In situ injectable bone repair adhesive material with dual sequential osteogenesis function, preparation method and application thereof

Abstract

The present invention discloses an in situ injectable bone repair adhesive material with dual sequential osteogenic function, its preparation method and application. The preparation method comprises: 1) sequentially using methacrylic anhydride and dopamine hydrochloride to perform dual chemical grafting modification on hyaluronic acid to obtain DA-HAMA biomacromolecules with UV curing and adhesion functions; 2) using a microfluidic system to prepare Ca-HAMA loaded with osteogenic drugs. 2+ Physically cross-linked sodium alginate microspheres; 3) Prepare a DA-HAMA solution in the dark and under heating conditions. Once cooled to room temperature, add the angiogenic factors and drug-loaded microspheres and mix thoroughly. After centrifugation and sterilization by irradiation, an in situ injectable bone repair adhesive material is obtained. This bone repair adhesive material combines biodegradability, injectability, wet adhesion, and the ability to promote angiogenesis and osteogenesis, providing a novel strategy for bone defect repair.

Description

Technical Field

[0001] The present invention relates to a preparation technology of bone repair materials, and in particular to an in-situ injectable bone repair adhesive material with dual sequential osteogenesis function, a preparation method thereof, and applications thereof. Background Art

[0002] The destruction of the integrity of the bone structure is called a bone defect, which is usually caused by factors such as traffic accidents and surgery. The materials currently used in clinical practice, such as autologous bone and titanium mesh, have problems such as the need for secondary surgery and poor biological activity. In recent years, tissue engineering technology has developed rapidly. With the advantages of being degradable and biocompatible, hydrogels and scaffold materials have been used in the treatment of bone defects. However, ordinary tissue engineering materials are difficult to match the complex and changeable shapes of bone defects, the materials do not have adhesion properties, resulting in loose fit with the defect area and easy displacement, and the timing of the material's effect on bone repair is uncontrollable. These problems make it difficult for most existing tissue engineering materials to fully meet the complex needs of bone defect repair.

[0003] Further analysis of these issues revealed that injectability can address the complex shape of bone defects; adhesion allows for relative fixation between the material and the defect. Furthermore, considering the presence of abundant tissue fluid in bone defects, wet adhesion is necessary. Natural bone repair primarily involves forming a vascular network to meet blood supply needs in the early stages, while new bone formation occurs in the later stages. Therefore, developing a novel tissue-engineered material that meets all of these requirements would significantly advance bone defect repair.

[0004] Based on this, the present invention designs an in situ injectable bone repair adhesive material with dual sequential osteogenesis, as well as its preparation method and application. By using hyaluronic acid as the primary raw material and subjecting it to dual chemical grafting modification to impart UV curing and wet adhesion properties, and simultaneously designing methods for direct loading of angiogenic drugs and physically cross-linking sodium alginate to load osteogenic drugs, dual sequential release is achieved, providing a novel therapeutic strategy for bone defect treatment. Summary of the Invention

[0005] The present invention aims to address the ineffectiveness and inconvenient application of traditional bone repair materials by providing an in situ injectable bone repair adhesive material with dual sequential osteogenesis, as well as its preparation method and application. This bone repair adhesive material is biodegradable, injectable, adheres to the skin in wet conditions, and promotes vascularization and osteogenesis, offering a novel strategy for bone defect repair.

[0006] The present invention is implemented by the following technical solution, and the specific steps include:

[0007] A method for preparing an in situ injectable bone repair adhesive material with dual sequential osteogenesis function comprises the following steps:

[0008] 1) Hyaluronic acid was double-grafted with methacrylic anhydride and dopamine hydrochloride to obtain DA-HAMA biomacromolecules with UV curing and adhesion functions;

[0009] 2) Preparation of Ca using microfluidic system 2+ Physically cross-linking sodium alginate microspheres, placing them in an osteogenic drug solution for immersion and adsorption to obtain drug-loaded microsphere materials;

[0010] 3) preparing a DA-HAMA solution, adding the angiogenic factor and the drug-loaded microspheres to the solution when the solution is cooled to room temperature, and mixing them evenly. After centrifugal degassing and irradiation sterilization, an in situ injectable bone repair adhesive material is obtained.

