Antibacterial osteogenic scaffold material as well as preparation method and application thereof

Abstract

The invention provides an antibacterial osteogenic scaffold material as well as a preparation method and application thereof, and relates to the technical field of biomedical materials. The antibacterial osteogenesis coating material is a stent material formed by loading a sound-sensitive agent material on a porous titanium stent loaded with hydroxyapatite, the sound-sensitive agent material is a piezoelectric material which can respond to ultrasonic stimulation, generate charge separation, generate active oxygen and generate hydrogen peroxide and hydroxyl free radicals through a piezoelectric effect to generate antibacterial activity and has biocompatibility. The antibacterial osteogenic scaffold material provided by the invention can provide excellent antibacterial performance and osteogenic ability, has good biocompatibility and operability, and has a wide clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular, to an antibacterial osteogenic scaffold material, a preparation method thereof, and an application thereof. Background Art

[0002] Implant-Associated Infections (IAIs) are common and challenging complications after orthopedic surgeries. Their occurrence is mainly related to the formation of biofilms by bacteria on the surface of implants. The formation of biofilms enables bacteria to effectively evade the penetration of antibiotics and the clearance of the host immune system, thereby leading to persistent inflammation and tissue damage, and ultimately causing implant failure. Due to the lack of oxygen and low bacterial metabolic activity within the biofilm, traditional antibiotic treatments often have poor effects and are vulnerable to drug-resistant bacteria during the treatment process, and the limitations of the treatment are becoming increasingly apparent. Therefore, how to effectively prevent and treat implant-associated infections, especially the treatment of biofilms, has become an urgent problem in the current medical field.

[0003] Existing treatment methods mainly rely on antibiotic treatment and debridement surgery. However, due to the special structure of biofilms, antibiotics are often difficult to effectively penetrate. In addition, the hypoxic environment within the biofilm reduces the metabolic activity of bacteria, further limiting the effect of antibiotics. In recent years, with the progress of intelligent coating technology, researchers have begun to explore the possibility of actively sensing and destroying biofilms through coating materials. Such technologies activate antibacterial effects remotely in order to break through the bottleneck of traditional antibiotic treatments. However, existing technologies still face many challenges in practical applications. For example, how to improve the persistence of antibacterial effects and the ability to combat drug-resistant bacteria remains an urgent problem to be solved.

[0004] Chemodynamic therapy (CDT) is a technique that uses hydrogen peroxide and variable-valent metal iron to generate hydroxyl radicals (·OH) through the Fenton reaction to kill bacteria and tumor cells. Although CDT has certain antibacterial and anti-tumor potential, it faces several significant problems in practical applications. First, the lack of active sites of the CDT reaction—Fe 2+ severely limits the generation of ROS. Since Fe 2+ is rapidly consumed during the reaction, it is impossible to continuously generate effective ROS, thus affecting the treatment effect. Second, the reaction substrate required for CDT—hydrogen peroxide, has a low concentration in the local microenvironment, which further limits the reaction efficiency and the generation of ROS. In addition, the lack of effective control during the reaction process makes it difficult to achieve the best effect of ROS generation, limiting the exertion of its antibacterial ability.

[0005] Sonodynamic therapy (SPT) based on the piezoelectric effect is an emerging treatment method that activates piezoelectric materials through ultrasound to generate reactive oxygen species (ROS), thereby triggering an oxidative stress response. It has the advantages of non-invasiveness and good biocompatibility. However, there are still many problems with current piezoelectric sonosensitizer materials in practical applications. Most materials have limited carrier concentrations, and the electron-hole pairs excited under ultrasound quickly recombine, resulting in unsatisfactory redox reaction effects, insufficient amounts of generated ROS, and ineffective sterilization. At the same time, the short lifespan of ROS further reduces its antibacterial efficiency, limiting its application in clinical treatment. Although recent studies have attempted to combine CDT with SPT to enhance the antibacterial effect, due to their independent actions, deep-level linkage and optimization have not been achieved, so the effect is only a simple addition and cannot significantly improve the ROS generation efficiency and antibacterial effect.

[0006] Generally speaking, there are several obvious deficiencies in the prior art in solving implant-related infections. The insufficient reactive sites and low substrate hydrogen peroxide concentration of CDT lead to low ROS generation efficiency and difficulty in continuously and effectively generating ROS. Although SPT generates ROS under ultrasound stimulation, due to the limitations of the carrier concentration of piezoelectric materials and the recombination rate of electron-hole pairs, its effect is difficult to meet clinical requirements. In addition, traditional antibacterial materials and osteogenic materials are usually used separately and cannot simultaneously exert the dual functions of antibacterial and osteogenic promotion on the same platform (material), which makes it difficult to comprehensively improve the treatment effect. Current treatment methods have not effectively solved these problems, so there is an urgent need to develop new technologies and materials to break through the limitations of the prior art.

[0007] In view of this, the present invention is specifically proposed. Summary of the Invention

[0008] The object of the present invention is to provide an antibacterial and osteogenic scaffold material, its preparation method and application. The antibacterial and osteogenic scaffold material can provide excellent antibacterial performance and osteogenic ability, and at the same time has good biocompatibility and operability, with broad clinical application prospects.

[0009] To achieve the above object of the present invention, the following technical solutions are specifically adopted:

[0010] In the first aspect, the present invention provides an antibacterial and osteogenic scaffold material, and the antibacterial and osteogenic coating material is a scaffold material composed of a sonosensitizer material carried on a porous titanium scaffold loaded with hydroxyapatite;

[0011] Among them, the sonosensitizer material is a biocompatible piezoelectric material that can respond to ultrasonic stimulation and generate charge separation, generate reactive oxygen species, and can produce hydrogen peroxide and hydroxyl radicals through the piezoelectric effect to produce antibacterial activity.

[0012] In an alternative embodiment, the photosensitizer material is a material having a variable-valence metal reaction site; in an alternative embodiment, the photosensitizer material includes at least one of lead-free piezoelectric materials containing variable-valence metal Fe-based or Mn-based; in an alternative embodiment, the photosensitizer material is (BiFe) x (BaTi) 1-x O 3 , and 0 < x < 1. In an alternative embodiment, the porous titanium scaffold is a material with a three-dimensional porous structure having a honeycomb-like surface; in an alternative embodiment, the pore size in the three-dimensional porous structure of the porous titanium scaffold is 200 nm to 400 nm.

[0013] Second, the present invention provides a method for preparing an antibacterial osteogenic scaffold material as described in any one of the foregoing embodiments, including: subjecting a solid titanium metal raw material to alkali heat treatment to obtain a porous titanium scaffold; loading hydroxyapatite in the porous titanium scaffold to obtain an intermediate scaffold material; and loading the photosensitizer material on the intermediate scaffold material by using polydopamine to obtain the antibacterial osteogenic scaffold material.

[0014] In an alternative embodiment, the photosensitizer material is (BiFe) x (BaTi) 1-x O 3 , and 0 < x < 1;

[0015] The method for preparing the photosensitizer material includes: mixing Bi 2 O 3 , Fe 2 O 3 , BaCO 3 and TiO 2 according to the stoichiometric ratio of the photosensitizer material for mixing treatment to obtain a mixed material; calcining the mixed material to obtain a composite oxide; and sanding the composite oxide to obtain the photosensitizer material.

