CN114904050A

CN114904050A - Preparation method and application of hydrogel for simulating spinal cord

Info

Publication number: CN114904050A
Application number: CN202210769310.4A
Authority: CN
Inventors: 林权; 冯钰斌; 关琳; 杨欣婷
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2022-06-30
Filing date: 2022-06-30
Publication date: 2022-08-16

Abstract

The invention provides a preparation method and application of hydrogel for simulating spinal cord. The hydrogels of the present invention can be prepared rapidly at room temperature. The hydrogel has the characteristics of excellent injectability, good conductivity, certain adhesiveness, mechanical strength and the like, simulates the structure and biophysical characteristics of natural spinal cord tissues, and has the characteristics of similar pore structure, modulus, conductivity and the like with the spinal cord tissues. The conductive hydrogel can effectively promote the differentiation of the neural stem cells to neurons and the growth of axons by combining with electrical stimulation after loading the neural stem cells, and has application prospects in the aspects of promoting the repair of spinal cord injury, the recovery of motor functions and the like.

Description

Preparation method and application of hydrogel for simulating spinal cord

Technical Field

The invention relates to a preparation method of a hydrogel material for simulating natural spinal cord tissue and application of the hydrogel material in the aspect of spinal cord injury repair.

Background

Repair of spinal cord injury has been a major research point worldwide and has not made breakthrough progress for a long time. The introduction of neural stem cells at the site of injury is currently accepted as a spinal cord repair procedure. The neural stem cells which can differentiate into neurons, astrocytes and oligodendrocytes can participate in the repair of injuries at multiple levels. However, the inhibitory microenvironment inside the spinal cord is not conducive to proliferation and differentiation of transplanted neural stem cells, and the differentiation direction is unreasonably controlled, so that excessive differentiation of astrocytes which is not conducive to injury repair affects repair of spinal cord injury.

In recent years, hydrogels have received much attention in the field of biomaterials due to their versatility and excellent biocompatibility. The characteristics of adjustability and cell loading of hydrogel are utilized to simulate the biophysical characteristics of spinal cord tissues to provide a better proliferation and differentiation microenvironment for neural stem cells. But the neural stem cells are not beneficial to the differentiation in the microenvironment with over-soft or over-hard; the spinal cord tissue is more favorable for differentiation to neurons in an environment with proper hardness (0.1-1 kPa), and the modulus of the natural spinal cord tissue is 0.1-3 kPa. In addition, the bioelectrical signals in the spinal cord tissues can promote the proliferation and differentiation of endogenous and exogenous cells, and the hydrogel can imitate the high conductivity of the natural spinal cord, so that the hydrogel is very beneficial to tissue repair. Therefore, it is necessary to design an injectable conductive hydrogel simulating spinal modulus and conductance to promote the directional differentiation of neural stem cells to neurons.

The conductive hydrogel is combined with electrical stimulation to form a promising treatment mode, and can be used for promoting the directional differentiation of the neural stem cells to the neurons and promoting the growth of the axons. Solves the problems of uncontrollable differentiation and excessive differentiation to astrocytes of the conventional neural stem cells. Therefore, the injectable conductive hydrogel loaded neural stem cells simulating the modulus and the conductivity of the spinal cord tissue has great potential in the spinal cord injury repair combined with an electrical stimulation treatment method.

Disclosure of Invention

The invention aims to provide a preparation method of a hydrogel material for simulating natural spinal cord tissue and application of the hydrogel material in spinal cord injury repair.

The method comprises the steps of firstly preparing modified chitosan CHIC, then preparing HA-ALD, and then preparing MXene nanosheets by an etching method. MXene nanosheets are mixed into an HA-ALD solution to react with a CHIC solution doped with a certain amount of ADH according to a certain proportion, and the injectable conductive hydrogel simulating the tissue structure, the modulus and the conductivity of a spinal cord can be obtained.

