Antibacterial bone adhesive as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of bone adhesives, and particularly relates to an antibacterial bone adhesive as well as a preparation method and application thereof. A callus-imitating bone adhesive is used as a main component and is mixed with an antibacterial component; the antibacterial components comprise sodium chloride, tea polyphenol, linoleic acid and linolenic acid; the mass ratio of the callus bone imitating adhesive to the sodium chloride to the tea polyphenol to the linoleic acid to the linolenic acid is 100: (0.5-0.6): (0.5-0.6): (0.1-0.5): (0.1-0.5); the antibacterial component is used for killing pathogenic bacteria. The antibacterial bone adhesive provided by the invention can selectively kill staphylococcus aureus, escherichia coli, pseudomonas aeruginosa and coagulase negative staphylococcus, does not influence the activity of normal osteoblasts, and is high in biological safety.

Description

Technical Field

[0001] This invention belongs to the field of bone adhesive technology, specifically relating to an antibacterial bone adhesive, its preparation method, and its application. Background Technology

[0002] The traditional treatment for fracture patients is internal fixation, which mainly uses intramedullary nails, metal plates and other methods to apply pressure to fix the fracture ends. Although the traditional fixation method can achieve good results, it still has the following defects: (1) It cannot be used for severe comminuted or complex fractures; (2) The use of screws and intramedullary nails can cause fracture stress problems and affect the growth of remaining tissues; (3) Metal is easy to wear and break, requiring a second surgery to remove it and it cannot be biodegraded.

[0003] Bone bonding agents are biomaterials used in orthopedic surgery to bond fracture fragments. They can overcome the defects of traditional fixation methods and have the following advantages: (1) Fast and effective bonding, with great potential to replace metal internal fixation devices; (2) Simple operation, such as simply dissolving the powder in the liquid phase and injecting it into the fracture surface, which improves the surgical effect and reduces the operation time; (3) Good biocompatibility, many bone bonding agent materials are biodegradable and can be naturally absorbed by the body after bone healing, which can avoid the risk of secondary surgery to remove internal fixation devices; (4) Highly minimally invasive, since bone bonding agent materials are injected, fewer surgical incisions are required, which can greatly reduce intraoperative trauma.

[0004] For the reasons mentioned above, existing technologies are dedicated to the research and development of bone adhesives, such as the imitation callus bone adhesive researched by the Air Force Medical University of the Chinese People's Liberation Army (see patent publication number CN120678984A), and the silk fibroin bone cement bio-adhesive researched by Fuzhou University (see patent publication number CN108744055A), etc.

[0005] However, bone bonding surgery is an invasive procedure, and germs in the environment may cause infection at the site of the surgery. Therefore, surgical patients usually need to undergo antibacterial and anti-inflammatory treatment. If an antibacterial bone repair agent could be developed, it would reduce the risk of surgical infection and the hassle of antibacterial and anti-inflammatory treatment. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides an antibacterial bone adhesive, its preparation method, and its application. In addition to meeting basic bone bonding and repair requirements, the antibacterial bone adhesive of this invention also has antibacterial effects, which can reduce the risk of surgical infection and the hassle of antibacterial and anti-inflammatory treatment.

[0007] The first objective of this invention is to provide an antibacterial bone adhesive, which uses a callus-like bone adhesive as the main component and is mixed with antibacterial ingredients; The antibacterial components are sodium chloride, tea polyphenols (CAS No. 84650-60-2), linoleic acid, and linolenic acid, which have good biosafety.

[0008] The mass ratio of the imitation callus bone adhesive, sodium chloride, tea polyphenols, linoleic acid, and linolenic acid is 100:0.5~0.6:0.5~0.6:0.1~0.5:0.1~0.5: the antibacterial component is used to fight pathogenic bacteria.

[0009] It should be noted that in the aforementioned antibacterial bone adhesive, the callus-like bone adhesive is obtained through a second polymerization reaction between hydrogen atoms in type I collagen fibers and isocyanate groups in the callus-like polyurethane bone adhesive prepolymer; wherein, the callus-like polyurethane bone adhesive prepolymer is obtained by a polyaddition reaction between hydroxyl groups in nano-hydroxyapatite and isocyanate groups in hexamethylene diisocyanate trimer. The specific method is described in our prior study (CN120678984A).

