Nanometer needle-like hydroxyapatite as well as preparation method and application thereof

Abstract

The invention discloses nano needle-like hydroxyapatite as well as a preparation method and application thereof. The preparation method comprises the following steps: firstly, uniformly loading bioactive glass nanoparticles synthesized by a sol-gel method into photosensitive resin, and synthesizing a bioactive material substrate with bioactive sites; and further performing simple low-temperature soaking treatment to generate nano needle-like hydroxyapatite with uniform morphology and high length-diameter ratio on the surface of the bioactive substrate material in situ. The nano needle-like hydroxyapatite can kill bacteria attached to the surface by utilizing surface mechanical stress, and meanwhile, the osteogenesis promoting performance is exerted. The nano needle-like hydroxyapatite makes up for bacterial drug resistance generated by antibacterial drugs, and has a very wide application prospect in the aspect of treating infectious bone defects.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and specifically relates to nano needle-shaped hydroxyapatite and a preparation method and application thereof. Background Art

[0002] Bone implant materials face many challenges in clinical applications, among which the osteogenic properties of materials have always been the core clinical requirement of orthopedic implantable medical devices. However, the infection problems caused by infected bone defects and bone implant materials, as bacterial infection often affects the normal healing of bone defects, pose a huge challenge to clinical treatment. Therefore, the development of a bone implant material with both antibacterial and osteogenic properties is of great significance for orthopedic clinical practice, especially for the treatment of infected bone defects.

[0003] As the main inorganic component of human bone tissue and dental hard tissue, nano-hydroxyapatite (HA) has excellent biocompatibility, osteogenic induction activity and bone integration ability due to its highly biomimetic properties with natural bone matrix. It is widely used in bone tissue engineering fields such as bone defect repair and regeneration. In addition, due to its high specific surface area and strong adsorption, nano-HA can load antibacterial drugs and exert antibacterial effects. Studies have shown that the application of antibiotic-loaded hydroxyapatite can effectively repair bone defects while releasing antibiotics to treat infections. However, long-term or excessive use of antibiotics can easily induce the formation of bacterial resistance, which not only significantly reduces the antibacterial efficacy, but also may accelerate the evolution of "super bacteria", bringing new challenges to the treatment of bone infections. In recent years, the emergence of antibacterial material surface design strategies has provided great possibilities for replacing antibiotics and preventing the development of bacterial resistance. Therefore, the development of a nano-hydroxyapatite that inhibits the attachment of bacteria on the surface of the material or directly kills bacteria is a technical problem that needs to be solved urgently. Summary of the invention

[0004] In order to solve the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a method for preparing nano needle-shaped hydroxyapatite. The preparation method firstly uniformly loads the bioactive glass nanoparticles synthesized by the sol-gel method into a photosensitive resin to synthesize a bioactive substrate material with bioactive sites. Further, a nano needle-shaped hydroxyapatite with uniform morphology and high aspect ratio is generated in situ on the surface of the bioactive substrate material through a simple low-temperature immersion treatment. The nano needle-shaped hydroxyapatite can kill bacteria attached to the surface by using surface mechanical stress, while exerting osteogenic properties. The nano needle-shaped hydroxyapatite makes up for the bacterial resistance produced by antibacterial drugs and has a very broad application prospect in the treatment of infected bone defects.

[0005] The second object of the present invention is to provide a nano-needle-shaped hydroxyapatite prepared by the above method, which has a unique morphology and function, can simultaneously achieve the dual effects of antibacterial and osteogenesis, and is suitable for the fields of infectious bone defect repair, bone tissue engineering, etc.

[0006] The third object of the present invention is to provide an application of the above-mentioned nano needle-shaped hydroxyapatite.

[0007] The primary purpose of the present invention is achieved through the following technical solutions:

[0008] A method for preparing nano needle-shaped hydroxyapatite comprises the following preparation steps:

[0009] (1) Preparation of bioactive glass nanoparticles: dissolving hexadecyltrimethylammonium bromide in deionized water, stirring and dissolving, adding ethyl acetate to form microemulsion droplets, then adding ammonia water and continuing to stir, mixing evenly, slowly adding tetraethyl orthosilicate and calcium nitrate tetrahydrate and stirring, centrifuging and washing, vortexing and dispersing, and drying to obtain bioactive glass nanoparticles;

[0010] (2) Preparation of a bioactive substrate material: stirring and mixing the bioactive glass nanoparticles obtained in step (1) and a photosensitive resin under light-proof conditions, and subjecting the mixture to ultraviolet light curing to obtain a bioactive substrate material having bioactive sites;

[0011] (3) The bioactive substrate material obtained in step (2) is subjected to low-temperature immersion treatment to generate nano-needle-shaped hydroxyapatite on the surface in situ.

