Fluorine and selenium co-doped hydroxyapatite implant coating and its preparation method and application

Abstract

The present invention discloses a fluorine and selenium co-doped hydroxyapatite implant coating and its preparation method and application. The chemical formula of the coating is Ca 10 (PO4) 6‑y (SeO3) y (OH) 2‑x F x The preparation method is as follows: step A, preparation of suspension A; step B, preparation of suspension B: adding fluorine-doped hydroxyapatite powder and dispersant B to ethanol solution B and mixing evenly; ball-milling the obtained mixed solution B, and sieving it after ball milling to obtain suspension B; step C, treatment of the substrate: pre-treating medical metal or medical alloy material as the substrate; step D, using liquid phase plasma spraying method, adopting synchronous double liquid feeding mode to simultaneously spray suspension A and suspension B onto the substrate; the coating is used to repair bone defects, repair bone defects in patients with osteosarcoma, inhibit the recurrence of osteosarcoma, and prevent infection during bone reconstruction.

Description

Technical Field

[0001] The present invention relates to the technical field of biological coatings, in particular to a fluorine and selenium co-doped hydroxyapatite implant coating and a preparation method and application thereof. Background Art

[0002] Hydroxyapatite [Ca 10 (PO4)6(OH)2, HA] is the main inorganic component of human and animal bones and teeth. It has good bioactivity and bone tissue guidance, and can form chemical bonds with human bone tissue. Therefore, it is widely used in titanium and titanium alloy implant surface coatings to increase the surface bioactivity of titanium and titanium alloys. However, from the perspective of long-term clinical implant effects, the performance of HA coatings still has some shortcomings: 1) Compared with other bioactive materials, the solubility of HA coatings is relatively high. After a period of time in the implant body, the coating partially dissolves, reducing the stability of the coating and the long-term effectiveness of the implant; 2) The antibacterial property of HA coatings is poor, and implant infections caused by orthopedic surgery (ie, "biomaterial-induced infection") occur frequently, causing great pain to patients. In addition, HA coatings lack specificity in repairing bone damage under pathological conditions and are not very effective.

[0003] Osteosarcoma is a primary bone tumor originating from bone-forming stem cells, often occurring in joints. Current clinical treatment primarily relies on surgery, which involves removing the tumor and recurrent sites, followed by the placement of artificial implants for reconstruction. Chemotherapy and radiotherapy are also required to kill any remaining osteosarcoma cells and prevent recurrence. Repairing this pathological bone injury requires not only that the coating material on the surface of the artificial implant possess the ability to rapidly induce bone regeneration but also that it possesses certain functional properties, such as anti-tumor activity.

[0004] Therefore, it is necessary to develop a new HA coating material with excellent biological activity, safe and long-lasting antibacterial and anti-tumor properties by doping and modifying the HA material based on its own advantages, so as to promote bone defect repair while inhibiting the recurrence of bone tumors and reducing the possibility of infection during bone reconstruction, so as to better meet the needs of clinical applications.

[0005] Existing experimental results have shown that the use of fluorine (F) element to dope and modify HA can improve the biological activity and antibacterial activity of HA materials while reducing its solubility. But the key is how to effectively control the release of F element in the complex microenvironment of the body. If the amount of F released is too high, it will inhibit multiple enzymes in the body, causing fluorosis, dental fluorosis and other fluorosis poisoning diseases. Selenium (Se) is one of the trace elements necessary for maintaining human health. It can regulate organ function, prevent cardiovascular disease, reduce inflammation, regulate bone tissue metabolism, and significantly reduce the possibility of tumors. Appropriate amounts of Se have no obvious effect on the growth and survival of normal tissue cells, but have a specific killing effect on tumor cells. In addition to its obvious anti-tumor effect, Se can also achieve antagonism to F by improving the integrity of cell membranes and immune function within a certain concentration range.

[0006] Therefore, in view of the good biological activity and bone tissue repair function of HA materials, the antibacterial activity of F elements and the anti-tumor and antagonistic fluorine effects of Se elements, the preparation of F and Se co-doped HA coating materials can promote bone defect repair while inhibiting the recurrence of osteosarcoma and reducing the possibility of infection during bone reconstruction, playing a certain preventive role.

[0007] Liquid phase plasma spraying is a type of plasma spraying technology. Liquid phase plasma spraying uses liquid feeding instead of powder feeding in traditional plasma spraying technology. The synthesized nano or submicron powder is dispersed in water or organic solvent by ultrasound or ball milling. The stability of the suspension is adjusted, and the suspension is heated by the plasma flame and then deposited on the substrate to form a coating. Summary of the Invention

[0008] To this end, the technical problem to be solved by the present invention is to provide a fluorine and selenium co-doped hydroxyapatite implant coating and its preparation method and application, fluorine and selenium are doped into HA material and sprayed onto the surface of orthopedic implant material, and the bone microenvironment of the bone damage site of osteosarcoma patients is improved through the coating, thereby achieving the purpose of bone damage repair, preventing tumor recurrence, and preventing infection during bone reconstruction.

[0009] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0010] Fluorine and selenium co-doped hydroxyapatite implant coating, the chemical formula of fluorine and selenium co-doped hydroxyapatite is Ca 10 (PO4) 6-y (SeO3) y (OH) 2-x F xwherein x is greater than 0.6 and less than or equal to 2, and y is greater than 0.06 and less than 2. The molar ratio of calcium and phosphorus in the hydroxyapatite implant coating co-doped with fluorine and selenium is close to that of calcium and phosphorus in hydroxyapatite, and Se and F are successfully doped, which helps optimize the performance of the coating.

