An injectable bone repair material and its preparation method

Abstract

This invention discloses an injectable bone repair material and its preparation method, belonging to the field of biomaterials technology. The material is composed of grafted chitosan obtained by reacting N-formylglycine, allyl lactate, and S-allyl cysteine ​​with chitosan. The grafted chitosan, methacrylamide gelatin, and icariin-naringenin liposomes are then photocured to form a stable three-dimensional interpenetrating network hydrogel. The preparation method includes: first synthesizing grafted chitosan, then preparing icariin-naringenin liposomes, and finally mixing the grafted chitosan solution, methacrylamide gelatin solution, and liposomes, adding a photoinitiator, and then ultrasonically degassing and ultraviolet curing to obtain the finished product. This material utilizes thioether bonds to achieve intelligent drug release in response to reactive oxygen species, and liposome encapsulation improves drug utilization. The dual-network structure endows it with excellent injectability, mechanical strength, and biocompatibility, effectively adapting to irregular bone defects and promoting bone regeneration.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, and in particular relates to an injectable bone repair material and its preparation method. Background Technology

[0002] Bone tissue is one of the most regenerative connective tissues in the human body. However, when large or critical-sized defects are caused by trauma, tumors, infections, or congenital diseases, the bone's self-repair ability is limited, making complete regeneration difficult. Currently, commonly used clinical bone repair strategies include autologous bone grafting, allogeneic bone grafting, and metal / ceramic artificial bone implantation. However, autologous bone grafting has drawbacks such as secondary damage to the donor site and limited availability; allogeneic bone carries the risk of immune rejection and disease transmission; and traditional metal or bioceramic materials often suffer from insufficient bioactivity, mismatched degradation properties, and difficulty in perfectly fitting irregular bone defect shapes.

[0003] Flavonoids, such as icariin and naringenin, possess a variety of pharmacological effects, including anti-inflammatory, antibacterial, antioxidant, and osteogenic differentiation-promoting effects. Studies have shown that icariin can significantly activate osteogenic-related signaling pathways such as BMP-2 / Smad, promoting osteogenic differentiation and mineralized nodule formation of bone marrow mesenchymal stem cells. Naringenin not only effectively inhibits osteoclast activity and reduces bone resorption, but also exerts a synergistic antioxidant effect with icariin, scavenging excess reactive oxygen species, reducing oxidative stress damage in the bone defect microenvironment, and breaking the vicious cycle of inflammation-oxidative stress that hinders bone healing. However, the poor water solubility, rapid metabolism in vivo, and low bioavailability of these two drugs limit their direct application.

[0004] Hydrogels possess inherent biocompatibility, biodegradability, and injectability. They can be injected with a syringe to form a complete object, sealing the complex contours of defects and achieving complete filling. Furthermore, hydrogels act as sustained-release carriers, enabling functional materials to exhibit more stable performance. In addition, the 3D microenvironment of hydrogels can support cell adhesion and proliferation, mimicking the function of the extracellular matrix, which is beneficial for cell ingrowth and tissue regeneration. However, currently available single-component hydrogels often struggle to simultaneously meet multiple requirements, including mechanical support, controlled drug release, and microenvironment regulation. For example, purely physically cross-linked hydrogels have relatively weak mechanical strength, while conventional chemically cross-linked hydrogels may lack intelligent responsiveness to the pathological microenvironment, leading to a mismatch between drug release behavior and the healing process.

[0005] Therefore, it is necessary to propose a novel bone repair material with good injectability, mechanical strength, and drug release properties to overcome the shortcomings of existing technologies. Summary of the Invention

[0006] In view of the above situation and to overcome the defects of the prior art, the purpose of this invention is to provide an injectable bone repair material and its preparation method. The material is composed of grafted chitosan, methacrylamide gelatin and icariin-naringenin liposomes, which form a stable three-dimensional interpenetrating network structure hydrogel through photocuring. It has excellent injectability, mechanical strength, reactive oxygen species responsive drug release and bone regeneration promotion capabilities.

