In vitro model for assessing the performance of pro-remineralizing materials, methods for its construction and uses thereof

Abstract

The application provides an in-vitro model for evaluating the performance of a remineralization-promoting material, a construction method and application thereof, and comprises the following steps: (1) gel preparation; (2) model assembly. The in-vitro model can effectively and accurately evaluate the performance of the remineralization-promoting material, has the characteristics of good evaluation effect, strong distinguishability, strong repeatability, good uniformity, stable and efficient evaluation system, and can complete remineralization of multiple test groups in a short time (5-7 days).

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of remineralization material research, and particularly relates to an in vitro model for evaluating the performance of a remineralization-promoting material, a construction method thereof and application, and especially relates to a stable and efficient in vitro model for evaluating the performance of a remineralization-promoting material, a construction method thereof and application. BACKGROUND

[0002] Remineralization, also known as recalcification or rehardening, refers to the phenomenon that an already mineralized tissue is partially demineralized and then re-mineralized, which is commonly seen in the repair process of teeth. The current common products such as fluoride and bioactive glass powder can effectively realize the remineralization process of teeth and achieve the repair of teeth. With the continuous research on tooth repair materials, the method for evaluating the performance of these materials has become an indispensable tool, and the use of in vitro remineralization technology model for the research on these materials has also become a common means.

[0003] The current in vitro remineralization technology model mainly includes simulated body fluid, double membrane system, single molecule membrane and agarose hydrogel. The simulated body fluid model test cycle is long, and in the absence of remineralization-promoting materials, the mineralization efficiency is extremely low, which is not conducive to the rapid evaluation of the performance of remineralization materials / products. The main shortcomings of the double membrane system are as follows: firstly, the double membrane system model is difficult to deposit a large amount of calcium phosphate crystals on the surface of the tooth piece; secondly, the stability of the double membrane system is poor, because the whole system is in a solution environment during the mineralization period, the double membrane is easy to fall off, affecting the mineralization efficiency; thirdly, the application range of the double membrane system model is small, and it can only evaluate inorganic or organic materials that are soluble in liquid, and it is difficult to directly evaluate the performance of remineralization products. The single molecule membrane model is generally suitable for evaluating the performance of a certain remineralization-promoting functional group, and is not suitable for evaluating the performance of multiple remineralization-promoting materials. The agarose hydrogel model generally uses an isolated tooth as the remineralization object, and due to the large difference between samples, the test uniformity is poor.

[0004] CN102172334B discloses a method for in vitro induction of dentin remineralization. The method uses a calcium ion solution and a phosphate agarose hydrogel to construct a biomimetic remineralization system, and realizes the remineralization of acid-etched dentin. The method successfully induces the growth of strip-shaped nanocrystals and the dense arrangement of the grown crystals. However, the use of isolated teeth as substrates for remineralization tests has poor uniformity, and it is difficult to obtain stable and repeatable test results.

[0005] CN106539693B discloses a method for preparing a high-molecular film-forming material loaded ACP biomimetic mineralization patch for enamel remineralization. The method is to mix high-molecular film-forming material, polyacrylic acid and glutamic acid to prepare a patch with good remineralization function for enamel. The method can induce the generation of needle-shaped hydroxyapatite crystals on the surface of enamel and arrange them in a woven manner. However, the remineralization cycle of this method is long, and it is difficult to establish a remineralization layer on the surface of enamel in a short period of time.

[0006] CN103342819A discloses a method for preparing a polyamide-amine dendrimer with a phosphate end group and its remineralization effect on enamel and dentin. The polyamide-amine dendrimer with a phosphate end group prepared by the method has good adsorption of hydroxyapatite and can induce hydroxyapatite remineralization. In a simulated body fluid, the polymer can generate a new hydroxyapatite mineralization layer on the surface of abraded enamel within 4 weeks. However, the method of using simulated body fluid as a remineralization medium is time-consuming, and it is difficult to obtain an ideal remineralization layer without a pro-remineralization material.

[0007] Because the current common models all have the problems of long cycle and poor uniformity. Therefore, how to provide a model with good uniformity, small sample difference, good evaluation effect and short cycle has become a problem to be solved. SUMMARY

[0008] In view of the deficiencies of the prior art, the purpose of the present application is to provide an in vitro model for evaluating the performance of pro-remineralization materials and a construction method and application thereof, especially to provide a stable and efficient in vitro model for evaluating the performance of pro-remineralization materials and a construction method and application thereof. The in vitro model provided by the present application can effectively and accurately evaluate the performance of pro-remineralization materials, has the characteristics of good evaluation effect, strong discrimination and strong repeatability, has good uniformity, and the evaluation system is stable and efficient, and can complete the remineralization of multiple test groups in a short time (5-7 days).

[0009] To achieve the purpose of the present application, the following technical solutions are adopted:

[0010] In a first aspect, the present application provides a construction method of an in vitro model for evaluating the performance of pro-remineralization materials, which comprises the following steps:

[0011] (1) Gel preparation: mix and melt agarose, gelatin and water to obtain a blank mixed hydrogel; mix and melt agarose, gelatin and Na2HPO4 aqueous solution to obtain a hydrogen phosphate hydrogel;

[0012] (2) Model assembly: open the hole at the bottom of the small tube, then temporarily seal it; add hydrogen phosphate hydrogel into the small tube, form the first hydrogen phosphate hydrogel layer after cooling; then put in hydroxyapatite sheet (HA), and add hydrogen phosphate hydrogel again, form the second hydrogen phosphate hydrogel layer after cooling; then add blank mixed hydrogel, form the blank gel layer after cooling; finally add calcium ion solution to form the calcium ion solution layer, and remove the temporary seal at the bottom of the small tube, complete the assembly of the small tube;

[0013] Insert the assembled small tube into the large tube which is closed at the bottom and contains Na2HPO4 aqueous solution, and the in vitro model is completed.

[0014] The in vitro model obtained by the above construction method can promote the formation of rod-shaped aggregated calcium phosphate by calcium and phosphate ions through the synergistic effect of agarose and gelatin, and shorten the experimental period; and the in vitro model can effectively improve the uniformity and repeatability of the experiment by using hydroxyapatite to replace the isolated tooth for remineralization test.

[0015] Preferably, the mass fraction of agarose in the blank mixed hydrogel and the hydrogen phosphate hydrogel in step (1) is independently 0.3-0.7%.

[0016] Preferably, the mass fraction of gelatin in the blank mixed hydrogel and the hydrogen phosphate hydrogel in step (1) is independently 2.5-3.5%.

[0017] Preferably, the concentration of the Na2HPO4 aqueous solution is 0.2-0.3M, and the pH is 7.0-7.3.

[0018] Preferably, the thickness of the first hydrogen phosphate hydrogel layer in step (2) is 0.8-1.2cm.

[0019] Preferably, the thickness of the second hydrogen phosphate hydrogel layer in step (2) is 1.5-2.5mm.

[0020] Preferably, the thickness of the blank gel layer in step (2) is 1.5-2.5mm.

[0021] Preferably, the thickness of the calcium ion solution layer in step (2) is 60-70mm.

[0022] Preferably, the concentration of calcium ions in the calcium ion solution in step (2) is 0.1-0.15M, and the pH is 5.5-6.5.