[0012] Preferably, the mass volume ratio of hexadecyltrimethylammonium bromide, deionized water, ethyl acetate, ammonia water, tetraethyl orthosilicate and calcium nitrate tetrahydrate in step (1) is 0.5-1.0 g: 30-40 mL: 8-12 mL: 0.4-0.6 mL: 3.0-4.0 mL: 2.0-3.0 g; the mass fraction of ammonia water is 28%-30%.

[0013] Preferably, in step (1), ethyl acetate and ammonia water are added dropwise and stirred for 10 to 30 minutes respectively, with a stirring rate of 100 to 300 rpm; in step (1), tetraethyl orthosilicate and calcium nitrate tetrahydrate are slowly added for 30 to 40 minutes, with a stirring time of 4 to 6 hours and a stirring rate of 100 to 300 rpm.

[0014] Preferably, the centrifugal washing in step (1) refers to alternating centrifugal washing with anhydrous ethanol and deionized water for 3 to 5 times, wherein the centrifugal speed is 5000 to 10000 rpm for 15 to 30 min; the vortex dispersion power is 30 to 90 W for 3 to 5 min; the drying refers to drying at 50 to 80 ° C for 12 to 24 h; the calcination temperature is 550 ° C to 650 ° C for 3 to 6 h, and the heating rate is 2 ° C / min to 5 ° C / min.

[0015] Preferably, in step (2), the mass percentage of the bioactive glass nanoparticles to the photosensitive resin is 25-35:65-75.

[0016] Preferably, the photosensitive resin in step (2) is prepared by mixing and stirring 40-60wt% of acrylate monomers, 1-5wt% of photoinitiator and 40-60wt% of diluent at room temperature in the dark; the mixing and stirring speed at room temperature in the dark is 200-500rpm, and the stirring time is 10-30min.

[0017] Preferably, the acrylic ester monomer is one of methyl methacrylate or hydroxyethyl acrylate; the photoinitiator is one of 2-hydroxy-2-methyl-1-phenyl-1-propanone (Irgacure 1173) or phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide (TPO); and the diluent is one of ethylene glycol dimethacrylate (EGDMA) or 1,6-hexanediol diacrylate (HDDA).

[0018] Preferably, the UV curing treatment in step (2) is performed using a radiation intensity of 50-100 mW / cm 2 , irradiate the surface of the biologically active substrate material at a distance of 0 to 1 mm for 10 to 30 seconds.

[0019] Preferably, the bioactive substrate material having bioactive sites in step (2) can be customized in shape and size.

[0020] Preferably, the low-temperature soaking treatment process in step (3) is as follows: soaking the bioactive substrate material in the treatment solution at a temperature of 37 to 37.5°C for 3 to 21 days, wherein the ratio of the bioactive substrate material to the treatment solution is 15 to 20 mm 2: 1mL; the treatment solution is composed of 1.50-3.00mmol / L CaCl2, 0.90-1.80mmol / L KH2PO4 and 50.00-80.00mmol / LKCl; the pH of the treatment solution is 7.4-8.5; the pH of the treatment solution is adjusted by 15.00-20.00mmol / L 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) or Tris-HCl buffer; the main components of Tris-HCl are tris(hydroxymethyl)aminomethane (Tris) and hydrochloric acid (HCl).

[0021] Further preferably, the nano needle-shaped hydroxyapatite crystals prepared in step (3) can be immersed in a 0.1-1.0 mol / L AgNO3 or Zn(NO3)2 solution for ion exchange to introduce Ag + or Zn 2+ After drying, nano-needle-shaped hydroxyapatite with better antibacterial properties is obtained.

[0022] The second object of the present invention is achieved by the following technical solutions:

[0023] A nano needle-shaped hydroxyapatite is prepared by the above preparation method.

[0024] The third object of the present invention is achieved by the following technical solutions:

[0025] The invention discloses an application of nano needle-shaped hydroxyapatite in the field of bone defect repair and bone tissue engineering.

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0027] (1) The present invention uses bioactive glass nanoparticles synthesized by the sol-gel method as the main body and photosensitive resin as the carrier to prepare a bioactive substrate material with certain bioactive sites, and further generates a nano-needle-shaped hydroxyapatite with uniform morphology and high aspect ratio in situ through a simple low-temperature immersion treatment.