[0011] The preparation method of the fluorine and selenium co-doped hydroxyapatite implant coating comprises the following steps to prepare the fluorine and selenium co-doped hydroxyapatite implant coating as described above:

[0012] Step A, preparation of suspension A: adding selenium-doped hydroxyapatite powder and dispersant A to ethanol solution A and mixing evenly; ball-milling the obtained mixed solution A, and sieving the suspension A after ball milling to obtain suspension A; and sieving the suspension A after ball milling through a 325-mesh sieve;

[0013] Step B, preparation of suspension B: adding fluorine-doped hydroxyapatite powder and dispersant B to ethanol solution B and mixing evenly; ball-milling the obtained mixed solution B, and sieving after ball milling to obtain suspension B;

[0014] Step C, substrate treatment: pre-treating the medical metal or medical alloy material as the substrate;

[0015] In step D, suspensions A and B are simultaneously sprayed onto the substrate using a liquid-phase plasma spraying method, employing a synchronous dual-liquid delivery mode. This forms the aforementioned fluorine- and selenium-co-doped hydroxyapatite implant coating on the substrate. This simultaneous dual-liquid delivery method simplifies the coating preparation process and is more efficient than traditional methods, allowing for easier control of the coating's composition and physical and chemical properties.

[0016] The preparation method of the above-mentioned fluorine and selenium co-doped hydroxyapatite implant coating, in step A: dispersant A is an ammonium citrate-polyacrylic acid composite dispersant, and the mass ratio of ammonium citrate to polyacrylic acid in dispersant A is 1:1-5, and the viscosity of polyacrylic acid is 1.2-3.5 mPa·s; the volume fraction of anhydrous ethanol in ethanol solution A is 50%-55%; ammonium citrate is an ionic dispersant, and polyacrylic acid is a non-ionic dispersant. When preparing suspension A, using a composite dispersant with a mass ratio of ammonium citrate to polyacrylic acid of 1:1-5 can not only obtain a better dispersion effect, but also a better spraying effect when suspension A and suspension B are sprayed simultaneously, thereby helping to improve the performance of the fluorine and selenium co-doped hydroxyapatite implant coating finally obtained; if the mass ratio of ammonium citrate to polyacrylic acid exceeds the above range, it will affect the dispersion effect and viscosity of suspension A, which is not conducive to the spraying process; the amount of ethanol solution used will affect the heat of the flame flow during plasma spraying, and ultimately affect the spraying effect;

[0017] The dosage of dispersant A is 0.5 wt.% to 5 wt.% of the selenium-doped hydroxyapatite powder; the mass fraction of the selenium-doped hydroxyapatite powder in the suspension A is 15 wt.% to 17 wt.%; if the dosage of the dispersant is too small, the dispersion effect cannot be achieved, while if the dosage of the dispersant is too large, the viscosity of the suspension A will be affected; when the mass fraction of the selenium-doped hydroxyapatite powder is within this range, the powder can be stably suspended, without causing difficulty in liquid delivery during spraying, and the resulting fluorine and selenium co-doped hydroxyapatite implant coating has a better effect;

[0018] During ball milling, the mass ratio of grinding balls to mixed solution A is 1:1-1.5; the ball milling time is 10-12 hours;

[0019] In step B, dispersant B is a sodium pyrophosphate-polyacrylic acid composite dispersant, wherein the mass ratio of sodium pyrophosphate to polyacrylic acid in dispersant B is 1:1-6, and the viscosity of polyacrylic acid is 1.2-3.5 mPa·s; the volume fraction of anhydrous ethanol in ethanol solution B is 50%-55%; sodium pyrophosphate is an ionic dispersant, and polyacrylic acid is a non-ionic dispersant. Using the composite dispersant with the above ratio helps to achieve a better dispersion effect;

[0020] The amount of dispersant B is 0.5 wt.% to 5 wt.% of the fluorine-doped hydroxyapatite powder; the mass fraction of the fluorine-doped hydroxyapatite powder in the suspension B is 15 wt.% to 17 wt.%. The amount of dispersant affects the viscosity of the suspension B. When the mass fraction of the fluorine-doped hydroxyapatite powder is within this range, the powder can be stably suspended without causing difficulty in liquid delivery during spraying, and the resulting fluorine-selenium co-doped hydroxyapatite implant coating has a better effect.

[0021] During ball milling, the mass ratio of grinding balls to mixed solution B is 1:1 to 1.5, and the ball milling time is 10 to 12 hours. By using specific dispersant selection, dispersant dosage, and ball milling, the viscosity and surface tension of the suspension are optimized, the zeta potential is negative, and the dispersion effect is significantly improved.

[0022] The preparation method of the above-mentioned fluorine and selenium co-doped hydroxyapatite implant coating and the preparation method of selenium-doped hydroxyapatite powder are as follows:

[0023] Step (A-1), mixing the diammonium hydrogen phosphate solution and the sodium selenite solution and then adding the mixture dropwise to the calcium nitrate solution to obtain a mixed system A;

[0024] Step (A-2), adjusting the pH of the mixed system A with an alkaline solution, then heating and stirring the mixture for reaction, and aging the mixture after the reaction is complete;

[0025] Step (A-3): After aging, the supernatant is discarded, and the obtained lower precipitate is washed with distilled water until the washing liquid is neutral. Finally, the washed precipitate is dried to obtain selenium-doped hydroxyapatite powder.

[0026] The method for preparing the fluorine and selenium co-doped hydroxyapatite implant coating comprises the following steps: in step (A-1): the molar concentration of the diammonium hydrogen phosphate solution is 0.4 to 0.6 mol / L; the molar concentration of the sodium selenite solution is 0.3 to 0.6 mol / L; and the molar concentration of the calcium nitrate solution is 0.3 to 0.6 mol / L. The molar ratio of calcium, phosphorus, and selenium in the mixed system A is 10:(6-n):n, where n is greater than 0.06 and less than 2. When the molar ratio of calcium, phosphorus, and selenium meets the above ratio, the biological properties and structural stability of the fluorine and selenium co-doped hydroxyapatite implant coating obtained are high. However, if this ratio is changed, especially when the selenium doping amount is increased, the biological properties and structural stability of the coating will be significantly reduced.

[0027] In step (A-2), the pH of the mixed system A is adjusted to 10-11 using aqueous ammonia. The heating and stirring reaction conditions are: stirring the reaction at a temperature of 60-65° C. for 12-14 hours; and the aging time is 12-14 hours. When the temperature, stirring time, and aging time exceed the above ranges, the purity and crystallinity of the target product, selenium-doped hydroxyapatite powder, will decrease. When the pH exceeds the above range, the target product cannot be obtained.

[0028] In step (A-3), the particle size of the selenium-doped hydroxyapatite powder is 10 to 80 nm. If the particle size is too large or too small, the biological properties of the final coating will be affected.

