Zirconium oxide surface functionalization modification method

Abstract

The invention provides a zirconium oxide surface functional modification method, and belongs to the technical field of biological materials. According to the method, an anti-fibronectin aptamer is used for modifying the surface of zirconium oxide so as to promote the formation of periimplant soft tissue attachment. According to the method, an anti-fibronectin aptamer is applied to zirconium oxide surface modification for the first time, the aptamer has high specificity and stability, and the aptamer serving as a molecular bridge can specifically recognize and combine with FN in the environment, so that directional enrichment and functionalization of the FN on the zirconium oxide surface are facilitated, and cell adhesion and soft tissue adhesion are promoted.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and particularly relates to a method for functional modification of zirconia surface. Background Art

[0002] Due to its excellent physical and chemical properties, biocompatibility and aesthetic effects, zirconia has become an ideal material for implant abutments. Especially in the anterior tooth area with high aesthetic requirements, the application of zirconia materials has increased significantly. However, zirconia is an inert material and lacks the ability to promote soft tissue adhesion and induction, resulting in poor integration between the abutment surface and soft tissue. Treating the zirconia surface to enhance its bioactivity is an effective method to improve the abutment-soft tissue seal. Previous physical, chemical and biological treatment methods for zirconia surface all have certain deficiencies. Therefore, it is urgent to find a more ideal method to improve the bioactivity of zirconia surface to enhance the adhesion between soft tissue and zirconia abutment.

[0003] The surface of zirconia can be modified by physical and chemical methods (such as UV treatment, plasma treatment, forming micro / nano structures, ion implantation method, etc.) to promote soft tissue adhesion. However, these treatment methods rely on specific equipment, and some treatment methods are time-consuming or require special conditions, which limit their convenient application to a certain extent.

[0004] Biological methods include constructing biomimetic coatings on the zirconia surface, such as the main adhesion glycoproteins laminin 332 and fibronectin (FN) in the extracellular matrix (ECM). The coatings of the two can promote the adhesion of epithelial cells and fibroblasts.

[0005] FN is an important extracellular matrix protein, rich in arginine-glycine-aspartic acid (RGD) sequence, which is the main binding site of integrin receptors (such as α5β1), and can promote cell adhesion and tissue healing. In implant treatment, FN can enhance the adhesion, proliferation and spreading of human gingival fibroblasts (HGFs) on the implant material surface and promote soft tissue healing. Therefore, fixing FN on the zirconia surface will effectively enhance the adhesion of HGFs.

[0006] Previous studies mostly fixed FN or FN fragments on the zirconia surface by methods such as physical adsorption or covalent fixation, but there are problems such as spontaneous desorption of FN, conformational changes or protein degradation, and the high molecular weight limits the enrichment of FN on the zirconia surface, affecting its stability and bioavailability. Summary of the Invention

[0007] The present invention provides a method for functional modification of the zirconia surface. For the first time, an anti-fibronectin aptamer is applied to the modification of the zirconia surface. This aptamer has high specificity and stability. As a molecular bridge, it can specifically recognize and bind FN in the environment, which is beneficial to the directional enrichment and functionalization of FN on the zirconia surface, thereby promoting cell adhesion and soft tissue attachment.

[0008] To achieve the above object, the present invention provides a method for functional modification of the zirconia surface, using an anti-fibronectin aptamer to modify the zirconia surface to promote the formation of soft tissue attachment around the implant.

[0009] Preferably, before modifying the zirconia surface, the anti-fibronectin aptamer is treated as follows:

[0010] One end of the anti-fibronectin aptamer is modified with an amino group, and then centrifuged at 3000 - 3500 rpm for 5 - 10 s to ensure that the lyophilized powder is at the bottom of the centrifuge tube;

[0011] Add 80 - 85 μl of amine resuspension buffer to 100 μg of the aptamer, and centrifuge at 3000 - 3500 rpm for 10 - 15 s to obtain a 100 μM aptamer solution;

[0012] Add folding buffer to the aptamer solution at a volume ratio of 1:10 - 1:15, heat the resulting solution to 90 - 95 °C and keep it for 3 - 5 min, and then cool it to room temperature to fold the aptamer.