[0028] (2) The bioactive substrate material prepared by the present invention can be customized in shape and size, and the nano-needle-shaped hydroxyapatite generated in situ on its surface has strong binding force, which significantly expands its application range in the fields of bone tissue engineering, dental restoration, drug sustained-release system and bionic materials.

[0029] (3) The nano-needle-shaped hydroxyapatite prepared by the present invention can better simulate the microstructure and biological activity of natural bone tissue, and has both antibacterial and osteogenic properties, and has a very broad application prospect in the treatment of infected bone defects.

[0030] (4) The preparation method of the present invention has simple process and low raw material cost, which is conducive to large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a characterization diagram of the bioactive glass nanoparticles prepared in Example 1; wherein, Figure 1 (A) is a scanning electron microscope (SEM) image; Figure 1 (B) is the X-ray diffraction (XRD) pattern;

[0032] Figure 2 Elemental map of the bioactive substrate material prepared in Example 1;

[0033] Figure 3 This is a characterization diagram of the nano-needle hydroxyapatite prepared in Example 1; wherein, Figure 3 (A) is the SEM image; Figure 3 (B) is the element distribution diagram; Figure 3 (C) is the Fourier transform infrared spectrum (FTIR); Figure 3 (D) is the XRD pattern;

[0034] Figure 4 This is a diagram showing the effect of the nano-needle-shaped hydroxyapatite prepared in Example 1 on anti-Gram-positive bacteria (S. mutans);

[0035] Figure 5 This is a diagram showing the effect of the nano-needle-shaped hydroxyapatite prepared in Example 1 on anti-Gram-negative bacteria (P.gingivalis);

[0036] Figure 6 This is a graph showing the osteogenic performance of the nano-needle-shaped hydroxyapatite prepared in Example 1;

[0037] Figure 7 This is a rendering of the nano-needle hydroxyapatite prepared in Example 2, wherein: Figure 7 (A) is the SEM image of the material surface morphology; Figure 7 (B) is the SEM image of the anti-S. mutans;

[0038] Figure 8 This is a rendering of the nano-needle-shaped hydroxyapatite prepared in Example 3, wherein: Figure 8 (A) is the SEM image of the material surface morphology; Figure 8 (B) is the SEM image of the anti-S. mutans;

[0039] Fig. 9 This is a rendering of the nano-needle hydroxyapatite prepared in Example 4, wherein: Fig. 9 (A) is the SEM image of the material surface morphology; Fig. 9 (B) is the SEM image of the anti-S. mutans;

[0040] Fig.10This is the SEM image of the nano-needle hydroxyapatite prepared in Example 5 against S. mutans;

[0041] Fig.11 is the SEM image of hydroxyapatite prepared in Comparative Example 1;

[0042] Fig.12 is the SEM image of hydroxyapatite prepared in Comparative Example 2;

[0043] Fig.13 This is the SEM image of hydroxyapatite prepared in Comparative Example 3. DETAILED DESCRIPTION

[0044] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0045] Example 1

[0046] The preparation method of the nano needle-shaped hydroxyapatite described in this embodiment includes the following preparation steps:

[0047] (1) Preparation of bioactive glass nanoparticles: 0.7 g of hexadecyltrimethylammonium bromide was dissolved in 33 mL of deionized water, and after stirring and dissolving, 10 mL of ethyl acetate was added to form microemulsion droplets, and further stirred for 30 min; then 0.47 mL of ammonia water was added and stirring was continued for 15 min; after mixing evenly, 3.6 mL of tetraethyl orthosilicate and 2.56 g of calcium nitrate tetrahydrate were slowly added and stirred for 4 h; after completion, anhydrous ethanol and deionized water were alternately centrifuged and washed at a speed of 10,000 rpm for 15 min, and vortex dispersed at a power of 80 W for 5 min. The collected particles were dried at 60°C overnight, and then calcined at 600°C for 6 h, with a heating rate of 2°C / min, to obtain bioactive glass nanoparticles;

[0048] (2) Preparation of bioactive substrate material: The bioactive glass nanoparticles obtained in step (1) and the photosensitive resin were stirred and mixed in a light-proof condition, with a mass fraction ratio of 20wt%:80wt%; the photosensitive resin was obtained by mixing 50wt% methyl methacrylate, 3wt% Irgacure 1173 and 47wt% EGDMA; and then the bioactive substrate material was prepared by using a radiation intensity of 100mW / cm 2 The ultraviolet light is used to irradiate the surface of the material for 20 seconds for light curing to obtain a bioactive substrate material with bioactive sites;

[0049] (3) The bioactive substrate material prepared in step (2) is immersed in 5 mL of a treatment solution for 14 days at a temperature of 37° C. to generate nano-needle-shaped hydroxyapatite on the surface in situ; the treatment solution is composed of 1.50 mmol / L CaCl2, 0.90 mmol / L KH2PO4 and 50.00 mmol / L KCl; the pH of the treatment solution is 7.4; and the pH of the treatment solution is adjusted by 20.00 mmol / L HEPES.

