A biomimetic mineralized hydrogel and a preparation method and application thereof

Abstract

This application provides a biomimetic mineralized hydrogel, relating to the field of medical polymer materials. The biomimetic mineralized hydrogel provided in this application uses a biodegradable polymer containing carboxylate groups as a mineralization template, and phosphate-containing small molecules as strong ligands and phosphate donors, inducing Fe... 3+ In-situ mineralization forms stable iron-bearing mineralized crystals, achieving Fe³⁺ + Long-term, low-dose sustained release, thereby delivering Fe 3+ The concentration is maintained within a safe window to allow it to exert its positive bioregulatory effects while avoiding the risks of iron overload-induced oxidative damage and ferroptosis. Simultaneously, it slows the release of Fe³⁺. + It can regulate cellular iron metabolism and activate antioxidant stress-related pathways, inhibit ferroptosis, and create a favorable repair microenvironment for osteochondral regeneration. Experimental results show that this hydrogel not only has excellent flexibility, biocompatibility, antibacterial properties, and angiogenesis-promoting properties, but also promotes osteogenic and chondrogenic differentiation of cells, thereby achieving an integrated osteochondral repair effect.

Description

Technical Field

[0001] This application relates to the field of medical polymer materials, and in particular to a biomimetic mineralized hydrogel, its preparation method, and its application. Background Technology

[0002] Joint injuries are increasingly common among the elderly and those engaging in high-intensity exercise, affecting over 300 million people worldwide. Early symptoms may only include swelling and pain, but severe or late-stage injuries can lead to joint defects that are difficult to heal. Clinically, based on lesion depth, joint defects are classified into three categories: partial-thickness, full-thickness, and osteochondral defects. The latter often develops from superficial cartilage defects and, if not treated properly, can eventually involve the subchondral bone. Unlike skin or bone tissue, which can heal spontaneously if the defect does not exceed a critical size, cartilage has extremely weak self-repair capabilities due to a lack of blood vessels and nerves, low cell density, and poor metabolic capacity. Furthermore, osteochondral units have a complex and delicate structure, composed of cartilage layers and subchondral bone with varying characteristics, bearing weight and transmitting force. Therefore, achieving integrated regeneration of osteochondral defects remains a significant clinical challenge.

[0003] Currently, clinical treatments for osteochondral defects mainly include microfracture surgery, autologous transplantation (such as autologous chondrocyte implantation), and allogeneic tissue transplantation. These conventional methods can usually relieve pain in the short term, but they cannot cure the disease and are difficult to completely repair the complex osteochondral tissue structure and restore its normal function in the long term. Poor long-term repair results may even aggravate tissue damage, expand the lesion area, and ultimately lead to poor clinical efficacy and treatment failure. In recent years, the development of advanced biomaterials has promoted extensive research on scaffold-based tissue engineering therapy techniques (such as matrix-induced autologous chondrocyte implantation) in the regeneration and repair of osteochondral defects. Tissue engineering combines cell biology and materials science to construct functional tissues and even organs in vivo to maintain, repair, or enhance diseased tissues. However, these cell-borne tissue constructs, which require in vitro culture before implantation, still face significant limitations: limited stem cell sources, low survival rate of exogenous cells, poor and uncontrollable stem cell differentiation, and complex procedures. These problems severely limit their clinical translation, and therefore they are currently mainly used for tissues with simpler structures (such as skin).

[0004] Inspired by biomineralization processes, mineralized hydrogels, as a typical representative of organic-inorganic composite systems, have become a research hotspot in osteochondral repair materials. These materials typically use polymeric hydrogels as a matrix, inducing in-situ mineralization through the introduction of metal ions to construct composite scaffolds with certain mechanical support capabilities. However, while most mineralized hydrogels currently possess high mechanical strength, they generally lack sufficient toughness, making them prone to brittle fracture under long-term dynamic loads on joints, thus failing to meet the comprehensive mechanical performance requirements of osteochondral repair materials. Furthermore, existing technologies largely rely on Ca²⁺... + Mg² +Conventional metal ions, such as Fe³⁺, lack the ability to actively regulate the cellular metabolic microenvironment. + While possessing potential advantages, its rapid coordination rate and tendency to aggregate make it difficult to prepare a uniform and stable gel system. More importantly, existing materials cannot simultaneously achieve multiple functions such as antibacterial activity, angiogenesis promotion, iron metabolism regulation, oxidative stress resistance, and osteochondral differentiation promotion, severely limiting their clinical application. Therefore, developing a biomimetic mineralized hydrogel with a uniform structure, well-defined functions, and good toughness and moderate strength is of great significance for overcoming the technical bottlenecks in the field of osteochondral repair. Summary of the Invention

[0005] In view of this, this application provides a biomimetic mineralized hydrogel, its preparation method and application. The biomimetic mineralized hydrogel provided by this application can achieve the integration of mineralization enhancement and biological function regulation, and improve the microenvironment for osteochondral regeneration.

[0006] This application provides a biomimetic mineralized hydrogel, which is mainly composed of a biodegradable polymer containing carboxyl groups, small molecules containing phosphate groups, and Fe. 3+ get; The phosphate-containing small molecules have one or more of the structures of formula (I-1), (I-2), (I-3), (I-4), (I-5), and (I-6); (Ⅰ-1); (Ⅰ-2); (Ⅰ-3); (Ⅰ-4); (Ⅰ-5); (Ⅰ-6).

[0007] In some specific implementations, the Fe 3+ It is selected from one or more of ferric chloride, ferric chloride hexahydrate, ferric nitrate, ferric nitrate nonahydrate, ferric sulfate, ferric sulfate nonahydrate, and ferric acetate.

[0008] In some specific implementations, the carboxylate groups of the degradable polymer containing carboxylate, the phosphate groups of small phosphate molecules, and Fe... 3+ The molar ratio is (2~20):(0.1~10):1.

[0009] In some specific implementations, the biomimetic mineralized hydrogel also includes bioactive factors.

[0010] In some specific implementations, the bioactive factor is selected from one or more of transforming growth factor-β (TGF-β), insulin-like growth factor-1 (IGF-1), and Kartogenin (KGN).

[0011] In some specific implementations, the carboxylate-containing biodegradable polymer has one or more of the structures of formula (II-1), (II-2), (II-3), (II-4), and (II-5); (Ⅱ-1); (Ⅱ-2); (Ⅱ-3); (Ⅱ-4); (Ⅱ-5); Wherein, R is H or CH2COO - ; n, m, x, y, and b are all degrees of aggregation; 30≤n≤7000, 50≤m≤8500, 20≤x≤6500, 100≤y≤6000, 20≤b≤6500.

[0012] This application also provides a method for preparing a biomimetic mineralized hydrogel, comprising the following steps: A) A precursor hydrogel is obtained by mixing a biodegradable polymer containing carboxyl groups and a small molecule containing phosphate groups. B) The precursor hydrogel was combined with Fe 3+ After the compounds are mixed, in-situ mineralization is carried out through ion diffusion under static conditions to obtain a biomimetic mineralized hydrogel.

