A 3D printing-based biological tooth root support and a preparation method and application thereof

Abstract

The application discloses a kind of biological tooth root support based on 3D printing and its preparation method and application, belong to the technical field of biomedical materials.The biological tooth root support includes 3D printing biological tooth root support matrix and CMC-BAPE@BMP7 nanoparticles, the interface of the 3D printing biological tooth root support matrix has activated nanowhisker structure, the CMC-BAPE@BMP7 nanoparticles are adsorbed and fixed in the 3D printing biological tooth root support matrix.The application combines 3D printing preparation high-precision bioceramics, excellent mechanical properties after high-temperature sintering, interface activity reconstruction promotes cell adhesion differentiation, and combines anti-inflammatory and promotes cell differentiation nano CMC-BAPE@BMP7 drug, realizes the individual customization of high-strength, high-precision, immunoregulatory activity biological tooth root support, and has 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, personalized recovery mechanical performance, 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 personalized biological teeth 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 3D printing research 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 and other methods 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 these methods are poor, the precision of the tooth root support is low, and the tooth root support does not have anti-inflammatory and 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 and immune regulation ability 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 and anti-inflammatory and 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 and immune regulation ability in the existing low-temperature deposition, ink direct writing method and other 3D printing technologies. The biological tooth root support combines high-precision biological ceramics prepared by 3D printing, excellent mechanical properties after high-temperature sintering, surface interface activity reconstruction to promote cell adhesion and differentiation, and anti-inflammatory and cell differentiation nano CMC-BAPE@BMP7 drugs to realize the personalized customization of a biological tooth root support with high strength, high precision and immune regulation activity, which has a broad application prospect in the field of tooth loss repair.

[0005] The present application is realized by the following technical solutions:

[0006] A 3D printing-based biological tooth root support comprises a 3D printed biological tooth root support base body and CMC-BAPE@BMP7 nanoparticles, the surface interface of the 3D printed biological tooth root support base body has an activated nanowhisker structure, 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 surface interface nanowhisker structure 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 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, so that the CMC-BAPE@BMP7 nanoparticles with anti-inflammatory immune regulation are compounded, so that 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.

[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 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, so as to make up for the defects of the brittle and weak biological tooth root support after high-temperature sintering in the prior art, which is difficult to meet the continuous functional chewing requirements of teeth.

[0008] 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 the high-strength and high-precision biological tooth root support is realized. As an option, the mass fraction of the inorganic oxide in the 3D printing ink is 5%-20%. Further, the mass ratio of the zirconia to the zinc oxide is in the range of 1:5 to 5:1.

[0009] As an optional mode, in the above-mentioned 3D printing-based biological tooth root support, the CMC-BAPE@BMP7 nanoparticle particle size range is 200-400 nm, and the unit surface area deposition rate thereof in the 3D printing biological tooth root support matrix is 10-25 / um 2 .

[0010] As an optional mode, in the above-mentioned 3D printing-based biological tooth root support, the activated nanowire structure is an ordered nanowire structure arranged on the surface interface of the 3D printing biological tooth root support matrix. Further, the nanowire structure is 500-700 nm long and 2-5 nm wide.

[0011] 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 modulus is 20-30 GPa.

[0012] The application also provides a preparation method of a 3D printing-based biological tooth root support, comprising the following steps:

[0013] (1) constructing a three-dimensional model of a biological tooth root;

[0014] (2) preparing 3D printing ink;

[0015] (3) preparing a biological tooth root support matrix by 3D printing and sintering;

[0016] (4) activating the nanowire structure on the surface interface of the biological tooth root support matrix;

[0017] (5) compounding CMC-BAPE@BMP7 nanoparticles.

[0018] As an optional mode, in the above-mentioned preparation method, the following steps are specifically included:

[0019] (1) constructing a three-dimensional model of a biological tooth root:

[0020] Obtaining imaging data of missing teeth, constructing an original three-dimensional data model of missing teeth, and obtaining a three-dimensional data model of individualized biological tooth roots through optimization of undercut structure and three-dimensional finite element analysis;

[0021] (2) preparing 3D printing ink:

[0022] The calcium phosphate bioceramic powder is mixed with inorganic oxide toughening ceramic powder and a photosensitive resin, a photoinitiator and a dispersant to prepare a 3D printing ink; as an option, the photosensitive resin is at least one of polyurethane acrylate, polyethylene glycol acrylate acryloyl morpholine, trimethylolpropane triacrylate, hyperbranched polyester acrylate; the photoinitiator is at least one of cyclohexyl vinyl ether, phenyl bis(2,4,6-trimethylbenzoyl) phosphine oxide, tripropylene glycol diacrylate, and the dispersant is at least one of castor oil phosphate, pyrrolidone, and sodium polyacrylate.