[0007] To achieve the above objectives, the following technical solution is adopted: This invention provides an injectable bone repair material and its preparation method, the preparation method comprising the following steps:

[0008] S1. N-formylglycine and allyl lactate were dissolved in DMF at a molar ratio of 1:1.1. Dicyclohexylcarbodiimide (5% by mass of the total reactants) and 4-dimethylaminopyridine (2% by mass of the total reactants) were added. The mixture was stirred at 35°C for 8 hours, filtered, and the filtrate was evaporated under reduced pressure to obtain an intermediate product. The intermediate product and S-allyl cysteine ​​were dissolved in DMF at a molar ratio of 1:1.05. Dicyclohexylcarbodiimide (5% by mass of the total reactants) and 4-dimethylaminopyridine (2% by mass of the total reactants) were added. The mixture was stirred at 32°C for 9 hours. Chitosan was added at a mass ratio of intermediate product to chitosan of 1:4. The pH of the system was adjusted to 5.0 by adding 1.5% dilute acetic acid solution. The mixture was stirred at 48°C for 14 hours. The reaction solution was dialyzed through a dialysis bag with a molecular weight cutoff of 8000-14000 Da for 3 days and then freeze-dried to obtain grafted chitosan.

[0009] S2. Icariin, naringenin, hydrogenated soybean lecithin, and cholesterol were dissolved in a mixed solvent of chloroform and methanol in a mass ratio of 1:1.5:20:5 and 3:1 (volume ratio). A uniform lipid film was formed by rotary evaporation in a water bath at 35°C. Phosphate buffer at pH 7.4 was added at a total lipid mass to PBS volume ratio of 1:10 g / mL. The mixture was hydrated by shaking at 37°C for 30 min. After sonication and centrifugation, icariin-naringenin liposomes were obtained.

[0010] S3. First, dissolve the grafted chitosan in 1% dilute acetic acid solution to prepare a 3% (w / w) grafted chitosan solution. Dissolve the methacrylamide gelatin in PBS at pH 7.4 to prepare a 12% (w / w) methacrylamide gelatin solution. Mix the grafted chitosan solution and the methacrylamide gelatin solution at a volume ratio of 5.5:1. Stir at 25°C for 30-60 min. Then add 4-8% (w / w) of icariin-naringenin liposomes to the mixed solution and continue stirring for 20 min. Subsequently, add 0.8% (w / w) of photoinitiator to the system. Degas by sonication and cure by ultraviolet light to obtain the injectable bone repair material.

[0011] Furthermore, in step S1, the degree of deacetylation of chitosan is ≥90%, and the molecular weight is 50-100 kDa.

[0012] Preferably, in step S1, the degree of deacetylation of chitosan is ≥90%, and the molecular weight is 80kDa.

[0013] Furthermore, in step S3, when preparing the grafted chitosan solution, the mixture is stirred at 200-300 rpm for 1.5-2 hours under a water bath at 30-35℃. After dissolution, it is naturally cooled to 25℃ and then filtered through a 0.22μm microporous membrane to obtain the final product.

[0014] Preferably, in step S3, when preparing the grafted chitosan solution, the mixture is stirred at 250 rpm for 1.8 h in a 32°C water bath, dissolved, and then naturally cooled to 25°C. The solution is then filtered through a 0.22 μm microporous membrane to obtain the final product.

[0015] Furthermore, in step S3, the degree of substitution of the methacrylamide gelatin is 60%-70%, and the molecular weight is 120-180 kDa.

[0016] Preferably, in step S3, the degree of substitution of the methacrylamide gelatin is 65%, and the molecular weight is 160 kDa.

[0017] Furthermore, in step S3, when preparing the methacrylamide gelatin solution, the mixture is stirred at 400-500 rpm for 1.5-2 hours under a water bath at 50-60°C, and then naturally cooled to 25°C after dissolution to obtain the final product.

[0018] Preferably, in step S3, the degree of substitution of the methacrylated gelatin is 65%, and the molecular weight is 160 kDa. In step S3, the methacrylated gelatin solution is prepared by stirring at 450 rpm for 1.8 hours in a 55°C water bath, and then naturally cooling to 25°C after dissolution to obtain the final product.