[0011] Furthermore, in step 1), the double chemical grafting modification of hyaluronic acid using methacrylic anhydride and dopamine hydrochloride in sequence is specifically performed by first modifying the hyaluronic acid with methacrylic anhydride to obtain HAMA, and then modifying the HAMA with dopamine hydrochloride.

[0012] Furthermore, in the process of modifying hyaluronic acid with methacrylic anhydride, the hyaluronic acid is heated and dissolved in a PBS solution, methacrylic anhydride is slowly added dropwise, and after reacting for 2-3 hours, an equal volume of deionized water is added, and then hydrogen peroxide is slowly added dropwise to the solution. After stirring for 30-60 minutes, it is dialyzed in the dark, and lyophilized to obtain HAMA; wherein the reaction temperature is 50-60°C, the stirring rate is 200-400 r / min, the hyaluronic acid has a molecular weight greater than 100,000 and a concentration of 3-8wt%, the methacrylic anhydride is added dropwise to a concentration of 2-6wt%, and the amount of hydrogen peroxide added dropwise is 1 / 10-1 / 5 of the total volume of the solution. The deionized water is dialyzed for 5-10 days, and the solution is changed 3-5 times a day.

[0013] Furthermore, in the process of modifying HAMA with dopamine hydrochloride, the HAMA and dopamine hydrochloride are dissolved in a DMSO / deionized water mixed solvent, EDC is added under nitrogen protection and stirred for activation for 30-60 minutes, and then NHS is added for coupling reaction. The obtained product is dialyzed in the dark and freeze-dried to obtain the DA-HAMA biomacromolecule; the concentration of dopamine hydrochloride is 0.2-0.8wt%; the concentration of HAMA is 5-10wt%; the ratio of the mixed solvent is 10wt% DMSO / 90wt% deionized water; the concentration of EDC is 0.02-0.1wt%; the concentration of NHS is 0.02-0.1wt%; the coupling reaction time is 12-24 hours; the stirring is performed by magnetic stirring at 400-600 rpm; after the coupling reaction, the product is dialyzed using deionized water in the dark for 5-7 days, and the liquid is changed 3-5 times a day.

[0014] Furthermore, in step 2), the microfluidic system prepares Ca 2+ During the physical cross-linking of sodium alginate microspheres, the microfluidic system channel diameter is 50-200 μm; the coagulation bath is 0.04-0.1 g / ml calcium chloride, and the solvent system volume ratio is 1:3-3:1 deionized water and anhydrous ethanol; the sodium alginate viscosity is 15-25 cP (1 wt% in H2O) and the concentration is 0.02-0.04 g / ml; the microfluidic device extrusion rate is 0.1-1.0 mL / h, the solution stirring rate is 20-40 r / min, the microsphere aging time is 5-15 minutes, the temperature is 20-25°C, and the pH is 6.5-7.5. After completion, the obtained microspheres are washed 3-5 times with deionized water, each time for 3-5 minutes;

[0015] During the immersion adsorption process, the osteogenic drug is one or more of bone morphogenetic protein-2 (BMP-2), osteogenic growth peptide (OGP), transforming growth factor β (TGF-β), danshensu, icariin, total flavonoids from Rhizoma Drynariae, paeoniflorin and total saponins from Panax notoginseng, with a concentration of 100 ng / ml-0.1 g / ml. The immersion adsorption time is 5-15 minutes. After rinsing with deionized water, the drug is air-dried and stored for later use.

