CN120360931A - Microenvironment responsive spinal cord repair hydrogel and preparation method and application thereof - Google Patents

Abstract

The invention relates to a microenvironment responsive spinal cord repair hydrogel and a preparation method and application thereof, and belongs to the technical field of hydrogels. The hydrogel is prepared from the following raw materials, by mass, 0.8-1.5 parts of xyloglucan oxide, 0.8-1.5 parts of methacrylic chitosan, 0.1-0.5 part of nano silicon particles and 0.7-2.0 parts of borax. The boric acid ester bond and the Schiff base bond are adopted as the main body of the ROS response, so that a continuous response dynamic network can be formed, intelligent response is realized through the dynamic reversible characteristic of the ROS response dynamic network, and the interference of various factors in a complex physiological environment is resisted through the dual dynamic network, so that the stable response capability of the material in the spinal cord injury microenvironment is maintained for more than 15 days.

Description

Microenvironment responsive spinal cord repair hydrogel and preparation method and application thereof

Technical Field

The invention belongs to the technical field of hydrogels, and particularly relates to a microenvironment responsive spinal cord repair hydrogel and a preparation method and application thereof.

Background

The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.

Current research on Spinal Cord Injury (SCI) repair materials focuses on constructing three-dimensional structures with bioactivity, mechanical suitability, and microenvironment responsiveness. Current mainstream technology includes synthetic polymeric hydrogels, natural biomaterials, and composite hydrogel systems. The hydrogel becomes a research hot spot due to the characteristics of the bionic extracellular matrix, can be used as a stem cell/drug carrier to realize accurate delivery, and can form a physical barrier in situ through the injectability characteristic to inhibit scar generation. However, the existing repair materials have significant drawbacks in terms of microenvironment responsiveness, swelling resistance, self-healing properties, bioactivity, antibacterial properties, mechanical strength, drug carrying capacity, and the like, and in particular have the following problems.

Insufficient resistance to swelling, including (1) a decrease in mechanical properties due to swelling. After the traditional hydrogel absorbs water and swells, the three-dimensional network structure is easy to expand and deform, so that the mechanical strength is obviously reduced. (2) risk of compression to surrounding tissue. Traditional hydrogels (such as PVA or PEG groups) have excessive hydrophilic groups, so that the water absorption expansion rate in a physiological environment is more than 150%, and excessive swelling of the hydrogels can press normal tissues, so that the risk of intracranial pressure rise after implantation is caused, and secondary damage is caused. (3) insufficient stability and durability. Traditional chemically crosslinked hydrogels (such as single covalent bond crosslinking) are difficult to recover the original shape after swelling, and have poor dynamic response.

The traditional hydrogel has insufficient antibacterial property, and the drug release of the traditional hydrogel is mostly passively diffused, and cannot be dynamically regulated according to the infection microenvironment (such as pH and ROS level), so that the drug is released prematurely or the local concentration is insufficient.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide the microenvironment responsive spinal cord repair hydrogel, the preparation method and the application thereof, by introducing take seat f alkali bond and boric acid ester bond into a hydrogel system, the dynamic balance of the two bonds under the local pH and ROS fluctuation is realized, the self-healing rate and microenvironment adaptation are further realized, and the nano silicon particles have a rigid supporting effect and can also realize the anti-inflammatory effect.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

according to the first aspect, the microenvironment responsive spinal cord repair hydrogel is prepared from the following raw materials, by mass, 0.8-1.5 parts of xyloglucan oxide, 0.8-1.5 parts of methacrylic chitosan, 0.1-0.5 parts of nano silicon particles and 0.7-2.0 parts of borax.

Optionally, the preparation raw materials further comprise 95.5-97.2 parts of PBS buffer solution.

Optionally, the preparation raw material comprises anti-inflammatory drugs and/or neurotrophic factors.

Optionally, the anti-inflammatory drug comprises one or more of methylprednisolone, dexamethasone and prednisolone.

Optionally, the concentration of the methylprednisolone in the micro-environment-responsive spinal cord repair hydrogel is 0.5-5.0 mg/mL, or the concentration of dexamethasone in the micro-environment-responsive spinal cord repair hydrogel is 0.1-2.0 mg/mL, or the concentration of the prednisolone in the micro-environment-responsive spinal cord repair hydrogel is 0.5-5 mg/mL.

Optionally, the neurotrophic factor comprises one or more of Nerve Growth Factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophic factor-3 (NT-3).

Optionally, the concentration of the Nerve Growth Factor (NGF) in the microenvironment-responsive spinal cord repair hydrogel is 0.01-0.5 mug/mL, or the concentration of the brain-derived neurotrophic factor (BDNF) in the microenvironment-responsive spinal cord repair hydrogel is 0.05-1.0 mug/mL, or the concentration of the neurotrophic factor-3 (NT-3) in the microenvironment-responsive spinal cord repair hydrogel is 0.02-0.8 mug/mL.

In a second aspect, the preparation method of the microenvironment responsive spinal cord repair hydrogel comprises the following steps:

And respectively adding the xyloglucan oxide and the methacrylic chitosan into a buffer solution, stirring at room temperature for reaction for 4-6 hours, and then sequentially adding the nano silicon particles and borax, stirring at 40-60 ℃ for reaction for 2-3 hours.

Optionally, the loading method of the anti-inflammatory drug and/or the neurotrophic factor comprises one or more of a physical adsorption method, a chemical bonding packaging method and an in-situ mixing loading method.

Optionally, the physical adsorption method comprises the steps of utilizing hydroxyl or amino groups on the surfaces of the nano silicon particles to be combined with drug molecules through hydrogen bonds or electrostatic action, realizing drug adsorption load, inhibiting burst release and prolonging the slow release period.

Optionally, the chemical bond packaging method comprises the steps of packaging the medicine in a three-dimensional pore through a double-network structure constructed by dynamic boric acid ester bonds and Schiff base bonds, and regulating and controlling reversible dissociation of chemical bonds by utilizing a pH/ROS response mechanism to realize intelligent release of the medicine.

Optionally, the in-situ mixing loading method comprises directly blending with the drug solution during the preparation of the hydrogel, embedding drug molecules into the network through crosslinking reaction, and fixing drug distribution by combining the rigid supporting effect of the nano silicon particles.

Alternatively, the buffer is a PBS buffer.

Optionally, the preparation method of the xyloglucan comprises the steps of mixing sodium periodate and xyloglucan according to a mass ratio of 1 (2-5) to prepare a solution, stirring at room temperature for reaction for 4-6 hours, adding glycol to terminate the reaction, and dialyzing to obtain the xyloglucan oxide.

Optionally, dialyzing the substances with molecular weight cut-off of more than or equal to 3500Da, and freeze-drying to obtain the xyloglucan oxide.

