Biodegradable bone glue and its preparation method and use

Abstract

The application provides biodegradable bone glue and a preparation method and use thereof, and belongs to the technical field of biological medicine. The bone glue is prepared from raw materials in a mass ratio of europium-doped polyphosphoric calcium: phosphorylated amino acid: polyacrylic acid: polyether glycol: metal-carboxyl-containing aromatic compound coordination complex=(1-3):(1-3):(2-5):(2-4):(0.1-0.3). The bone glue has significantly improved adhesion in a humid biological environment, and has excellent injectability, adhesion and low interfacial adhesion, and also has excellent biocompatibility, antibacterial and anti-inflammatory activity and osteogenesis promoting activity, effectively improves inflammation and oxidative stress problems in the bone healing process, and provides a more efficient and reliable repair material for clinical bone repair.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a biodegradable bone glue, its preparation method, and its uses. Background Technology

[0002] As the core supporting structure of the human musculoskeletal system, the skeleton not only undertakes key physiological functions such as weight-bearing, levering movement, and protecting organs, but also possesses a unique multi-level hierarchical structure and inorganic-organic composite interface characteristics. The skeleton is mainly composed of inorganic mineral phases such as hydroxyapatite, organic matrices such as collagen, and small amounts of water and bioactive factors. The inorganic phase forms a tight interfacial bond with the organic phase, giving the skeleton excellent mechanical strength and toughness. More importantly, bone tissue has a strong self-repair and regeneration capacity. In the event of minor injury, it can achieve wound healing through physiological processes such as osteocyte proliferation and differentiation, and bone matrix deposition and remodeling, maintaining its structural integrity and load-bearing stability.

[0003] However, in clinical practice, bone defects or fractures caused by accidents, illnesses, and other factors often exceed the body's own repair capabilities, requiring intervention with artificial repair techniques. Traditional bone repair methods primarily rely on internal fixation devices such as metal plates, screws, and intramedullary nails. While these offer reliable fixation and ease of postoperative disassembly and repair, they also have some technical limitations: due to inherent differences in biocompatibility with human bone tissue and a mismatch in elastic modulus, they can easily cause foreign body reactions and aseptic loosening, affecting long-term bone healing. Furthermore, for repairs of irregular bone defects, microcracks, and complex areas around joints, metal internal fixation devices struggle to achieve precise fit and adequate fixation, failing to effectively induce bone regeneration and repair. Therefore, there is an urgent need to develop a new repair technique to overcome the shortcomings of traditional bone repair methods.

[0004] In recent years, bone adhesives have gained significant attention in the field of bone repair as a novel alternative. They can fill bone defects in liquid or semi-solid form, rapidly solidify to form a prosthesis that adheres tightly to bone tissue, achieving precise filling and fixation of bone defects. Furthermore, they allow for personalized repair based on defect morphology. Currently, the most widely used traditional bone adhesives in clinical practice mainly include polymethyl methacrylate (PMMA) bone cement and calcium phosphate (CPC) bone cement. However, their practical application still faces many unresolved technical challenges, limiting their repair efficacy and application scope. PMMA bone cement suffers from poor biodegradability and weak adhesion to bone tissue, easily triggering chronic inflammatory reactions and potentially affecting bone regeneration and healing. It is also prone to interfacial delamination in moist physiological environments, leading to prosthesis loosening and failure. Calcium phosphate bone cement suffers from low mechanical strength and slow curing speed, making it difficult to meet the needs of load-bearing bone repair. It is prone to fracture under stress, and its curing process is easily disrupted in moist physiological environments, resulting in incomplete curing and affecting the structural stability of the prosthesis.

[0005] Therefore, current clinically used bone repair materials suffer from defects such as poor biocompatibility, mismatched mechanical properties, insufficient bone adhesion, and poor stability, making it difficult to fully meet the actual needs of clinical bone repair. Especially in the moist physiological environment of the human body, the adhesive properties of bone adhesives are easily affected by factors such as moisture erosion and ion interference, leading to a significant decrease in interfacial bonding strength, which in turn causes problems such as loosening of the repair and failure of bone healing. There is an urgent need to develop a bone adhesive with strong bone adhesion, good biocompatibility, suitable biodegradability, excellent mechanical properties, and stability in moist environments, which is of great significance to the development of clinical bone repair. Summary of the Invention

[0006] The purpose of this invention is to provide a biodegradable bone glue, its preparation method, and its uses.

[0007] The present invention provides a bone glue made from raw materials comprising the following mass ratio: europium-doped calcium polyphosphate: phosphorylated amino acids: polyacrylic acid: polyether diol: metal-carboxyl-containing aromatic compound coordination complex = (1~3): (1~3): (2~5): (2~4): (0.1~0.3).

[0008] Furthermore, it is prepared from raw materials comprising the following mass ratio: europium-doped calcium polyphosphate: phosphorylated amino acids: polyacrylic acid: polyether diol: metal-carboxyl-containing aromatic compound coordination complex = 2: 2: 3.75: 3: 0.2.

