A dual-network hydrogel containing drug-loaded ACC nanoparticles, its preparation method and application

Abstract

This invention provides a dual-network hydrogel containing drug-loaded ACC nanoparticles, its preparation method, and its applications. The dual-network hydrogel includes a LAMG compound, sodium alginate, and drug-loaded ACC nanoparticles. The LAMG compound is prepared by a first reaction of a mixture of gelatin aqueous solution and thioctic acid solution. The drug-loaded ACC nanoparticles are prepared by a second reaction of a raw material system including nicotinamide mononucleotide aqueous solution, calcium source solution, and ammonium bicarbonate. The LAMG compound, sodium alginate, and drug-loaded ACC nanoparticles are then mixed to obtain the dual-network hydrogel. This dual-network hydrogel exhibits excellent adhesion strength and ROS scavenging ability, providing a new therapeutic approach for the repair of bone defects or fractures, and has significant medical implications.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a dual-network hydrogel containing drug-loaded ACC nanoparticles, its preparation method, and its applications. Background Technology

[0002] Osteoporosis (OP), a metabolic bone disease, is characterized by low bone mass, high bone fragility, low bone density, damaged bone microstructure, and slow bone healing. It is commonly seen in postmenopausal women, the elderly, and those with nutritional imbalances or deficiencies. Studies have shown that the development of osteoporosis is related to oxidative stress caused by increased reactive oxygen species (ROS). High ROS levels not only cause oxidative stress in osteoblasts, osteoclasts, and bone marrow mesenchymal stem cells (BMSCs), reducing cell activity, but also damage mitochondria, affecting normal cellular metabolic processes. With an aging population, osteoporosis has become a global health problem. According to relevant statistics, it is estimated that by 2050, the number of osteoporosis patients in my country will reach 202 million. Osteoporosis patients are prone to bone defects or fractures, therefore, there is a high clinical demand for the repair and treatment of bone defects or fractures.

[0003] Clinically, metal implants (bone repair materials) such as bone plates and artificial joints are used to repair bone defects or fractures. Among them, titanium implants are widely used for the repair of bone defects or fractures due to their excellent mechanical and biological properties. Commonly used titanium implant materials include pure titanium, Ti6Al4V, and Ti15Zr. Although titanium implants can provide mechanical support, they are prone to loosening.

[0004] To address this, existing technologies typically involve artificially constructing a transitional material on the surface of titanium implants that interacts benignly with bone tissue to mitigate implant loosening. For example, patent CN 104911674A discloses a bioactive coating on the surface of a porous metal material and its preparation method. This bioactive coating (transitional material) has a three-layer structure comprising a dense film on the metal surface, a porous gel layer in the middle, and a loose layer on the coating surface, exhibiting good adhesion stability. However, this bioactive coating (transitional material) still has some problems: (1) the adhesion strength between the bioactive coating and bone tissue is poor, and the repair site is prone to detachment, affecting the repair effect; (2) the bioactive coating cannot effectively reduce the ROS level at the repair site, and the repair site still has the problem of excessively high ROS levels, making it difficult to repair damaged mitochondria.

[0005] Therefore, exploring a material that combines excellent adhesion strength and ROS removal capability is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] This invention provides a dual-network hydrogel containing drug-loaded amorphous calcium carbonate (ACC) nanoparticles. This dual-network hydrogel containing drug-loaded ACC nanoparticles has excellent adhesion strength and ROS scavenging ability, which can reduce ROS levels, effectively repair mitochondrial damage, and promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). It provides a new treatment approach for the repair and treatment of bone defects or fractures, which has important medical significance.

[0007] The present invention also provides a method for preparing a dual-network hydrogel containing drug-loaded ACC nanoparticles, which is used to prepare the above-mentioned dual-network hydrogel with excellent adhesion strength and ROS scavenging ability.

[0008] The present invention also provides a bone repair material comprising the above-mentioned dual-network hydrogel containing drug-loaded ACC nanoparticles and aminated titanium. Therefore, the bone repair material has excellent adhesion strength and ROS scavenging ability, and can also promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).

[0009] A first aspect of the present invention provides a dual-network hydrogel containing drug-loaded ACC nanoparticles, comprising a LAMG compound, sodium alginate, and drug-loaded ACC nanoparticles;

[0010] The LAMG compound is formed by mixing gelatin aqueous solution and thioctic acid solution and then undergoing a first reaction. The drug-loaded ACC nanoparticles are formed by a second reaction of a raw material system including nicotinamide mononucleotide aqueous solution, calcium source solution and ammonium bicarbonate. After mixing the LAMG compound, the sodium alginate and the drug-loaded ACC nanoparticles, the dual-network hydrogel containing the drug-loaded ACC nanoparticles is obtained.

[0011] Gelatin is a well-known biomaterial, a partially hydrolyzed product of natural collagen, and has good biocompatibility.

[0012] Lipoic acid is a hydrophobic substance with excellent antioxidant properties, which can reduce cell damage caused by oxidative stress, bovine head disease, and ferrodexia.

[0013] Nicotinamide mononucleotide (NMN) is a cofactor of the longevity protein nicotinamide adenine dinucleotide (NAD). + The precursor of NAD, and NAD + It is an important coenzyme for redox reactions and the center of energy metabolism. Its mediated mitochondrial oxidative phosphorylation is a necessary process for osteogenic differentiation and bone repair of bone marrow mesenchymal stem cells (BMSCs).

[0014] Through extensive research, the inventors have discovered that the dual-network hydrogel containing drug-loaded ACC nanoparticles (the dual-network hydrogel containing drug-loaded ACC nanoparticles (GSC) in this embodiment of the invention) exhibits excellent adhesion strength. The inventors' research shows that the dual-network hydrogel containing drug-loaded ACC nanoparticles can continuously and stably promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) at different stages (early, middle, and late stages), and can reduce ROS levels within BMSCs, effectively repairing mitochondrial damage.

[0015] In this invention, the thioctic acid solution is prepared by mixing thioctic acid with anhydrous ethanol.

[0016] The dual-network hydrogel containing drug-loaded ACC nanoparticles as described above, wherein the mass ratio of LAMG compound, sodium alginate and drug-loaded ACC nanoparticles in the dual-network hydrogel is (0.5-2):(0.05-1):(0.005-0.015).

