Copper-doped coral / hydroxyapatite artificial bone meal as well as preparation method and application thereof

Abstract

The invention discloses copper-doped coral / hydroxyapatite artificial bone meal as well as a preparation method and application thereof. The preparation method comprises the following steps: pretreating coral; the pretreatment comprises ball milling and calcining treatment; the pretreated coral is soaked in a mixed solution for a hydrothermal reaction, and the copper-doped coral / hydroxyapatite artificial bone meal is obtained; the mixed solution is a mixed solution of a phosphorus source solution and a copper source solution. The coral is converted into artificial bone powder which can be potentially applied to bone repair after osteomyelitis operation for the first time, waste recycling is achieved, the preparation method is simple, the doping efficiency is high, the structure is stable, and the porous structure of the powder is well reserved. Meanwhile, the artificial bone meal is beneficial to adhesion, proliferation and differentiation of cells in the artificial bone meal, has good biocompatibility and better antibacterial performance, and is expected to play an important role in the field of bone tissue engineering.

Description

Technical Field

[0001] The invention relates to the technical field of ion-doped hydroxyapatite materials, and in particular to a copper-doped coral / hydroxyapatite artificial bone powder and a preparation method and application thereof. Background Art

[0002] Corals are coral polyps that absorb Ca from the external marine environment during their growth. 2+ With carbon dioxide, and secrete limestone as its own living shell. The main component of coral is calcium carbonate, which exists in the form of calcite microcrystals, accounting for about 95-99%, and there are also certain components of organic matter, such as sulfated polysaccharides, and trace ions. Among them, the trace elements such as magnesium and strontium contained in coral are beneficial to bone growth. Coral has a porous structure with a pore size of 100-500μm, which is similar to the pore structure of cancellous bone. This structure is conducive to cell adhesion and has the characteristics of promoting blood vessel growth. It can be used as bone transplantation and bone repair material.

[0003] Calcium carbonate, the main component of coral, has poor stability and is easily dissolved in weak acid. Hydroxyapatite (HAp) is the main component of natural bone tissue and has good biocompatibility and osteoconductivity. If the reaction conditions are controlled, a layer of hydroxyapatite is transformed and prepared on the surface of the coral to obtain a coral / hydroxyapatite material containing hydroxyapatite on the surface, which can not only retain the porous structure well, but also make it have good biocompatibility. However, these scaffolds currently lack antibacterial properties, so it is necessary to develop new coral-based artificial bone powder.

[0004] With the help of ion doping modification technology, different ions are doped into the lattice of hydroxyapatite, which can give hydroxyapatite new functions. Common doping cations include Sr 2+ 、Ag + 、Zn 2+ etc., easily replace Ca in the lattice 2+ ; Common anions include F - 、SiO4 4- , Cl - etc., can replace PO4 in the lattice 3- or OH - .

[0005] Copper (Cu) is an essential nutrient element in animals and plants, and has a significant effect in antibacterial applications. 2+ Replace Ca 2+Entering the lattice of hydroxyapatite, under aerobic conditions, copper ions can react with proteins or fatty acids in the bacterial cell membrane to produce oxidative damage. Osteomyelitis is mainly caused by Staphylococcus aureus infection. Osteomyelitis surgery often causes bone defects. Coral is similar to cancellous bone, but there is no report on the application of copper doping in coral / hydroxyapatite artificial bone powder. Whether copper can be added to coral / hydroxyapatite artificial bone powder, what effect does it have on the lattice and morphology after addition, and whether the obtained composite material is stable are all issues that need to be studied in depth and solved. Summary of the invention

[0006] In order to solve the above problems, the purpose of the present invention is to provide a copper-doped coral / hydroxyapatite artificial bone powder and a preparation method and application thereof; currently there is no coral-based artificial bone powder material used in the treatment of osteomyelitis. The present invention prepares a functional artificial bone powder that not only has good biological activity but also has a porous structure, high doping efficiency and good stability.

[0007] To achieve the above objectives, the present invention adopts the following technical solutions.