[0029] The preparation method of the fluorine- and selenium-co-doped hydroxyapatite implant coating and the preparation method of the fluorine-doped hydroxyapatite powder are as follows:

[0030] Step (B-1), mixing the diammonium hydrogen phosphate solution and the ammonium fluoride solution and then adding the mixture dropwise to the calcium nitrate solution to obtain a mixed system B;

[0031] Step (B-2), adjusting the pH of the mixed system B with an alkaline solution, then heating and stirring the mixture for reaction, and aging the mixture after the reaction is completed;

[0032] Step (B-3): After aging, the supernatant is discarded, and the obtained lower precipitate is washed with distilled water until the washing liquid is neutral. Finally, the washed precipitate is dried to obtain fluorine-doped hydroxyapatite powder.

[0033] The method for preparing the fluorine- and selenium-co-doped hydroxyapatite implant coating comprises the following steps: in step (B-1): the molar concentration of the diammonium hydrogen phosphate solution is 0.4 to 0.6 mol / L; the molar concentration of the ammonium fluoride solution is 0.4 to 0.6 mol / L; and the molar concentration of the calcium nitrate solution is 0.4 to 0.6 mol / L. The molar ratio of calcium, phosphorus, and fluorine in the mixed system B is 10:6:m, where m is greater than 0.6 and less than or equal to 2. When the molar ratio of calcium, phosphorus, and fluorine meets the above ratio, the resulting fluorine- and selenium-co-doped hydroxyapatite implant coating has high biological properties and structural stability, and the fluorine content is appropriate, so it will not be toxic to the body.

[0034] In step (B-2), the pH of the mixed system B is adjusted to 9-10 using aqueous ammonia. The heating and stirring reaction conditions are: stirring at 60-65° C. for 12-14 hours; and aging for 12-14 hours. When the temperature, stirring time, and aging time exceed the above ranges, the purity and crystallinity of the target product, fluorine-doped hydroxyapatite powder, will decrease. When the pH exceeds the above range, the target product cannot be obtained.

[0035] In step (B-3), the particle size of the fluorine-doped hydroxyapatite powder is 10 to 80 nm. If the particle size is too large or too small, the biological properties of the final coating will be affected.

[0036] In the above-mentioned method for preparing fluorine and selenium co-doped hydroxyapatite implant coating, in step D: during spraying, suspension A is delivered by radial delivery, and suspension B is delivered by axial delivery; compared with radial delivery, axial delivery allows suspension B to stay in the flame flow longer, the material obtains more energy, and melts more fully.

[0037] The spraying conditions for the synchronous dual-liquid delivery mode are as follows: spray power of 30-120 kW, spray distance of 30-150 mm, axial liquid delivery rate of 20-100 mL / min, radial liquid delivery rate of 20-100 mL / min, and axial liquid delivery atomization pressure of 0.8-2.0 bar. The substrate preheat temperature is 150-400°C, the spray gun travel speed is 500-700 mm / s, and the stepping distance is 1.5 mm / step. The working gases are hydrogen and argon, with an argon flow rate of 20-80 standard liters / min and a hydrogen flow rate of 2-20 standard liters / min. The number of spray gun passes can be adjusted according to specific needs. When using the synchronous dual-liquid feeding mode for spraying, suspension A and suspension B are mixed in the plasma flame, which can ensure that the droplets in the suspension react when they are in a molten state, the reaction is more complete, and the final coating texture is more uniform; in addition, compared with premixing suspension A and suspension B first and then spraying the premixed liquid, the above-mentioned synchronous dual-liquid feeding mode spraying can independently control the spraying process parameters of the two spraying liquids, which is conducive to obtaining a coating with an ideal structure.

[0038] In the aforementioned method for preparing a fluorine- and selenium-co-doped hydroxyapatite implant coating, in step C, the medical metal is pure titanium, and the medical alloy material is a titanium alloy, stainless steel, or a cobalt-chromium-molybdenum alloy. The pretreatment method comprises ultrasonically cleaning the substrate with gasoline and then anhydrous ethanol, followed by sandblasting the dried substrate with corundum. Sandblasting improves the bonding strength between the coating and the metal substrate. This improvement enhances the bonding stability between the coating and the substrate and reduces the risk of coating detachment.

[0039] Application of fluorine and selenium co-doped hydroxyapatite implant coating, the fluorine and selenium co-doped hydroxyapatite implant coating prepared by the above method is used for any of the following purposes: for repairing bone defects; for repairing bone defects in patients with osteosarcoma and inhibiting the recurrence of osteosarcoma; for preventing infection during bone reconstruction.

[0040] The technical solution of the present invention achieves the following beneficial technical effects:

[0041] 1. The present invention sprays fluorine and selenium co-doped hydroxyapatite onto the surface of orthopedic implant materials, thereby improving the bone microenvironment of the bone damage site of osteosarcoma patients through the coating, thereby achieving the purpose of bone damage repair; although ordinary fluorine-doped hydroxyapatite coatings have certain antibacterial properties, the release of the fluorine element it contains in the patient's body is usually uncontrolled, which can easily cause problems such as fluorine poisoning; selenium can enhance the body's antagonistic ability to fluorine and has anti-tumor effects, and the fluorine and selenium co-doped hydroxyapatite coating prepared by the present invention has a targeted and good repair effect on osteosarcoma patients, can be used to repair their bone damage, and inhibit the proliferation of bacteria and cancer cells without inhibiting the activity of normal cells, and the coating has an appropriate dissolution rate, and the fluorine release rate is effectively controlled, which is not easy to cause fluorine poisoning.

[0042] 2. In the present invention, when preparing suspension A and suspension B, ammonium citrate and polyacrylic acid with a mass ratio of 1:1 to 5 are selected as composite dispersants for homemade selenium-doped hydroxyapatite powder, and sodium pyrophosphate and polyacrylic acid with a mass ratio of 1:1 to 6 are selected as composite dispersants for homemade fluorine-doped hydroxyapatite powder, and the contents of selenium-doped hydroxyapatite powder in suspension A and fluorine-doped hydroxyapatite powder in suspension B are controlled respectively, so that the viscosity and surface tension of the finally prepared suspension A and suspension B are appropriate, and their Zeta potentials are both negative; the above-mentioned suspension A and suspension B are respectively sprayed synchronously using radial and axial liquid delivery methods to obtain a fluorine and selenium co-doped hydroxyapatite implant coating with good density and appropriate selenium and fluorine release rate. When used for bone repair in osteosarcoma patients, the hydroxyapatite implant coating not only has excellent biocompatibility, but also selenium and fluorine can better exert their repair effects, avoiding the problem of fluorine poisoning.

