A glucose solution-stable calcium phosphate ion cluster, its preparation method and application

Abstract

This invention relates to the field of dental restoration technology, providing a glucose solution-stabilized calcium phosphate ion cluster, its preparation method, and its application. The invention utilizes a glucose solution to stabilize calcium phosphate ion clusters. In a high-concentration glucose aqueous solution, calcium ions and phosphate groups aggregate to form ion clusters. Upon contact with saliva, the glucose dissolves, releasing the calcium phosphate ion clusters, which then attach to the enamel prism structure of the tooth, triggering a mineralization reaction. This promotes the deposition and growth of calcium phosphate crystals along a specific orientation, restoring the original morphology of the tooth and improving the strength and hardness of the enamel. Furthermore, the glucose solution used in this invention stabilizes the calcium phosphate ion clusters, resulting in clusters with a size <2 nm, allowing them to penetrate nanoscale demineralization pores and easily penetrate enamel micropores. This results in high mineralization efficiency, high enamel restoration strength, and the mineralization process does not require high temperature, high pressure, or ultraviolet irradiation, exhibiting good biosafety and high clinical translation feasibility, thus possessing broad application prospects in the field of dental restoration.

Description

Technical Field

[0001] This invention relates to the field of dental restoration technology, and in particular to a glucose solution-stabilized calcium phosphate ion cluster, its preparation method, and its application. Background Technology

[0002] Tooth enamel is the outermost layer of tissue covering the surface of teeth, mainly composed of hydroxyapatite crystals (Ca). 10 (PO4)6(OH)2 is formed by highly ordered stacking, while also containing a small amount of organic matrix and water. As the hardest tissue in the human body, tooth enamel not only protects the dentin and pulp from external physical and chemical stimuli, but also undertakes the important physiological functions of chewing and grinding food. Its integrity and mechanical properties directly determine oral health.

[0003] However, in daily life, tooth enamel is susceptible to damage from various factors, leading to structural destruction and functional degeneration. For example, acidic substances produced by bacterial metabolism in the mouth can trigger enamel demineralization, causing micropores to form on the enamel surface, reducing its hardness, and eventually leading to tooth decay. Long-term mechanical wear, alternating hot and cold stimuli, and improper oral care can also cause enamel wear, cracks, and other defects. In addition, as we age, the natural mineralization of enamel decreases, which also exacerbates the risk of damage. Because tooth enamel itself does not have the ability to regenerate, once substantial damage occurs, if it is not repaired in time, the damage will continue to progress, seriously affecting the normal function and aesthetics of the teeth, and even threatening the health of the dental pulp.

[0004] Since the main component of tooth enamel is hydroxyapatite, current enamel restoration materials mostly focus on mimicking the mineralization process of hydroxyapatite. This involves supplementing the surface of damaged enamel with mineralizing ions such as calcium and phosphorus to promote the formation of hydroxyapatite crystals, thus achieving repair and remineralization. However, existing restoration materials have low mineralization efficiency; calcium and phosphorus ions tend to aggregate rapidly within the restoration system, failing to efficiently penetrate the micropores and cracks in the damaged enamel, resulting in poor enamel recovery strength. Summary of the Invention

[0005] In view of this, the present invention provides a glucose solution-stabilized calcium phosphate ion cluster, its preparation method, and its application. The glucose solution-stabilized calcium phosphate ion cluster provided by the present invention has high mineralization efficiency, good enamel restoration strength, and good biocompatibility.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: A glucose solution-stable calcium phosphate ion cluster, comprising glucose, water, and calcium phosphate ion clusters; wherein the glucose mass fraction in the glucose solution-stable calcium phosphate ion cluster is 20-60%.

[0007] Preferably, the average size of the calcium phosphate ion clusters is <2 nm.

[0008] This invention also provides a method for preparing glucose solution-stabilized calcium phosphate ion clusters as described above, comprising the following steps: A calcium chloride aqueous solution and glucose are mixed to obtain a glucose-calcium chloride mixed solution; A phosphate aqueous solution and glucose are mixed to obtain a glucose-phosphate mixed solution; The glucose-calcium chloride mixture and the glucose-phosphate mixture are mixed to obtain stable calcium phosphate ion clusters in the glucose solution.

[0009] Preferably, the concentration of calcium chloride in the calcium chloride aqueous solution is 10~70 mmol / L.

[0010] Preferably, the concentration of phosphate in the phosphate aqueous solution is 6~42 mmol / L.

[0011] Preferably, the phosphate is trisodium phosphate.

[0012] Preferably, the glucose mass fraction in the glucose-calcium chloride mixed solution is 20-60%.