[0017] In some embodiments, the mass ratio of LAMG compound, sodium alginate, and drug-loaded ACC nanoparticles in the dual-network hydrogel is preferably 0.75:0.1:0.0025.

[0018] The dual-network hydrogel containing drug-loaded ACC nanoparticles as described above, wherein the concentration of gelatin in the gelatin aqueous solution is 0.04 g / mL to 0.07 g / mL;

[0019] And / or, in the lipoic acid solution, the concentration of lipoic acid is 0.01 g / mL to 0.03 g / mL;

[0020] And / or, in the aqueous solution of nicotinamide mononucleotide, the concentration of nicotinamide mononucleotide is 11 mg / mL to 15 mg / mL;

[0021] And / or, in the calcium source solution, the concentration of the calcium source is 0.5 mol / L to 2 mol / L.

[0022] The present invention does not impose any particular limitation on the mass of gelatin and thioctic acid, and the mass can be selected according to actual needs. In some embodiments, the mass ratio of gelatin in the gelatin aqueous solution to thioctic acid in the thioctic acid solution is (4-8):(0.8-2).

[0023] This invention does not impose any particular limitations on the mass of nicotinamide mononucleotide and the volume of the calcium source solution; these can be selected according to actual needs.

[0024] The dual-network hydrogel containing drug-loaded ACC nanoparticles as described above, wherein the calcium source is at least one of calcium chloride, calcium chloride dihydrate, and calcium carbonate.

[0025] The first reaction of the dual-network hydrogel containing drug-loaded ACC nanoparticles, as described above, is carried out at a temperature of 38°C to 42°C for 5 to 7 hours.

[0026] And / or, the temperature of the second reaction is 38℃~42℃ and the time is 12h~18h.

[0027] In some embodiments, the temperature of the first reaction is preferably 40°C and the time is preferably 6 hours.

[0028] In some embodiments, the temperature of the second reaction is preferably 40°C and the time is preferably 14 hours.

[0029] A second aspect of the present invention provides a method for preparing the dual-network hydrogel containing drug-loaded ACC nanoparticles, comprising:

[0030] A gelatin aqueous solution and a thioctic acid solution were prepared separately. After the thioctic acid solution was first stirred, a catalyst was added and then stirred a second time to obtain a first mixture. The gelatin aqueous solution and the first mixture were mixed and subjected to a first reaction to obtain a first reaction solution. The first reaction solution was subjected to double distillation and a first drying treatment to obtain the LAMG compound.

[0031] An aqueous solution of nicotinamide mononucleotide (NMN) was prepared and placed in a reaction vessel. A calcium source solution was added to the NMN aqueous solution to obtain a mixed solution. Anhydrous ethanol was added to the mixed solution to obtain a milky white solution. The reaction vessel was then placed in a sealed container with ammonium bicarbonate particles at the bottom to obtain a reaction system. The reaction system was subjected to a second reaction to obtain a second reaction solution. The second reaction solution was subjected to a first centrifugation, washing, and a second centrifugation to obtain a precipitate. The precipitate was subjected to a second drying treatment to obtain drug-loaded ACC nanoparticles.

[0032] The LAMG compound was mixed with water, sodium alginate was added, and a second mixture was obtained after a third stirring. The drug-loaded ACC nanoparticles were added to the second mixture, and a double-network hydrogel precursor solution was obtained after a fourth stirring. The double-network hydrogel precursor solution was irradiated under ultraviolet light to obtain the double-network hydrogel containing drug-loaded ACC nanoparticles.

[0033] The present invention does not impose any particular limitation on the reaction vessel, and it can be selected as needed. In some embodiments, the reaction vessel can be a reaction flask.

[0034] The present invention does not impose particular limitations on the specific methods of the first drying treatment and the second drying treatment, and the methods can be selected according to actual needs. In some embodiments, the first drying treatment may be a freeze-drying treatment at -20°C, and the second drying treatment may be a freeze-drying treatment at -20°C.

[0035] In some embodiments, the activating stabilizer is prepared by mixing 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide and N-hydroxysuccinimide.

[0036] In the preparation method of the dual-network hydrogel containing drug-loaded ACC nanoparticles as described above, the temperature of the first stirring is 35℃~38℃ and the time is 25min~40min;

[0037] And / or, the second stirring time is 3 min to 6 min;

[0038] And / or, the temperature of the double-distilled water dialysis is 48℃~52℃ and the time is 6d~8d;

[0039] And / or, the temperature of the first centrifugation treatment is 3℃~5℃ and the rotation speed is 11000rpm~16000rpm;

[0040] And / or, the temperature of the second centrifugation treatment is 3℃~5℃ and the rotation speed is 11000rpm~16000rpm.

[0041] In the preparation method of the dual-network hydrogel containing drug-loaded ACC nanoparticles as described above, the third stirring temperature is 48℃~52℃ and the time is 2.5h~3.5h;

[0042] And / or, the temperature of the fourth stirring is 38℃~42℃ and the time is 10h~14h.

[0043] A third aspect of the present invention provides a bone repair material comprising the aforementioned dual-network hydrogel containing drug-loaded ACC nanoparticles.

[0044] The bone repair material described above also includes aminated titanium, the preparation process of which includes:

[0045] After titanium is immersed in nitric acid for a third reaction, the product is obtained. The product is then washed and incubated in an APTES ethanol solution to obtain the aminated titanium.

[0046] This invention does not impose any particular limitation on the specific form of titanium. In some embodiments, the titanium may be titanium sheet, titanium foil, or titanium rod.

[0047] In the bone repair material described above, the temperature of the third reaction is 58℃~62℃ and the time is 35min~45min;

[0048] And / or, the incubation treatment is carried out at a temperature of 35°C to 40°C for a time of 1.5h to 2.5h.

[0049] The bone repair material described above, wherein the APTES ethanol solution is prepared by adding 3-aminopropyltriethoxysilane to anhydrous ethanol.

[0050] In the bone repair material described above, the volume ratio of 3-aminopropyltriethoxysilane to anhydrous ethanol is (1-4):(90-120), and the volume ratio of 3-aminopropyltriethoxysilane to anhydrous ethanol is preferably 2:98.

