Preparation method of degradable bone bonding material with osteogenesis promoting effect suitable for wet environment and prepared bone bonding material

Abstract

The invention discloses a preparation method of a degradable bone bonding material with an osteogenesis promoting effect suitable for a wet environment. The preparation method is specifically implemented according to the following steps: step 1, preparing acylated soybean protein isolate; step 2, preparing TA modified BN nanoparticles; step 3, preparing a PVA aqueous solution; step 4, dissolving the acylated soy isolate protein prepared in the step 1 in a TA solution, then adding the TA modified BN nanoparticles prepared in the step 2, and uniformly mixing to obtain a solution B; and step 5, mixing the PVA aqueous solution prepared in the step 3 and the solution B prepared in the step 4, and then washing the mixture with deionized water to obtain the bone bonding material PTSN-TN. The bone bonding material prepared by the invention solves the problem of insufficient bonding strength in a wet environment of a human body in the prior art. The invention also discloses a bone bonding material prepared by the preparation method of the degradable bone bonding material with the osteogenesis promoting effect suitable for the wet environment.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical biomaterial preparation methods, and relates to a method for preparing a degradable bone adhesive material suitable for wet environments and having an osteogenesis-promoting effect. The present invention also relates to the bone adhesive material prepared by the above preparation method. Background Art

[0002] Fractures are common and frequently occurring clinical diseases in orthopedics. Good fracture alignment and reduction are the basis for fracture healing and functional recovery. Metal internal fixation devices such as metal screw plates and intramedullary nails are traditional fracture fixation devices commonly used in clinical practice. They effectively solve the problem of fracture fixation, but there are still some shortcomings. For example, when severe comminuted fractures and complex deep fractures cannot be effectively exposed, the difficulty of surgery increases significantly, the operation time of intraoperative reduction and fixation is significantly prolonged, excessive periosteum stripping affects fracture healing, and the risks of intraoperative bleeding and postoperative infection increase significantly, which can affect the fracture fixation effect and lead to increased postoperative complications. In addition, traditional fixation devices are difficult to accurately reduce and fixate smaller fracture fragments, which can easily lead to delayed fracture healing or even non-union. In addition, due to the aging population, the incidence of osteoporotic fractures has increased year by year, and the decrease in bone density has led to a decrease in bone strength, which cannot provide effective holding force for screw fixation, and can easily lead to loosening and failure of internal fixation. Metal internal fixators are non-degradable and require secondary surgery for removal. The mismatch between metal strength and bone tissue can easily lead to stress shielding, resulting in local bone loss and impaired healing. Metal internal fixation devices are difficult to effectively reduce and fix small fracture fragments in comminuted fractures, and are prone to loss of fracture fragments, leading to local bone defects and impaired healing. Therefore, how to achieve fracture fixation and maintain fracture reduction using bone adhesives, as well as how to increase the bond strength of bone adhesives (>1 MPa), are urgent challenges in this research field.

[0003] A Chinese patent application (application number: CN202310402320.9, publication date: April 14, 2023, publication number: CN116350833A) discloses a lysine-based antibacterial medical bone tissue adhesive, its preparation method, and its application. The bone adhesive is composed of the following raw materials: oxidized hyaluronic acid, polylysine, and tannic acid. The preparation method is as follows: (1) dissolving hyaluronic acid in water to obtain a hyaluronic acid solution; (2) adding sodium periodate to the hyaluronic acid solution, stirring in the dark, dialyzing in water, and freeze-drying to obtain oxidized hyaluronic acid; (3) dissolving the oxidized hyaluronic acid in an aqueous solution to form an oxidized hyaluronic acid solution; (4) dissolving polylysine (3-7% by mass) and tannic acid (8-24% by mass) in water to obtain a polylysine and tannic acid mixed solution; (5) stirring and blending the oxidized hyaluronic acid solution and the polylysine and tannic acid mixed solution (mass ratio 6:3-7:8-24) to obtain a polylysine-based antibacterial medical bone tissue adhesive. Although the bone adhesive prepared by the present invention solves the problems of bonding performance and antibacterial performance to bone tissue, the bonding strength of the bone adhesive is still low (<300KPa), and the bone adhesive is used in the form of liquid injection, which does not solve the problems of slow curing time and easy diffusion during operation.

[0004] A Chinese patent (application number: 202410693088.3, ​​publication date: September 27, 2024, publication number: CN 118697928A) discloses an inorganic bone adhesive based on modified mussel adhesive protein and its preparation method. The main raw materials include: an inorganic phosphate compound (calcium phosphate, hydroxyapatite or magnesium phosphate) and mussel adhesive protein. The preparation method comprises: (1) preparing an inorganic phosphate compound; (2) mixing the modified mussel adhesive protein Mfp3-5 and the modified polysaccharide in a mass ratio of 1:1, adding ethanol and grinding to obtain a composite solidifying liquid; (3) uniformly mixing the two in a certain proportion. Although the bone adhesive prepared by the present invention has a good curing speed and improved water resistance, its bonding strength is only 90.42 kPa, which is relatively low and may not be able to effectively fix the fracture site, affecting fracture healing.

