Preparation method of antibacterial and anti-inflammatory bone repair composite material

Abstract

The invention discloses an antibacterial and anti-inflammatory bone repair composite material and a preparation method thereof. The composite material is formed by compounding (S)-lactic acid hydrazone-based polyamide, 2, 5-dihydroxy phenyl acetic acid gamma-lactone polyester particles and modified hydroxyapatite according to the mass ratio of 0.5: 0.2: 1-2: 2: 1, and a porous structure with the porosity of 40%-60% is obtained through ultrasonic dispersion and vacuum freeze drying forming. Wherein the modified hydroxyapatite provides biocompatibility and osteogenesis promoting performance, the polyester microparticles reserve 2, 5-dihydroxyphenyl active groups to achieve the anti-inflammatory function, and the surface of the modified hydroxyapatite is grafted with pyrimidine derivatives containing phenolic hydroxyl groups and (S)-lactic acid hydrazone-based polyamide to enhance the antibacterial activity and osteoconductivity. The antibacterial rate of the composite material to staphylococcus aureus and escherichia coli is larger than or equal to 90%, the compression strength is 5-20 MPa, the in-vitro osteoblast proliferation rate is increased by 43% or above compared with pure hydroxyapatite, and the composite material is suitable for infectious bone defect repair scaffolds, antibacterial orthopedic implants and periodontitis adjuvant therapy filling materials and has the advantages of being simple and convenient in process, synergistic in function and high in biological safety.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical materials, in particular, to a composite material with antibacterial, anti-inflammatory and bone repair functions and a preparation method thereof, which is especially suitable for the repair of infected bone defects, antibacterial orthopedic implants and the auxiliary treatment of periodontitis. BACKGROUND

[0002] Bone defect repair is one of the important challenges in clinical practice. Traditional bone repair materials (such as autologous bone, allogeneic bone or metal implants) have limited donor sources, immune rejection, mechanical performance mismatch or secondary surgery risks. Therefore, the development of artificial bone repair materials with good biocompatibility, osteoinduction and mechanical properties has become a research hotspot.

[0003] However, bacterial infection and inflammatory response often occur during bone repair, which seriously affects the effect of bone tissue regeneration. Existing antibacterial bone repair materials (such as silver-loaded hydroxyapatite and antibiotic slow-release scaffolds) can inhibit bacterial growth, but have problems such as narrow antibacterial spectrum, drug resistance, cytotoxicity or unstable combination with the material matrix. At the same time, the overuse of antibiotics may destroy the local microenvironment and delay bone healing. In addition, some materials lack anti-inflammatory function and cannot effectively regulate the inflammatory response after implantation, leading to fibrous encapsulation or failure of bone integration.

[0004] At present, most researches focus on single antibacterial or osteogenic materials, while composite materials with efficient antibacterial, anti-inflammatory and bone repair functions still have technical bottlenecks such as complex preparation process, easy inactivation of active ingredients or uncontrollable slow release. Therefore, the development of a composite material with simple process, high biological safety and synergistic antibacterial, anti-inflammatory and bone regeneration effects is of great significance for clinical bone defect treatment. SUMMARY

[0005] In view of the problems of poor antibacterial performance, lack of anti-inflammatory function, insufficient functional synergy and complex preparation process of existing bone repair materials, the purpose of the present application is to provide an antibacterial and anti-inflammatory bone repair composite material and a preparation method thereof. By reasonably designing the raw material composition and preparation process, the antibacterial, anti-inflammatory and osteogenic functions of the material are synergistically exerted, while the biocompatibility, mechanical properties and process feasibility of the material are ensured, meeting the clinical needs of infected bone defect repair.

[0006] In order to achieve the purpose of the present application, the technical scheme of the present application is as follows:

[0007] The core idea of the present application is that three kinds of biocompatible raw materials with specific functions are selected, and a porous composite material is constructed through synergistic compounding and simple process. Among them, (S)-lactyl hydrazone-based polyamide provides good biocompatibility and promotes bone activity, 2,5-dihydroxyphenylacetic acid γ-lactone polyester microparticles realize active anti-inflammatory function, and modified hydroxyapatite endows the material with high-efficiency antibacterial performance and bone conductivity, and the synergistic effect of the three solves the functional short board of the existing materials.

[0008] Specifically, the preparation method of the antibacterial and anti-inflammatory bone repair composite material includes three core steps of raw material mixing, suspension preparation, and porous composite material forming, and is specifically as follows:

[0009] (1) Raw material mixing

[0010] (S)-lactyl hydrazone-based polyamide, 2,5-dihydroxyphenylacetic acid γ-lactone polyester microparticles and modified hydroxyapatite are mixed in a mass ratio of 0.5:0.2:1~2:2:1. The mass ratio is determined through a large number of experiments and can ensure the antibacterial and anti-inflammatory performance while making the mechanical strength and pore characteristics of the material meet the requirements of cancellous bone repair. If the proportion of (S)-lactyl hydrazone-based polyamide is too high, the mechanical strength of the material will decrease; if the proportion of modified hydroxyapatite is too high, the degradability and cell compatibility of the material may be reduced.

[0011] Among them, (S)-lactyl hydrazone-based polyamide has an alternating structure of hydrazone bond and amide bond, which not only endows the material with good degradability and antibacterial property, but also can regulate the adhesion and proliferation of osteoblasts through intermolecular interaction, and promote bone tissue regeneration; 2,5-dihydroxyphenylacetic acid γ-lactone polyester microparticles retain 2,5-dihydroxyphenyl active groups, which can realize active anti-inflammatory function by inhibiting the release of inflammatory factors (such as IL-6 and TNF-α), and reduce the fiber wrapping after the material is implanted; the modified hydroxyapatite is grafted with pyrimidine derivatives containing phenolic hydroxyl groups, and the synergistic effect of phenolic hydroxyl groups and pyrimidine rings can damage the bacterial cell membrane to achieve high-efficiency antibacterial property, and the bone conductivity of hydroxyapatite can guide the growth of bone tissue into the material.

