Coating for modifying dental restoration material, application and dental restoration material

Abstract

The invention relates to the technical field of biological materials, and particularly provides a coating for modifying a mouth rehabilitation material, application and the mouth rehabilitation material.The coating for modifying the mouth rehabilitation material is formed by adsorbing cationic oligopeptide on the surface of the mouth rehabilitation material, the amino acid sequence of the cationic oligopeptide comprises H (K) nS, and n is 1-20. The coating for modifying the dental restoration material has good binding capacity to materials such as zirconium oxide, zirconium phosphate or titanium, so that biological activity modification of inert materials such as zirconium oxide, phosphorylated zirconium oxide or titanium or zirconium phosphate serving as surface components is possible, and the coating has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a coating for modifying an oral restoration material, an application thereof, and an oral restoration material. Background Art

[0002] Zirconia (ZrO2), as a high-performance bioceramic material, has become the mainstream choice in the field of oral restoration (such as implants, all-ceramic crowns and bridges, abutments) and orthopedic implants due to its excellent mechanical strength, chemical inertness, biocompatibility and aesthetic properties similar to natural tooth tissue. However, its clinical application still has the following problems: 1) Surface biological inertness: Due to the lack of active groups on its surface, it is difficult to form effective chemical bonds with the surrounding bone tissue, resulting in low bone integration efficiency and prolonged postoperative healing period. 2) Insufficient antibacterial performance: The oral environment is complex, and biofilms are easily formed on the surface of zirconia, causing infection risks such as peri-implantitis, while the material itself does not have antibacterial function. 3) Difficulty in functional modification: The high chemical stability of zirconia makes it difficult to introduce cationic molecules (such as antibacterial agents and growth factors) into its surface through conventional methods (such as chemical coupling and physical deposition), which limits its multifunctional application.

[0003] The current solutions to the above problems mainly include: 1) Chemical coating method: coating hydroxyapatite (HA) or antibacterial metals (such as Ag, ZnO) on the surface of zirconia through sol-gel, electrochemical deposition, etc., but there are disadvantages such as easy peeling of the coating due to weak interfacial bonding, complex process and high cost; 2) Plasma treatment: using plasma to bombard the material surface to increase the roughness or introduce active groups, but this method is highly dependent on equipment and the modification effect decays over time, making it difficult to achieve long-term stability; 3) Silane coupling agent modification: grafting functional molecules on the surface through silanization reaction, but the reaction needs to be carried out at high temperature and in an anhydrous environment. The conditions are harsh and the coupling agent may cause cytotoxicity.

[0004] However, implants and abutments made of zirconium phosphate and titanium also have many shortcomings that limit their clinical application. For example, titanium itself is a biologically inert material, and its surface cannot directly chemically bond with bone tissue. Early bone healing is slow, especially in patients with metabolic abnormalities such as osteoporosis or diabetes, where bone integration may be delayed. Surface modification is required to enhance activity. If modification is performed by sandblasting, acid etching, hydroxyapatite coating, anodizing, etc., there is not only a risk of coating shedding, but also a complex surface treatment process, which increases production costs and quality control difficulties.

[0005] In recent years, peptides have become a research hotspot for biomaterial surface functionalization due to their high biocompatibility, programmability, and specific recognition capabilities. For example, the RGD peptide (arginine-glycine-aspartic acid) can promote cell adhesion through integrin receptors, and antimicrobial peptides such as LL-37 can be used to disrupt bacterial membrane structures. However, existing peptide coatings are mostly targeted at titanium alloys or polymers, with low adsorption efficiency on materials such as zirconium oxide and zirconium phosphate (ZrP). Furthermore, due to the unique surface charge distribution of zirconium oxide (isoelectric point approximately pH 6-7), conventional anions or peptides have difficulty achieving stable binding through electrostatic interactions.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The problem solved by the present invention is that existing oral restoration materials made of materials such as zirconium oxide, zirconium phosphate or titanium have disadvantages such as surface biological inertness, insufficient antibacterial performance, and difficulty in functional modification, which limit their use.

[0008] To solve the above problems, the present invention provides a coating for modifying oral restoration materials. The coating is formed by adsorbing a cationic short peptide on the surface of the oral restoration material. The amino acid sequence of the cationic short peptide includes H(K)nS, where n is 1-20.

[0009] Preferably, n is 1 to 10. Preferably, n is 1 to 5. Preferably, n is 1 to 3.

