Electric-eel-imitating hydrogel for peripheral nerve repair and preparation method of electric-eel-imitating hydrogel
By using highly biocompatible imitation eel hydrogels, the bioelectric signals of electric eels are simulated, and the surgical complexity, equipment dependence and biocompatibility challenges of traditional neurorepair materials are solved, achieving neural regeneration without external equipment.
Patent Information
- Application Number
- CN202510370313.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-27
- Publication Date
- 2025-06-27
AI Technical Summary
Traditional implantable electrically stimulating nerve repair materials have surgical complexity, device dependence and biocompatibility challenges, making it difficult to effectively promote the regeneration of peripheral nerves.
Using a highly biocompatible imitation electric eel hydrogel, the permeability imitation of salt ions and anions are driven through the gel through the inter-salt ion permeation imbalance, simulate the bioelectric signal of the electric eel and promote nerve regeneration.
The electrical signal control is achieved without external equipment, reducing surgical complexity and equipment dependence, and improving biocompatibility and neural regeneration effects.
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Figure CN120204468A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biomedical materials, and particularly relates to an electric eel - mimicking hydrogel for peripheral nerve repair and a preparation method thereof. Background Art
[0002] Peripheral nerve injury is one of the common clinical diseases, seriously affecting the quality of life of patients. Traditional nerve repair methods such as nerve transplantation and the use of nerve - guiding materials still face many challenges, such as unsatisfactory repair effects and difficult regeneration.
[0003] Implantable electrical - stimulation nerve - repair materials usually involve complex surgical operations and delicate implantation techniques. The surgical process is complex, requiring high professional skills and experience of doctors, which increases the risk and difficulty of the surgery. Current materials usually need to be used in conjunction with external devices (such as power sources, controllers, etc.) to achieve the electrical - stimulation function. This device - dependence may cause certain restrictions on patients in daily life and requires regular maintenance and replacement of the devices. Although modern materials science has made significant progress, implantable electrical - stimulation materials still face certain challenges in biocompatibility. The materials may cause adverse reactions such as tissue reactions, infections, or rejections, affecting the health status of patients.
[0004] Bionic hydrogels have become a research hotspot for nerve - repair materials due to their excellent biocompatibility and ability to simulate the in - vivo environment. Electric eels have unique electrophysiological characteristics, and their bio - electrical signals can stimulate the biological reactions of surrounding tissues. Therefore, the application of electric - eel - mimicking hydrogels in nerve repair has important potential. Summary of the Invention
[0005] The present invention provides an electric eel - mimicking hydrogel for peripheral nerve repair and a preparation method thereof. It is prepared from a gel material with high biocompatibility. By driving cations and anions to pass through the gel through the osmotic imbalance between salt ions to generate voltage, it mimics the characteristics of electric eels and achieves the effect of promoting nerve regeneration.
[0006] The solution of the present invention to the above - mentioned technical problems is as follows: An electric eel - mimicking hydrogel for peripheral nerve repair includes a plurality of high - concentration salt - ion hydrogels arranged at intervals, and an intermediate gel unit is connected in series between any two adjacent high - concentration salt - ion hydrogels. The intermediate gel unit includes an anion - selective hydrogel, a low - concentration salt - ion hydrogel, and a cation - selective hydrogel connected in series along the arrangement direction.
[0007] Preferably, the high-concentration salt ion hydrogel, anion-selective hydrogel, low-concentration salt ion hydrogel, and cation-selective hydrogel are all cylindrical, with a diameter of 1.3 to 1.8 mm and a height of 2 to 6 mm. The electrocyte-mimicking hydrogel has a certain effect on nerve growth. If it is too large, it is likely to cause insufficient sealing of the local microenvironment, and other tissues will grow in. If it is too small, it will hinder nerve growth.
[0008] Preferably, the raw materials of the high-concentration salt ion hydrogel include water-soluble inorganic salts and a gel substrate solution, and the mass-volume ratio of the water-soluble inorganic salts to the gel substrate solution is 0.02 to 0.12 g:1 mL;
[0009] The raw materials of the low-concentration salt ion hydrogel include water-soluble inorganic salts and a gel substrate solution, and the mass-volume ratio of the water-soluble inorganic salts to the gel substrate solution is 0.0004 to 0.005 g:1 mL;
[0010] The raw materials of the cation-selective hydrogel include a cation substrate and a gel substrate solution, and the mass-volume ratio of the cation substrate to the gel substrate solution is 0.004 to 0.02 g:1 mL. Preferably, the raw materials of the gel substrate solution include lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, a gel substrate material, and water; the weight ratio of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, the gel substrate material, and water is 2.5:1 to 15:500;
[0011] The water-soluble inorganic salt is one or any combination of LiCl, MgCl2, SrCl2, ZnCl2, KCl, NaCl, Ca(NO3)2;
[0012] The cation substrate is one of 3-sulfopropyl ester and double-bonded chondroitin sulfate;
[0013] The gel substrate material is one or any combination of double-bonded chitosan, double-bonded hydroxyethyl cellulose, acrylamide, and polyvinyl alcohol 1799.
[0014] Lithium phenyl(2,4,6-trimethylbenzoyl)phosphate is used as a photoinitiator.
[0015] Preferably, the raw materials of the anion-selective hydrogel include lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, quaternized double-bonded chitosan, and water; the weight ratio of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, quaternized double-bonded chitosan, and water is 2.5:1 to 15:500.
