Bionic polypeptide cp20k-p3 for inducing remineralization of demineralized dentin and application thereof

Abstract

The invention discloses a biomimetic polypeptide cp20k-p3 for inducing remineralization of demineralized dentin and application of the biomimetic polypeptide cp20k-p3, the biomimetic polypeptide disclosed by the invention is a polypeptide cp20k-p3 derived from barnacle gum protein 20K, and the amino acid sequence of the biomimetic polypeptide cp20k-p3 is CNQKHPCWRRHGKKHGLHRKFHGNACNC. According to the bionic polypeptide disclosed by the invention, amorphous calcium phosphate can be rapidly generated in simulated body fluid and can be well adsorbed on the surface of a demineralized dentin matrix, and newly generated hydroxyapatite crystals can deeply seal exposed demineralized dentin tubules. A brand new biomineralization polypeptide is provided for bone demineralization dentin repair.

Description

Technical Field

[0001] The present invention belongs to the technical field of materials for repairing demineralized dentin, and particularly relates to a biomimetic polypeptide cp20k-p3 for inducing remineralization of demineralized dentin and its application. Background Art

[0002] Tooth wear, fracture, and caries can all lead to tooth defects and exposure of dentin. There are a large number of radially arranged dentinal tubules in dentin. When the dentinal tubules are exposed to various external stimuli, the liquid components, flow, and pressure in the dentin suddenly change, stimulating the pulp nerve endings, thus causing dentin hypersensitivity. The incidence of dentin hypersensitivity in the population is approximately 33.5%.

[0003] Currently, the materials used to seal dentinal tubules have certain limitations, such as limited penetration depth, slow remineralization, and lack of long-term stability.

[0004] There are already relevant studies. For example, CN 114605516 A discloses a barnacle glue protein cp20k-derived polypeptide cp20k-p3 with biomineralization function, which can significantly promote the proliferation and osteogenic induction differentiation of mouse bone marrow mesenchymal stem cells, but cp20k-p3 has not been applied to the field of inducing remineralization of demineralized dentin. Summary of the Invention

[0005] Aiming at the problems of slow dentin remineralization rate and poor mineralization effect in the prior art, the present invention provides a biomimetic polypeptide cp20k-p3 for inducing remineralization of demineralized dentin and its application. The biomimetic polypeptide cp20k-p3 can selectively adsorb on the surface of demineralized dentin and perform remineralization on the surface and inside of the demineralized tooth, ultimately restoring the mechanical strength of the tooth surface.

[0006] The object of the present invention is achieved through the following technical solutions: A biomimetic polypeptide cp20k-p3 for inducing remineralization of demineralized dentin, the biomimetic polypeptide cp20k-p3 is derived from the barnacle glue protein 20K-derived polypeptide, and the amino acid sequence of this polypeptide is: CNQKHPCWRRHGKKHGLHRKFHGNACNC.

[0007] Furthermore, the biomimetic polypeptide cp20k-p3 is synthesized by chemical synthesis method.

[0008] Furthermore, the demineralized dentin is obtained by cross-cutting an ex vivo tooth and etching the dentin surface with 37% phosphoric acid for 30 s.

[0009] The present invention also relates to the application of the above-mentioned biomimetic polypeptide cp20k-p3 for inducing remineralization of demineralized dentin, specifically for preparing a repair material for inducing remineralization of demineralized dentin. The above-mentioned biomimetic polypeptide cp20k-p3 for inducing remineralization of demineralized dentin is added to simulated body fluid. The simulated body fluid added with the biomimetic polypeptide cp20k-p3 can be directly mineralized in vitro. In addition, the biomimetic polypeptide cp20k-p3 can also adsorb on the surface of demineralized dentin, which is beneficial for the polypeptide to induce remineralization on the surface of demineralized dentin, and the formed remineralized layer can seal the exposed dentinal tubules.

[0010] The application of a biomimetic polypeptide cp20k-p3 for inducing remineralization of demineralized dentin includes the following steps: S1. Prepare simulated body fluid with the concentration of each component as follows: 137.5 mM NaCl, 3.0 mM KCl, 1.0 mM K2HPO4, 1.5 mM MgCl2, 2.5 mM CaCl2, 0.5 mM Na2SO4, 10.0 mM HEPES, 0.77 mM NaF; S2. Dissolve the biomimetic polypeptide cp20k-p3 in the simulated body fluid with the concentration of the biomimetic polypeptide cp20k-p3 being 0.01 mg / mL - 1 mg / mL, mix well to obtain the simulated body fluid containing cp20k-p3, and the obtained simulated body fluid containing cp20k-p3 is used for inducing remineralization of demineralized dentin.

