Viscous calcified protein hydrogel for inducing dentinal tubule depth remineralization as well as construction method and application thereof

Abstract

The invention discloses viscous calcified protein hydrogel for inducing dentinal tubule depth remineralization and a construction method and application thereof, and the construction method comprises the following steps: dissolving bovine serum albumin (BSA) in deionized water, adding calcium oxide (CaO), and stirring and mixing at normal temperature to form a suspension, with the mass ratio of BSA to CaO being (1-2.5): 0.4. The calcified hydrogel (BCaH) with a loose and porous structure is successfully prepared through reaction of BSA and CaO in an aqueous solution at normal temperature. It is proved that the constructed calcified hydrogel has good adhesion performance, can induce biomimetic mineralization to seal dentinal tubules, can improve enzymolysis tolerance of collagen, has good tissue biocompatibility, actively responds to a microenvironment of dentin in an oral environment to obtain a deep stable DTs sealing effect, and is easy to operate and remarkable in effect.

Description

Technical Field

[0001] The present invention belongs to the field of materials for treating dentin hypersensitivity, and particularly relates to an adhesive calcified protein hydrogel for inducing deep remineralization of dentinal tubules, and a construction method and application thereof. Background Art

[0002] Dentin hypersensitivity (DH) is a common symptom in oral clinical diagnosis and treatment. Its pathogenesis is closely related to the external stimulation of exposed dentinal tubules (DTs). At present, the desensitization strategy based on the "fluid dynamics theory" mainly relieves pain by blocking the dentinal tubules to reduce fluid flow. Commonly used clinical methods include oxalate, fluoride precipitation or laser melting technology, which seal the dentinal tubules through mineral deposition or the formation of insoluble substances. However, existing materials have the following limitations in the complex dynamic environment of the oral cavity: (1) The sealing depth is insufficient, and it is difficult to penetrate deep into the dentinal tubules to form a long-term seal; (2) The interfacial bonding force with the dentin matrix is weak, resulting in the sealing layer being easily affected by mechanical wear or acid corrosion and falling off.

[0003] Biomimetic remineralization strategies deliver mineral ions and mineralized templates deep within the dentinal tubules, inducing the formation of internal mineralized products, theoretically providing a more stable seal. However, existing biomimetic mineralized materials still face challenges in practical application: for example, insufficient delivery efficiency of the mineralized templates and limited adhesion of the materials to dentin make it difficult to effectively initiate mineralization deep within the tubules. Furthermore, the complex synthesis steps or specific reaction conditions of some materials limit their clinical applicability.

[0004] Calcium oxide (CaO) has potential value in tooth restoration due to its high calcium ion release capacity and biocompatibility. Bovine serum albumin (BSA), a widely available, low-cost natural protein, is often used in biomimetic mineralization research. However, how to combine the two through simple methods to construct a hydrogel system with both adhesion, sustained calcium ion release, and mineralization template function to achieve deep remineralization and sealing of dentinal tubules remains a technical challenge. Summary of the Invention

[0005] Technical problem to be solved: In view of the fact that dentin hypersensitivity is a common clinical symptom in dental clinics, the present invention provides an adhesive calcified protein hydrogel for inducing deep remineralization of dentinal tubules, as well as a construction method and application thereof.

[0006] Technical solution: A method for constructing an adhesive calcified protein hydrogel that induces deep remineralization of dentinal tubules, comprising the following steps: dissolving bovine serum albumin (BSA) in deionized water, adding calcium oxide (CaO), and stirring and mixing at room temperature to form a suspension, wherein the mass ratio of BSA to CaO is (1-2.5):0.4.

[0007] Preferably, the mass ratio of BSA to CaO is selected from any one of the following combinations: 100 mg BSA to 40 mg CaO, 80 mg BSA to 40 mg CaO, 60 mg BSA to 40 mg CaO, and 40 mg BSA to 40 mg CaO.

[0008] The hydrogel forms a loose porous structure through electrostatic interaction and disulfide bonds, and the gelation time is to form a stable gel within 30 seconds after stirring at room temperature.

[0009] The adhesive calcified protein hydrogel prepared by the above method contains uniformly distributed calcium ions and has the in-situ gelling property of adhering to the dentin surface and the dentin tubules.

[0010] The adhesive strength of the hydrogel is measured by a lap shear tensile test and is 0.1 MPa to 1.5 MPa.

[0011] Application of the above-mentioned adhesive calcified protein hydrogel in the preparation of materials for treating dentin hypersensitivity.

