Biophysical and biochemical double-gradient bionic hydrogel, preparation and application of biophysical and biochemical double-gradient bionic hydrogel in promoting osteochondral regeneration

Abstract

The invention discloses biophysical and biochemical double-gradient bionic hydrogel, preparation and application of the biophysical and biochemical double-gradient bionic hydrogel in promoting osteochondral regeneration, and belongs to the field of biomedical materials. The hydrogel is composed of a double structure of hydrogel and a functional drug nanocapsule, and the hydrogel has a concentration gradual change in the overall structure so as to form a soft-hard gradual change biophysical mechanical gradient; the functional drug nanocapsule has a biochemical gradient with gradually changed concentration from high concentration to low concentration; the biophysical mechanical gradient change direction of the hydrogel is the same as or different from the biochemical gradient change direction of the functional drug nanocapsule. The low electric field aligns the charged matrix network and the biochemically packaged nanoparticles to the electrode, and double gradients are formed on the network structure and spatial biochemical distribution. By adjusting the used monomer and cross-linking agent, the release rate and the surface charge of the biodegradable polymer nanoparticles can be accurately controlled, and the programmable biochemical gradient can be realized in the hydrogel. The application prospect of realizing clinical transformation is realized.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and particularly relates to a biophysical and biochemical dual-gradient biomimetic hydrogel, a preparation method thereof, and an application thereof in promoting osteochondral regeneration. Background Art

[0002] Articular tissue is a multi-layered structure composed of cartilage and subchondral bone, showing various anisotropic characteristics to guide site-specific differentiation of different chondrocytes and osteocytes. Articular cartilage, as an elastic connective tissue in joints, is vulnerable to acute trauma and chronic strain. However, the low metabolic rate and avascularity of chondrocytes hinder the self-repair of cartilage. Clinical studies also show that articular cartilage injuries often extend to the subchondral bone, resulting in full-thickness osteochondral defects of the knee joint, indicating the importance of comprehensive osteochondral constructs. The challenge of osteochondral repair is to mimic the spatial morphogenetic gradients in connective tissues, including biochemical gradients such as extracellular matrix (ECM) components, metabolism-related biomolecules, and various cell types with different phenotypes, and biophysical gradients including factors such as mechanical strength, pore size, and physiological signals. Standard clinical treatment methods for osteochondral repair, such as total joint replacement, microfracture, autologous osteochondral transplantation and allogeneic bone transplantation, stem cell therapy, and growth factor injection, aim to relieve pain and restore function. However, these methods face limitations, including low efficiency of cartilage regeneration, formation of fibrocartilage instead of hyaline cartilage, and the risk of immune rejection or donor site morbidity. These limitations mainly stem from the lack of advanced platforms for precise and sustained therapeutic delivery, which hinders the effective regeneration and integration of osteochondral tissues. Biomimetic synthetic cartilage scaffolds provide another strategy for osteochondral regeneration.

[0003] Previous studies have utilized techniques such as 3D printing and supramolecular crosslinking to mimic the biomechanical characteristics of natural osteochondral tissue, and applied external electric or magnetic fields to fabricate gradient hydrogel matrices, which serve as biophysical cues to promote chondrocyte differentiation. Meanwhile, biological components such as metal ions, growth factors, or stem cells are introduced as biochemical cues to construct implantable full-thickness cartilage regeneration scaffolds. However, implantable hydrogel scaffolds face problems such as cumbersome processing, low load-bearing mechanical strength, and challenges in scaling up to match the real tissue structure. In addition, constructing specific growth factor gradients and achieving sustained release of biochemical substances in a controllable manner are important issues in scaffold fabrication. Therefore, it is crucial to develop a biomimetic implantable scaffold with biomechanical and biochemical gradients for osteochondral regeneration. Summary of the Invention

[0004] Based on the above scientific understanding and clinical needs, inspired by electrophoresis, we developed an electric field-driven method to fabricate hydrogels with mechanical and spatiotemporal biochemical gradients for full-thickness osteochondral regeneration. This technology can control the net charge and biochemical properties of the matrix during the fabrication of the hydrogel scaffold. A low electric field aligns the charged matrix network and biochemically encapsulated nanoparticles with the electrodes, forming dual gradients in the network structure and spatial biochemical distribution. By adjusting the monomers and crosslinkers used, the release rate and surface charge of biodegradable polymer nanoparticles can be precisely controlled, enabling programmable biochemical gradients within the hydrogel.

[0005] The present invention provides the following technical solutions:

[0006] A dual-gradient biomimetic hydrogel with biophysical and biochemical gradients, consisting of a dual structure of a hydrogel and functional drug nanocapsules. The hydrogel has a gradually changing concentration in the overall structure, forming a soft-hard gradually changing biophysical and mechanical gradient; the functional drug nanocapsules have a high-low gradually changing biochemical gradient; the direction of change of the biophysical and mechanical gradient of the hydrogel may be the same as or different from the direction of change of the biochemical gradient of the functional drug nanocapsules, which can be specifically adjusted according to needs during preparation. See Figure 9 。

