Hydroxyapatite-collagen microspheres for oral repair and preparation method thereof

Abstract

The present invention relates to the field of bone material filling, a method for preparing hydroxyapatite-collagen microspheres for oral repair, including: selecting hydroxyapatite (HA) particles; surface treating the selected hydroxyapatite (HA); stirring hydroxyapatite (HA) and collagen solution to form a first mixed solution; adding a photoinitiator to the first mixed solution and mixing it with the first mixed solution and stirring it into a second mixed solution; using microfluidics to prepare microspheres, the second mixed solution can be used as a dispersed phase, and the continuous phase uses an oil phase; the dispersed phase flow rate is 0.8-2ul / min; the continuous phase flow rate is 8-15ul / min, and microspheres are made; the prepared microspheres are exposed to ultraviolet rays, and a photocuring reaction is initiated by a photoinitiator to crosslink collagen to form stable gel microspheres; the microspheres are cleaned and dried to improve their mechanical properties and biological activity, and the prepared microspheres are packaged for use. The filler obtained by this method has strong biocompatibility, mechanical properties and biological activity.

Description

Technical Field

[0001] The invention relates to the field of bone material filling, and in particular to hydroxyapatite-collagen microspheres for oral repair and a preparation method thereof. Background Art

[0002] Hydroxyapatite (HAP or HA for short), also known as hydroxyapatite, or basic calcium phosphate, has a chemical formula of Ca 10 (PO 4 ) 6 (OH) 2 Hydroxyapatite has the same chemical composition as natural bone inorganic matter, has good biocompatibility and bioactivity, can participate in the body's metabolism, has a stimulating or inducing effect on bone hyperplasia, can promote the growth of bone tissue and the repair of defective tissue, and has good bone conductivity and adsorption properties. Therefore, hydroxyapatite, as a widely studied inorganic non-metallic biomaterial, can be used as a bone substitute, calcium supplement and plastic surgery material, and is widely used in the field of oral restoration.

[0003] However, the hydroxyapatite material itself has the disadvantages of slow degradation, high brittleness and difficulty in forming, which limits its wide application in oral restoration. There are many technologies that attempt to improve its performance by compounding hydroxyapatite with natural or synthetic polymer materials. For example, the authorization announcement number is CN116036372B "A method for preparing a bone filler for oral cavity", which obtains a bone filler through the steps of raw material selection - dissolving collagen - preparing a cross-linking agent - freeze-drying solid particles. However, the biocompatibility of the bone material filler obtained by this method is general, and the mechanical properties and biological activity are poor, and there is still a lot of room for improvement.

[0004] Therefore, it is necessary to provide a hydroxyapatite-collagen microsphere for oral restoration and a preparation method thereof to solve the above technical problems. Summary of the invention

[0005] In order to overcome the problems of general biocompatibility, poor mechanical properties and bioactivity of the above-mentioned existing bone material fillers, the present invention provides hydroxyapatite-collagen microspheres for oral restoration and a preparation method thereof.

[0006] To solve the above technical problems, the embodiments of the present invention provide the following technical solutions:

[0007] A method for preparing hydroxyapatite-collagen microspheres for oral restoration comprises the following steps:

[0008] (1) Selecting hydroxyapatite (HA) particles with a particle size of 100-300 μm, wherein the hydroxyapatite (HA) is derived from animal bones or is artificially synthesized;

[0009] (2) silanizing the selected hydroxyapatite (HA) with a silane coupling agent, or chemically adsorbing or physically embedding the hydroxyapatite (HA) with dopamine, or physically adsorbing or chemically coupling with polyethylene glycol to perform a first stirring to surface treat the hydroxyapatite (HA);

[0010] (3) The surface-treated hydroxyapatite (HA) and the collagen solution are stirred and evenly mixed to form a first mixed solution: the concentration of the hydroxyapatite (HA) is 7wt%-12wt%; the collagen is animal-derived or recombinant collagen; the collagen is dissolved in a phosphate buffer solution to a concentration of 1%-3%; the first mixed solution is stirred for a second time using a homogenizer at 2-8°C;

