Preparation method of composite hydrogel scaffold, composite hydrogel scaffold and application thereof

Abstract

The application provides a preparation method of a composite hydrogel scaffold, the composite hydrogel scaffold and application thereof, and the method comprises the following steps: placing a specific mold containing a mixed solution of a hydrogel precursor liquid and a plurality of superparamagnetic particles in a non-uniform magnetic field, driving the superparamagnetic particles in the mixed solution to arrange in a concentration gradient in a set direction through a magnetic field induction technology, and performing radiation on the high-concentration side of the superparamagnetic particles in the mixed solution through a radiation cross-linking technology to obtain a first hydrogel scaffold; adding the hydrogel precursor liquid to the low-concentration side of the superparamagnetic particles in the specific mold, and forming a second hydrogel scaffold which is cross-linked with the first hydrogel scaffold through an interface, so as to obtain the composite hydrogel scaffold after demolding. The composite hydrogel scaffold prepared by the application not only can adapt to the natural chemical component gradient of the cartilage tissue, but also can improve the gap problem caused by material stacking, so as to promote bone repair.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological materials, in particular to a preparation method of a composite hydrogel scaffold, the composite hydrogel scaffold and application thereof. BACKGROUND

[0002] Osteoarthritis is a chronic joint disease that can cause damage to the cartilage and surrounding tissues. Current research shows that hydrogels can be used for cartilage repair caused by osteoarthritis. Considering that the cartilage tissue is formed by multiple layers such as the cartilage layer on the outer surface and the subchondral bone at the bottom, the treatment of osteoarthritis often needs to consider the treatment and repair of both the supracartilage (cartilage layer) and the subchondral bone.

[0003] The Chinese patent application with the application number 2024106554692 and the title "Magnetic hydroxyapatite-based composite hydrogel scaffold and its preparation method and application" proposes a hydrogel scaffold for repairing subchondral bone. However, the chemical composition and its distribution in each layer of the subchondral bone are not the same, for example, there is a natural chemical composition gradient of hydroxyapatite in the cartilage tissue, and the bone repair effect of the prepared hydrogel scaffold is limited. In view of the chemical gradient existing in the cartilage tissue, the current main method is to stack different materials to adapt to the complex structure of the cartilage tissue, which greatly increases the difficulty of cartilage tissue repair and reduces the treatment effect. At the same time, the use of multiple materials for stacking also greatly increases the difficulty of surgery during implantation.

[0004] Further, the supracartilage and the subchondral bone have completely different compositions and thus completely different properties, which also brings great challenges to the design and use of implant materials for repair. The current common solution is to implant two materials during bone treatment, i.e., the upper layer uses a material that can promote the repair of the supracartilage, and the lower layer uses a material that can support the regeneration and repair of the subchondral bone. Although this scheme can take into account the respective needs of the upper and lower bone repair, the two materials are usually implanted by simple stacking, which inevitably causes interfaces and gaps between the materials during use, which is extremely unfavorable for bone repair. At the same time, the implantation of two materials separately also increases the difficulty of surgery and increases the risk faced during the surgical process, making it a major obstacle for hydrogel materials to move towards actual clinical application.

[0005] Therefore, there is an urgent need for a new type of hydrogel scaffold for bone treatment and its preparation method, so that the prepared hydrogel scaffold can not only adapt to the natural chemical composition gradient of the cartilage tissue, but also improve the gap problem caused by material stacking, thereby improving the bone repair effect while reducing the difficulty of implantation repair. SUMMARY

[0006] In view of this, the application provides a preparation method of a composite hydrogel scaffold, the composite hydrogel scaffold and application thereof, which can adapt to the chemical component gradient of the cartilage tissue, solve the gap problem caused by material stacking, and promote bone repair.

[0007] One aspect of the application provides a preparation method of a composite hydrogel scaffold, which comprises the following steps: placing a specific mold containing a mixed solution of a hydrogel precursor liquid and a plurality of superparamagnetic particles in a non-uniform magnetic field, driving the superparamagnetic particles in the mixed solution to arrange in a concentration gradient in a set direction through a magnetic field induction technology, and performing radiation on the high-concentration side of the superparamagnetic particles in the mixed solution through a radiation crosslinking technology to obtain a first hydrogel scaffold in which the high-concentration side of the superparamagnetic particles is completely crosslinked and the low-concentration side of the superparamagnetic particles is not completely crosslinked.

[0008] The hydrogel precursor liquid is added to the low-concentration side of the superparamagnetic particles in the specific mold, and the added hydrogel precursor liquid is completely crosslinked through the radiation crosslinking technology to form a second hydrogel scaffold that is crosslinked with the first hydrogel scaffold at an interface, so that the composite hydrogel scaffold is demolded.

[0009] In some embodiments of the application, before the mixed solution is formed, the method further comprises: performing drug loading treatment on the superparamagnetic particles.

[0010] Before the hydrogel precursor liquid is added to the low-concentration side of the superparamagnetic particles in the specific mold, the method further comprises: adding a drug to the added hydrogel precursor liquid; and

[0011] Before the specific mold containing the mixed solution is placed in the non-uniform magnetic field, the method further comprises: performing defoaming treatment on the mixed solution containing the hydrogel precursor liquid and the plurality of superparamagnetic particles.