[0007] The purpose of the present invention to provide a dual-gradient biomimetic hydrogel with biophysical and biochemical gradients is to use an electric field-driven method to achieve a soft-hard biophysical mechanics and functional drug nanocapsule biochemical dual-gradient gel for promoting full-thickness osteochondral regeneration. It is particularly suitable for cartilage repair problems caused by acute trauma, chronic strain, and the low metabolic rate of chondrocytes. By aligning the electrodes, the charged hydrogel matrix network and biochemically encapsulated drugs such as transforming growth factor β1 nanocapsules n(TGF-β1) are under the action of an electric field, forming dual gradients in the network structure and spatial biochemical distribution; after the biodegradable polymer shell layer in the nanocapsules encapsulates the drug, the release rate and surface charge of the drug in the nanocapsules can be precisely controlled by adjusting the monomers and crosslinkers used (i.e., the polymer shell layer on the surface of the capsule), and the mechanical gradient of the hydrogel can be precisely controlled by the type of external electric field, its voltage magnitude, and crosslinking strength, providing a biophysical and biochemical mechanical dual-gradient hydrogel with simple preparation and programmability.

[0008] The preparation method of the above dual-gradient biomimetic hydrogel with biophysical and biochemical gradients includes the following steps:

[0009] (1) Mix the nano-functional drug, neutral monomer, charged monomer, and dual crosslinkers (i.e., non-hydrolyzable crosslinker and pH-responsive hydrolyzable crosslinker) evenly in an aqueous phase to obtain a mixed solution, and then add the corresponding initiator and catalyst to the mixed solution for free radical polymerization reaction to obtain a charged functional drug nanocapsule solution;

[0010] (2) Mix the functional drug nanocapsule solution in step (1) with the charged hydrogel matrix material solution evenly to form a hydrogel matrix material / functional drug nanocapsule mixed solution. Further, add the catalytic crosslinking mixture solution to the hydrogel matrix material / drug mixed solution to obtain a pre-gel solution of hydrogel matrix material / drug nanocapsule / catalytic crosslinking mixture.

[0011] (3) Pour the pre-gel solution of hydrogel matrix material / functional drug nanocapsule / catalytic crosslinking mixture into a mold, and there are conductive graphite electrodes at any two ends of the mold, serving as the anode and cathode.

[0012] (4) After the pre-gel solution of hydrogel matrix material / functional drug nanocapsule / catalytic crosslinking mixture is completely gelated to form a uniform drug-loaded gel (HS), through an externally applied electric field, induce the charged gel network and charged functional drug nanocapsules in the uniform drug-loaded gel (HS) to migrate towards the electrodes. Apply the electric field for a period of time to obtain a biophysical and biochemical mechanical double-gradient biomimetic hydrogel.

[0013] Further, the functional drug in step (1) is selected from one or more of TGF-β1, bovine serum albumin (BSA), bone morphogenetic protein 2

[0014] (BMP-2), anti-vascular endothelial growth factor receptor 2 antibody, anti-programmed death receptor 1 antibody, vascular endothelial growth factor, platelet-derived growth factor, etc.; preferably, the selected functional drug is TGF-β1.

[0015] Further, the neutral monomer in step (1) is selected from one or more of acrylamide (AAM) monomer, methyl acrylate, ethyl acrylate, butyl acrylate, methacrylic acid, 2-methacryloyloxyethyl phosphorylcholine, etc.; preferably, the selected neutral monomer is AAM.

[0016] Further, the charged monomer in step (1) is positively charged or negatively charged, and is selected from one or more of (3-aminopropyl) methacrylamide hydrochloride (APM), methacryloyloxyethyl trimethyl ammonium chloride, dimethyldiallyl ammonium chloride, vinyl pyridine, methacrylic acid, and N,N-dimethylaminoethyl methacrylate lactone. If the charged monomer is positively charged, the obtained nano-functional drug capsule is positively charged; if the charged monomer is negatively charged, the obtained nano-functional drug capsule is negatively charged; it can be selected according to needs; preferably, the selected charged monomer is the positively charged monomer APM.

[0017] In the further step (1), the non-hydrolyzable crosslinking agent is selected from divinyl compounds, epoxy compounds, isocyanate compounds, aldehyde compounds, siloxane compounds, and carboxylic acetic acid compounds. For example, the non-hydrolyzable crosslinking agent is one or more of GDMA, bisphenol A epoxy resin, toluene diisocyanate (TDI), glutaraldehyde, methyltrimethoxysilane, or maleic anhydride; preferably, the selected non-hydrolyzable crosslinking agent is GDMA.

[0018] In the further step (1), the pH-responsive hydrolyzable crosslinking agent is selected from one or more of poly(DL-lactide)-b-poly(ethylene glycol)-b-poly(DL-lactide)-diacrylate (AI102), polyethylene glycol diacrylate, N,N'-dacryloyl cystine, methacrylic anhydride, or acrylic acid; preferably, the selected pH-responsive hydrolyzable crosslinking agent is AI102.

[0019] In the further step (1), the initiator is selected from peroxide initiators, azo initiators, organic peroxides, photoinitiators, and ketone initiators. For example, it is one or more of ammonium persulfate (APS), benzoyl peroxide, diisopropylbenzene peroxide, azobisisobutyronitrile, azobis(cyclohexyl) disulfide, benzoyl peroxide, benzophenone, and 2-chloro-1,3-dimethylbenzofuran; preferably, the selected initiator is APS.

[0020] In the further step (1), the catalyst is selected from metal catalysts and catalysts used in combination with peroxide initiators, such as one or more of copper catalysts, cobalt catalysts, and N,N,N',N'-tetramethylethylenediamine (TEMED); preferably, the selected catalyst is TEMED.