[0010] The second objective of this invention is to provide a method for preparing an antibacterial bone adhesive, wherein the simulated callus bone adhesive, sodium chloride, tea polyphenols, linoleic acid and linolenic acid are mixed evenly in a mass ratio of 100:0.5~0.6:0.5~0.6:0.1~0.5:0.1~0.5 to obtain the antibacterial bone adhesive.

[0011] A third objective of this invention is to provide an application of an antibacterial bone adhesive, which is used to prepare materials for bone repair, anti-pathogenic bacteria, or inhibition of osteoblast apoptosis, and the materials are suitable for in vivo or in vitro environments.

[0012] Preferably, in the application of the above-mentioned antibacterial bone adhesive, the anti-pathogenic bacteria refers to the ability to kill Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and coagulase-negative staphylococci.

[0013] Preferably, in the application of the above-mentioned antibacterial bone adhesive, the anti-pathogenic bacteria refers to selective bactericidal effect, that is, the bactericidal rate against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and coagulase-negative staphylococci is greater than the bactericidal rate against osteoblasts.

[0014] Preferably, in the application of the above-mentioned antibacterial bone adhesive, the inhibition of osteoblast apoptosis refers to the inhibition of osteoblast apoptosis caused by inflammatory factors.

[0015] Preferably, in the application of the above-mentioned antibacterial bone adhesive, the inflammatory factor is TNF-α.

[0016] Preferably, in the application of the above-mentioned antibacterial bone adhesive, the bone cells are osteoblasts.

[0017] Preferably, in the application of the above-mentioned antibacterial bone adhesive, the bone repair material is a material used for fracture fixation.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The antibacterial bone adhesive of the present invention uses a simulated callus bone adhesive as the main component, mixed with antibacterial ingredients; the antibacterial ingredients are sodium chloride, tea polyphenols, linoleic acid and linolenic acid; the mass ratio of the simulated callus bone adhesive, sodium chloride, tea polyphenols, linoleic acid and linolenic acid is 100:0.5~0.6:0.5~0.6:0.1~0.5:0.1~0.5; the antibacterial ingredients are used to kill pathogenic bacteria.

[0019] Sodium chloride has the function of regulating osmotic pressure and can reduce the water activity in the environment, thereby inhibiting the growth of pathogens. The higher the concentration, the stronger the bactericidal ability. However, it should be noted that excessively high sodium chloride concentrations can interfere with normal physiological metabolism. Therefore, this invention limits the mass of sodium chloride to 0.5% to 0.6% of the bone callus adhesive.

[0020] The antibacterial components of this invention also include tea polyphenols, which can disrupt the cell membrane integrity of pathogens to achieve antibacterial effects. In addition, they can also inhibit the decomposition and metabolism of collagenase and other enzymes, thereby inhibiting osteoblast apoptosis.

[0021] Linoleic acid and linolenic acid play an antibacterial role in this invention and inhibit osteoblast apoptosis.

[0022] This invention incorporates antibacterial components after preparing the callus-like bone adhesive, thus achieving both bone adhesion and antibacterial effects. In addition to meeting basic bone adhesion and repair needs, it reduces the risk of surgical infection and the hassle of postoperative antibacterial and anti-inflammatory treatment.

[0023] The antibacterial bone adhesive provided by this invention can selectively kill Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and coagulase-negative staphylococci without affecting the activity of normal osteoblasts. It can also inhibit osteoblast apoptosis induced by the inflammatory factor TNF-α. It has high safety. Attached Figure Description

[0024] Figure 1 This is a photograph of cranioplasty repair using the antibacterial bone adhesive of Example 1 of this invention.

[0025] Figure 2 This is a photograph of cranioplasty repair using the antibacterial bone adhesive of Example 2 of this invention.

[0026] Figure 3 This is a photograph of cranioplasty repair using the antibacterial bone adhesive of Example 3 of this invention.