[0011] (4) using a microfluidic device to prepare the first mixed solution into microspheres: adding a photoinitiator to the first mixed solution formed by mixing hydroxyapatite (HA) and collagen, mixing with the first mixed solution and stirring evenly to form a second mixed solution; the initiator is benzoin, 2-hydroxybenzophenone, -methylbenzoin, 2,2-dimethyl-1-[4-(1-methacryloyloxy)phenyl]-1,2-diphenyl-1-butanone (Irgacure651), 2,2-dimethyl-1-[4-(1-isoacryloyloxy)phenyl]-1,2-diphenyl-1-butanone (Irgacure819); the concentration of the initiator is 1%-3%; microfluidics is used to prepare microspheres, the second mixed liquid can be used as a dispersed phase, the continuous phase uses an oil phase, and the oil phase is one of paraffin oil, sunflower oil, olive oil, and methyl silicone oil; the dispersed phase flow rate is 0.8-2ul / min; the continuous phase flow rate is 8-15ul / min, and the microspheres are prepared; the prepared microspheres are exposed to ultraviolet rays, and the photoinitiator is used to initiate a photocuring reaction to cross-link the collagen to form stable gel microspheres;

[0012] (5) Processing the stabilized microspheres: washing and drying the microspheres to improve their mechanical properties and biological activity, and packaging the prepared microspheres, such as filling them into pre-filled filling needles for use.

[0013] As a further solution of the present invention: the surface treatment adopts a silane coupling agent.

[0014] As a further solution of the present invention: the concentration of the silane coupling agent is 1-5%.

[0015] As a further solution of the present invention: the first stirring time is 2-8h.

[0016] As a further solution of the present invention: the second stirring time is 4-6h.

[0017] As a further solution of the present invention: the first mixed solution is stirred for a second time using a homogenizer at 1200 rpm.

[0018] As a further embodiment of the present invention: the collagen is animal-derived type I collagen.

[0019] A hydroxyapatite-collagen microsphere for oral restoration is prepared by any of the above-mentioned preparation methods of the hydroxyapatite-collagen microsphere for oral restoration.

[0020] As a further solution of the present invention: the hydroxyapatite-collagen microspheres for oral repair are used in the preparation of medicinal products or medical devices for repairing surgical bone defects.

[0021] The present invention relates to a hydroxyapatite-collagen microsphere for oral repair and a preparation method thereof, and its beneficial effects:

[0022] Select hydroxyapatite of appropriate size and perform surface treatment on it, control the concentration of hydroxyapatite (HA) and collagen and control the time to stir the two into a first mixed solution, control the concentration of initiator and add it to the first mixed solution to stir into a second mixed solution, and use microfluidics to prepare microspheres by controlling the dispersed phase flow rate and ultraviolet exposure. The biocompatibility of microsphere particles can be improved. Cleaning and drying the microspheres can improve the mechanical properties and biological activity of the microsphere particles. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the present invention more clear, the following is a further detailed description of the hydroxyapatite-collagen microspheres for oral restoration and the preparation method thereof in combination with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0024] A method for preparing hydroxyapatite-collagen microspheres for oral restoration comprises the following steps:

[0025] (1) selecting hydroxyapatite (HA) particles with a particle size of 100-300 μm, wherein the hydroxyapatite (HA) is derived from animal bones or is artificially synthesized, and has an irregular shape or a spherical shape, preferably derived from animal bones, so as to maintain a natural porous structure, and repeatedly washing the selected hydroxyapatite (HA) particles with water;

[0026] (2) Silane coupling agent is used to silanize the selected hydroxyapatite (HA), or dopamine is used to chemically adsorb or physically embed the hydroxyapatite (HA), or polyethylene glycol is used to physically adsorb or chemically couple the hydroxyapatite (HA) to perform surface treatment, so as to improve the compatibility of the hydroxyapatite (HA) with collagen. Preferably, the surface treatment is performed using a silane coupling agent, the concentration of which is 1-5%, and the first stirring time of the reaction is 2-8 hours;

[0027] (3) The surface-treated hydroxyapatite (HA) and the collagen solution are stirred and evenly mixed to form a first mixed solution: the concentration of the hydroxyapatite (HA) is 7wt%-12wt%; the collagen is animal-derived or recombinant collagen, preferably animal-derived type I collagen; the collagen is dissolved in a phosphate buffer to a concentration of 1%-3%; the first mixed solution is stirred for a second time at 1200 rpm using a homogenizer at 2-8°C, and the second stirring time is 4-6h;