[0013] Preferably, the glucose-phosphate mixed solution contains 20-60% glucose by mass.

[0014] Preferably, the volume ratio of the glucose-calcium chloride mixed solution to the glucose-phosphate mixed solution is 0.1 to 10:1.

[0015] The present invention also provides the application of glucose solution-stabilized calcium phosphate ion clusters as described in the above-described scheme or glucose solution-stabilized calcium phosphate ion clusters prepared by the above-described preparation method in the preparation of dental restorative products.

[0016] This invention provides a glucose solution-stabilized calcium phosphate ion cluster, comprising glucose, water, and calcium phosphate ion clusters; the glucose mass fraction in the glucose solution-stabilized calcium phosphate ion cluster is 20-60%. This invention utilizes glucose solution to stabilize calcium phosphate ion clusters. In a high-concentration glucose aqueous solution, calcium ions and phosphate ions aggregate to form ion clusters (also known as glucose-stabilized calcium phosphate pre-nucleation clusters, abbreviated as G-CPIC). These ion clusters are stable amorphous structures. Upon contact with saliva, glucose dissolves, releasing calcium phosphate ion clusters, which attach to the enamel prism structure of hydroxyapatite in teeth, triggering a mineralization reaction. The surface of the G-CPIC is rich in active calcium. 2+ / PO4 3-This invention can induce the orderly growth of ion clusters, lower the nucleation energy barrier, and promote crystal deposition along specific orientations. After crystallization, calcium phosphate adheres and grows directionally along the enamel prism structure, restoring the original morphology of the tooth, and significantly improving the strength and hardness of the enamel. Furthermore, this invention uses glucose solution to stabilize calcium phosphate ion clusters, with cluster sizes <2 nm, allowing them to penetrate deep into nanoscale demineralization pores and easily penetrate enamel micropores, resulting in high mineralization efficiency and high enamel restoration strength. The mineralization process does not require high temperature, high pressure, or ultraviolet irradiation, exhibiting good biocompatibility and broad application prospects in the field of dental restoration (such as oral rinsing and chairside treatment), with high feasibility for clinical translation. Attached Figure Description

[0017] Figure 1 The particle size distribution of calcium phosphate ion clusters obtained in Example 1 of this invention; Figure 2 The X-ray diffraction pattern of the calcium phosphate ion cluster gel obtained in Example 1 of this invention; Figure 3 This is an example of how calcium phosphate ion clusters obtained in Example 1 of the present invention repair the surface of tooth enamel after demineralization treatment. Figure 4 This is the product obtained by centrifuging the calcium phosphate ion clusters obtained in Example 2 of the present invention in methanol; Figure 5 This is a transmission electron microscope image of the distribution of calcium phosphate ion clusters prepared in anhydrous ethanol in Example 3 of the present invention. Figure 6 The X-ray diffraction patterns of the calcium phosphate ion clusters obtained in anhydrous ethanol after vacuum drying and water treatment are shown in Example 3 of this invention. Figure 7 This is a laser confocal fluorescence image of calcium phosphate ion clusters labeled with calcein obtained in Example 1 of the present invention on demineralized tooth enamel after treatment. Figure 8 This is a comparison chart of tooth hardness after restoration using different products. Detailed Implementation

[0018] This invention provides a glucose solution-stable calcium phosphate ion cluster, comprising glucose, water, and calcium phosphate ion clusters; wherein the glucose mass fraction in the glucose solution-stable calcium phosphate ion cluster is 20-60%.

[0019] In this invention, the glucose aqueous solution contains 20-60% glucose by mass, specifically 20%, 30%, 40%, 50% or 60%; the average size of the calcium phosphate ion clusters is <2nm, and in the example it is 1.48nm.

[0020] This invention also provides a method for preparing glucose solution-stabilized calcium phosphate ion clusters as described above, comprising the following steps: A calcium chloride aqueous solution and glucose are mixed to obtain a glucose-calcium chloride mixed solution; A phosphate aqueous solution and glucose are mixed to obtain a glucose-phosphate mixed solution; The glucose-calcium chloride mixture and the glucose-phosphate mixture are mixed to obtain stable calcium phosphate ion clusters in the glucose solution.

[0021] This invention mixes an aqueous solution of calcium chloride and glucose to obtain a glucose-calcium chloride mixed solution. In this invention, the concentration of calcium chloride in the aqueous solution is preferably 10-70 mmol / L, specifically 30 mmol / L, 60 mmol / L, or 70 mmol / L; the mass fraction of glucose in the glucose-calcium chloride mixed solution is preferably 20-60%, specifically 50%. In a specific embodiment of this invention, the aqueous solution of calcium chloride and glucose can be mixed in equal weights to obtain a glucose-calcium chloride mixed solution with a glucose mass fraction of 50%.