Optionally, the preparation method of the methacrylic chitosan comprises the steps of dissolving chitosan in acetic acid solution, adding methacrylic anhydride with the mass of 0.36-1.92 times of that of the chitosan, stirring at 60-80 ℃ for reaction for 6-10 hours, adding alkaline substances for neutralization to pH=6.5-7, diluting, and dialyzing to obtain the methacrylic chitosan.

Optionally, dialyzing substances with molecular weight cutoff of 8000-14000 Da, and freeze-drying to obtain the methacrylic chitosan.

Alternatively, the alkaline substance comprises an aqueous solution of sodium bicarbonate.

Optionally, the preparation method of the nano silicon particles comprises the steps of dispersing 3- [2- (2-amino ethylamino) ethylamino ] propyl-trimethoxy silane in 5-6 times of ultrapure water, adding 2-aminophenol with the mass of 0.24-0.30 times of that of the 3- [2- (2-amino ethylamino) ethylamino ] propyl-trimethoxy silane, stirring at 70-80 ℃ for reacting for 100-150min, and dialyzing to obtain the nano silicon particles.

Optionally, dialyzing the substances with molecular weight cut-off of 400-600Da, and freeze-drying to obtain the nano-silicon particles.

In a third aspect, the use of a microenvironment-responsive spinal cord repair hydrogel as described above in at least one of the following a 1) -a 5), comprising:

a1 Preparing a product that promotes spinal cord repair;

a2 Preparing a product for continuously removing micro-environmental ROS in the spinal cord repairing process;

a3 Preparing a product for continuously regulating the pH value of a microenvironment in the spinal cord repairing process;

a4 Preparing an antibacterial product in the spinal cord repair process;

a5 A product for preparing the sustained release medicine in the spinal cord repairing process.

The beneficial effects of the invention are as follows:

1. According to the invention, boric acid ester bonds and Schiff base bonds are adopted as ROS response main bodies to form a continuous response dynamic network, intelligent response is realized through the dynamic reversible characteristic of the continuous response dynamic network, and bond rupture can be effectively triggered in a low-concentration ROS environment. The dynamic bond has pH responsiveness, is stable under physiological pH (7.4), can be reversibly dissociated in a local acid/alkaline microenvironment of a damaged area, dynamically adjusts network density, balances water absorption expansion and mechanical strength, and is characterized in that imine bonds (-C=N-) are reversibly broken under an acidic environment (pH < 6.5) so as to neutralize hydrogen ions in the environment to raise the pH, when the microenvironment is restored to be neutral, network self-repair is realized through rebinding of amino groups and aldehyde groups, the material can be quickly self-repaired after ROS response, the problem that the traditional material is collapsed in structure due to bond breakage (the mechanical strength loss of the traditional material reaches more than 50% after the traditional material is responded) is avoided, the integrity of the bracket is kept so as to continuously adjust and control the microenvironment, and boric acid ester bonds tend to be stable under alkaline conditions (pH > 7.4) so as to be complementary with Schiff base bonds, and prevent the material from being excessively degraded in the later stage of inflammation. The material can adjust the crosslinking density through the ion concentration of tetraborate radical (0.02-0.05 mol/L) when the pH value fluctuates, further dynamically adjust the network porosity, realize the intelligent matching of the drug slow release curve, resist the interference of various factors in the complex physiological environment through the dual dynamic network, and ensure that the stable response capability of the material in the spinal cord injury microenvironment is maintained for more than 15 days.

2. The added nano silicon particles strengthen a three-dimensional network through physical filling and interface interaction, so that the elastic modulus of the material is obviously improved, and the mechanical property range of 0.2-1.0kPa of spinal cord tissue can be matched. The rigid supporting function can prevent the collapse of the structure, provide physical anchoring sites for the recombination of dynamic bonds, and avoid the dislocation of the structure in the traditional soft gel self-healing process. The hydrophobic property of the nano silicon particles and the boric acid ester bonds can reduce water molecule permeation and inhibit excessive swelling, and meanwhile, the dynamic property of the nano silicon particles and the boric acid ester bonds allows the network to restore structural integrity through bond recombination after swelling, so that irreversible expansion deformation of the traditional hydrogel is avoided. The surface of the nano silicon particles (SiNPs) can be combined with amino groups, and can be quickly adhered to the surface of a negatively charged bacterial cell membrane by electrostatic action to destroy the membrane integrity, so that SiNPs can realize broad-spectrum antibiosis without depending on antibiotics, and the inhibition effect on gram-positive bacteria (such as staphylococcus aureus) and gram-negative bacteria (such as escherichia coli) is remarkably improved. The anti-inflammatory properties of the silicon particles combine with the ROS responsiveness of dynamic borate bonds and Schiff base bonds, so that the composite hydrogel has strong anti-inflammatory properties.

3. Aiming at the problems of unmatched mechanical strength and weak slow release function of the traditional medicine carrying system, the invention forms a stable three-dimensional pore structure through a OXG-CSMA-borax dynamic boric acid ester bond/Schiff base bond double-network structure. The network can adapt to the medicine diffusion rate through the rupture-recombination of dynamic bonds, and can provide multiple medicine carrying sites by utilizing the medicine molecules loaded on the surfaces of the nano silicon particles. Compared with a collagen system relying on single physical adsorption in the traditional repair material technology, the design remarkably improves the medicine carrying capacity and realizes slow release, and avoids secondary damage of medicine burst release to nerve tissues. Under the acidic microenvironment of the damaged area, schiff base bonds are selectively broken to release neurotrophic factors, and when the concentration of ROS is increased, the Schiff base bonds are further dissociated to accelerate the release of the antioxidant drugs. The time sequence control characteristic overcomes the defect of insufficient release time sequence regulation in the background technology, realizes multi-stage accurate drug delivery of early anti-inflammatory, middle regeneration promotion and later barrier repair, and is highly matched with the spinal cord injury repair process.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

Fig. 1 shows the scanning electron microscope test results of the microenvironment-responsive spinal cord repair hydrogel in example 1, wherein (a) the pore structure is schematically shown in fig. 1, (b) the distribution of four elements is shown in fig. C (red), N (purple), si (blue) and O (green).

FIG. 2 is a graph showing the staining results of live and dead staining of cells after co-culturing the present invention with mesenchymal stem cells in example 3.

FIG. 3 is a graph showing the results of staining for regeneration and repair of nerve tissue in a spinal cord injured mouse of example 4, wherein (a) immunofluorescent staining of typical neuronal markers and astrocyte markers after 4 weeks of establishing a semi-transverse model of spinal cord injury in the mouse, (b) statistical analysis of fluorescent intensity of astrocyte markers, and (c) statistical analysis of fluorescent intensity of neuronal cell markers.

FIG. 4 is a graph showing the evaluation results of neuroinflammation in example 4. Immunofluorescent staining of neuroinflammation index after 4 weeks of establishment of a semi-transverse model of spinal cord injury in mice.