[0028] (4) using a microfluidic device to prepare the first mixed solution into microspheres: adding a photoinitiator to the first mixed solution of hydroxyapatite (HA) and collagen, mixing with the first mixed solution and stirring evenly to form a second mixed solution; the initiator is benzoin, 2-hydroxybenzophenone, -methylbenzoin, 2,2-dimethyl-1-[4-(1-methacryloyloxy)phenyl]-1,2-diphenyl-1-butanone (Irgacure 651), 2,2-dimethyl-1-[4-(1-isoacryloyloxy)phenyl]-1,2-diphenyl-1-butanone (Irgacure 651), e819), preferably benzoin; the concentration of the initiator is 1%-3%; microfluidics is used to prepare microspheres, the second mixed liquid can be used as a dispersed phase, the continuous phase uses an oil phase, the oil phase is one of paraffin oil, sunflower oil, olive oil, and methyl silicone oil, preferably methyl silicone oil; the dispersed phase flow rate is 0.8-2ul / min; the continuous phase flow rate is 8-15ul / min, and the microspheres are prepared; the prepared microspheres are exposed to ultraviolet rays, and the photoinitiator initiates a photocuring reaction to cross-link the collagen to form stable gel microspheres;

[0029] (5) Processing the stabilized microspheres: Clean and dry the microspheres to improve their mechanical properties and biological activity, and package the prepared microspheres, such as filling them into a pre-filled filling needle for use. The microspheres prepared by microfluidic technology are similar in size, which is conducive to their wide application in oral restoration. The microspheres can be prepared into pre-filled oral restoration materials and filled into the pre-filled filling needle. When used, the friction between the particles and the tube wall can be reduced, making it easier to extrude the microspheres, and the operation is convenient.

[0030] The hydroxyapatite particles are wrapped in the collagen, which can improve the biological activity of the material.

[0031] A hydroxyapatite-collagen microsphere for oral restoration is prepared by any of the above-mentioned preparation methods of the hydroxyapatite-collagen microsphere for oral restoration.

[0032] As a further solution of the present invention: the hydroxyapatite-collagen microspheres for oral repair are used in the preparation of pharmaceutical products or medical devices for repairing periodontal bone defects and other surgical bone defects.

[0033] Table 1: Hydroxyapatite surface treatment conditions

[0034] serial number Hydroxyapatite particle size (um) APTES concentration (wt) Stirring time (h) H1 100-300um 1％ 2h H2 100-300um 1％ 8h H3 100-300um 5％ 2h H4 100-300um 5％ 8h

[0035] First embodiment

[0036] Select multiple animal bone-derived hydroxyapatite particles with a particle size of 100-300 um, repeatedly wash the hydroxyapatite particles with deionized water to remove impurities and organic matter on the surface of the hydroxyapatite particles, and dry the washed particles in an oven to remove moisture; dissolve aminopropyltriethoxysilane (APTES) in ethanol at a concentration shown in Table 1, add the dried hydroxyapatite particles to the ethanol solvent containing aminopropyltriethoxysilane (APTES), ensure that the hydroxyapatite particles are completely immersed, and stir at room temperature for a time shown in Table 1 to promote the reaction of the silane coupling agent with the hydroxyl groups on the surface of the hydroxyapatite; after the reaction is completed, wash the hydroxyapatite particles with ethanol to remove the organic solvent;

[0037] Experiment 1: CCK-8 cell activity assay to evaluate the proliferation and survival status of human mesenchymal stem cells. Blank control group: no material; Experimental group: hydroxyapatite (HA) particles in the first embodiment, a total of 4 groups;

[0038] According to the test results, after 2 days of culture, the cell proliferation of the blank control group was better than that of the experimental group. After 7 days of culture, the cell proliferation of the experimental group was better than that of the control group. There was no significant difference among the experimental groups (p=0.64), indicating that there was no difference in the hydroxyapatite particles of the experimental groups. See Table 2 for details.

[0039] Table 2: CCK8 detection results of human mesenchymal stem cells after different culture times.