[0013] Preferably, the anti-fibronectin aptamer is ATW008 Anti-fibronectin aptamer, the amine resuspension buffer is RTW0002, and the folding buffer is RTW0003, all of which are purchased from Base Pair Biotechnologies.

[0014] Preferably, before use, the aptamer solution is mixed with an equal volume of 10 mM tris(2-carboxyethyl)phosphine (TCEP) solution and incubated at room temperature for 30 - 60 min to reduce the thiolated aptamer.

[0015] Preferably, the modification of the zirconia surface is specifically as follows:

[0016] Place the pretreated zirconia sheet in a reaction vessel, add the reduced aptamer solution, and then slowly drop NHS solution and EDC solution successively at a volume ratio of 1:1.5 - 1:2, react at room temperature for 20 - 24 hours, connect the aptamer to the zirconia surface through an amidation reaction, and wash with PBS to obtain the surface-functionalized modified zirconia.

[0017] Preferably, the NHS solution and the EDC solution are 0.01 M solutions respectively prepared with 0.1 M MES buffer solution.

[0018] Preferably, for a zirconia sheet with a diameter of 15 mm, the volume of the aptamer solution added is 20 - 80 μl, and the corresponding mass of the aptamer is 1 - 4 μg.

[0019] Preferably, pretreatment is carried out by polishing the zirconia sheet and ultrasonically washing it with absolute ethanol and deionized water for 20 - 30 min respectively.

[0020] The present invention also provides a surface-functionalized and modified zirconia prepared by the method according to any one of the above technical solutions.

[0021] The present invention also provides the use of the surface-functionalized and modified zirconia according to the above technical solution in enhancing the attachment of implant soft tissue to the zirconia abutment.

[0022] Preferably, the surface of the zirconia sheet treated with 80 μl / 4 μg aptamer promotes the inoculation of human gingival fibroblasts.

[0023] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0024] Compared with the FN or FN fragment-based biomimetic coatings in the prior art, the present invention for the first time applies an anti-fibronectin aptamer to the surface modification of zirconia. This aptamer has high specificity and can specifically recognize and bind FN in the environment as a molecular bridge. At the same time, as a DNA fragment, the aptamer has stability, avoiding problems such as spontaneous desorption, conformational change or protein degradation that may occur when directly connecting FN or FN fragments to the zirconia surface. This technology is beneficial to the directional enrichment and functionalization of FN on the zirconia surface, thereby promoting cell adhesion and soft tissue attachment. Description of the Drawings

[0025] Figure 1 In a - c: X-ray photoelectron spectroscopy (XPS) detection results, XPS analysis (P 2p) of the zirconia surface in the control group (a) and the aptamer treatment group (b) and the surface P element ratio (c); d - e: Laser confocal microscope images (d) and fluorescence area quantitative analysis (e) after SYBR Safe staining of zirconia treated with 0, 1, 2, 4 μg aptamer.

[0026] Figure 2 In a: Biolayer interferometry (BLI) detection of the affinity between the anti-fibronectin aptamer and fibronectin; b - c: Laser confocal microscope images (b) and fluorescence area quantitative analysis (c) after immunofluorescence staining of zirconia sheets treated with 0, 4 μg aptamer incubated in fibronectin solution.

