Bionic remineralized polypeptide as well as preparation method and application thereof

Abstract

The invention provides a bionic remineralized polypeptide and a preparation method and application thereof, and relates to the technical field of polypeptides, the bionic remineralized polypeptide comprises at least one of a first peptide chain, a second peptide chain and a third peptide chain; the amino acid sequence of the first peptide chain is as shown in SEQ ID NO. 1; the amino acid sequence of the second peptide chain is as shown in SEQ ID NO. 2; and the amino acid sequence of the third peptide chain is as shown in SEQ ID NO.3. The biomimetic remineralized polypeptide is adopted, the biomimetic remineralized polypeptide has the advantages of being small in molecular weight, low in cost, easy to synthesize, good in biocompatibility and the like, the modified polypeptide improves the adsorption capacity of mother peptide to type I collagen, a calcium-phosphorus solution can be stabilized, an amorphous mineralized precursor can be formed, remineralization of demineralized dentin can be promoted, and the biomimetic remineralized dentin is obtained. The oligopeptide disclosed by the invention is convenient to synthesize, relatively low in cost and good in remineralization effect, and provides an ideal new way for preventing and treating caries and dentin hypersensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptides, and in particular to a bionic remineralization polypeptide and a preparation method and application thereof. Background Art

[0002] Teeth are important organs in the human body. Under normal physiological conditions, the demineralization and remineralization of teeth are balanced. However, under pathological conditions, the demineralization effect will be greater than the remineralization, resulting in the destruction of the hard tissue of the tooth. The remineralization of dentin is an important way to prevent and treat carious or non-carious damage to the hard tissue of the tooth. The current preparations that promote the remineralization of the hard tissue of the tooth include fluoride, mineralization precursors such as amorphous calcium phosphate (ACP) and silicates, etc. However, the remineralization caused by most of the existing remineralization preparations does not cause spontaneous nucleation of mineral particles and mineral precipitation in the matrix. Only the growth of the residual crystal nuclei of the demineralized tooth in the calcium-phosphorus ion environment can occur. Therefore, the research on bionic remineralization based on the non-classical mineralization crystallization theory has aroused widespread interest among scholars, and the search for new bionic remineralization materials has become a common concern in the field of oral medicine and biomaterials.

[0003] Bionic peptides refer to peptides with the same sequence and important physiological functions obtained by first screening out peptide sequences with specific functions from human proteins and then synthesizing them in vitro. In recent years, such peptides have been mainly used as functional analogs of stabilizers to regulate crystal growth and promote biomineralization. Based on a large number of studies, non-collagenous proteins (NCPs) account for less than 10% of the dentin matrix, but they have strong calcium ion binding ability and can be adsorbed to collagen fibers, playing a vital role in regulating mineralization and the mechanical integrity of bones. Scholars have gradually focused on peptides derived from NCPs and found that peptides derived from dentin phosphoproteins and dentin matrix protein-1 have a good effect on promoting dentin remineralization.

[0004] At present, the remineralized dentin obtained in the field of bionics still has a large gap with natural tooth hard tissue in terms of mineralization degree and orderliness of mineralized particle arrangement, and the mineralizing peptides based on the design concept of NCPs mainly focus on their interaction with calcium ions and initiating hydroxyapatite nucleation or its stable amorphous mineralized precursors to gradually guide mineralization within the fiber. However, there are few studies on bionic peptides acting on adsorbed collagen fiber design. Summary of the invention

[0005] The main purpose of the present invention is to provide a bionic remineralization polypeptide and a preparation method and application thereof, aiming at promoting the remineralization of demineralized dentin.

[0006] To achieve the above object, the present invention provides a bionic remineralization polypeptide, comprising at least one of a first peptide chain, a second peptide chain and a third peptide chain;

[0007] The amino acid sequence of the first peptide chain is shown in SEQ ID NO.1;

[0008] The amino acid sequence of the second peptide chain is shown in SEQ ID NO.2;

[0009] The amino acid sequence of the third peptide chain is shown in SEQ ID NO.3.