[0011] Further, in S2, the concentration of the biomimetic polypeptide cp20k-p3 is 0.3 mg / mL.

[0012] Further, after incubating the simulated body fluid containing cp20k-p3 in a culture dish and sampling, the experimental results of cp20k-p3-induced in vitro mineralization of the simulated body fluid can be obtained; Add fluorescein isothiocyanate (FITC)-labeled cp20k-p3 (named FITC / cp20k-p3) to phosphate buffer at pH = 7.4, and use this solution to incubate demineralized teeth to study the adsorption effect of cp20k-p3 on demineralized dentin; Add demineralized tooth slices to the simulated body fluid containing cp20k-p3, incubate at 37 °C for 3 to 6 days to repair demineralized dentin, and it can induce the formation of a remineralized layer on the surface of demineralized dentin to seal the dentinal tubules.

[0013] Further, for the added fluorescein isothiocyanate (FITC)-labeled cp20k-p3, the fluorescein isothiocyanate (FITC) is labeled on the lysine at the 20th position in the cp20k-p3 sequence.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. The biomimetic polypeptide cp20k-p3 for inducing remineralization of demineralized dentin according to the present invention can rapidly induce the formation of amorphous calcium phosphate in simulated body fluid, and then promote the formation of hydroxyapatite crystals. In addition, it can selectively adsorb on the surface of the organic matrix exposed by demineralized dentin, form a mineralized layer on the surface of demineralized dentin, and seal the dentinal tubules.

[0015] 2. The present invention provides a biomimetic polypeptide cp20k-p3 for inducing remineralization of demineralized dentin, which has great application prospects in the repair of demineralized dentin. The application scenario of the biomimetic polypeptide cp20k-p3 according to the present invention is different from that of existing polypeptides. The existing polypeptide of the biomimetic polypeptide cp20k-p3 is used as a bone repair bioactive factor to promote the osteogenic induction and differentiation of bone marrow mesenchymal stem cells. The application of the biomimetic polypeptide cp20k-p3 for inducing remineralization of demineralized dentin according to the present invention is to adhere the biomimetic polypeptide cp20k-p3 to the surface of demineralized teeth. Under the action of this biomimetic polypeptide, the demineralized dentin directly undergoes remineralization without the participation of cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The drawings are used to provide a further understanding of the present invention, and constitute a part of the specification. They are used together with the embodiments of the present invention to explain the present invention, and do not constitute a limitation to the present invention.

[0017] Figure 1 Transmission electron micrographs of simulated body fluid and simulated body fluid supplemented with cp20k-p3 incubated for 15 minutes and 6 days in Example 1 of the present invention (a is the transmission electron micrograph of simulated body fluid without cp20k-p3 incubated at 37°C for 15 minutes; b is the transmission electron micrograph of simulated body fluid containing cp20k-p3 incubated at 37°C for 15 minutes; c is the transmission electron micrograph of simulated body fluid without cp20k-p3 incubated at 37°C for 6 days; d is the transmission electron micrograph of simulated body fluid containing cp20k-p3 incubated at 37°C for 6 days; e is the diffraction image of the yellow small box area in c in SAED mode; f is the diffraction image of the yellow small box area in d in SAED mode); Figure 2 Confocal fluorescence images taken after treating demineralized teeth with FITC and cp20k-p3 loaded with FITC in Example 2 of the present invention (a is the image after treating demineralized teeth with FITC; b is the image after treating demineralized teeth with cp20k-p3 loaded with FITC); Figure 3Scanning electron microscope images of the acid-etched dentin sections in Example 3 of the present invention and the demineralized tooth sections after incubation in simulated body fluid containing cp20k-p3 (a is the scanning electron microscope image of the demineralized dentin surface after acid etching magnified 1000 times; b is the scanning electron microscope image of the demineralized dentin surface after acid etching magnified 5000 times; c is the scanning electron microscope image of the demineralized dentin surface after treatment with cp20k-p3 magnified 1000 times; d is the scanning electron microscope image of the demineralized dentin surface after treatment with cp20k-p3 magnified 5000 times); Figure 4 Scanning electron microscope images of the cross-sections of the acid-etched dentin sections in Example 4 of the present invention and the demineralized tooth sections after incubation in simulated body fluid containing cp20k-p3, and the calcium and phosphorus element distribution maps obtained by scanning energy-dispersive X-ray spectroscopy respectively (a is the scanning electron microscope image of the dentin after acid etching; c is the energy-dispersive X-ray spectroscopy image of calcium in the dentin after acid etching; e is the energy-dispersive X-ray spectroscopy image of phosphorus in the dentin after acid etching; b is the scanning electron microscope image of the dentin after acid etching treated with simulated body fluid containing cp20k-p3; d is the energy-dispersive X-ray spectroscopy image of calcium in the dentin after acid etching treated with simulated body fluid containing cp20k-p3; f is the energy-dispersive X-ray spectroscopy image of phosphorus in the dentin after acid etching treated with simulated body fluid containing cp20k-p3). Detailed implementation manners