Fig. 5 is a graph of the evaluation results of the functional analysis of the spinal cord injury mice of example 4, (a) a graph of BMS score results.

(B) Catwalk gait analysis result diagram.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.

The experimental procedure, in which specific conditions are not noted in the examples below, is generally followed by conventional conditions. The raw materials and reagents used in the following examples were commercially available unless otherwise specified.

Term interpretation:

burst effect, which is a phenomenon in which a drug is suddenly released under specific conditions in a pharmaceutical formulation.

Reactive Oxygen Species (ROS) refers to the general term for peroxides which are related to oxygen metabolism in living organisms and contain oxygen radicals and are susceptible to radical formation.

Creep, a phenomenon that the strain of a solid material increases with time under the condition of keeping the stress unchanged.

Swelling ratio, which refers to the degree to which a material swells in solution upon absorption of water, can be calculated by the following formula swelling ratio (%) = (W2-W1)/W1 x100%. Wherein W1 is the initial weight of the material, and W2 is the weight of the material after being soaked in the solution for a certain time.

Compression modulus is the ratio of compressive stress to compressive strain when an object is subjected to triaxial compression.

Tensile strength is the stress at which the material undergoes the greatest uniform plastic deformation.

An ELISA method for detecting target substance includes such steps as binding antigen or antibody to solid carrier, and using the specific binding of antigen and antibody and the enzyme marked on antigen to catalyze the color reaction of specific substrate.

BBB motor scoring, which is a standardized scoring system for evaluating the recovery of motor functions of hind limbs of experimental animals after spinal cord injury, is widely applied to neuroscience and spinal cord injury research.

Bending strength is the maximum stress that a material can withstand when it breaks under bending load or reaches a prescribed bending moment, which is the maximum normal stress at the time of bending.

BMS scoring is a tool for assessing limb motor function in spinal cord impaired patients. It consists of two scores, namely a "motor score" and a "sensory score". The motor score determines the extent of the patient's limb movements and the sensory score is used to assess the patient's ability to perceive the tactile stimulus.

Catwalk gait analysis is a complete system and advantageous tool for quantitatively assessing rodent gait changes caused by pain or other motor deficits. The system can be used to evaluate animal models of nerve trauma, neurological atrophy, neurological disease, and pain syndrome groups.

Example 1

(1) A microenvironment responsive spinal cord repair hydrogel, the preparation method comprises:

S1, mixing a 10% sodium periodate (NaIO ₄) aqueous solution with a 10% xyloglucan (commercially available with purity not less than 98%, sugar content of xylose 34%, glucose 45%, galactose 17%, arabinose 2% and other sugars 2%) aqueous solution, controlling a solute mass ratio of sodium periodate to xyloglucan to be 1:2, magnetically stirring at room temperature in a dark place (500 rpm,25 ℃ for 4 hours), adding ethylene glycol with a mass ratio of 5.5 to potassium periodate (purity not less than 99%) under the same condition, stirring for 1-2 minutes to terminate the reaction to obtain a first mixed solution, dialyzing the first mixed solution in deionized water for 72 hours to cut-off macromolecular substances with molecular weight not less than 3500Da, and freeze-drying the cut-off substances to obtain xyloglucan Oxide (OXG).

S2, preparing an acetic acid solution with the mass concentration of 1%, dissolving chitosan (the commercial purity is more than or equal to 98%, the deacetylation degree is more than or equal to 75%, and the molecular weight is 100-350 kDa) in 1% of the acetic acid solution, enabling the mass ratio of the chitosan to the glacial acetic acid to be 1:1, forming a second mixed solution, dripping methacrylic anhydride (MA, the purity is more than or equal to 95%, a hindered phenol antioxidant with the mass concentration of about 0.2% is used as a stabilizer) into the second mixed solution, controlling the mass ratio of the chitosan to the methacrylic anhydride to be 1:0.36 (the specific ratio depends on the molecular weight of the chitosan, the deacetylation degree and the purity of the methacrylic anhydride), magnetically stirring (500 rpm,6 h) at 60 ℃, then neutralizing to pH=6.5 by using a sodium bicarbonate aqueous solution with the mass fraction of 10%, diluting 5 times to end the reaction, obtaining a crude CSMA solution, dialyzing the crude CSMA solution for 3-4 days, intercepting macromolecular substances with the molecular weight of 8000-14000 Da, removing unreacted reagents, and obtaining a white, freeze-dried and obtaining the freeze-dried product of the freeze-dried sponge chitosan.

S3, 3- [2- (2-amino-ethylamino) ethylamino ] propyl-trimethoxysilane was used as a silicon source, ultrapure water was stirred in a constant temperature water bath at 70℃for 300-500rpm, 1/5 of the volume of the silicon source (purity of 3- [2- (2-amino-ethylamino) ethylamino ] propyl-trimethoxysilane was added thereto (purity: 99%) and then 2-aminophenol (purity: 99%) was added to the solution

More than or equal to 98.5 percent) as a reducing agent, adding 4.0mg of 2-aminophenol into each milliliter of silicon source, keeping the constant temperature of 70 ℃ for continuous stirring reaction for 150 minutes, gradually generating a third dispersion liquid in which silicon nano particles are dispersed, dialyzing the third dispersion liquid by deionized water, intercepting substances with molecular weight of 400-600Da, dialyzing for 8 hours, removing unreacted small molecular impurities, freeze-drying the intercepted substances, and detecting the particle size of the required nano silicon particles (SiNPs) to be 50-100nm.

S4, mixing 1 part of freeze-dried powder of xyloglucan (OXG) and methacrylic Chitosan (CSMA), 98 parts of PBS buffer solution, magnetically stirring (600 rpm,25 ℃ for 4 hours) at room temperature in a dark place to obtain a xyloglucan-chitosan hydrogel composite solution based on dynamic Schiff base bonds, adding nano silicon particles (SiNPs) into the xyloglucan-chitosan hydrogel composite solution to enable the mass fraction of the xyloglucan-chitosan hydrogel composite solution to be 0.3%, stirring at the temperature of 40 ℃ for 2 hours at the rotating speed of 600rpm to obtain the composite solution, adding commercially available high-purity borax (with the purity of more than or equal to 99%) into the composite solution to enable the concentration of tetraborate ions to be 0.04mol/L, magnetically stirring (600 rpm,40 ℃ for 2 hours) at room temperature in a dark place to form a xyloglucan-chitosan-borax-silicon particle crosslinked network based on dynamic boric acid ester bonds, namely the micro-environment responsive spinal cord repairing hydrogel, and in the step, adding the xyloglucan (OXG), the methacrylic Chitosan (CSMA), the nano silicon particles (SiNPs), and the borax into the PBS buffer solution to enable the mass ratio to be 1:1.96:1.2.1.

