A method for preparing porous hydroxyapatite

Abstract

The application provides a preparation method of porous hydroxyapatite, which comprises the following steps: dissolving an organic phosphorus source in a water-miscible organic solvent to obtain solution A, dissolving an inorganic phosphorus source in water to obtain solution B, uniformly mixing solution A and solution B, adding an aqueous solution of a calcium source, adjusting the pH to 4-6, adding a high-molecular-pore-forming agent, uniformly stirring, heating to 100-130 DEG C, continuously stirring for hydrothermal reaction for 6-10 h, cooling, standing for aging for 3-5 h, washing the obtained precipitate, and calcining to obtain porous hydroxyapatite; the organic phosphorus source is polyoxyethylene nonyl phenol phosphate ester. The method uses a compound phosphorus source of an organic phosphorus source and an inorganic phosphorus source, and the compound phosphorus source has a synergistic effect, so that the prepared porous hydroxyapatite has high sphericity and good special micro-morphology, is suitable for being used as a filler for medical and beauty skin injection, can maintain a natural and full skin filling effect for a long time within an effective period after injection, and does not affect biodegradation.

Description

Technical Field

[0001] This invention relates to the field of medical aesthetic injection microsphere materials, specifically to a method for preparing porous hydroxyapatite. Background Technology

[0002] Hydroxyapatite, with the chemical formula Ca 10 (PO4)6(OH)2, possessing excellent biocompatibility and biodegradability, and being non-toxic and non-immunogenic, is widely used as a biocompatible material in bone repair and cosmetic injection products. As an injectable facial filler, hydroxyapatite requires an appropriate particle size. If the particle size is too small, it may easily penetrate the dermis or be phagocytosed by macrophages, failing to achieve the desired filling effect; if the particle size is too large, the degradation rate decreases, posing safety risks. Furthermore, hydroxyapatite particles need a narrow particle size distribution to improve product uniformity and stability. Currently, the market has set high quality requirements for hydroxyapatite microparticles used in cosmetic injections.

[0003] There are many methods for preparing hydroxyapatite microspheres, such as hydrothermal methods, emulsion crosslinking methods, microwave methods, and spray drying methods. However, some challenges remain, such as expensive and complex equipment, cumbersome and difficult experimental processes, high preparation costs, and poor product structure, size, dispersibility, and uniformity, which hinder their further application in the medical aesthetics field.

[0004] CN116946993A discloses an injectable high-purity medical-grade hydroxyapatite microsphere. First, a primary product is prepared via wet chemical precipitation. A supporting electrolyte is added, and pulsed reverse electrodeposition is performed to obtain a composite coating. Then, the coating is separated at high temperature to separate the hydroxyapatite microspheres, which are then sieved to obtain the final injectable high-purity medical-grade hydroxyapatite microsphere. The hydroxyapatite microspheres prepared by this patent have high purity, good particle size distribution, and good sphericity. However, the preparation process is complex and costly, lacking feasibility for commercialization.

[0005] CN115040689A discloses a highly fluid hydroxyapatite nanomaterial, which involves adding calcium chloride dropwise to disodium hydrogen phosphate to adjust the pH to 5, followed by the addition of cyclohexanehexacarboxylic acid and pretreated carbon microspheres. The reaction is then carried out at high temperature to remove the pretreated carbon microspheres, yielding hollow hydroxyapatite. This patent uses pretreated carbon microspheres as a template agent to prepare hollow hydroxyapatite. However, the high-temperature treatment requires a large amount of energy, making it neither environmentally friendly nor economical. Furthermore, the high-temperature calcination process reduces the dispersibility of the hydroxyapatite.

[0006] CN107572494A discloses a method for preparing hollow hydroxyapatite, which involves a hydrothermal reaction using rosin-based phosphate as the phosphorus source and inorganic calcium salt as the calcium source to obtain hydroxyapatite with a hierarchical porous structure, high specific surface area, and good drug loading capacity. However, testing revealed that rosin-based phosphate has a large molecular weight and volume, resulting in a slow hydrolysis rate. The resulting hollow hydroxyapatite is a nanoflower-structured microparticle with poor sphericity, making it unsuitable for use as a dermal filler in the medical aesthetics field.