[0010] Application of the above-mentioned coating for modifying oral restoration materials in the preparation of oral restoration materials.

[0011] Preferably, the oral restoration material is used to prepare a dental implant or abutment, and the material of the dental implant or abutment is zirconium oxide, phosphated zirconium oxide or titanium, or the surface component of the dental implant or abutment is zirconium phosphate.

[0012] Preferably, the amino acid sequence of the cationic short peptide is H(K)nS-X, wherein n is 1-20, and X is an active amino acid sequence.

[0013] Preferably, the active amino acid sequence is any one of antimicrobial peptides, cell adhesion peptides, and osteogenic peptides. Preferably, the active amino acid sequence is any one of SLIGRL, RGD, and YIGSR.

[0014] The present invention also provides an oral restoration material for preparing dental implants or abutments. The material of the oral restoration material is zirconium oxide, phosphated zirconium oxide or titanium, or the surface component of the oral restoration material is zirconium phosphate, and the surface of the oral restoration material forms the above-mentioned coating for modifying the oral restoration material.

[0015] Compared with the existing technology, the coating for modifying oral restoration materials described in the present invention has good bonding ability to materials such as zirconium oxide, zirconium phosphate or titanium, which provides the possibility of biologically active modification of the surfaces of dental implants and abutments made of the same material, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a chromatogram of a cationic short peptide prepared for surface modification in Example 1 of the present invention; Figure 2 The mass spectrum of the cationic short peptide prepared for surface modification in Example 1 of the present invention; Figure 3 These are laser confocal fluorescence images of three cationic short peptides used for surface modification in Example 2 of the present invention adsorbed on zirconium oxide, zirconium phosphate, and titanium surfaces, respectively; Figure 4 The following are histograms of water contact angles of three cationic short peptides used for surface modification in Example 2 of the present invention adsorbed on zirconium oxide, zirconium phosphate, and titanium surfaces, respectively; Figure 5 This is a graph showing the stability of three cationic short peptides used for surface modification in Example 3 of the present invention after adsorption on zirconium oxide, zirconium phosphate and titanium surfaces after 30 minutes of ultrasonic treatment; Figure 6 This is a graph showing the toxic effects of the complexes of the cationic short peptide used for surface modification, three adhesion functional peptides, and six scrambled cationic short peptides on L929 cells in Example 4 of the present invention; Figure 7 This is a diagram showing the early adhesion results of the complexes of the cationic short peptide used for surface modification and three adhesion functional peptides and six scrambled cationic short peptides to L929 cells in Example 4 of the present invention; Figure 8 This is a statistical result graph of cell counting of L929 cells using complexes of the cationic short peptide used for surface modification and three adhesion functional peptides and six scrambled cationic short peptides thereof in Example 4 of the present invention; Figure 9 This is a fluorescence imaging image of the complex of the cationic short peptide used for surface modification and three adhesion functional peptides and six scrambled cationic short peptides after adsorption of L929 cells in Example 4 of the present invention; Figure 10 Statistical results of the average cell morphology of L929 cells by complexes of the cationic short peptide used for surface modification in Example 4 of the present invention with three adhesion functional peptides and six scrambled cationic short peptides. DETAILED DESCRIPTION

[0017] To make the above-mentioned objects, features and advantages of the present invention more clearly understood, the following describes the specific embodiments of the present invention in detail with reference to the accompanying drawings. The technical features of the embodiments of the present invention may be combined with each other without conflict.

[0018] Example 1 Preparation of cationic short peptides A method for synthesizing a cationic short peptide for surface modification, wherein the cationic short peptide is HKS, comprising: S1. Resin pretreatment Rink resin (solid support, degree of substitution 0.6 mmol / g) was weighed and swelled with dichloromethane (DCM, 10 mL / g resin) for 1 h. It was then washed with N,N-dimethylformamide (DMF, 10 mL × 3 times) to remove impurities. S2, Fmoc deprotection Add 20% piperidine / DMF solution (10 mL / g resin) to the resin treated with S1, stir at room temperature for 10 min twice to completely remove the Fmoc protecting group at the end of the resin, and then wash with DMF until neutral. S3, lysine coupling Take 1 equivalent (eq) of Fmoc-Lys(dde)-OH (to protect the amino group of the lysine side chain), activate it with 1.5 eq of hydroxybenzotriazole (HOBT) and 1.5 eq of diisopropylcarbodiimide (DIC) in DMF (0.3 mmol / L) for 5 minutes, add it to the resin, and react at room temperature under nitrogen for 2 hours. After the reaction, drain the solvent and wash with DMF three times. S4, amino blocking Acetic anhydride (5 eq) and N,N-diisopropylethylamine (DIEA, 2 eq) in DMF were added and reacted at room temperature for 1 hour to block unreacted amino groups and prevent subsequent side reactions.