[0043] 3. The present invention utilizes a synchronized dual-liquid delivery mode during spraying. Radial delivery is external to the plasma torch, delivering the liquid phase radially into the plasma flame; axial delivery is internal to the plasma torch, delivering the liquid phase axially into the plasma flame. This allows the powder material to obtain higher energy and melt more fully. Suspension A is delivered radially, while suspension B is delivered axially. This promotes a thorough reaction of the different powder materials within the plasma flame. Both suspensions contain 50% ethanol by volume, which increases the heat of the plasma flame and further promotes the reaction. This results in coatings made from suspensions A and B, each containing uniformly dispersed powders, being more stable during application.

[0044] Suspension A containing selenium-doped hydroxyapatite powder is delivered radially, and suspension B containing fluorine-doped hydroxyapatite powder is delivered synchronously with suspension A by axial delivery. By controlling the viscosity of suspension A and suspension B and the content of the powder, under specific spraying conditions, fluorine and selenium can be distributed more reasonably in the formed hydroxyapatite coating, thereby making the doped hydroxyapatite coating have a more suitable dissolution rate and density. When the dissolution rate and density of the fluorine and selenium co-doped hydroxyapatite coating in the organism are suitable, the release rates of the chemical components fluorine and selenium contained in the coating will be balanced. While the fluorine and selenium in the coating jointly exert their biological anti-cancer and antibacterial effects, the selenium released into the body is sufficient to antagonize the toxicity of fluorine. After the hydroxyapatite coating prepared by the above-mentioned preparation method of the present invention is doped with Se and F elements, the ratio of the four elements calcium, phosphorus, selenium and fluorine is appropriate and close to the theoretical Ca / P ratio of hydroxyapatite. This shows that the coating prepared by the preparation method of fluorine and selenium co-doped hydroxyapatite implant coating provided by the present invention has a higher purity.

[0045] 4. The fluorine and selenium co-doped hydroxyapatite coating prepared on the surface of medical metals and alloys by liquid phase plasma spraying technology in the present invention not only has excellent biocompatibility, but also has antibacterial and anti-tumor activity, and can promote bone repair in bone defects in patients with bone tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 A schematic structural diagram of a plasma spraying device according to an embodiment of the present invention;

[0047] Figure 2 SEM photograph of the cross section of the fluorine and selenium co-doped hydroxyapatite coating obtained in Example 1 of the present invention;

[0048] Figure 3 XRD spectrum of the fluorine and selenium co-doped hydroxyapatite coating in Example 1 of the present invention;

[0049] Figure 4 EDS spectrum of the fluorine and selenium co-doped hydroxyapatite coating in Example 1 of the present invention;

[0050] Figure 5 The full XPS spectrum of the fluorine and selenium co-doped hydroxyapatite coating in Example 1 of the present invention;

[0051] Figure 6 SEM photograph of the morphology of MC3T3-E1 cells adhered to the surface of the fluorine and selenium co-doped hydroxyapatite coating in Example 1 of the present invention;

[0052] Figure 7 Graph showing the OD value measurement results of MC3T3-E1 cells at 570 nm detected by the MTT assay in Example 1 of the present invention;

[0053] Figure 8 The graph shows the OD value measurement results of MG63 cells at 450 nm detected by MTT assay in Example 1 of the present invention;

[0054] Figure 9 Graph showing the results of the antibacterial test on the surface of the fluorine- and selenium-coated hydroxyapatite coating in Example 1 of the present invention. DETAILED DESCRIPTION

[0055] Example 1

[0056] The chemical formula of the fluorine and selenium co-doped hydroxyapatite coating in this embodiment is Ca 10 (PO4)5(SeO3)(OH)F, the preparation method of which is described as follows.

[0057] 1. Synthesize fluorine-doped hydroxyapatite nanopowder according to chemical reaction formula (1).

[0058] Ca(NO2)2+(NH4)2HPO4+NH4F→Ca 10 (PO4)6(OH) 2-x F x (0 <x≤2) (1)

[0059] A 0.5 mol / L (NH4)2HPO4 and NH4F solution was mixed and then added dropwise to a 0.5 mol / L Ca(NO3)2·4H2O solution to ensure that the Ca / P molar ratio was 1.67. In this embodiment, the theoretical substitution amount of F for OH was 50% (i.e., x=1). The reaction temperature was controlled at 60°C, and the pH was adjusted to 9 using aqueous ammonia (concentration of 14 mol / L). After continuous stirring for 12 hours, the mixture was aged for 12 hours. The supernatant was directly discarded without filtration, and the powder was washed with distilled water several times until the supernatant was neutral, thereby obtaining fluorine-doped hydroxyapatite powder with a particle size of 10 to 80 nm. To achieve a 15 wt.% solid content in the suspension, anhydrous ethanol and distilled water (1:1) were added as the dispersion medium. A sodium pyrophosphate-polyacrylic acid composite dispersant (sodium pyrophosphate: polyacrylic acid mass ratio of 1:3, polyacrylic acid viscosity of 2 mPa·s) was used as the dispersant. The dispersant concentration (equivalent to the mass fraction of the fluorine-doped hydroxyapatite) was 1.0 wt.%. To further enhance the dispersion, ball milling was used. After 12 hours of ball milling, the suspension was passed through a 325-mesh sieve to obtain a fluorine-doped hydroxyapatite suspension (suspension B) for later use. The ball:liquid mass ratio during ball milling was 1:1. The resulting fluorine-doped hydroxyapatite suspension had a viscosity of 1.98 mPa·s, a surface tension of 66.7 mN / m, and a zeta potential of -23.3 mV.

[0060] 2. Synthesize selenium-doped hydroxyapatite nanopowder according to chemical reaction formula (2).

