A biomimetic enamel composite material, preparation method and application

Abstract

The present invention discloses a biomimetic enamel composite material, which is a composite of ceramic hydroxyapatite nanowires and a polymer organic polymer assembled in an oriented manner; the biomimetic enamel composite material has a multi-level, multi-scale, and multi-component overall orientation structure; the hardness of the biomimetic enamel composite material is 2.5 - 5.5 GPa, the modulus is 40 - 60 GPa, the viscoelastic loss factor is tanδ = 0.03 - 0.04, and the viscoelastic quality factor is 1.5 - 2.5. The biomimetic enamel composite material is used as an upper restoration material for oral medical implants and is used for the integrated preparation of oral implant abutments and crowns. The present invention also discloses a preparation method of the biomimetic enamel composite material. The present invention has broad application prospects in clinical dental crowns, upper healing abutments of oral implants, repair abutments, and abutment integrated crowns in oral medicine.

Description

Technical Field

[0001] The present invention belongs to the field of dental implant materials, and relates to a biomimetic enamel composite material, a preparation method and an application thereof. Background Art

[0002] Implant restoration is an important means for the treatment of tooth loss. According to statistics, the demand for dental implants in China is as high as 40 million per year, and oral and maxillofacial health is restored through implant restoration. During the process of implant restoration of tooth loss, the upper part of the implant (abutment, crown) is the key to the success or failure of implant restoration. At present, the commonly used abutment materials in clinics are titanium and titanium alloy materials, and the commonly used crown restoration materials are zirconia, alumina or lithium disilicate all-ceramic materials. Metal materials are not conducive to aesthetic restoration of teeth. All-ceramic materials have high elastic modulus, high hardness, high brittleness and low viscoelasticity. Their mechanical properties have poor compatibility with natural teeth, and there is a risk of wear and fracture of the opposing natural teeth. Moreover, their structural compositions mostly focus on a single scale and a single component, resulting in insufficient comprehensive properties of the materials, a functional gap with natural teeth, and the inability to meet the requirements of an integrated restoration of the abutment + crown with one material. The traditional method for making crowns is to process the abutment and the crown independently after an integrated design and then bond them together, which may lead to common clinical complications such as insufficient space at the bonding interface, crown detachment, crown or abutment fracture, and adhesive overflow. In addition, for patients with limited implant restoration space, separate restoration of the two often requires a larger occlusal space and is difficult to complete. Therefore, it is difficult to achieve true biomimesis in the upper part of the implant restoration, which is not conducive to the long-term effect of the upper part of the implant restoration, and in severe cases, it may even ultimately lead to implant failure. In summary, it is of great scientific significance and engineering value to develop a functional material for the upper part of the implant restoration that is consistent with the component structure and mechanical properties of natural teeth and realizes the integrated design of the abutment and the crown. Summary of the Invention

[0003] In order to solve the deficiencies existing in the prior art, the purpose of the present invention is to provide a biomimetic enamel composite material, a preparation method and an application thereof, which are used as upper part restoration materials for dental implants and are used for the integrated preparation of dental implant abutments and crowns. The present invention prepares and synthesizes a three-dimensional ceramic hydroxyapatite nanowire / polymeric organic polymer bulk composite material with a multi-level, multi-scale and multi-component overall orientation structure, and is used for the production of upper part crowns and abutment materials of implants based on its high strength, high toughness, high hardness and high viscoelastic mechanical property effects; subsequently, taking advantage of its surface topography structure, the influence of the material surface microenvironment on soft tissue healing and sealing is explored, so as to provide a design idea and scientific basis for the "integrated" upper part implant restoration scheme of the biomimetic enamel material for crown restoration and soft tissue sealing.

[0004] In the present invention, the multi-level refers to a matrix structure based on the oriented arrangement of ceramic hydroxyapatite nanowires; a reinforcing structure based on ceramic hydroxyapatite nanowires; a crystal / amorphous interface structure based on crystalline ceramic hydroxyapatite nanowires and amorphous polymer organic polymers.

[0005] The multi-scale refers to the nano-scale design: the diameter of the ceramic hydroxyapatite nanowires is 20 - 30 nm; the crystal / amorphous interface (nano-mechanical reinforcement unit) constructed by crystalline ceramic hydroxyapatite nanowires / amorphous polymer organic polymers; the micro-scale design: the micron-length ceramic hydroxyapatite nanowires are orientationally assembled with the polymer organic polymer, and the thickness of the prepared composite film is 20 - 100 μm (micro-mechanical reinforcement unit); the macro-scale design: the composite film is modularly assembled by a hot pressing process to prepare a ceramic hydroxyapatite nanowire / polymer organic polymer composite material with a macro-scale thickness.