[0023] (3) Preparing a biological root support base body by 3D printing and sintering:

[0024] According to the three-dimensional data model of the personalized biological root obtained in step (1), a 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;

[0025] (4) Surface and interface nanowhisker structure activation of the biological root support base body:

[0026] Depositing an activated nanowhisker structure on the surface and interface of the biological root support base body obtained in step (3);

[0027] (5) Composite CMC-BAPE@BMP7 nanoparticles:

[0028] The biological root support base body after surface and interface whisker activation prepared in step (4) is compounded with CMC-BAPE@BMP7 nanoparticles.

[0029] As an optional mode, in the above preparation method, the following steps are specifically included:

[0030] (1) Constructing a three-dimensional model of a biological root:

[0031] 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;

[0032] (2) Preparing a 3D printing ink:

[0033] The polyurethane acrylate and polyethylene glycol acrylate are mixed uniformly to prepare a photosensitive resin, the hydroxyapatite powder and the zirconia / zinc oxide mixture powder are mixed uniformly in a ball mill with the photosensitive resin, a photoinitiator and a dispersant to prepare a 3D printing ink, the mass fraction of the hydroxyapatite powder in the 3D printing ink is more than 60%, the mass fraction of the zirconia / zinc oxide mixture is 5%-20%, and the viscosity of the 3D printing ink is between 400 and 800 Pas under the action of a shearing rate of 0.1 / S; as an option, the mass ratio of the polyurethane acrylate and the polyethylene glycol acrylate ranges from 3:1 to 5:1.

[0034] (3) Preparing a biological root support base body by 3D printing and sintering:

[0035] 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 the biological root support blank body is obtained by 3D printing through layer-by-layer solidification; after printing, the unsolidified printing ink in the blank body is removed by using an ultrasonic dental treatment instrument; the blank body is gradually debound and sintered to form a biological root support base body, which is maintained at 300°C and 1100°C for 6 hours, respectively, and finally maintained at 1450°C-1550°C for 2-4 hours, and then cooled to room temperature in the furnace, to obtain a biological root support base body;

[0036] (4) Activation of the surface interface nanowhisker structure of the biological root support base body:

[0037] The biological root support base body obtained in step (3) is put into a hydroxyapatite whisker deposition system containing 1:1.5 NaH2PO4 and EDTA, ammonia and nitric acid are used to maintain the reaction concentration pH value at 8.0, and the system is maintained at 200°C for 8 hours, so that the ordered nanowhisker structure is formed on the surface interface of the base body through hydrothermal reaction;

[0038] (5) Composite CMC-BAPE@BMP7 nanoparticles:

[0039] After the biological root support base body prepared in step (4) is activated on the surface interface whisker and freeze-dried, it is soaked in a 1mg / ml CMC-BAPE@BMP7 nanoparticle colloidal solution at room temperature for 12 hours to obtain a composite CMC-BAPE@BMP7 nanoparticle biological root support.

[0040] The application also provides an application of the biological root support based on 3D printing, characterized in that the biological root support is used as a tooth loss repair material.

[0041] All features disclosed in this specification, and all steps of any methods or processes disclosed, may be combined in any combination, except combinations where at least some of the features and / or steps are mutually exclusive.

[0042] Advantages of the present application:

[0043] 1. The 3D printing-based biological tooth root support according to the present application has high strength, high precision, immunomodulatory activity, and can realize personalized and precise customization.