[0050] Figure 1 is a characterization diagram of the bioactive glass nanoparticles prepared in this embodiment. It can be seen from the figure that the bioactive glass nanoparticles were successfully prepared in this embodiment, and the particle size distribution is uniform and the dispersibility is good ( Figure 1 A); XRD spectrum shows a diffraction peak with a wide peak width and strong dispersion at 20°~35°, which has a typical amorphous structure of bioactive glass ( Figure 1 B).

[0051] Figure 2 : is the element map of the bioactive substrate material prepared in this embodiment, in which the Ca element is evenly distributed in the material.

[0052] Figure 3 is a characterization diagram of the nano needle-shaped hydroxyapatite prepared in this embodiment. It can be seen from the figure that the nano needle-shaped hydroxyapatite was successfully prepared in this embodiment. The width of these needle-shaped structures is about 70nm and the length is about 8μm ( Figure 3 A); the main elements are Ca, P, Si, O ( Figure 3 B); XRD pattern ( Figure 3 C) shows specific peaks at 2θ=25.9°, 28.1°, 31.7°, 34.0°, and 46.7°, which are attributed to the (002), (102), (211), (202), and (222) crystal planes of HA; FTIR spectrum ( Figure 3 D) Strong absorption bands were observed at 558 cm-1 and 1000-1100 cm-1, corresponding to the ν4 and ν3 absorption bands of the PO stretching vibration mode, respectively, indicating the presence of PO4 3- Functional groups.

[0053] Figure 4 This is a diagram showing the anti-Gram-positive bacteria (S. mutans) effect of the nano-needle-shaped hydroxyapatite prepared in this embodiment. The results show that there are fewer bacteria on the surface of the experimental group, and almost all of them are dead bacteria (red), and the bacteria have lost their original chain shape. It can be seen from the figure that the nano-needle-shaped hydroxyapatite prepared in this embodiment has effective antibacterial adhesion and bactericidal properties.

[0054] Figure 5This is a diagram showing the anti-Gram-negative bacteria (P.gingivalis) effect of the nano-needle-shaped hydroxyapatite prepared in this embodiment. The results show that the surface bacteria in the experimental group were significantly reduced, and the bacteria appeared concave. It can be seen from the figure that the nano-needle-shaped hydroxyapatite prepared in this embodiment has effective antibacterial adhesion and bactericidal properties.

[0055] Figure 6 3 is a graph showing the in vitro osteogenic performance of the nano-needle-shaped hydroxyapatite prepared in this example. It can be seen from the graph that the nano-needle-shaped hydroxyapatite has good expression of osteogenic-related genes.

[0056] Example 2

[0057] The preparation method of the nano needle-shaped hydroxyapatite described in this embodiment includes the following preparation steps:

[0058] (1) Preparation of bioactive glass nanoparticles: 0.7 g of hexadecyltrimethylammonium bromide was dissolved in 33 mL of deionized water, and after stirring and dissolving, 10 mL of ethyl acetate was added to form microemulsion droplets, and further stirred for 30 min; then 0.47 mL of ammonia water was added and stirring was continued for 15 min; after mixing evenly, 3.6 mL of tetraethyl orthosilicate and 2.56 g of calcium nitrate tetrahydrate were slowly added and stirred for 4 h; after completion, anhydrous ethanol and deionized water were alternately centrifuged and washed at a speed of 10,000 rpm for 15 min, and vortex dispersed at a power of 80 W for 5 min. The collected particles were dried at 60°C overnight, and then calcined at 600°C for 6 h, with a heating rate of 2°C / min, to obtain bioactive glass nanoparticles;

