Preparation method of a composite hydroxyapatite filler for injection

Abstract

The present invention relates to the technical field of biomedical materials, and specifically to a preparation method of a composite hydroxyapatite filler for injection. A preparation method of a composite hydroxyapatite filler for injection includes the following steps: preparation of fluorine-containing collagen; preparation of unidirectional nano-hydroxyapatite composite material; preparation of anti-inflammatory extract solution; mixing preparation and treatment of injectable hydrogel. By placing the pre-frozen solution in a strong magnetic field for reaction, collagen fibers and hydroxyapatite crystals arranged orderly are initially formed under the action of the strong magnetic field, and then unidirectional freezing from bottom to top of the reaction system and application of a strong magnetic field are carried out by liquid nitrogen, so that the internal structure of the unidirectional nano-hydroxyapatite composite material is more compact and stable, with better anti-swelling performance and higher anti-compression strength. During the process of bone defect repair, it can have excellent space maintenance ability and enhance the restoration effect of the external contour of the bone defect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and specifically relates to a preparation method of a composite hydroxyapatite filler for injection. Background Art

[0002] Hydroxyapatite is the main component of bones and teeth and has excellent bioactivity and biocompatibility. Mineralized hydrogels prepared mainly from hydroxyapatite and organic components are similar to natural bone tissues in chemical composition and structural characteristics and have good effects in the treatment of bone defects.

[0003] Existing hydroxyapatite mineralized hydrogels are generally prepared by mixing hydroxyapatite with various morphologies with organic substances such as collagen, hyaluronic acid or sodium alginate. The morphologies of hydroxyapatite mainly include hydroxyapatite microspheres and porous hydroxyapatite. Hydroxyapatite microspheres have good fluidity and specific surface area due to their spherical appearance, which can enhance the filling effect of bone repair. Porous hydroxyapatite has a high porosity and good drug release property, which is beneficial to enhancing the speed of bone repair.

[0004] However, in the preparation process of hydroxyapatite microspheres, there are defects such as easy aggregation of microspheres and wide particle size distribution, which easily lead to uneven dispersion of the obtained components and affect the tensile strength and toughness of the mineralized hydrogel. Porous hydroxyapatite has the disadvantages of difficult control of pore size and porosity and easy generation of cracks in the preparation process, which reduces the strength of porous hydroxyapatite, both of which will result in poor space maintenance ability of the mineralized hydrogel and reduce the repair effect on bone defects. Summary of the Invention

[0005] In order to solve the above technical defects, the present invention has developed a preparation method of a composite hydroxyapatite filler for injection, which can make the product have good anti-swelling performance, high compressive strength and excellent repair effect on bone defects.

[0006] A preparation method of a composite hydroxyapatite filler for injection includes the following steps:

[0007] S1: Preparation of fluorine-containing collagen

[0008] Mix ethylene glycol and ultrapure water and add polyvinylpyrrolidone. After heating in a water bath and cooling, add acrylamide and stir, then add Tween 80 and stir evenly to obtain a reaction solution. After ultrasonic treatment of the reaction solution, a homogeneous emulsion is obtained. Add calcium carbonate, sodium fluoride, collagen and ultrapure water to the homogeneous emulsion and stir to obtain fluorine-containing collagen;

[0009] S2: Preparation of unidirectional nano-hydroxyapatite composite material

[0010] Prepare a mixed solution of fluorinated collagen and phosphate buffer solution. Mix the mixed solution with calcium chloride dihydrate solution and then slowly add NaOH solution to adjust the pH to obtain a pre-frozen solution. First, place the pre-frozen solution in a unidirectional strong magnetic field for reaction, and then place it in a unidirectional strong magnetic field while undergoing unidirectional freezing until the pre-frozen solution is completely frozen solid to obtain a freeze-dried solid. Immerse the freeze-dried solid in ammonium persulfate solution, filter, perform dialysis with deionized water, and then perform freeze-drying dehydration and isostatic pressing to obtain a unidirectional nano-hydroxyapatite composite material;

[0011] S3: Preparation of anti-inflammatory extract solution

[0012] Stir-fry ginkgo leaves and then crush them together with passionflower and tea polyphenols to obtain plant powder. Mix the plant powder with deionized water and perform ultrasonic treatment. After filtration, concentrate the filtrate to obtain an anti-inflammatory extract solution;

[0013] S4: Mixing preparation and treatment of injectable hydrogel

[0014] Add sodium hyaluronate to deionized water and stir evenly. Add anesthetic and unidirectional cross-linked hydroxyapatite composite material, and then stir evenly. Finally, add the anti-inflammatory extract solution and stir evenly to obtain an injectable hydrogel. Fill the injectable hydrogel and perform moist heat sterilization to obtain an injectable hydroxyapatite filler.

