CN115177792A - Preparation method of photo-crosslinking '4D' IPN magnetic response cartilage repair gradient hydrogel - Google Patents
Preparation method of photo-crosslinking '4D' IPN magnetic response cartilage repair gradient hydrogel Download PDFInfo
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Abstract
The invention relates to a preparation method of photo-crosslinking '4D' IPN magnetic-response cartilage repair gradient hydrogel, which takes methacrylic acid anhydride gelatin GelMA and natural high molecular polypeptide fibroin SF as main raw materials, embeds drug-loaded magnetic nanochains, makes the magnetic nanochains directionally migrate and arrange in an SF/GelMA composite solution system under the action of a constant magnetic field, makes the SF/GelMA composite solution system quickly crosslink and solidify into gel through a light driving system, and further crosslinks SF by micromolecules to prepare the photo-crosslinking '4D' IPN magnetic-response cartilage repair gradient hydrogel. The method is favorable for preparing the photo-crosslinking '4D' IPN magnetic response cartilage repair gradient hydrogel which has good biocompatibility, conforms to the mechanical strength of osteochondral and can promote osteochondral defect repair.
Description
Technical Field
The invention belongs to the field of composite materials, and particularly relates to a preparation method of photo-crosslinked '4D' IPN magnetic response cartilage repair gradient hydrogel.
Background
Cartilage damage caused by acute trauma, chronic strain, degenerative diseases, etc. is a common clinical condition. The cartilage tissue has a complex structure and does not contain structures such as blood vessels and lymphatic vessels, so that the self-repairing capacity of the cartilage is limited. At present, the main methods for treating cartilage defects are to adopt cartilage simulation implants or microfracture surgeries to accelerate tissue repair and regeneration, but the methods are invasive, the repair process is mostly uncontrollable, and the serious side effects are accompanied. Therefore, there is a need to further explore new cartilage repair or replacement materials.
In recent years, the development of new hydrogel materials based on the research of traditional hydrogel materials has become a research hotspot for repairing cartilage injuries. The hydrogel is a cross-linked network material formed by hydrophilic polymer after water swelling, has high water content, elasticity, flexibility, mechanical strength and porosity, and is suitable for loading drugs and cells. In the past, due to the limitation of technical conditions, the advantages of the hydrogel material in repairing osteochondral injuries cannot be effectively exerted. At present, new hydrogel materials prepared by new technologies such as modification and hybridization are increasingly gaining attention.
GelMA is one of the most commonly used hydrogel materials, has the characteristics of natural and synthetic biomaterials, has a three-dimensional structure suitable for cell growth and differentiation, excellent biocompatibility and cell reaction characteristics, and has good temperature-sensitive gel characteristics, degradability and mechanical property adjustability. The GelMA hydrogel can promote cell adhesion and chondrocyte proliferation, and induce the mesenchymal stem cells to differentiate into the chondrocytes. GelMA is cooperated with other materials (such as natural polymers, small molecules, metal ions and the like) to construct a multifunctional composite carrier under the physical, chemical and photo-crosslinking actions, so that the regeneration of cartilage can be promoted, and the method is one of the current strategies for cartilage repair.
SF is derived from degumming of silk and is a high molecular polypeptide composed of various amino acids. SF has the characteristics of good biocompatibility, biodegradability, high tensile strength and the like, and has been used in various biomedical fields, while photo-crosslinked fibroin hydrogels exhibit more stable mechanical properties.
The SF/GelMA interpenetrating network (IPN) hydrogel prepared by the improved method has greatly improved mechanical strength and degradation performance, and simultaneously shows high swelling ratio, excellent mechanical property and resistance to enzymatic degradation of collagenase. The IPN hydrogel has the performance which cannot be presented by a single polymer network, and the material performance can be regulated and controlled by regulating and controlling the proportion of the polymer. Therefore, the SF/GelMA IPN hydrogel is prepared by initiating crosslinking under a light driving system, the complementation of the properties of SF and GelMA can be realized, and an unexpected synergistic gain effect is achieved, so that the SF/GelMA IPN hydrogel has great potential in osteochondral repair.
In practical applications, a problem exists in geometrically static scaffolds made of either metallic or degradable materials, in that the formed tissue cannot dynamically change shape, which results in the inability to interact with dynamically changing adjacent tissues. Foreign researchers developed a new type of 4D hydrogel material, and the 4D material is similar to the 3D material, but the difference between them is that the 4D material can realize dynamic change of shape in a specific environment (such as water, light, temperature, etc.), i.e. the 4D material is a 3D material that can be geometrically changed.