[0021] In the further step (1), the molar ratio range between the corresponding functional drug and the neutral monomer is 1:300 - 12000; preferably, the molar ratio of the selected functional drug TGF-β1 to the neutral monomer AAM is 1:4000.

[0022] In the further step (1), the molar ratio range between the corresponding functional drug and the charged monomer is 1:30 - 1200; preferably, the molar ratio of the selected functional drug TGF-β1 to the charged monomer APM is 1:400.

[0023] In the further step (1), the molar ratio range between the corresponding functional drug and the double crosslinking agent is 1:30 - 1200; preferably, the molar ratio of the selected functional drug TGF-β1 to the double crosslinking agent is 1:500.

[0024] Among the double crosslinking agents in the further step (1), the molar ratio between the non-hydrolyzable crosslinking agent and the pH-responsive hydrolyzable crosslinking agent is 1:0 - 800; preferably, the molar ratio between the selected non-hydrolyzable crosslinking agent GDMA and the pH-responsive hydrolyzable crosslinking agent AI102 is 1:400;

[0025] In the further step (1), the molar ratio range between the corresponding functional drug and the initiator is 1:1 - 800; preferably, the molar ratio between the selected functional drug TGF-β1 and the initiator APS is 1:320;

[0026] In the further step (1), the mass ratio range between the corresponding initiator and the catalyst is 1:1 - 100; preferably, the mass ratio between the selected initiator APS and the catalyst TEMED is 1:2;

[0027] The temperature range of the free radical polymerization reaction in the further step (1) is 0 - 60 °C, and the reaction time range is 30 min - 48 h; preferably, the selected free radical polymerization reaction temperature is 4 °C, and the reaction time is 2 h;

[0028] The hydrogel charged matrix material in the further step (2) is selected from one or more of hyaluronic acid grafted with double bonds, silk fibroin, sodium alginate, carboxymethyl cellulose, polyacrylic acid, chitosan, and gelatin, etc.; preferably, the selected hydrogel matrix material is a negatively charged silk fibroin solution;

[0029] The concentration of the charged hydrogel charged matrix material in the further step (2) is 2% - 20%; preferably, the concentration of the silk fibroin solution, which is the selected hydrogel matrix material, is 6%;

[0030] In the further step (2), the dosage relationship between the functional drug and the hydrogel matrix material can be adjusted according to needs. For example, the mass ratio range between the nano-functional drug and the hydrogel charged matrix material is 1:1 - 2000000; preferably, the mass ratio between the selected functional drug TGF-β1 and the hydrogel charged matrix material silk fibroin is 1:100;

[0031] In the further step (2), the catalytic crosslinking mixture is selected from one or more of horseradish peroxidase / hydrogen peroxide (HRP / H 2 O 2 ) mixture, phenolic compound / H 2 O 2 mixture, benzimidazole compound / HRP mixture, or photoinitiator; preferably, the selected catalytic crosslinking mixture is HRP / H 2 O 2 ;

[0032] In the further step (2), the mass (mg) of the charged matrix material of the hydrogel: the range of the HRP enzyme activity (U) in the catalytic cross-linking mixture is 1:1 - 5000. Preferably, the selected mass (mg) of silk fibroin: the HRP enzyme activity (U) is 1:140;

[0033] In the further step (2), the H in the catalytic cross-linking mixture 2 O 2 The final concentration range in the pre-gel solution is 0.1 - 50 mM; preferably, the H in the catalytic cross-linking mixture 2 O 2 The final concentration in the pre-gel solution is 5 mM;

[0034] In the further step (3), the mold can be a mold of any shape and size as needed, such as a cuboid, an irregular shape, etc.; preferably, the selected mold is a cuboid with a length x width x height of 4 x 3 x 3 cm.

[0035] In the further step (4), the external electric field is selected from one or more of a direct current electric field, an alternating current electric field, a pulsed electric field, a rotating electric field, a non-uniform electric field, and a high-frequency electric field; preferably, the selected external electric field is a direct current electric field.

[0036] In the further step (4), the magnitude range of the external electric field is 2 - 20 V / mm -1 , and the application time range of the electric field is 1 - 30 min; preferably, the selected electric field magnitude of the direct current electric field is 10 V / mm -1 , and the application time of the electric field is 5 min. A double-gradient structure with biophysical and biochemical gradients can be formed between the anode and the cathode; the process of applying the electric field induces the negatively charged gel network or / and the negatively charged functional drug nanocapsules in the uniformly drug-loaded gel (HS) to migrate from the cathode to the anode, and the positively charged functional drug nanocapsules or / and the gel network to migrate from the anode to the cathode. After applying the electric field for a period of time, a double-gradient biomimetic hydrogel with biophysical and biochemical mechanics can be obtained.

[0037] Generally speaking, compared with the prior art, the above technical solution conceived by the present invention has the following advantages:

[0038] (1) The double-gradient biomimetic hydrogel with biophysical and biochemical gradients provided by the present invention has high safety of its biological matrix material and growth factors encapsulated by nanocapsules, and has the application prospect of realizing clinical transformation. Using an external electric field to integrate the hydrogel to form a mechanical gradient, simulating the anisotropic structure of natural osteochondral tissue, and functional drugs such as the growth factor TGF-β1 provide precise biochemical gradients to effectively promote the osteogenic and chondrogenic differentiation of bone marrow mesenchymal stem cells.

