Hydroxyapatite microspheres for dermal injection and method for preparing the same

Abstract

The application provides a hydroxyapatite microsphere for skin injection filling and a preparation method thereof. The preparation method comprises the following steps: step 1: dissolving a calcium source reagent, a phosphorus source reagent and sodium chloride to obtain a high-concentration supersaturated calcium-phosphorus solution; step 2: adding tromethamine into the calcium-phosphorus solution to establish a buffer system, adjusting the pH value of the reaction system to 5.0-9.0, and reacting for 1-24 hours under the condition of constant temperature and low-speed stirring, and then standing for 0.2-4 hours to obtain a hydroxyapatite micro-particle precipitate; step 3: washing the precipitate, and then spray drying to obtain a hydroxyapatite microsphere crude product; and step 4: screening the hydroxyapatite microsphere crude product to obtain the hydroxyapatite microsphere. The obtained hydroxyapatite microsphere product has high sphericity and low crystallinity, and the defects of poor degradability caused by high-temperature sintering and high crystallinity of the microsphere are overcome.

Description

Technical Field

[0001] This invention relates to the field of medical biomaterials technology, and in particular to a hydroxyapatite microsphere for skin injection filling and its preparation method. Background Technology

[0002] Regenerative cosmetic fillers combine regenerative medicine with aesthetic medicine, using injections and other methods to repair, replace, or stimulate the body's own tissue regeneration. This achieves the repair, enhancement, and reshaping of the body's appearance, form, and function, thus achieving anti-aging effects. Hydroxyapatite microspheres (CaHA) have the chemical formula Ca... 10 (PO4)6(OH)2, with a hexagonal crystal system, is an important biomedical material. Its excellent biocompatibility, safety, and ability to stimulate collagen regeneration make it an indispensable material in the field of cosmetic filling.

[0003] Currently, the mechanism by which CaHA stimulates collagen regeneration has been studied by many scholars, among which calcium plays a key role in the biological function of myofibroblasts. 2+ CaHA is a universal second messenger in fibroblasts, influencing cell proliferation, cell division, cell differentiation, and collagen synthesis. Calcium ions at the cell-matrix junction, entering the cell via the TRPV4 channel, have been shown to be crucial for collagen remodeling. It promotes the formation of essential cellular protrusions by regulating the interaction between flightless-1 protein and non-muscle myosin IIA. Therefore, CaHA's favorable degradation properties can promote the formation of essential protrusions. 2+ The release of these substances helps maintain calcium-mediated biochemical signaling pathways.

[0004] Currently, the main methods for preparing hydroxyapatite microspheres include hydrothermal methods, sol-gel methods, template methods, and spray drying methods. Hydrothermal reactions, occurring in closed containers, are difficult to observe and control, and are currently only suitable for small-scale production and scientific research. The sol-gel method is mostly used for preparing nano-sized hydroxyapatite powders, but obtaining hydroxyapatite with uniform particle size and controllable dimensions presents certain difficulties, making it unsuitable for medical aesthetic fillings. Template methods suffer from the problem of incomplete removal of the oil phase, surfactants, and template from the particle surface. Spray drying, on the other hand, has many advantages such as simple equipment and easy process control, making it a commonly used method for preparing hydroxyapatite microspheres, especially suitable for large-scale industrial production. However, existing spray drying methods also have certain drawbacks. Various wet chemical precipitation methods result in excessively high pH values ​​in the reaction solution, leading to primary hydroxyapatite precipitates containing many impurities, requiring long aging times, and the precipitates exhibiting flocculent behavior in solution, resulting in poor sphericity of the final product. Furthermore, in order to achieve specific shapes and mechanical strength, the primary hydroxyapatite precipitates obtained by these preparation methods need to be dried, ground, and then re-prepared into a slurry for spray drying. Finally, the crude hydroxyapatite microspheres are calcined at high temperatures of 800–1400°C. The process is too complicated and not conducive to continuous production. Moreover, the products have defects such as agglomeration due to high-temperature sintering, excessive crystallinity, and extremely slow degradation of the products in vivo, which leads to the obstruction of calcium ion release.