[0016] Preferably, the double-bonded chitosan is prepared by dissolving chitosan in deionized water, reacting with methylacrylic anhydride, and then undergoing purification and freeze-drying, as follows:
[0017] Dissolve 2 g of chitosan in 200 mL of deionized water, stir to dissolve at room temperature, pipette 1 mL of methacrylic anhydride, and slowly add it dropwise to the chitosan aqueous solution within 1 h under the action of a peristaltic pump. Continue stirring and reacting for 8 h. After the reaction is completed, dialyze for 3 days and freeze-dry for 24 h to obtain double-bonded chitosan.
[0018] The double-bonded hydroxyethyl cellulose is prepared by dissolving hydroxyethyl cellulose in deionized water, reacting with methacrylic anhydride, and then undergoing purification and freeze-drying, specifically as follows:
[0019] Dissolve 2 g of hydroxyethyl cellulose in 200 mL of deionized water, stir to dissolve at room temperature, pipette 1 mL of methacrylic anhydride, and adjust the pH to 8.0. Slowly add it dropwise to the hydroxyethyl cellulose aqueous solution within 1 h under the action of a peristaltic pump. Continue stirring and reacting for 8 h. After the reaction is completed, dialyze for 3 days and freeze-dry for 24 h to obtain the product, double-bonded hydroxyethyl cellulose.
[0020] The quaternized double-bonded chitosan is prepared by dissolving chitosan and glacial acetic acid in water, reacting with an aqueous solution of 2,3-epoxypropyltrimethylammonium chloride, undergoing purification and freeze-drying to obtain quaternized chitosan. The quaternized chitosan is dissolved in deionized water and reacted with methacrylic anhydride, and then undergoes purification and freeze-drying, specifically as follows:
[0021] Weigh 5 g of chitosan and 1 ml of glacial acetic acid, dissolve them in 450 mL of water, place them in a constant temperature water bath at 55 °C, and stir for 1 h to completely dissolve. Then weigh 4.7 g of 2,3-epoxypropyltrimethylammonium chloride, dissolve it in 50 mL of water to form a solution, and use a peristaltic pump to slowly add the aqueous solution of 2,3-epoxypropyltrimethylammonium chloride dropwise to the chitosan acetic acid aqueous solution within 30 min. The mixed solution continues to stir and react at 55 °C for 18 h, then stop the reaction. Dialyze the obtained solution for 3 days to remove impurities, and freeze-dry for 24 h to obtain quaternized chitosan.
[0022] Weigh 2 g of quaternized chitosan, add it to 200 mL of deionized water, stir to dissolve at room temperature, pipette 1 mL of methacrylic anhydride, and slowly add it dropwise to the quaternized chitosan aqueous solution within 1 h under the action of a peristaltic pump. Continue stirring and reacting for 8 h. After the reaction is completed, dialyze for 3 days and freeze-dry for 24 h to obtain quaternized double-bonded chitosan.
[0023] Preferably, the mass ratio of the inorganic salt in the high-concentration salt ionic hydrogel to that in the low-concentration salt ionic hydrogel is 5 - 200:1.
[0024] The preparation method of the electric eel-like hydrogel for peripheral nerve repair as described above is characterized by including the following steps;
[0025] 1) The gel base material is dissolved in deionized water, and lithium phenyl(2,4,6-trimethylbenzoyl)phosphate is added to obtain a base gel solution.
[0026] 2) The cationic substrate is dissolved in the base gel solution to obtain a cation-selective hydrogel solution.
[0027] 3) Quaternized double-bonded chitosan is dissolved in deionized water, and lithium phenyl(2,4,6-trimethylbenzoyl)phosphate is added to obtain an anion-selective hydrogel solution.
[0028] 4) Water-soluble inorganic salts are dissolved in the base gel solution to prepare high-concentration salt ion hydrogel solutions and low-concentration salt ion hydrogel solutions respectively.
[0029] 5) When there is 1 group of intermediate gel units, the gel solutions prepared in steps 2) - 4) are correspondingly poured into the mold in the order of high-concentration salt ion hydrogel, anion-selective hydrogel, low-concentration salt ion hydrogel, cation-selective hydrogel, and high-concentration salt ion hydrogel. Before pouring each gel solution, the upper layer of gel solution is cured by ultraviolet light and then poured until the curing of the topmost high-concentration salt ion hydrogel is completed, thus obtaining an electric eel-like hydrogel with 1 group of intermediate gel units.
[0030] Or, when there are N groups of intermediate gel units, with the preset number of intermediate gel units, the gel solutions prepared in steps 2) - 4) are correspondingly poured into the mold in the order of high-concentration salt ion hydrogel, anion-selective hydrogel, low-concentration salt ion hydrogel, cation-selective hydrogel. Before pouring each gel solution, the upper layer of gel solution is cured by ultraviolet light and then poured; subsequent N - 1 groups of intermediate gel units are cured repeatedly according to the above order of high-concentration salt ion hydrogel, anion-selective hydrogel, low-concentration salt ion hydrogel, cation-selective hydrogel, and finally the topmost high-concentration salt ion hydrogel solution is poured and cured, thus obtaining an electric eel-like hydrogel with N groups of intermediate gel units.
[0031] Preferably, in step 1), the concentration of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate in the base gel solution is 0.005 g / mL; the concentration of the gel base material in the base gel solution is 0.002 - 0.03 g / mL; in step 2), the concentration of the cationic substrate in the cation-selective hydrogel solution is 0.004 - 0.02 g / mL. In step 3), the concentration of quaternized double-bonded chitosan in the anion-selective hydrogel solution is 0.005 - 0.03 g / mL; in step 4), the concentration of the inorganic salt in the high-concentration salt ion hydrogel solution is 0.02 - 0.12 g / mL, and the concentration of the inorganic salt in the low-concentration salt ion hydrogel solution is 0.0004 - 0.005 g / mL.
