A hydroxyapatite-based facial filler material and a preparation process thereof

Abstract

The application relates to the technical field of biomedical materials, in particular to a hydroxyapatite-based facial filling material and a preparation process thereof. The preparation process of the hydroxyapatite-based facial filling material comprises the following steps: preparing strontium-doped hydroxyapatite; preparing composite hydroxyapatite; preparing hyaluronic acid-coated hydroxyapatite; preparing aloe extract and moringa seed extract; and preparing the facial filling material. The application prepares sulfhydrylated hyaluronic acid and acrylated hyaluronic acid respectively, mixes the two with the composite hydroxyapatite, crosslinks the hyaluronic acid in the absence of a crosslinking agent through click chemistry, and prepares the hyaluronic acid-coated hydroxyapatite through an organic phase solidification ball method, so that the crosslinked hyaluronic acid is wrapped on the surface of the composite hydroxyapatite to form a core-shell structure, the potential toxicity risk caused by the residual crosslinking agent is eliminated from the source, and the wrapping rate of the hyaluronic acid to the hydroxyapatite is effectively improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a hydroxyapatite-based facial filler and its preparation process. Background Technology

[0002] In the field of facial fillers, hydroxyapatite, as a bioactive material similar to the inorganic components of human bones and teeth, possesses good osteoconductivity and biocompatibility. However, due to its hard texture and significant difference in elastic modulus compared to human tissue, hydroxyapatite can easily cause a strong foreign body sensation after injection, affecting the natural presentation of facial expressions. Furthermore, the uneven distribution of pure inorganic materials under the skin can easily lead to the formation of local lumps, triggering an immune response and potentially causing adverse reactions such as granulomas, placing a dual burden on patients both physiologically and psychologically. While physically mixing hydroxyapatite with hyaluronic acid is currently the mainstream application method, the hard texture of hydroxyapatite and the lack of effective bonding and buffering between particles require a high level of clinical experience and skill from the doctor during injection. Improper operation, such as inaccurate injection depth or uneven injection speed, can easily lead to unevenness at the injection site, or even the formation of nodules, affecting not only the cosmetic or treatment results but also potentially causing additional health risks and psychological burdens for the patient.

[0003] Currently, there are methods for coating hydroxyapatite with cross-linked hyaluronic acid. The elastic network of cross-linked hyaluronic acid can effectively buffer the rigidity of hydroxyapatite, making the new material soft and greatly reducing the injection resistance of the material, thus significantly improving the convenience and safety of clinical operation. However, most existing technologies rely on adding cross-linking agents to cross-link the hyaluronic acid and then coat it on the surface of hydroxyapatite. This not only poses a potential toxicity risk due to the residue of the cross-linking agent, but also easily leads to inconsistent thickness of the microsphere coating layer and a low coating rate.

[0004] Furthermore, hydroxyapatite only has "mechanical contact" with the surrounding soft / bone tissue, without forming a tight chemical bond or tissue ingrowth. The hyaluronic acid coating can only temporarily isolate hydroxyapatite, which will rapidly degrade under the action of hyaluronidase in the body. In addition, there are tiny gaps between the filler material and the tissue. When the face is under dynamic stress, the interface is prone to peeling and loosening, and is eventually flushed away by body fluids or metabolized and excreted by the immune system, resulting in a short filling time.

[0005] Therefore, there is a need to propose a hydroxyapatite-based facial filler material and its preparation process that can prolong the filling time. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a hydroxyapatite-based facial filler material and its preparation process.

[0007] This invention provides a preparation process for a hydroxyapatite-based facial filler material, comprising the following steps:

[0008] S1: Preparation of Strontium-doped hydroxyapatite

[0009] S1.1: Add calcium nitrate tetrahydrate and strontium nitrate to anhydrous ethanol at a mass ratio of (20-22):1:(160-170), stir thoroughly to dissolve, and then add 0.1 mol / L nitric acid solution to adjust the pH to 2-3 to obtain a metal mixed solution;

[0010] S1.2: Add triethyl phosphate to anhydrous ethanol at a ratio of 1g:(8-10)mL, stir and mix thoroughly to obtain a phosphorus source solution, and then add the phosphorus source solution to the above metal mixed solution while stirring, and continue stirring for 2-3 hours to obtain strontium-doped sol;

[0011] S1.3: Dry the above strontium-doped sol at 40-50℃ for 10-12h, and then keep it at 60-70℃ for 8-10h to obtain a dry gel block;

[0012] S1.4: After grinding the above-mentioned dry gel block, heat it at 80-90℃ for 3-4 hours, then place it in a muffle furnace, heat it at 5℃ / min to 300-350℃, hold it for 2-3 hours, then heat it to 680-720℃, hold it for 2-3 hours, cool it, grind it and sieve it to obtain strontium-doped hydroxyapatite;

[0013] S2: Preparation of composite hydroxyapatite

[0014] After dissolving vascular endothelial growth factor, the above-mentioned strontium-doped hydroxyapatite was added for loading to obtain composite hydroxyapatite.

[0015] S3: Preparation of hyaluronic acid-coated hydroxyapatite

[0016] First, thiolized hyaluronic acid and acrylic hyaluronic acid were prepared separately. Then, the two were dissolved and mixed separately, and the above-mentioned composite hydroxyapatite was added. After being fully dispersed, the mixture was added to xylene and stirred to carry out the reaction. Then, the mixture was centrifuged using chitosan solution as the centrifugation medium to obtain hyaluronic acid-coated hydroxyapatite.

[0017] S4: Preparation of aloe vera extract and moringa seed extract

[0018] Aloe vera and moringa seeds were extracted separately to obtain aloe vera extract and moringa seed extract;

[0019] S5: Preparation of facial filler materials

[0020] The above-mentioned hyaluronic acid-coated hydroxyapatite, aloe vera extract, moringa seed extract, hyaluronic acid and buffer solution are mixed and then sterilized to obtain a facial filler material.