[0018] The technical solutions of the present invention will be clearly and comprehensively described below in conjunction with the embodiments and the accompanying drawings. The following described embodiments are only a part of the embodiments of the present invention, not all embodiments. All other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0019] Example 1: The application of a biomimetic polypeptide cp20k-p3 for inducing the remineralization of demineralized dentin is an in vitro mineralization induction test of cp20k-p3, including the following steps: (1) Prepare simulated body fluid, and its formula is: 137.5 mM NaCl, 3.0 mM KCl, 1.0 mM K2HPO4, 1.5 mM MgCl2, 2.5 mM CaCl2, 0.5 mM Na2SO4, 10.0 mM HEPES, 0.77 mM NaF; (2) Add cp20k-p3 to the simulated body fluid so that the concentration of the polypeptide in the simulated body fluid is 0.3 mg / mL; (3) Incubate the simulated body fluid and the simulated body fluid added with cp20k-p3 at 37 °C respectively. When the incubation time is 15 minutes and 6 days, take 10 μL of the solution and place it on a copper grid, and dry it at 37 °C. Results and analysis: Figure 1 They are the transmission electron microscope images of the simulated body fluid and the simulated body fluid added with cp20k-p3 in Example 1 after incubating for 15 minutes and 6 days. The study introduced cp20k-p3 into the simulated body fluid, incubated it at 37 °C, and monitored the transformation process of calcium phosphate from amorphous to crystalline state in the solution by transmission electron microscopy. According to Figure 1 b, it was found that a large number of small granular aggregates were formed in the simulated body fluid added with cp20k-p3 when incubated for 15 min; when the two simulated body fluids were incubated for 6 days, a large number of flaky crystals appeared in the simulated body fluid added with cp20k-p3 ( Figure 1 d), and its diffraction image conforms to the diffraction characteristics of a single crystal with hexagonal symmetry ( Figure 1 f).

[0020] The results show that: At the initial stage of hydroxyapatite mineralization, cp20k-p3 can serve as a nucleation site to promote the aggregation of calcium ions and phosphate ions to form composite nanoparticles; the flaky crystals formed when incubated for 6 days have the typical characteristics of hydroxyapatite crystals, indicating the formation of hydroxyapatite crystals.

[0021] Example 2: Selective adhesion of cp20k-p3: Dissolve FITC / cp20k-p3 and FITC in phosphate buffer; immerse the demineralized tooth sections in the two solutions with the same molar concentration respectively, incubate at room temperature for 5 minutes, and obtain fluorescence images using a laser confocal microscope.

[0022] Results and analysis: Figure 2 They are the confocal fluorescence images taken after treating the demineralized teeth with FITC and FITC-loaded cp20k-p3 respectively in Example 2. The study found that the surface of dentin treated with FITC / cp20k-p3 emitted strong green fluorescence, and almost no fluorescence was seen on the enamel surface ( Figure 2 b).

[0023] This shows that FITC / cp20k-p3 can recognize and selectively adhere to the surface of demineralized dentin. Therefore, we infer that CCP has the ability to adsorb on the organic matrix of the demineralized dentin surface.

[0024] Example 3: Sealing of demineralized dentinal tubules: The dentin slices were demineralized by soaking in 37% phosphoric acid for 30 seconds to obtain acid-etched dentin slices. These dentin slices were then soaked in a simulated body fluid containing cp20k-p3 and incubated at 37 °C for 3 days. Scanning electron microscopy was used to photograph the dentinal tubules after acid etching and the demineralized dentinal tubules after treatment.