[0059] (2) Preparation of bioactive substrate material: The bioactive glass nanoparticles obtained in step (1) and the photosensitive resin were stirred and mixed in a light-proof condition, with a mass fraction ratio of 20wt%:80wt%; the photosensitive resin was obtained by mixing 50wt% methyl methacrylate, 3wt% Irgacure 1173 and 47wt% EGDMA; and then the bioactive substrate material was prepared by using a radiation intensity of 100mW / cm 2 The ultraviolet light is used to irradiate the surface of the material for 20 seconds for light curing to obtain a base material with biologically active sites;

[0060] (3) Soaking the bioactive substrate material prepared in step (2) in 5 mL of a treatment solution for 14 days at a temperature of 37° C. to generate nano-needle hydroxyapatite on the surface in situ; the treatment solution is composed of 1.50 mmol / L CaCl2, 0.90 mmol / L KH2PO4 and 50.00 mmol / L KCl; the pH of the treatment solution is 7.4; and the pH of the treatment solution is adjusted by 20.00 mmol / L Tris buffer;

[0061] Figure 7The effect diagram of the nano-needle-shaped hydroxyapatite prepared in this example is shown in FIG. 1 . The width of these needle-like structures is about 100 nm and the length is about 8 μm ( Figure 7 A), the surface bacteria lose their original chain shape ( Figure 7 B), it can be seen from the figure that the nano needle-shaped hydroxyapatite prepared in this example has effective antibacterial properties.

[0062] Example 3

[0063] The preparation method of the nano needle-shaped hydroxyapatite described in this embodiment includes the following preparation steps:

[0064] (1) Preparation of bioactive glass nanoparticles: 0.7 g of hexadecyltrimethylammonium bromide was dissolved in 33 mL of deionized water, and after stirring and dissolving, 10 mL of ethyl acetate was added to form microemulsion droplets, and further stirred for 30 min; then 0.47 mL of ammonia water was added and stirring was continued for 15 min; after mixing evenly, 3.6 mL of tetraethyl orthosilicate and 2.56 g of calcium nitrate tetrahydrate were slowly added and stirred for 4 h; after completion, anhydrous ethanol and deionized water were alternately centrifuged and washed at a speed of 10,000 rpm for 15 min, and vortex dispersed at a power of 80 W for 5 min. The collected particles were dried at 60°C overnight, and then calcined at 600°C for 6 h, with a heating rate of 2°C / min, to obtain bioactive glass nanoparticles;

[0065] (2) Preparation of bioactive substrate material: The bioactive glass nanoparticles obtained in step (1) and the photosensitive resin were stirred and mixed in a light-proof condition, with a mass fraction ratio of 30 wt %:70 wt %; the photosensitive resin was obtained by mixing 50 wt % methyl methacrylate, 3 wt % Irgacure 1173 and 47 wt % EGDMA; and then the bioactive substrate material was prepared by using a radiation intensity of 100 mW / cm 2 The ultraviolet light is used to irradiate the surface of the material for 20 seconds for light curing to obtain a base material with biologically active sites;

[0066] (3) The bioactive substrate material prepared in step (2) is immersed in 5 mL of a treatment solution for 14 days at a temperature of 37° C. to generate nano-needle-shaped hydroxyapatite on the surface in situ; the treatment solution is composed of 1.50 mmol / L CaCl2, 0.90 mmol / L KH2PO4 and 50.00 mmol / L KCl; the pH of the treatment solution is 7.4; and the pH of the treatment solution is adjusted by 20.00 mmol / L HEPES buffer.

[0067] Figure 8 The effect diagram of the nano-needle-shaped hydroxyapatite prepared in this example is shown in FIG. 1 . The width of these needle-like structures is about 150 nm and the length is about 8 μm ( Figure 8 A), the surface bacteria lose their original chain shape ( Figure 8 B), it can be seen from the figure that the nano needle-shaped hydroxyapatite prepared in this example has effective antibacterial properties.

[0068] Example 4

[0069] The preparation method of the nano needle-shaped hydroxyapatite described in this embodiment includes the following preparation steps:

[0070] (1) Preparation of bioactive glass nanoparticles: 0.7 g of hexadecyltrimethylammonium bromide was dissolved in 33 mL of deionized water, and after stirring and dissolving, 10 mL of ethyl acetate was added to form microemulsion droplets, and further stirred for 30 min; then 0.47 mL of ammonia water was added and stirring was continued for 15 min; after mixing evenly, 3.6 mL of tetraethyl orthosilicate and 2.56 g of calcium nitrate tetrahydrate were slowly added and stirred for 4 h; after completion, anhydrous ethanol and deionized water were alternately centrifuged and washed at a speed of 10,000 rpm for 15 min, and vortex dispersed at a power of 80 W for 5 min. The collected particles were dried at 60°C overnight, and then calcined at 600°C for 6 h, with a heating rate of 2°C / min, to obtain bioactive glass nanoparticles;