[0032] Use of the electric eel - mimicking hydrogel for peripheral nerve repair in the preparation of a nerve repair bionic scaffold as described above.
[0033] The present invention also provides a nerve repair bionic scaffold, which includes the electric eel - mimicking hydrogel for peripheral nerve repair as described above. A hole for accommodating a polycaprolactone catheter is formed on one side of the electric eel - mimicking hydrogel; one end of the polycaprolactone catheter is inserted into the electric eel - mimicking hydrogel, and the other end is used to connect to nerve tissue. The surface of the electric eel - mimicking hydrogel is coated with a polycaprolactone electrospun membrane.
[0034] The beneficial effects of the present invention are as follows: The electric eel - mimicking hydrogel prepared by the present invention is assembled from pure hydrogel, has good biocompatibility, and can avoid problems of mechanical mismatch and poor biocompatibility to the greatest extent. The purpose of electrical stimulation is achieved by separating positive and negative ions, without the need for external equipment to control electrical signals. At the same time, when implanted into the body, it does not require complicated operations and can be achieved only through simple minimally invasive suturing.
[0035] The above description is only an overview of the technical solution of the present invention. In order to be able to understand the technical means of the present invention more clearly and implement it in accordance with the content of the specification, the following takes the preferred embodiments of the present invention and combines with the attached drawings to elaborate in detail as follows. The specific implementation manners of the present invention are given in detail by the following embodiments and their attached drawings. Brief Description of the Drawings
[0036] The attached drawings described herein are used to provide a further understanding of the present invention, form a part of this application, and the schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:
[0037] Figure 1 It is a working principle diagram of an electric eel - mimicking hydrogel for peripheral nerve repair provided by the present invention;
[0038] Figure 2 It is a schematic diagram of an electric eel - mimicking hydrogel for peripheral nerve repair with a single middle gel unit in Example 1;
[0039] Figure 3 It is a schematic diagram of an electric eel - mimicking hydrogel for peripheral nerve repair with multiple middle gel units in Examples 2 - 5;
[0040] Figure 4 It is an open - circuit voltage and short - circuit current diagram of the electric eel - mimicking hydrogel prepared in Examples 1 - 5;
[0041] Figure 5 It is an open - circuit voltage diagram of the electric eel - mimicking hydrogel scaffold prepared in Examples 6 - 11;
[0042] Figure 6Cation migration rate diagram of the electrocyte-mimicking hydrogel scaffolds prepared in Examples 12 to 16;
[0043] Figure 7 Anion migration rate diagram of the electrocyte-mimicking hydrogel scaffolds prepared in Examples 16 to 20;
[0044] Figure 8 Test diagram of the effect of the gel substrate material concentration on the mechanical properties of the hydrogel in Comparative Example 1;
[0045] Figure 9 Linear equation of the conductivity and conductivity diagram at different inorganic salt concentrations in the high-concentration salt ion hydrogel in Comparative Example 2;
[0046] Figure 10 Schematic diagram of the nerve repair bionic scaffold provided in Example 21. Detailed implementation manners
[0047] The principles and features of the present invention will be described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention.
[0048] Working principle of the present invention: As Figure 1 shown, the osmotic imbalance between the high-concentration salt ion hydrogel and the low-concentration salt ion hydrogel drives cations and anions to diffuse through the gel boundary from the high-concentration salt ion hydrogel to the low-concentration salt ion hydrogel, and then the positive and negative suspended charges of the membrane selectively transfer anions and cations through the hopping mechanism, resulting in electrode polarization and generating a voltage in the external circuit. At the same time, the magnitude of the voltage can be adjusted as needed by connecting different repeating units in series.
[0049] Example 1
[0050] This example provides a preparation method of an electrocyte-mimicking hydrogel for peripheral nerve repair, including the following steps:
[0051] 1) Take 2 g of chitosan and dissolve it in 200 mL (i.e., 200 g) of deionized water, stir and dissolve at room temperature. Use a pipette to suck 1 mL of methacrylic anhydride and slowly drip it into the chitosan aqueous solution within 1 h under the action of a peristaltic pump, and continue to stir and react for 8 h. After the reaction, dialyze for 3 days and freeze-dry for 24 h to obtain double-bonded chitosan.
[0052] Weigh 1 g of double-bonded chitosan and dissolve it in 50 mL of deionized water, place it in a constant temperature water bath at 55 °C and stir until dissolved, and add 0.25 g of phenyl(2,4,6-trimethylbenzoyl)phosphate lithium salt (photoinitiator) to obtain a substrate gel solution.
[0053] 2) Weigh 2g of chondroitin sulfate and add it to 200mL of deionized water, stir and dissolve at room temperature, use a pipette to draw 1mL of methacrylic anhydride, and adjust the pH to 8.0. Then slowly add it dropwise to the chondroitin sulfate aqueous solution within 1 hour under the action of a peristaltic pump, continue stirring and reacting for 8 hours, dialyze for 3 days after the reaction is completed, and freeze-dry for 24 hours to obtain the product double-bonded chondroitin sulfate.
[0054] Weigh 0.2 g of double-bonded chondroitin sulfate and dissolve it in 10 mL of base gel solution. Stir evenly at room temperature to prepare a cationic selective hydrogel base solution.
