Preparation method of high-dispersity low-crystallinity antibacterial hydroxyapatite microspheres

Abstract

The invention discloses a preparation method of a hydroxyapatite microsphere with high dispersity, low crystallinity and antibacterial property. The preparation method of the high-dispersity, low-crystallinity and antibacterial hydroxyapatite microspheres is characterized by comprising the following steps: firstly, uniformly mixing a calcium nitrate solution and a phytic acid solution, then adding a diammonium hydrogen phosphate solution, keeping the molar ratio of Ca to P at 1.67, then adjusting the pH value to 2-4 by using a 10% NaOH solution, then adding urea, and uniformly stirring, so as to obtain the high-dispersity, low-crystallinity and antibacterial hydroxyapatite microspheres with the content of introduced phytic acid being 0.25-1.5 times of the mass of HA; the mass of the introduced urea is 3 times that of the HA. And sealing the mixed solution, standing in a blast oven at 80-95 DEG C for 24 hours, filtering the obtained product, washing with water until the product is neutral, and drying. Compared with the prior art, the hydroxyapatite microspheres prepared by the method are mild in preparation condition, have good dispersity, relatively low crystallinity and relatively good antibacterial property, so that the hydroxyapatite microspheres can be used as a high polymer filler for preparing a high-performance composite material, can also be directly used for bone filling particles, and can also be used for preparing bone filling materials. The material has a good application prospect in orthopedic materials.

Description

Technical Field

[0001] The invention relates to a method for preparing hydroxyapatite microspheres with high dispersibility, low crystallinity and antibacterial property, and belongs to the field of biomedical materials. Background Art

[0002] Hydroxyapatite [Ca 10 (PO4)6(OH)2, HA], contains mineral components and structures similar to those of human bones, has good biocompatibility, osteoconductivity and high wear resistance, and is slightly alkaline. It is considered to be an inorganic material with good bioactivity in orthopedic materials. However, it is brittle when used alone as an orthopedic material, and usually requires the use of the toughness of polymers to improve its processability. However, studies have shown that the easy agglomeration characteristics of HA inorganic particles are not conducive to composite with polymers. At the same time, there are many morphologies of hydroxyapatite, the main morphologies are needle (thread) shape, whisker, spherical, sheet (ribbon) shape, flower shape and rod shape. Among them, spherical HA has the characteristics of good fluidity, high packing density, high strength, and not easy to agglomerate, which is more conducive to composite with polymers. However, the HA microspheres prepared by the traditional common template method and spray drying method have their own shortcomings, and it is difficult to obtain an ideal morphology. On the other hand, high crystallinity HA is not conducive to its degradation in the body, and HA itself lacks antibacterial properties, making it difficult to prevent the recurrence and delay of bacterial infections in orthopedic surgical areas. Traditional Ag + 、Zn 2+ The research on plasma doping or adsorption of bioactive substances such as vancomycin and antimicrobial peptides has disadvantages such as limited doping amount, easy burst release, high cost, etc. Therefore, it is of great significance to obtain a HA microsphere with regular morphology, controllable particle size, high dispersibility, low crystallinity and antibacterial properties in the research of orthopedic materials.

[0003] Chinese patent CN 117185266 A discloses that sodium citrate is introduced and porous hydroxyapatite microspheres with uniform morphology and a particle size range of 50 nm~50 μm can be prepared by hydrothermal method. However, the hydrothermal method uses a polytetrafluoroethylene-lined hydrothermal kettle, which is placed in a blast drying oven, and the reaction temperature is set to 150~200 ℃, and the hydrothermal reaction time is 1~20 hours. Obviously, this preparation method has certain requirements for the reaction equipment, and long-term high-temperature reaction is not conducive to large-scale production. Phytic acid is a natural, non-toxic and unique biologically active small molecule widely found in plant seeds and fruits and vegetables. It has a very wide range of applications in the fields of chemical industry and food. Due to its unique structure (the cyclic molecule contains six phosphate groups and 12 free hydrogens), phytic acid has super strong chelating properties with metal ions such as calcium and magnesium. The literature [J Mater Sci-Mater M, 2024, 35: 36] reported calcium phosphate bone cement cured with phytic acid, which confirmed that it can improve its physical properties such as compressive strength and curing time, and also greatly improve its biocompatibility. The literature [Polymers 2019, 11, 1721] confirmed that phytic acid can strongly chelate with divalent metal ions, destroy the bacterial outer membrane, and cause organelle, DNA and protein dysfunction, thus having an antibacterial effect. Therefore, if phytic acid is introduced into the preparation process of HA, its chelation with calcium ions is utilized, and phytic acid hybridized HA microspheres with regular morphology can be obtained at a lower temperature, and the steric hindrance of phytic acid molecules is used to improve its dispersibility, reduce its crystallinity, and give it antibacterial properties, it is expected to be better compounded with polymers for better application in orthopedic materials. Summary of the invention

[0004] The present invention provides a method for preparing hydroxyapatite microspheres with high dispersibility, low crystallinity and antibacterial properties. The hydroxyapatite microspheres prepared by the method not only have good dispersibility and low crystallinity, but also have good antibacterial properties. Therefore, the novel nanoapatite can be used as a polymer filler to prepare high-performance composite materials, and can also be directly used to prepare bone filling particles, and has good application prospects in orthopedic materials.