[0015] Furthermore, the preparation of fluorinated collagen in step S1 includes the following steps:

[0016] S1.1: Mix 4 - 5 parts by weight of ethylene glycol and 8 - 10 parts by weight of ultrapure water in a container, add 1 - 2 parts by weight of polyvinylpyrrolidone, heat in a water bath at a temperature of 85 - 90 °C for 10 - 15 minutes, cool to room temperature, add 2 - 3 parts by weight of acrylamide, stir until the acrylamide is completely dissolved, and then add 0.1 - 0.2 parts by weight of Tween 80 and stir evenly to obtain a reaction solution;

[0017] S1.2: Place the reaction solution in an ultrasonic crusher and perform ultrasonic treatment at a power of 300 - 350 W for 20 - 25 minutes to obtain a homogeneous emulsion. Add 0.3 - 0.4 parts by weight of calcium carbonate, 0.2 - 0.3 parts by weight of sodium fluoride, 3 - 4 parts by weight of collagen, and 8 - 10 parts by weight of ultrapure water to the homogeneous emulsion and stir until a homogeneous colloidal liquid is formed to obtain fluorinated collagen.

[0018] Furthermore, the preparation of the unidirectional nano-hydroxyapatite composite material in step S2 includes the following steps:

[0019] S2.1: Mix fluorinated collagen and phosphate buffer solution to prepare a mixed solution with a fluorinated collagen content of 8 - 10 mg / ml and a phosphoric acid concentration of 0.06 - 0.08 mol / L. Mix the mixed solution and calcium chloride dihydrate solution in a volume ratio of 1:(6 - 8) and place it in a polytetrafluoroethylene container. Then slowly add NaOH solution to adjust the pH to 7.8 - 8.2 to obtain a pre-frozen solution;

[0020] S2.2: Place the polytetrafluoroethylene container filled with the pre-frozen solution into a magnetizer, adjust the magnetic field direction from bottom to top, with a magnetic field strength of 8 - 10 T, and let it stand for 2 - 3 hours. Then immerse the polytetrafluoroethylene container filled with the pre-frozen solution in liquid nitrogen, with liquid nitrogen submerging 1 / 5 - 1 / 4 of the polytetrafluoroethylene container. Liquid nitrogen serves as a unidirectional cold source to promote the unidirectional growth of ice crystals in the pre-frozen solution from bottom to top. Quickly place the polytetrafluoroethylene container in the liquid nitrogen into the magnetizer, adjust the magnetic field direction from bottom to top, with a magnetic field strength of 8 - 10 T, until the pre-frozen solution in the polytetrafluoroethylene container is completely frozen solid to obtain a freeze-dried solid;

[0021] S2.3: Place the freeze-dried solid in an ammonium persulfate solution with a mass fraction of 0.8 - 1%, let it stand for 4 - 5 hours, filter, and then dialyze the obtained filtrate with deionized water. Then place it in a vacuum freeze dryer for freeze-drying dehydration, and then perform isostatic pressing at a pressure of 180 - 200 MPa to obtain a unidirectional nano-hydroxyapatite composite material.

[0022] Further, the preparation of the anti-inflammatory extract solution in step S3 includes the following steps:

[0023] S3.1: Stir-fry ginkgo leaves in a casserole for 15 - 20 minutes, and then place them together with passionflower and tea polyphenols in a mass ratio of 1:(0.8 - 1):(0.1 - 0.2) into a pulverizer for pulverization to obtain plant powder;

[0024] S3.2: Add the plant powder and deionized water in a mass ratio of 1:(6 - 8) to an ultrasonic water bath, adjust the ultrasonic frequency to 20 - 25 KHz, the temperature to 85 - 90 °C, and perform ultrasonic treatment for 25 - 30 minutes. Then filter to remove the filter residue, and concentrate the filtrate in a concentrator to 1 / 4 - 1 / 3 of the original volume to obtain an anti-inflammatory extract solution.

[0025] Further, the mixing preparation and treatment of the injectable hydrogel in step S4 includes the following steps:

[0026] S4.1: Add 8 - 10 parts by weight of sodium hyaluronate to 50 - 60 parts by weight of deionized water, stir evenly at a stirring speed of 450 - 500 rpm, add 0.2 - 0.3 parts by weight of anesthetic and 4 - 5 parts by weight of unidirectionally crosslinked hydroxyapatite composite material, then stir evenly at a stirring speed of 800 - 900 rpm, and finally add the anti - inflammatory extract solution and stir evenly at a stirring speed of 200 - 250 rpm to obtain the injectable hydrogel;

[0027] S4.2: After filling the injectable hydrogel, place it in a moist heat sterilizer, adjust the temperature to 121 - 123 °C, and sterilize for 15 - 20 minutes to obtain the injectable hydroxyapatite filler.

[0028] Further, the collagen in step S1.2 is type I collagen from bovine Achilles tendon.

[0029] Further, the concentration of the calcium chloride dihydrate solution in step S2.1 is 0.1 - 0.2 mol / L.

[0030] Further, the base of the polytetrafluoroethylene container in step S2.1 is made of Cu material.

[0031] Further, the method of deionized water dialysis in step S2.3 is to put the obtained filtrate into a semi - permeable membrane bag and soak it in distilled water for 6 - 7 days.

[0032] Further, the anesthetic in step S4.1 is lidocaine hydrochloride.