[0039] (2) Eliminates the need for additional dedicated equipment and minimizes the risk of growth factor inactivation. By providing a restorative microenvironment compatible with native tissue, this bionic system has great potential in advancing osteochondral repair and broader regenerative medicine applications.

[0040] (3) Cationic nanocapsules with degradable crosslinkers establish biochemical gradients, enabling spatially selective delivery of growth factors in local areas. This one-step manufacturing method addresses the challenges of traditional multi-step manufacturing, preserves the bioactivity of the encapsulated formulation, and provides sustained release over 12 weeks to promote osteochondral regeneration.

[0041] (4) In addition to osteochondral repair, the dual-gradient hydrogel demonstrates scalability and potential for application in various complex anisotropic interfaces and targeted delivery in regenerative medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is a schematic diagram of the preparation process of the biophysical and biochemical dual-gradient bionic silk fibroin hydrogel of the present invention.

[0043] Figure 2 is a physical image of the biophysical and biochemical dual-gradient bionic silk fibroin hydrogel of the present invention

[0044] Figure 3 is a scanning electron microscope image of the biophysical and biochemical dual-gradient bionic silk fibroin hydrogel of the present invention

[0045] Figure 4 is a mechanical property image of the biophysical and biochemical dual-gradient bionic silk fibroin hydrogel of the present invention

[0046] Figure 5 is an IVIS drug-loaded fluorescence image of the biophysical and biochemical dual-gradient bionic silk fibroin hydrogel of the present invention

[0047] Figure 6 is a drug loading rate image of the biophysical and biochemical dual-gradient bionic silk fibroin hydrogel of the present invention

[0048] Figure 7 is an in vitro drug release curve of the biophysical and biochemical dual-gradient bionic silk fibroin hydrogel of the present invention

[0049] Figure 8 is a 3D micro-CT image of the biophysical and biochemical dual-gradient bionic silk fibroin hydrogel of the present invention for rabbit osteochondral defect repair in Example 1 after 12 weeks

[0050] Figure 9Schematic diagram of a dual-gradient biomimetic hydrogel with different or identical biophysical and biochemical mechanical properties formed by charged gel matrix materials and charged functional drug nanocapsules in the same electric field direction DETAILED DESCRIPTION

[0051] The following is further described in conjunction with specific implementation cases and drawings of the specification, but the present invention is not limited to the following embodiments.

[0052] Example 1

[0053] The used biomimetic silk protein hydrogel has biophysical and biochemical dual gradients and is a cylinder with a diameter of 3.5 mm and a height of 3 mm.

[0054] This example provides a method for preparing a biomimetic silk protein hydrogel with biophysical and biochemical dual gradients and investigates the therapeutic effect of the biomimetic silk protein hydrogel with biophysical and biochemical dual gradients on osteochondral defects in New Zealand white rabbits, including:

[0055] 1. Preparation of TGF-β1 nanocapsule solution: TGF-β1, AAM, APM, GDMA, AI102, and APS were mixed uniformly at a molar ratio of 1:4000:400:100:400:320, and TEMED was added to the mixed solution at a mass ratio of TEMED:APS=2:1. The mixed solution was subjected to free radical polymerization at 4°C for 2 hours to obtain TGF-β1 nano (n(TGF-β1)) solution A;

[0056] 2. Preparation of drug-loaded silk protein pre-gel solution: First, 600ug of TGF-β1 in n(TGF-β1) solution was mixed with 1mL of 6wt% silk protein solution, and then 140U of HRP was added to the silk / n(TGF-β1) mixed solution according to the ratio of silk protein (mg): HRP enzyme activity (U) of 1:140, that is, 140U of HRP was added per mg of silk protein. 2 O 2 The solution was added to the silk / n(TGF-β1) / HRP mixture to make it 2 O 2 The final concentration in the solution was 5 mM. After thorough mixing, silk / n(TGF-β1) / HRP / H 2 O 2 Pregel solution B;

[0057] 3. Preparation of biomimetic silk protein hydrogels with biophysical and biochemical dual gradients Figure 1As shown: Various components in the precursor polymer solution A undergo a radical polymerization reaction at 4 °C to form a nano-polymer shell layer, encapsulating TGF-β1 to obtain n(TGF-β1). AI102 in n(TGF-β1) has pH responsiveness and hydrolyzes in the sub-acidic microenvironment of osteochondral defects, further disintegrating the nano-polymer shell layer structure and releasing the encapsulated TGF-β1( Figure 1 a). Pour the precursor solution B into a cuboid mold with dimensions of 4 x 2 x 3 cm. There are two cuboid conductive graphite electrodes with dimensions of 4 x 1.7 x 3 cm at both ends of the cuboid mold, serving as the anode and cathode. After the precursor solution B is completely gelled to form a uniform HS, apply a constant DC electric field of 10 V / mm -1 . Induce the negatively charged silk fibroin network in HS to migrate from the cathode to the anode, and the positively charged n(TGF-β1) to migrate from the anode to the cathode. After applying the electric field for 5 minutes, a biophysical and biochemical dual-gradient biomimetic silk fibroin hydrogel (GS / n(TGF-β1)) can be obtained. Name the region near the cathode of the dual-gradient hydrogel as GS(-) / n(TGF-β1), and vice versa, the region near the anode as GS(+) / n(TGF-β1). The volume ratio of these two regions is approximately 1:1. GS(-) / n(TGF-β1) has the characteristics of a loose silk fibroin gel network structure and a high content of n(TGF-β1), while GS(-) / n(TGF-β1) has a dense silk fibroin gel network structure and a low content of n(TGF-β1)( Figure 1 b).

