A method for the preparation of a temperature-sensitive injectable shapeable mineralized material

Abstract

The application discloses a preparation method and application of a temperature-sensitive injectable plastic mineralization material, and the material can be stored for a long time and is used for inducing tooth mineralization. By combining amorphous calcium phosphate with an amphiphilic surfactant, dispersion and stability of the amorphous calcium phosphate are promoted. The mineralization material obtained by loading synthetic and dried amorphous calcium phosphate particles on the amphiphilic surfactant is in a solid state and cannot be plasticized at room temperature, can be injected and plasticized when heated to a certain temperature, and has a better mineralization effect compared with the dried amorphous calcium phosphate particles. The solid bulk material at room temperature is beneficial to packaging, storage and use. When coated on teeth in the oral cavity, the material gradually melts and dissolves at the normal oral cavity temperature, spontaneously forms high-concentration micelle-loaded amorphous calcium phosphate nanoparticles in a solution on the tooth surface, and plays a mineralization role.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials engineering technology, particularly the field of biomineralization. Specifically, it relates to a method for preparing a temperature-sensitive, injectable, and malleable mineralized material, which can be applied to the mineralization of type I collagen and dentin. Background Technology

[0002] Dental caries is a common chronic oral disease, and demineralization of dentin collagen is one of its manifestations. During dentin bonding, the interstitial spaces within the collagen fibers of the demineralized dentin layer cannot be fully penetrated by the bonding agent. These spaces become filled with water and endogenous matrix metalloproteinases, leading to collagen degradation and reduced dentin bond durability. Therefore, achieving the remineralization of demineralized dentin collagen has significant practical value in the field of dentistry.

[0003] Biomimetic mineralization of dentin has been widely recognized by researchers as a minimally invasive method for treating dental caries. In biological growth and development, dentin mineralization is a typical form of biomineralization, where amorphous calcium phosphate nanoparticles induce odontoblasts to generate mineralized tissue with hydroxyapatite (HAp) crystals as the main inorganic component, using collagen fibers as a template and under the regulation of non-collagenous proteins. To simulate this mineralization process, researchers have prepared inexpensive polyelectrolytes to replace non-collagenous proteins and induce collagen remineralization. Gower et al. used polyaspartic acid (PAsp) as a non-collagenous protein analog to achieve intrafibrillary mineralization of collagen fibers, proposing the polymer-induced liquid precursor (PILP) theory. In solution, the polyelectrolyte acts as a nucleation inhibitor, chelating calcium ions with carboxyl groups, which then attract phosphate groups, forming an amorphous calcium phosphate liquid precursor under the stabilization of the polyelectrolyte. In vivo, the amorphous calcium phosphate is transported to the collagen fiber scaffold via vesicles, where it is mineralized.

[0004] Most published studies currently report on the use of solutions or pastes as carriers for amorphous calcium phosphate to remineralize demineralized dentin or reconstituted type I collagen fibers. However, amorphous calcium phosphate cannot be delivered stably to the tooth surface in solutions (such as mouthwash) or pastes (dental protectants) for extended periods. Using Portland Cement as a slow-release calcium ion source, immersion in a solution containing phosphorus and polyelectrolytes can remineralize demineralized dentin. However, this method takes four months and has limited clinical applicability. Placing amorphous calcium phosphate in adhesives can also induce dentin remineralization and dentin sealing, but this requires several weeks. In patent CN106539693A, loading amorphous calcium phosphate onto a polymeric film-forming material can also remineralize experimentally demineralized dentin; however, the film preparation process is complex, the final shape is fixed, and it needs to be replaced daily. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems of dental mineralizing fluid being difficult to store for a long time, having few solid application modes, and requiring frequent replenishment of mineralizing sources, and to provide a method for preparing a temperature-sensitive, injectable, and malleable mineralizing material.