[0040] sample Culture for 2 days Culture for 7 days Experimental group 1 (H1) 0.1633±0.067 0.3711±0.019 Experimental group 2 (H2) 0.1640±0.028 0.38331±0.051 Experimental group 3 (H3) 0.1627±0.079 0.3762±0.061 Experimental group 4 (H4) 0.1661±0.088 0.3811±0.049 Blank control group 0.1891±0.0043 0.3489±0.0051

[0041] Second embodiment

[0042] According to the results in the first embodiment, there is no difference in the particles of each experimental group. The porous animal bone-derived hydroxyapatite particles with a particle size of 100-300 um are selected, and the hydroxyapatite particles are repeatedly washed with deionized water to remove impurities and organic matter on the surface of the hydroxyapatite particles. The washed particles are dried in an oven to remove moisture; aminopropyltriethoxysilane (APTES) is dissolved in ethanol at a concentration of 1%, and the dried hydroxyapatite particles are added to the ethanol solvent containing aminopropyltriethoxysilane (APTES) to ensure that the hydroxyapatite particles are completely immersed, and stirred at room temperature for 2 hours to promote the reaction between the silane coupling agent and the hydroxyl groups on the surface of the hydroxyapatite; after the reaction is completed, the hydroxyapatite particles are washed with ethanol to remove the organic solvent;

[0043] The lyophilized collagen was added to a PBS buffer solution, and the mixture was stirred at 2-8°C for 8 hours at a speed of 1200 rpm, and the collagen concentration was 1-3%. The treated hydroxyapatite particles were added to the collagen solution, and the hydroxyapatite concentration was 7%-12% by weight. The mixture was stirred for 4 hours using a magnetic stirrer to obtain the first hydroxyapatite (HA)-collagen mixed solution.

[0044] Benzoin was dissolved in dimethyl sulfoxide (DMSO), and the dissolved solution was added to the first mixed solution, the final concentration of benzoin was 1.5%, and a magnetic stirrer was used to stir at room temperature for 0.5h at a speed of 300rpm to obtain a dispersed phase solution for microfluidics. The microspheres were prepared using a microfluidic device at a dispersed phase flow rate of 0.9ul / min and a dimethyl silicone oil continuous phase flow rate of 10ul / min; the microspheres were exposed to ultraviolet light with a wavelength of 365nm and cured for 5 minutes. After 10 minutes, the microspheres were taken out to form gel microspheres; the solidified microspheres were cleaned and dried, and the gel microspheres were washed with ethanol water for 3 times in turn to remove the residual dimethyl silicone oil on the gel microspheres; the washed gel microspheres were vacuum dried; the freeze dryer parameters were set as follows: pre-freezing temperature -20°C, pre-freezing cooling rate of 1°C / min, pre-freezing time of 3h, and after pre-freezing, the temperature was cooled to -40°C at a cooling rate of 2°C / min, and the freeze-drying time was 12h, and finally hydroxyapatite-collagen microspheres were obtained, as shown in Table 3 for details.

[0045] Table 3: Preparation conditions of hydroxyapatite-collagen microparticles

[0046]

[0047] Experiment 2: CCK-8 cell activity assay to evaluate the proliferation and survival of human mesenchymal stem cells. Blank control group: no material; Experiment groups 1-9: hydroxyapatite-collagen microspheres in the second embodiment; Experiment group 10: commercially available alveolar bone repair material.

[0048] According to the test results, after 2 days of culture, the cell proliferation of each experimental group was better than that of other groups. After 7 days of culture, the cell proliferation of the experimental group was better than that of the control group. There was no significant difference between experimental groups 1-9, but they were all better than experimental group 10, indicating that the microsphere particles have better biocompatibility, see Table 4 for details.

[0049] Table 4: CCK8 detection results of human mesenchymal stem cells after different culture times.

[0050]

[0051]

[0052] Experiment 3: Osteoblasts express alkaline phosphatase (ALP) during differentiation. Alkaline phosphatase (ALP) detection evaluates the differentiation of human mesenchymal stem cells during the culture process. Blank control group: no material; Experiment groups 1-9: hydroxyapatite-collagen microspheres in the second embodiment; Experiment group 10: commercially available alveolar bone repair material.

[0053] According to the test results, after 7 days of culture, the cell differentiation of the experimental group was better than that of the blank control group. After 14 days of culture, the cell differentiation of the experimental group was better than that of the control group. The experimental groups 1-9 were better than the experimental group 10, indicating that the microsphere particles can better promote the differentiation of human mesenchymal stem cells into bones, as shown in Table 5 for details.