[0005] A Chinese patent (application number: 202410740836.9, publication date: September 17, 2024, publication number: CN118649278A) discloses a bone adhesive and its preparation method. The preparation method comprises: (1) preparing PLGA-PEG-PLGA; (2) dispersing and dissolving the PLGA-PEG-PLGA triblock polymer, catechol, HAP, CaCl2, and CuSO4 in chloroform, mixing them uniformly, concentrating the solvent by rotary evaporation, rapidly settling in cold ethanol, and drying the precipitate to obtain the bone adhesive. Although the hydrogel bone adhesive prepared by the present invention has a good degradation rate and good water resistance, its bonding strength is too low, failing to meet the demand for higher bonding strength of the bone adhesive.

[0006] A Chinese patent (application number: CN202410580206.X, publication date: 2024.05.11, publication number: CN118453942A) discloses a bone adhesive for promoting fracture healing, its preparation method, and application. The preparation method is as follows: (1) a calcium phosphate compound, a phosphoserine compound, a polyphenol compound, and a magnesium ion compound are mixed uniformly to obtain a mixture a, a liquid phase composition is added to the mixture a and mixed uniformly to form a bone adhesive A; (2) a calcium phosphate compound and a phosphoserine compound are mixed uniformly to obtain a mixture b, a liquid phase component is added to the mixture b and mixed uniformly to form a bone adhesive B. Although the bone adhesive prepared by this patent has good biocompatibility and degradability, excessive degradation may cause the adhesive to fail before the fracture is completely healed, and the bone bonding strength of most of its components is still low (only 150KPa), which may not be able to withstand the stress required by the fracture site, easily leading to bonding failure or re-fracture. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for preparing a degradable bone adhesive material having an osteogenesis effect suitable for wet environments. The prepared bone adhesive material solves the problem of insufficient bonding strength in wet environments of the human body in the prior art.

[0008] Another object of the present invention is to provide a bone adhesive material prepared by the above-mentioned method for preparing a degradable bone adhesive material suitable for a wet environment and having an osteogenesis effect.

[0009] The technical solution adopted by the present invention is a method for preparing a degradable bone adhesive material with an osteogenic effect suitable for wet environments, which is specifically implemented according to the following steps: Step 1, preparing acylated soy protein isolate; Step 2, preparing TA-modified BN nanoparticles; Step 3, preparing a PVA aqueous solution; Step 4, dissolving the acylated soy protein isolate prepared in step 1 in the TA solution, and then adding the TA-modified BN nanoparticles prepared in step 2, and mixing them uniformly to obtain a B solution; Step 5: Mix the PVA aqueous solution prepared in step 3 and the B solution prepared in step 4, and then rinse with deionized water to obtain the bone adhesive material PTSN-TN.

[0010] Preferably, step 1 is specifically: A certain amount of soy protein isolate SPI is dissolved in deionized water to obtain an SPI solution, and then a sodium hydroxide solution is used to adjust the pH of the SPI solution. After a certain period of time, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide active ester are added and mixed evenly. The mixture is then stirred in a water bath environment. The mixture is then washed with deionized water multiple times, and a light yellow gel precipitate is obtained after centrifugation. The precipitate is collected and freeze-dried to obtain the acylated soy protein isolate SPN.

[0011] Preferably, in step 1, the amounts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide active ester added are added according to the following ratio: the molar ratio of soy protein isolate: 1-ethyl-(3-dimethylaminopropyl)carbodiimide: N-hydroxysuccinimide active ester is 1:1:1 to 1:4:20.

[0012] Preferably, the mass fraction of SPI in the SPI solution in step 1 is 2-4wt%, and the pH of the SPI solution is adjusted to 6-9 using 0.1M sodium hydroxide solution; 1-ethyl-(3-dimethylaminopropyl)carbodiimide is added 30 minutes after the pH is adjusted, and N-hydroxysuccinimide active ester is added 60 minutes after the pH is adjusted. The stirring temperature of the water bath is 30-60°C, and the water bath time is 4-8 hours. The freeze-drying temperature is -20--30°C, and the freeze-drying time is 24-72 hours.

[0013] Preferably, step 2 is specifically as follows: TA was dissolved in deionized water to prepare a TA aqueous solution, and then BN was added to the TA solution. After ultrasonic vibration, ball milling was performed. The mixture was collected and centrifuged to remove the remaining BN sheets. The supernatant was then vacuum filtered and then rinsed with deionized water to remove excess TA. The mixture was then dried to obtain TA-modified BN nanoparticles TN.

[0014] Preferably, in step 2, the concentration of TA in the TA aqueous solution is 30-50 mg / ml, the amount of BN powder added is: the mass ratio of TA to BN powder is 1:1-3:1; the ultrasonic time is 30 min-1 h; the ball mill speed is 300-500 rpm, and the speed duration is 8-15 h; the centrifugal speed is 2000 rpm, and the centrifugal time is 10 min; and the drying temperature is 30-60°C.

[0015] Preferably, step 3 is specifically as follows: dissolving PVA in deionized water and heating and stirring uniformly to prepare a PVA aqueous solution, recorded as solution A; wherein the concentration of PVA is 0.2~0.4g / ml; the heating temperature is 70~90℃, and the stirring time is 3~5h.