[0006] This invention provides a method for preparing a temperature-sensitive, injectable, and malleable mineralized material, comprising the following steps:

[0007] 1) Mix the calcium salt solution containing polyelectrolytes with the phosphate solution under stirring, adjust the pH to 9-10; remove the supernatant by centrifugation, wash, centrifuge and dry, grind and pulverize to obtain dry amorphous calcium phosphate nanoparticles;

[0008] 2) Dry amorphous calcium phosphate nanoparticles are uniformly mixed and ground with molten amphiphilic surfactant, shaped and then cooled and solidified to obtain a mineralized material; the amphiphilic surfactant has a melting point range of 30~99 ℃, can form micelles in aqueous solution, and has at least one or more groups of ether / hydroxy / amino / carboxyl groups.

[0009] As a preferred embodiment of the present invention, in step 1), the polyelectrolyte is one or more of polyacrylic acid, polyaspartic acid, polyglutamic acid, and carboxymethyl chitosan, and its concentration in the calcium salt solution is 100 μg / ml-50 g / L; the calcium salt is one or more of calcium chloride, calcium acetate, calcium nitrate, and calcium gluconate, and its concentration in the calcium salt solution is 1 mM to 1 M; the phosphate is one or more of disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, trisodium phosphate, and tripotassium phosphate, and its concentration in the phosphate solution is 0.6 mM to 0.6 M.

[0010] As a preferred embodiment of the present invention, in step 1), the molar concentration ratio of Ca:P after mixing the calcium salt solution and the phosphate solution is 10:(5~6).

[0011] As a preferred embodiment of the present invention, in step 1), the washing is performed using water and anhydrous ethanol, and the drying method is one or more of the following: freeze-drying at low temperature, vacuum drying at room temperature, drying in an oven at 50~70℃, and spray drying.

[0012] As a preferred embodiment of the present invention, the amphiphilic surfactant is one or more of vitamin E polyethylene glycol succinate (TPGS), carboxyl-modified vitamin E polyethylene glycol succinate (TPGS-COOH), and Pluronic copolymer; the mass ratio of the amphiphilic surfactant to amorphous calcium phosphate powder particles is 2-5:1.

[0013] Furthermore, this invention first synthesizes and dries amorphous calcium phosphate nanoparticles, then encapsulates these nanoparticles with an amphiphilic surfactant to form a temperature-sensitive, injectable, and shapeable mineralized material. This material is applied in a form that is solid at room temperature and gradually dissolves and releases the amorphous calcium phosphate nanoparticles at oral temperatures, thereby achieving the remineralization of demineralized dentin and the mineralization of collagen.

[0014] Furthermore, in the preparation of the temperature-sensitive injectable and malleable mineralized material, sweeteners such as sucrose, glucose, saccharin, potassium butyrate, cyclohexanesulfonate, etc., one or more, can be added, with the amount added accounting for 0.1-1% of the material mass fraction.

[0015] Furthermore, in the preparation of the thermosensitive injectable and malleable mineralized material, an appropriate amount of wetting agent may be added, such as one or more of glycerol, xylitol, propylene glycol, glycerol (glycerol), sorbitol, erythritol, lactitol, and mannitol.

[0016] This invention also provides a thermosensitive, injectable, and shape-forming mineralized material prepared by the above method. The amphiphilic surfactant is vitamin E polyethylene glycol succinate (TPGS). The mineralized material is shape-forming and injectable when heated to above 38 °C, and can revert to a solid state below 34 °C. Vitamin E polyethylene glycol succinate (TPGS) acts as a carrier, being solid at room temperature and liquid above 38 °C, and spontaneously forming micelles in aqueous solution. Previous research results have found that the thermosensitive, injectable, and shape-forming mineralized material mentioned in this patent can maintain an amorphous state for at least one year when stored in a sealed tube at 4 °C, exhibiting mineralization capability. TPGS is used as a carrier for dried amorphous calcium phosphate nanoparticles, serving as a calcium reservoir for long-term release of calcium and phosphorus. After slow dissolution, TPGS forms micelles that encapsulate the amorphous calcium phosphate nanoparticles. Furthermore, the interaction between TPGS and amorphous calcium phosphate achieves effective dispersion and stability of the amorphous calcium phosphate, making it easier to penetrate collagen fibers and exert a better mineralization effect within the collagen fibers.

[0017] The present invention further provides the application of the above-mentioned thermosensitive, injectable, and malleable mineralized material in the induced in vitro recombinant type I collagen mineralization.