[0023] Preferably, in the large tube in step (2), the height of the Na2HPO4 aqueous solution above the bottom of the small tube is 5-10mm.

[0024] The mass fraction of agarose can be 0.3%, 0.4%, 0.5%, 0.6% or 0.7%, etc., the mass fraction of gelatin can be 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4% or 3.5%, etc., the concentration of Na2HPO4 aqueous solution can be 0.2M, 0.21M, 0.22M, 0.23M, 0.24M, 0.25M, 0.26M, 0.27M, 0.28M, 0.29M or 0.3M, etc., the pH can be 7.0, 7.1, 7.2 or 7.3, etc., the thickness of the first hydrogen phosphate hydrogel layer can be 0.8cm, 0.9cm, 1cm, 1.1cm or 1.2cm, etc., the thickness of the second hydrogen phosphate hydrogel layer can be 1.5mm, 1.6mm, 1.7mm, 1.8mm, 1.9mm, 2mm, 2.1mm, 2.2mm, 2.3mm, 2.4mm or 2.5mm, etc., the thickness of the blank gel layer can be 1.5mm, 1.6mm, 1.7mm, 1.8mm, 1.9mm, 2mm, 2.1mm, 2.2mm, 2.3mm, 2.4mm or 2.5mm, etc., the thickness of the calcium ion solution layer can be 60mm, 61mm, 62mm, 63mm, 64mm, 65mm, 66mm, 67mm, 68mm, 69mm or 70mm, etc.; the concentration of calcium ions can be 0.1M, 0.11M, 0.12M, 0.13M, 0.14M or 0.15M, etc., the pH can be 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4 or 6.5, etc., the height of the Na2HPO4 aqueous solution over the bottom of the tubule can be 5mm, 6mm, 7mm, 8mm, 9mm or 10mm, etc., but is not limited to the above listed values, other values within the above ranges are also applicable.

[0025] In a second aspect, the present application provides an in vitro model for evaluating the performance of a pro-remineralization material, which is constructed according to the construction method described above.

[0026] In a third aspect, the present application further provides a method for evaluating the performance of a pro-remineralization material, which comprises the following steps: soaking the hydroxyapatite sheet in a solution of the material to be tested, then taking it out, assembling the in vitro model for evaluating the performance of a pro-remineralization material according to the construction method described above, then placing the model for a plurality of mineralization periods, updating the solution and gel in the in vitro model after each mineralization period, and soaking the hydroxyapatite sheet in the solution of the material to be tested again before taking it out, observing the morphology of the hydroxyapatite sheet and performing composition detection after the end of the test, scoring the results, thereby completing the evaluation.

[0027] Preferably, the time of the mineralization period is 20-28h.

[0028] Preferably, the several mineralization cycles are at least 5 mineralization cycles.

[0029] Preferably, the soaking time is 2.5-3.5 min.

[0030] Preferably, the scoring criteria are as follows:

[0031] The surface morphology and cross-sectional morphology of the hydroxyapatite sheet are observed by scanning electron microscopy, in the surface morphology, if it presents sheet shape and a small amount of loose, it is recorded as 1 point; if it presents sheet shape and a large amount of dense, it is recorded as 2 points; if it presents granular or fused shape and is scattered and disordered, it is recorded as 2 points; if it presents granular or fused shape and is densely aggregated, it is recorded as 3 points; if it presents fibrous or rod shape and a small amount, it is recorded as 3 points; if it presents fibrous or rod shape and a large amount, it is recorded as 4 points.

[0032] In the cross-sectional morphology, if it is thin and loose, it is recorded as 1 point; if it is thick and loose, it is recorded as 2 points; if it is thin and dense, it is recorded as 3 points; if it is thick and dense, it is recorded as 4 points; if it is thin and ordered, it is recorded as 5 points; if it is thick and ordered, it is recorded as 6 points.

[0033] The atomic concentration of the generated crystals on the surface of the hydroxyapatite sheet is analyzed by energy spectrum analysis, and the Ca / P value is calculated, if the Ca / P value is lower than 1.1, it is recorded as 1 point; if the Ca / P value is 1.1-1.6, it is recorded as 2 points; if the Ca / P value is higher than 1.6, it is recorded as 3 points.

[0034] The phase of the generated crystals on the surface of the hydroxyapatite sheet is determined by XRD, if it is all hydroxyapatite precursor, it is recorded as 1 point; if it is a mixture of hydroxyapatite precursor and hydroxyapatite, it is recorded as 2 points; if it is all hydroxyapatite, it is recorded as 3 points; if it is a mixture of fluorapatite and its precursor, it is recorded as 4 points; if it is all fluorapatite, it is recorded as 5 points.

[0035] Compared with the prior art, the present application has the following beneficial effects:

[0036] The present application provides a construction method of an in vitro model for evaluating the performance of a remineralization material, by using agarose and gelatin in combination, the synergistic effect can promote calcium and phosphorus ions to form rod-shaped aggregated calcium phosphate, shorten the experimental period; and by using hydroxyapatite to replace the in vitro tooth for remineralization test, the uniformity and repeatability of the test can be effectively improved. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is a high-definition microscope image of the hydroxyapatite sheet before and after acid etching in Example 1;

[0038] Figure 2 is a structural schematic diagram of the in vitro model provided in Example 1;

[0039] Figure 3 is a surface morphology SEM image in Example 1;

[0040] Figure 4 is a cross-sectional morphology SEM image in Example 1 ;

[0041] Figure 5 is a plot of mineralization depth significance analysis for Examples 1-5 and Comparative Example 1 ;

[0042] Figure 6 is an XRD diffractogram of Example 1 ;

[0043] Figure 7 is a surface morphology SEM image in Example 2;

[0044] Figure 8 is a cross-sectional morphology SEM image in Example 2;

[0045] Figure 9 is an XRD diffractogram of Example 2;

[0046] Figure 10 is a surface morphology SEM image in Example 3;

[0047] Figure 11 is a cross-sectional morphology SEM image in Example 3;

[0048] Figure 12 is an XRD diffractogram of Example 3;

[0049] Figure 13 is a surface morphology SEM image in Example 4;

[0050] Figure 14 is a cross-sectional morphology SEM image in Example 4;

[0051] Figure 15 is an XRD diffractogram of Example 4;

[0052] Figure 16 is a surface morphology SEM image in Example 5;

[0053] Figure 17 is a cross-sectional morphology SEM image in Example 5;

[0054] Figure 18 is an XRD diffractogram of Example 5;

[0055] Figure 19 is a surface morphology SEM image in Comparative Example 1 ;

[0056] Figure 20 is a cross-sectional morphology SEM image in Comparative Example 1 ;

[0057] Figure 21 is an XRD diffractogram of Comparative Example 1 ;

[0058] Figure 22 is a surface morphology SEM image in Comparative Example 2;

[0059] Figure 23 is a cross-sectional morphology SEM image in Comparative Example 2;

[0060] Figure 24 is an XRD diffraction pattern of Comparative Example 2;

[0061] Figure 25 is a surface morphology SEM image in Comparative Example 3;

[0062] Figure 26 is a cross-sectional morphology SEM image in Comparative Example 3;

[0063] Figure 27 is an XRD diffraction pattern of Comparative Example 3;

[0064] Figure 28 is a surface morphology SEM image in Comparative Example 4;

[0065] Figure 29 is a cross-sectional morphology SEM image in Comparative Example 4;

[0066] Figure 30 is an XRD diffraction pattern of Comparative Example 4;

[0067] Figure 31 is a surface morphology SEM image in Comparative Example 5;

[0068] Figure 32 is a cross-sectional morphology SEM image in Comparative Example 5;

[0069] Figure 33 is an XRD diffraction pattern of Comparative Example 5. DETAILED DESCRIPTION

[0070] The technical solutions of the present application will be further illustrated by the specific embodiments. Those skilled in the art should understand that the embodiments are only to help understand the present application and should not be regarded as specific limitations of the present application.

