Preparation method of rare earth ion doped antibacterial modified hydroxyapatite whisker material

Abstract

The invention discloses a preparation method of a rare earth ion doped antibacterial modified hydroxyapatite whisker material, and relates to the technical field of medical biomaterials.The preparation method comprises the steps that acetamide is dissolved in deionized water, and a solution A is obtained; dissolving calcium salt, cerium salt and ammonium salt in a certain proportion in the solution A to obtain a solution B; stirring the solution B at room temperature, performing ultrasonic dispersion, and adjusting the pH value to be acidic to obtain a solution C; and putting the solution C into a high-pressure reaction kettle, heating and reacting, aging, cleaning, carrying out suction filtration, and drying to obtain the cerium-doped hydroxyapatite whisker material. The preparation process is simple, the product is stable, and the prepared modified hydroxyapatite whisker has an obvious antibacterial effect on escherichia coli and staphylococcus aureus and can be applied to the field of bone repair antibacterial materials.

Description

Technical Field

[0001] The invention relates to the technical field of medical biomaterials, and in particular to a method for preparing a rare earth ion-doped antibacterial modified hydroxyapatite whisker material. Background Art

[0002] Among the clinical failure cases of bone defect repair surgery, postoperative infection accounts for a significant proportion (clinical statistics show that the infection rate can reach 10%-20%), becoming a key bottleneck restricting the efficacy. Although traditional bone repair materials such as autologous bone and allogeneic bone have good biocompatibility, they have natural defects: the former is limited by insufficient bone supply and donor site complications (such as pain and fracture risk), and the latter faces the risk of immune rejection and potential pathogen transmission (such as virus and prion contamination). More importantly, both lack inherent antibacterial ability and cannot resist the adhesion, colonization and biofilm formation of bacteria (such as Staphylococcus aureus and Escherichia coli) around the surgical wound. The extracellular polysaccharide matrix formed by bacteria on the surface of the material will significantly enhance drug resistance, making conventional antibiotic treatment ineffective.

[0003] Hydroxyapatite whiskers, due to their homology with bone components and the mechanical enhancement brought about by their high aspect ratio, offer unique advantages in bone repair. Metal ion-doped hydroxyapatite is a new research direction for improving the performance of bone repair materials through modification. Existing technologies include magnesium and strontium, which promote osteoblast activity and bone mineralization. Antimicrobial ions such as silver and zinc can impart antibacterial properties to bone repair materials, inhibiting postoperative infection. However, silver ions have low biosafety and high toxicity, while zinc ions have drawbacks such as a narrow antimicrobial spectrum, potential biological and environmental toxicity, environmentally sensitive stability, poor material compatibility, and potential for drug resistance.

[0004] Therefore, it is necessary to propose a preparation method and application of rare earth ion doped antibacterial modified hydroxyapatite whisker material to solve the above technical problems. Summary of the Invention

[0005] The main purpose of the present invention is to provide a preparation method of rare earth ion doped antibacterial modified hydroxyapatite whisker material to solve the technical problems of low biosafety, inability to continuously sterilize, drug resistance, and lack of broad spectrum of antibacterial bone repair materials in the prior art, and to form a multifunctional bone repair material that integrates antibacterial, bone induction, and mechanical support.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A method for preparing a rare earth ion-doped antibacterial modified hydroxyapatite whisker material comprises the following steps:

[0008] (1) Dissolve acetamide in deionized water to obtain solution A;

[0009] (2) dissolving a certain proportion of calcium salt, cerium salt and ammonium salt in solution A to obtain solution B;

[0010] (3) Stirring Solution B at room temperature, ultrasonically dispersing, and adjusting the pH to acidic to obtain Solution C;

[0011] (4) Liquid C is placed in a high-pressure reactor for heating and reaction, and cerium-doped hydroxyapatite whisker material is obtained after aging, washing, filtering, and drying.

[0012] Specifically, the concentration of acetamide in solution A in step (1) is 0.9 to 1.1 mol / L.

[0013] Specifically, in step (2), the molar ratio of the total molar amount of calcium salt and cerium salt to ammonium salt is 1.60-1.72:1 based on atomic weight; wherein the ratio of calcium salt to cerium salt is 0.92-0.97:0.03-0.08.