[0012] In some embodiments of the application, the drug loading treatment on the superparamagnetic particles comprises:

[0013] The superparamagnetic particles are soaked in a solution containing a drug, and after a set soaking time, centrifugal, washing and drying treatments are performed to obtain superparamagnetic particles loaded with the drug.

[0014] In some embodiments of the application, the radiation crosslinking technology comprises a light crosslinking technology or a temperature crosslinking technology.

[0015] For the light crosslinking technology, the wavelength of the radiation light is 100-500 nm, the radiation time is 0-3600 s and is not 0; for the temperature crosslinking technology, the radiation temperature ranges from 0 to 100 DEG C and is not 0, and the radiation time is 0-3600 s and is not 0.

[0016] In some embodiments of the present application, the superparamagnetic particles include magnetic nanoparticles with superparamagnetism, or hollow porous magnetic hydroxyapatite microsphere particles formed by hydroxyapatite particles and magnetic nanoparticles with superparamagnetism; wherein the magnetic nanoparticles with superparamagnetism include ferroferric oxide nanoparticles, ferrous oxide nanoparticles, manganese ferrite nanoparticles or manganese oxide nanoparticles, the mass percentage of hydroxyapatite in the magnetic hydroxyapatite microsphere particles is 50-95%, the mass percentage of the magnetic nanoparticles with superparamagnetism is 5-50%, and the particle size of the magnetic hydroxyapatite microsphere particles is 1 nm-10 μm.

[0017] In some embodiments of the present application, the height of the second hydrogel scaffold is 1.5-2 mm.

[0018] The ratio of the hydrogel precursor solution to the superparamagnetic particles in the mixed solution is 1 mL: 0-15 mg and not 0; and

[0019] The non-uniform magnetic field is provided by a neodymium-iron-boron magnet or an electromagnet.

[0020] Another aspect of the present application provides a composite hydrogel scaffold prepared by the preparation method of any of the above embodiments, comprising a first hydrogel scaffold and a second hydrogel scaffold which is interfacially crosslinked with the first hydrogel scaffold; the first hydrogel scaffold comprises a first hydrogel scaffold body and superparamagnetic particles arranged in a concentration gradient in a set direction inside the first hydrogel scaffold body; wherein the superparamagnetic particles arranged in a concentration gradient are formed by placing a plurality of superparamagnetic particles in a non-uniform magnetic field using a magnetic field induction technique; the first hydrogel scaffold is formed by irradiating the high-concentration side of the superparamagnetic particles using a radiation crosslinking technique;

[0021] The second hydrogel scaffold is located on the low-concentration side of the superparamagnetic particles of the first hydrogel scaffold.

[0022] In some embodiments of the present application, the superparamagnetic particles and the second hydrogel scaffold are loaded with a drug to achieve sustained release of the drug.

[0023] In some embodiments of the present application, the radiation crosslinking technique includes a photo-crosslinking technique or a temperature-crosslinking technique.

[0024] In the case of the photo-crosslinking technique as the radiation crosslinking technique, the hydrogel precursor solution includes a photo-initiator and a hydrogel polymer for photo-crosslinking mixed in a set ratio; in the case of the temperature-crosslinking technique as the radiation crosslinking technique, the hydrogel precursor solution is a modified hydrogel polymer for temperature-crosslinking.

[0025] The water gel polymer for photo-crosslinking includes glycidyl methacrylate modified hyaluronic acid GMHA, silk fibroin, chitosan or agarose; and the modified water gel polymer for temperature crosslinking includes poly N-isopropyl acrylamide or chitosan.

[0026] Another aspect of the present application provides the use of the composite hydrogel scaffold prepared by the preparation method of any one of the above embodiments in cartilage repair in bone treatment or the use of nuclear magnetic resonance for observing the repair condition of the implant site in bone treatment.

[0027] The preparation method of the composite hydrogel scaffold, the composite hydrogel scaffold and the application thereof provided by the present application form the first hydrogel scaffold with the superparamagnetic particles arranged in a concentration gradient by using the magnetic field induction technology, so as to adapt to the natural chemical component gradient in the cartilage tissue, and obtain the second hydrogel scaffold which is interfacially crosslinked with the first hydrogel scaffold by controlling the radiation crosslinking technology, so as to improve the gap and friction caused by the material stacking, thereby improving the repair effect of bone treatment and reducing the difficulty of surgical implantation of the repair material.

[0028] Additional advantages, objects, and features of the application will be set forth in part by the description that follows, and will become apparent to those skilled in the art upon examination of the following detailed description and drawings in which

[0029] Those skilled in the art will appreciate that the objects and advantages of the application can be implemented without regard to the specific details of the following description and drawings, and that the application can be implemented in various ways, as will be apparent to those skilled in the art upon examination of the following detailed description and drawings. BRIEF DESCRIPTION OF DRAWINGS

[0030] The drawings described herein are intended to provide a further understanding of the present application, form a part of the specification, and are not intended to limit the present application. In the drawings:

[0031] Figure 1 The flowchart of the preparation of the water gel with a concentration gradient by the stacking method in an embodiment of the present application.

[0032] Figure 2 The flowchart of the preparation method of the composite hydrogel scaffold in an embodiment of the present application.

[0033] Figure 3 The schematic diagram of the composite hydrogel scaffold in an embodiment of the present application.