[0005] Therefore, the field of medical aesthetic filling urgently needs to develop a simple and efficient method for preparing hydroxyapatite microspheres, and the resulting hydroxyapatite microspheres need to have high sphericity, low crystallinity, good biosafety and biodegradability. Summary of the Invention

[0006] This invention addresses the problems of slow calcium-phosphorus crystallization deposition, overly cumbersome processes, easy product agglomeration, excessive crystallinity, and poor degradation in the existing preparation of hydroxyapatite microspheres. It provides a method for preparing hydroxyapatite precipitates based on an improved wet chemical deposition technique. According to this method, by adjusting the ionic strength of key ions in a supersaturated solution, hydroxyapatite precipitates can be obtained under neutral or near-neutral reaction conditions, significantly shortening the calcium-phosphorus crystallization time. After the precipitate reaction, it can be directly washed and spray-dried without the need for drying, grinding, and re-preparation into a slurry. The spray-dried hydroxyapatite microspheres also do not require high-temperature sintering, greatly improving production efficiency. The resulting hydroxyapatite microspheres are high in purity, non-agglomerated, and have low crystallinity, overcoming the defect of poor degradation caused by increased crystallinity due to high-temperature sintering. The product exhibits good biosafety and biodegradability, and can stimulate collagen regeneration after subcutaneous injection, showing broad application prospects in the medical aesthetics field.

[0007] 1. A method for preparing hydroxyapatite microspheres for skin injection filling, characterized by comprising the following steps:

[0008] Step 1: Dissolve the calcium source reagent, phosphorus source reagent, and sodium chloride with acid to prepare a high-concentration supersaturated calcium-phosphorus solution;

[0009] Step 2: Add tromethamine to the calcium phosphate solution to establish a buffer system, and adjust the pH of the reaction system to 5.0-9.0. Stir the reaction at a constant temperature for 1-24 hours, let it stand, and remove the supernatant after the precipitate has clearly separated into layers to obtain hydroxyapatite microparticle precipitate.

[0010] Step 3: After washing the precipitated hydroxyapatite particles, purified water is added to prepare a hydroxyapatite suspension, which is then spray-dried to obtain the crude hydroxyapatite microspheres.

[0011] Step 4: The crude hydroxyapatite microspheres are sieved using a vibrating grading sieve with mesh sizes of 325 mesh and 500 mesh to obtain the hydroxyapatite microspheres.

[0012] Preferably, in step 1, the acid is one or more of hydrochloric acid or sulfuric acid; the concentration of the acid is 1-10 mol / L, more preferably 1-3 mol / L; the calcium source reagent is one or more of calcium chloride or calcium chloride dihydrate; the phosphorus source reagent is selected from one or more of phosphate ions, monohydrogen phosphate ions, or dihydrogen phosphate ions; the molar ratio of the calcium source reagent to the phosphorus source reagent is (1-3):1, more preferably (2-3):1.

[0013] Preferably, in step 1, the concentration range of the high-concentration supersaturated solution is (1–5X) supersaturated solution, wherein the optimal concentration is 1–5X supersaturated solution; the optimal inorganic ion composition in the 5X supersaturated solution is shown in the table below:

[0014]

[0015] Preferably, in step 2, the pH value is 5.0–9.0, more preferably 6.5–7.5; the reaction is carried out under stirring conditions, with a stirring speed of 20–300 rpm, more preferably 40–100 rpm; the reaction temperature is 25–50°C, more preferably 30–40°C, and most preferably 35–37°C; the reaction time is 1–24 h, more preferably 18–24 h; and the standing time is 0.2–4 h, more preferably 1–3 h.

[0016] Preferably, in step 3, the hydroxyapatite microparticle precipitate is washed with purified water, and the washing is performed 3 times or more, preferably 5 to 8 times; the concentration of the hydroxyapatite suspension precipitate is 5 to 50 g / L.

[0017] Preferably, in step 3, the feed rate for spray drying is 0.5–20 L / h, more preferably 1–5 L / h; the air intake volume for spraying is 50–310 m³ / h. 3 / min, preferably 100-200m 3 / min; the inlet air temperature of the spray is 110-250℃, preferably 180-250℃; the centrifugal discharge speed of the spray dryer is 15000-30000rpm, preferably 17000-25000rpm.

[0018] The hydroxyapatite for skin injection filling of the present invention belongs to low-crystallinity hydroxyapatite microspheres, with an average crystallinity of 70-80% and an average sphericity greater than 85%.