[0006] The multi-component refers to an inorganic hydroxyapatite component, a polymer component, and an inorganic crystal / polymer amorphous interface component.

[0007] The overall oriented structure refers to the single orientation of hydroxyapatite in the polymer matrix.

[0008] A biomimetic enamel composite material for the integrated fabrication of oral implant abutments and crowns according to the present invention, wherein the biomimetic enamel composite material contains ceramic hydroxyapatite nanowires as a crystalline reinforcing phase and a polymer organic polymer as an amorphous toughening phase: the ceramic hydroxyapatite nanowires are a kind of micro-nano ceramic nanowires, with a length of 5 - 10 μm and a diameter of 20 - 30 nm; the amorphous polymer organic polymer is polymethyl methacrylate (PMMA), polyvinyl alcohol (PVA), or silk fibroin, etc.

[0009] The hardness of the biomimetic enamel composite material for the integrated fabrication of oral implant abutments and crowns is 2.5 - 5.5 GPa, the modulus can reach 40 - 60 GPa, the viscoelastic loss factor is tanδ = 0.03 - 0.04, and the viscoelastic quality factor is 1.5 - 2.5.

[0010] The present invention also provides a preparation method for a biomimetic enamel composite material with a multi-level, multi-scale, multi-component overall oriented structure for the integrated fabrication of oral implant abutments and crowns, including the following steps:

[0011] Step 1: Preparation of ceramic hydroxyapatite nanowires: mixing calcium salts and phosphates in a strong base solution, heating, cooling, centrifuging, and freeze-drying.

[0012] Step 2. Preparation of the polymer organic polymer solution: Dissolve polyvinyl alcohol or silk fibroin in water; and / or dissolve polymethyl methacrylate in N, N-dimethylformamide or acetone to obtain a polymer organic polymer solution;

[0013] Step 3. Add the ceramic hydroxyapatite nanowires obtained in Step 1 to the polymer organic polymer solution obtained in Step 2, stir to form a suspension, pour the suspension onto a glass substrate, and brush it unidirectionally to form a composite film. Cut, stack, and hot-press the composite film to obtain the biomimetic enamel composite material.

[0014] Specifically, Step 1 further includes the following steps:

[0015] Step 1.1. Mix oleic acid, water, and ethanol and stir to obtain a mixed solution;

[0016] Step 1.2. Dissolve sodium hydroxide, calcium chloride, and sodium dihydrogen phosphate in water respectively, and drop them into the mixed solution obtained in Step 1.1 in sequence after dissolution;

[0017] Step 1.3. Heat and react, cool and then centrifuge, add the centrifuged sample to an alkaline solution, and heat and stir to react;

[0018] Step 1.4. Centrifuge the product sample obtained in Step 1.3 and freeze-dry it to obtain ceramic hydroxyapatite nanowires.

[0019] In Step 1.1, the volume ratio of oleic acid, water, and ethanol is (5 - 15):(5 - 15):(5 - 7); the stirring time is 15 - 45 minutes, and the stirring speed is 100 - 500 r / min; preferably, the volume ratio of oleic acid, water, and ethanol is 10:5:5, the stirring time is 15 minutes, and the stirring speed is 200 r / min; and / or,

[0020] In step 1.2, the mass concentration of the sodium hydroxide solution is 50 - 150 mg / mL, the mass concentration of the calcium chloride solution is 20 - 50 mg / mL, and the mass concentration of the sodium dihydrogen phosphate solution is 30 - 80 mg / mL; the dropping amount of the sodium hydroxide solution is 10 - 15 mL, the dropping amount of the calcium chloride solution is 10 - 15 mL, and the dropping amount of the sodium dihydrogen phosphate solution is 10 - 20 mL; after the dropping is completed, stir for 10 - 30 min respectively, and the stirring speed is 100 - 500 r / min; preferably, the mass concentration of the sodium hydroxide solution is 95 mg / mL, the mass concentration of the calcium chloride solution is 30 mg / mL, and the mass concentration of the sodium dihydrogen phosphate solution is 50 mg / mL; the dropping amount of the calcium chloride solution is 10 mL, the dropping amount of the sodium dihydrogen phosphate solution is 10 mL, after the dropping is completed, stir for 10 min respectively, and the stirring speed is 200 r / min; and / or,

