Coating for modifying a dental restorative material, use and dental restorative material

Abstract

The present application relates to the technical field of biological materials, and specifically provides a coating for modifying an oral repair material, application and oral repair material, wherein the coating for modifying the oral repair material is formed by adsorbing cationic short peptides on the surface of the oral repair material, and the amino acid sequence of the cationic short peptides comprises H(K)nS, wherein n is 1-20.The coating for modifying the oral repair material has good binding capacity for materials such as zirconium oxide, zirconium phosphate or titanium, thereby making it possible to modify the biological activity of materials such as zirconium oxide, zirconium phosphate, zirconium oxide or titanium or inert materials such as zirconium phosphate on the surface, and having a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and more specifically, to a coating for modifying oral restorative materials, its application, and oral restorative materials. Background Technology

[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, and 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 faces the following challenges: 1) Surface biological inertness: Due to the lack of active groups on its surface, it is difficult to form effective chemical bonds with surrounding bone tissue, resulting in low osseointegration efficiency and prolonged postoperative healing time. 2) Insufficient antibacterial properties: The oral environment is complex, and biofilms easily form on the surface of zirconia, leading to the risk of infections such as peri-implantitis, while the material itself does not possess antibacterial functions. 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) onto its surface through conventional methods (such as chemical coupling and physical deposition), limiting its multifunctional applications.

[0003] Currently, the main solutions to the above problems include: 1) Chemical coating method: Hydroxyapatite (HA) or antibacterial metals (such as Ag, ZnO) are coated on the surface of zirconium oxide through sol-gel, electrochemical deposition, etc., but the coating is easy to peel off due to weak interfacial bonding and the process is complicated and costly; 2) Plasma treatment: Plasma is used to bombard the surface of the material to increase 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: Functional molecules are grafted onto the surface through silanization reaction, but the reaction needs to be carried out in a high temperature and anhydrous environment, which is 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 bioinert material, and its surface cannot directly form chemical bonds with bone tissue, resulting in a slower early bone healing rate. In particular, in patients with osteoporosis or metabolic abnormalities such as diabetes, bone integration may be delayed. Surface modification is required to enhance activity. If modification is carried out by means of sandblasting, acid etching, hydroxyapatite coating, anodizing, etc., there is not only a risk of coating peeling off, but the surface treatment process is also complicated, increasing production costs and quality control difficulties.

[0005] In recent years, peptides have become a research hotspot in the functionalization of biomaterial surfaces due to their high biocompatibility, programmability, and specific recognition capabilities. For example, 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 peptides for coatings are mostly targeted at titanium alloys or polymer materials, and their adsorption efficiency for materials such as zirconium oxide and zirconium phosphate (ZrP) is low. In addition, due to the special surface charge distribution of zirconium oxide (isoelectric point of about pH 6-7), conventional anions or peptides are also difficult to achieve stable binding through electrostatic interactions.

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

[0007] The problem solved by this invention is that existing oral restorative materials such as zirconium oxide, zirconium phosphate, or titanium have drawbacks such as surface biological inertness, insufficient antibacterial properties, and difficulty in functional modification, which limit their use.

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

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

[0010] The above-mentioned coatings for modifying oral restorative materials are used in the preparation of oral restorative materials.

[0011] Preferably, the oral restoration material is used to prepare dental implants or abutments, wherein the dental implants or abutments are made of zirconium oxide, phosphorylated zirconium oxide, or titanium, or the surface composition of the dental implants or abutments is zirconium phosphate.

[0012] Preferably, the amino acid sequence of the cationic short peptide is H(K)nS-X, where 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, wherein the oral restoration material is made of zirconium oxide, phosphorylated zirconium oxide or titanium, or the surface component of the oral restoration material is zirconium phosphate, and the surface of the oral restoration material is formed with the above-mentioned modified coating.