[0061] Ca(NO3)2+(NH4)2HPO4+Na2SeO3→Ca 10 (PO4) 6-y (SeO3) y (OH)2(0 <y≤3) (2)

[0062] In this embodiment, the value of y is 1. A 0.5 mol / L (NH4)2HPO4 and Na2SeO3 solution was mixed and then added dropwise to a 0.5 mol / L Ca(NO3)2·4H2O solution, ensuring a Ca / (P+Se) molar ratio of 1.67. The reaction temperature was controlled at 60°C, and the pH was adjusted to 10 using aqueous ammonia (14 mol / L concentration). After continuous stirring for 12 hours, the mixture was aged for 12 hours. The supernatant was directly discarded without filtration, and the powder was washed with distilled water several times until the supernatant was neutral, thereby obtaining selenium-doped hydroxyapatite powder with a particle size of 10 to 80 nm. Anhydrous ethanol and distilled water (1:1) were added as a dispersion medium, so that the solid content of the suspension was 15 wt.%. An ammonium citrate-polyacrylic acid composite dispersant (ammonium citrate to polyacrylic acid mass ratio of 1:2, polyacrylic acid viscosity of 2 mPa·s) was selected as the dispersant, with a dispersant concentration (equivalent to the mass fraction of selenium-doped hydroxyapatite) of 1.0 wt.%. To further enhance the dispersion effect, ball milling was used. After 12 hours of ball milling, the suspension was passed through a 325-mesh sieve to obtain a selenium-doped hydroxyapatite suspension (suspension A) for later use. The ball:liquid mass ratio during ball milling was 1:1. The resulting selenium-doped hydroxyapatite suspension had a viscosity of 1.84 mPa·s, a surface tension of 61.6 mN / m, and a zeta potential of -28.8 mV.

[0063] 3. Before plasma spraying, the metal Ti sheet is first cut into a size of 10mm×10mm×1mm, and then ultrasonically cleaned with gasoline and anhydrous ethanol in sequence to remove surface oil stains. The cleaned and dried substrate is sandblasted with corundum to improve the bonding strength between the coating and the substrate.

[0064] In this embodiment, the substrate is a metal Ti sheet. In other embodiments, the substrate can be pure titanium, titanium alloy, stainless steel or cobalt-chromium-molybdenum alloy.

[0065] 4. Use liquid plasma spraying technology with synchronous double liquid feeding mode to prepare coating on the substrate surface after sandblasting. Figure 1As shown, during spraying, the selenium-doped hydroxyapatite suspension (suspension A) is transported into the atomizer for atomization, while the fluorine-doped hydroxyapatite suspension (suspension B) is transported into the liquid inlet of the plasma spray gun. At the same time, hydrogen and argon are also drawn into the plasma spray gun. The hydrogen and argon are ignited at the nozzle of the plasma spray gun, generating a plasma flame. The atomizer sprays the atomized selenium-doped hydroxyapatite suspension into the plasma flame. Simultaneously, the fluorine-doped hydroxyapatite suspension is also ejected from the nozzle of the plasma spray gun. The selenium-doped hydroxyapatite suspension and the fluorine-doped hydroxyapatite suspension mix at the nozzle of the plasma spray gun 4 and are burned together by the plasma flame. They are then sprayed together by the plasma spray gun onto the surface of the substrate, ultimately forming a fluorine- and selenium-co-doped hydroxyapatite coating on the surface of the substrate. The spraying process parameters are as follows: spraying power 80kW; spraying distance 70mm; axial liquid feeding rate 50mL / min; radial liquid feeding rate 40mL / min, axial liquid feeding atomization pressure 1.2bar; substrate preheating temperature 250℃, spray gun moving speed 700mm / s, stepping 1.5mm / step. The working gases of the plasma spray gun are hydrogen and argon, the gas flow rate of argon is 60 standard liters / min, and the gas flow rate of hydrogen is 10 standard liters / min. The number of times the spray gun is scanned is 10 times, and the coating thickness is about 30μm. Figure 2 shown.

[0066] The structure and performance of the coating prepared in this example were analyzed and tested using methods including XRD diffraction peak analysis, EDS spectral analysis, XPS spectral analysis, observation of osteoblast adhesion morphology on the coating surface, biocompatibility testing, human osteosarcoma cell apoptosis induction testing, and antibacterial testing. The results of these tests or experiments are shown below.

[0067] (1) XRD diffraction peak analysis of fluorine and selenium co-doped hydroxyapatite coating

[0068] The results of XRD diffraction peak analysis of the coating are as follows Figure 3 As shown. Figure 3 The main phase of the coating is hydroxyapatite (HA), which is relatively pure. However, decomposed phases of tricalcium phosphate (TCP), tetracalcium phosphate (TTCP), and calcium oxide (CaO) also appear. This is mainly due to the partial decomposition of the hydroxyapatite phase at the extremely high temperature of the plasma.

[0069] (2) EDS and XPS spectrum analysis of fluorine and selenium co-doped hydroxyapatite coating

[0070] Figure 4 is the EDS spectrum of the coating, Figure 5 The XPS spectrum of the coating is shown in Figure 2. Figure 4 and Figure 5Analysis shows that Ca, P, Se and F elements are present in the coating, and the molar ratio of Ca / (P+Se) is 1.68, which is close to the theoretical Ca / P molar ratio of hydroxyapatite of 1.67, indicating that the fluorine and selenium co-doped hydroxyapatite coating has been successfully prepared.

[0071] (3) Observation of osteoblast adhesion morphology on the surface of fluorine and selenium co-doped hydroxyapatite coating

[0072] MC3T3-E1 cells (osteoblasts) were cultured at a volume of 5 × 10 4 The cells were seeded at a density of 1 / well on the sterilized coating material and cultured for 1 day. The culture medium was then discarded and the samples were washed twice with PBS. After treating the samples with 2% glutaraldehyde at 4°C for 4 hours, they were dehydrated and dried in different concentrations of alcohol. After gold spraying, the cell morphology was observed using a scanning electron microscope (SEM). The SEM photos are shown in the figure. Figure 6 As shown. Figure 6 It can be found that the cells on the surface of the fluorine and selenium co-doped hydroxyapatite coating extend in multiple directions and are tightly bound to the coating, indicating that the coating is conducive to the formation and adhesion and spreading of osteoblast lamellipodia.