[0007] Existing technologies also use polymers or carbon materials as pore-forming agents to prepare porous hydroxyapatite, such as CN107140617A and CN110538347A. While these methods produce hydroxyapatite with good sphericity, the complete volatilization of these pore-forming agents requires high-temperature treatment, which negatively impacts the strength of the hydroxyapatite microspheres. Furthermore, they are prone to leaving carbon residues, which, even in very low concentrations, preclude their use as a medical-grade dermal filler.

[0008] As injectable fillers for cosmetic dermatology, the goal is for them to maintain skin support and elasticity throughout their effective period, while simultaneously ensuring complete biodegradability. However, this presents a contradiction: achieving the required support strength within the effective period necessitates slow degradation. Therefore, there is a need to develop a cosmetic injectable filler that can maintain skin elasticity and plumpness throughout its effective period, while also being safe to degrade and not remaining in the body for an extended period. Summary of the Invention

[0009] To address the shortcomings of existing technologies in preparing porous hydroxyapatite for injection in the medical aesthetics field, which suffers from insufficient performance and cannot simultaneously satisfy the contradictory requirements of cosmetic effects and safe biodegradability, this invention proposes a method for preparing porous hydroxyapatite. By combining organic and inorganic phosphorus sources, a layered porous hydroxyapatite is obtained, which can maintain its shape within its shelf life while simultaneously satisfying the safety requirement of complete biodegradability. This invention achieves the above objectives through the following technical solutions.

[0010] A method for preparing porous hydroxyapatite includes the following steps:

[0011] The organic phosphorus source is dissolved in an organic solvent miscible with water to obtain solution A, and the inorganic phosphorus source is dissolved in water to obtain solution B. After solutions A and B are mixed evenly, an aqueous solution of calcium source is added to adjust the pH to 4-6. A polymeric pore-forming agent is added, and the mixture is stirred evenly. The temperature is raised to 100-130℃ and stirring is continued for hydrothermal reaction for 6-10 hours. After cooling, the mixture is allowed to stand for 3-5 hours. The resulting precipitate is washed and calcined to obtain porous hydroxyapatite.

[0012] The porous hydroxyapatite prepared by this invention has a particle size of 20-60 μm, preferably 30-50 μm; a sphericity of 80% or more, preferably 85% or more, more preferably 90% or more; and a porosity of 30-45%, preferably 35-40%.

[0013] The porous hydroxyapatite prepared according to the above method has a porous structure and a large specific surface area, which is beneficial for skin tissue absorption and utilization, and slows down its loss. It also has excellent sphericity, reaching over 80%, with preferred embodiments exceeding 85%, providing excellent skin support. Furthermore, it has suitable porosity, allowing it to load beneficial components such as growth factors. More importantly, the prepared porous hydroxyapatite exhibits a layered, spherical morphology, providing good support. When used as a filler in cosmetic skin injections, it lubricates and fills wrinkles, resulting in a full and natural skin filling effect.

[0014] Further, the organophosphorus source is polyoxyethylene nonylphenol phosphate, and the water-miscible organic solvent is selected from at least one of ethanol, glycerol, tetrahydrofuran, acetone, dimethyl ether, and mannitol; in solution A, the molar concentration of the organophosphorus source, calculated as P, is 0.2-0.3 mol / L; the inorganic phosphorus source is selected from at least one of disodium hydrogen phosphate, sodium dihydrogen phosphate, diammonium hydrogen phosphate, and ammonium dihydrogen phosphate; in solution B, the molar concentration of the inorganic phosphorus source, calculated as P, is 0.5-1 mol / L.

[0015] Furthermore, the amounts of solution A and solution B satisfy the molar ratio of P in the organic phosphorus source to P in the inorganic phosphorus source as 2-3:7-10; preferably 2:10.

[0016] This invention selects polyoxyethylene nonylphenol phosphate as the organophosphorus source, which combines the functions of a phosphorus source and a surfactant. It eliminates the need for additional surfactants and avoids introducing new impurities. Furthermore, the inventors discovered that among numerous organophosphorus sources, only polyoxyethylene nonylphenol phosphate produces porous hydroxyapatite with a layered morphology. This is likely due to a synergistic effect between its hydrolysis rate and the inorganic phosphorus source, resulting in the preparation of porous hydroxyapatite with a suitable microstructure.