[0033] The beneficial effects are as follows: 1. By mixing the fluorine - containing collagen and hydroxyapatite reaction system in the present invention, a pre - frozen solution is obtained. Then, the pre - frozen solution is first placed in a strong magnetic field for reaction. Under the action of the strong magnetic field, collagen fibers and hydroxyapatite crystals arranged in an orderly manner are initially formed. Then, by applying a strong magnetic field while performing unidirectional freezing of the reaction system from bottom to top with liquid nitrogen, the collagen fibers and hydroxyapatite in the fluorine - containing collagen can be better oriented along the ice crystal growth direction, greatly reducing the intermolecular spacing. At the same time, the subsequent cross - linking treatment with ammonium persulfate can form a more dense and regular network structure, making the internal structure of the unidirectional nano - hydroxyapatite composite material more dense and stable, having better anti - swelling performance and higher anti - compression strength. During the process of bone defect repair, it can have excellent space - maintaining ability and enhance the restoration effect of the bone defect contour.

[0034] 2. The present invention prepares fluorine-containing collagen and then reacts with a hydroxyapatite reaction system to generate a unidirectional nano-hydroxyapatite composite material. Fluoride ions are introduced into the finally prepared injectable composite hydroxyapatite filler, enabling the injectable composite hydroxyapatite filler to release fluoride ions during tooth repair. The diffusion of fluoride ions to the tooth surface can not only reconstruct demineralized tooth enamel, promote bone tissue regeneration, but also form a fluorapatite protective layer to prevent further tooth demineralization, thereby protecting the deep dental tissue and achieving the prevention and treatment of dental caries lesions.

[0035] 3. The present invention uniformly disperses acrylamide into fluorine-containing collagen. After obtaining a freeze-dried solid from the reaction system of fluorine-containing collagen and hydroxyapatite, the freeze-dried solid is immersed in an ammonium persulfate solution. Ammonium persulfate acts as an initiator to polymerize acrylamide to generate polyacrylamide, which then entangles with the hydrogen bonds in the collagen to obtain a cross-linked network structure, making the formed nano-hydroxyapatite more uniformly dispersed in the unidirectional nano-hydroxyapatite composite material, enhancing the compressive strength and toughness of the unidirectional nano-hydroxyapatite composite material, further improving the space maintenance ability of the injectable composite hydroxyapatite filler, and obtaining a better bone defect repair effect.

[0036] 4. The present invention prepares an anti-inflammatory extract solution and adds it to the preparation of the injectable composite hydroxyapatite filler, enabling the injectable composite hydroxyapatite filler to prevent and treat inflammation and swelling of dental tissues during the repair of tooth bone defects, and enhancing the safety and practicality of the injectable composite hydroxyapatite filler. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is a flowchart of a preparation method of an injectable composite hydroxyapatite filler adopted in an embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0038] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0039] Example 1

[0040] A preparation method of an injectable composite hydroxyapatite filler, as Figure 1 shown, includes the following steps:

[0041] S1: Preparation of fluorine-containing collagen

[0042] S1.1: Mix 4 parts by weight of ethylene glycol and 8 parts by weight of ultrapure water in a container. Add 1 part by weight of polyvinylpyrrolidone, heat in a water bath at 85 °C for 10 minutes, cool to room temperature, then add 2 parts by weight of acrylamide, stir until the acrylamide is completely dissolved, and then add 0.1 part by weight of Tween 80 and stir evenly to obtain a reaction solution;

[0043] S1.2: Place the reaction solution in an ultrasonic crusher and perform ultrasonic treatment at a power of 300 W for 20 minutes to obtain a homogeneous emulsion. Add 0.3 part by weight of calcium carbonate, 0.2 part by weight of sodium fluoride, 3 parts by weight of collagen, and 8 parts by weight of ultrapure water to the homogeneous emulsion, stir until a homogeneous colloidal liquid is formed to obtain a fluorine-containing collagen, enabling the injectable composite hydroxyapatite filler to release fluoride ions during tooth repair. The fluoride ions diffuse to the tooth surface, which can not only reconstruct the demineralized enamel, promote bone tissue regeneration, but also form a fluorapatite protective layer to prevent further tooth demineralization, thereby protecting the deep dental tissue and achieving the prevention and treatment of dental caries lesions.

[0044] S2: Preparation of unidirectional nano-hydroxyapatite composite material

[0045] S2.1: Mix the fluorine-containing collagen and phosphate buffer solution to prepare a mixed solution with a fluorine-containing collagen content of 8 mg / ml and a phosphoric acid concentration of 0.06 mol / L. Mix the mixed solution and a 0.1 mol / L calcium chloride dihydrate solution in a volume ratio of 1:8 and place them in a polytetrafluoroethylene container. The base of the polytetrafluoroethylene container is made of Cu material. Then slowly add NaOH solution to adjust the pH to 7.8 to obtain a pre-frozen solution;