[0014] Specifically, in step (2), the calcium salt is one of calcium nitrate and calcium oxide; the ammonium salt is one of diammonium hydrogen phosphate and ammonium dihydrogen phosphate; and the cerium salt is one of cerium nitrate and cerium chloride.

[0015] The method uses calcium nitrate, calcium oxide, diammonium hydrogen phosphate, and ammonium dihydrogen phosphate as calcium and phosphorus sources; cerium nitrate hexahydrate and cerium chloride heptahydrate as sources of rare earth cerium; and acetamide as a pH buffer during whisker growth, to grow hydroxyapatite into hydroxyapatite whiskers with a hexagonal cross-section under high pressure and high temperature conditions. The method is simple to prepare the rare earth cerium ion-doped antibacterial modified hydroxyapatite whiskers, resulting in a stable finished product and stable production. Furthermore, the prepared rare earth cerium ion-doped antibacterial modified hydroxyapatite whiskers maintain a good whisker shape, have uniform cerium ion distribution, and exhibit excellent antibacterial effects, making them suitable as a novel antibacterial material for bone repair.

[0016] Specifically, calcium salt, cerium salt and ammonium salt are added to solution A in sequence with an interval of 8 to 15 minutes.

[0017] Specifically, in step (3), the stirring time is 40 to 50 minutes; the ultrasonic time is 5 to 8 minutes, and the ultrasonic frequency is 40 kHz to 50 kHz.

[0018] Specifically, the pH in step (3) is adjusted by adding nitric acid to adjust the pH value to 2-3; the concentration of nitric acid is 5-10 mol / L.

[0019] Specifically, in step (4), the pressure of the high-pressure reactor is 3-6 MPa, the reaction time is 4-6 h, and the reaction temperature is 150° C.-180° C.

[0020] Specifically, the aging time in step (4) is 20 to 30 hours, and the washing is to reduce the pH to 7 to 8.

[0021] Specifically, the drying in step (4) is carried out by forced air drying at 30°C to 80°C for 8 to 10 hours. The beneficial effects of the present invention are:

[0022] 1. The rare earth cerium-doped hydroxyapatite whiskers prepared by the present invention still maintain the morphological characteristics of hydroxyapatite whiskers, and the cross-section is a hexagonal structure. The hexagonal structure makes its crystal structure stable and the mechanical properties are improved; and the cerium ions are evenly distributed and have excellent antibacterial properties. It can be used as an antibacterial material for bone repair, for example, to reduce wound infection after bone implant surgery, and as an antibacterial bone nail.

[0023] 2. The modified hydroxyapatite whiskers prepared by the present invention have obvious antibacterial effects on Escherichia coli and Staphylococcus aureus, and can be used in the field of bone repair antibacterial materials.

[0024] 3. The preparation method of the present invention has simple process, stable finished product, can be produced on a large scale, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is an electron micrograph of 5% Ce-doped hydroxyapatite whiskers prepared in Example 2 of the present invention;

[0026] Figure 2 This is an electron microscope image of 5% Ce-doped hydroxyapatite whiskers prepared in Comparative Example 2 of the present invention;

[0027] Figure 3 This is an electron micrograph of 3% Ce-doped hydroxyapatite whiskers prepared in Example 1 of the present invention;

[0028] Figure 4 This is the EDS element distribution diagram of 5% Ce-doped hydroxyapatite whiskers prepared in Example 2 of the present invention.

[0029] Figure 5 XRD patterns of 3% Ce-doped hydroxyapatite whiskers prepared in Example 1 of the present invention and hydroxyapatite whiskers prepared in Comparative Example 1.

[0030] Figure 6 These are diagrams showing bacterial biofilm formation for Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) in Example 1, Example 2, and Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0032] Example 1

[0033] The preparation of rare earth cerium doped hydroxyapatite whiskers (3% Ce) comprises the following steps:

[0034] (1) 5.927 g of acetamide was dissolved in 100 ml of deionized water to obtain solution A. 3.82 g of calcium nitrate tetrahydrate, 0.21 g of cerium nitrate hexahydrate, and 1.32 g of diammonium hydrogen phosphate were added to solution A in sequence at intervals of 8 min to obtain solution B.