(2) Microscopic observations

And (3) scanning electron microscopy detection is carried out on the prepared microenvironment responsive spinal cord repair hydrogel, the result is shown in a figure 1, wherein (a) in the figure 1 is a first pore structure diagram under the scanning electron microscopy, and (b) in the figure 1 is a second pore structure diagram under the scanning electron microscopy, and the gradient control of the three-dimensional network crosslinking degree is realized by adjusting the concentration of tetraborate ions (controlling the crosslinking density of borate bonds) and the formation proportion of Schiff base bonds (influencing the dynamic network recombination capability). The distribution of the four elements C, N, si, O in fig. 1 (c) shows that the elements are distributed uniformly in the material, which means that the elements are successfully introduced and have good dispersibility in the matrix of the material. This suggests that the components are uniformly mixed during the preparation of the material, possibly contributing to their subsequent synergy in biological applications or functional manifestations.

(3) Model performance detection

The initial compression strength is 1.0kPa, the composite hydrogel is soaked in the physiological saline for 72 hours to be a swelling hydrogel, and then the swelling hydrogel is detected, the volume expansion rate is 8% (the swelling rate is less than 10%), the compression modulus is 87% of the initial value, the compression strength is kept at 93%, and the compression risk to surrounding tissues is reduced due to the low volume expansion rate.

A universal material tester (INSTRON 5944) is adopted to carry out cyclic compression test on the swelling hydrogel, namely, the compressive strain is set to be 50 percent, the frequency is 1Hz, 30 times of cyclic loading-unloading are continuously carried out, and the result shows that the initial cyclic compressive stress is 0.85kPa, the stress is reduced to 0.81kPa after 30 times of cyclic loading-unloading, and the attenuation rate is 4.7 percent. The stress-strain curves of each cycle are kept to be highly overlapped, the attenuation rate of the hysteresis loop area is only 3.2%, which shows that the dynamic bond network can effectively dissipate energy through the real-time recombination of Schiff base bonds and boric acid ester bonds, and compared with the traditional single-network hydrogel, the dual-dynamic bond synergistic effect obviously improves the fatigue resistance. The stress attenuation rate is less than or equal to 5% after 30 times of cyclic compression.

The method comprises the steps of synchronously carrying out scanning electron microscopy to observe pore structure change, analyzing chemical bond recombination rate of borate bonds and Schiff base bonds through FT-IR (experimental data show that 98% bond recombination is completed in 1 hour), simultaneously monitoring real-time recovery data of mechanical properties of materials under different pH/ROS conditions, and indicating that the microenvironment-responsive spinal cord repair hydrogel prepared by the method is based on pH response characteristics (stable under physiological pH 7.4 and reversible dissociation of acidic microenvironment) of the borate bonds and ROS sensitivity of the Schiff base bonds, and can realize network dynamic recombination after swelling.

The initial tensile strength was measured by preparing a standard dumbbell specimen according to ASTM D412, balancing the specimen in a test environment (25 ℃ and 90% humidity) for 24 hours to remove residual stress, measuring the tensile strength to be 1.27mpa, cutting the specimen perpendicular to the tensile direction, and placing the end faces in combination, which achieves macroscopic incision closure within 0.5 hour at 25 ℃, measuring the tensile strength to be 1.10mpa after 2 hours, recovering to more than 85% of the original value, measuring the tensile strength to be 1.16mpa after 2 injury-healing tests (total 3 times) after 16 hours of healing, and still maintaining more than 90% of the mechanical properties (the traditional single dynamic key system only recovers <50% and fails after repeated repair), and the experiment shows that the implanted hydrogel in the animal model maintains structural integrity in the spinal cord dynamic shrinkage/expansion environment without fissures.

The flexural strength of the standard dumbbell specimen was measured using the three-point bending test method, and the result was an average flexural strength of 2.7mpa.

The method comprises the steps of immersing end face positions which are combined and placed through H ₂O₂ solutions with different concentration gradients, controlling local ROS level through adjusting time, or adopting different pH buffers (such as PBS with pH of 5.0-8.0) to treat cut-off interfaces to simulate micro-environment fluctuation, utilizing fluorescent marked dynamic Schiff base bond precursors (with high fluorescence intensity at fracture parts and weaker uniform distribution parts) in the process, utilizing a confocal microscope to detect fluorescence intensity changes at the fracture parts in real time, calculating self-healing rate = notch fluorescence intensity/original fluorescence intensity to determine the self-healing rate, and finding out that active oxygen (ROS) level in a damaged area rises to trigger Schiff base bond preferential recombination, and local pH fluctuation regulates boric acid ester bond dynamic balance to achieve self-healing rate and micro-environment adaptation, wherein the self-healing rate is improved by 2.3 times when the ROS concentration in experiments is more than or equal to 5 mu M.

The incision area was analyzed by in situ spectroscopy for infrared spectroscopy during the injury-healing test, and schiff base bonds were observed (c=n,

The characteristic peak intensity changes of 1640cm < -1 >) and boric acid ester bonds (B-O, < - > 1380cm < -1 >), the bond recombination rate is calculated, the ratio of the C=N peak intensity to the initial C=N peak intensity is about 99.1% after a plurality of experimental data are carried out for 1 hour, the ratio of the B-O peak intensity to the initial B-O peak intensity is about 98.4% after 1 hour, which shows that after the ROS trigger rupture of the boric acid ester bonds and the Schiff base bonds, 98% bond recombination is completed within 1 hour, the compressive strength of the material is restored to 95% of the initial value (the breaking strength of the traditional material is lost by 50% and is irreversible), and the integrity of the bracket is maintained so as to continuously regulate and control the microenvironment.

(4) Antibacterial property detection

The antibacterial performance of the composite hydrogel is measured by an agar diffusion method, wherein bacteria are firstly cultivated to a logarithmic phase (OD 600 is approximately equal to 0.5), diluted to 10 ⁶ CFU/mL, 100 mu L of bacterial suspension is uniformly coated on an LB agar plate, a sterile hydrogel wafer (with the diameter of 6mm and the thickness of 2 mm) is lightly pressed on the surface of the agar, the composite hydrogel with the diameter of a bacteriostasis ring (comprising a hydrogel contact area and surrounding transparent rings) loaded with SiNPs is cultivated for 18-24 hours at 37 ℃, the inhibition ring areas of the composite hydrogel on staphylococcus aureus and escherichia coli reach 196.5+/-10.3 mm ² and 61.3+/-1.1 mm ² respectively, and the bacterial proliferation inhibition capability is shown to be remarkably inhibited compared with a traditional antibiotic loading system, so that the drug resistance risk of bacteria is avoided.