[0021] Furthermore, S2 specifically includes the following steps:

[0022] S2.1: Add vascular endothelial growth factor to PBS buffer at a concentration of (310-320) mg / L, and dissolve by shaking in an ice bath at 1-3℃ to obtain a vascular endothelial growth factor solution;

[0023] S2.2: Add the strontium-doped hydroxyapatite obtained in step S1.4 to the above vascular endothelial growth factor solution at a ratio of 1g:(10-12)mL, stir and load for 10-12h under ice bath conditions at 1-3℃, and then centrifuge, wash and freeze dry to obtain composite hydroxyapatite.

[0024] Furthermore, S3 specifically includes the following steps:

[0025] S3.1: Under an ice bath at 0-2℃, add hyaluronic acid powder to MES buffer at a ratio of 1g:(180-220)mL, stir thoroughly to dissolve, then add EDC and NHS, and continue stirring to activate for 1-2 hours to obtain an activated hyaluronic acid solution.

[0026] S3.2: Add cysteine ​​hydrochloride to ultrapure water at a ratio of 1g:(15-25)mL, stir thoroughly to dissolve, and then add the above activated hyaluronic acid solution. Under nitrogen protection, heat and stir at 30-35℃ for 2-4 hours, and then obtain thiolated hyaluronic acid by alcohol precipitation, dialysis and freeze drying.

[0027] S3.3: Dissolve hyaluronic acid powder in a mixed solvent at 50-60℃ at a ratio of 1g:(90-100)mL to obtain a hyaluronic acid solution, and then dissolve acryloyl chloride in anhydrous dimethyl sulfoxide at a volume ratio of 1:(18-20) to obtain an acryloyl chloride solution.

[0028] S3.4: Under ice bath conditions at 0-4℃, triethylamine and the above acryloyl chloride solution were added to the above hyaluronic acid solution at a volume ratio of 1:(10-12):(45-50). Then the ice bath was removed, and the reaction was stirred for 8-10 hours under nitrogen protection. After the reaction was completed, the precipitate was poured into ice acetone to precipitate. The precipitate was collected by centrifugation, dialyzed, and freeze-dried to obtain acrylic hyaluronic acid.

[0029] S3.5: Dissolve thiolized hyaluronic acid and acrylic hyaluronic acid separately in deionized water at a ratio of 1g:(10-20)mL to obtain thiolized hyaluronic acid solution and acrylic hyaluronic acid solution, and then mix them thoroughly to obtain a mixed hyaluronic acid solution;

[0030] S3.6: Add the composite hydroxyapatite prepared in step S2.2 to the above mixed hyaluronic acid solution at a ratio of 1g:(30-40)mL while stirring. After ultrasonic dispersion for 20-30min, the aqueous phase is obtained.

[0031] S3.7: While stirring at a rate of 600-800 rpm, add the above aqueous phase to xylene at a volume ratio of 1:(4-5), and then stir at 800-1000 rpm for 12-16 h. After the reaction is completed, wash with ethanol and water 3-5 times respectively, and use 0.5% chitosan solution as centrifugation medium. First, centrifuge at 200g for 10 min and discard the precipitate, then centrifuge at 1000g for 10 min and collect the precipitate to obtain hyaluronic acid coated hydroxyapatite.

[0032] Furthermore, S4 specifically includes the following steps:

[0033] S4.1: Wash, dry, and peel disease-free and spotless aloe vera. After blanching, drying, pulverizing, and sieving, aloe vera powder is obtained. Then, aloe vera powder is added to 70% ethanol solution at a ratio of 1g:(20-30)mL. After microwave treatment for 20-30min, the filtrate is filtered and recovered. The ethanol is then concentrated under reduced pressure to obtain the first concentrate. Then, water-saturated n-butanol solution is added to the filter residue at a ratio of 1g:(20-30)mL. After ultrasonic extraction for 10-20min, the filtrate is filtered and recovered. The n-butanol is then concentrated under reduced pressure to obtain the second concentrate. Finally, the first and second concentrates are mixed and dried to obtain aloe vera extract.

[0034] S4.2: Crush moringa seeds into powder, add them to deionized water at a ratio of 1g:(15-20)mL, heat and stir at 40-50℃ for 3-4 hours, cool, centrifuge to collect the supernatant, and then freeze-dry to obtain moringa seed extract.

[0035] Furthermore, the mass ratio of triethyl phosphate to calcium nitrate tetrahydrate is (2-2.1):1.

[0036] Furthermore, the amount of EDC added is 50% of the mass of hyaluronic acid powder, the amount of NHS added is 30% of the mass of hyaluronic acid powder, and the concentration of MES buffer is 0.1 mol / L with a pH of 5.5-6.0.

[0037] Furthermore, the molar ratio of cysteine ​​hydrochloride to hyaluronic acid uronic acid units in the activated hyaluronic acid solution is (1.3-1.5):1, and the mixed solvent is composed of anhydrous dimethyl sulfoxide and anhydrous N,N-dimethylformamide mixed in equal volumes.

[0038] Furthermore, the molar ratio of thiol groups in the thiolized hyaluronic acid solution to that of acrylic acid groups in the acrylated hyaluronic acid solution is equal.

[0039] Furthermore, by mass percentage, the raw material composition of the facial filler is: 10-12% hyaluronic acid-coated hydroxyapatite, 3-5% aloe vera extract, 1-2% moringa seed extract, 6-8% hyaluronic acid, with the remainder being a buffer solution, wherein the buffer solution is a 0.02 mol / L disodium hydrogen phosphate solution.

[0040] A hydroxyapatite-based facial filler material, prepared by the preparation process of a hydroxyapatite-based facial filler material as described in any one of the above claims.

[0041] The present invention has the following advantages:

[0042] 1. In this invention, compared to the method of using a crosslinking agent to encapsulate hydroxyapatite with hyaluronic acid, thiolized hyaluronic acid and acrylic hyaluronic acid are first prepared separately, and then the two are mixed with composite hydroxyapatite. The hyaluronic acid is crosslinked without a crosslinking agent through click chemistry, and the hyaluronic acid is encapsulated in hydroxyapatite by an organic phase solidification spheroidization method. The crosslinked hyaluronic acid is encapsulated on the surface of the composite hydroxyapatite to form a core-shell structure. This not only eliminates the potential toxicity risk caused by the residue of the crosslinking agent from the source and improves the biosafety of the hyaluronic acid-coated hydroxyapatite material, but also the hyaluronic acid-coated hydroxyapatite has a high spheroidization efficiency, thereby effectively improving the encapsulation rate of hyaluronic acid on hydroxyapatite.