Disclosure of Invention
The invention aims to provide a preparation method of photo-crosslinking '4D' IPN magnetic response cartilage repair gradient hydrogel, which is favorable for preparing the photo-crosslinking '4D' IPN magnetic response cartilage repair gradient hydrogel which has good biocompatibility, accords with the mechanical strength of osteochondral and can promote osteochondral defect repair.
In order to realize the purpose, the invention adopts the technical scheme that: a preparation method of photo-crosslinking '4D' IPN magnetic response cartilage repair gradient hydrogel comprises the steps of taking methacrylic acid anhydrization gelatin GelMA and natural high-molecular polypeptide fibroin SF as main raw materials, embedding a drug-loaded magnetic nano-chain, enabling the magnetic nano-chain to directionally migrate and arrange in an SF/GelMA composite solution system under the action of a constant magnetic field, enabling the SF/GelMA composite solution system to be rapidly crosslinked and solidified into gel through a light driving system, and further crosslinking SF by small molecules to prepare the photo-crosslinking '4D' IPN magnetic response cartilage repair gradient hydrogel.
Further, the method comprises the following steps:
(1) Treating silkworm cocoon with NaCO 3 Degumming the solution at 80-100 deg.C for 2-3 times, cleaning, and drying to obtain silk fiber; dissolving silk fiber in the ternary solution, stirring in a constant temperature pot for 2-4 hours to obtain a first mixed solution, and dialyzing the first mixed solution in a dialysis bag for 2-4 days; dissolving gelatin in PBS buffer solution, magnetically stirring for 2-4 hours, adding methacrylic anhydride to obtain a second mixed solution, and dialyzing the second mixed solution in a dialysis bag for 5-7 days; respectively carrying out freeze drying on the two mixed solutions to obtain SF and GelMA sponges;
(2) Constructing magnetic mesoporous silica microspheres Fe consisting of rough porous shells and movable magnetic cores by adopting an interface deposition and co-assembly method 3 O 4 @nSiO 2 Applying a constant magnetic field around the reaction vessel for 50-100s without mechanical stirring to induce the Fe obtained 3 O 4 @nSiO 2 Arranging the microspheres to form a one-dimensional magnetic nanochain;
(3) Mixing and stirring the mesoporous magnetic nanochain in the step (2) with a small molecular traditional Chinese medicine quercetin solution for 12-24 hours to obtain a quercetin-loaded magnetic nanochain; according to the mass ratio of 1: 100. 1:300 or 1:500 dissolving the nano-particles in ultrapure water and performing ultrasonic dispersion to obtain a drug-loaded magnetic nanochain solution;
(4) Dissolving the SF sponge in the step (1) in ultrapure water to obtain an SF solution, and dissolving GelMA sponge in a photocrosslinking agent LAP to obtain a GelMA solution;
(5) Mixing the SF solution and the GelMA solution obtained in the step (4) according to the proportion of 1: 2. 1:1 or 2:1, adding 75 mu L, 150 mu L or 225 mu L of the medicine-carrying magnetic nanochain solution uniformly dispersed by ultrasonic in the step (3); on the basis, 70-250 mu L of cross-linking agent ethylene glycol glycidyl ether EGDE is added, and the mixed solution is stirred uniformly;
(6) Adding a magnetic field to induce the directional migration arrangement of the magnetic nanochains in the mixed solution;
(7) Irradiating the mixed solution by blue light for 10-30 minutes, and then placing the mixed solution at normal temperature for crosslinking for 12-24 hours to obtain the photo-crosslinking 4D IPN magnetic response cartilage repair gradient hydrogel.
Further, in step (1), 0.5% NaCO was used 3 The mass ratio of the components in the solution and the ternary liquid is LiBr: c 2 H 5 OH：H 2 0=44:45:11, dissolving silk fiber in the ternary solution, and stirring in a constant-temperature water bath kettle for 2-4 hours.
Further, in the step (2), a constant magnetic field 80s was applied around the reaction vessel to induce Fe to be obtained 3 O 4 @nSiO 2 And arranging the microspheres to form a one-dimensional magnetic nano chain.