[0008] A method for preparing copper-doped coral / hydroxyapatite artificial bone powder comprises the following steps:

[0009] (1) pre-treating the coral; the pre-treatment includes ball milling and calcination;

[0010] (2) Immersing the pretreated coral in a mixed solution for hydrothermal reaction to obtain copper-doped coral / hydroxyapatite artificial bone powder; the mixed solution is a mixed solution of a phosphorus source solution and a copper source solution.

[0011] Preferably, the coral in step (1) is first ball-milled, soaked in hydrogen peroxide solution, and washed before being calcined;

[0012] Further preferably, the concentration of the hydrogen peroxide solution is 15-30wt%; the soaking time is 6-10 hours; the washing is ultrasonic washing in deionized water;

[0013] Preferably, the skeleton of the coral in step (1) is in the shape of branches, so as to facilitate ball milling to obtain coral granular powder with regular particle size.

[0014] Preferably, the average particle size of the coral after ball milling in step (1) is 1-2 mm; and the ball mill used for the ball milling is a steel ball mill.

[0015] Preferably, the calcination treatment temperature in step (1) is 450°C-650°C, the heating and cooling rate is 2.5-3°C / min, and the holding time is 50min-80min.

[0016] Further preferably, the calcination temperature is 600° C., the heating and cooling rates are 2.5° C. / min, and the holding time is 1 h.

[0017] Preferably, the molar ratio Cu / Ca of the copper in the mixed solution and the calcium in the pretreated coral in step (2) is 0.01-0.1:1;

[0018] Further preferably, the molar ratio Cu / Ca of the copper in the mixed solution to the calcium in the pretreated coral is 0.02:1.

[0019] Preferably, the molar ratio of calcium in the pretreated coral to copper and phosphorus in the mixed solution (Ca+Cu) / P is 1.5-2:1;

[0020] Further preferably, the molar ratio of calcium in the pretreated coral to copper and phosphorus in the mixed solution (Ca+Cu) / P is 1.67.

[0021] Preferably, the mass volume ratio of the pretreated coral and the mixed solution in step (2) is 0.1-1 g:50 mL.

[0022] Further preferably, the mass volume ratio of the pretreated coral and the mixed solution is 0.5 g:50 mL.

[0023] Preferably, the temperature of the hydrothermal reaction in step (2) is 140-180° C., and the reaction time is ≥16 h.

[0024] Further preferably, the temperature of the hydrothermal reaction is 180° C. and the reaction time is 16 h.

[0025] Preferably, the copper source solution in step (2) is a copper nitrate solution, and the phosphorus source solution is a diammonium hydrogen phosphate solution;

[0026] Preferably, before immersing the pretreated coral in the mixed solution in step (2), the pH of the mixed solution is adjusted to 9-11 with aqueous ammonia;

[0027] Further preferably, before immersing the pretreated coral in the mixed solution, the pH of the mixed solution is adjusted to 10 with ammonia water.

[0028] Further preferably, the concentration of the ammonia water is 35-40wt%.

[0029] Preferably, the hydrothermal reaction in step (2) is followed by ultrasonic washing and drying;

[0030] Further preferably, the ultrasonic washing is ultrasonic washing in water, and the sign of the completion of washing is that the pH of the last washing liquid is in the pH range of pure water 6.8-7.2.

[0031] Further preferably, the drying process is carried out at 50-60° C. for 12-24 hours.

[0032] The copper-doped coral / hydroxyapatite artificial bone powder is prepared by the above preparation method.

[0033] Preferably, the artificial bone powder is a granular powder with an average particle diameter of 1.5 mm and a pore size distribution of 50-200 μm.

[0034] Application of the copper-doped coral / hydroxyapatite artificial bone powder in the preparation of bone transplant materials or bone repair materials.

[0035] In the copper-doped coral / hydroxyapatite artificial bone powder of the present invention, copper can be effectively doped into the hydroxyapatite layer on the surface of the coral granular powder. The doping amount of copper can be easily controlled according to different doping ratios, and the doping effect is stable.