[0046] S2.2: After placing the pre-frozen solution in a polytetrafluoroethylene container, put it into a magnetizer, adjust the magnetic field direction from bottom to top, and the magnetic field strength is 8 T. Let it stand for 2 hours to initially form orderly arranged collagen fibers and hydroxyapatite crystals. Then immerse the polytetrafluoroethylene container containing the pre-frozen solution in liquid nitrogen, with the liquid nitrogen submerging 1 / 5 of the polytetrafluoroethylene container. The liquid nitrogen serves as a unidirectional cold source to promote the unidirectional growth of ice crystals in the pre-frozen solution from bottom to top. Quickly place the polytetrafluoroethylene container in the liquid nitrogen into the magnetizer, adjust the magnetic field direction from bottom to top, and the magnetic field strength is 8 T until the pre-frozen solution in the polytetrafluoroethylene container is completely frozen solid, enabling the collagen fibers and hydroxyapatite in the fluorine-containing collagen to better align along the ice crystal growth direction, greatly reducing the intermolecular spacing to obtain a freeze-dried solid;

[0047] S2.3: Place the freeze-dried solid in an ammonium persulfate solution with a mass fraction of 0.8%, and let it stand for 4 hours. Ammonium persulfate acts as an initiator to polymerize acrylamide to form polyacrylamide, which then entangles with the hydrogen bonds in collagen to obtain a cross-linked network structure, enabling the formed nano-hydroxyapatite to be more evenly dispersed in the unidirectional nano-hydroxyapatite composite material. After filtration, put the obtained filtrate into a semi-permeable membrane bag and soak it in distilled water for 6 days, then place it in a vacuum freeze-dryer for freeze-drying and dehydration, and then perform isostatic pressing at a pressure of 180 MPa to obtain the unidirectional nano-hydroxyapatite composite material.

[0048] S3: Preparation of anti-inflammatory extract solution

[0049] S3.1: Put ginkgo leaves in a casserole and stir-fry for 15 minutes, then place them in a pulverizer together with passionflower and tea polyphenols in a mass ratio of 1:0.8:0.1 to obtain plant powder;

[0050] S3.2: Add the plant powder and deionized water in a mass ratio of 1:6 to an ultrasonic water bath, adjust the ultrasonic frequency to 20 KHz and the temperature to 85 °C, perform ultrasonic treatment for 25 minutes, then filter to remove the residue, and concentrate the filtrate to 1 / 4 of the original volume in a concentrator to obtain the anti-inflammatory extract solution, which is added to the preparation of the injectable composite hydroxyapatite filler, enabling the injectable composite hydroxyapatite filler to prevent and treat dental tissue inflammation and swelling during the repair of dental bone defects, and enhancing its safety and practicality.

[0051] S4: Mixing preparation and treatment of injectable hydrogel

[0052] S4.1: Add 8 parts by weight of sodium hyaluronate to 50 parts by weight of deionized water, stir evenly at a stirring speed of 450 rpm, add 0.2 parts by weight of anesthetic and 4 parts by weight of unidirectional cross-linked hydroxyapatite composite material, then stir evenly at a stirring speed of 800 rpm, and finally add the anti-inflammatory extract solution and stir evenly at a stirring speed of 200 rpm to obtain the injectable hydrogel;

[0053] S4.2: After filling the injectable hydrogel, place it in a moist heat sterilizer, adjust the temperature to 121 °C, and sterilize for 20 minutes to obtain the injectable hydroxyapatite filler.

[0054] Example 2

[0055] A preparation method of an injectable composite hydroxyapatite filler, as Figure 1 shown, includes the following steps:

[0056] S1: Preparation of fluorine-containing collagen

[0057] S1.1: Mix 5 parts by weight of ethylene glycol and 10 parts by weight of ultrapure water in a container, add 2 parts by weight of polyvinylpyrrolidone, heat in a water bath at 85 °C for 10 minutes, cool to room temperature, then add 3 parts by weight of acrylamide, stir until the acrylamide is completely dissolved, and then add 0.2 parts by weight of Tween 80 and stir evenly to obtain a reaction solution;

[0058] S1.2: Place the reaction solution in an ultrasonic crusher and perform ultrasonic treatment at a power of 300 W for 20 minutes to obtain a homogeneous emulsion. Add 0.4 parts by weight of calcium carbonate, 0.3 parts by weight of sodium fluoride, 4 parts by weight of collagen, and 10 parts by weight of ultrapure water to the homogeneous emulsion, stir until a homogeneous colloidal liquid is formed to obtain fluorine-containing collagen, enabling the injectable composite hydroxyapatite filler to release fluoride ions during tooth repair. The fluoride ions diffuse to the tooth surface, which can not only reconstruct the demineralized enamel, promote bone tissue regeneration, but also form a fluorapatite protective layer to prevent further tooth demineralization, thereby protecting the deep dental tissue and achieving the prevention and treatment of dental caries lesions.