[0016] In an alternative embodiment, the mixing treatment is ball milling using ZrO 2 balls; in an alternative embodiment, the mixing treatment time is at least 24 hours; in an alternative embodiment, the mixing treatment temperature is room temperature; in an alternative embodiment, the ball milling speed in the mixing treatment is 120 rpm; in an alternative embodiment, the calcining treatment temperature is 850 °C; in an alternative embodiment, the calcining treatment time is not less than 6 hours; in an alternative embodiment, the sanding treatment speed is 2000 rpm; in an alternative embodiment, the sanding treatment time is not less than 8 hours.

[0017] In an alternative embodiment, the alkali heat treatment of the solid titanium metal raw material to obtain the porous titanium scaffold includes: performing surface polishing treatment and cleaning treatment on the solid titanium metal raw material to obtain a pretreated raw material; performing alkali heat treatment on the pretreated raw material in a sodium hydroxide solution to obtain the porous titanium scaffold with a three-dimensional porous structure having a honeycomb-like surface.

[0018] In an alternative embodiment, the surface polishing treatment includes: polishing the surface of the solid titanium metal raw material with SiC sandpapers of different coarseness degrees in the order of decreasing sandpaper grit size to obtain the solid titanium metal raw material with a smooth surface; in an alternative embodiment, the grit size of the SiC sandpaper is 400# to 1500#; in an alternative embodiment, the cleaning treatment includes: ultrasonically cleaning the solid titanium metal raw material with a smooth surface with acetone, ethanol, and distilled water for 15 minutes respectively, and then obtaining the pretreated raw material after air drying.

[0019] In an alternative embodiment, the alkali heat treatment includes: putting the pretreated raw material into a treatment container and performing alkali heat treatment with a sodium hydroxide solution for at least 24 hours; adding distilled water to the treatment container and performing vacuum drying to obtain the porous titanium scaffold.

[0020] In an alternative embodiment, the temperature of the alkali heat treatment is 60°C; in an alternative embodiment, the concentration of the sodium hydroxide solution is 5M.

[0021] In an alternative embodiment, loading hydroxyapatite in the porous titanium scaffold to obtain an intermediate scaffold material includes: forming a polydopamine coating on the porous titanium scaffold by spontaneous polymerization of dopamine under alkaline conditions; soaking the porous titanium scaffold with the formed polydopamine coating in a hydroxyapatite solution to obtain the intermediate scaffold material loaded with the hydroxyapatite.

[0022] In an alternative embodiment, forming a polydopamine coating on the porous titanium scaffold by spontaneous polymerization of dopamine under alkaline conditions includes: putting the porous titanium scaffold into an alkaline solution containing dopamine and performing a polymerization reaction at room temperature.

[0023] In an alternative embodiment, the alkaline solution containing dopamine is a Tris-HCl alkaline solution containing dopamine; in an alternative embodiment, the concentration of dopamine is 3 mg / mL; in an alternative embodiment, the concentration of the Tris-HCl alkaline solution is 10 mM.

[0024] In an alternative embodiment, the method of loading the photosensitizer material onto the scaffold intermediate material using polydopamine to obtain the antibacterial osteogenic scaffold material includes: soaking the scaffold intermediate material in a solution of the photosensitizer material for at least 12 hours at room temperature to obtain the antibacterial osteogenic scaffold material.

[0025] In a third aspect, the present invention provides an application of the antibacterial osteogenic scaffold material according to any one of the foregoing embodiments in an antibacterial repair material for bone defects.

[0026] The present invention provides an antibacterial osteogenic scaffold material, a preparation method thereof, and an application thereof. Among them, the photosensitizer material in the antibacterial osteogenic scaffold material can respond to ultrasonic stimulation and generate charge separation through the piezoelectric effect, and then generate reactive oxygen species (ROS) such as hydrogen peroxide (H 2 O 2 ) and hydroxyl radicals (·OH). These ROS have strong antibacterial effects and can effectively destroy the cell structure and biofilm of bacteria, and remove possible bacterial infections on the surface of implants. Compared with traditional antibacterial materials, this ultrasonic activation-based antibacterial mechanism can continuously generate ROS in a dynamic environment, improving the persistence of antibacterial effects and treatment efficiency.

[0027] The photosensitizer material not only has antibacterial activity but also has good biocompatibility and can be compatible with human tissues without causing immune rejection reactions. This makes the material suitable for long-term implants and can maintain stable functions for a long time after being implanted into the human body. At the same time, ultrasonic stimulation, as an external activation method, has the advantages of no radiation, simple operation, and good biosafety, further enhancing the clinical application potential of the material.

[0028] The porous titanium scaffold used in the antibacterial osteogenic scaffold material has good mechanical properties and biocompatibility and can provide stable support for the regeneration of bone tissue. Titanium material itself has the property of promoting the attachment of bone cells. By loading hydroxyapatite (HA), the osteogenic function of the material is further enhanced. As a natural bone mineral component, HA can effectively promote the proliferation and differentiation of bone cells and accelerate the repair of bone tissue.

[0029] The antibacterial osteogenic scaffold material combines antibacterial and osteogenic functions on one scaffold, achieving a dual treatment effect. The ultrasonic-activated antibacterial effect and the osteogenic effect of the titanium scaffold can be carried out simultaneously, solving the problem that traditional materials usually need to separately set antibacterial functions and osteogenic functions. Such a design improves the comprehensive treatment effect of the material, can simultaneously achieve anti-infection and promote bone healing, thereby shortening the recovery time of patients and reducing complications during the treatment process.

[0030] In summary, the antibacterial osteogenic scaffold material provided in the present invention can provide excellent antibacterial performance and osteogenic ability, and at the same time has good biocompatibility and operability, showing broad prospects for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0032] Figure 1 Morphology diagram of BFBT (antibacterial osteogenic scaffold material) prepared in Example 1 of this application;

[0033] Figure 2 Piezoelectric properties of BFBT prepared in Example 1 of this application;

[0034] Figure 3 Band gap value (left) and flat band potential (right) of BFBT prepared in Example 1 of this application;

[0035] Figure 4 Energy band diagram (left) and schematic diagram of cascade reaction under ultrasound (right) of BFBT prepared in Example 1 of this application;

[0036] Figure 5 Schematic diagram of the characterization result of oxygen vacancies of BFBT prepared in Example 1 of this application;

[0037] Figure 6 Schematic diagram of the change results of the content and valence state of Fe in Example 1 of this application;

[0038] Figure 7 Schematic diagram of the morphology of the porous titanium scaffold prepared in Example 1 of this application (left before alkali-heat treatment, right after alkali-heat treatment);

[0039] Figure 8 Schematic diagram of the microscopic morphology of the porous titanium scaffold with a polydopamine coating prepared in Example 1 of this application;

[0040] Figure 9 Schematic diagram of the morphology of the porous titanium scaffold loaded with hydroxyapatite on the europolydopamine coating prepared in Example 1 of this application;

[0041] Figure 10 Schematic diagram of the morphology of TH-BFBT prepared in Example 1 of this application;

[0042] Figure 11Schematic diagram of the investigation results of ·OH generation in Test Experimental Example 1 of this application;

[0043] Figure 12 Schematic diagram of the investigation results of hydrogen peroxide generation of TH-BFBT in Test Experimental Example 1 of this application;

[0044] Figure 13 For Fe in Test Experimental Example 1 of this application 2+ Schematic diagram of the process of increasing content (illustrated that in the presence of hydrogen peroxide H 2 O 2 , ultrasonic US stimulation can maintain the Fe content of TH-BFBT 2+ content);