[0009] Further, the metal is copper; the carboxyl-containing aromatic compound is selected from trimellitic acid, terephthalic acid, phthalic acid, trimellitic acid, or pyromellitic acid; the phosphorylated amino acid is selected from phosphoserine, phosphothreonine, phostyrosine, or phosphohistidine; the polyether diol is selected from polyethylene glycol or polypropylene glycol; and the europium-doped calcium polyphosphate has a particle size of less than 5 μm.

[0010] Further, the europium-doped calcium polyphosphate is prepared by the following method: calcium carbonate and europium carbonate are added to phosphoric acid, the solvent is removed after reaction, and a precipitate is obtained; then it is washed, dried, calcined, melted and quenched to obtain an amorphous melt, which is then ground and sieved; the metal-carboxyl-containing aromatic compound coordination complex is prepared by the following method: the metal-containing compound is dissolved in an organic solvent, and then the carboxyl-containing aromatic compound is dissolved in water; subsequently, the metal-containing compound solution and the carboxyl-containing aromatic compound solution are mixed, reacted, centrifuged, washed, and dried to obtain the final product.

[0011] Furthermore, the polyacrylic acid is composed of low molecular weight polyacrylic acid with a molecular weight of 2kDa to 7kDa, medium and high molecular weight polyacrylic acid with a molecular weight of 200kDa to 300kDa, and high molecular weight polyacrylic acid with a molecular weight of 1000 to 1500kDa.

[0012] Furthermore, the polyacrylic acid is composed of a mixture of low molecular weight polyacrylic acid with a molecular weight of 5 kDa, medium-to-high molecular weight polyacrylic acid with a molecular weight of 240 kDa, and high molecular weight polyacrylic acid with a molecular weight of 1250 kDa.

[0013] Further, the mass ratio of the low molecular weight polyacrylic acid, the medium-to-high molecular weight polyacrylic acid, and the high molecular weight polyacrylic acid is (0.5~1.5):(0.5~1.5):(0.5~1.5);

[0014] Furthermore, the mass ratio of the low molecular weight polyacrylic acid, medium-to-high molecular weight polyacrylic acid, and high molecular weight polyacrylic acid is 1:1:1.

[0015] The present invention also provides a method for preparing bone glue, which involves mixing and stirring europium-doped calcium polyphosphate, phosphorylated amino acids, polyacrylic acid, a metal-carboxyl-containing aromatic compound coordination complex and polyether diol to form bone glue through self-assembly.

[0016] The present invention also provides the use of bone glue as a bone repair material.

[0017] This invention prepares a bone glue by using europium-doped calcium polyphosphate, phosphorylated amino acids, polyacrylic acid of different molecular weights, polyether diol, and metal-carboxyl-containing aromatic compound coordination complex. Compared with PMMA, it has significantly improved adhesion strength under humid conditions. At the same time, it has excellent injectability, adhesion, and low interfacial bonding, with a lower risk of adhesive failure and is more suitable for complex clinical environments. It also has excellent biocompatibility, antibacterial and anti-inflammatory properties, and osteopromoting activity.

[0018] Experiments have shown that Cu-BTC (copper-pyromellitic acid) has significantly better scavenging ability for reactive oxygen species (ROS) and DPPH free radicals than Co-BTC and Zn-BTC. Furthermore, bone glue with added Cu-BTC exhibits superior antibacterial properties and osteopromoting activity, with antibacterial rates of 94.3% and 92.7% against Escherichia coli and Staphylococcus aureus, respectively. Compared with bone glue without added Cu-BTC, its antibacterial rates against Escherichia coli and Staphylococcus aureus increased by 45.8% and 57.9%, respectively. It is also superior in promoting the production of COL-1 and OPN.

[0019] The bone glue of this invention improves adhesion in moist biological environments, effectively alleviates inflammation and oxidative stress during bone healing, and ensures good biocompatibility and osteogenic capacity, providing a more efficient and reliable new repair material for clinical bone repair.

[0020] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0021] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0022] Figure 1 The images show SEM (A), FTIR (B), and XRD (C) images of Cu-BTC, Zn-BTC, and Co-BTC nanozymes.

[0023] Figure 2 These are the ECPP XRD (A), SEM (B), EDS (C), and element mapping intensity (D).

[0024] Figure 3The characteristics of POPEM are: (A) adhesion at regular bone adhesion interfaces and injectability of bone glue; (B) load-bearing capacity after adhesion at regular bone adhesion interfaces; (C) adhesion at irregular bone adhesion interfaces; (D) FTIR test of bone glue; (E) SEM image of adhesion at irregular bone adhesion interfaces.