[0027] Figure 4This is a photograph of cranioplasty repair using the antibacterial bone adhesive of Example 4 of this invention. Detailed Implementation

[0028] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0029] Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0030] This invention provides an antibacterial bone adhesive, including the following embodiments.

[0031] Example 1 An antibacterial bone adhesive, with a callus-like bone adhesive as the main component, mixed with antibacterial components; the antibacterial components are sodium chloride, tea polyphenols, linoleic acid and linolenic acid; the mass ratio of the callus-like bone adhesive, sodium chloride, tea polyphenols, linoleic acid and linolenic acid is 100:0.5:0.5:0.1:0.1.

[0032] The preparation method of the imitation callus bone adhesive includes the following steps: (1) Mix 0.5g of Tri-HDI and 0.33g of nHAP evenly, add 2.5μL of acetyl chloride and 2.5μL of dibutyltin dilaurate as catalysts, stir evenly, and react at 75℃ for 3min under nitrogen to obtain a viscous prepolymer, which is denoted as Pre-CLA.

[0033] (2) Weigh 0.01 g of Col I and add it to the prepared Pre-CLA at room temperature. Mix evenly to obtain a callus-like bone adhesive with a collagen content of 2% of Tri-HDI. This method is performed according to Example 1 of our prior art CN120678984A.

[0034] The preparation method of the antibacterial bone adhesive is as follows: The antibacterial bone adhesive is obtained by mixing the simulated callus bone adhesive, sodium chloride, tea polyphenols, linoleic acid and linolenic acid evenly according to the mass ratio of 100:0.5:0.5:0.1:0.1.

[0035] Example 2 An antibacterial bone adhesive, with a callus-like bone adhesive as the main component, mixed with antibacterial components; the antibacterial components are sodium chloride, tea polyphenols, linoleic acid and linolenic acid; the mass ratio of the callus-like bone adhesive, sodium chloride, tea polyphenols, linoleic acid and linolenic acid is 100:0.6:0.5:0.1:0.1.

[0036] The preparation method of the imitation callus bone adhesive includes the following steps: (1) Mix 0.5g of Tri-HDI and 0.33g of nHAP evenly, add 2.5μL of acetyl chloride and 2.5μL of dibutyltin dilaurate as catalysts, stir evenly, and react at 75℃ for 3min under nitrogen to obtain a viscous prepolymer, which is denoted as Pre-CLA.

[0037] (2) Weigh 0.01g of Col I and add it to the prepared Pre-CLA at room temperature. Mix evenly to obtain a callus-like bone adhesive with a collagen content of 2% of Tri-HDI.

[0038] The preparation method of the antibacterial bone adhesive is as follows: The antibacterial bone adhesive is obtained by mixing the imitation callus bone adhesive, sodium chloride, tea polyphenols, linoleic acid and linolenic acid evenly according to the mass ratio of 100:0.6:0.5:0.1:0.1.

[0039] Example 3 An antibacterial bone adhesive, with a callus-like bone adhesive as the main component, mixed with antibacterial components; the antibacterial components are sodium chloride, tea polyphenols, linoleic acid and linolenic acid; the mass ratio of the callus-like bone adhesive, sodium chloride, tea polyphenols, linoleic acid and linolenic acid is 100:0.5:0.6:0.1:0.1.

[0040] The preparation method of the imitation callus bone adhesive includes the following steps: (1) Mix 0.5g of Tri-HDI and 0.33g of nHAP evenly, add 2.5μL of acetyl chloride and 2.5μL of dibutyltin dilaurate as catalysts, stir evenly, and react at 75℃ for 3min under nitrogen to obtain a viscous prepolymer, which is denoted as Pre-CLA.

[0041] (2) Weigh 0.01g of Col I and add it to the prepared Pre-CLA at room temperature. Mix evenly to obtain a callus-like bone adhesive with a collagen content of 2% of Tri-HDI.

[0042] The preparation method of the antibacterial bone adhesive is as follows: The antibacterial bone adhesive is obtained by mixing the simulated callus bone adhesive, sodium chloride, tea polyphenols, linoleic acid and linolenic acid evenly according to the mass ratio of 100:0.5:0.6:0.1:0.1.

