h. AMPEG750 (18.76 g, 25.02 mmol) in dried THF was added to the reaction mixture and stirred at room temperature for 24 h and then at 40-50 °C for 24 h. The reaction mixture was filtered; the filtrate was concentrated and poured into n-hexane to obtain a precipitate,..."
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"text": "Several structural changes may explain how neural function for locomotion was recovered by I-5 injection. We found that filling cavity spaces with fibrotic matrix resulted in preservation of motor neurons and myelination. In animals with control PBS injection, myelin immunoreactivity was severely reduced especially in the spared white matter surrounding cavity spaces. We speculate that fluid within the cystic spaces may contain elevated levels of toxic substances that can contribute to secondary degeneration of motor neurons and/or demyelination of the white matter. Filling cystic spaces with fibrotic matrix may minimize exposure to toxic materials in the cystic fluid, leading to preservation of myelin and motor neurons. Another possibility is that the formation of fibrotic matrix in the lesion center may increase the stability of post-injury spinal cord. Spinal cord tissue with a lar..."
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"text": "Step 3: [NP(IleOEt) 1.45 (Succinic acid) 0.13 (Imi) 0.01 (AMPEG750) 0.41 ] n (I-5). The carboxylic acid terminated polymer II (10 g, 16.55 mmol) was dissolved in dried THF. After cooling to 0 °C, diisopropylcarbodiimide (DIC; 3.58 ml, 23.17 mmol) and N -hydroxysuccinimide (NHS; 2.67 g, 23.16 mmol) were added and the mixture was stirred for 30 min in order to activate the carboxyl groups of the polymer. The mixture was moved to 1-3-aminopropylimidazole (1.45 g, 11.54 mmol) dissolved in distilled THF. The reaction mixture was stirred at 0 °C for 6 h and then at room temperature for 18 h. Afterwards, the solution was concentrated and purified by precipitation with 1 M potassium fluoride solution. The precipitate was dialyzed with a dialysis membrane (Spectra/Por, Spectrum Laboratories, mol..."
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"text": "All animal protocols were approved by the Institutional Animal Care and Use Committee of Ajou University School of Medicine. Adult female Sprague Dawley rats (250-300 g) were used in this study. Spinal contusion injury was inflicted using the Infinite Horizon impactor (Precision Systems and Instrumentation). Animals were anesthetized with 4% chloral hydrate (10 ml/kg, injected intraperitoneally), followed by a dorsal laminectomy at the 10th thoracic vertebral level (T10-11) to expose the dorsal surface of the spinal cord. Then, standardized contusion at a force of 200 kdyn was performed with the Infinite Horizon impactor. After the injury, muscles were sutured in layer and the skin was stapled. Bladder care was provided twice daily until spontaneous voiding resumed. I-5 injection was performed 1 week after contusion injury to avoid potential tissue damage relat..."
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"identifier": "10.1038/s41467-017-00583-8"
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