[0012] (2) Suspension preparation

[0013] The above mixture is dispersed in N,N-dimethylformamide (DMF) or hexafluoroisopropanol, and is treated with ultrasonic waves of 300~500W power for 30~60 minutes to form a uniform suspension. N,N-dimethylformamide or hexafluoroisopropanol is selected as the dispersion medium because both of them have good solubility / dispersibility for the three kinds of raw materials, and can be easily removed completely in the subsequent freeze-drying process without affecting the biocompatibility of the material; ultrasonic treatment can effectively avoid the agglomeration of raw materials, ensure the uniform distribution of the three components in the suspension, and further ensure the uniformity of the function of the composite material.

[0014] (3) Porous composite material forming

[0015] The suspension is cast in a mold of a predetermined shape, and vacuum freeze-drying treatment is performed to obtain a porous composite material. The conditions for vacuum freeze-drying are: freezing temperature -50~ -40℃, vacuum degree ≤10 Pa, and drying time 24~48 hours. This process forms a connected porous structure in the material by freezing the dispersion medium at low temperature and sublimating ice crystals under vacuum. The porosity is controlled to be 40%~60%, and the pore size distribution is 50~300 μm. This porosity facilitates cell infiltration, nutrient transport and metabolic product discharge, and meets the microenvironmental requirements for bone tissue regeneration. At the same time, freeze-drying can avoid the destruction of active ingredients (such as 2,5-dihydroxyphenyl groups) in the material by high temperature, ensuring the functional stability of the material.

[0016] Further, the application also provides specific preparation methods for the three functional raw materials:

[0017] The preparation of (S)-lactic acid hydrazone-based polyamide includes three steps of (S)-lactic acid hydrazine synthesis, (S)-lactic acid hydrazone synthesis, and (S)-lactic acid hydrazone-based polyamide synthesis, specifically:

[0018] (1) (S)-lactic acid hydrazine synthesis: Under nitrogen protection, equimolar amounts of (S)-lactic acid ethyl ester and hydrazine hydrate are added to a three-necked flask, and heated to reflux at 80~90℃ for 3~5 hours. After cooling, it is transferred to a beaker, and then distilled under reduced pressure (≤5 kPa) at room temperature for 2~4 days to remove water and ethanol, obtaining (S)-lactic acid hydrazine. Nitrogen protection can avoid oxidation of raw materials, and reduced pressure distillation can effectively remove reaction byproducts, improving the purity of (S)-lactic acid hydrazine.

[0019] (2) (S)-lactic acid hydrazone synthesis: (S)-lactic acid hydrazine and 3,5-diaminobenzaldehyde are dissolved in methanol at a 1:1 molar ratio, heated to reflux at 60~70℃ under nitrogen protection for 3~5 hours, and then concentrated to 1 / 3 of the original volume. After cooling to 0~4℃, crystals are precipitated, filtered, and the filter cake is washed with ice-cold methanol and freeze-dried (-50℃, ≤10 Pa) to obtain (S)-lactic acid hydrazone. The amino group of 3,5-diaminobenzaldehyde reacts with the hydrazine group of (S)-lactic acid hydrazine to form a hydrazone bond, laying the foundation for the alternating structure of hydrazone-amide of the subsequent polyamide. Low-temperature crystallization and freeze-drying can ensure the purity and activity of the product.

[0020] (3) Synthesis of (S)-Lactohydrazone-based polyamide: (S)-lactohydrazone, sebacic acid, CaCl2, and triphenyl phosphate were mixed in a molar ratio of 1:1:0.05:0.1. A small amount of pyridine (5%~10% of the mass of the reaction system) was added and dissolved in N-methyl-2-pyrrolidone. The mixture was slowly heated to 70~80℃ and stirred for 4~6 hours (to form amide bonds). After cooling to room temperature, 2 times the volume of methanol was added to precipitate the product. After filtration, the product was vacuum dried at 60℃ for 12 hours to obtain (S)-lactohydrazone-based polyamide. Sebacic acid provides a carboxyl group, which reacts with the amino group of (S)-lactohydrazone to form an amide bond. CaCl2 is a catalyst, triphenyl phosphate is a cosolvent, and pyridine is an acid-binding agent. The three work together to promote the polycondensation reaction and form a high molecular weight polyamide. Methanol precipitation can remove unreacted monomers and additives to ensure the purity of the product.

[0021] The preparation of 2,5-dihydroxyphenylacetic acid γ-lactone polyester microparticles includes four steps: lactone dissolution, ring-opening reaction, polyesterification reaction, purification, and molding. Specifically:

[0022] (1) Dissolution of lactone: γ-lactone of 2,5-dihydroxyphenylacetic acid is dissolved in distilled water and dissolved with ultrasonic assistance at 300W. The concentration is controlled at 0.5~1.5mol / L. Ultrasonic assistance can accelerate the dissolution rate and ensure complete dissolution of lactone.

[0023] (2) Ring-opening reaction: Add 1 mol / L NaOH solution to adjust the pH to 9.0~10.0, and stir at 25~30℃ for 12~36 hours. Under strong alkaline conditions, the lactone undergoes ring-opening to generate sodium carboxylate, which provides active groups for the subsequent polyesterification reaction.

[0024] (3) Polyesterification reaction: Adjust the pH to 3.0-4.0 by adding 1 mol / L HCl solution dropwise, add p-toluenesulfonic acid (polyesterification catalyst) to a concentration of 0.01-0.03 mol / L, and stir at 30-40℃ for 24-48 hours (intermolecular ester bond condensation occurs). Under acidic conditions, sodium carboxylate is converted to carboxylic acid, and p-toluenesulfonic acid catalyzes intermolecular ester bond condensation to form polyester; the mild reaction temperature can avoid oxidation of the 2,5-dihydroxyphenyl group.

[0025] (4) Purification and molding: Unreacted monomers are removed by ultrafiltration through a 3000 Da ultrafiltration membrane, washed with distilled water until neutral, dried under vacuum at 60°C, and ground through a 200-mesh sieve. Ultrafiltration can remove unreacted small molecule monomers, ensuring the purity of polyester microparticles; passing through a 200-mesh sieve controls the particle size to 20~50μm, resulting in polyester microparticles with a particle size of 20~50μm, ensuring their uniform dispersion in the composite material.