[0058] Investigation of the therapeutic effect on promoting the healing of osteochondral defects in the trochlear surface of the knees of New Zealand white rabbits: Select several 8-month-old adult male New Zealand white rabbits and randomly divide them into 5 experimental groups: blank group (no treatment), GS / n(TGF-β1) group, HS / n(TGF-β1) group, GS / TGF-β1 group, and GS group (5 rabbits in each group, and two knees of each rabbit are used). Use isoflurane at a concentration of 2.5% (air: isoflurane, v / v) to induce New Zealand white rabbits into an anesthetized state. Make an anteromedial incision beside the patella to expose the knee joint, and strip the patella to enter the distal femoral trochlear groove. After creating a cylindrical osteochondral defect (diameter 3.5 mm, depth 3 mm) using a corneal punch, implant the dedicated scaffold for each group and suture the knee joint with 4-0 thread. After the operation, use prophylactic antibiotics to prevent the rabbits from being infected, and keep each rabbit alone in a cage without restricting its activities, so that it can freely obtain food and water.

[0059] At the 12th week after treatment, the New Zealand white rabbits were euthanized, the femoral joints were removed and photographed, and then the fixed samples were scanned and imaged using a Micro-CT scanner. The data was visualized and three-dimensionally reconstructed using NRecon software (version 1.6). Then, CTAn software (version 1.14) was used for the analysis of new bone regeneration, including the ratio of new bone volume to total volume (BV / TV), bone mineral density (BMD), trabecular bone thickness (Tb.Th), and trabecular bone number (Tb.N).

[0060] In this embodiment, TGF-β1 can be replaced or further include one or more of BSA, BMP-2, anti-vascular endothelial growth factor receptor 2 antibody, anti-programmed death receptor 1 antibody, pro-vascular endothelial growth factor, and platelet-derived growth factor.

[0061] In this example, the neutral AAM monomer can be replaced or further include one or more of methyl acrylate, ethyl acrylate, butyl acrylate, methacrylic acid, and 2-methacryloyloxyethyl phosphorylcholine.

[0062] In this example, the charged monomer APM can be replaced or further include one or more of methacryloyloxyethyl trimethyl ammonium chloride, dimethyldiallyl ammonium chloride, vinyl pyridine, methacrylic acid, and N,N-dimethylaminoethyl methacrylate lactone.

[0063] In this example, the non-hydrolyzable crosslinker GDMA can be replaced or further include one or more of bisphenol A epoxy resin, toluene diisocyanate (TDI), glutaraldehyde, N,N'-methylenebisacrylamide (BIS), methyltrimethoxysilane, or maleic anhydride.

[0064] In this example, the hydrolyzable pH-responsive crosslinker AI102 can be replaced or further include one or more of polyethylene glycol diacrylate, N,N'-dacryloyl cystine, methacrylic anhydride, or acrylic acid.

[0065] In this example, the initiator APS can be replaced or further include peroxide initiators, azo initiators, organic peroxides, photoinitiators, ketone initiators, for example, one or more of benzoyl peroxide, diisopropylbenzene peroxide, azobisisobutyronitrile, azo-bis(cyclohexyl) disulfide, benzoyl peroxide, benzophenone, and 2-chloro-1,3-dimethylbenzofuran;

[0066] In this example, the catalyst TEMED can be replaced or further include metal catalysts such as copper catalysts and cobalt catalysts.

[0067] In this embodiment, the silk fibroin solution can be replaced or further include one or more of gelatin, chitosan, sodium alginate, hyaluronic acid grafted with double bonds, carboxymethyl cellulose, or polyacrylic acid.

[0068] In this embodiment, HRP / H 2 O 2 The catalytic cross-linking mixture can be replaced or further include one or more of phenolic compounds / H 2 O 2 , benzimidazole compounds / HRP, or photoinitiators.

[0069] In this embodiment, the applied DC electric field can be replaced or further include one or more of an AC electric field, a pulsed electric field, a rotating electric field, a non-uniform electric field, and a high-frequency electric field.