[0018] The present invention further provides the application of the above-mentioned thermosensitive, injectable, and malleable mineralizing material in in vitro induced demineralized dentin remineralization.

[0019] The present invention further provides a method for preparing a mineralized material containing antibacterial function. In the above preparation method, an antibacterial functional component is added in step 1) and / or step 2) to obtain a temperature-sensitive injectable and malleable mineralized material with antibacterial mineralization function.

[0020] The method for adding antibacterial functional components in step 1) is as follows: during the mixing process in step 1), one or more of zinc, silver, magnesium, and rare earth elements are incorporated.

[0021] The method for adding antibacterial functional components in step 2) is as follows: add one or more antibacterial oxides from nano zinc oxide, nano silver oxide, nano magnesium oxide, and nano cerium oxide to the amphiphilic surfactant in the molten state in step 2).

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1) This invention uses amphiphilic surfactants (such as TPGS, TPGS-COOH, Pluronic copolymers, etc.) as carriers for dried amorphous calcium phosphate nanoparticles, which have high drug loading capacity and bioavailability. They are solid at room temperature and can reach their melting point at a low temperature. They have advantages such as simple mixing, injectability, convenient application, and suitability for long-term storage.

[0024] 2) The amphiphilic surfactant forms high-concentration micelles loaded with amorphous calcium phosphate locally on the tooth surface, and can disperse and stabilize amorphous calcium phosphate, which is conducive to the penetration of amorphous calcium phosphate into collagen fibers, realizing remineralization with dentin demineralized collagen fibers as templates, and restoring the special striped hierarchical structure of mineralized collagen.

[0025] 3) Compared with the original dentin mineralization solution, the solid application method is more convenient to use, and the amorphous calcium phosphate encapsulated by the amphiphilic surfactant has a stable slow-release function, which does not require the calcium and phosphorus source to be replaced regularly, thus having an application advantage.

[0026] 4) The mineralized material obtained by this invention has good biocompatibility.

[0027] 5) The mineralized material obtained by this invention can achieve mineralization of about 5 micrometers of demineralized dentin within 6 days after being soaked in artificial saliva, and has a relatively fast mineralization speed. Attached Figure Description

[0028] Figure 1 This thermosensitive, injectable, and malleable mineralized material can be molded into various shapes.

[0029] Figure 2 Fourier transform infrared (FTIR) spectra of thermosensitive, injectable, and malleable mineralized materials.

[0030] Figure 3 The morphology observed by transmission electron microscope (TEM) after the dried amorphous calcium phosphate nanoparticles (A) and the thermosensitive injectable and malleable mineralized material described in this invention were dissolved in an aqueous solution for 1 day.

[0031] Figure 4 The Raman spectra are for TPGS solution (solid line) and TPGS + calcium chloride (dashed line).

[0032] Figure 5 TEM image of mineralized recombinant type I collagen from the thermosensitive injectable and malleable mineralized material used in Case 1.

[0033] Figure 6 TEM and SAED images of the gradual mineralization of dentin after demineralization in the implementation case. Detailed Implementation

[0034] The technical solution of this invention is only applicable to the processing of recombinant type I collagen and extracted teeth separated from living biological tissue. This method is for non-therapeutic purposes only.

[0035] Implementation Case 1:

[0036] 1) Synthesis of amorphous calcium phosphate particles: 500 ml of 10 mM CaCl2·H2O was mixed with 480 μg / ml of polyaspartic acid and an equal amount of 6 mM K2HPO4 under vigorous stirring until homogeneous. The pH was adjusted to 9.5 using 5 M NaOH. After centrifugation to obtain the precipitate, the precipitate was washed with water and ethanol, and then vacuum dried overnight in a vacuum drying oven at room temperature. The precipitate was then ground to obtain amorphous calcium phosphate powder.