[0054] Table 5: Alkaline phosphatase (ALP) test results of human mesenchymal stem cells after different culture time (nmol / ug / h)

[0055] sample Culture for 7 days Cultivation for 14 days Blank control group 0.1456±0.0221 0.1772±0.0256 Experimental Group 1 0.1635±0.0245 0.2458±0.0565 Experimental Group 2 0.1713±0.0354 0.2664±0.0681 Experimental Group 3 0.1778±0.0154 0.2741±0.0481 Experimental Group 4 0.1808±0.0154 0.3005±0.0713 Experimental Group 5 0.1656±0.0154 0.2521±0.0533 Experimental Group 6 0.1732±0.0154 0.2811±0.0601 Experimental Group 7 0.1648±0.0154 0.2561±0.0413 Experimental Group 8 0.1811±0.0154 0.3105±0.0511 Experimental Group 9 0.1611±0.0276 0.2430±0.0448 Experimental Group 10 0.1489±0.0223 0.2152±0.0036

[0056] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing hydroxyapatite-collagen microspheres for oral restoration, characterized in that: The following steps are involved: (1) Selecting hydroxyapatite (HA) particles with a particle size of 100-300 μm, wherein the hydroxyapatite (HA) is derived from animal bones or is artificially synthesized; (2) silanizing the selected hydroxyapatite (HA) with a silane coupling agent, or chemically adsorbing or physically embedding the hydroxyapatite (HA) with dopamine, or physically adsorbing or chemically coupling with polyethylene glycol to perform a first stirring to surface treat the hydroxyapatite (HA); (3) The surface-treated hydroxyapatite (HA) and the collagen solution are stirred and evenly mixed to form a first mixed solution: the concentration of the hydroxyapatite (HA) is 7wt%-12wt%; the collagen is animal-derived or recombinant collagen; the collagen is dissolved in a phosphate buffer solution to a concentration of 1%-3%; the first mixed solution is stirred for a second time using a homogenizer at 2-8°C; (4) using a microfluidic device to prepare the first mixed solution into microspheres: adding a photoinitiator to the first mixed solution formed by mixing hydroxyapatite (HA) and collagen, mixing with the first mixed solution and stirring evenly to form a second mixed solution; the initiator is benzoin, 2-hydroxybenzophenone, -methylbenzoin, 2,2-dimethyl-1-[4-(1-methacryloyloxy)phenyl]-1,2-diphenyl-1-butanone (Irgacure651), 2,2-dimethyl-1-[4-(1-isoacryloyloxy)phenyl]-1,2-diphenyl-1-butanone (Irgacure819); the concentration of the initiator is 1%-3%; microfluidics is used to prepare microspheres, the second mixed liquid can be used as a dispersed phase, the continuous phase uses an oil phase, and the oil phase is one of paraffin oil, sunflower oil, olive oil, and methyl silicone oil; the dispersed phase flow rate is 0.8-2ul / min; the continuous phase flow rate is 8-15ul / min, and the microspheres are prepared; the prepared microspheres are exposed to ultraviolet rays, and the photoinitiator is used to initiate a photocuring reaction to cross-link the collagen to form stable gel microspheres; (5) Processing the stabilized microspheres: washing and drying the microspheres to improve their mechanical properties and biological activity, and packaging the prepared microspheres, such as filling them into pre-filled filling needles for use.

2. The method for preparing hydroxyapatite-collagen microspheres for oral restoration according to claim 1, characterized in that: The surface treatment adopts a silane coupling agent.

3. The method for preparing hydroxyapatite-collagen microspheres for oral restoration according to claim 2, characterized in that: The concentration of the silane coupling agent is 1-5%.

4. The method for preparing hydroxyapatite-collagen microspheres for oral restoration according to any one of claims 1 to 3, characterized in that: The first stirring time is 2-8 hours.

5. The method for preparing hydroxyapatite-collagen microspheres for oral restoration according to claim 1, characterized in that: The second stirring time is 4-6h.

6. The method for preparing hydroxyapatite-collagen microspheres for oral restoration according to claim 1, characterized in that: The first mixed solution was stirred for the second time using a homogenizer at 1200 rpm.

7. The method for preparing hydroxyapatite-collagen microspheres for oral restoration according to claim 1, characterized in that: The collagen is animal-derived type I collagen.

8. A hydroxyapatite-collagen microsphere for oral restoration, characterized in that: The hydroxyapatite-collagen microspheres for oral restoration are prepared by the preparation method of any one of claims 1-7.

9. Use of the hydroxyapatite-collagen microspheres for oral repair according to claim 8 in the preparation of medicinal products or medical devices for repairing surgical bone defects.

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