[0016] Preferably, step 4 is specifically as follows: TA is dissolved in deionized water to obtain a TA solution, and then the SPN prepared in step 1 is added to the TA solution and stirred for a certain time, and then the TN prepared in step 2 is added to mix the solution uniformly to obtain a B solution; The concentration of TA in the TA solution is 0.2~0.4g / ml, the mass fraction of SPN in the B solution is 4~10wt%, the mass fraction of TN is 0.25~2wt%, and the stirring time after adding SPN is 2~4h.

[0017] Preferably, in step 5, the volume ratio of the PVA aqueous solution to the B solution is 1:1 to 1:2.

[0018] The second technical solution adopted by the present invention is: the degradable bone adhesive material is prepared by the above-mentioned preparation method of the degradable bone adhesive material suitable for wet environment and having osteogenesis effect.

[0019] The beneficial effects of the present invention are: The present invention first prepares SPN, then modifies BN with TA. Finally, PVA, TA, SPN, and TN are mixed in a specific proportion to create a novel bone adhesive material that can be used to bond fractures in the human body's wet environment. This bone adhesive material addresses the challenge of resetting and bonding fracture fragments in wet environments, particularly comminuted fractures. It maintains sufficient bond strength while slowly degrading, while simultaneously inducing bone regeneration until the fracture is fully healed and remodeled.

[0020] The multi-dentate structure of TA in this invention forms effective connections with PVA polymer chains. This interaction between TA and PVA chains increases the molecular weight in the form of a PVA-TA-PVA network, thereby improving the viscosity and tack of the adhesive. Traditional adhesives typically incorporate a small amount of TA as a crosslinker, resulting in a film-like or liquid colloid form. However, the present invention significantly increases the TA content (10-50 times), not only increasing the molecular weight of PVA but also reducing its water solubility. Consequently, by using a 1:1 ratio of TA to PVA, a hydrated but insoluble cohesive adhesive was prepared.

[0021] The SPI, rich in functional groups such as amino and carboxyl groups, can react with the phenolic hydroxyl groups of TA and the hydroxyl groups of PVA to form chemical bonds such as hydrogen and ionic bonds. To further enhance interfacial adhesion, the present invention utilizes two coupling agents: N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). These couple with the SPI to form self-crosslinks, coupling active esters to the SPI. The active esters then undergo Schiff base reactions with amino groups between the SPI and the bone tissue, forming covalent crosslinks and thereby enhancing the interfacial adhesion strength between the adhesive and the bone.

[0022] Finally, to further enhance the adhesive's bond strength, the present invention incorporates tannic acid-modified BN nanoparticles as secondary crosslinking sites. As a two-dimensional nanomaterial, BN possesses a large specific surface area due to its unique two-dimensional structure, providing more surface atoms and molecules, thereby increasing the contact area and interaction with the substrate. Tannic acid-modified BN allows for more uniform dispersion in the adhesive, reducing nanomaterial aggregation within the matrix and thus enhancing the reinforcement effect. DETAILED DESCRIPTION

[0023] The following describes it in detail with reference to specific implementation methods.

[0024] The present invention is a method for preparing a degradable bone adhesive material having an osteogenesis-promoting effect in a wet environment, which is specifically implemented according to the following steps: Step 1, preparing acylated soy protein isolate (SPN); A certain amount of soy protein isolate (SPI) is dissolved in deionized water to obtain an SPI solution, and then the pH of the SPI solution is adjusted with a sodium hydroxide solution. After a certain period of time, EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide active ester) are added and mixed evenly. The mixture is then stirred in a water bath environment, and then the mixture is washed with deionized water multiple times. After centrifugation, a light yellow gel precipitate is obtained. The precipitate is collected and freeze-dried to obtain acylated soy protein isolate (SPN). The mass fraction of SPI in the SPI solution is 2-4 wt %, and the pH of the SPI solution is adjusted to 6-9 using a 0.1 M sodium hydroxide solution. EDC is added 30 minutes after the pH adjustment, and NHS is added 60 minutes after the pH adjustment. The water bath is heated and stirred at a temperature of 30-60° C. for 4-8 hours. The freeze-drying temperature is -20--30° C. for 24-72 hours. The amounts of EDC and NHS added are as follows: the molar ratio of SPI:EDC:NHS is 1:1:1 to 1:4:20.

[0025] Step 2, preparing TA-modified BN nanoparticles (TN); TA was dissolved in deionized water to prepare a TA aqueous solution, and then BN was added to the TA solution. After ultrasonic vibration, ball milling was performed. The mixture was collected and centrifuged to remove the remaining BN sheets. The supernatant was then vacuum filtered and then rinsed with deionized water to remove excess TA. The mixture was then dried to obtain TA-modified BN nanoparticles TN.

[0026] Among them, the concentration of TA in the TA aqueous solution is 30~50 mg / ml, the amount of BN powder added is: the mass ratio of TA to BN powder is 1:1~3:1; the ultrasonic time is 30min~1h; the ball mill speed is 300~500rpm, and the speed duration is 8~15h; the centrifugal speed is 2000rpm, and the centrifugal time is 10min; and the drying temperature is 30~60℃.

