Biological tooth root stent based on 3D printing as well as preparation method and application of biological tooth root stent

Abstract

The invention discloses a biological tooth root scaffold based on 3D printing and a preparation method and application thereof, and belongs to the technical field of biomedical materials. The biological tooth root scaffold comprises a 3D printed biological tooth root scaffold base body and CMC-BAPE at BMP7 nano-particles, the surface interface of the 3D printed biological tooth root scaffold base body is provided with an activated nano-whisker structure, and the CMC-BAPE at BMP7 nano-particles are adsorbed and fixed in the 3D printed biological tooth root scaffold base body. According to the preparation method disclosed by the invention, high-precision biological ceramic is prepared by combining 3D printing, excellent mechanical properties after high-temperature sintering are realized, surface and interface activity is reconstructed to promote cell adhesion and differentiation, and an anti-inflammatory cell differentiation promoting nano CMC-BAPE-coated BMP7 medicine is combined, so that personalized customization of the biological tooth root scaffold with high strength, high precision and immunomodulatory activity is realized; the method has a wide application prospect in the field of tooth loss repair.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and particularly relates to a 3D-printed biological tooth root support and a preparation method and application thereof. BACKGROUND

[0002] Tooth loss is a common and frequently-occurring disease. According to the Fourth National Oral Health Epidemiological Survey Report, 67.7% of people in the 35-44 age group have complete dentition in China, and the rate of tooth loss is higher in people over 45 years old. At present, the common implant denture repair after tooth loss has defects such as lack of nerve occlusion sensation, weak anti-infection ability, high maintenance cost, strict indications, etc. There is an urgent need for biological tooth repair that is similar to natural tooth structure, nerve, and nutritional function, has personalized recovery mechanical properties, and has no rejection risks, especially for congenital multiple tooth loss or post-natal caries in adolescents and children who cannot be repaired by implantation. Therefore, exploring a strategy for constructing a personalized biological tooth has great market and economic demand.

[0003] With the development of 3D printing technology and stem cell in-vivo treatment, the biological tooth root constructed by seed cells combined with personalized biological scaffold material provides a feasible solution for physiological and functional biological tooth regeneration. At present, important breakthroughs have been made in the research of 3D printing of biological tooth roots, but the mechanical properties of the biological tooth roots prepared by the current methods are poor. Although some researchers use low-temperature deposition, DLP hydrogel printing, etc. to obtain biological tooth roots with certain biological induction efficiency, the mechanical properties such as stiffness and elastic modulus of the biological tooth roots prepared by this method are poor, the precision of the tooth root support is low, and the tooth root support does not have anti-inflammatory immune regulation ability to the initial implant microenvironment, resulting in a low success rate of functional biological tooth root regeneration. Therefore, how to obtain a personalized biological tooth root support with high strength, high precision, and anti-inflammatory immune regulation has become a technical bottleneck that needs to be solved for the application of biological tooth roots in clinical demand for tooth loss. Therefore, how to prepare a personalized, high-precision, high-strength, anti-inflammatory immune regulation type biological tooth root that meets the requirements of clinical treatment has important scientific significance for the final realization of the clinical transformation of biological tooth roots. SUMMARY

[0004] The present application provides a 3D-printed biological tooth root support to solve the problems of low strength, low precision, and poor anti-inflammatory immune regulation ability in the existing low-temperature deposition, ink direct writing method, etc. The biological tooth root support combines 3D printing to prepare high-precision biological ceramics, excellent mechanical properties after high-temperature sintering, surface interface activity reconstruction to promote cell adhesion and differentiation, and anti-inflammatory drug CMC-BAPE@BMP7 to promote cell differentiation, realizes the personalized customization of a biological tooth root support with high strength, high precision, and immune regulation activity, and has a broad application prospect in the field of tooth loss repair.

[0005] The present application is realized by the following technical solutions: A 3D printing-based biological tooth root support, comprising a 3D printed biological tooth root support base body and CMC-BAPE@BMP7 nanoparticles, the 3D printed biological tooth root support base body has an activated nanowhisker structure on the surface interface, and the CMC-BAPE@BMP7 nanoparticles are adsorbed and fixed in the 3D printed biological tooth root support base body. Since the surface of the high-temperature sintered bioceramic is too smooth, it is not conducive to cell adhesion and growth, the present application modifies the nanowhisker structure on the surface interface of the sintered biological tooth root support, thereby promoting stem cell adhesion, attachment, and periodontal membrane cell differentiation. Benzene boronic acid pinacol ester (BAPE) forms CMC-BAPE amphiphilic polymer nanoparticles through a dehydrogenation condensation reaction with carboxymethyl chitosan (CMC) which has good biocompatibility, and then is compounded with recombinant human bone morphogenetic protein-7 (BMP7) to form CMC-BAPE@BMP7 nanoparticles. Through the compounding of CMC-BAPE@BMP7 nanoparticles with anti-inflammatory immune regulation, the biological tooth root support can consume active oxygen free radicals (ROS) and other inflammatory factors in the implanted microenvironment, promote the M2 differentiation of M1 macrophages, and promote the formation of local microenvironment immune homeostasis, thereby obtaining a biological tooth root with high strength, high precision, and anti-inflammatory immune regulation.

[0006] As an optional mode, in the above-mentioned 3D printing-based biological tooth root support, the 3D printed biological tooth root support base body is prepared by using a digital light processing (DLP) 3D printing technology. Based on the DLP 3D printing, corresponding technical iteration improvement is made after high-temperature debinding and sintering, which makes up for the defects of the brittle and weak resistance of the high-temperature sintered biological tooth root support in the prior art, and the biological tooth root support cannot meet the continuous functional chewing requirements of teeth.