[0021] In step 1.3, the temperature of the heating reaction is 90 - 200 °C, and the time of the heating reaction is 12 - 24 h; the alkaline solution includes a sodium hydroxide solution or a potassium hydroxide solution; the reaction temperature of the heating and stirring reaction is 50 - 90 °C, the reaction time of the heating and stirring reaction is 5 - 12 h, and the stirring speed is 100 - 500 r / min; preferably, the temperature of the heating reaction is 90 °C, and the time of the heating reaction is 12 h; the alkaline solution includes a sodium hydroxide solution; the reaction temperature of the heating and stirring reaction is 50 °C, the reaction time of the heating and stirring reaction is 5 h; the stirring speed is 200 r / min; and / or,

[0022] In step 1.4, the sample in step 1.3 is centrifuged and washed with water at a rotation speed of 5000 - 10000 r / min, washed 3 - 8 times each time, and each time for 3 - 5 minutes. The centrifuged sample is freeze-dried for 24 - 48 h and then collected. Preferably, the sample is centrifuged and washed with water at a rotation speed of 8000 r / min, washed 3 times each time, and each time for 3 minutes; the centrifuged sample is freeze-dried for 48 h and then collected.

[0023] In step two, the dissolution temperature of the polyvinyl alcohol or the silk fibroin is 25 - 50 °C; and / or, the dissolution temperature of the polymethyl methacrylate is 70 - 90 °C; preferably, the dissolution temperature of the polyvinyl alcohol is 30 °C; the dissolution temperature of the silk fibroin is 30 °C; the dissolution temperature of the polymethyl methacrylate is 90 °C; and / or,

[0024] The concentration of the polymer organic polymer solution is 20 mg / mL - 100 mg / mL; preferably, the concentration of the polyvinyl alcohol solution or the silk fibroin solution is 40 mg / mL; the concentration of the polymethyl methacrylate solution is 20 mg / mL; and / or,

[0025] In the third step, the suspension is obtained by stirring at a temperature of 20 - 50 °C, the stirring speed is 200 - 600 r / min, the stirring time is 12 - 48 h, and the concentration of the ceramic hydroxyapatite nanowires in the polymer organic polymer solution is 0.01 g / mL - 0.1 g / mL; preferably, the suspension formed by adding the ceramic hydroxyapatite nanowires to the polyvinyl alcohol solution or the silk fibroin solution is obtained by stirring at a temperature of 30 °C, the stirring speed is 200 r / min, the stirring time is 48 h, and the concentration of the ceramic hydroxyapatite nanowires in the polyvinyl alcohol solution or the silk fibroin solution is 0.04 g / mL; and / or, the suspension formed by adding the ceramic hydroxyapatite nanowires to the polymethyl methacrylate is obtained by stirring at a temperature of 25 °C, the stirring speed is 250 r / min, the stirring time is 24 h, and the final concentration of the ceramic hydroxyapatite nanowires in the polymethyl methacrylate solution is 0.05 g / mL; and / or,

[0026] The temperature of the glass substrate is 70 - 120 °C, and the speed of the one-way brush coating is 1 - 3 cm / s; preferably, when the suspension components are the polyvinyl alcohol or the silk fibroin and the ceramic hydroxyapatite nanowires, the temperature of the glass substrate is 90 °C, and the speed of the one-way brush coating is 2 cm / s; and / or, when the suspension components are the polymethyl methacrylate and the ceramic hydroxyapatite nanowires, the temperature of the glass substrate is 100 °C, and the speed of the one-way brush coating is 1 cm / s; and / or,

[0027] The cutting size of the composite film is (2 - 10) cm × (2 - 10) cm; the temperature of the hot pressing is 40 - 60 °C, the pressure of the hot pressing is 5 - 20 MPa, and the time of the hot pressing is 12 - 24 h. Preferably, the cutting size of the composite film is a 3 cm × 3 cm square; when the composite film is prepared from the polyvinyl alcohol or the silk fibroin and the ceramic hydroxyapatite nanowires, the initial hot pressing temperature is 40 °C, the hot pressing pressure is 5 MPa, and the hot pressing time is 12 h, and then it is adjusted to a hot pressing temperature of 60 °C, a hot pressing pressure of 20 MPa, and a hot pressing time of 24 h; and / or, when the composite film is prepared from the polymethyl methacrylate and the ceramic hydroxyapatite nanowires, the temperature of the hot pressing is 40 °C, the pressure of the hot pressing is 20 MPa, and the time of the hot pressing is 24 h.

