An injectable double-bionic bone adhesive for promoting fracture healing and its preparation method

Abstract

The present invention discloses an injectable dual - biomimetic bone adhesive for promoting fracture healing and its preparation method, belonging to the field of biomaterial preparation. Inspired by the underwater adhesion mechanisms of marine mussels and barnacles, it uses biomimetic marine mussel substances (such as tannic acid), biomimetic marine barnacle substances (such as sodium alginate), silk fibroin, calcium chloride, and magnesium particles to form a dual - biomimetic bone adhesive with wet adhesion. Based on the hydrogen bonds, metal - phenol coordination bonds, electrostatic interactions, and β β - sheet protein folding conformations formed among the components, the obtained dual - biomimetic bone adhesive not only has good biocompatibility, excellent wet adhesion, and mechanical properties matching bone tissue strength, can be used for fracture bone fixation and repair, but also has the ability of in - situ gelation and can be directly injected at the injury site. The raw materials used in the adhesive prepared by the present invention are safe and widely sourced. Therefore, it is expected to achieve potential clinical application value in the fixation and repair of bone injuries.

Description

Technical Field

[0001] The present invention belongs to the field of biocompatible material preparation, and particularly relates to an injectable double-bionic bone adhesive for promoting fracture healing and a preparation method thereof. Background Art

[0002] All along, fracture problems caused by traffic accidents, accidental injuries, sports injuries, osteoporosis, etc. have attracted much attention. For the treatment strategy of fractures, currently, the bone structure is mainly restored by fixing methods such as embedding metal plates, nails, screws, etc. in the body to promote the self-regeneration and repair of body tissues. However, these non-degradable devices or materials need to be removed by a second operation after bone repair is completed. Moreover, due to stress shielding, metal fixation devices may have high-risk problems such as infection, damage to surrounding tissues, and secondary fractures. In addition, metal implant materials will be corroded by the in-vivo physiological environment, thereby producing toxic and side effects, and the corroded metal implant materials will cause the degradation or failure of material functions, thus triggering adverse histological reactions such as edema, infection, tissue necrosis, etc. For example, the difference in elastic modulus between metal implant materials such as cobalt alloys and titanium alloys and bones will lead to local tension and bone atrophy at the fixation site. Therefore, there is an urgent need to develop new biomedical materials for clinical fracture surgery.

[0003] As a new type of biomedical material, bone adhesives can provide adhesion between bone and bone and / or bone and implant by forming a strong and durable bearing force in a wet environment, and will ultimately metabolize into non-toxic materials after new bone formation. Compared with traditional invasive fixation materials such as steel plates or screws, the prepared bone adhesives have been proven not to cause problems of secondary tissue damage and microbial infection. In the past few decades, researchers have sought inspiration from nature, including mimicking organisms or natural materials with strong adhesion in nature, to develop strong bioactive adhesives for bone tissues. An ideal bionic bone adhesive should meet the requirements of various parameters of the bone tissue environment, specifically including: maintaining strong adhesion in a wet environment, maintaining mechanical stability under physiological conditions, good biocompatibility, an elastic modulus similar to that of bone tissue, and biodegradability.

[0004] Patent CN 111973804 A discloses an injectable bone repair adhesive loaded with stem cells that highly mimics living active bone tissue. The adhesive is composed of a polymer material, type I collagen, micron-sized spherical hydroxyapatite, and dopamine-modified hyaluronic acid, and is loaded with stem cells. While improving tissue adhesion, this bone repair adhesive can promote the adhesion and proliferation of stem cells, and enhance the angiogenesis and osteogenesis of stem cells. However, the grafting rate of dopamine in this material is relatively low, the preparation process is complex, and the adhesion strength of this adhesive is poor, unable to be fixed at the bone injury site, thus affecting the bone repair efficiency. For another example, Patent CN 110947026 A discloses a bone adhesive bio-glue for accelerating fracture healing, which is composed of a natural biopolymer modified by o-nitrobenzyl, a photoinitiator, and nano-osteogenic particles. By activating the o-nitrobenzyl type trigger through light irradiation to generate aldehyde groups, chemical bonding occurs with the amino groups in the tissue, thereby bonding bone fragments to the surrounding bone tissue. Subsequently, the nano-osteogenic particles enter the molecular interstitial pores of the natural biopolymer modified by o-nitrobenzyl, accumulate and mineralize, accelerating new bone formation. However, this method of forming a gel and bonding by relying on an external light source device increases the complexity of the operation and the limitations of the use conditions, and is not conducive to application in emergency situations. For another example, Patent CN 102307941 A discloses a viscous colloidal substance formed by the reaction of tetracalcium phosphate and a substance effectively structurally similar to phosphoserine. This colloidal substance can bond bone defect sites and fix bones. Although this colloidal substance has strong adhesion in a dry environment and can effectively bond defective bones in in vitro experiments, in a wet environment, the adhesion performance of this colloidal substance decreases, and the degradation rate of this adhesive in animals within one year is only 70%, and its degradation rate does not match the osteogenesis rate, so the application of this adhesive in the body is severely limited.