1) Firstly, chitosan (CHIC) modified by catechol group is prepared. Dissolving 0.5-0.8 g of chitosan in deionized water, adjusting the pH value to 5.0-6.0 by HCl, and dispersing for 20-30 min by using ultrasonic oscillation to finally form a 1.0 wt% chitosan solution. And then, dissolving 255-455 mg of dihydrocaffeic acid HCA in 5.0-8.0 mL of deionized water, fully stirring and dissolving, and then adding into the chitosan liquid. Dissolving 240-300 mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide EDC and 150-200 mg of n-hydroxy thiosuccinimide NHS in 50.0mL of a mixed solution of ethanol and deionized water in a ratio of 1: 1-1: 2, and keeping the pH value within a range of 6-7. And slowly dripping the chitosan/HCA mixed solution into the mixture, reacting in an environment of 20-25 ℃, sealing and violently stirring for 10-12 h, keeping the pH value at 5.0-6.0, and then placing in an oscillator for fast shaking reaction for 10-15 min. Dialyzing the solution in acidified deionized water for 2-3 days, dialyzing in deionized water for half a day, and freeze-drying in a freeze-dryer to obtain a yellow solid, namely preparing the catechol group-modified chitosan CHIC.

2) Preparation of aldehyde radicalHyaluronic acid HA-ALD. Dissolving 2.0-3.0 g of hyaluronic acid HA in 200-300 mL of deionized water, and sealing and rapidly stirring for 24-28 h at 20-25 ℃ until the hyaluronic acid HA is completely dissolved. 1.0-1.5 g NaIO ₄ Adding the HA solution into the solution, stirring the solution for 4 to 7 hours in a dark place, adding 1.5 to 2.3mL of ethylene glycol, stirring the solution for 1 to 2 hours, and then carrying out ultrasonic oscillation reaction for 10 to 20 min. Putting the solution into a dialysis bag, dialyzing with deionized water for 3-4 days, and keeping the pH of the solution within a range of 7-8. And (3) putting the reactant into a freeze dryer for drying, and finally obtaining a white solid, namely the aldehyde hyaluronic acid HA-ALD.

3) Preparing MXene nano-sheets. Adding 1-4 g of LiF into 20-80 mL of hydrochloric acid, and stirring for 5-10 min. Then adding 1-4 g of Ti ₃ AlC ₂ Slowly adding the solution and stirring for 24-26 h at 35-40 ℃. Washing the acidic suspension with deionized water, and centrifuging at 3500-4500 rpm for 5-8 min until the pH value of the supernatant reaches 6-7. And then collecting the deposit, adding deionized water into the deposit, introducing Ar into the dispersion liquid for 10-20 min, and dispersing for 1-1.5 h by using ultrasonic oscillation while ensuring the temperature to be within 30 ℃. And finally, centrifuging at the rotating speed of 4500-5000 for 10-15 minutes, and freeze-drying at the temperature of-50 to-30 ℃ for 2-3 days to obtain black solid powder MXene nanosheets.

4) Preparing a 4-6 wt% solution of the CHIC solid by using deionized water, and adding 1.0-2.0 wt% of adipic dihydrazide ADH to prepare a light yellow solution A. And then adding the HA-ALD into deionized water to prepare a 4-6 wt% solution, adding 0.1-0.15% wtMXene nanosheet powder, and performing ultrasonic oscillation for 10-15 min to obtain a black solution B. And mixing the solution A and the solution B according to the volume ratio of 1: 2-3: 1, and quickly mixing and reacting for 10-60 seconds by using an oscillator. The injectable conductive hydrogel which simulates the structure, modulus and conductivity of spinal cord tissue can be obtained.

The invention has the following advantages: 1. the synthesized hydrogel has the advantages of good main raw material and biocompatibility, and is beneficial to commercialization of products; 2. the conductive hydrogel simulates the structure and biophysical characteristics of natural spinal cord tissue, has the characteristics of similar pore structure, modulus, conductivity and the like with the spinal cord tissue, and is beneficial to promoting the directional differentiation of neural stem cells; 4, the hydrogel has the characteristics of injectability, adhesiveness, conductivity, mechanical strength and the like, and can close irregular defects after spinal cord injury; 5. the hydrogel has a hemostatic function, and can effectively avoid inflammatory reaction caused by bleeding at the spinal cord injury part; 6. the conductive hydrogel can effectively promote the differentiation of the neural stem cells to neurons and the growth of axons by combining with electrical stimulation after loading the neural stem cells; 7. when the injectable conductive hydrogel loaded with the neural stem cells is injected into the spinal cord injury part and is assisted by electrical stimulation, the spinal cord injury repair and motor function recovery can be effectively promoted.