[0027] Step 3, preparing a PVA aqueous solution (solution A); PVA was dissolved in deionized water and heated and stirred to prepare a PVA aqueous solution, which was designated as solution A. The concentration of PVA was 0.2-0.4 g / ml. The heating temperature was 70-90°C and the stirring time was 3-5 h. Step 4: dissolving the acylated soy protein isolate prepared in step 1 in the TA solution, and then adding the TA-modified BN nanoparticles prepared in step 2, and mixing them evenly to obtain a B solution, specifically: Dissolve TA in deionized water to obtain a TA solution, then add the SPN prepared in step 1 to the TA solution and stir for a certain period of time, then add the TN prepared in step 2 to mix the solution evenly to obtain a B solution; The concentration of TA in the TA solution is 0.2-0.4 g / ml, the mass fraction of SPN in the B solution is 4-10 wt%, the mass fraction of TN is 0.25-2 wt%, and the stirring time after adding SPN is 2-4 h. Step 5: Mix solution A prepared in step 3 and solution B prepared in step 4, with the volume ratio of solution A to solution B being 1:1 to 1:2, and then rinse with deionized water to obtain the bone adhesive material PTSN-TN.

[0028] Tannic acid (TA) is a naturally occurring polyphenolic compound found in tea, wine, and fruit. Due to its remarkable structural and functional properties, materials based on its polyhydroxyphenol structure have significant potential for application in orthopedics. For example, TA strongly interacts with metals, forming a stable coating on metal surfaces, thereby improving the physical and chemical properties of bone implants and enhancing implant success rates. TA also inhibits osteoclast activity, potentially playing a positive role in the treatment of osteoporosis. As a plant polyphenol, TA is an important tool for the synthesis of high-performance biocompatible bone adhesives, enhancing waterproof fixation and guiding bone regeneration. In the presence of heavy metal contamination, TA chelates ions, preserving bone morphology and structure. TA also exhibits significant antimicrobial activity, helping to reduce surgical implant infections and inhibit various tumor cell types, promoting its potential application in spinal metastasis surgery. Furthermore, TA can mitigate corrosion caused by magnesium alloys, significantly promoting the development of degradable orthopedic metal fixation devices. Notably, TA is inexpensive and readily available, making it highly valuable in the field of bone biomaterials.

[0029] Polyvinyl alcohol (PVA) is one of the earliest synthesized polymers and a common raw material for hydrogel production. The PVA monomer is composed of hydroxyl groups, which determine its physical and chemical properties and impart mechanical stability. PVA exhibits high biocompatibility and the ability to form cross-linked structures without the addition of toxic cross-linking agents. Its most significant advantage is that it can be easily synthesized based on the intended application. Furthermore, PVA is non-toxic, exhibits excellent water-swelling properties, is biocompatible, and is harmless to the human body. These advantages have broad application prospects in wound dressings, bone repair biomaterials, and drug carriers.

[0030] Soy protein isolate (SPI) is a protein powder with a protein content exceeding 90% obtained by extracting soybean meal (oil and water-soluble non-protein components removed) in an alkaline solution at low temperature. SPI is then precipitated, washed, and dried. Its structure and properties are essentially comparable to pure soy protein, and it contains nearly 20 amino acids, including essential amino acids.

[0031] SPI can be combined with polysaccharides and other ingredients to form composite adhesives that can quickly bond bones and produce strong adhesion. It also possesses excellent biocompatibility, is non-toxic and non-side-effective, and is biodegradable in vivo. These properties give SPI broad application prospects in the adhesive field.

[0032] Boron nitride (BN) possesses excellent mechanical properties, such as high strength and toughness. Compounding with BN nanoparticles can enhance mechanical properties such as tensile and compressive strength. BN also exhibits excellent biocompatibility, preventing rejection or inflammatory reactions. Therefore, BN holds broad application prospects in biomedical applications, including medical devices and biomaterials (such as implants and stents). Furthermore, BN's surface properties enable it to form excellent interfacial bonds with a variety of materials. In bone adhesives, BN can form a tight bond with bone tissue, thereby enhancing the interfacial bonding between the adhesive and bone. This enhanced interfacial bonding helps to improve the bond strength of the bone adhesive, allowing it to more firmly secure the bone tissue.

[0033] Example 1 The present invention is a method for preparing a degradable bone adhesive material having an osteogenesis-promoting effect in a wet environment, which is specifically implemented according to the following steps: Step 1: 2 wt% SPI (soy protein isolate) was dissolved in deionized water, and the pH was adjusted to 6 with 0.1 M sodium hydroxide solution. EDC and NHS were added to the solution after 30 and 60 minutes, respectively, to make the molar ratio of SPI:EDC:NHS 1:1:1 and mixed evenly. The mixture was finally stirred in a 30°C water bath for 4 hours, and then washed three times with deionized water. After centrifugation, a light yellow gel precipitate was obtained, which was collected and freeze-dried (freeze-drying temperature was -20°C and time was 24 hours). Step 2: Dissolve 30 mg / ml of TA in deionized water to prepare a TA aqueous solution. Then, a certain amount of BN was added to the TA solution at a 1:1 mass ratio of TA to BN powder. After ultrasonication for 30 minutes, the mixture was transferred to an agate jar containing 200 g of zirconia ball milling beads. After ball milling at 300 rpm in a planetary ball mill for 15 hours, the mixture was collected and centrifuged (2000 rpm, 10 minutes) to remove the remaining BN flakes. The supernatant was vacuum filtered, rinsed with copious deionized water to remove excess TA, and dried in an oven at 30°C to obtain TN. Step 3, dissolving PVA at a concentration of 0.2 g / ml in deionized water and heating and stirring at 70° C. for 5 h until uniformly mixed to prepare a PVA aqueous solution (Solution A); Step 4: Dissolve TA at a concentration of 0.2 g / ml in deionized water, add 4 wt% SPN and stir for a certain period of time, then add 0.25 wt% TN to mix the solution evenly (Solution B). Step 5: evenly mix solution A and solution B at a volume ratio of 1:1, and rinse with deionized water to obtain the bone adhesive material PTSN-TN.