[0028] In a specific embodiment of the present invention,

[0029] 1) Ceramic hydroxyapatite nanowires (crystal component synthesis), with the nanowire size ranging from 20 - 30 nm in diameter and 5 - 10 μm in length, of crystalline hydroxyapatite. The synthesis process is as follows:

[0030] 1. Pipette 5 - 15 mL of oleic acid, measure 5 - 15 mL of water and 5 - 7 mL of ethanol with a measuring cylinder and add them to a beaker. Stir at room temperature for 15 - 45 min at a rotation speed of 200 r / min;

[0031] 2. Weigh 10 - 22.5 g of sodium hydroxide and dissolve it in 150 - 200 mL of water. Stir ultrasonically until completely dissolved, then measure 15 mL of the solution with a measuring cylinder, transfer it to a funnel and start dropping. After all the dropping is completed, stir for 10 - 30 min at a rotation speed of 200 r / min;

[0032] 3. Weigh 4 - 5 g of calcium chloride and dissolve it in 100 - 200 mL of water. Stir ultrasonically until completely dissolved, then measure 10 - 15 mL of the solution with a measuring cylinder, transfer it to a funnel and start dropping. After all the dropping is completed, stir for 10 - 30 min at a rotation speed of 200 r / min;

[0033] 4. Weigh 7.5 - 16 g of sodium dihydrogen phosphate and dissolve it in 200 - 250 mL of water. Stir ultrasonically until completely dissolved, then measure 10 - 20 mL of the solution with a measuring cylinder, transfer it to a funnel and start dropping. After all the dropping is completed, stir for 10 - 30 min at a rotation speed of 200 r / min;

[0034] 5. After the stirring is completed, transfer the sample to a 100 mL hydrothermal autoclave and react at 90 - 200 °C for 12 - 24 h. After completion, let it cool naturally;

[0035] 6. Centrifuge the cooled sample, pour off the floating matter on the upper layer of the inner lining of the hydrothermal autoclave, rinse it with water and pour it into a 50 mL centrifuge tube for centrifugation. After completion, transfer the sample to a solution of sodium hydroxide or potassium hydroxide with a small amount of water and stir at 50 - 90 °C for 5 - 12 h at a rotation speed of 200 r / min;

[0036] 7. After the stirring is completed, centrifuge the sample, then transfer it to a beaker with a small amount of water, stir at room temperature, make up the volume, and freeze-dry.

[0037] 2) A preparation method of a biomimetic enamel composite material for the integrated fabrication of oral implant abutments and crowns, comprising the following steps:

[0038] 1. Dissolve the polymer PVA or silk fibroin in an aqueous solution at 25 - 50 °C; and / or dissolve PMMA in N, N - dimethylformamide at 70 - 90 °C, and stir until completely dissolved to obtain a colorless transparent solution; the concentration of the obtained polymer organic polymer is 20 mg / mL - 100 mg / mL;

[0039] 2. Take the colorless transparent solution obtained in step 1, add the pre - prepared crystalline ceramic hydroxyapatite nanowires, and stir at 20 - 50 °C until a uniform white suspension is obtained. The concentration range of the ceramic hydroxyapatite nanowires in the colorless transparent polymer organic polymer solution is 0.01 g / mL to 0.1 g / mL;

[0040] 3. Pour the white suspension obtained in step 2 onto a pre - heated glass substrate, and use a commercial brush to perform a unidirectional brushing and sliding on the glass substrate at a temperature range of 70 - 120 °C at a speed of about 1 - 3 cm / s. During the sliding process, as the high - temperature solution evaporates, a composite film is finally formed on the glass substrate;

[0041] 4. Cut the composite film obtained in step 3 to a certain size, with the size being (2 - 10) cm × (2 - 10) cm. Spray a small amount of water or N, N - dimethylformamide onto the surface of the film, stack the films, and hot - press for 12 - 24 h under the conditions of a hot - press temperature of 40 - 60 °C and a hot - press pressure of 5 - 20 MPa.

[0042] 5. Prepare a biomimetic enamel material in which the nanowires are oriented parallel to each other, forming a micro - nano - sized topological structure and a clear spatial anisotropy, similar to the crystal - amorphous structure of natural teeth, for the production of the upper abutment and dental crown of an implant.

[0043] The present invention also provides the application of the above - mentioned biomimetic enamel composite material, or the preparation method of the biomimetic enamel composite material, in the preparation of clinical dental crowns, upper healing abutments of oral implants, restoration abutments, and abutment - integrated crowns in oral medicine.