[0026] The preparation of modified hydroxyapatite includes six steps: cross-linked pyrimidine derivative synthesis, purification, dissolution, Schiff base reaction, coordination grafting, purification, and drying. Specifically:

[0027] (1) Synthesis of cross-linked pyrimidine derivatives: 2,5-diamino-4,6-dihydroxypyrimidine and diisocyanate were dissolved in DMF at a molar ratio of 1:(1~2). The concentration of 2,5-diamino-4,6-dihydroxypyrimidine was controlled at 0.1~0.3 mol / L. After stirring at room temperature for 30 minutes, the temperature was slowly increased to 60~70℃ and stirred for 4~6 hours. The reaction was monitored by FTIR at 2270 cm⁻¹. -1 The disappearance of the -NCO peak confirms the completeness of the reaction. The -NCO group of the diisocyanate reacts with the amino group of the pyrimidine to form a urea bond, achieving cross-linking of the pyrimidine derivative; FTIR monitoring ensures the completeness of the reaction and avoids residual -NCO groups affecting biocompatibility.

[0028] (2) Purification: Add 0.1g methanol to quench the remaining -NCO, stir for 30 minutes, add 2 times the volume of diethyl ether, centrifuge (8000rpm, 10 minutes) to collect the precipitate, repeat the washing with diethyl ether 3 times, and freeze-dry to obtain the diisocyanate crosslinked pyrimidine derivative. Methanol reacts with the residual -NCO to generate carbamate, and the diethyl ether precipitation can remove unreacted diisocyanate and auxiliaries.

[0029] (3) Dissolution: Dissolve 2.0~5.0g of diisocyanate crosslinked pyrimidine derivative in 10mL of anhydrous DMF / dimethyl sulfoxide (DMSO) mixed solvent (volume ratio 1:1) and stir at 60~80℃ for 30 minutes until completely dissolved.

[0030] (4) Schiff base reaction: Add 10 mL of anhydrous DMF / DMSO (1:1) solution containing 0.6~1.2 g of 4-hydroxybenzaldehyde, adjust the pH to 4~5 by adding acetic acid dropwise under ice bath (0~4℃), and stir for 2~4 hours (to form a Schiff base structure). The amino group of the pyrimidine derivative reacts with the aldehyde group of 4-hydroxybenzaldehyde to form a Schiff base structure, introducing a phenolic hydroxyl group to enhance the antibacterial properties.

[0031] (5) Coordination grafting: 1.0~2.0g of hydroxyapatite nanoparticles (particle size 50~100nm) were added to 5mL of DMF / DMSO (1:1), and dispersed by ultrasonication (300W) for 30 minutes. The mixture was then transferred to the reaction system of step (4) and ultrasonically stirred (200W) at 25℃ for 24 hours to allow the nanoparticle surface to bind pyrimidine derivatives through coordination. The Ca on the surface of the hydroxyapatite... 2+ It forms coordinate bonds with the nitrogen and oxygen atoms of pyrimidine derivatives to achieve stable grafting of pyrimidine derivatives on the particle surface; ultrasonic stirring can promote the uniformity of the grafting reaction.

[0032] (6) Purification and drying: The solid was collected by centrifugation (10,000 rpm, 10 minutes), and washed successively with ethanol-DMF (50:50, v / v), ethanol-water (80:20, v / v), and pure ethanol. The solid was then freeze-dried to obtain modified hydroxyapatite. Multi-step washing can remove ungrafted pyrimidine derivatives and ensure the purity of the modified hydroxyapatite.

[0033] Preferably, the diisocyanate is 4,4'-diphenylmethane diisocyanate (MDI) or hexamethylene diisocyanate (HDI).

[0034] On the other hand, the present invention also provides an antibacterial and anti-inflammatory bone repair composite material prepared according to the above method. The composite material has the following properties:

[0035] (1) Antibacterial rate against Staphylococcus aureus and Escherichia coli ≥90%;

[0036] (2) The proliferation rate of osteoblasts (MC3T3-E1) in vitro was more than 43% higher than that of pure hydroxyapatite;

[0037] (3) The compressive strength is 5~20MPa, which meets the mechanical requirements for cancellous bone repair;

[0038] (4) It has good anti-inflammatory effects.

[0039] On the other hand, the present invention also provides the application of the above-mentioned antibacterial and anti-inflammatory bone repair composite material in the preparation of scaffolds for repairing infected bone defects, antibacterial orthopedic implants, or filling materials for adjuvant treatment of periodontitis.

[0040] Compared with the prior art, the present invention has the following significant advantages:

[0041] 1. Strong functional synergy: The three raw materials respectively undertake antibacterial, anti-inflammatory, and osteogenic functions, and promote each other. Modified hydroxyapatite can accelerate bone regeneration and provide a clean environment for bone regeneration; the anti-inflammatory function of polyester microparticles reduces inflammatory interference and promotes the integration of bone tissue and materials; at the same time, the antibacterial properties of modified hydroxyapatite and (S)-lactic acid hydrazone polyamide reduce the risk of infection and provide a clean environment for bone regeneration. The three work together to achieve an integrated effect of "infection control-inflammation regulation-bone repair".

[0042] 2. Excellent and safe antibacterial properties: The phenolic hydroxyl group and pyrimidine ring on the surface of modified hydroxyapatite work together to fight bacteria, and (S)-lactate hydrazone polyamide has an antibacterial rate of ≥90% against Staphylococcus aureus and Escherichia coli. It does not rely on antibiotics or heavy metal ions, avoiding drug resistance and cytotoxicity problems, and has high biosafety.

[0043] 3. Active and controllable anti-inflammatory function: The 2,5-dihydroxyphenyl active groups retained in the polyester microparticles can actively inhibit the release of inflammatory factors, rather than passively inhibiting inflammation simply by relying on the physical structure of the material. The anti-inflammatory effect is long-lasting and stable.

[0044] 4. Outstanding bone regeneration capacity: The hydrazone-amide bond structure of (S)-lactic acid hydrazone polyamide synergistically promotes osteoblast proliferation with the osteoconductivity of modified hydroxyapatite. The in vitro cell proliferation rate is more than 30% higher than that of pure hydroxyapatite, and the porous structure of the material is conducive to bone tissue ingrowth.