[0037] 2) Obtaining a temperature-sensitive, injectable, and malleable mineralized material: TPGS block solid was heated to 40 °C to melt, and then mixed evenly with amorphous calcium phosphate powder. The mass ratio of TPGS to amorphous calcium phosphate powder was 2:1. Before use, Fourier transform infrared spectroscopy (FTIR) was used to characterize whether the amorphous calcium phosphate underwent a phase transition. Its morphology under a TEM microscope was observed one day after dissolving in aqueous solution. Raman spectra of TPGS and TPGS + calcium chloride solution were used to investigate the interaction mechanism between TPGS and calcium.

[0038] Figure 1 shows the thermosensitive, injectable, and malleable mineralized material obtained in Implementation Case 1, demonstrating that the material can be molded into various shapes.

[0039] Figure 2 This is the Fourier transform infrared (FTIR) spectrum of the thermosensitive, injectable, and malleable mineralized material obtained in Case Study 1. The spectrum is located at 560 cm⁻¹. -1 The peak at the point did not show any bifurcation, which is a characteristic peak of amorphous calcium phosphate, indicating that the calcium phosphate in this mineralized material is still in an amorphous state and has mineralization activity.

[0040] Figure 3The morphology of dried amorphous calcium phosphate nanoparticles (A) and the thermosensitive injectable and malleable mineralized material described in this invention, after being dissolved in an aqueous solution for 1 day, was observed by TEM. This result indicates that the amorphous calcium phosphate in the thermosensitive injectable and malleable mineralized material is more dispersed and has a smaller particle size. Smaller amorphous calcium phosphate particles generally penetrate the gap regions of collagen more easily, thereby inducing faster intrafibrillary mineralization of collagen fibers.

[0041] Figure 4 The Raman spectra of TPGS solution (solid line) and TPGS + calcium chloride (dashed line) are shown. The results indicate that the characteristic peak of TPGS in the 1200–800 nm range (corresponding to CO) changed after the addition of calcium chloride, suggesting that TPGS can interact with calcium ions. Therefore, the hypothetical mechanism is that the CO group in TPGS can interact with calcium ions in amorphous calcium phosphate, and the steric hindrance of surfactant molecules promotes the dispersion and reduction of particle size of amorphous calcium phosphate.

[0042] Implementation Case 2:

[0043] 1) Synthesis of amorphous calcium phosphate particles: 100 ml of 20 mM CaCl2·H2O was mixed with 2000 μg / ml polyacrylic acid and an equal amount of 12 mM K2HPO4 under vigorous stirring until homogeneous. The pH was adjusted to 9.5 using 5 M NaOH. After centrifugation to obtain the precipitate, the precipitate was washed with water and ethanol, and then vacuum dried overnight in a vacuum drying oven at room temperature. The precipitate was then ground to obtain amorphous calcium phosphate powder.

[0044] 2) Obtaining a thermosensitive, injectable, and malleable mineralized material: The Pluronic copolymer: poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) is heated to 55 ℃ and melted, and then mixed evenly with amorphous calcium phosphate powder for later use. The mass ratio of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol):amorphous calcium phosphate powder is 3:1.

[0045] Implementation Case 3:

[0046] 1) Synthesis of amorphous calcium phosphate particles: 200 ml of 10 mM CaCl2·H2O, 1 mM silver chloride, 500 μg / ml polyglutamic acid, and an equal amount of 6 mM K2HPO4 were mixed uniformly under vigorous stirring, and the pH was adjusted to 9.5 with 5 M NaOH. After centrifugation to obtain the precipitate, the precipitate was washed with water and ethanol, and vacuum dried overnight in a vacuum drying oven at room temperature. The precipitate was then ground to obtain silver-doped amorphous calcium phosphate powder.

[0047] 2) Obtain a thermosensitive, injectable, and malleable mineralized material with antibacterial properties: Heat TPGS to 40 ℃ to melt it, and mix it evenly with silver-doped amorphous calcium phosphate powder for later use. The mass ratio of TPGS to silver-doped amorphous calcium phosphate powder is 3:1.

[0048] Implementation Case 4:

[0049] 1) Synthesis of amorphous calcium phosphate particles: 10 ml of 100 mM CaCl2·H2O was mixed with 5000 μg / ml polyaspartic acid and an equal amount of 60 mM K2HPO4 under vigorous stirring until homogeneous. The pH was adjusted to 9.5 using 5 M NaOH. After centrifugation to obtain the precipitate, the precipitate was washed with water and ethanol, and then vacuum dried overnight in a vacuum drying oven at room temperature. The precipitate was then ground to obtain amorphous calcium phosphate powder.