Drawings

FIG. 1: phase change diagram of hydrogel prepared for example 1. As can be seen from the reagent bottle inclination experiment, the injectable conductive hydrogel is formed after the solution A and the solution B are mixed into a black solution for ten seconds.

FIG. 2: is an electron micrograph of the hydrogel prepared in example 1. As can be seen, the conductive hydrogel has a three-dimensional network pore structure similar to the spinal cord.

FIG. 3: a graph of the rheological data for the hydrogel prepared in example 1. As can be seen, the modulus of the hydrogel is about 950kPa, is close to the modulus of the natural spinal cord tissue, and is favorable for the differentiation of neural stem cells to neurons.

FIG. 4: after the hydrogel prepared in example 1 is loaded with neural stem cells, the differentiation of the neural stem cells to neurons can be effectively promoted by combining with electrical stimulation.

FIG. 5: the injectable conductive hydrogel loaded with the neural stem cells prepared in example 1 is injected into a rat spinal cord total-section model and then is combined with electrical stimulation treatment to obtain a rat gait condition. It can be seen from the figure that the motor function of the group of rats was significantly restored as observed by gait after combination with electrical stimulation.

Detailed Description

Example 1

(1)0.5g of chitosan was dissolved in deionized water, the pH was adjusted to 5.5 by HCl, and then dispersed for 20min using ultrasonic agitation to form a 1.0% wt chitosan solution. Then 300mg of HCA was dissolved in 5.0mL of deionized water, and added to the chitosan solution after fully stirred and dissolved. Dissolving 240mg of EDC and 150mg of NHS in 50.0mL of mixed solution of ethanol and deionized water in a ratio of 1:1, and keeping the pH value between 6 and 7. Then slowly dropping the chitosan/HCA mixed solution into the mixed solution, sealing and stirring vigorously for 10h in an environment with the temperature of 20 ℃, keeping the pH value at 5.5, and then placing the mixed solution in an oscillator to shake rapidly for 10 min. Dialyzing the solution in acidified deionized water for 2 days, then dialyzing in deionized water for half a day, and freeze-drying in a freeze dryer to obtain yellow solid.

(2) 2.0g HA was dissolved in 200mL deionized water and sealed under rapid stirring at 20 ℃ for 24h to dissolve completely. 1.0g NaIO ₄ Adding the HA solution into the solution, stirring the solution for 5 hours in a dark place, adding 1.5mL of glycol, stirring the solution for 1.5 hours, and carrying out ultrasonic oscillation reaction for 15 min. Putting the solution into a dialysis bag, dialyzing with deionized water for 3 days, and keeping the pH of the solution between 7 and 8. The reaction mass is put into a freeze dryer for freeze drying to finally obtain white solid.

(3) 1g of LiF was added to 20mL of hydrochloric acid and stirred for 5 min. 1g of Ti ₃ AlC ₂ The above solution was slowly added and stirred at 35 ℃ for 26h to give a black suspension. The acidic suspension was then washed with deionized water and centrifuged at 4500 rpm for 5min until the supernatant reached pH 6.0. And then collecting the deposit, adding deionized water into the deposit, introducing Ar into the dispersion liquid for 10min, dispersing for 1h by using ultrasonic oscillation, and adding and ensuring the temperature to be within 30 ℃. And finally, centrifuging at the rotating speed of 5000 for 10min, and freeze-drying at the temperature of 50 ℃ below zero for 3 days to obtain fluffy black powder A of the MXene nanosheets.