[0045] Figure 14 For Fe in Test Experimental Example 1 of this application 2+ Schematic diagram of the principle of the process of increasing content (ultrasound-driven self-circulating Fenton reaction realizes continuous ROS generation);

[0046] Figure 15 Schematic diagram of the verification results of the antibacterial performance of the bio-coated stent in Test Experimental Example 2 of this application;

[0047] Figure 16 Schematic diagram of the verification results of the osteogenic performance of the bio-coated stent in Test Experimental Example 3 of this application;

[0048] Figure 17 Schematic diagram of the verification results of the immunomodulatory performance of the bio-coated stent in Test Experimental Example 4 of this application (Merge is the merged result of CD206 and iNOS);

[0049] Figure 18 Schematic diagram of the finite element modeling of BF and BFBT in Test Experimental Example 4 of this application (A) Simulates the piezopotential distribution of BF and BFBT samples under 10 nN; (B) Simulates the dependence of piezopotential on cavitation force;

[0050] Figure 19 Schematic diagram of the detection of intracellular calcium levels by Fluo-4 staining in Test Experimental Example 4 of this application. Detailed implementation manners

[0051] The following will describe the implementation schemes of the present invention in detail in conjunction with the embodiments. However, those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the embodiments, they are carried out under conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified in the manufacturer are all conventional products that can be obtained through commercial purchase.

[0052] In the embodiments of the present application, an antibacterial osteogenic scaffold material is provided. The antibacterial osteogenic coating material is a scaffold material formed by loading a photosensitizer material on a porous titanium scaffold loaded with hydroxyapatite.

[0053] Among them, the photosensitizer material is a biocompatible piezoelectric material that can respond to ultrasonic stimulation and generate charge separation, generate reactive oxygen species, and produce antibacterial activity by generating hydrogen peroxide and hydroxyl radicals through the piezoelectric effect.

[0054] The antibacterial osteogenic scaffold material provided in this embodiment is a scaffold material formed by using a photosensitizer material (piezoelectric material) as the base material and loading it on a porous titanium scaffold (T). Among them, the porous titanium scaffold is a material loaded with hydroxyapatite (HA).

[0055] It should be noted that a photosensitizer material refers to a material that can respond to ultrasonic stimulation and undergo physical or chemical changes through a certain mechanism. These materials usually undergo deformation or trigger specific reactions under the action of ultrasonic waves, such as generating reactive oxygen species (ROS), and thus have functions such as antibacterial and anti-tumor. In the field of biomedical applications, photosensitizer materials are widely used in the treatment of tumors, infections, bone tissue regeneration, etc. For example, under ultrasonic stimulation, photosensitizer materials can separate electrons and holes through the piezoelectric effect or other mechanisms, thereby generating hydrogen peroxide and hydroxyl radicals with antibacterial or anti-tumor effects.

[0056] A piezoelectric material is a material that can generate charge separation (voltage) under mechanical stress, or generate deformation under an electric field. In short, a piezoelectric material can convert mechanical energy into electrical energy, or convert electrical energy into mechanical energy. Common piezoelectric materials include titanium dioxide (TiO), lead zirconate titanate (PZT), quartz, etc. In the field of biomedicine, piezoelectric materials are used to prepare ultrasonic sensors, drive devices, or as photosensitizer materials, which can respond to external ultrasonic excitation to generate charges, thereby triggering a series of biological reactions, such as the generation of reactive oxygen species.

[0057] The antibacterial osteogenic scaffold material provided in this embodiment has significant beneficial effects. First, the photosensitizer material can respond to ultrasonic stimulation and generate charge separation through the piezoelectric effect, producing reactive oxygen species (ROS) such as hydrogen peroxide and hydroxyl radicals, effectively exerting an antibacterial effect, destroying bacterial cell structures and biofilms, and enhancing the persistence of the antibacterial effect and the treatment efficiency. Second, the material has good biocompatibility and is compatible with human tissues, ensuring that no immune rejection reaction will be triggered during long-term implantation, and the ultrasonic activation method is radiation-free and easy to operate, further enhancing the feasibility of its clinical application. At the same time, the hydroxyapatite (HA) loaded on the porous titanium scaffold not only provides stable mechanical support, but also promotes the attachment and differentiation of bone cells, accelerates bone tissue repair, and supports osteogenic function. This material effectively combines the dual functions of antibacterial and osteogenic, realizes the synchronous progress of anti-infection and bone healing during the treatment process, significantly improves the overall treatment effect, shortens the recovery time of patients, reduces complications, and has broad clinical application prospects.

[0058] In some embodiments, the photosensitizer material is a material having variable-valence metal reaction sites.

[0059] In some embodiments, the photosensitizer material includes at least one of lead-free piezoelectric materials containing variable-valence metal Fe-based or Mn-based.

[0060] In some embodiments, the photosensitizer material is (BiFe) x (BaTi) 1-x O 3 , and 0 < x < 1.

[0061] As described above, the photosensitizer material can be abbreviated as BFBT in this embodiment. The prepared antibacterial osteogenic scaffold material can be TH-BFBT.

[0062] In this embodiment, for the photosensitizer material, it is required to have excellent piezoelectric properties and be able to produce effective electron-hole separation under ultrasound, so as to be able to utilize substances in the environment (such as H 2 O or O 2 ) to generate ROS. If the piezoelectric properties are weak, effective electrons and holes cannot be generated, and thus the reaction cannot occur.

[0063] The photosensitizer material BFBT needs to have appropriate valence band and conduction band positions so as to be sufficient to satisfy the utilization of H 2 O and O 2 to generate H 2 O 2 , and combined with the band tilting under ultrasound caused by excellent piezoelectricity, further enhance the self-supply of H 2 O 2 , providing sufficient raw materials for CDT.

[0064] In addition, the sonosensitizer material is required to be rich in oxygen vacancies, and under ultrasonic stimulation, the electron-rich oxygen vacancies can continuously supply electrons to the reaction center Fe. The Fenton reaction converts Fe 2+ to Fe 3+ , and the continuous electron supply converts Fe 3+ to Fe 2+ . Fe 2+ is the highly reactive state of Fe, which can continuously catalyze the Fenton reaction to occur CDT. If there is no power supply from oxygen vacancies to the Fe site, the content of Fe 3+ increases, and in the absence of additional electrons, the energy barrier for the re-conversion of Fe 3+ to Fe 2+ is high, resulting in a slowdown in the Fenton reaction rate, which is not conducive to the conversion of self-supplied H 2 O 2 to hydroxyl radicals.

[0065] In some embodiments, the porous titanium scaffold is a material with a three-dimensional porous structure having a honeycomb-like surface.

[0066] In some embodiments, the pore size in the three-dimensional porous structure of the porous titanium scaffold is 200 nm to 400 nm. For example, the pore size can be 200 nm, 300 nm, 400 nm, and so on.

[0067] In the embodiments of the present application, a preparation method of the antibacterial osteogenic scaffold material as described in any one of the foregoing embodiments is provided, including:

[0068] Step S1, subjecting the solid titanium metal raw material to alkaline heat treatment to obtain a porous titanium scaffold.