[0027] Figure 3 a: Laser confocal microscopy images of HGF cultured on zirconia slices treated with 0, 1, 2, and 4 μg aptamers for 3 h and 24 h. Green fluorescence indicates the cytoskeleton, blue indicates the cell nucleus, and red indicates vinculin; b: Quantitative analysis of the adhesion number and spreading area of HGF after culturing for 3 h and 24 h in different groups; c: Cell proliferation of HGF cultured on zirconia slices in each group for 1, 3, 5, and 7 days, determined by CCK-8 assay; d: Western Blot detection results of HGF cultured on zirconia slices in each group for 24 h. Detailed implementation manners

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0029] Example 1 Fabrication of zirconia slices

[0030] Use computer-aided design / computer-aided manufacturing (CAD / CAM) to fabricate circular zirconia slices with a diameter of 15 mm and a thickness of 2 mm. After cutting and sintering, use SiC sandpaper to polish step by step until the surface is uniform. Detect the surface roughness and control it at 0.10 ± 0.02 μm to meet the surface roughness of clinical implant abutments. Ultrasonically clean the specimens with absolute ethanol and deionized water for 20 minutes each, and dry them naturally.

[0031] Immerse the zirconia slices in 2.5 M NaOH solution and heat to 60 °C for 24 hours to introduce hydroxyl groups on the zirconia surface. Take out and wash with PBS. Then immerse the zirconia slices in an aqueous solution of 1% (w / v) p-vinylbenzoic acid (pVBA), adjust the pH to 10.8 with NaOH, and soak overnight at room temperature to react the hydroxyl groups on the zirconia surface to carboxyl groups. Take out and wash with PBS for standby.

[0032] Example 2 Functionalization of zirconia surface

[0033] One end of the anti-fibronectin aptamer (ATW008 Anti-fibronectin aptamer, Base Pair Biotechnologies, Pearland, TX) was modified with an amino group to facilitate subsequent grafting. Centrifuge at 3000 - 3500 rpm for 5 - 10 s before the first use to ensure that the lyophilized powder is at the bottom of the centrifuge tube. Add 80 - 85 μl of amine resuspension buffer (RTW0002, Base Pair Biotechnologies, Pearland, TX) to 100 μg of the aptamer, and centrifuge at 3000 - 3500 rpm for 10 - 15 s to obtain a 100 μM aptamer solution. Add folding buffer (RTW0003, Base Pair Biotechnologies, Pearland, TX) to the aptamer solution at a volume ratio of 1:10 - 1:15, heat the solution to 90 - 95 °C for 5 min, and then cool to room temperature to fold the aptamer. To ensure the best performance of the aptamer, before use, mix the aptamer solution with an equal volume of 10 mM tris(2-carboxyethyl)phosphine (TCEP) solution and incubate at room temperature for 30 - 60 min to reduce the thiolated aptamer.

[0034] Use N-hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) as catalysts. NHS and EDC are respectively prepared into 0.01 M solutions with 0.1 M MES buffer.

[0035] Place the treated zirconia slices in a 24-well plate, add 20 - 80 μl of the aptamer solution to the wells, and then slowly drop the NHS solution and the EDC solution successively at a volume ratio of 1:1.5 - 1:2. React at room temperature for 20 - 24 hours, and connect the aptamer to the zirconia surface through the amidation reaction between the carboxyl group on the zirconia surface and the amino group at one end of the aptamer, and wash with PBS.

[0036] Example 3 Evaluation of the Connection of Anti-fibronectin Aptamer

[0037] Use X-ray photoelectron spectroscopy to determine the elemental distribution on the surface of the aptamer-modified zirconia. Figure 1 As shown in a - c, after treatment with the aptamer, a peak of the characteristic element P of the aptamer can be detected on the surface of the zirconia slices; by analyzing the distribution of surface chemical elements, the proportion of element P on the surface of the aptamer-treated zirconia slices is 10.47%, while no P element is detected in the untreated group.

[0038] Stain the zirconia slices with SYBR Safe DNA fluorescent dye, observe under a laser confocal microscope, and quantitatively analyze the fluorescent images with ImageJ software. Figure 1As shown in d-e, as the dosage of aptamer treatment increased, the fluorescence area on the zirconium sheet surface increased from 0.005% to 0.437%. The results indicated that the anti-fibronectin aptamer was successfully conjugated to the zirconia surface.

[0039] Example 4 Fibronectin can be adsorbed on the surface of zirconia modified by aptamer

[0040] The affinity between the anti-fibronectin aptamer and fibronectin was determined by bio-layer interferometry (BLI) experiment. Figure 2 As shown in a, the measured KD value was 173.6 nM, indicating a strong affinity between the two.