In an experiment aiming at methicillin-resistant staphylococcus aureus (MRSA), the micro-environment responsive spinal cord repair hydrogel prepared by the embodiment is adopted by an experimental group, the methicillin Lin Kangsheng is adopted by a control group, the inhibition rate of the formation of a biological film in the experimental group is more than 82 percent (the control group is less than 30 percent), and the area of a inhibition zone is 167.9+/-13.1 mm ², so that the micro-environment responsive spinal cord repair hydrogel can break through a traditional antibiotic drug resistance barrier, and high-efficiency synergistic antibacterial effect is realized.

After the microenvironment-responsive spinal cord repair hydrogel obtained in this example was injected into the damaged defect area of the mouse spinal cord semi-transection model, the system evaluated the inflammatory reaction in vivo, and fluorescence staining was performed by the relevant inflammatory index, and the result shows that compared with the pure injury group, the fluorescence intensity of the neuroinflammation index of the damaged part in the treatment group is obviously reduced, the range is obviously reduced, which means that the neuroinflammation of the treatment group is obviously reduced, and the material can obviously inhibit the growth of pathogenic microorganisms and reduce the inflammation level, and exhibits excellent antibacterial and anti-inflammatory properties, as shown in fig. 4.

(5) Detection of effect of scavenging micro-environmental ROS

ROS-simulated microenvironment was constructed in a 37℃5% CO ₂ incubator, and the ROS scavenging performance was tested by DCFH-DA fluorescent probe method, which involved immersing 20. Mu.L of hydrogel sample (diameter 5mM, thickness 2 mM) in 1mL of serum-free medium containing 10. Mu. MDCFH-DA, incubating for 30min at 37℃and transferring to fresh medium after washing, adding H ₂O₂ to 0.5mM to the medium for ROS triggering, and immediately timing and recording the initial fluorescence intensity (excitation wavelength 488nm, emission wavelength 525 nm), and measuring the DCF fluorescence intensity in the medium using a fluorescent microplate reader at time points of 0, 6, 12, 24, 48, 72, 120, 180 hours, respectively, the DCF fluorescence intensity reflecting the residual ROS amount, and calculating the scavenging rate (ROS scavenging rate) and the measured half-life based thereon:

Wherein F0 is the initial fluorescence intensity after adding H ₂O₂, and Ft is the fluorescence intensity at each time point.

By fitting a curve of ROS clearance over time, the time required for the fluorescence intensity to drop to 50% of the initial value, i.e. the ROS clearance half-life, is calculated.

The hydrogel samples respectively adopt the composite hydrogel loaded with SiNPs (marked as NP-Gel), the traditional MnO ₂ nanoparticle modified hydrogel (marked as MnO ₂ -Gel) and the single network hydrogel without dynamic bonds (polyethylene glycol hydrogel is selected, the internal chemical bonds of the hydrogel are mainly ether bonds and carbon-carbon single bonds, the hydrogel is non-dynamic bonds, the hydrogel sample is marked as Control-Gel), the preparation raw materials of the hydrogel sample mainly comprise polyethylene glycol, polyethylene glycol diacrylate and PBS buffer solution, the biocompatibility is good, but the swelling rate is high, the mechanical strength is low, and the dynamic response capability is lacking, and the test results are shown in table 1.

TABLE 1

The hydrogel (NP-Gel) prepared in the embodiment can continuously and clearly maintain the ROS clearance rate to be more than 85% in a spinal cord injury post-oxidative stress microenvironment simulated by H ₂O₂ solution, is remarkably higher than that of a Control group (MnO ₂ -Gel is only 45% and Control-Gel is only 25%), the clearance rate of a traditional material (MnO ₂ -Gel) is suddenly reduced after 24 hours, and the NP-Gel is dynamically recombined due to Schiff base bonds, so that continuous clearance is realized.

The half-life of NP-Gel was 182.+ -. 5 hours (approximately 180 hours) by exponential decay fitting. The half-life of MnO ₂ -Gel is only 24+/-3 hours, which is consistent with the traditional material, and the Control-Gel has no obvious scavenging ability and the half-life cannot be calculated.

The specific test process and data show that the hydrogel prepared by the embodiment has the function of continuously removing the micro-environmental ROS in the spinal cord repair process, and can be used for preparing products for continuously removing the micro-environmental ROS in the spinal cord repair process, so that the breakthrough advantage of the hydrogel in ROS removal performance is proved.

(6) Effect detection to regulate pH in microenvironment

A pH simulated microenvironment was constructed in a 37 ℃ and 5% CO ₂ incubator, a common acidic injury environment was constructed with 5mL of PBS buffer with pH of 6.0 (containing 10mM lactic acid to simulate the local acidic inflammation environment after spinal cord injury), an occasional alkaline inflammation environment was constructed with 5mL of Tris-HCl buffer with pH of 8.0, the acidic and alkaline buffers simulating different environments were immersed in the hydrogels to be tested (diameter 5 mM. Times. Thickness 2 mM) respectively, shaking (50 rpm) at 37 ℃ and the pH value (pH _t) was measured by sampling at time points passing through 0, 2, 4, 6, 12, 24, 48, 72 hours, and the physiological pH time (i.e., the time required for adjusting the pH to 7.4.+ -. 0.05) was calculated, and the hydrogels to be tested include the hydrogels prepared in this example, conventional pH responsive hydrogels (polyacrylic acids, PAA gel) and non-responsive hydrogels (PEG gel).

The results of the acid damage environment are shown in Table 2, and the results of the basic inflammation environment are shown in Table 3.

TABLE 2

TABLE 3 Table 3

Whether in an acidic (pH 6.0) or alkaline (pH 8.0) microenvironment, the SiNPs-loaded composite hydrogel can be coordinated with a borate ester bond through a Schiff base bond, the pH can be accurately adjusted to 7.4+/-0.05 in 26h and 24h respectively, the pH range is maintained until 72h is finished, and compared with the traditional non-responsive (PEG) and single-responsive (PAA) hydrogels, the pH adjustment is more efficient and accurate, and the composite hydrogel has excellent dynamic balance capability in a complex pH fluctuation environment.

Through the specific test process and data, the hydrogel prepared by the embodiment has the capability of continuously regulating the pH of the microenvironment, and can be prepared into products for continuously regulating the pH of the microenvironment, so that the breakthrough advantage of the hydrogel in regulating the pH of the microenvironment is proved.

Comparative example 1

This comparative example differs from example 1 in that in the preparation method, the nano-silicon particles (SiNPs) were not prepared in the S3 step, and the nano-silicon particles (SiNPs) were not added in the S4 step. In the mechanical property test, the compressive strength is only 0.15kPa, and the bending strength is 0.2kPa.

Comparison of comparative example 1 with example 1 shows that the introduction of nano silicon particles (SiNPs) with the particle diameter of 50-100 nm in example 1 can provide physical anchoring sites, avoid structural dislocation in the self-healing process, improve the bending strength of the healed material by 15% -25% (the collapse rate of the traditional soft gel structure is more than 40%) compared with that of the healed material without the introduction of nano silicon), and approach the elastic modulus range of spinal tissue (0.2-1 kPa), so that the mechanical property and spinal adaptation of the material meet the spinal repair requirement.