[0069] In the above step, the titanium metal raw material is treated by alkaline heat treatment (alkaline hydrothermal treatment) to change its surface properties and obtain a porous structure. Alkaline heat treatment is a surface modification technology that promotes the formation of a porous structure on the surface of titanium materials at high temperature by treating titanium materials in a strong alkaline solution, improving the biocompatibility and cell adhesion ability of titanium materials.

[0070] After alkaline heat treatment, the surface of the titanium scaffold forms a surface with a honeycomb-like or porous structure, and these pores can effectively increase the surface area of the scaffold, contributing to the subsequent loading of materials such as hydroxyapatite (HA).

[0071] This step is used to increase the surface roughness and porosity of the titanium scaffold, enhance the cell adhesion ability, and is beneficial to promoting bone tissue growth. The formation of the porous structure helps to provide a better habitat space for osteoblasts and promote bone regeneration.

[0072] In this step, a suitable alkaline solution (such as NaOH) and treatment time should be selected, and the temperature and solution concentration should be controlled to optimize the surface pores and chemical composition.

[0073] Step S2: Load hydroxyapatite into the porous titanium scaffold to obtain an intermediate scaffold material.

[0074] In this step, hydroxyapatite (HA) is loaded onto the porous titanium scaffold to form an intermediate of the osteogenic scaffold. Hydroxyapatite is the main component of natural bone and has good biocompatibility and the ability to promote bone regeneration. Therefore, it can support the osteogenic process.

[0075] In this step, HA loading can be carried out by the immersion method. The porous titanium scaffold is immersed in the HA solution, and HA is deposited on the scaffold surface through physical adsorption or chemical bonding. According to needs, the HA loading amount can be adjusted by conditions such as the concentration of HA in the solution, immersion time, and temperature. Thus, the surface of the obtained intermediate scaffold material is loaded with hydroxyapatite, providing a good habitat for osteocytes and enhancing the osteogenic ability of the scaffold.

[0076] Hydroxyapatite can effectively promote the attachment and proliferation of osteocytes, accelerate bone healing, provide a chemical environment suitable for the growth of osteocytes, and at the same time maintain the mechanical strength of the titanium material.

[0077] Methods such as the immersion method and coating method can be used for HA loading. By controlling the immersion time and HA concentration, its loading amount on the titanium scaffold can be adjusted.

[0078] Step S3: Use polydopamine to carry the photosensitizer material on the intermediate scaffold material to obtain an antibacterial osteogenic scaffold material.

[0079] In this step, polydopamine (pDA) is used as an adhesive to carry the photosensitizer material onto the surface of the porous titanium scaffold loaded with hydroxyapatite. Due to its ability to spontaneously polymerize in an alkaline environment and its good biocompatibility and strong adhesion, polydopamine can stably fix the photosensitizer material (such as piezoelectric material) on the scaffold surface.

[0080] In this step, spontaneous polymerization of polydopamine (pDA) is required. The porous titanium scaffold is immersed in a Tris-HCl alkaline solution containing dopamine (DA) and stirred at room temperature for a certain time to promote the polymerization of dopamine molecules on the scaffold surface to form a polydopamine coating. Then, the photosensitizer material is loaded, that is, through the adhesion of the polydopamine coating, the photosensitizer material (such as piezoelectric material) is fixed to the scaffold surface. Thus, the obtained material is an antibacterial osteogenic scaffold material, in which the photosensitizer material is fixed on the porous titanium scaffold through polydopamine, can respond to ultrasonic stimulation and generate reactive oxygen species, and has antibacterial properties.

[0081] Polydopamine provides strong adhesion properties, enabling the sonosensitizer material to stably adhere to the surface of the scaffold. Polydopamine also has biocompatibility, ensuring the long-term stability of the material and its compatibility with human tissues. Utilizing the piezoelectric effect of the sonosensitizer material and ultrasound activation to generate ROS enhances the antibacterial effect.

[0082] By adjusting the concentration of dopamine, the soaking time, and the pH value of the solution, the thickness of the polydopamine coating and the loading amount of the sonosensitizer material can be controlled.

[0083] These steps work together, and the final antibacterial osteogenic scaffold material integrates antibacterial and osteogenic functions, can effectively promote the repair and regeneration of bone tissue, and prevent infection. Each step has its specific significance and advantages, ensuring the excellent performance of the scaffold material in terms of antibacterial, osteogenic, and compatibility with biological tissues. By finely regulating the processing conditions of each step, the performance of the scaffold can be optimized to achieve precise treatment.

[0084] In some embodiments, the sonosensitizer material is (BiFe) x (BaTi) 1-x O 3 , and 0 < x < 1;

[0085] The preparation method of the sonosensitizer material includes:

[0086] Step S100, mixing Bi 2 O 3 , Fe 2 O 3 , BaCO 3 and TiO 2 according to the stoichiometric ratio of the sonosensitizer material to obtain a mixture.

[0087] The above step is to mix different metal oxide raw materials (Bi 2 O 3 , Fe 2 O 3 , BaCO 3 and TiO 2 ) according to a certain stoichiometric ratio to synthesize the target sonosensitizer material (BiFe) x (BaTi) 1-x O 3 . The proportional relationship of these metal oxides determines the physicochemical properties and piezoelectric properties of the final composite oxide.

[0088] In this embodiment, in the stoichiometric ratio, it refers to the ratio of the components in the synthesis of the sonosensitizer material such as (BiFe) x (BaTi) 1-x O 3At this time, the ratio between different metal oxides (Bi 2 O 3 、Fe 2 O 3 、BaCO 3 and TiO 2 ). By reasonably selecting the stoichiometric ratio, the structure, properties, and performance of the finally synthesized material can be controlled to ensure that the obtained composite material has the required piezoelectric properties and antibacterial functions.

[0089] In the calculation of the stoichiometric ratio, BiFeO 3 (BF) with x = 1 is used as a reference for the calculation of the ratio of BiFeO 3 -BaTiO 3 . For example, (BiFe) 0.9 (BaTi) 0.1 O 3 : indicates that BiFeO 3 accounts for 90%, and BaTiO 3 accounts for 10%. (BiFe) 0.5 (BaTi) 0.5 O 3 : indicates that BiFeO 3 and BaTiO 3 each account for 50%. (BiFe) 0.1 (BaTi) 0.9 O 3 : indicates that BiFeO 3 accounts for 10%, and BaTiO 3 accounts for 90%.

[0090] By the correct stoichiometric ratio, it can be ensured that the amounts of Bi 2 O 3 , Fe 2 O 3 , BaCO 3 and TiO 2 are appropriate, so that the target composite material such as (BiFe) 0.9 (BaTi) 0.1 O 3 is formed during the calcination process.

[0091] The mixing method can be to make different powder raw materials fully and uniformly mixed by means of ball milling, etc., so that the obtained mixture is uniform and ready for the next calcination treatment. In some preferred ways, the mixing treatment is to use ZrO 2 balls for ball milling; the time of the mixing treatment is at least 24 hours; the temperature of the mixing treatment is room temperature; the speed of ball milling in the mixing treatment is 120 rpm.

[0092] Ensure the uniform dispersion of different metal oxides to provide a uniform precursor material for subsequent reactions. By adjusting the stoichiometric ratio, the structure and properties of the final material can be precisely controlled.

[0093] Step S200: Calcinate the mixture to obtain a composite oxide.

[0094] The above step is to subject the mixture to high-temperature calcination, enabling different metal oxides to react and form the desired composite oxide. Calcination is a crucial step in converting metal oxides into composite materials with a specific crystal structure. Place the mixture in a high-temperature furnace for calcination.