[0005] A method for preparing hydroxyapatite microspheres with high dispersibility, low crystallinity and antibacterial properties comprises the following steps: First, mix the calcium nitrate solution and the phytic acid solution evenly, then add the diammonium hydrogen phosphate solution, keep the Ca / P molar ratio at 1.67, then adjust the pH to 2-4 with 10% NaOH solution, and then add urea, wherein the introduced phytic acid content is 0.25-1.5 times the mass of HA, and the introduced urea mass is 3 times the mass of HA. After sealing the mixed solution, place it in a blast oven at 80-95 ° C for 24 hours, filter the obtained product, wash it with water until it is neutral, and dry it. The advantages of the preparation method of hydroxyapatite microspheres with high dispersibility, low crystallinity and antibacterial properties provided by the present invention are: (1) In terms of material performance, the hydroxyapatite microspheres prepared by the present invention have phytic acid molecules that combine with calcium ions through chelation, giving them a steric hindrance and achieving high dispersibility. At the same time, the introduced phytic acid is connected to hydroxyapatite through chemical bonding, so that the phytic acid can be slowly released, which can give them antibacterial properties. At the same time, due to the introduction of phytic acid molecules, the hydroxyapatite structure is distorted and its crystallinity is reduced, which is conducive to its degradation in the body. It can be seen that the hydroxyapatite microspheres have high dispersibility, low crystallinity and antibacterial properties.

[0006] (2) The various raw materials used in the present invention are easily available, the preparation steps are simple, the reaction conditions are mild, no equipment such as a reaction kettle and a high-temperature oven is required, and the reaction time is short. At the same time, the reagents used are environmentally friendly and suitable for large-scale production.

[0007] (3) The hydroxyapatite microspheres prepared by the present invention can have their diameter and biological properties regulated by reaction temperature and phytic acid content. They can not only be directly used as bone defect filling particles, but also can be used as polymer fillers to prepare high-performance composite materials. They have great application prospects in various orthopedic fields such as bone tissue engineering scaffold materials, guided bone tissue regeneration membranes, and drug-loaded materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 The following are SEM photos of different powders in the examples and their dispersion in dichloromethane. DETAILED DESCRIPTION

[0009] Example 1: Weigh 0.5 g of phytic acid solution (the phytic acid concentration used is 70% and needs to be converted to 100% phytic acid by mass fraction), add 40 ml of water to dilute. Weigh 4.76 g of calcium nitrate tetrahydrate and dissolve it in 40 ml of water, mix the two solutions obtained, weigh 1.58 g of diammonium hydrogen phosphate and dissolve it in 40 ml of water, stir it with a glass rod to dissolve it, drop the dissolved diammonium hydrogen phosphate solution into the above-mentioned calcium nitrate tetrahydrate solution, adjust the pH value to 4 with 10% NaOH solution, add 6 g of urea, seal the beaker and transfer it to a 95°C oven for 24 hours. The obtained product is filtered, washed with water until neutral, and dried. The sample was observed by SEM to have a microsphere diameter of 5~10 μm, and the XRD test showed a crystallinity of 40%. It was well dispersed in dichloromethane for 1 hour, and the antibacterial rate was 75%.

[0010] Example 2: Weigh 2 grams of phytic acid solution (the phytic acid concentration used is 70% and needs to be converted to phytic acid with a mass fraction of 100%), and add 40 ml of water to dilute. Weigh 4.76 grams of calcium nitrate tetrahydrate and dissolve it in 40 ml of water, mix the two solutions obtained, weigh 1.58 grams of diammonium hydrogen phosphate and dissolve it in 40 ml of water, stir it with a glass rod to dissolve it, drop the dissolved diammonium hydrogen phosphate solution into the calcium nitrate tetrahydrate solution, adjust the pH value to 2 with 10% NaOH solution, add 6 grams of urea, seal the beaker and transfer it to an 80°C oven for 24 hours. The obtained product is filtered, washed with water until neutral, and dried. The sample was observed by SEM to have a microsphere diameter of 30~50 μm, and the XRD test showed a crystallinity of 30%. It was well dispersed in dichloromethane for 1 hour, and the antibacterial rate was 80%.