[0010] The present invention also provides a method for preparing a biomimetic remineralization polypeptide, comprising the following steps:

[0011] Providing a resin, and soaking the resin in a solvent to swell it, thereby obtaining a resin carrier;

[0012] According to the amino acid sequence, the amino acid residues with protective groups are dehydrated and condensed onto the resin carrier, and after the condensation is completed, the protective groups are removed, and the amino acid residues are added in sequence, and then a cleavage reagent is added to obtain a biomimetic remineralized polypeptide;

[0013] Wherein, the amino acid sequence includes at least one of the sequences shown in SEQ ID NO.1-3.

[0014] In one embodiment, the resin comprises dichloro resin or CTC resin; and / or,

[0015] The solvent comprises at least one of dichloromethane and N,N-dimethylformamide; and / or,

[0016] The protecting group includes fluorenylmethoxycarbonyl.

[0017] The present invention also proposes an application of a bionic remineralization polypeptide in dentin remineralization.

[0018] In one embodiment, the application comprises the following steps:

[0019] Providing an ex vivo dental crown, preparing the ex vivo dental crown into a dentin block, etching the surface of the dentin block with acid, and immersing the block in an active short peptide solution;

[0020] The dentin block is taken out, ultrasonically cleaned in water, and then immersed in artificial saliva for water bath mineralization for 7 to 14 days, with artificial saliva replaced every day to complete the remineralization of the demineralized dentin block;

[0021] In which, in the active short peptide solution, the active short peptide includes at least one of a first peptide chain, a second peptide chain and a third peptide chain, the amino acid sequence of the first peptide chain is shown as SEQ ID NO.1, the amino acid sequence of the second peptide chain is shown as SEQ ID NO.2, and the amino acid sequence of the third peptide chain is shown as SEQ ID NO.3.

[0022] In one embodiment, in the step of etching the surface of the dentin block, a phosphoric acid solution is used for etching, the mass proportion of phosphoric acid in the phosphoric acid solution is 30% to 40%, and the etching time is 20 to 40 seconds; and / or,

[0023] In the active short peptide solution, the concentration of the active short peptide is 0.1-0.2 mM.

[0024] The present invention adopts at least one of the first peptide chain, the second peptide chain and the third peptide chain as a bionic remineralization polypeptide, which has a small molecular weight, low cost, simple synthesis, good biocompatibility and the like. The modified polypeptides all improve the adsorption capacity of the parent peptide to type I collagen, stabilize the calcium-phosphate solution, form an amorphous mineralization precursor, and further promote the remineralization of demineralized dentin. It has considerable application prospects in common oral clinical diseases such as caries and dentin sensitivity, such as being used to repair exposed dentinal tubules and improve dentin sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 The cross-sectional SEM images of the demineralized dentin after in vitro mineralization for 14 days provided in Examples 1 to 3 and Comparative Example 1 of the present invention;

[0027] Figure 2 The longitudinal cross-sectional SEM images of the demineralized dentin after in vitro mineralization for 14 days provided in Examples 1 to 3 and Comparative Example 1 of the present invention;

[0028] Figure 3 A statistical diagram of the nanoparticle size after the MDP peptide provided in Examples 1 to 3 of the present invention is mixed with a saturated calcium phosphate solution;

[0029] Figure 4 This is a TEM image of the MDP peptide provided in Examples 1 to 3 of the present invention after being mixed with a saturated calcium phosphate solution for 2 hours;

[0030] Figure 5This is a TEM image of the saturated calcium-phosphorus solution of the control group of the present invention after mixing for 2 hours;

[0031] Figure 6 This is the energy spectrum analysis diagram after the MDP peptide provided in Examples 1 to 3 of the present invention was mixed with a saturated calcium phosphate solution for 2 hours.