[0045] 5. Matching of mechanical properties and biocompatibility: The compressive strength of the material is 5~20MPa, which matches the mechanical properties of human cancellous bone, avoiding material fracture or bone resorption caused by mechanical mismatch after implantation; all raw materials are biocompatible components, degradable and non-cytotoxic, meeting the requirements for clinical application.

[0046] 6. Simple and feasible preparation process: The entire preparation process does not require high-temperature sintering, complex chemical modification and other steps. It mainly relies on ultrasonic dispersion and freeze drying. The process is simple, energy-efficient, easy to scale up industrially, and can effectively retain the active components of the material, which is conducive to clinical translation. Attached Figure Description

[0047] Figure 1 The composite material promotes the proliferation of osteoblasts and endothelial cells.

[0048] Figure 2 This is to demonstrate the anti-inflammatory effect of the composite material. Detailed Implementation

[0049] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0050] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0051] Example 1

[0052] 1. Preparation of (S)-Lactohydrazone Polyamide

[0053] (1) Under nitrogen protection, 0.05 mol of (S)-ethyl lactate and 0.05 mol of hydrazine hydrate were added to a three-necked flask and heated under reflux for 3 hours (reaction temperature 80℃). After cooling, the mixture was transferred to a beaker and distilled under reduced pressure (≤5 kPa) at room temperature for 2 days to remove the generated water and ethanol, and (S)-hydrazine lactate was obtained with a yield of 98% and a mass of about 5.1 g. The molecular weight of (S)-hydrazine lactate was 104.11 g / mol.

[0054] (2) Dissolve 2.082 g of (S)-lactate hydrazine and 2.723 g of 3,5-diaminobenzaldehyde in 20 mL of methanol, heat under nitrogen protection and reflux for 3 hours (reaction temperature 60℃), concentrate the reaction solution to 1 / 3 of the original volume and cool to 0~4℃, filter, wash the filter cake 3 times with ice-cold methanol, freeze-dry under vacuum (-50℃, ≤10Pa) to obtain (S)-lactate hydrazone, with a yield of 95%, an estimated molecular weight of 222.24 g / mol, and a weight of 4.257 g.

[0055] (3) Dissolve 2.222g of (S)-lactohydrazone, 2.023g of sebacic acid, 0.055g of CaCl2, 0.326g of triphenyl phosphate and 0.143g of pyridine in 15mL of N-methyl-2-pyrrolidone, slowly heat to 70℃, stir for 4 hours, cool to room temperature, add 2 times the volume of methanol to precipitate, filter, freeze dry under vacuum at 60℃ for 12 hours to obtain (S)-lactohydrazone polyamide.

[0056] 2. Preparation of 2,5-dihydroxyphenylacetic acid γ-lactone polyester microparticles

[0057] (1) Dissolve 1.66g of γ-lactone 2,5-dihydroxyphenylacetic acid in 20mL of distilled water and sonicate (300W) to dissolve to a concentration of 0.5mol / L;

[0058] (2) Add 1 mol / L NaOH solution to adjust the pH to 9.0~10.0, stir at 25~30℃ for 12 hours (ring-opening reaction) to obtain sodium 2,5-dihydroxyphenylacetate solution;

[0059] (3) Add 1 mol / L HCl solution to adjust the pH to 3.0, add p-toluenesulfonic acid (polyesterification catalyst) to a concentration of 0.01 mol / L, and stir at 30~40℃ for 24 hours;

[0060] (4) The unreacted monomers were removed by ultrafiltration through a 3000 Da ultrafiltration membrane, washed with distilled water until neutral, dried under vacuum at 60°C, and ground through a 200-mesh sieve to obtain polyester microparticles with a particle size of 50±12μm.

[0061] 3. Preparation of modified hydroxyapatite

[0062] (1) 2,5-Diamino-4,6-dihydroxypyrimidine was dried under vacuum at 80℃ for 12 hours. 0.284 g of the dried 2,5-diamino-4,6-dihydroxypyrimidine and 0.6 g of 4,4'-diphenylmethane diisocyanate (MDI) were weighed and dissolved in 20 mL of anhydrous DMF. The mixture was stirred at room temperature for 30 minutes, then slowly heated to 60℃ and stirred for 4 hours. The reaction was monitored by FTIR at 2270 cm⁻¹. -1The disappearance of the -NCO peak confirms that the reaction is complete;

[0063] (2) Add 0.1g of methanol to quench the remaining -NCO, stir for 30 minutes, add 2 times the volume of diethyl ether, centrifuge (8000rpm, 10 minutes) to collect the precipitate, repeat the washing with diethyl ether 3 times, and freeze dry to obtain 5.83g of diisocyanate crosslinked pyrimidine derivative;

[0064] (3) Dissolve 2.0g of the above cross-linked pyrimidine derivative in 10mL of anhydrous DMF / DMSO mixed solvent (volume ratio 1:1) and stir at 60~80℃ for 30 minutes until completely dissolved;

[0065] (4) Add 10 mL of anhydrous DMF / DMSO (volume ratio 1:1) solution containing 0.6 g 4-hydroxybenzaldehyde, and adjust the pH to 4.5 by adding acetic acid dropwise under ice bath (0~4℃), and stir the reaction for 3 hours (to form a Schiff base structure).

[0066] (5) Add 1.0g of hydroxyapatite nanoparticles (particle size 50~100nm) to 5mL of anhydrous DMF / DMSO (volume ratio 1:1), disperse by ultrasonication (300W) for 30 minutes, transfer to the reaction system of step (4), and stir by ultrasonication (200W) at 25℃ for 24 hours to allow the nanoparticle surface to bind pyrimidine derivatives through coordination.

[0067] (6) After the reaction, the solid was collected by centrifugation (10,000 rpm, 10 minutes), and washed twice each with ethanol-DMF (50:50, v / v), ethanol-water (80:20, v / v), and pure ethanol. The modified hydroxyapatite was obtained by freeze drying.