[0013] In some specific implementations, the settling temperature is 0℃~80℃, and the settling time is 5h~12 days.

[0014] In some specific implementations, the biomimetic mineralized hydrogel is immersed in an external environment to obtain a biomimetic iron phosphate hydrogel. The external environment is one or more of the following: phosphate buffer solution, bone, and body fluids.

[0015] This application also provides a bone and cartilage repair material, including the biomimetic mineralized hydrogel described in any of the above technical solutions or the biomimetic mineralized hydrogel prepared by the preparation method described in any of the above technical solutions.

[0016] This application provides a biomimetic mineralized hydrogel, which is composed of a biodegradable polymer containing carboxyl groups, small molecules containing phosphate groups, and Fe. 3+The phosphate-containing small molecules are obtained; they possess one or more of the structures of formulas (I-1), (I-2), (I-3), (I-4), (I-5), and (I-6). The biomimetic mineralized hydrogel provided in this application uses a carboxylate-containing biodegradable polymer as a three-dimensional network matrix and mineralization template, with phosphate-containing small molecules acting as strong ligands and phosphate donors, inducing Fe... 3+ In-situ mineralization forms stable coordination nodes, and the Fe formed by mineralization 3+ Fe exists in the form of nanoparticles in the hydrogel network to achieve 3+ Sustained release; and simultaneously utilizing sustained-release Fe 3+ This hydrogel activates cellular iron metabolism and antioxidant stress pathways, inhibits ferroptosis, and creates a superior microenvironment for osteochondral regeneration. Experimental results show that the hydrogel not only possesses excellent flexibility, blood compatibility, biocompatibility, antibacterial properties, and angiogenesis-promoting properties, but also promotes osteogenic differentiation, in vitro mineralization deposition of cells, cartilage matrix synthesis, and integrated osteochondral repair. Furthermore, the preparation method provided in this application yields a biomimetic mineralized hydrogel with a uniform and stable structure, and is simple to prepare with readily available raw materials, showing broad clinical translational prospects in the field of minimally invasive repair of osteochondral defects. Attached Figure Description

[0017] Figure 1 A schematic diagram of the construction technology route for the multifunctional biomimetic mineralized hydrogel provided in this application; Figure 2 This is a cross-sectional morphology diagram of the hydrogel described in Example 1 of this application; Figure 3 This is an internal morphology diagram of the hydrogel after molding as described in Example 1 of this application; Figure 4 The strain-modulus test results are for the hydrogels described in Example 1 and Comparative Examples 1-5 of this application; Figure 5 These are stability images of the hydrogel described in Example 1 of this application in different media environments; Figure 6 The results of the hemolysis rate test of the hydrogel described in Example 2 of this application; Figure 7 This is a morphological image of red blood cells after co-incubation of the hydrogel described in Example 2 of this application with rabbit red blood cells for 3 hours; Figure 8 The results of the cytotoxicity test of the hydrogel described in Example 3 of this application; Figure 9 The results are the antibacterial performance test results of the hydrogel described in Example 4 of this application; Figure 10 The results of the hydrogel ALP staining test described in Example 5 of this application; Figure 11The results of the hydrogel ARS staining test described in Example 6 of this application; Figure 12 The results of the Sox9 hydrogel immunofluorescence assay described in Example 7 of this application; Figure 13 This is a tube-forming image of the hydrogel described in Example 8 of this application; Figure 14 This is a macroscopic photograph of the hydrogel treatment group described in Example 10 of this application. Detailed Implementation

[0018] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0019] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0020] It should be understood that the order of steps or the sequence of actions is not important as long as this application remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0021] The use of any and all instances or exemplary language such as “e.g.” or “including” herein is merely intended to better illustrate the application and does not constitute a limitation on the scope of the application unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of this application.

[0022] Furthermore, the numerical ranges and parameters used to define this application are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0023] This application provides a biomimetic mineralized hydrogel, which is mainly composed of a biodegradable polymer containing carboxyl groups, small molecules containing phosphate groups, and Fe. 3+ get; The phosphate-containing small molecules have one or more of the structures of formula (I-1), (I-2), (I-3), (I-4), (I-5), and (I-6); (Ⅰ-1); (Ⅰ-2); (Ⅰ-3); (Ⅰ-4); (Ⅰ-5); (Ⅰ-6).

[0024] This application uses a biodegradable polymer containing carboxylate groups as a mineralization template, and functional small molecules containing phosphate groups crystallize themselves while simultaneously acting as crystal nuclei to induce Fe... 3+ Depositional mineralization, coupled with the negative charge of the mineralization template, can slow down the crystallization rate, resulting in more compact and regular crystals. Ultimately, the negatively charged groups, phosphate groups, and Fe in the mineralization template contribute to this process. 3+ Co-coordination and crystallization of the system form a hydrogel cross-linked with mineralized crystals. The mineralized Fe... 3+ Fe exists in the form of nanoparticles in the hydrogel network to achieve 3+ Sustained release; and simultaneously utilizing sustained-release Fe 3+ It activates cellular iron metabolism and antioxidant stress pathways, inhibits ferroptosis, and creates a high-quality microenvironment for osteochondral regeneration.

[0025] The specific mechanism by which iron metabolism is regulated is: the sustained release of Fe by the hydrogel. 3+ Fe can be actively recognized and taken up by cells through transferrin receptors (TFRs) on the cell membrane surface, and enter the cell. 3+ First, it is reduced to ferrous ions (Fe2+). Then, some of these ferrous ions are safely stored under the action of ferritin, achieving controlled storage and buffering of iron ions and preventing the accumulation of free iron ions in the cell, which could lead to oxidative stress damage. The remaining ferrous ions participate in normal physiological metabolic processes such as hemoglobin synthesis and electron transport, meeting the iron supply required for cell growth, proliferation, and differentiation. Simultaneously, Fe... 3+ The sustained release of iron can activate intracellular iron metabolism regulatory pathways. By upregulating the expression of iron metabolism-related genes (such as Ferritin, TFR, and iron regulatory proteins), it dynamically balances the uptake, storage, and utilization of intracellular iron ions, maintains intracellular iron homeostasis, avoids cellular metabolic disorders caused by iron deficiency, or lipid peroxidation and ferroptosis caused by iron excess, thereby ensuring the survival and functional activity of chondrocytes, osteoblasts, and mesenchymal stem cells. It effectively regulates the oxidative stress microenvironment in the defect area, inhibits ferroptosis, and significantly promotes the integrated repair and reconstruction of osteochondral tissue.