[0050] 2) Obtain a thermosensitive, injectable, and malleable mineralized material with antibacterial properties: Heat TPGS to 40 ℃ to melt it, and mix it evenly with amorphous calcium phosphate powder and zinc oxide nanoparticles (10 nm diameter) for later use (the mass ratio of TPGS: amorphous calcium phosphate powder: zinc oxide is 40:15:1).

[0051] Implementation Case 5: Construction and Mineralization of Monolayer Reconstructed Collagen Fibers:

[0052] 1) Mix 8.3 μl of rat tail collagen stock solution with 0.5 ml of collagen buffer solution, let stand at room temperature for 20 min, then drop 3 μl onto a nickel grid, incubate overnight at 37°C, crosslink with glutaraldehyde, rinse with deionized water, and air dry for later use. The collagen buffer solution contains 50 mM glycine, 200 mM potassium chloride, and the pH is adjusted to 9.2.

[0053] 2) Mineralization of monolayer reconstituted collagen fibers: A thermosensitive, injectable, and malleable mineralizing material was melted at 40°C and coated onto the surface of a nickel mesh containing reconstituted collagen fibers. The mesh was then inverted and placed in 600 μl of artificial saliva; alternatively, the thermosensitive, injectable, and malleable mineralizing material was dissolved in 600 μl of artificial saliva, and the reconstituted collagen fiber nickel mesh was floated inverted in the artificial saliva. The mesh was then incubated at 37°C for 1–4 days, and the mineralization of the collagen was observed using TEM combined with SAED (Self-Improving Electrode Array) observation.

[0054] Figure 5 TEM image of mineralized recombinant type I collagen from the thermosensitive injectable and malleable mineralized material used in Case 1. Figure 5Images A and B represent the control group, showing the effect of using only amorphous calcium phosphate particles to mineralize collagen for 4 days (B is a magnified view of A). Some collagen fibers turned black, with a significant amount of exposed, transparent, unmineralized collagen and considerable external mineralization. Images C and D show the effect of using a thermosensitive, injectable, and malleable mineralizing material to mineralize and reconstitute type I collagen for 4 days. In these images, the collagen fibers all turned noticeably black, with crystalline substances forming inside. These results indicate that the thermosensitive, injectable, and malleable mineralizing material is more effective in mineralizing and reconstituted type I collagen compared to the control group using only amorphous calcium phosphate particles.

[0055] Implementation Case 6: Preparation and Mineralization of In Vitro Demineralized Dentin:

[0056] 1) Under continuous water flow, use an Isomet low-speed cutter to cut dentin slices perpendicular to the long axis of the crown of freshly extracted caries-free third molars. Prepare Class I cavities with a bur, treat with 37% phosphoric acid for 15 seconds, and rinse with plenty of deionized water for later use.

[0057] 2) Mineralization of demineralized dentin: Thermosensitive injectable and malleable mineralizing material is heated to malleable (38°C) and injected or filled into Class I cavities of dentin slices. The samples are then placed in artificial saliva for 1-7 days and placed in a 37°C constant temperature incubator to prepare dentin TEM observation slices. The remineralization of demineralized dentin is observed under an electron microscope.

[0058] The steps for preparing the dentin TEM slides are as follows:

[0059] Dentin samples were collected, rinsed with deionized water, dehydrated using an alcohol gradient, and treated with acetone:embedding medium in a 1:1 ratio for 1 hour, then with acetone:embedding medium in a 3:1 ratio for 3 hours. After embedding with pure embedding medium, the samples were dried overnight in a 70°C oven. The embedded dentin samples were then cut into ultrathin sections, and TEM was used to observe the mineralization.