[0070] As Figure 2 and Figure 3 shown, under the action of the applied electric field, the silk fibroin network migrates from the cathode to the anode, making the silk fibroin gel GS(-) on the cathode side appear translucent, while the silk fibroin gel GS(+) on the anode side appears milky white. By observing the cross-section of GS with an electron scanning microscope, as Figure 3 shown, the gradient protein network distribution in the GS hydrogel. A denser pore structure with a diameter of about 10 μm was observed near GS(+), while a looser pore structure with a diameter of about 50 μm was observed in the region of GS(-). Subsequently, the rheological test results Figure 4 showed that the storage modulus (G') of the GS(+) region was 29.6 kPa, significantly higher than that of the HS and GS(-) parts, which were 0.84 kPa and 0.37 kPa respectively. These results fully confirmed the formation of the biophysical gradient gel. Immediately afterwards, using BSA as a model protein for TGF-β1, it was fluorescently modified with fluorescein isothiocyanate (FITC) for easy tracing in the gel. And using the same method as preparing n(TGF-β1), the cross-linking agents GDMA and AI102 were replaced with the non-degradable cross-linking agent BIS, and n(BSA-FITC) BIS was synthesized. After incorporating it into the preformed silk fibroin gel solution and applying an electric field, the GS / n(BSA-FITC) BIS gel was obtained. The in vivo imaging system images Figure 5 showed that the positively charged n(BSA-FITC) BIS migrated towards the cathode, and an obvious fluorescence intensity gradient was generated in the GS / n(BSA-FITC) BIS gel, which was in sharp contrast to the uniform intensity distribution observed in HS / n(BSA-FITC) BIS . And Figure 6 showed that more than 60% of n(BSA-FITC)BIS Accumulated in the cathode region of the gradient hydrogel (GS(-) / n(BSA-FITC) BIS ). In addition Figure 7 The release curve shows that the cumulative release amount of GS(-) / n(BSA-FITC) BIS in n(BSA-FITC) BIS is about 60% of the loading content, while in the anode region (GS(+) / n(BSA-FITC) BIS ), the cumulative release amount is about 26% of the loading content. These results indicate the formation of a biochemical gradient gel. Combining the above results, a biophysical and biochemical dual-gradient silk fibroin gel was successfully prepared in this example. Finally, the osteochondral defects of New Zealand white rabbits were treated. The GS / n(TGF-β1) group showed dense bone tissue filling the defect area and complete closure of the bone plate. In contrast, the blank group retained more than 60% of the bone defect. Further, the bone volume / total volume (BV / TV, %), bone mineral density (BMD, g / cm 3 ) and trabecular thickness (Tb.Th, mm) of the defect sites of New Zealand white rabbits in each treatment group were quantitatively evaluated. The results showed that GS / n(TGF-β1) had the highest new bone volume, bone mineral density and trabecular thickness. It indicates that this biophysical and biochemical dual-gradient biomimetic silk fibroin hydrogel has excellent osteochondral repair ability( Figure 8 ).

[0071] Example 2

[0072] This example provides a method for preparing a biophysical and biochemical dual-gradient biomimetic hyaluronic acid hydrogel, including: 1. Preparation of BMP-2 nanocapsules n(BMP-2): Mix BMP-2, neutral monomer acrylamide (AAM), positively charged monomer (3-aminopropyl) methacrylamide hydrochloride (APM), non-hydrolyzable crosslinking agent glycerol dimethacrylate (GDMA), pH-responsive hydrolyzable crosslinking agent poly(DL-lactide)-b-poly(ethylene glycol)-b-poly(DL-lactide)-diacrylate (AI102), ammonium persulfate (APS) in a molar ratio of 1:3600:400:100:300:320, and then add N,N,N′,N′-tetramethylethylenediamine (TEMED) to the mixed solution at a mass ratio of TEMED:APS = 3:1. The mixed solution is subjected to a free radical polymerization reaction at 4°C for 2 hours to obtain the n(BMP-2) solution A

[0073] 2. Preparation of drug-loaded hyaluronic acid pre-gel solution: First, hyaluronic acid grafted with double bonds, GDMA, and photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-benzophenone (I2959) were mixed at a mass ratio of 1:1:4. Subsequently, solution A containing 600 μg of BMP-2 was mixed evenly with this mixed solution to obtain drug-loaded hyaluronic acid pre-gel solution B;

[0074] 3. Pour drug-loaded hyaluronic acid pre-gel solution B into a cuboid mold with a length x width x height of 4 x 2 x 3 cm. There are two cuboid conductive graphite electrodes with a length x width x height of 4 x 1.7 x 3 cm at both ends of this cuboid mold, serving as the anode and cathode. After the pre-gel solution B is completely gelled under ultraviolet light irradiation for 30 s to form a uniform drug-loaded hyaluronic acid gel, a DC electric field of 5 V / mm -1 is applied for 10 min to induce the formation of a biophysical and biochemical dual-gradient hyaluronic acid gel.

[0075] In this embodiment, BMP-2 can be replaced or further include one or more of bovine serum albumin, anti-vascular endothelial growth factor receptor 2 antibody, anti-programmed death receptor 1 antibody, vascular endothelial growth factor, and platelet-derived growth factor.

[0076] In this embodiment, neutral AAM monomer can be replaced or further include one or more of methyl acrylate, ethyl acrylate, butyl acrylate, methacrylic acid, and 2-methacryloyloxyethyl phosphorylcholine.

[0077] In this embodiment, charged monomer APM can be replaced or further include one or more of methacryloyloxyethyl trimethyl ammonium chloride, dimethyldiallyl ammonium chloride, vinyl pyridine, methacrylic acid, and N,N-dimethylaminoethyl methacrylate lactone.

[0078] In this embodiment, non-hydrolyzable cross-linker GDMA can be replaced or further include one or more of bisphenol A epoxy resin, toluene diisocyanate (TDI), glutaraldehyde, N,N'-methylenebisacrylamide (BIS), methyltrimethoxysilane, or maleic anhydride.