[0032] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings in conjunction with the embodiments. DETAILED DESCRIPTION

[0033] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0034] It should be noted that, in the embodiments, those without specifying specific conditions are carried out according to normal conditions or conditions recommended by the manufacturer. Those without specifying the manufacturer of reagents or instruments used are conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, and "A and / or B" is taken as an example, including schemes A, B, or A and B that meet the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of the technical schemes is contradictory or cannot be achieved, it should be considered that the combination of such technical schemes does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work, all belong to the scope of protection of the present invention.

[0035] At present, the remineralized dentin obtained in the field of bionics still has a large gap with natural tooth hard tissue in terms of mineralization degree and orderliness of mineralized particle arrangement, and the mineralizing peptides based on the design concept of NCPs mainly focus on their interaction with calcium ions and initiating hydroxyapatite nucleation or its stable amorphous mineralized precursors to gradually guide mineralization within the fiber. However, there are few studies on bionic peptides acting on adsorbed collagen fiber design.

[0036] In view of the deficiencies in the prior art, the purpose of the present invention is to solve one or more problems in the above-mentioned prior art. The present invention modifies natural proteins with the help of the concept of bionic mineralization. On the one hand, the amphiphilicity of the mother peptide is enhanced, which can enhance the adsorption capacity of the short peptide to substances, and innovatively provides an amphiphilic short peptide based on enhanced collagen adsorption capacity. On the other hand, it combines the non-classical mineralization crystallization theory, combines the collagen adsorption peptide segment with the calcium binding functional peptide segment to synthesize a new short peptide, and designs a polypeptide with remineralization function.

[0037] For this reason, please see Figures 1 to 5 The present invention proposes a bionic remineralization polypeptide, comprising at least one of a first peptide chain, a second peptide chain and a third peptide chain; the amino acid sequence of the first peptide chain is shown as SEQ ID NO.1; the amino acid sequence of the second peptide chain is shown as SEQ ID NO.2; the amino acid sequence of the third peptide chain is shown as SEQ ID NO.3.

[0038] Biomimetic peptides refer to peptides with the same sequence and important physiological functions obtained by first screening out peptide sequences with specific functions from human proteins and then synthesizing them in vitro. In recent years, such peptides have been mainly used as functional analogs of stabilizers to regulate crystal growth and promote biomineralization. Based on a large number of studies, non-collagenous proteins (NCPs) account for less than 10% of the dentin matrix, but they have strong calcium ion binding ability and can be adsorbed to collagen fibers, playing a vital role in regulating mineralization and the mechanical integrity of bones. Scholars have gradually focused on peptides derived from NCPs and found that peptides derived from dentin phosphoproteins and dentin matrix protein-1 have a good effect on promoting dentin remineralization. However, the remineralized dentin obtained in the field of biomimetic research still has a large gap with natural tooth hard tissue in terms of mineralization degree and orderliness of mineralized particle arrangement, and mineralized peptides based on the design concept of NCPs mainly focus on their interaction with calcium ions and initiating hydroxyapatite nucleation or their stable amorphous mineralized precursors to gradually guide mineralization within the fiber. However, there are few studies on biomimetic peptides designed for adsorption of collagen fibers.

[0039] The bionic remineralization polypeptide of the present invention is mainly based on the high affinity binding of the CBP1495 domain in the matrix metalloproteinase 2 (MMP2) precursor to type I collagen. It is designed to be modified based on the theory of the MMP2 precursor CBP1495 sequence as the mother peptide combined with the non-classical mineralization pathway, and is designed as MMP2 derived peptides (MDP).