Further, in the step (3), 0.01g of quercetin is fully dissolved in 100mL of absolute ethyl alcohol under the condition of keeping out of the sun, then 0.01g of mesoporous magnetic nanochain is added, the mixture is stirred for 24 hours, and the drug-loaded magnetic nanochain is obtained after magnetic absorption and drying; according to the mass ratio of 1:3000 the drug-loaded magnetic nanochain is dissolved in ultrapure water and dispersed by ultrasound to obtain a drug-loaded magnetic nanochain solution for later use.
Further, in the step (4), SF sponge is dissolved in ultrapure water to obtain 15wt% SF, and GelMA sponge is dissolved in 0.25% LAP to obtain 15wt% GelMA.
Further, in step (5), 15wt% SF and 15wt% GelMA were mixed in a ratio of 2:1, mixing to obtain a 1mL mixed hydrogel system, and adding 75 mu L of medicine-carrying magnetic nanochain solution which is uniformly dispersed by ultrasonic; on this basis, 233. Mu.L of ethylene glycol glycidyl ether EGDE was added, and the mixed solution was stirred uniformly.
Further, in the step (7), the mixed solution is irradiated by blue light for 25 minutes and then is placed at the normal temperature for crosslinking for 12 hours, so that the photo-crosslinked "4D" IPN magnetic response cartilage repair gradient hydrogel is obtained.
Compared with the prior art, the invention has the following beneficial effects:
(1) Compared with the cartilage repair hydrogel with a single component, the SF/GelMA IPN hydrogel scaffold prepared by the method has greatly improved mechanical strength and degradation performance, and simultaneously shows high swelling rate, excellent mechanical property and resistance to enzymatic degradation of collagenase. The IPN hydrogel has the performance which cannot be presented by a single polymer network, can realize the regulation and control of the material performance by regulating and controlling the proportion of the polymer, can realize the complementation of the performance between SF and GelMA, achieves an unexpected synergistic effect, and further exerts great potential in the bone cartilage repair.
(2) According to the invention, the magnetic nano-chain is embedded in the SF/GelMA IPN hydrogel bracket, so that the SF/GelMA IPN hydrogel bracket generates a magnetic response dynamic deformation characteristic under the action of a certain magnetic field, and the defect that the traditional 3D material can not fully play a role in coordinating with surrounding healing tissues is overcome.
(3) According to the invention, the magnetic nanochains are embedded in the SF/GelMA IPN hydrogel support, and under the action of a constant magnetic field, the magnetic nanochains in the SF/GelMA IPN hydrogel system are subjected to directional migration, arrangement and aggregation, so that the support material presents magnetic responsiveness in different degrees and mechanical strength with different gradients, the mechanical properties of different structural regions of osteochondral tissues can be well simulated, and the difficulty that the microenvironment and the mechanical strength of different structural layers are difficult to be considered in the traditional osteochondral materials is overcome.
(4) The magnetic nanochain embedded in the SF/GelMA IPN hydrogel scaffold has a mesoporous structure, can be used as a drug controlled slow release system, prolongs the drug release time, and thus has a better treatment effect. The micromolecule traditional Chinese medicine quercetin capable of promoting proliferation and differentiation of cartilage cells is loaded in the magnetic nano-chain, so that compared with the traditional single cartilage support material, the cartilage defect repair can be accelerated and promoted.
Drawings
FIG. 1 is a flow chart of a method implementation of an embodiment of the present invention.
FIG. 2 is SEM images of different parts of photo-crosslinked "4D" IPN magnetic-responsive cartilage repair gradient hydrogel without drug-loaded magnetic nanochains prepared in the example of the present invention.
FIG. 3 is SEM images of different parts of photo-crosslinked "4D" IPN magnetic-responsive cartilage repair gradient hydrogel with drug-loaded magnetic nanochains prepared in the example of the invention.
FIG. 4 is a bacteriostatic effect diagram of photo-crosslinked "4D" IPN magnetic-response cartilage repair gradient hydrogel prepared in the example of the present invention.
Detailed Description
The invention is further explained below with reference to the drawings and the embodiments.