[0005] The mussel adhesive protein (Mfp) secreted by marine mussels is rich in 3 , 4 -dihydroxyphenylalanine (DOPA) can expel water molecules and ionic salts between tissue interfaces and form strong covalent and non-covalent bonds, thereby exhibiting strong adhesion ability. Barnacles can adhere to interfaces by producing a specific mixture of adhesive proteins (CPs). This mixture of adhesive proteins is mainly composed of the protein CP19K, which contains a large number of cationic lysine and hydrophobic amino acids. It is speculated that when barnacles come into contact with the interface, under the synergistic action of adjacent hydrophobic amino acids, strong electrostatic interactions are formed between the cations and the negatively charged surface, resulting in strong wet adhesion. At the same time, the CP19K protein is rich in amyloid β -sheet, which is oriented perpendicular to the fiber axis and connected by a dense hydrogen bond network, and can continuously extend into a supermacromolecule of more than thousands of molecular units, increasing the underwater adhesion of barnacles to the interface.

[0006] Based on this, the present invention provides an injectable double - biomimetic bone adhesive for promoting fracture healing and its preparation method. The double - biomimetic bone adhesive mainly consists of biomimetic marine mussel substances such as tannic acid, biomimetic marine barnacle substances such as sodium alginate, and silk fibroin, and calcium ions (Ca 2+ ), and magnesium particles are introduced for self - assembly to stabilize the internal structure of the adhesive. When in use, after simple mixing, the double - biomimetic bone adhesive can be directly injected at the injury site and gelate in situ. Experimental results show that the double - biomimetic bone adhesive has rapid curing, good biocompatibility, strong wet - state tissue adhesion, and mechanical properties matching the strength of bone tissue; and based on the mechanism of hydrogen and magnesium ions (Mg 2+ ) generated by the reaction of magnesium particles with water, the adhesive also has the potential to reduce inflammation and regulate the differentiation of mesenchymal stem cells to promote bone repair, and can play a good fixing role and potential bone regeneration - promoting ability in the early repair of bone injuries. Summary of the Invention

[0007] The object of the present invention is to provide an injectable double - biomimetic bone adhesive for promoting fracture healing and its preparation method. Among them, the introduced biomimetic marine mussel substances such as tannic acid can form π - π stacking with catechol groups to induce the transformation of silk fibroin from a random coil conformation to a more stable β -sheet protein folding conformation; at the same time, biomimetic marine mussel substances such as tannic acid can also form metal - phenolic coordination bonds with Ca 2+ and Mg 2+ . The stable metal coordination bonds endow the adhesive with a dynamic network structure with good mechanical properties and mechanical strength, which can play a role in fixing the fracture site, protect the bone morphology and structure, thereby improving the implantation rate of allografts and promoting the repair efficiency of the injury site. And based on the mechanism of hydrogen and magnesium ions generated by the reaction of magnesium particles with water, the adhesive also has the function of reducing inflammation and regulating the differentiation of mesenchymal stem cells to promote angiogenesis. Therefore, the bone adhesive of the present invention exhibits the advantages of strong adhesion, strong plasticity, good biocompatibility, excellent mechanical properties, wide application range, safety, non - toxicity, and easy operation, and is expected to be a potential clinical material for stabilizing bones and repairing defect sites.

[0008] To achieve the above - mentioned invention object, the present invention adopts the following technical solutions:

[0009] An injectable double - biomimetic bone adhesive for promoting fracture healing, and its preparation method includes the following steps:

[0010] (1) adding anhydrous sodium carbonate to boiling deionized water, stirring and mixing, and then adding silk to degumming; after washing and drying, the product is dissolved in lithium bromide solution or calcium chloride / anhydrous ethanol / water ternary solution, and then dialyzed in deionized water; the dialyzed solution is centrifuged and filtered to obtain solution S1;

[0011] (2) adding the bionic marine barnacle biological material into deionized water, stirring and mixing at a certain temperature to obtain a solution S2;

[0012] (3) Add calcium chloride powder to deionized water, and stir to mix at room temperature to obtain solution S3;

[0013] (4) dissolving the bionic marine mussel biological material in deionized water, then adding magnesium powder thereto, stirring and mixing evenly to obtain a mixed solution S4 of bionic marine mussel biological material-magnesium particles;

[0014] (5) At room temperature, solutions S1, S2, and S3 are mixed and added into the mixed solution S4, and the mixture is stirred thoroughly to obtain the injectable dual-bionic bone adhesive material.

[0015] Furthermore, the degumming time in step (1) is 5-120 min.

[0016] Furthermore, the drying time in step (1) is 1-50 h and the temperature is 5-100 °C.

[0017] Furthermore, the dialysis time in step (1) is 36-100 h.

[0018] Furthermore, the mass concentration of the solution S1 obtained in step (1) is 0.1-50.0%, preferably 1-15.0%.