[0060] Figure 6 TEM and SAED images of the gradual remineralization of dentin after demineralization in the implementation case. Figure 6 In the images, A, B, and C show TEM sections of demineralized dentin samples treated with a thermosensitive, injectable, and malleable mineralizing material for 1, 4, and 6 days, respectively (scale bar = 1 μm). In section A, after 1 day of treatment, a small amount of scattered mineralization is visible at the base of the demineralized dentin, with a noticeable, lighter-colored demineralized layer still present. In section B, after 4 days of treatment, a remineralized layer is visible, but its color is lighter compared to the unmineralized dentin. After 6 days of treatment, the demineralized layer is completely mineralized, showing no significant difference from the unmineralized dentin. Selected area electron diffraction (SAED) results show 002, 211, and 004 crystal rings, consistent with the characteristic parameters of hydroxyapatite. These results indicate that the thermosensitive, injectable, and malleable mineralizing material can induce remineralization of demineralized dentin in vitro.

[0061] The above-described embodiments are merely one preferred embodiment of the present invention and are not intended to limit the invention. Various adjustments and modifications can be made without departing from the scope of the invention. Therefore, all technical solutions obtained by equivalent substitution and equivalent transformation are within the protection scope of the present invention.

Claims

1. A method of preparing a temperature-sensitive injectable shapeable dental mineralizing material, characterized in that, The method comprises the following steps: 1) mixing a calcium salt solution containing a polyelectrolyte with a phosphate solution under stirring, adjusting the pH to 9-10; removing the supernatant by centrifugation, washing the centrifugate, drying, grinding, and obtaining dry amorphous calcium phosphate nanoparticles; 2) uniformly mixing the dry amorphous calcium phosphate nanoparticles with a melting state amphiphilic surfactant, uniformly grinding, shaping, and cooling and solidifying to obtain a warm-sensitive injectable and plastic-shaped tooth mineralization material; wherein the amphiphilic surfactant has a melting point range of 30-99 ℃, can form micelles in an aqueous solution, and has at least one or more of ether groups, hydroxyl groups, amino groups, and carboxyl groups; the amphiphilic surfactant is one or more of vitamin E polyethylene glycol succinate (TPGS) and carboxyl-modified vitamin E polyethylene glycol succinate (TPGS-COOH); and the mass ratio of the amphiphilic surfactant to the amorphous calcium phosphate powder particles is 2-5:

1.

2. The production method according to claim 1, characterized by, In step 1), the polyelectrolyte is one or more of polyacrylic acid, polyaspartic acid, polyglutamic acid, and carboxymethyl chitosan, and the concentration in the calcium salt solution is 100 μg / ml-50 g / L; the calcium salt is one or more of calcium chloride, calcium acetate, calcium nitrate, and calcium gluconate, and the concentration in the calcium salt solution is 1 mM-1 M; and the phosphate is one or more of disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, trisodium phosphate, and tripotassium phosphate, and the concentration in the phosphate solution is 0.6 mM-0.6 M.

3. The preparation method according to claim 1, characterized in that, In step 1), the molar concentration ratio of Ca:P after mixing the calcium salt solution and the phosphate solution is =10:(5-6).

4. The preparation method according to claim 1, characterized in that, In step 1), the washing uses water and anhydrous ethanol, and the drying method is one or more of freeze low-temperature vacuum drying, room-temperature vacuum drying, 50-70 ℃ oven drying, and spray drying.

5. A temperature-sensitive injectable moldable mineralized material prepared by the method of any one of claims 1-4, wherein, The amphiphilic surfactant is vitamin E polyethylene glycol succinate (TPGS), and the mineralization material can be plastic-shaped and injectable when heated to above 38 ℃, and can be restored to a solid material when below 34 ℃.

6. A method for preparing a tooth mineralization material with antibacterial function, characterized in that, In the preparation method of any one of claims 1-4, the warm-sensitive injectable and plastic-shaped tooth mineralization material with antibacterial mineralization function is obtained by adding an antibacterial functional component in step 1) and / or step 2); In step 1), the method for adding the antibacterial functional component is: incorporating one or more of zinc, silver, magnesium, and rare earth elements during the mixing process of step 1); In step 2), the method for adding the antibacterial functional component is: adding one or more of nano-zinc oxide, nano-silver oxide, nano-magnesium oxide, and nano-cerium oxide antibacterial oxides to the melting state amphiphilic surfactant in step 2).

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