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. 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 application 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 example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As shown in fig. 1, this example provides a preparation method of a photo-crosslinked "4d" ipn magnetic-response cartilage repair gradient hydrogel, which uses methacrylic acid anhydrified gelatin GelMA and natural high-molecular polypeptide fibroin SF as main raw materials, embeds drug-loaded magnetic nanochains, makes the magnetic nanochains migrate and arrange in a SF/GelMA composite solution system directionally under the action of a constant magnetic field, makes the SF/GelMA composite solution system rapidly crosslink and solidify into gel through a light-driven system, and further crosslinks the SF with small molecules, thereby preparing the photo-crosslinked "4d" ipn magnetic-response cartilage repair gradient hydrogel which has good biocompatibility, meets the mechanical strength of osteochondral and can promote osteochondral defect repair.
Example 1
The photo-crosslinking '4D' IPN magnetic response cartilage repair gradient hydrogel provided by the embodiment comprises the following steps:
(1) High quality silkworm cocoon is treated with 0.5% NaCO 3 Degumming the solution for 3 times at 100 ℃, cleaning and drying to obtain silk fiber; dissolving silk fiber in ternary liquid (mass ratio LiBr: C) 2 H 5 OH：H 2 0=44:45:11 Stirring for 4 hours in a constant-temperature water bath to prepare a first mixed solution, and putting the first mixed solution into a dialysis bag for dialysis for 3 days. Dissolving gelatin in PBS buffer solution, magnetically stirring for 4 hours, adding methacrylic anhydride to obtain a second mixed solution, and dialyzing the second mixed solution in a dialysis bag for 6 days; and respectively carrying out freeze drying on the two mixed solutions to obtain SF and GelMA sponges.
(2) Constructing magnetic mesoporous silica microspheres (Fe) consisting of rough porous shells and movable magnetic cores by adopting an interface deposition and co-assembly method 3 O 4 @nSiO 2 ) Applying a constant magnetic field 80s around the reaction vessel to induce the Fe obtained, without mechanical stirring 3 O 4 @nSiO 2 And arranging the microspheres to form a one-dimensional magnetic nano chain.
(3) And (3) sufficiently dissolving 0.01g of quercetin in 100mL of absolute ethyl alcohol in dark, then adding 0.01g of magnetic nanochain, uniformly dispersing by ultrasonic, mixing and stirring the solution for 24 hours, and performing magnetic absorption and drying to obtain the quercetin-loaded magnetic nanochain. 0.01g of drug-loaded magnetic nanochain is dissolved in 3mL of deionized water and is dispersed uniformly by ultrasonic to obtain a drug-loaded magnetic nanochain solution.
(4) SF sponge is dissolved in ultrapure water in the dark to obtain 15wt% SF solution, and GelMA sponge is dissolved in 0.25% LAP to obtain 15wt% GelMA solution.
(5) 15wt% SF and 15wt% GelMA were mixed in 2:1, and adding 75 mu L of medicine-carrying magnetic nanochain solution which is uniformly dispersed by ultrasonic. On this basis, 233. Mu.L of ethylene glycol glycidyl ether (EGDE) was added, and the mixed solution was stirred uniformly. And (3) adding a magnetic field to induce the oriented arrangement of the magnetic nanochains in the mixed solution. And irradiating the mixed solution by blue light for 25 minutes, and then crosslinking for 12 hours at normal temperature to obtain the photo-crosslinking '4D' IPN magnetically-responsive cartilage repair gradient hydrogel.
Example 2
The photo-crosslinking '4D' IPN magnetic response cartilage repair gradient hydrogel provided by the embodiment comprises the following steps:
(1) High quality silkworm cocoon is treated with 0.5% NaCO 3 Degumming the solution for 3 times at 100 ℃, cleaning and drying to obtain silk fiber; dissolving silk fiber in ternary liquid (mass ratio LiBr: C) 2 H 5 OH：H 2 0=44:45:11 Stirring for 4 hours in a constant-temperature water bath to prepare a first mixed solution, and putting the first mixed solution into a dialysis bag for dialysis for 3 days. Dissolving gelatin in PBS buffer solution, magnetically stirring for 4 hours, adding methacrylic anhydride to obtain a second mixed solution, and dialyzing the second mixed solution in a dialysis bag for 6 days; and respectively carrying out freeze drying on the two mixed solutions to obtain SF and GelMA sponges.
(2) Constructing magnetic mesoporous silica microspheres (Fe) consisting of rough porous shells and movable magnetic cores by adopting an interface deposition and co-assembly method 3 O 4 @nSiO 2 ) Applying a constant magnetic field 80s around the reaction vessel to induce the Fe obtained, without mechanical stirring 3 O 4 @nSiO 2 And arranging the microspheres to form a one-dimensional magnetic nano chain.