(4) Preparing a 5 wt% solution of the CHIC solid by using deionized water, and adding 1.0 wt% ADH to prepare a light yellow solution A; and then adding the HA-ALD solid into deionized water to prepare a 5 wt% solution, adding 0.1% wtMXene nanosheet powder, and performing ultrasonic oscillation for 10min to prepare a black solution B. Mixing the solution A and the solution B according to the volume ratio of 1:1, mixing and reacting for 10 seconds by a vibrator to finally obtain the injectable conductive hydrogel simulating spinal cord tissue.

Example 2

(1)0.8g of chitosan was dissolved in deionized water, the pH was adjusted to 5 by HCl, and then dispersed for 25min using ultrasonic agitation to form a 1.0% wt chitosan solution. Then, 420mg of HCA was dissolved in 6.0mL of deionized water, and the solution was added to the chitosan solution after being sufficiently stirred and dissolved. Dissolving 280mg of EDC and 190mg of NHS in 50.0mL of mixed solution of ethanol and deionized water in a ratio of 1:2, and keeping the pH value between 6 and 7. Then slowly dropping the chitosan/HCA mixed solution into the mixed solution, sealing and stirring vigorously for 12h in an environment with the temperature of 25 ℃, keeping the pH value at 5, and then placing the mixed solution in an oscillator to shake rapidly for 15 min. Dialyzing the solution in acidified deionized water for 3 days, then dialyzing in deionized water for half a day, and freeze-drying in a freeze dryer to obtain yellow solid.

(2) 3.0g HA was dissolved in 300mL deionized water and stirred under sealed rapid stirring at 25 ℃ for 24h to dissolve completely. 1.2g NaIO ₄ Adding the HA solution into the solution, stirring the solution for 4 hours in a dark place, adding 2mL of glycol, stirring the solution for 2 hours, and carrying out ultrasonic oscillation reaction for 20 min. Putting the solution into a dialysis bag, dialyzing with deionized water for 3 days, and keeping the pH of the solution between 7 and 8. The reaction mass is put into a freeze dryer for freeze drying to finally obtain white solid.

(3) 1g of LiF was added to 20mL of hydrochloric acid and stirred for 5 min. Mixing 1g of Ti ₃ AlC ₂ The solution was slowly added and stirred at 40 ℃ for 24h to give a black suspension. The acidic suspension was then washed with deionized water and centrifuged at 3500 rpm for 8min until the supernatant reached a pH of 6.0. Then collecting the deposit, adding deionized water into the deposit, introducing Ar into the dispersion liquid for 20min, and dispersing for 1.5h by using ultrasonic oscillation while ensuring the temperature to be within 30 ℃. And finally, centrifuging at the rotating speed of 4500 for 10min, and freeze-drying at-50 ℃ for 3 days to obtain fluffy black powder A of the MXene nanosheet.

(4) Preparing a 6 wt% solution of the CHIC solid by using deionized water, and adding 1.5 wt% of adipic dihydrazide ADH to prepare a light yellow solution A. And then adding a proper amount of deionized water into the HA-ALD solid to prepare 6 wt% of solution, adding 0.12% of wtMXene nanosheet powder, and performing ultrasonic oscillation for 15min to prepare a black solution B. Mixing the solution A and the solution B according to a volume ratio of 2: 3, mixing, and rapidly mixing and reacting for 60 seconds by using an oscillator to finally obtain the injectable conductive hydrogel simulating spinal cord tissue.

Claims

1. A preparation method of hydrogel simulating spinal cord is characterized in that: the method comprises the following specific steps:

1) firstly, preparing catechol group modified chitosan (CHIC); dissolving 0.5-0.8 g of chitosan in deionized water, adjusting the pH to 5.0-6.0 by HCl, and then dispersing for 20-30 min by using ultrasonic oscillation to finally form a 1.0 wt% chitosan solution; then, dissolving 255-455 mg of dihydrocaffeic acid HCA in 5.0-8.0 mL of deionized water, fully stirring and dissolving, and then adding into chitosan liquid; dissolving 240-300 mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide EDC and 150-200 mg of n-hydroxy thiosuccinimide NHS in 50.0mL of a mixed solution of ethanol and deionized water in a ratio of 1: 1-1: 2, and keeping the pH value within a range of 6-7; slowly dripping the chitosan/HCA mixed solution into the mixed solution, reacting in an environment of 20-25 ℃, sealing and violently stirring for 10-12 h, keeping the pH value at 5.0-6.0, and then placing in an oscillator for fast shaking reaction for 10-15 min; dialyzing the solution in acidified deionized water for 2-3 days, dialyzing in deionized water for half a day, and freeze-drying in a freeze dryer to obtain a yellow solid, namely preparing the catechol group-modified chitosan CHIC;