[0095] It should be noted that in some preferred embodiments, during the calcination process, the temperature can be 850 °C and maintained for at least 6 hours to ensure complete reaction of the metal oxides and the formation of a stable crystal structure.

[0096] Solid-phase changes may occur during the calcination process, especially the interaction between iron and bismuth oxides to form the desired composite oxide. The obtained composite oxide is the preliminary form of the target material and has piezoelectric properties, allowing it to enter the subsequent processing stage.

[0097] Calcination can promote the solid-state reaction of metal oxides to form a composite material with an ideal structure and properties. High-temperature calcination helps improve the crystal structure quality of the material and enhance its piezoelectric properties.

[0098] Ensure accurate control of the calcination temperature and time to prevent over-calcination or incomplete reaction. Use a high-temperature furnace for uniform calcination to ensure the quality consistency of the samples.

[0099] Step S300: Grind the composite oxide to obtain the sonosensitizer material.

[0100] In some embodiments, the speed of the grinding process is 2000 rpm;

[0101] In some embodiments, the time of the grinding process is not less than 8 hours.

[0102] This step grinds the calcined composite oxide to further refine the particles and optimize its particle size and dispersibility. The grinding process helps improve the piezoelectric properties of the material and ensures effective excitation of the reaction under ultrasonic waves. Add the composite oxide obtained by calcination to a grinding medium (such as ZrO 2 balls).

[0103] Grinding can effectively improve the uniformity and dispersibility of the particles and optimize the properties of the material. It improves the responsiveness of the sonosensitizer material under ultrasonic waves and enhances the antibacterial effect.

[0104] Select an appropriate sanding speed (e.g., 2000 RPM) and time (e.g., at least 8 hours) to ensure that the particles reach the desired particle size. Ensure the use of an appropriate medium (e.g., ZrO 2 balls) during sanding to avoid contamination of the material.

[0105] In some embodiments, step S1 of subjecting the solid titanium metal raw material to alkali heat treatment to obtain a porous titanium scaffold includes:

[0106] Step S11 of performing surface polishing treatment and cleaning treatment on the solid titanium metal raw material to obtain a pretreated raw material.

[0107] In the above steps, performing surface polishing and cleaning treatment aims to remove impurities, oxides or other contaminants on the surface of the titanium metal, so as to provide higher adhesion and reactivity for subsequent treatments (such as alkali heat treatment and loading materials).

[0108] In some embodiments, the surface polishing treatment includes: polishing the surface of the solid titanium metal raw material with SiC sandpapers of different thicknesses based on the order of the sandpaper grit from coarse to fine to obtain the solid titanium metal raw material with a smooth surface;

[0109] In some embodiments, the grit of the SiC sandpaper is 400# - 1500#; for example, the grits are 400# (coarse), 600# (medium thickness), 800# (finer), 1000# (fine sandpaper), 1200# (very fine), 1500# (extremely fine), etc.

[0110] Gradually polish the surface of the titanium metal with SiC sandpapers of different grits (such as 400# - 1500#). This polishing treatment can make the surface smoother and increase its adhesion ability to subsequent coatings (such as polydopamine, hydroxyapatite, sonosensitizer materials).

[0111] In some embodiments, the cleaning treatment includes: ultrasonically cleaning the solid titanium metal raw material with a smooth surface with acetone, ethanol and distilled water for 15 minutes respectively, and then obtaining the pretreated raw material after air drying;

[0112] In the above steps, ultrasonically clean the titanium metal raw material with acetone, ethanol and distilled water respectively for 15 minutes each time to remove surface impurities. After cleaning, the titanium metal raw material needs to be air dried to ensure that there is no residual solvent or moisture on the surface. The cleaning treatment improves the purity and activity of the material surface, ensuring that subsequent chemical reactions are more efficient. The smooth surface of the titanium metal is more likely to form a stable bond with subsequent coating materials (such as polydopamine, hydroxyapatite, etc.).

[0113] Step S12: subject the pretreated raw material to alkali heat treatment in a sodium hydroxide solution to obtain the porous titanium scaffold with a three-dimensional porous structure having a honeycomb-like surface.

[0114] The above alkali heat treatment is a treatment method that removes surface materials by chemical corrosion means and generates a porous structure through reactions.

[0115] Alkali heat treatment can generate a porous structure with a high surface area, which is crucial for the subsequent loading of materials (such as hydroxyapatite and sonosensitizer materials). The porous structure helps to promote the growth and bonding of bone tissue, improving the biocompatibility and bone regeneration ability of the scaffold.

[0116] After alkali heat treatment, the pretreated raw material can exhibit a porous structure. Compared with a smooth structure, the pretreated raw material can ensure sufficient loading of HA and BFBT, and the porous structure is more conducive to bone ingrowth, increasing the osteogenic ability of the scaffold.

[0117] In some embodiments, in step S12, the alkali heat treatment includes:

[0118] Step S121: place the pretreated raw material in a treatment container and subject it to alkali heat treatment with a sodium hydroxide solution for at least 24 hours.

[0119] Among them, in some embodiments, the temperature of the alkali heat treatment is 60°C; the concentration of the sodium hydroxide solution is 5M.

[0120] As described above, the sodium hydroxide solution can be a 5M sodium hydroxide solution. Use the sodium hydroxide solution to subject the pretreated raw material placed in the treatment container to alkali heat treatment at 60°C for 24 hours.

[0121] Step S122: add distilled water to the treatment container and perform vacuum drying to obtain the porous titanium scaffold.

[0122] Remove excess water and solvent by washing with distilled water and vacuum drying, thereby obtaining a porous titanium scaffold.

[0123] In some embodiments, step S2: load hydroxyapatite in the porous titanium scaffold to obtain an intermediate scaffold material, including:

[0124] Step S21: under alkaline conditions, form a polydopamine coating on the porous titanium scaffold by the spontaneous polymerization of dopamine.

[0125] In this step, forming a polydopamine coating on the porous titanium scaffold is to enhance the adhesion of sonosensitizer materials (such as hydroxyapatite) and provide a stable adhesion basis for subsequent materials (such as hydroxyapatite). The polydopamine coating can also improve the biocompatibility and adhesion performance of the scaffold.

[0126] Specifically, under alkaline conditions, dopamine (DA) can be dissolved in an alkaline solution. Usually, a solution such as Tris-HCl solution (e.g., pH 8.5 - 9.0) is used to promote the spontaneous polymerization reaction of dopamine. Dopamine will rapidly polymerize in the alkaline solution to form polydopamine (pDA), which attaches to the surface of the porous titanium scaffold in the form of a coating.

[0127] In some embodiments, the concentration of dopamine is 3 mg / mL; in some embodiments, the concentration of the Tris-HCl alkaline solution is 10 mM.

[0128] Polydopamine has good biocompatibility, has good adsorption properties for bone cells and other biomolecules, and can effectively promote the loading of subsequent materials. The polydopamine coating can enhance the bioadhesion performance of the scaffold and provide a stable attachment basis for subsequent materials (such as hydroxyapatite).

[0129] The polymerization rate and the thickness of the polydopamine coating can be adjusted by controlling conditions such as the concentration of dopamine, temperature, pH, etc. Generally, the stronger the temperature and alkaline conditions, the more rapid the polymerization reaction and the thicker the coating.