[0019] Furthermore, the bionic marine barnacle biological material in step (2) is one or more of sodium alginate, polyacrylic acid-hydroxysuccinimide ester, polyasparagine, Trx-Balcp19k fusion protein, thiolated hyaluronic acid and globulin, preferably sodium alginate.

[0020] Furthermore, in step (2), the stirring temperature is 10-80°C, preferably 15-80°C, and the stirring time is 5-72h, preferably 8-24h.

[0021] Furthermore, the mass concentration of the solution S2 obtained in step (2) is 0.1-80.0%, preferably 0.1-20.0%.

[0022] Furthermore, the stirring time in step (3) is 1-120 min, preferably 5-15 min.

[0023] Furthermore, the mass concentration of the solution S3 obtained in step (3) is 0.01 - 20.0%, preferably 0.1 - 10.0%.

[0024] Furthermore, the biomimetic marine mussel biomaterial in step (4) is one or more of phenolic hydroxyl chitosan, dopamine, tannic acid, gallic acid, and phenolic hydroxylated alanine, preferably tannic acid.

[0025] Furthermore, the mass concentration of the mixed solution S4 obtained in step (4) is 0.1 - 60.0%, preferably 1 - 50.0%, and the mass of the magnesium powder contained therein is 0.001 - 10.0%.

[0026] Furthermore, the volume ratio of the solution S1 to the solution S2 used in step (5) is 1:1000 - 1000:1, preferably 1:15 - 15:1; the volume ratio of the solution S1 to the solution S3 is 1:1000 - 1000:1, preferably 1:15 - 15:1; the volume ratio of the used mixed solution S4 to the mixed solution of the solutions S1, S2, and S3 is 1:1000 - 1000:1, preferably 1:20 - 20:1.

[0027] Furthermore, the stirring and mixing time in step (5) is 1 - 180 min, preferably 1 - 20 min, and the temperature is 5 - 50 °C, preferably 15 - 30 °C.

[0028] Compared with the prior art, inspired by the underwater adhesion mechanisms of marine mussels and barnacles, the present invention introduces biomimetic marine mussel biomaterials such as tannic acid and biomimetic marine barnacle biomaterials such as sodium alginate to form metal - phenolic coordination bonds with Ca 2+ and Mg 2+ to prepare an adhesive with strong mechanical properties and a dynamic network structure. Among them, the carboxyl groups on the side chains of the biomimetic marine barnacle biomaterials can absorb the moisture in the interface to break the combination of the interface and the water layer, improving the binding strength between the dynamic network and the interface, thereby enhancing the adhesion performance of the adhesive. At the same time, through π - π stacking interactions, the biomimetic marine mussel biomaterials can induce the silk fibroin in the adhesive to transform from a random coil conformation to a more stable β -sheet protein folding conformation to improve the cohesion of the dynamic network, and further enhance the adhesion strength of the adhesive. In addition, as an essential component in the bone healing process, Mg 2+ has the functions of anti - inflammation and regulating the differentiation of mesenchymal stem cells to promote angiogenesis, and can promote the repair of bone injuries. Therefore, the dual - biomimetic bone adhesive designed and prepared by the present invention has the advantages of strong wet adhesion, good toughness, wide application range, safety and non - toxicity, and good biocompatibility, and is expected to be applied to the fixation and repair of in - vivo bone injuries in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Scanning electron microscope image of the double bionic bone adhesive prepared in Example 2.

[0030] Figure 2 Fourier transform infrared spectroscopy comparison chart of the double bionic bone adhesives prepared in Example 2 and Comparative Example 1.

[0031] Figure 3 Mechanical property comparison chart of the double bionic bone adhesives prepared in Example 2 and Comparative Example 1, where A. Tensile stress-strain curve diagram, B. Tensile strength, C. Tensile Young's modulus, D. Compressive stress-strain curve diagram, E. Compressive strength. F. Compressive Young's modulus.

[0032] Figure 4 Adhesion property diagram of the double bionic bone adhesive prepared in Example 2, where A. Adhesion behavior on various substrate surfaces. B. Peeling process on the substrate. C. Ability of underwater adhesion. D. Stress-strain of the lap shear test of the adhesion to porcine skin tissue. E. Average lap shear strength of porcine skin tissue.

[0033] Figure 5 Application situation diagram of the double bionic bone adhesive prepared in Example 2 in the 3D printed model of the simulated fractured human femur.

[0034] Figure 6 CCK-8 result diagrams of the double bionic bone adhesives prepared in Example 2 and Comparative Example 1 co-cultured with NIH / 3T3 cells for 1, 3, and 7 days.