[0025] Figure 4 Activity assessment of Cu-BTC, Zn-BTC, and Co-BTC nanozymes: (A) Superoxide dismutase (SOD) simulated activity assessment; (B) •O2 activity assessment in the absence of Cu-BTC nanozymes and in the presence of 20 μg / mL Cu-BTC nanozymes. - (C) Absorption spectrum of the chromogenic reaction; (D) Absorption spectrum of H2O2 in the absence of Cu-BTC nanozyme and in the presence of 100 μg / mL Cu-BTC nanozyme; (E) DPPH elimination efficiency; (F) Absorption spectrum of DPPH in the absence of Cu-BTC nanozyme and in the presence of 20 μg / mL Cu-BTC nanozyme.

[0026] Figure 5 The adhesion strength tests for POPE and POPEM are as follows: (A) measured by head-to-head bonding; (B) measured by bridging bonding.

[0027] Figure 6 The antibacterial properties of POPE and POPEM were tested: (A) the inhibitory effects of POPE and POPEM on Escherichia coli and Staphylococcus aureus; (B) the inhibition rate of POPE and POPEM on Escherichia coli; and (C) the inhibition rate of POPE and POPEM on Staphylococcus aureus.

[0028] Figure 7 A is the biocompatibility test for POPE and POPEM; Figure 7 B~C are tests of POPE and POPEM on the secretion of inflammatory factors TNF-α and IL-10 by Raw 264.7; Figure 7 D represents the role of POPE and POPEM in clearing ROS from BMSCs cells; Figure 7 E is a test of the viability of BMSCs affected by ROS.

[0029] Figure 8 Osteogenic capacity tests for POPE and POPEM: (A) ALP staining on day 14 and ARS staining on day 21; (B) Quantitative analysis of ARS; (C) Quantitative analysis of ALP; (D~G) ELISA assay for osteogenic cytokines BMP-2, COL-1, OPN, and RUNX-2.

[0030] p<0.05, p<0.01, p<0.001, p<0.0001. Detailed Implementation

[0031] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0032] The raw materials used in the embodiments of this invention, including copper nitrate trihydrate, zinc nitrate hexahydrate, cobalt nitrate hexahydrate, anhydrous ethanol, sodium hydroxide, and phosphoric acid, were purchased from Chengdu Kelong Chemical Co., Ltd.; polyethylene glycol (PEG, molecular weight: 2000) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; and phosphoseserine, calcium carbonate, europium carbonate, trimesolic acid (BTC), and polyacrylic acid (PAA, 5kDa, 240kDa, 1250kDa) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0033] The PMMA used in the experimental example was purchased from Tianjin Synthetic Materials Industry Research Institute Co., Ltd., product name: acrylic resin bone cement, national medical device registration number 20183651624.

[0034] Example 1: Preparation and Characterization of Bone Glue

[0035] 1.1 Preparation of bone glue

[0036] (1) Preparation of europium-doped calcium polyphosphate (ECPP)

[0037] Preparation of ECPP with an Eu / Ca molar ratio of 5:95: CaCO3 and Eu2(CO3)3 were slowly added to phosphoric acid (mass fraction 85%) and the reaction was stirred continuously at 25℃ for 12 h; the solvent was evaporated by a rotary evaporator to obtain a precipitate, which was then thoroughly washed with ethanol until the pH of the filtrate was 7; after drying for 24 h, the precipitate was calcined at 500℃ for 10 h for polymerization, and then heated to 1100℃ at a heating rate of 15℃ / min to melt it. Subsequently, it was quenched in frozen distilled water to obtain an amorphous melt, which was then ground and screened to obtain ECPP with a particle size of less than 5 μm for later use.

[0038] (2) Preparation of Cu-BTC nanozymes

[0039] 2 mmol of copper nitrate trihydrate was dissolved in 3 mL of anhydrous ethanol, and then 1 mmol of BTC was dissolved in 3 mL of pure water. Subsequently, the copper nitrate solution and BTC solution were added to a 10 mL reactor and mixed. The mixture was heated at 110 °C for 18 hours. The resulting blue crystals were collected by centrifugation, washed alternately with ethanol and pure water, and finally dried at 60 °C to obtain Cu-BTC nanozyme.

[0040] (3) Preparation of bone glue

[0041] Preparation of POPEM: 0.2 g ECPP, 0.2 g phosphoserine (OPLS), 0.5 mL 25% (w / v) PAA (5 kDa), 0.5 mL 25% (w / v) PAA (240 kDa), 0.5 mL 25% (w / v) PAA (1250 kDa), 0.02 g Cu-BTC and 0.6 mL 50% (w / v) PEG were mixed and stirred to form POPEM bone glue by self-assembly; where w / v is g / mL.

[0042] Comparative Example 1: Preparation of Zn-BTC and Co-BTC nanozymes

[0043] The preparation of Zn-BTC and Co-BTC nanozymes is the same as that of Cu-BTC nanozymes, except that copper nitrate trihydrate is replaced with zinc nitrate hexahydrate and cobalt nitrate hexahydrate, respectively.