[0079] In this embodiment, initiator APS can be replaced or further include peroxide initiators, azo initiators, organic peroxides, and ketone initiators. For example, one or more of benzoyl peroxide, diisopropylbenzene peroxide, azobisisobutyronitrile, azo-bis(cyclohexyl) disulfide, benzoyl peroxide, benzophenone, and 2-chloro-1,3-dimethylbenzofuran;

[0080] In this embodiment, catalyst TEMED used in combination with peroxide initiators can be replaced or further include one or more of metal catalysts and copper catalysts or cobalt catalysts;

[0081] In this embodiment, the hydrolyzable pH-responsive crosslinking agent AI102 can be replaced or further include one or more of polyethylene glycol diacrylate, N,N'-diallylcystine, methacrylic anhydride, or acrylic acid.

[0082] In this embodiment, the hyaluronic acid grafted with double bonds can be replaced or further include one or more of gelatin, carboxymethyl chitosan, or quaternized chitosan.

[0083] In this embodiment, the photoinitiator I2959 can be replaced or further include one or more of benzoyl peroxide, aluminum chloride, potassium, sodium, 1-hydroxycyclohexyl phenyl ketone, H 2 O 2 -sodium thiosulfate system, H 2 O 2 or benzoyl peroxide.

Claims

1. A biophysical and biochemical dual gradient biomimetic hydrogel, characterized in that: It consists of a dual structure of hydrogel and functional drug nanocapsules. The hydrogel has a concentration gradient in the overall structure to form a soft-hard gradient biophysical mechanical gradient; the functional drug nanocapsules have a high-low concentration gradient biochemical gradient; the direction of change of the biophysical mechanical gradient of the hydrogel is the same as or different from the direction of change of the biochemical gradient of the functional drug nanocapsules.

2. The method for preparing a biophysical and biochemical dual gradient biomimetic hydrogel according to claim 1, characterized in that: The following steps are involved: (1) mixing a nanofunctional drug, a neutral monomer, a charged monomer, and a dual cross-linking agent (i.e., a non-hydrolyzable cross-linking agent and a pH-responsive hydrolyzable cross-linking agent) in an aqueous phase to obtain a mixed solution, and then adding a corresponding initiator and a catalyst to the mixed solution to perform a free radical polymerization reaction to obtain a charged functional drug nanocapsule solution; (2) uniformly mixing the functional drug nanocapsule solution in step (1) with the charged hydrogel matrix material solution to form a hydrogel matrix material / functional drug nanocapsule mixed solution, and further adding the catalytic cross-linking mixture solution to the hydrogel matrix material / drug mixed solution to obtain a pre-gelled solution of the hydrogel matrix material / drug nanocapsule / catalytic cross-linking mixture; (3) pouring a pre-gelled solution of a hydrogel matrix material / functional drug nanocapsule / catalytic cross-linking mixture into a mold, with conductive graphite electrodes at either end of the mold serving as an anode and a cathode; (4) After the pre-gelled solution of the hydrogel matrix material / functional drug nanocapsules / catalytic cross-linking mixture is completely gelled to form a uniform drug-loaded gel (HS), an external electric field is applied to induce the charged gel network and the charged functional drug nanocapsules in the uniform drug-loaded gel (HS) to migrate toward the electrode. After applying the electric field for a period of time, a dual-gradient biomimetic hydrogel with biophysical and biochemical mechanical properties can be obtained.

3. The method according to claim 2, characterized in that In step (1), the functional drug is selected from one or more of TGF-β1, bovine serum albumin (BSA), bone morphogenetic protein 2 (BMP-2), anti-vascular endothelial growth factor receptor 2 antibody, anti-programmed death receptor 1 antibody, vascular endothelial growth factor and platelet growth factor, etc.; preferably, the selected functional drug is TGF-β1; In step (1), the neutral monomer is selected from one or more of acrylamide (AAM) monomer, methyl acrylate, ethyl acrylate, butyl acrylate, methacrylic acid and 2-methacryloyloxyethyl phosphorylcholine; preferably, the selected neutral monomer is AAM; In step (1), the charged monomer is positively charged or negatively charged, and is selected from one or more of the charged monomers (3-aminopropyl) methacrylamide hydrochloride (APM), methacryloyloxyethyl trimethylammonium chloride, dimethyl diallyl ammonium chloride, vinyl pyridine, methacrylic acid, and N,N-dimethylaminoethyl methacrylate lactone; if the charged monomer is positively charged, the obtained nano-functional drug capsule is positively charged, and if the charged monomer is negatively charged, the obtained nano-functional drug capsule is negatively charged; the charged monomer can be selected according to needs; preferably, the selected charged monomer is the positively charged monomer APM; In step (1), the non-hydrolyzable cross-linking agent is selected from divinyl compounds, epoxy compounds, isocyanate compounds, aldehyde compounds, siloxane compounds and carboxylic acid acetic acid compounds, for example, one or more of the non-hydrolyzable cross-linking agent GDMA, bisphenol A epoxy resin, toluene diisocyanate (TDI), glutaraldehyde, methyltrimethoxysilane or maleic anhydride; preferably, the non-hydrolyzable cross-linking agent is GDMA; In step (1), the pH-responsive hydrolyzable cross-linking agent is selected from one or more of poly(DL-lactide)-b-poly(ethylene glycol)-b-poly(DL-lactide)-diacrylate (AI102), polyethylene glycol diacrylate, N,N'-diacrylcystine, methacrylic anhydride or acrylic acid; preferably, the selected pH-responsive hydrolyzable cross-linking agent is AI102; The initiator in step (1) is selected from peroxide initiators, azo initiators, organic peroxides, photoinitiators, and ketone initiators, for example, one or more of ammonium persulfate (APS), benzoyl peroxide, diisophorone peroxide, azobisisobutyronitrile, azobis(cyclohexyl) disulfide, dibenzoyl peroxide, benzophenone, and 2-chloro-1,3-dimethylbenzofuran; preferably, the selected initiator is APS; In step (1), the catalyst is selected from a metal catalyst and a catalyst used in conjunction with a peroxide initiator, such as one or more of a copper catalyst, a cobalt catalyst and N,N,N',N'-tetramethylethylenediamine (TEMED); preferably, the selected catalyst is TEMED.