[0035] (2) Solution B was stirred at room temperature for 40 min, then ultrasonicated for 8 min at a frequency of 45 kHz. The pH was adjusted to 2.8 with 10 mol / L nitric acid solution to obtain Solution C.

[0036] (3) Liquid C was placed in a high-pressure reactor at 3 MPa and 160°C and heated for 4 h. After cooling to room temperature, it was taken out and aged for 20 h. The pH value was washed to 7.4, filtered, and dried at 60°C for 10 h to obtain 3% Ce-doped hydroxyapatite whiskers.

[0037] Example 2

[0038] The preparation of rare earth cerium doped hydroxyapatite whiskers (5% Ce) comprises the following steps:

[0039] (1) 5.334 g of acetamide was dissolved in 100 ml of deionized water to obtain solution A. 3.74 g of calcium nitrate tetrahydrate, 0.36 g of cerium nitrate hexahydrate, and 1.32 g of diammonium hydrogen phosphate were added to solution A in sequence at intervals of 9 min to obtain solution B.

[0040] (2) Solution B was stirred at room temperature for 40 min, then ultrasonicated for 8 min at a frequency of 45 kHz. The pH was adjusted to 3 with 10 mol / L nitric acid solution to obtain Solution C.

[0041] (3) Liquid C was placed in a high-pressure reactor at 3 MPa and 160°C and heated for 4 h. After cooling to room temperature, it was taken out and aged for 20 h. The pH value was washed to 7.4, filtered, and dried at 60°C for 10 h to obtain 5% Ce-doped hydroxyapatite whiskers.

[0042] Example 3

[0043] The preparation of rare earth cerium doped hydroxyapatite whiskers (8% Ce) comprises the following steps:

[0044] (1) 6.5197 g of acetamide was dissolved in 100 ml of deionized water to obtain solution A. 3.63 g of calcium nitrate tetrahydrate, 0.58 g of cerium nitrate hexahydrate, and 1.32 g of diammonium hydrogen phosphate were added to solution A at intervals of 9 minutes to obtain solution B.

[0045] (2) Solution B was stirred at room temperature for 50 min, then ultrasonicated for 8 min at a frequency of 45 kHz. The pH was adjusted to 3 with 8 mol / L nitric acid solution to obtain Solution C.

[0046] (3) Liquid C was placed in a high-pressure reactor at 4 MPa and 180°C and heated for 6 h. After cooling to room temperature, it was taken out and aged for 20 h. The pH value was washed to 7.4, filtered, and dried at 60°C for 10 h to obtain 8% Ce-doped hydroxyapatite whiskers.

[0047] Example 4

[0048] The preparation of rare earth cerium doped hydroxyapatite whiskers (5% Ce) comprises the following steps:

[0049] (1) 5.334 g of acetamide was dissolved in 100 ml of deionized water to obtain solution A. 1.75 g of calcium chloride, 0.308 g of cerium chloride heptahydrate, and 1.53 g of ammonium dihydrogen phosphate were added to solution A in sequence at intervals of 9 min to obtain solution B.

[0050] (2) Solution B was stirred at room temperature for 40 min, then ultrasonicated for 5 min at a frequency of 40 kHz. The pH was adjusted to 2.8 with 10 mol / L hydrochloric acid solution to obtain Solution C.

[0051] (3) Liquid C was placed in a high-pressure reactor at 6 MPa and 160°C and heated for 4 h. After cooling to room temperature, it was taken out and aged for 25 h. The pH value was washed to 8, filtered, and dried at 30°C for 9 h to obtain 5% Ce-doped hydroxyapatite whiskers.

[0052] Example 5

[0053] The preparation of rare earth cerium doped hydroxyapatite whiskers (8% Ce) comprises the following steps:

[0054] (1) 6.5197 g of acetamide was dissolved in 100 ml of deionized water to obtain solution A. 1.705 g of calcium chloride, 0.58 g of cerium chloride heptahydrate, and 1.53 g of ammonium dihydrogen phosphate were added to solution A in sequence at intervals of 15 min to obtain solution B.