[0055] 3) Weigh 5g chitosan and 1ml glacial acetic acid and dissolve them in 450mL water, place in a constant temperature water bath at 55°C and stir for 1h to completely dissolve. Then weigh 4.7g 2,3-epoxypropyltrimethylammonium chloride and dissolve in 50mL water to prepare a solution. Use a peristaltic pump to slowly drop the 2,3-epoxypropyltrimethylammonium chloride aqueous solution into the chitosan acetic acid aqueous solution within 30min. Continue stirring the mixed solution at 55°C for 18h before stopping the reaction. The resulting solution is dialyzed for 3 days to remove impurities and freeze-dried for 24h to obtain quaternary ammonium salt chitosan.
[0056] Weigh 2 g of quaternized chitosan and add it to 200 mL of deionized water. Stir and dissolve it at room temperature. Use a pipette to draw 1 mL of methacrylic anhydride and slowly add it dropwise to the ammonium-salted chitosan aqueous solution within 1 hour under the action of a peristaltic pump. Continue stirring to react for 8 hours. After the reaction is completed, dialyze for 3 days and freeze-dry for 24 hours to obtain the quaternized double-bonded chitosan.
[0057] 1 g of quaternized double-bonded chitosan was weighed and dissolved in 50 mL of deionized water, stirred at room temperature until dissolved, and 0.25 g of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate was added to prepare an anion selective hydrogel solution.
[0058] 4) Weigh 0.5 g and 0.005 g of LiCl respectively, and dissolve them in 10 mL of the base solution to prepare high-concentration salt ion hydrogel and low-concentration salt ion hydrogel solutions.
[0059] 5) If Figure 2 As shown, a transparent mold with a diameter of 1.3-1.8 mm is selected to inject solutions in the order of high-concentration salt ion hydrogel solution-anion selective hydrogel solution-low-concentration salt ion hydrogel solution-cation selective hydrogel solution-high-concentration salt ion hydrogel solution, and each layer of solution has the same height. Before pouring each gel solution, the previous layer of gel solution is cured by ultraviolet light for 20 seconds and then poured in again until the top layer of high-concentration salt ion hydrogel is cured. The electric eel-like hydrogel with a height of 2-6 mm for each layer of gel and 1 intermediate gel unit is obtained.
[0060] Example 2
[0061] As Figure 3 shown, this example provides a preparation method of an electric eel - mimicking hydrogel for peripheral nerve repair. Its preparation is basically the same as that of Example 1, except for step 5). After injecting the solutions into a transparent mold in the order of high - concentration salt - ion hydrogel solution - anion - selective hydrogel solution - low - concentration salt - ion hydrogel solution - cation - selective hydrogel solution and repeating this 2 times, then inject the high - concentration salt - ion hydrogel solution. The height of each layer of solution is the same. Before pouring each gel solution, cure the upper - layer gel solution with ultraviolet light for 20 s and then pour. The height of each layer of gel obtained is 2 - 6 mm, and an electric eel - mimicking hydrogel with 2 intermediate gel units is obtained.
[0062] Example 3
[0063] This example provides a preparation method of an electric eel - mimicking hydrogel for peripheral nerve repair. Its preparation is basically the same as that of Example 1, except for step 5). After injecting the solutions into a transparent mold in the order of high - concentration salt - ion hydrogel solution - anion - selective hydrogel solution - low - concentration salt - ion hydrogel solution - cation - selective hydrogel solution and repeating this 3 times, then inject the high - concentration salt - ion hydrogel solution. The height of each layer of solution is the same. Before pouring each gel solution, cure the upper - layer gel solution with ultraviolet light for 20 s and then pour. The height of each layer of gel obtained is 2 - 6 mm, and an electric eel - mimicking hydrogel with 3 intermediate gel units is obtained.
[0064] Example 4
[0065] This example provides a preparation method of an electric eel - mimicking hydrogel for peripheral nerve repair. Its preparation is basically the same as that of Example 1, except for step 5). After injecting the solutions into a transparent mold in the order of high - concentration salt - ion hydrogel solution - anion - selective hydrogel solution - low - concentration salt - ion hydrogel solution - cation - selective hydrogel solution and repeating this 4 times, then inject the high - concentration salt - ion hydrogel solution. The height of each layer of solution is the same. Before pouring each gel solution, cure the upper - layer gel solution with ultraviolet light for 20 s and then pour. The height of each layer of gel obtained is 2 - 6 mm, and an electric eel - mimicking hydrogel with 4 intermediate gel units is obtained.
[0066] Example 5
[0067] This example provides a method for preparing an electric eel - like hydrogel for peripheral nerve repair. Its preparation is basically the same as that of Example 1, except for step 5). After repeating the injection of solutions in the order of high - concentration salt - ion hydrogel solution - anion - selective hydrogel solution - low - concentration salt - ion hydrogel solution - cation - selective hydrogel solution 5 times in a transparent mold, then inject the high - concentration salt - ion hydrogel solution, and the height of each layer of solution is the same. Before pouring each gel solution, cure the upper - layer gel solution with ultraviolet light for 20 s and then pour. The height of each layer of gel obtained is 2 - 6 mm, and an electric eel - like hydrogel with 5 intermediate gel units is obtained.
[0068] Table 1 Number of repeating units in Examples 1 - 5
[0069] Number of intermediate gel units Example 1 1 Example 2 2 Example 3 3 Example 4 4 Example 5 5
[0070] The electric eel - like hydrogels with different numbers of gel units prepared in Examples 1 - 5 were clamped on an electrometer with Ag / AgCl electrodes (Keithley 2636b, Tektronix, USA), and the open - circuit voltage and short - circuit current were measured. The results are as Figure 4 shown. As the number of repeating units increases, the open - circuit voltage and short - circuit current of the electric eel - like hydrogel also increase.