[0073] (4) Osteoblast biocompatibility experiment on fluorine and selenium co-doped hydroxyapatite coating surface

[0074] MC3T3-E1 cells were cultured at a volume of 5 × 10 4 The density of 100 μg / well was inoculated on the surface of the sterilized selenium and fluorine co-doped hydroxyapatite coating material and the control hydroxyapatite coating, and cultured for 2 days and 4 days respectively. The OD value was measured at 570 nm using the MTT method. The measurement results are shown in Figure 2. Figure 7 As shown. Figure 7 It can be seen that the cell activity on the surface of the fluorine and selenium co-doped hydroxyapatite coating is higher than that of the hydroxyapatite coating in the control group, indicating that osteoblasts can proliferate better on the surface of the fluorine and selenium co-doped hydroxyapatite coating in this embodiment.

[0075] (5) Experiment on induction of apoptosis of human osteosarcoma cells on fluorine and selenium co-doped hydroxyapatite coating surface

[0076] MG63 cells (a type of cancer cell) were cultured at a rate of 2×10 4 The cells were inoculated at a density of 100 μg / well on the surface of sterilized selenium and fluorine co-doped hydroxyapatite coating material and the control hydroxyapatite coating and cultured for 4 and 7 days respectively. The OD value was measured at 450 nm using the MTT method. The results are shown in Figure 2. Figure 8 As shown. Figure 8The results show that the activity of MG63 cells on the fluorine- and selenium-coated hydroxyapatite coating was significantly lower than that on the control hydroxyapatite coating. After 7 days of culture, the activity of MG63 cells on the fluorine- and selenium-coated hydroxyapatite coating was reduced by about 25% compared with the control.

[0077] (6) Antibacterial test on the surface of fluorine and selenium co-doped hydroxyapatite coating

[0078] After culturing Staphylococcus aureus in the logarithmic growth phase with fluorine and selenium co-doped hydroxyapatite coating materials (control group hydroxyapatite coating materials) for 24 hours, the bacterial solution was applied to the plate, the number of colonies on the plate surface was counted, and the logarithmic value was obtained. Figure 9 It can be seen that the fluorine and selenium co-doped hydroxyapatite coating material showed obvious antibacterial properties. Compared with the control group, the number of Staphylococcus aureus colonies decreased by 38% after 24 hours of treatment.

[0079] Example 2

[0080] The chemical formula of the fluorine and selenium co-doped hydroxyapatite coating in this embodiment is Ca 10 (PO4) 4.01 (SeO3) 1.99 (OH) 0.5 F 1.5 , and its preparation method is as follows.

[0081] 1. Synthesize fluorine-doped hydroxyapatite nanopowder according to chemical reaction formula (1).

[0082] Ca(NO3)2+(NH4)2HPO4+NH4F→Ca 10 (PO4)6(OH)2- x F x (0 <x≤2) (1)

[0083] A 0.5 mol / L solution of (NH4)2HPO4 and NH4F was mixed and then added dropwise to a 0.5 mol / L solution of Ca(NO3)2·4H2O, ensuring a Ca / P molar ratio of 1.67. In this example, the theoretical substitution of F for OH was 75% (i.e., x = 1.5). The reaction temperature was controlled at 60°C, and the pH was adjusted to 9 using aqueous ammonia (14 mol / L). Stirring was continued for 12 hours, followed by aging for 12 hours. The supernatant was discarded without filtration, and the powder was washed with distilled water several times until the supernatant was neutral, yielding fluorine-doped hydroxyapatite powder with a particle size of 10 to 80 nm. Anhydrous ethanol and distilled water (1:1) were added as the dispersion medium, maintaining a solids content of 15% in the suspension. A sodium pyrophosphate-polyacrylic acid composite dispersant (sodium pyrophosphate:polyacrylic acid mass ratio of 1:3, polyacrylic acid viscosity of 2.5 mPa·s) was used as the dispersant. The dispersant concentration (dispersant equivalent to the mass fraction of fluorine-doped hydroxyapatite) was 1.0 wt.%. To further enhance the dispersion effect, ball milling was used. After 12 hours of ball milling, the suspension was passed through a 325-mesh sieve to obtain a fluorine-doped hydroxyapatite suspension (suspension B) for later use. The ball:liquid mass ratio during ball milling was 1:1. The resulting fluorine-doped hydroxyapatite suspension had a viscosity of 1.66 mPa·s, a surface tension of 61.0 mN / m, and a zeta potential of -28.1 mV.

[0084] 2. Synthesize selenium-doped hydroxyapatite nanopowder according to chemical reaction formula (2).

[0085] Ca(NO3)2+(NH4)2HPO4+Na2SeO3→Ca 10 (PO4) 6-y (SeO3) y (OH)2(0 <y≤3) (2)

[0086] In this embodiment, the value of y is 1.99. 0.5 mol / L (NH4)2HPO4 and Na2SeO3 solutions were mixed and then added dropwise to a 0.5 mol / L Ca(NO3)2·4H2O solution to ensure that the Ca / (P+Se) molar ratio was 1.67. The reaction temperature was controlled to 60°C, and the pH value was adjusted to 10 using aqueous ammonia (concentration of 14 mol / L). After continuous stirring for 12 hours, the mixture was aged for 12 hours. The supernatant was directly discarded without filtration, and distilled water was added to wash the powder several times until the supernatant was neutral to obtain a selenium-doped hydroxyapatite powder with a particle size of 10 to 80 nm. Anhydrous ethanol and distilled water (1:1) were added as a dispersion medium according to a solid content of 15% in the suspension. An ammonium citrate-polyacrylic acid composite dispersant (ammonium citrate to polyacrylic acid mass ratio of 1:2, polyacrylic acid viscosity of 2.5 mPa·s) was selected as the dispersant, with a dispersant concentration (equivalent to the mass fraction of selenium-doped hydroxyapatite) of 1.0 wt.%. To further enhance the dispersion effect, ball milling was used. After 12 hours of ball milling, the suspension was passed through a 325-mesh sieve to obtain a selenium-doped hydroxyapatite suspension (suspension A) for later use. The ball:liquid mass ratio during ball milling was 1:1. The resulting selenium-doped hydroxyapatite suspension had a viscosity of 1.96 mPa·s, a surface tension of 69.2 mN / m, and a zeta potential of -18.8 mV.