[0034] Figure 4 The comparison diagram of the bone repair effect of the composite hydrogel scaffold in an embodiment of the present application. DETAILED DESCRIPTION

[0035] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be described in further detail below with reference to the embodiments and drawings. Here, the illustrative embodiments of the present application and their descriptions are used to explain the present application but are not intended to limit the present application.

[0036] It should also be noted that, in order to avoid the present application being obscured by unnecessary details, only structures and / or processing steps closely related to the solutions according to the present application are shown in the drawings, and other details less related to the present application are omitted.

[0037] It should be emphasized that the term "comprises / comprising" when used in this text specifies the presence of stated features, elements, steps or components but does not preclude the presence or addition of one or more other features, elements, steps or components.

[0038] Hereinafter, embodiments of the present application will be described with reference to the accompanying drawings. In the drawings, the same reference numerals represent the same or similar components, or the same or similar steps.

[0039] Since cartilage tissue has a natural chemical composition gradient, hydrogel materials with a concentration gradient can be used to treat osteoarthritis. One of the commonly used methods for preparing hydrogels with a concentration gradient is the stacking method, which does not require additional equipment, only needs to configure pre-prepared liquids with different solute concentrations to prepare hydrogel materials for each layer, and then stacks them layer by layer to obtain materials with a preset structure. The preparation process is as shown in Figure 1 However, the hydrogel prepared by the stacking method is prone to produce interfaces and voids between the stacked layers, not only limiting the repair effect, but also increasing the difficulty of surgery.

[0040] In view of the positions of the upper and lower bones of the cartilage in the treatment of osteoarthritis and their respective requirements for materials, the application designs a composite hydrogel scaffold (CHS) and a preparation method thereof. Before gel formation (i.e., cross-linking and solidification), the superparamagnetic particles in the hydrogel precursor liquid (or hydrogel precursor solution) are induced by an external magnetic field to form a concentration gradient, and a first hydrogel scaffold is formed by a modified cross-linking method, thereby solving the problem that the regeneration of the cartilage layer in the cartilage tissue is affected by the chemical component gradient such as hydroxyapatite. At the same time, the application adds a second hydrogel scaffold which is cross-linked with the first hydrogel scaffold at the interface on one side of the first hydrogel scaffold which is completely cross-linked on the high-concentration side of the superparamagnetic particles and incompletely cross-linked on the low-concentration side of the superparamagnetic particles (which can be arranged opposite to the high-concentration side of the superparamagnetic particles), so as to improve the gap problem caused by the stacking of different repair materials for the upper and lower bones of the cartilage. A new composite hydrogel scaffold with a concentration gradient can be prepared by the above process, which can be used for the treatment and repair of osteoarthritis. In addition, the composite hydrogel scaffold prepared by the application can achieve good cartilage repair effect while providing the function of real-time observation of the hydrogel scaffold implantation by using magnetic resonance imaging (Magnetic Resonance Imaging, MRI, also known as magnetic resonance imaging).

[0041] Concentration gradient is a phenomenon widely existing in nature, which represents the difference in the concentration of solute or dispersed particles in space. For example, the oxygen and ATP in cells, the migration of white blood cells in the inflammation process, and the migration of sperm in the process of fertilization of ova or embryonic development are all triggered by concentration gradient. In addition to the concentration gradient in solution, concentration gradient also appears in the "solid environment" in nature. For example, the spatial difference in the degree of mineralization and the amount of collagen plays a core role in the structure of tissues such as tendons, human dentin and cartilage. This gradient plays a mediating role between materials of different moduli, and has significant crack propagation resistance and contact deformation capacity. Based on this concept, researchers have designed and prepared many new composite materials, which are generally referred to as "concentration gradient materials".

[0042] The novel composite hydrogel scaffold proposed in the present application can be divided into a first hydrogel scaffold (including a first hydrogel scaffold body and a plurality of superparamagnetic particles) and a second hydrogel scaffold. Considering that the concentration gradient of the hydrogel material itself is difficult to control, and previous studies have shown that magnetic particles can help promote cartilage growth, therefore, in order to adapt to the natural chemical component gradient existing in the subchondral bone, the present application can use magnetic field induction technology to make the first hydrogel scaffold for promoting subchondral bone repair contain superparamagnetic particles with a concentration gradient; and the second hydrogel scaffold does not contain superparamagnetic particles, and only uses hydrogel material to promote suprachondral bone repair. That is, the first hydrogel scaffold can be prepared from a mixed solution including a hydrogel precursor solution and a plurality of superparamagnetic particles, and the first hydrogel scaffold can be prepared from only a hydrogel precursor solution. In addition, by improving the cross-linking method, the present application can establish an interfacial cross-linking between the first hydrogel scaffold and the second hydrogel scaffold, thereby reducing the difficulty of surgery while promoting the repair of subchondral and suprachondral bones.