[0055] (2) Solution B was stirred at room temperature for 45 min, then ultrasonicated for 6 min at a frequency of 50 kHz. The pH was adjusted to 2 with 5 mol / L hydrochloric acid solution to obtain Solution C.

[0056] (3) Liquid C was placed in a high-pressure reactor at 6 MPa and 150°C and heated for 5 h. After cooling to room temperature, it was taken out and aged for 30 h. The pH value was washed to 7, filtered, and dried at 80°C for 8 h to obtain 8% Ce-doped hydroxyapatite whiskers.

[0057] Comparative Example 1

[0058] This comparative example provides a method for preparing hydroxyapatite whiskers, comprising the following steps:

[0059] (1) Dissolve 5.305 g of acetamide in 100 ml of deionized water to obtain solution A. Add 3.94 g of calcium nitrate tetrahydrate and 1.32 g of diammonium hydrogen phosphate to solution A at intervals of 8 min to obtain solution B.

[0060] (2) Solution B was stirred at room temperature for 40 min, then ultrasonicated for 8 min at a frequency of 45 kHz. The pH was adjusted to 2.8 with 10 mol / L nitric acid solution to obtain Solution C.

[0061] (3) Liquid C was placed in a high-pressure reactor at 3 MPa and 160°C and heated for 4 h. After cooling to room temperature, it was taken out and aged for 20 h. The pH value was washed to 7.4, filtered, and dried at 60°C for 10 h to obtain hydroxyapatite whiskers.

[0062] Comparative Example 2

[0063] A method for preparing rare earth cerium-doped hydroxyapatite whiskers (5% Ce) comprises the following steps:

[0064] (1) 4.5 g of urea and 0.2 g of sorbitol were dissolved in 100 ml of deionized water to obtain solution A. 3.74 g of calcium nitrate tetrahydrate, 0.36 g of cerium nitrate hexahydrate, and 1.32 g of diammonium hydrogen phosphate were added to solution A at intervals of 9 min to obtain solution B.

[0065] (2) Solution B was stirred at room temperature for 40 min, then ultrasonicated for 8 min at a frequency of 45 kHz. The pH was adjusted to 3 with 10 mol / L nitric acid solution to obtain Solution C.

[0066] (3) Liquid C was placed in a high-pressure reactor at 3 MPa and 160°C and heated for 4 h. After cooling to room temperature, it was taken out and aged for 20 h. The pH value was washed to 7.4, filtered, and dried at 60°C for 10 h to obtain hydroxyapatite whiskers.

[0067] Comparative Example 3

[0068] The preparation of rare earth cerium doped hydroxyapatite whiskers (5% Ce) comprises the following steps:

[0069] (1) 5.334 g of acetamide was dissolved in 100 ml of deionized water to obtain solution A. 3.74 g of calcium nitrate tetrahydrate, 0.36 g of cerium nitrate hexahydrate, and 1.32 g of diammonium hydrogen phosphate were added to solution A in sequence at intervals of 9 min to obtain solution B.

[0070] (2) Solution B was stirred at room temperature for 40 min, then ultrasonicated for 8 min at a frequency of 45 kHz. The pH was adjusted to 3 with 10 mol / L nitric acid solution to obtain Solution C.

[0071] (3) Place liquid C in a water bath at 90°C for 12 hours, cool it to room temperature, take it out, age it for 20 hours, adjust the pH value to 7.4, filter it, and air-dry it at 60°C for 10 hours to obtain hydroxyapatite whiskers.

[0072] The SEM images show that the cerium-doped hydroxyapatite whiskers prepared in Comparative Example 3 also exhibit short, flat shapes, with some sea urchin-like hydroxyapatite present. This indicates that regardless of whether the reaction temperature of Solution C is high or low, cerium-doped hydroxyapatite whiskers with a hexagonal cross-section cannot be obtained.

[0073] Test Example 1:

[0074] Figure 1 The figure shows the SEM image of the cerium-doped hydroxyapatite whisker (5% Ce) obtained in Example 2. It can be seen that the cross section of the prepared cerium-doped hydroxyapatite whisker presents a hexagonal shape.