2) preparing aldehyde hyaluronic acid HA-ALD; dissolving 2.0-3.0 g of hyaluronic acid HA in 200-300 mL of deionized water, and sealing and rapidly stirring for 24-28 h in an environment at 20-25 ℃ until the hyaluronic acid HA is completely dissolved; 1.0-1.5 g NaIO ₄ Adding the HA solution into the solution, stirring the solution for 4 to 7 hours in a dark place, adding 1.5 to 2.3mL of ethylene glycol, stirring the solution for 1 to 2 hours, and then carrying out ultrasonic oscillation reaction for 10 to 20 min; putting the solution into a dialysis bag, dialyzing with deionized water for 3-4 days, and keeping the pH of the solution within a range of 7-8; putting the reactant into a freeze dryer for drying to finally obtain a white solid of aldehyde hyaluronic acid HA-ALD;

3) preparing MXene nanosheets; adding 1-4 g of LiF into 20-80 mL of hydrochloric acid, and stirring for 5-10 min; then adding 1-4 g of Ti ₃ AlC ₂ Slowly adding the solution and stirring for 24-26 h at 35-40 ℃; washing the acidic suspension with deionized water, and centrifuging at 3500-4500 rpm for 5-8 min until the pH value of the supernatant reaches 6-7; then collecting the deposit, adding deionized water into the deposit, introducing Ar into the dispersion liquid for 10-20 min, and dispersing for 1-1.5 h by using ultrasonic oscillation while ensuring the temperature to be within 30 ℃; finally, centrifuging at the rotating speed of 4500-5000 for 1Freeze-drying for 0-15 minutes at-50 to-30 ℃ for 2-3 days to obtain black solid powder as MXene nanosheets;

4) preparing a CHIC solid into a 4-6 wt% solution by using deionized water, and adding 1.0-2.0 wt% of adipic dihydrazide ADH into the solution to prepare a light yellow solution A; adding HA-ALD into deionized water to prepare a 4-6 wt% solution, adding 0.1-0.15% wtMXene nanosheet powder, and performing ultrasonic oscillation for 10-15 min to obtain a black solution B; mixing the solution A and the solution B according to the volume ratio of 1: 2-3: 1, and quickly mixing and reacting for 10-60 seconds by using an oscillator; the injectable conductive hydrogel which simulates the structure, modulus and conductivity of spinal cord tissue can be obtained.

2. The hydrogel prepared by the method for preparing the hydrogel simulating the spinal cord according to claim 1, wherein the method comprises the following steps: the hydrogel has the properties of a three-dimensional network porous structure, excellent injectability, good conductivity, certain adhesion, mechanical strength and the like, simulates the structure and biophysical characteristics of natural spinal cord tissues, and has the characteristics of similar pore structure, modulus, conductivity and the like as the spinal cord tissues.

3. The hydrogel prepared by the method for preparing the hydrogel simulating the spinal cord according to claim 1, wherein the method comprises the following steps: the hydrogel can effectively promote the differentiation of the neural stem cells to the neurons and the growth of the axons by combining with the electrical stimulation, and can be applied as an auxiliary material for repairing spinal cord injuries and regenerating tissues.

4. The hydrogel prepared by the method for preparing the hydrogel simulating the spinal cord according to claim 1, wherein the method comprises the following steps: the hydrogel has the characteristic of simulating spinal cord, can be used as an injectable and conductive filling material in the process of spinal cord injury repair, and has application prospects in the aspects of promoting spinal cord injury repair, motor function recovery and the like.