[0044] 2. The preparation method of the biological tooth root support according to the present application adopts light-cured 3D printing technology, designs a three-dimensional model of a personalized digital biological tooth root based on the three-dimensional shape of the missing tooth of a patient, uses calcium phosphate ceramic with tooth-forming biological activity as the main body, composites photosensitive resin and mechanical toughening ceramic powder, etc., configures a biological printing ink with certain fluidity, obtains a personalized biological tooth root support embryo through layer-by-layer solidification after exposure to ultraviolet light or other light sources, and then high-temperature debinding and sintering, and then constructs a surface interface nanowhisker through a hydrothermal method, and then composites an anti-inflammatory and regenerative immunomodulatory CMC-BAPE@BMP7 nanoparticle, thereby realizing functional biological tooth root construction. The technology combines 3D printing to prepare high-precision biological ceramics, high-temperature sintering to obtain excellent mechanical properties, surface interface activity reconstruction to promote cell adhesion and differentiation, and the combination of anti-inflammatory and cell differentiation nanometer CMC-BAPE@BMP7 drugs, realizing the personalized customization of a biological tooth root support with high strength, high precision, and immunomodulatory activity, and having a broad application prospect in the field of tooth loss repair. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 The preparation process of the biological tooth root support according to the present application;

[0046] Figure 2 The biological tooth root support prepared in the embodiment of the present application is shown in the figure;

[0047] Figure 3 The figure shows the preparation of the implanted biological tooth root support according to the extraction of the natural tooth in the embodiment of the present application;

[0048] Figure 4 The mechanical property test results of the biological tooth root support prepared in the embodiment of the present application are shown. The left figure is an in-vitro pressure-bearing photo of the support, and the right figure is a stress-strain curve of different supports;

[0049] Figure 5 The scanning electron microscope photo of the biological tooth root support blank prepared in the embodiment of the present application after high-temperature sintering is shown;

[0050] Figure 6 The scanning electron microscope photo of the biological tooth root support substrate deposited with nanowhisker structure prepared in the embodiment of the present application is shown;

[0051] Figure 7A scanning electron microscope photo of the bio-root-scaffold composite CMC-BAPE@BMP7 nanoparticles prepared in the embodiment of the present application;

[0052] Figure 8 A graph of the in-vivo biological regeneration performance test results of the bio-root-scaffold described in the functional comparative example 1 of the present application. Wherein A is a bio-root with good morphological structure prepared by the present application, specifically, after the DLP 3D-printed root-scaffold composite CMC-BAPE@BMP7 / GelMA and dental follicle stem cell (DFSC) membrane are transplanted into the kidney dorsal membrane of a rat for 3 months, a DFSC root parenchyma / interstitial cell differentiation biological root is obtained, B is a biological root with short shape and sparse cells formed after the DLP 3D-printed root-scaffold composite CMC-BAPE and DFSC membrane are transplanted under the same conditions;

[0053] Figure 9 A photo of the regenerated sample obtained after the bio-root-scaffold described in the functional comparative example 2 of the present application is transplanted into the kidney dorsal membrane of a rat;

[0054] Figure 10 A graph of the performance test results of the bio-root-scaffold described in the functional comparative example 2 of the present application transplanted into the kidney dorsal membrane of a rat. DETAILED DESCRIPTION

[0055] In order to make the purpose, technical solutions and advantages of the present application clearer and more apparent, 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.

[0056] A bio-root-scaffold based on 3D printing, the preparation method thereof comprises the following steps:

[0057] (1) Constructing a three-dimensional model of a bio-root:

[0058] Obtain the imaging data of the missing tooth, construct the original three-dimensional data model of the missing tooth, and obtain the three-dimensional data model of the personalized bio-root through optimization of the undercut structure and three-dimensional finite element analysis;

[0059] (2) Preparing 3D printing ink:

[0060] Mix the calcium phosphate bioceramic powder, inorganic oxide toughening ceramic powder, photosensitive resin, photoinitiator and dispersant uniformly to prepare the 3D printing ink;

[0061] (3) Preparing a bio-root-scaffold matrix by 3D printing and sintering:

[0062] According to the three-dimensional data model of the personalized biological tooth root obtained in step (1), a 3D printing ink prepared in step (2) is used to obtain a biological tooth root support blank by 3D printing, sintering forming, and obtaining a biological tooth root support base;

[0063] (4) Surface and interface nanowhisker structure activation of the biological tooth root support base obtained in step (3);

[0064] The surface and interface of the biological tooth root support base obtained in step (3) are deposited with activated nanowhisker structures;

[0065] (5) CMC-BAPE@BMP7 nanoparticles are compounded;

[0066] The biological tooth root support base with surface and interface whisker activation prepared in step (4) is compounded with CMC-BAPE@BMP7 nanoparticles.