[0016] Furthermore, in step 3), during the preparation of the DA-HAMA solution, the DA-HAMA solution contains a photoinitiator, and under dark and heating conditions, the DA-HAMA preparation temperature is 60-80° C., the concentration is 5-15 wt %, the photoinitiator is one or more of LAP, Irgacure, and photoinitiator 2959, the concentration is 0.25-0.5 wt %, and the solvent is PBS;

[0017] The angiogenic drug is one or more of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), QK peptide, deferoxamine, salvianolic acid B, astragaloside, ginsenoside, and paeoniflorin, with a concentration of 100 ng / ml-0.1 g / ml;

[0018] The concentration of the drug-loaded microspheres is 0.5-5wt%;

[0019] During the centrifugal degassing process, the rotation speed is 500-1000 rpm for 3-10 minutes.

[0020] An in-situ injectable bone repair adhesive material with dual sequential osteogenesis function is obtained by using any of the above preparation methods.

[0021] A bone repair material comprises the bone repair adhesive material.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] 1) The present invention uses HAMA, sodium alginate, dopamine hydrochloride, methacrylic anhydride, osteogenic drugs, and angiogenic drugs as raw materials, and combines dual chemical grafting modification, blending, microfluidics technology, injectability, and in-situ UV curing to produce an in-situ injectable bone repair adhesive material with dual sequential osteogenic function. The above raw material selection and process combination are original to the present invention.

[0024] 2) In the present invention, a dual chemical grafting modification technique is sequentially used to modify hyaluronic acid to obtain DA-HAMA biomacromolecules, which can be used to prepare a bone repair gel pre-solution with injectable, in-situ UV curing and wet adhesion functions. First, hyaluronic acid, the main component of the extracellular matrix, is selected as the macromolecular backbone. It is widely distributed in the human body and participates in various physiological processes such as tissue hydration, wound repair, and cell migration, and is of great significance for tissue repair. Secondly, methacrylic anhydride is used for the first grafting modification to impart UV curing ability to hyaluronic acid. Combined with the high solubility of the hyaluronic acid backbone in water, the injectable performance of DA-HAMA can be guaranteed. Then, dopamine hydrochloride is used through an EDC coupling reaction to achieve a reaction between dopamine hydrochloride and the carboxyl groups on the HAMA backbone, further endowing the DA-HAMA molecule with wet adhesion properties.

[0025] 3) The present invention successfully prepared Ca-containing osteogenic drug-loaded Ca-containing ... 2+ Physically cross-linked sodium alginate microspheres provide the possibility for the second stage of double-order osteogenesis. 2+ Physical cross-linking molding, and Ca 2+Cross-linking is very easy to occur in solutions rich in phosphate, hydrogen phosphate, dihydrogen phosphate and other ions such as PBS, SBF, and human body fluids. 2+ Cross-linking to Ca 2+ The transfer of cross-linking with the above-mentioned phosphate, hydrogen phosphate, dihydrogen phosphate and other ions causes the originally formed microspheres to gradually dissolve, and because the concentrations of phosphate, hydrogen phosphate, dihydrogen phosphate and other ions in PBS, SBF, human body fluids, etc. are not particularly high, the dissolution rate of the microspheres is relatively slow. Therefore, it is equivalent to the applicant preparing a long-term self-dissolving drug carrier, and the osteogenic drugs inside can be released for a long time as the microspheres dissolve, achieving a promoting effect on bone defect repair. Moreover, the dissolution of sodium alginate microspheres provides physical space for cell growth, further providing support for new bone formation. The above-mentioned Ca 2+ Although physically cross-linked sodium alginate microsphere materials have been widely used, the present invention is the first to utilize this property and organically cooperate with other materials with different drug release rates to achieve the purpose of efficient osteogenesis.