(3) And (3) sufficiently dissolving 0.01g of quercetin in 100mL of absolute ethyl alcohol in dark, then adding 0.01g of magnetic nanochain, uniformly dispersing by ultrasonic, mixing and stirring the solution for 24 hours, and performing magnetic absorption and drying to obtain the quercetin-loaded magnetic nanochain. 0.01g of the drug-loaded magnetic nanochain is dissolved in 3mL of deionized water and dispersed uniformly by ultrasound to obtain a drug-loaded magnetic nanochain solution.
(4) And dissolving SF sponge in ultrapure water in the dark to obtain a 15wt% SF solution, and dissolving GelMA sponge in 0.25% LAP to obtain a 15wt% GelMA solution.
(5) 15wt% SF and 15wt% GelMA were mixed in 2:1, and adding 125 mu L of medicine-carrying magnetic nanochain solution which is uniformly dispersed by ultrasonic. On this basis, 233. Mu.L of ethylene glycol glycidyl ether (EGDE) was added, and the mixed solution was stirred uniformly. And (3) adding a magnetic field to induce the oriented arrangement of the magnetic nanochains in the mixed solution. And irradiating the mixed solution for 25 minutes by blue light, and then crosslinking for 12 hours at normal temperature to obtain the photo-crosslinked 4D IPN magnetically-responsive cartilage repair gradient hydrogel.
Example 3
The photo-crosslinking '4D' IPN magnetic response cartilage repair gradient hydrogel provided by the embodiment comprises the following steps:
(1) High quality silkworm cocoon is treated with 0.5% NaCO 3 Degumming the solution at 100 deg.C for 3 times, cleaning, and drying to obtain silk fiber; dissolving silk fiber in ternary liquid (mass ratio LiBr: C) 2 H 5 OH：H 2 0=44:45:11 Stirring for 4 hours in a constant-temperature water bath to prepare a first mixed solution, and putting the first mixed solution into a dialysis bag for dialysis for 3 days. Dissolving gelatin in PBS buffer solution, magnetically stirring for 4 hours, adding methacrylic anhydride to obtain a second mixed solution, and dialyzing the second mixed solution in a dialysis bag for 6 days; and respectively carrying out freeze drying on the two mixed solutions to obtain SF and GelMA sponges.
(2) Constructing magnetic mesoporous silica microspheres (Fe) consisting of rough porous shells and movable magnetic cores by adopting an interface deposition and co-assembly method 3 O 4 @nSiO 2 ) Applying a constant magnetic field 80s around the reaction vessel to induce the Fe obtained, without mechanical stirring 3 O 4 @nSiO 2 And arranging the microspheres to form a one-dimensional magnetic nano chain.
(3) And (3) sufficiently dissolving 0.01g of quercetin in 100mL of absolute ethyl alcohol in dark, then adding 0.01g of magnetic nanochain, uniformly dispersing by ultrasonic, mixing and stirring the solution for 24 hours, and performing magnetic absorption and drying to obtain the quercetin-loaded magnetic nanochain. 0.01g of drug-loaded magnetic nanochain is dissolved in 3mL of deionized water and is dispersed uniformly by ultrasonic to obtain a drug-loaded magnetic nanochain solution.
(4) And dissolving SF sponge in ultrapure water in the dark to obtain a 15wt% SF solution, and dissolving GelMA sponge in 0.25% LAP to obtain a 15wt% GelMA solution.
(5) 15wt% SF and 15wt% GelMA were mixed in 2:1, and adding 225 mu L of medicine-carrying magnetic nanochain solution which is uniformly dispersed by ultrasonic. On this basis, 233. Mu.L of ethylene glycol glycidyl ether (EGDE) was added, and the mixed solution was stirred uniformly. And (3) adding a magnetic field to induce the oriented arrangement of the magnetic nanochains in the mixed solution. And irradiating the mixed solution for 25 minutes by blue light, and then crosslinking for 12 hours at normal temperature to obtain the photo-crosslinked 4D IPN magnetically-responsive cartilage repair gradient hydrogel.