[0067] 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.

[0068] Example 1:

[0069] 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:

[0070] (1) Constructing a three-dimensional model of a biological tooth root:

[0071] 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 a gray threshold segmentation method is used to segment and construct an original three-dimensional data model of the missing teeth. Through optimization of the undercut structure and three-dimensional finite element analysis, the completed tooth root model is exported in STL format to obtain a three-dimensional data model of a personalized biological tooth root;

[0072] (2) Preparing a 3D printing ink:

[0073] 90g of polyurethane acrylate and 30g of polyethylene glycol acrylate are uniformly mixed to prepare a photosensitive resin, 180g of hydroxyapatite powder, 36g of zirconia / zinc oxide mixture powder (mass ratio of 1:1), and the above photosensitive resin, 6g of a photoinitiator, and 5g of a dispersant are uniformly mixed in a ball mill to prepare a 3D printing ink. The mass fraction of hydroxyapatite powder in the 3D printing ink is more than 60%, and the viscosity of the 3D printing ink is 800 mPa·s under the action of a shear rate of 0.1 / S;

[0074] (3) Preparing a biological tooth root support base by 3D printing and sintering forming:

[0075] 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 device, and the printing parameters are set as a thickness of 50 microns per layer, a light curing time of 1200 ms, and a light curing light intensity of 2000 mW / cm 2 , layer by layer solidification, and a biological tooth 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 true natural teeth in size and precision are printed, respectively), and the uncured printing ink in the blank is removed by an ultrasonic dental treatment instrument after printing; the blank is gradually debound and sintered to form a biological tooth root support body (as shown in Figure 3 , the implantable biological tooth root support is designed based on the morphology of the extracted mandibular first molar); the sintered biological tooth 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 tooth root support are higher than those of the natural tooth root of the same size in the development period (as shown in Figure 4 right), and the possible reason is that after high-temperature sintering, a large number of HAp crystals in the DLP 3D printed support are fused, and the addition of zirconia / zinc oxide and other materials plays a second-phase toughening effect (as shown in Figure 5 ).

[0076] (4) Activation of the nano-whisker structure on the surface interface of the biological tooth root support body:

[0077] The biological tooth 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 nano-whisker structure is formed on the surface interface of the body through hydrothermal reaction (as shown in Figure 6 , the smooth DLP 3D printed biological tooth root surface in Figure 5 forms a structured and uniform length hydroxyapatite whisker, which is about 500-700 nm long and 2-5 nm wide);

[0078] (5) Composite CMC-BAPE@BMP7 nanoparticles:

[0079] 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, after complete dissolution, vacuum filtration pump filtration is carried out using a double-layer filter paper with a pore size of 20 um, and then 2% clear CMC solution is obtained. Then 2.97 g of 4-bromomethylphenylboronic acid pinacol ester is dissolved in 10 ml of acetonitrile, and then 5% Na2CO3 solution is added to adjust the solution to pH 9, and then 20 ml of 2% CMC solution is added, and the reaction is carried out at room temperature for 24 h under light protection and nitrogen protection. CMC-BAPE 0.726 g (1 mmol) is obtained, 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 prepared in PBS with pH 7.2 is added, and the reaction is carried out at low temperature for 4 h. The uncoupled reagent is washed by low-temperature ultracentrifugation with deionized water with pH 7.4, 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 in step (4) is freeze-dried, and 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 Figure 7 shown, the particle size of the obtained CMC-BAPE@BMP7 nanoparticles in the biological tooth root support is in the range of 200-400 nm, the deposition rate per unit surface area 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 in a 1 cm x 1 cm area is in the range of 0.4-0.6 um, the compressive strength is 30-50 Mpa, and the Young's modulus is 20-30 GPa)

[0080] Example 2:

[0081] The biological tooth root support is prepared according to the method described in Example 1, and the difference from Example 1 is only that:

[0082] In the step (2):

[0083] 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;

[0084] In the step (3), the printing parameters are set as follows:

[0085] 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 : The sintering is finally maintained at 1550℃ for 4h

[0086] 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 of the nanoparticles per unit surface area 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 a 1 cm*1 cm area of the sample surface is in the range of 0.4-0.6 um, the compressive strength is 30-50 Mpa, and the Young's modulus of elasticity is 20-30 GPa.