[0012] The hydrogel enhances the enzymatic resistance of collagen through physical or chemical interaction with the collagen fibers on the surface of dentin.

[0013] A reagent composition for sealing dentinal tubules comprises the above-mentioned adhesive calcified protein hydrogel and a pharmaceutically acceptable carrier.

[0014] The above-mentioned carrier includes artificial saliva or phosphate buffer.

[0015] A method for evaluating the sealing effect of dentinal tubules comprises the following steps: immersing the hydrogel-treated dentin slice in artificial saliva, observing the mineralized sealing morphology of the dentinal tubule cross-section and longitudinal section by scanning electron microscopy (SEM), and analyzing the mineralization depth by xylenol orange staining combined with laser confocal microscopy.

[0016] Beneficial Effects: A calcified hydrogel (BCaH) with a loose, porous structure was successfully prepared by reacting BSA and CaO in aqueous solution at room temperature. Its gelation process relies primarily on electrostatic interactions and disulfide bond formation, endowing it with excellent biocompatibility and biodegradability. The calcified hydrogel exhibits excellent adhesion properties, adhering to the dentin surface and within the dentin dentine (DTs) through in situ gelation. In an artificial saliva environment, it attracts mineral ions and serves as a mineralization template, inducing mineralization to tightly seal the DTs to a depth of over 100 μm. The resulting remineralization is tightly integrated with natural tooth hard tissue and resists mechanical stimulation and acid attack to a certain degree. The calcified hydrogel adheres to the surface of demineralized dentin matrix and, through interaction with collagen, improves its resistance to enzymatic degradation. During the mineralization process, it promotes dentin remineralization and protects collagen fibers. The calcified hydrogel has no inhibitory effect on the proliferation of human dental pulp stem cells and gingival fibroblasts, and has no significant irritation to the oral mucosa, demonstrating excellent tissue biocompatibility. In the complex oral environment in vivo, the calcified hydrogel can actively respond to the microenvironment of the dentin (such as liquid flow, ion concentration, etc.), triggering a biomimetic mineralization reaction, and obtaining a deep and stable DTs sealing effect, further confirming the clinical operability of the calcified hydrogel, which is simple to operate and effective, and has huge application potential in the field of DH treatment. The present invention successfully prepared a calcified hydrogel (BCaH) with a loose porous structure by reacting BSA and CaO in an aqueous solution at room temperature. It was confirmed that the constructed calcified hydrogel has good adhesion properties, can induce biomimetic mineralization to seal dentinal tubules, can improve the enzymatic tolerance of collagen, has good tissue biocompatibility, actively responds to the microenvironment of dentin in the oral environment to obtain a deep and stable DTs sealing effect, is simple to operate, and has a significant effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Gelation of hydrogels synthesized with different BSA / CaO mass ratios.

[0018] Figure 2 Morphological characterization of hydrogels synthesized with different BSA / CaO mass ratios.

[0019] Figure 3 Cumulative release rates of BSA from hydrogels synthesized with different BSA / CaO mass ratios.

[0020] Figure 4 (A) Adhesion of hydrogel to different substrates; (B) Schematic diagram and actual image of hydrogel adhesion to substrate surface; (C) BCaH 2.5 Adhesion strength of BCaH2 hydrogel on glass and plastic surfaces after curing for 24 h at room temperature.

[0021] Figure 5Sealing effect of dentinal tubules on the surface (A) and longitudinal section (B) after 7 days of mineralization in artificial saliva.

[0022] Figure 6 SEM images of the surface and cross-section of dentin slices under conditions simulating daily mechanical wear in the oral cavity.

[0023] Figure 7 Longitudinal cross-sectional CLSM images of calcification formation in dentinal tubules after dentin slices were incubated in artificial saliva for 1, 4, and 7 days.

[0024] Figure 8 Hydroxyproline release (A) and dry mass loss (B) of demineralized dentin after cross-linking with different solutions and digestion with type I collagenase for 48 hours.

[0025] Figure 9 CCK-8 was used to detect the effect of BCaH2 hydrogel on the proliferation of human gingival fibroblasts (HGFs).

[0026] Figure 10 Histological sections of irritated oral mucosa of golden hamsters treated with BCaH2 hydrogel and saline (control group).