[0040] On the one hand, the present invention enhances the amphiphilicity of the mother peptide "CDWWSCWLDVLKD" (SEQ ID NO.01) and names it MDP1; on the other hand, the mother peptide has an amino acid sequence "CPKESCNLFVLKD" that specifically binds to type I collagen, combined with the amino acid sequence "ESQES" that binds to calcium in dentin matrix protein-1 or with the amino acid sequence "DSSDSSDSS" that binds to calcium in dentin phosphoprotein (named MDP2 and MDP3, respectively) to simulate non-collagen proteins (NCPs) to play a regulatory role.

[0041] The present invention adopts at least one of the first peptide chain, the second peptide chain and the third peptide chain as a bionic remineralization polypeptide, which has a small molecular weight, low cost, simple synthesis, good biocompatibility and the like. The modified polypeptides all improve the adsorption capacity of the parent peptide to type I collagen, stabilize the calcium-phosphate solution, form an amorphous mineralization precursor, and further promote the remineralization of demineralized dentin. The short peptide of the present invention is easy to synthesize, has a low cost, and has a good remineralization effect, thereby providing an ideal new approach for the prevention and treatment of caries and dentin sensitivity.

[0042] The present invention also proposes a method for preparing a biomimetic remineralization polypeptide, comprising the following steps: providing a resin, soaking the resin in a solvent to swell it, and obtaining a resin carrier; dehydrating and condensing amino acid residues with protective groups onto the resin carrier according to the amino acid sequence, removing the protective groups after the condensation is completed, adding amino acid residues in sequence, and then adding a cleavage reagent to obtain a biomimetic remineralization polypeptide; wherein the amino acid sequence includes at least one of the sequences shown in SEQ ID NO.1 to 3.

[0043] The technical solution of the present invention uses a solid-phase peptide synthesis method to synthesize a biomimetic remineralization peptide, which helps to reduce unnecessary side reactions, is simpler to operate, and does not require purification after each reaction. Solvents and by-products can be removed by simple filtration or centrifugation; a high yield can be obtained even at a low dilution, and the yield can be optimized by selecting a suitable resin and other synthesis parameters; the reaction progress can be easily monitored during the synthesis process, and the coupling efficiency of each amino acid can be checked through simple tests; synthesis from milligram to gram levels is relatively easy to achieve, and is suitable for large-scale production and commercial applications.

[0044] In one embodiment, the resin includes dichlororesin or CTC resin; and / or the solvent includes at least one of dichloromethane and N,N-dimethylformamide; and / or the protecting group includes fluorenylmethoxycarbonyl.

[0045] The technical solution of the present invention uses CTC resin or dichloro resin as a carrier. These resins have good chemical stability and mechanical strength, can effectively carry and release polypeptide chains during the synthesis process, thereby significantly improving the efficiency of the polypeptide synthesis process; by using specific solvents such as dichloromethane and N, N-dimethylformamide, the reaction conditions can be improved, the formation of by-products can be reduced, thereby improving the purity of the final product; by using fluorenylmethyloxycarbonyl as a protecting group, the synthesis steps can be simplified, the selectivity in the synthesis process can be improved, the possibility of side reactions can be reduced, and the purity and yield of the polypeptide product can be improved. Wherein, CTC resin refers to chlorotribenzoyl chloride resin.

[0046] The present invention also proposes an application of a bionic remineralization polypeptide in dentin remineralization.

[0047] Since the present application adopts all the technical solutions of all the above embodiments, it has at least all the beneficial effects brought by the technical solutions of the above embodiments, which will not be described one by one here.

[0048] In one embodiment, the application includes the following steps: providing an ex vivo dental crown, preparing the ex vivo dental crown into a dentin block, etching the surface of the dentin block, and immersing it in an active short peptide solution; taking out the dentin block, placing it in water for ultrasonic cleaning, and then immersing it in artificial saliva, mineralizing it in a water bath for 7 to 14 days, replacing the artificial saliva every day, and completing the remineralization of the demineralized dentin block; wherein, in the active short peptide solution, the active short peptide includes at least one of a first peptide chain, a second peptide chain and a third peptide chain, the amino acid sequence of the first peptide chain is shown in SEQ ID NO.1, the amino acid sequence of the second peptide chain is shown in SEQ ID NO.2, and the amino acid sequence of the third peptide chain is shown in SEQ ID NO.3.