[0035] Figure 7 Live / dead staining diagrams of the double bionic bone adhesive prepared in Example 2 co-cultured with NIH / 3T3 cells for 1, 3, and 7 days. Embodiment

[0036] An injectable double bionic bone adhesive for promoting fracture healing, and its preparation method includes the following steps:

[0037] (1) Add anhydrous sodium carbonate to boiling deionized water, stir and mix evenly, then add degummed silk for 5 - 120 min; after cleaning, dry at 5 - 100 °C for 1 - 50 h, dissolve the product in 9 mol / L lithium bromide solution or a ternary solution of calcium chloride / absolute ethanol / water (molar ratio 1:8:2) for 1 - 80 h, and then dialyze in deionized water for 36 - 100 h; the dialyzed solution is centrifuged and filtered to obtain a solution S1 with a mass concentration of 0.1 - 50.0%.

[0038] (2) Add the bionic marine barnacle biomaterial into deionized water, stir at 10 - 80 °C for 5 - 72 h to make it evenly mixed, and obtain solution S2 with a mass concentration of 0.1 - 80.0%;

[0039] (3) Add calcium chloride powder into deionized water, stir at room temperature for 1 - 120 min to make it evenly mixed, and obtain solution S3 with a mass concentration of 0.01 - 20.0%;

[0040] (4) Dissolve the bionic marine mussel biomaterial in deionized water, then add magnesium powder into it, stir and mix evenly to obtain a mixed solution S4 of bionic marine mussel biomaterial - magnesium particles with a mass concentration of 0.1 - 60.0%, where the mass of magnesium powder is 0.001 - 10.0%;

[0041] (5) At room temperature, measure solutions S1, S2, and S3 respectively according to the volume ratio of solution S1 to solution S2 being 1:1000 - 1000:1 and the volume ratio of solution S1 to solution S3 being 1:1000 - 1000:1. Then mix solutions S1, S2, and S3, and add mixed solution S4 according to the volume ratio of the mixed solution to mixed solution S4 being 1000:1 - 1:1000. Stir and mix well at 5 - 50 °C for 1 - 180 min to obtain the injectable double - bionic bone adhesive material.

[0042] Among them, the bionic marine barnacle biomaterial is one or more of sodium alginate, polyacrylic acid - hydroxysuccinimide ester, polyasparagine, Trx - Balcp19k fusion protein, thiolated hyaluronic acid, and globulin.

[0043] The bionic marine mussel biomaterial is one or more of phenolic - hydroxyl chitosan, dopamine, tannic acid, gallic acid, and phenolic - hydroxylated alanine.

[0044] Through in - vivo and in - vitro experiments, it is confirmed that this material not only has good cell and blood biocompatibility, but also combines the advantages of strong wet - state adhesion and high mechanical properties. With timely adhesion and curing and high - strength hardness support, the material can withstand the pressure at the bone site and form a strong binding force with bone tissue. In addition, this new - generation composite material that can replace bone nails and metal plates to promote fracture healing can solve the problems of complications such as infection and necrosis caused by improper postoperative care from a technical level, and has a positive effect on fixing the fracture site and inducing bone cell regeneration.

[0045] To further understand the present invention, the preferred embodiments of the present invention will be described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention. In addition, it should be understood that after reading the content described in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application. Example

[0046] (1) Add 8 g of sodium carbonate to boiling deionized water (5 L), stir and mix evenly, add 15 g of silk and degum it for 20 min; after the product is washed 5 times with deionized water, place it in an oven at 60 °C and dry for 6 h. Then put the dried degummed silk fibroin fiber into 42 mL of 9 mol / L lithium bromide solution and dissolve it at 60 °C for 5 h, and then dialyze it in deionized water for 72 h; the dialyzed solution is filtered and centrifuged to obtain a silk fibroin (SF) solution with a mass fraction of about 3%;

[0047] (2) Dissolve 1.5 g of sodium alginate (SA) in 50 mL of deionized water, stir overnight at 50 °C to obtain a 3% SA solution by mass fraction;

[0048] (3) Dissolve 0.4 g of calcium chloride (CaCl2) powder in 20 mL of deionized water, stir at room temperature for 10 min to mix evenly to obtain a 2% CaCl2 solution by mass fraction;

[0049] (4) Dissolve 6 g of tannic acid (TA) in 20 mL of deionized water, add 20 mg of magnesium powder, stir and mix evenly at room temperature to obtain a 30% tannic acid-magnesium particle (TA-Mg) solution by mass concentration;

[0050] (5) At room temperature, add the TA-Mg solution to the mixed solution of the SF solution, SA solution, and CaCl2 solution according to a volume ratio of 4:8:3:1, stir and mix well to obtain an injectable STSA-Mg double-bionic bone adhesive material for promoting fracture healing. Example

[0051] (1) Add 8 g of sodium carbonate to boiling deionized water (5 L), stir and mix evenly, add 15 g of silk and degum it for 20 min; after the product is washed 5 times with deionized water, place it in an oven at 60 °C and dry for 6 h. Then put the dried degummed silk fibroin fiber into 42 mL of 9 mol / L lithium bromide solution and dissolve it at 60 °C for 5 h, and then dialyze it in deionized water for 72 h; the dialyzed solution is filtered and centrifuged to obtain an SF solution with a mass fraction of about 3%;

[0052] (2) Dissolve 2.5 g of SA in 50 mL of deionized water and stir overnight at 50 °C to obtain a 5% SA solution.