[0071] (2) Preparation of bioactive substrate material: The bioactive glass nanoparticles obtained in step (1) and the photosensitive resin were stirred and mixed in a light-proof condition, with a mass fraction ratio of 30 wt %:70 wt %; the photosensitive resin was obtained by mixing 50 wt % methyl methacrylate, 3 wt % Irgacure 1173 and 47 wt % EGDMA; and then the bioactive substrate material was prepared by using a radiation intensity of 100 mW / cm 2 The ultraviolet light is used to irradiate the surface of the material for 20 seconds for light curing to obtain a base material with biologically active sites;

[0072] (3) The bioactive substrate material prepared in step (2) is immersed in 5 mL of a treatment solution for 14 days at a temperature of 37° C. to generate nano-needle-shaped hydroxyapatite on the surface in situ; the treatment solution is composed of 1.50 mmol / L CaCl2, 0.90 mmol / L KH2PO4 and 50.00 mmol / L KCl; the pH of the treatment solution is 7.4; and the pH of the treatment solution is adjusted by 20.00 mmol / L Tris buffer.

[0073] Fig. 9 The effect diagram of the nano-needle-shaped hydroxyapatite prepared in this example is shown in FIG. 1 . The width of these needle-like structures is about 200 nm and the length is about 8 μm ( Fig. 9 A), the surface bacteria lose their original chain shape ( Fig. 9 B), it can be seen from the figure that the nano needle-shaped hydroxyapatite prepared in this example has effective antibacterial properties.

[0074] Example 5

[0075] The nano needle-shaped hydroxyapatite crystals prepared in Example 1 were immersed in a 0.1-1.0 mol / L AgNO3 or Zn(NO3)2 solution for ion exchange, and Ag was introduced. + or Zn 2+ After drying, nano-needle-shaped hydroxyapatite with better antibacterial properties is obtained.

[0076] Fig.10 This is the SEM image of the nano-needle-shaped hydroxyapatite prepared in this example against S. mutans. The cell wall and cell membrane of the bacteria on the surface of the material are completely deformed and ruptured, and "dissolution death" occurs.

[0077] Comparative Example 1

[0078] The preparation method of hydroxyapatite described in this comparative example comprises the following steps:

[0079] (1) Preparation of bioactive glass nanoparticles: 0.7 g of hexadecyltrimethylammonium bromide was dissolved in 33 mL of deionized water, and after stirring and dissolving, 10 mL of ethyl acetate was added to form microemulsion droplets, and further stirred for 30 min; then 0.47 mL of ammonia water was added and stirring was continued for 15 min; after mixing evenly, 3.6 mL of tetraethyl orthosilicate and 2.56 g of calcium nitrate tetrahydrate were slowly added and stirred for 4 h; after completion, anhydrous ethanol and deionized water were alternately centrifuged and washed at a speed of 10,000 rpm for 15 min, and vortex dispersed at a power of 80 W for 5 min. The collected particles were dried at 60°C overnight, and then calcined at 600°C for 6 h, with a heating rate of 2°C / min, to obtain bioactive glass nanoparticles;

[0080] (2) Preparation of bioactive substrate material: The bioactive glass nanoparticles obtained in step (1) and the photosensitive resin were stirred and mixed in a light-proof condition, with a mass fraction ratio of 10 wt %:90 wt %; the photosensitive resin was obtained by mixing 50 wt % methyl methacrylate, 3 wt % Irgacure 1173 and 47 wt % EGDMA; and then the bioactive substrate material was prepared by using a radiation intensity of 100 mW / cm 2 The ultraviolet light is used to irradiate the surface of the material for 20 seconds for light curing to obtain a base material with biologically active sites;

[0081] (3) The bioactive substrate material prepared in step (2) is immersed in 5 mL of a treatment solution for 14 days at a temperature of 37° C. to generate nano-needle-shaped hydroxyapatite on the surface in situ; the treatment solution is composed of 1.50 mmol / L CaCl2, 0.90 mmol / L KH2PO4 and 50.00 mmol / L KCl; the pH of the treatment solution is 7.4; and the pH of the treatment solution is adjusted by 20.00 mmol / L HEPES buffer.