[0019] The beneficial effects of this invention are as follows:

[0020] The preparation method provided by this invention can significantly accelerate the deposition rate of hydroxyapatite crystals, resulting in high production efficiency. The product does not require high-temperature sintering, the process steps are simple, and the reaction conditions are easy to achieve, making it suitable for large-scale production. The obtained hydroxyapatite microspheres have high sphericity and low crystallinity, compensating for the defect of poor degradability caused by increased crystallinity due to high-temperature sintering. The product exhibits good biosafety and biodegradability, and can stimulate collagen regeneration after subcutaneous injection, showing broad application prospects in the medical aesthetics field. Attached Figure Description

[0021] Figure 1 A macroscopic digital photograph of the hydroxyapatite microparticle precipitate obtained according to step (3) of Example 1;

[0022] Figure 2 The image shows a scanning electron microscope (50,000x) image of the hydroxyapatite microparticle precipitate obtained according to step (3) of Examples 1 and 2.

[0023] Figure 3 FT-IR image of hydroxyapatite microspheres for skin injection prepared according to Example 1;

[0024] Figure 4 This is a low-magnification SEM image of hydroxyapatite microspheres for skin injection prepared according to Example 1;

[0025] Figure 5 XRD pattern of hydroxyapatite microspheres for skin injection prepared according to Example 2;

[0026] Figure 6 This is a low-magnification SEM image of hydroxyapatite microspheres for skin injection prepared according to Example 2;

[0027] Figure 7The image shows a low-magnification SEM image of hydroxyapatite microspheres for skin injection prepared according to Comparative Example 2. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0029] Example 1

[0030] A method for preparing hydroxyapatite microspheres for skin injection filling includes the following steps:

[0031] (1) Dissolve disodium hydrogen phosphate, calcium chloride dihydrate, and sodium chloride in hydrochloric acid, stir thoroughly until all solids are dissolved, and then add purified water to prepare a 5X high-concentration supersaturated calcium phosphate solution A. The inorganic ionic composition of supersaturated calcium phosphate solution A is shown in the table below:

[0032]

[0033] (2) Add tromethorphan to the supersaturated calcium phosphate solution A and adjust the pH to 7.4.

[0034] (3) Under a constant temperature of 37℃, stirring was started at a speed of 50 rpm, and the reaction was carried out for 24 hours. After standing for 1 hour to separate the solid and liquid phases, the supernatant was drained to obtain hydroxyapatite microparticle precipitate. The precipitate yield was 99.8%. The formula for calculating the precipitate yield is as follows:

[0035]

[0036] (4) After washing the precipitate with purified water 8 times, add an appropriate amount of purified water to obtain a hydroxyapatite microparticle suspension B with a precipitate concentration of 10 g / L.

[0037] (5) The suspension B is spray-dried using a centrifugal spray dryer. The spray drying process parameters are as follows: the feed rate of the suspension is 2 L / h, and the air intake volume of the spray is 120 m³ / h. 3 The spray drying inlet air temperature is 200℃, and the spray drying outlet centrifugation rate is 20000rpm.

[0038] (6) The spray-dried crude hydroxyapatite microspheres were sieved using a vibrating grading sieve with mesh sizes of 325 mesh and 500 mesh to obtain the hydroxyapatite microspheres.

[0039] Technical effects:

[0040] Testing revealed that the hydroxyapatite microspheres prepared in Example 1 were of low crystallinity. FI-IR analysis of these microspheres showed that the -OH peak appeared at 3562 cm⁻¹, the O-P-O bending vibration peaks appeared at 565 and 603 cm⁻¹, the P-O symmetric stretching vibration peak appeared at 962 cm⁻¹, and the P-O asymmetric stretching vibration peaks appeared at 1035 and 1096 cm⁻¹. This confirms that the substance is hydroxyapatite (e.g., ...). Figure 3 (As shown). By measuring the ratio of the microsphere's circumference equivalent diameter to its area equivalent diameter, the sphericity of the microspheres in this embodiment was calculated to be approximately 91.2%, and the average particle size was approximately 25–45 μm (e.g., ...). Figure 4 (As shown).