[0044] The beneficial effects of the present invention include: The present invention provides a biomimetic enamel composite material, which has the following advantages: 1) The prepared ceramic hydroxyapatite nanowires have uniform size and good crystallinity. 2) The crystalline ceramic hydroxyapatite nanowires and the amorphous polymer organic polymer are compounded to construct a crystal / amorphous interface. 3) By the method of unidirectional brushing, a large-sized composite film with a nanowire orientation structure can be prepared. 4) Through a simple and large-scale hot pressing process, the composite film is assembled into a macroscopic three-dimensional bulk material with a multi-level, multi-scale, and multi-component overall orientation structure, and is used for the integrated design of the upper abutment and crown of the implant. At the same time, large bulk materials (three-dimensional dimensions reaching the centimeter level) can be macroscopically prepared. The mechanical strength of the biomimetic enamel composite material is close to that of natural enamel, and it can be used for the upper abutment of the implant (more aesthetically pleasing and having a stronger promoting effect on soft tissue healing than the widely used metal abutments currently). BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those skilled in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0046] Figure 1 It is a morphology diagram of the crystalline hydroxyapatite nanowires in Example 1.

[0047] Figure 2 It is a schematic diagram of the unidirectional brushing process of the present invention.

[0048] Figure 3 The XRD results show that the characteristic crystal peaks of the original crystalline hydroxyapatite nanowires are retained after adding the polymer organic polymer to the crystalline hydroxyapatite nanowire material.

[0049] Figure 4 It is an optical photograph of the biomimetic enamel composite material sample obtained by the present invention.

[0050] Figure 5 It is a corresponding fluorescence staining result diagram of immunofluorescence detection after culturing fibroblasts on the surfaces of the used materials (pure titanium, non-oriented hybrid materials, and oriented biomimetic enamel materials).

[0051] Figure 6 It is the elastic modulus and hardness of the two-dimensional mechanical data of the biomimetic enamel composite material obtained in Example 2 of the present invention for the integrated production of oral implant abutments and crowns.

[0052] Figure 7The tanδ value of the two-dimensional mechanical data of the biomimetic enamel composite material for the integrated fabrication of oral implant abutments and crowns obtained in Example 2 of the present invention.

[0053] Figure 8 Scanning electron microscope photograph of the cross-section of the biomimetic enamel composite material for the integrated fabrication of oral implant abutments and crowns obtained in Example 3 of the present invention after compression.

[0054] Figure 9 The two-dimensional mechanical data of the biomimetic enamel composite material for the integrated fabrication of oral implant abutments and crowns obtained in Example 3 of the present invention.

[0055] Figure 10 The two-dimensional mechanical data of the biomimetic enamel composite material for the integrated fabrication of oral implant abutments and crowns obtained in Example 3 of the present invention.

[0056] Figure 11 The microscopic scanning electron microscope of the biomimetic enamel composite material for the integrated fabrication of oral implant abutments and crowns obtained in Example 3 of the present invention.

[0057] Figure 12 Comparison of the replication amounts of cell transcriptome RNA extracted after culturing fibroblasts with the synthetic material (oriented biomimetic enamel material) of the present invention compared to non-oriented materials and pure titanium materials.

[0058] Figure 13 Comparison of the wound healing conditions after subcutaneous implantation of the material of the present invention into mouse wounds. Detailed implementation manners

[0059] In combination with the following specific embodiments and drawings, the present invention will be further described in detail. The processes, conditions, experimental methods, etc. for implementing the present invention, except for the specifically mentioned content below, are all common knowledge and well-known common sense in the art, and the present invention has no particularly restricted content.

[0060] Next, the technical solutions in the embodiments of the present disclosure will be clearly and completely described in conjunction with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are only a part of the embodiments of the present disclosure, rather than all of the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and in no way constitutes a limitation on the present disclosure and its application or use. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present disclosure without creative efforts shall fall within the scope of protection of the present disclosure.

[0061] In all the examples shown and discussed here, any specific value should be construed as merely exemplary and not as a limitation. Therefore, other examples of the exemplary embodiments may have different values.

[0062] Characterize and detect the biomimetic enamel composite material prepared in the present invention

[0063] 1) Performance detection of the prepared biomimetic enamel composite material

[0064] 1. Prepare nanohydroxyapatite nanowires by the above method, and confirm the crystal structure through X-ray diffraction technology and transmission electron microscopy technology; characterize the surface structure and morphology of the composite material by scanning electron microscopy, and preliminarily determine the arrangement of nanowires on the surface of the composite material; determine the ordered orientation structure of nanowires inside the structure of the enamel-like composite material by Nano-CT testing; characterize the chemical bond changes after the regulation of the crystal-amorphous phase interface of the composite material by Fourier transform infrared spectroscopy and Raman spectroscopy.

[0065] 2. The synthesized crown / abutment integrated biomimetic enamel composite material needs to meet the following mechanical property requirements: the hardness of the composite material is 2.5 - 5.5 GPa, the modulus can reach 40 - 60 GPa, the viscoelastic loss factor is tanδ = 0.03 - 0.04, and the viscoelastic quality factor is 1.5 - 2.5.