[0019] Furthermore, the photoinitiator is selected from Irgacure 2959 and Irgacure 1173.

[0020] Preferably, the photoinitiator is Irgacure2959.

[0021] Furthermore, the ultrasonic degassing power in step S3 is 200-300W, and the ultrasonic treatment is carried out at 25°C for 5-8 minutes.

[0022] Preferably, the ultrasonic degassing power in step S3 is 250W, and the ultrasonic treatment is carried out at 25°C for 6 minutes.

[0023] Furthermore, the ultraviolet curing process in step S3 needs to be carried out under a nitrogen protective atmosphere, with the distance between the light source and the system surface controlled at 5-8 cm, and a wavelength of 365 nm and a light intensity of 15 mW / cm². 2 Cured under ultraviolet light for 25 seconds.

[0024] Preferably, the ultraviolet curing process in step S3 is carried out in a nitrogen protective atmosphere, the distance between the light source and the system surface is controlled at 6 cm, and the curing is carried out for 25 seconds under ultraviolet light with a wavelength of 365 nm and a light intensity of 15 mW / cm2.

[0025] The beneficial effects of this invention are:

[0026] (1) Both naringenin and icariin have significant antioxidant activity, which can effectively remove excessive ROS generated during bone healing, reduce the damage of oxidative stress to osteoblasts, break the vicious cycle of inflammation-oxidative stress, and create a favorable microenvironment for bone regeneration. Encapsulating the two active ingredients, icariin and naringenin, in liposomes can not only effectively protect the drug activity and prevent it from being rapidly metabolized and degraded in vivo, but also achieve controlled sustained release of the drug through the lipid bilayer structure, thereby improving bioavailability.

[0027] (2) By functionalizing chitosan through grafting, a variety of active groups such as double bonds, amides, carboxyl groups and thioether bonds are introduced. The introduction of double bonds enables it to form a stable three-dimensional interpenetrating network structure with methacrylamide gelatin through photocuring, which improves the mechanical properties of the hydrogel. The hydrogel network formed by photocuring crosslinking has excellent mechanical strength and stability, and can maintain structural integrity under external force, adapt to the mechanical requirements of bone load-bearing parts, and provide necessary physical support for defect sites. The introduction of amide bonds and carboxyl groups significantly enhances the hydrophilicity and swelling properties of the material, which is conducive to absorbing tissue exudate and maintaining a local moist environment. The introduction of thioether bonds gives it reactive oxygen species response characteristics, which can selectively break under the oxidative stress environment common in bone defect sites, and realize the intelligent triggering release of drugs.

[0028] (3) The synthetic components of this material include amino acid derivatives, chitosan and gelatin, all of which have good biocompatibility and degradability. The degradation products are non-toxic and can be absorbed and metabolized by the human body, with no risk of foreign body reaction. Chitosan itself has broad-spectrum antibacterial properties. After grafting modification, its antibacterial activity is retained or even enhanced, which helps to prevent postoperative infection at bone defect sites. Attached Figure Description

[0029] Figure 1 This is the equation for the preparation process of grafted chitosan in this invention;

[0030] Figure 2Infrared characterization images of chitosan and grafted chitosan during the preparation process of this invention;

[0031] Figure 3 The figure shows the cytotoxicity test results of the material prepared in this invention;

[0032] Figure 4 The graph shows the test results of the swelling properties of the material prepared in this invention.

[0033] Figure 5 The graph shows the test results of the mass residue rate of the material prepared in this invention after degradation.

[0034] Figure 6 The graph shows the cumulative drug release rate test results of the material prepared in this invention;

[0035] Figure 7 This is a graph showing the results of alkaline phosphatase activity detection in the osteogenic differentiation performance test of this invention;

[0036] Figure 8 This is a graph showing the results of osteogenic differentiation performance testing of the present invention, specifically the detection of osteogenic-related gene expression.

