A method for preparing a titanium-based prosthetic coating with both antibacterial and cell burial regulation functions and its application.

Abstract

This invention belongs to the field of biotechnology, specifically relating to a method for preparing a titanium-based prosthetic coating with both antibacterial and cell burial regulation functions, and its application. The preparation method of the titanium-based prosthetic coating in this invention includes the following steps: adding (DOPA)6-PEG5-DBCO lyophilized powder to Tris-HCl buffer to prepare a mixture; immersing a pretreated titanium rod in the mixture to obtain a titanium rod with (DOPA)6-PEG5-DBCO covalently bonded to its surface; immersing the obtained titanium rod in a metal ion aqueous solution and soaking it in the dark for 6-24 hours to obtain a surface-loaded (DOPA)6-PEG5-DBCO / Mn solution. 2+ / Cu 2+ A titanium rod with a bimetallic coordination coating is then immersed in an N3-GLP-1RA solution, and after a light-protected reaction, the titanium-based prosthesis coating is obtained. The coating of this invention exhibits excellent biocompatibility and can promote osteogenic differentiation of bone marrow mesenchymal stem cells.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for preparing a titanium-based prosthetic coating with both antibacterial and cell burial regulation functions and its application. Background Technology

[0002] Current surface engineering strategies for infectious implants mostly focus on single antibacterial functions, including antibiotic loading, single-metal ion delivery, and photothermal / photocatalytic sterilization. While these can reduce bacterial load to some extent, they have significant limitations: they can only eliminate pathogens and cannot improve post-infection host immune dysfunction and bone regeneration defects; they are difficult to simultaneously and efficiently kill both airborne bacteria and mature biofilms, and long-term use can easily induce bacterial resistance; the burst release effect of high-dose antibacterial agents can also cause dose-dependent cytotoxicity, damaging normal tissue activity. To solve the problem of bone regeneration disorders, bone immunomodulatory biomaterials have become a research hotspot in recent years. Most existing research focuses on regulating macrophage polarization towards the M2 anti-inflammatory phenotype, attempting to promote osteogenic repair through phenotypic conversion. However, this research paradigm has not fully explained the fundamental reason for the persistence of inflammation in aging or osteoporotic tissues. Recent research confirms that the defective cytotoxic function of aging macrophages is the core driving factor for the persistence of chronic inflammation and the deterioration of the regenerative microenvironment. In aging, macrophage TAM receptor (MerTK, Axl) signaling pathways are impaired, hindering the effective clearance of apoptotic cells and pathogen remnants. This leads to persistent activation of inflammatory signals, and even inducing macrophage polarization towards the M2 phenotype fails to fundamentally terminate inflammation and rebuild a regenerative microenvironment. Currently, few orthopedic implant coatings are designed to repair the cytotoxic function of aging macrophages. This critical technological gap severely restricts the improvement of implant osseointegration in infectious osteoporosis.

[0003] Glucagon-like peptide-1 receptor agonists (GLP-1RAs), classic drugs for treating type 2 diabetes, have recently been found to have significant anti-inflammatory and tissue-protective effects, showing potential application value in orthopedics. However, current orthopedic applications of GLP-1RAs mostly rely on systemic administration or simple physical embedding methods, which have problems such as high risk of systemic side effects, low local bioavailability, easy peptide degradation and detachment, and short duration of action. Furthermore, existing implant coatings with both antibacterial and osteogenic functions are mostly just simple physical mixtures of antibacterial components and osteogenic factors, failing to achieve organic coupling of functions at the pathological mechanism level, and thus unable to simultaneously break the vicious cycle of "infection-immunoaging-bone regeneration disorder." At the same time, there are significant deficiencies in the stable anchoring and controlled release of functional molecules; osteogenic peptides are prone to rapid loss, and the release of metal ions is uncontrollable, making it difficult to balance long-term bioactivity and safety. In summary, current technologies cannot meet the clinical needs for treatment of infected osteoporotic bone implants, and there is an urgent need to develop a modular implant surface modification technology that is structurally stable, functionally synergistic, and biosafe. Based on this, the present invention aims to provide a method for preparing a titanium-based prosthetic coating with both antibacterial and cell burial regulation functions, and its application. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing a titanium-based prosthesis coating with both antibacterial and cell burial regulation functions and its application. This functional titanium surface modification material can regulate the metabolism of senescent macrophages and promote osteogenic dual functions, thereby meeting the clinical needs for regulating the immune microenvironment and osteointegration of orthopedic implants.