[0043] Example 4 An antibacterial bone adhesive, with a callus-like bone adhesive as the main component, mixed with antibacterial components; the antibacterial components are sodium chloride, tea polyphenols, linoleic acid and linolenic acid; the mass ratio of the callus-like bone adhesive, sodium chloride, tea polyphenols, linoleic acid and linolenic acid is 100:0.5:0.5:0.5:0.1.

[0044] The preparation method of the imitation callus bone adhesive includes the following steps: (1) Mix 0.5g of Tri-HDI and 0.33g of nHAP evenly, add 2.5μL of acetyl chloride and 2.5μL of dibutyltin dilaurate as catalysts, stir evenly, and react at 75℃ for 3min under nitrogen to obtain a viscous prepolymer, which is denoted as Pre-CLA.

[0045] (2) Weigh 0.01g of Col I and add it to the prepared Pre-CLA at room temperature. Mix evenly to obtain a callus-like bone adhesive with a collagen content of 2% of Tri-HDI.

[0046] The preparation method of the antibacterial bone adhesive is as follows: The antibacterial bone adhesive is obtained by mixing the simulated callus bone adhesive, sodium chloride, tea polyphenols, linoleic acid and linolenic acid evenly according to the mass ratio of 100:0.5:0.5:0.5:0.1.

[0047] Example 5 An antibacterial bone adhesive, with a callus-like bone adhesive as the main component, mixed with antibacterial components; the antibacterial components are sodium chloride, tea polyphenols, linoleic acid and linolenic acid; the mass ratio of the callus-like bone adhesive, sodium chloride, tea polyphenols, linoleic acid and linolenic acid is 100:0.5:0.5:0.1:0.5.

[0048] The preparation method of the imitation callus bone adhesive includes the following steps: (1) Mix 0.5g of Tri-HDI and 0.33g of nHAP evenly, add 2.5μL of acetyl chloride and 2.5μL of dibutyltin dilaurate as catalysts, stir evenly, and react at 75℃ for 3min under nitrogen to obtain a viscous prepolymer, which is denoted as Pre-CLA.

[0049] (2) Weigh 0.01g of Col I and add it to the prepared Pre-CLA at room temperature. Mix evenly to obtain a callus-like bone adhesive with a collagen content of 2% of Tri-HDI.

[0050] The preparation method of the antibacterial bone adhesive is as follows: The antibacterial bone adhesive is obtained by mixing the simulated callus bone adhesive, sodium chloride, tea polyphenols, linoleic acid and linolenic acid evenly according to the mass ratio of 100:0.5:0.5:0.1:0.0.5.

[0051] Control group 1 (blank control - imitation callus bone adhesive) An antibacterial bone adhesive, with a callus-like bone adhesive as its sole component.

[0052] The preparation method of the imitation callus bone adhesive includes the following steps: (1) Mix 0.5g of Tri-HDI and 0.33g of nHAP evenly, add 2.5μL of acetyl chloride and 2.5μL of dibutyltin dilaurate as catalysts, stir evenly, and react at 75℃ for 3min under nitrogen to obtain a viscous prepolymer, which is denoted as Pre-CLA.

[0053] (2) Weigh 0.01g of Col I and add it to the prepared Pre-CLA at room temperature. Mix evenly to obtain a callus-like bone adhesive with a collagen content of 2% of Tri-HDI.

[0054] Control group 2 (high concentration of sodium chloride) An antibacterial bone adhesive, with a callus-like bone adhesive as the main component, mixed with antibacterial components; the antibacterial components are sodium chloride, tea polyphenols, linoleic acid and linolenic acid; the mass ratio of the callus-like bone adhesive, sodium chloride, tea polyphenols, linoleic acid and linolenic acid is 100:0.9:0.1:0.1.

[0055] The preparation method of the imitation callus bone adhesive includes the following steps: (1) Mix 0.5g of Tri-HDI and 0.33g of nHAP evenly, add 2.5μL of acetyl chloride and 2.5μL of dibutyltin dilaurate as catalysts, stir evenly, and react at 75℃ for 3min under nitrogen to obtain a viscous prepolymer, which is denoted as Pre-CLA.