[0087] 3. Before plasma spraying, the metal Ti sheet was first cut into a size of 10mm×10mm×1mm, and then ultrasonically cleaned with gasoline and anhydrous ethanol in sequence to remove surface oil stains. The cleaned and dried substrate was sandblasted with corundum to improve the bonding strength between the coating and the substrate.

[0088] 4. Liquid-phase plasma spraying technology in a dual-liquid delivery mode was used to prepare a coating on the sandblasted substrate surface. Suspension A was delivered radially, and suspension B was delivered axially. The spraying process parameters were: spray power 90 kW; spray distance 80 mm; axial liquid delivery rate 50 mL / min; radial liquid delivery rate 40 mL / min, and axial liquid delivery atomization pressure 1.2 bar; substrate preheating temperature 250°C, spray gun movement speed 700 mm / s, and stepping 1.5 mm / step. The working gases of the plasma spray gun were hydrogen and argon, with an argon flow rate of 65 standard liters / min and a hydrogen flow rate of 15 standard liters / min. The spray gun was scanned 12 times, and the coating thickness was approximately 36 μm.

[0089] The structure and properties of the coating prepared in this example were analyzed and tested using methods including XRD diffraction peak analysis, EDS spectrum analysis, XPS spectrum analysis, biocompatibility testing, human osteosarcoma cell apoptosis induction testing, and antibacterial testing. The results of these tests or experiments are as follows.

[0090] (1) XRD diffraction peak analysis of fluorine and selenium co-doped hydroxyapatite coating

[0091] By analyzing the XRD diffraction peaks of the prepared coating, it was found that the main phase of the coating was still hydroxyapatite phase, and the decomposed phases included tricalcium phosphate (TCP), tetracalcium phosphate (TTCP) and calcium oxide (CaO).

[0092] (2) EDS and XPS spectrum analysis of fluorine and selenium co-doped hydroxyapatite coating

[0093] EDS and XPS analysis results show that the coating contains Ca, P, Se and F elements, and Ca / (P+Se)=1.65, which indicates that the prepared coating is a fluorine and selenium co-doped hydroxyapatite coating.

[0094] (3) Osteoblast biocompatibility experiment on fluorine and selenium co-doped hydroxyapatite coating surface

[0095] The experimental method was the same as that in Example 1. The results of cell adhesion morphology and MTT experiments showed that the fluorine and selenium co-doped hydroxyapatite coating material had excellent biocompatibility and could promote the adhesion and proliferation of MC3T3-E1 cells, but inhibited the cell activity of osteosarcoma cells.

[0096] (4) Experimental study on apoptosis induction of human osteosarcoma cells on fluorine and selenium co-doped hydroxyapatite coatings

[0097] The experimental method was the same as that of Example 1. After 7 days of culture, the activity of MG63 cells on the surface of the fluorine- and selenium-co-doped hydroxyapatite coating decreased by about 36% compared with the control group.

[0098] (6) Antibacterial test on the surface of fluorine and selenium co-doped hydroxyapatite coating

[0099] The experimental method was the same as that in Example 1. The antibacterial test results showed that the number of Staphylococcus aureus colonies decreased by 49% after 24 hours of treatment compared with the control group.

[0100] The performance of the fluorine- and selenium-co-doped hydroxyapatite coatings in the two examples above was compared. In terms of cell activity, Example 2 showed that after 7 days of culture, the activity of MG63 cells on the fluorine- and selenium-co-doped hydroxyapatite coating surface was reduced by approximately 36% compared to the control group. This data was superior to the approximately 25% reduction in Example 1, indicating that the coating in Example 2 was more effective in inhibiting cancer cell activity.

[0101] In terms of antibacterial performance, the antibacterial test results of Example 2 showed that compared with the control group, the number of Staphylococcus aureus colonies decreased by 49% after 24 hours of treatment. Compared with the 38% reduction in Example 1, the coating of Example 2 showed higher antibacterial efficiency.

[0102] Comprehensive comparison shows that Example 2 exhibits superior effects in terms of cell activity and antibacterial performance compared to Example 1. This may be due to the combined effects of the specific fluorine and selenium doping levels in the coating of Example 2, the viscosity and dispersion of the suspension, and the liquid delivery method during spraying.

[0103] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the claims of this patent application.

Claims

1. A method for preparing a fluorine and selenium co-doped hydroxyapatite implant coating, characterized in that: The chemical formula of fluorine and selenium co-doped hydroxyapatite is Ca 10 (PO4) 6-y (SeO3) y (OH) 2-x F x ; Wherein, x is greater than 0.6 and less than or equal to 2, and y is greater than 0.06 and less than 2. The fluorine and selenium co-doped hydroxyapatite implant coating is prepared by the following steps: Step A, preparation of suspension A: adding selenium-doped hydroxyapatite powder and dispersant A to ethanol solution A and mixing evenly; ball-milling the obtained mixed solution A, and sieving after ball milling to obtain suspension A; Step B, preparation of suspension B: adding fluorine-doped hydroxyapatite powder and dispersant B to ethanol solution B and mixing evenly; ball-milling the obtained mixed solution B, and sieving after ball milling to obtain suspension B; Step C, substrate treatment: pre-treating the medical metal or medical alloy material as the substrate; Step D: Using a liquid-phase plasma spraying method, suspension A and suspension B are simultaneously sprayed onto the substrate in a synchronous dual-liquid delivery mode to form the fluorine-selenium co-doped hydroxyapatite implant coating on the substrate; wherein suspension A is delivered radially and suspension B is delivered axially. Suspensions A and B are mixed at the injection port of the plasma spray gun and burned together by the plasma flame flow, and then sprayed together by the plasma spray gun onto the surface of the substrate; In step A, dispersant A is an ammonium citrate-polyacrylic acid composite dispersant, wherein the mass ratio of ammonium citrate to polyacrylic acid in dispersant A is 1:1-5, and the viscosity of polyacrylic acid is 1.2-3.5 mPa·s; the volume fraction of anhydrous ethanol in ethanol solution A is 50%-55%; The amount of dispersant A is 0.5wt.% to 5wt.% of the selenium-doped hydroxyapatite powder; the mass fraction of the selenium-doped hydroxyapatite powder in the suspension A is 15wt.% to 17wt.%; During ball milling, the mass ratio of grinding balls to mixed solution A is 1:1-1.5; the ball milling time is 10-12 hours; In step B, dispersant B is a sodium pyrophosphate-polyacrylic acid composite dispersant, wherein the mass ratio of sodium pyrophosphate to polyacrylic acid in dispersant B is 1:1-6, and the viscosity of polyacrylic acid is 1.2-3.5 mPa·s; and the volume fraction of anhydrous ethanol in ethanol solution B is 50%-55%; The amount of dispersant B is 0.5 wt.% to 5 wt.% of the fluorine-doped hydroxyapatite powder; the mass fraction of the fluorine-doped hydroxyapatite powder in suspension B is 15 wt.% to 17 wt.%; During ball milling, the mass ratio of the grinding balls to the mixed liquid B is 1:1 to 1.5; the ball milling time is 10 to 12 hours.