[0037] Figure 9 This is a CT scan of the skull during the repair of critical-sized skull defects in rats according to the present invention.

[0038] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. Detailed Implementation

[0039] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the experimental materials used in the following examples are all purchased from commercial channels.

[0042] Chitosan and methacrylamide gelatin were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; naringenin, icariin, hydrogenated soybean lecithin, cholesterol, DMF, dicyclohexylcarbodiimide, 4-dimethylaminopyridine, photoinitiator, and lysozyme were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; N-formylglycine was purchased from Shanghai Mairui Biochemical Technology Co., Ltd.; and allyl lactate was purchased from Shanghai Hans Chemical Co., Ltd. Mouse fibroblast cell line L929 and rat bone marrow mesenchymal stem cells were purchased from Shanghai Binsui Biotechnology Co., Ltd.; and SD rats were purchased from Liaoning Changsheng Biotechnology Co., Ltd.

[0043] Example 1:

[0044] A method for preparing an injectable bone repair material includes the following steps:

[0045] S1. N-Formylglycine and allyl lactate were dissolved in DMF at a molar ratio of 1:1.1. 5% dicyclohexylcarbodiimide and 2% 4-dimethylaminopyridine were added to the mixture, and the mixture was stirred at 35°C for 8 hours. After filtration, the filtrate was evaporated under reduced pressure to obtain an intermediate product. The intermediate product and S-allyl cysteine ​​were dissolved in DMF at a molar ratio of 1:1.05. 5% dicyclohexylcarbodiimide and 2% 4-dimethylaminopyridine were added to the mixture, and the mixture was reacted at 32°C for 9 hours. Chitosan was added at a mass ratio of intermediate product to chitosan of 1:4, and the pH of the system was adjusted to 5.0 with 1.5% dilute acetic acid solution. The mixture was stirred at 48°C for 14 hours. The reaction solution was dialyzed through a dialysis bag with a molecular weight cutoff of 12000 Da for 3 days, and then freeze-dried to obtain grafted chitosan. The equations for the above preparation process are shown in [reference needed]. Figure 1 ;

[0046] S2. Icariin, naringenin, hydrogenated soybean lecithin, and cholesterol were dissolved in a mixed solvent of chloroform and methanol in a mass ratio of 1:1.5:20:5 and 3:1 (volume ratio). A uniform lipid film was formed by rotary evaporation in a water bath at 35°C. Phosphate buffer at pH 7.4 was added at a total lipid mass to PBS volume ratio of 1:10 g / mL. The mixture was hydrated by shaking at 37°C for 30 min. After sonication and centrifugation, icariin-naringenin liposomes were obtained.

[0047] S3. First, dissolve the grafted chitosan in 1% dilute acetic acid solution to prepare a 3% (w / w) grafted chitosan solution. Dissolve the methacrylamide gelatin in PBS at pH 7.4 to prepare a 12% (w / w) methacrylamide gelatin solution. Mix the grafted chitosan solution and the methacrylamide gelatin solution at a volume ratio of 5.5:1. Stir at 25°C for 45 min. Then add 5% (w / w) of icariin-naringenin liposomes to the mixed solution and continue stirring for 20 min. Subsequently, add 0.8% (w / w) of photoinitiator to the system. Degas by sonication and cure by ultraviolet light to obtain the injectable bone repair material.

[0048] In step S1, the degree of deacetylation of chitosan is ≥90%, and the molecular weight is 80 kDa. In step S3, when preparing the grafted chitosan solution, it is stirred at 250 rpm for 1.8 hours in a 32°C water bath, dissolved, and then naturally cooled to 25°C. It is then filtered through a 0.22 μm microporous membrane to obtain the final product. In step S3, the degree of substitution of methacryloyl gelatin is 65%, and the molecular weight is 160 kDa. In step S3, when preparing the methacryloyl gelatin solution, it is stirred at 450 rpm for 1.8 hours in a 55°C water bath, dissolved, and then naturally cooled to 25°C to obtain the final product. The photoinitiator is Irgacure 2959. In step S3, the ultrasonic degassing power is 250 W, and ultrasonic treatment is performed at 25°C for 6 minutes. In step S3, the ultraviolet curing process must be carried out under a nitrogen protective atmosphere, with the distance between the light source and the system surface controlled at 6 cm, using a wavelength of 365 nm and a light intensity of 15 mW / cm². 2 Cured under ultraviolet light for 25 seconds.