[0026] 4) The bone repair adhesive material of the present invention has the functions of injectability, in-situ UV curing, wet adhesion, and dual-sequence osteogenesis, which can meet the complex requirements of bone defect repair. Among them, since the hyaluronic acid main chain has abundant carboxyl groups, it still maintains good water solubility even after being modified with methacrylic anhydride and dopamine hydrochloride, and will not physically solidify at room temperature after being dissolved into a solution, so it has good injectability and can well match bone defects of various shapes to obtain excellent filling effects; secondly, due to the modification of methacrylic anhydride, a photocurable group is introduced, and when injected into the bone defect area, in-situ UV curing can be achieved; and the modification of dopamine hydrochloride introduces a catechol group, which can form good wet adhesion with the tissue, keeping the solution from moving under the influence of surgery or exercise after solidification. , always maintaining a good fit with the bone defect area. Otherwise, once the position shifts, it is very easy to cause heterotopic ossification in other areas, and the bone defect area cannot be well treated. Finally, through the slow release of the angiogenic drug in the adhesive matrix, a vascularized network is formed to meet the nutritional and vascular needs of the early stage of bone repair. The osteogenic drug in the sodium alginate microspheres will be slowly released as the calcium ions in the microspheres are chelated and transferred by other phosphate ions. Due to the physical barrier between the microspheres and the adhesive body, the release time of the osteogenic drug will be later than that of the angiogenic drug, which is consistent with the timing of natural bone repair: angiogenesis first and then osteogenesis, and angiogenesis synergistically promotes osteogenesis. It is precisely because of the above-mentioned functional synergy of the bone repair composite adhesive that the ideal effect of bone defect treatment is achieved.

[0027] 5) The bone repair adhesive material of the present invention is easy to use and operate. It only requires the preparation of the required gel pre-solution and the use of a sterile injection device for in situ injection and curing. It is suitable for small bone defects of any shape and has obvious universality. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The figure is a schematic diagram of the preparation process of the in situ injectable bone repair adhesive material with dual sequential osteogenesis function of the present invention. DETAILED DESCRIPTION

[0029] The present invention is further described below with reference to specific examples.

[0030] Comparative Example 1:

[0031] 1) Hyaluronic acid was modified with methacrylic anhydride to obtain a HAMA molecule. The specific process was as follows: Hyaluronic acid with a molecular weight greater than 100,000 (concentration of 5 wt%) was dissolved in a PBS solution at 60°C under magnetic stirring at 200 r / min. Methacrylic anhydride was slowly added dropwise until the concentration in the solution reached 2.5 wt%. After reacting for 3 hours, an equal volume of deionized water was added. 30% hydrogen peroxide was slowly added dropwise, accounting for 1 / 10 of the total volume of the solution. After stirring for 60 minutes, the solution was dialyzed in the dark (dialysis with deionized water for 10 days, with the solution changed four times daily). The desired HAMA was obtained after lyophilization.

[0032] 2) Prepare a 7.5 wt% HAMA solution (0.25 wt% LAP, solvent: PBS) in a dark place at 60°C. After cooling to room temperature, add 300 ng / ml VEGF and mix thoroughly to obtain a mixed solution.

[0033] 4) After degassing by low-speed centrifugation (500 rpm, 3 min), the irradiated and sterilized mixed solution, i.e., the adhesive, was injected into the bone defect using a sterile injection device. In situ UV curing (UV curing wavelength: 405 nm, power: 20 W, time: 1 min) was performed using a handheld UV pen to achieve adhesion to the defect area and achieve bone repair.

[0034] An extract of the above material was prepared and co-cultured with bone marrow mesenchymal stem cells (BMSCs) for 7 days. The CCK-8 results showed that the cell survival rate was about 89.5%; it was injected into a skull defect model, and initially adhered well to the defect area. After surgical suture and feeding, sampling 4 weeks later found that about 50% of the gel had shifted, the bone repair effect was general, and the shifted gel led to heterotopic ossification.

[0035] Example 1:

[0036] 1) Hyaluronic acid is double-grafted using methacrylic anhydride and dopamine hydrochloride in sequence to obtain a DA-HAMA biomacromolecule with UV curing and adhesion functions.