[0087] Example 3:

[0088] The biological root support is prepared according to the method described in Example 1, and the difference from Example 1 is only that:

[0089] In the step (2), the printing parameters are set as follows:

[0090] 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;

[0091] In the step (3), the printing parameters are set as follows:

[0092] 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 / cm2 sintering, finally maintained at 1450℃ for 2h

[0093] The whiskers in the interface of the final prepared biological root support are 500-700nm long and 2-5nm wide, and the CMC-BAPE@BMP7 nanoparticles have a particle size range of 200-400nm, and the unit surface area deposition rate thereof 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 a 1cm×1cm area of the sample surface 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.

[0094] Example 4:

[0095] The biological root support is prepared according to the method described in Example 1, and the only difference from Example 1 is that:

[0096] In the step (2):

[0097] 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-mentioned 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 the zirconia / zinc oxide mixture is 2:1, and the viscosity of the 3D printing ink is 500mPa·s under the action of a shear rate of 0.1 / S;

[0098] In the step (3):

[0099] The printing parameters are set as follows: the thickness of each layer is 25 microns, the light curing time is 1100ms, and the light curing light intensity is 1800mW / cm 2 sintering, finally maintained at 1450℃ for 2h

[0100] The whiskers in the interface of the final prepared biological root support are 500-700nm long and 2-5nm wide, and the CMC-BAPE@BMP7 nanoparticles have a particle size range of 200-400nm, and the unit surface area deposition rate thereof 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 a 1cm×1cm area of the sample surface 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.

[0101] Example 5:

[0102] The biological tooth root support was prepared according to the method described in Example 1, and the only difference from Example 1 was that:

[0103] In the step (2), the hydroxyapatite was replaced by:

[0104] The hydroxyapatite was replaced by tricalcium phosphate.

[0105] In the step (3), the printing parameters were set as follows:

[0106] The printing parameters were 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 was to maintain the temperature at 1500 °C for 3 h

[0107] The final biological tooth root support had whiskers at the interface with a length of 500-700 nm and a width of 2-5 nm, and the CMC-BAPE@BMP7 nanoparticles had a particle size range of 200-400 nm, and the deposition rate of the nanoparticles per unit surface area in the 3D-printed biological tooth root support matrix was 10-25 per um 2 , the porosity of the support was 5-10%, the surface roughness Ra of the sample surface in a 1 cm x 1 cm area was 0.4-0.6 um, the compressive strength was 30-50 Mpa, and the Young's modulus was 20-30 GPa.

[0108] Example 6:

[0109] The biological tooth root support was prepared according to the method described in Example 1, and the only difference from Example 1 was that:

[0110] In the step (2), the hydroxyapatite was replaced by:

[0111] The hydroxyapatite was replaced by biphasic calcium phosphate (HA:TCP mass ratio 1:1).

[0112] In the step (3), the printing parameters were set as follows:

[0113] The printing parameters were 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 was to maintain the temperature at 1520 °C for 2 h.

[0114] The final biological tooth root support had whiskers at the interface with a length of 500-700 nm and a width of 2-5 nm, and the CMC-BAPE@BMP7 nanoparticles had a particle size range of 200-400 nm, and the deposition rate of the nanoparticles per unit surface area in the 3D-printed biological tooth root support matrix was 10-25 per um 2, the porosity of the scaffold 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.

[0115] Comparative Example 1:

[0116] The biological tooth root scaffold was prepared according to the method of Example 1, and the difference from Example 1 was only that step (4) was omitted.

[0117] The interfacial whisker structure in the finally prepared biological tooth root scaffold was not activated, the CMC-BAPE@BMP7 nanoparticle particle size range was 200-400 nm, and the unit surface area deposition rate thereof in the 3D printed biological tooth root scaffold matrix was 10-25 / um 2 , the porosity of the scaffold is 5-10%, the surface roughness Ra of the sample surface 1 cm x 1 cm area based on the needle tracing method is 0.1-0.2 um, the compressive strength is 30-50 Mpa, and the Young's modulus is 20-30 GPa.

[0118] Comparative Example 2:

[0119] The biological tooth root scaffold was prepared according to the method of Example 1, and the difference from Example 1 was only that step (5) was omitted.