[0011] Example 3: Weigh 3 grams of phytic acid solution (the phytic acid concentration used is 70%, which needs to be converted to 100% phytic acid by mass fraction), and add 40 ml of water to dilute. Weigh 4.76 grams of calcium nitrate tetrahydrate and dissolve it in 40 ml of water, mix the two solutions obtained, weigh 1.58 grams of diammonium hydrogen phosphate and dissolve it in 40 ml of water, stir it with a glass rod to dissolve it, drop the dissolved diammonium hydrogen phosphate solution into the calcium nitrate tetrahydrate solution, adjust the pH value to 3 with 10% NaOH solution, add 6 grams of urea, seal the beaker and transfer it to a 90 ° C oven, and let it stand for 24 hours. The obtained product is filtered, washed with water until neutral, and dried. The sample was observed by SEM to have a microsphere diameter of 15~30 μm, and the XRD test showed a crystallinity of 27%. It was well dispersed in dichloromethane for 1 hour, and the antibacterial rate was 82%.

[0012] Comparative Example 1: Weigh 2 grams of citric acid and add 40 milliliters of water to dilute. Weigh 4.76 grams of calcium nitrate tetrahydrate and dissolve it in 40 milliliters of water, mix the two solutions evenly, weigh 1.58 grams of diammonium hydrogen phosphate and dissolve it in 40 milliliters of water, stir it with a glass rod to dissolve it, drop the dissolved diammonium hydrogen phosphate solution into the calcium nitrate tetrahydrate solution, add 6 grams of urea to it, seal the beaker and transfer it to a 90 ℃ oven and let it stand for 24 hours. The product is filtered, washed with water until neutral, and dried. The sample was observed by SEM to obtain irregular microspheres with a diameter of 10~20 μm, and the crystallinity tested by XRD was 35%. It began to agglomerate after standing in dichloromethane for 30 minutes, and had no antibacterial effect.

[0013] Comparative Example 2: Weigh 4.76 grams of calcium nitrate tetrahydrate and dissolve it in 40 milliliters of water, mix the two solutions evenly, weigh 1.58 grams of diammonium hydrogen phosphate and dissolve it in 40 milliliters of water, stir it with a glass rod to dissolve it, drop the dissolved diammonium hydrogen phosphate solution into the calcium nitrate tetrahydrate solution, adjust the pH value to 3 with HCl solution, add 6 grams of urea, seal the beaker and transfer it to a 90°C oven, and let it stand for 24 hours. The product is filtered, washed with water until neutral, and dried. The sample was observed by SEM to be whisker-like HA with a length of 5~10 μm and a diameter of about 0.5 μm. The XRD test showed that the crystallinity was 70%. It began to agglomerate in dichloromethane for 5 minutes and the upper layer was a clear liquid with no antibacterial effect.

[0014] Comparative Example 3: Weigh 4.76 grams of calcium nitrate tetrahydrate and dissolve it in 40 milliliters of water, mix the two solutions evenly, weigh 1.58 grams of diammonium hydrogen phosphate and dissolve it in 40 milliliters of water, stir it with a glass rod to dissolve it, drop the dissolved diammonium hydrogen phosphate solution into the calcium nitrate tetrahydrate solution, adjust the pH value to about 10 with 10% NaOH solution, seal the beaker and transfer it to a 90°C oven and let it stand for 24 hours. The obtained product is filtered, washed with water until neutral, and dried. The sample was observed by SEM to be needle-shaped HA with a length of 0.6~0.8 μm and a diameter of about 0.2 μm. The XRD test showed that the crystallinity was 80%. It was allowed to stand in dichloromethane for 30 minutes and the upper layer of the agglomerate was a clear liquid with no antibacterial effect.

[0015] Crystallinity calculation: X-ray diffractometer was used to test the crystal structure of HA samples. The crystallinity was calculated according to the following formula:

[0016] Where Xc is the crystallinity; I 300 is the reflection intensity of the (300) crystal plane; V 112 / 300 is the reflection intensity of the valley between two adjacent peaks corresponding to the (112) and (300) crystal planes.

[0017] Antibacterial activity assay: Escherichia coli (DH5a) was cultured in lysogeny broth (LB) in an incubator for 3 h, at which time 5 mL of bacterial suspension (10 5 CFU / mL) and HA extracts were incubated in test tubes at 37°C in a shaking incubator for 24 h. Finally, the bacteria retained in the suspension were counted. The bactericidal rate was calculated using the following formula: .

Claims

1. A method for preparing hydroxyapatite microspheres with high dispersibility, low crystallinity and antibacterial properties, Its characteristics are First, the calcium nitrate solution and the phytic acid solution are mixed evenly, and then the diammonium hydrogen phosphate solution is added to maintain the Ca / P molar ratio of 1.67, and then the pH is adjusted to 2-4 with 10% NaOH solution, and then urea is added, wherein the introduced phytic acid content is 0.25-1.5 times the mass of HA, and the introduced urea mass is 3 times the mass of HA. The mixed solution is sealed and placed in a blast oven at 80-95 ° C for 24 hours. The obtained product is filtered, washed with water until neutral, and dried.

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