[0007] As an optional mode, in the above-mentioned 3D printing-based biological tooth root support, the 3D printed biological tooth root support base body is prepared by adding inorganic oxides to calcium phosphate bioceramics. As an optional mode, the calcium phosphate bioceramics is at least one of hydroxyapatite (HA), tricalcium phosphate (TCP), and biphasic calcium phosphate (BCP). As an optional mode, the inorganic oxide is not only white but also can improve the mechanical properties, and the color and mechanical property requirements of the tooth root are met, and the inorganic oxide is preferably a zirconia / zinc oxide mixture or yttrium-stabilized tetragonal zirconia (Y-TZP). By adding inorganic oxide toughening materials, the toughness and strength of the biological tooth root support are improved, and the preparation of a high-strength and high-precision biological tooth root support is realized. As an option, the mass fraction of inorganic oxides in the 3D printing ink is 5%-20%. Further, the mass ratio of zirconia to zinc oxide is in the range of 1:5 to 5:1.

[0008] As an optional mode, in the above-mentioned 3D printing-based biological tooth root support, the CMC-BAPE@BMP7 nanoparticle has a particle size range of 200-400 nm, and a unit surface area deposition rate of 10-25 / um in the 3D printing-based biological tooth root support matrix. 2 .

[0009] As an optional mode, in the above-mentioned 3D printing-based biological tooth root support, the activated nanowhisker structure is an ordered nanowhisker structure arranged on the surface interface of the 3D printing-based biological tooth root support matrix. Further, the nanowhisker structure has a length of 500-700 nm and a width of 2-5 nm.

[0010] As an optional mode, in the above-mentioned 3D printing-based biological tooth root support, the support porosity is 5-10%, the surface roughness Ra is 0.4-0.6 um, the compressive strength is 30-50 Mpa, and the Young's elastic modulus is 20-30 GPa.

[0011] The application also provides a preparation method of a 3D printing-based biological tooth root support, comprising the following steps: (1) constructing a three-dimensional model of a biological tooth root; (2) preparing 3D printing ink; (3) preparing a biological tooth root support matrix by 3D printing and sintering; (4) activating the nanowhisker structure on the surface interface of the biological tooth root support matrix; (5) compounding CMC-BAPE@BMP7 nanoparticles.

[0012] As an optional mode, in the above-mentioned preparation method, the following steps are specifically included: (1) constructing a three-dimensional model of a biological tooth root: Obtaining imaging data of a missing tooth, constructing an original three-dimensional data model of the missing tooth, and obtaining a three-dimensional data model of a personalized biological tooth root through optimization of undercut structure and three-dimensional finite element analysis; (2) preparing 3D printing ink: Mixing calcium phosphate bioceramic powder, inorganic oxide toughening ceramic powder, photosensitive resin, photoinitiator and dispersant uniformly to prepare 3D printing ink. As an optional mode, the photosensitive resin is at least one of polyurethane acrylate, polyethylene glycol acrylate acryloyl morpholine, trimethylolpropane triacrylate and hyperbranched polyester acrylate; the photoinitiator is at least one of cyclohexyl vinyl ether, phenyl bis(2,4,6-trimethylbenzoyl) phosphine oxide and tripropylene glycol diacrylate; and the dispersant is at least one of castor oil phosphate, pyrrolidone and sodium polyacrylate.

[0013] (3) preparing a biological root support base body by 3D printing and sintering: According to the three-dimensional data model of the personalized biological root obtained in step (1), the 3D printing ink prepared in step (2) is used to obtain a biological root support blank body by 3D printing, and sintering is performed to obtain a biological root support base body; (4) surface and interface nano-whisker structure activation of the biological root support base body: The surface and interface of the biological root support base body obtained in step (3) are deposited with activated nano-whisker structures; (5) CMC-BAPE@BMP7 nanoparticle composite: The biological root support base body with surface and interface whisker activation prepared in step (4) is composited with CMC-BAPE@BMP7 nanoparticles.

[0014] As an optional mode, in the above preparation method, the following steps are specifically included: (1) constructing a three-dimensional model of a biological root: Based on cone beam computed tomography (CBCT), all imaging data of the missing tooth are obtained, and then a gray threshold segmentation method is used to segment and construct an original three-dimensional data model of the missing tooth. Through optimization of the undercut structure and three-dimensional finite element analysis, the completed root model is exported in STL format to obtain a three-dimensional data model of a personalized biological root; (2) preparing a 3D printing ink: Polyurethane acrylate and polyethylene glycol acrylate are mixed uniformly to prepare a photosensitive resin. Hydroxyapatite powder and zirconia / zinc oxide mixture powder are mixed uniformly with the photosensitive resin, a photoinitiator and a dispersant in a ball mill to prepare a 3D printing ink. The mass fraction of hydroxyapatite powder in the 3D printing ink is 60% or more, and the mass fraction of the zirconia / zinc oxide mixture is 5%-20%. The viscosity of the 3D printing ink is between 400 and 800 Pas under the action of a shear rate of 0.1 / S. Optionally, the mass ratio of the polyurethane acrylate and the polyethylene glycol acrylate ranges from 3:1 to 5:1.