[0051] In summary, the solution of the present invention has at least the following effects:

[0052] This invention provides a dual-network hydrogel containing drug-loaded ACC nanoparticles. The dual-network hydrogel comprises a LAMG compound, sodium alginate, and drug-loaded ACC nanoparticles. The LAMG compound is formed by a first reaction of a mixture of gelatin aqueous solution and thioctic acid solution. The drug-loaded ACC nanoparticles are formed by a second reaction of a raw material system comprising nicotinamide mononucleotide aqueous solution, calcium source solution, and ammonium bicarbonate. The LAMG compound, sodium alginate, and drug-loaded ACC nanoparticles are then mixed to obtain the dual-network hydrogel containing drug-loaded ACC nanoparticles. Studies have shown that the dual-network hydrogel containing drug-loaded ACC nanoparticles provided by this invention exhibits excellent adhesion strength and ROS scavenging ability, can reduce ROS levels, effectively repair mitochondrial damage, and can also promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), providing a new therapeutic approach for the repair of bone defects or fractures, and has significant medical implications. Attached Figure Description

[0053] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0054] Figure 1 Morphological characteristics of drug-loaded ACC nanoparticles;

[0055] in, Figure 1 ab is a SEM image containing drug-loaded ACC nanoparticles; Figure 1The cd is a TEM image containing drug-loaded ACC nanoparticles; Figure 1 Ej is the EDS diagram of drug-loaded ACC nanoparticles.

[0056] Figure 2 The Fourier transform infrared (FT-IR) spectrum of the LAMG compound is shown.

[0057] Figure 3 Scanning electron microscope (SEM) and atomic force microscope (AFM) images of different titanium materials;

[0058] in, Figure 3 Image a is a scanning electron microscope (SEM) image of titanium material (Ti), titanium material after nitric acid etching treatment (Ti-H), titanium material modified with amylation (Ti-NH2), and titanium material modified with double network hydrogel (Ti-GSC). Figure 3 Image b shows atomic force microscopy (AFM) images of titanium (Ti), aminated titanium (Ti-NH2), and double-network hydrogel-modified titanium (Ti-GSC).

[0059] Figure 4 Adhesion strength tests were conducted on different hydrogels (Ti-G, NH2-G, NH2-GS, NH2-GSC).

[0060] Figure 5 To detect the early osteogenic differentiation effect of dual-network hydrogels;

[0061] in, Figure 5 Figure a shows the results of quantitative detection of ALP expression levels in BMSCs of each group after 7 days using an ALP activity assay kit. Figure 5 Figure b is a statistical graph showing the quantitative detection of ALP expression levels in BMSCs of each group after 7 days using an ALP activity assay kit.

[0062] Figure 6 To detect the mid-term osteogenic differentiation effect of dual-network hydrogels;

[0063] in, Figure 6 Figure a shows the results of Sirius red staining to show the secretion of extracellular collagen (type I collagen) in BMSCs of each treatment group at 14 days. Figure 6 Figure b is a statistical graph showing the secretion of extracellular collagen (type I collagen) in BMSCs of each treatment group at 14 days by Sirius red staining.

[0064] Figure 7 To detect the late-stage osteogenic differentiation effect of dual-network hydrogels;

[0065] in, Figure 7Figure a shows the results of qualitative and quantitative detection of the mineralization level of BMSCs in each treatment group at 14 days (d) and 21 days (d) by alizarin red staining method; Figure 7 Figure b is a statistical graph showing the mineralization level of BMSCs in each treatment group at 14 and 21 days, determined qualitatively and quantitatively by alizarin red staining.

[0066] Figure 8 For the detection of reactive oxygen species (ROS) levels in dual-network hydrogels;

[0067] in, Figure 8 Figure a shows the fluorescence intensity results of femoral tissue sections from rats in each group (control group (Ti), Ti-GS group (GS), Ti-GSN group (GSN), Ti-GSC group (GSC)) under a fluorescence microscope; Figure 8 Figure b is a statistical graph of the fluorescence intensity of femoral tissue sections of rats in each group (control group (Ti), Ti-GS group (GS), Ti-GSN group (GSN), Ti-GSC group (GSC)) under a fluorescence microscope. Detailed Implementation

[0068] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this art or in accordance with the product instructions. Reagents or instruments used without specified manufacturers are all conventional products that can be obtained commercially.

[0069] The following detailed description, in conjunction with embodiments, illustrates a dual-network hydrogel containing drug-loaded ACC nanoparticles, its preparation method, and its applications. However, these descriptions should not be construed as limiting the scope of protection of this invention.

[0070] Example 1: Drug-loaded ACC nanoparticles and their preparation

[0071] I. Experimental Reagents and Materials

[0072] Nicotinamide mononucleotide (NMN), ultrapure water, 1 mol / L calcium chloride solution, anhydrous ethanol, reaction vessel, sealed container.

[0073] II. Preparation of drug-loaded ACC nanoparticles

[0074] 33.4 mg of nicotinamide mononucleotide (NMN) was added to a reaction flask containing 2.5 mL of ultrapure water. The mixture was stirred until NMN was fully dissolved, yielding an aqueous solution of nicotinamide mononucleotide (NMN). 0.5 mL of a 1 mol / L calcium chloride solution was added to the NMN aqueous solution, and the mixture was stirred for 5 min to obtain a mixed solution. 40 mL of anhydrous ethanol was added to the mixed solution, which turned milky white. Upon laser irradiation, a clear light path was observed, and the Tyndall effect was also present. The reaction flask was then placed at the bottom and covered with ammonium bicarbonate granules. The reaction system was obtained in a sealed container; the reaction system was transferred to a heated stirring table at 40℃ and 500 rpm for 14 h to obtain a reaction solution; the reaction solution was centrifuged at 4℃ and 11000 rpm to obtain a product; the product was washed twice with anhydrous ethanol and then centrifuged at 4℃ and 11000 rpm to obtain a precipitate; the precipitate was freeze-dried at -20℃ under vacuum to obtain drug-loaded ACC nanoparticles, which were stored at 4℃ for later use.

[0075] III. Test

[0076] (1) Morphological characteristics

[0077] The morphology of the drug-loaded ACC nanoparticles was observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and the elemental composition of the drug-loaded ACC nanoparticles was determined by X-ray energy dispersive spectroscopy (EDS) of TEM.