[0068] 4. Preparation of antibacterial and anti-inflammatory bone repair composite materials

[0069] (1) Mix 0.5g (S)-lactohydrazone polyamide, 0.2g 2,5-dihydroxyphenylacetic acid γ-lactone polyester microparticles with 1.0g modified hydroxyapatite;

[0070] (2) Disperse the mixture in DMF or hexafluoroisopropanol and sonicate for 30 minutes to form a uniform suspension. The ultrasonic power is 300W.

[0071] (3) The suspension was poured into a mold and then freeze-dried under vacuum to obtain a porous composite material with a compressive strength of 20 MPa and a porosity of about 40%.

[0072] Example 2

[0073] 1. Preparation of (S)-Lactohydrazone Polyamide

[0074] (1) Under nitrogen protection, 0.1 mol of (S)-ethyl lactate and 0.1 mol of hydrazine hydrate were added to a three-necked flask and heated under reflux for 3 hours (reaction temperature 80℃). After cooling, the mixture was transferred to a beaker and distilled under reduced pressure (≤5 kPa) at room temperature for 2 days to remove the generated water and ethanol, and (S)-hydrazine lactate was obtained with a yield of 97%.

[0075] (2) Dissolve 5.206 g of (S)-lactic acid hydrazine and 6.808 g of 3,5-diaminobenzaldehyde in 30 mL of methanol, heat under nitrogen protection and reflux for 5 hours (reaction temperature 60℃), concentrate the reaction solution to 1 / 3 of the original volume and cool to 0~4℃, filter, wash the filter cake 3 times with ice-cold methanol, freeze-dry under vacuum (-50℃, ≤10Pa) to obtain (S)-lactic acid hydrazone, with a yield of 90%.

[0076] (3) Dissolve 4.444g of (S)-lactohydrazone, 4.046g of sebacic acid, 0.11g of CaCl2, 0.652g of triphenyl phosphate and 0.286g of pyridine in 15mL of N-methyl-2-pyrrolidone, slowly heat to 80℃, stir for 5 hours, cool to room temperature, add 2 times the volume of methanol to precipitate, filter, freeze dry under vacuum at 60℃ for 12 hours to obtain (S)-lactohydrazone polyamide.

[0077] 2. Preparation of 2,5-dihydroxyphenylacetic acid γ-lactone polyester microparticles

[0078] (1) Dissolve 3.32g of 2,5-dihydroxyphenylacetic acid γ-lactone in 20mL of distilled water and sonicate (300W) to dissolve to a concentration of 1.0mol / L;

[0079] (2) Add 1 mol / L NaOH solution to adjust the pH to 9.0~10.0, stir at 25~30℃ for 30 hours (ring-opening reaction) to obtain sodium 2,5-dihydroxyphenylacetate solution;

[0080] (3) Add 1 mol / L HCl solution to adjust the pH to 3.0, add p-toluenesulfonic acid (polyesterification catalyst) to a concentration of 0.02 mol / L, and stir at 30~40℃ for 40 hours;

[0081] (4) The unreacted monomers were removed by ultrafiltration through a 3000 Da ultrafiltration membrane, washed with distilled water until neutral, dried under vacuum at 60°C, and ground through a 200-mesh sieve to obtain polyester microparticles with a particle size of 20±7μm.

[0082] 3. Preparation of modified hydroxyapatite

[0083] (1) 2,5-Diamino-4,6-dihydroxypyrimidine was dried under vacuum at 80℃ for 12 hours. 0.854 g of the dried 2,5-diamino-4,6-dihydroxypyrimidine and 1.8 g of 4,4'-diphenylmethane diisocyanate (MDI) were weighed and dissolved in 20 mL of anhydrous DMF. The mixture was stirred at room temperature for 30 minutes, then slowly heated to 70℃ and stirred for 6 hours. The reaction was monitored by FTIR at 2270 cm⁻¹. -1 The disappearance of the -NCO peak confirms that the reaction is complete;

[0084] (2) Add 0.1g of methanol to quench the remaining -NCO, stir for 30 minutes, add 2 times the volume of diethyl ether, centrifuge (8000rpm, 10 minutes) to collect the precipitate, repeat the washing with diethyl ether 3 times, and freeze dry to obtain 17.8g of diisocyanate crosslinked pyrimidine derivative.

[0085] (3) Dissolve 4.0g of the above cross-linked pyrimidine derivative in 10mL of anhydrous DMF / DMSO mixed solvent (volume ratio 1:1) and stir at 60~80℃ for 30 minutes until completely dissolved;

[0086] (4) Add 10 mL of anhydrous DMF / DMSO (volume ratio 1:1) solution containing 0.88 g 4-hydroxybenzaldehyde, adjust the pH to 5.0 by adding acetic acid dropwise under ice bath (0~4℃), and stir the reaction for 4 hours (to form a Schiff base structure).

[0087] (5) Add 1.5g of hydroxyapatite nanoparticles (particle size 50~100nm) to 5mL of anhydrous DMF / DMSO (volume ratio 1:1), disperse by ultrasonication (300W) for 30 minutes, transfer to the reaction system of step (4), and stir ultrasonically (200W) at 25℃ for 24 hours to allow the nanoparticle surface to bind pyrimidine derivatives through coordination.

[0088] (6) After the reaction, the solid was collected by centrifugation (10,000 rpm, 10 minutes), and washed twice each with ethanol-DMF (50:50, v / v), ethanol-water (80:20, v / v), and pure ethanol. The modified hydroxyapatite was obtained by freeze drying.

[0089] 4. Preparation of antibacterial and anti-inflammatory bone repair composite materials

[0090] (1) Mix 4.0g (S)-lactohydrazone polyamide, 4.0g 2,5-dihydroxyphenylacetic acid γ-lactone polyester microparticles with 2.0g modified hydroxyapatite;

[0091] (2) Disperse the mixture in DMF or hexafluoroisopropanol and sonicate for 60 minutes to form a uniform suspension. The ultrasonic power is 500W.

[0092] (3) The suspension was poured into a mold and then freeze-dried under vacuum to obtain a porous composite material with a compressive strength of 14.5 MPa and a porosity of about 48%.