[0043] In some embodiments of the present application, the present application combines the advantages of the stacking method and the magnetic field induction method, and designs an improved preparation method of a composite hydrogel scaffold with a concentration gradient, which can include the following steps:

[0044] A specific mold containing a mixed solution (or mixed suspension) of a hydrogel precursor solution and a plurality of superparamagnetic particles is placed in a non-uniform magnetic field, and the superparamagnetic particles in the mixed solution are arranged in a concentration gradient in a set direction by magnetic field induction technology, and radiation cross-linking technology is used to irradiate the high-concentration side of the superparamagnetic particles in the mixed solution, to obtain a first hydrogel scaffold in which the high-concentration side of the superparamagnetic particles is completely cross-linked and the low-concentration side of the superparamagnetic particles is not completely cross-linked; the hydrogel precursor solution is added to the low-concentration side of the superparamagnetic particles in the specific mold, and the added hydrogel precursor solution is completely cross-linked by using radiation cross-linking technology, to form a second hydrogel scaffold with interfacial cross-linking with the first hydrogel scaffold, thereby obtaining a composite hydrogel scaffold.

[0045] Specifically, the process of the method for preparing the composite hydrogel scaffold according to any one of the above embodiments is as follows: as shown in FIG. 1, a specific mold is prepared, and a mixed solution (or mixed suspension) of a hydrogel precursor solution and a plurality of superparamagnetic particles is prepared. Figure 2As shown, the mixed hydrogel precursor liquid and a plurality of superparamagnetic particles are mixed, and the mixture is added to a specific mold; an external magnetic field source is used to provide a non-uniform magnetic field, and the specific mold loaded with the mixture is placed behind the non-uniform magnetic field, so that the superparamagnetic particles in the mixture move in the uncrosslinked mixture under the action of the magnetic force (different magnetic field strengths at different positions) and are arranged in a concentration gradient in a set direction; after the arrangement of the superparamagnetic particles reaches the expectation, an external radiation source is used to radiate the high-concentration side of the superparamagnetic particles, and a first hydrogel scaffold in which the high-concentration side of the superparamagnetic particles is completely crosslinked and the low-concentration side of the superparamagnetic particles is not completely crosslinked is obtained by crosslinking and solidification. The hydrogel precursor liquid is added to the low-concentration side of the superparamagnetic particles in the first hydrogel scaffold (the hydrogel precursor liquid is added to the specific mold), and the uncrosslinked bonds in the low-concentration side of the superparamagnetic particles in the first hydrogel scaffold can be crosslinked and solidified by the radiation crosslinking technology, and finally the composite hydrogel scaffold of the present application is obtained after demolding. That is, the composite hydrogel scaffold proposed in the present application can include a first hydrogel scaffold, and a second hydrogel scaffold that is crosslinked with the first hydrogel scaffold at the interface. The first hydrogel scaffold includes a first hydrogel scaffold body and superparamagnetic particles arranged in a concentration gradient in a set direction inside the first hydrogel scaffold body; and the second hydrogel scaffold is located at the low-concentration side of the superparamagnetic particles in the first hydrogel scaffold.

[0046] Specifically, for the mixture formed by the magnetic component and the hydrogel precursor liquid, the movement of the magnetic component can be controlled by using an external magnetic field to affect the arrangement of the magnetic particles, thereby realizing the preparation of a concentration gradient material, that is, a hydrogel with a concentration gradient can be prepared by using a magnetic field induction method. Therefore, in the present application, a plurality of superparamagnetic particles are placed in a non-uniform magnetic field, the movement of the superparamagnetic particles is controlled by using a magnetic field induction technology, the superparamagnetic particles are arranged in a concentration gradient, and after the arrangement of the superparamagnetic particles reaches the expectation, the hydrogel is partially formed by crosslinking (a first hydrogel scaffold in which the high-concentration side of the superparamagnetic particles is completely crosslinked and the low-concentration side of the superparamagnetic particles is not completely crosslinked) by using a crosslinking method, and at this time the superparamagnetic particles can be fixed by the first hydrogel scaffold and no longer move. The composite hydrogel scaffold proposed in the present application can be as shown. Figure 3 In addition, the standing time of the mixture for preparing the first hydrogel scaffold in the non-uniform magnetic field in the present application needs to be determined according to various factors such as the magnetic field strength, for example, the standing time can be 2 min, 5 min or 30 min, etc., which is not limited in the present application.

[0047] As an example, the inhomogeneous magnetic field in the present application is provided by a neodymium-iron-boron magnet or an electromagnet when inducing the magnetic field, for example, the neodymium-iron-boron magnet can be placed on either side of a specific mold (loaded with a mixed solution including a hydrogel precursor solution and a plurality of superparamagnetic particles) so that the superparamagnetic particles in the specific mold move due to the different magnetic field strengths at different positions of the inhomogeneous magnetic field. The above is only an example, and the present application does not specifically limit the external magnetic field source providing the inhomogeneous magnetic field, and any magnetic field source capable of providing an inhomogeneous magnetic field is suitable for the magnetic field induction of the present application. The concentration of the superparamagnetic particles in each position in the present application can be the same as or different from the concentration of the corresponding position in the cartilage tissue, and a concentration gradient arrangement is formed.