[0049] The technical solution of the present invention is to acid-etch the dentin block to make holes and then immerse it in an active short peptide solution, so that the short peptide can enter the dentin block and deposit in the pores of the dentin block. The original peptide is modified to enhance its amphiphilicity and increase its calcium binding functional area, which can promote dentin remineralization. In one embodiment of the present invention, refer to Figure 2 The longitudinal section showed that the experimental group had denser neoplasms blocking the small tube openings than the control group, and there was a thicker sediment layer on the surface. The sealing effect of MDP1, MDP2, and MDP3 was better than that of the control group.

[0050] In one embodiment, in the step of etching the surface of the dentin block, a phosphoric acid solution is used for etching, the mass proportion of phosphoric acid in the phosphoric acid solution is 30% to 40%, and the etching time is 20 to 40 seconds; and / or, in the active short peptide solution, the concentration of the active short peptide is 0.1 to 0.2 mM.

[0051] The technical solution of the present invention uses a 30% to 40% phosphoric acid solution to perform acid etching on the dentin surface for 20 to 40 seconds. This treatment can effectively remove the smear layer on the dentin surface, demineralize and dissolve the peritubular dentin, increase the diameter of the dentin tubule opening, and expose the collagen fiber network, which is beneficial to the subsequent performance research of bionic remineralization peptide repair materials; by using an active short peptide solution containing a concentration of 0.1 to 0.2 mM, it can effectively combine with type I collagen and stabilize amorphous calcium phosphate mineralization precursors, thereby promoting the remineralization of demineralized dentin, providing new ideas and methods for the research of remineralization repair materials.

[0052] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention, and are not used to limit the present invention.

[0053] Example 1

[0054] This embodiment provides a biomimetic remineralization polypeptide, which comprises an amino acid sequence as shown in SEQ ID NO.1 and is named MDP1.

[0055] SEQ ID NO. 1: CDWWSCWLDVLKD.

[0056] Example 2

[0057] This embodiment provides a biomimetic remineralization polypeptide, which comprises an amino acid sequence as shown in SEQ ID NO.2 and is named MDP2.

[0058] SEQ ID NO. 2: CPKESCNLFVLKDESQES.

[0059] Example 3

[0060] This embodiment provides a biomimetic remineralization polypeptide, which comprises an amino acid sequence as shown in SEQ ID NO.3 and is named MDP3.

[0061] SEQ ID NO.3: CPKESCNLFVLKDDSSDSSDSS.

[0062] Example 4

[0063] This embodiment provides an application of a biomimetic remineralization polypeptide in dentin remineralization, wherein the biomimetic remineralization polypeptide is the biomimetic remineralization polypeptide provided in Embodiments 1 to 3;

[0064] The application of dentin remineralization is a demineralized dentin block remineralization test, including the following process:

[0065] One-third of the collected ex vivo crowns were prepared into dentin blocks, and the surface was etched with 35% phosphoric acid for 30 seconds. After etching, the samples in the experimental group were immersed in a 0.1 mM active short peptide solution overnight. On the second day, the samples were ultrasonically bathed in deionized water at 37°C for 10 minutes, changed to EP tubes containing artificial saliva, and placed in a constant temperature water bath for remineralization. The artificial saliva was changed every day, and SEM examination of cross-sections and longitudinal sections was performed on the 14th day of the remineralization experiment.

[0066] Comparative Example 1

[0067] This comparative example provides a dentin block remineralization test, including the following process:

[0068] One-third of the collected ex vivo crowns were prepared into dentin blocks, and the surface was etched with 35% phosphoric acid for 30 seconds. After etching, it was immersed in pure water overnight. On the second day, the sample was ultrasonically bathed in deionized water at 37°C for 10 minutes, changed to an EP tube containing artificial saliva, and placed in a constant temperature water bath for remineralization. The artificial saliva was replaced every day.