[0036] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0037] (1) The method for preparing copper-doped coral / hydroxyapatite artificial bone powder of the present invention is simple. The raw material is discarded corals from the seaside, which achieves waste recycling. The main component is calcium carbonate, and the porosity and pore size are similar to those of cancellous bone. Moreover, copper can be stably incorporated into the hydroxyapatite layer on the surface of the coral, the doping rate is high and controllable, and the obtained artificial bone powder has a stable structure.

[0038] (2) The copper-doped coral / hydroxyapatite artificial bone powder of the present invention retains the porous structure of the coral while undergoing transformation, which is beneficial to the adhesion, proliferation and differentiation of cells.

[0039] (3) Utilizing the antibacterial effect of copper, the copper-doped coral / hydroxyapatite artificial bone powder of the present invention can be combined with other fields such as antibacterial, anti-inflammatory, and osteogenesis to exert a synergistic effect.

[0040] (4) The copper-doped coral / hydroxyapatite artificial bone powder prepared by the present invention has good biocompatibility and highly effective antibacterial and anti-inflammatory effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 2. XRD diagram of different Cu / Ca-doped copper coral / hydroxyapatite artificial bone powder in Example 1.

[0042] Figure 2 This is the FTIR distribution diagram of different Cu / Ca-doped copper coral / hydroxyapatite artificial bone powder in Example 1.

[0043] Figure 3 FESEM images of ball-milled coral granular powder (a) and copper-doped coral / hydroxyapatite artificial bone powder samples 1-5 (bf).

[0044] Figure 4 The colony statistics of copper-doped coral / hydroxyapatite artificial bone powder samples 1-5 were co-cultured with Staphylococcus aureus (a) or Escherichia coli (b) for 12 hours.

[0045] Figure 5 The absorbance results of cck8 were tested after ball-milled coral granular powder and copper-doped coral / hydroxyapatite artificial bone powder samples 1-5 were co-cultured with mBMSCs for 1d (a) or 3d (b).

[0046] Figure 6 The results of PCR analysis of the pro-inflammatory gene IL-6 (a) and the anti-inflammatory gene TGF-β (b) after co-culture of ball-milled coral granular powder and copper-doped coral / hydroxyapatite artificial bone powder samples 1 and 3 with polarized macrophages for 1 day. DETAILED DESCRIPTION

[0047] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

[0048] Example 1

[0049] Copper-doped coral / hydroxyapatite artificial bone powder with Cu / Ca molar ratios of 0, 0.01, 0.02, 0.05, and 0.1 were prepared.

[0050] (1) The ball-milled coral granular powder with an average particle size of 1.5 mm was immersed in a 15 wt% hydrogen peroxide solution for 6 hours, ultrasonically cleaned, and calcined in a muffle furnace for 1 hour. The calcination temperature was 600°C, and the heating rate and cooling rate were both 2.5°C / min. Too fast temperature change or too long insulation time will cause the granular powder to be heated unevenly and break.

[0051] (2) According to (Ca+Cu) / P of 1.67, Cu / Ca molar ratio of 0, 0.01, 0.02, 0.05, 0.1, and the mass of coral granular powder (0.5g), calculate the required mass of (NH4)2HPO4 and Cu(NO3)2, dissolve (NH4)2HPO4 (analytical grade) and Cu(NO3)2 (analytical grade) in deionized water, respectively, and adjust the solution to 50mL. After adjusting the pH of the two solutions to 10 with concentrated ammonia (37wt%), the phosphorus source solution is titrated into the copper source solution while stirring to form a 100mL mixed solution. The coral granular powder and the mixed solution are transferred to a 150ml reactor and hydrothermally reacted at 180℃ for 16h. After the reaction, the coral powder was taken out and immersed in deionized water for ultrasonic cleaning. After each ultrasonic treatment for 3 minutes, it was replaced with fresh deionized water until the pH of the last washing solution was in the pH range of pure water (6.8-7.2) and then washed again with anhydrous ethanol. After drying at 60°C for 24 hours, different Cu / Ca-doped copper coral / hydroxyapatite artificial bone powder samples were obtained, as shown in Table 1.