[0056] (2) Weigh 0.01g of Col I and add it to the prepared Pre-CLA at room temperature. Mix evenly to obtain a callus-like bone adhesive with a collagen content of 2% of Tri-HDI.

[0057] The preparation method of the antibacterial bone adhesive is as follows: The antibacterial bone adhesive is obtained by mixing the imitation callus bone adhesive, sodium chloride, linoleic acid and linolenic acid evenly according to the mass ratio of 100:0.9:0.1:0.1.

[0058] Control group 3 (simulated callus bone bonding agent + sodium chloride) An antibacterial bone adhesive, with a callus-like bone adhesive as the main component, mixed with an antibacterial component; the antibacterial component is sodium chloride; the mass ratio of the callus-like bone adhesive to sodium chloride is 100:0.5.

[0059] The preparation method of the imitation callus bone adhesive includes the following steps: (1) Mix 0.5g of Tri-HDI and 0.33g of nHAP evenly, add 2.5μL of acetyl chloride and 2.5μL of dibutyltin dilaurate as catalysts, stir evenly, and react at 75℃ for 3min under nitrogen to obtain a viscous prepolymer, which is denoted as Pre-CLA.

[0060] (2) Weigh 0.01g of Col I and add it to the prepared Pre-CLA at room temperature. Mix evenly to obtain a callus-like bone adhesive with a collagen content of 2% of Tri-HDI.

[0061] The preparation method of the antibacterial bone adhesive is as follows: The imitation callus bone adhesive and sodium chloride are mixed evenly at a mass ratio of 100:0.5 to obtain the antibacterial bone adhesive.

[0062] Control group 4 (simulated callus bone bonding agent + tea polyphenols) An antibacterial bone adhesive, with a callus-like bone adhesive as the main component, mixed with an antibacterial component; the antibacterial component is tea polyphenol; the mass ratio of the callus-like bone adhesive to the tea polyphenol is 100:0.5.

[0063] The preparation method of the imitation callus bone adhesive includes the following steps: (1) Mix 0.5g of Tri-HDI and 0.33g of nHAP evenly, add 2.5μL of acetyl chloride and 2.5μL of dibutyltin dilaurate as catalysts, stir evenly, and react at 75℃ for 3min under nitrogen to obtain a viscous prepolymer, which is denoted as Pre-CLA.

[0064] (2) Weigh 0.01g of Col I and add it to the prepared Pre-CLA at room temperature. Mix evenly to obtain a callus-like bone adhesive with a collagen content of 2% of Tri-HDI.

[0065] The antibacterial bone adhesive is prepared by mixing the simulated callus bone adhesive and tea polyphenols evenly.

[0066] Control group 5 (simulated callus bone bonding agent + linoleic acid) An antibacterial bone adhesive, with a callus-like bone adhesive as the main component, mixed with an antibacterial component; the antibacterial component is linoleic acid; the mass ratio of the callus-like bone adhesive to linoleic acid is 100:0.1.

[0067] The preparation method of the imitation callus bone adhesive includes the following steps: (1) Mix 0.5g of Tri-HDI and 0.33g of nHAP evenly, add 2.5μL of acetyl chloride and 2.5μL of dibutyltin dilaurate as catalysts, stir evenly, and react at 75℃ for 3min under nitrogen to obtain a viscous prepolymer, which is denoted as Pre-CLA.

[0068] (2) Weigh 0.01g of Col I and add it to the prepared Pre-CLA at room temperature. Mix evenly to obtain a callus-like bone adhesive with a collagen content of 2% of Tri-HDI.

[0069] The preparation method of the antibacterial bone adhesive is as follows: The imitation callus bone adhesive and linoleic acid are mixed evenly at a mass ratio of 100:0.1 to obtain the antibacterial bone adhesive.

[0070] Control group 6 (simulated callus bone bonding agent + linolenic acid) An antibacterial bone adhesive, with a callus-like bone adhesive as the main component, mixed with an antibacterial component; the antibacterial component is linolenic acid; the mass ratio of the callus-like bone adhesive to linolenic acid is 100:0.1.