[0043] 2. In this invention, strontium-doped hydroxyapatite is first prepared using calcium nitrate tetrahydrate, strontium nitrate, and triethyl phosphate as raw materials. Then, vascular endothelial growth factor is loaded onto the strontium-doped hydroxyapatite to form a composite hydroxyapatite. After coating its surface with hyaluronic acid and preparing it as a facial filler material, strontium can specifically activate calcium-sensitive receptors on osteoblasts, promoting the signaling pathways for osteoblast proliferation, differentiation, and bone matrix synthesis, while inhibiting osteoclast activity and differentiation. This slows down the rate of material absorption and decomposition, inducing a strong chemical bond between hydroxyapatite and bone tissue. The loaded vascular endothelial growth factor, after being slowly released from the composite hydroxyapatite, attracts endothelial cells and stimulates their proliferation, forming a new capillary network around the filler material. This ensures that the osteogenic process induced by strontium ions can proceed smoothly and be maintained for a long time, thereby effectively prolonging the filling time of the facial filler material.

[0044] 3. In this invention, aloe vera extract contains aloe tincture, which can disrupt the integrity of bacterial cell membranes, causing bacterial contents to leak out and die. Flavonoids can inhibit bacterial nucleic acid synthesis and metabolic enzyme activity, preventing bacterial reproduction. Moringin in Moringa seed extract can target and bind to bacterial cell wall synthases, inhibiting cell wall formation and causing bacteria to rupture due to osmotic pressure imbalance. Polyphenols can interfere with the formation of bacterial biofilms, reducing bacterial colonization on puncture wounds and hydroxyapatite surfaces. When used in combination, the two can synergistically inhibit bacteria and reduce the risk of secondary infections caused by bacterial invasion. Attached Figure Description

[0045] Figure 1 This is a flowchart illustrating the preparation process of the hydroxyapatite-based facial filler material used in an embodiment of the present invention. Detailed Implementation

[0046] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of this invention.

[0047] Example 1: A preparation process for a hydroxyapatite-based facial filler, such as... Figure 1 As shown, it includes the following steps:

[0048] S1: Preparation of Strontium-doped hydroxyapatite

[0049] S1.1: Add calcium nitrate tetrahydrate and strontium nitrate to anhydrous ethanol at a mass ratio of 20:1:160, stir thoroughly to dissolve, and then add 0.1 mol / L nitric acid solution to adjust the pH to 2 to obtain a mixed metal solution;

[0050] S1.2: Add triethyl phosphate to anhydrous ethanol at a ratio of 1g:8mL, stir and mix thoroughly to obtain a phosphorus source solution, and then add the phosphorus source solution to the above metal mixed solution while stirring, and continue stirring for 2h to obtain a strontium-doped sol, wherein the mass ratio of triethyl phosphate to calcium nitrate tetrahydrate is 2:1;

[0051] S1.3: The above strontium-doped sol was dried at 40°C for 10 hours and then kept at 60°C for 8 hours to obtain a dry gel block;

[0052] S1.4: After grinding the above dry gel block, heat it at 80°C for 3 hours, then place it in a muffle furnace, heat it to 300°C at 5°C / min, keep it at that temperature for 2 hours, then heat it to 680°C, keep it at that temperature for 2 hours, cool it, grind it and sieve it to obtain strontium-doped hydroxyapatite.

[0053] S2: Preparation of composite hydroxyapatite

[0054] S2.1: Add vascular endothelial growth factor to PBS buffer at a concentration of 310 mg / L, and dissolve by shaking in an ice bath at 1°C to obtain a vascular endothelial growth factor solution;

[0055] S2.2: The strontium-doped hydroxyapatite obtained in step S1.4 was added to the above vascular endothelial growth factor solution at a ratio of 1g:10mL. The mixture was stirred and loaded for 10h under ice bath conditions at 1℃. Then, it was centrifuged, washed, and freeze-dried to obtain composite hydroxyapatite.

[0056] S3: Preparation of hyaluronic acid-coated hydroxyapatite

[0057] S3.1: Under 0℃ ice bath conditions, add hyaluronic acid powder to MES buffer at a ratio of 1g:180mL, stir thoroughly to dissolve, then add EDC and NHS, and continue stirring to activate for 1h to obtain activated hyaluronic acid solution. The amount of EDC added is 50% of the mass of hyaluronic acid powder, the amount of NHS added is 30% of the mass of hyaluronic acid powder, and the concentration of MES buffer is 0.1mol / L and the pH is 5.5.

[0058] S3.2: Add cysteine ​​hydrochloride to ultrapure water at a ratio of 1g:15mL, stir thoroughly to dissolve, and then add the cysteine ​​hydrochloride solution to the above activated hyaluronic acid solution. Under nitrogen protection, heat and stir at 30°C for 2 hours. Then, after alcohol precipitation, dialysis and freeze drying, thiolized hyaluronic acid is obtained. The molar ratio of cysteine ​​hydrochloride to hyaluronic acid glucuronic acid units in the activated hyaluronic acid solution is 1.3:1.

[0059] S3.3: Dissolve hyaluronic acid powder in a mixed solvent of anhydrous dimethyl sulfoxide and anhydrous N,N-dimethylformamide at 50℃ at a ratio of 1g:90mL to obtain a hyaluronic acid solution. Then dissolve acryloyl chloride in anhydrous dimethyl sulfoxide at a volume ratio of 1:18 to obtain an acryloyl chloride solution.

[0060] S3.4: Under 0℃ ice bath conditions, triethylamine and the above acryloyl chloride solution were added to the above hyaluronic acid solution at a volume ratio of 1:10:45. Then the ice bath was removed, and the reaction was stirred for 8 hours under nitrogen protection. After the reaction was completed, the precipitate was poured into ice acetone to precipitate. The precipitate was collected by centrifugation, dialyzed, and freeze-dried to obtain acrylic hyaluronic acid.