[0052] Table 1

[0053] sample Cu / Ca 1 0 2 0.01 3 0.02 4 0.05 5 0.1

[0054] Figure 1 XRD diagram of copper-doped coral / hydroxyapatite artificial bone powder obtained by controlling different Cu / Ca. Figure 1 As shown, when the Cu / Ca ratio is low (0-0.05), the samples all show characteristic peaks of hydroxyapatite. When the Cu / Ca ratio is 0.1, new diffraction peaks appear at around 40° and 50°, and copper phosphate will be produced on the sample surface.

[0055] Figure 2 The FTIR images of copper-doped coral / hydroxyapatite artificial bone powder obtained by controlling different Cu / Ca ratios are shown below. Figure 2 As shown, CO3 appears in each image 2- and PO4 3- The characteristic peaks prove that the copper-doped hydroxyapatite layer is only formed on the surface of the coral granular powder, and the interior is still calcium carbonate.

[0056] Figure 3 Figure a is the FESEM image of the coral granular powder after ball milling. Figure 3 bf in the figure are the surface FESEM images of samples 1 to 5. It can be seen that after the hydroxyapatite transformation, the calcite morphology on the coral surface gradually transforms into a rod-like hydroxyapatite structure. As the Cu doping content increases further, the rod-like structure gradually transforms into a needle-like structure.

[0057] In summary, copper is doped into the hydroxyapatite lattice, and the doping efficiency increases first and then stabilizes with the increase of the doping amount, among which the optimal Cu / Ca is 0.05.

[0058] Example 2

[0059] Antimicrobial effect of copper-doped coral / hydroxyapatite artificial bone powder.

[0060] The artificial bone powder (samples 1-5) prepared in Example 1 was sterilized at 121°C for 30 min under high pressure and then mixed with 1.5 mL 10 4 -10 5 After co-culture of CFU of S. aureus (Staphylococcus aureus) and E. coli (Escherichia coli) for 6-12 hours, 10-20 μL of the co-culture solution was taken to plate, and then cultured in a 37°C bacterial incubator for 6 hours, and the number of colonies was counted; no artificial bone powder was added to the control group.

[0061] Figure 4 a in the figure is the statistical result of the plate coating after the copper-doped coral / hydroxyapatite artificial bone powder obtained with different Cu / Ca was co-cultured with Staphylococcus aureus. Figure 4 b in the figure is the result of the plate coating after the copper-doped coral / hydroxyapatite artificial bone powder obtained with different Cu / Ca was co-cultured with Escherichia coli. It can be found that with the increase of the doping amount, the number of colonies decreased. When Cu / Ca is greater than or equal to 0.02, the antibacterial effect of the sample reaches more than 90%.

[0062] Example 3

[0063] Biocompatibility of copper-doped coral / hydroxyapatite artificial bone powder.

[0064] The ball-milled coral granular powder and the artificial bone powder (samples 1-5) prepared in Example 1 were sterilized at 121°C for 30 min under high pressure and high temperature, and then placed in a 96-well plate and co-cultured with 5000-6000 mBMSCs cells for 1 day and 3 days, respectively. After the culture medium was aspirated, the cck8 reagent was added, and the mixture was placed in a 37°C incubator in a dark place and cultured for 1.5h-2h. After that, 70 μL of the cck8 dye solution was taken and the absorbance was tested using a microplate reader. No coral or artificial bone powder was added to the control group.

[0065] Figure 5 a in the figure is the cck8 result of co-culture of copper-doped coral / hydroxyapatite artificial bone powder and mBMSCs for 1 day obtained with different Cu / Ca. Figure 5Figure b shows the CCK8 results of 3 days of co-culture of copper-doped coral / hydroxyapatite artificial bone powder and mBMSCs obtained with different Cu / Ca. It can be found that when the Cu / Ca is low (0-0.02), the sample has good biocompatibility, and when the Cu / Ca is high (0.05-0.1), the sample begins to show obvious cytotoxicity. It can be seen that the optimal Cu / Ca is 0.02.