[0041] Zirconium oxide sheets treated with 80 μl / 4 μg anti-fibronectin aptamer were used, and untreated zirconium oxide sheets were used as the control group. The two groups of zirconium oxide sheets were incubated in 50 μg / ml fibronectin solution for 24 h, and washed three times with PBS to remove unbound proteins. Immunofluorescence staining of the two groups of zirconium oxide sheets was performed using anti-fibronectin monoclonal antibody and the corresponding fluorescent secondary antibody, observed under a laser confocal microscope, and the area covered by FN on the zirconia surface was quantified using ImageJ software to evaluate the effect of anti-fibronectin aptamer modification on the amount of fibronectin adsorbed on the zirconia surface. Figure 2 As shown in b-c, the FN fluorescence area was 0.04% on the surface of untreated zirconia, and increased to 19.08% after aptamer treatment, indicating that the amount of FN adsorbed on the zirconium sheet increased significantly after aptamer modification.

[0042] Example 5 Anti-fibronectin aptamer treatment can promote the adhesion, spreading and proliferation of HGF

[0043] Human gingival fibroblasts (HGFs) were seeded on zirconium oxide sheets treated with aptamer at different dosages (20 μl / 1 μg, 40 μl / 2 μg, 80 μl / 4 μg) and untreated zirconium oxide surfaces, and the following experiments were carried out respectively:

[0044] Cell adhesion and spreading

[0045] By using laser confocal microscopy combined with fluorescence staining technology, the early adhesion and spreading behaviors of HGFs on zirconia surfaces in each group were systematically studied. At 3 hours (initial adhesion stage) and 24 hours (stable spreading stage) after inoculation, the cell cytoskeleton F-actin was labeled with FITC-Phalloidin, the cell nuclei were stained with DAPI, and the focal adhesion structures were labeled with anti-focal adhesion protein antibody and fluorescent secondary antibody. Fluorescent images were quantitatively analyzed using ImageJ software to count the number of cell adhesions per unit area (based on DAPI-positive cell counting) and the cell spreading area (calculated based on the F-actin-covered area), revealing the regulatory effects of different doses of aptamer treatment on the adhesion efficiency and spreading ability of HGFs at the morphological level.

[0046] Figure 3 As shown in a-b, after 3 hours of culture, more HGFs adhered to the zirconia slices treated with aptamer, and partial cell spreading began to occur. The fluorescence signal of focal adhesion protein increased, and the promoting effect enhanced with the increase in the aptamer treatment dose. Among them, the number of cell adhesions in the 4 μg group increased by 145.5% compared with the control group, and the cell spreading area increased by 325.0%. After 24 hours of culture, the number of HGFs adhering to the zirconia slices treated with aptamer was higher, the spreading area was larger, and the cell protrusions were more numerous compared with the control group. The promoting effect enhanced with the increase in the aptamer treatment dose. Among them, the number of cells in the 4 μg group increased by 110.6% compared with the control group, and the spreading area increased by 101.8%.

[0047] Cell proliferation

[0048] HGFs were inoculated on the zirconia slices in each group, and the proliferation activity of HGFs was detected by the CCK-8 method after 1, 3, 5, and 7 days of culture. By comparing the differences in absorbance values between the aptamer treatment group and the control group, the effect of aptamer modification on the proliferation of HGFs was evaluated. Figure 3 As shown in c, the cell proliferation activity on the aptamer-modified zirconia surface increased, and the effect enhanced with the increase in the aptamer treatment dose. After 7 days of culture, the proliferation activity of HGFs in the 4 μg group increased by 45.3% compared with the control group, indicating that the aptamer-modified zirconia surface has good biosafety and can promote the proliferation of HGFs.