[0048] In some embodiments of the present application, the superparamagnetic particles are magnetic particles with superparamagnetic properties, such as iron oxide particles, magnetite particles, manganese ferrite particles, or manganese oxide particles, etc. The superparamagnetic particles arranged in a concentration gradient in the present application can be composed of any substance with superparamagnetic properties, and the above-mentioned iron oxide is only an example, and the present application is not limited thereto. Moreover, in order to facilitate the regulation of the arrangement position of the particles by the applied inhomogeneous magnetic field, the superparamagnetic particles can be nanoscale particles, for example, the particle size of the superparamagnetic particles in the present application can be in the range of 1-100 nm. In addition, there is a concentration gradient of hydroxyapatite in the subchondral bone, and in order to conform to the composition of the current cartilage tissue as much as possible, the superparamagnetic particles in the present application can be hollow porous magnetic hydroxyapatite microsphere particles formed by hydroxyapatite particles and magnetic nanometer particles with superparamagnetic properties. The present application does not specifically limit the preparation method of the magnetic hydroxyapatite microsphere particles, which can be obtained by using existing preparation techniques, for example, co-precipitation or biomimetic mineralization, etc. The mass percentage of hydroxyapatite in the magnetic hydroxyapatite microsphere particles can be 50-95%, the mass percentage of the magnetic nanometer particles with superparamagnetic properties in the magnetic hydroxyapatite microsphere particles can be 5-50%, and the magnetic hydroxyapatite microsphere particles have a spherical or spherical-like structure, and the particle size thereof can be 1 nm-10 μm. In order to ensure the effect of subsequent bone repair, the superparamagnetic particles in the present application can use magnetic materials with good biocompatibility.

[0049] As an example, the cross-linking method in the present application can be a radiation cross-linking technology, or other existing cross-linking methods. Since the composite hydrogel scaffold prepared in the present application is used for bone repair, in order to precisely regulate the composition of the hydrogel to promote cartilage regeneration, the present application can use a radiation cross-linking technology which has less effect on the composition and materials of the hydrogel scaffold to form the hydrogel. The following is described by taking the radiation cross-linking technology as an example.

[0050] In some embodiments of the present application, to prepare a second hydrogel scaffold which is cross-linked at the interface with the first hydrogel scaffold, the present application can use the radiation cross-linking technology to irradiate the side with high concentration of superparamagnetic particles in the first hydrogel scaffold, so that the side with high concentration of superparamagnetic particles is completely cross-linked and the side with low concentration of superparamagnetic particles is not completely cross-linked. This is because the closer to the radiation source, the higher the radiation energy and the faster the gel forming speed. When irradiating from the side with high concentration of superparamagnetic particles, there can be more un-cross-linked hydrogen bonds, free radicals or ions in the side with low concentration of superparamagnetic particles. When the second hydrogel scaffold is prepared by adding the hydrogel precursor solution to the side with low concentration of superparamagnetic particles of the first hydrogel scaffold, cross-linking can be achieved between the added hydrogel precursor solution and the first hydrogel scaffold.

[0051] Further, the radiation cross-linking technology can use various radiation to initiate cross-linking reaction between long polymer chains, including light radiation, gamma radiation or temperature radiation. If the present application uses light cross-linking technology, a specific mold for loading the mixture of hydrogel precursor solution and superparamagnetic particles can be used in the process of forming the first hydrogel scaffold, so that the light can irradiate from the side with high concentration of superparamagnetic particles (for example, the specific mold is made of light-transmitting material on the opposite sides in a certain direction). The radiation source can be a light source (such as ultraviolet light source or other light source), and the hydrogel precursor solution can include light initiator and hydrogel polymer for light cross-linking mixed in a certain proportion. If temperature cross-linking technology is used, the specific mold for loading the mixture of hydrogel precursor solution and superparamagnetic particles cannot be made of heat insulation material during the cross-linking process of the first hydrogel scaffold, and a material that can transmit heat should be used to make the specific mold. The radiation source is a heat source, and the hydrogel precursor solution can be a modified hydrogel polymer for temperature cross-linking, for example, the hydrogel precursor solution can be a precursor solution of temperature-sensitive hydrogel. If gamma radiation technology is used, the specific mold should be made of material that can be penetrated by gamma rays. In addition, the shape of the specific mold is not specifically limited in the present application, and can be determined according to the treatment method and treatment effect.

[0052] As an example, the set ratio of the photoinitiator and the hydrogel polymer in the hydrogel precursor liquid can be 0-30%wt and not 0. The photoinitiator is used to generate free radicals or cations, etc. based on the radiation light in the ultraviolet light region or the visible light region, thereby initiating the monomer polymerization cross-linking and curing compound, which can include lithium phenyl (2, 4, 6-trimethylbenzoyl) phosphinate (Lithium Phenyl (2, 4, 6-trimethylbenzoyl) phosphinate, LAP) or photoinitiator I2959, etc. The present application does not specifically limit the material type of the photoinitiator, which can be any substance with photoinitiating properties. In addition, the hydrogel polymer mentioned in the present application refers to an intermediate before the hydrogel is cross-linked and cured, which can be used to form a hydrogel. The hydrogel polymer for photo-crosslinking includes glycidyl methacrylate-modified hyaluronic acid (glycidyl methacrylate-modified hyaluronic acid, GMHA), silk fibroin, chitosan or agarose, etc. High molecular materials that can form hydrogels by photo-crosslinking, and modified hydrogel polymers for temperature cross-linking can include poly-N-isopropyl acrylamide or chitosan, etc. The specific materials of the above-mentioned hydrogel polymers for photo-crosslinking and hydrogel polymers for temperature cross-linking are only examples, and the present application is not limited thereto.