Example 4 (magnetic nanochain without drug loading)
The photo-crosslinking '4D' IPN magnetic response cartilage repair gradient hydrogel provided by the embodiment comprises the following steps:
(1) High quality silkworm cocoon is treated with 0.5% NaCO 3 Degumming the solution at 100 deg.C for 3 times, cleaning, and drying to obtain silk fiber; dissolving silk fiber in ternary liquid (mass ratio LiBr: C) 2 H 5 OH：H 2 0=44:45:11 Stirring for 4 hours in a constant-temperature water bath to prepare a first mixed solution, and putting the first mixed solution into a dialysis bag for dialysis for 3 days. Dissolving gelatin in PBS buffer solution, magnetically stirring for 4 hours, adding methacrylic anhydride to obtain a second mixed solution, and dialyzing the second mixed solution in a dialysis bag for 6 days; respectively carrying out freeze drying on the two mixed solutions to obtain SF and GelMA sponges;
(2) And dissolving SF sponge in ultrapure water in the dark to obtain a 15wt% SF solution, and dissolving GelMA sponge in 0.25% LAP to obtain a 15wt% GelMA solution.
(3) 15wt% SF and 15wt% GelMA were mixed in 2:1, adding 233 mu L of ethylene glycol glycidyl ether (EGDE), and stirring the mixed solution uniformly. And irradiating the mixed solution by blue light for 25 minutes, and then crosslinking for 12 hours at normal temperature to obtain the photo-crosslinking '4D' IPN magnetically-responsive cartilage repair gradient hydrogel.
Example 4 gel SEM images of different sites of the resulting photo-crosslinked "4d" ipn magnetic-responsive cartilage repair gradient hydrogel were prepared as shown in fig. 2. As can be seen from fig. 2, the positions where no nanochain exists form a uniform, irregular microporous structure.
Example 2 gel SEM images of different sites of the resulting photo-crosslinked "4d" ipn magnetic-responsive cartilage repair gradient hydrogel were prepared as shown in fig. 3. As can be seen from FIG. 3, the number of pores at the position where the nanochain is deposited becomes smaller, the pore size becomes smaller, the surface becomes rough, and growth factors can also be released, which is favorable for the adhesion and growth of cells.
The bacteriostatic effect of the photo-crosslinked "4d" ipn magnetic-responsive cartilage repair gradient hydrogel prepared in example 2 is shown in fig. 4. FIG. 4 shows that the photo-crosslinked "4D" IPN magnetic-responsive cartilage repair gradient hydrogel (SG 2) prepared in example 2 can effectively inhibit Staphylococcus aureus: (S.aureus) ((S.aureus))S) And Escherichia coli (E) (ii) a Under the condition of an external magnetic field, the SG2 carries out magnetic response drug release by drug-loaded magnetic nanochains, so that the two bacteria are more remarkably inhibited.
The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.
Claims (8)
1. A preparation method of photo-crosslinking '4D' IPN magnetic response cartilage repair gradient hydrogel is characterized in that methacrylic acid anhydrified gelatin GelMA and natural high molecular polymer polypeptide fibroin SF are used as main raw materials, drug-loaded magnetic nano-chains are embedded, the magnetic nano-chains are directionally migrated and arranged in an SF/GelMA composite solution system under the action of a constant magnetic field, the SF/GelMA composite solution system is rapidly crosslinked and cured to form gel through a light driving system, small molecules are further crosslinked with SF, and the photo-crosslinking '4D' IPN magnetic response cartilage repair gradient hydrogel is prepared.