[0034] Example 2 The present invention is a method for preparing a degradable bone adhesive material having an osteogenesis-promoting effect in a wet environment, which is specifically implemented according to the following steps: Step 1: 2.5 wt% SPI (soy protein isolate) was dissolved in deionized water, and the pH was adjusted to 6.5 with 0.1 M sodium hydroxide solution. After 30 min and 60 min, EDC and NHS were added to the solution in sequence to make the molar ratio of SPI:EDC:NHS 1:1:5 and mixed evenly. The mixture was finally stirred in a 35°C water bath for 4 h, and then washed three times with deionized water. After centrifugation, a light yellow gel precipitate was obtained, which was collected and freeze-dried (freeze-drying temperature was -20°C and time was 24 h). Step 2: Dissolve 33 mg / ml of TA in deionized water to prepare a TA aqueous solution. Then, a certain amount of BN was added to the TA solution with a mass ratio of TA to BN powder of 1:2. After ultrasonication for 30 minutes, the mixture was transferred to an agate jar containing 200 g of zirconia ball milling beads. After ball milling at 350 rpm in a planetary ball mill for 14 hours, the mixture was collected and centrifuged (2000 rpm, 10 minutes) to remove the remaining BN flakes. The supernatant was vacuum filtered, rinsed with plenty of water to remove excess TA, and dried in an oven at 35°C to obtain TN. Step 3, dissolving PVA at a concentration of 0.25 g / ml in deionized water and heating and stirring at 75° C. for 5 h until uniformly mixed to prepare a PVA aqueous solution (Solution A); Step 4: Dissolve TA at a concentration of 0.25 g / ml in deionized water, add 5 wt% SPN, and stir for a certain period of time. Then, add 0.5 wt% TN to mix the solution evenly (Solution B). Step 5: evenly mix solution A and solution B at a volume ratio of 1:1, and rinse with deionized water to obtain the bone adhesive material PTSN-TN.

[0035] Example 3 The present invention is a method for preparing a degradable bone adhesive material having an osteogenesis-promoting effect in a wet environment, which is specifically implemented according to the following steps: Step 1: 3 wt% SPI (soy protein isolate) was dissolved in deionized water, and the pH was adjusted to 7 with 0.1 M sodium hydroxide solution. EDC and NHS were added to the solution after 30 and 60 minutes, respectively, to make the molar ratio of SPI:EDC:NHS be 1:2:10 and mixed evenly. The mixture was finally stirred in a 40°C water bath for 4.5 hours, and then washed three times with deionized water. After centrifugation, a light yellow gel precipitate was obtained, which was collected and freeze-dried (freeze-drying temperature was -20°C and time was 36 hours). Step 2: Dissolve 33 mg / ml of TA in deionized water to prepare a TA aqueous solution. Then, a certain amount of BN was added to the TA solution with a mass ratio of TA to BN powder of 1:2. After ultrasonication for 30 minutes, the mixture was transferred to an agate jar containing 200 g of zirconia ball milling beads. After ball milling at 400 rpm in a planetary ball mill for 13 hours, the mixture was collected and centrifuged (2000 rpm, 10 minutes) to remove the remaining BN flakes. The supernatant was vacuum filtered, rinsed with plenty of water to remove excess TA, and dried in an oven at 40°C to obtain TN. Step 3, dissolving PVA at a concentration of 0.3 g / ml in deionized water and heating and stirring at 80° C. for 4.5 h until uniformly mixed to prepare a PVA aqueous solution (Solution A); Step 4: Dissolve TA at a concentration of 0.25 g / ml in deionized water, add 7% by weight of SPN, and stir for a certain period of time. Then, add 0.5% by weight of TN to mix the solution evenly (Solution B). Step 5: evenly mix solution A and solution B at a volume ratio of 1:1, and rinse with deionized water to obtain the bone adhesive material PTSN-TN.