[0026] In the biomimetic mineralized hydrogel provided in this application, the flexible segments of the carboxylate-containing biodegradable polymer provide a good flexible framework for the hydrogel, giving the gel basic elasticity. In some specific implementations, the carboxylate-containing biodegradable polymer has one or more of the structures (II-1), (II-2), (II-3), (II-4), and (II-5); (Ⅱ-1); (Ⅱ-2); (Ⅱ-3); (Ⅱ-4); (Ⅱ-5); Wherein, R is H or CH2COO - ; The n, m, x, y, and b are all degrees of polymerization, with 30≤n≤7000, preferably 1000≤n≤7000, and more preferably 3000≤n≤5000; 50≤m≤8500, preferably 1000≤m≤8500, and more preferably 3000≤m≤6000; 20≤x≤6500, preferably 1000≤x≤6500, and more preferably 3000≤x≤5000; 100≤y≤6000, preferably 1000≤y≤6000, and more preferably 3000≤y≤5000; and 20≤b≤6500, preferably 1000≤b≤6500, and more preferably 3000≤b≤5000.

[0027] In some specific implementations, the carboxylate-containing biodegradable polymer is preferably structured as shown in formulas (II-2), (II-3), (II-4), or (II-5): (Ⅱ-2); (Ⅱ-3); (Ⅱ-4); (Ⅱ-5); A more preferred structure is the one shown in formula (II-5): (Ⅱ-5); The values ​​m, x, y, and b represent the degree of polymerization, with 50 ≤ m ≤ 8500, preferably 1000 ≤ m ≤ 8500, and more preferably 3000 ≤ m ≤ 6000; 20 ≤ x ≤ 6500, preferably 1000 ≤ x ≤ 6500, and more preferably 3000 ≤ x ≤ 5000; 100 ≤ y ≤ 6000, preferably 1000 ≤ y ≤ 6000, and more preferably 3000 ≤ y ≤ 5000; and 20 ≤ b ≤ 6500, preferably 1000 ≤ b ≤ 6500, and more preferably 3000 ≤ b ≤ 5000.

[0028] In some specific implementations, the viscosity of sodium carboxymethyl cellulose shown in formula (II-3) is 500 mPa·s to 15000 mPa·s, more preferably 500 mPa·s to 5000 mPa·s. In some specific implementations, the viscosity of carboxymethyl chitosan shown in formula (II-4) is 10 mPa·s to 100 mPa·s, more preferably 40 mPa·s to 80 mPa·s.

[0029] In the biomimetic mineralized hydrogel provided in this application, the phosphate-containing small molecule serves as a strong ligand and phosphate donor. The phosphate-containing small molecule described in this application has one or more of the structures of formulas (I-1), (I-2), (I-3), (I-4), (I-5), and (I-6); (Ⅰ-1); (Ⅰ-2); (Ⅰ-3); (Ⅰ-4); (Ⅰ-5); (Ⅰ-6).

[0030] In some specific implementations, the phosphate-containing small molecule is preferably the structure shown in formula (I-1), (I-2), or (I-3): (Ⅰ-1); (Ⅰ-2); (Ⅰ-3); A more preferred structure is the one shown in formula (Ⅰ-2): (Ⅰ-2).

[0031] In the biomimetic mineralized hydrogel provided in this application, through Fe 3+ This mediates in-situ coordination mineralization, forming a uniform and stable soft elastic network structure. In some specific implementations, the Fe... 3+ The ferric chloride is selected from one or more of ferric chloride, ferric chloride hexahydrate, ferric nitrate, ferric nitrate nonahydrate, ferric sulfate, ferric sulfate nonahydrate, and ferric acetate, preferably ferric chloride hexahydrate, ferric nitrate, or ferric acetate, and more preferably ferric chloride hexahydrate. In some specific implementations, the carboxylate group of the degradable polymer containing carboxylate, the phosphate group of the small phosphate molecule, and Fe... 3+ The molar ratio is (2~20):(0.1~10):1, preferably (2~15):(0.2~6):1, and more preferably (2~12):(0.2~2):1.

[0032] The biomimetic mineralized hydrogel provided in this application also includes bioactive factors. In some specific implementations, the bioactive factors are selected from one or more of transforming growth factor-β (TGF-β), insulin-like growth factor-1 (IGF-1), and Kartogenin (KGN). In some specific implementations, the carboxyl group of the degradable polymer containing carboxyl groups, the phosphate group of small molecules containing phosphate groups, and Fe... 3+ The molar ratio of bioactive factors is (2~20):(0.1~10):1:(10) -9 ~10 -2 The preferred order is (2~15):(0.2~6):1:(10). -7 ~10 -2 More preferably, it is (2~12):(0.2~2):1:(10) -6 ~10 -3 ).

[0033] The biomimetic mineralized hydrogel provided in this application preferably has a water content of 50% to 99%.

[0034] The biomimetic mineralized hydrogel provided in this application uses an electronegatively charged biodegradable polymer containing carboxylate groups as a mineralization template, a functional small molecule containing phosphate groups as a phosphate donor, and Fe is added. 3+ As mineralizing ions, through Fe 3 + Mineralization occurs through coordination with small molecules containing phosphate groups, leading to the formation of nanoparticles and thus realizing Fe 3+ It also features sustained release of small phosphate molecules. Furthermore, this hydrogel exhibits excellent flexibility, blood compatibility, biocompatibility, antibacterial properties, angiogenesis-promoting properties, and good osteochondral repair effects.

[0035] The hydrogel exhibits excellent toughness, primarily due to the three-dimensional network formed by the carboxylate-containing biodegradable polymer and the phosphate-containing small-molecule -Fe. 3+ Ion-constructed coordination mineralization nodes form a synergistic effect. Flexible segments of carboxylate-containing biodegradable polymers provide a good flexible framework, imparting elasticity to the gel base; while phosphate-containing small molecules and Fe... 3+ Iron phosphate particles formed through in-situ mineralization act as rigidity-enhancing sites, uniformly dispersed within the network, effectively dispersing external forces and preventing crack propagation; furthermore, Fe... 3+The dynamic coordination between the hydrogel and the carboxyl groups of carboxylate-containing biodegradable polymers and the phosphate groups of phosphate-containing small molecules allows the coordination bonds to break and recombine rapidly when the gel is subjected to external stretching or compression, absorbing energy and restoring its original structure, thus significantly improving the hydrogel's tear resistance and toughness. The hydrogel also exhibits unique environmental responsiveness. This responsiveness is significantly reflected in its differentiated behavior in different media: in a constant temperature and humidity environment of 37°C, the hydrogel gradually thins over time, exhibiting flexible state tunability; while in PBS or culture media simulating physiological environments, it maintains structural stability, ensuring its supporting role during the repair process.

[0036] This precise response to the environment and the dynamic control of the aforementioned mechanical properties make it more suitable for the complex needs of osteochondral repair.