[0071] In the following examples, the hydroxyapatite tablets were purchased from Jiangyin Dianyu New Material Technology Co., Ltd.;

[0072] The bioactive glass powder (BGA) was purchased from Shanghai Aladdin Biochem Technology Co., Ltd.;

[0073] The bovine enamel tablets were purchased from Tao Bao merchant—Yimi Sunshine Angle Shop;

[0074] In the following examples, the pH of the solution was adjusted by hydrochloric acid;

[0075] The surface morphology and cross-sectional morphology of the generated crystals of the samples were observed by scanning electron microscopy (SEM).

[0076] The atomic concentration of the crystals generated by the sample was analyzed by energy dispersive spectroscopy (EDS), and the Ca / P value was calculated;

[0077] The identity of the crystals generated by the sample was determined by XRD;

[0078] The scoring criteria are as follows:

[0079] Table 1

[0080]

[0081] The mineralization system, substrate, and evaluation of the pro- remineralization material of Examples 1-5 and Comparative Examples 1-5 are shown in Table 2:

[0082] Table 2

[0083] Mineralizing system Mineralizing substrate Remineralization promoting material Example 1 0.5% agarose and 3% gelatin Synthetic hydroxyapatite / Example 2 0.5% agarose and 3% gelatin Synthetic hydroxyapatite 6000 ppm sodium fluoride Example 3 0.5% agarose and 2% gelatin Synthetic hydroxyapatite 12,300 ppm sodium fluoride Example 4 0.5% agarose and 3% gelatin Synthetic hydroxyapatite 6000 ppm of olaflur Example 5 0.5% agarose and 3% gelatin Synthetic hydroxyapatite 4% bioactive glass powder Comparative Example 1 0.5% agarose Synthetic hydroxyapatite / Comparative Example 2 0.5% agarose and 3% gelatin Bovine enamel chips 6000 ppm sodium fluoride Comparative Example 3 Simulated body fluid (SBF) Synthetic hydroxyapatite chips / Comparative Example 4 3.5% agarose Synthetic hydroxyapatite 6000 ppm sodium fluoride Comparative Example 5 3.5% gelatin Synthetic hydroxyapatite 6000 ppm sodium fluoride

[0084] Example 1

[0085] The present example provides an in vitro model for evaluating the performance of a pro-remineralization material and a method for evaluating the performance of a pro-remineralization material, the specific steps of which are as follows:

[0086] (1) Acid etching of hydroxyapatite tablets:

[0087] Hydroxyapatite tablets with an outer diameter of 15 mm and a thickness of 1.2 mm were immersed in a 37% phosphoric acid solution for acid etching for 1 min, and then rinsed with a large amount of ultrapure water. The acid-etched tablets were immersed in ultrapure water for 3 min, and then left to dry naturally at 20°C for 30 min or more. As shown in FIG. 1 (A is a high-definition microscope image of a blank hydroxyapatite tablet without any treatment; B is a high-definition microscope image of a hydroxyapatite tablet after acid etching with 37% phosphoric acid), the acid-etched hydroxyapatite tablets appeared loose and porous under a high-definition microscope. Figure 1

[0088] (2) Preparation of mixed gels:

[0089] A blank gel and a hydrogen phosphate gel were prepared, respectively, and the gel was a mixture of 0.5 wt% agarose and 3 wt% gelatin. The hydrogen phosphate gel was prepared from a 0.26 M, pH = 7 Na2HPO4solution. The blank gel was prepared from ultrapure water. The preparation method of the gel was to mix agarose and gelatin with a solvent, then place them in a 80°C water bath to dissolve completely to a molten state, and then prepare for use.

[0090] (3) Remineralization of hydroxyapatite tablets:

[0091] ​First, pour 1cm thick hydrogen phosphate gel into a small tube (open 5 holes with a diameter of 6mm at the bottom, temporarily seal it, and remove the seal after the calcium ion solution is added). After it cools and solidifies, put in a small piece of hydroxyapatite that has been acid-etched. Then pour in 2mm thick hydrogen phosphate gel, and after it cools and solidifies, pour in a 2mm thick blank gel. After all the gels are cooled and solidified, add a 65mm thick, 0.13M, pH=6 CaCl2 solution into the system. Insert the transparent tube containing the calcium ion solution and the mixed gel into the large tube with the lower tube closed and containing the Na2HPO4 solution (0.26M, pH=7). It is required that after the transparent tube is inserted, the hydrogen phosphate solution in the large tube submerges the transparent tube by 10mm. Put the model (the schematic diagram of the overall model structure is shown in the figure) Figure 2 The samples were placed in a 37°C incubator (as shown) for a 24-hour mineralization cycle. The solution and gel in the system were refreshed every 24 hours. After each mineralization cycle, the samples were immersed in sterile water for 3 minutes for 7 days. A blank control of hydroxyapatite without any treatment and a negative control of hydroxyapatite with acid etching without mineralization were also prepared.

[0092] (4) Remineralization effect evaluation:

[0093] 4.1) Surface morphology: Figure 3 (A is a blank control group (untreated chips), B is a negative control group (chips treated only with acid etching), and C is a 2000x surface SEM image of chips from the mixed gel system mineralization group in Example 1.) The chips in the blank control group were smooth and had no obvious cracks, while the chips in the negative control group had a rough surface with uneven pores or cracks. The chips mineralized with the mixed gel system showed flaky and molten crystals on their surfaces. The surface morphology score was 2.

[0094] 4.2) Cross-sectional morphology: Figure 4 (A is the blank control group (small piece without any treatment), B is the negative control group (small piece treated with acid etching only), and C is a 1000x cross-sectional SEM image of the small piece of the mixed gel system mineralization group in Example 1, where the area above the white dotted line is the remineralized layer and the area below the dotted line is the non-remineralized layer) As shown, compared with the negative control group, after the mixed gel mineralization, a thicker and denser remineralized layer is formed on the surface of the small piece. Figure 5 As shown, Example 1 generated a thick and dense remineralized layer, and therefore the cross-sectional morphology score was 4. Except for the mineralization thickness of Comparative Example 1, which showed no significant difference compared with Example 1, the mineralization thicknesses of the other examples were significantly lower or higher than that of Example 1, with P < 0.01.

[0095] 4.3) Calcium-to-phosphorus ratio: As shown in Table 3, the average calcium-to-phosphorus ratio of the crystals generated by the mixed gel system was 1.52, which was between 1.1 and 1.6, and was scored 2 points.