[0044] Comparative Example 2: Preparation of bone glue POPE

[0045] Preparation of POPE: 0.2g ECPP, 0.2g phosphoserine (OPLS), 0.5mL 25% (w / v) PAA (5kDa), 0.5mL 25% (w / v) PAA (240kDa), 0.5mL 25% (w / v) PAA (1250kDa) and 0.6mL 50% (w / v) PEG were mixed and stirred to form POPE bone glue.

[0046] 1.2 Characterization of bone glue

[0047] (1) Characterization of Co / Cu / Zn-BTC nanozymes

[0048] like Figure 1 As shown in Figure A, Co-BTC exhibits a spindle-shaped structure, Cu-BTC displays an irregularly shaped, sheet-like accumulation, while Zn-BTC is larger and elongated. Figure 1 As shown in B, 492cm -1 and 721cm -1 The bands at 663 cm⁻¹ are due to the bending and stretching modes of Cu-O, respectively; -1 ~766cm -1 The peaks observed between these peaks are in-ring and out-of-plane bending vibrations of the aromatic ring; at 827 cm⁻¹ -1 ~1153cm -1 The absorption band between them is attributed to the symmetric and asymmetric stretching modes of OC=O and the unreacted CO stretching vibrations of BTC; 1368 cm⁻¹ -1 1445cm -1 and 1640cm -1The strong absorption peaks at these locations are related to the stretching mode, asymmetric mode, and symmetric mode of CO, respectively. For example... Figure 1 As shown in Figure C, Cu-BTC, Zn-BTC, and Co-BTC all exhibit sharp peaks, indicating that they all have good crystallization morphology. The different peak positions are attributed to their different spatial folding modes and crystallization morphologies.

[0049] (2) Characterization of ECPP

[0050] like Figure 2 As shown in Figure A, by comparing with the standard PDF card (JCPD, 77-1953), the europium-doped calcium polyphosphate in this invention is β-crystalline. The three characteristic peaks of calcium phosphate between 20° and 30° indicate that it has good crystal morphology and is β-crystalline. Figure 2 As shown in B, the microstructure of ECPP is that of small, elongated flakes, with a length of approximately 1 μm; Figure 2 C presents the EDS spectra of different elements in ECPP, showing that these elements are uniformly distributed, indicating the successful preparation of ECPP; combined with Figure 2 The elemental mapping intensity of D clearly shows that Eu element was successfully incorporated, indicating that europium ions were successfully incorporated into the CPP lattice by replacing some calcium ions.

[0051] (3) Characterization of POPEM

[0052] Figure 3 A shows the effect of regular bone adhesion, demonstrating that POPEM has excellent injectability; at the same time, Figure 3 The load-bearing capacity of the three circular bone segments of B after bonding shows that it can easily lift 2.5 kg of water, indicating excellent adhesion. Figure 3 The irregular bone adhesion interface of C shows the low interfaciality of bone glue bonding.

[0053] like Figure 3 SEM testing of E showed that the irregular bone adhesion interface was 500 μm, which is lower than that of PMMA commonly used in clinical practice; a lower interface means a lower risk of adhesive failure.

[0054] like Figure 3 FTIR spectra of the various components of D, and FTIR measurements of ECPP and OPLS powders revealed two main peak clusters at 900 cm⁻¹. -1 Up to 1160cm -1 and 520cm -1 Up to 600cm -1 Both correspond to the main absorption mode associated with the presence of phosphate (PO4). 3- In the spectrum of collagen, a broad peak is observed between 3000 and 3500 cm⁻¹. -1The peak corresponding to the absorbed water is at 2698 cm⁻¹, and the peak corresponding to the carboxyl group is at 2698 cm⁻¹. -1 The disappearance is attributed to the reaction of the carboxyl group with the amino group in OPLS, or with the Ca in ECPP. 2+ Formation of coordination complexes. Appears at 1642 cm⁻¹ in POPE and POPEM. -1 The characteristic peak at 1451 cm⁻¹ corresponds to -COO- from the COOH group, or C=O stretched from the amide (formed via the reaction of carboxyl and amino groups in OPLS); -1 Peak value at CO3 2- The addition of Cu-BTC resulted in a leftward shift of the POPEM peak relative to POPE, which is related to the formation of hydrogen bonds. However, no new characteristic peaks appeared, indicating that Cu-BTC did not form new chemical bonds with other components of the bone glue.

[0055] The above characterization results show that the present invention has successfully prepared Cu-BTC, Zn-BTC, Co-BTC, ECPP, POPE and POPEM. At the same time, the POPEM of the present invention has excellent injectability, adhesion and low interfacial properties of adhesion, with a lower risk of adhesive failure, and can be applied in complex clinical environments.