[0037] The hydrogel also exhibits rich bioactivity, containing Fe. 3+ The complex system formed with phosphate-containing small molecules can disrupt the integrity of bacterial cell membranes and inhibit bacterial metabolism and reproduction, giving the gel excellent antibacterial properties and effectively reducing the risk of infection in areas of osteochondral defects; at the same time, Fe 3+ It can activate cellular iron metabolism pathways and Nrf2 antioxidant pathways, synergistically regulate the expression of related genes such as vascular endothelial growth factor, thereby promoting vascular endothelial cell proliferation and tube formation, achieving efficient angiogenesis, and providing sufficient nutrient supply for subchondral bone regeneration.

[0038] This application also provides a method for preparing a biomimetic mineralized hydrogel, comprising the following steps: A) A precursor hydrogel is obtained by mixing small molecules containing phosphate and degradable polymers containing carboxylate. B) The precursor hydrogel was combined with Fe 3+ After the compounds are mixed, in-situ mineralization is carried out by ion diffusion under completely static conditions to obtain a biomimetic mineralized hydrogel.

[0039] The preparation method provided in this application is through Fe 3+ Hydrogels are prepared using a free diffusion method, where functional small molecules containing phosphate groups act as nucleation sites. Metal ions deposit and crystal nuclei grow, simultaneously forming negatively charged groups, phosphate groups, and Fe from biodegradable polymer compounds. 3+ Co-coordination, preparation process see Figure 1 , Figure 1 A schematic diagram of the construction technology route for the multifunctional biomimetic mineralized hydrogel provided in this application.

[0040] This application first mixes small molecules containing phosphate groups with a biodegradable polymer containing carboxylate groups to obtain a precursor hydrogel. Specifically, the small molecules containing phosphate groups are dissolved in an alkaline solution to obtain a solution of small molecules containing phosphate groups. The solution of small molecules containing phosphate groups is then mixed with the biodegradable polymer containing carboxylate groups, and the mixture is stirred to obtain the precursor hydrogel.

[0041] The specific types of the phosphate-containing small molecules and carboxylate-containing biodegradable polymers are as described above and will not be repeated here. In some specific implementations, the pH value of the alkaline solution is preferably 8-10, more preferably 8.5-9.5, and the alkaline solution includes, but is not limited to, sodium hydroxide solution, sodium carbonate-sodium bicarbonate buffer solution, and alkaline phosphate buffer solution, and may be one or more of these. In some specific implementations, the dissolution temperature is preferably room temperature. In some specific implementations, the molar ratio of carboxylate groups in the carboxylate-containing biodegradable polymer to phosphate groups in the phosphate-containing small molecules is (2-20):(0.1-10), preferably (2-15):(0.2-6), more preferably (2-12):(0.2-2). In some specific implementations, the mixing is preferably carried out under stirring conditions, and the stirring time is 10 min-40 min, preferably 10 min-30 min. In some specific implementations, the water content of the precursor hydrogel is preferably 50%-99%.

[0042] After obtaining the precursor hydrogel, the precursor hydrogel and Fe... 3+ After the compounds are mixed, in-situ mineralization occurs via ion diffusion under completely static conditions to obtain a biomimetic mineralized hydrogel. The gelation principle of the hydrogels described in this application is based on physical interactions.

[0043] The Fe 3+ The specific types are as described above, and will not be repeated here. In some specific implementations, the carboxylate groups of the degradable polymer containing carboxylate, the phosphate groups of small molecules containing phosphate, and Fe... 3+ The molar ratio is (2~20):(0.1~10):1, preferably (2~15):(0.2~6):1, and more preferably (2~12):(0.2~2):1. In some specific implementations, the in-situ mineralization is preferably carried out in a constant temperature and humidity incubator, wherein the constant temperature is 0℃~80℃, preferably 20℃~70℃, and the time is 5h~12 days, preferably 10h~10 days. In some specific implementations, the water content of the biomimetic mineralized hydrogel is preferably 50%~99%.

[0044] After obtaining the precursor hydrogel, it is preferable to combine the precursor hydrogel with Fe...3+ After mixing the compound and bioactive factors, in-situ mineralization is carried out via ion diffusion under completely static conditions to obtain a biomimetic mineralized hydrogel. The specific types of bioactive components are as described above and will not be repeated here. In some specific implementations, the carboxylate groups of the degradable polymer containing carboxylate, the phosphate groups of small phosphate molecules, and Fe... 3+ The molar ratio of bioactive factors is (2~20):(0.1~10):1:(10) -9 ~10 -2 The preferred order is (2~15):(0.2~6):1:(10). -7 ~10 -2 More preferably, it is (2~12):(0.2~2):1:(10) -6 ~10 -3 The preparation method provided in this application is based on ion autonomous diffusion. Under completely static conditions, in-situ mineralization is achieved through ion diffusion to obtain a biomimetic mineralized hydrogel with a uniform and stable structure. The preparation method provided in this application effectively solves the Fe... 3+ The method avoids the problem of uneven aggregation caused by excessively rapid coordination. In addition, the preparation method is simple and mild, requires no additional chemical cross-linking agents, has a wide range of raw material sources, and is cost-controllable. It is easy to achieve large-scale preparation and has great potential for clinical translation.

[0045] After obtaining the biomimetic mineralized hydrogel, this application allows the hydrogel to be immersed in an external environment to undergo ion exchange, resulting in a biomimetic iron phosphate hydrogel. This step demonstrates that the gel can eventually transform into a bio-bone-like structure in a simulated body fluid environment. Furthermore, during use, the hydrogel can slowly transform into a bio-bone-like structure and eventually degrade through body fluid exchange, further illustrating its excellent biocompatibility. In some specific implementations, the external environment includes, but is not limited to, phosphate buffer solution, bone, body fluid, etc., and can be one or more of these. In some specific implementations, the time for ion exchange to reach equilibrium is preferably 10 to 30 days, more preferably 15 to 25 days. In some specific implementations, the ambient temperature for ion exchange is preferably 10°C to 60°C, more preferably 15°C to 40°C.

[0046] The hydrogel provided in this application can transform into a biomimetic phosphate saline gel in the later stages of osteochondral repair. During this later repair phase, phosphate ions in the body will exchange with the drug within the hydrogel, and the phosphate ions will displace the Fe ions within the hydrogel. 3+ The combination transforms the hydrogel into a phosphate-containing hydrogel. Its structure is similar to that of human bone, further promoting the repair of bone wounds, while also exhibiting excellent biocompatibility and biodegradability.

[0047] This application also provides a bone and cartilage repair material, including the biomimetic mineralized hydrogel described in any of the above technical solutions or the biomimetic mineralized hydrogel prepared by any of the above preparation methods.

[0048] This biomimetic mineralized hydrogel uses a biodegradable polymer containing carboxylate groups as a natural matrix and small molecules containing phosphate groups as functional ligands, through Fe... 3+ It mediates in-situ coordination mineralization to form a uniform and stable soft elastic network structure that can directly adapt to and closely fit irregular osteocartilage defects, achieving precise filling without injection.