[0082] Fig.11 3 is a SEM image of the hydroxyapatite prepared in this comparative example. It can be seen from the image that this comparative example failed to successfully generate nano-needle-shaped hydroxyapatite in situ.

[0083] Comparative Example 2

[0084] The preparation method of hydroxyapatite described in this comparative example comprises the following steps:

[0085] (1) Preparation of bioactive glass nanoparticles: 0.7 g of hexadecyltrimethylammonium bromide was dissolved in 33 mL of deionized water, and after stirring and dissolving, 10 mL of ethyl acetate was added to form microemulsion droplets, and further stirred for 30 min; then 0.47 mL of ammonia water was added and stirring was continued for 15 min; after mixing evenly, 3.6 mL of tetraethyl orthosilicate and 2.56 g of calcium nitrate tetrahydrate were slowly added and stirred for 4 h; after completion, anhydrous ethanol and deionized water were alternately centrifuged and washed at a speed of 10,000 rpm for 15 min, and vortex dispersed at a power of 80 W for 5 min. The collected particles were dried at 60°C overnight, and then calcined at 600°C for 6 h, with a heating rate of 2°C / min, to obtain bioactive glass nanoparticles;

[0086] (2) Preparation of bioactive substrate material: The bioactive glass nanoparticles obtained in step (1) and the photosensitive resin were stirred and mixed in a light-proof condition, with a mass fraction ratio of 40wt%:60wt%; the photosensitive resin was obtained by mixing 50wt% methyl methacrylate, 3wt% Irgacure 1173 and 47wt% EGDMA; and then the bioactive substrate material was prepared by using a radiation of 100mW / cm 2 The ultraviolet light is used to irradiate the surface of the material for 20 seconds for light curing to obtain a base material with biologically active sites;

[0087] (3) The bioactive substrate material prepared in step (2) is immersed in 5 mL of a treatment solution for 14 days at a temperature of 37° C. to generate nano-needle-shaped hydroxyapatite on the surface in situ; the treatment solution is composed of 1.50 mmol / L CaCl2, 0.90 mmol / L KH2PO4 and 50.00 mmol / L KCl; the pH of the treatment solution is 7.4; and the pH of the treatment solution is adjusted by 20.00 mmol / L HEPES buffer.

[0088] Fig.12 This is a SEM image of the hydroxyapatite prepared in this comparative example. It can be seen from the image that the hydroxyapatite prepared in this comparative example is severely fused, resulting in the inability to obtain individually dispersed nano-needle structures.

[0089] Comparative Example 3

[0090] The preparation method of hydroxyapatite described in this comparative example comprises the following steps:

[0091] (1) Preparation of bioactive substrate material: Commercial hydroxyapatite and photosensitive resin were stirred and mixed in a light-proof condition, with a mass fraction ratio of 20 wt %:80 wt %; the photosensitive resin was obtained by mixing 50 wt % methyl methacrylate, 3 wt % Irgacure 1173 and 47 wt % EGDMA; and then the irradiance was 100 mW / cm 2 The ultraviolet light is used to irradiate the surface of the material for 20 seconds for light curing to obtain a base material with biologically active sites;

[0092] (1) Soaking the bioactive substrate material prepared in step (2) in 5 mL of a treatment solution for 14 days at a temperature of 37° C. to generate nano-needle hydroxyapatite on the surface in situ; the treatment solution is composed of 1.50 mmol / L CaCl2, 0.90 mmol / L KH2PO4 and 50.00 mmol / L KCl; the pH of the treatment solution is 7.4; the pH of the treatment solution is adjusted by 20.00 mmol / L HEPES buffer;

[0093] Fig.13 3 is a SEM image of the hydroxyapatite prepared in this comparative example. It can be seen from the image that the hydroxyapatite prepared in this comparative example exhibits a flaky structure.

[0094] It can be seen from Examples 1 to 5 and Comparative Examples 1 to 3 that the present invention uses bioactive glass nanoparticles synthesized by the sol-gel method as the main body, uses photosensitive resin as a carrier to prepare a bioactive substrate material with certain bioactive sites, and further generates a nano-needle-shaped hydroxyapatite with uniform morphology and high aspect ratio in situ through a simple low-temperature immersion treatment; the nano-needle-shaped hydroxyapatite prepared by the present invention can better simulate the microstructure and bioactivity of natural bone tissue, has both antibacterial properties and osteogenic properties, and has very broad application prospects in the treatment of infectious bone defects.