[0053] (3) Dissolve 0.4 g of CaCl2 powder in 20 mL of deionized water and stir at room temperature for 10 min to obtain a 2% CaCl2 solution.

[0054] (4) Dissolve 6 g of TA in 20 mL of deionized water, add 20 mg of magnesium powder, and stir at room temperature to obtain a TA-Mg solution with a mass concentration of 30%;

[0055] (5) At room temperature, the TA-Mg solution is added to a mixture of the SF solution, the SA solution, and the CaCl2 solution in a volume ratio of 4:8:3:1, and the mixture is stirred thoroughly to obtain an injectable STSA-Mg dual-bionic bone adhesive material for promoting fracture healing. Example

[0056] (1) Add 8 g of sodium carbonate to boiling deionized water (5 L), stir and mix, add 15 g of silk degumming treatment for 20 min; wash the product with deionized water for 5 times, and dry it in a 60 ℃ oven for 6 h. Then, dissolve the dried degummed silk fibroin fiber in 42 mL of 9 mol / L lithium bromide solution at 60 ℃ for 5 h, and then dialyze it in deionized water for 72 h; the dialyzed solution is filtered and centrifuged to obtain a SF solution with a mass fraction of about 3%;

[0057] (2) Dissolve 2.5 g of SA in 50 mL of deionized water and stir overnight at 50 °C to obtain a 5% SA solution.

[0058] (3) Dissolve 0.2 g of CaCl2 powder in 20 mL of deionized water and stir at room temperature for 10 min to obtain a CaCl2 solution with a mass fraction of 1%;

[0059] (4) Dissolve 6 g of TA in 20 mL of deionized water, add 20 mg of magnesium powder, and stir at room temperature to obtain a TA-Mg solution with a mass concentration of 30%;

[0060] (5) At room temperature, the TA-Mg solution is added to a mixture of the SF solution, the SA solution, and the CaCl2 solution in a volume ratio of 4:8:3:1, and the mixture is stirred thoroughly to obtain an injectable STSA-Mg dual-bionic bone adhesive material for promoting fracture healing. Example

[0061] (1) Add 8 g of sodium carbonate to 5 L of boiling deionized water, stir to mix evenly, add 15 g of silk and degum for 20 min; after the product is washed 5 times with deionized water, place it in an oven at 60 °C and dry for 6 h. Then put the dried degummed silk fibroin fiber into 42 mL of 9 mol / L lithium bromide solution and dissolve at 60 °C for 5 h, and then dialyze in deionized water for 72 h; the dialyzed solution is filtered and centrifuged to obtain an SF solution with a mass fraction of about 3%;

[0062] (2) Dissolve 2.5 g of SA in 50 mL of deionized water and stir overnight at 50 °C to obtain a 5% SA solution;

[0063] (3) Dissolve 0.4 g of CaCl2 powder in 20 mL of deionized water and stir at room temperature for 10 min to mix evenly to obtain a 2% CaCl2 solution;

[0064] (4) Dissolve 3 g of TA in 20 mL of deionized water, add 20 mg of magnesium powder, and stir and mix evenly at room temperature to obtain a 15% TA-Mg solution;

[0065] (5) At room temperature, add the TA-Mg solution to the mixed solution of the SF solution, SA solution, and CaCl2 solution according to a volume ratio of 4:8:3:1, stir and mix well to obtain an injectable STSA-Mg double-bionic bone adhesive material for promoting fracture healing. Example

[0066] (1) Add 8 g of sodium carbonate to 5 L of boiling deionized water, stir to mix evenly, add 15 g of silk and degum for 20 min; after the product is washed 5 times with deionized water, place it in an oven at 60 °C and dry for 6 h. Then put the dried degummed silk fibroin fiber into 42 mL of 9 mol / L lithium bromide solution and dissolve at 60 °C for 5 h, and then dialyze in deionized water for 72 h; the dialyzed solution is filtered and centrifuged to obtain an SF solution with a mass fraction of about 3%;

[0067] (2) Dissolve 2.5 g of SA in 50 mL of deionized water and stir overnight at 50 °C to obtain a 5% SA solution;

[0068] (3) Dissolve 0.4 g of CaCl2 powder in 20 mL of deionized water and stir at room temperature for 10 min to mix evenly to obtain a 2% CaCl2 solution;

[0069] (4) Dissolve 6 g of TA in 20 mL of deionized water, add 40 mg of magnesium powder, and stir and mix evenly at room temperature to obtain a TA-Mg solution with a mass concentration of 30%.