[0093] Example 3

[0094] 1. Preparation of (S)-Lactohydrazone Polyamide

[0095] (1) Under nitrogen protection, 0.08 mol of (S)-ethyl lactate and 0.08 mol of hydrazine hydrate were added to a three-necked flask and heated under reflux for 3 hours (reaction temperature 80℃). After cooling, the mixture was transferred to a beaker and distilled under reduced pressure (≤5 kPa) at room temperature for 2 days to remove the generated water and ethanol, and (S)-hydrazine lactate was obtained with a yield of 95%.

[0096] (2) Dissolve 4.164 g of (S)-hydrazine lactate and 5.446 g of 3,5-diaminobenzaldehyde in 20 mL of methanol, heat under nitrogen protection and reflux for 4 hours (reaction temperature 70 °C), concentrate the reaction solution to 1 / 3 of the original volume and cool to 0~4 °C, filter, wash the filter cake 3 times with ice-cold methanol, freeze-dry under vacuum (-50 °C, ≤10 Pa) to obtain (S)-hydrazone lactate with a yield of 95%.

[0097] (3) Dissolve 6.667g (S)-lactohydrazone, 6.068g sebacic acid, 0.166g CaCl2, 0.979g triphenyl phosphate and 0.731g pyridine (8% of the reaction system mass) in 30mL N-methyl-2-pyrrolidone, slowly heat to 80℃, stir for 6 hours, cool to room temperature, add 2 times the volume of methanol to precipitate, filter and freeze dry under vacuum to obtain (S)-lactohydrazone polyamide.

[0098] 2. Preparation of 2,5-dihydroxyphenylacetic acid γ-lactone polyester microparticles

[0099] (1) Dissolve 4.98g of γ-lactone 2,5-dihydroxyphenylacetic acid in 20mL of distilled water and sonicate (300W) to dissolve to a concentration of 1.5mol / L;

[0100] (2) Add 1 mol / L NaOH solution to adjust the pH to 9.0~10.0, stir at 25~30℃ for 36 hours (ring-opening reaction) to obtain sodium 2,5-dihydroxyphenylacetate solution;

[0101] (3) Add 1 mol / L HCl solution to adjust the pH to 3.0, add p-toluenesulfonic acid (polyesterification catalyst) to a concentration of 0.03 mol / L, and stir at 30~40℃ for 48 hours;

[0102] (4) The unreacted monomers were removed by ultrafiltration through a 3000 Da ultrafiltration membrane, washed with distilled water until neutral, dried under vacuum at 60°C, and ground through a 200-mesh sieve to obtain polyester microparticles with a particle size of 38±6μm.

[0103] 3. Preparation of modified hydroxyapatite

[0104] (1) 2,5-Diamino-4,6-dihydroxypyrimidine was dried under vacuum at 80 °C for 12 hours. 0.568 g of the dried 2,5-diamino-4,6-dihydroxypyrimidine and 1.32 g of hexamethylene diisocyanate (HDI) were weighed and dissolved in 20 mL of anhydrous DMF. The mixture was stirred at room temperature for 30 minutes, then slowly heated to 70 °C and stirred for 5 hours. The reaction was monitored by FTIR at 2270 cm⁻¹. -1 The disappearance of the -NCO peak confirms that the reaction is complete;

[0105] (2) Add 0.1g of methanol to quench the remaining -NCO, stir for 30 minutes, add 2 times the volume of diethyl ether, centrifuge (8000rpm, 10 minutes) to collect the precipitate, repeat the washing with diethyl ether 3 times, and freeze dry to obtain 11.85g of diisocyanate crosslinked pyrimidine derivative.

[0106] (3) Dissolve 5.0g of the above cross-linked pyrimidine derivative in 10mL of anhydrous DMF / DMSO mixed solvent (volume ratio 1:1) and stir at 60~80℃ for 30 minutes until completely dissolved;

[0107] (4) Add 10 mL of anhydrous DMF / DMSO (volume ratio 1:1) solution containing 1.2 g of 4-hydroxybenzaldehyde, adjust the pH to 4.0 by adding acetic acid dropwise under ice bath (0~4℃), and stir the reaction for 2 hours;

[0108] (5) Add 2.0g of hydroxyapatite nanoparticles (particle size 50~100nm) to 5mL of DMF / DMSO (volume ratio 1:1), disperse by ultrasonication (300W) for 30 minutes, transfer to the reaction system of step (4), and stir by ultrasonication (200W) at 25℃ for 24 hours to allow the nanoparticle surface to bind pyrimidine derivatives through coordination.

[0109] (6) After the reaction, the solid was collected by centrifugation (10,000 rpm, 10 minutes), and washed twice each with ethanol-DMF (50:50, v / v), ethanol-water (80:20, v / v), and pure ethanol. The modified hydroxyapatite was obtained by freeze drying.

[0110] 4. Preparation of antibacterial and anti-inflammatory bone repair composite materials

[0111] (1) Mix 3.0g (S)-lactohydrazone polyamide, 2.4g 2,5-dihydroxyphenylacetic acid γ-lactone polyester microparticles with 3.0g modified hydroxyapatite;

[0112] (2) Disperse the mixture in DMF or hexafluoroisopropanol and sonicate for 40 minutes to form a uniform suspension. The ultrasonic power is 400W.

[0113] (3) The suspension was poured into a mold and then freeze-dried under vacuum to obtain a porous composite material with a compressive strength of 5 MPa and a porosity of about 60%.

[0114] Example 4

[0115] Biocompatibility analysis was performed on the composite materials prepared in Examples 1-3 (according to GB / T 16886.5-2017 standard).

[0116] 1. Experimental Materials

[0117] (1) Samples: Composite materials prepared in Examples 1-3 (sterilized by γ-ray, soaked in DMEM medium at a ratio of 0.2 g / L, stood at 37°C for 24 h, filtered through a 0.22 μm filter membrane for later use, and diluted to prepare a series of concentrations of 0.1 g / L and 0.05 g / L).

[0118] (2) Control: Pure hydroxyapatite material (same specifications, same concentration of extract), blank DMEM culture medium;

[0119] (3) Cells: mouse osteoblasts MC3T3-E1, human umbilical vein endothelial cells HUVEs.