[0071] The preparation method of the imitation callus bone adhesive includes the following steps: (1) Mix 0.5g of Tri-HDI and 0.33g of nHAP evenly, add 2.5μL of acetyl chloride and 2.5μL of dibutyltin dilaurate as catalysts, stir evenly, and react at 75℃ for 3min under nitrogen to obtain a viscous prepolymer, which is denoted as Pre-CLA.

[0072] (2) Weigh 0.01g of Col I and add it to the prepared Pre-CLA at room temperature. Mix evenly to obtain a callus-like bone adhesive with a collagen content of 2% of Tri-HDI.

[0073] The preparation method of the antibacterial bone adhesive is as follows: The imitation callus bone adhesive and linolenic acid are mixed evenly at a mass ratio of 100:0.1 to obtain the antibacterial bone adhesive.

[0074] It should be noted that this invention does not directly conduct single-factor experiments on sodium chloride, tea polyphenols, linoleic acid, and linolenic acid because these substances alone cannot form bone adhesion and do not meet the basic requirements of the adhesive repair of this invention. This invention focuses on how to improve the antibacterial and repair properties of the imitation callus bone adhesive. Therefore, the imitation callus bone adhesive alone is a blank control, while the types of antibacterial ingredients added are used as control experimental groups to compare the performance differences of different antibacterial bone adhesives.

[0075] I. Fracture Repair Results Figure 1 This is a photograph of cranioplasty repair using the antibacterial bone adhesive of Example 1 of this invention. Figure 2 This is a photograph of cranioplasty repair using the antibacterial bone adhesive of Example 2 of this invention. Figure 3 This is a photograph of cranioplasty repair using the antibacterial bone adhesive of Example 3 of this invention. Figure 4 This is a photograph of cranioplasty repair using the antibacterial bone adhesive of Example 4 of this invention.

[0076] Fracture Repair Experimental Procedure: A cranial ring fracture model of the skull was established using 6-week-old SD rats. Anesthesia was administered via intraperitoneal injection of sodium pentobarbital (30 mg / kg). After thorough disinfection, a scalp incision was made along the midline of the rat's scalp, and the periosteum of the skull was fully dissected to expose the surgical area. A complete circular bone fragment was then cut out using a 4 mm diameter trephine to form the fracture model. During the procedure, the fracture fragment was kept moist to prevent tissue dehydration from affecting subsequent healing. Adhesive was evenly applied around the circular fracture fragment to connect it with the surrounding bone tissue. Results showed that the fracture repair method prepared in this invention can fix and repair fractured bone. After two days of routine postoperative disinfection and antibacterial treatment, no infection occurred, demonstrating practical application value. Figures 1-4 In the image, the circled area represents the fractured bone that has been fixed and repaired.

[0077] II. Antibacterial Test Studies show that bacterial infections are the most common among implant-related infections, with Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and coagulase-negative staphylococci or their related species being the most prevalent. Therefore, this invention conducts antibacterial experiments on all of these species to meet the antibacterial requirements for implant-based applications.

[0078] The antibacterial test results are as follows: The sterilization rate was calculated using existing techniques. The following bacterial strains were prepared: Staphylococcus aureus ATCC25923, Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC 27853, and coagulase-negative staphylococci (CNS). After one activation of the bacterial strains, a concentration of 10 was prepared. 6For each 100 mL of bacterial solution containing 1 cell / mL, 1 g of test material was added and allowed to react for 30 min. The viable cell concentration before and after the reaction was statistically analyzed using the viable cell technique, and the sterilization rate was calculated.

[0079] Sterilization rate (%) = 10 × [(Viable bacteria concentration before treatment - Viable bacteria concentration after treatment) / Cell concentration before treatment].

[0080] Table 1. Statistical results of bacterial sterilization rate Control group 1 demonstrates the antibacterial effect of the original imitation callus bone adhesive. As shown in Table 1, the antibacterial effect of the imitation callus bone adhesive alone is very weak, almost nonexistent; its main function is... Figures 1-4 The bone bonding effect is shown.