[0059] S2: Preparation of unidirectional nano-hydroxyapatite composite material

[0060] S2.1: Mix the fluorine-containing collagen and phosphate buffer solution to prepare a mixed solution with a fluorine-containing collagen content of 10 mg / ml and a phosphoric acid concentration of 0.08 mol / L. Mix the mixed solution and a 0.2 mol / L calcium chloride dihydrate solution in a volume ratio of 1:6 and place them in a polytetrafluoroethylene container. The base of the polytetrafluoroethylene container is made of Cu material, and then slowly add NaOH solution to adjust the pH to 7.8 to obtain a pre-frozen solution;

[0061] S2.2: After placing the pre-frozen solution in the polytetrafluoroethylene container, put it into a magnetizer, adjust the magnetic field direction from bottom to top, and the magnetic field strength is 8 T. Let it stand for 2 hours to initially form orderly arranged collagen fibers and hydroxyapatite crystals. Then immerse the polytetrafluoroethylene container containing the pre-frozen solution in liquid nitrogen, with the liquid nitrogen submerging 1 / 5 of the polytetrafluoroethylene container. The liquid nitrogen serves as a unidirectional cold source to promote the unidirectional growth of ice crystals in the pre-frozen solution from bottom to top. Quickly place the polytetrafluoroethylene container in the liquid nitrogen into the magnetizer, adjust the magnetic field direction from bottom to top, and the magnetic field strength is 8 T until the pre-frozen solution in the polytetrafluoroethylene container is completely frozen solid, enabling the collagen fibers and hydroxyapatite in the fluorine-containing collagen to better align along the ice crystal growth direction, significantly reducing the intermolecular spacing, and obtaining a freeze-dried solid;

[0062] S2.3: Place the freeze-dried solid in an ammonium persulfate solution with a mass fraction of 0.8%, and let it stand for 4 hours. Ammonium persulfate acts as an initiator to polymerize acrylamide to form polyacrylamide, which then entangles with the hydrogen bonds in collagen to obtain a cross-linked network structure, enabling the formed nano-hydroxyapatite to be more uniformly dispersed in the unidirectional nano-hydroxyapatite composite material. After filtration, the obtained filtrate is placed in a semi-permeable membrane bag and soaked in distilled water for 6 days, then placed in a vacuum freeze dryer for freeze-drying dehydration, and then isostatically pressed at a pressure of 180 MPa to obtain the unidirectional nano-hydroxyapatite composite material.

[0063] S3: Preparation of the anti-inflammatory extract solution

[0064] S3.1: Put the ginkgo leaves in a casserole and stir-fry for 15 minutes, then place them in a pulverizer together with passionflower and tea polyphenols in a mass ratio of 1:1:0.2 to obtain plant powder;

[0065] S3.2: Add the plant powder and deionized water in a mass ratio of 1:8 to an ultrasonic water bath, adjust the ultrasonic frequency to 20 KHz and the temperature to 85 °C, perform ultrasonic treatment for 25 minutes, then filter to remove the filter residue, and concentrate the filtrate in a concentrator to 1 / 4 of the original volume to obtain the anti-inflammatory extract solution, which is added to the preparation of the injectable composite hydroxyapatite filler, enabling the injectable composite hydroxyapatite filler to prevent and treat dental tissue inflammation and swelling during the repair of dental bone defects, and enhancing its safety and practicality.

[0066] S4: Mixing preparation and treatment of the injectable hydrogel

[0067] S4.1: Add 10 parts by weight of sodium hyaluronate to 60 parts by weight of deionized water, stir evenly at a stirring speed of 450 rpm, add 0.3 parts by weight of anesthetic and 5 parts by weight of the unidirectional cross-linked hydroxyapatite composite material, then stir evenly at a stirring speed of 800 rpm, and finally add the anti-inflammatory extract solution and stir evenly at a stirring speed of 200 rpm to obtain the injectable hydrogel;

[0068] S4.2: After filling the injectable hydrogel, place it in a moist heat sterilizer, adjust the temperature to 121 °C, and sterilize for 20 minutes to obtain the injectable hydroxyapatite filler.

[0069] Example 3

[0070] A preparation method of an injectable composite hydroxyapatite filler, as Figure 1 shown, includes the following steps:

[0071] S1: Preparation of fluorine-containing collagen

[0072] S1.1: Mix 4 parts by weight of ethylene glycol and 8 parts by weight of ultrapure water in a container, add 1 part by weight of polyvinylpyrrolidone, heat in a water bath at 90 °C for 15 minutes, cool to room temperature, then add 2 parts by weight of acrylamide, stir until the acrylamide is completely dissolved, and then add 0.1 part by weight of Tween 80 and stir evenly to obtain a reaction solution;

[0073] S1.2: Place the reaction solution in an ultrasonic crusher and perform ultrasonic treatment at a power of 350 W for 25 minutes to obtain a homogeneous emulsion. Add 0.3 part by weight of calcium carbonate, 0.2 part by weight of sodium fluoride, 3 parts by weight of collagen, and 8 parts by weight of ultrapure water to the homogeneous emulsion, and stir until a homogeneous colloidal liquid is formed to obtain fluorine-containing collagen, enabling the injectable composite hydroxyapatite filler to release fluoride ions during tooth repair. The fluoride ions diffuse to the tooth surface, which can not only reconstruct the demineralized enamel, promote bone tissue regeneration, but also form a fluorapatite protective layer to prevent further tooth demineralization, thereby protecting the deep dental tissue and achieving the prevention and treatment of dental caries lesions.