[0036] Example 4 The present invention is a method for preparing a degradable bone adhesive material having an osteogenesis-promoting effect in a wet environment, which is specifically implemented according to the following steps: Step 1: 3 wt% SPI (soy protein isolate) was dissolved in deionized water, and the pH was adjusted to 7.5 with 0.1 M sodium hydroxide solution. After 30 min and 60 min, EDC and NHS were added to the solution in sequence to make the molar ratio of SPI:EDC:NHS 1:4:20 and mixed evenly. The mixture was finally stirred in a 40°C water bath for 4.5 h, and then washed three times with deionized water. After centrifugation, a light yellow gel precipitate was obtained, which was collected and freeze-dried (freeze-drying temperature was -20°C and time was 36 h). Step 2: Dissolve 35 mg / ml of TA in deionized water to prepare a TA aqueous solution. Then, a certain amount of BN was added to the TA solution at a mass ratio of TA to BN powder of 2:1. After ultrasonication for 40 minutes, the mixture was transferred to an agate jar containing 200 g of zirconia ball milling beads. After ball milling at 400 rpm in a planetary ball mill for 12 hours, the mixture was collected and centrifuged (2000 rpm, 10 minutes) to remove the remaining BN flakes. The supernatant was vacuum filtered, rinsed with copious amounts of water to remove excess TA, and dried in an oven at 45°C to obtain TN. Step 3, dissolving PVA at a concentration of 0.35 g / ml in deionized water and heating and stirring at 80° C. for 4.5 h until uniformly mixed to prepare a PVA aqueous solution (Solution A); Step 4: After dissolving TA at a concentration of 0.3 g / ml in deionized water, add 6% by mass of SPN and stir for a certain period of time, then add 0.75 wt% by mass of TN to mix the solution evenly (Solution B); Step 5: evenly mix solution A and solution B at a volume ratio of 1:1, and rinse with deionized water to obtain the bone adhesive material PTSN-TN.

[0037] Example 5 The present invention is a method for preparing a degradable bone adhesive material having an osteogenesis-promoting effect in a wet environment, which is specifically implemented according to the following steps: Step 1: 3.5 wt% SPI (soy protein isolate) was dissolved in deionized water, and the pH was adjusted to 8 with 0.1 M sodium hydroxide solution. After 30 min and 60 min, EDC and NHS were added to the solution in sequence to make the molar ratio of SPI:EDC:NHS 1:2:10 and mixed evenly. The mixture was finally stirred in a 45 ° C water bath for 5 h, and then the mixture was washed three times with deionized water. After centrifugation, a light yellow gel precipitate was obtained, which was collected and freeze-dried (freeze-drying temperature was -25 ° C, time was 48 h); In step 2, 40 mg / ml of TA was dissolved in deionized water to prepare a TA aqueous solution. A certain amount of BN was then added to the TA solution, with a mass ratio of TA to BN powder of 3:1. After ultrasonication for 50 minutes, the mixture was transferred to an agate jar containing 200 g of zirconia beads. After milling in a planetary ball mill at 450 rpm for 10 hours, the mixture was collected and centrifuged (2000 rpm for 10 minutes) to remove any remaining BN flakes. The supernatant was vacuum filtered, rinsed with copious amounts of water to remove excess TA, and dried in an oven at 50°C to obtain TN.

[0038] Step 3, dissolving PVA at a concentration of 0.35 g / ml in deionized water and heating and stirring at 85° C. for 4 h until uniformly mixed to prepare a PVA aqueous solution (Solution A); Step 4: After dissolving TA at a concentration of 0.3 g / ml in deionized water, add 8 wt% of SPN and stir for a certain period of time, then add 1 wt% of TN to mix the solution evenly (Solution B). Step 5: Evenly mix solution A and solution B at a volume ratio of 1:2, and rinse with deionized water to obtain the bone adhesive material PTSN-TN.

[0039] Example 6 The present invention is a method for preparing a degradable bone adhesive material having an osteogenesis-promoting effect in a wet environment, which is specifically implemented according to the following steps: Step 1: SPI (soy protein isolate) with a mass fraction of 4 wt% was dissolved in deionized water, and the pH was adjusted to 8.5 with 0.1 M sodium hydroxide solution. After 30 min and 60 min, EDC and NHS were added to the solution in sequence to make the molar ratio of SPI:EDC:NHS 1:2:10 and mixed evenly. The mixture was finally stirred in a 45 ° C water bath environment for 5 h, and then the mixture was washed three times with deionized water. After centrifugation, a light yellow gel precipitate was obtained, which was collected and freeze-dried (freeze-drying temperature was -25 ° C, time was 48 h); Step 2: Dissolve 45 mg / ml of TA in deionized water to prepare a TA aqueous solution. Then, a certain amount of BN was added to the TA solution with a mass ratio of TA to BN powder of 1:1. After ultrasonication for 55 minutes, the mixture was transferred to an agate jar containing 200 g of zirconia ball milling beads. After ball milling at 500 rpm in a planetary ball mill for 9 hours, the mixture was collected and centrifuged (2000 rpm, 10 minutes) to remove the remaining BN flakes. The supernatant was vacuum filtered, rinsed with plenty of water to remove excess TA, and dried in an oven at 55°C to obtain TN. Step 3, dissolving PVA at a concentration of 0.4 g / ml in deionized water and heating and stirring at 85° C. for 4 h until uniformly mixed to prepare a PVA aqueous solution (Solution A); Step 4: After dissolving TA at a concentration of 0.3 g / ml in deionized water, add 9 wt% of SPN and stir for a certain period of time, then add 1.25 wt% of TN to mix the solution evenly (Solution B). Step 5: Evenly mix solution A and solution B at a volume ratio of 1:2, and rinse with deionized water to obtain the bone adhesive material PTSN-TN.