[0049] Test Example 1:

[0050] The chemical structure of chitosan and grafted chitosan was characterized by Fourier transform infrared spectroscopy. The specific steps are as follows: The dried sample was mixed with potassium bromide and compressed into a pellet using the KBr pellet method at 4000-5000 cm⁻¹. -1 The absorption peaks of characteristic functional groups were analyzed by scanning within the wavelength range, and the images are shown below. Figure 2 .from Figure 2 It can be seen from the grafted chitosan at 1735cm -1 A new strong absorption peak appears at 1650 cm⁻¹, and the transmittance decreases significantly. This is characteristic of the C=O stretching vibration of the ester bond. -1 The enhanced amide I band and decreased transmittance in the vicinity indicate the formation of new amide bonds. (1550 cm⁻¹) -1 A new intermediate-intensity absorption peak appears nearby, corresponding to the amide II band, at 1590 cm⁻¹. -1The characteristic amino peaks of chitosan were significantly weakened and the transmittance increased, proving that the amino groups were consumed in the reaction. This indicates that the amino groups on chitosan participated in the amidation reaction, thus proving that the present invention successfully carried out the grafting reaction of chitosan and obtained grafted chitosan.

[0051] Test Example 2:

[0052] The specific steps for assessing the cytotoxicity of materials and ensuring their biosafety are as follows:

[0053] Preparation of material extract: The cured hydrogel material prepared in Example 1 was immersed in cell culture medium (DMEM containing 10% fetal bovine serum) under aseptic conditions, with a material surface area to culture medium volume ratio of 3 cm². 2 / mL, and extract in a 37℃, 5% CO2 incubator for 24h. Collect the supernatant as 100% extract and dilute with fresh culture medium to 50% and 25% concentrations.

[0054] Cell culture: Mouse fibroblast cell line L929 was used, and cells were cultured at a concentration of 1×10⁻⁶ cells / cells. 4 Cells were seeded at a density of 1 / well in 96-well plates and cultured at 37°C and 5% CO2 for 24 hours to allow the cells to adhere.

[0055] Cell treatment: Remove the original culture medium and add 100%, 50%, and 25% concentrations of material extract and fresh culture medium (as negative controls), respectively. Add culture medium containing 10% dimethyl sulfoxide (DMSO) as a positive control. Set up 6 replicates for each group.

[0056] Cell viability assay: After culturing for 24 h and 48 h, 10 μL CCK-8 solution was added to each well and incubated for 2 h. The absorbance (OD value) of each well was measured at 450 nm using a microplate reader.

[0057] Relative cell proliferation rate (RGR) calculation: RGR(%) = (OD value of experimental group / OD value of negative control group) × 100%.

[0058] The above test results are shown in Figure 3 .

[0059] According to GB / T16886.5 standard: RGR ≥ 100% is Grade 0 (non-toxic), 75%-99% is Grade 1 (slightly toxic), 50%-74% is Grade 2 (moderately toxic), 25%-49% is Grade 3 (severely toxic), and 1%-24% is Grade 4 (extremely toxic). Figure 3 As can be seen from the data, the relative proliferation rate of the injectable bone repair material extract prepared in this invention on L929 cells is higher than 90%, and the cytotoxicity level is 1, indicating that the material has good cell compatibility.

[0060] Test Example 3:

[0061] Swelling performance testing evaluates the material's ability to absorb water and swell under physiological conditions. The specific steps are as follows:

[0062] Sample preparation: The hydrogel precursor solution prepared in Example 1 was injected into a mold (10 mm in diameter and 5 mm in height), and a cylindrical hydrogel sample was obtained after UV curing. The sample was freeze-dried and its dry weight (W0) was measured.