[0074] S2: Preparation of unidirectional nano-hydroxyapatite composite material

[0075] S2.1: Mix the fluorine-containing collagen and phosphate buffer solution to prepare a mixed solution with a fluorine-containing collagen content of 8 mg / ml and a phosphoric acid concentration of 0.06 mol / L. Mix the mixed solution and a 0.1 mol / L calcium chloride dihydrate solution in a volume ratio of 1:6 and place them in a polytetrafluoroethylene container. The base of the polytetrafluoroethylene container is made of Cu material, and then slowly add NaOH solution to adjust the pH to 8.2 to obtain a pre-frozen solution;

[0076] S2.2: After placing the pre-frozen solution in the polytetrafluoroethylene container, put it into a magnetizer, adjust the magnetic field direction from bottom to top, with a magnetic field strength of 10 T, and let it stand for 3 hours to initially form orderly arranged collagen fibers and hydroxyapatite crystals. Then immerse the polytetrafluoroethylene container containing the pre-frozen solution in liquid nitrogen, with the liquid nitrogen submerging 1 / 4 of the polytetrafluoroethylene container. The liquid nitrogen serves as a unidirectional cold source to promote the unidirectional growth of ice crystals in the pre-frozen solution from bottom to top. Quickly place the polytetrafluoroethylene container in the liquid nitrogen into the magnetizer, adjust the magnetic field direction from bottom to top, with a magnetic field strength of 10 T, until the pre-frozen solution in the polytetrafluoroethylene container is completely frozen solid, enabling the collagen fibers and hydroxyapatite in the fluorine-containing collagen to better align along the ice crystal growth direction, significantly reducing the intermolecular spacing, and obtaining a freeze-dried solid;

[0077] S2.3: Place the freeze-dried solid in an ammonium persulfate solution with a mass fraction of 1%, let it stand for 5 hours. Ammonium persulfate acts as an initiator to polymerize acrylamide to form polyacrylamide, which then entangles with the hydrogen bonds in collagen to obtain a cross-linked network structure, enabling the formed nano-hydroxyapatite to be more evenly dispersed in the unidirectional nano-hydroxyapatite composite material. After filtration, put the obtained filtrate into a semi-permeable membrane bag and soak it in distilled water for 7 days, then place it in a vacuum freeze dryer for freeze-drying and dehydration, and then perform isostatic pressing at a pressure of 200 MPa to obtain the unidirectional nano-hydroxyapatite composite material.

[0078] S3: Preparation of the anti-inflammatory extract solution

[0079] S3.1: Put ginkgo leaves in a casserole and stir-fry for 20 minutes, then place them in a pulverizer together with passionflower and tea polyphenols in a mass ratio of 1:0.8:0.1 to obtain plant powder.

[0080] S3.2: Add the plant powder and deionized water in a mass ratio of 1:6 to an ultrasonic water bath, adjust the ultrasonic frequency to 25KHz and the temperature to 90°C, perform ultrasonic treatment for 30 minutes, then filter to remove the residue, and concentrate the filtrate in a concentrator to 1 / 3 of the original volume to obtain the anti-inflammatory extract solution, which is added to the preparation of the injectable composite hydroxyapatite filler, enabling the injectable composite hydroxyapatite filler to prevent and treat dental tissue inflammation and swelling during the repair of dental bone defects, and enhancing its safety and practicality.

[0081] S4: Mixing preparation and treatment of the injectable hydrogel

[0082] S4.1: Add 8 parts by weight of sodium hyaluronate to 50 parts by weight of deionized water, stir evenly at a stirring speed of 500 rpm, add 0.2 parts by weight of anesthetic and 4 parts by weight of the unidirectional cross-linked hydroxyapatite composite material, then stir evenly at a stirring speed of 900 rpm, and finally add the anti-inflammatory extract solution and stir evenly at a stirring speed of 250 rpm to obtain the injectable hydrogel.

[0083] S4.2: After filling the injectable hydrogel, place it in a moist heat sterilizer, adjust the temperature to 123°C, and sterilize for 15 minutes to obtain the injectable hydroxyapatite filler.

[0084] Comparative Example 1

[0085] Compared with Example 1, the difference in Comparative Example 1 is that in step S2.2, the pre-frozen solution was not placed in a strong magnetic field, but directly allowed to stand for 3 hours and then subjected to unidirectional freeze crystallization with liquid nitrogen. The other steps are the same as in Example 1. The prepared injectable hydroxyapatite filler is denoted as Comparative Example 1.

[0086] Comparative Example 2

[0087] Compared with Example 1, the difference in Comparative Example 2 is that in Comparative Example 2, the pre-frozen solution was not subjected to unidirectional freeze crystallization with liquid nitrogen in step S2.2, but was directly placed in a strong magnetic field and allowed to stand for 6 hours. The remaining steps were the same as those in Example 1. The prepared hydroxyapatite filler for injection was designated as Comparative Example 2.