[0015] (3) preparing a biological root support base body by 3D printing and sintering: According to the three-dimensional data model of the personalized biological root obtained in step (1), the 3D printing ink prepared in step (2) is added to a 3D printing device, printing parameters are set, and layer-by-layer solidification is performed to obtain a biological root support blank body. After printing, the unsolidified printing ink in the blank body is removed using an ultrasonic dental treatment instrument. The blank body is gradually debound and sintered to form a shape, maintained at 300°C and 1100°C for 6 hours, respectively, and finally maintained at 1450°C-1550°C for 2-4 hours. After that, the furnace is cooled to room temperature, and a biological root support base body is obtained. (4) Bio-root scaffold base surface interface nanowhisker structure activation: The bio-root scaffold base obtained in step (3) is put into a hydroxyapatite whisker deposition system containing 1:1.5 NaH2PO4 and EDTA, ammonia water and nitric acid are used to maintain the pH value of the reaction concentration at 8.0, and the ordered nanowhisker structure is formed on the base surface interface through hydrothermal reaction at 200°C for 8 hours; (5) Composite CMC-BAPE@BMP7 nanoparticles: The bio-root scaffold with the surface interface whisker activated in step (4) is freeze-dried, then soaked in a 1mg / ml CMC-BAPE@BMP7 nanoparticle colloidal solution at room temperature for 12 hours to obtain a bio-root scaffold with composite CMC-BAPE@BMP7 nanoparticles.

[0016] The application also provides an application of the bio-root scaffold based on 3D printing, characterized in that it is used as a tooth loss repair material.

[0017] All features disclosed in this specification, or all steps of any methods or processes disclosed, may be combined in any manner, except where features or steps are mutually exclusive.

[0018] Advantages of the application: 1. The bio-root scaffold based on 3D printing has high strength, high precision, and immunomodulatory activity, and can realize personalized and precise customization.

[0019] 2. The preparation method of the bio-root scaffold adopts a light-cured 3D printing technology, designs a three-dimensional model of a personalized digital bio-root based on the three-dimensional shape of the missing tooth of a patient, uses calcium phosphate ceramics with tooth-forming biological activity as the main body, composites photosensitive resin and mechanical toughening ceramic powder, etc., configures bio-printing ink with certain fluidity, obtains a personalized bio-root scaffold embryo after layer-by-layer solidification after exposure to ultraviolet light or other light sources, and then performs high-temperature debinding and sintering, and then performs surface interface nanowhisker construction by using a hydrothermal method, and then composites immunomodulatory CMC-BAPE@BMP7 nanoparticles with anti-inflammatory and regenerative effects, so as to realize functional bio-root construction. This technology combines 3D printing to prepare high-precision bioceramics, high-temperature sintering to obtain excellent mechanical properties, surface interface activation to promote cell adhesion and differentiation, and the combination of anti-inflammatory and cell differentiation nano CMC-BAPE@BMP7 drugs, realizes the personalized customization of the bio-root scaffold with high strength, high precision, and immunomodulatory activity, and has a broad application prospect in the field of tooth loss repair. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1The preparation process of the biological tooth root support according to the present application; Figure 2 The physical photos of the biological tooth root support prepared in the embodiments of the present application; Figure 3 The photos of the biological tooth root support prepared according to the extraction of natural teeth in the embodiments of the present application; Figure 4 The mechanical property test results of the biological tooth root support prepared in the embodiments of the present application. The left photo is the in-vitro pressure-bearing photo of the support, and the right photo is the stress-strain curve of different supports; Figure 5 The scanning electron microscope photos of the green body of the biological tooth root support prepared in the embodiments of the present application after high-temperature sintering; Figure 6 The scanning electron microscope photos of the biological tooth root support prepared in the embodiments of the present application after the deposition of nanowhisker structure on the substrate; Figure 7 The scanning electron microscope photos of the biological tooth root support prepared in the embodiments of the present application after the deposition of CMC-BAPE@BMP7 nanoparticles and; Figure 8 The in-vivo biological regeneration performance test results of the biological tooth root support in the functional comparative example 1. A is the biological tooth root prepared by the present application, specifically the DLP 3D-printed tooth root support combined with CMC-BAPE@BMP7 / GelMA and DFSC membrane, and the obtained DFSC tooth root parenchyma / interstitial cell differentiation biological tooth root after the transplantation of the rat kidney dorsal membrane for 3 months, B is the biological tooth root with short shape and sparse cells formed after the DLP 3D-printed tooth root support combined with CMC-BAPE and DFSC membrane under the same conditions; Figure 9 The regenerated sample photos of the biological tooth root support in the functional comparative example 2 after the transplantation of the rat kidney dorsal membrane; Figure 10 The performance test results of the biological tooth root support in the functional comparative example 2 after the transplantation of the rat kidney dorsal membrane. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the present application more clear and explicit, the present application will be further described in detail below with specific embodiments, but it should not be understood that the scope of the above-mentioned subject matter of the present application is limited to the following embodiments.