[0066] Example 4

[0067] Anti-inflammatory effects of copper-doped coral / hydroxyapatite artificial bone powder.

[0068] The ball-milled coral granular powder and the artificial bone powder prepared in Example 1 (sample 1 and sample 3) were sterilized at 121°C for 30 min under high pressure and placed in a 6-well plate and co-cultured with 20,000-30,000 polarized macrophages for 1 day, and then the cell RNA was extracted for PCR experiments; no coral and artificial bone powder were added to the control group.

[0069] Figure 6 a in the figure is the PCR result of the pro-inflammatory gene IL-6 obtained by co-culturing polarized macrophages with copper-doped coral / hydroxyapatite artificial bone powder with a Cu / Ca molar ratio of 0.02 for 1 day. Figure 6 b in the figure is the PCR result of the anti-inflammatory gene TGF-β after 1 day of co-culture of copper-doped coral / hydroxyapatite artificial bone powder with polarized macrophages at a Cu / Ca molar ratio of 0.02. It can be found that the anti-inflammatory effect of coral hydroxyapatite is significantly improved after copper addition.

[0070] 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 made without departing from the spirit and principle of the present invention shall be equivalent replacement modes and shall be included in the protection scope of the present invention.

Claims

1. A method for preparing copper-doped coral / hydroxyapatite artificial bone powder, characterized in that: The following steps are involved: (1) pre-treating the coral; the pre-treatment includes ball milling and calcination; (2) Immersing the pretreated coral in a mixed solution for hydrothermal reaction to obtain copper-doped coral / hydroxyapatite artificial bone powder; the mixed solution is a mixed solution of a phosphorus source solution and a copper source solution.

2. The preparation method according to claim 1, characterized in that: The coral in step (1) is ball-milled, soaked in hydrogen peroxide solution, and washed before being calcined; The average particle size of the coral after ball milling in step (1) is 1-2 mm; The calcination temperature in step (1) is 450°C-650°C, the heating and cooling rate is 2.5-3°C / min, and the holding time is 50min-80min.

3. The preparation method according to claim 2, characterized in that: The calcination temperature is 600° C., the heating and cooling rates are 2.5° C. / min, and the holding time is 1 h.

4. The preparation method according to claim 1, characterized in that: The molar ratio of copper in the mixed solution and calcium in the pretreated coral in step (2) is 0.01-0.1:

1. The molar ratio of calcium in the pretreated coral to copper and phosphorus in the mixed solution (Ca+Cu) / P is 1.5-2:1 in step (2); The mass volume ratio of the pretreated coral and the mixed solution in step (2) is 0.1-1 g:50 mL.

5. The preparation method according to claim 4, characterized in that: The molar ratio Cu / Ca of the copper in the mixed solution and the calcium in the pretreated coral is 0.02:

1.

6. The preparation method according to claim 1, characterized in that: The temperature of the hydrothermal reaction in step (2) is 140-180° C., and the reaction time is ≥16 h.

7. The preparation method according to claim 6, characterized in that: The temperature of the hydrothermal reaction is 180° C. and the reaction time is 16 h.

8. The preparation method according to claim 1, characterized in that: The copper source solution in step (2) is a copper nitrate solution, and the phosphorus source solution is a diammonium hydrogen phosphate solution; Step (2) Before immersing the pretreated coral in the mixed solution, the pH of the mixed solution is adjusted to 9-11 with ammonia water; In step (2), the hydrothermal reaction is followed by ultrasonic washing and drying; the ultrasonic washing is carried out in water, and the completion of washing is indicated by the pH of the washing liquid at the last washing being in the pH range of 6.8-7.2 of pure water.

9. Copper-doped coral / hydroxyapatite artificial bone powder prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the copper-doped coral / hydroxyapatite artificial bone powder according to claim 9 in preparing bone transplant materials or bone repair materials.

Images