[0019] S5, side chain deprotection Treat with 2% hydrazine hydrate / DMF solution (10 mL / g resin) for 30 minutes to selectively remove the dde protecting group of the lysine side chain, and then wash with DMF to obtain the product.

[0020] It should be noted that the preparation method of the cationic short peptide HKKS and HKKKS is the same as that of HKS. The only difference is that S2-S4 need to be repeated 1-2 times, which will not be repeated here.

[0021] In order to facilitate subsequent experimental detection, the following scheme was used to fluorescently couple the cationic short peptide HKS: 3 eq of fluorescein isothiocyanate (FITC) and 3 eq of DIEA were added and reacted in DMF in the dark for 4 hours to achieve fluorescent labeling of the C-terminus of the peptide. A cleavage solution was used to react at room temperature for 2 hours to release the peptide and remove the side chain protecting groups. The cleavage solution consisted of 95% trifluoroacetic acid (TFA) + 2% triisopropylsilane (Tis), 2% ethanedithiol (EDT) + 1% H2O. The filtrate was precipitated with glacial ether and centrifuged. The crude product was purified by high-performance liquid chromatography (HPLC, C18 column, acetonitrile / water gradient elution). After lyophilization, the fluorescently labeled cationic short peptide Ac-HKSGGK(FITC)-NH2 was obtained with a purity of >95%. The corresponding chromatogram and mass spectrum are shown in Figure 2, respectively. Figure 1-2 .

[0022] Ac-HKSGGK(FITC)-NH2 Example 2 Coating preparation experiment 2.1 Experimental methods: Zirconium oxide, zirconium phosphate, and pure titanium polished to 2000 mesh were placed in 24-well plates. FITC-labeled cationic peptides were diluted to 10 mM in PBS (pH 7.4) and incubated at room temperature (25 ± 1°C) for 24 h. The samples were washed three times with PBS (pH 7.4) for 5 min each time to completely remove unadsorbed peptides. An equal amount of PBS solution without FITC-labeled cationic peptides was added to the wells as a control. After the sample was dried, the sample surface and three-dimensional (3D) fluorescence images were taken under a laser confocal microscope. Figure 3 ; Use a microsyringe to drop 10 μL of ultrapure water vertically on the center of the dried sample surface, record the water drop morphology with a high-speed camera (0 second instantaneous image), and the software automatically fits the droplet profile and calculates the contact angle (θ). The results are shown in Figure 4 ; It should be noted that: Ac-HKSGGK (FITC) -NH2, Ac-HKKSGGK (FITC) -NH2, and Ac-HKKKSGGK (FITC) -NH2 were used for experiments separately.

[0023] 2.2 Experimental Results 2.2.1 Three-dimensional fluorescence images The greater the fluorescence intensity of the sample surface, the more cationic peptides adhere to the surface of zirconium oxide or zirconium phosphate, which may be related to the significant improvement of the electrostatic interaction between the cation and the sample surface. Figure 3 It can be seen that the three-dimensional images of the sample surface show that the three peptides are attached to the surface of the zirconium oxide sample and the zirconium phosphate sample, and the fluorescence intensity is statistically significant compared with the control group. Figure 3 The results are consistent.

[0024] 2.2.2 Contact angle Depend on Figure 4 It can be seen that the contact angle of the control sample is 75.3±2.5°, indicating that the unmodified zirconium oxide surface is moderately hydrophobic. However, after the adsorption of cationic peptides, the contact angle is significantly reduced, among which HKS is 42.1±3.1°; HKKS is 38.5±2.8°; HKKKS is 35.2±2.6°, and the hydrophilicity is significantly improved, indicating that the cationic peptides have been successfully adsorbed on the surfaces of the zirconium oxide sample and the zirconium phosphate sample. Specifically, the surface of the zirconium oxide sample adsorbed with cationic peptides becomes more hydrophilic, and the hydrophilic surface can inhibit the adsorption of hydrophobic bacteria, which will be beneficial to promote cell adhesion and reduce bacterial colonization; while the surface of the zirconium phosphate sample adsorbed with cationic peptides becomes more hydrophobic, and the hydrophobic interface delays the diffusion of hydrophilic drugs, which has broad application prospects in the application of drug sustained-release carriers.