[0022] This invention involves mixing an aqueous phosphate solution and glucose to obtain a glucose-phosphate mixed solution. In this invention, the concentration of phosphate in the aqueous phosphate solution is preferably 6-42 mmol / L, specifically 18 mmol / L, 36 mmol / L, or 42 mmol / L; the phosphate is preferably trisodium phosphate; the glucose mass fraction in the glucose-phosphate mixed solution is 20-60%, specifically 50%; in a specific embodiment of this invention, the aqueous phosphate solution and glucose can be mixed in equal weights to obtain a glucose-phosphate mixed solution with a glucose mass fraction of 50%; in a specific embodiment of this invention, the glucose concentration is the same in both the glucose-calcium chloride mixed solution and the glucose-phosphate mixed solution.

[0023] After obtaining the glucose-calcium chloride mixed solution and the glucose-phosphate mixed solution, the present invention mixes the glucose-stable calcium phosphate ion clusters with the glucose-calcium chloride mixed solution and the glucose-phosphate mixed solution to obtain the glucose-stable calcium phosphate ion clusters; the volume ratio of the glucose-calcium chloride mixed solution and the glucose-phosphate mixed solution is preferably 0.1~10:1, specifically 1:1; in a specific embodiment of the present invention, it is preferable to rapidly mix the glucose-calcium chloride mixed solution and the glucose-phosphate mixed solution and then let them stand.

[0024] This invention also provides the application of glucose solution-stabilized calcium phosphate ion clusters as described in the above-described scheme, or glucose solution-stabilized calcium phosphate ion clusters prepared by the above-described preparation method, in the preparation of dental restorative products. In this invention, the dental restorative product specifically promotes tooth mineralization. The glucose solution-stabilized calcium phosphate ion clusters provided by this invention can deliver a large number of amorphous, non-crystalline hydroxyapatite pre-formed nucleation clusters to the tooth surface, where, under the stimulation of saliva, in-situ crystallization and mineralization occur, forming enamel prism structures similar to the original enamel surface. Furthermore, the calcium phosphate ion clusters can effectively improve enamel demineralization lesions, thereby restoring enamel hardness. In summary, the calcium phosphate ion clusters provided by this invention can simultaneously achieve tooth tissue regeneration and tooth demineralization repair effects, and have broad application prospects in the field of dental restoration.

[0025] In a specific embodiment of the present invention, the glucose solution-stabilized calcium phosphate ion clusters can be dropped onto the surface of a tooth enamel sample for 5-60 minutes. After simple rinsing with simulated saliva, the sample is incubated in simulated saliva at a constant temperature of 25-40°C for 6-96 hours. During this period, a shaker is used to simulate human activity, with an oscillation frequency of 20-300 rpm. The mineralization effect is then tested.

[0026] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0027] The tooth enamel samples used in this embodiment of the invention are extracted teeth from patients collected clinically. The entire tooth was cut into 5mm pieces using a tooth-cutting machine. 5mm For small enamel pieces of about 3mm, use sandpaper to polish and remove tartar, pigments, impurities, etc. from the enamel surface to form a relatively flat surface. Then, use deionized water and ethanol for ultrasonic cleaning, followed by acid etching with 20-40% phosphoric acid solution for 10-60 seconds. After that, use deionized water and ethanol alternately for ultrasonic cleaning again, and then dry in the air for later use.

[0028] The simulated saliva used in the following examples is an inorganic salt buffer solution prepared to mimic the ionic strength, pH value, and buffer capacity of human oral saliva. The specific preparation method is as follows: 1.5 mmol / L calcium chloride, 0.9 mmol / L dipotassium hydrogen phosphate, 15 ppm sodium fluoride, 130 mmol / L potassium chloride, 1 mmol / L sodium azide, and 20 mmol / L Hepes buffer powder are added to sterile deionized water, the pH is adjusted to 7.0, and then stored at 4°C for later use.

[0029] Example 1 To prepare a 50% glucose-calcium chloride solution, add 10g of glucose to 10g of a 30mmol / L calcium chloride aqueous solution (solution A). To prepare a 50% glucose-calcium chloride solution, add 10g of glucose to 10g of a 18mmol / L trisodium phosphate aqueous solution (solution B). Mix equal volumes of solutions A and B rapidly with stirring, then allow to stand to obtain stable calcium phosphate ion clusters in the glucose solution.