[0025] Results and analysis Figure 3 Figure 6 shows scanning electron micrographs of the surface of the acid-etched dentin slices in Example 3 and the demineralized tooth slices after incubation in a simulated body fluid containing cp20k-p3. Figure 3 a shows that the dentinal tubules on the surface of demineralized dentin are fully exposed. Figure 3 c, Figure 3 d shows that the demineralized dentinal tubules are significantly sealed after incubation in a simulated body fluid containing cp20k-p3. This indicates that a significant remineralized layer is formed on the surface of the dentin after incubation in a simulated body fluid containing cp20k-p3, which can effectively seal the demineralized dentinal tubules.

[0026] Example 4: Characterization of remineralization products: The acid-etched dentin slices in Example 3 and the demineralized tooth slices after incubation in a simulated body fluid containing cp20k-p3 were taken. They were broken in half, and the cross-sections were photographed using scanning electron microscopy, and the calcium phosphate distribution in the cross-sections was scanned using energy-dispersive X-ray spectroscopy.

[0027] Results and analysis: Figure 4 Figure 7 shows scanning electron micrographs of the cross-sections of the acid-etched dentin slices in Example 4 and the demineralized tooth slices after incubation in a simulated body fluid containing cp20k-p3, as well as the calcium and phosphorus element distribution maps obtained by scanning energy-dispersive X-ray spectroscopy respectively. An obvious covering layer is formed on the surface of the demineralized dentinal tubules after incubation in a simulated body fluid containing cp20k-p3. The covering layer penetrates into the interior of the dentinal tubules, and there are obvious calcium and phosphorus element signals in this covering layer ( Figure 4 b, Figure 4 d, Figure 4 f).

[0028] The results show that a dense calcium phosphate mineral product is formed on the surface of the demineralized dentinal tubules after incubation in a simulated body fluid containing cp20k-p3.

[0029] Comparative Example 1: The difference between Comparative Example 1 and Example 1 is that the biomimetic polypeptide cp20k-p3 was not added, and other test conditions were the same as those in Example 1.

[0030] The simulated body fluid was incubated at 37 °C. The transformation process of calcium phosphate from amorphous to crystalline state in the solution was monitored by transmission electron microscopy. According to Figure 1 a, it was found that at 15 min of incubation, only a very small amount of large particle aggregates could be observed in the m-SBF group; at day 6, no crystal characteristics were exhibited by the nanoparticles in the simulated body fluid, that is, the amorphous morphology ( Figure 1 c, Figure 1 e).

[0031] The results showed that: It was difficult for the simulated body fluid to nucleate and form amorphous nanoparticles in the short term. At day 6 of incubation, it was difficult for the nanoparticles to transform into hydroxyapatite crystals.

[0032] Comparative Example 2: The difference between Comparative Example 2 and Example 2 was that FITC was labeled on the biomimetic polypeptide cp20k-p3, and other test conditions were the same as those in Example 2; It was found in the study that there was almost no green fluorescence on the surface of the demineralized teeth after FITC treatment ( Figure 2 a).

[0033] The results showed that: FITC did not have the ability to adsorb on the surface of demineralized dentin.

[0034] Comparative Example 3: The difference between Comparative Example 3 and Example 3 was that only phosphoric acid etching was performed for 30 s without other treatments, and other conditions were the same as those in Example 1; Figure 3 a and Figure b showed that the dentinal tubules on the surface of demineralized dentin were fully exposed.

[0035] This indicated that the test protocol of phosphoric acid etching for 30 s could effectively expose the dentinal tubules.

[0036] Comparative Example 4: The difference between Comparative Example 4 and Example 4 was that the observed cross-section was the cross-section of the acid-etched dentinal tubules without treatment, and Example 4 was the cross-section of the dentinal tubules treated with the simulated body fluid added with cp20k-p3, and the experimental conditions were the same for both; The minerals at the orifice of the acid-etched dentinal tubules were dissolved, and the calcium and phosphorus element signals disappeared ( Figure 4 a, Figure 4 c, Figure 4 e).

[0037] The results showed that the minerals on the surface of the acid-etched dentin were partially dissolved by phosphoric acid, and the organic matrix was exposed.