[0037] ① The process for modifying hyaluronic acid with methacrylic anhydride is as follows: under magnetic stirring at 200 r / min, hyaluronic acid with a molecular weight greater than 100,000 (concentration of 5wt%) is heated at 60°C and dissolved in a PBS solution. Methacrylic anhydride is slowly added dropwise until the concentration in the solution reaches 2.5wt%. After reacting for 3 hours, an equal volume of deionized water is added. 30% concentration hydrogen peroxide, which is 1 / 10 of the total volume of the solution, is slowly added dropwise. After stirring for 60 minutes, dialyze in the dark (dialysis with deionized water for 10 days, with the solution changed 4 times a day). The desired HAMA is obtained after freeze-drying.

[0038] ② The process for modifying HAMA with dopamine hydrochloride is as follows: 5wt% HAMA and 0.4wt% dopamine hydrochloride are dissolved in a 10wt% DMSO / 90wt% deionized water mixed solvent, 0.02wt% EDC is added for stirring and activation for 30 minutes under nitrogen protection and magnetic stirring at 400r / min, and then 0.03wt% NHS is added for coupling reaction for 24 hours. The obtained product is dialyzed in the dark (5 days, with the liquid changed 3 times a day) and freeze-dried to obtain the desired DA-HAMA.

[0039] 2) Preparation of Ca using microfluidic system 2+ Physically cross-linked sodium alginate microspheres (microfluidic system channel diameter 50 μm; coagulation bath is 0.1 g / ml calcium chloride, solvent system volume ratio is 1:1 deionized water and anhydrous ethanol; microsphere preparation process parameters: sodium alginate viscosity 15-25 cP (1wt% in H2O), concentration 0.03 g / ml, microfluidic device extrusion rate 0.3 mL / h, solution stirring rate 40 r / min, microsphere aging time 5 min, temperature 25 ° C, pH 7.2, after the end of the microspheres with deionized water washing 5 times, each time time 3 min) were placed in 300 ng / ml BMP-2 solution for 10 min for immersion adsorption to obtain drug-loaded microsphere material, rinsed with deionized water, dried, and stored at 4 ° C for use;

[0040] 3) Prepare a 7.5 wt% DA-HAMA solution (0.25 wt% LAP in PBS) in the dark and at 60°C. After cooling to room temperature, add 300 ng / ml VEGF and 0.5 wt% drug-loaded microspheres and mix thoroughly.

[0041] 4) After degassing by low-speed centrifugation (500 rpm, 3 min), the irradiated and sterilized mixed solution, i.e., the in situ injectable bone repair adhesive, was injected into the bone defect area using a sterile injection device. A handheld UV pen was used for in situ UV curing (UV curing wavelength: 405 nm, power: 20 W, time: 1 min) to achieve adhesion to the defect area and achieve bone repair.

[0042] Compared with Comparative Example 1, this embodiment adds a second modification of the HAMA molecule and a drug-loaded physically cross-linked sodium alginate microsphere material.

[0043] The extract of the above material was prepared and co-cultured with BMSCs for 7 days. The results of CCK-8 showed that the cell survival rate was about 92.6% and the adhesion strength was about 32.4kPa. It was injected into the skull defect model and adhered well to the defect area. After surgical suture and feeding, sampling 4 weeks later found that the defect area was obviously vascularized and the bone repair effect was good.

[0044] Example 2:

[0045] 1) Hyaluronic acid is double-grafted using methacrylic anhydride and dopamine hydrochloride in sequence to obtain a DA-HAMA biomacromolecule with UV curing and adhesion functions.

[0046] ① The process for modifying hyaluronic acid with methacrylic anhydride is as follows: under magnetic stirring at 200 r / min, hyaluronic acid with a molecular weight greater than 100,000 (concentration of 5wt%) is heated at 60°C and dissolved in a PBS solution. Methacrylic anhydride is slowly added dropwise until the concentration in the solution reaches 2.5wt%. After reacting for 3 hours, an equal volume of deionized water is added. 30% concentration hydrogen peroxide, which is 1 / 10 of the total volume of the solution, is slowly added dropwise. After stirring for 60 minutes, dialyze in the dark (dialysis with deionized water for 10 days, with the solution changed 4 times a day). The desired HAMA is obtained after freeze-drying.