[0027] Figure 11 SEM images of the surface and cross-section of New Zealand White rabbit dentin sections. DETAILED DESCRIPTION

[0028] The following examples may enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0029] Example 1

[0030] Take a 1.5mL centrifuge tube, weigh different masses of BSA (100mg, 80mg, 60mg, 40mg) and dissolve them in 1mL of deionized water. Add 40mg of CaO to each tube, stir and mix at room temperature to form a suspension, and observe the gelation of the hydrogel with the naked eye. According to the mass ratio of BSA and CaO, the resulting calcified hydrogel is named BCaH 2.5 、BCaH2、BCaH 1.5 The gelation reaction of the suspension was evaluated by the inverted bottle method, that is, the prepared suspension was transferred to a 2 mL glass bottle. If the solution did not flow within 30 s when inverted, it was judged to be a gel. The reaction time at this time was defined as the gelation time. Figure 1 The hydrogel structure was observed by scanning electron microscopy. Figure 2 .

[0031] Example 2: In vitro degradation experiment of calcified hydrogel.

[0032] The protein degradation and release performance of the calcified hydrogel was tested by in vitro degradation experiments. Four 1.5 mL centrifuge tubes were prepared. According to the method of 2.2.1.1, calcified hydrogel solutions with different protein concentrations were prepared. After cross-linking for 1 hour, 1 mL of artificial saliva was added to each centrifuge tube and placed in a 37 ° C incubator. The following release time points were set: 2h, 4h, 8h, 12h, 24h, 2 days, 3 days, 4 days, 5 days, 6 days and 7 days. At each time point, the supernatant of each sample was removed with a pipette, and an equal amount of fresh artificial saliva was added to each tube. The protein concentration at each time point was determined using the BCA protein concentration assay kit and the data was analyzed. The Origin2024 software was used for graphics, as shown below. Figure 3 .

[0033] Example 3: Adhesion detection of calcified hydrogel.

[0034] The adhesion of the calcified hydrogel was tested by overlapping shear tensile testing using a universal material testing machine. The dentin slices were tested by overlapping two dentin slices soaked in gel solution. The diameter of the dentin slices was 8 mm (the overlapping area was 50.27 mm 2 ), after curing at room temperature for 1 hour, the overlap shear tensile test was carried out by a microcomputer-controlled electronic universal testing machine equipped with a 100N sensor. The adhesion strength was calculated by dividing the maximum adhesion force by the initial bonding area. The tensile rate of the test was 5mm / min. The test of other substrates was done by overlapping them in pairs, injecting the prepared gel liquid directly between the two matrices (the overlap area was 25mm×8mm), and then using a 200g weight to press on the overlap part until a gel was formed. After curing at room temperature for 24 hours, the adhesion strength was tested. At least 3 independent samples were tested in each experimental group. Relevant data analysis was carried out and the Origin2024 software was used for drawing. Figure 4 .

[0035] Example 4: Evaluation of the effect of calcified hydrogel-induced biomimetic mineralization to seal dentinal tubules.

[0036] The treated dentin slice samples were immersed in 5 mL of artificial saliva and placed in a 37°C incubator. Fresh artificial saliva was replaced every 24 hours for a total of 7 days. After 7 days, the dentin slices were rinsed with deionized water and dried naturally. The control group, CaO group, BSA group and BCaH2 group were split along the grooves of the pulp cavity. Half of them were used to observe the cross-sectional morphology of the dentinal tubules, and the other half were used to observe the longitudinal morphology of the dentinal tubules. The dentin slice samples were vacuum-sprayed with gold, and the effect of the calcified hydrogel group on remineralization and sealing of the dentinal tubules was evaluated under a scanning electron microscope (SEM). Figure 5 .

[0037] Example 5: Evaluation of the sealing effect of dentinal tubules in a simulated daily oral environment.

[0038] The treated dentin slice samples were immersed in 5 mL of artificial saliva and placed in a 37°C incubator. According to previous literature reports, the recommended normal tooth brushing time is 2 minutes per time, 2 times per day, so the maximum contact time on any tooth surface is usually 5 seconds per time, 2 times per day. In this study, we tried to maintain a design similar to daily tooth brushing. Both groups received mechanical brushing with a soft-bristled toothbrush for 10 seconds per day (applying a constant force of about 150 g perpendicular to the occlusal plane, a frequency of 120 times per minute), twice a day. All dentin slices were placed in artificial saliva on a daily basis, and fresh artificial saliva was replaced every day. After 7 days, the dentin slices were rinsed with deionized water and dried naturally. The dentin slices of each group were split along the grooves, half of which was used to observe the surface morphology of the dentinal tubules, and the other half was used to observe the longitudinal cross-sectional morphology of the dentinal tubules. The dentin slice samples were vacuum-sprayed with gold, and the effects of different treatments on remineralization and sealing of the dentinal tubules were observed under a scanning electron microscope, such as Figure 6 .