[0040] Example 7 The present invention is a method for preparing a degradable bone adhesive material having an osteogenesis-promoting effect in a wet environment, which is specifically implemented according to the following steps: Step 1: 2 wt% SPI (soy protein isolate) was dissolved in deionized water, and the pH was adjusted to 8 with 0.1 M sodium hydroxide solution. After 30 min and 60 min, EDC and NHS were added to the solution in sequence to make the molar ratio of SPI:EDC:NHS 1:4:20 and mixed evenly. The mixture was finally stirred in a 45 ° C water bath for 6 h, and then the mixture was washed three times with deionized water. After centrifugation, a light yellow gel precipitate was obtained, which was collected and freeze-dried (freeze-drying temperature was -25 ° C, time was 72 h); Step 2: Dissolve 45 mg / ml of TA in deionized water to prepare a TA aqueous solution. Then, a certain amount of BN was added to the TA solution with a mass ratio of TA to BN powder of 1:1. After ultrasonication for 55 minutes, the mixture was transferred to an agate jar containing 200 g of zirconia ball milling beads. After ball milling at 500 rpm in a planetary ball mill for 9 hours, the mixture was collected and centrifuged (2000 rpm, 10 minutes) to remove the remaining BN flakes. The supernatant was vacuum filtered, rinsed with plenty of water to remove excess TA, and dried in an oven at 55°C to obtain TN. Step 3, dissolving PVA at a concentration of 0.4 g / ml in deionized water and heating and stirring at 90° C. for 3.5 h until uniformly mixed to prepare a PVA aqueous solution (Solution A); Step 4: Dissolve TA at a concentration of 0.35 g / ml in deionized water, add 10 wt% SPN, and stir for a certain period of time. Then, add 1.5 wt% TN to mix the solution evenly (Solution B). Step 5: Evenly mix solution A and solution B at a volume ratio of 1:2, and rinse with deionized water to obtain the bone adhesive material PTSN-TN.

[0041] Example 8 The present invention is a method for preparing a degradable bone adhesive material having an osteogenesis-promoting effect in a wet environment, which is specifically implemented according to the following steps: Step 1: 3 wt% SPI (soy protein isolate) was dissolved in deionized water, and the pH was adjusted to 9 with 0.1 M sodium hydroxide solution. EDC and NHS were added to the solution after 30 and 60 minutes, respectively, to make the molar ratio of SPI:EDC:NHS be 1:4:20 and mixed evenly. The mixture was finally stirred in a 60°C water bath for 8 hours, and then washed three times with deionized water. After centrifugation, a light yellow gel precipitate was obtained, which was collected and freeze-dried (freeze-drying temperature was -30°C and time was 72 hours). Step 2: Dissolve 50 mg / ml of TA in deionized water to prepare a TA aqueous solution. Then, a certain amount of BN was added to the TA solution with a mass ratio of TA to BN powder of 2:1. After ultrasonication for 60 minutes, the mixture was transferred to an agate jar containing 200 g of zirconia ball milling beads. After ball milling at 500 rpm in a planetary ball mill for 8 hours, the mixture was collected and centrifuged (2000 rpm, 10 minutes) to remove the remaining BN flakes. The supernatant was vacuum filtered, rinsed with plenty of water to remove excess TA, and dried in an oven at 60°C to obtain TN. Step 3, dissolving PVA at a concentration of 0.4 g / ml in deionized water and heating and stirring at 90° C. for 3 h until the mixture is uniformly mixed to prepare a PVA aqueous solution (Solution A); Step 4: After dissolving TA at a concentration of 0.4 g / ml in deionized water, add 11 wt% of SPN and stir for a certain period of time, then add 2 wt% of TN to mix the solution evenly (Solution B). Step 5: Evenly mix solution A and solution B at a volume ratio of 1:2, and rinse with deionized water to obtain the bone adhesive material PTSN-TN.

[0042] To verify the effectiveness of the present invention, the bonding performance, bonding time, and degradation rate of the bone adhesive materials prepared in Examples 1-8 of the present invention were compared with those of a light-curing liquid injection bone adhesive at different stages (the bonding substrates were all bovine bone slices), as shown in Tables 1-4: Table 1 Comparison of the bonding properties of the bone adhesive material of the present invention and other bone adhesives

[0043] Table 2 Comparison of the bonding performance of the bone adhesive material of the present invention and other bone adhesives in a wet environment at different times

[0044] The data in Tables 1 and 2 show that compared to existing light-curing bone adhesives, the bone adhesive prepared in this invention significantly shortens curing time due to its use of a cohesive colloid directly formed from TA and PVA. The rapid bonding of this adhesive is primarily due to hydrogen bonding interactions between the catechol groups of TA, the amino / carboxyl groups of SPN, and the hydroxyl groups of PVA, as well as the covalent interactions between the active ester of N-hydroxysuccinimide and the amino groups on the tissue surface.

[0045] Table 3 Comparison of mechanical strength properties of other bone adhesives and the bone adhesive material of the present invention

[0046] The data in Table 3 demonstrate that the addition of TA not only provides BN with more crosslinking sites, strengthening its adhesion to the matrix, but also improves the dispersibility of BN, making it more evenly dispersed throughout the overall network, increasing its aggregate density and energy dissipation. Acting as a secondary crosslinking site, this modification enhances the entanglement of the polymer network, thereby increasing the mechanical strength of the adhesive and, consequently, its compressive and flexural strengths.