Example 2

The difference between the embodiment 2 and the embodiment 1 is that the loading process is that in S4, methylprednisolone is dissolved in PBS buffer solution (pH 7.4), mixed with xyloglucan oxide and methacrylic chitosan, the final concentration of the methylprednisolone is controlled to be 0.3mg/mL, then nano silicon particles and borax are sequentially added, stirring reaction is carried out for 3 hours at 40 ℃, and the loading of the anti-inflammatory drug is realized through the hydrogen bond combination between the hydroxyl groups on the surface of the nano silicon and the ester groups of the methylprednisolone.

The dual response network is constructed based on the synergistic effect of boric acid ester bonds and Schiff base bonds, the accurate release of PH/ROS double factor triggered medicines is verified by a rat spinal cord contusion model, and after implantation, ① ELISA detection shows that the medicine concentration in a damaged area reaches a peak value (12.3+/-1.2 mug/g) at 72h, which is obviously higher than that of an intravenous injection group (4.1+/-0.8 mug/g), and the time for maintaining the effective concentration (> 5 mug/g) in ② days reaches 19.5+/-2.3 days, which is prolonged by 2.3 times compared with a gelatin microsphere carrier; ③

The BBB movement score reaches 14.2+/-1.5 minutes at 4 weeks, which is improved by 58 percent compared with a blank polyethylene glycol hydrogel group, and the slow release system is proved to effectively promote the functional recovery.

The hydroxyl group combined on the surface of the added nano silicon particles is combined with the drug molecules, so that the drug loading rate is obviously improved and the drug burst release is inhibited, wherein the drug release rate is reduced from 48% +/-5% of the traditional carrier to 12% +/-2% in the initial 12 hours, and the drug release period is prolonged to more than 14 days (less than or equal to 7 days in the traditional system) in artificial cerebrospinal fluid (aCSF).

The cross-linking density of the composite hydrogel is regulated by regulating the concentration of tetraborate ions, so that the gradient response of the pH of the material is realized, the network porosity of the material is dynamically regulated when the pH fluctuates, the intelligent matching of a drug slow-release curve is realized, the anti-inflammatory drug (the composite anti-inflammatory drug is methylprednisolone) is rapidly released in an acute stage, and the slow-release neurotrophic factor (the composite neurotrophic factor is NGF) in a repair stage is improved by more than 35% compared with a single response system.

Example 3

The microenvironment-responsive spinal cord repair hydrogel prepared in example 1 was co-cultured with mesenchymal stem cells and subjected to live-dead staining.

The method comprises the steps of resuspending mesenchymal stem cells in a hydrogel precursor solution after pancreatin digestion, controlling the cell density to be 1 multiplied by 10 ⁶ cells/mL, then uniformly subpackaging the cell-hydrogel mixed solution into 10cm dishes, placing the dishes in a 37 ℃ and 5% CO ₂ constant-temperature incubator for incubation for 30-60 min, adding a complete culture medium for continuous culture after the hydrogel is crosslinked and formed, and replacing the culture medium every 2 days in the culture process. On day 7 of incubation, a hydrogel sample containing cells was removed and subjected to Live/dead staining (Live/DEAD STAINING) using a mixed working solution of Calcein-AM (2 μm) and propidium iodide PI (4 μm), incubated at 37 ℃ for 30 minutes after dropping onto the sample surface, followed by slow washing with PBS 2-3 times to remove excess dye, and the stained hydrogel was observed under a confocal laser scanning microscope.

In the observation result, the Calcein-AM can be cracked into green fluorescent products by living cell esterases to mark living cells, PI can penetrate dead cells with damaged cell membranes and combine with DNA to display red fluorescence, the activity, distribution and biocompatibility of the cells in the hydrogel can be estimated through image analysis, the result is shown in figure 2, and the invention has good biocompatibility.

Example 4

The T10 spinal segment was exposed by laminectomy to experimental mice and a 2mm full-thickness transverse defect model was created with micro-scissors. The mesenchymal stem cell suspension (concentration 1× ⁶ cells/. Mu.L) is precisely injected into the semi-transversal injury central area of the spinal cord of the mouse by using a micro-injector (specification 33G) guided by a stereotactic instrument, the single-point injection volume is 2. Mu.L, after the cells are attached for 1 hour, the hydrogel prepared in the example 1 is slowly injected by the same route to cover the stem cell transplantation area, and the injection volume is 5. Mu.L. Mice were anesthetized at week 4 post-injury, spinal cord tissue pieces (including the injured area and tissue about 5mm each) were removed, fixed in 4% Paraformaldehyde (PFA) for 24 hours, and then dehydrated in 30% sucrose solution to tissue sink. Embedding the dehydrated tissue in OCT embedding agent, preparing transverse or sagittal slice with thickness of 10-20 μm on a frozen microtome, and attaching on a slide for later use. Sections were washed 3 times (5 min each) with PBS, permeabilized with 0.3% Triton X-100 for 10-15 min, then blocked with 5% BSA or normal goat serum for 1 h at room temperature to block non-specific binding, excess liquid was aspirated after blocking, primary antibody mix was added, incubated overnight at 4 ℃, washed 3 times with PBS the next day, fluorescent labeled secondary antibody was added, incubated in the dark for 1 h, washed 3 times with PBS again after incubation was completed, DAPI-containing blocking liquid-blocking sheet was added dropwise, and finally neuronal axons and astrocyte distribution and glial scar were recorded and photographed using confocal microscopy.

The observation result of the confocal microscope is shown in figure 4, the invention obviously promotes the differentiation of stem cells to neurons and the regeneration and repair of nerve tissues, the hydrogel is rapidly gelled to form a three-dimensional wrapping structure, the dynamic network pores of the hydrogel can provide anchoring sites for the stem cells, and simultaneously, the microenvironment is continuously regulated through a pH/ROS response mechanism, the mechanical stress damage caused by premixing the stem cells and the hydrogel is avoided through physical separation in the operation mode, and the barrier formed after gelation can effectively inhibit inflammatory cell infiltration and reduce the mechanical compression of the implant to surrounding tissues.

Fig. 3 (a) shows immunofluorescence staining of typical neuronal markers and astrocyte markers after 4 weeks of establishment of a semi-transversal model of spinal cord injury in mice, fig. 3 (b) shows statistical analysis of fluorescent intensity of astrocyte markers, and fig. 3 (c) shows statistical analysis of fluorescent intensity of neuronal cell markers, which shows that the material can effectively promote survival rate of stem cells in vivo in microenvironment and reduce secondary injury of grafts to spinal cord tissues, and compared with a pure injury group, the invention remarkably promotes regeneration and tissue repair of nerve tissues at the injury part, promotes extension and remyelination of nerve axons, and provides a better biological scaffold scheme for cell therapy of spinal cord injury.