[0039] Example 6: Laser confocal microscopy observation of the mineralization process in dentinal tubules.

[0040] The dentin slices were divided into blank control group, BSA group, CaO group and BCaH2 group. After treatment, they were soaked for 20 minutes, dried naturally and then soaked in artificial saliva. The solution was changed every 24 hours. A dentin slice was taken from each group on the 1st, 4th and 7th day respectively. The dentin slices of each group were placed in a 0.5wt.% xylenol orange solution at 37°C and soaked for 24 hours. The dentin slices were divided into two parts. One part was used to observe the effect of remineralization on the surface of the dentin slice to seal the dentinal tubules. The other part of the dentin slices was cut into strips and polished with a hard tissue grinder according to the same SiC sandpaper gradient to prepare the specimens with a thickness of 50μm. After polishing, they were ultrasonically cleaned for 5 minutes. The remineralization sealing of the dentinal tubules in the dentin slices of each group was observed from the longitudinal section using a laser confocal microscope. Among them, xylenol orange is excited at 570nm to emit a red spectrum of 610nm, such as Figure 7 .

[0041] Example 7: Exploring the effect of calcified hydrogel on collagen fibers in demineralized dentin.

[0042] 1. Determination of drying mass loss

[0043] (1) Under running water cooling, a 1 mm thick dentin slice was cut perpendicular to the long axis near the pulp. The enamel layer was removed using a high-speed turbine diamond drill. After decalcification in 10% phosphoric acid solution for 24 h, a 2 × 6 × 1 mm dentin block was cut with a scalpel and decalcified for another 24 h. After demineralization, the demineralized dentin matrix was thoroughly rinsed with distilled water three times and hydrated with distilled water for 1 h before use.

[0044] (2) Experimental grouping: The demineralized dentin matrix was randomly divided into 5 groups, with 5 pieces in each group, and immersed in the prepared distilled water, 10% GA, BSA solution, CaO solution and BCaH2 group calcified hydrogel solution for 20 minutes.

[0045] (3) Drying and weighing: Remove each treatment solution, rinse with ddH2O three times, freeze-dry for 12 hours, and weigh. Use an analytical balance to weigh three times and obtain the average value m0.

[0046] (4) Type I collagenase digestion: Add 1 mL of ddH2O to rehydrate for 1 hour, then transfer the dentin slices to 1 mL of type I collagenase preheated at 37°C for digestion and digest in a 37°C incubator for 48 hours.

[0047] (5) Dry quality of dentin after digestion: After digestion, the supernatant was transferred to a new EP tube for subsequent measurement of hydroxyproline release. The remaining dentin block was rinsed three times with ddH2O, freeze-dried for 12 h, and weighed three times to obtain the average m L , m0 is the dry mass of dentin before digestion, mL is the dry mass of dentin after digestion.

[0048] Calculation formula: Drying mass loss (%) = (m0-m L ) / m0×100%

[0049] 2. Hydroxyproline determination

[0050] 500 μL of the supernatant from each of the dentin digests was taken and alkaline hydrolyzed according to the hydroxyproline assay kit. The pH was adjusted and the volume was adjusted to 10 mL. After carbon powder adsorption and centrifugation, 0.5 mL of the sample was collected for hydroxyproline content determination. 0.5 mL of the standard (5 μg / mL) and distilled water were also collected as controls. The subsequent steps were: add 0.25 mL of reagent 1, mix, and let stand for 10 minutes; add 0.25 mL of reagent 2, mix, and let stand for 5 minutes; add 0.25 mL of reagent 3, mix, incubate at 60°C for 15 minutes, cool, and centrifuge at 3500 rpm for 10 minutes. 200 μL of the supernatant was collected and the OD550 value was determined.

[0051] The formula for calculating the release amount of hydroxyproline in μg / mg is:

[0052]

[0053] The experimental results were analyzed using GraphPad Prism9 software and graphed using Origin2024 software. Figure 8 .

[0054] Example 8: Effect of calcified hydrogel on the proliferation activity of human dental pulp stem cells.