[0075] Figure 2 The SEM image of the cerium-doped hydroxyapatite whiskers (5% Ce) prepared in Comparative Example 2 shows that the cerium-doped hydroxyapatite whiskers are short and flat, with some sea urchin-like hydroxyapatite particles present. It can be seen that other dissolution methods cannot produce whisker materials with a hexagonal cross-section.

[0076] Figure 3 The figure shows the SEM image of the cerium-doped hydroxyapatite whiskers (3% Ce) obtained in Example 1. It can be seen that the prepared cerium-doped hydroxyapatite whiskers are whisker-like hydroxyapatite with a high aspect ratio.

[0077] The average aspect ratios of the cerium-doped hydroxyapatite whiskers (3% Ce) in Example 1, the cerium-doped hydroxyapatite whiskers (5% Ce) in Example 2, the cerium-doped hydroxyapatite whiskers (8% Ce) in Example 3, and the cerium-doped hydroxyapatite whiskers (5% Ce) in Example 4, the hydroxyapatite whiskers prepared in Comparative Example 1, and the rare earth cerium-doped hydroxyapatite whiskers (5% Ce) in Comparative Example 2 are analyzed as shown in Table 1 below. It can be seen that as the amount of cerium incorporated increases, the aspect ratio decreases, but the cerium-doped hydroxyapatite whiskers prepared by the present invention still maintain a high aspect ratio, which is beneficial to the enhancement and toughening of biological bone tissue scaffold engineering materials.

[0078] Table 1

[0079] sample Average aspect ratio Comparative Example 1 31.695 Example 1 25.356 Comparative Example 2 15.624 Example 2 20.435 Example 3 15.461 Example 4 21.524

[0080] The doping success rates of the cerium-doped hydroxyapatite whiskers (5% Ce) in Example 2 and the cerium-doped hydroxyapatite whiskers (5% Ce) in Comparative Example 2 were compared. As shown in Table 2, it can be seen that the doping success rate of the cerium-doped hydroxyapatite whiskers prepared using the present invention is greatly improved. The doping success rate formula is as follows:

[0081]

[0082] Table 2

[0083] sample Doping success rate Comparative Example 2 25.6％ Example 2 81.2％

[0084] Figure 4 Shown is the EDS surface element distribution diagram of the cerium-doped hydroxyapatite whiskers (5% Ce) in Example 2. The results show that the cerium element is evenly distributed on the hydroxyapatite, and the prepared cerium-doped hydroxyapatite whiskers maintain the hexagonal structure of the hydroxyapatite crystals, proving that the preparation method adopted in the present invention successfully prepared cerium-doped hydroxyapatite whiskers.

[0085] Figure 5 Shown are the XRD patterns of the cerium-doped hydroxyapatite whiskers (3% Ce) in Example 1 and the hydroxyapatite whiskers in Comparative Example 1. The results show that the cerium-doped hydroxyapatite whiskers are still the hydroxyapatite whisker phase, but compared with Comparative Example 1, the diffraction peak of Example 1 decreases and the size of the doped whiskers is reduced, which is confirmed by the results obtained in Table 1.

[0086] Test Example 2

[0087] The antibacterial effect of the obtained samples was tested as follows:

[0088] A 316L stainless steel sheet (1 cm × 1 cm × 0.3 cm) was placed at the bottom of a 24-well plate, and Escherichia coli and Staphylococcus aureus bacterial liquids (1 × 10 7CFU / mL) were co-cultured with suspensions (1 mg / mL) of hydroxyapatite whiskers in Comparative Example 1, cerium-doped hydroxyapatite whiskers (3% Ce) in Example 1, and cerium-doped hydroxyapatite whiskers (5% Ce) in Example 2 in a well plate. The cells were incubated under anaerobic conditions at 37°C for 24 hours, washed with PBS, and fixed with methanol. The bacteria adhered to the surface of the stainless steel sheet were stained with 0.1% crystal violet staining solution. The dye attached to the biofilm was eluted with anhydrous ethanol and transferred to a 96-well plate. The absorbance at 600 nm was detected by a microplate reader. The formula for the inhibition rate of biofilm formation is as follows:

[0089]