[0049] Expression of adhesion-related proteins

[0050] The expression changes of adhesion-related molecules were detected by Western Blot method. Integrin β1 and Focal Adhesion Kinase (FAK) were selected as key indicators. By comparing the expression differences of the above molecules between the aptamer treatment groups with different concentrations and the control group, the molecular mechanism of aptamer modification affecting the biological behavior of HGFs was determined. Figure 3It can be seen that the expression levels of Integrinβ1 and FAK in the aptamer treatment group of HGFs were both increased, and increased with the increase of the aptamer dose.

[0051] Comparative Example 1

[0052] In the technical route of zirconia surface functionalization, the zirconia sheet was immersed in an aqueous solution of 1% (w / v) p-vinylbenzoic acid (pVBA), and the pH was adjusted to 10.8 with NaOH, and immersed overnight at room temperature to react the hydroxyl groups on the zirconia sheet surface into carboxyl groups. If NaOH was not used to adjust the pH, or the reaction time was too short (less than 8 h), the reaction could not proceed or was incomplete, and effective surface functionalization could not be achieved.

[0053] Comparative Example 2

[0054] In the technical route of zirconia surface functionalization, zirconia sheets were treated with aptamers at different doses (20 μl / 1 μg, 40 μl / 2 μg, 80 μl / 4 μg). Among them, the promotion effect of the 4 μg group on the adhesion, spreading and proliferation of HGFs was the most obvious. The promotion effects of the 1 μg and 2 μg groups were worse than those of the 4 μg group. Continuing to increase the aptamer dose was not conducive to cost control.

Claims

1. A method for functionalizing the surface of zirconium oxide, characterized in that: Zirconia surfaces were modified with anti-fibronectin aptamers to promote peri-implant soft tissue attachment formation.

2. The method according to claim 1, characterized in that: Before modifying the zirconia surface, the anti-fibronectin aptamer was treated as follows: One end of the anti-fibronectin aptamer was modified with an amino group, and then centrifuged at 3000-3500 rpm for 5-10 s to ensure that the lyophilized powder was at the bottom of the centrifuge tube; Add 80-85 μl of amine resuspension buffer to 100 μg of aptamer and centrifuge at 3000-3500 rpm for 10-15 s to obtain a 100 μM aptamer solution; Folding buffer was added to the aptamer solution at a volume ratio of 1:10-1:15, and the resulting solution was heated to 90-95°C for 3-5 min, and then cooled to room temperature to fold the aptamer.

3. The method according to claim 2, characterized in that Before use, the aptamer solution was mixed with an equal volume of 10 mM trichloroethyl phosphate solution and incubated at room temperature for 30-60 min to reduce the thiolated aptamer.

4. The method according to claim 3, characterized in that The modification of the zirconium oxide surface is specifically as follows: The pretreated zirconia sheet was placed in a reaction container, and the reduced aptamer solution was added. Then, NHS solution and EDC solution were slowly dripped in a volume ratio of 1:1.5-1:

2. The reaction was carried out at room temperature for 20-24 hours. The aptamer was connected to the zirconia surface through an amidation reaction, and the aptamer was washed with PBS to obtain surface functionalized zirconia.

5. The method according to claim 4, characterized in that The NHS solution and EDC solution were prepared into 0.01 M solutions using 0.1 M MES buffer respectively.

6. The method according to claim 4, characterized in that For a zirconium oxide sheet with a diameter of 15 mm, the volume of the aptamer solution added is 20-80 μl, and the corresponding mass of the aptamer is 1-4 μg.

7. The method according to claim 4, characterized in that The zirconium oxide sheet was pretreated by polishing and ultrasonically washing with anhydrous ethanol and deionized water for 20-30 minutes respectively.

8. Surface functionalized zirconium oxide prepared by the method according to any one of claims 1 to 7.

9. Use of the surface functionalized modified zirconium oxide according to claim 8 in enhancing the attachment of implant soft tissue to the zirconium oxide abutment.

10. The use according to claim 9, characterized in that The surface of the zirconia sheet treated with 80 μl / 4 μg of aptamers promoted the seeding of human gingival fibroblasts.

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