[0005] This invention provides a method for preparing a titanium-based prosthetic coating with both antibacterial and cell burial regulation functions, comprising the following steps:

[0006] (1) Take (DOPA)6-PEG5-DBCO lyophilized powder and add it to Tris-HCl buffer to prepare a mixture; immerse the titanium rod after the pretreatment step in the mixture and soak it in the dark for 12-24 hours to obtain a titanium rod with (DOPA)6-PEG5-DBCO covalently bonded to its surface.

[0007] (2) Prepare an aqueous solution of metal ions using manganese and copper salts, immerse the titanium rod with (DOPA)6-PEG5-DBCO covalently bonded to its surface in the solution, and soak it in the dark for 6-24 hours to obtain surface-loaded (DOPA)6-PEG5-DBCO / Mn. 2+ / Cu 2+ Titanium rods with bimetallic coordination coating;

[0008] (3) The GLP-1RA peptide was modified with an azide group to obtain N3-GLP-1RA, and then an N3-GLP-1RA solution was prepared using phosphate buffer as a solvent; the surface-loaded (DOPA) 6-PEG5-DBCO / Mn 2+ / Cu 2+ The titanium rod with the bimetallic coordination coating is immersed in N3-GLP-1RA solution, and the titanium-based prosthesis coating is obtained after the reaction is carried out in the dark.

[0009] Furthermore, the concentration of the Tris-HCl buffer is 5-15 mmol / L, and the pH is 8.4-8.6.

[0010] Furthermore, the pretreatment step includes: polishing the titanium rod with sandpaper, sonicating it with deionized water, treating it with sodium hydroxide aqueous solution for 20-28 hours, removing it, cleaning it until neutral, and drying it with nitrogen gas.

[0011] Furthermore, the metal ion aqueous solution is a mixed aqueous solution containing 4-6 mmol / L MnCl2 and 4-6 mmol / L CuCl2.

[0012] Furthermore, in step (2), the pH of the aqueous solution of metal ions is pre-adjusted to 7.1-7.3 before immersing the titanium rod with (DOPA)6-PEG5-DBCO covalently bonded to its surface.

[0013] Furthermore, the preparation method of N3-GLP-1RA includes: dissolving GLP-1RA peptide in phosphate buffer to prepare a 2-10 mg / mL solution, adding N-hydroxysuccinimide azide acetate in an ice bath protected from light, stirring the reaction in the dark for 6-8 hours, removing impurities, and freeze-drying to obtain the final product.

[0014] Furthermore, the molar ratio of GLP-1RA to N-hydroxysuccinimide azide acetate is 1:1.2-1:2.0.

[0015] Further, the N3-GLP-1RA solution is an N3-GLP-1RA solution with a concentration of 4-6 mg / mL, using 8-12 mmol / L phosphate buffer solution at pH 7.3-7.5 as the solvent.

[0016] Furthermore, the phosphate buffer solution is prepared by weighing sodium dihydrogen phosphate and disodium hydrogen phosphate, dissolving them in deionized water, and adjusting the pH of the system to 7.3-7.5 using sodium hydroxide aqueous solution or hydrochloric acid aqueous solution.

[0017] On the other hand, the present invention also provides the application of the titanium-based prosthesis coating prepared by the preparation method in the preparation of materials for regulating the metabolism of aging macrophages and osteointegration.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] This invention, for the first time, constructs a modular multifunctional titanium surface modification platform based on biomimetic adhesion-metal coordination-bioorthogonal click chemistry. Stable bonding with the titanium substrate is achieved through a (DOPA)6-PEG5-DBCO anchoring layer, utilizing Mn... 2+ / Cu 2+ A bimetallic-phenol coordination network enables the controlled release of antibacterial ions, and an azide-acetylene cycloaddition reaction is used to precisely immobilize GLP-1RA ligands, solving the core problems of traditional coatings such as single function, unstable binding, and loss of activity. This platform has high versatility and scalability, providing a new technical paradigm for the functionalization of orthopedic implant surfaces, and achieving the optimal balance between physicochemical stability and bioactivity.

[0020] This invention innovatively proposes Mn 2+ / Cu 2+ The bimetallic synergistic antibacterial mechanism overcomes the technical bottleneck of limited antibacterial effect and easy development of drug resistance by single metal ions. 2+ Mn disrupts biofilm barrier structure by blocking bacterial quorum sensing systems. 2+ By activating the host's innate immune system to clear planktonic bacteria, the two work synergistically to inhibit key pathways such as peptidoglycan synthesis, oxidative phosphorylation, and DNA replication at the molecular level. This bimetallic strategy achieves comprehensive clearance of both planktonic bacteria and mature biofilms, significantly reducing the cytotoxicity and drug resistance risks associated with high-dose application of single metals, and providing a safer and more efficient solution for the design of antibacterial surfaces for orthopedic implants.