[0120] 2. Cytotoxicity test (CCK-8 assay)

[0121] (1) Take MC3T3-E1 cells in the logarithmic growth phase and adjust the concentration to 5×10⁻⁶. 4 Cells were seeded at a density of 100 μL / mL in 96-well plates and cultured for 24 h to allow the cells to adhere.

[0122] (2) Discard the original culture medium and add the extraction solution of Examples 1-3, the control extraction solution, and the blank culture medium (3 replicates per group), 100 μL per well, and continue to culture for 24 h, 48 h, and 72 h respectively;

[0123] (3) Add 10 μL of CCK-8 reagent to each well, incubate at 37°C in the dark for 2 h, and measure the absorbance (OD value) at 450 nm using an ELISA reader.

[0124] (4) Calculate the relative cell proliferation rate (RGR): RGR = (OD value of experimental group / OD value of blank group) × 100%, and grade according to RGR (≥100% is grade 0, 80%~99% is grade 1, 60%~79% is grade 2, <60% is grade 3~4, and grade 0~1 is judged as no cytotoxicity).

[0125] 3. Results

[0126] The osteoblasts in the composite materials used in Examples 1-3 all exhibited good biocompatibility. At extraction concentrations of 0.2, 0.1, and 0.05 g / L, compared to the untreated control, the osteoblast proliferation-promoting effect ranged from 93% to 157%, and the endothelial cell proliferation-promoting effect ranged from 97% to 139%, demonstrating potential for promoting angiogenesis and bone repair. Figure 1 The in vitro osteoblast proliferation rate (MC3T3-E1) can be increased by more than 43% compared with pure hydroxyapatite.

[0127] Example 5

[0128] The antibacterial effects of the composite materials prepared in Examples 1-3 were analyzed (according to GB / T 20944.3-2008 standard).

[0129] 1. Experimental Materials

[0130] (1) Sample: Composite materials of Examples 1-3 (sterilized and ground into powder, passed through a 100-mesh sieve);

[0131] (2) Strains: Staphylococcus aureus (ATCC 6538) and Escherichia coli (ATCC 8739) (both in logarithmic growth phase, concentration adjusted to 1×10⁻⁶). 6 (CFU / mL)

[0132] (3) Culture medium: LB liquid medium.

[0133] 2. Antibacterial rate test (shaking flask method)

[0134] (1) Take a 250mL Erlenmeyer flask, add 100mL LB medium, add the sample powder of Examples 1-3 (final concentration 0.1g / L), and add an equal amount of sterile physiological saline to the control group. Each group has 3 replicates.

[0135] (2) Inoculate with 1 mL of bacterial culture (1×10⁻⁶) 6 (CFU / mL), co-cultured on a shaker at 37℃ for 24 h;

[0136] (3) Take the cultured bacterial solution and dilute it serially to 10. -4 ~10 -6 Take 100 μL of the solution and spread it onto LB solid medium. Incubate at 37°C for 16 h and count the number of colonies (CFU / mL) using a colony counter.

[0137] (4) Calculate the antibacterial rate: Antibacterial rate = (number of colonies in the control group - number of colonies in the experimental group) / number of colonies in the control group × 100%. An antibacterial rate ≥ 90% is considered highly effective antibacterial.

[0138] 3. Results

[0139] The osteoblasts in the composite materials in Examples 1-3 all exhibit good biocompatibility. At a concentration of 0.1 g / L, the inhibition rates against Staphylococcus aureus were 78.3%, 95.5%, and 97.2%, respectively, and the inhibition rates against Escherichia coli were 74.7%, 89.4%, and 98%, respectively. Compared with pure hydroxyapatite (antibacterial inhibition rate <25%), they have strong antibacterial properties.

[0140] Example 6

[0141] The composite materials prepared in Examples 1-3 were analyzed for pro-inflammatory and anti-inflammatory factors (ELISA method).

[0142] 1. Experimental Materials

[0143] (1) Sample: Extract of composite materials from Examples 1-3 (same as Example 5) (0.2 g / L);

[0144] (2) Cells: Mouse macrophages RAW264.7;

[0145] (3) Reagents: Mouse TNF-α ELISA kit (Thermo Fisher Scientific) and mouse IL-10 ELISA kit (Bio-Techne).

[0146] 2. Experimental Procedure

[0147] (1) Take 5th generation RAW264.7 cells and use 5×10 4 Inoculate one cell per well into a 24-well plate and incubate for 12 hours, then discard the culture medium.

[0148] (2) Blank control group: containing only cells and basal culture medium, without any stimulants or test substances, used to determine the "basal cell secretion level". Inflammation model group (positive control 1, pro-inflammatory reference): cells + basal culture medium + inflammatory stimulant (such as LPS, 10 μg / mL), used to simulate the "inflammatory state" as a reference for high expression of pro-inflammatory factors. Anti-inflammatory positive control group (positive control 2, anti-inflammatory reference): cells + inflammatory stimulant + known anti-inflammatory substance (recombinant IL-10, 10 ng / mL), used to verify the "effectiveness of anti-inflammatory effect" as a reference for increased anti-inflammatory factors / decreased pro-inflammatory factors. Experimental group: cells + inflammatory stimulant + extract from Examples 1-3 (0.2 mg / mL) or hydroxyapatite solution (0.2 mg / mL).

[0149] (3) Add the experimental group (extracts from Examples 1-3), positive control group 1, positive control group 2, and blank control group, with 5 replicates for each group.

[0150] (4) Incubate at 37℃ for 24 hours, collect the supernatant, centrifuge at 3000 rpm for 10 minutes, and take the supernatant.

[0151] (5) Strictly follow the instructions of the kit, add the standard, supernatant sample, detection antibody and enzyme conjugate in sequence, incubate in the dark, add substrate for color development, measure the OD value at 450nm with an ELISA reader, and calculate the concentration of each factor based on the standard curve.

[0152] 3. Results

[0153] The TNF-α concentrations in Examples 1-3 were significantly lower than those in the inflammation model group (*p<0.05, **p<0.05), slightly higher than the anti-inflammatory positive control group, and higher than the blank control group. The IL-10 concentrations in Examples 1-3 were significantly higher than those in the inflammation model group (*p<0.05, **p<0.05), lower than the anti-inflammatory positive control group, and higher than the blank control group. In contrast, the TNF-α concentration in the hydroxyapatite group was slightly higher than that in the blank control, while IL-10 showed no difference compared to the blank control. Furthermore, the cell viability in the experimental groups was ≥80%, excluding toxicity interference, indicating that Examples 1-3 possess anti-inflammatory effects. Figure 2 ).