[0071] Example 6
[0072] This example provides a method for preparing an electric eel - like hydrogel for peripheral nerve repair. Its preparation is basically the same as that of Example 1, except for step 4). Weigh 0.5 g and 0.005 g of MgCl2 respectively, dissolve them in 10 mL of the base solution, and prepare a high - concentration salt - ion hydrogel solution and a low - concentration salt - ion hydrogel solution.
[0073] Example 7
[0074] This example provides a method for preparing an electric eel - like hydrogel for peripheral nerve repair. Its preparation is basically the same as that of Example 1, except for step 4). Weigh 0.5 g and 0.005 g of SrCl2 respectively, dissolve them in 10 mL of the base solution, and prepare a high - concentration salt - ion hydrogel and a low - concentration salt - ion hydrogel solution.
[0075] Example 8
[0076] This example provides a method for preparing an electric eel - like hydrogel for peripheral nerve repair. Its preparation is basically the same as that of Example 1, except for step 4). Weigh 0.5 g and 0.005 g of ZnCl2 respectively, dissolve them in 10 mL of the base solution, and prepare a high - concentration salt - ion hydrogel solution and a low - concentration salt - ion hydrogel solution.
[0077] Example 9
[0078] This example provides a method for preparing an electric eel - mimicking hydrogel for peripheral nerve repair. Its preparation is basically the same as that of Example 1, except for step 4). Weigh 0.5 g and 0.005 g of KCl respectively, dissolve them in 10 mL of the base solution to prepare a high - concentration salt - ion hydrogel solution and a low - concentration salt - ion hydrogel solution.
[0079] Example 10
[0080] This example provides a method for preparing an electric eel - mimicking hydrogel for peripheral nerve repair. Its preparation is basically the same as that of Example 1, except for step 4). Weigh 0.5 g and 0.005 g of NaCl respectively, dissolve them in 10 mL of the base solution to prepare a high - concentration salt - ion hydrogel solution and a low - concentration salt - ion hydrogel solution.
[0081] Example 11
[0082] This example provides a method for preparing an electric eel - mimicking hydrogel for peripheral nerve repair. Its preparation is basically the same as that of Example 1, except for step 4). Weigh 0.5 g and 0.005 g of Ca(NO3)2 respectively, dissolve them in 10 mL of the base solution to prepare a high - concentration salt - ion hydrogel solution and a low - concentration salt - ion hydrogel solution.
[0083] Table 2 Inorganic salt types in high - concentration salt - ion hydrogels and low - concentration salt - ion hydrogels in Examples 6 - 11
[0084]
[0085]
[0086] The open - circuit voltages of the electric eel - mimicking hydrogels with different salt - ion types prepared in Examples 1, 6 - 11 were tested. The results are as Figure 5 shown. The open - circuit voltage of the electric eel - mimicking hydrogel prepared with MgCl2 as the inorganic salt is the largest.
[0087] Example 12
[0088] This example provides a method for preparing an electric eel - mimicking hydrogel for peripheral nerve repair. The preparation steps are as follows:
[0089] 1) Take 2 g of hydroxyethyl cellulose and dissolve it in 200 mL of deionized water, stir and dissolve at room temperature. Use a pipette to suck 1 mL of methacrylic anhydride and adjust the pH to 8.0. Under the action of a peristaltic pump, slowly drip it into the hydroxyethyl cellulose aqueous solution within 1 h, continue stirring and reacting for 8 h. After the reaction, dialyze for 3 days and freeze - dry for 24 h to obtain the product, double - bond - modified hydroxyethyl cellulose.
[0090] Weigh 1 g of double-bonded hydroxyethyl cellulose and dissolve it in 50 mL of deionized water. Place it in a constant temperature water bath at 50 °C and stir until dissolved. Then add 0.25 g of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate to obtain a base gel solution.
[0091] 2) Weigh 2 g of chondroitin sulfate and dissolve it in 200 mL of deionized water. Stir to dissolve at room temperature. Use a pipette to aspirate 1 mL of methacrylic anhydride and adjust the pH to 8.0. Then, under the action of a peristaltic pump, slowly drip it into the chondroitin sulfate aqueous solution within 1 h, and continue to stir and react for 8 h. After the reaction, dialyze for 3 days and freeze-dry for 24 h to obtain double-bonded chondroitin sulfate.
[0092] Weigh 0.04 g of double-bonded chondroitin sulfate and dissolve it in 10 mL of the base gel solution. Stir evenly at room temperature to prepare a cation-selective hydrogel base solution.
[0093] 3) Weigh 5 g of chitosan and 1 mL of glacial acetic acid and dissolve them in 450 mL of water. Place it in a constant temperature water bath at 55 °C and stir for 1 h until completely dissolved. Then weigh 4.7 g of 2,3-epoxypropyltrimethylammonium chloride and dissolve it in 50 mL of water to form a solution. Use a peristaltic pump to slowly drip the 2,3-epoxypropyltrimethylammonium chloride aqueous solution into the chitosan acetic acid aqueous solution within 30 min. The mixed solution continues to stir and react at 55 °C for 18 h, then stop the reaction. Dialyze the obtained solution for 3 days to remove impurities and freeze-dry for 24 h to obtain quaternized chitosan.
[0094] Weigh 2 g of quaternized chitosan and dissolve it in 200 mL of deionized water. Stir to dissolve at room temperature. Use a pipette to aspirate 1 mL of methacrylic anhydride and slowly drip it into the quaternized chitosan aqueous solution within 1 h under the action of a peristaltic pump. Continue to stir and react for 8 h. After the reaction, dialyze for 3 days and freeze-dry for 24 h to obtain quaternized double-bonded chitosan.