[0021] This invention reveals and utilizes for the first time a novel mechanism by which the GLP-1R-TAM signaling axis regulates the necrolysis function of senescent macrophages, breaking through the limitation of traditional orthopedic materials that only focus on macrophage phenotypic polarization. Surface-anchored GLP-1RA, through specific binding to receptors on the surface of senescent macrophages, significantly upregulates the expression of key TAM family molecules such as MerTK and Axl, restoring their ability to phagocytose apoptotic cells, and fundamentally solving the persistent inflammation problem caused by defective necrolysis function in the aging microenvironment.

[0022] This invention establishes an innovative theory of synergistic osteogenic development through a dual pathway of "direct induction and immune regulation," solving the clinical challenge of coexisting osteoblast dysfunction and inflammatory osteolysis in the microenvironment of infectious osteoporosis. The coating surface directly upregulates the expression of osteogenic markers such as RUNX2 and OCN in BMSCs via GLP-1RA, while indirectly enhancing osteogenic differentiation by reshaping the anti-inflammatory and regenerative immune microenvironment. This synergistic dual-pathway mechanism significantly reversed infection-induced osteolysis in in vivo experiments, increasing bone mineral density and bone volume fraction, achieving highly efficient integration of the implant-bone interface.

[0023] This invention pioneers a three-pronged functional coupling strategy of "antibacterial defense, immune regeneration, and bone integration," achieving for the first time simultaneously on a single implant surface three major functions: infection control, reversal of immune aging, and bone regeneration and repair, overcoming the limitations of existing technologies that target single pathological stages. Through multi-target synergistic action, this strategy effectively breaks the vicious cycle formed by bacterial infection, macrophage aging, and impaired bone regeneration, demonstrating remarkable therapeutic effects in the complex clinical scenario of elderly patients with osteoporosis and periprosthetic infection. This innovation not only establishes a new paradigm for orthopedic implant surface engineering but also provides important scientific evidence and translational prospects for immune regulation strategies for aging-related tissue regeneration. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly described below.

[0025] Figure 1 These are the full and high-resolution X-ray photoelectron spectra of the coating surface, among which... Figure 1 In the image, A represents the full spectrum scan. Figure 1 B and C in the diagram are spectral scans;

[0026] Figure 2 These are atomic force microscopy (AFM) images and energy-dispersive X-ray spectroscopy (EDS) spectra of surfaces modified with different grouped coatings; among them, Figure 2 In the image, A represents the atomic force microscope (AFM) image. Figure 2 B in the diagram represents the energy dispersive X-ray spectrum (EDS). Figure 2 In the diagram, C represents the elemental analysis quantization plot in the EDS spectrum;

[0027] Figure 3 These are graphs showing the water contact angle measurements of surfaces modified with different grouped coatings;

[0028] Figure 4 This is a graph showing the analysis of the CCK-8 biocompatibility experiment results on days 1, 3, and 5; among them, Figure 4 A in the figure represents the quantitative analysis of CCK-8 in RAW264.7 cells. Figure 4In the figure, B represents the quantitative analysis of CCK-8 in BMSCs cells.

[0029] Figure 5 This is an immunofluorescence staining image of the BMSCs cytoskeleton;

[0030] Figure 6 This is an immunofluorescence staining image of macrophages for iNOS and Arg-1; among them, Figure 6 In the image, A represents the iNOS immunofluorescence staining image, a marker for macrophage M1. Figure 6 In the image, B represents the immunofluorescence staining image of Arg-1, a marker for macrophage M2.

[0031] Figure 7 This is an immunofluorescence image of Arg-1 macrophages compared to the existing anti-inflammatory peptide K23;

[0032] Figure 8 These are Western blot images and quantification results of macrophage burial-related markers; among them, Figure 8 In the image, A represents a Western blot image of macrophage burial-related markers; Figure 8 In the figure, B represents the quantification result of the protein blot experiment image;

[0033] Figure 9 Images of bacteria from different groups on smear plates and transmission electron micrographs of the bacteria; among them... Figure 9 A in the image represents a bacterial plating experiment of Staphylococcus aureus and Pseudomonas aeruginosa. Figure 9 In the figure, B represents the quantitative analysis of the number of colony-forming units (CFU) of Staphylococcus aureus in each group; Figure 9 In this context, C represents the quantitative analysis of the number of colony-forming units (CFU) of Pseudomonas aeruginosa in each group. Figure 9 D in the image represents a transmission electron microscope (TEM) image of a Staphylococcus aureus smear. Figure 9 E in the image is a transmission electron microscope image of a smear of Pseudomonas aeruginosa.