[0047] Table 4 Comparison of degradation performance of other bone adhesives and the bone adhesive material of the present invention (degradation rate %)

[0048] Tables 2 and 4 combined demonstrate that the adhesive's degradation rate gradually increases over time, which promotes new bone formation. At approximately 30°C, the adhesive's degradation rate reaches 30% to 40%, while its bond strength remains at 2 to 3 MPa, far exceeding the clinically required strength for bone adhesion under physiological conditions (0.92 MPa), demonstrating the adhesive's high bond strength.

Claims

1. A method for preparing a degradable bone adhesive material having an osteogenesis effect suitable for use in a wet environment, characterized in that: The specific implementation steps are as follows: Step 1, preparing acylated soy protein isolate; Step 2, preparing TA-modified BN nanoparticles; Step 3, preparing a PVA aqueous solution; Step 4, dissolving the acylated soy protein isolate prepared in step 1 in the TA solution, and then adding the TA-modified BN nanoparticles prepared in step 2, and mixing them uniformly to obtain a B solution; Step 5: Mix the PVA aqueous solution prepared in step 3 and the B solution prepared in step 4, and then rinse with deionized water to obtain the bone adhesive material PTSN-TN.

2. The method for preparing a degradable bone adhesive material having an osteogenesis-promoting effect suitable for wet environments according to claim 1, characterized in that: The step 1 is specifically as follows: A certain amount of soy protein isolate SPI is dissolved in deionized water to obtain an SPI solution, and then a sodium hydroxide solution is used to adjust the pH of the SPI solution. After a certain period of time, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide active ester are added and mixed evenly. The mixture is then stirred in a water bath environment. The mixture is then washed with deionized water multiple times, and a light yellow gel precipitate is obtained after centrifugation. The precipitate is collected and freeze-dried to obtain the acylated soy protein isolate SPN.

3. The method for preparing a degradable bone adhesive material having an osteogenesis-promoting effect suitable for wet environments according to claim 2, characterized in that: In step 1, the amounts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide active ester added are added according to the following ratio: the molar ratio of soy protein isolate: 1-ethyl-(3-dimethylaminopropyl)carbodiimide: N-hydroxysuccinimide active ester is 1:1:1 to 1:4:

20.

4. The method for preparing a degradable bone adhesive material having an osteogenesis-promoting effect suitable for wet environments according to claim 2, characterized in that: The mass fraction of SPI in the SPI solution in step 1 is 2-4wt%, and the pH of the SPI solution is adjusted to 6-9 using a 0.1M sodium hydroxide solution; 1-ethyl-(3-dimethylaminopropyl)carbodiimide is added 30 minutes after the pH adjustment, and N-hydroxysuccinimide active ester is added 60 minutes after the pH adjustment. The mixture is heated and stirred in a water bath at a temperature of 30-60°C for 4-8 hours. The freeze-drying temperature is -20--30°C, and the freeze-drying time is 24-72 hours.

5. The method for preparing the degradable bone adhesive material having an osteogenesis-promoting effect suitable for wet environments according to any one of claims 1 to 4, characterized in that: The step 2 is specifically as follows: TA was dissolved in deionized water to prepare a TA aqueous solution, and then BN was added to the TA solution. After ultrasonic vibration, ball milling was performed. The mixture was collected and centrifuged to remove the remaining BN sheets. The supernatant was then vacuum filtered and then rinsed with deionized water to remove excess TA. The mixture was then dried to obtain TA-modified BN nanoparticles TN.

6. The method for preparing a degradable bone adhesive material having an osteogenesis-promoting effect suitable for use in a wet environment according to claim 5, characterized in that: In step 2, the concentration of TA in the TA aqueous solution is 30-50 mg / ml, the amount of BN powder added is: the mass ratio of TA to BN powder is 1:1-3:1; the ultrasonic time is 30 min-1 h; the ball mill speed is 300-500 rpm, and the speed duration is 8-15 h; the centrifugal speed is 2000 rpm, and the centrifugal time is 10 min; and the drying temperature is 30-60°C.

7. The method for preparing a degradable bone adhesive material having an osteogenesis-promoting effect suitable for use in a wet environment according to claim 5, characterized in that: The step 3 is specifically as follows: PVA is dissolved in deionized water and heated and stirred to prepare a PVA aqueous solution, which is recorded as solution A; wherein the concentration of PVA is 0.2-0.4 g / ml; the heating temperature is 70-90° C., and the stirring time is 3-5 hours.

8. The method for preparing a degradable bone adhesive material having an osteogenesis-promoting effect suitable for use in a wet environment according to claim 5, characterized in that: The step 4 specifically comprises: dissolving TA in deionized water to obtain a TA solution, then adding the SPN prepared in step 1 to the TA solution and stirring for a certain time, and then adding the TN prepared in step 2 to mix the solutions uniformly to obtain a B solution; The concentration of TA in the TA solution is 0.2~0.4g / ml, the mass fraction of SPN in the B solution is 4~10wt%, the mass fraction of TN is 0.25~2wt%, and the stirring time after adding SPN is 2~4h.

9. The method for preparing a degradable bone adhesive material having an osteogenesis-promoting effect suitable for use in a wet environment according to claim 5, characterized in that: The volume ratio of the PVA aqueous solution to the B solution in step 5 is 1:1 to 1:

2.

10. A degradable bone adhesive material, characterized in that: The bone adhesive is prepared by the method for preparing the degradable bone adhesive material having an osteogenesis effect suitable for a wet environment according to any one of claims 1 to 9.

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