[0047] ② The process for modifying HAMA with dopamine hydrochloride is as follows: 5wt% HAMA and 0.4wt% dopamine hydrochloride are dissolved in a 10wt% DMSO / 90wt% deionized water mixed solvent, 0.02wt% EDC is added for stirring and activation for 30 minutes under nitrogen protection and magnetic stirring at 400r / min, and then 0.03wt% NHS is added for coupling reaction for 24 hours. The obtained product is dialyzed in the dark (5 days, with the liquid changed 3 times a day) and freeze-dried to obtain the desired DA-HAMA.

[0048] 2) Preparation of Ca using microfluidic system 2+Physically cross-linked sodium alginate microspheres (microfluidic system channel diameter 50 μm; coagulation bath is 0.1 g / ml calcium chloride, solvent system volume ratio is 1:1 deionized water and anhydrous ethanol; microsphere preparation process parameters: sodium alginate viscosity 15-25 cP (1wt% in H2O), concentration 0.03 g / ml, microfluidic device extrusion rate 0.3 mL / h, solution stirring rate 40 r / min, microsphere aging time 5 min, temperature 25 ° C, pH 7.2, after the end of the microspheres with deionized water washing 5 times, each time time 3 min) were placed in 300 ng / ml BMP-2 solution for 10 min for immersion adsorption to obtain drug-loaded microsphere material, rinsed with deionized water, dried, and stored at 4 ° C for use;

[0049] 3) Prepare a 7.5 wt% DA-HAMA solution (0.25 wt% LAP in PBS) in the dark and at 60°C. After cooling to room temperature, add 300 ng / ml VEGF and 5 wt% drug-loaded microspheres and mix thoroughly.

[0050] 4) After degassing by low-speed centrifugation (500 rpm, 3 min), the irradiated and sterilized mixed solution, i.e., the in situ injectable bone repair adhesive, was injected into the bone defect area using a sterile injection device. A handheld UV pen was used for in situ UV curing (UV curing wavelength: 405 nm, power: 20 W, time: 1 min) to achieve adhesion to the defect area and achieve bone repair.

[0051] Compared to Example 1, this example increased the amount of drug-loaded sodium alginate microspheres. An extract of the above material was prepared and co-cultured with BMSCs for 7 days. CCK-8 assay results showed a cell survival rate of approximately 91.5% and an adhesion strength of approximately 28.2 kPa. Injection into a skull defect model revealed good adhesion to the defect area. Following surgical suturing and subsequent feeding, sampling 4 weeks later revealed significant vascularization in the defect area and superior bone repair efficacy compared to Example 1.

[0052] Example 3:

[0053] 1) Hyaluronic acid is double-grafted using methacrylic anhydride and dopamine hydrochloride in sequence to obtain a DA-HAMA biomacromolecule with UV curing and adhesion functions.

[0054] ① The process for modifying hyaluronic acid with methacrylic anhydride is as follows: under magnetic stirring at 200 r / min, hyaluronic acid with a molecular weight greater than 100,000 (concentration of 5wt%) is heated at 60°C and dissolved in a PBS solution. Methacrylic anhydride is slowly added dropwise until the concentration in the solution reaches 2.5wt%. After reacting for 3 hours, an equal volume of deionized water is added. 30% concentration hydrogen peroxide, which is 1 / 10 of the total volume of the solution, is slowly added dropwise. After stirring for 60 minutes, dialyze in the dark (dialysis with deionized water for 10 days, with the solution changed 4 times a day). The desired HAMA is obtained after freeze-drying.

[0055] ② The process for modifying HAMA with dopamine hydrochloride is as follows: 5wt% HAMA and 0.4wt% dopamine hydrochloride are dissolved in a 10wt% DMSO / 90wt% deionized water mixed solvent, 0.02wt% EDC is added for stirring and activation for 30 minutes under nitrogen protection and magnetic stirring at 400r / min, and then 0.03wt% NHS is added for coupling reaction for 24 hours. The obtained product is dialyzed in the dark (5 days, with the liquid changed 3 times a day) and freeze-dried to obtain the desired DA-HAMA.