[0130] Step S22: Immerse the porous titanium scaffold with the formed polydopamine coating in a hydroxyapatite solution to obtain an intermediate scaffold material loaded with the hydroxyapatite.

[0131] In this step, by immersing the porous titanium scaffold with the polydopamine coating in a hydroxyapatite solution, with the aid of the adhesion of polydopamine, the hydroxyapatite is effectively loaded on the surface of the scaffold. This step enables the scaffold to have osteogenic ability, which is beneficial to the growth of bone cells and the repair of bone tissue.

[0132] Immerse the porous titanium scaffold that has been coated with polydopamine in a solution containing hydroxyapatite (HA). Usually, an aqueous hydroxyapatite solution is used and immersed at room temperature for 12 hours or longer to ensure that the hydroxyapatite can be evenly loaded on the surface and within the pores of the scaffold.

[0133] Hydroxyapatite is a very important osteogenic material, which can promote the adhesion and differentiation of bone cells and has excellent biocompatibility. By being loaded on the surface of the scaffold, it can provide the necessary support for bone repair. The polydopamine coating not only promotes the loading of hydroxyapatite but also stabilizes its attachment on the scaffold, preventing it from falling off during subsequent processing.

[0134] During the soaking process, controlling the solution concentration, soaking time, and temperature can further optimize the loading amount and uniformity of apatite. In addition, if it is necessary to improve the stability of the coating, further cross-linking treatment can be considered to enhance the structural stability of polydopamine and ensure its stronger binding to hydroxyapatite.

[0135] In some embodiments, step S21 of forming a polydopamine coating on the porous titanium scaffold by the spontaneous polymerization of dopamine under alkaline conditions includes:

[0136] Step S211: Put the porous titanium scaffold into an alkaline solution containing dopamine and carry out a polymerization reaction at room temperature.

[0137] Among them, in some embodiments, the alkaline solution containing dopamine is a Tris-HCl alkaline solution containing dopamine.

[0138] In the above steps, the porous titanium scaffold after alkali-heating is stirred at room temperature for 24 hours in a Tris-HCl alkaline solution (10 mM, sigma, USA) containing 3 mg / ml DA (sigma, USA) to obtain Ti@pDA (Tp), that is, a porous titanium scaffold with a polydopamine coating formed. Dopamine DA can spontaneously polymerize into polydopamine (pDA) under alkaline conditions. Polydopamine is a general adhesion material with good biosecurity and adhesion performance. It can stably adhere HA and BFBT to the scaffold and maintain its biosecurity.

[0139] In some embodiments, step S3 of loading the photosensitizer material on the scaffold intermediate material by using polydopamine to obtain an antibacterial osteogenic scaffold material includes:

[0140] Step S31: Immerse the scaffold intermediate material in the solution of the photosensitizer material at room temperature for no less than 12 hours to obtain the antibacterial osteogenic scaffold material.

[0141] In the above steps, the Tp scaffold is immersed in the HA solution at room temperature for 12 h to obtain Tp@HA (TH). Finally, the TH-BFBT scaffold is prepared by immersing the TH scaffold in the BFBT (photosensitizer material) solution at room temperature for 12 h.

[0142] In the embodiments of the present application, there is provided an application of the antibacterial osteogenic scaffold material as described in any one of the foregoing embodiments in an antibacterial repair material for bone defects.

[0143] The present invention will be further described below through specific embodiments. However, it should be understood that these embodiments are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0144] Example 1

[0145] In this example, a preparation of an antibacterial osteogenic scaffold material (TH - BFBT) was carried out.

[0146] Experimental method:

[0147] (1) Preparation of BFBT:

[0148] In this example, taking BiFeO 3 (BF) with x = 1 as a reference, for the nanomaterial (BiFe) x (BaTi) 1-x O 3 (x = 9) as the target BFBT, its specific material ratio is as follows: Bi 2 O 3 (99%, 21.18 g), Fe 2 O 3 (98%, 7.33 g), BaCO 3 (99%, 1.99 g) and TiO 2 (98%, 0.82 g).

[0149] Mix alcohol and ZrO 2 balls with BiO (99%), FeO (98%), BaCO (99%) and TiO (98%) in the above stoichiometric ratio in a nylon jar and carry out ball milling at room temperature (120 RPM, 24 hours). Then calcine the mixture at 850 °C for 6 hours, and then carry out sand milling at room temperature at a speed of 2000 RPM for 8 hours to obtain (BiFe) 0.9 (BaTi) 0.1 O 3 obtained, which is BFBT nanomaterial with a particle size of 200 nanometers ( Figure 1 ).

[0150] It should be noted that the BFBT nanomaterial prepared in this example needs to have the following characteristics:

[0151] A. Excellent piezoelectric properties: It can produce effective electron - hole separation under ultrasound, so as to utilize substances in the environment (such as H 2 O or O 2 ) to generate ROS. If the piezoelectric property is weak, effective electron - holes cannot be generated, and thus the reaction cannot occur. The piezoelectric property is characterized as follows. Second - harmonic generation (SHG) is often used to evaluate the symmetry and piezoelectric property of piezoelectric materials. At 532 nm, the oscilloscope waveforms of the SHG signals of BF and BFBT samples are shown in the figure, indicating the existence of piezoelectricity. It should be noted that the SHG polar plot shows that the BFBT sample has a higher degree of anisotropy, indicating its more superior piezoelectric response (Figure 2 )。

[0152] B. Appropriate valence band and conduction band positions are sufficient to utilize H in the microenvironment 2 O and O 2 to generate H 2 O 2 , and combined with the band tilting under ultrasound induced by excellent piezoelectricity, further enhance the self - supply of H2O2, providing sufficient raw materials for CDT. It is the energy level calculation and comparison of BF and BFBT. The band gap (E g ) is determined by ultraviolet - visible diffuse reflectance spectroscopy. The E g of BF and BFBT are 1.98 eV and 2.07 eV respectively. The flat - band potential (E fb ) is calculated by the Mott - Schottky plot. The E fb of BF and BFBT are - 0.09 V and - 0.28 V (versus NHE) respectively. Further calculation gives the conduction band energy levels (ECB) of - 0.19 eV (BF) and - 0.38 eV (BFBT). According to the formula E VB = E CB + E g , the valence band energy levels (EVB) are 1.79 eV (BF) and 1.69 eV (BFBT) ( Figure 3 ). The conduction band potential of BFBT (- 0.38 eV) is more negative than that of BF and can reach the redox potential of O 2 / ·O 2 - (- 0.33 V), indicating that its conduction band electrons are beneficial to redox reactions. The ECB of both BF and BFBT satisfy the reaction conditions of ·O 2 - / H 2 O 2 (0.35 V) and HO / ·OH (1.49 V). In addition, the band - edge bending of BFBT also satisfies the reaction condition of HO / HO (1.76 V), which is beneficial to promoting the occurrence of tandem redox reactions ( Figure 4 ).

[0153] C. Rich in oxygen vacancies, and under ultrasonic stimulation, the electron - rich oxygen vacancies can continuously supply electrons to the reaction center Fe. The Fenton reaction converts Fe 2+ to Fe 3+ , and the continuous electron supply further converts Fe3+ to Fe2+. Fe2+ is the highly reactive state of Fe and can continuously catalyze the Fenton reaction to occur CDT. If there is no power supply from oxygen vacancies to the Fe site, the content of Fe 3+ increases, and in the absence of additional electrons, Fe 3+ is reconverted to Fe2+ The high energy barrier leads to a slowdown in the Fenton reaction rate, which is not conducive to the conversion of self-supplied H2O2 into hydroxyl radicals. The following is the characterization of oxygen vacancies in BFBT ( Figure 5 ), as well as the change in the Fe valence state of BFBT under ultrasonic stimulation ( Figure 6 ).