[0120] The whisker in the interfacial interface of the finally prepared biological tooth root scaffold was 500-700 nm long and 2-5 nm wide, the porosity of the scaffold was 5-10%, the surface roughness Ra of the sample surface 1 cm x 1 cm area based on the needle tracing method was 0.4-0.6 um, the compressive strength was 30-50 Mpa, and the Young's modulus was 20-30 GPa.

[0121] Comparative Example 3:

[0122] The biological tooth root scaffold was prepared according to the method of Example 1, and the difference from Example 1 was only that:

[0123] In the step (2):

[0124] 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, 21 g of yttrium oxide stabilized tetragonal zirconium oxide (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.

[0125] The final biological tooth root scaffold was prepared by adding 55 wt% HA and 10 wt% Y-TZP.

[0126] Comparative Example 4:

[0127] Biological tooth root scaffolds were prepared according to the method described in Example 1, with the only difference from Example 1 being:

[0128] In step (2):

[0129] A photosensitive resin was prepared by uniformly mixing 60g of polyurethane acrylate and 20g of polyethylene glycol acrylate. 120g of hydroxyapatite powder, the above photosensitive resin, 5g of photoinitiator and 5g of dispersant were ball-milled and mixed uniformly in a ball mill to prepare 3D printing ink. The mass fraction of hydroxyapatite powder in the 3D printing ink was 65%, and the viscosity of the 3D printing ink was 600mPa·s at a shear rate of 0.1 / s.

[0130] The final biological tooth root scaffold was prepared using 65wt%HA + 0wt% Y-TZP.

[0131] Functional comparison experiment 1:

[0132] Using this invention to prepare DLP 3D printed tooth root scaffolds, and after 3 months of rat kidney capsule transplantation with CMC-BAPE@BMP7 / GelMA (50 ng / ml) composite membranes inside the root canal and peripheral composite dental follicle stem cell (DFSC) membranes, it was found that compared with the control group with composite CMC-BAPE and DFSC membranes, the CMC-BAPE@BMP7 group had larger and more cellularly abundant tooth roots. Figure 8 a1), with abundant DSP-positive expression of neodentin on the inner side of the framework ( Figure 8 a2-a3: ND: new dentin), with BSP-positive expression of new cementum visible on the lateral side. Figure 8 a4-a5), and there are obvious blood vessels in the root canal ( Figure 8 (a6 red arrow) and CD31-positive angiogenesis-NF-positive nerve tract formation ( Figure 8 a7) and obvious calcified parenchymal structures are visible within the medullary cavity ( Figure 8 a8), while the outer side of the root relative to the pulp cavity has a large number of inflammatory cell infiltrations ( Figure 8 a9) also indicates that the CMC-BAPE@BMP7 compounded on the inner side of the medullary cavity has a good effect on transplant inflammation, while in the CMC-BAPE group ( Figure 8 B), short biological tooth roots with sparse cells ( Figure 8 b1), with fewer mesenchymal blood vessels and nerves ( Figure 8 b2) Simultaneously, the newly formed solid structure—dentin—is irregular and thin. Figure 8(b3). The above results demonstrate that the DLP 3D printed root framework composite CMC-BAPE@BMP7 of the present invention can promote the differentiation of the DFSC into a solid / mesenchymal type in the root modality and has a certain anti-inflammatory effect.

[0133] Functional comparison experiment 2:

[0134] The periodontal ligament regeneration effect of this invention was further verified using an in vivo biological tooth root regeneration model of a large animal, the beagle dog. Specifically, the first premolar tooth germ of a 6-month-old beagle dog was obtained ( Figure 10 A), extract dental follicle tissue from the outer layer of the tooth germ during its developmental stage ( Figure 10 B), further isolated and cultured in vitro into dental follicle stem cell sheet-DFSC ( Figure 10 C), and then using the bio-ink based on hydroxyapatite-zirconia / zinc oxide as the main component in this invention, a bioceramic tooth root framework with a height of 6mm, a thickness of 1.5mm, and a hollow center of 1mm was prepared by DLP-3D printing. Figure 10 D), with 5% pure GelMA (control group), GelMA mixture containing 1 mg / ml BMP7 (BMP7 group), and GelMA mixture containing 1 mg / ml CMC-BAPE@BMP7 (CMC-BAPE@BMP7 group) as different experimental groups. Figure 10 E) After soaking the 3D-printed bioceramic tooth root framework in various drugs, a composite canine DFSC membrane is then applied to its outer layer to form a composite bio-tooth root model implanted in the body. Figure 10 F), after extracting the mandibular fourth premolar of a 1-year-old adult beagle, different composite biological roots (4 samples per group) were immediately implanted. X-ray images showed that the composite biological roots were in relatively good position after implantation. Figure 10 G), three months later, the implanted tooth root area healed well, and X-ray showed no bone resorption around the biological tooth root framework. Figure 10 H). Further painless euthanasia of the beagle dogs to obtain regenerative samples ( Figure 9 Histological examination was performed using hard tissue sections (30µm thick). Figure 10 I), the results are as follows: In the control group, the scaffold was embedded below the gum line and surrounded by alveolar bone (I), Figure 10 i), HE staining results further showed obvious cell adhesion on the outer side (i), Figure 10 ii) On the inner side, homogeneous new tissue formation is visible ( Figure 10 (iii) White pentagrams), Masson staining results showed that these cells belonged to collagen fibroblasts, and lacked obvious regenerated microvessels, while the homogeneous blue staining indicated that DFSCs formed collagen components secreted on the scaffold surface. Figure 10 iv). Meanwhile, the BMP7 group showed significant adhesion between the scaffold and the alveolar bone ( Figure 10v), and the formation of new eosinophilic material can also be seen in the stent Figure 10 vi white pentagram), and the adhesion of the stent to the alveolar bone can be seen on the outside of the stent Figure 10 vii white triangle), and the Masson staining result shows that the eosinophilic new material is collagen component Figure 10 viii white pentagram), and a small amount of blood vessel formation (red arrow) can be seen inside, indicating that after direct combination with BMP7, the over-secretion of collagen by DFSC is promoted, and the matrix mineralization and angiogenic induction ability are also increased; in contrast, the CMC-BAPE@BMP7 group is characterized by the presence of a relatively obvious eosinophilic periodontal ligament-like gap structure around the implanted 3D printed material Figure 10 ix), and further HE magnification shows that whether it is the upper part of the tooth root Figure 10 x) or the lower part of the tooth root Figure 10 xi), a large number of periodontal fiber attachment structures can be seen (white arrow), and the periodontal fibers are parallel and orderly arranged (green dotted arrow), and there are also a large number of microvascular structures (red arrow) between them, and the Masson result Figure 10 xii) also shows a similar regeneration trend, indicating that compared with pure BMP7 induction, the CMC-BAPE@BMP7 combination with the DLP-3D printed biological tooth root support prepared by the present application can promote the controllable differentiation of DFSC into periodontal ligament-like structures, thereby promoting the formation of periodontal attachment structures and microvessels.

Claims

1. A 3D-printed biological tooth root scaffold, characterized in that, The invention comprises a 3D-printed bio-dental root scaffold matrix and CMC-BAPE@BMP7 nanoparticles. The CMC-BAPE@BMP7 nanoparticles are carboxylated chitosan-pinacol ester-recombinant human bone morphogenetic protein-7 nanoparticles. The surface of the 3D-printed bio-dental root scaffold matrix has an activated nanocrystal structure. The CMC-BAPE@BMP7 nanoparticles are adsorbed and immobilized within the 3D-printed bio-dental root scaffold matrix. The 3D-printed bio-dental root scaffold matrix is ​​prepared using DLP 3D printing technology. The 3D-printed bio-dental root scaffold matrix is ​​prepared using calcium phosphate bioceramics with added inorganic oxides, specifically a zirconium oxide / zinc oxide mixture.

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

3. The 3D-printed biological tooth root scaffold according to claim 1, characterized in that, The activated nanowhisker structure is an ordered nanowhisker structure formed on the surface of a 3D-printed biological tooth root scaffold matrix, with a length of 500-700 nm and a width of 2-5 nm.

4. The 3D-printed biological tooth root scaffold according to claim 1, characterized in that, The scaffold has a porosity of 5-10%, a surface roughness Ra of 0.4-0.6 μm, a compressive strength of 30-50 MPa, and a Young's modulus of 20-30 GPa.