[0078] SEM results (e.g.) Figure 1 (as shown in ab) and TEM results (as shown in ab) Figure 1 The results (as shown in the CD image) indicate that the drug-loaded ACC nanoparticles are uniform in size and have a morphology similar to reported amorphous calcium carbonate (ACC), being spherical particles. EDS results (as shown in the CD image) further support this finding. Figure 1 As shown in the figure (ej), phosphorus (P), nitrogen (N), and other elements unique to NMN are uniformly distributed throughout the drug-loaded ACC nanoparticles, while calcium, a unique element of calcium carbonate, is enriched on the drug-loaded ACC nanoparticles.

[0079] Example 2: Preparation of LAMG compounds

[0080] I. Experimental Reagents and Materials

[0081] Gelatin, ultrapure water, 1 mol / L hydrochloric acid solution, lipoic acid, and anhydrous ethanol.

[0082] II. Preparation of LAMG Compounds

[0083] Add 6g of gelatin to 100mL of ultrapure water and stir at 50℃ until the gelatin is completely dissolved to obtain a gelatin aqueous solution. Adjust the pH of the gelatin aqueous solution to 5.5 using a 1mol / L hydrochloric acid solution to obtain the adjusted gelatin aqueous solution.

[0084] 1.2 g of lipoic acid was added to 60 mL of anhydrous ethanol and mixed to obtain a lipoic acid solution. The solution was then stirred at 37 °C for 30 min until it turned yellow and transparent. Subsequently, 1.0 g of EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide) and 1.0 g of NHS (N-hydroxysuccinimide) were added to the lipoic acid solution and stirred for 5 min to obtain a lipoic acid mixture. EDC has the function of activating carboxyl groups, while NHS maintains the stability of the activated product after the carboxyl groups are activated.

[0085] At a temperature of 40℃ and a rotation speed of 500 rpm, a mixture of thioctic acid was added dropwise to the adjusted gelatin aqueous solution and mixed. After the addition was complete, the reaction was carried out for 6 hours to obtain a reaction solution. The reaction solution was dialyzed against double-distilled water at 50℃ for 7 days and then freeze-dried under vacuum at -20℃ to obtain the LAMG compound.

[0086] III. Test

[0087] (1) Fourier transform infrared spectroscopy (FT-IR)

[0088] Gelatin (Gel), lipoic acid (LA), and LAMG compound (LAMG) were analyzed using Fourier transform infrared spectroscopy (FT-IR). The infrared spectra of gelatin (Gel), lipoic acid (LA), and LAMG compound (LAMG) were obtained, and the changes in chemical composition were analyzed. The FT-IR results are shown below. Figure 2 As shown.

[0089] Depend on Figure 2 It can be seen that, compared with gelatin (Gel) and lipoic acid (LA), the LAMG compound (LAMG) has a higher concentration at 1557 cm⁻¹. -1 The absorption peak at 567 cm⁻¹ is due to the in-plane bending vibration of the amide bond, indicating an amidation reaction between LA and gel; compared to gel, the peak at 567 cm⁻¹ is... -1 The presence of a disulfide bond (SS) absorption peak unique to lipoic acid proves that lipoic acid has been successfully grafted onto gelatin.

[0090] Example 3: Preparation of different hydrogels

[0091] I. Preparation of LAMG hydrogel (G)

[0092] 0.75 g of the above LAMG compound was added to 5 mL of ultrapure water and stirred at 50 °C and 1000 rpm for 30 min to obtain a LAMG hydrogel precursor solution. The LAMG hydrogel (G) precursor solution was irradiated with 365 nm ultraviolet light for 10 min to form a gel, thus obtaining LAMG hydrogel (G).

[0093] II. Preparation of Composite Hydrogels (GS)

[0094] 0.75 g of the above LAMG compound was added to 5 mL of ultrapure water and stirred at 50 °C and 1000 rpm for 30 min. Then, 0.1 g of sodium alginate powder was added and stirred at 50 °C and 50 rpm for 3 h to obtain a composite hydrogel precursor solution. The composite hydrogel (GS) precursor solution was irradiated with 365 nm ultraviolet light for 10 min to form a gel, thus obtaining the composite hydrogel (GS).

[0095] III. Preparation of NMN-containing hydrogels (GSN)

[0096] 0.75g of the above LAMG compound was added to 5mL of ultrapure water and stirred at 50℃ and 1000rpm for 30min. Then, 0.1g of sodium alginate powder was added and stirred at 50℃ and 50rpm for 3h to obtain a mixture. NMN was added to the mixture and stirred at 40℃ and 50rpm for 12h to prepare a hydrogel (GSN) precursor solution containing 0.01wt% NMN. The hydrogel (GSN) precursor solution containing NMN was irradiated with 365nm ultraviolet light for 10min to form a gel, thus obtaining a hydrogel (GSN) containing NMN.

[0097] IV. Preparation of dual-network hydrogels (GSCs) containing drug-loaded ACC nanoparticles

[0098] 0.75 g of the above LAMG compound was added to 5 mL of ultrapure water and stirred at 50 °C and 1000 rpm for 30 min. Then, 0.1 g of sodium alginate powder was added and stirred at 50 °C and 50 rpm for 3 h to obtain a mixture. 2.5 mg of the above drug-loaded ACC nanoparticles were added to the mixture and stirred at 40 °C and 50 rpm for 12 h to prepare a dual-network hydrogel (GSC) precursor solution containing drug-loaded ACC nanoparticles (content of 0.05 wt%). The dual-network hydrogel precursor solution was irradiated under ultraviolet light at a wavelength of 365 nm for 10 min to obtain a dual-network hydrogel (GSC) containing drug-loaded ACC nanoparticles.

[0099] Example 4: Preparation of different titanium materials

[0100] I. Experimental Reagents and Materials

[0101] Pure titanium sheet, ethanol, distilled water, nitric acid, 2 wt% APTES ethanol solution.

[0102] II. Titanium Materials (Ti)

[0103] Pure titanium sheets were ultrasonically cleaned by immersing them in ethanol and distilled water in sequence, and then dried at 60°C for 6 hours to obtain titanium material (Ti).

[0104] III. Titanium materials after nitric acid etching treatment (Ti-H)

[0105] Pure titanium sheets were ultrasonically cleaned by immersing them in ethanol and distilled water in sequence, and then dried at 60°C for 6 hours. The dried pure titanium sheets were then immersed in nitric acid (CAS No.: 7697-37-2) and reacted at 60°C for 40 minutes to obtain titanium material (Ti-H) after nitric acid corrosion treatment.