[0081] Although the sodium chloride concentration in control group 2 was the same as that of physiological saline, the results in Table 1 show that, under the premise of the antibacterial requirements of this invention, this concentration of physiological saline was not suitable and could not achieve the antibacterial effect. It needed to be reduced to the concentration of the example and used in combination with tea polyphenols, linoleic acid, and linolenic acid to demonstrate a superior antibacterial effect. The reason for this consideration is that a 0.9% mass fraction of physiological saline provides suitable physiological potential signals and electrolytes, which is not conducive to destroying the cell membrane of pathogens and does not meet the antibacterial requirements of this invention.

[0082] Control groups 3-6 showcased the antibacterial effects of combinations of the simulated callus bone adhesive and single-factor formulations. Table 1 shows that the single-factor formulations, due to their limited and singular selection of substances, while still maintaining the basic requirements for bone adhesion, failed to produce significant antibacterial effects. This is likely because when tea polyphenols, linoleic acid, and linolenic acid were individually combined with the simulated callus bone adhesive, no synergistic effect was observed; rather, each substance acted independently.

[0083] Compared with the control groups, Examples 1 to 4 contain a rich variety of substances and more elements that regulate physiological potential and the membrane structure of pathogenic bacteria, thus producing a synergistic effect that can rapidly destroy the membrane structure of pathogenic bacteria and achieve a significant antibacterial effect.

[0084] It should be noted that some existing antibacterial materials, while inhibiting pathogens such as Staphylococcus aureus, may also kill a small number of normal cells, which can have adverse side effects on fracture repair and may delay recovery. The antibacterial material provided by this invention can selectively kill Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and coagulase-negative staphylococci, while having no inhibitory effect on ordinary somatic cells, such as osteoblasts, exhibiting high biosafety. Osteoblasts, with different genome sequences than pathogenic bacteria, have their physiological metabolism regulated by different signaling pathways, making them more resistant to antibacterial components and unaffected by these components, thus maintaining basic physiological activity.

[0085] III. Inflammatory Factor-Induced Osteoblast Injury Test (1) Osteoblasts were collected using an enzymatic method: neonatal C57BL / 6 suckling mice (≤24 h). After euthanizing the suckling mice, they were disinfected by soaking in 75% alcohol, rinsed twice with sterile phosphate buffer (pH=7.2), and the skull bone was cut off in a laminar flow hood. The periosteum and blood were removed, and the bone material was rinsed twice with sterile PBS to obtain bone material. The bone material was cut into small pieces and transferred to sterile tubes containing 0.25% trypsin-EDTAEP (preheated at 37°C). The tubes were incubated for 20 min in a cell incubator containing 5% CO2 at 37°C. Then, the tubes were centrifuged at 1500 r / min for 5 min, and 0.1% type II collagenase was added. The tubes were digested at 37°C for 30 min. The digestion was repeated twice. All digestion liquid was collected, centrifuged at 300g for 5 min, and the supernatant was discarded. After resuspending and mixing the osteoblasts, they were seeded in T25 cell culture flasks. The cell culture medium was α-MEM containing 10% fetal bovine serum and 1% penicillin and streptomycin (0.5% each). Primary osteoblasts were obtained by culturing.

[0086] (2) Treatment, CCK-8 method: Primary osteoblasts were seeded at a concentration of 50,000 per well in 96-well plates. 100 μL of α-MEM medium containing 10% (v / v) fetal bovine serum and 1% (w / v) penicillin and streptomycin (0.5% each) was added to each well. TNF-α was then added to bring the final concentration to 10 ng / mL, followed by the test reagent to bring the final concentration to 10 μg / mL. The plates were incubated for 48 h. The old medium was discarded, and 100 μL of fresh α-MEM medium containing 10% (v / v) fetal bovine serum and 1% (w / v) penicillin and streptomycin (0.5% each) and 10 μL of CCK-8 were added to each well. The plates were incubated at 37°C in the dark for 2 h. The absorbance at 450 nm was measured, and osteoblast viability was calculated.

[0087] The control group consisted of no experimental reagents added, serving as the model group. The control group consisted of no experimental reagents added and TNF-α added.