[0055] Select P3-P4 generation human dental pulp stem cells with good growth status and use 2×10 4 The cells were seeded at a density of 100 cells / well in a 24-well plate. The trans-dentin slice devices were installed in the wells of the 24-well plate. 500 μL of culture medium was added to each well. The entire Transwell culture plate was placed in a 37°C, 5% CO2 cell culture incubator for one day. On the second day, when the cells were well attached, 100 μL of BCaH2 hydrogel solution was added to the dentin slice in the Transwell chamber of the BCaH2 group. The cells were allowed to stand for 20 minutes and then placed back in the 24-well plate. The control group used PBS buffer. Three samples were placed in each group and cultured in the cell culture incubator. The medium was changed every two days. Figure 9 .

[0056] Example 9: Mucosal irritation test of calcified hydrogel solution

[0057] Dentin slices soaked in calcified hydrogel were placed in the left cheek pouch of a golden hamster for 20 minutes, and dentin slices soaked in saline were placed in the right cheek pouch as a self-control. Afterwards, both cheek pouches were rinsed with saline. Figure 10 .

[0058] Example 10: Establishing a New Zealand White Rabbit Tooth Sensitivity Experimental Model

[0059] A high-speed turbine handpiece was used to remove the lower 1 / 3 of the enamel on the labial side of the maxillary and mandibular incisors under water cooling to expose the dentin. The exposed dentin was etched with 0.5M EDTA for 2 minutes to remove the smear layer, and then rinsed thoroughly with an air gun to expose the dentinal tubules to simulate dentin hypersensitivity. The experiment was divided into a blank control group and a BCaH2 hydrogel group. For the same New Zealand white rabbit, the maxillary incisors were treated with BCaH2 gel for 20 minutes as the experimental group, and the mandibular incisors were treated with deionized water for 20 minutes as the control group. All rabbits were killed by overdose of anesthesia after 7 days, and the maxillary and mandibular incisors were extracted with dental extraction forceps. After rinsing with deionized water, the teeth were dried naturally, vacuum dried, and then gold-sprayed. The surface morphology and longitudinal cross-sectional morphology of the dentin were observed using a field emission SEM, as shown in the following figure. Figure 11 .

Claims

1. A method for constructing a calcified protein hydrogel that can induce deep remineralization of dentinal tubules, characterized in that: The following steps are involved: Bovine serum albumin (BSA) was dissolved in deionized water, calcium oxide (CaO) was added, and the mixture was stirred at room temperature to form a suspension, wherein the mass ratio of BSA to CaO was (1-2.5):0.

4.

2. The construction method according to claim 1, characterized in that The mass ratio of BSA to CaO is selected from any one of the following combinations: 100 mg BSA to 40 mg CaO, 80 mg BSA to 40 mg CaO, 60 mg BSA to 40 mg CaO, and 40 mg BSA to 40 mg CaO.

3. The construction method according to claim 1 or 2, characterized in that The hydrogel forms a loose porous structure through electrostatic interaction and disulfide bonds, and the gelation time is to form a stable gel within 30 seconds after stirring at room temperature.

4. The adhesive calcified protein hydrogel prepared by the method of claim 3, characterized in that: The hydrogel contains uniformly distributed calcium ions and has the in-situ gelling property of adhering to the dentin surface and the dentin tubules.

5. The adhesive calcified protein hydrogel according to claim 4, characterized in that: The adhesion strength of the hydrogel was determined to be 0.1 MPa to 1.5 MPa by lap shear tensile test.

6. Use of the adhesive calcified protein hydrogel according to claim 4 or 5 in preparing a material for treating dentin hypersensitivity.

7. The use according to claim 6, characterized in that The hydrogel interacts with the collagen fibers on the surface of dentin physically or chemically to enhance the enzymatic degradation resistance of the collagen.

8. A reagent composition for sealing dentinal tubules, characterized in that: The invention comprises the adhesive calcified protein hydrogel according to claim 4 and a pharmaceutically acceptable carrier.

9. The reagent composition according to claim 8, characterized in that The carrier includes artificial saliva or phosphate buffered saline.

10. A method for evaluating the sealing effect of dentinal tubules, characterized in that: The method comprises the following steps: immersing the dentin slice treated with the hydrogel according to claim 4 in artificial saliva, observing the mineralized sealing morphology of the cross section and longitudinal section of the dentinal tubules by scanning electron microscopy (SEM), and analyzing the mineralization depth by xylenol orange staining combined with laser confocal microscopy.

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