4. The method according to claim 2, characterized in that The molar ratio between the corresponding functional drug and the neutral monomer in step (1) is in the range of 1:300-12000; preferably, the molar ratio between the selected functional drug TGF-β1 and the neutral monomer AAM is 1:4000; The molar ratio between the corresponding functional drug and the charged monomer in step (1) is in the range of 1:30-1200; preferably, the molar ratio between the selected functional drug TGF-β1 and the charged monomer APM is 1:400; The molar ratio between the corresponding functional drug and the double cross-linking agent in step (1) is in the range of 1:30-1200; preferably, the molar ratio between the selected functional drug TGF-β1 and the double cross-linking agent is 1:500; In the double cross-linking agent of step (1), the molar ratio between the non-hydrolyzable cross-linking agent and the pH-responsive hydrolyzable cross-linking agent is 1:0-800; preferably, the molar ratio between the selected non-hydrolyzable cross-linking agent GDMA and the pH-responsive hydrolyzable cross-linking agent AI102 is 1:400; The molar ratio between the corresponding functional drug and the initiator in step (1) is in the range of 1:1-800; preferably, the molar ratio between the selected functional drug TGF-β1 and the initiator APS is 1:320; The mass ratio between the corresponding initiator and the catalyst in step (1) ranges from 1:1 to 100; preferably, the mass ratio between the selected initiator APS and the catalyst TEMED is 1:

2.

5. The method according to claim 2, characterized in that The free radical polymerization reaction temperature range of step (1) is 0-60°C, and the reaction time range is 30min-48h; preferably, the selected free radical polymerization reaction temperature is 4°C, and the reaction time is 2h.

6. The method according to claim 2, characterized in that The hydrogel charged matrix material in step (2) is selected from one or more of natural biomacromolecules such as hyaluronic acid, silk protein, sodium alginate, carboxymethyl cellulose, polyacrylic acid, chitosan and gelatin; preferably, the selected hydrogel matrix material is a negatively charged silk protein solution; the catalytic cross-linking mixture in step (2) is selected from one or more of a horseradish peroxidase / hydrogen peroxide (HRP / H2O2) mixture, a phenolic compound / H2O2 mixture, a benzimidazole compound / HRP mixture or a photoinitiator; preferably, the selected catalytic cross-linking mixture is HRP / H2O2.

7. The method according to claim 2, characterized in that The concentration of the charged hydrogel charged matrix material in step (2) is 2%-20%; preferably, the concentration of the selected hydrogel matrix material silk protein solution is 6%; The dosage relationship between the functional drug and the hydrogel matrix material in step (2) can be adjusted as needed, for example, the mass ratio of the nano-functional drug to the hydrogel charged matrix material is in the range of 1:1-2000000; preferably, the mass ratio of the selected functional drug TGF-β1 to the hydrogel charged matrix material silk protein is 1:100; In step (2), the mass (mg) of the hydrogel charged matrix material: the HRP enzyme activity (U) in the catalytic cross-linking mixture is in the range of 1:1-5000, preferably, the mass (mg) of the selected silk protein: the HRP enzyme activity (U) is 1:140; In step (2), the final concentration of H2O2 in the pre-gelling solution of the catalytic cross-linking mixture is in the range of 0.1-50 mM; preferably, the final concentration of H2O2 in the pre-gelling solution of the catalytic cross-linking mixture is 5 mM.

8. The method according to claim 2, characterized in that In step (3), the mold can be of any shape and size as required, such as a rectangular or irregular shape; in step (4), the externally applied electric field is selected from one or more of a direct current electric field, an alternating current electric field, a pulsed electric field, a rotating electric field, a non-uniform electric field and a high-frequency electric field; preferably, the selected externally applied electric field is a direct current electric field.

9. The method according to claim 2, characterized in that The range of the applied electric field in step (4) is 2-20V mm -1 The electric field application time range is 1-30min; preferably, the DC electric field is selected to be 10V mm -1 , the electric field is applied for 5 minutes; a dual gradient structure of biophysical and biochemical gradients can be formed between the anode and the cathode; the process of applying the electric field induces the negatively charged gel network or / and the negatively charged functional drug nanocapsules in the uniform drug-loaded gel (HS) to migrate from the cathode to the anode, and the positively charged functional drug nanocapsules or / and the gel network to migrate from the anode to the cathode. After applying the electric field for a period of time, a dual gradient biomimetic hydrogel with biophysical and biochemical mechanics can be obtained.

10. Use of the biophysical and biochemical dual gradient biomimetic hydrogel according to claim 1 in the medical field, especially as a material for promoting osteochondral regeneration.

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