[0049] This application provides a biomimetic mineralized hydrogel, which is composed of a biodegradable polymer containing carboxyl groups, small molecules containing phosphate groups, and Fe. 3+ The phosphate-containing small molecules are obtained; they possess one or more of the structures of formulas (I-1), (I-2), (I-3), (I-4), (I-5), and (I-6). The biomimetic mineralized hydrogel provided in this application uses a carboxylate-containing biodegradable polymer as a three-dimensional network matrix and mineralization template, with phosphate-containing small molecules acting as strong ligands and phosphate donors, inducing Fe... 3+ In-situ mineralization forms stable coordination nodes, and the Fe formed by mineralization 3+ Fe exists in the form of nanoparticles in the hydrogel network to achieve 3+ Sustained release; and simultaneously utilizing sustained-release Fe 3+ This hydrogel activates cellular iron metabolism and antioxidant stress pathways, inhibits ferroptosis, and creates a superior microenvironment for osteochondral regeneration. Experimental results show that the hydrogel not only possesses excellent flexibility, blood compatibility, biocompatibility, and antibacterial properties, but also promotes osteogenic differentiation, in vitro mineralization and deposition of cells, maintains chondrocyte phenotype, promotes cartilage matrix synthesis, promotes angiogenesis, and facilitates integrated osteochondral repair. Furthermore, the preparation method provided in this application yields a biomimetic mineralized hydrogel with a uniform and stable structure, and is simple to prepare with readily available raw materials, showing broad clinical translational prospects in the field of minimally invasive repair of osteochondral defects.

[0050] The present invention is further illustrated below with reference to the embodiments. The scope of protection of the present invention is not limited to the following embodiments.

[0051] Example 1

[0052] a. At room temperature, add 18 mg of alendronate sodium trihydrate (Formula I-2) to 1 mL of sodium hydroxide solution with pH = 9.5, dissolve completely, and prepare a solution; b. Add 150 mg of sodium hyaluronate (Formula II-5, Mn=1000 kDa) to the final solution obtained in step a and stir for 15 min to fully dissolve it and prepare the precursor hydrogel; c. 1.2 g / mL ferric chloride hexahydrate solution: Add 18 μL of ferric chloride hexahydrate solution to the system obtained in step b and allow it to undergo sufficient ion diffusion. d. The system obtained in step c was placed in a 40℃ constant temperature and humidity incubator and left to stand for 24 hours to successfully prepare a hydrogel; e. After final solidification, the hydrogel is immersed in PBS (a simulated body fluid) at room temperature for 15 days to allow for sufficient ion exchange inside and outside the body, thus preparing a biomimetic ferric phosphate hydrogel.

[0053] Example 2

[0054] a. Add 35 mg of alendronate sodium trihydrate (Formula I-2) to 2 mL of sodium carbonate-sodium bicarbonate buffer solution (pH = 9.4), dissolve completely, and prepare a solution; b. Add 300 mg of carboxymethyl chitosan (Formula II-4, Mn=1000 kDa, η=80 mPa·s) to the final solution obtained in step a and stir for 20 min to fully dissolve it and prepare the precursor hydrogel; c. Prepare a 1 g / mL ferric chloride hexahydrate solution. Add 40 μL of the ferric chloride hexahydrate solution to the system obtained in step b and allow it to undergo sufficient ion diffusion. d. The hydrogel can be successfully prepared by placing the system obtained in step c at 60°C for 12 hours; e. After final solidification, the hydrogel is immersed in PBS (a simulated body fluid) at room temperature for 15 days to allow for sufficient ion exchange inside and outside the body, thus preparing a biomimetic ferric phosphate hydrogel.

[0055] Example 3

[0056] a. Add 35 mg of zoledronic acid disodium (Formula I-3) to 2 mL of sodium carbonate-sodium bicarbonate buffer solution (pH = 9.4), dissolve completely, and prepare a solution; b. Add 300 mg of carboxymethyl chitosan (Formula II-4, Mn=800 kDa, η=60 mPa·s) to the final solution obtained in step a and stir for 20 min to fully dissolve it and prepare the precursor hydrogel; c. Prepare a 750 mg / mL ferric chloride hexahydrate solution and a 10 μg / μL TGF-β solution. Add 80 μL of ferric chloride hexahydrate and 3 μL of TGF-β solution to the system obtained in step b and allow it to undergo sufficient ion diffusion. d. The hydrogel can be successfully prepared by placing the system obtained in step c at 24°C for 24 hours; e. The final hydrogel was immersed in PBS, a simulated body fluid, at room temperature for 15 days to allow for sufficient ion exchange inside and outside the body, thus preparing a biomimetic ferric phosphate hydrogel.

[0057] Example 4

[0058] a. Add 45 mg of dexamethasone sodium phosphate (Formula I-1) to 2 mL of sodium carbonate-sodium bicarbonate buffer solution (pH = 9.4), dissolve completely, and prepare a solution; b. Add 180 mg of sodium alginate (Formula II-2, Mn = 300 kDa) to the final solution obtained in step a and stir for 20 minutes to ensure complete dissolution; c. Prepare an 800 mg / mL ferric chloride hexahydrate solution and a 10 μg / μL IGF-1 solution. Add 70 μL of the ferric chloride hexahydrate solution and 2 μL of the IGF-1 solution to the system obtained in step b and allow it to undergo sufficient ion diffusion. d. The hydrogel can be successfully prepared by placing the system obtained in step c at 15°C for 7 days; e. The final hydrogel was immersed in PBS, a simulated body fluid, at room temperature for 15 days to allow for sufficient ion exchange inside and outside the body, thus preparing a biomimetic ferric phosphate hydrogel.

[0059] Example 5

[0060] a. Add 48 mg of dexamethasone sodium phosphate (Formula I-1) to 2 mL of PBS buffer solution (pH = 7.5), and adjust the pH of the solution to about 9 using 100 mg / mL NaOH to prepare the solution; b. Add 200 mg of sodium carboxymethyl cellulose (Formula II-3, Mn = 1200 kDa, η = 2000 mPa·s) to the final solution obtained in step a and stir for 15 min to ensure complete dissolution; c. Prepare a 1 g / mL ferric chloride hexahydrate solution. Add 78 μL of the ferric chloride hexahydrate solution to the system obtained in step b and allow it to undergo sufficient ion diffusion. d. The hydrogel can be successfully prepared by placing the system obtained in step c at 25°C for 2 days; e. The final hydrogel was immersed in PBS (a simulated body fluid) at 37°C for 15 days to allow for sufficient ion exchange between the inside and outside of the body, thus preparing a biomimetic ferric phosphate hydrogel.