[0056] 2) Preparation of Ca using microfluidic system 2+ Physically cross-linked sodium alginate microspheres (microfluidic system channel diameter 50 μm; coagulation bath is 0.1 g / ml calcium chloride, solvent system volume ratio is 1:1 deionized water and anhydrous ethanol; microsphere preparation process parameters: sodium alginate viscosity 15-25 cP (1wt% in H2O), concentration 0.03 g / ml, microfluidic device extrusion rate 0.3 mL / h, solution stirring rate 40 r / min, microsphere aging time 5 min, temperature 25 ° C, pH 7.2, after the end of the microspheres with deionized water washing 5 times, each time time 3 min) were placed in 300 ng / ml BMP-2 solution for 10 min for immersion adsorption to obtain drug-loaded microsphere material, rinsed with deionized water, dried, and stored at 4 ° C for use;

[0057] 3) Prepare a 7.5 wt% DA-HAMA solution (0.25 wt% LAP in PBS) in a dark place and heat at 60°C. After cooling to room temperature, add 0.5 wt% drug-loaded microspheres and mix thoroughly.

[0058] 4) After degassing by low-speed centrifugation (500 rpm, 3 min), the irradiated and sterilized mixed solution, i.e., the adhesive, was injected into the bone defect using a sterile injection device. In situ UV curing (UV curing wavelength: 405 nm, power: 20 W, time: 1 min) was performed using a handheld UV pen to achieve adhesion to the defect area and achieve bone repair.

[0059] Compared to Example 1, this example reduces the use of VEGF. An extract of the above-mentioned material was prepared and co-cultured with BMSCs for 7 days. CCK-8 assay results showed a cell survival rate of approximately 88.7% and an adhesion strength of approximately 31.1 kPa. This extract was injected into a skull defect model, where it adhered well to the defect area. Following surgical suturing and culture, sampling 4 weeks later revealed moderate vascularization and bone repair in the defect area, with new bone formation even worse than in Example 1.

Claims

1. A method for preparing an in situ injectable bone repair adhesive material with dual sequential osteogenesis function, characterized in that: The following steps are involved: 1) Hyaluronic acid was double-grafted with methacrylic anhydride and dopamine hydrochloride to obtain DA-HAMA biomacromolecules with UV curing and adhesion functions; 2) Preparation of Ca using microfluidic system 2+ Physically cross-linking sodium alginate microspheres, placing them in an osteogenic drug solution for immersion and adsorption to obtain drug-loaded microsphere materials; 3) preparing a DA-HAMA solution, adding the angiogenic factor and the drug-loaded microspheres to the solution when the solution is cooled to room temperature, and mixing them evenly. After centrifugation for degassing and irradiation sterilization, an in situ injectable bone repair adhesive material is obtained; In step 3), during the preparation of the DA-HAMA solution, the DA-HAMA solution contains a photoinitiator. Under dark and heating conditions, the DA-HAMA preparation temperature is 60-80° C., the concentration is 5-15 wt %, the photoinitiator is one or more of LAP, Irgacure, and photoinitiator 2959, the concentration is 0.25-0.5 wt %, and the solvent is PBS; The angiogenic drug is one or more of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), QK peptide, deferoxamine, salvianolic acid B, astragaloside, ginsenoside, and paeoniflorin, with a concentration of 100 ng / ml-0.1 g / ml; The concentration of the drug-loaded microspheres is 0.5-5wt%; During the centrifugal degassing process, the rotation speed is 500-1000 rpm for 3-10 minutes.

2. The method for preparing the in situ injectable bone repair adhesive material with dual sequential osteogenesis function according to claim 1, characterized in that: In step 1), the double chemical grafting modification of hyaluronic acid using methacrylic anhydride and dopamine hydrochloride in sequence is specifically performed by first modifying the hyaluronic acid with methacrylic anhydride to obtain HAMA, and then modifying the HAMA with dopamine hydrochloride.