[0056] Experimental Example 1: Cu-BTC's effect on reactive oxygen species (ROS) (superoxide radicals (•O2)) - Scavenging performance of hydrogen peroxide (H2O2) and DPPH free radicals

[0057] 1. Experimental Methods

[0058] (1) •O2 - Scavenging efficiency was determined using nitroblue tetrazolium chloride (NBT) as a probe: NBT and •O2 - The reaction-generated solution exhibited a characteristic peak at 560 nm (forming a blue substance, phenazine); the detection solution contained riboflavin (6.67 μM), methionine (4.33 mM), and NBT (25 μM), as well as different concentrations (2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL) of Co-BTC, Cu-BTC, or Zn-BTC samples, dissolved in PBS buffer (pH=7.35, 0.01 M); subsequently, the mixture was irradiated with a 30 W UV lamp for 5 minutes; finally, the absorbance of pyridine was measured using a UV-Vis spectrophotometer; •O2 - The removal efficiency is calculated using the following formula: [(A1-A2) / (A1-A0)]×100%, where A0 is the absorbance of a single NBT, and A1 and A2 are the absorbances of the sample (Co-BTC, Cu-BTC, or Zn-BTC) in the presence and absence of the sample, respectively.

[0059] (2) H2O2 scavenging ability: Different concentrations (10 μg / mL, 25 μg / mL, 50 μg / mL, 75 μg / mL, 100 μg / mL, 150 μg / mL) of samples (Co-BTC, Cu-BTC or Zn-BTC) were added to 10 mL of H2O2 solution (100 μM) and reacted for 2 h; subsequently, the supernatant of each group was mixed with 2 mL of Ti(SO4)2 solution (composed of 1.33 mL of 5% Ti(SO4)2 and 16.66 mL of 2M solution). (H2SO4 was prepared by mixing in 100 mL of deionized water) and mixed for 30 minutes; then the absorbance of the supernatant at 405 nm was measured using a UV-Vis spectrophotometer; the hydrogen peroxide removal rate was calculated according to the following formula: hydrogen peroxide removal rate = [(A0-A1) / A0]×100%, where A0 represents the absorbance of the mixture without the sample (Co-BTC, Cu-BTC or Zn-BTC, control sample), and A1 is the absorbance of the experimental group sample.

[0060] (3) DPPH free radical scavenging ability: Samples (Co-BTC, Cu-BTC or Zn-BTC) of different concentrations (2.5 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL) were added to DPPH ethanol solution (0.1 mM, 10 mL) and then incubated in the dark at 37 °C for 40 minutes; the absorbance of the solution at 517 nm was measured using a UV-Vis spectrophotometer and the DPPH scavenging ability was calculated according to the following formula: DPPH scavenging rate = [(D0-D1) / D0] × 100%, where D0 is the absorbance of the experimental group and D1 is the absorbance of DPPH.

[0061] 2. Experimental Results

[0062] like Figure 4 The simulated catalytic activity results of superoxide dismutase (SOD) shown in Figure A indicate that an increase in Cu-BTC concentration leads to a decrease in •O2. - The enhanced scavenging ability indicates a concentration-dependent scavenging effect. Furthermore, at the same concentration, the order of SOD-simulated catalytic activity was: Cu-BTC > Co-BTC > Zn-BTC; among them, at a concentration of 20 μg / mL, Cu-BTC nanozymes effectively scavenged over 60% of superoxide radicals, demonstrating superior SOD-simulated activity.

[0063] like Figure 4 The absorption spectrum of B shows that, under UV light, the absorption peak intensity of Cu-BTC at 550 nm is significantly lower than that under pure UV light, indicating that it has excellent SOD mimicry activity and can efficiently remove •O2. - .

[0064] like Figure 4 The CAT simulation results shown in C indicate that the H2O2 level decreases with increasing Cu-BTC concentration, suggesting that the CAT activity of Cu-BTC is concentration-dependent, reaching a relative activity of 68.12% at 100 μg / mL; at the same time, Cu-BTC exhibits the highest CAT simulation activity.

[0065] like Figure 4 The absorption spectrum shown in D shows that pure H2O2 exhibits a characteristic absorption peak at 405 nm. In the presence of 100 μg / mL Cu-BTC, the absorption intensity is significantly reduced, indicating that Cu-BTC has excellent H2O2 scavenging activity.

[0066] like Figure 4 The DPPH elimination efficiency results shown in E indicate that Cu-BTC has a significantly better elimination ability than Co-BTC and Zn-BTC. Figure 4 The absorption spectrum of F also shows that DPPH+Cu-BTC does not have the characteristic absorption peaks of pure DPPH, indicating that Cu-BTC has excellent DPPH scavenging ability.

[0067] The above experimental results show that Cu-BTC has excellent scavenging ability of ROS and DPPH free radicals, and is significantly better than Co-BTC and Zn-BTC.