[0030] The particle size of calcium phosphate ion clusters was characterized using a dynamic light scattering nanoparticle size analyzer, and the results are as follows: Figure 1 As shown. According to Figure 1 It can be seen that the calcium phosphate ion clusters prepared in this embodiment are uniform in size and have good dispersibility.

[0031] The glucose solution-stabilized calcium phosphate ion clusters prepared in Example 1 were centrifuged at 40,000 rcf to obtain a transparent gel-like product, denoted as G-CPIC Gel. The structure of G-CPIC Gel was characterized by X-ray diffraction, and the results are as follows: Figure 2 As shown. By Figure 2 It can be seen that the calcium phosphate ion clusters prepared in this embodiment have amorphous characteristic diffraction peaks at around 25 degrees; however, when G-CPIC Gel is dissolved in deionized water and glucose is dissolved in water, and then characterized by X-ray diffraction again, amorphous characteristic diffraction peaks at around 30 degrees, similar to those of amorphous calcium phosphate ACP, are obtained. Figure 2 (G-CPIC-Gel+H2O).

[0032] The glucose solution-stabilized calcium phosphate ion clusters prepared in Example 1 were dropped onto the surface of the enamel sample to be repaired. After standing for 20 minutes, the calcium phosphate ion clusters adhered to the enamel prism structure. Subsequently, the sample was placed in simulated saliva at 37°C for 24 hours for a phase transition. The mineralized enamel grew a new enamel structure along the original structure. Scanning electron microscopy revealed hydroxyapatite nanorod structures growing along the c-axis of the enamel prisms. Figure 3 As shown, there is no clear boundary with the original glaze column structure, and it appears to be an extensional growth.

[0033] Example 2 To prepare a 50% glucose-calcium chloride solution, add 10g of glucose to 10g of a 40mmol / L calcium chloride aqueous solution (solution A). To prepare a 50% glucose-calcium chloride solution, add 10g of glucose to 10g of a 24mmol / L trisodium phosphate aqueous solution (solution B). Mix equal volumes of solutions A and B rapidly with stirring, then allow to stand to obtain stable calcium phosphate ion clusters in the glucose solution.

[0034] When the stable calcium phosphate ion clusters prepared in Example 2 were rapidly added to 50 mL of methanol from 2 mL of glucose solution, the stable calcium phosphate ion clusters showed good dispersibility in methanol, remained stable in the solution, and exhibited a very pale blue color. Centrifugation at 40,000 rcf yielded a transparent gel-like product (e.g., ...). Figure 4 (As shown).

[0035] Example 3 To prepare a 50% glucose-calcium chloride solution, add 10g of glucose to 10g of a 70mmol / L calcium chloride aqueous solution (solution A). To prepare a 50% glucose-calcium chloride solution, add 10g of glucose to 10g of a 42mmol / L trisodium phosphate aqueous solution (solution B). Mix equal volumes of solutions A and B rapidly with stirring, then allow to stand to obtain stable calcium phosphate ion clusters in the glucose solution.

[0036] After rapidly adding the stable calcium phosphate ion clusters prepared in Example 3 (2 mL glucose solution) to 50 mL anhydrous ethanol, the morphology of the calcium phosphate ion clusters was characterized using transmission electron microscopy. The results are as follows: Figure 5 As shown. According to Figure 5 It can be seen that the calcium phosphate ion clusters prepared in Example 3 are uniform in size and well-dispersed. Selected area electron diffraction (SED) patterns show no obvious crystal diffraction rings, proving that amorphization has occurred. After adding the stable calcium phosphate ion clusters from 2 mL of glucose solution to 50 mL of anhydrous ethanol, centrifuging at 15000 rcf, and drying the resulting precipitate under vacuum at room temperature, the structure was characterized by X-ray diffraction. The results are as follows... Figure 6 As shown, by Figure 6 It can be seen that the calcium phosphate ion clusters prepared in this embodiment are a mixture of glucose and amorphous calcium phosphate characteristic diffraction peaks. After adding water to the precipitate to dissolve the glucose, the diffraction peaks are restored to the amorphous characteristic diffraction peaks.