[0041] Example 2

[0042] (1) Dissolve disodium hydrogen phosphate, calcium chloride dihydrate, and sodium chloride in hydrochloric acid, stir thoroughly until all solids are dissolved, and then add purified water to prepare a 5X high-concentration supersaturated calcium phosphate solution A. The inorganic ionic composition of supersaturated calcium phosphate solution A is shown in the table below:

[0043]

[0044] (2) Add tromethorphan to the supersaturated calcium phosphate solution A and adjust the pH to 7.2.

[0045] (3) Under constant temperature of 37℃, the stirring was turned on at a speed of 50 rpm, and the reaction was carried out for 24 hours. After standing for 1 hour, the solid and liquid separated into layers, and the supernatant was drained to obtain hydroxyapatite microparticle precipitate with a yield of 99.2%.

[0046] (4) After washing the precipitate with purified water 8 times, add an appropriate amount of purified water to obtain a hydroxyapatite microparticle suspension B with a concentration of 10 g / L.

[0047] (5) The suspension B is spray-dried using a centrifugal spray drying device.

[0048] The process parameters are as follows: the feed rate of the suspension is 1.5 L / h, and the air intake volume of the spray is 120 m³ / h. 3 The spray drying inlet air temperature is 220℃, and the spray drying outlet centrifugation rate is 20000rpm.

[0049] (6) The spray-dried crude hydroxyapatite microspheres were sieved using a vibrating grading sieve with mesh sizes of 325 mesh and 500 mesh to obtain the hydroxyapatite microspheres.

[0050] Technical effects:

[0051] Testing revealed that the hydroxyapatite microspheres prepared in Example 2 exhibited low crystallinity. XRD patterns showed that the peak positions of the prepared hydroxyapatite microspheres matched the characteristic peak positions of the standard hydroxyapatite PDF diffraction card, and the diffraction peak intensities were relatively weak, indicating that the hydroxyapatite microspheres possessed low crystallinity, with an average crystallinity of 70-80% (e.g., ...). Figure 5 (As shown). By measuring the ratio of the microsphere's circumference equivalent diameter to its area equivalent diameter, the sphericity of the microspheres in this embodiment was calculated to be approximately 90.7%. The average particle size of the microspheres is approximately 25–45 μm (e.g., ...). Figure 6 (As shown).

[0052] Comparative Example 1 (Comparison of the time required for the formation of hydroxyapatite microparticles)

[0053] This comparative example differs from Example 1 only in that the ionic strength of the 5X supersaturated calcium phosphorus solution A prepared in step (1) of Example 1 is adjusted to that of a 1X supersaturated calcium phosphorus solution; all other steps are the same as in Example 1. The inorganic ion composition of the supersaturated calcium phosphorus solution A in this comparative example is shown in the table below:

[0054]

[0055] The time for the formation of hydroxyapatite microparticles in step (3) of Example 1 was compared with the time for the formation of hydroxyapatite microparticles in step (3) of Comparative Example 1. The results are shown in the table below:

[0056] Hydroxyapatite microparticle precipitation formation time Yield Example 1 1h 99.2％ Comparative Example 1 72h 98.9％

[0057] As shown in the table above, the precipitation time of hydroxyapatite in step (3) of Example 1 was significantly shorter than that in Comparative Example 1. Our research revealed that this was mainly due to the higher ionic strength of the supersaturated calcium phosphate solution A in Example 1 compared to Comparative Example 1, and the lower Zeta potential of the calcium phosphate particles in the supersaturated calcium phosphate solution A in Example 1 compared to Comparative Example 1. 100 mL of supersaturated calcium phosphate solution A from both Example 1 and Comparative Example 1 (step (3)) after 24 hours of isothermal reaction without settling was taken, and the Zeta potential was measured using a Zeta potential analyzer (Malvern Zetasizer Pro, UK). The measurement showed that the Zeta potential of the calcium phosphate particles in the supersaturated calcium phosphate solution A from Example 1 (step (3)) after 24 hours of isothermal reaction without settling was approximately 2.27 mV, while the Zeta potential of the calcium phosphate particles in Comparative Example 1 was approximately 12.21 mV. The lower Zeta potential of the calcium phosphate particles in Example 1 indicates a weaker electrostatic repulsion between particles, making them more likely to approach each other and increasing the likelihood of deposition. The calcium phosphate particles are also more likely to aggregate under the influence of van der Waals forces.