[0063] Swelling experiment: The dried sample was immersed in an excess of pH 7.4 phosphate buffer solution and swollen in a constant temperature water bath at 37°C.

[0064] Weighing: Samples were taken out at preset time points (0.5, 1, 2, 4, 6, 8, 12, 24h), and excess surface moisture was gently absorbed with filter paper. The wet weight (W) was then immediately measured. t ).

[0065] Swelling rate calculation: The formula for calculating the swelling rate is: SR (%) = (W t -W0) / W0×100%.

[0066] The above test results are shown in Figure 4 .

[0067] from Figure 4 As can be seen, the hydrogel material exhibits rapid swelling behavior in PBS, reaching swelling equilibrium within 8 hours, with a final swelling rate of approximately 22.8%. This indicates that the material possesses excellent hydrophilicity and water retention capacity, providing a moist 3D microenvironment for cells.

[0068] Test Example 4:

[0069] The specific steps for evaluating the degradation behavior of materials under simulated physiological conditions are as follows:

[0070] Sample preparation and initial mass: Same as in test example 3, prepare and freeze-dry the hydrogel sample, and weigh the initial dry weight (W). i ).

[0071] Degradation experiment: The sample was immersed in PBS (pH 7.4) containing lysozyme (1 mg / mL) and degraded at 60 rpm in a 37°C constant temperature shaker. The degradation solution was changed every 3 days to maintain enzyme activity.

[0072] Sampling and Weighing: Samples (3 replicates per group) were collected at preset time points (1, 3, 7, 14, 21, 28 days), gently rinsed with deionized water, freeze-dried, and weighed to determine the dry weight (W) after degradation. d ).

[0073] Calculation of residual mass: Residual mass (%) = (Wd / W i )×100%.

[0074] The above test results are shown in Figure 5 .

[0075] from Figure 4 The study shows that the hydrogel material degraded by approximately 57.7% within 4 weeks, exhibiting a relatively gradual degradation trend. Its degradation rate matches the early to mid-stage process of bone defect repair, providing gradual support for new bone tissue.

[0076] Test Example 5:

[0077] The release behavior of liposome-encapsulated drugs under normal physiological conditions and reactive oxygen species (ROS) stimulation was investigated to verify their ROS responsiveness. The specific steps are as follows:

[0078] Sample preparation: Place the drug-loaded hydrogel sample (10 mm in diameter and 2 mm in height) in a dialysis bag (molecular weight cutoff 8000-14000 Da).

[0079] Release medium:

[0080] Normal group: Immerse the dialysis bag in 50 mL of pH 7.4 PBS.

[0081] ROS group: The dialysis bag was immersed in 50 mL of pH 7.4 PBS containing 100 μM H2O2 (simulating ROS environment).

[0082] Release conditions: Place all release vials in a 37°C constant temperature shaker (100 rpm).

[0083] Sampling and Detection: At predetermined time points (4, 12, 24, 48, 72, 96 h), 2 mL of the release medium was collected and replenished with an equal volume of fresh release medium at the same temperature. The concentrations of icariin and naringenin in the release medium were determined by high performance liquid chromatography (HPLC).

[0084] Chromatographic conditions: C18 column; mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution, gradient elution; flow rate: 1.0 mL / min; detection wavelength: 270 nm (icariin) and 290 nm (naringenin); column temperature: 30 ℃.

[0085] Cumulative release rate calculation: Based on the measured drug concentration and sampling volume, calculate the cumulative drug release rate at each time point.

[0086] The above measurement results are shown in Figure 6 .

[0087] exist Figure 6The results show that in normal PBS, the drug is released slowly and continuously, and the release of icariin and naringenin is not complete within 8 days. However, in a simulated ROS environment containing H2O2, the drug release is significantly accelerated and is basically complete within 96 hours. This proves that the thioether bonds in the grafted chitosan are responsive to ROS and can intelligently accelerate drug release, thereby targeting the release of drugs during the inflammatory / oxidative stress phase of bone defects and exerting anti-inflammatory and antioxidant effects.