5. The method for preparing a 3D-printed biological tooth root scaffold according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Construct a three-dimensional model of a biological tooth root; (2) Preparation of 3D printing ink; (3) Prepare biological tooth root scaffold matrix by 3D printing and sinter it; (4) Activation of nanocrystal structure at the interface of biological tooth root scaffold matrix; (5) Composite CMC-BAPE@BMP7 nanoparticles.

6. The preparation method according to claim 5, characterized in that, Specifically, the following steps are included: (1) Constructing a three-dimensional model of a biological tooth root: By acquiring imaging data of missing teeth, constructing the original three-dimensional data model of the missing teeth, and optimizing the undercut structure and performing three-dimensional finite element analysis, a personalized three-dimensional data model of the biological tooth root is obtained. (2) Preparation of 3D printing ink: 3D printing ink is prepared by uniformly mixing calcium phosphate bioceramic powder with inorganic oxide toughened ceramic powder, photosensitive resin, photoinitiator and dispersant. (3) Fabrication and sintering of biological tooth root scaffold matrix by 3D printing: Based on the three-dimensional data model of the personalized biological tooth root obtained in step (1), the biological tooth root framework blank is obtained by 3D printing using the 3D printing ink prepared in step (2), and then sintered to obtain the biological tooth root framework matrix. (4) Activation of nanocrystal structure at the interface of biological tooth root scaffold matrix: The surface and interface of the biological tooth root scaffold matrix obtained in step (3) are deposited with activated nanocrystal whisker structures; (5) Composite CMC-BAPE@BMP7 nanoparticles: The bio-root scaffold matrix with activated surface whiskers prepared in step (4) was combined with CMC-BAPE@BMP7 nanoparticles.

7. The preparation method according to claim 5, characterized in that, Specifically, the following steps are included: (1) Constructing a three-dimensional model of a biological tooth root: All imaging data of the missing tooth were acquired using cone-beam CT. Then, the original three-dimensional data model of the missing tooth was constructed by segmenting using the gray-scale thresholding method. By optimizing the undercut structure and performing three-dimensional finite element analysis, the completed tooth root model was exported to STL format to obtain a personalized three-dimensional data model of the biological tooth root. (2) Preparation of 3D printing ink: A photosensitive resin is prepared by uniformly mixing polyurethane acrylate and polyethylene glycol acrylate. Hydroxyapatite powder, a zirconium oxide / zinc oxide mixture powder, the photosensitive resin, a photoinitiator, and a dispersant are ball-milled and mixed uniformly in a ball mill to prepare 3D printing ink. The 3D printing ink contains more than 60% hydroxyapatite powder by mass, and the zirconium oxide / zinc oxide mixture by mass is 5%-20%. The viscosity of the 3D printing ink is between 400 and 800 Pas at a shear rate of 0.1 / s. (3) Fabrication and sintering of biological tooth root scaffold matrix by 3D printing: Based on 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 ink is cured layer by layer. The biological tooth root framework blank is obtained by 3D printing. After printing, the uncured printing ink in the blank is removed by an ultrasonic dental treatment instrument. The blank is gradually degreased and sintered, and maintained at 300℃ and 1100℃ for 6 hours respectively. Finally, it is maintained at 1450℃-1550℃ for 2-4 hours, and then cooled to room temperature with the furnace to obtain the biological tooth root framework matrix. (4) Activation of nanocrystal structure at the interface of biological tooth root scaffold matrix: The biological tooth root scaffold matrix obtained in step (3) was put into a hydroxyapatite whisker deposition system containing 1:1.5 NaH2PO4 and EDTA. Ammonia and nitric acid were used to maintain the reaction concentration pH at 8.0 and the system was kept at 200°C for 8 hours. Through hydrothermal reaction, an ordered nano-whisker structure was formed on the surface of the matrix. (5) Composite CMC-BAPE@BMP7 nanoparticles: After freeze-drying the biological tooth root scaffold matrix prepared in step (4) with activated surface whiskers, it was immersed at room temperature in a colloidal solution of 1 mg / mL CMC-BAPE@BMP7 nanoparticles for 12 h to obtain a biological tooth root scaffold composite with CMC-BAPE@BMP7 nanoparticles.

8. The application of the 3D-printed biological tooth root scaffold according to any one of claims 1-4, characterized in that, It is used to prepare materials for tooth loss restoration.

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