[0034] Figure 10 It is similar to existing antibacterial metal ions Zn 2+ Comparative scanning electron microscope images of bacterial biofilms; among which, Figure 10 In the image, A represents an experimental image of Staphylococcus aureus. Figure 10 B in the image represents an experimental image of Pseudomonas aeruginosa.

[0035] Figure 11 The results of ALP and ARS staining are from BMSCs cells, which promote bone differentiation; among them, Figure 11 In the image, A represents ARS staining images of BMSCs cells cultured on titanium surfaces with different surface modifications. Figure 11 In the image, B represents ALP staining images of BMSCs cells cultured on titanium surfaces with different surface modifications.

[0036] Figure 12 These are CT three-dimensional reconstruction and quantitative analysis images of the osteointegration of the femoral titanium implant bone interface in rats 4 weeks post-surgery; among them, Figure 12 In the image, A represents a 3D CT reconstruction of the bone-integration interface of a rat femoral titanium implant. Figure 12 In the image, B represents the bone mineral density (BMD) data analysis image of the bone interface of the rat femoral titanium implant, based on CT three-dimensional reconstruction. Figure 12 C in the image represents the bone volume fraction (BV / TV) data analysis image of the bone interface of the rat femoral titanium implant and its osseointegration in CT three-dimensional reconstruction. Figure 12 In the image, D represents the trabecular thickness (Tb.Th) data analysis image of the bone interface and osteointegration of the rat femoral titanium implant in CT three-dimensional reconstruction. Figure 12 E in the image represents the trabecular bone number (Tb.N) data analysis image of the bone interface of the rat femoral titanium implant bone integration CT three-dimensional reconstruction;

[0037] Figure 13 These are images of immunohistochemical staining and quantitative analysis of macrophage M1 / M2 phenotypes 5 days post-surgery; among them, Figure 13 Image A in the image is an immunohistochemical staining image of CD68 / CD86 / DAPI in the bone tissue surrounding the implanted titanium rod; Figure 13 Image B in the image is an immunohistochemical staining image of CD68 / CD206 / DAPI in the bone tissue surrounding the implanted titanium rod; Figure 13 C in the image represents a quantitative analysis of the CD68 / CD86 / DAPI immunohistochemical staining-positive area in the bone tissue surrounding the implanted titanium rod. Figure 13 In the image, D represents a quantitative analysis of the CD68 / CD206 / DAPI immunohistochemical staining-positive area in the bone tissue surrounding the implanted titanium rod.

[0038] Figure 14 These are the results of continuous osteogenic fluorescent staining of bone tissue; among them, Figure 14 Image A in the image represents a continuous osteogenic fluorescence staining image of Calcein / ARS around the implanted titanium rod. Figure 14 B in the image represents the quantized resolution of the positive region in the continuous osteogenic fluorescence staining image. Detailed Implementation

[0039] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] It should also be noted that (DOPA)6-PEG5-DBCO (C 81 H 91 N7O 26 , Mw 1570.62, Qiangyao Biotechnology, Shanghai, China); GLP-1RA (C 187 H2 91 N 45 O 59 CAS No.: 910463-68-2 Mw 4113.64, Qiangyao Biotechnology, Shanghai, China); Fmoc-3,4-Isopropylidene dioxyphenylalanine (C 27 H 25 NO6, CAS No.: 852288-18-7 Mw 459.49, Sigma-Aldrich, St. Louis, USA); Fmoc-glycine (C 17 H 15 NO4, CAS No.: 29022-11-5 Mw 297.31, Allah Man Shanghai, China); Maleimide-pentaethylene glycol-carboxylic acid (C 20 H 31 NO 10 CAS No.: 1286755-26-7 Mw 389.40, Broad Pharmaceuticals, San Diego, USA); N,N-Diisopropylethylamine (C8H 19 N, CAS No.: 7087-68-5 Mw 129.24, Allah Man (Shanghai, China); dibenzocyclooctylmercaptopropionic acid (C 26 H 22 N2O3S, CAS No.: 1384870-47-4 Mw 430.51, Conjuprob, San Diego, USA) Fmoc-8-amino-3,6-dioxanoic acid (C 22 H 25 NO6, CAS No.: 166108-71-0 Mw 385.41, Allah Man (Shanghai, China); N-hydroxysuccinimide azide acetate (C6H6N4O4, CAS No.: 824426-32-6, Mw 198.14, Sigma Aldrich, St. Louis, USA.