[0022] A biological tooth root support based on 3D printing, the preparation method thereof comprises the following steps: (1) constructing a three-dimensional model of a biological tooth root; Obtaining the imaging data of the missing tooth, constructing the original three-dimensional data model of the missing tooth, and obtaining the three-dimensional data model of the personalized biological tooth root through optimization of the undercut structure and three-dimensional finite element analysis; (2) Preparation of 3D printing ink: The calcium phosphate bioceramic powder, the inorganic oxide toughening ceramic powder, the photosensitive resin, the photoinitiator and the dispersant are uniformly mixed to prepare the 3D printing ink; (3) Preparation of biological tooth root support matrix by 3D printing and sintering: According to the three-dimensional data model of the personalized biological tooth root obtained in step (1), the 3D printing ink prepared in step (2) is used to obtain the biological tooth root support blank by 3D printing, and the biological tooth root support matrix is obtained by sintering; (4) Surface and interface nanowhisker structure activation of biological tooth root support matrix: The surface and interface of the biological tooth root support matrix obtained in step (3) are deposited with activated nanowhisker structures; (5) CMC-BAPE@BMP7 nanoparticles: The biological tooth root support matrix with activated surface and interface whiskers prepared in step (4) is compounded with CMC-BAPE@BMP7 nanoparticles.

[0023] The following listed are several best embodiments of the present application, and it should be understood that these embodiments are only for illustrative purposes, and by no means limit the protection scope of the present application.

[0024] Example 1: A biological tooth root support based on 3D printing, the preparation method thereof is as shown in Figure 1 , and specifically includes the following steps: (1) Construction of three-dimensional model of biological tooth root: Based on cone beam computed tomography (CBCT), all imaging data of different types of missing teeth (including single-rooted teeth, double-rooted teeth and triple-rooted teeth) are obtained, and then the original three-dimensional data model of the missing tooth is constructed by gray threshold segmentation method. Through optimization of the undercut structure and three-dimensional finite element analysis, the completed tooth root model is exported as an STL format, and the three-dimensional data model of the personalized biological tooth root is obtained; (2) Preparation of 3D printing ink: A photosensitive resin is prepared by uniformly mixing 90 g of polyurethane acrylate and 30 g of polyethylene glycol acrylate, and a 3D printing ink is prepared by uniformly ball-milling 180 g of hydroxyapatite powder, 36 g of zirconia / zinc oxide mixture powder (mass ratio of 1:1), and the photosensitive resin, 6 g of a photoinitiator, and 5 g of a dispersant in a ball mill, the mass fraction of the hydroxyapatite powder in the 3D printing ink being 60% or more, and the viscosity of the 3D printing ink being 800 mPa·s under the action of a shear rate of 0.1 / S; (3) Preparing a biological root support body by 3D printing and sintering: According to the three-dimensional data model of the personalized biological root obtained in step (1), the 3D printing ink prepared in step (2) is added to a 3D printing device, and the printing parameters are set as a thickness of 50 microns per layer, a photocuring time of 1200 ms, and a photocuring light intensity of 2000 mW / cm 2 , layer by layer solidification, and a biological root support blank is obtained by 3D printing (as shown in Figure 2 from left to right: three maxillary molars, two mandibular molars, incisors, and premolars of a real natural tooth in size and precision are printed, respectively), and the uncured printing ink in the blank is removed by using an ultrasonic dental treatment instrument after printing; the blank is gradually debound and sintered to form a biological root support body (as shown in Figure 3 , the implantable biological root support is designed based on the morphology of the extracted mandibular first molar), and the biological root support body is obtained by maintaining at 300°C and 1100°C for 6 hours, respectively, and finally maintaining at 1450°C for 2 hours, and then cooling to room temperature in the furnace; the sintered biological root support can withstand a pressure of 500 g without obvious deformation (as shown in Figure 4 left), and the corresponding stress-strain curve result shows that the mechanical properties of the sintered DLP 3D printed root support are higher than those of a natural tooth root of the same size in the development stage (as shown in Figure 4 right), and the possible reason is that a large number of HAp crystals are fused in the DLP 3D printed support after high-temperature sintering, and the second-phase toughening effect is played by the addition of zirconia / zinc oxide and the like (as shown in Figure 5 ). (4) Activation of the nanowhisker structure on the surface and interface of the biological root support body: The biological root support body obtained in step (3) is put into a hydroxyapatite whisker deposition system containing 1:2 NaH2PO4 and EDTA, ammonia and nitric acid are used to maintain the reaction concentration pH value at 8.0, and the system is kept at 200°C for 8 hours, so that an ordered nanowhisker structure is formed on the surface and interface of the body by hydrothermal reaction (as shown in Figure 6 , and Figure 5The smooth DLP 3D printing of the biological tooth root surface in the Central Plains forms a structured hydroxyapatite whisker with consistent length, which is about 500-700 nm in length and 2-5 nm in width. (5) CMC-BAPE@BMP7 nanoparticles: The synthesis route of CMC-BAPE@BMP7 nanoparticles is as follows: 200 ml of 1% (v / v) acetic acid solution, 4 g of carboxymethyl chitosan is stirred at room temperature at 1000 r / min, and after complete dissolution, vacuum filtration pump filtration is carried out using a double-layer filter paper with a pore size of 20 um to obtain a 2% clear CMC solution. Then 2.97 g of 4-bromomethylphenylboronic acid pinacol ester is dissolved in 10 ml of acetonitrile, and 5% Na2CO3 solution is added to adjust the solution to pH 9, then 20 ml of 2% CMC solution is added, and the reaction is carried out at room temperature in the dark under nitrogen protection for 24 h to obtain CMC-BAPE 0.726 g (1 mmol). CMC-BAPE is dissolved in 10 ml of MES solution with pH 5, and about 1.5 times the molar amount of catalyst EDC 0.288 g and NHS 0.172 g are added to activate the carboxyl group for 1 h, then the waste liquid is removed, 10 ml of 1 ug / ml BMP7 recombinant protein is added to PBS with pH 7.2, and low-temperature stirring reaction is carried out for 4 h. The uncoupled reagent is washed by low-temperature ultracentrifugation with pH 7.4 deionized water, the precipitate is resuspended in PBS, and after thin film dialysis, the CMC-BAPE-BMP7 nanomicelle solution is obtained by filtering through a 0.45 um microporous filter. The biological tooth root support matrix activated by the surface whisker prepared in step (4) is freeze-dried, then soaked in 1 mg / ml CMC-BAPE@BMP7 nanoparticle colloidal solution at room temperature for 12 h to obtain a biological tooth root support combined with CMC-BAPE@BMP7 nanoparticles. (As shown in Figure 7 The particle size range of CMC-BAPE@BMP7 nanoparticles in the obtained biological tooth root support is 200-400 nm, the unit surface deposition rate of CMC-BAPE@BMP7 nanoparticles in the 3D printed biological tooth root support matrix is 10-25 / um 2 , the porosity of the support is 5-10%, the surface roughness Ra of the sample surface 1 cm x 1 cm area based on the needle tracing method is 0.4-0.6 um, the compressive strength is 30-50 Mpa, and the Young's modulus is 20-30 GPa) Example 2:

[0025] The biological tooth root support is prepared according to the method described in Example 1, and the difference from Example 1 is only that: In the step (2): The 3D printing ink is prepared by mixing 60 g of polyurethane acrylate and 20 g of polyethylene glycol acrylate uniformly to prepare a photosensitive resin, and mixing 120 g of hydroxyapatite powder, 6 g of zirconia / zinc oxide mixture powder, and the photosensitive resin, 4 g of a photoinitiator, and 3 g of a dispersant uniformly in a ball mill, the mass fraction of the hydroxyapatite powder in the 3D printing ink is 60% or more, the mass ratio of the zirconia / zinc oxide mixture is 5:1, and the viscosity of the 3D printing ink is 400 mPa·s under the action of a shear rate of 0.1 / S; In the step (3), the printing parameters are set as follows: The printing parameters are set as follows: the thickness of each layer is 20 microns, the light curing time is 900 ms, and the light curing light intensity is 1000 mW / cm 2 : When sintering, the last time is 1550℃ for 4h The whiskers at the interface of the finally prepared biological root support have a length of 500-700 nm and a width of 2-5 nm, the CMC-BAPE@BMP7 nanoparticles have a particle size range of 200-400 nm, the deposition rate per unit surface area of the nanoparticles in the 3D printed biological root support matrix is 10-25 per um 2 , the porosity of the support is 5-10%, the surface roughness Ra of the sample surface in a 1 cm*1 cm area is 0.4-0.6 um, the compressive strength is 30-50 Mpa, and the Young's modulus is 20-30 GPa.

[0026] Example 3: The biological root support is prepared according to the method of Example 1, and the difference from Example 1 is only that: In the step (2), the printing parameters are set as follows: The 3D printing ink is prepared by mixing 60 g of polyurethane acrylate and 20 g of polyethylene glycol acrylate uniformly to prepare a photosensitive resin, and mixing 120 g of hydroxyapatite powder, 6 g of zirconia / zinc oxide mixture powder, and the photosensitive resin, 4 g of a photoinitiator, and 3 g of a dispersant uniformly in a ball mill, the mass fraction of the hydroxyapatite powder in the 3D printing ink is 60% or more, the mass ratio of the zirconia / zinc oxide mixture is 5:1, and the viscosity of the 3D printing ink is 400 mPa·s under the action of a shear rate of 0.1 / S; In the step (3), the printing parameters are set as follows: The printing parameters are set as follows: the thickness of each layer is 30 microns, the light curing time is 1000 ms, and the light curing light intensity is 1500 mW / cm 2 . When sintering, the last time is 1450℃ for 2h The length of the whisker in the interface of the final prepared biological tooth root support is 500-700 nm, the width is 2-5 nm, the particle size range of CMC-BAPE@BMP7 nanoparticles is 200-400 nm, and the unit surface area deposition rate of the nanoparticles in the 3D printed biological tooth root support matrix is 10-25 / um 2 The porosity of the support is 5-10%, the surface roughness Ra of the sample surface based on the needle tracing method is 0.4-0.6 um, the compressive strength is 30-50 Mpa, and the Young's elastic modulus is 20-30 GPa.

[0027] Example 4: The biological tooth root support is prepared according to the method described in Example 1, and the only difference from Example 1 is that: In the step (2): 75g of polyurethane acrylate and 30g of polyethylene glycol acrylate are uniformly mixed to prepare a photosensitive resin, 160g of hydroxyapatite powder and 30g of zirconia / zinc oxide mixture powder are uniformly mixed in a ball mill with the above photosensitive resin, 4g of a photoinitiator and 3g of a dispersant to prepare a 3D printing ink, the mass fraction of hydroxyapatite powder in the 3D printing ink is more than 60%, the mass ratio of zirconia / zinc oxide mixture is 2:1, and the viscosity of the 3D printing ink is 500 mPa·s under the action of a shear rate of 0.1 / s; In the step (3): The printing parameters are set as follows: the thickness of each layer is 25 microns, the light curing time is 1100 ms, and the light curing light intensity is 1800 mW / cm 2 When sintering, finally maintain at 1450℃ for 2h The length of the whisker in the interface of the final prepared biological tooth root support is 500-700 nm, the width is 2-5 nm, the particle size range of CMC-BAPE@BMP7 nanoparticles is 200-400 nm, and the unit surface area deposition rate of the nanoparticles in the 3D printed biological tooth root support matrix is 10-25 / um 2 The porosity of the support is 5-10%, the surface roughness Ra of the sample surface based on the needle tracing method is 0.4-0.6 um, the compressive strength is 30-50 Mpa, and the Young's elastic modulus is 20-30 GPa.

