Hydroxyapatite microspheres with low crystallinity and preparation method thereof

Abstract

The invention provides a low-crystallinity hydroxyapatite microsphere for implantation and a preparation method thereof. The preparation method comprises the following steps: preparing a mixed solution of a calcium source reagent and a phosphorus source reagent, adding an inorganic acid, an alkali and a buffer agent into the mixed solution, then adding a surfactant, and carrying out a constant-temperature reaction to obtain a hydroxyapatite particle precipitate; cleaning the hydroxyapatite particle precipitate, and adding water to prepare a hydroxyapatite particle suspension; and carrying out spray drying on the hydroxyapatite particle suspension to obtain the hydroxyapatite microspheres, wherein the surfactant is one or more of PEG (Polyethylene Glycol) and PVP (Polyvinyl Pyrrolidone). The hydroxyapatite microsphere obtained by the invention is stable in structure, belongs to spherical large particles, is easy to inject and not easy to agglomerate, has a good supporting effect after being injected into a human body, and is simple in preparation method, few in process steps, easy in realization of reaction conditions and suitable for large-scale production.

Description

Technical Field

[0001] The present invention relates to the technical field of medical biomaterials, and in particular to a hydroxyapatite microsphere with low crystallinity and a preparation method thereof. Background Art

[0002] Hydroxyapatite is the main inorganic component of human and animal bones. It can achieve chemical bonding with body tissues at the interface, has a certain solubility in the body, can release ions harmless to the body, and can participate in body metabolism. As an ideal bone repair material, hydroxyapatite has the advantages of good osteoconductivity, bioactivity, and biocompatibility. Porous hydroxyapatite microspheres have a wide range of applications in the fields of tissue engineering and drug separation due to their low relative density, high specific surface area, and light weight.

[0003] At present, the main methods for preparing hydroxyapatite microspheres are hydrothermal method, microemulsion method, and spray drying method. Chinese Patent (Application No. 201580022238.7) discloses a spherical porous hydroxyapatite adsorbent and its method, which is to first prepare a suspension of hydroxyapatite primary particles, then spray-dry the suspension of hydroxyapatite primary particles to obtain hydroxyapatite microspheres, and finally perform a complex screening method for classification to obtain spherical hydroxyapatite. Chinese Patent (Application No. 201710225034.4) discloses a hydroxyapatite microsphere and its preparation method, which is to first prepare hydroxyapatite primary particles, then filter, wash, and dry the particles, then grind the particles into powder, re-make them into an aqueous solution, and finally spray-dry them. The process is too complex and not suitable for mass production.

[0004] There are many problems in the current methods for preparing hydroxyapatite microspheres, such as uneven particle size distribution, low yield, and complex preparation process. There is no process for specifically preparing hydroxyapatite with low crystallinity in the current methods for preparing hydroxyapatite microspheres.

[0005] Therefore, developing a new preparation method for hydroxyapatite microspheres with low crystallinity and capable of industrial production is an urgent problem to be solved in current research. Summary of the Invention

[0006] The purpose of the present invention is to provide a hydroxyapatite microsphere, and the obtained hydroxyapatite microsphere has a uniform particle size distribution and low crystallinity, is suitable for bone tissue repair and filling, and has a simple preparation process.

[0007] In the first aspect, the present invention provides a preparation method for a hydroxyapatite microsphere with low crystallinity, including the following steps: S1: Prepare a mixed solution of a calcium source reagent and a phosphorus source reagent; S2: Add inorganic acid, base and buffer into the mixed solution, then add surfactant, and carry out a constant-temperature reaction to obtain hydroxyapatite particle precipitate; S3: Wash the hydroxyapatite particle precipitate, and add water to prepare a hydroxyapatite particle suspension; S4: Spray-dry the hydroxyapatite particle suspension, and the spray-drying parameters are a feeding rate of 0.5 - 5 L / h, a temperature of 120 - 150 °C, and a centrifugal rate of 15000 - 30000 rpm to obtain the hydroxyapatite microspheres; Among them, the surfactant is one or more of PEG and PVP.

[0008] By adopting the above technical solution, through the synergy of adding PEG and / or PVP and specific spray-drying conditions, the performance limitations of traditional high-crystallinity hydroxyapatite microspheres are broken through, and the bioactivity of the implant material is improved; in addition, directly forming in one step by spray-drying can omit steps such as sieving and secondary dispersion, and the process steps are reduced by more than 50%.