[0095] Weigh 1 g of quaternized double-bonded chitosan and dissolve it in 50 mL of deionized water. Stir until dissolved at room temperature, and add 0.25 g of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate to prepare an anion-selective hydrogel solution.
[0096] 4) Weigh 0.5 g and 0.005 g of LiCl respectively, and dissolve them in 10 mL of the base solution to prepare a high-concentration salt ion hydrogel and a low-concentration salt ion hydrogel solution.
[0097] 5) Inject the solutions in sequence of high-concentration salt ion hydrogel solution - anion-selective hydrogel solution - low-concentration salt ion hydrogel solution - cation-selective hydrogel solution - high-concentration salt ion hydrogel solution into a transparent mold with a diameter of 1.3 - 1.5 mm. The height of each layer of solution is the same. Before pouring each gel solution, cure the upper layer of gel solution with ultraviolet light for 20 s and then pour it until the curing of the high-concentration salt ion hydrogel in the uppermost layer is completed. An electric eel-like hydrogel with a height of 2 - 6 mm for each layer of gel and having 1 intermediate gel unit is obtained.
[0098] Example 13
[0099] This example provides a preparation method of an electric eel-like hydrogel for peripheral nerve repair. The difference lies in step 2). Weigh 0.08 g of double-bonded chondroitin sulfate and dissolve it in 10 mL of base gel solution, stir evenly at room temperature, and prepare a cation-selective hydrogel base solution.
[0100] Example 14
[0101] This example provides a preparation method of an electric eel-like hydrogel for peripheral nerve repair. Its preparation is basically the same as that of Example 1. The difference lies in step 2). Weigh 0.12 g of double-bonded chondroitin sulfate and dissolve it in 10 mL of base gel solution, stir evenly at room temperature, and prepare a cation-selective hydrogel base solution.
[0102] Example 15
[0103] This example provides a preparation method of an electric eel-like hydrogel for peripheral nerve repair. Its preparation is basically the same as that of Example 1. The difference lies in step 2). Weigh 0.16 g of double-bonded chondroitin sulfate and dissolve it in 10 mL of base gel solution, stir evenly at room temperature, and prepare a cation-selective hydrogel base solution.
[0104] Example 16
[0105] This example provides a preparation method of an electric eel-like hydrogel for peripheral nerve repair. Its preparation is basically the same as that of Example 1. The difference lies in step 2). Weigh 0.2 g of double-bonded chondroitin sulfate and dissolve it in 10 mL of base gel solution, stir evenly at room temperature, and prepare a cation-selective hydrogel base solution.
[0106] Table 3 Concentrations of double-bonded hydroxyethyl cellulose in the cation-selective hydrogels in Examples 12 - 16
[0107]
[0108] The cation migration rates of the electrocyte-mimicking hydrogels with different mass ratios of double-bonded hydroxyethyl cellulose and double-bonded chondroitin sulfate prepared in Examples 12 - 16 were tested. First, 1 mL of 1 M CuCl2 solution was dropped onto cation-selective hydrogels with different ratios. Below it, there was a low-salt gel of the same volume connected. After 5 h, the low-salt gel was ground and broken, soaked in deionized water for 1 h, and then the leachate was collected and the Cu 2+ concentration was detected by ICP-MS. The results are as Figure 6 shown. As the mass ratio of double-bonded chondroitin sulfate increases, the cation migration rate of the electrocyte-mimicking hydrogel increases.
[0109] Example 17
[0110] This example provides a method for preparing an electrocyte-mimicking hydrogel for peripheral nerve repair. The preparation steps are basically the same as those in Example 16, except for step 3). Weigh 0.25 g of quaternized double-bonded chitosan and dissolve it in 50 mL of deionized water. Stir at room temperature until dissolved, and then add 0.25 g of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate to prepare an anion-selective hydrogel solution.
[0111] Example 18
[0112] This example provides a method for preparing an electrocyte-mimicking hydrogel for peripheral nerve repair. The preparation is basically the same as that in Example 16, except for step 3). Weigh 0.5 g of quaternized double-bonded chitosan and dissolve it in 50 mL of deionized water. Stir at room temperature until dissolved, and then add 0.25 g of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate to prepare an anion-selective hydrogel solution.
[0113] Example 19
[0114] This example provides a method for preparing an electrocyte-mimicking hydrogel for peripheral nerve repair. The preparation is basically the same as that in Example 16, except for step 3). Weigh 0.75 g of quaternized double-bonded chitosan and dissolve it in 50 mL of deionized water. Stir at room temperature until dissolved, and then add 0.25 g of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate to prepare an anion-selective hydrogel solution.
[0115] Example 20
[0116] This example provides a method for preparing an electrocyte-mimicking hydrogel for peripheral nerve repair. The preparation is basically the same as that in Example 16, except for step 3). Weigh 1.5 g of quaternized double-bonded chitosan and dissolve it in 50 mL of deionized water. Stir at room temperature until dissolved, and then add 0.25 g of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate to prepare an anion-selective hydrogel solution.
[0117] Table 4 Quaternized double-bonded chitosan content in the anionic selective hydrogel in Examples 16 - 20
[0118]
[0119] The eel-mimicking hydrogels with different quaternized double-bonded chitosan contents prepared in Examples 16 - 20 were tested for anion migration rate. First, 1 mL of 1 M KMnO4 solution was dropped above the anionic selective hydrogels with different ratios. The lower part was connected to a low-salt gel of the same volume. After 5 h, the low-salt gel was ground and broken, soaked in deionized water for 1 h, and then the leachate was collected. The MnO - 4 concentration was detected by a high-performance ion chromatograph (HPIC). The results are as Figure 7 shown. As the quaternized double-bonded chitosan content increases, the anion migration rate of the eel-mimicking hydrogel decreases.