[0028] Example 5: The biological tooth root support is prepared according to the method described in Example 1, and the only difference from Example 1 is that: In the step (2): The hydroxyapatite is replaced by tricalcium phosphate.

[0029] In the step (3): The printing parameters are set as follows: thickness of each layer, 20-50 microns; photocuring time, 900-1200 ms; photocuring light intensity, 1000-2000 mW / cm 2 : During sintering, the last step is to maintain at 1500℃ for 3h The whiskers in the interface of the final prepared biological root support have a length of 500-700 nm and a width of 2-5 nm, and the CMC-BAPE@BMP7 nanoparticles have a particle size range of 200-400 nm, and the deposition rate per unit surface area of the nanoparticles in the 3D-printed biological root support matrix is 10-25 / um 2 The porosity of the support is 5-10%, the surface roughness Ra of the sample surface in a 1cm×1cm area based on the needle tracing method is in the range of 0.4-0.6um, the compressive strength is 30-50Mpa, and the Young's elastic modulus is 20-30GPa.

[0030] Example 6 The biological root support is prepared according to the method described in Example 1, and the only difference from Example 1 is that: In the step (2): The hydroxyapatite is replaced by biphasic calcium phosphate (HA and TCP in a mass ratio of 1:1).

[0031] In the step (3): The printing parameters are set as follows: thickness of each layer, 20-50 microns; photocuring time, 900-1200 ms; photocuring light intensity, 1000-2000 mW / cm 2 : During sintering, the last step is to maintain at 1520℃ for 2h.

[0032] The whiskers in the interface of the final prepared biological root support have a length of 500-700 nm and a width of 2-5 nm, and the CMC-BAPE@BMP7 nanoparticles have a particle size range of 200-400 nm, and the deposition rate per unit surface area of the nanoparticles in the 3D-printed biological root support matrix is 10-25 / um 2 The porosity of the support is 5-10%, the surface roughness Ra of the sample surface in a 1cm×1cm area based on the needle tracing method is in the range of 0.4-0.6um, the compressive strength is 30-50Mpa, and the Young's elastic modulus is 20-30GPa) Comparative Example 1

[0033] The biological root support is prepared according to the method described in Example 1, and the only difference from Example 1 is that step (4) is omitted.

[0034] The final biological root support has no activated whisker structure in the interface, the CMC-BAPE@BMP7 nanoparticles have a particle size range of 200-400 nm, and the deposition rate per unit surface area of the nanoparticles in the 3D-printed biological root support matrix is 10-25 per um 2 The porosity of the support is 5-10%, the surface roughness Ra of the sample surface based on the needle tracing method is 0.1-0.2 um in a 1 cm x 1 cm area, the compressive strength is 30-50 Mpa, and the Young's elastic modulus is 20-30 GPa.

[0035] Comparative Example 2: The biological root support was prepared according to the method of Example 1, and the only difference from Example 1 was that step (5) was omitted.

[0036] The final biological root support has whiskers with a length of 500-700 nm and a width of 2-5 nm in the interface, the porosity of the support is 5-10%, the surface roughness Ra of the sample surface based on the needle tracing method is 0.4-0.6 um in a 1 cm x 1 cm area, the compressive strength is 30-50 Mpa, and the Young's elastic modulus is 20-30 GPa.

[0037] Comparative Example 3: The biological root support was prepared according to the method of Example 1, and the only difference from Example 1 was that: In the step (2): The 3D printing ink was prepared by uniformly mixing 60 g of polyurethane acrylate and 20 g of polyethylene glycol acrylate, uniformly mixing 120 g of hydroxyapatite powder with 21 g of yttrium-stabilized tetragonal zirconia (Y-TZP) powder, and the above-mentioned photosensitive resin, 5 g of a photoinitiator and 5 g of a dispersant in a ball mill. The mass fraction of hydroxyapatite powder in the 3D printing ink is 55%, and the viscosity of the 3D printing ink is 600 mPa·s under the action of a shear rate of 0.1 / S.

[0038] The final biological root support has 55 wt% HA + 10 wt% Y-TZP.

[0039] Comparative Example 4: The biological root support was prepared according to the method of Example 1, and the only difference from Example 1 was that: In the step (2): A photosensitive resin is prepared by uniformly mixing 60 g of polyurethane acrylate and 20 g of polyethylene glycol acrylate, 120 g of hydroxyapatite powder is uniformly mixed with the photosensitive resin, 5 g of a photoinitiator and 5 g of a dispersant in a ball mill to prepare a 3D printing ink, the mass fraction of the hydroxyapatite powder in the 3D printing ink is 65%, and the viscosity of the 3D printing ink is 600 mPa·s under the action of a shear rate of 0.1 / S.

[0040] The final prepared biological root support is 65wt%HA+0wt%Y-TZP.