[0009] Optionally, the calcium source reagent is calcium chloride; the phosphorus source reagent is selected from at least one of phosphate ion, monohydrogen phosphate ion or dihydrogen phosphate ion.

[0010] Optionally, the inorganic acid is sulfuric acid or hydrochloric acid, the base is sodium hydroxide, and the buffer is PBS buffer.

[0011] Optionally, the molecular weight of the PEG is 3000 - 6000, and the molecular weight of the PVP is 20000 - 50000.

[0012] By adopting the above technical solution, adding PEG and PVP with specific molecular weights can effectively control the growth morphology of hydroxyapatite particles, make them form spherical precipitates, and at the same time reduce their crystallinity.

[0013] Optionally, in step S2, the temperature of the constant-temperature reaction is 80 - 90 °C, and the reaction time is 2 - 4 h.

[0014] Optionally, in step S2, the temperature of the constant-temperature reaction is 50 - 60 °C, and the reaction time is 18 - 24 h.

[0015] By adopting the above technical solution, adjusting the reaction temperature (50 - 60 °C) and time (18 - 24 h) can inhibit the excessive growth of HA crystal nuclei and reduce the crystallinity.

[0016] In the second aspect, the present invention provides a hydroxyapatite microsphere obtained by the preparation method as described above, and the particle size of the hydroxyapatite microsphere is 25 μm - 50 μm.

[0017] By adopting the above technical solution, the hydroxyapatite microspheres in this particle size range have good filling effect and cell compatibility in bone repair and tissue engineering applications.

[0018] Optionally, the crystallinity of the hydroxyapatite microspheres is less than 50%.

[0019] By adopting the above technical solution, the degradation rate of the low-crystallinity microspheres in vivo is significantly increased, enhancing the safety of human use; under the same conditions, the smaller the crystallinity, the greater the solubility and the higher the concentration of calcium and phosphorus released, thus showing stronger reactivity; moreover, the lower the crystallinity of hydroxyapatite, the larger the specific surface area and the better the bioactivity.

[0020] Optionally, the tapped density of the hydroxyapatite microspheres is 0.6 - 1.0 g / mL.

[0021] In a third aspect, the present invention provides an application of the hydroxyapatite microspheres as described above in bone tissue repair and filling, drug sustained-release carriers or tissue engineering scaffolds.

[0022] The beneficial effects of the present invention are as follows: 1. The prepared hydroxyapatite has low crystallinity, breaking through the performance limitations of traditional high-crystallinity hydroxyapatite microspheres, being conducive to cell adhesion, proliferation and tissue ingrowth, and improving the bioactivity of the implant material; 2. The prepared hydroxyapatite microspheres have uniform particle size, high sphericity and good dispersibility, and have good filling effect and cell compatibility in bone repair and tissue engineering applications; 3. The entire preparation method has relatively simple process, does not require complex equipment and operations, reduces production costs, and is conducive to large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the scanning electron microscope image of the hydroxyapatite microspheres obtained in Example 1 of the present invention; Figure 2 It is the scanning electron microscope image of the hydroxyapatite microspheres obtained in Example 2 of the present invention; Figure 3 It is the scanning electron microscope image of the hydroxyapatite microspheres obtained in Comparative Example 1 of the present invention; Figure 4 It is the scanning electron microscope image of the hydroxyapatite microspheres obtained in Comparative Example 2 of the present invention; Figure 5 It is the XRD detection image of the hydroxyapatite microspheres obtained in Example 1 of the present invention; Figure 6 It is the XRD detection image of the hydroxyapatite microspheres obtained in Comparative Example 2 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0024] To better illustrate the purpose, technical solution and advantages of the present invention, the following will further illustrate the present application with specific embodiments.

[0025] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention.

[0026] Example 1

[0027] A method for preparing hydroxyapatite microspheres, comprising the following steps: S1: Weigh 71 g of disodium hydrogen phosphate and 122 g of calcium chloride dihydrate, dissolve them in 40 L of water, and mix to obtain solution A; S2: Add 10 L of hydrochloric acid with a concentration of 1 mol / L to solution A and stir to mix. After the reaction solution becomes clear, add 192 g of sodium hydroxide to neutralize the reaction, then add PBS buffer, and adjust the pH to 7.0. Then add 2.51 g of PEG (brand: Macklin, specification: average Mn 4000) and 0.94 g of PVP (brand: Macklin, specification: average molecular weight 24000, K23 - 27). Keep the reaction temperature at 50 °C and end the constant-temperature reaction after 20 hours. When the reaction is completed, all the hydroxyapatite particles precipitate.