[0070] (5) At room temperature, add the TA-Mg solution to the mixed solution of SF solution, SA solution, and CaCl2 solution according to a volume ratio of 4:8:3:1, and stir and mix well to obtain an injectable STSA-Mg double-bionic bone adhesive material for promoting fracture healing. Example

[0071] (1) Add 8 g of sodium carbonate to boiling deionized water (5 L), stir and mix evenly, add 15 g of silk for degumming treatment for 20 min; after the product is washed 5 times with deionized water, place it in an oven at 60 °C and dry for 6 h. Then put the dried degummed silk fibroin fiber into 42 mL of 9 mol / L lithium bromide solution and dissolve it at 60 °C for 5 h, and then dialyze it in deionized water for 72 h; the dialyzed solution is filtered and centrifuged to obtain an SF solution with a mass fraction of about 3%.

[0072] (2) Dissolve 2.5 g of SA in 50 mL of deionized water, stir overnight at 50 °C to obtain a SA solution with a mass fraction of 5%.

[0073] (3) Dissolve 0.4 g of CaCl2 powder in 20 mL of deionized water, stir and mix evenly at room temperature for 10 min to obtain a CaCl2 solution with a mass fraction of 2%.

[0074] (4) Dissolve 6 g of TA in 20 mL of deionized water, add 60 mg of magnesium powder, and stir and mix evenly at room temperature to obtain a TA-Mg solution with a mass concentration of 30%.

[0075] (5) At room temperature, add the TA-Mg solution to the mixed solution of SF solution, SA solution, and CaCl2 solution according to a volume ratio of 4:8:3:1, and stir and mix well to obtain an injectable STSA-Mg double-bionic bone adhesive material for promoting fracture healing. Example

[0076] (1) Add 8 g of sodium carbonate to boiling deionized water (5 L), stir and mix evenly, add 15 g of silk for degumming treatment for 20 min; after the product is washed 5 times with deionized water, place it in an oven at 60 °C and dry for 6 h. Then put the dried degummed silk fibroin fiber into 42 mL of 9 mol / L lithium bromide solution and dissolve it at 60 °C for 5 h, and then dialyze it in deionized water for 72 h; the dialyzed solution is filtered and centrifuged to obtain an SF solution with a mass fraction of about 3%.

[0077] (2) Dissolve 2.5 g of SA in 50 mL of deionized water and stir overnight at 50 °C to obtain a 5% SA solution by mass;

[0078] (3) Dissolve 0.4 g of CaCl₂ powder in 20 mL of deionized water and stir at room temperature for 10 min to mix evenly, obtaining a 2% CaCl₂ solution by mass;

[0079] (4) Dissolve 6 g of TA in 20 mL of deionized water, add 20 mg of magnesium powder, and stir and mix evenly at room temperature to obtain a 30% TA-Mg solution by mass concentration;

[0080] (5) At room temperature, add the TA-Mg solution to the mixed solution of SF solution, SA solution, and CaCl₂ solution according to a volume ratio of 4:8:7:1, and stir and mix well to obtain an injectable STSA-Mg double-bionic bone adhesive material for promoting fracture healing. Example

[0081] (1) Add 8 g of sodium carbonate to boiling deionized water (5 L), stir and mix evenly, add 15 g of silk for degumming treatment for 20 min; after the product is washed 5 times with deionized water, place it in an oven at 60 °C and dry for 6 h. Then put the dried degummed silk fibroin fiber into 42 mL of 9 mol / L lithium bromide solution and dissolve it at 60 °C for 5 h, and then dialyze it in deionized water for 72 h; the dialyzed solution is filtered and centrifuged to obtain an SF solution with a mass fraction of about 3%;

[0082] (2) Dissolve 2.5 g of SA in 50 mL of deionized water and stir overnight at 50 °C to obtain a 5% SA solution by mass;

[0083] (3) Dissolve 0.4 g of CaCl₂ powder in 20 mL of deionized water and stir at room temperature for 10 min to mix evenly, obtaining a 2% CaCl₂ solution by mass;

[0084] (4) Dissolve 6 g of TA in 20 mL of deionized water, add 20 mg of magnesium powder, and stir and mix evenly at room temperature to obtain a 30% TA-Mg solution by mass concentration;

[0085] (5) At room temperature, add the TA-Mg solution to the mixed solution of SF solution, SA solution, and CaCl₂ solution according to a volume ratio of 8:8:3:1, and stir and mix well to obtain an injectable STSA-Mg double-bionic bone adhesive material for promoting fracture healing.

[0086] (1) Add 8 g of sodium carbonate to boiling deionized water (5 L), stir and mix well, add 15 g of silk for degumming treatment for 20 min; after the product is washed 5 times thoroughly with deionized water, place it in an oven at 60 °C for drying for 6 h. Then put the dried degummed silk fibroin fiber into 42 mL of 9 mol / L lithium bromide solution and dissolve it at 60 °C for 5 h, and then dialyze it in deionized water for 72 h; the dialyzed solution is filtered and centrifuged to obtain an SF solution with a mass fraction of about 3%;

[0087] (2) Dissolve 2.5 g of SA in 50 mL of deionized water and stir overnight at 50 °C to obtain a SA solution with a mass fraction of 5%;

[0088] (3) Dissolve 40 mg of CaCl₂ powder in 20 mL of deionized water and stir at room temperature for 10 min to mix well to obtain a CaCl₂ solution with a mass fraction of 2%;

[0089] (4) Dissolve 6 g of TA in 20 mL of deionized water and stir and mix evenly at room temperature to obtain a TA solution with a mass concentration of 30%;

[0090] (5) At room temperature, add the TA solution to the mixed solution of the SF solution, SA solution, and CaCl₂ solution according to a volume ratio of 4:8:3:1, stir and mix well, namely the STSA double bionic bone adhesive material.