[0088] Comparative Example 3

[0089] Compared with Example 1, the difference in Comparative Example 3 is that in Comparative Example 3, acrylamide was not added to the reaction solution in step S1.1. The remaining steps were the same as those in Example 1. The prepared hydroxyapatite filler for injection was designated as Comparative Example 3.

[0090] Take 100 g of the hydroxyapatite filler for injection prepared in the example, 100 g of Comparative Example 1, and 100 g of Comparative Example 2, then soak them separately in PBS solution and place them in an incubator at 37°C. Take them out after 6 h, 18 h, and 30 h, dip off the moisture on the surface of the material with filter paper, and then use an analytical balance to test and record their mass W respectively. Calculate the swelling ratio according to the formula swelling ratio = (W - 100) / 100, record the data and make a table as shown in Table 1. It can be seen that the swelling ratios of the hydroxyapatite filler for injection prepared in the example are all smaller than those of Comparative Example 1 and Comparative Example 2, which can prove that subjecting the pre-frozen solution to both unidirectional freeze crystallization and strong magnetic field treatment in the example can enhance the anti-swelling performance of the hydroxyapatite filler for injection.

[0091] Swelling ratio 6h 18h 30h Example 1 0.46 0.49 0.53 Example 2 0.45 0.48 0.51 Example 3 0.48 0.51 0.55 Comparative Example 1 0.91 1.45 1.49 Comparative Example 2 0.78 1.03 1.08

[0092] Table 1

[0093] Take three weight parts each of the hydroxyapatite filler for injection prepared in the example, Comparative Example 1, Comparative Example 2, and Comparative Example 3, and use a microcomputer-controlled electronic universal testing machine equipped with a 100 N sensor to conduct compression tests respectively. The compression displacement rate is 1 mm / min. Record the data and make a table as shown in Table 2. It can be seen that the compression strengths of the hydroxyapatite filler for injection prepared in the example are all greater than those of Comparative Example 1, Comparative Example 2, and Comparative Example 3, which can prove that subjecting the pre-frozen solution to both unidirectional freeze crystallization and strong magnetic field treatment in the example can enhance the anti-compression performance of the hydroxyapatite filler for injection. At the same time, it can be proved that introducing acrylamide into the fluorine-containing collagen and cooperating with the initiation cross-linking of ammonium persulfate can further enhance the anti-compression performance of the hydroxyapatite filler for injection.

[0094]

[0095] Table 2

[0096] The above embodiments are only illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A preparation method of a composite hydroxyapatite filler for injection, characterized in that, It includes the following steps: S1: Preparation of fluorinated collagen Mix ethylene glycol and ultrapure water, add polyvinylpyrrolidone, heat in a water bath and cool, then add acrylamide and stir, and then add Tween 80 and stir evenly to obtain a reaction solution. After ultrasonic treatment of the reaction solution, a homogeneous emulsion is obtained. Add calcium carbonate, sodium fluoride, collagen and ultrapure water to the homogeneous emulsion and stir to obtain fluorinated collagen; S2: Preparation of unidirectional nano-hydroxyapatite composite Prepare a mixed solution of fluorinated collagen and phosphate buffer solution. Mix the mixed solution and calcium chloride dihydrate solution, and then slowly add NaOH solution to adjust the pH to obtain a pre-frozen solution. First, place the pre-frozen solution in a unidirectional strong magnetic field for reaction, and then place it in a unidirectional strong magnetic field while unidirectionally freezing until the pre-frozen solution is completely frozen to obtain a freeze-dried solid. Immerse the freeze-dried solid in ammonium persulfate solution, filter, perform dialysis with deionized water, and then perform freeze-drying dehydration and isostatic pressing to obtain a unidirectional nano-hydroxyapatite composite; S3: Preparation of anti-inflammatory extract solution Stir-fry ginkgo leaves and then crush them together with passion fruit and tea polyphenols to obtain plant powder. Mix the plant powder with deionized water and perform ultrasonic treatment. After filtration, concentrate the filtrate to obtain an anti-inflammatory extract solution; S4: Mixing preparation and treatment of injectable hydrogel Add sodium hyaluronate to deionized water and stir evenly. Add anesthetic and unidirectionally crosslinked hydroxyapatite composite, and then stir evenly. Finally, add the anti-inflammatory extract solution and stir evenly to obtain an injectable hydrogel. Fill the injectable hydrogel and perform moist heat sterilization to obtain an injectable hydroxyapatite filler.

2. The preparation method of a composite hydroxyapatite filler for injection according to claim 1, wherein, The preparation of fluorinated collagen in step S1 includes the following steps: S1.1: Mix 4-5 parts by weight of ethylene glycol and 8-10 parts by weight of ultrapure water in a container, add 1-2 parts by weight of polyvinylpyrrolidone, heat in a water bath at 85-90 °C for 10-15 minutes, cool to room temperature, add 2-3 parts by weight of acrylamide, stir until the acrylamide is completely dissolved, and then add 0.1-0.2 parts by weight of Tween 80 and stir evenly to obtain a reaction solution; S1.2: Place the reaction solution in an ultrasonic crusher and perform ultrasonic treatment at a power of 300-350 W for 20-25 minutes to obtain a homogeneous emulsion. Add 0.3-0.4 parts by weight of calcium carbonate, 0.2-0.3 parts by weight of sodium fluoride, 3-4 parts by weight of collagen and 8-10 parts by weight of ultrapure water to the homogeneous emulsion, and stir until a homogeneous colloidal liquid is formed to obtain fluorinated collagen.