[0106] IV. Amination-modified titanium materials (Ti-NH2)

[0107] Pure titanium sheets were ultrasonically cleaned by sequentially immersing them in ethanol and distilled water, and then dried at 60°C for 6 hours. The dried pure titanium sheets were then immersed in nitric acid and reacted at 60°C for 40 minutes. Afterward, the reacted pure titanium sheets were placed in a plasma cleaner for 5 minutes. Immediately after cleaning, they were immersed in a 2 wt% APTES ethanol solution (3-aminopropyltriethoxysilane (APTES) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., obtained by adding 2 mL of APTES to 98 mL of anhydrous ethanol) and incubated in a shaker at 37°C and 60 rpm for 2 hours to obtain the amino-modified titanium material (Ti-NH2).

[0108] V. Titanium materials modified with dual-network hydrogels (Ti-GSC)

[0109] 20 μL of a dual-network hydrogel precursor solution (the same as the dual-network hydrogel precursor solution prepared in Example 3, hereinafter the same) was uniformly coated on the surface of the above-mentioned aminated titanium material (Ti-NH2), and then irradiated under ultraviolet light with a wavelength of 365 nm for 10 min to obtain the titanium material modified by the dual-network hydrogel.

[0110] VI. Testing

[0111] (1) Scanning electron microscopy (SEM) and atomic force microscopy (AFM)

[0112] After freezing and fixing at -80℃, freeze-drying and liquid nitrogen fixing, the samples were cut open and their internal structure was observed using SEM; the changes in the microscopic surface structure of the samples were observed using atomic force microscopy (AFM).

[0113] Titanium (Ti), nitric acid-etched titanium (Ti-H), aminated titanium (Ti-NH2), and double-network hydrogel-modified titanium (Ti-GSC) were used as samples and subjected to scanning electron microscopy (SEM) analysis according to the above method. The results are as follows. Figure 3 As shown in Figure a; titanium material (Ti), aminated titanium material (Ti-NH2), and double-network hydrogel modified titanium material (Ti-GSC) were used as samples and subjected to atomic force microscopy (AFM) tests, and the results are as follows. Figure 3 As shown in b.

[0114] Depend on Figure 3 As shown in a and b, compared with the surface of titanium material (Ti), the surface roughness of titanium material (Ti-H) after nitric acid etching treatment and titanium material (Ti-NH2) after amylation modification is significantly increased. However, the surface smoothness of titanium material (Ti-GSC) modified by double network hydrogel is significantly higher than that of titanium material (Ti) and titanium material (Ti-NH2).

[0115] Example 5 Adhesion Strength Test

[0116] I. Experimental Materials

[0117] Pure titanium foil (1cm*4cm), quartz sheet (1cm*4cm), LAMG hydrogel (G) precursor solution prepared in Example 3, composite hydrogel (GS) precursor solution prepared in Example 3, and dual-network hydrogel (GSC) precursor solution prepared in Example 3.

[0118] II. Amination-modified titanium foil

[0119] Pure titanium foil (1cm*4cm) was ultrasonically cleaned by sequentially immersing it in ethanol and distilled water, and then dried at 60℃ for 6 hours. The dried pure titanium foil (1cm*4cm) was then immersed in nitric acid and reacted at 60℃ for 40 minutes. Subsequently, the reacted pure titanium foil (1cm*4cm) was placed in a plasma cleaner for 5 minutes. After cleaning, it was immediately immersed in a 2wt% APTES ethanol solution (the preparation process is the same as above) and incubated in a shaker at 37℃ and 60 rpm for 2 hours to obtain the amination-modified titanium foil (1cm*4cm).

[0120] III. Aminated Quartz Plates

[0121] Quartz sheets (1cm*4cm) were ultrasonically cleaned by immersing them in ethanol and distilled water in sequence, and then dried at 60℃ for 6 hours. After drying, the quartz sheets (1cm*4cm) were placed in a plasma cleaner for 5 minutes. Immediately after cleaning, they were immersed in a 2wt% APTES ethanol solution (the preparation process is the same as above) and incubated in a shaker at 37℃ and 60 rpm for 2 hours to obtain aminated quartz sheets (1cm*4cm).

[0122] IV. Test

[0123] (1) 20 μL of LAMG hydrogel (G) precursor solution (the same as the LAMG hydrogel (G) precursor solution prepared in Example 3) was dropped onto the upper surface of a pure titanium foil (1cm*4cm). The lower surface of an aminated quartz sheet (1cm*4cm) was then placed in contact with the LAMG hydrogel (G) precursor solution. The overlapping area of ​​the titanium foil (1cm*4cm) and the aminated quartz sheet (1cm*4cm) was controlled to be a cube with a thickness of 1cm*1cm and 1.5mm to obtain the test sample (Ti-G). In the test sample (Ti-G), the titanium foil (1cm*4cm), the LAMG hydrogel (G) precursor solution, and the aminated quartz sheet (1cm*4cm) were stacked in sequence. The test sample (Ti-G) was irradiated under ultraviolet light with a wavelength of 365nm for 10min. Then, at 37°C, an overlap shear test was performed using a universal testing machine at a tensile speed of 0.1mm / s, and the adhesion strength was calculated.

[0124] (2) 20 μL LAMMG hydrogel (G) precursor solution was dropped onto the upper surface of the above-mentioned aminated titanium foil (1cm*4cm). The lower surface of the aminated quartz sheet (1cm*4cm) was brought into contact with the LAMMG hydrogel (G) precursor solution. The overlapping area of ​​the aminated titanium foil (1cm*4cm) and the aminated quartz sheet (1cm*4cm) was controlled to be a cube with a thickness of 1cm*1cm and 1.5mm to obtain the test sample (NH2-G). In the test sample (NH2-G), the aminated titanium foil (1cm*4cm), the LAMMG hydrogel (G) precursor solution and the aminated quartz sheet (1cm*4cm) were stacked in sequence. The test sample (NH2-G) was irradiated under ultraviolet light with a wavelength of 365nm for 10min. Then, at 37℃, an overlap shear test was performed using a universal testing machine at a tensile speed of 0.1mm / s, and the adhesion strength was calculated.