[0017] Furthermore, the calcium source is selected from at least one of calcium chloride and calcium nitrate.

[0018] Furthermore, phosphoric acid or ammonia is used to adjust the pH; preferably, the pH is adjusted to 4.5-5.5. Within this pH range, the impurity content is lowest.

[0019] The inventors discovered that the type of phosphorus source directly affects the sphericity and surface morphology of porous hydroxyapatite. This invention employs a combination of organic and inorganic phosphorus sources. The inorganic phosphorus source serves as the primary source of phosphorus, exhibiting rapid hydrolysis; the organic phosphorus source simultaneously functions as a phosphorus source, surfactant, and pore-modifying agent. This synergistic effect yields porous hydroxyapatite with excellent properties, high sphericity, and suitable microstructure. It also exhibits a suitable degradation time and a relatively long disintegration time, minimizing the difference between disintegration and degradation times, thus simultaneously satisfying both cosmetic effects and safety requirements.

[0020] Furthermore, the amounts of phosphorus source (the sum of organic and inorganic phosphorus sources) and calcium source are adjusted so that the molar ratio of P to Ca in the system is 1.6-1.8:1, preferably 1.65-1.7:1, such as 1.66:1, 1.67:1, 1.68:1, or 1.69:1.

[0021] Further, the polymeric pore-forming agent is selected from at least one of polyvinyl alcohol, hydroxypropyl methylcellulose, chitosan, polydextrose, polyethylene, polypropylene, and polycarbonate, preferably polyvinyl alcohol. The ratio of the polymeric pore-forming agent to Ca in the calcium source is 30-40 g: 1 mol. Even further, the polyvinyl alcohol is a blend of polyvinyl alcohol with a number average molecular weight of 5000-8000 g / mol and polyvinyl alcohol with a number average molecular weight of 20000-30000 g / mol in a mass ratio of 4-7:1. The porous hydroxyapatite prepared using the above-mentioned blend of low and high molecular weight polyvinyl alcohol has a more suitable pore structure and a greater growth factor loading.

[0022] Further washing involves washing with water until the pH of the supernatant reaches 7-7.5, followed by washing with ethanol.

[0023] Furthermore, the calcination is carried out in an oxygen-containing atmosphere (such as air or oxygen) at a temperature above 320°C, for example, 320-360°C for 6-10 hours. This invention does not use pore-forming agents requiring high-temperature calcination; polyvinyl alcohol can be completely removed by calcination at temperatures above 320°C. Since it contains no carbon materials, it can be safely used as a filler for skin injection.

[0024] Compared with existing technologies, the present invention has the following technical advantages:

[0025] I. The method of this invention uses a compound phosphorus source of organic phosphorus source and inorganic phosphorus source, which works synergistically to prepare porous hydroxyapatite with high sphericity and good special microstructure, which is suitable as a filler for medical aesthetic skin injection, to fill skin defects such as wrinkles after injection.

[0026] Second, the porous hydroxyapatite of this invention has a moderate degradation rate of 3-4 months, which can meet the needs of consumers who want to maintain the effect for a long time after a single injection, without the potential risks of staying in the human body for a long time.

[0027] Third, this invention uses low molecular weight and high molecular weight polyvinyl alcohol. The combined effect of the two makes the porous hydroxyapatite morphology more suitable for skin injection, and the pore structure is more developed, which is conducive to the loading of beneficial ingredients to the skin, such as growth factors such as peptides and amino acids, resulting in better skin condition after injection. Attached Figure Description

[0028] Figure 1 Here is a SEM image of the porous hydroxyapatite prepared in Example 1;

[0029] Figure 2 This is a SEM image of porous hydroxyapatite prepared in Example 2;

[0030] Figure 3 The image shows a SEM image of porous hydroxyapatite prepared in Comparative Example 1.

[0031] Figure 4 This is a SEM image of porous hydroxyapatite prepared in Comparative Example 2. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. The following embodiments are provided to better understand this invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods.

[0033] Unless otherwise specified, the "parts" mentioned in the embodiments of the present invention refer to parts by mass, and the "%" refers to a percentage by mass.