[0090] Table 3

[0091] Test items Inhibition rate of Escherichia coli biofilm (%) Inhibition rate of Staphylococcus aureus biofilm (%) Comparative Example 1 5.3 7.6 Example 1 61.2 62.3 Example 2 86.5 88.5

[0092] The results show that the rare earth ion cerium-doped hydroxyapatite whiskers prepared by the method of the present invention have excellent inhibitory effects on the growth of Escherichia coli and Staphylococcus aureus. Figure 6 As shown, it further demonstrates its excellent antibacterial effect. Among them, the aspect ratio of cerium-doped hydroxyapatite whiskers (8% Ce) in Example 3 is relatively not excellent. Regardless of its antibacterial property, the antibacterial effect of cerium-doped hydroxyapatite whiskers (5% Ce) in Example 2 is better than that of cerium-doped hydroxyapatite whiskers (3% Ce) in Example 1.

[0093] The above detailed description of the specific embodiments of the invention is intended to be illustrative only, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications or substitutions to the invention are also within the scope of the present invention. Therefore, equivalent changes, modifications, and improvements made without departing from the spirit and scope of the present invention are also encompassed within the scope of the present invention.

Claims

1. A method for preparing rare earth ion-doped antibacterial modified hydroxyapatite whisker material, characterized in that: The steps include: (1) Dissolve acetamide in deionized water to obtain solution A; (2) dissolving a certain proportion of calcium salt, cerium salt and ammonium salt in solution A to obtain solution B; (3) Stirring solution B at room temperature, ultrasonically dispersing, and adjusting the pH to acidic to obtain solution C; (4) Liquid C is placed in a high-pressure reactor for heating and reaction, and cerium-doped hydroxyapatite whisker material is obtained after aging, washing, filtering, and drying.

2. The method for preparing the rare earth ion doped antibacterial modified hydroxyapatite whisker material according to claim 1, wherein: The concentration of acetamide in solution A of step (1) is 0.9-1.1 mol / L.

3. The method for preparing the rare earth ion doped antibacterial modified hydroxyapatite whisker material according to claim 1, wherein: In step (2), the molar ratio of the total molar amount of the calcium salt and the cerium salt to the ammonium salt is 1.60-1.72:1 based on atomic weight; wherein the ratio of the calcium salt to the cerium salt is 0.92-0.97:0.03-0.

08.

4. The method for preparing the rare earth ion doped antibacterial modified hydroxyapatite whisker material according to claim 3, wherein: In step (2), the calcium salt is one of calcium nitrate and calcium chloride; the ammonium salt is one of diammonium hydrogen phosphate and ammonium dihydrogen phosphate; and the cerium salt is one of cerium nitrate and cerium chloride.

5. The method for preparing the rare earth ion doped antibacterial modified hydroxyapatite whisker material according to claim 4, wherein: Calcium salt, cerium salt and ammonium salt are added to solution A in sequence with an interval of 8 to 15 minutes.

6. The method for preparing the rare earth ion doped antibacterial modified hydroxyapatite whisker material according to claim 1, wherein: In step (3), the stirring time is 40 to 50 minutes; the ultrasonic time is 5 to 8 minutes, and the ultrasonic frequency is 40 kHz to 50 kHz.

7. The method for preparing the rare earth ion doped antibacterial modified hydroxyapatite whisker material according to claim 1, wherein: The pH in step (3) is adjusted by adding one of nitric acid or hydrochloric acid to adjust the pH value to 2-3; the concentration of the acid is 5-10 mol / L.

8. The method for preparing the rare earth ion doped antibacterial modified hydroxyapatite whisker material according to claim 1, wherein: In step (4), the pressure of the high-pressure reactor is 3-6 MPa, the reaction time is 4-6 h, and the reaction temperature is 150° C.-180° C.

9. The method for preparing the rare earth ion-doped antibacterial modified hydroxyapatite whisker material according to claim 8, wherein: The aging time in step (4) is 20 to 30 hours, and the cleaning is to reduce the pH to 7 to 8.

10. The method for preparing the rare earth ion doped antibacterial modified hydroxyapatite whisker material according to claim 9, characterized in that: The drying in step (4) is carried out by forced air drying at 30°C to 80°C for 8 to 10 hours.

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