[0088] Test Example 6:

[0089] The specific steps for verifying the promoting effect of the material extract on osteogenic differentiation of bone marrow mesenchymal stem cells are as follows:

[0090] Cells and grouping: Rat bone marrow mesenchymal stem cells (rBMSCs) were used.

[0091] Control group: using conventional osteogenic induction medium.

[0092] Control group A: Osteogenic induction culture medium containing 50% material extract (prepared from a blank hydrogel without drugs).

[0093] Control group B: Osteogenic induction culture medium containing 50% material extract (hydrogel prepared by replacing grafted chitosan and methacrylamide gelatin with equal amounts of chitosan and gelatin).

[0094] Drug-loaded experimental group: osteogenic induction culture medium containing 50% drug-loaded hydrogel material extract.

[0095] Alkaline phosphatase activity assay: rBMSCs were seeded in 24-well plates and cultured in different media for 7 and 14 days. After the cells were lysed, ALP activity was measured using an ALP assay kit and the total protein concentration was standardized by BCA method.

[0096] Osteogenesis-related gene expression: After 14 days of culture, total RNA was extracted from cells and reverse transcribed into cDNA. The expression levels of key osteogenic genes (Runx2, Osterix, OCN, COL1) were detected by real-time quantitative PCR. GAPDH was used as an internal reference gene, and the relative expression levels of the genes were calculated using the 2^(-ΔΔCt) method.

[0097] The above measurement results are shown in Figure 7 and Figure 8 .

[0098] from Figure 7 , Figure 8As can be seen, compared with the blank group, control group A, and control group B, the ALP activity and the expression level of osteogenic related genes in the drug-loaded experimental group were significantly upregulated. This indicates that the drug-loaded bone repair material prepared in this invention can effectively promote the osteogenic differentiation of rBMSCs, and its effect is significantly better than that of the blank hydrogel and conventional induction culture medium. This proves that the scheme of this invention, which uses grafted chitosan and methacrylamide gelatin to make hydrogel and combines it with icariin-naringenin liposomes, has a synergistic effect on promoting osteoogenesis.

[0099] Test Example 7:

[0100] In a rat model of critical-sized skull defects, the in vivo bone repair effect of the material was evaluated. The specific steps are as follows:

[0101] A critical-sized cranial defect model was established in 8-week-old male SD rats (180-200g) to evaluate the osteogenic capacity of the hydrogel. After general anesthesia and disinfection, critical-sized bone defects with a diameter of 8mm were prepared at the center of each skull using a microcranial drill. After defect stabilization, hydrogels were implanted into the bone defect sites according to the following groups:

[0102] Blank group: No material is implanted at the defect site;

[0103] Control group A: Implanted with blank hydrogel containing no drug;

[0104] Control group B: Implanted with a hydrogel material prepared by replacing grafted chitosan and methacrylamide gelatin with equal amounts of chitosan and gelatin;

[0105] Experimental group: Implanted with injectable hydrogel material carrying icariin-naringenin liposomes.

[0106] Twelve weeks after hydrogel implantation, rats were euthanized and their skulls were harvested. The skulls were cleaned with physiological saline and then preserved by soaking in 4% paraformaldehyde. CT scans were performed on the harvested skulls to assess bone regeneration.

[0107] In the blank group, only a small amount of discontinuous new bone grew along the edge of the defect area. In the control group A, a small amount of new bone tissue grew. In the control group B, a large amount of new bone tissue grew. In the experimental group, the amount of new bone was the largest, and it connected into a sheet, almost completely covering the defect area. This proves that the drug-loaded injectable bone repair material prepared in this invention can significantly promote the repair of critical-size skull defects in rats, demonstrating its excellent bone regeneration ability in vivo.