[0028] S3: After washing the precipitate 6 times with purified water, add an appropriate amount of purified water to obtain a hydroxyapatite particle suspension B with a concentration of 10 g / L.

[0029] S4: Spray-dry the suspension B. The centrifugal spray-drying equipment is used for spray-drying. The process parameters of the spray are as follows: the feed flow rate of the suspension is 2 L / h, the centrifugal rate of the spray-drying discharge is 18000 rpm, and the spray-drying temperature is 120 °C.

[0030] The particle size of the hydroxyapatite microspheres prepared in this Example 1 is 25 - 50 μm, D50 is 35.06 μm, the tapped density is 0.73 g / mL, the crystallinity is 22%, which belongs to low-crystallinity spherical large particles, the spherical structure is stable, and the sphericity is high (roundness ≥ 0.9), and the dispersibility is good (agglomeration index ≤ 0.1), which is suitable for subcutaneous injection filling.

[0031] Example 2

[0032] A method for preparing hydroxyapatite microspheres, comprising the following steps: S1: Weigh 71 g of disodium hydrogen phosphate and 122 g of calcium chloride dihydrate, dissolve them in 40 L of water, and mix to obtain solution A; S2: Add 10 L of 1 mol / L hydrochloric acid to solution A and stir to mix. After the reaction solution becomes clear, add 192 g of sodium hydroxide to neutralize the reaction, then add PBS buffer and adjust the pH to 7.0. Then add 2.51 g of PEG (brand: Macklin, specification: average Mn 4000) and 0.94 g of PVP (brand: Macklin, specification: average molecular weight 24000, K23 - 27). Keep the reaction temperature at 80 °C and end the constant-temperature reaction after 3 hours. When the reaction is completed, all the hydroxyapatite particles precipitate.

[0033] S3: After washing the precipitate 6 times with purified water, add an appropriate amount of purified water to obtain a 10 g / L suspension B of hydroxyapatite particles.

[0034] S4: Spray-dry suspension B using a centrifugal spray-drying device. The process parameters of the spray are as follows: the feed flow rate of the suspension is 4 L / h, the centrifugal rate of the spray-drying discharge is 25000 rpm, and the spray-drying temperature is 150 °C.

[0035] The particle size of the hydroxyapatite microspheres prepared in Example 2 is 25 - 50 μm, D 50 is 31.15 μm, the tapped density is 0.79 g / mL, and the crystallinity is 30%, belonging to low-crystallinity spherical large particles with a stable spherical structure.

[0036] Comparative Example 1

[0037] A method for preparing hydroxyapatite microspheres includes the following steps: S1: Weigh 71 g of disodium hydrogen phosphate and 122 g of calcium chloride dihydrate, dissolve them in 40 L of water, and mix to prepare solution A; S2: Add 10 L of 1 mol / L hydrochloric acid to solution A and stir to mix. After the reaction solution becomes clear, add 192 g of sodium hydroxide to neutralize the reaction, then add PBS buffer and adjust the pH to 7.0. Keep the reaction temperature at 80 °C and end the constant-temperature reaction after 3 hours. When the reaction is completed, all the hydroxyapatite particles precipitate.

[0038] S3: After washing the precipitate 6 times with purified water, add an appropriate amount of purified water to obtain a 10 g / L suspension B of hydroxyapatite particles.

[0039] S4: Spray-dry suspension B using a centrifugal spray-drying device. The process parameters of the spray are as follows: the feed flow rate of the suspension is 4 L / h, the centrifugal rate of the spray-drying discharge is 25000 rpm, and the spray-drying temperature is 150 °C.

[0040] The particle size of the hydroxyapatite microspheres prepared in Comparative Example 1 is 5 - 30 μm, D 50 is 15.18 μm, the tapped density is 1.2 g / ml, the crystallinity is 65%, and it belongs to spherical particles with a wide particle size distribution range.

[0041] Comparative Example 2

[0042] A method for preparing hydroxyapatite microspheres includes the following steps: S1: Weigh 71 g of disodium hydrogen phosphate and 122 g of calcium chloride dihydrate, dissolve them in 40 L of water, and mix to obtain solution A; S2: Add 10 L of hydrochloric acid with a concentration of 1 mol / L to solution A and stir to mix. After the reaction solution becomes clear, add 192 g of sodium hydroxide to neutralize the reaction, then add PBS buffer solution, and adjust the pH to 7.0. Keep the reaction temperature at 80 °C and end the constant-temperature reaction for 3 hours. When the reaction is completed, all the hydroxyapatite particles precipitate.