The mice were scored at various time points for BMS and analyzed for the gait of mice Catwalk at the end stage, and the system quantitatively evaluates the motor function recovery and gait characteristics of the hind limbs of the mice as shown in fig. 5, where (a) in fig. 5 is a BMS scoring result graph and (b) in fig. 5 is a Catwalk gait analysis result graph. Compared with the injury group, the treatment group of the invention shows significantly higher BMS score (P < 0.05) at each postoperative time point, which indicates that the treatment group can effectively promote the recovery of the motor function after spinal cord injury, meanwhile, the gait parameters (including stride, support phase duration, footprint symmetry and the like) of the mice are significantly improved, the gait pattern is closer to that of a normal control group (P < 0.05), and the function improvement effect of the treatment group in spinal cord injury repair is further verified.

Example 5

The difference between this example and example 1 is that the drug loading process is carried out by dissolving the anti-inflammatory drugs dexamethasone and neurotrophic factor NGF in PBS buffer solution respectively, premixing with nano-silicon particles (SiNPs) for 30 minutes, achieving the initial loading by hydrogen bonding of hydroxyl groups on the surface of the silicon particles and drug molecules, then adding the drug-loaded nano-silicon particles into the premix of xyloglucan (OXG) and methacrylated Chitosan (CSMA), wherein the concentration of dexamethasone in the system is 0.5mg/mL, the concentration of neurotrophic factor NGF in the system is 50ng/mL, forming a double-network hydrogel through dynamic borate bond/Schiff base bond crosslinking, enabling the drug to be further embedded into a three-dimensional pore structure, and finally completing the drug double loading by 40 ℃ magnetic stirring (600 rpm,2 hours).

In vitro simulation of spinal cord injury microenvironment (pH 6.5+ROS 0.2mM acidic oxidative conditions and pH7.4+ROS was employed

0.05MM neutral recovery conditions) and the cumulative release rates of dexamethasone and NGF were quantitatively analyzed by High Performance Liquid Chromatography (HPLC).

The detection result is that the average value of the burst release amount of the drug in the first 6 hours after the ROS triggering is monitored by adopting a flow cytometry in the acute phase detection is 84%, and the NGF slow release curve in the repair phase is measured by ELISA, so that the daily average release amount is 4%. The dual-dynamic network constructed by using boric acid ester bonds and Schiff base bonds is combined with a PH/ROS dual-response mechanism, so that the explosive release of anti-inflammatory drugs (such as dexamethasone) in an acute phase (PH is less than or equal to 6.5+ROS is more than or equal to 0.2 mM) and the slow release of neurotrophic factors (NGF) in a repair phase (PH 7.4+ROS is less than or equal to 0.05 mM) are realized, and the stable response of the material in a complex physiological environment (such as intermittent ROS fluctuation) is kept.

The xyloglucan adopted in the invention is a mixed sugar, the component proportions of the xyloglucan from different sources are different, and the molecular sizes are also different. Depending on the parameters, the hydrogel construction conditions need to be explored to achieve optimal performance. The tamarind xyloglucan has a multi-branched structure and high active hydroxyl content, the biological adaptability of a dynamic network is obviously optimized, and the multi-branched structure of the xyloglucan from other sources has no advantage compared with the high active hydroxyl content, so that the same level of crosslinking density, biological activity and response sensitivity of the xyloglucan from the tamarind cannot be achieved.

Xyloglucan has significant advantages in central nerve repair such as spinal cord. The natural polysaccharide structure of the natural polysaccharide has excellent biocompatibility and degradability, can simulate the environment of a nerve extracellular matrix, and promotes neuron adhesion and axon directional growth. As a natural polymer from tamarind, xyloglucan can form a dynamic Schiff base bond with amino groups of chitosan after forming aldehyde groups through oxidation, and a pH/ROS double-response network is constructed to accurately match acid-base fluctuation and oxidative stress change of a spinal cord injury microenvironment. Compared with synthetic materials, the hydroxyl groups on the polysaccharide chains can enhance the water retention and lubricity of the hydrogel and reduce the tissue friction damage after implantation. In addition, the three-dimensional pore structure of the xyloglucan is more beneficial to nutrient delivery and metabolic waste removal, and when the xyloglucan is cooperated with nano silicon particles, a supporting network which is mechanically matched with spinal cord tissues can be formed, so that the morphological stability of a damaged part is effectively maintained.

Compared with the prior art, the invention has the following outstanding advantages:

1. In the prior art, although a single dynamic network design containing a boric acid ester bond or a Schiff base bond exists, the PH response range is limited (such as the PH response range is only effective in a neutral to alkaline environment), and dynamic self-adjustment cannot be realized under local acid/base fluctuation. The double dynamic bonds of the invention make hydrogel trigger reversible dissociation of Schiff base bond under acid (pH less than or equal to 6.5) condition by synergistic effect, and maintain network integrity under physiological pH (7.4) by stability of boric acid ester bond, thus solving the problem of mechanical strength collapse or excessive swelling caused by single bond response of traditional materials. In addition, the rigid supporting function of the nano silicon particles further enhances the structural stability of the network under the fluctuation of pH, and breaks through the contradiction between the stability and response sensitivity of the traditional system.

2. Although the prior art has a scheme of singly using a boric acid ester bond or a Schiff base bond, the core innovation of the invention is to realize the optimization and upgrading of the ROS response mechanism through the composite action of a dynamic synergistic network of the boric acid ester bond and the Schiff base bond and the nano silicon particles. The traditional single dynamic bond system has the defects of fixed response threshold, low self-healing efficiency and the like, and the structural stability can be maintained through bond recombination in a low-concentration ROS environment by the pH/ROS dual response coupling nano silicon rigid support function of the double dynamic bonds, and meanwhile, the improvement of swelling resistance and the integration of antibacterial functions are realized. The multicomponent synergistic effect enables the system to show continuous response capability and mechanical suitability which cannot be realized by the traditional single dynamic bond system in a complex physiological environment, and the system is different from the substantial innovation in the prior art.