[0053] In some embodiments of the present application, for the photo-crosslinking technology, the wavelength of the radiation light can be 100-500 nm, and the radiation time is 0-3600 s and not 0. For example, the present application can use ultraviolet light of 100-400 nm as a radiation light source for radiation, or visible light of 400-500 nm (for example, blue light with a wavelength of 405 nm) as a radiation light source, and the radiation time can be 30 s, 60 s, 2 min, 5 min, 15 min, 30 min, 1 h, 1.5 h or 2 h, etc. For the temperature cross-linking technology, the radiation temperature ranges from 0-100°C and is not 0, and the radiation time is 0-3600 s and is not 0, for example, the radiation temperature can be 50°C, 60°C, 80°C or 100°C, etc., and the radiation time can be 30 s, 60 s, 2 min, 5 min, 15 min, 30 min, 1 h, 1.5 h or 2 h, etc.

[0054] As an example, for repairing the supracartilage bone by using the composite hydrogel scaffold prepared in the present application, the height of the second hydrogel scaffold in the composite hydrogel scaffold can be designed as 1.5-2 mm. The height of the second hydrogel scaffold mentioned above is only an example, and can be set according to individual differences or based on the height ratio of the first hydrogel scaffold to the second hydrogel scaffold, and the present application is not limited thereto. For example, according to the specific situation of the required repair site and the defect, the height of the second hydrogel scaffold can be 2 mm, 1.7 mm and 1.5 mm, respectively. Further, although the first hydrogel scaffold body and the second hydrogel scaffold can both be formed from the hydrogel precursor solution, the hydrogel precursor solution used for preparing the first hydrogel scaffold and the hydrogel precursor solution used for preparing the second hydrogel scaffold can be made of different components, and the first hydrogel scaffold and the second hydrogel scaffold can be cured by using different radiation methods when the radiation crosslinking technology is used, that is, even if the first hydrogel scaffold and the second hydrogel scaffold are crosslinked by using the same radiation method, different radiation parameters can be used, and the present application does not make specific limitations thereto.

[0055] In some embodiments of the present application, the superparamagnetic particles and the second hydrogel scaffold are loaded with drugs to achieve sustained release of the drugs. The drugs loaded in the superparamagnetic particles and the second hydrogel scaffold can be different, the superparamagnetic particles are loaded with drugs for promoting the regeneration of the subchondral bone, such as vascular endothelial growth factor (VEGF); and the second hydrogel scaffold is loaded with drugs for promoting the regeneration of the supracartilage bone, such as metformin. The types of drugs mentioned above are only examples, and other types of drugs can also be used, and the drug loading can be based on the use of the composite hydrogel scaffold. In addition, the hollow porous structure of the magnetic hydroxyapatite microsphere particles is beneficial to the loading and sustained release of the drugs.

[0056] For example, the first hydrogel scaffold in the composite hydrogel scaffold mentioned in the present application can be a hydrogel material combined with superparamagnetic hydroxyapatite microsphere particles and GMHA, and studies have shown that this material can have a good repairing effect on the subchondral bone and promote the repair of cartilage tissue, and has a positive effect on the treatment of osteoarthritis. For example, Figure 4As shown, compared with the composite hydrogel scaffold with uniformly arranged superparamagnetic particles proposed in the application number 2024106554692 and the application name "Magnetic hydroxyapatite-based composite hydrogel scaffold and its preparation method and application", the repair effect of the hydrogel scaffold proposed in the present application is better in the same bone repair period, and the score of the ICRS (International Cartilage Repair Society) scoring system is also higher, that is, the design of realizing the concentration gradient based on the same material system can help more in the repair of cartilage tissue and the treatment of osteoarthritis. In addition, the composite hydrogel scaffold of the present application can also realize in-situ MRI monitoring of the affected area and can better realize the evaluation of the treatment effect.

[0057] As an example, Figure 2 In the present application, a neodymium iron boron magnet can be arranged as an external magnetic field source at the bottom of a specific mold, and a high concentration side of superparamagnetic particles can be formed on the side close to the neodymium iron boron magnet. When preparing a second hydrogel scaffold by adding a hydrogel precursor liquid, an external irradiation source can be arranged at the top of the specific mold for irradiation, so that the second hydrogel scaffold is formed. The composite hydrogel scaffold can also be prepared by the following irradiation method: the external magnetic field source is arranged at the top of the specific mold, and the mixed liquid in the specific mold can form a concentration gradient with gradually decreasing concentration from top to bottom. At this time, the external irradiation source is arranged at the top of the specific mold, and a first hydrogel scaffold with a high concentration side of superparamagnetic particles completely cross-linked and a low concentration side of superparamagnetic particles incompletely cross-linked is obtained by cross-linking and solidification. The first hydrogel scaffold is demolded and placed in another mold, a hydrogel precursor liquid is added to the low concentration side of superparamagnetic particles of the first hydrogel scaffold, and a composite hydrogel scaffold is obtained after cross-linking and solidification. In addition, the first hydrogel scaffold and the second hydrogel scaffold can also be prepared separately and then assembled together and interface cross-linked by irradiation cross-linking technology, for example, the irradiation time is minimized so that the first hydrogel scaffold and the second hydrogel scaffold are preliminarily formed (i.e., the high concentration side of superparamagnetic particles in the first hydrogel scaffold is completely cross-linked and the low concentration side of superparamagnetic particles is incompletely cross-linked; one side of the second hydrogel scaffold is completely cross-linked, and the opposite side is in an incompletely cross-linked state), and then the interface cross-linking is realized by irradiation again after stacking the two.