[0154] (2) Preparation of Tp:

[0155] A. Pretreatment: The solid titanium metal raw material in the shape of a cylinder with a diameter of 4 mm and a height of 3 mm was gradually polished with different SiC sandpapers from 400# to 1500# (surface polishing treatment), ultrasonically cleaned with acetone, ethanol, and distilled water for 15 minutes each (cleaning treatment), and then air-dried.

[0156] B. Alkali heat treatment: The disk was subjected to alkali heat treatment with 5M NaOH at 60 °C for 24 hours, immersed in distilled water, and dried under vacuum to obtain a porous titanium scaffold. The porous titanium scaffold presents a honeycomb-like porous surface morphology with a pore size of about 200 - 400 nm and a surface roughness of 0.69. Compared with the smooth structure, the porous structure of the porous titanium scaffold, first, can ensure sufficient loading of HA and BFBT, and second, is more conducive to bone ingrowth, increasing the osteogenic ability of the scaffold ( Figure 7 ).

[0157] (3) Loading of hydroxyapatite:

[0158] A. The alkali-heated porous titanium scaffold was stirred at room temperature in a Tris-HCl alkaline solution (10 mM, sigma, USA) containing 3 mg / ml DA (dopamine, sigma, USA) for 24 h to obtain Ti@pDA (Tp), that is, a polydopamine coating was formed on the porous titanium scaffold. Dopamine DA can spontaneously polymerize into polydopamine (pDA) under alkaline conditions. Polydopamine is a general adhesion material with good biosecurity and adhesion performance. It can stably adhere HA and BFBT to the scaffold and maintain their biosecurity. ( Figure 8 ).

[0159] B. The Tp scaffold was immersed in an HA solution (hydroxyapatite solution) at room temperature for 12 h to obtain Tp@HA (TH, the scaffold intermediate material).

[0160] (4) Preparation of TH-BFBT:

[0161] The TH-BFBT scaffold (antibacterial osteogenic scaffold material) was prepared by immersing the TH scaffold (scaffold intermediate material) in a BFBT solution (solution of the photosensitizer material) at room temperature for 12 hours. The morphology of the final product is referred to Figure 9 and Figure 10 .

[0162] Comparative Example 1

[0163] In this comparative example, a scaffold material (TH-BF) was prepared. Among them, TH-BF is a control material without doping defect engineering.

[0164] Experimental method:

[0165] The method used in this comparative example is basically the same as that in Example 1, except that:

[0166] (1) The sonosensitizer material is BiFeO 3 (BF), which is an ABO 3 type perovskite structure without element doping. Due to the absence of doping defects, this material has low piezoelectric performance and low oxygen vacancy content, which in turn leads to low ability to generate electron-hole pairs in response to ultrasound, low ability of oxygen vacancies to provide electrons to the reaction center, and ultimately low ability to generate ROS.

[0167] (2) The synthesis process is similar to that in Example 1, except that the raw materials are Bi 2 O 3 (99%, 21.18 g), Fe 2 O 3 (98%, 7.33 g).

[0168] Test Experimental Example 1: Investigation of scaffold performance

[0169] (1) Experiment 1: Absorbance measurement before and after ultrasonic stimulation

[0170] In this experiment, in order to evaluate the generation amount of ·OH, the research mixed different samples composed of Example 1 (TH-BFBT) and Comparative Example 1 (TH-BF) with a solution containing MB (Methylene Blue), and performed ultrasonic stimulation (1 MHz, 1.0 W / cm2, 50% duty cycle) in the presence or absence of H 2 O 2 . By measuring the change in MB absorbance at 664 nm before and after ultrasonic stimulation, the generation of ROS was evaluated.

[0171] Reference Figure 11 , the generation of ROS was characterized by the degradation of methylene blue MB. A decrease in absorbance indicates that MB was degraded, and since MB was degraded due to the generation of ROS, the more ROS was generated, the lower the absorbance. The results showed that under ultrasonic conditions, TH-BFBT has a stronger ability to generate ROS than TH-BF, and if hydrogen peroxide H 2 O 2 exists, its ability to generate ROS will be further enhanced.

[0172] (2) Experiment 2: Potassium Permanganate Test

[0173] In this experiment, the presence of hydrogen peroxide was verified by using a potassium permanganate test at 525 nm.

[0174] Reference Figure 12 , the results showed that the ROS production of the TH-BFBT sample increased continuously over time, indicating its significant bactericidal potential.

[0175] (3) Experiment 3: XPS Analysis

[0176] In this experiment, XPS analysis was performed.

[0177] Reference Figure 13 and Figure 14 , after ultrasonic treatment, the proportion of Fe 2+ increased, revealing the role of oxygen vacancies in promoting the Fe 3+ / Fe 2+ cycle, which is a key factor for the performance improvement of TH-BFBT.

[0178] Test Experimental Example 2: Antibacterial Performance Test

[0179] In this test experimental example, the effects of TH-BFBT on Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) with and without US irradiation were evaluated by the spread plate method.

[0180] Experimental method:

[0181] The scaffolds were exposed to a bacterial suspension (2×10 7 CFU / mL) in a 48-well plate for a specified time and were irradiated with US at 1 W / cm2 for 5 minutes or left untreated.

[0182] The spread plate method was used to culture on agar plates at 37 °C for 18 h to quantify the colony-forming units (cfu).

[0183] To further verify, after culturing the bacteria on different scaffolds for 5 days, bacterial morphology was detected by scanning electron microscopy (SEM).

[0184] Experimental results:

[0185] As Figure 15 shown, the obtained TH-BFBT scaffolds had good antibacterial ability in the simulated in vivo environment.

[0186] Test Experimental Example 3: Osteogenic Performance Detection

[0187] In this test experimental example, in order to evaluate the osteogenic ability of TH-BFBT, osteogenic performance was investigated.

[0188] Experimental method:

[0189] First, ALP activity was detected. MC3T3-E1 cells were seeded in 12-well plates. After the cells reached approximately 70% confluence, the culture medium was replaced with osteogenic induction medium containing 10 mM β-glycerophosphate, 50 μg / mL ascorbic acid, and 10 nM dexamethasone (all purchased from Sigma) to promote osteogenic differentiation of the cells.

[0190] The culture medium and scaffold materials were renewed every 3 days. On the 7th and 14th days, the BCIP / NBT ALP color development kit (Beyotime, China) was used to detect ALP activity.

[0191] Experimental results:

[0192] Reference Figure 16 , and the staining results showed that the scaffold materials exhibited significant osteogenic potential.

[0193] Test experimental example 4: Verification of immunomodulatory performance

[0194] In this test experimental example, the immunomodulatory performance was evaluated.

[0195] Experimental method:

[0196] In the experiment, to evaluate macrophage polarization, RAW 264.7 cells were cultured with 10 μg / mL of LPS (L4391, Sigma, USA) for 24 hours and co-cultured with different treatment groups.

[0197] When performing immunofluorescence staining, primary antibodies were used to detect inducible nitric oxide synthase (iNOS) and cluster of differentiation 206 (CD206).