[0025] Example 3 Coating mechanical stability test The sample adsorbed with cationic peptides was immersed in deionized water and ultrasonically treated for 30 minutes (power 100 W, frequency 40 kHz). After being taken out, it was blown dry with nitrogen and the image was taken with a fluorescence microscope. The results are shown in Figure 5 .

[0026] Depend on Figure 5 It can be seen that the fluorescence images confirm that under physical conditions, cationic short peptides can also be stably adsorbed on the surface of zirconium oxide samples and zirconium phosphate samples. The possible reason is that the binding strength with the material surface through electrostatic interaction and hydrogen bonds can resist physical shear force.

[0027] Example 4 Preparation of functional cationic short peptide complex 4.1 Experimental methods Taking SLIGRL, RGD and YIGSR as examples, the three cell adhesion peptides were chemically synthesized to add the three cationic sequences of SLIGRL, RGD and YIGSR to the sequence of HK(n)S, respectively. After HPLC purification and mass spectrometry verification, they were ready for use. SLIGRL: protease-activated receptor agonist peptide, used to promote cell migration, RGD is an integrin-binding peptide, used to enhance cell adhesion, and YIGSR is a laminin-derived peptide. pep1-3 is a complex of HKKS and the three adhesion functional peptides SLIGRL, RGD and YIGSR, and pep4-6 is a scrambled sequence of pep1-3 as a control. The specific sequences are SLIGRL-GG-HKKS-NH2, Ac-HKKSGG-RGD-NH2, Ac-HKKSGG-YIGSR-NH, SKRLGGLKHSGI-NH2, Ac-GKHSRGKDG-NH2, and Ac-GKHSRGKYSGI-NH2; Zirconium oxide and zirconium phosphate samples were immersed in a 100 μM cationic peptide solution and adsorbed at 37°C for 24 hours (120 rpm). After removal, nitrogen was blown dry and UV sterilized for 30 minutes. L929 cells were then cultured at 5×10 4 cells / mL density was inoculated on the sample surface and cultured for 72 h. CCK values ​​were measured at 24 h and 72 h respectively. The results are shown in Figure 6 At the same time, the cells were fixed and stained after 24 hours of culture, and the cell morphology and adhesion were photographed under an inverted fluorescence microscope. Figure 7-10 .

[0028] 4.2 Experimental Results Depend on Figure 6 It can be seen that the CCK values ​​of 24h and 72h showed that the six synthesized peptides were non-toxic to L929 cells at a concentration gradient of 0-100μM, and the cell survival rate exceeded 80%, indicating good biocompatibility. Figure 7-9 It can be seen that the number of cells adhering and the spreading morphology on the surface of the peptide-coated zirconium phosphate sample are better than those in the control group of zirconium oxide and zirconium phosphate samples, especially the sequence pep2:Ac-HKKSGG-RGD-NH2.

[0029] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the scope defined by the claims.

Claims

1. A coating for modifying oral restoration materials, characterized in that: The coating is formed by adsorbing a cationic short peptide on the surface of the oral restoration material. The amino acid sequence of the cationic short peptide includes H(K)nS, wherein n is 1-20.

2. The coating for modifying oral restoration materials according to claim 1, characterized in that: Said n is 1-10.

3. The coating for modifying oral restoration materials according to claim 2, characterized in that: The n is 1-3.

4. Use of the coating for modifying oral restoration materials according to any one of claims 1 to 3 in the preparation of oral restoration materials.

5. The use according to claim 4, characterized in that The oral restoration material is used for preparing a dental implant or a base. The material of the dental implant or the base is zirconium oxide, phosphated zirconium oxide or titanium, or the surface component of the dental implant or the base is zirconium phosphate.

6. The use according to claim 5, characterized in that The amino acid sequence of the cationic short peptide is H(K)nS-X, wherein n is 1-20, and X is an active amino acid sequence.

7. The use according to claim 6, characterized in that The active amino acid sequence is any one of antimicrobial peptides, cell adhesion peptides and osteogenic peptides.

8. An oral restoration material for preparing a dental implant or abutment, characterized in that: The oral restoration material is made of zirconium oxide, phosphated zirconium oxide or titanium, or the surface component of the oral restoration material is zirconium phosphate, and the surface of the oral restoration material forms the coating for modifying the oral restoration material according to any one of claims 1 to 3.

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