[0154] The above detailed description is a specific description of the embodiments of the present invention. These embodiments are not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included in the patent scope of this case.

Claims

1. A method for preparing an antibacterial and anti-inflammatory bone repair composite material, characterized in that, Includes the following steps: (1) Mix (S)-lactohydrazone polyamide, 2,5-dihydroxyphenylacetic acid γ-lactone polyester microparticles and modified hydroxyapatite at a mass ratio of 0.5:0.2:1~2:2:1; (2) Disperse the mixture in N,N-dimethylformamide or hexafluoroisopropanol and sonicate for 30-60 minutes to form a uniform suspension; (3) The suspension is poured into a mold and then freeze-dried under vacuum to obtain a porous composite material.

2. The preparation method according to claim 1, characterized in that, The (S)-lactic acid hydrazone polyamide is prepared by the following steps: (1) Under nitrogen protection, add equimolar amounts of (S)-ethyl lactate and hydrazine hydrate to a three-necked flask, heat to reflux at 80~90℃ for 3~5 hours, cool and transfer to a beaker, distill under reduced pressure at room temperature for 2~4 days to remove the generated water and ethanol, and obtain (S)-hydrazine lactate. (2) Dissolve (S)-lactic acid hydrazine and 3,5-diaminobenzaldehyde in methanol in an equimolar ratio, heat under nitrogen protection at 60-70°C for 3-5 hours under reflux, concentrate the reaction solution and cool it, filter to collect the solid, wash the filter cake with ice-cold methanol, and then freeze dry to obtain (S)-lactic acid hydrazone. (3) Dissolve (S)-lactogen, sebacic acid, CaCl2, triphenyl phosphate and a small amount of pyridine in N-methyl-2-pyrrolidone, slowly heat to 70~80℃, stir for 4~6 hours, cool to room temperature after the reaction is completed, add methanol to precipitate, filter to collect the solid, and vacuum dry to obtain (S)-lactogen polyamide.

3. The preparation method according to claim 2, characterized in that, In step (3), the molar ratio of (S)-lactohydrazone, sebacic acid, CaCl2 and triphenyl phosphate is 1:1:0.05:0.1, and the pyridine accounts for 5% to 10% of the mass of the reaction system.

4. The preparation method according to claim 1, characterized in that, The 2,5-dihydroxyphenylacetic acid γ-lactone polyester microparticles were prepared by the following steps: (1) Dissolve γ-lactone of 2,5-dihydroxyphenylacetic acid in distilled water and sonicate to a concentration of 0.5~1.5 mol / L; (2) Add NaOH solution dropwise to adjust the pH to 9.0~10.0, stir at 25~30℃ for 12~36 hours to obtain sodium 2,5-dihydroxyphenylacetic acid solution; (3) Adjust the pH to 3.0-4.0 by adding HCl solution dropwise, add p-toluenesulfonic acid to a concentration of 0.01-0.03 mol / L, and stir at 30-40℃ for 24-48 hours; (4) The unreacted monomers were removed by ultrafiltration through an ultrafiltration membrane, washed with distilled water until neutral, dried under vacuum, ground and sieved to obtain polyester microparticles with a particle size of 20~50μm.

5. The preparation method according to claim 1, characterized in that, The modified hydroxyapatite is prepared through the following steps: (1) Dissolve 2,5-diamino-4,6-dihydroxypyrimidine and diisocyanate in anhydrous DMF, stir and mix at room temperature, slowly raise the temperature to 60~70℃, stir and react for 4~6 hours, and monitor the reaction at 2270 cm⁻¹ by FTIR. -1 The disappearance of the -NCO peak confirms that the reaction is complete; (2) Add methanol to quench the remaining -NCO, add diethyl ether, centrifuge to collect the precipitate, wash with diethyl ether repeatedly, and freeze dry to obtain the diisocyanate crosslinked pyrimidine derivative; (3) Dissolve the above-mentioned cross-linked pyrimidine derivatives in anhydrous DMF / DMSO mixed solvent and stir at 60~80℃ until completely dissolved; (4) Add 4-hydroxybenzaldehyde solution dissolved in anhydrous DMF / DMSO mixed solvent, adjust the pH to 4-5 by adding acetic acid dropwise under ice bath at 0-4℃, and stir the reaction for 2-4 hours; (5) Add hydroxyapatite nanoparticles to anhydrous DMF / DMSO mixed solvent, disperse them by ultrasonication, and then transfer them to the reaction system in step (4). Stir them by ultrasonication so that the nanoparticles are bound to pyrimidine derivatives through coordination. (6) After the reaction, the solid was collected by centrifugation and washed successively with ethanol / DMF mixed solvent, ethanol / water mixed solvent and pure ethanol, and then freeze-dried to obtain modified hydroxyapatite.

6. The preparation method according to claim 5, characterized in that, In step (1), the molar ratio of 2,5-diamino-4,6-dihydroxypyrimidine to diisocyanate is 1:(1~2).

7. The preparation method according to claim 5, characterized in that, The mass ratio of the crosslinked pyrimidine derivative, 4-hydroxybenzaldehyde, and hydroxyapatite nanoparticles is (2.0~5.0):(0.6~1.2):(1.0~2.0).

8. The preparation method according to claim 5, characterized in that, The volume ratio of DMF to DMSO in the anhydrous DMF / DMSO mixed solvent is 1:1, the volume ratio of ethanol to DMF in the ethanol / DMF mixed solvent is 1:1, and the volume ratio of ethanol to water in the ethanol / water mixed solvent is 4:

1.

9. An antibacterial and anti-inflammatory bone repair composite material prepared according to any one of claims 1-8.

10. The application of the antibacterial and anti-inflammatory bone repair composite material of claim 9 in the preparation of scaffolds for repairing infected bone defects, antibacterial orthopedic implants, or filling materials for adjuvant treatment of periodontitis.

Images