[0066] 3. The layer thickness of the sample after hot pressing and compression is 0.5 - 1.5 cm. The length and width are 2 - 3 cm respectively.

[0067] 2) Biological effects of the material

[0068] Verify through in vivo experiments using the synthetic material that the material has the phenotype of promoting soft tissue in vivo when applied as an implant abutment; or confirm through in vitro experiments that the material surface has a positive effect on the high expression of related genes and proteins such as early cell adhesion and migration.

[0069] Example 1

[0070] Prepare one-dimensional hydroxyapatite nanowires for the preparation of a biomimetic enamel composite material for the integrated production of oral implant abutments and crowns. The synthesis process is as follows:

[0071] 1. Use a pipette to measure 10 mL of oleic acid, use a graduated cylinder to measure 5 mL of water and 5 mL of ethanol and add them to a beaker, and stir at room temperature for 15 min at a rotation speed of 200 r / min;

[0072] 2. Weigh 15 g of sodium hydroxide and dissolve it in 175 mL of water. Stir ultrasonically until completely dissolved, then use a graduated cylinder to measure 15 mL of the solution and transfer it to a funnel and start dropping. After all the dropping is completed, stir for 15 min at a rotation speed of 200 r / min;

[0073] 3. Weigh 2 g of calcium chloride and dissolve it in 100 mL of water. Stir ultrasonically until completely dissolved, then measure 10 mL of the solution with a measuring cylinder, transfer it to a funnel and start dropping. After all the dropping is completed, stir for 10 min at a rotation speed of 200 r / min;

[0074] 4. Weigh 10 g of sodium dihydrogen phosphate and dissolve it in 200 mL of water. Stir ultrasonically until completely dissolved, then measure 10 mL of the solution with a measuring cylinder, transfer it to a funnel and start dropping. After all the dropping is completed, stir for 10 min at a rotation speed of 200 r / min;

[0075] 5. After the stirring is completed, transfer the sample to a 100 mL hydrothermal reactor and react at 90 °C for 12 h. After completion, cool it naturally;

[0076] 6. Centrifuge the cooled sample, pour off the floating matter on the upper layer of the inner lining of the hydrothermal reactor, rinse it with water and pour it into a 50 mL centrifuge tube for centrifugation. After completion, transfer the sample to a sodium hydroxide solution with a small amount of water and stir at 50 °C for 5 h at a rotation speed of 200 r / min;

[0077] 7. After the stirring is completed, centrifuge the sample, perform centrifugal water washing at a rotation speed of 8000 r / min, wash 3 times each time, 3 minutes for each time; then transfer it to a beaker with a small amount of water and stir at room temperature at a rotation speed of 400 r / min, make up the volume, and freeze-dry for 48 h.

[0078] In the present invention, hydroxyapatite nanowires prepared in Example 1 are used. The test results show that the width of the nanowires prepared in Example 1 is 20 nm and the length is 8 μm (as Figure 1 ). Through XRD characterization, it is a nanowire with good crystallinity (as Figure 3 shown above).

[0079] Example 2

[0080] Preparation method of a biomimetic enamel composite material for integrally fabricating an oral implant abutment and a dental crown based on the directional assembly of one-dimensional hydroxyapatite nanowires and polymethyl methacrylate.

[0081] 1. Dissolve 1 g of polymethyl methacrylate in 50 mL of N, N-dimethylformamide in a water bath at 80 °C and stir for 12 h at a stirring rate of 200 revolutions per minute to obtain a uniformly mixed colorless and transparent solution with a concentration of 20 mg / mL;

[0082] 2. Take 20 mL of the solution obtained in the first step, add 0.5 g of hydroxyapatite nanowires (with a length of about 10 microns), and continuously stir in a water bath at 25 °C and a stirring rate of 250 revolutions per minute for 24 h to form a uniform white suspension;

[0083] 3. Dip a commercial brush into the 50 mg / mL suspension prepared in Step 2 and perform a unidirectional brushing and sliding on a glass substrate at a temperature of 100 °C at a speed of about 1 cm / s (as Figure 2 shown). During the sliding process, with the evaporation of the high-temperature solution, a composite film is finally formed on the glass substrate. The phase analysis of the hydroxyapatite composite PMMA can refer to Figure 3 (below). It should be particularly noted that compared with pure HA, a weak peak appears at 2θ = 30° in the composite material, which may be the peak of the amorphous polymer. Therefore, the composite material is a crystal / amorphous composite product;