[0061] S3.5: Dissolve thiolized hyaluronic acid and acrylic hyaluronic acid separately in deionized water at a ratio of 1g:10mL to obtain thiolized hyaluronic acid solution and acrylic hyaluronic acid solution, and then mix them thoroughly to obtain a mixed hyaluronic acid solution, wherein the molar ratio of thiol groups in the thiolized hyaluronic acid solution to acrylic groups in the acrylic hyaluronic acid solution is equal.

[0062] S3.6: Add the composite hydroxyapatite prepared in step S2.2 to the above mixed hyaluronic acid solution at a ratio of 1g:30mL while stirring. After ultrasonic dispersion for 20min, the aqueous phase is obtained.

[0063] S3.7: While stirring at 600 rpm, the above aqueous phase was added to xylene at a volume ratio of 1:4, and then stirred at 800 rpm for 12 h. After the reaction was completed, the mixture was washed three times with ethanol and water respectively. Using 0.5% chitosan solution as the centrifugation medium, the mixture was first centrifuged at 200g for 10 min and the precipitate was discarded. Then, it was centrifuged at 1000g for 10 min and the precipitate was collected to obtain hyaluronic acid-coated hydroxyapatite.

[0064] S4: Preparation of aloe vera extract and moringa seed extract

[0065] S4.1: Wash, dry, and peel disease-free and spotless aloe vera. After blanching, drying, pulverizing, and sieving, aloe vera powder is obtained. Then, aloe vera powder is added to 70% ethanol solution at a ratio of 1g:20mL. After microwave treatment for 20min, the filtrate is collected by filtration and the ethanol is recovered by vacuum concentration to obtain the first concentrate. Then, water-saturated n-butanol solution is added to the filter residue at a ratio of 1g:20mL. After ultrasonic extraction for 10min, the filtrate is collected by filtration and the n-butanol is recovered by vacuum concentration to obtain the second concentrate. Finally, the first and second concentrates are mixed and dried to obtain aloe vera extract.

[0066] S4.2: Moringa seeds are crushed into powder, and then added to deionized water at a ratio of 1g:15mL. The mixture is heated and stirred at 40℃ for 3 hours. After cooling, the supernatant is collected by centrifugation and then freeze-dried to obtain Moringa seed extract.

[0067] S5: Preparation of facial filler materials

[0068] By weight percentage, the above-mentioned 10% hyaluronic acid-coated hydroxyapatite, 3% aloe vera extract, 1% moringa seed extract, 6% hyaluronic acid, and 80% disodium hydrogen phosphate solution with a concentration of 0.02 mol / L are mixed and then sterilized by irradiation to obtain a facial filler material.

[0069] Example 2: A preparation process for a hydroxyapatite-based facial filler, as follows: Figure 1 As shown, it includes the following steps:

[0070] S1: Preparation of Strontium-doped hydroxyapatite

[0071] S1.1: Add calcium nitrate tetrahydrate and strontium nitrate to anhydrous ethanol at a mass ratio of 21:1:165, stir thoroughly to dissolve, and then add 0.1 mol / L nitric acid solution to adjust the pH to 2.5 to obtain a mixed metal solution;

[0072] S1.2: Add triethyl phosphate to anhydrous ethanol at a ratio of 1 g: 9 mL, stir thoroughly to obtain a phosphorus source solution, and then add the phosphorus source solution to the above metal mixed solution while stirring. Continue stirring for 2.5 h to obtain a strontium-doped sol, wherein the mass ratio of triethyl phosphate to calcium nitrate tetrahydrate is 2.05:1;

[0073] S1.3: The above strontium-doped sol was dried at 45°C for 11 hours and then kept at 65°C for 9 hours to obtain a dry gel block;

[0074] S1.4: After grinding the above-mentioned dry gel block, heat it at 85℃ for 3.5h and then place it in a muffle furnace. Heat it to 325℃ at 5℃ / min and hold it for 2.5h. Then heat it to 700℃ and hold it for 2.5h. After cooling, grind it and sieve it to obtain strontium-doped hydroxyapatite.

[0075] S2: Preparation of composite hydroxyapatite

[0076] S2.1: Add vascular endothelial growth factor to PBS buffer at a concentration of 315 mg / L, and dissolve by shaking in an ice bath at 2°C to obtain a vascular endothelial growth factor solution;

[0077] S2.2: The strontium-doped hydroxyapatite obtained in step S1.4 was added to the above vascular endothelial growth factor solution at a ratio of 1g:11mL. The mixture was stirred and loaded for 11h under ice bath conditions at 2℃. After centrifugation, washing and freeze-drying, composite hydroxyapatite was obtained.

[0078] S3: Preparation of hyaluronic acid-coated hydroxyapatite

[0079] S3.1: Under an ice bath at 1℃, add hyaluronic acid powder to MES buffer at a ratio of 1g:200mL, stir thoroughly to dissolve, then add EDC and NHS, and continue stirring to activate for 1.5h to obtain an activated hyaluronic acid solution. The amount of EDC added is 50% of the mass of hyaluronic acid powder, the amount of NHS added is 30% of the mass of hyaluronic acid powder, and the concentration of MES buffer is 0.1mol / L and the pH is 5.5.

[0080] S3.2: Cysteine ​​hydrochloride was added to ultrapure water at a ratio of 1 g: 20 mL and stirred thoroughly to dissolve, thus obtaining a cysteine ​​hydrochloride solution. This solution was then added to the above activated hyaluronic acid solution. Under nitrogen protection, the mixture was heated and stirred at 32.5 °C for 3 h. After alcohol precipitation, dialysis, and freeze-drying, thiolized hyaluronic acid was obtained. The molar ratio of cysteine ​​hydrochloride to hyaluronic acid glucuronic acid units in the activated hyaluronic acid solution was 1.4:1.

[0081] S3.3: Dissolve hyaluronic acid powder in a mixed solvent of anhydrous dimethyl sulfoxide and anhydrous N,N-dimethylformamide at 55℃ at a ratio of 1g:95mL to obtain a hyaluronic acid solution. Then dissolve acryloyl chloride in anhydrous dimethyl sulfoxide at a volume ratio of 1:19 to obtain an acryloyl chloride solution.