[0125] (3) 20 μL of a composite hydrogel (GS) precursor solution (consistent with the composite hydrogel (GS) precursor solution prepared in Example 3) was dropped onto the upper surface of the above-mentioned aminated titanium foil (1 cm * 4 cm). The lower surface of the above-mentioned aminated quartz sheet (1 cm * 4 cm) was then brought into contact with the composite hydrogel (GS) precursor solution. The overlap area between the aminated titanium foil (1 cm * 4 cm) and the aminated quartz sheet (1 cm * 4 cm) was controlled to be 1 cm * 1 cm and the thickness to be 1.5 μm. A cube of size m was used to obtain the test sample (NH2-GS). In the test sample (NH2-GS), an aminated titanium foil (1cm*4cm), a composite hydrogel (GS) precursor solution, and an aminated quartz sheet (1cm*4cm) were stacked in sequence. The test sample (NH2-GS) was irradiated under ultraviolet light with a wavelength of 365nm for 10min. Then, at 37℃, an lap shear test was performed using a universal testing machine at a tensile speed of 0.1mm / s, and the adhesion strength was calculated.

[0126] (4) 20 μL of a double-network hydrogel (GSC) precursor solution (consistent with the double-network hydrogel (GSC) precursor solution prepared in Example 3) was dropped onto the upper surface of the above-mentioned aminated titanium foil (1 cm * 4 cm). The lower surface of the above-mentioned aminated quartz sheet (1 cm * 4 cm) was then brought into contact with the double-network hydrogel (GSC) precursor solution. The overlap area between the aminated titanium foil (1 cm * 4 cm) and the aminated quartz sheet (1 cm * 4 cm) was controlled to be 1 cm * 1 cm and the thickness to be 1.5 μm. A cube of m was used to obtain the test sample (NH2-GSC). In the test sample (NH2-GSC), an aminated titanium foil (1cm*4cm), a double-network hydrogel (GSC) precursor solution, and an aminated quartz sheet (1cm*4cm) were stacked in sequence. The test sample (NH2-GSC) was irradiated under ultraviolet light with a wavelength of 365nm for 10min. Then, at 37℃, an lap shear test was performed using a universal testing machine at a tensile speed of 0.1mm / s, and the adhesion strength was calculated.

[0127] like Figure 4As shown, NH2-G exhibits better adhesion strength compared to Ti-G, which is attributed to the hydrogen bonding interaction between disulfide bonds and amino groups, as well as the hydrogen bonding interaction between carboxyl groups on the LAMG hydrogel matrix and amino groups on the aminated titanium foil surface. NH2-GS shows better adhesion strength than NH2-G, possibly due to the higher viscosity of sodium alginate, which enhances the adhesion of the composite hydrogel (GS). NH2-GSC shows even higher adhesion strength than NH2-GS, indicating that the addition of drug-loaded ACC nanoparticles introduces calcium ions into the hydrogel system, forming a sodium alginate-calcium hydrogel network with sodium alginate and a dual-network hydrogel with gelatin, further improving the hydrogel's adhesion strength. In summary, the dual-network hydrogel (GSC) provided by this invention possesses excellent adhesion strength.

[0128] Example 6: Performance Testing of Dual-Network Hydrogel

[0129] I. Osteogenic Differentiation Effect Detection

[0130] (1) Experimental Grouping

[0131] There are a total of 4 groups: control group (Ti), Ti-GS group (GS), Ti-GSN group (GSN), and Ti-GSC group (GSC).

[0132] Control group (Ti): Untreated titanium (Ti) sheets;

[0133] Ti-GS group (GS): 20 μL of composite hydrogel (GS) precursor solution was uniformly coated on the surface of the aminated titanium sheet (which is the same as the aminated titanium material prepared in Example 4, the same below), and then placed under ultraviolet light with a wavelength of 365 nm for 10 min to obtain the composite hydrogel modified titanium sheet.

[0134] Ti-GSN group (GSN): 20 μL of hydrogel (GSN) precursor solution containing NMN was uniformly coated on the surface of the aminated titanium sheet and irradiated under ultraviolet light with a wavelength of 365 nm for 10 min to obtain a titanium sheet modified with hydrogel containing NMN.

[0135] Ti-GSC group (GSC): 20 μL of dual-network hydrogel (GSC) precursor solution was uniformly coated on the surface of the aminated titanium sheet and then irradiated under ultraviolet light at a wavelength of 365 nm for 10 min to obtain titanium sheet modified with dual-network hydrogel.

[0136] (2) Osteogenic differentiation effect detection

[0137] Titanium sheets from each group (control group, Ti-GS group, Ti-GSN group, and Ti-GSC group) were placed in 24-well plates in low-glucose DMEM medium containing 10% FBS (medium formulation: 4 mM L-glutamine, 3700 mg / L sodium bicarbonate, 1000 mg / L D-glucose, 1 mM sodium pyruvate). Normal BMSCs (2 × 10⁻⁶) were then inoculated. 4 BMSCs were co-cultured (cells / well) for 7, 14, and 21 days, with the culture medium changed every 3 days, to obtain BMSCs from each group (BMSCs treated in the control group, BMSCs treated in the Ti-GS group, BMSCs treated in the Ti-GSN group, and BMSCs treated in the Ti-GSC group), which were used to detect osteogenic differentiation levels at different stages (early, middle, and late).

[0138] ① To investigate the effects of different treatments on early osteogenic differentiation of bone MSCs, the ALP activity of BMSCs in each treatment group was qualitatively detected at 7 days using an alkaline phosphatase (ALP) colorimetric reaction. The ALP expression level of BMSCs in each treatment group was quantitatively detected at 7 days using an ALP activity assay kit. The results are as follows: Figure 5 As shown in a and b.

[0139] Previous studies have shown that the higher the ALP expression level in early osteogenic bone marrow mesenchymal stem cells (BMSCs), the higher their differentiation potential for later osteogenic formation. Figure 5 The results of a and b showed that the ALP activity of BMSCs treated with Ti-GSC (GSC) was significantly increased. Compared with BMSCs treated with the control group (Ti), the ALP staining of BMSCs treated with Ti-GSC (GSC) was darker and the area was larger. BMSCs treated with Ti-GSN (GSN) did not show a higher ALP expression level than BMSCs treated with the control group (Ti). This may be because NMN in GSN has no significant effect on the osteogenic differentiation rescue of BMSCs under osteoporotic conditions. However, the ALP expression level of BMSCs treated with Ti-GSC (GSC) was significantly higher than that of BMSCs treated with Ti-GSN (GSN). This may be because the introduction of calcium ions in GSC can promote the early osteogenic differentiation of BMSCs.