[0061] Example 6

[0062] a. Add 48 mg of dexamethasone sodium phosphate (Formula I-1) to 2 mL of PBS buffer solution (pH = 7.5), and adjust the pH of the solution to about 9 using 100 mg / mL NaOH to prepare the solution; b. Add 200 mg of sodium carboxymethyl cellulose (Formula II-3, Mn = 500 kDa, η = 1600 mPa·s) to the final solution obtained in step a and stir for 15 min to ensure complete dissolution; c. Prepare a 900 mg / mL ferric chloride hexahydrate solution and a 10 μg / μL KGN solution. Add 90 μL of the ferric chloride hexahydrate solution and 10 μL of the KGN solution to the system obtained in step b and allow it to undergo sufficient ion diffusion. d. The hydrogel can be successfully prepared by placing the system obtained in step c at 20°C for 3 days; e. The final hydrogel was immersed in PBS (a simulated body fluid) at 25°C for 20 days to allow for sufficient ion exchange inside and outside the body, thus preparing a biomimetic iron phosphate hydrogel.

[0063] Example 7

[0064] a. Add 30 mg of dexamethasone sodium phosphate (Formula I-1) to 2 mL of sodium carbonate-sodium bicarbonate buffer solution (pH = 9.4), dissolve completely, and prepare a solution; b. Add 500 mg of sodium carboxymethyl cellulose (Formula II-3, Mn = 1300 kDa, η = 5000 mPa·s) to the final solution obtained in step a and stir for 15 min to ensure complete dissolution; c. Prepare an 800 mg / mL ferric nitrate solution by adding 100 μL of the ferric nitrate solution to the system obtained in step b, allowing for sufficient ion diffusion. d. The hydrogel can be successfully prepared by placing the system obtained in step c at 37°C for 24 hours; e. The final hydrogel was immersed in PBS, a simulated body fluid, at room temperature for 25 days to allow for sufficient ion exchange inside and outside the body, thus preparing a biomimetic ferric phosphate hydrogel.

[0065] Example 8

[0066] a. Add 60 mg of dexamethasone sodium phosphate (Formula I-1) to 2 mL of PBS (pH = 7.5), and adjust the pH of the system to approximately 9 using 50 mg / mL NaOH solution to prepare the solution; b. Add 600 mg of sodium hyaluronate (Formula II-5, Mn=1500 kDa) to the final solution obtained in step a and stir for 10 min to fully dissolve it and prepare the precursor hydrogel; c. Prepare a 500 mg / mL ferric chloride hexahydrate solution, add 90 μL of the ferric chloride hexahydrate solution to the system obtained in step b, and allow it to undergo sufficient ion diffusion; d. The hydrogel can be successfully prepared by placing the system obtained in step c at 30°C for 24 hours; e. The final hydrogel was immersed in PBS, a simulated body fluid, at room temperature for 15 days to allow for sufficient ion exchange inside and outside the body, thus preparing a biomimetic ferric phosphate hydrogel.

[0067] Example 9

[0068] a. Add 60 mg of dexamethasone sodium phosphate (Formula I-1) to 2 mL of PBS (pH = 7.5), and adjust the pH of the system to approximately 9 using 100 mg / mL KOH solution to prepare the solution; b. Add 650 mg of carboxymethyl chitosan (Formula II-4, Mn=1100 kDa, η=90 mPa·s) to the final solution obtained in step a and stir for 10 min to fully dissolve it and prepare the precursor hydrogel; c. Prepare a 650 mg / mL ferric acetate solution, add 80 μL of the ferric acetate solution to the system obtained in step b, and allow it to undergo sufficient ion diffusion; d. The hydrogel can be successfully prepared by placing the system obtained in step c at 40℃ for 24 hours; e. The final hydrogel was immersed in PBS (a simulated body fluid) at room temperature for 20 days to allow for sufficient ion exchange inside and outside the body, thus preparing a biomimetic iron phosphate hydrogel.

[0069] Example 10

[0070] a. Add 50 mg of zoledronic acid disodium salt tetrahydrate (Formula I-3) to 2 mL of PBS (pH = 7.5), and adjust the pH of the system to about 9 using 50 mg / mL triethylamine solution to prepare the solution; b. Add 100 mg of sodium hyaluronate (Formula II-1, Mn = 2000 kDa) to the final solution obtained in step a and stir for 10 min to fully dissolve it and prepare the precursor hydrogel; c. Prepare a 1 g / mL ferric chloride hexahydrate solution and a 10 μg / μL KGN solution. Add 15 μL of the ferric chloride hexahydrate solution and 4 μL of the KGN solution to the system obtained in step b and allow it to undergo sufficient ion diffusion. d. The hydrogel can be successfully prepared by placing the system obtained in step c at 37°C for 24 hours; e. The final hydrogel was immersed in PBS, a simulated body fluid, at room temperature for 15 days to allow for sufficient ion exchange inside and outside the body, thus preparing a biomimetic ferric phosphate hydrogel.

[0071] Comparative Example 1

[0072] The only difference from Example 1 is that the ferric chloride hexahydrate solution is replaced with a calcium chloride solution.

[0073] Comparative Example 2

[0074] The only difference from Example 1 is that the ferric chloride hexahydrate solution is replaced with a magnesium chloride solution.

[0075] Comparative Example 3

[0076] The only difference from Example 1 is that the ferric chloride hexahydrate solution is replaced with a manganese chloride solution.

[0077] Comparative Example 4

[0078] The only difference from Example 1 is that the ferric chloride hexahydrate solution is replaced with a copper chloride solution.

[0079] Comparative Example 5

[0080] The only difference from Example 1 is that the ferric chloride hexahydrate solution is replaced with a strontium chloride solution.

[0081] Experimental Example 1

[0082] The morphological characteristics of the hydrogel after molding were tested.

[0083] The hydrogels prepared in Example 1 were each frozen at -20°C for 24 hours and then freeze-dried in a freeze dryer to remove water. The resulting freeze-dried hydrogels were then rapidly frozen in liquid nitrogen and quickly broken apart. The surface morphology of the fracture surface was observed using a scanning electron microscope.

[0084] Results reference Figure 2 and Figure 3 ,in Figure 2 This is a cross-sectional morphology diagram of the hydrogel described in Embodiment 1 of this application. It can be seen that its three-dimensional skeleton has a macroporous structure and a relatively thick network pore wall. Figure 3 This is an internal morphology diagram of the hydrogel after molding as described in Embodiment 1 of this application. It can be observed that a large number of cubic particles formed by in-situ mineralization are distributed on the pore walls.

[0085] Experimental Example 2

[0086] The change in hydrogel modulus is evaluated by testing the rheological behavior of the hydrogel after molding.

[0087] Results reference Figure 4 , Figure 4 The strain-modulus test results are for the hydrogels described in Example 1 and Comparative Examples 1-5 of this application. Figure 4 It can be seen that, compared with other metal ions, the addition of Fe to the system... 3+The gel's toughness increases to some extent, and its modulus also improves significantly. This phenomenon is due to the presence of Fe³⁺ in the system. + As a cross-linking center, it undergoes stronger in-situ coordination cross-linking with phosphate-containing small molecules and negatively charged biodegradable polymers in the hydrogel system, forming more stable coordination nodes, further densifying the three-dimensional network structure of the hydrogel, enhancing the network's rigidity and stability, thus leading to a certain degree of increase in the gel's elastic modulus; simultaneously, the increase in the number of cross-linking nodes also improves the network's connectivity, resulting in a significant improvement in the gel's toughness. Since the core function of cartilage in the body is to serve as a tough, low-friction load-bearing pad, gel materials used to replace or repair it must possess matching high toughness; otherwise, they will be unable to withstand the complex, high-intensity cyclic loads within the joint, leading to early fracture, wear, or separation from the bone, resulting in repair failure. Therefore, compared to other metal ions, Fe-containing... 3+ The mineralized hydrogel has better cartilage repair potential.