[0120] Comparative Example 1
[0121] In this comparative example, the effect of the concentration of the gel substrate material in the gel substrate solution on the mechanical properties of the hydrogel was explored as follows:
[0122] 2 g of hydroxyethyl cellulose was dissolved in 200 mL of deionized water and stirred at room temperature until dissolved. 1 mL of methacrylic anhydride was aspirated with a pipette, and the pH was adjusted to 8.0. Under the action of a peristaltic pump, it was slowly dropped into the hydroxyethyl cellulose aqueous solution within 1 h, and stirring was continued for 8 h. After the reaction, dialysis was carried out for 3 days, and freeze-drying was carried out for 24 h to obtain the product, double-bonded hydroxyethyl cellulose.
[0123] 0.1 g, 0.25 g, 0.5 g, 1.0 g, and 1.5 g of double-bonded hydroxyethyl cellulose were respectively dissolved in 50 mL of deionized water, placed in a constant temperature water bath at 55 °C and stirred until dissolved, and 0.25 g of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate was added to prepare gel substrate materials with concentrations of 0.2%, 0.5%, 1%, 2%, and 3% (unit: g / mL).
[0124] The prepared gel substrate solutions with different concentrations were respectively placed in cylindrical molds with a diameter of 10 mm and a height of 5 mm, and irradiated with 405 nm ultraviolet light for 20 s to obtain hydrogel samples with the same volume and shape. Compression performance tests (rate: 2 mm / min) were carried out by a universal testing machine (Instron MTS, USA). Every 5 parallel samples were taken as a group, and the calculation results were averaged. The results are as Figure 8 shown. As the double-bonded hydroxyethyl cellulose content increases, the mechanical strength of the eel-mimicking hydrogel increases.
[0125] Comparative Example 2
[0126] In this comparative example, the effect of adjusting the inorganic salt concentration in the high-concentration salt ion hydrogel on its electrical conductivity was investigated as follows:
[0127] Take 2 g of chitosan and dissolve it in 200 mL of deionized water. Stir to dissolve at room temperature. Use a pipette to aspirate 1 mL of methacrylic anhydride and slowly add it dropwise to the chitosan aqueous solution within 1 h under the action of a peristaltic pump. Continue stirring and reacting for 8 h. After the reaction, dialyze for 3 days and freeze-dry for 24 h to obtain double-bonded chitosan.
[0128] Weigh 1 g of double-bonded chitosan and dissolve it in 50 mL of deionized water. Place it in a constant temperature water bath at 55 °C and stir until dissolved, and add 0.25 g of phenyl(2,4,6-trimethylbenzoyl)phosphate lithium salt as the base gel solution.
[0129] Respectively take 0.21 g, 0.42 g, 0.63 g, 0.84 g, 1.05 g of LiCl and dissolve them in 10 mL of the base solution respectively to prepare high-concentration salt ion hydrogel solutions with inorganic salt concentrations of 0.5 M, 1 M, 1.5 M, 2 M, and 2.5 M.
[0130] The prepared high-concentration salt ion hydrogel solutions with different LiCl concentrations were molded in a square mold with a side length of 3 cm and a height of 0.5 cm, and then they were respectively placed in a four-probe detector (Haierpa Electronic Technology Co., Ltd., model HPS58002). Three groups were measured for each concentration, and three samples were measured for each group. The conductivity was measured, and the results are as Figure 9 shown. As the concentration of the inorganic salt LiCl in the high-concentration salt ion hydrogel increases, the conductivity increases accordingly.
[0131] Example 21
[0132] This example provides a nerve repair bionic scaffold, and its preparation steps are as follows:
[0133] As Figure 10 shown, use the electrocyte-like hydrogel prepared in Example 1 to dig out holes in the center along the arrangement direction of the electrocyte-like hydrogel large enough to accommodate a polycaprolactone catheter. Insert the polycaprolactone catheter into the hole, and the end of the polycaprolactone catheter protrudes relative to the surface of the electrocyte-like hydrogel. The protruding end is used to connect nerve tissue, and a polycaprolactone electrospun membrane is coated on the surface of the electrocyte-like hydrogel to obtain a nerve repair bionic scaffold.
[0134] By implanting the nerve repair bionic scaffold at the wound and monitoring the electrophysiological signals at the nerve injury site, the progress of nerve regeneration can be evaluated. The scaffold continuously releases electrical stimulation to promote the growth and regeneration of nerve cells, and its structural design also helps to guide the correct regeneration direction of nerve tissue.
[0135] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention; any ordinary technician in the industry can smoothly implement the present invention as shown in the accompanying drawings of the specification and as described above; however, any minor changes, modifications, and equivalent variations made by those skilled in the art within the scope of the technical solution of the present invention by using the technical content disclosed above are equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications, and evolutions made to the above embodiments based on the substantial technology of the present invention still fall within the protection scope of the technical solution of the present invention.
Claims
1. An electric eel-like hydrogel for peripheral nerve repair, characterized in that: The invention comprises a plurality of high-concentration salt ion hydrogels arranged at intervals, wherein an intermediate gel unit is connected in series between any two adjacent high-concentration salt ion hydrogels, and the intermediate gel unit comprises an anion selective hydrogel, a low-concentration salt ion hydrogel and a cation selective hydrogel connected in series along the arrangement direction.