[0198] The primary antibodies used were rabbit anti-CD206 (1:200, Abcam, USA) and mouse anti-iNOS (1:200, Abcam, USA). The secondary antibodies included donkey anti-rabbit Alexa Fluor 594 (1:500, Invitrogen, USA) and donkey anti-mouse Alexa Fluor 488 (1:500, Invitrogen, USA).

[0199] After staining, DAPI was used to label the cell nuclei, and the fluorescence intensity was measured and images were obtained through the Opera Phenix Plus system.

[0200] Experimental results:

[0201] Reference Figure 17, The fluorescence results showed that the scaffold material exhibited significant immunomodulatory ability, promoting the polarization of macrophages from M1 to M2 type.

[0202] The osteogenic and immunomodulatory functions of TH-BFBT are attributed to its ability to generate bioelectric signals under cell adhesion, thereby regulating cell behavior. The BFBT on TH-BFBT has excellent piezoelectric properties and can well respond to the action of external forces, even cell adhesion forces. Finite element modeling was used to calculate the piezoelectric potential generated by BF and BFBT when subjected to the mechanical stresses typically exerted by cells (ranging from 0.1 to 10 nN). The analysis showed that compared with BF under the same mechanical force, the piezoelectric potential generated by BFBT was significantly higher, ranging from 92.30 μV to 9.23 mV( Figure 18 ).

[0203] In addition, the intracellular Ca 2+ concentration was evaluated using the fluorescent probe Fluo-4AM, which represents the mechanism of promoting osteogenesis through electrical stimulation. The Ca 2+ level of TH-BFBT increased significantly, indicating that the electrical stimulation generated by BFBT led to an increase in intracellular calcium concentration( Figure 19 ). If the piezoelectric properties of the material are not strong enough to generate sufficient electrical signals under cell adhesion forces, it will not be able to regulate cell behavior, promote osteogenesis, or regulate macrophage polarization.

[0204] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antibacterial osteogenic scaffold material, characterized in that: The antibacterial osteogenic coating material is a scaffold material composed of a sonosensitizer material carried on a porous titanium scaffold loaded with hydroxyapatite; The sonosensitizer material is a biocompatible piezoelectric material that can respond to ultrasonic stimulation and produce charge separation, generate active oxygen, and produce hydrogen peroxide and hydroxyl radicals through piezoelectric effect to produce antibacterial activity.

2. The antibacterial osteogenic scaffold material according to claim 1, characterized in that: The sonosensitizer material is a material having a variable valence metal reaction site; Preferably, the acoustic sensitizer material includes at least one of lead-free piezoelectric materials containing variable valence metal Fe-based or Mn-based; Preferably, the sonosensitizer material is (BiFe) x (BaTi) 1-x O3, and 0 <x<1。 3. The antibacterial osteogenic scaffold material according to claim 1, characterized in that: The porous titanium support is a material with a three-dimensional porous structure having a honeycomb surface; Preferably, the pore size in the three-dimensional porous structure of the porous titanium scaffold is 200 nm to 400 nm.

4. A method for preparing the antibacterial osteogenic scaffold material according to any one of claims 1 to 3, characterized in that: include: Performing alkaline heat treatment on the titanium metal raw material solid to obtain a porous titanium stent; Loading hydroxyapatite in the porous titanium stent to obtain a stent intermediate material; The sonosensitizer material is loaded on the scaffold intermediate material using polydopamine to obtain an antibacterial osteogenic scaffold material.

5. The method for preparing the antibacterial osteogenic scaffold material according to claim 4, characterized in that: The sonosensitizer material is (BiFe) x (BaTi) 1-x O3, and 0 <x<1; The preparation method of the sonosensitizer material comprises: Bi2O3, Fe2O3, BaCO3 and TiO2 are mixed according to the stoichiometric ratio of the sonosensitizer material to obtain a mixture; calcining the mixture to obtain a composite oxide; The composite oxide is subjected to sand milling treatment to obtain the sonosensitizer material; Preferably, the mixing process is ball milling using ZrO2 balls; Preferably, the mixing treatment time is at least 24 hours; Preferably, the temperature of the mixing process is room temperature; Preferably, the ball milling speed in the mixing process is 120 rpm; Preferably, the calcination temperature is 850°C; Preferably, the calcination treatment time is not less than 6 hours; Preferably, the sanding speed is 2000 rpm; Preferably, the sanding treatment time is not less than 8 hours.

6. The method for preparing the antibacterial osteogenic scaffold material according to claim 4, characterized in that: The method of subjecting the titanium metal raw material solid to alkaline heat treatment to obtain the porous titanium stent comprises: Performing surface polishing and cleaning treatment on the titanium metal raw material solid to obtain a pretreated raw material; The pretreated raw material is subjected to alkaline heat treatment in a sodium hydroxide solution to obtain the porous titanium scaffold having a three-dimensional porous structure with a honeycomb surface; Preferably, the surface polishing treatment comprises: polishing the surface of the titanium metal raw material solid using SiC sandpaper of different coarseness and fineness in the order of sandpaper particle size from coarse to fine, so as to obtain the titanium metal raw material solid with a smooth surface; Preferably, the particle size of the SiC sandpaper is 400# to 1500#; Preferably, the cleaning treatment comprises: ultrasonically cleaning the titanium metal raw material solid with a smooth surface using acetone, ethanol and distilled water for 15 minutes respectively, and then air-drying to obtain the pretreated raw material; Preferably, the alkali heat treatment comprises: placing the pretreated raw material in a treatment container and performing alkaline heat treatment with sodium hydroxide solution for at least 24 hours; Adding distilled water into the processing container and performing vacuum drying to obtain the porous titanium stent; Preferably, the temperature of the alkali heat treatment is 60°C; Preferably, the concentration of the sodium hydroxide solution is 5M.

7. The method for preparing the antibacterial osteogenic scaffold material according to claim 4, characterized in that: The method of loading hydroxyapatite in the porous titanium stent to obtain a stent intermediate material comprises: Under alkaline conditions, forming a polydopamine coating on the porous titanium scaffold by utilizing spontaneous polymerization of dopamine; The porous titanium stent with the polydopamine coating is immersed in a hydroxyapatite solution to obtain a stent intermediate material loaded with the hydroxyapatite.

8. The method for preparing the antibacterial osteogenic scaffold material according to claim 7, characterized in that: The method of forming a polydopamine coating on the porous titanium stent by spontaneous polymerization of dopamine under alkaline conditions comprises: The porous titanium scaffold is placed in an alkaline solution containing dopamine to perform a polymerization reaction at room temperature; Preferably, the alkaline solution containing dopamine is a Tris-HCl alkaline solution containing dopamine; Preferably, the concentration of dopamine is 3 mg / mL; Preferably, the concentration of the Tris-HCl alkaline solution is 10 mM.

9. The method for preparing the antibacterial osteogenic scaffold material according to claim 4, characterized in that: The method of loading the sonosensitizer material onto the scaffold intermediate material using polydopamine to obtain the antibacterial osteogenic scaffold material comprises: Under normal temperature conditions, the scaffold intermediate material is immersed in the solution of the sonosensitizer material for no less than 12 hours to obtain the antibacterial osteogenic scaffold material.

10. Use of the antibacterial osteogenic scaffold material according to any one of claims 1 to 3 in antibacterial repair materials for bone defects.

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