[0058] As an example, the mixing ratio of the hydrogel precursor solution and the superparamagnetic particles is 1 mL: 0-15 mg and not 0 (for example, the mixing ratio can be 5 mg / mL, 8 mg / mL, 10 mg / mL or 15 mg / mL, etc.), and before the mixed solution including the hydrogel precursor solution and the superparamagnetic particles is placed in the non-uniform magnetic field, the mixed solution also needs to be defoamed. For precise control of the bone repair effect of the hydrogel scaffold, a physical defoaming treatment method that does not introduce other chemical components can be used, such as standing, vacuum extraction or ultrasonic treatment, etc.

[0059] In some embodiments of the present application, before forming the mixed suspension, the method further comprises: performing drug loading treatment on the superparamagnetic particles; and before adding the hydrogel precursor solution on the low concentration side of the superparamagnetic particles of the first hydrogel scaffold, the method further comprises: adding a drug in the added hydrogel precursor solution. Specifically, the process of performing drug loading treatment on the superparamagnetic particles can be: soaking the superparamagnetic particles in a solution containing a drug, and after a set soaking time, performing centrifugation, washing and drying treatment to obtain drug-loaded superparamagnetic particles. The set soaking time is not more than 72 h. The step of loading a drug in the hydrogel precursor solution for preparing the second hydrogel scaffold is as follows: adding a set amount of a drug in the hydrogel precursor solution for preparing the second hydrogel scaffold, and stirring to dissolve it.

[0060] In a specific embodiment of the present application, the superparamagnetic particles are hollow porous magnetic hydroxyapatite microsphere particles, the hydrogel polymer used to prepare the first hydrogel scaffold and the second hydrogel scaffold is GMHA that can be photo-crosslinked, and the photoinitiator is LAP. The preparation steps are specifically as follows:

[0061] (1) 0.25 mg of magnetic hydroxyapatite microspheres particles were added into 500 μL of hydrogel precursor solution including GMHA and LAP to form a mixed suspension according to the concentration standard of 5 mg / mL; (2) after defoaming treatment of the mixed suspension by ultrasonic, it was added into a transparent cylindrical PMMA (Poly (methyl methacrylate) ) mold with a cylindrical Nd-Fe-B magnet placed at the bottom of the mold to provide an external inhomogeneous magnetic field, and the particles were allowed to move for 2 min; (3) after the concentration distribution of the magnetic hydroxyapatite microspheres particles reached the expectation, the external magnetic field source was removed, and the bottom of the mold was irradiated by a UV light source for 1 min; (4) after the irradiation was completed, the UV light source was removed, and the top of the mold was added with the hydrogel precursor solution including GMHA and LAP, and the height was controlled to be 1.5-2 mm, and the top of the mold was irradiated by a UV light source for 1 min; (5) after the irradiation was completed, the mold was removed to obtain a composite hydrogel scaffold with a concentration gradient.

[0062] The composite hydrogel scaffold and the preparation method thereof proposed in the present application have the following advantages:

[0063] (1) The new process has the advantages of combining the magnetic field induction method and the stacking method to prepare the composite hydrogel scaffold with a concentration gradient, and at the same time, eliminates the possible gap between the pure GMHA hydrogel and the lower composite hydrogel scaffold, and reduces the potential influence of the interface gap on the performance of the composite hydrogel scaffold with a concentration gradient.

[0064] (2) Compared with the overall uniform composite hydrogel scaffold, the composite hydrogel scaffold with a concentration gradient proposed in the present application still contains a certain amount of superparamagnetic particles on the low concentration side of the superparamagnetic particles, but the concentration is much lower than that of the uniform composite hydrogel scaffold, especially under the condition that there is a second hydrogel scaffold with the same thickness as the cartilage layer and completely free of superparamagnetic particles, theoretically, the influence of superparamagnetic particles on cartilage repair has been weakened to the maximum extent.

[0065] (3) The first hydrogel scaffold and the second hydrogel scaffold can be loaded with drugs to promote cartilage repair, and the loaded drugs can independently act on the respective target treatment areas, thereby achieving the best treatment effect.

[0066] The present application proposes an application of the composite hydrogel scaffold prepared by the preparation method described in any one of the above embodiments or the composite hydrogel scaffold prepared by the preparation method described in any one of the above embodiments in cartilage repair in bone treatment or an application of observing the repair condition of the implantation site in bone treatment by nuclear magnetic resonance.

[0067] It is to be expressly understood that the invention is not limited to the specific configurations and process described above and illustrated in the accompanying drawings. For the sake of clarity, detailed descriptions of known methods are omitted. In the above-described embodiments, several specific steps are described and illustrated as examples. However, the method processes of the present invention are not limited to the specific steps described and illustrated, and various changes, modifications and additions can be made thereto by one of ordinary skill in the art without departing from the spirit of the present invention, and the order of the steps can be changed.

[0068] In the present invention, features described and / or illustrated with respect to one embodiment can be used in the same or a similar way in one or more other embodiments, and / or in combination with or instead of features of other embodiments.