[0068] Experimental Example 2: Test of Bone Glue Adhesion Performance

[0069] 1. Experimental Methods

[0070] The adhesion performance of the bone glue was tested using an Instron 5967 electronic universal testing machine (USA): Fresh bovine bones were purchased from a slaughterhouse, and bovine bone strips with a length of 50 mm, a width of 12 mm, and a thickness of 3 mm were prepared. The bovine bone strips were then bridged (area: 12 mm). 12mm) and "head-to-head" (area: 3 The 12mm bond was subjected to tensile testing at a rate of 20mm / min.

[0071] 2. Experimental Results

[0072] Under air-conditioning, the bond strengths measured head-to-head were as follows: PMMA bond sample 828.78±45.10 kPa, POPE bond sample 420.87±15.01 kPa, and POPEM bond sample 418.46±2.69 kPa. Figure 5A) When biomaterials are cured in a moist environment, the bond strength measured using a head-to-head configuration is significantly reduced, with the bond strength of samples bonded with PMMA, POPE, and POPEM decreasing by 97.55%, 83.14%, and 82.9%, respectively.

[0073] Under dry conditions, the bond strengths measured using the lap shear configuration were: PMMA bond sample 1797.92±142.72 kPa, POPE bond sample 1015.89±117.97 kPa, and POPEM bond sample 968.78±41.22 kPa. Figure 5 B) When biomaterials are cured in a humid environment, the bond strength measured using the lap shear configuration is significantly reduced, with the bond strength of samples bonded with PMMA, POPE and POPEM decreasing by 97.93%, 86.46% and 85.52%, respectively.

[0074] The above experimental results show that commercial adhesive PMMA has extremely low adhesion strength under wet conditions, while bone glue POPE and POPEM can still maintain high adhesion strength under wet conditions, making them more suitable for practical application scenarios. Among them, POPEM has better adhesion strength under wet conditions.

[0075] Experiment Example 3: Antibacterial Properties Test of Bone Glue

[0076] 1. Experimental Methods

[0077] The inhibitory effects of bone glue (POPE, POPEM) on Escherichia coli (typical Gram-negative bacteria) and Staphylococcus aureus (typical Gram-positive bacteria) were tested using the disc diffusion method: First, 120 μL of bacterial suspension (10 6 CFU / mL was inoculated onto an agar plate; then, bone glue (POPE or POPEM, diameter: 9 mm) was placed on the surface of the agar plate, and after incubation at 37°C for 24 hours, the size of the inhibition zone was recorded.

[0078] The antibacterial activity of bone glue was assessed using the plate count method: First, ultraviolet-sterilized bone glue samples were immersed in a bacterial suspension containing 1000 μL (10 6 The bacteria were co-cultured with bone glue in agar plates (CFU / mL) and then gently shaken at 180 rpm for 4 hours at 37°C. The morphology of the bacteria was observed using scanning electron microscopy (SEM) (the bacteria were fixed with 5% glutaraldehyde and dehydrated with ethanol before observation) to verify the antibacterial effect of the bone glue. Subsequently, 120 μL of the co-culture solution was spread on agar plates and photographed after 24 hours.

[0079] 2. Experimental Results

[0080] like Figure 6As shown in Figure A, the antibacterial effects of different bone glues on Escherichia coli and Staphylococcus aureus are as follows: with the increase of Cu-BTC content, the number of bacterial colonies in the culture dish co-cultured with POPEM is significantly reduced, indicating that the antibacterial activity of POPEM is significantly improved. Figure 6 Quantitative analysis of B-C showed that POPE had antibacterial rates of 64.7% and 58.7% against Escherichia coli and Staphylococcus aureus, respectively, while POPEM had antibacterial rates of 94.3% and 92.7% against Escherichia coli and Staphylococcus aureus, respectively. Compared with POPE, POPEM increased the antibacterial rates against Escherichia coli and Staphylococcus aureus by 45.8% and 57.9%, respectively. This fully demonstrates that the introduction of Cu-BTC significantly improved the antibacterial properties of bone glue, giving it a stronger bactericidal ability.

[0081] Experiment Example 4: Study on the cell compatibility, ROS scavenging, and anti-inflammatory effects of bone glue

[0082] 1. Experimental subjects

[0083] Cell culture: The culture medium consisted of 89% basal medium, 10% fetal bovine serum, and 1% penicillin-streptomycin (all from Gibco, USA); the cells were cultured in a 37°C incubator containing 5% carbon dioxide and 95% relative humidity; the culture medium was changed every 2 days, and the cells were washed with PBS before each change; human umbilical vein endothelial cells (HUVECs), RAW264.7 cells, and rat bone marrow mesenchymal stem cells (BMSCs) were all obtained from West China Hospital of Sichuan University.