[0108] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0109] The present invention and its embodiments have been described above. This description is not restrictive, and the embodiments shown are only one of the embodiments of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for preparing an injectable bone repair material, characterized by: Comprising the following steps: S1. N-formyl glycine and allyl lactate were dissolved in DMF at a molar ratio of 1:1.1, 5% of dicyclohexyl carbodiimide and 2% of 4-dimethylamino pyridine were added according to the total mass of the reactants, and the reaction was stirred at 35°C for 8h. The filtrate was obtained by filtration and rotary evaporation under reduced pressure to obtain an intermediate product; the intermediate product and S-allyl cysteine were dissolved in DMF at a molar ratio of 1:1.05, 5% of dicyclohexyl carbodiimide and 2% of 4-dimethylamino pyridine were added according to the total mass of the reactants, and the reaction was carried out at 32°C for 9h. Chitosan was added according to the mass ratio of the intermediate product to chitosan of 1:4, and 1.5% dilute acetic acid solution was added to adjust the pH of the system to 5.

0. The reaction was carried out at 48°C for 14h. The reaction solution was dialyzed in a dialysis bag with a molecular weight cutoff of 8000-14000Da for 3 days, and freeze-dried to obtain grafted chitosan; S2. Icaritin, naringenin, hydrogenated soy lecithin, and cholesterol were dissolved in a mixed solvent of chloroform and methanol at a volume ratio of 3:1 at a mass ratio of 1:1.5:20:

5. A uniform lipid film was formed by rotary evaporation at 35°C water bath. PBS with pH 7.4 was added according to the total mass of the lipids to PBS volume ratio of 1:10g / mL, and the mixture was hydrated at 37°C for 30min. The Icaritin-naringenin liposomes were obtained by ultrasonic treatment and centrifugation; S3. First, the grafted chitosan was dissolved in 1% dilute acetic acid solution to prepare a 3% mass fraction grafted chitosan solution. The methacrylated gelatin was dissolved in PBS with pH 7.4 to prepare a 12% mass fraction methacrylated gelatin solution. The grafted chitosan solution and the methacrylated gelatin solution were mixed at a volume ratio of 5.5:1, stirred at 25°C for 30-60min, and then 4-8% Icaritin-naringenin liposomes were added according to the total mass of the mixed solution. The mixture was stirred for another 20min, and then 0.8% photoinitiator was added according to the total mass of the system. The mixture was ultrasonically degassed and subjected to UV curing to obtain the injectable bone repair material.

2. The method of claim 1, wherein: The degree of deacetylation of the chitosan in step S1 is ≥90%, and the molecular weight is 50-100kDa.

3. The method for preparing the injectable bone repair material according to claim 2, characterized in that: In the preparation of the grafted chitosan solution in step S3, the solution was stirred at a speed of 200-300rpm under water bath conditions at 30-35°C for 1.5-2h, and then naturally cooled to 25°C. The solution was filtered through a 0.22μm microporous filter to obtain the grafted chitosan solution.

4. The method for preparing the injectable bone repair material according to claim 3, characterized in that: The degree of substitution of the methacrylated gelatin in step S3 is 60%-70%, and the molecular weight is 120-180kDa.

5. The method for preparing the injectable bone repair material according to claim 4, characterized in that: In the preparation of the methacrylated gelatin solution in step S3, the solution was stirred at a speed of 400-500rpm under water bath conditions at 50-60°C for 1.5-2h, and then naturally cooled to 25°C to obtain the methacrylated gelatin solution.

6. The method for preparing the injectable bone repair material according to claim 5, characterized in that: The photoinitiator is selected from one of Irgacure 2959 and Irgacure 1173.

7. The method for preparing the injectable bone repair material according to claim 6, characterized in that: The ultrasonic degassing power in step S3 is 200-300W, and the ultrasonic treatment is carried out at 25°C for 5-8min.

8. The method for preparing the injectable bone repair material according to claim 7, characterized in that: The UV curing process in step S3 is carried out under nitrogen atmosphere, the distance between the light source and the surface of the system is controlled at 5-8 cm, and the system is cured under the irradiation of UV light with wavelength of 365 nm and light intensity of 15 mW / cm 2 for 25 s.

9. An injectable bone repair material, characterized by: Prepared by the method of any one of claims 1-8.

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