[0091] (1) Add 8 g of sodium carbonate to boiling deionized water (5 L), stir and mix well, add 15 g of silk for degumming treatment for 20 min; after the product is washed 5 times thoroughly with deionized water, place it in an oven at 60 °C for drying for 6 h. Then put the dried degummed silk fibroin fiber into 42 mL of 9 mol / L lithium bromide solution and dissolve it at 60 °C for 5 h, and then dialyze it in deionized water for 72 h; the dialyzed solution is filtered and centrifuged to obtain an SF solution with a mass fraction of about 3%;

[0092] (2) Dissolve 1.5 g of SA in 50 mL of deionized water and stir overnight at 50 °C to obtain a SA solution with a mass fraction of 3%;

[0093] (3) Dissolve 40 mg of CaCl₂ powder in 20 mL of deionized water and stir at room temperature for 10 min to mix well to obtain a CaCl₂ solution with a mass fraction of 2%;

[0094] (4) Dissolve 6 g of TA in 20 mL of deionized water and stir and mix evenly at room temperature to obtain a TA solution with a mass concentration of 30%;

[0095] (5) At room temperature, add the TA solution to the mixed solution of the SF solution, SA solution, and CaCl₂ solution according to a volume ratio of 4:8:3:1, and stir well to mix, then the STSA double biomimetic bone adhesive material is obtained.

[0096] Perform mechanical property tests on the double biomimetic bone adhesives prepared in the examples and comparative examples, and the results are shown in Table 1.

[0097] Table 1 Test results of tensile / compressive mechanical strength and adhesion strength of the prepared double biomimetic bone adhesives

[0098]

[0099] It can be seen from Table 1 that: compared with Example 2, the wet adhesion of the material prepared in Example 1 is not ideal. This is because as the concentration of SA decreases, the content of side-chain carboxyl groups decreases, thus reducing the ability of the material to remove interfacial water; at the same time, since SA can also form metal-phenolic coordination bonds with Ca 2+ and Mg 2+ resulting in a weakening of the mechanical properties of the material in Example 1. The results show that the concentration of SA has a great influence on the mechanical properties and adhesion properties of the material.

[0100] Compared with Example 2, the mechanical properties of the material prepared in Example 3 are weaker. This is because Ca 2+ can form metal-phenolic coordination bonds and ionic networks with TA and SA respectively, which is beneficial to improving the mechanical properties of the material, while the decrease in the content of Ca 2+ will lead to a weakening of the mechanical properties of the material. This proves that the content of Ca 2+ also has a certain influence on the mechanical properties of the material.

[0101] Compared with Example 2, the mechanical properties of the materials prepared in Examples 4-6 are all reduced, and the adhesion properties of the materials obtained in Examples 5 and 6 are also weakened. This is because TA forms metal-phenolic coordination bonds with Ca 2+ and Mg 2+ while the decrease in the content of TA in Example 4 leads to a reduction in coordination bonds, thus slightly reducing the mechanical properties of the above materials compared with Example 2. The addition of magnesium particles will form Mg 2+ and hydrogen with the water molecules in the material, generating more bubbles, resulting in the collapse of the internal structure of the material, thus causing a decrease in both the adhesion properties and mechanical properties of the materials obtained in Examples 5 and 6.

[0102] Compared with Example 2, since the content of SA was increased in Example 7, the mechanical properties of the prepared material decreased significantly and it could not be applied to fracture repair; while in Example 8, due to the increase in the content of TA-Mg, the structure of the prepared material was loose, the hardness and adhesion decreased, and the pH value of the material decreased due to the too high proportion of TA added, which was also not conducive to bone injury repair and fracture area healing.

[0103] Figure 1 Figure 4 is the scanning electron microscope image of the STSA-Mg double bionic bone adhesive prepared in Example 2. It was found by observing under the microscope at high and low magnifications that the obtained STSA-Mg double bionic bone adhesive had a highly porous network structure, which was beneficial to improving the mechanical properties of the bone adhesive and could provide a scaffold for cell growth and proliferation, facilitating the acceleration of the repair process.

[0104] Figure 2 Figure 5 is the Fourier transform infrared spectroscopy analysis of the double bionic bone adhesive materials prepared in Example 2 and Comparative Example 1. As can be seen from the spectrogram, the characteristic peak of the STSA-Mg double bionic bone adhesive at 1641 cm −1 was attributed to the stretching vibration of C=O in the amide I band of SF, which was caused by the "competitive crosslinking" resulting from the formation of metal-phenol coordination bonds between Mg 2+ and TA and hydrogen bonds between TA (catechol group) and SF (amide group), indicating the successful preparation of the STSA-Mg double bionic bone adhesive.