2. The method for preparing a fluorine and selenium co-doped hydroxyapatite implant coating according to claim 1, characterized in that: The preparation method of selenium-doped hydroxyapatite powder is as follows: Step (A-1), mixing the diammonium hydrogen phosphate solution and the sodium selenite solution and then adding the mixture dropwise to the calcium nitrate solution to obtain a mixed system A; Step (A-2): adjusting the pH of the mixed system A with an alkaline solution, then heating and stirring the mixture for reaction, and aging the mixture after the reaction is completed; Step (A-3): After aging, the supernatant is discarded, and the obtained lower precipitate is washed with distilled water until the washing liquid is neutral. Finally, the washed precipitate is dried to obtain selenium-doped hydroxyapatite powder.

3. The method for preparing a fluorine and selenium co-doped hydroxyapatite implant coating according to claim 2, characterized in that: In step (A-1), the molar concentration of the diammonium hydrogen phosphate solution is 0.4 to 0.6 mol / L; the molar concentration of the sodium selenite solution is 0.3 to 0.6 mol / L; and the molar concentration of the calcium nitrate solution is 0.3 to 0.6 mol / L. The molar ratio of calcium, phosphorus, and selenium in the mixed system A is 10:(6-n):n, where n is greater than 0.06 and less than 2. In step (A-2), the pH of the mixed system A is adjusted to 10-11 with aqueous ammonia. The heating and stirring reaction conditions are: stirring the reaction at a temperature of 60-65° C. for 12-14 hours; and the aging time is 12-14 hours. In step (A-3), the particle size of the selenium-doped hydroxyapatite powder is 10 to 80 nm.

4. The method for preparing a fluorine and selenium co-doped hydroxyapatite implant coating according to claim 1, characterized in that: The preparation method of fluorine-doped hydroxyapatite powder is as follows: Step (B-1), mixing the diammonium hydrogen phosphate solution and the ammonium fluoride solution and then adding the mixture dropwise to the calcium nitrate solution to obtain a mixed system B; Step (B-2): adjusting the pH of the mixed system B with an alkaline solution, then heating and stirring the mixture for reaction, and aging the mixture after the reaction is completed; Step (B-3): After aging, the supernatant is discarded, and the obtained lower precipitate is washed with distilled water until the washing liquid is neutral. Finally, the washed precipitate is dried to obtain fluorine-doped hydroxyapatite powder.

5. The method for preparing a fluorine and selenium co-doped hydroxyapatite implant coating according to claim 4, characterized in that: In step (B-1), the molar concentration of the diammonium hydrogen phosphate solution is 0.4 to 0.6 mol / L; the molar concentration of the ammonium fluoride solution is 0.4 to 0.6 mol / L; and the molar concentration of the calcium nitrate solution is 0.4 to 0.6 mol / L. The molar ratio of calcium, phosphorus, and fluorine in the mixed system B is 10:6:m, where m is greater than 0.6 and less than or equal to 2. In step (B-2), the pH of the mixed system B is adjusted to 9-10 with aqueous ammonia. The heating and stirring reaction conditions are: stirring the reaction at a temperature of 60-65° C. for 12-14 hours; and the aging time is 12-14 hours. In step (B-3), the particle size of the fluorine-doped hydroxyapatite powder is 10 to 80 nm.

6. The method for preparing a fluorine and selenium co-doped hydroxyapatite implant coating according to any one of claims 1 to 5, characterized in that: In step D: during spraying, suspension A is delivered in a radial direction, and suspension B is delivered in an axial direction; The spraying conditions of the synchronous dual-liquid feeding mode are as follows: spraying power is 30-120 kW, spraying distance is 30-150 mm; axial liquid feeding rate is 20-100 mL / min; radial liquid feeding rate is 20-100 mL / min, and axial liquid feeding atomization pressure is 0.8-2.0 bar; substrate preheating temperature is 150-400°C, spray gun moving speed is 500-700 mm / s, and stepping is 1.5 mm / step; working gases are hydrogen and argon, with argon gas flow rate of 20-80 standard liters / min and hydrogen gas flow rate of 2-20 standard liters / min.

7. The method for preparing a fluorine and selenium co-doped hydroxyapatite implant coating according to claim 6, characterized in that: In step C: the medical metal is pure titanium, and the medical alloy material is titanium alloy, stainless steel or cobalt-chromium-molybdenum alloy; the pretreatment method is: ultrasonically cleaning the substrate with gasoline and anhydrous ethanol in sequence; then sandblasting the ultrasonically cleaned and dried substrate with corundum.

8. Application of fluorine and selenium co-doped hydroxyapatite implant coating, characterized in that: The fluorine and selenium co-doped hydroxyapatite implant coating prepared by the method of claim 1 is used to manufacture an orthopedic implant having any of the following functions: for repairing bone defects; for repairing bone defects in patients with osteosarcoma and inhibiting the recurrence of osteosarcoma; Used to prevent infection during bone remodeling.

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