[0095] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing nano-acicular hydroxyapatite, characterized in that: The method comprises the following preparation steps: (1) Preparation of bioactive glass nanoparticles: dissolving hexadecyltrimethylammonium bromide in deionized water, stirring and dissolving, adding ethyl acetate to form microemulsion droplets, then adding ammonia water and continuing to stir, mixing evenly, slowly adding tetraethyl orthosilicate and calcium nitrate tetrahydrate and stirring, centrifuging and washing, vortexing and dispersing, and drying to obtain bioactive glass nanoparticles; (2) Preparation of a bioactive substrate material: stirring and mixing the bioactive glass nanoparticles obtained in step (1) and a photosensitive resin under light-proof conditions, and subjecting the mixture to ultraviolet light curing to obtain a bioactive substrate material having bioactive sites; (3) The bioactive substrate material obtained in step (2) is subjected to low-temperature immersion treatment to generate nano-needle-shaped hydroxyapatite on the surface in situ.

2. The method for preparing nano needle-shaped hydroxyapatite according to claim 1, characterized in that: The mass volume ratio of hexadecyltrimethylammonium bromide, deionized water, ethyl acetate, ammonia water, tetraethyl orthosilicate and calcium nitrate tetrahydrate in step (1) is 0.5-1.0 g: 30-40 mL: 8-12 mL: 0.4-0.6 mL: 3.0-4.0 mL: 2.0-3.0 g; the mass fraction of ammonia water is 28%-30%.

3. The method for preparing nano needle-shaped hydroxyapatite according to claim 1, characterized in that: The centrifugal washing in step (1) refers to alternating centrifugal washing with anhydrous ethanol and deionized water for 3 to 5 times, wherein the centrifugal speed is 5000 to 10000 rpm for 15 to 30 min; the vortex dispersion power is 30 to 90 W for 3 to 5 min; the drying refers to drying at 50 to 80° C. for 12 to 24 h; the calcination temperature is 550 to 650° C. for 3 to 6 h, and the heating rate is 2° C. / min to 5° C. / min.

4. The method for preparing nano needle-shaped hydroxyapatite according to claim 1, characterized in that: The mass percentage of the bioactive glass nanoparticles to the photosensitive resin in step (2) is 25-35:65-75.

5. The method for preparing nano needle-shaped hydroxyapatite according to claim 1, characterized in that: The photosensitive resin in step (2) is prepared by mixing and stirring 40-60wt% of acrylate monomer, 1-5wt% of photoinitiator and 40-60wt% of diluent at room temperature in the dark; the mixing and stirring speed at room temperature in the dark is 200-500rpm, and the stirring time is 10-30min.

6. The method for preparing nano needle-shaped hydroxyapatite according to claim 5, characterized in that: The acrylic ester monomer is one of methyl methacrylate or hydroxyethyl acrylate; the photoinitiator is one of 2-hydroxy-2-methyl-1-phenyl-1-propanone or phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide; and the diluent is one of ethylene glycol dimethacrylate or 1,6-hexanediol diacrylate.

7. The method for preparing nano needle-shaped hydroxyapatite according to claim 1, characterized in that: The low temperature soaking treatment process in step (3) is as follows: soaking the bioactive substrate material in the treatment solution at a temperature of 37 to 37.5°C for 3 to 21 days, wherein the ratio of the bioactive substrate material to the treatment solution is 15 to 20 mm 2 : 1mL; the treatment solution is composed of a mixture of 1.50-3.00mmol / L CaCl2, 0.90-1.80mmol / L KH2PO4 and 50.00-80.00mmol / L KCl; the pH of the treatment solution is 7.4-8.5; the pH of the treatment solution is adjusted by 15.00-20.00mmol / L 4-hydroxyethylpiperazineethanesulfonic acid or Tris-HCl buffer; the main components of Tris-HCl are tris(hydroxymethyl)aminomethane and hydrochloric acid.

8. The method for preparing nano needle-shaped hydroxyapatite according to claim 1, characterized in that: The nano-needle-shaped hydroxyapatite crystals prepared in step (3) can also be immersed in a 0.1-1.0 mol / L AgNO3 or Zn(NO3)2 solution for ion exchange to introduce Ag+ or Zn2+, and after drying, nano-needle-shaped hydroxyapatite with better antibacterial properties can be obtained.

9. A nano-needle hydroxyapatite, characterized in that: Prepared according to the preparation method according to any one of claims 1 to 8.

10. Use of the nano needle-shaped hydroxyapatite according to claim 9 in the field of bone defect repair and bone tissue engineering.

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