3. The method for preparing the in situ injectable bone repair adhesive material with dual sequential osteogenesis function according to claim 2, characterized in that: In the process of modifying hyaluronic acid with methacrylic anhydride, hyaluronic acid is heated and dissolved in a PBS solution, methacrylic anhydride is slowly added dropwise, and after reacting for 2-3 hours, an equal volume of deionized water is added, and then hydrogen peroxide is slowly added dropwise to the solution. After stirring for 30-60 minutes, it is dialyzed in the dark, and lyophilized to obtain HAMA. The reaction temperature is 50-60°C, the stirring rate is 200-400 r / min, the hyaluronic acid has a molecular weight greater than 100,000 and a concentration of 3-8wt%, the methacrylic anhydride is added dropwise to a concentration of 2-6wt%, and the amount of hydrogen peroxide added dropwise is 1 / 10-1 / 5 of the total volume of the solution. The deionized water is dialyzed for 5-10 days, and the solution is changed 3-5 times a day.

4. The method for preparing the in situ injectable bone repair adhesive material with dual sequential osteogenesis function according to claim 2, characterized in that: In the process of modifying HAMA with dopamine hydrochloride, the HAMA and dopamine hydrochloride are dissolved in a DMSO / deionized water mixed solvent, EDC is added under nitrogen protection and stirred for activation for 30-60 minutes, and then NHS is added for coupling reaction. The obtained product is dialyzed in the dark and freeze-dried to obtain a dopamine-modified methacryloyl hyaluronic acid (DA-HAMA) biomacromolecule; the concentration of dopamine hydrochloride is 0.2-0.8wt%; the concentration of HAMA is 5-10wt%; the mixed solvent is stirred for 30-60 minutes, and the mixture is stirred for 30-60 minutes. The ratio of the reagent is 10wt% dimethyl sulfoxide (DMSO) / 90wt% deionized water; the concentration of the 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) is 0.02-0.1wt%; the concentration of the N-hydroxysuccinimide (NHS) is 0.02-0.1wt%; the coupling reaction time is 12-24 hours; the stirring is magnetic stirring at 400-600 rpm; after the coupling reaction is completed, the product is dialyzed with deionized water in the dark for 5-7 days, and the solution is changed 3-5 times a day.

5. The method for preparing the in situ injectable bone repair adhesive material with dual sequential osteogenesis function according to claim 1, characterized in that: In step 2), the microfluidic system prepares Ca 2+ In the process of physically cross-linking sodium alginate microspheres, the microfluidic system channel diameter is 50-200 μm; the coagulation bath is 0.04-0.1 g / ml calcium chloride, and the solvent system volume ratio is 1:3-3:1 deionized water and anhydrous ethanol; the sodium alginate has a viscosity of 15-25 cP and a concentration of 0.02-0.04 g / ml in a 1 wt% aqueous solution; the microfluidic device extrusion rate is 0.1-1.0 mL / h, the solution stirring rate is 20-40 r / min, the microsphere aging time is 5-15 minutes, the temperature is 20-25°C, and the pH is 6.5-7.

5. After completion, the obtained microspheres are washed 3-5 times with deionized water, each time lasting 3-5 minutes; During the immersion adsorption process, the osteogenic drug is one or more of bone morphogenetic protein-2 (BMP-2), osteogenic growth peptide (OGP), transforming growth factor β (TGF-β), danshensu, icariin, total flavonoids from Rhizoma Drynariae, paeoniflorin and total saponins from Panax notoginseng, with a concentration of 100 ng / ml-0.1 g / ml. The immersion adsorption time is 5-15 minutes. After rinsing with deionized water, the drug is air-dried and stored for later use.

6. An in situ injectable bone repair adhesive material with dual sequential osteogenesis function, characterized in that: The method is described in any one of claims 1 to 5.

7. A bone repair material, characterized in that: Comprising the bone repair adhesive material as claimed in claim 6.

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