[0084] 4. Cut the composite film obtained in Step 3 into a certain size, and the size after cutting is a 3 cm × 3 cm square. Then, spray a small amount of N,N-dimethylformamide onto the surface of the film, stack the films in the same direction according to the scraping direction, and hot press at 40 °C and about 20 MPa for 24 h (as Figure 4 shown);

[0085] 5. The synthetic material is used to culture fibroblasts, and the spreading of fibroblasts and the expression of related cell membrane receptors are significantly better than those of the smooth pure titanium and non-oriented material groups. (as Figure 5 )

[0086] In the present invention, quasi-static nanoindentation is used to test the biomimetic enamel composite material prepared in Example 2, and the indentation depth ranges from 100 to 600 nm. The test results show that the two-dimensional area hardness of the composite material prepared in Example 2 can reach 3 GPa, and the modulus can reach 60 GPa, which is comparable to the mechanical properties of real enamel (see Figure 6 shown), and its viscoelastic loss factor tanδ = 0.04 (as Figure 7 ). Observing its surface and cross-sectional morphologies, it is proved that it has a highly oriented structure. Without adopting the method of directional scraping, the surface morphology of the composite material shows a randomly arranged state (see Figure 8 , the left is the ordered structure and the right is the disordered structure).

[0087] Example 3

[0088] A preparation method of a biomimetic enamel composite material for the integrated production of oral implant abutments and crowns based on the directional assembly of one-dimensional hydroxyapatite nanowires and polyvinyl alcohol.

[0089] 1. Dissolve 2 g of polyvinyl alcohol in 50 mL of deionized water in a water bath at 30 °C and stir for 8 h at a stirring rate of 250 revolutions per minute to obtain a uniformly mixed colorless and transparent solution with a concentration of 40 mg / mL;

[0090] 2. Take 10 mL of the solution obtained in the first step, add 0.4 g of hydroxyapatite nanowires (with a length of about 8 microns), and continuously stir for 48 h in a water bath at 30 °C with a stirring rate of 200 revolutions per minute to form a uniform white suspension;

[0091] 3. Dip a commercial brush into the 40 mg / mL suspension prepared in the second step and perform a unidirectional brushing and sliding on a glass substrate at a temperature of 90 °C at a speed of about 2 cm / s. During the sliding process, with the evaporation process of the high-temperature solution, a composite film is finally formed on the glass substrate;

[0092] 4. Cut the composite film obtained in step 3 to a certain size, and the size after cutting is a 3 cm × 3 cm square. Then, spray a small amount of deionized water onto the surface of the film, stack the films in the same direction according to the scraping direction, and perform a preliminary hot pressing at about 5 MPa at 40 °C for 12 h. Then, adjust the heating temperature to 60 °C and continue to hot press at 20 MPa for 24 h;

[0093] 5. In the present invention, quasi-static nanoindentation is used to test the biomimetic enamel composite material prepared in Example 3, and the range of the indentation depth is 50 - 650 nm. The test results show that the hardness of the composite material prepared in Example 3 can reach 2.5 GPa, and the modulus can reach 55 GPa, which is comparable to the mechanical properties of real enamel (such as Figure 9 ), and its viscoelastic quality factor E'tanδ = 1.8 (such as Figure 10 ). Observing its cross-sectional morphology, it is proved that it has a highly oriented structure, such as Figure 11 shown.

[0094] 6. When using the material to culture fibroblasts, the visible transcriptome RNA expression is as shown in Figure 12 . Genes ITGβ1 (integrin protein 1), COL 1 (type I collagen), and VCL (vinculin) related to cell spreading and tissue healing show high expression on the surface of the oriented biomimetic enamel material (dark gray: titanium material; light gray: non-oriented material; black: oriented biomimetic enamel composite material).

[0095] 7. When different materials are implanted subcutaneously in mice, it can be seen that the oriented biomimetic enamel composite material has a better ability to promote epidermal tissue healing (such as Figure 13 ).

[0096] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this invention belongs. The terms used in the specification of this invention herein are only for the purpose of describing specific embodiments

[0097] As used in the present invention, the term "comprising" is an open-ended expression, that is, it includes the content specified in the present invention, but does not exclude other aspects of the content.

[0098] As used in the present invention, the term "about", when applied to a value, means that there is some slight imprecision in the calculation or measurement (by a certain method approaching the accuracy of the value; approximating or reasonably approaching the value; almost). If for some reason the imprecision specified by "about" is not understood in this conventional sense in the art, then "about" as used in the present invention at least represents the variation that may be caused by the conventional methods of measuring or using such parameters.

[0099] As used in the present invention, the term "and / or" includes any and all combinations of one or more of the related listed items. It is not intended to limit the present invention.