[0037] In vitro mineralization experiment In vitro mineralization experiments were conducted on the glucose solution-stabilized calcium phosphate ion clusters obtained in Example 1. The experimental method was as follows: 100 μL of glucose solution-stabilized calcium phosphate ion clusters were mixed with calcein to label the calcium ions in the clusters with green fluorescence. This mixture was then dropped onto the surface of acid-etched tooth enamel samples. After incubation at room temperature for 20 min, excess solution was simply rinsed away with simulated saliva. The treated tooth enamel samples were then placed in 20 mL of artificial saliva and incubated at 100 rpm in a 37°C constant temperature shaker for 24 hours. Afterward, the samples were removed and ultrasonically cleaned alternately with deionized water and ethanol. The cross-sectional morphology of the enamel was then photographed using a laser confocal microscope. The detection results are as follows: Figure 7As shown, the enamel treated with calcium phosphate ion clusters exhibits a distinct green fluorescent edge, demonstrating the growth of new enamel on the original sample.

[0038] Commercially available products and artificial saliva were used to mineralize acid-etched enamel samples using the same method to conduct a comparative experiment. The hardness of the enamel before and after restoration was measured using a Vickers hardness tester, and the results are as follows: Figure 8 As shown (sample size = 5), Figure 8 In this context, Sound represents healthy tooth enamel that has not been etched, Etched represents etched tooth enamel, SSF represents an etched tooth enamel sample repaired only by artificial saliva, CPP-ACP represents an etched tooth enamel sample repaired using commercially available tooth care products, and G-CPIC represents an etched tooth enamel sample repaired using the calcium phosphate ion clusters of this invention. Figure 8 The results showed that, compared with the clinically used dental prostaglandin (CPP-ACP), the hardness of teeth repaired with the calcium phosphate ion clusters of the present invention was significantly improved, and the hardness was closer to that of unetched enamel.

[0039] The results of the above embodiments demonstrate that the calcium phosphate ion clusters provided by this invention can deliver a large number of amorphous, non-crystalline hydroxyapatite pre-formed nuclei to the tooth surface, where they undergo in-situ crystallization and mineralization under the stimulation of simulated saliva, forming enamel prism structures similar to the original enamel surface. Furthermore, the calcium phosphate ion clusters can effectively improve enamel demineralization lesions, thereby restoring enamel hardness. In summary, the calcium phosphate ion clusters provided by this invention can simultaneously achieve tooth tissue regeneration and tooth demineralization repair. Moreover, by mixing glucose with the calcium phosphate ion cluster solution, this invention overcomes the bottlenecks of low mineralization efficiency and poor strength recovery in traditional techniques, achieving high-strength biomimetic remineralization of enamel with a morphology close to healthy enamel. Compared with existing technologies, this invention achieves significant improvements in mineralization efficiency, mechanical property restoration, and biocompatibility. It is also gentle, easy to operate, and inexpensive, with high clinical patient acceptance and broad clinical application prospects, providing a novel non-destructive restorative technology pathway for the treatment of early enamel caries such as leukoplakia and tooth sensitivity.

[0040] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A glucose solution-stable calcium phosphate ion cluster, characterized in that, The components include glucose, water, and calcium phosphate ion clusters; the glucose solution contains 20-60% glucose by mass in the stable calcium phosphate ion clusters.

2. The glucose solution-stabilized calcium phosphate ion cluster according to claim 1, characterized in that, The average size of the calcium phosphate ion clusters is <2 nm.

3. The method for preparing glucose solution-stabilized calcium phosphate ion clusters according to claim 1 or 2, characterized in that, Includes the following steps: A calcium chloride aqueous solution and glucose are mixed to obtain a glucose-calcium chloride mixed solution; A phosphate aqueous solution and glucose are mixed to obtain a glucose-phosphate mixed solution; The glucose-calcium chloride mixture and the glucose-phosphate mixture are mixed to obtain stable calcium phosphate ion clusters in the glucose solution.

4. The preparation method according to claim 3, characterized in that, The concentration of calcium chloride in the calcium chloride aqueous solution is 10~70 mmol / L.

5. The preparation method according to claim 3, characterized in that, The concentration of phosphate in the phosphate aqueous solution is 6~42 mmol / L.

6. The preparation method according to claim 3 or 5, characterized in that, The phosphate is trisodium phosphate.

7. The preparation method according to claim 3, characterized in that, The glucose-calcium chloride mixed solution contains 20-60% glucose by mass.

8. The preparation method according to claim 3, characterized in that, The glucose-phosphate mixed solution contains 20-60% glucose by mass.

9. The preparation method according to claim 3, characterized in that, The volume ratio of the glucose-calcium chloride mixture and the glucose-phosphate mixture is 0.1 to 10:

1.

10. The application of the glucose solution-stabilized calcium phosphate ion clusters according to claim 1 or 2, or the glucose solution-stabilized calcium phosphate ion clusters prepared by any one of claims 3 to 9, in the preparation of dental restorative products.

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