[0096] 4.4) Material analysis: As shown in Figure 6 XRD results showed that Ca(HPO4)2·2H2O, a precursor of hydroxyapatite, was generated on the surface of the hydroxyapatite small pieces after mixed gel mineralization, which scored 1 point.

[0097] In summary, based on the re-mineralization evaluation scheme established in the present application, the mineralization score of the mixed gel system in Example 1 was 9 points.

[0098] Example 2

[0099] The present embodiment provides an in vitro model for evaluating the performance of a re-mineralization-promoting material and a method for evaluating the performance of a re-mineralization-promoting material, and the specific steps are as follows:

[0100] (1) Acid etching and treatment of hydroxyapatite small pieces:

[0101] Except for immersing the acid-etched small pieces in a 6000 ppm sodium fluoride solution for 3 min, and then placing the small pieces at 20°C for 30 min instead of immersing them in a 37% phosphoric acid solution, the rest is the same as Example 1.

[0102] (2) Preparation of mixed gel:

[0103] The preparation method of the mixed gel is the same as that of Example 1.

[0104] (3) Re-mineralization of hydroxyapatite pieces:

[0105] The re-mineralization method is the same as that of Example 1, however, after the end of each mineralization cycle, the small pieces are immersed in a 6000 ppm sodium fluoride solution for 3 min, and are placed at 20°C for 30 min or more, instead of being immersed in sterile water.

[0106] (4) Evaluation of re-mineralization-promoting effect:

[0107] 4.1) Surface morphology: As shown in Figure 7 (A is a negative control group (small pieces without any treatment) 2000 times SEM image; B is a blank control group (small pieces only treated by acid etching) 2000 times SEM image; C is a 6000 ppm sodium fluoride treatment group of Example 2 2000 times SEM image; D is a 6000 ppm sodium fluoride treatment group of Example 2 40000 times surface SEM image) As shown in, compared with the control group, a large number of fibrous crystals were generated on the surface of the hydroxyapatite small pieces after treatment with 6000 ppm sodium fluoride, and there was obvious directionality, which scored 4 points.

[0108] 4.2) Cross-sectional morphology: As shown in Figure 8(A is negative control (only acid-etched piece) 1000 times SEM image; B is 2000 times SEM image of Example 2 6000 ppm sodium fluoride treatment group; C is 10000 times cross-section SEM image of Example 2 6000 ppm sodium fluoride treatment group, in which the white dotted line above is the remineralization layer and the white dotted line below is the non-remineralization layer) as shown, compared with the control group, after the surface of Example 2 is treated with 6000 ppm sodium fluoride, a layer of ordered and dense crystals is generated on the surface of the hydroxyapatite piece. The generated crystals can be clearly observed under high magnification electron microscope to grow perpendicular to the piece plane. As shown in Figure 5 , the average mineralization thickness of Example 2 is significantly higher than that of Example 1, P < 0.01. Therefore, it is considered that Example 2 generates a thick and ordered remineralization layer, and the score is 6 points.

[0109] 4.3) Calcium-phosphorus ratio: As shown in Table 3, the calcium-phosphorus ratio of the crystals generated in the 6000 ppm sodium fluoride group is > 1.6. The score is 3 points.

[0110] 4.4) Material analysis: As shown in Figure 9 , the diffraction peaks of the newly generated crystals of Example 2 analyzed by XRD correspond to the characteristic peaks on the hydroxyapatite standard card one by one, proving that the newly generated crystals are hydroxyapatite. In addition, the 002 (2 θ ≈25.9°) crystal face characteristic peak of the newly generated hydroxyapatite crystals of Example 2 is significantly higher than that of the 300 (2 θ ≈33.0°) crystal face, indicating that the newly generated crystals grow along the C-axis direction. The score is 3 points.

[0111] In summary, based on the remineralization effect evaluation scheme established in the present application, the 6000 ppm sodium fluoride mineralization score of Example 2 is 16 points.

[0112] Example 3

[0113] The present embodiment provides an in vitro model for evaluating the performance of a remineralization-promoting material and a method for evaluating the performance of a remineralization-promoting material, and the specific steps are as follows:

[0114] (1) Acid etching and treatment of hydroxyapatite pieces:

[0115] Except that the acid-etched pieces are immersed in a 12300 ppm sodium fluoride solution for 3 minutes, and then the pieces are placed at 20°C for 30 minutes instead of being immersed in a 37% phosphoric acid solution, the rest is consistent with Example 1.

[0116] (2) Preparation of mixed gel:

[0117] The preparation method of the mixed gel is the same as that of Example 1, and the ratio of gelatin is 2 wt% instead of 3 wt%.

[0118] (3) Remineralization of hydroxyapatite sheets:

[0119] The remineralization method was the same as in Example 1, however, after each mineralization cycle, the pieces were immersed in a 12300 ppm sodium fluoride solution for 3 minutes and then left to rest at 20°C for 30 minutes instead of being immersed in sterile water.

[0120] (4) Evaluation of the effect of promoting remineralization

[0121] 4.1) Surface morphology: Figure 10 (A is a 2000x SEM image of the negative control (untreated tablet); B is a 2000x SEM image of the group treated with 12,300 ppm sodium fluoride in Example 3; C is a 40,000x surface SEM image of the group treated with 12,300 ppm sodium fluoride in Example 2.) As shown, compared with the negative control, the mineralized tablets treated with 12,300 ppm sodium fluoride exhibited a large number of short, clustered crystals on their surfaces, but the resulting crystals lacked obvious directionality. The score was 4.

[0122] 4.2) Cross-sectional morphology: Figure 11 (A is a 1000x SEM image of the negative control group (a small piece without any treatment); B is a 2000x SEM image of the group treated with 12300ppm sodium fluoride in Example 3; C is a 10000x cross-sectional SEM image of the group treated with 12300ppm sodium fluoride in Example 2, wherein the area above the white dotted line is the remineralized layer, and the area below the white dotted line is the non-remineralized layer) As shown, compared with the negative control group, a dense and orderly mineralized layer is generated on the surface of the small piece after the 12300ppm sodium fluoride treatment and mineralization. In the high-magnification SEM image, it can be clearly observed that there are crystals growing perpendicular to the surface of the small piece and growing parallel to the surface of the small piece. Figure 5 As shown, compared with Example 1, the average mineralization thickness of Example 3 is significantly lower than that of Example 1, P < 0.01, that is, Example 3 is considered to have generated a thin and orderly remineralization layer, with a score of 5 points.

[0123] 4.3) Calcium-to-phosphorus ratio: As shown in Table 3, the calcium-to-phosphorus ratio of the crystals produced in Example 3 after treatment with 12,300 ppm sodium fluoride was >1.6, resulting in a score of 3.

[0124] 4.4) Item Analysis: Figure 12 As shown in FIG. 3 , the diffraction peaks of the newly generated crystals of Example 3 correspond to the characteristic peaks on the hydroxyapatite labeling card through XRD analysis, proving that the newly generated crystals are hydroxyapatite. In addition, the 002(2 θ ≈25.9°) crystal plane characteristic peak is significantly higher than 300(2 θ The characteristic peak of the crystal plane (≈33.0°) indicates that the newly formed crystal grows along the C axis. The score is 3 points.

[0125] In summary, the re-mineralization effect evaluation scheme established based on the present application, the re-mineralization score of 12300 ppm sodium fluoride in Example 3 is 15.