[0041] Functional comparison experiment 1: After 3 months of rat renal capsule transplantation of the DLP 3D printed root support prepared by the application, the CMC-BAPE@BMP7 group, and the DFSC membrane piece control group, it can be found that: compared with the CMC-BAPE and DFSC membrane piece control group, the CMC-BAPE@BMP7 group biological tooth root is thick and large, and the cells are rich (Fig. Figure 8 a1), a large number of DSP positive expression of new dentin in the inner side of the support (Fig. Figure 8 a2-a3: ND: new dentin- new dentin), BSP positive expression of new cementum can be seen on the outside (Fig. Figure 8 a4-a5), and there are obvious blood vessels in the root canal (Fig. Figure 8 a6 red arrow) and CD31 positive blood vessels-NF positive nerve bundle formation (Fig. Figure 8 a7) and obvious calcified substance structure in the pulp cavity (Fig. Figure 8 a8), and a large number of inflammatory cells infiltrate the outside of the tooth root relative to the pulp cavity (Fig. Figure 8 a9) also shows that the CMC-BAPE@BMP7 compounded in the inner side of the pulp cavity has good anti-inflammatory effect, and in the CMC-BAPE group (Fig. Figure 8 B), the biological tooth root is short and small, and the cells are sparse (Fig. Figure 8 b1), mesenchymal blood vessels and nerves are less (Fig. Figure 8 b2) and the irregular and thin new substance structure-dentin (Fig. Figure 8 b3). The above results show that the DLP 3D printed root support of the application compounded with CMC-BAPE@BMP7 can promote DFSC to differentiate into substance / interstitial type in the root mode and has certain anti-inflammatory effect.

[0042] Functional comparison experiment 2: The periodontal membrane regeneration effect in the application is further verified by using a large animal beagle dog in vivo biological tooth root regeneration model, as follows: a first premolar tooth germ of a 6-month-old beagle dog is obtained (Fig. Figure 10A), extraction of tooth germ outer layer development period tooth capsule tissue ( Figure 10 B), further in vitro isolation and culture into dental follicle stem cell membrane-DFSC ( Figure 10 C), and then using the hydroxyapatite-zirconium / zinc oxide-based bio-ink as the main component in the application, a 6mm high, 1.5mm thick, 1mm hollow bioceramic root support is prepared by DLP-3D printing ( Figure 10 D), 5% pure GelMA (control group), GelMA mixed solution containing 1mg / ml BMP7 (BMP7 group) and GelMA mixed solution containing 1mg / ml CMC-BAPE@BMP7 (CMC-BAPE@BMP7 group) as different experimental groups, Figure 10 E), after soaking the 3D printed bioceramic root support in each group of drugs, the outer layer is compounded with dog DFSC membrane to form a composite biological root model for implantation in vivo ( Figure 10 F), after the mandibular fourth premolar of a 1-year-old adult beagle dog is removed, the composite biological root of each group is immediately implanted (4 samples per group), and the X-ray image shows that the position of the composite biological root after implantation is correct ( Figure 10 G), after three months, the implanted root area heals well, and X-ray shows that there is no bone inflammation absorption around the bioceramic root support ( Figure 10 H). Further, the beagle dog is painlessly killed to obtain the regenerated sample ( Figure 9 ), hard tissue sections (thickness 30um) are used for histological detection ( Figure 10 I), the results are as follows: in the control group, the support is buried under the gum and surrounded by alveolar bone ( Figure 10 i), HE staining results further show that there is obvious cell adhesion on the outside ( Figure 10 ii) and new tissue formation can be seen on the inside ( Figure 10 iii white pentagram), Masson staining results show that these cells are collagen fiber cells, and there is also a lack of obvious regenerated microvessels, and the blue-stained uniform tissue indicates that DFSC forms collagen component secretions on the surface of the support ( Figure 10 iv). At the same time, the BMP7 group shows that the support and the alveolar bone are obviously adhered ( Figure 10 v), the support also shows that the new material is formed by eosin staining ( Figure 10 vi white pentagram), and the support outside the support can be seen to be adhered to the alveolar bone ( Figure 10 vii white triangle), Masson staining results show that the eosin-stained new material is collagen component ( Figure 10viii), and a small amount of blood vessel formation can be seen inside (red arrows), indicating that direct compounding with BMP7 promotes excessive collagen secretion of DFSC, while increasing matrix mineralization and angiogenesis induction ability; in contrast, the CMC-BAPE@BMP7 group showed a relatively obvious eosinophilic periodontal ligament-like space structure around the implanted 3D printing material ( Figure 10 ix), further HE magnification shows that both the upper part of the root ( Figure 10 x) or the lower part of the root ( Figure 10 xi), a large number of periodontal fiber attachment structures were formed (white arrows), and the periodontal fibers were arranged in parallel and orderly (green dotted arrows), and there were also a large number of tiny blood vessel-like structures (red arrows) between them. The Masson result ( Figure 10 xii) A similar regeneration trend was also observed, indicating that compared with pure BMP7 induction, the DLP-3D printed biological root scaffold prepared by the CMC-BAPE@BMP7 composite of the present invention can promote the controllable differentiation of DFSC into periodontal ligament-like structures, thereby promoting the formation of periodontal attachment structures and microvascularization.

Claims

1. A biological tooth root scaffold based on 3D printing, characterized in that: It includes a 3D-printed biological root scaffold matrix and CMC-BAPE@BMP7 nanoparticles. The surface of the 3D-printed biological root scaffold matrix has an activated nanowhisker structure, and the CMC-BAPE@BMP7 nanoparticles are adsorbed and fixed in the 3D-printed biological root scaffold matrix.

2. The biological tooth root scaffold based on 3D printing according to claim 1, characterized in that: The 3D-printed biological tooth root scaffold matrix is ​​prepared using DLP 3D printing technology.