[0082] S3.4: Under ice bath conditions at 2℃, triethylamine and the above acryloyl chloride solution were added to the above hyaluronic acid solution at a volume ratio of 1:11:47.5. Then the ice bath was removed, and the reaction was stirred for 9 hours under nitrogen protection. After the reaction was completed, the precipitate was poured into ice acetone to precipitate. The precipitate was collected by centrifugation, dialyzed, and freeze-dried to obtain acrylic hyaluronic acid.

[0083] S3.5: Dissolve thiolized hyaluronic acid and acrylic hyaluronic acid separately in deionized water at a ratio of 1g:15mL to obtain thiolized hyaluronic acid solution and acrylic hyaluronic acid solution, and then mix them thoroughly to obtain a mixed hyaluronic acid solution, wherein the molar ratio of thiol groups in the thiolized hyaluronic acid solution to acrylic groups in the acrylic hyaluronic acid solution is equal;

[0084] S3.6: Add the composite hydroxyapatite prepared in step S2.2 to the above mixed hyaluronic acid solution at a ratio of 1g:35mL while stirring. After ultrasonic dispersion for 25min, the aqueous phase is obtained.

[0085] S3.7: While stirring at 700 rpm, the above aqueous phase was added to xylene at a volume ratio of 1:4.5, and then stirred at 900 rpm for 14 h. After the reaction was completed, the mixture was washed 4 times with ethanol and water respectively. Using 0.5% chitosan solution as the centrifugation medium, the mixture was first centrifuged at 200g for 10 min and the precipitate was discarded. Then, it was centrifuged at 1000g for 10 min and the precipitate was collected to obtain hyaluronic acid-coated hydroxyapatite.

[0086] S4: Preparation of aloe vera extract and moringa seed extract

[0087] S4.1: Wash, dry, and peel disease-free and spotless aloe vera. After blanching, drying, pulverizing, and sieving, aloe vera powder is obtained. Then, aloe vera powder is added to 70% ethanol solution at a ratio of 1g:25mL. After microwave treatment for 25min, the filtrate is collected by filtration and the ethanol is recovered by vacuum concentration to obtain the first concentrate. Then, water-saturated n-butanol solution is added to the filter residue at a ratio of 1g:25mL. After ultrasonic extraction for 15min, the filtrate is collected by filtration and the n-butanol is recovered by vacuum concentration to obtain the second concentrate. Finally, the first and second concentrates are mixed and dried to obtain aloe vera extract.

[0088] S4.2: Grind the Moringa seeds into powder, add them to deionized water at a ratio of 1g:17.5mL, heat and stir at 45℃ for 3-4 hours, cool, centrifuge to collect the supernatant, and then freeze-dry to obtain Moringa seed extract;

[0089] S5: Preparation of facial filler materials

[0090] By weight percentage, the above-mentioned 11% hyaluronic acid-coated hydroxyapatite, 4% aloe vera extract, 1.5% moringa seed extract, 7% hyaluronic acid, and 76.5% disodium hydrogen phosphate solution with a concentration of 0.02 mol / L are mixed and then sterilized by irradiation to obtain a facial filler material.

[0091] Example 3: A preparation process for a hydroxyapatite-based facial filler, such as... Figure 1 As shown, it includes the following steps:

[0092] S1: Preparation of Strontium-doped hydroxyapatite

[0093] S1.1: Add calcium nitrate tetrahydrate and strontium nitrate to anhydrous ethanol at a mass ratio of 22:1:170, stir thoroughly to dissolve, and then add 0.1 mol / L nitric acid solution to adjust the pH to 3 to obtain a mixed metal solution;

[0094] S1.2: Add triethyl phosphate to anhydrous ethanol at a ratio of 1g:10mL, stir thoroughly to obtain a phosphorus source solution, and then add the phosphorus source solution to the above metal mixed solution while stirring. Continue stirring for 3 hours to obtain a strontium-doped sol, wherein the mass ratio of triethyl phosphate to calcium nitrate tetrahydrate is 2.1:1.

[0095] S1.3: The above strontium-doped sol was dried at 50°C for 12 hours and then kept at 70°C for 10 hours to obtain a dry gel block;

[0096] S1.4: After grinding the above dry gel block, heat it at 90℃ for 4 hours, then place it in a muffle furnace, heat it to 350℃ at 5℃ / min, keep it at that temperature for 3 hours, then heat it to 720℃, keep it at that temperature for 3 hours, cool it, grind it and sieve it to obtain strontium-doped hydroxyapatite.

[0097] S2: Preparation of composite hydroxyapatite

[0098] S2.1: Add vascular endothelial growth factor to PBS buffer at a concentration of 320 mg / L, and dissolve by shaking in an ice bath at 3°C ​​to obtain a vascular endothelial growth factor solution;

[0099] S2.2: The strontium-doped hydroxyapatite obtained in step S1.4 was added to the above vascular endothelial growth factor solution at a ratio of 1g:12mL. The mixture was stirred and loaded for 12h under ice bath conditions at 3℃. Then, it was centrifuged, washed and freeze-dried to obtain composite hydroxyapatite.

[0100] S3: Preparation of hyaluronic acid-coated hydroxyapatite

[0101] S3.1: Under an ice bath at 2℃, add hyaluronic acid powder to MES buffer at a ratio of 1g:220mL, stir thoroughly to dissolve, then add EDC and NHS, and continue stirring to activate for 2h to obtain activated hyaluronic acid solution. The amount of EDC added is 50% of the mass of hyaluronic acid powder, the amount of NHS added is 30% of the mass of hyaluronic acid powder, and the concentration of MES buffer is 0.1mol / L and the pH is 6.0.

[0102] S3.2: Cysteine ​​hydrochloride was added to ultrapure water at a ratio of 1 g: 25 mL and stirred thoroughly to dissolve, thus obtaining a cysteine ​​hydrochloride solution. This solution was then added to the above activated hyaluronic acid solution. The mixture was heated and stirred at 35 °C for 4 h under nitrogen protection. After alcohol precipitation, dialysis, and freeze-drying, thiolized hyaluronic acid was obtained. The molar ratio of cysteine ​​hydrochloride to hyaluronic acid glucuronic acid units in the activated hyaluronic acid solution was 1.5:1.