2. The preparation method of the photo-crosslinked "4D" IPN magnetic-responsive cartilage repair gradient hydrogel according to claim 1, which is characterized by comprising the following steps:
(1) Treating silkworm cocoon with NaCO 3 Degumming the solution at 80-100 deg.C for 2-3 times, cleaning, and drying to obtain silk fiber; dissolving silk fiber in the ternary solution, stirring in a constant temperature pot for 2-4 hours to obtain a first mixed solution, and dialyzing the first mixed solution in a dialysis bag for 2-4 days; dissolving gelatin in PBS buffer solution, magnetically stirring for 2-4 hours, adding methacrylic anhydride to obtain a second mixed solution, and dialyzing the second mixed solution in a dialysis bag for 5-7 days; respectively carrying out freeze drying on the two mixed solutions to obtain SF and GelMA sponges;
(2) Constructing magnetic mesoporous silica microspheres Fe consisting of rough porous shells and movable magnetic cores by adopting an interface deposition and co-assembly method 3 O 4 @nSiO 2 Applying a constant magnetic field around the reaction vessel for 50-100s without mechanical stirring to induce the Fe obtained 3 O 4 @nSiO 2 Arranging the microspheres to form a one-dimensional magnetic nanochain;
(3) Mixing and stirring the mesoporous magnetic nanochain in the step (2) with a small molecular traditional Chinese medicine quercetin solution for 12-24 hours to obtain a quercetin-loaded magnetic nanochain; according to the mass ratio of 1: 100. 1:300 or 1:500 dissolving the nano-particles in ultrapure water and performing ultrasonic dispersion to obtain a drug-loaded magnetic nanochain solution;
(4) Dissolving the SF sponge in the step (1) in ultrapure water to obtain an SF solution, and dissolving GelMA sponge in a photocrosslinking agent LAP to obtain a GelMA solution;
(5) Mixing the SF solution and the GelMA solution obtained in the step (4) according to the proportion of 1: 2. 1:1 or 2:1, adding 75 mu L, 150 mu L or 225 mu L of the drug-loaded magnetic nanochain solution uniformly dispersed by the ultrasonic wave in the step (3); on the basis, 70-250 mu L of cross-linking agent ethylene glycol glycidyl ether EGDE is added, and the mixed solution is stirred uniformly;
(6) Adding a magnetic field to induce the directional migration arrangement of the magnetic nanochains in the mixed solution;
(7) Irradiating the mixed solution by blue light for 10-30 minutes, and then placing the mixed solution at normal temperature for crosslinking for 12-24 hours to obtain the photo-crosslinking 4D IPN magnetic response cartilage repair gradient hydrogel.
3. The method for preparing the photo-crosslinked "4D" IPN magnetically-responsive cartilage repair gradient hydrogel according to claim 2, wherein in the step (1), 0.5% NaCO is used 3 The mass ratio of the components in the solution and the ternary liquid is LiBr: c 2 H 5 OH：H 2 0=44:45:11, dissolving silk fiber in the ternary solution, and stirring in a constant-temperature water bath kettle for 2-4 hours.
4. The method for preparing photo-crosslinked "4D" IPN magnetically-responsive cartilage repair gradient hydrogel according to claim 2, wherein in the step (2), a constant magnetic field is applied for 80s around the reaction vessel to induce the obtained Fe 3 O 4 @nSiO 2 And arranging the microspheres to form a one-dimensional magnetic nano chain.
5. The preparation method of the photo-crosslinked "4D" IPN magnetic-responsive cartilage repair gradient hydrogel according to claim 2, wherein in the step (3), 0.01g of quercetin is fully dissolved in 100mL of absolute ethyl alcohol under the condition of keeping out of the sun, 0.01g of mesoporous magnetic nanochain is added, the mixture is stirred for 24 hours, and the drug-loaded magnetic nanochain is obtained after magnetic absorption and drying; according to the mass ratio of 1:3000 the drug-loaded magnetic nanochain is dissolved in ultrapure water and dispersed by ultrasound to obtain a drug-loaded magnetic nanochain solution for later use.
6. The method for preparing the photo-crosslinked "4D" IPN magnetic-responsive cartilage repair gradient hydrogel according to claim 2, wherein in the step (4), SF sponge is dissolved in ultra-pure water to obtain 15wt% SF, and GelMA sponge is dissolved in 0.25% LAP to obtain 15wt% GelMA.
7. The method for preparing the photo-crosslinked "4D" IPN magnetic-responsive cartilage repair gradient hydrogel according to claim 2, wherein in the step (5), 15wt% SF and 15wt% GelMA are mixed according to a ratio of 2:1, mixing to obtain a 1mL mixed hydrogel system, and adding 75 mu L of medicine-carrying magnetic nanochain solution which is uniformly dispersed by ultrasonic; on this basis, 233. Mu.L of ethylene glycol glycidyl ether EGDE was added, and the mixed solution was stirred uniformly.
8. The method for preparing the photo-crosslinked "4D" IPN magnetically-responsive cartilage repair gradient hydrogel according to claim 2, wherein in the step (7), the photo-crosslinked "4D" IPN magnetically-responsive cartilage repair gradient hydrogel is obtained by irradiating the mixed solution with blue light for 25 minutes and then crosslinking the solution at room temperature for 12 hours.
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