2. According to claim 1, an electric eel-like hydrogel for peripheral nerve repair, the high-concentration salt ion hydrogel, anion-selective hydrogel, low-concentration salt ion hydrogel, and cation-selective hydrogel are all cylindrical, with diameters of 1.3 to 1.8 mm and heights of 2 to 6 mm.
3. The electric eel-like hydrogel for peripheral nerve repair according to claim 1, characterized in that: The raw materials of the high-concentration salt ion hydrogel include water-soluble inorganic salt and gel base solution, and the mass volume ratio of the water-soluble inorganic salt to the gel base solution is = 0.02-0.12g:1mL; The raw materials of the low-concentration salt ion hydrogel raw material include a water-soluble inorganic salt and a gel base solution, and the mass volume ratio of the water-soluble inorganic salt to the gel base solution is 0.0004-0.005g:1mL; The raw materials of the cationic selective hydrogel include a cationic substrate and a gel base solution, and the mass volume ratio of the cationic substrate to the gel base solution is 0.004-0.02g:1mL.
4. The electric eel-like hydrogel for peripheral nerve repair according to claim 1, characterized in that: The raw materials of the gel base solution include phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, gel base material and water; the weight ratio of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, gel base material and water is 2.5:1 to 15:500; The gel base material is one of double-bonded chitosan, double-bonded hydroxyethyl cellulose, acrylamide, polyvinyl alcohol 1799 or any combination thereof; The water-soluble inorganic salt is one or a combination of any of LiCl, MgCl2, SrCl2, ZnCl2, KCl, NaCl, Ca(NO3)2; The cationic substrate is one of 3-sulfopropyl ester and double-bonded chondroitin sulfate.
5. The electric eel-like hydrogel for peripheral nerve repair according to claim 3, characterized in that: The raw materials of the anion selective hydrogel include phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate, quaternized double-bonded chitosan and water; the weight ratio of phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate, quaternized double-bonded chitosan and water is 2.5:1-15:
500.
6. The electric eel-like hydrogel for peripheral nerve repair according to claim 3, characterized in that: The mass ratio of the inorganic salt in the high-concentration salt ion hydrogel to the low-concentration salt ion hydrogel is 5 to 200:
1.
7. A method for preparing the electric eel-like hydrogel for peripheral nerve repair according to any one of claims 1 to 6, characterized in that: The steps include: 1) dissolving the gel base material in deionized water and adding phenyl (2,4,6-trimethylbenzoyl) lithium phosphate to prepare a base gel solution; 2) the cationic substrate is dissolved in the base gel solution to prepare a cationic selective hydrogel solution; 3) dissolving the quaternized double-bonded chitosan in deionized water and adding phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate to prepare an anion selective hydrogel solution; 4) dissolving a water-soluble inorganic salt in the base gel solution to prepare a high-concentration salt ion hydrogel solution and a low-concentration salt ion hydrogel solution respectively; 5) When the intermediate gel unit is one group, the gel solutions prepared in steps 2) to 4) are poured into the mold in the order of high-concentration salt ion hydrogel, anion-selective hydrogel, low-concentration salt ion hydrogel, cation-selective hydrogel and high-concentration salt ion hydrogel, and each time the gel solution is poured in, the upper layer of gel solution is cured by ultraviolet light and then poured in again until the uppermost layer of high-concentration salt ion hydrogel is completely cured, thereby obtaining the electric eel-like hydrogel with one intermediate gel unit; Alternatively, when the intermediate gel units are N groups, the gel solutions prepared in steps 2) to 4) are poured into the mold according to the preset number of intermediate gel units in the order of high-concentration salt ion hydrogel, anion-selective hydrogel, low-concentration salt ion hydrogel, and cation-selective hydrogel. Before pouring the gel solution each time, the upper layer of gel solution is cured by ultraviolet light before pouring it again; subsequently, N-1 groups of intermediate gel units are repeatedly cured in the order of high-concentration salt ion hydrogel, anion-selective hydrogel, low-concentration salt ion hydrogel, and cation-selective hydrogel, and finally the uppermost layer of high-concentration salt ion hydrogel solution is poured and cured, so as to obtain the electric eel-like hydrogel with N groups of intermediate gel units.
8. The method for preparing the electric eel-like hydrogel for peripheral nerve repair according to claim 7, characterized in that: In the step 1), the concentration of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate in the base gel solution is 0.005 g / mL; the concentration of the gel base material in the base gel solution is 0.002-0.03 g / mL; In the step 2), the concentration of the cationic substrate in the cation selective hydrogel solution is 0.004-0.02 g / mL; In the step 3), the concentration of the quaternized double-bonded chitosan in the anion selective hydrogel solution is 0.005-0.03 g / mL; In the step 4), the concentration of the inorganic salt in the high-concentration salt ion hydrogel solution is 0.02-0.12 g / mL, and the concentration of the inorganic salt in the low-concentration salt ion hydrogel solution is 0.0004-0.005 g / mL.
9. Use of the electric eel-like hydrogel for peripheral nerve repair as described in any one of claims 1 to 6 in preparing a bionic scaffold for nerve repair.
10. A bionic scaffold for nerve repair, characterized in that: It comprises the electric eel-like hydrogel for peripheral nerve repair as described in any one of claims 1 to 6, wherein one side of the electric eel-like hydrogel is provided with a hole for accommodating a polycaprolactone catheter; one end of the polycaprolactone catheter is inserted into the electric eel-like hydrogel, and the other end is used to connect to nerve tissue, and the surface of the electric eel-like hydrogel is coated with a polycaprolactone electrospinning membrane.