[0043] S3: After washing the precipitate 6 times with purified water, add an appropriate amount of purified water to obtain a 10 g / L suspension of hydroxyapatite particles B.

[0044] S4: Spray-dry the suspension B. The centrifugal spray-drying equipment is used for spray-drying. The process parameters of the spray are as follows: the feed flow rate of the suspension is 4 L / h, the centrifugal rate of the spray-drying discharge is 30000 rpm, and the spray-drying temperature is 300 °C.

[0045] The particle size of the hydroxyapatite microspheres prepared in Comparative Example 2 is 3 - 30 μm, D 50 is 15.05 μm, the tapped density is 1.8 g / mL, the crystallinity is 90%, and some of the microspheres are not spherical, with a wide particle size distribution range.

[0046] Performance testing

[0047] 1. According to the method of "GB / T 23101.3 - 2023 Surgical implants - Hydroxyapatite - Part 3: Chemical analysis and characterization of crystalline phase and phase purity", the crystallinity of the microspheres in Example 1 and Comparative Example 2 was detected respectively. The results are as Figure 5 and Figure 6 shown. It can be clearly seen from the figure that Figure 5 the peak width of the diffraction peak increases, while Figure 6 is basically a sharp diffraction peak, indicating that the crystallinity of the microspheres in Example 1 is significantly lower than that in Comparative Example 2.

[0048] 2. Use GeminiSEM 300 to detect the microspheres obtained in Examples 1 - 2 and Comparative Examples 1 - 2. The obtained scanning electron microscope images are respectively as Figures 1-4 shown. It can be seen from the figure thatFigure 3 and Figure 4 The microspheres are small and unevenly distributed, Figure 4 and there are also some that do not present a spherical shape, indicating that the microspheres prepared in Comparative Examples 1-2 are not suitable for the application of this application; Figure 1 and Figure 2 the microspheres have appropriate particle size and uniform distribution, and high sphericity, and are very suitable for subcutaneous injection filling or bone repair.

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing hydroxyapatite microspheres with low crystallinity, characterized in that, It includes the following steps: S1: Prepare a mixed solution of a calcium source reagent and a phosphorus source reagent; S2: Add an inorganic acid, a base, and a buffer to the mixed solution, then add a surfactant, and carry out a constant-temperature reaction to obtain hydroxyapatite particle precipitate; S3: Wash the hydroxyapatite particle precipitate and add water to prepare a hydroxyapatite particle suspension; S4: Spray-dry the hydroxyapatite particle suspension, and the spray-drying parameters are a feeding rate of 0.5 - 5 L / h, a temperature of 120 - 150 °C, and a centrifugal rate of 15000 - 30000 rpm to obtain the hydroxyapatite microspheres; Among them, the surfactant is one or more of PEG and PVP.

2. The preparation method of hydroxyapatite microspheres according to claim 1, characterized in that: The calcium source reagent is calcium chloride; the phosphorus source reagent is selected from at least one of phosphate ion, hydrogen phosphate ion, or dihydrogen phosphate ion.

3. The method for preparing hydroxyapatite microspheres according to claim 1, characterized in that: The inorganic acid is sulfuric acid or hydrochloric acid, the base is sodium hydroxide, and the buffer is PBS buffer.

4. The method for preparing hydroxyapatite microspheres according to claim 1, wherein: The molecular weight of the PEG is 3000 - 6000, and the molecular weight of the PVP is 20000 - 50000.

5. The method for preparing hydroxyapatite microspheres according to claim 1, wherein: In step S2, the temperature of the constant-temperature reaction is 80 - 90 °C, and the reaction time is 2 - 4 h.

6. The method for preparing hydroxyapatite microspheres according to claim 1, wherein: In step S2, the temperature of the constant-temperature reaction is 50 - 60 °C, and the reaction time is 18 - 24 h.

7. A hydroxyapatite microsphere obtained by the method for preparing hydroxyapatite microspheres according to any one of claims 1-6, characterized in that: The particle size of the hydroxyapatite microspheres is 25 μm - 50 μm.

8. The hydroxyapatite microspheres according to claim 7, characterized in that: The crystallinity of the hydroxyapatite microspheres is less than 50%.

9. The hydroxyapatite microspheres according to claim 7, wherein: The tapped density of the hydroxyapatite microspheres is 0.6 - 1.0 g / mL.

10. Use of the hydroxyapatite microspheres according to any one of claims 7 - 9 in bone tissue repair and filling, drug sustained-release carriers, or tissue engineering scaffolds.

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