[0034] Example 1

[0035] Polyoxyethylene nonylphenol phosphate was dissolved in ethanol solution A, with a concentration of 0.2 mol / L. Disodium hydrogen phosphate was dissolved in water to obtain solution B, with a concentration of 1 mol / L. 100 mL of solution A and 100 mL of solution B were mixed thoroughly. Then, an aqueous solution of CaCl2 with a concentration of 0.5 mol / L was added. The amount of CaCl2 solution used was adjusted so that the P:Ca ratio in the system was 1.68. The pH was adjusted to 4.5. Polyvinyl alcohol (polyvinyl alcohol with a number average molecular weight of 5000 g / mol and a number average molecular weight of...) was added. The mixture was prepared using 20,000 g / mol polyvinyl alcohol (PVA). The PVA content was such that the mass ratio of PVA to calcium source Ca was 20 g: 1 mol. After stirring evenly, the mixture was heated to 120°C and stirred for 10 h for hydrothermal reaction. After cooling, the mixture was allowed to stand for 5 h for aging. The resulting precipitate was first washed with water until the pH of the supernatant was 7, then washed with anhydrous ethanol and dried in a vacuum oven at 60°C for 10 h. The material was then transferred to a muffle furnace and calcined at 350°C in air atmosphere for 10 h to obtain porous hydroxyapatite.

[0036] Example 2

[0037] The other conditions are the same as in Example 1, except that 150 mL of solution A and 70 mL of solution B are mixed thoroughly.

[0038] Example 3

[0039] The other conditions are the same as in Example 1, except that the pH is adjusted to 5.5.

[0040] Example 4

[0041] The other conditions are the same as in Example 1, except that the polyvinyl alcohol is a blend of polyvinyl alcohol with a number average molecular weight of 8000 g / mol and polyvinyl alcohol with a number average molecular weight of 30000 g / mol, and the amount of polyvinyl alcohol used meets the ratio of the mass of polyvinyl alcohol to the calcium source Ca of 30 g: 1 mol.

[0042] Example 5

[0043] The other conditions are the same as in Example 1, except that all polyvinyl alcohol is polyvinyl alcohol with a number average molecular weight of 5000 g / mol.

[0044] Example 6

[0045] The other conditions are the same as in Example 1, except that all the polyvinyl alcohol is polyvinyl alcohol with a number average molecular weight of 20,000 g / mol.

[0046] Comparative Example 1

[0047] Other conditions are the same as in Example 1, except that no organic phosphorus source is added, and all phosphorus sources are inorganic phosphorus sources, such as disodium hydrogen phosphate, keeping the Ca:P ratio of the system constant.

[0048] Comparative Example 2

[0049] Other conditions are the same as in Example 1, except that the organophosphorus source is replaced with disodium hydroxyethylidene diphosphate, while keeping the Ca:P ratio of the system constant.

[0050] Figure 1 Here is a SEM image of the porous hydroxyapatite prepared in Example 1; Figure 2 This is a SEM image of porous hydroxyapatite prepared in Example 2; Figure 3 The image shows a SEM image of porous hydroxyapatite prepared in Comparative Example 1. Figure 4 This is a SEM image of porous hydroxyapatite prepared in Comparative Example 2.

[0051] Application examples

[0052] The porous hydroxyapatite prepared in the above examples and comparative examples was subjected to the following performance tests, and the results are shown in Table 1.

[0053] Sphericity is measured using a scanning electron microscope and calculated using image processing software.

[0054] Cytotoxicity was tested using GB / T 16886.5-2017.

[0055] The residual carbon content was measured using XPS energy dispersive spectroscopy.

[0056] The stability test involves preparing a 5 wt% aqueous solution of the prepared porous hydroxyapatite with physiological saline and storing it at 25 ± 2 °C for 60 days, observing for any aggregation. A uniform and stable single phase indicates success in the stability test; the appearance of visible phase separation or precipitation indicates failure.

[0057] The disintegration time and degradation time were determined by preparing a 1 wt% aqueous solution of porous hydroxyapatite in PBS buffer solution at pH 7.3 and testing it at a constant temperature of 37°C. The disintegration time was the time when the sphericity of the hydroxyapatite microspheres was less than 70%, at which point the hydroxyapatite microspheres could no longer support the skin. The degradation time was the time when the sphericity of the hydroxyapatite microspheres completely disintegrated.