[0015] Compared with existing technologies, the coating for modifying oral restorative materials described in this invention has a good bonding ability with materials such as zirconium oxide, zirconium phosphate, or titanium. This makes it possible to perform bioactive modification on the surface of dental implants and abutments made of the same material, and has broad application prospects. Attached Figure Description

[0016] Figure 1 The chromatogram of the cationic short peptides prepared for surface modification in Example 1 of the present invention;

[0017] Figure 2 Mass spectrum of cationic short peptides for surface modification prepared in Example 1 of this invention;

[0018] Figure 3 The images show the laser confocal fluorescence patterns of three cationic short peptides used for surface modification adsorbed onto the surfaces of zirconium oxide, zirconium phosphate, and titanium, respectively, in Example 2 of this invention.

[0019] Figure 4 The above are bar charts showing the water contact angles of three cationic short peptides used for surface modification adsorbed onto the surfaces of zirconium oxide, zirconium phosphate, and titanium, respectively, in Example 2 of this invention.

[0020] Figure 5 The graph shows the stability results of three cationic short peptides used for surface modification in Example 3 of the present invention after adsorbing zirconium oxide and zirconium phosphate onto titanium surfaces and after ultrasonic treatment for 30 min.

[0021] Figure 6 This is a graph showing the toxic effects of the cationic short peptides used for surface modification, three adhesion functional peptides, and six cationic short peptide complexes of disordered composition on L929 cells in Example 4 of the present invention.

[0022] Figure 7 This is a diagram showing the early adhesion results of the cationic short peptides used for surface modification, three adhesion functional peptides, and six cationic short peptide complexes of disordered composition to L929 cells in Example 4 of the present invention.

[0023] Figure 8 This is a graph showing the statistical results of cell counting in L929 cells using the cationic short peptides used for surface modification, three adhesion functional peptides, and six cationic short peptide complexes of disordered composition, as described in Example 4 of the present invention.

[0024] Figure 9 This is a fluorescence imaging image of the six cationic short peptide complexes used for surface modification and three adhesion functional peptides and their disordered composition, which were adsorbed onto L929 cells in Example 4 of the present invention.

[0025] Figure 10The statistical results of the average cell morphology of L929 cells in the complex of the surface-modified cationic short peptides, three adhesion functional peptides, and six cationic short peptides in disordered order in Example 4 of this invention. Detailed Implementation

[0026] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Without conflict, the technical features of the embodiments of the present invention can be combined with each other.

[0027] Example 1: Preparation of cationic short peptides

[0028] A method for synthesizing a cationic short peptide for surface modification, wherein the cationic short peptide is HKS, comprising:

[0029] S1, Resin Pretreatment

[0030] Weigh Rink resin (solid support, degree of substitution 0.6 mmol / g), add dichloromethane (DCM, 10 mL / g resin) to swell for 1 hour, and then wash with N,N-dimethylformamide (DMF, 10 mL × 3 times) to remove impurities;

[0031] S2, Fmoc deprotection

[0032] Add 20% piperidine / DMF solution (10 mL / g resin) to the resin treated with S1, and stir at room temperature for 10 min × 2 times to completely remove the Fmoc protecting group at the end of the resin. Then wash with DMF until neutral.

[0033] S3, lysine coupling

[0034] One equivalent (eq) of Fmoc-Lys(dde)-OH (protected lysine side chain amino group) was activated for 5 minutes in DMF (0.3 mmol / L) with 1.5 eq of hydroxybenzotriazole (HOBT) + 1.5 eq of diisopropylcarbodiimide (DIC). The mixture was then added to the resin and reacted at room temperature for 2 hours under nitrogen protection. After the reaction, the solvent was removed by vacuum drying, and the mixture was washed three times with DMF.

[0035] S4, amino-terminated

[0036] Add acetic anhydride (5 eq) and a DMF solution of N,N-diisopropylethylamine (DIEA, 2 eq), and react at room temperature for 1 hour to block unreacted amino groups and prevent subsequent side reactions.

[0037] S5, Side Chain Detachment Protection

[0038] The resin was treated 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, followed by DMF washing to obtain the final product.