[0140] ② To further investigate the effects of each treatment group on the intermediate osteogenic differentiation of BMSCs, Sirius red staining was used to show the secretion of extracellular collagen (type I collagen) in BMSCs of each treatment group at 14 days. The results are as follows: Figure 6 As shown in a and b.

[0141] Depend on Figure 6Results a and b showed that, compared to other BMSCs, the control group (Ti) had the lowest type I collagen secretion. The Ti-GS group (GS) showed a significant increase in type I collagen secretion compared to the control group (Ti) (P<0.05). This may be because gelatin and sodium alginate, two natural macromolecules, act as extracellular matrix (ECM) mimics, providing ECM-like support for BMSCs and stimulating them to secrete more type I collagen to enhance ECM formation and remodeling. The Ti-GSN group (GSN) had a higher secretion level than the Ti-GS group (GS), possibly because the addition of NMN upregulated SIRT1, increasing the type I collagen secretion capacity of BMSCs in an osteoporotic environment. The GSC group had the highest secretion level, indicating that the combined effect of NMN and calcium ions in drug-loaded ACC nanoparticles significantly increased type I collagen secretion in BMSCs, effectively promoting mid-stage osteogenic differentiation of BMSCs.

[0142] ③ To investigate the effects of each group on late osteogenic differentiation of BMSCs, the mineralization level (absorbance @ 405 nm) of BMSCs in each group was qualitatively and quantitatively detected at 14 days (14d) and 21 days (21d) using alizarin red staining, and the results were statistically analyzed. Figure 7 As shown in ac.

[0143] Depend on Figure 7 According to the results, at 14 days, the control group (Ti) showed the lowest mineralization level of BMSCs. In contrast, the BMSCs treated in the GS and GSN groups showed improved mineralization levels, consistent with early osteogenic differentiation detection results. This indicates that the presence of gelatin and sodium alginate, two natural macromolecules, can promote osteogenic differentiation to some extent, while the effect of NMN alone on promoting osteogenic differentiation is not significant. The BMSCs treated in the GSC group showed the highest mineralization level, significantly different from other groups, further demonstrating the significant effect of drug-loaded ACC nanoparticles on promoting osteogenic differentiation. At 21 days, the mineralization levels of BMSCs in all groups were further improved compared to 14 days, indicating that the osteogenic differentiation level of BMSCs increased with prolonged culture time. The differences between groups were similar to those at 14 days, but the differences were more significant. Furthermore, the BMSCs treated in the GSC group showed a more sustained and stable promotion of osteogenic differentiation over time.

[0144] II. Detection of Reactive Oxygen Species (ROS) Levels

[0145] (1) Construction of animal models

[0146] Experimental animals: female SD rats, purchased from Chongqing Enswell Company.

[0147] Establishment of a rat model of osteoporosis: Three months after bilateral ovariectomy, 40 rats (n=40) were randomly divided into 4 groups (n=10 per group) and given the following different treatments:

[0148] Control group (Ti): Untreated titanium (Ti) rods (1.2mm*10mm) were used as implants, and rats underwent implantation surgery. The specific procedure of the implantation surgery is as follows:

[0149] 1) Prepare a sodium pentobarbital solution with a concentration of 0.02 g / mL by mixing sodium pentobarbital with pure water;

[0150] 2) Anesthetize rats by injecting sodium pentobarbital solution (intraperitoneal injection, 0.2 mL / 100 g);

[0151] 3) Place the anesthetized rats in a sterile, clean operating table;

[0152] 4) After disinfecting the rat's shaved hind legs with povidone-iodine, make an incision in the skin at the knee joint with a sterile scalpel, and then carefully cut open the skin and muscle layers on the outer side of the knee joint (be careful not to scratch the knee joint) and make a mark at the femoral epiphysis to determine the implantation site.

[0153] 5) Use a surgical drill to drill an implantation hole (1.2 mm in diameter and 1 cm in length) along the femur. Then, implant an untreated titanium (Ti) rod.

[0154] 6) The muscle layer and skin layer are sutured together using absorbable sutures;

[0155] Ti-GS group (GS): 200 μL of composite hydrogel (GS) precursor solution (consistent with the composite hydrogel (GS) precursor solution prepared in Example 3) was injected into a mold containing an aminated titanium rod (1.2 mm * 10 mm) (consistent with the aminated titanium material prepared in Example 4, except that the titanium rod is 1.2 mm * 10 mm, the same below). The mold was then placed under ultraviolet light at a wavelength of 365 nm for cross-linking for 30 min to obtain the GS-treated titanium rod implant. The implantation surgery was then performed on rats (the specific procedure of the implantation surgery was basically the same as that of the control group (Ti), except that the GS-treated titanium rod implant was implanted).

[0156] Ti-GSN group (GSN): 200 μL of NMN-containing hydrogel (GSN) precursor solution (consistent with the NMN-containing hydrogel (GSN) precursor solution prepared in Example 3) was injected into a mold containing an aminated titanium rod (1.2 mm * 10 mm), and then placed under ultraviolet light at a wavelength of 365 nm for cross-linking for 30 min to obtain GSN-treated titanium rod implants. Then, rats underwent implantation surgery (the specific process of implantation surgery was basically the same as that of the control group (Ti), except that the GSN-treated titanium rod implants were implanted).

[0157] Ti-GSC group (GSC): 200 μL of dual-network hydrogel (GSC) precursor solution (consistent with the dual-network hydrogel (GSC) precursor solution prepared in Example 3) was injected into a mold containing an aminated titanium rod (1.2 mm * 10 mm), and then placed under ultraviolet light at a wavelength of 365 nm for cross-linking for 30 min to obtain GSC-treated titanium rod implants. Then, rats underwent implantation surgery (the specific process of implantation surgery was basically the same as that of the control group (Ti), except that GSC-treated titanium rod implants were implanted).

[0158] (2) ROS level detection

[0159] To assess the levels of reactive oxygen species (ROS) in rats of each group, fluorescent staining was performed using dihydroethidium (DHE) solution.

[0160] Seven days after the implantation surgery, rats in each group were euthanized by intraperitoneal injection of dihydroethidium (DHE) solution (10 μM, 100 μL). Twenty-four hours later, each group was euthanized by intraperitoneal injection of sodium pentobarbital solution (0.02 g / mL, 200 mg / kg). Femoral tissue was collected from each group. The sodium pentobarbital solution was prepared by mixing sodium pentobarbital with pure water.