[0126] Example 4

[0127] The present embodiment provides an in vitro model for evaluating the performance of a re-mineralization-promoting material and a method for evaluating the performance of a re-mineralization-promoting material, and the specific steps are as follows:

[0128] (1) Acid etching and treatment of hydroxyapatite small pieces:

[0129] Except that the small pieces after acid etching are immersed in a 6000 ppm Oraflur solution for 3 min, and then the small pieces are placed at 20°C for 30 min instead of being immersed in a 37% phosphoric acid solution, the rest is the same as Example 1.

[0130] (2) Preparation of mixed gels:

[0131] The preparation method of the mixed gel is the same as that of Example 1.

[0132] (3) Re-mineralization of hydroxyapatite pieces:

[0133] The re-mineralization method is the same as that of Example 1, however, after the end of each mineralization cycle, the small pieces are immersed in a 6000 ppm Oraflur solution for 3 min, and are placed at 20°C for 30 min instead of being immersed in sterile water.

[0134] (4) Evaluation of re-mineralization-promoting effect:

[0135] 4.1) Surface morphology: As shown in Figure 13 (A is a 2000 times SEM image of the negative control group (small pieces without any treatment); B is a 2000 times SEM image of the 6000 ppm Oraflur treatment group of Example 4; C is a 40000 times surface SEM image of the 6000 ppm Oraflur treatment group of Example 4), compared with the negative control group, a large number of rod-shaped crystals are generated on the surface of the small pieces after 6000 ppm Oraflur treatment. The score is 4.

[0136] 4.2) Cross-sectional morphology: As shown in Figure 14 (A is a 1000 times SEM image of the negative control group (small pieces without any treatment); B is a 1000 times SEM image of the 6000 ppm Oraflur treatment group of Example 4; C is a 5000 times cross-sectional SEM image of the 6000 ppm Oraflur treatment group of Example 4, in which the white / black dashed line above is the re-mineralization layer, and the dashed line below is the non-re-mineralization layer), compared with the negative control group, a thin but dense and disordered rod-shaped crystal mineralization layer is generated on the surface of the small pieces after 6000 ppm Oraflur treatment. AsFigure 5 As shown in Table 3, the average mineralization thickness of Example 4 was significantly lower than that of Example 1 (P<0.01), which indicated that Example 4 generated a thin and dense remineralization layer, and scored 3 points.

[0137] 4.3) Calcium to phosphorus ratio: As shown in Table 3, the calcium to phosphorus ratio of the crystals generated after Example 4 was treated with 6000 ppm of Oraflur was >1.6. Scored 3 points.

[0138] 4.4) Material analysis: As shown in Table 3, the diffraction peaks of the crystals generated after Example 4 was treated with 6000 ppm of Oraflur were basically consistent with the characteristic diffraction peaks of fluorapatite, indicating that the newly generated crystals belonged to fluorapatite, and scored 5 points. Figure 15

[0139] In summary, based on the remineralization effect evaluation scheme established by the present application, the mineralization score of Example 4 treated with 6000 ppm of Oraflur was 15 points.

[0140] Example 5

[0141] The present embodiment provides an in vitro model for evaluating the performance of a remineralization-promoting material and a method for evaluating the performance of a remineralization-promoting material, and the specific steps are as follows:

[0142] (1) Acid etching and treatment of hydroxyapatite small pieces:

[0143] Except that the small pieces after acid etching were immersed in a 4% BAG solution for treatment for 3 min, and then the small pieces were placed at 20°C for 30 min instead of being immersed in a 37% phosphoric acid solution, the rest was consistent with Example 1.

[0144] (2) Preparation of mixed gel:

[0145] The preparation method of the mixed gel was the same as that of Example 1.

[0146] (3) Remineralization of hydroxyapatite pieces:

[0147] The method of remineralization was the same as that of Example 1, however, after the end of each mineralization cycle, the small pieces were immersed in a 4% BAG solution for treatment for 3 min, and were placed at 20°C for 30 min instead of being immersed in sterile water.

[0148] (4) Evaluation of mineralization-promoting effect:

[0149] 4.1) Surface morphology: As shown in Table 3, Figure 16 ​As shown in FIG. 5B, a large number of irregular fused crystals were generated on the surface of the hydroxyapatite tablets after the 4% BAG treatment in Example 5, and the score was 3.

[0150] 4.2) Cross-sectional morphology: As shown in FIG. 6A, the surface of the hydroxyapatite tablets in Example 1 was covered with a large number of irregular fused crystals, and the score was 3. Figure 17 As shown in FIG. 6B, a relatively thick dense mineralized layer composed of sheet crystals and fused crystals was generated on the surface of the hydroxyapatite tablets after the 4% BAG treatment in Example 5, and the white dashed line above was the mineralized layer, and the white dashed line below was the non-mineralized layer. As shown in FIG. 6C, the average mineralization thickness of Example 5 was significantly higher than that of Example 1, P<0.01, which indicated that Example 5 generated a thick and dense mineralized layer, and the score was 4. Figure 5

[0151] 4.3) Calcium-phosphorus ratio: As shown in Table 3, the calcium-phosphorus ratio of the crystals generated after the 4% BAG treatment in Example 5 was >1.6. The score was 3.

[0152] 4.4) Material analysis: As shown in FIG. 7, the diffraction peaks of the newly generated crystals in Example 5 basically corresponded to the characteristic peaks of dihydrate and calcium hydrogen phosphate, which indicated that the crystals generated in Example 5 were dihydrate and calcium hydrogen phosphate, which belonged to hydroxyapatite precursor, and the score was 1. Figure 18

[0153] In summary, based on the re-mineralization effect evaluation scheme established in the present application, the mineralization score of Example 5 4% bioactive glass was 11.

[0154] Comparative Example 1

[0155] The present comparative example provides an in vitro model for evaluating the performance of a re-mineralization-promoting material and a method for evaluating the performance of a re-mineralization-promoting material, and the specific steps are as follows:

[0156] (1) Acid etching and treatment of hydroxyapatite tablets:

[0157] The same as Example 1.

[0158] (2) Preparation of mixed gel:

[0159] Except that only 0.5% agarose was used without using gelatin, the rest was the same as Example 1.

[0160] (3) Re-mineralization of hydroxyapatite tablets:

[0161] The same as Example 1. ​​

[0162] (4) Remineralization effect evaluation:

[0163] 4.1) Surface morphology: As shown in Figure 19 (A is the negative control group (unprocessed small pieces) 2000 times SEM image; B is the surface SEM image of the mineralization group of the gel system of Comparative Example 1), the crystals generated on the surface of the hydroxyapatite small pieces by the 0.5% agarose mineralization system are mainly in the form of plates, and a small amount of fibrous crystals are contained on the surface and in the gaps of the plate crystals, with a score of 2.

[0164] 4.2) Cross-sectional morphology: As shown in Figure 20 (A is the negative control group (unprocessed small pieces) 1000 times SEM image; B is the cross-sectional SEM image of the mineralization group of the gel system of Comparative Example 1, in which the white dashed line above is the remineralization layer, and the white dashed line below is the non-remineralization layer), the 0.5% agarose mineralization system can generate a relatively thick but loose plate crystal mineralization layer on the surface of the small pieces.