[0100] The protection scope of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept of the present invention, the changes and advantages that can be conceived by those skilled in the art are included in the present invention, and the scope of protection is defined by the appended claims.

Claims

1. A preparation method of a biomimetic enamel composite material, characterized in that, The preparation method includes the following steps: Step 1. Preparation of ceramic hydroxyapatite nanowires: A calcium salt and a phosphate are mixed, heated, cooled, centrifuged, and freeze-dried in a strong base solution; Step 1 further includes the following steps: Step 1.

1. Mix oleic acid, water, and ethanol and stir to obtain a mixed solution; Step 1.

2. Dissolve sodium hydroxide, calcium chloride, and sodium dihydrogen phosphate in water respectively, and drop them into the mixed solution obtained in Step 1.1 in sequence after dissolution; Step 1.

3. Heat for reaction, cool and then centrifuge, add the centrifuged sample to an alkaline solution, and heat and stir for reaction; Step 1.

4. Centrifuge the product sample obtained in Step 1.3 and freeze-dry to obtain the ceramic hydroxyapatite nanowires; Step 2. Preparation of a polymer organic polymer solution: Dissolve polyvinyl alcohol or silk fibroin in water; or dissolve polymethyl methacrylate in N,N-dimethylformamide or acetone to obtain a polymer organic polymer solution; Step 3. Add the ceramic hydroxyapatite nanowires obtained in Step 1 to the polymer organic polymer solution obtained in Step 2, stir to form a suspension, pour the suspension onto a glass substrate, and brush it unidirectionally to form a composite film. Cut, stack, and hot-press the composite film to obtain the biomimetic enamel composite material.

2. The preparation method according to claim 1, wherein in Step 1.1, the volume ratio of oleic acid, water, and ethanol is (5-15):(5-15):(5-7); the stirring time is 15-45 minutes; and / or, in Step 1.2, the mass concentration of the sodium hydroxide solution is 50-150 mg / mL, the mass concentration of the calcium chloride solution is 20-50 mg / mL, and the mass concentration of the sodium dihydrogen phosphate solution is 30-80 mg / mL; the dropping amount of the sodium hydroxide solution is 10-15 mL, the dropping amount of the calcium chloride solution is 10-15 mL, and the dropping amount of the sodium dihydrogen phosphate solution is 10-20 mL; after dropping, stir for 10-30 min respectively; and / or, in Step 1.3, the temperature of the heating reaction is 90-200 °C, and the time of the heating reaction is 12-24 h; the alkaline solution includes a sodium hydroxide solution or a potassium hydroxide solution; the reaction temperature of the heating and stirring reaction is 50-90 °C, and the reaction time of the heating and stirring reaction is 5-12 h; the rotation speed of the stirring speed is 100-500 r / min; and / or, in Step 1.4, the sample of Step 1.3 is centrifuged and washed with water at a rotation speed of 5000-10000 r / min, washed 3-8 times each time, and each time for 3-5 minutes; the centrifuged sample is collected after freeze-drying for 24-48 h.

3. The preparation method according to claim 1, wherein in Step 2, the dissolution temperature of polyvinyl alcohol or silk fibroin is 25-50 °C, and the dissolution temperature of polymethyl methacrylate is 70-90 °C; and / or, The concentration of the polymer organic polymer solution is 20 mg / mL - 100 mg / mL.

4. The preparation method according to claim 1, characterized in that, In the third step, the suspension is obtained by stirring at a temperature of 20 - 50 °C; the stirring speed is 200 - 600 r / min; the stirring time is 12 - 48 h; the concentration of the ceramic hydroxyapatite nanowires in the polymer organic polymer solution is 0.01 g / mL - 0.1 g / mL; and / or, The temperature of the glass substrate is 70 - 120 °C, and the speed of the one-way brushing is 1 - 3 cm / s; and / or, the temperature of the hot pressing is 40 - 60 °C, the pressure of the hot pressing is 5 - 20 MPa, and the time of the hot pressing is 12 - 24 h; and / or, The cutting size of the composite film is (2 - 10) cm × (2 - 10) cm.

5. The biomimetic enamel composite material obtained by the preparation method according to any one of claims 1-4, characterized in that, The hardness of the biomimetic enamel composite material is 2.5 - 5.5 GPa, the modulus is 40 - 60 GPa, the viscoelastic loss factor is tanδ = 0.03 - 0.04, and the viscoelastic quality factor is 1.5 - 2.

5.

6. The application of the biomimetic enamel composite material according to claim 5, or the preparation method according to any one of claims 1 - 4 in the preparation of clinical dental crowns, upper healing abutments of oral implants, repair abutments, and abutment integrated crowns in oral medicine.

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