[0105] Figure 3 Figure 6 is the comparison chart of the mechanical properties of the double bionic bone adhesives prepared in Example 2 and Comparative Example 1. The results showed that the tensile and compressive strengths of the STSA-Mg double bionic bone adhesive formed after adding magnesium particles were both improved, which was due to the metal-phenol coordination and ionic bonds formed by the introduced Mg 2+ with TA and SA respectively, improving the mechanical properties of the material.

[0106] Figure 4 Figure 7 is the adhesion performance diagram of the double bionic bone adhesive prepared in Example 2. The results showed that the STSA-Mg double bionic bone adhesive was applicable to various wet adhesion scenarios: the surfaces of plastic, rubber, metal, wood, glass, magnet, as well as the surfaces of plastic and pigskin under water flow scouring. The lap shear test of the adhesion to pig skin showed that the STSA-Mg double bionic bone adhesive exhibited excellent wet adhesion strength. The above results indicated that this material could achieve strong adhesion to bone tissue in the in-vivo liquid environment.

[0107] Figure 5The figure shows the application of the STSA-Mg dual bionic bone adhesive prepared in Example 2 in a 3D printed model of a simulated fractured human femur. That is, the STSA-Mg dual bionic bone adhesive was adhered between two fractured femurs for fixation, and a load of about 30 g was applied to it. The results showed that the STSA-Mg dual bionic bone adhesive could be fixed at the bone fracture site and withstand a certain load.

[0108] Figure 6 The figure shows the CCK-8 results of the co-culture of the dual bionic bone adhesives prepared in Example 2 and Comparative Example 1 with NIH / 3T3 cells for 1, 3, and 7 days. As can be seen from the figure, the NIH / 3T3 cells co-cultured in each material group showed a good cell proliferation trend on the 3rd day and the 7th day. Among them, the STSA-Mg bionic bone adhesive group showed better cell proliferation.

[0109] Figure 7 The figure shows the live / dead staining of the co-culture of the dual bionic bone adhesives prepared in Example 2 and Comparative Example 1 with NIH / 3T3 cells for 1, 3, and 7 days. The test results were consistent with the CCK-8 quantitative results. That is, after the STSA-Mg bone adhesive was co-cultured with NIH / 3T3 cells, most of the NIH / 3T3 cells were stained green (live cells), indicating that the material had good biocompatibility.

[0110] The above description of the embodiments is only used to help understand the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

[0111] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A preparation method of an injectable double-bionic bone adhesive for promoting fracture healing, characterized in that: It includes the following steps: (1) Add anhydrous sodium carbonate into boiling deionized water, stir and mix evenly, then add silk for degumming; after washing and drying, dissolve the product, and then perform dialysis in deionized water; the solution after dialysis is centrifuged and filtered to obtain solution S1, and the mass concentration of the obtained solution S1 is 1-15.0%; (2) Add the biomimetic marine barnacle biomaterial into deionized water, stir and mix evenly at a certain temperature to obtain solution S2, and the mass concentration of the obtained solution S2 is 0.1-20.0%; (3) Add calcium chloride powder into deionized water, stir and mix evenly at room temperature to obtain solution S3, and the mass concentration of the obtained solution S3 is 0.1-10.0%; (4) Dissolve the biomimetic marine mussel biomaterial in deionized water, then add magnesium powder thereto, stir and mix evenly to obtain a mixed solution S4 of the biomimetic marine mussel biomaterial-magnesium particles, and the mass concentration of the obtained mixed solution S4 is 1-50.0%, and the mass of the magnesium powder contained therein is 0.001-10.0%; (5) At room temperature, mix solution S1, S2, and S3 and add the mixed solution S4, stir and mix well to obtain the injectable double biomimetic bone adhesive; The biomimetic marine barnacle biomaterial in step (2) is sodium alginate; The biomimetic marine mussel biomaterial in step (4) is one or more of phenol-hydroxy chitosan, dopamine, tannic acid, gallic acid, and phenol-hydroxylated alanine; In step (5), the volume ratio of solution S1 to solution S2 used is 1:15-15:1; the volume ratio of solution S1 to solution S3 used is 1:15-15:1; the volume ratio of the mixed solution S4 to the mixed solution of solution S1, S2, and S3 is 1:20-20:

1.

2. The preparation method of an injectable dual-bionic bone adhesive for promoting fracture healing according to claim 1, wherein: The degumming time in step (1) is 5-120 min.

3. The preparation method of an injectable dual-bionic bone adhesive for promoting fracture healing according to claim 1, characterized in that: The stirring temperature in step (2) is 10-80 °C, and the time is 5-72 h.

4. An injectable double biomimetic bone adhesive for promoting fracture healing prepared by any of the methods as claimed in claims 1-3.

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