[0069] Performance Testing

[0070] Figure 1 Figure 2 is a cross-sectional SEM image of demineralized dentin after 14 days of in vitro mineralization, wherein Figure A is a cross-sectional SEM image of the control group (comparative example 1), Figure B is a cross-sectional SEM image of MDP1 (example 1), Figure C is a cross-sectional SEM image of MDP2 (example 2), and Figure D is a cross-sectional SEM image of MDP3 (example 3). Figure a is a partial enlarged image of the white arrow in Figure A, Figure b is a partial enlarged image of the white arrow in Figure B, Figure c is a partial enlarged image of the white arrow in Figure C, and Figure d is a partial enlarged image of the white arrow in Figure D.

[0071] Scanning electron microscopy results showed that at 14 days of remineralization, mineral deposition was observed on the surface of the cross-section of the demineralized dentin blocks in the MDP1-3 experimental groups, and more tubules were closed in the experimental groups.

[0072] Figure 2 These are longitudinal section SEM images of demineralized dentin after 14 days of in vitro mineralization, among which Figure A is the longitudinal section SEM image of the control group, Figure B is the longitudinal section SEM image of MDP1, Figure C is the longitudinal section SEM image of MDP2, and Figure D is the longitudinal section SEM image of MDP3. Figure a is a local enlarged image of the white arrow in Figure A, Figure b is a local enlarged image of the white arrow in Figure B, Figure c is a local enlarged image of the white arrow in Figure C, and Figure d is a local enlarged image of the white arrow in Figure D.

[0073] Scanning electron microscopy results showed that at 14 days of remineralization, the longitudinal section showed that the tubules in the MDP1-3 experimental group had denser neoplasms blocking the tubule openings than those in the control group, and there was a thicker sediment layer on the surface.

[0074] The binding of MDP-derived peptides to collagen was detected by surface ion resonance method. The results are shown in Table 1.

[0075] According to Table 1, the affinity KD values ​​of the four short peptides are all above 10 -3 ~10 -6 M, indicating that the four short peptides have good affinity with type I. And the lower the KD value, the stronger the affinity. The KD values ​​of the modified peptides MDP1, MDP2, and MDP3 are lower than those of the original peptides, indicating that the collagen adsorption capacity is enhanced.

[0076] Table 1 Affinity analysis of MDP and type I collagen

[0077] aisle Fitting the model Ligand Analytes kd(1 / s) KD(M) Rmax(RU) tc 1 1:1 combination Collagen Type I Propeptide <![CDATA[6.24×10 -1 ]]> <![CDATA[5.90×10 -5 ]]> 190942.7 <![CDATA[1.32×10 10 ]]> 2 1:1 combination Collagen Type I MDP1 <![CDATA[5.94×10 -1 ]]> <![CDATA[1.80×10 -5 ]]> 53152.0 <![CDATA[1.26×10 10 ]]> 3 1:1 combination Collagen Type I MDP2 <![CDATA[6.16×10 -1 ]]> <![CDATA[5.00×10 -5 ]]> 254269.1 <![CDATA[2.87×10 10 ]]> 4 1:1 combination Collagen Type I MDP3 <![CDATA[5.82×10 -1 ]]> <![CDATA[1.89×10 -5 ]]> 65526.7 <![CDATA[1.25×10 10 ]]>

[0078] Figure 3 The particle size distribution of the MDP peptide provided in Examples 1 to 3 after mixing with a saturated calcium-phosphorus solution in an aqueous dispersion was measured by a nanoparticle size analyzer. A calcium solution (3.90 mM, PH=7.0) and a phosphorus solution (3.83 mM, PH=7.0) were prepared, and the calcium-phosphorus solution was mixed in equal volumes as a control group, and the experimental group was a calcium-phosphorus mixed solution to which peptide lyophilized powder was added to make the peptide concentration 0.1 mM. Before the peptide was mixed with the calcium-phosphorus solution, immediately after mixing, for 1 h, and for 2 h, 1 mL of the calcium-phosphorus mixed solution and a separate peptide solution (0.1 mM, PH=7.0, prepared with pure water) were taken for measuring the particle size distribution by a nanoparticle size analyzer.