[0084] 2. Experimental Methods

[0085] (1) In vitro BMSCs cell compatibility assay: All samples were sterilized by γ-ray; 50 mg of sterilized bone glue (POPE or POPEM) was placed in a well plate and 5 mL of culture medium was added for 72 h. The supernatant was taken as the bone glue extract; then 1 × 10⁻⁶ cells were added to each well. 4 BMSCs were seeded at a density of [number] cells per well, and conditioned medium was prepared at a ratio of extract:medium of 1:2. The viability of BMSCs at different time points was assessed using CCK-8 assay. The relative growth rate (RGR) percentage was used to represent the cell compatibility of the samples, calculated as: RGR (%) = OD1 / OD0 × 100%, where OD1 is the optical density of cells cultured with different bone glue, and OD0 is the optical density of cells in bone glue-free medium. BMSCs seeded in 24-well plates were co-cultured with the bone glue extract. On days 1 and 3, 10 μg / mL fluorescein diacetate (FDA) was added, and the cells were incubated for 30 minutes and photographed under an inverted fluorescence microscope (Nikon, Japan).

[0086] (2) Effect of bone glue on Raw 264.7 cells: The experiment was conducted according to the ELISA kit provided by Beyotime Biotechnology Co., Ltd. (China); RAW 264.7 macrophages (1×10 5 Cells ( / mL) were first pretreated with lipopolysaccharide (LPS, 1 μg / mL) for 2 hours, and then co-cultured with bone glue extract in 24-well culture plates for 72 hours; then the total content of pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine IL-10 was detected using an enzyme-linked immunosorbent assay (ELISA) kit.

[0087] (3) Intracellular reactive oxygen species (ROS) scavenging activity: Intracellular ROS production was measured using a ROS detection kit (Beyotime, China) according to the manufacturer's instructions. H2O2 was used as an exogenous oxidative stress inducer in BMSCs. After culturing with bone glue for 12 hours, the culture medium was replaced with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, 10 μM), which can be oxidized by ROS to produce fluorescence. The cells were then co-cultured for 25 minutes. Cells were observed under an inverted fluorescence microscope. All images were taken under the same conditions, and the fluorescence was quantitatively analyzed using ImageJ software. At the same time, the effect of ROS on BMSC cell viability was detected using a CCK-8 assay kit: cell viability = OD 处理组 / OD 对照组 100%.

[0088] 3. Experimental Results

[0089] like Figure 7 As shown in Figure A, the absorbance (OD value) of BMSCs co-cultured with CCK-8 at different time points showed no statistically significant difference in the absorbance of BMSCs cultured in all bone glue extracts, indicating excellent biocompatibility. Meanwhile, the OD value increased significantly with time, indicating that bone glue provided a favorable environment for cell growth.

[0090] like Figure 7 As shown in B~C, after LPS treatment, the expression level of the pro-inflammatory M1 marker TNF-α secreted by macrophages increased by 4.18 times, while the expression level of the anti-inflammatory M2 marker IL-10 decreased. After bone glue treatment, the expression level of TNF-α decreased significantly, while the expression level of IL-10 increased significantly. Compared with the LPS+POPE group, the expression level of TNF-α in the LPS+POPEM group was significantly decreased, while the expression level of IL-10 was significantly increased. This indicates that Cu-BTC can effectively scavenge reactive oxygen species and reduce stimulation of macrophages, and POPEM has excellent anti-inflammatory activity.

[0091] like Figure 7D shows the ROS clearance capacity test results of bone glue in BMSCs. POPEM significantly reduced the ROS level in BMSCs; Figure 7 As shown in Figure E, calculations showed that the viability of BMSCs cultured with POPEM extract was above 90%, while the viability of BMSCs in the H2O2 treatment group decreased to 53.20%, indicating that POPEM can effectively protect cells from H2O2-induced oxidative stress damage.

[0092] The above experimental results show that POPEM not only has good biocompatibility, but also effectively scavenges ROS, protects BMSCs from oxidative damage, and has excellent anti-inflammatory activity.

[0093] Experimental Example 5: In vitro osteogenic differentiation test

[0094] 1. Experimental Methods

[0095] BMSCs were 1×10 5 Cells were seeded at a density of 10 cells / well in 12-well plates. After 24 hours of culture, the cells were stimulated with ROS reagent (Beyotime, China) to simulate the oxidative stress microenvironment. The culture medium was replaced with the cell supernatant from co-culture of bone glue and RAW264.7 in Experiment 4 (2), and a 1:2 mixture of osteogenic medium (containing 10mM β-glycerophosphate and 50mg / mL ascorbic acid) for osteogenic studies.

[0096] After co-culturing and induction for 14 days, cells were fixed with 4% (w / v) paraformaldehyde for 30 minutes; ALP activity was then detected using an alkaline phosphatase (ALP) staining kit (Beyotime, China); BMSCs were incubated with ALP chromogenic agent under light-protected conditions for 30 minutes, and the results were observed under a microscope.

[0097] After co-culturing for 21 days, the cells were washed with PBS, fixed with 4% (w / v) paraformaldehyde for 30 minutes, and then stained with Alizarin Red S (ARS) for 15 minutes. The calcium nodules were then observed under a microscope.