[0103] S3.3: Dissolve hyaluronic acid powder in a mixed solvent of anhydrous dimethyl sulfoxide and anhydrous N,N-dimethylformamide at 60℃ at a ratio of 1g:100mL to obtain a hyaluronic acid solution. Then dissolve acryloyl chloride in anhydrous dimethyl sulfoxide at a volume ratio of 1:20 to obtain an acryloyl chloride solution.

[0104] S3.4: Under ice bath conditions at 4℃, triethylamine and the above acryloyl chloride solution were added to the above hyaluronic acid solution at a volume ratio of 1:12:50. Then the ice bath was removed, and the reaction was stirred for 10 hours under nitrogen protection. After the reaction was completed, the precipitate was poured into ice acetone to precipitate. The precipitate was collected by centrifugation, dialyzed, and freeze-dried to obtain acrylic hyaluronic acid.

[0105] S3.5: Dissolve thiolized hyaluronic acid and acrylic hyaluronic acid separately in deionized water at a ratio of 1g:20mL to obtain thiolized hyaluronic acid solution and acrylic hyaluronic acid solution, and then mix them thoroughly to obtain a mixed hyaluronic acid solution, wherein the molar ratio of thiol groups in the thiolized hyaluronic acid solution to acrylic groups in the acrylic hyaluronic acid solution is equal.

[0106] S3.6: Add the composite hydroxyapatite prepared in step S2.2 to the above mixed hyaluronic acid solution at a ratio of 1g:40mL while stirring. After ultrasonic dispersion for 30min, the aqueous phase is obtained.

[0107] S3.7: While stirring at 800 rpm, the above aqueous phase was added to xylene at a volume ratio of 1:5, and then stirred at 1000 rpm for 16 h. After the reaction was completed, the mixture was washed 5 times with ethanol and water respectively. Using 0.5% chitosan solution as the centrifugation medium, the mixture was first centrifuged at 200g for 10 min and the precipitate was discarded. Then, it was centrifuged at 1000g for 10 min and the precipitate was collected to obtain hyaluronic acid-coated hydroxyapatite.

[0108] S4: Preparation of aloe vera extract and moringa seed extract

[0109] S4.1: Wash, dry, and peel disease-free and spotless aloe vera. After blanching, drying, pulverizing, and sieving, aloe vera powder is obtained. Then, aloe vera powder is added to 70% ethanol solution at a ratio of 1g:30mL. After microwave treatment for 30min, the filtrate is collected by filtration and the ethanol is recovered by vacuum concentration to obtain the first concentrate. Then, water-saturated n-butanol solution is added to the filter residue at a ratio of 1g:30mL. After ultrasonic extraction for 20min, the filtrate is collected by filtration and the n-butanol is recovered by vacuum concentration to obtain the second concentrate. Finally, the first and second concentrates are mixed and dried to obtain aloe vera extract.

[0110] S4.2: Moringa seeds are crushed into powder, and then added to deionized water at a ratio of 1g:20mL. The mixture is heated and stirred at 50℃ for 4 hours. After cooling, the supernatant is collected by centrifugation and then freeze-dried to obtain Moringa seed extract.

[0111] S5: Preparation of facial filler materials

[0112] The above-mentioned 12% hyaluronic acid-coated hydroxyapatite, 5% aloe vera extract, 2% moringa seed extract, 8% hyaluronic acid, and 73% disodium hydrogen phosphate solution with a concentration of 0.02 mol / L were mixed by weight percentage and then sterilized by irradiation to obtain a facial filler material.

[0113] Comparative Example 1 differs from Example 1 in that step S3 is modified as follows:

[0114] Sodium hyaluronate was dissolved in a 1% sodium hydroxide solution at a mass ratio of 1:20. After complete dissolution, 1,4-butanediol glycidyl ether (5% of the mass of sodium hyaluronate) was added and mixed thoroughly. Then, composite hydroxyapatite was added at a ratio of 1g:30mL and mixed thoroughly. The mixture was heated at 50℃ for 8 hours for cross-linking reaction. After the reaction was complete, dialysis was performed. After removing the dialysate, 0.5% chitosan solution was used as the centrifugation medium. The mixture was first centrifuged at 200g for 10 minutes and the precipitate was discarded. Then, it was centrifuged at 1000g for 10 minutes and the precipitate was collected to obtain hyaluronic acid-coated hydroxyapatite.

[0115] Comparative Example 2 differs from Example 1 in that: strontium nitrate in step S1.1 is removed, and step S2 is also removed, i.e., hydroxyapatite is directly added to the mixed hyaluronic acid solution to prepare an aqueous phase.

[0116] Comparative Example 3 differs from Example 1 in that strontium nitrate in step S1.1 is removed, i.e., strontium is not doped.

[0117] Comparative Example 4 differs from Example 1 in that step S2 is removed and the composite hydroxyapatite in step S3.6 is replaced with an equal amount of strontium-doped hydroxyapatite, i.e., it is not loaded with vascular endothelial growth factor.

[0118] Comparative Example 5 differs from Example 1 in that the aloe vera extract in step S5 is replaced with an equal amount of moringa seed extract.

[0119] Comparative Example 6 differs from Example 1 in that the Moringa seed extract in step S5 is replaced with an equal amount of Aloe vera extract.

[0120] Test example:

[0121] Test 1: The mass of composite hydroxyapatite in the washing liquid after ethanol and water washing in steps S3.7 of Examples 1-3, the precipitate discarded after the first centrifugation, and the supernatant discarded after the second centrifugation were measured respectively, and recorded as the mass of unencapsulated composite hydroxyapatite. The mass of composite hydroxyapatite in the precipitate discarded after the first centrifugation and the supernatant discarded after the second centrifugation in Comparative Example 1 were measured, and recorded as the mass of unencapsulated composite hydroxyapatite. Then the encapsulation rate was calculated, and the calculation formula is as follows:

[0122] Encapsulation rate = (total amount of initially added composite hydroxyapatite - mass of unencapsulated composite hydroxyapatite) / total amount of initially added composite hydroxyapatite × 100%. The measurement was repeated three times, and the average value was taken. The results are shown in Table 1.