[0038] Results and Discussion: Example 1 and Comparative Example 1 were compared in terms of the mineralization and crystallization processes of hydroxyapatite crystals. The results showed that the simulated body fluid added with cp20k-p3 could promote the nucleation and crystallization of amorphous calcium phosphate.

[0039] Example 2 and Comparative Example 2 verified the feasibility of the interaction between cp20k-p3 and the surface of demineralized dentin. FITC could not adhere to demineralized dentin, while FITC / cp20k-p3 adhered to the surface of demineralized dentin, indicating that cp20k-p3 could interact with demineralized dentin.

[0040] Example 3, Example 4, Comparative Example 3, and Comparative Example 4 verified that the simulated body fluid added with cp20k-p3 could form a dense mineralized layer on the surface of demineralized dentinal tubules, and this mineralized layer effectively sealed the demineralized dentinal tubules.

[0041] The above experiments showed that cp20k-p3 had the ability to promote the formation of hydroxyapatite crystals. It could selectively adhere to the surface of demineralized dentin and generate dense mineralized products on its surface, thereby achieving the sealing of dentinal tubules.

[0042] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A biomimetic polypeptide cp20k-p3 for inducing the remineralization of demineralized dentin, characterized in that: The bionic polypeptide cp20k-p3 is derived from the 20K-derived polypeptide of barnacle glue protein. The amino acid sequence of this polypeptide is: CNQKHPCWRRHGKKHGLHRKFHGNACNC.

2. The biomimetic polypeptide cp20k-p3 for inducing remineralization of demineralized dentin according to claim 1, wherein: The bionic polypeptide cp20k-p3 is synthesized by chemical synthesis.

3. The biomimetic polypeptide cp20k-p3 for inducing remineralization of demineralized dentin according to claim 1, characterized in that: The demineralized dentin is obtained by cross-cutting extracted teeth and etching the dentin surface with 37% phosphoric acid for 30 s.

4. Use of a biomimetic polypeptide cp20k-p3 according to any one of claims 1-3 for inducing remineralization of demineralized dentin, characterized in that: It includes the following steps: S1. Prepare simulated body fluid with the following component concentrations: 137.5 mM NaCl, 3.0 mM KCl, 1.0 mM K2HPO4, 1.5 mM MgCl2, 2.5 mM CaCl2, 0.5 mM Na2SO4, 10.0 mM HEPES, 0.77 mM NaF; S2. Dissolve the bionic polypeptide cp20k-p3 in the simulated body fluid at a concentration of 0.01 mg / mL - 1 mg / mL, mix well to obtain the simulated body fluid containing cp20k-p3, and the obtained simulated body fluid containing cp20k-p3 is used to induce the remineralization of demineralized dentin.

5. Use of a biomimetic polypeptide cp20k-p3 for inducing remineralization of demineralized dentin according to claim 4, characterized in that: In S2, the concentration of the bionic polypeptide cp20k-p3 is 0.3 mg / mL.

6. Use of a biomimetic polypeptide cp20k-p3 for inducing remineralization of demineralized dentin according to claim 4, characterized in that: After incubating the simulated body fluid containing cp20k-p3 in a culture dish and sampling, the results of the in vitro mineralization test of the simulated body fluid induced by cp20k-p3 can be obtained.

7. Use of a biomimetic polypeptide cp20k-p3 for inducing remineralization of demineralized dentin according to claim 4, characterized in that: Add fluorescein isothiocyanate (FITC)-labeled cp20k-p3 to phosphate buffer at pH = 7.4, and use this solution to incubate demineralized teeth to study the adsorption effect of cp20k-p3 on demineralized dentin.

8. Use of a biomimetic polypeptide cp20k-p3 for inducing remineralization of demineralized dentin according to claim 4, characterized in that: Add demineralized tooth slices to the simulated body fluid containing cp20k-p3, incubate at 37 °C for 3 to 6 days to repair demineralized dentin, and induce the formation of a remineralized layer on the surface of demineralized dentin to seal the dentinal tubules.

9. Use of a biomimetic polypeptide cp20k-p3 for inducing remineralization of demineralized dentin according to claim 4, characterized in that: For the added fluorescein isothiocyanate (FITC)-labeled cp20k-p3, the fluorescein isothiocyanate is labeled on the lysine at the 20th position in the cp20k-p3 sequence.

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