[0098] The treated cells were co-incubated with 10% (v / v) acetic acid for 12 hours; the co-culture supernatant was then collected and centrifuged to remove solid particles, and then 10% (v / v) ammonia was added, and the absorbance of the supernatant at 405 nm was measured; after a 14-day incubation period, the levels of osteogenic-related cytokines (morphogenetic protein-2 (BMP-2), type I collagen (COL-1), osteoprotegerin (OPN), and RUNX-2) were determined using enzyme-linked immunosorbent assay (ELISA, Beyotime, China).

[0099] 2. Experimental Results

[0100] like Figure 8As shown in A~C, ALP expression in the POPE and POPEM groups was significantly higher than that in the control group, indicating that POPE and POPEM have excellent early osteogenic induction activity; both the POPE and POPEM groups showed significant mineralized calcium nodules and the deepest ARS staining on day 21, indicating that POPE and POPEM have excellent late osteogenic activity.

[0101] like Figure 8 The expression levels of osteogenic-related factors in BMSCs shown in D~G indicate that, compared with the control group, the expression levels of four key osteogenic markers, BMP-2, COL-1, OPN, and RUNX-2, were significantly increased in the POPE and POPEM groups, indicating that ECPP has a significant promoting effect on osteogenic differentiation. Compared with the POPE group, the expression levels of the four key osteogenic markers in the POPEM group were also significantly increased, proving that Cu-BTC also has a significant promoting effect on osteogenic differentiation. It is worth noting that Cu-BTC is more superior in promoting the production of COL-1 and OPN.

[0102] In summary, screening experiments revealed that Cu-BTC exhibits significantly superior ROS and DPPH radical scavenging capabilities compared to Co-BTC and Zn-BTC. Adhesion strength tests demonstrated that, compared to PMMA, POPEM of this invention exhibits significantly improved adhesion strength under humid conditions. Furthermore, it possesses excellent injectability, adhesion, and low interfacial adhesion, resulting in a lower risk of adhesive failure and making it more suitable for complex clinical environments. In antibacterial performance tests, POPEM demonstrated excellent antibacterial activity against Escherichia coli and Staphylococcus aureus. The antibacterial rates against bacteria were 94.3% and 92.7%, respectively. Compared with POPE, POPEM showed an increase of 45.8% and 57.9% in antibacterial rates against Escherichia coli and Staphylococcus aureus, respectively, demonstrating that the introduction of Cu-BTC significantly improved the antibacterial properties of the bone glue. In addition, POPEM also exhibited excellent biocompatibility. In the anti-inflammatory test, POPEM showed superior reactive oxygen species scavenging ability and anti-inflammatory activity compared to POPE. POPEM also showed excellent osteogenic activity, especially in promoting the production of COL-1 and OPN, which was significantly better than POPE.

Claims

1. A type of bone glue, characterized in that, It is prepared from raw materials comprising the following mass ratio: europium-doped calcium polyphosphate: phosphorylated amino acids: polyacrylic acid: polyether diol: metal-carboxyl-containing aromatic compound coordination complex = 2:2:3.75:3:0.2; wherein the metal is copper; wherein the carboxyl-containing aromatic compound is trimesic acid; wherein the polyacrylic acid is a mixture of low molecular weight polyacrylic acid with a molecular weight of 5 kDa, medium-to-high molecular weight polyacrylic acid with a molecular weight of 240 kDa, and high molecular weight polyacrylic acid with a molecular weight of 1250 kDa; wherein the mass ratio of the low molecular weight polyacrylic acid, medium-to-high molecular weight polyacrylic acid, and high molecular weight polyacrylic acid is 1:1:

1.

2. The bone glue according to claim 1, characterized in that, The phosphorylated amino acid is selected from phosphoserine, phosphothreonine, phosphotyrosine, or phosphohistidine; the polyether diol is selected from polyethylene glycol or polypropylene glycol; and the europium-doped calcium polyphosphate has a particle size of less than 5 μm.

3. The bone glue according to any one of claims 1 to 2, characterized in that, The europium-doped calcium polyphosphate is prepared by adding calcium carbonate and europium carbonate to phosphoric acid, reacting and removing the solvent to obtain a precipitate; then washing, drying, calcining, melting and quenching to obtain an amorphous melt, which is then ground and sieved. The metal-carboxyl-containing aromatic compound coordination complex is prepared by dissolving the metal compound in an organic solvent, then dissolving the carboxyl-containing aromatic compound in water; subsequently, mixing the metal compound solution and the carboxyl-containing aromatic compound solution, reacting, centrifuging, washing, and drying to obtain the final product.

4. The method for preparing bone glue according to any one of claims 1 to 3, characterized in that, Bone glue is formed by mixing and stirring europium-doped calcium polyphosphate, phosphorylated amino acids, polyacrylic acid, metal-carboxyl-containing aromatic compound coordination complex and polyether diol.

5. Use of the bone glue according to any one of claims 1 to 3 in the preparation of bone repair materials for curing in a moist biological environment.

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