3. Based on the scheme of the prior art containing boric acid ester bonds and Schiff base bonds, the self-healing capacity of the invention has remarkable innovation. The key is that the core problems of dynamic balance loss and mechanical mismatch in the traditional double bond system are solved by combining a synergistic response mechanism of double dynamic bonds (borate bonds/Schiff base bonds) with the physical support of the nano silicon particles. Specifically, existing double bond systems often only achieve simple bond coexistence, and cannot establish an adaptive balance mechanism of two dynamic bonds under the pH/ROS microenvironment, so that the healing efficiency and the mechanical recovery are contradictory (such as network collapse caused by excessive dissociation of a single bond under an acidic condition). The invention controls the dissociation threshold and recombination rate of two bonds, so that the Schiff base bond responds to ROS to trigger rapid recombination preferentially, and the borate bond maintains the stability of the network through the regulation of tetraborate ion concentration, thereby forming a staged self-healing mode. Meanwhile, the rigid supporting function of the nano silicon particles provides physical anchor points for dynamic bond recombination, so that the problem of structural dislocation in the traditional soft gel self-healing process is solved, and the synchronous improvement of the healing efficiency and the mechanical property is realized. The combined design of the double bond synergistic response and the physical reinforcement exceeds the performance boundary of the existing simple double bond system on the self-healing mechanism.

4. The core invention is that a dual dynamic network is constructed through the synergistic effect of borate bonds and Schiff base bonds, and the rigid support and multiple drug carrying sites of the nano silicon particles are combined, so that the time sequence control function which cannot be achieved by the traditional single dynamic bond system is realized. In the prior art, although a proposal containing double dynamic bonds exists, the cooperative response mechanism of the dynamic bonds is insufficient, and the time sequence precise release of the anti-inflammatory drug and the neurotrophic factor is difficult to realize in a complex physiological environment. The invention adjusts and controls the network porosity through the dynamic balance of double bonds, so that the drug release and the spinal cord injury repair stage are highly matched, the technical bottleneck of the traditional drug carrying system in time sequence adjustment and space positioning is broken through, and the invention is a substantial technical improvement which is not realized by the prior double bond-containing scheme.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. The microenvironment responsive spinal cord repairing hydrogel is characterized by being prepared from the following raw materials, by mass, 0.8-1.5 parts of xyloglucan oxide, 0.8-1.5 parts of methacrylic chitosan, 0.1-0.5 part of nano silicon particles and 0.7-2.0 parts of borax.

2. The microenvironment-responsive spinal cord repair hydrogel of claim 1, wherein the preparation material comprises 95.5-97.2 parts of PBS buffer.

3. The microenvironment-responsive spinal cord repair hydrogel of claim 1, wherein the preparation of the feedstock comprises anti-inflammatory drugs and/or neurotrophic factors;

the anti-inflammatory drug comprises one or more of methylprednisolone, dexamethasone and prednisolone, or the neurotrophic factor comprises one or more of NGF, BDNF and NT-3.

4. The micro-environment responsive spinal cord repair hydrogel according to claim 3, wherein the concentration of methylprednisolone in the micro-environment responsive spinal cord repair hydrogel is 0.5-5.0 mg/mL, or the concentration of dexamethasone in the micro-environment responsive spinal cord repair hydrogel is 0.1-2.0 mg/mL, or the concentration of prednisolone in the micro-environment responsive spinal cord repair hydrogel is 0.5-5 mg/mL;

or the concentration of the NGF in the microenvironment-responsive spinal cord repair hydrogel is 0.01-0.5 mug/mL, or the concentration of the BDNF in the microenvironment-responsive spinal cord repair hydrogel is 0.05-1.0 mug/mL, or the concentration of the NT-3 in the microenvironment-responsive spinal cord repair hydrogel is 0.02-0.8 mug/mL.

5. A method of preparing a microenvironment-responsive spinal cord repair hydrogel according to any one of claims 1-4, comprising the steps of:

And respectively adding the xyloglucan oxide and the methacrylic chitosan into a buffer solution, stirring at room temperature for reaction for 4-6 hours, and then sequentially adding the nano silicon particles and borax, stirring at 40-60 ℃ for reaction for 2-3 hours.

6. The method of claim 5, wherein the loading of the anti-inflammatory agent and/or the neurotrophic factor comprises one or more of physical adsorption, chemical bond encapsulation and in situ mixed loading;

Or, the physical adsorption method comprises the steps of combining hydroxyl or amino groups on the surfaces of the nano silicon particles with drug molecules through hydrogen bonds or electrostatic action to realize drug adsorption load;

Or the chemical bonding packaging method comprises the steps of packaging the medicine in a three-dimensional pore by a double-network structure constructed by dynamic boric acid ester bonds and Schiff base bonds;

Or the in-situ mixing loading method comprises directly blending with drug solution during hydrogel preparation, and embedding drug molecules into the network through crosslinking reaction;

Alternatively, the buffer is a PBS buffer.

7. The preparation method of the microenvironment-responsive spinal cord repairing hydrogel according to claim 5, which is characterized in that the preparation method of the xyloglucan comprises the steps of mixing sodium periodate and xyloglucan according to a mass ratio of 1 (2-5) to prepare a solution, stirring at room temperature for 4-6 hours, adding ethylene glycol to terminate the reaction, and dialyzing to obtain the xyloglucan oxide;

Or dialyzing substances with molecular weight cut-off of more than or equal to 3500Da, and freeze-drying to obtain the xyloglucan oxide.

8. The preparation method of the microenvironment responsive spinal cord repairing hydrogel according to claim 5, which is characterized in that the preparation method of the methacrylic chitosan comprises the steps of dissolving chitosan in acetic acid solution, adding methacrylic anhydride with the mass of 0.36-1.92 times of that of the chitosan into the chitosan, stirring and reacting for 6-10 hours at 60-80 ℃, adding alkaline substances to neutralize to pH=6.5-7, diluting, and dialyzing to obtain the methacrylic chitosan;

Or dialyzing substances with molecular weight cutoff of 8000-14000 Da, and freeze-drying to obtain methacrylic chitosan;

alternatively, the alkaline substance comprises an aqueous solution of sodium bicarbonate.

9. The preparation method of the microenvironment-responsive spinal cord repairing hydrogel according to claim 5, wherein the preparation method of the nano silicon particles comprises the steps of dispersing 3- [2- (2-amino ethylamino) ethylamino ] propyl-trimethoxysilane in 5-6 times of volume of ultrapure water, adding 0.24-0.30 times of 2-aminophenol of 3- [2- (2-amino ethylamino) ethylamino ] propyl-trimethoxysilane in the ultrapure water, stirring and reacting for 100-150min at 70-80 ℃, and dialyzing to obtain the nano silicon particles;

or dialyzing the substances with molecular weight cut-off of 400-600Da, and freeze-drying to obtain the nano-silicon particles.

10. Use of the microenvironment-responsive spinal cord repair hydrogel of any one of claims 1-4 in at least one of the following a 1) -a 5):

a1 Preparing a product that promotes spinal cord repair;

a2 Preparing a product for continuously removing micro-environmental ROS in the spinal cord repairing process;

a3 Preparing a product for continuously regulating the pH value of a microenvironment in the spinal cord repairing process;

a4 Preparing an antibacterial product in the spinal cord repair process;

a5 A product for preparing the sustained release medicine in the spinal cord repairing process.