[0079] according to Figure 3 It can be seen that the particle size of the peptide solution alone is larger after being dissolved in water, but the particle size decreases after adding calcium phosphate ions, indicating that calcium phosphate ions help the peptide to disperse in water and make it less likely to agglomerate. In addition, 1-2 hours after the peptide and calcium phosphate are mixed, the particle size of the particles in the solution is smaller than that of the pure calcium phosphate solution in the control group, indicating that the peptide in the experimental group can stabilize a certain amount of calcium phosphate ions, inhibit the nucleation and crystallization of hydroxyapatite in the aqueous phase, and maintain the stable state of calcium phosphate ions. In the calcium phosphate solution without peptide, there is a trend of increasing the particle size of calcium phosphate ions, which can lead to accelerated nucleation.

[0080] Figure 4 To detect the particle morphology and crystal properties of the MDP peptide provided in Examples 1 to 3 mixed with a saturated calcium-phosphorus solution for 2 hours by transmission electron microscopy (TEM) and selected area electron diffraction (SAED). Prepare a calcium solution (3.90mM, PH=7.0) and a phosphorus solution (3.83mM, PH=7.0), mix the calcium-phosphorus solution in equal volumes, and then add the peptide lyophilized powder to make the peptide concentration 0.1mM. After mixing for 2 hours, take 10uL of the mixed solution and drop it on the front of a 200-mesh copper mesh. After natural drying, observe the particle morphology and properties by TEM and SAED.

[0081] In the TEM images after the MDP peptide was mixed with the saturated calcium phosphate solution for 2 hours, the magnification of Figures A to C is 28,500 times, and Figures a to c correspond to the local enlarged images in the white frames in Figures A to C, with a magnification of 120,000 times. The upper right corner of Figures a to c is the SAED detection diffraction pattern.

[0082] Figure 5The TEM image of the control group after 2 hours of mixing of saturated calcium and phosphorus solutions. The method is as follows: calcium solution (3.90mM, PH=7.0) and phosphorus solution (3.83mM, PH=7.0) were prepared, and after mixing for 2 hours, 10uL of the mixed solution was dropped on the front of a 200-mesh copper grid. After natural drying, the particle morphology and properties were observed by TEM and SAED.

[0083] Combination Figures 4 to 5 TEM observation of the peptide-calcium phosphate mixture after 2 hours of mixing, under low magnification, it can be seen that the MDP1 group formed evenly dispersed, dense spherical aggregated particles, while the MDP2 and MDP3 groups formed loosely chained aggregated particles. SAED analysis showed that the nanoparticles were all amorphous mineralized precursors without crystals; under high magnification, the nanoparticles formed in the experimental group were measured, and the diameter of a single particle was about 16-30mm, which was smaller than the 100nm of the control group. This shows that the peptide can inhibit its particle size, stabilize ACP, and inhibit its growth and nucleation.

[0084] Figure 6 To detect the nanoparticles of the peptide-calcium-phosphate mixture provided by Examples 1 to 3 after the MDP peptide and the saturated calcium-phosphate solution were mixed for 2 hours by energy dispersive spectrometer (EDS), the method is as follows: prepare calcium solution (3.90mM, pH=7.0) and phosphorus solution (3.83mM, pH=7.0), mix the calcium-phosphate solution in equal volumes, and then add the peptide lyophilized powder to make the peptide concentration 0.1mM. After mixing for 2 hours, take 10uL of the mixed solution and drop it on the front of a 200-mesh copper mesh, and perform energy dispersive spectrometer analysis after natural drying.