3. The biological tooth root scaffold based on 3D printing according to claim 1, characterized in that: The 3D-printed biological tooth root scaffold matrix is ​​prepared by using calcium phosphate bioceramics added with inorganic oxides, and the inorganic oxides are a zirconium oxide / zinc oxide mixture.

4. The biological tooth root scaffold based on 3D printing according to claim 1, characterized in that: The CMC-BAPE@BMP7 nanoparticles have a particle size range of 200-400 nm, and a deposition rate of 10-25 particles per μm per unit surface area in the 3D-printed biological root scaffold matrix. 2 .

5. The biological tooth root scaffold based on 3D printing according to claim 1, characterized in that: The activated nano whisker structure is an orderly arranged nano whisker structure formed on the surface of the 3D printed biological root scaffold matrix. The nano whisker structure is 500-700 nm long and 2-5 nm wide.

6. The biological tooth root scaffold based on 3D printing according to claim 1, characterized in that: The porosity of the stent is 5-10%, the surface roughness Ra is 0.4-0.6 μm, the compressive strength is 30-50 MPa, and the Young's elastic modulus is 20-30 GPa.

7. The method for preparing a biological root scaffold based on 3D printing according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) Construct a three-dimensional model of the biological tooth root; (2) Preparation of 3D printing ink; (3) Prepare the biological root scaffold matrix by 3D printing and sinter it; (4) Activation of nano-whisker structure on the surface of biological root scaffold matrix; (5) Composite CMC-BAPE@BMP7 nanoparticles.

8. The preparation method according to claim 7, characterized in that The specific steps include: (1) Constructing a three-dimensional model of biological tooth roots: Obtain imaging data of missing teeth, construct the original 3D data model of missing teeth, and obtain a personalized 3D data model of biological tooth roots through optimization of undercut structure and 3D finite element analysis; (2) Preparation of 3D printing ink: Calcium phosphate bioceramic powder, inorganic oxide toughened ceramic powder, photosensitive resin, photoinitiator and dispersant are mixed evenly to prepare 3D printing ink; (3) Prepare biological root scaffold matrix by 3D printing and sintering: According to the three-dimensional data model of the personalized biological tooth root obtained in step (1), the 3D printing ink prepared in step (2) is used to obtain a biological tooth root scaffold blank by 3D printing, and sintering is performed to obtain a biological tooth root scaffold matrix; (4) Activation of nano-whisker structure on the surface of biological root scaffold matrix: Depositing activated nano whisker structures on the surface and interface of the biological root scaffold matrix obtained in step (3); (5) Composite CMC-BAPE@BMP7 nanoparticles: The biological root scaffold matrix after surface whisker activation prepared in step (4) is compounded with CMC-BAPE@BMP7 nanoparticles.

9. The preparation method according to claim 7, characterized in that The specific steps include: (1) Constructing a three-dimensional model of biological tooth roots: The complete imaging data of the missing tooth is acquired using cone-beam CT. The original 3D data model of the missing tooth is then constructed using the grayscale threshold segmentation method. The completed root model is exported to STL format through optimization of the undercut structure and 3D finite element analysis to obtain a personalized 3D data model of the biological root. (2) Preparation of 3D printing ink: Polyurethane acrylate and polyethylene glycol acrylate are uniformly mixed to prepare a photosensitive resin, and hydroxyapatite powder, zirconium oxide / zinc oxide mixture powder, photosensitive resin, photoinitiator, and dispersant are uniformly ball-milled in a ball mill to prepare a 3D printing ink, wherein the mass fraction of hydroxyapatite powder in the 3D printing ink is greater than 60%, the mass fraction of the zirconium oxide / zinc oxide mixture is 5%-20%, and the viscosity of the 3D printing ink is between 400 and 800 Pas at a shear rate of 0.1 / s; (3) Prepare biological root scaffold matrix by 3D printing and sintering: According to the three-dimensional data model of the personalized biological tooth root obtained in step (1), the 3D printing ink prepared in step (2) is added to the 3D printing equipment, the printing parameters are set, and the blank of the biological tooth root scaffold is solidified layer by layer. After the printing is completed, the unsolidified printing ink in the blank is removed by using an ultrasonic dental treatment device; the blank is gradually degreased and sintered, respectively, at 300°C and 1100°C for 6 hours, and finally at 1450°C-1550°C for 2-4 hours, and then cooled to room temperature with the furnace to obtain the biological tooth root scaffold matrix; (4) Activation of nano-whisker structure on the surface of biological root scaffold matrix: The biological root scaffold matrix obtained in step (3) is placed in a hydroxyapatite whisker deposition system containing NaH2PO4 and EDTA in a ratio of 1:1.5, and ammonia and nitric acid are used to maintain the reaction concentration pH at 8.0, and maintained at 200°C for 8 hours to form an ordered nano-whisker structure on the surface of the matrix through a hydrothermal reaction; (5) Composite CMC-BAPE@BMP7 nanoparticles: The biological root scaffold matrix after surface whisker activation prepared in step (4) was freeze-dried and then immersed in a 1 mg / ml CMC-BAPE@BMP7 nanoparticle colloidal solution at room temperature for 12 hours to obtain a biological root scaffold composited with CMC-BAPE@BMP7 nanoparticles.

10. The use of the biological tooth root scaffold based on 3D printing according to any one of claims 1 to 6, characterized in that: It is used as a restorative material for missing teeth.

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