[0088] Experimental Example 3

[0089] The environmental responsiveness of the mineralized hydrogel was characterized by placing it in different media environments.

[0090] 300 mg of the mineralized hydrogel from Example 1 was weighed and placed in 3 mL of phosphate-buffered saline (PBS). The control group contained only hydrogel. The hydrogel was photographed after 20 days.

[0091] Results reference Figure 5 When the gel was placed in a phosphate buffer, it remained stable, while the control group had become liquid. This indicates that the gel is stable in a simulated body fluid environment, while the control group, whose main medium was water, easily disintegrated, demonstrating that the gel has a certain degree of environmental responsiveness.

[0092] Test Example 4

[0093] The blood compatibility of hydrogels was evaluated using a hemolysis test.

[0094] First, 2 mL of fresh rabbit blood was added to 20 mL of physiological saline and centrifuged at 1500 rpm for 10 min. The supernatant was discarded, and 20 mL of physiological saline was added to the lower layer of blood cells. This process was repeated three times. The lower layer of blood cells was collected. The lower layer of blood cells was prepared into a 2% blood cell suspension and co-cultured with a hydrogel at 37°C. The negative control group was pure physiological saline, and the positive control group was 1% Triton X-100. After 3 hours, the supernatant was collected, and the absorbance at 545 nm was measured using a UV spectrophotometer to obtain the hemolysis rate.

[0095] Hemolysis rate % = (Abs gel group - Abs negative group) / (Abs positive group - Abs negative group) × 100%

[0096] Results reference Figure 6 , Figure 6 The hemolysis rate test results of the hydrogel described in Example 2 of this application show that the hemolysis rate is less than 5%, which proves that the hydrogel has excellent blood compatibility.

[0097] Remove most of the supernatant from the centrifuged blood cells, leaving about 50 μL of supernatant to disperse the cells. Then, take 10 μL of the liquid and drop it onto a glass slide. Finally, place the glass slide under a fluorescence inverted microscope for observation and take a photograph.

[0098] Results reference Figure 7 , Figure 7 This is a morphological image of red blood cells after co-incubation of the hydrogel described in Example 2 of this application with rabbit red blood cells for 3 hours. The red blood cells in the field of view exhibit a biconcave disc-shaped structure, indicating that the gel has good blood compatibility.

[0099] Experimental Example 5

[0100] The biocompatibility of the hydrogel was evaluated using cytotoxicity assays.

[0101] First, bone mesenchymal stem cells (BMSCs) were cultured at 8000 cells per well in a 96-well plate. After 24 hours of culture at 37°C and 5% CO2, the original culture medium was discarded, and cell culture medium containing gel extraction medium was added. The cells were then cultured for another 24 hours at 37°C and 5% CO2. The negative control group used pure cell culture medium. Subsequently, the culture medium containing gel extraction medium was discarded, and 100 μL of serum-free culture medium containing CCK8 was added to each well. After incubation at 37°C and 5% CO2 for 2 hours, the absorbance spectrum at 450 nm was measured using a microplate reader. The final cell viability was calculated using the following formula.

[0102] Cell viability % = OD gel group / OD negative group 100%

[0103] Results reference Figure 8 , Figure 8 The cytotoxicity test results of the hydrogel described in Example 3 of this application show that the cell survival rate is close to 100%, proving that the hydrogel has excellent biocompatibility.

[0104] Experimental Example 6

[0105] The test demonstrated the antibacterial properties of the hydrogel, proving whether it can effectively inhibit bacterial growth in wounds and reduce the risk of infection.

[0106] The antibacterial properties of the hydrogel were tested using the plate count method. Target bacteria in the logarithmic growth phase (such as Staphylococcus aureus and Escherichia coli) were diluted with sterile physiological saline to a concentration of 1 × 10⁻⁶. 6 For CFU / mL, 100 μL of the bacterial suspension was mixed evenly with 64 mg / mL hydrogel extract and incubated at 37°C in a 5% CO2 incubator for 24 h. A blank control group (containing only the bacterial suspension, without hydrogel extract) was also set up under identical incubation conditions. After incubation, the mixture was serially diluted, and 100 μL of each dilution was evenly spread onto LB agar plates. After incubation at 37°C for 18–24 h, the number of colonies (CFU) on the medium was counted. The dilution gradient was 10-10. -3 10 -4 10 -5 .

[0107] Results reference Figure 9 , Figure 9 The results of the antibacterial performance test of the hydrogel described in Example 4 of this application show that the hydrogel has excellent antibacterial properties against Staphylococcus aureus and Escherichia coli, and can effectively inhibit the growth of common pathogenic bacteria in wounds, reducing the risk of infection in osteochondral defect areas. It is worth noting that the Fe in the hydrogel... 3+ The coordination mineralization structure formed with phosphate-containing small molecules significantly enhances its antibacterial activity without affecting the biocompatibility of the material itself.

[0108] Experimental Example 7

[0109] By testing the effect of hydrogels on the activity of cellular alkaline phosphatase (ALP), this study demonstrates whether hydrogels can effectively promote early osteogenic differentiation of cells.

[0110] The ALP staining method was used to test the regulatory effect of hydrogel on early cell differentiation. BMSCs were seeded in confocal dishes containing hydrogel extract and cultured at 37°C in a 5% CO2 incubator for 7 days. The culture medium was discarded, the cells were gently washed with PBS, fixed with 4% paraformaldehyde, and incubated with staining working solution in the dark according to the ALP staining kit procedure. After washing with PBS, the cells were observed with the naked eye and under a microscope.

[0111] Results reference Figure 10 , Figure 10 The results of the hydrogel ALP staining test described in Example 5 of this application show that the cell staining intensity in the hydrogel group is significantly higher, ALP activity is significantly upregulated, and it has excellent ability to promote early differentiation, which can effectively initiate the differentiation process of osteochondral repair.

[0112] Experimental Example 8

[0113] By testing the effect of hydrogels on the formation of mineralized nodules in cells, this study demonstrates whether hydrogels can effectively promote in vitro mineralization deposition of cells.

[0114] Alizarin Red (ARS) staining was used to assess the hydrogel-mediated cell mineralization capacity. BMSCs were seeded in confocal dishes containing hydrogel extract and cultured at 37°C with 5% CO2 for 14 days. The culture medium was discarded, cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min, washed with PBS, and then stained with Alizarin Red in the dark. After rinsing thoroughly with deionized water to remove excess stain, macroscopic imaging and microscopic observation were performed. The regulatory effect of the hydrogel on the formation of mineralized nodules in cells was qualitatively evaluated based on the staining results.