[0058] Table 1

[0059]

[0060] Table 1 shows that the type and proportion of organophosphorus sources, as well as the selection of pore-forming agents, all affect the morphology of porous hydroxyapatite. Without the addition of an organophosphorus source, porous hydroxyapatite cannot be successfully obtained. However, excessive use of organophosphorus sources, or substitution with other organophosphorus sources, also fails to produce porous hydroxyapatite with high sphericity, suitable pore structure, long disintegration time, and a short difference between disintegration and degradation times.

Claims

1. A method for preparing porous hydroxyapatite, characterized in that, Includes the following steps: An organophosphorus source was dissolved in a water-miscible organic solvent to obtain solution A, and an inorganic phosphorus source was dissolved in water to obtain solution B. After solutions A and B were mixed evenly, an aqueous solution of a calcium source was added to adjust the pH to 4-6. A polymeric pore-forming agent was added, and the mixture was stirred evenly. The temperature was then raised to 100-130℃ and stirring was continued for a hydrothermal reaction for 6-10 hours. After cooling, the mixture was allowed to stand and age for 3-5 hours. The resulting precipitate was washed and calcined to obtain porous hydroxyapatite. The organophosphorus source was polyoxyethylene nonylphenol phosphate. In solution A, the molar concentration of the organic phosphorus source, calculated as phosphorus (P), is 0.2-0.3 mol / L; the inorganic phosphorus source is selected from at least one of disodium hydrogen phosphate, sodium dihydrogen phosphate, diammonium hydrogen phosphate, and ammonium dihydrogen phosphate. In solution B, the molar concentration of the inorganic phosphorus source, calculated as phosphorus (P), is 0.5-1 mol / L. The amounts of P in solutions A and B satisfy the molar ratio of P in the organic phosphorus source to P in the inorganic phosphorus source as 2-3:7-10. The amounts of phosphorus and calcium sources used make the molar ratio of P:Ca in the system 1.6-1.8:1; the phosphorus source is the sum of organic and inorganic phosphorus sources.

2. The preparation method according to claim 1, characterized in that, The water-miscible organic solvent is selected from at least one of ethanol, glycerol, tetrahydrofuran, acetone, dimethyl ether, and mannitol.

3. The preparation method according to claim 1, characterized in that, The calcium source is selected from at least one of calcium chloride and calcium nitrate.

4. The preparation method according to claim 1, characterized in that, Adjust the pH using phosphoric acid or ammonia to a value of 4.5-5.

5.

5. The preparation method according to claim 1, characterized in that, The amounts of phosphorus and calcium sources used resulted in a P:Ca molar ratio of 1.65-1.7:1 in the system.

6. The preparation method according to claim 1, characterized in that, The polymeric pore-forming agent is selected from at least one of polyvinyl alcohol, hydroxypropyl methylcellulose, chitosan, polydextrose, polyethylene, polypropylene, and polycarbonate; the ratio of the polymeric pore-forming agent to Ca in the calcium source is 30-40 g: 1 mol.

7. The preparation method according to claim 6, characterized in that, The polyvinyl alcohol is a blend of polyvinyl alcohol with a number average molecular weight of 5000-8000 g / mol and polyvinyl alcohol with a number average molecular weight of 20000-30000 g / mol in a mass ratio of 4-7:

1.

8. The preparation method according to claim 1, characterized in that, The washing process involves washing with water until the pH of the supernatant reaches 7-7.5, followed by washing with ethanol. The calcination process involves calcining at 320-360℃ for 6-10 hours in an oxygen-containing atmosphere.

9. A porous hydroxyapatite, prepared by the method according to any one of claims 1-8, characterized in that, The porous hydroxyapatite has a particle size of 20-60 μm, a sphericity of over 80%, and a porosity of 30-45%.

10. The porous hydroxyapatite according to claim 9, characterized in that, The porous hydroxyapatite has a particle size of 30-50 μm, a sphericity of over 85%, and a porosity of 35-40%.

11. The porous hydroxyapatite according to claim 9, characterized in that, The porous hydroxyapatite has a sphericity of over 90%.

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