[0161] After decalcifying the femoral tissue of each group of rats at room temperature for 24 hours using a rapid decalcification solution (water:formaldehyde:nitric acid = 750:150:100, 40 mL / rat), the femoral tissue of each group of rats was frozen using OCT (Optimal Cutting Temperature, 4583, Sakura). The tissue was then embedded and placed in a liquid nitrogen freeze bath to rapidly freeze the femoral tissue. Subsequently, femoral tissue sections (4 μm thick) were cut from each group of rats at -30℃. The fluorescence intensity of the femoral tissue sections of each group of rats was observed under a fluorescence microscope, and statistical analysis was performed. The results are as follows: Figure 8 As shown in a and b.

[0162] Depend on Figure 8As shown in a and b, the addition of dual-network hydrogel (GSC) can significantly improve the problem of excessive ROS around the implant, indicating that GSC itself has excellent ROS scavenging ability. The addition of GSN did not improve ROS scavenging, possibly because GSN has low stability in vivo and its mitochondrial damage repair effect is not significant. The addition of GSC improved the implant's ROS scavenging ability, possibly because the addition of drug-loaded ACC nanoparticles effectively repaired mitochondrial damage in bone marrow mesenchymal stem cells around the implant, reducing ROS levels within the bone marrow mesenchymal stem cells.

[0163] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A double network hydrogel containing drug-loaded ACC nanoparticles, characterized in that, The LAMG compound, sodium alginate, and the drug-loaded ACC nanoparticle; The LAMG compound is obtained by mixing a gelatin aqueous solution with a lipoic acid solution and then performing a first reaction, and the drug-loaded ACC nanoparticle is obtained by performing a second reaction on a raw material system including a nicotinamide mononucleotide aqueous solution, a calcium source solution, and ammonium bicarbonate, and then mixing the LAMG compound, the sodium alginate, and the drug-loaded ACC nanoparticle and irradiating under ultraviolet light to obtain the double-network hydrogel containing the drug-loaded ACC nanoparticle.

2. The dual network hydrogel containing drug loaded ACC nanoparticles as claimed in claim 1, wherein, In the double-network hydrogel, the mass ratio of the LAMG compound, the sodium alginate, and the drug-loaded ACC nanoparticle is (0.5-2):(0.05-1):(0.005-0.015).

3. The dual network hydrogel containing drug loaded ACC nanoparticles as claimed in claim 1, wherein, In the gelatin aqueous solution, the concentration of gelatin is 0.04 g / mL-0.07 g / mL. In the lipoic acid solution, the concentration of lipoic acid is 0.01 g / mL-0.03 g / mL. In the nicotinamide mononucleotide aqueous solution, the concentration of nicotinamide mononucleotide is 11 mg / mL-15 mg / mL. In the calcium source solution, the concentration of the calcium source is 0.5 mol / L-2 mol / L.

4. The dual network hydrogel containing drug loaded ACC nanoparticles as claimed in claim 3, wherein, The calcium source is at least one of calcium chloride and calcium chloride dihydrate.

5. The dual network hydrogel containing drug loaded ACC nanoparticles as claimed in claim 1, wherein, The temperature of the first reaction is 38-42°C, and the time is 5-7 h. The temperature of the second reaction is 38-42°C, and the time is 12-18 h.

6. A method of preparing a double network hydrogel containing drug-loaded ACC nanoparticles according to any one of claims 1 to 5, characterized in that, The method comprises the following steps: Preparation of a gelatin aqueous solution and a lipoic acid solution; After the lipoic acid solution is stirred for a first time, an activating stabilizer is added and stirred for a second time to obtain a first mixture; The gelatin aqueous solution is mixed with the first mixture to perform a first reaction, and the first reaction liquid is subjected to dialysis with double-distilled water and a first drying treatment in sequence to obtain the LAMG compound; A nicotinamide mononucleotide aqueous solution is prepared and placed in a reaction container, a calcium source solution is added to the nicotinamide mononucleotide aqueous solution to obtain a mixed solution, anhydrous ethanol is added to the mixed solution to obtain a milky white solution, and then the reaction container is placed in a sealed container with ammonium bicarbonate particles on the bottom to obtain a reaction system; the reaction system is subjected to a second reaction to obtain a second reaction liquid; the second reaction liquid is subjected to a first centrifugal treatment, cleaning, and a second centrifugal treatment in sequence to obtain a precipitate; and the precipitate is subjected to a second drying treatment to obtain the drug-loaded ACC nanoparticle; The LAMG compound is mixed with water, and then sodium alginate is added and stirred for a third time to obtain a second mixture; the drug-loaded ACC nanoparticle is added to the second mixture and stirred for a fourth time to obtain a double-network hydrogel precursor solution; The double-network hydrogel precursor solution is irradiated under ultraviolet light to obtain the double-network hydrogel containing the drug-loaded ACC nanoparticle.

7. The dual network hydrogel containing drug loaded ACC nanoparticles as claimed in claim 6, wherein, The temperature of the first stirring is 35-38°C, and the time is 25-40 min. And / or, the second stirring time is 3 min-6 min; And / or, the temperature of the second dialysis is 48 ℃-52 ℃, and the time is 6 d-8 d; And / or, the temperature of the first centrifugal treatment is 3 ℃-5 ℃, and the speed is 11000 rpm-16000 rpm; And / or, the temperature of the second centrifugal treatment is 3 ℃-5 ℃, and the speed is 11000 rpm-16000 rpm.

8. The dual network hydrogel containing drug loaded ACC nanoparticles as claimed in claim 6, wherein, The temperature of the third stirring is 48 ℃-52 ℃, and the time is 2.5 h-3.5 h; And / or, the temperature of the fourth stirring is 38 ℃-42 ℃, and the time is 10 h-14 h.

9. A bone repair material, characterized by, The double-network hydrogel containing the drug-loaded ACC nanoparticles according to any one of claims 1-5.

10. The bone repair material of claim 9, wherein, The preparation process of the aminated titanium comprises: After the third reaction of soaking the titanium in nitric acid, a product is obtained, and after the product is cleaned and soaked in an APTES ethanol solution for incubation treatment, the aminated titanium is obtained.

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