[0085] The results are as follows Figure 6 As shown, the nanoparticles of the peptide-calcium-phosphorus mixture after 2 hours of mixing were detected by EDS, and the results showed that they were aggregates of calcium-phosphorus ions. Figure 4 The SAED results in further prove that it is an amorphous calcium phosphate mineralization precursor, indicating that the peptide can stabilize ACP and inhibit its nucleation and crystallization.

[0086] In summary, at 14 days of remineralization, the surface mineral deposition of demineralized dentin cross-section samples further blocked the dentinal tubules, and more tubules were closed in the MDP1, MDP2, and MDP3 experimental groups. The longitudinal section showed that the tubules in the experimental group had denser neoplasms blocking the tubule orifices than those in the control group, and there was a thicker deposition layer on the surface. The sealing effect of MDP1, MDP2, and MDP3 was better than that of the control group. In addition, through SPR, nanoparticle size analyzer, TEM, EDS and other analyses, the amphiphilic enhancement modification of the original peptide and the increase of the calcium binding functional area can enhance the adsorption of the original peptide to type I collagen, stabilize the calcium-phosphate solution, and form an amorphous mineralization precursor, which can promote the remineralization of demineralized dentin.

[0087] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification under the inventive concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A biomimetic remineralization polypeptide, characterized in that: comprising at least one of a first peptide chain, a second peptide chain and a third peptide chain; The amino acid sequence of the first peptide chain is shown in SEQ ID NO.1; The amino acid sequence of the second peptide chain is shown in SEQ ID NO.2; The amino acid sequence of the third peptide chain is shown in SEQ ID NO.

3.

2. A method for preparing a biomimetic remineralization polypeptide, characterized in that: The following steps are involved: Providing a resin, and soaking the resin in a solvent to swell it, thereby obtaining a resin carrier; According to the amino acid sequence, the amino acid residues with protective groups are dehydrated and condensed onto the resin carrier, and after the condensation is completed, the protective groups are removed, and the amino acid residues are added in sequence, and then a cleavage reagent is added to obtain a biomimetic remineralized polypeptide; Wherein, the amino acid sequence includes at least one of the sequences shown in SEQ ID NO.1-3.

3. The method for preparing the biomimetic remineralization polypeptide according to claim 2, characterized in that: The resin comprises dichloro resin or CTC resin; and / or, The solvent comprises at least one of dichloromethane and N,N-dimethylformamide; and / or, The protecting group includes fluorenylmethoxycarbonyl.

4. Application of a bionic remineralizing polypeptide in dentin remineralization.

5. The use according to claim 4, characterized in that The application comprises the following steps: Providing an ex vivo dental crown, preparing the ex vivo dental crown into a dentin block, etching the surface of the dentin block with acid, and immersing the block in an active short peptide solution; The dentin block is taken out, ultrasonically cleaned in water, and then immersed in artificial saliva for water bath mineralization for 7 to 14 days, with artificial saliva replaced every day to complete the remineralization of the demineralized dentin block; In which, in the active short peptide solution, the active short peptide includes at least one of a first peptide chain, a second peptide chain and a third peptide chain, the amino acid sequence of the first peptide chain is shown as SEQ ID NO.1, the amino acid sequence of the second peptide chain is shown as SEQ ID NO.2, and the amino acid sequence of the third peptide chain is shown as SEQ ID NO.

3.

6. The use according to claim 5, characterized in that In the step of etching the surface of the dentin block, a phosphoric acid solution is used for etching, wherein the mass proportion of phosphoric acid in the phosphoric acid solution is 30% to 40%, and the etching time is 20 to 40 seconds; and / or, In the active short peptide solution, the concentration of the active short peptide is 0.1-0.2 mM.

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