[0039] It should be noted that the preparation methods of cationic short peptides HKKS and HKKKS are the same as those of HKS. The only difference is that S2-S4 needs to be repeated 1-2 times, which will not be elaborated here.

[0040] To facilitate subsequent experimental detection, the following method was used for fluorescence conjugation of the cationic short peptide HKS:

[0041] Adding 3 eq fluorescein isothiocyanate (FITC) and 3 eq DIEA, and reacting in DMF in the dark for 4 hours, fluorescent labeling of the peptide's C-terminus was achieved. The peptide was then reacted at room temperature for 2 hours using a cleavage buffer to release the peptide and remove side-chain protecting groups. The cleavage buffer consisted of 95% trifluoroacetic acid (TFA) + 2% triisopropylsilane (Tis), 2% ethylenedithiol (EDT) + 1% H2O. The filtrate was precipitated with ice-cold ether, centrifuged, and 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 with a purity >95% was obtained. The corresponding chromatograms and mass spectra are shown below. Figure 1-2 .

[0042]

[0043] Ac-HKSGGK (FITC) - NH2

[0044] Example 2 Coating Preparation Experiment

[0045] 2.1 Experimental Methods:

[0046] Zirconium oxide, zirconium phosphate, and pure titanium polished to 2000 mesh were placed in 24-well plates, respectively. The FITC-labeled cationic short peptides were diluted to 10 mM with PBS (pH 7.4) solution and incubated at room temperature (25±1℃) 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 short peptides was added to the wells as a control.

[0047] After drying, the sample surface and three-dimensional (3D) fluorescence images were captured using a laser confocal microscope. The results are shown in the figure. Figure 3 Using a microsyringe, 10 μL of ultrapure water was vertically dropped onto the center of the dried sample surface. The droplet morphology was recorded using a high-speed camera (0-second instantaneous image). The software automatically fitted the droplet profile and calculated the contact angle (θ). The results are shown in [Figure Number]. Figure 4It should be noted that: Ac-HKSGGK(FITC)-NH2, Ac-HKKSGGK(FITC)-NH2, and Ac-HKKKSGGK(FITC)-NH2 were tested separately.

[0048] 2.2 Experimental Results

[0049] 2.2.1 Three-dimensional fluorescence image

[0050] A higher fluorescence intensity on the sample surface indicates a greater amount of cationic short peptides adhering to the surface of zirconium oxide or zirconium phosphate, which may be related to a significant increase in the electrostatic interaction between the cations and the sample surface. Figure 3 As can be seen, the three-dimensional images of the sample surface show that all three peptides adhered to the surfaces of the zirconia and zirconium phosphate samples, and the fluorescence intensity was statistically significant compared with the control group. Figure 3 The results are consistent.

[0051] 2.2.2 Contact Angle

[0052] Depend on Figure 4 The contact angle of the control group sample was 75.3±2.5°, indicating that the unmodified zirconia surface was moderately hydrophobic. However, the contact angle significantly decreased after adsorption of cationic short peptides, with HKS at 42.1±3.1°, HKKS at 38.5±2.8°, and HKKKS at 35.2±2.6°, showing a significant increase in hydrophilicity. This indicates that the cationic short peptides were successfully adsorbed onto the surfaces of both zirconia and zirconium phosphate samples. Specifically, the zirconia sample surface adsorbed with cationic short peptides became more hydrophilic, and this hydrophilic surface can inhibit the adsorption of hydrophobic bacteria, which will help promote cell adhesion and reduce bacterial colonization. Conversely, the zirconium phosphate surface adsorbed with cationic short peptides became more hydrophobic, and the hydrophobic interface delayed the diffusion of hydrophilic drugs, which has broad application prospects in drug sustained-release carriers.

[0053] Example 3: Coating Mechanical Stability Test

[0054] The sample containing adsorbed cationic short peptides was immersed in deionized water and sonicated for 30 minutes (100 W, 40 kHz). After removal, it was dried with nitrogen gas, and images were captured using a fluorescence microscope. The results are shown below. Figure 5 .