[0058] Comparative Example 2 (Comparison of microsphere sphericity)

[0059] This comparative example differs from Example 2 only in the following ways: 1. The amount of hydrochloric acid used in step (1) of Example 2 is reduced, thus lowering the chloride ion concentration; 2. The order of adding hydrochloric acid in step (1) of Example 2 is adjusted. In this comparative example, disodium hydrogen phosphate, calcium chloride dihydrate, and sodium chloride are first dissolved in purified water before hydrochloric acid is slowly added dropwise. The remaining steps are the same as in Example 2. The inorganic ion composition of the supersaturated calcium phosphorus A solution in this comparative example is shown in the table below:

[0060]

[0061] The results showed that, compared with Example 2, the chloride ion concentration in the reaction system of Comparative Example 2 was lower, and the resulting hydroxyapatite microparticle precipitate became more porous, thus affecting the sphericity of the spray-dried hydroxyapatite microspheres. By measuring the ratio of the microsphere's perimeter equivalent diameter to its area equivalent diameter, the sphericity of the microspheres in this example was calculated to be approximately 75%, which is considered low (e.g., ...). Figure 7 (As shown).

[0062] The preparation method provided by this invention can significantly accelerate the deposition rate of hydroxyapatite crystals, resulting in high production efficiency. The product does not require high-temperature sintering, the process steps are simple, and the reaction conditions are easy to achieve, making it suitable for large-scale production. The obtained hydroxyapatite microspheres have high sphericity and low crystallinity, compensating for the defect of poor degradability caused by increased crystallinity due to high-temperature sintering. The product exhibits good biosafety and biodegradability, and can stimulate collagen regeneration after subcutaneous injection, showing broad application prospects in the medical aesthetics field.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing hydroxyapatite microspheres for skin injection filling, characterized in that: Includes the following steps: Step 1: Dissolve the calcium source reagent, phosphorus source reagent, and sodium chloride in acid to prepare a high-concentration supersaturated calcium-phosphorus solution; the ion concentration of the inorganic ions in the high-concentration supersaturated calcium-phosphorus solution is: Na + :700.0mM,Ca 2+ :20.0mM,Cl - :920.0mM,HPO4 2- :10.0mM; Step 2: Add tromethamine to the calcium phosphate solution to establish a buffer system, and adjust the pH of the reaction system to 5.0~9.

0. Stir the reaction at a constant temperature for 1~24 hours, let it stand, and remove the supernatant after the precipitate has clearly separated into layers to obtain hydroxyapatite microparticle precipitate. Step 3: After washing the precipitated hydroxyapatite particles, purified water is added to prepare a hydroxyapatite suspension, which is then spray-dried to obtain crude hydroxyapatite microspheres. Step 4: The crude hydroxyapatite microspheres are sieved using a vibrating grading sieve with mesh sizes of 325 mesh and 500 mesh to obtain the hydroxyapatite microspheres.

2. The preparation method according to claim 1, characterized in that: In step 1, the acid is one or more of hydrochloric acid or sulfuric acid; the concentration of the acid is 1~10 mol / L; the calcium source reagent is one or more of calcium chloride or calcium chloride dihydrate; the phosphorus source reagent is selected from one or more of phosphate ions, monohydrogen phosphate ions, or dihydrogen phosphate ions; the molar ratio of the calcium source reagent to the phosphorus source reagent is (1~3):

1.

3. The preparation method according to claim 1, characterized in that: In step 2, the pH value is 5.0~9.0; the reaction is carried out under stirring conditions, the stirring speed is 20~300 rpm; the reaction temperature is 25~50℃; the reaction time is 1~24h; and the standing time is 0.2~4h.

4. The preparation method according to claim 1, characterized in that: In step 3, the hydroxyapatite microparticle precipitate is washed with purified water, and the washing is performed more than 3 times; the concentration of the hydroxyapatite suspension precipitate is 5~50g / L.

5. The preparation method according to claim 1, characterized in that: In step 3, the feed rate for spray drying is 0.5~20 L / h; the air intake volume for spraying is 50~310 m³ / h. 3 / min; the inlet air temperature of the spray is 110~250℃; the centrifugation speed of the spray-dried material is 15000~30000rpm.

6. A type of hydroxyapatite microsphere for skin injection filling prepared by the preparation method according to any one of claims 1-5, characterized in that: It belongs to low-crystallinity hydroxyapatite microspheres, with an average crystallinity of 70-80% and an average sphericity greater than 85%.

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