[0165] As shown in Figure 5 , there is no significant difference in the average mineralization depth between Example 1 and Comparative Example 1, so it is determined that Comparative Example 1 generates a thick and loose remineralization layer, with a score of 2.

[0166] 4.3) Calcium to phosphorus ratio: As shown in Table 3, the calcium to phosphorus ratio of the crystals generated by the 0.5% agarose mineralization system is between 1.1-1.6, with a score of 2.

[0167] 4.4) Material analysis: As shown in Figure 21 , the diffraction peaks of the newly generated crystals of Comparative Example 1 correspond to the characteristic peaks of dicalcium phosphate dihydrate and calcium hydrogen phosphate, indicating that the crystals generated by the 0.5% agarose mineralization system are dicalcium phosphate dihydrate and calcium hydrogen phosphate, which belong to hydroxyapatite precursors, with a score of 1.

[0168] In summary, based on the remineralization effect evaluation scheme established by the present application, the 0.5% agarose mineralization of Comparative Example 1 scores 7 points.

[0169] Comparative Example 2

[0170] This comparative example provides an in vitro model for evaluating the performance of a remineralization-promoting material and a method for evaluating the performance of a remineralization-promoting material, with the specific steps as follows:

[0171] (1) Acid etching and treatment of bovine enamel pieces:

[0172] The enamel pieces with the size of 4 mm x 4 mm and the thickness of 2 mm were cut from the buccal side of bovine teeth using a polishing mobile phone. The enamel pieces were polished and polished from 400 mesh, 800 mesh and 2000 mesh sandpaper in turn, so that the enamel surface showed mirror effect. The treated enamel pieces were immersed in a 37% phosphoric acid solution for 1 min, and then rinsed with a large amount of ultrapure water. The acid-etched enamel pieces were immersed in a 6000 ppm sodium fluoride solution for 3 min, and then the enamel pieces were placed at 20°C for more than 30 min.

[0173] (2) Preparation of mixed gels:

[0174] The preparation method of the mixed gel was the same as that of Example 1.

[0175] (3) Remineralization of bovine enamel pieces:

[0176] The method of remineralization was the same as that of Example 1, however, after the end of each mineralization cycle, the enamel pieces were immersed in a 6000 ppm sodium fluoride solution for 3 min, and were placed at 20°C for more than 30 min, instead of being immersed in sterile water. The enamel pieces without any treatment were set as blank control and the enamel pieces only treated by acid etching were set as negative control.

[0177] (4) Evaluation of the effect of promoting remineralization:

[0178] 4.1) Surface morphology: As shown in Figure 22 (A is the blank control (the enamel pieces without any treatment) 20000 times SEM image; B is the negative control (the enamel pieces only treated by acid etching) 20000 times SEM image; C is the comparative example 2 (the enamel pieces treated by 6000 ppm for mineralization) 20000 times SEM image of ordered rod-shaped crystals in bundles; D is the comparative example 2 10000 times SEM image of disordered crystal cells), the surface of the blank control enamel pieces was smooth and flat, and the enamel surface after acid etching with a 37% phosphoric acid solution showed a fish scale structure. The enamel surface of the comparative example 2 treated by 6000 ppm sodium fluoride for mineralization had a large number of columnar deposits in some areas and obvious directionality, while in some areas a large number of disordered crystal cells gathered, and the uniformity was poor.

[0179] 4.2) Cross-sectional morphology: As shown in Figure 23(A is a 2000x cross-sectional SEM image of the blank control (enamel slice without any treatment); B is a 2000x cross-sectional SEM image of the negative control (enamel slice treated only with acid etching); C is a 2000x cross-sectional SEM image of comparative example 2 (enamel slice treated with 6000ppm for mineralization) with a large number of columnar crystals growing between the enamel pillars; D is a 2000x cross-sectional SEM image of comparative example 2 (enamel slice treated with 6000ppm for mineralization) with only a thin mineralized layer covering the surface of the enamel pillars after acid etching) As shown, the enamel cross-section of the blank control is relatively smooth, but after etching with phosphoric acid solution, the enamel pillars can be seen to be fully exposed. After mineralization with 6000ppm sodium fluoride, it can be seen that a large number of ordered columnar crystals fill the spaces between the enamel pillars to form an ordered and dense remineralized layer in some cross-sections, but some cross-sections only cover the acid-etched enamel surface with a thin mineralized layer, and the mineralization effect is uneven.

[0180] 4.3) Calcium-to-phosphorus ratio: As shown in Table 3, the calcium-to-phosphorus ratio of the newly formed rod-shaped crystals in Comparative Example 2 was >1.6.

[0181] 4.4) Item Analysis: Figure 24 As shown in FIG. 2 , the diffraction peaks of the newly generated rod-shaped crystals of Comparative Example 2 correspond to the characteristic peaks on the hydroxyapatite labeling card through XRD analysis, proving that the newly generated crystals are hydroxyapatite. In addition, the 002(2 θ ≈25.9°) crystal plane characteristic peak is significantly higher than 300(2 θ The characteristic peaks of the crystal plane (≈33.0°) indicate that the newly generated crystals grow along the C-axis.

[0182] Comparative Example 3

[0183] This comparative example provides an in vitro model for evaluating the performance of a remineralization-promoting material and a method for evaluating the performance of a remineralization-promoting material. The specific steps are as follows:

[0184] (1) Acid etching and treatment of hydroxyapatite flakes:

[0185] Same as Example 1.

[0186] (2) Preparation of SBF solution:

[0187] SBF was prepared as follows: 700 mL of deionized water at 36.5 ± 1.5°C was prepared. To the water, 8.035 g of NaCl, 0.355 g of NaHCO₃, 0.225 g of KCl, 0.231 g of K₂HPO₄·3H₂O, 0.311 g of MgCl₂·6H₂O, 39 mL of 1 M hydrochloric acid, 0.292 g of CaCl₂, 0.072 g of Na₂SO₄, and 6.118 g of Tris were added, sequentially adding each reagent until fully dissolved before adding the next. Ensure that no precipitate forms after the last reagent is added. After all reagents have been added, the pH of the solution was adjusted to 7.40 using 1 M hydrochloric acid. The solution was then diluted to 1000 mL, transferred to a Teflon bottle, and stored at 4°C until further use.

[0188] (3) Remineralization of hydroxyapatite sheets in SBF:

[0189] The treated hydroxyapatite pieces were immersed in SBF solution and placed in a 37°C incubator. Each mineralization cycle was 24 hours, with the pieces removed and the SBF solution refreshed every 24 hours for seven cycles.

[0190] (4) Evaluation of remineralization effect:

[0191] 4.1) Surface morphology: Figure 25 (A is a 2000x surface SEM image of a hydroxyapatite piece in the negative control group (acid etching treatment only); B is a 2000x surface SEM image of a piece in the SBF system mineralization group of comparative example 3) as shown in FIG. 3 . After 7 mineralization cycles in SBF, a small amount of flake crystals appeared on the surface of the piece in comparative example 3 compared with the negative group, indicating a poor mineralization effect and a score of 1.