3. The preparation method of a composite hydroxyapatite filler for injection according to claim 2, characterized in that, The preparation of unidirectional nano-hydroxyapatite composite in step S2 includes the following steps: S2.1: Mix fluorinated collagen and phosphate buffer solution to prepare a mixed solution with a fluorinated collagen content of 8-10 mg / mL and a phosphoric acid concentration of 0.06-0.08 mol / L. Mix the mixed solution and calcium chloride dihydrate solution in a volume ratio of 1:(6-8) in a polytetrafluoroethylene container, and then slowly add NaOH solution to adjust the pH to 7.8-8.2 to obtain a pre-frozen solution; S2.2: Place the polytetrafluoroethylene container filled with the pre-frozen solution into a magnetizer, adjust the magnetic field direction from bottom to top, with the magnetic field strength being 8 - 10 T, let it stand for 2 - 3 hours, then immerse the polytetrafluoroethylene container filled with the pre-frozen solution in liquid nitrogen, with liquid nitrogen submerging 1 / 5 - 1 / 4 of the polytetrafluoroethylene container. Liquid nitrogen serves as a unidirectional cold source to promote the unidirectional growth of ice crystals in the pre-frozen solution from bottom to top. Quickly place the polytetrafluoroethylene container in the liquid nitrogen into the magnetizer, adjust the magnetic field direction from bottom to top, with the magnetic field strength being 8 - 10 T, until the pre-frozen solution in the polytetrafluoroethylene container is completely frozen solid to obtain a freeze-dried solid; S2.3: Place the freeze-dried solid in an ammonium persulfate solution with a mass fraction of 0.8 - 1%, let it stand for 4 - 5 hours, filter the obtained filtrate, perform dialysis with deionized water, then place it in a vacuum freeze dryer for freeze-drying dehydration, and then perform isostatic pressing at a pressure of 180 - 200 MPa to obtain a unidirectional nano-hydroxyapatite composite material.

4. The preparation method of a composite hydroxyapatite filler for injection according to claim 3, characterized in that, Step S3 Preparation of the anti-inflammatory extract solution, including the following steps: S3.1: Stir-fry ginkgo leaves in a casserole for 15 - 20 minutes, then place them together with passionflower and tea polyphenols in a pulverizer at a mass ratio of 1: (0.8 - 1):(0.1 - 0.2) for pulverization to obtain plant powder; S3.2: Add the plant powder and deionized water at a mass ratio of 1:(6 - 8) to an ultrasonic water bath, adjust the ultrasonic frequency to 20 - 25 KHz, the temperature to 85 - 90 °C, perform ultrasonic treatment for 25 - 30 minutes, then filter to remove the filter residue, and concentrate the filtrate in a concentrator to 1 / 4 - 1 / 3 of the original volume to obtain the anti-inflammatory extract solution.

5. The preparation method of a composite hydroxyapatite filler for injection according to claim 4, characterized in that, Step S4 Mixing preparation and treatment of the injectable hydrogel, including the following steps: S4.1: Add 8 - 10 parts by weight of sodium hyaluronate to 50 - 60 parts by weight of deionized water, stir evenly at a stirring speed of 450 - 500 rpm, add 0.2 - 0.3 parts by weight of anesthetic and 4 - 5 parts by weight of the unidirectional cross-linked hydroxyapatite composite material, then stir evenly at a stirring speed of 800 - 900 rpm, and finally add the anti-inflammatory extract solution and stir evenly at a stirring speed of 200 - 250 rpm to obtain the injectable hydrogel; S4.2: After filling the injectable hydrogel, place it in a moist heat sterilizer, adjust the temperature to 121 - 123 °C, and sterilize for 15 - 20 minutes to obtain the injectable hydroxyapatite filler.

6. The preparation method of a composite hydroxyapatite filler for injection according to claim 2, characterized in that, The collagen in step S1.2 is type I collagen from bovine Achilles tendon.

7. The preparation method of a composite hydroxyapatite filler for injection according to claim 3, characterized in that, The concentration of the calcium chloride dihydrate solution in step S2.1 is 0.1 - 0.2 mol / L.

8. The preparation method of a composite hydroxyapatite filler for injection according to claim 3, characterized in that, The base of the polytetrafluoroethylene container in step S2.1 is made of Cu material.

9. The preparation method of a composite hydroxyapatite filler for injection according to claim 3, characterized in that, The method of deionized water dialysis in step S2.3 is to put the obtained filtrate into a semi-permeable membrane bag and immerse it in distilled water for 6 - 7 days.

10. The preparation method of a composite hydroxyapatite filler for injection according to claim 5, characterized in that, The anesthetic in S4.1 is lidocaine hydrochloride.

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