[0088] Each experiment was conducted in triplicate, and the average value was taken.

[0089] Table 2 Results of osteoblast viability test Osteoblasts are responsible for secreting osteoid, a matrix rich in type I collagen, and for inducing hydroxyapatite crystal deposition through the release of alkaline phosphatase (ALP) and osteocalcin (OCN) from vesicles, thus completing bone matrix mineralization. Therefore, they play a crucial role in regulating bone remodeling balance. TNF-α may impair both the number and function of osteoblasts through a mechanism of "pro-apoptosis + inhibition of differentiation + disruption of RANKL / OPG balance." Therefore, this invention designs experiments targeting the influence of this inflammatory factor and osteoblasts to identify reagents that can resist TNF-α interference, aiming to repair bone fractures while mitigating the risks of inflammatory factors.

[0090] As shown in Table 2, Examples 1-5 can maintain osteoblast viability and alleviate the problem of decreased cell viability caused by TNF-α. The results of Control Group 2 are similar to those of Example 5, likely because the sodium chloride concentration is at the physiological saline level. The other control groups showed poorer results and did not effectively alleviate the problem of decreased cell viability caused by TNF-α. This is likely because these control groups primarily consist of bone adhesives, which are simpler in composition and therefore have a significant bonding effect, but cannot address the weakening of osteoblast viability caused by external factors.

[0091] Furthermore, the antibacterial bone bonding materials provided in Examples 1-5 of this invention exhibit antibacterial effects in in vitro antibacterial experiments and can be used as in vitro antibacterial materials. Following in vivo cranioplasty, routine anti-inflammatory treatment for 2 days resulted in no infection. In contrast, the materials in Control Groups 1-6, after routine anti-inflammatory treatment for 2 days, showed an infection rate of 5%-10%, requiring at least 3 days of routine anti-inflammatory treatment to prevent infection. For user safety, it is still recommended to administer anti-inflammatory treatment after bone repair surgery to avoid or reduce inflammatory responses.

[0092] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described in this invention to avoid redundancy. Although preferred embodiments of this invention have been described, those skilled in the art, once they understand the inventive concept of this invention, can make other changes and modifications to these embodiments, and all such changes and modifications fall within the scope of this invention.

[0093] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. If such modifications and variations fall within the scope of equivalents of this invention, then this invention is also intended to include these modifications and variations.

Claims

1. An antibacterial bone adhesive, characterized in that, It uses a callus-like bone adhesive as the main component, mixed with antibacterial ingredients; The antibacterial components are sodium chloride, tea polyphenols, linoleic acid, and linolenic acid; The mass ratio of the imitation callus bone adhesive, sodium chloride, tea polyphenols, linoleic acid, and linolenic acid is 100:0.5~0.6:0.5~0.6:0.1~0.5:0.1~0.5; The antibacterial component is used to fight pathogenic bacteria.

2. A method for preparing the antibacterial bone adhesive according to claim 1, characterized in that, The antibacterial bone adhesive is obtained by mixing the imitation callus bone adhesive, sodium chloride, linoleic acid and linolenic acid evenly according to the mass ratio of 100:0.5~1:0.1~0.5:0.1~0.

5.

3. The application of the antibacterial bone adhesive according to claim 1, characterized in that, The antibacterial bone adhesive is used to prepare materials for bone repair or to inhibit osteoblast apoptosis.

4. The application of the antibacterial bone adhesive according to claim 3, characterized in that, The term "anti-pathogenic bacteria" refers to bacteria that kill Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and coagulase-negative staphylococci.

5. The application of the antibacterial bone adhesive according to claim 3, characterized in that, The inhibition of osteoblast apoptosis refers to the inhibition of osteoblast apoptosis caused by inflammatory factors.

6. The application of the antibacterial bone adhesive according to claim 5, characterized in that, The inflammatory factor is TNF-α.

7. The application of the antibacterial bone adhesive according to claim 3, characterized in that, The bone cells mentioned are osteoblasts.

8. The application of the antibacterial bone adhesive according to claim 3, characterized in that, The bone repair material is used for fracture fixation.

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