[0192] 4.2) Cross-sectional morphology: Figure 26 (A is a 1000x cross-sectional SEM image of a hydroxyapatite flake in the negative control group (only acid etching treatment); B is a 2000x cross-sectional SEM image of a flake in the SBF system mineralization group in comparative example 3. The layer above the white dotted line is the remineralized layer, and the layer below is the non-remineralized layer.) As shown, similar to the surface morphology, only a small amount of crystals are generated in the cross-sectional area of ​​the flake that has been mineralized in SBF for 7 cycles. Figure 5 As shown in FIG1 , the thickness of the remineralized layer of Comparative Example 3 is significantly lower than that of Example 1, P < 0.01. Therefore, it is determined that Comparative Example 3 produces a thin and loose remineralized layer and is scored as 1 point.

[0193] 4.3) Calcium-phosphorus ratio: As shown in Table 3, the average calcium-phosphorus ratio of the small amount of crystals produced in Comparative Example 3 was 1.40, ranging from 1.1 to 1.6. It was speculated that it was a hydroxyapatite precursor and scored 2 points.

[0194] 4.4) Item Analysis: Figure 27The crystals generated from the apatite small pieces mineralized in SBF for 7 cycles were dicalcium phosphate dihydrate and calcium hydrogen phosphate, which belonged to hydroxyapatite precursor, and scored 1.

[0195] In summary, based on the re-mineralization effect evaluation scheme established in the present application, the mineralization score of the apatite small pieces in SBF in Comparative Example 3 was 5.

[0196] Comparative Example 4

[0197] The present comparative example provides an in vitro model for evaluating the performance of a re-mineralization promoting material and a method for evaluating the performance of a re-mineralization promoting material, and the specific steps are as follows:

[0198] (1) Acid etching and treatment of hydroxyapatite small pieces:

[0199] The same as Example 2.

[0200] (2) Preparation of mixed gel:

[0201] Except that only 3.5% agarose was used without gelatin, the rest was the same as Example 2.

[0202] (3) Re-mineralization of hydroxyapatite pieces:

[0203] The same as Example 2.

[0204] (4) Evaluation of re-mineralization effect:

[0205] 4.1) Surface morphology: as shown in Figure 28 (A is the surface SEM image of the hydroxyapatite small pieces at 2000 times of the negative control group (only acid etching treatment); B is the surface SEM image at 2000 times of Comparative Example 4 after 7 cycles of mineralization of 6000 ppm sodium fluoride in 3.5% agarose gel; C is the surface SEM image at 10000 times of Comparative Example 4) compared with the negative control group, the surface of Comparative Example 4 generated sparse granular crystals, and the spacing between the granular crystals could be seen after 40000 magnification. According to the established evaluation system, the surface morphology score of Comparative Example 4 was 2.

[0206] 4.2) Cross-sectional morphology: as shown in Figure 29 (A is the cross-sectional SEM image of the hydroxyapatite small pieces at 1000 times of the negative control group (only acid etching treatment); B is the cross-sectional SEM image at 5000 times of Comparative Example 4 after 7 cycles of mineralization of 6000 ppm sodium fluoride in 3.5% agarose gel, wherein the white dashed line above is the re-mineralization layer and the below is the non-re-mineralization layer) compared with the negative control group, Comparative Example 4 generated a thin and loose re-mineralization layer on the surface of the apatite small pieces. As shown in Figure 5As shown, the thickness of the remineralized layer generated by Comparative Example 4 was significantly lower than that of Example 1, P<0.01. Therefore, it was determined that Comparative Example 4 generated a thin and loose remineralized layer, and the cross-sectional morphology score of Comparative Example 4 was 1 according to the established evaluation system.

[0207] 4.3) Calcium to phosphorus ratio: As shown in Table 3, the average calcium to phosphorus ratio of the rod-shaped crystals generated by Comparative Example 4 was 1.59 > 1.6, which was speculated to be a mixture of hydroxyapatite and its precursor, and the score was 2.

[0208] 4.4) Material analysis: As shown in Table 3, the newly generated crystals of Comparative Example 4 contained both hydroxyapatite and dicalcium phosphate dihydrate according to XRD analysis, and the diffraction intensity of dicalcium phosphate dihydrate was stronger, and the score was 2. Figure 30

[0209] In summary, based on the established remineralization effect evaluation scheme of the present application, the mineralization score of Comparative Example 4 was 7.

[0210] Comparative Example 5

[0211] This comparative example provides an in vitro model for evaluating the performance of a remineralization-promoting material and a method for evaluating the performance of a remineralization-promoting material, and the specific steps are as follows:

[0212] (1) Acid etching and treatment of hydroxyapatite small pieces:

[0213] The same as Example 2.

[0214] (2) Preparation of mixed gel:

[0215] Except that only 3.5% gelatin was used instead of agarose, the rest was the same as Example 2.

[0216] (3) Remineralization of hydroxyapatite pieces:

[0217] The same as Example 2.

[0218] (4) Evaluation of remineralization effect:

[0219] 4.1) Surface morphology: As shown in Figure 31 (A is the surface SEM image of the negative control group (acid etching treatment only) hydroxyapatite small pieces 2000 times; Figure B is the surface SEM image of Comparative Example 5 6000 ppm sodium fluoride in 3.5% gelatin mineralized for 7 cycles 2000 times; Figure C is the surface SEM image of Comparative Example 5 40000 times) Compared with the negative control group, Comparative Example 5 generated a large number of densely aggregated granular crystals on the apatite small pieces, and the surface morphology score of Comparative Example 5 was 3 according to the established evaluation system.

[0220] 4.2) Cross-sectional morphology: As shown in Figure 32 ​(A is the negative control group (acid etching treatment only) hydroxyapatite flake 1000 times section SEM image; B is the comparative example 5 6000 ppm sodium fluoride in 3.5% gelatin mineralization after 7 cycles section 2000 times SEM image, which is the white dotted line above the re-mineralization layer, below the non-remineralization layer) as shown, compared with the negative control group, the comparative example 5 in the apatite flake generated a thick and dense re-mineralization layer. As shown in Figure 5 The mineralization thickness of the crystal generated by comparative example 5 was significantly higher than that of example 1, P<0.01. Therefore, it is concluded that comparative example 5 generates a thick and dense re-mineralization layer, with a score of 4.

[0221] 4.3) Calcium to phosphorus ratio: As shown in Table 3, the average calcium to phosphorus ratio of the rod-shaped crystals generated by comparative example 5 is 1.69>1.6, which is speculated to be hydroxyapatite, with a score of 3.

[0222] 4.4) Item analysis: As shown in Figure 33 The diffraction peaks of the newly generated granular crystals of comparative example 5 analyzed by XRD correspond to the characteristic peaks on the hydroxyapatite standard card one by one, proving that the newly generated crystals are hydroxyapatite, with a score of 3

[0223] In summary, based on the re-mineralization effect evaluation scheme established by the present application, the mineralization score of comparative example 4 is 13.

[0224] Table 3

[0225]

[0226] The re-mineralization evaluation effects of examples 1-5 and comparative examples 1-5 above are summarized in Table 4:

[0227] Table 4

[0228]

[0229] As can be seen from Table 4, the hydroxyapatite flake-hybrid gel re-mineralization system established by the present application successfully evaluates the re-mineralization effect of sodium fluoride, olaflur and bioactive glass.