[0115] Results reference Figure 11 , Figure 11 The results of the hydrogel ARS staining test described in Example 6 of this application show that the hydrogel group has more mineralized nodules, deeper staining, and significantly improved calcium deposition level, demonstrating excellent cell mineralization promotion ability and effectively promoting the mineralization and maturation of osteochondral tissue.

[0116] Experimental Example 9

[0117] By testing the effect of hydrogel on the expression of Sox9, a characteristic transcription factor of cartilage, this study demonstrates whether hydrogel can effectively maintain chondrocyte phenotype and promote cartilage matrix synthesis.

[0118] The regulatory effect of hydrogel on Sox9 expression was tested using immunofluorescence staining. Chondrocytes were seeded in confocal microscopy dishes containing hydrogel extract and incubated for 14 days at 37°C with 5% CO2. Afterward, the culture medium was discarded, cells were washed with PBS, fixed with 4% paraformaldehyde, and non-specific sites were blocked with blocking buffer after permeabilization. Sox9 primary antibody was added and incubated overnight at 4°C. The primary antibody was collected, washed with PBS, and then incubated with fluorescently labeled secondary antibody in the dark for 2 hours. The secondary antibody was discarded, and Actin-tracker red-594 antibody was added and incubated at room temperature in the dark for 2 hours. Nuclei were then stained with DAPI, and images were acquired using a confocal microscope after mounting.

[0119] Results reference Figure 12 , Figure 12 The results of the Sox9 immunofluorescence assay using the hydrogel described in Example 7 of this application show that the Sox9 fluorescence signal in the hydrogel group is stronger and the positive expression area is larger, which can significantly promote the expression of the cartilage characteristic transcription factor Sox9 and effectively maintain the normal phenotype of chondrocytes.

[0120] Experimental Example 10

[0121] The gel's ability to promote angiogenesis in vitro was evaluated using a cell tube formation assay.

[0122] First, human umbilical vein endothelial cells (HUVECs) were cultured. 50 µL of Matrigel was added to each well of a 96-well plate and allowed to stand for 0.5 h. Then, 100 µL of gel extract containing 8000 cells was seeded onto the Matrigel. After 6 hours of incubation, the tube-forming ability of the HUVECs was observed using a fluorescence microscope.

[0123] Results reference Figure 13 , Figure 13 This is a tube formation image of the hydrogel in Example 8 of this application. It can be seen from the image that the gel has a good ability to promote angiogenesis.

[0124] Experimental Example 11

[0125] The effect of hydrogels on promoting integrated osteochondral repair in vivo was evaluated using a rabbit knee joint osteochondral defect model.

[0126] Healthy adult New Zealand white rabbits (3-4 months old, 2.0-2.5 kg) were randomly assigned to groups. A standard full-thickness osteochondral defect with a diameter of 4 mm and a depth of 2 mm was constructed at the trochlear joint of the rabbit femur using a drill. An appropriate amount of hydrogel was used to fill and repair the osteochondral defect area. Twelve weeks postoperatively, all experimental rabbits were sacrificed, and femoral specimens from the knee joint were collected and immersed in 4% paraformaldehyde solution at 4°C for 24 hours. The integrated osteochondral repair effect of the hydrogel was evaluated by gross observation, CT scan, and histological staining.

[0127] Results reference Figure 14 , Figure 14 This is a macroscopic photograph of the hydrogel treatment group described in Example 10 of this application. The results show that the hydrogel can simultaneously repair the interface between cartilage and subchondral bone, and has a good integrated osteochondral repair effect.

[0128] The above description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in this application, based on the technical solution and inventive concept of this application, should be included within the scope of protection of this application.

Claims

1. A biomimetic mineralized hydrogel, characterized in that, It mainly consists of biodegradable polymers containing carboxyl groups, small molecules containing phosphate groups, and Fe. 3+ get; The phosphate-containing small molecules have one or more of the structures of formula (I-1), (I-2), (I-3), (I-4), (I-5), and (I-6); （Ⅰ-1）； （Ⅰ-2）； （Ⅰ-3）； （Ⅰ-4）； （Ⅰ-5）； （Ⅰ-6）。 2. The biomimetic mineralized hydrogel according to claim 1, characterized in that, The Fe 3+ It is selected from one or more of ferric chloride, ferric chloride hexahydrate, ferric nitrate, ferric nitrate nonahydrate, ferric sulfate, ferric sulfate nonahydrate, and ferric acetate.

3. The biomimetic mineralized hydrogel according to claim 1, characterized in that, The carboxylate-containing biodegradable polymer contains carboxylate groups, phosphate groups in small phosphate molecules, and Fe... 3+ The molar ratio is (2~20):(0.1~10):

1.

4. The biomimetic mineralized hydrogel according to claim 1, characterized in that, The biomimetic mineralized hydrogel also includes bioactive factors.

5. The biomimetic mineralized hydrogel according to claim 4, characterized in that, The bioactive factor is selected from one or more of transforming growth factor-β (TGF-β), insulin-like growth factor-1 (IGF-1), and Kartogenin (KGN).

6. The biomimetic mineralized hydrogel according to claim 1, characterized in that, The degradable polymer containing carboxylate groups has one or more of the structures (II-1), (II-2), (II-3), (II-4), and (II-5); （Ⅱ-1）； （Ⅱ-2）； （Ⅱ-3）； （Ⅱ-4）； （Ⅱ-5）； Wherein, R is H or CH2COO - ; n, m, x, y, and b are all degrees of aggregation, with 30≤n≤7000, 50≤m≤8500, 20≤x≤6500, 100≤y≤6000, and 20≤b≤6500.

7. A method for preparing a biomimetic mineralized hydrogel, characterized in that, Includes the following steps: A) A precursor hydrogel is obtained by mixing a biodegradable polymer containing carboxyl groups and a small molecule containing phosphate groups. B) The precursor hydrogel was combined with Fe 3+ After the compounds are mixed, in-situ mineralization is carried out through ion diffusion under static conditions to obtain a biomimetic mineralized hydrogel.

8. The preparation method according to claim 7, characterized in that, The settling temperature is 0℃~80℃, and the settling time is 5h~12 days.

9. The preparation method according to claim 7, characterized in that, The biomimetic mineralized hydrogel was immersed in an external environment to obtain a biomimetic iron phosphate hydrogel. The external environment is one or more of the following: phosphate buffer solution, bone, and body fluids.

10. A bone and cartilage repair material, characterized in that, The biomimetic mineralized hydrogel includes the biomimetic mineralized hydrogel according to any one of claims 1 to 6 or the biomimetic mineralized hydrogel prepared by the preparation method according to any one of claims 7 to 9.

Images