[0069] The above description is merely illustrative of the application, and is not intended to limit the scope of the application. Various modifications and changes can be made by one of ordinary skill in the art without departing from the spirit and scope of the application. Any modification, equivalent replacement, improvement, and the like made within the spirit and principle of the application should be included in the scope of the application.

Claims

1. A method of preparing a composite hydrogel scaffold, characterized in that, The method comprises the following steps: placing a special mold containing a mixed solution of a hydrogel precursor solution and a plurality of superparamagnetic particles in a non-uniform magnetic field, driving the superparamagnetic particles in the mixed solution to arrange in a concentration gradient in a set direction by a magnetic field induction technology, and performing radiation on the high-concentration side of the superparamagnetic particles in the mixed solution by a radiation cross-linking technology to obtain a first hydrogel scaffold in which the high-concentration side of the superparamagnetic particles is completely cross-linked and the low-concentration side of the superparamagnetic particles is not completely cross-linked; adding the hydrogel precursor solution to the low-concentration side of the superparamagnetic particles in the special mold, and completely cross-linking the added hydrogel precursor solution by the radiation cross-linking technology to form a second hydrogel scaffold that is cross-linked with the first hydrogel scaffold at an interface, thereby obtaining a composite hydrogel scaffold after demolding; the radiation cross-linking technology is selected from a light cross-linking technology or a temperature cross-linking technology; for the light cross-linking technology, the wavelength of the radiation light is 100-500 nm, the radiation time is 0-3600 s and is not 0; for the temperature cross-linking technology, the radiation temperature ranges from 0-100℃ and is not 0, and the radiation time is 0-3600 s and is not 0; in the case where the radiation cross-linking technology is the light cross-linking technology, the hydrogel precursor solution comprises a light initiator and a hydrogel polymer for light cross-linking mixed in a set proportion; in the case where the radiation cross-linking technology is the temperature cross-linking technology, the hydrogel precursor solution is a modified hydrogel polymer for temperature cross-linking; wherein the hydrogel polymer for light cross-linking is selected from glycidyl methacrylate modified hyaluronic acid GMHA, silk fibroin, chitosan or agarose; and the modified hydrogel polymer for temperature cross-linking is selected from poly-N-isopropyl acrylamide or chitosan.

2. The production method according to claim 1, characterized by, Before forming the mixed solution, the method further comprises: performing drug loading treatment on the superparamagnetic particles; Before adding the hydrogel precursor solution to the low-concentration side of the superparamagnetic particles in the special mold, the method further comprises: adding a drug to the added hydrogel precursor solution; and Before placing the special mold containing the mixed solution in the non-uniform magnetic field, the method further comprises: performing defoaming treatment on the mixed solution containing the hydrogel precursor solution and the plurality of superparamagnetic particles.

3. The production method according to claim 2, characterized by, The drug loading treatment on the superparamagnetic particles comprises: immersing the superparamagnetic particles in a solution containing a drug, and performing centrifugation, washing and drying treatment after a set soaking time to obtain superparamagnetic particles loaded with the drug.

4. The method of claim 1, wherein, The superparamagnetic particles are selected from magnetic nanoparticles with superparamagnetic properties, or hollow porous magnetic hydroxyapatite microsphere particles formed by hydroxyapatite particles and magnetic nanoparticles with superparamagnetic properties; The magnetic nanoparticles with superparamagnetism are selected from ferriferrous oxide nanoparticles, ferroferric oxide nanoparticles, manganese ferrite nanoparticles or manganese oxide nanoparticles, the mass percentage of hydroxyapatite in the magnetic hydroxyapatite microspheres is 50-95%, the mass percentage of the magnetic nanoparticles with superparamagnetism is 5-50%, and the particle size of the magnetic hydroxyapatite microspheres is 1 nm-10 μm.

5. The preparation method according to claim 1, characterized in that, The height of the second hydrogel support is 1.5-2 mm; The ratio of the hydrogel precursor solution to the superparamagnetic particles in the mixed solution is 1 mL: 0-15 mg and not 0; and The inhomogeneous magnetic field is provided by a neodymium-iron-boron magnet or an electromagnet.

6. A composite hydrogel scaffold, characterized in that, The composite hydrogel support is prepared by the preparation method of any one of claims 1-5, comprising a first hydrogel support and a second hydrogel support which is interfacially crosslinked with the first hydrogel support; The first hydrogel support comprises a first hydrogel support body and superparamagnetic particles arranged in a concentration gradient in a set direction inside the first hydrogel support body; wherein the superparamagnetic particles arranged in a concentration gradient are formed by placing a plurality of superparamagnetic particles in an inhomogeneous magnetic field using magnetic field induction technology; and the first hydrogel support is formed by irradiating the high-concentration side of the superparamagnetic particles using radiation crosslinking technology; The second hydrogel support is located on the low-concentration side of the superparamagnetic particles of the first hydrogel support.

7. The composite hydrogel scaffold of claim 6, wherein, The superparamagnetic particles and the second hydrogel support are loaded with drugs to achieve sustained release of the drugs.

8. Use of a composite hydrogel support prepared by the preparation method of any one of claims 1-5 in the preparation of a drug for cartilage repair in bone treatment.

9. Use of a composite hydrogel support prepared by the preparation method of any one of claims 1-5 in the preparation of a drug for observing repair conditions of a bone treatment implant site by nuclear magnetic resonance.

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