[0230] By analyzing comparative example 1 and example 1, it can be seen that the re-mineralization effect of the hybrid gel system of example 1 is obviously better than that of comparative example 1 (0.5% agarose system). Specifically, the crystal surface morphology of the hybrid gel system is more aggregated, the section morphology is more dense, and the calcium to phosphorus ratio of the generated crystal is higher.

[0231] By analyzing example 2 and example 3, it can be seen that reducing the gelatin concentration results in a slightly worse re-mineralization effect of 12300 ppm sodium fluoride than that of 6000 ppm.

[0232] From the analysis of Comparative Example 2 and Example 2, it can be seen that the hydroxyapatite sheet can replace the tooth enamel sheet to a certain extent to evaluate the effect of the remineralization material. Specifically, 6000 ppm sodium fluoride can exhibit the characteristics of promoting the conversion of hydroxyapatite crystals to generate and orderly grow on the enamel sheet and the synthetic hydroxyapatite sheet.

[0233] From the analysis of Example 1 and Comparative Example 3, it can be seen that the mixed gel system is much better than the simulated body fluid for the mineralization of the hydroxyapatite sheet. Specifically, in the same mineralization period, the mixed gel system generates more crystals, the mineralization layer is thicker, and the result is more convenient for scoring and evaluation.

[0234] From the analysis of Example 2 and Comparative Examples 4-5, it can be seen that the evaluation effect of the model is effectively improved by using gelatin and agarose in combination. Specifically, in the evaluation of 6000 ppm, the crystals generated by Example 2 are orderly fibrous, while Comparative Example 4 has a larger concentration of agarose and smaller hydrogel pores, resulting in a much poorer mineralization effect than Example 2; Comparative Example 5 lacks the effect of agarose, resulting in the generation of granular crystal agglomeration, i.e., the generated crystals have no obvious orientation. From the analysis of Example 2 and Comparative Examples 4-5, it can be seen that the superiority of the combination of gelatin and agarose is used in the present application.

[0235] The applicant declares that the in vitro model for evaluating the performance of the remineralization material and the construction method and application thereof of the present application are illustrated by the above examples, but the present application is not limited to the above examples, i.e., it does not mean that the present application must rely on the above examples to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc., all fall within the protection scope and disclosure scope of the present application.

[0236] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application.

[0237] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination manners.

Claims

1. A method of constructing an in vitro model to assess the performance of a remineralization-promoting material, characterized in that, The construction method comprises the following steps: (1) Gel preparation: mix and melt agarose, gelatin and water to obtain a blank mixed hydrogel; mix and melt agarose, gelatin and Na2HPO4 aqueous solution to obtain a hydrogen phosphate hydrogel; the mass fraction of agarose in the blank mixed hydrogel and the hydrogen phosphate hydrogel is independently 0.3-0.7%, and the mass fraction of gelatin is independently 2.5-3.5%; The concentration of the Na2HPO4 aqueous solution is 0.2-0.3M, and the pH is 7.0-7.3; (2) Model assembly: open a hole at the bottom of the small tube, and then temporarily seal it; add the hydrogen phosphate hydrogel into the small tube, and form a first hydrogen phosphate hydrogel layer after cooling; then put in the hydroxyapatite sheet, and add the hydrogen phosphate hydrogel again, and form a second hydrogen phosphate hydrogel layer after cooling; then add the blank mixed hydrogel, and form a blank gel layer after cooling; finally, add the calcium ion solution to form a calcium ion solution layer, and remove the temporary seal at the bottom of the small tube to complete the assembly of the small tube; the concentration of calcium ions in the calcium ion solution is 0.1-0.15M, and the pH is 5.5-6.5; Insert the assembled small tube into the large tube which is sealed at the bottom and contains the Na2HPO4 aqueous solution, and the in-vitro model assembly is completed.

2. The construction method according to claim 1, characterized in that, The thickness of the first hydrogen phosphate hydrogel layer in step (2) is 0.8-1.2cm.

3. The construction method of claim 1, wherein, The thickness of the second hydrogen phosphate hydrogel layer in step (2) is 1.5-2.5mm.

4. The construction method of claim 1, wherein, The thickness of the blank gel layer in step (2) is 1.5-2.5mm.

5. The construction method of claim 1, wherein, The thickness of the calcium ion solution layer in step (2) is 60-70mm.

6. The construction method of claim 1, wherein, In the large tube in step (2), the height of the Na2HPO4 aqueous solution above the bottom of the small tube is 5-10mm.

7. An in-vitro model for evaluating the performance of a pro-remineralization material, which is constructed by the construction method according to any one of claims 1-6.

8. A method of assessing the performance of a pro- remineralization material, characterized by, The method comprises the following steps: immerse the hydroxyapatite sheet in a solution of the material to be tested, then take it out, and assemble the in-vitro model for evaluating the performance of a pro-remineralization material according to the construction method of any one of claims 1-6; then place the model for several mineralization periods, update the solution and gel in the in-vitro model after each mineralization period, and immerse the hydroxyapatite sheet in the solution of the material to be tested again before taking it out; observe the morphology of the hydroxyapatite sheet and perform composition detection after the test is completed, score the results, and the evaluation is completed.

9. The method of assessing the performance of a pro- remineralisation material according to claim 8, wherein, The time of the mineralization period is 20-28h.

10. The method of assessing the performance of a pro- remineralisation material according to claim 8, wherein, The number of the mineralization periods is at least 5.

11. The method of assessing the performance of a pro- remineralisation material according to claim 8, wherein, The immersion time is 2.5-3.5min.

12. The method of assessing the performance of a pro- remineralisation material according to claim 8, wherein, The scoring standard is as follows: Observe the surface morphology and cross-sectional morphology of the hydroxyapatite sheet by scanning electron microscopy; in the surface morphology, if it presents a sheet shape and a small amount of loose, score 1; if it presents a sheet shape and a large amount of dense, score 2; if it presents a granular or fused shape and is scattered and disordered, score 2; if it presents a granular or fused shape and is densely aggregated, score 3; if it presents a fibrous or rod shape and is in a small amount, score 3; if it presents a fibrous or rod shape and is in a large amount, score 4. The cross-sectional morphology is thin and loose, and is scored 1 point; The cross-sectional morphology is thick and loose, and is scored 2 points; The cross-sectional morphology is thin and dense, and is scored 3 points; The cross-sectional morphology is thick and dense, and is scored 4 points; The cross-sectional morphology is thin and ordered, and is scored 5 points; The cross-sectional morphology is thick and ordered, and is scored 6 points; The atomic concentration of the generated crystal on the surface of the hydroxyapatite sheet is analyzed by energy spectrum analysis, and the Ca / P value is calculated. If the Ca / P value is lower than 1.1, it is scored 1 point; if the Ca / P value is 1.1-1.6, it is scored 2 points; and if the Ca / P value is higher than 1.6, it is scored 3 points; The phase of the generated crystal on the surface of the hydroxyapatite sheet is determined by XRD. If the phase is all hydroxyapatite precursor, it is scored 1 point; if the phase is a mixture of hydroxyapatite precursor and hydroxyapatite, it is scored 2 points; if the phase is all hydroxyapatite, it is scored 3 points; if the phase is a mixture of fluorapatite and its precursor, it is scored 4 points; and if the phase is all fluorapatite, it is scored 5 points.

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