[0123] Table 1: Package Rate Test Results

[0124] Parcel rate (%) Example 1 94.4 Example 2 94.7 Example 3 95.1 Comparative Example 1 82.6

[0125] As shown in Table 1 above, when a crosslinking agent was directly added in Comparative Example 1 to crosslink hyaluronic acid onto the composite hydroxyapatite, the encapsulation rate of the hyaluronic acid-coated hydroxyapatite was lower than that in Example 1. Therefore, compared with the method of using a crosslinking agent to encapsulate hydroxyapatite with hyaluronic acid, the method of first preparing thiolized hyaluronic acid and acrylic hyaluronic acid separately, then mixing them with the composite hydroxyapatite, and using click chemistry to crosslink the hyaluronic acid without a crosslinking agent, and then preparing hyaluronic acid-coated hydroxyapatite by solidifying it into spheres using an organic phase, so that the crosslinked hyaluronic acid is encapsulated on the surface of the composite hydroxyapatite to form a core-shell structure, can effectively improve the encapsulation rate of hyaluronic acid on hydroxyapatite.

[0126] Test 2: Sixty 8-10 week old female BALB / c nude mice were divided into 6 groups of 10 mice each: Examples 1-3 and Comparative Examples 2-3. Mice in Examples 1-3 and Comparative Examples 2-4 were subcutaneously injected with 100 μL of the facial filler material prepared in Examples 1-3 and Comparative Examples 2-4, respectively. The mass of hyaluronic acid-coated hydroxyapatite in the facial filler material was recorded as m0. Mice were sacrificed at week 12 after injection, the filler material was removed, dried, and weighed, recorded as m1. The residual rate was calculated using the following formula:

[0127] The residual rate is calculated as (m0-m1) / m0×100%, and the results are shown in Table 2.

[0128] Table 2: Results of Residual Mass Percentage Test

[0129] Residual mass percentage (%) Example 1 87.6 Example 2 87.9 Example 3 88.2 Comparative Example 2 54.8 Comparative Example 3 69.3 Comparative Example 4 72.5

[0130] As shown in Table 2 above, in Comparative Example 2, the residual rate of the facial filler material prepared by directly adding hydroxyapatite was much lower than that in Example 1. In Comparative Example 3, the residual rate of the facial filler material prepared without doping strontium in hydroxyapatite was higher than that in Comparative Example 2 but still lower than that in Example 1. In Comparative Example 4, the residual rate of the facial filler material prepared without loading vascular endothelial growth factor in strontium-doped hydroxyapatite was also higher than that in Comparative Example 1 but lower than that in Example 1. Therefore, it can be seen that by first preparing strontium-doped hydroxyapatite using calcium nitrate tetrahydrate, strontium nitrate and triethyl phosphate as raw materials, then loading vascular endothelial growth factor onto strontium-doped hydroxyapatite to prepare composite hydroxyapatite, and then coating its surface with hyaluronic acid to prepare facial filler material, the filling time of facial filler material can be effectively extended.

[0131] Test 3: Staphylococcus aureus and Escherichia coli were used as test bacteria to test the antibacterial rate of the facial filler materials prepared in Examples 1-3 and Comparative Examples 5-6, respectively. The tests were conducted in parallel three times, and the average value was taken. The results are shown in Table 3.

[0132] Table 3: Results of Antibacterial Rate Test

[0133]

[0134] As shown in Table 3 above, when only one of Moringa seed extract or Aloe extract was added in Comparative Examples 5 and 6, the antibacterial rate of the facial filler materials against Staphylococcus aureus and Escherichia coli was lower than that in Example 1. This shows that when Aloe extract and Moringa seed extract are used together, they can synergistically inhibit bacteria and reduce the risk of secondary infection caused by bacterial invasion.

[0135] It should be understood that those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims. Parts not described in detail in this specification are prior art known to those skilled in the art.

Claims

1. A process for the preparation of a hydroxyapatite-based facial filler material, characterized in that, Comprising the following steps: S1: preparing strontium-doped hydroxyapatite S1.1: adding calcium nitrate tetrahydrate and strontium nitrate into anhydrous ethanol according to the mass ratio (20-22):1:(160-170), fully stirring and dissolving, then adding 0.1 mol / L nitric acid solution to adjust the pH to 2-3, to obtain a metal mixed solution; S1.2: adding triethyl phosphate into anhydrous ethanol according to 1g:(8-10)mL, fully stirring and mixing to obtain a phosphorus source solution, then adding the phosphorus source solution into the above metal mixed solution while stirring, continuing to stir for 2-3h to obtain a strontium-doped sol; S1.3: drying the above strontium-doped sol at 40-50℃ for 10-12h, then incubating at 60-70℃ for 8-10h to obtain a dry gel block; S1.4: grinding the above dry gel block, heating and aging at 80-90℃ for 3-4h, then placing it in a muffle furnace, heating at 5℃ / min to 300-350℃, incubating for 2-3h, then heating to 680-720℃, incubating for 2-3h, cooling, grinding and sieving to obtain strontium-doped hydroxyapatite; S2: preparing composite hydroxyapatite After dissolving the vascular endothelial growth factor, adding the above strontium-doped hydroxyapatite for loading to obtain composite hydroxyapatite; S3: preparing hyaluronic acid-coated hydroxyapatite First, preparing thiolated hyaluronic acid and acrylated hyaluronic acid respectively, then dissolving and mixing them, adding the above composite hydroxyapatite, fully dispersing, adding dimethylbenzene for stirring and reaction, then centrifuging with chitosan solution as the centrifugation medium to obtain hyaluronic acid-coated hydroxyapatite; S4: preparing aloe extract and moringa seed extract Extracting aloe and moringa seeds respectively to obtain aloe extract and moringa seed extract; S5: preparing a facial filling material Mixing the above hyaluronic acid-coated hydroxyapatite, aloe extract, moringa seed extract, hyaluronic acid and buffer, then sterilizing to obtain a facial filling material.