[0055] Depend on Figure 5 As can be seen from the fluorescence images, cationic short peptides can also be stably adsorbed on the surface of zirconium oxide and zirconium phosphate samples under physical conditions. The possible reason is that the binding strength with the material surface through electrostatic interaction and hydrogen bonding can resist physical shear force.

[0056] Example 4 Preparation of functional cationic short peptide complexes

[0057] 4.1 Experimental Methods

[0058] Taking SLIGRL, RGD, and YIGSR, three cell adhesion peptides, as examples, a chemical synthesis method was used to attach the three cationic sequences SLIGRL, RGD, and YIGSR to the sequence of HK(n)S, respectively. After purification by HPLC and verification by mass spectrometry, they were ready for use. Among them, SLIGRL is a 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. Among them, pep1-3 are complexes of HKKS with the three adhesion functional peptides SLIGRL, RGD, and YIGSR, and pep4-6 are randomized pep1-3 as controls. Their specific sequences are SLIGRL-GG-HKKS-NH2, Ac-HKKSGG-RGD-NH2, Ac-HKKSGG-YIGSR-NH, SKRLGGLKHSGI-NH2, Ac-GKHSRGKDG-NH2, and Ac-GKHSRGKYSGI-NH2, respectively.

[0059] Zirconia and zirconium phosphate samples were immersed in a 100 μM cationic short peptide solution and adsorbed by shaking at 37°C for 24 hours (120 rpm). After removal, they were dried under nitrogen and sterilized under UV light for 30 minutes; then L929 cells were incubated at 5 × 10⁻⁶ cells / day. 4 Cells / mL were seeded on the sample surface and cultured for 72 h. CCK values ​​were measured at 24 h and 72 h, respectively. Results are shown in the figure. Figure 6 Simultaneously, after 24 hours of culture, the cells were fixed and stained, and cell morphology and adhesion were photographed under an inverted fluorescence microscope. The results are shown in [Figure number missing]. Figure 7-10 .

[0060] 4.2 Experimental Results

[0061] Depend on Figure 6 The CCK values ​​at 24h and 72h showed that the six synthesized peptides were not toxic to L929 cells at concentration gradients of 0-100μM, with cell viability exceeding 80%, indicating good biocompatibility. Figure 7-9 It can be seen that the number of cells adhering to and the spreading morphology on the surface of zirconium phosphate samples coated with peptides are better than those of the control groups of zirconium oxide and zirconium phosphate samples, especially the pep2:Ac-HKKSGG-RGD-NH2 sequence.

[0062] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A coating for modifying oral restorative materials, characterized in that, The coating is formed by adsorbing cationic short peptides onto the surface of oral restorative materials, the amino acid sequence of which includes H(K). n S, where n is 1-3, the oral restorative material is used to prepare dental implants or abutments, the material of the oral restorative material is zirconium oxide, titanium or phosphorylated zirconium oxide, or the surface component of the oral restorative material is zirconium phosphate.

2. The application of the coating for modifying oral restorative materials as described in claim 1 in the preparation of oral restorative materials.

3. The application according to claim 2, characterized in that, The oral restoration material is used to prepare dental implants or abutments, wherein the dental implants or abutments are made of zirconium oxide, titanium or phosphorylated zirconium oxide, or the surface composition of the dental implants or abutments is zirconium phosphate.

4. The application according to claim 3, characterized in that, The amino acid sequence of the cationic short peptide is H(K). n SX, where n is 1-3 and X is the active amino acid sequence.

5. The application according to claim 4, characterized in that, The active amino acid sequence is any one of antimicrobial peptides, cell adhesion peptides, or osteogenic peptides.

6. An oral restorative material for preparing dental implants or abutments, characterized in that, The oral restoration material is made of zirconium oxide, titanium, or phosphorylated zirconium oxide, or the surface component of the oral restoration material is zirconium phosphate, and the oral restoration material further includes a coating for modifying the oral restoration material as described in claim 1 formed on the surface.

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