2. The process for preparing a hydroxyapatite-based facial filler material according to claim 1, characterized in that, S2 specifically comprises the following steps: S2.1: adding vascular endothelial growth factor into PBS buffer according to (310-320)mg / L, dissolving under 1-3℃ ice bath conditions, to obtain a vascular endothelial growth factor solution; S2.2: adding the strontium-doped hydroxyapatite prepared in step S1.4 into the above vascular endothelial growth factor solution according to 1g:(10-12)mL, stirring and loading at 1-3℃ ice bath conditions for 10-12h, then centrifuging, washing and freeze-drying to obtain composite hydroxyapatite.

3. The process for preparing a hydroxyapatite-based facial filler material according to claim 2, characterized in that, S3 specifically comprises the following steps: S3.1: under 0-2℃ ice bath, adding hyaluronic acid powder into MES buffer according to 1g:(180-220)mL, fully stirring and dissolving, then adding EDC and NHS, continuing to stir and activate for 1-2h to obtain an activated hyaluronic acid solution; S3.2: Cysteamine hydrochloride is added into ultrapure water at a mass ratio of 1 g: (15-25) mL, and stirred to dissolve to obtain a cysteamine hydrochloride solution, which is then added into the activated hyaluronic acid solution, and heated and stirred at 30-35°C for 2-4 h under nitrogen protection, followed by alcohol precipitation, dialysis and freeze-drying to obtain thiolated hyaluronic acid; S3.3: Hyaluronic acid powder is dissolved in a mixed solvent at 50-60°C to obtain a hyaluronic acid solution, and acryloyl chloride is dissolved in anhydrous dimethyl sulfoxide at a volume ratio of 1: (18-20) to obtain an acryloyl chloride solution; S3.4: Under ice bath conditions at 0-4°C, triethylamine and the acryloyl chloride solution are added into the hyaluronic acid solution at a volume ratio of 1: (10-12): (45-50), and then the ice bath is removed, and the reaction is stirred for 8-10 h under nitrogen protection, after which the reaction is completed, the mixture is poured into ice acetone to precipitate, and the precipitate is collected by centrifugation, dialyzed and freeze-dried to obtain acrylated hyaluronic acid; S3.5: Thiolated hyaluronic acid and acrylated hyaluronic acid are dissolved in deionized water at a mass ratio of 1 g: (10-20) mL to obtain a thiolated hyaluronic acid solution and an acrylated hyaluronic acid solution, respectively, and then the two solutions are fully stirred and mixed to obtain a mixed hyaluronic acid solution; S3.6: The composite hydroxyapatite prepared in step S2.2 is added into the mixed hyaluronic acid solution at a mass ratio of 1 g: (30-40) mL while stirring, and ultrasonic dispersion is performed for 20-30 min to obtain an aqueous phase; S3.7: The aqueous phase is added into xylene at a volume ratio of 1: (4-5) while stirring at a speed of 600-800 rpm, and then the reaction is stirred at a speed of 800-1000 rpm for 12-16 h, after which the reaction is completed, the mixture is washed with ethanol and water for 3-5 times, respectively, and then 0.5% chitosan solution is used as a centrifugation medium, and the mixture is first centrifuged at 200 g for 10 min, and then the precipitate is discarded, and the mixture is centrifuged at 1000 g for 10 min to collect the precipitate to obtain hyaluronic acid-coated hydroxyapatite.

4. The process for preparing a hydroxyapatite-based facial filler material according to claim 3, characterized in that, S4 specifically comprises the following steps: S4.1: After being washed, dried, peeled, fixed, dried, crushed and sieved, aloe vera powder is obtained, and then the aloe vera powder is added into 70% ethanol solution at a mass ratio of 1 g: (20-30) mL, and microwave treatment is performed for 20-30 min, after which the filtrate is recovered by filtration, and ethanol is recovered by concentration under reduced pressure to obtain a first concentrated solution, and then water-saturated n-butanol solution is added into the residue at a mass ratio of 1 g: (20-30) mL, and ultrasonic extraction is performed for 10-20 min, after which the filtrate is recovered by filtration, and n-butanol is recovered by concentration under reduced pressure to obtain a second concentrated solution, and finally the first concentrated solution and the second concentrated solution are mixed and dried to obtain aloe vera extract; S4.2: The jatropha seed is crushed into powder, and then added into deionized water at a mass ratio of 1 g: (15-20) mL, and heated and stirred for extraction at 40-50°C for 3-4 h, after which the supernatant is collected by centrifugation after cooling, and then freeze-dried to obtain jatropha seed extract.

5. The process for preparing a hydroxyapatite-based facial filler material according to claim 1, wherein, The mass ratio of triethyl phosphate to calcium nitrate tetrahydrate is (2-2.1):

1.

6. The process for preparing a hydroxyapatite-based facial filler material according to claim 3, wherein, The EDC is added in an amount of 50% of the mass of the hyaluronic acid powder, the NHS is added in an amount of 30% of the mass of the hyaluronic acid powder, and the MES buffer solution has a concentration of 0.1 mol / L and a pH of 5.5-6.

0.

7. The process for preparing a hydroxyapatite-based facial filler material according to claim 3, wherein, The molar ratio of cysteamine hydrochloride to the hyaluronic acid uronic acid units in the activated hyaluronic acid solution is (1.3-1.5):1, and the mixed solvent is obtained by mixing equal volumes of anhydrous dimethyl sulfoxide and anhydrous N,N-dimethylformamide.

8. The process for preparing a hydroxyapatite-based facial filler material according to claim 3, wherein, The molar ratio of the thiol groups in the thiolated hyaluronic acid solution to the acrylic acid groups in the acrylic acid-modified hyaluronic acid solution is equal.

9. The process for preparing a hydroxyapatite-based facial filler material according to claim 1, wherein, The raw material composition of the facial filling material is 10-12% hyaluronic acid-coated hydroxyapatite, 3-5% aloe extract, 1-2% Moringa oleifera seed extract, 6-8% hyaluronic acid, and the balance is a buffer solution, wherein the buffer solution is a sodium phosphate dibasic solution with a concentration of 0.02 mol / L.

10. A hydroxyapatite-based facial filler material, characterized by, It is prepared by the preparation process of the hydroxyapatite-based facial filling material according to any one of claims 1-9.

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