[0041] Example 1

[0042] This invention provides a titanium-based prosthetic coating with both antibacterial and cell burial regulation functions, namely (DOPA)6-PEG5-DBCO / Mn 2+ / Cu 2+ The preparation method of the / GLP-1RA biomimetic multifunctional titanium surface modification system includes the following steps:

[0043] (1) Bonding (DOPA)6-PEG5-DBCO to the surface of a titanium rod:

[0044] Using the purchased (DOPA)6-PEG5-DBCO polypeptide, the Fmoc solid-phase peptide synthesis method was employed, with 2-chlorotriphenylmethyl chloride resin as the solid-phase support. Fmoc-DOPA (acetonide)-OH and Fmoc-Gly-OH were sequentially coupled to construct the Ac-(DOPA)-G-(DOPA)-G-(DOPA)-K (Fmoc)-(DOPA)-G-(DOPA)-G-(DOPA)-G-(DOPA) peptide backbone.

[0045] The titanium rod was polished in the order of 400#, 800#, 1600# to 2000# sandpaper. After ultrasonic treatment with anhydrous ethanol and deionized water for 15 minutes each, it was treated with 5 mol / L sodium hydroxide aqueous solution at 60℃ for 24 hours. After removal, it was rinsed with deionized water until neutral and dried with nitrogen gas for later use.

[0046] First, prepare a 10 mmol / L Tris-HCl buffer solution with a pH of 8.5 (weigh 1.211 g of Tris base and dissolve it in approximately 900 mL of ultrapure water, slowly adjust the pH to 8.5 with concentrated hydrochloric acid, bring the volume to 1000 mL and mix well, filter through a 0.22 μm filter membrane for sterilization, and store at 4 ℃ for later use); take (DOPA)6-PEG5-DBCO lyophilized powder, add Tris-HCl buffer solution at a ratio of 5 mg of lyophilized powder per 1 mL of buffer solution, vortex to completely dissolve, and prepare a 5 mg / mL (DOPA)6-PEG5-DBCO solution. Avoid high temperature and vigorous shaking. After preparation, aliquot and store at -20 ℃ protected from light.

[0047] The pretreated titanium rod was completely immersed in a 5 mg / mL (DOPA)6-PEG5-DBCO solution and soaked at 37°C in the dark for 18 hours. After being removed, it was thoroughly rinsed with Tris-HCl buffer and deionized water to remove unbound peptides. After drying with nitrogen, a titanium rod with (DOPA)6-PEG5-DBCO covalently bound to its surface was obtained. Its surface forms stable bidentate coordination bonds with titanium oxide through catechol groups, while retaining DBCO groups for subsequent copper-free click chemistry reactions.

[0048] (2) Synthesis of (DOPA)6-PEG5-DBCO / Mn 2+ / Cu 2+ :

[0049] A titanium rod with surface covalently bonded (DOPA)6-PEG5-DBCO was completely immersed in a mixed aqueous solution containing 5 mmol / L MnCl2 and 5 mmol / L CuCl2.

[0050] The following steps were taken to prepare a mixed aqueous solution of 5 mmol / L MnCl2 and 5 mmol / L CuCl2: First, calculate the required mass of each chloride (0.990 g is needed to prepare 1000 mL of the mixture; 0.852 g is needed to prepare 1000 mL of the mixture); then, place the weighed MnCl2·4H2O and CuCl2·2H2O solids in a clean beaker, add about 800 mL of ultrapure water, and stir with a glass rod until completely dissolved; after dissolution, transfer the solution to a 1000 mL volumetric flask, wash the beaker three times with a small amount of ultrapure water, and transfer the washings into the volumetric flask as well; finally, add ultrapure water to make up to 1000 mL, shake well, filter through a 0.22 μm filter membrane, and store in a sealed container at 4 ℃ after preparation.

[0051] The mixed aqueous solution was pre-adjusted to pH 7.2 with 0.1 mol / L HCl or NaOH, and then subjected to horizontal reciprocating oscillation at 150 r / min in a constant temperature environment at 30°C under light-protected conditions for 15 hours. This allowed the catechol groups of the (DOPA)6 peptide chain to coordinate with Mn through bidentate coordination. 2+ Cu 2+ Forming stable metal-ligand complexes; each catechol unit can bind 1-2 metal ions, and Mn 2+ Cu preferentially forms an octahedral coordination structure with deprotonated phenolic hydroxyl groups. 2+ It then coordinates with both the phenolic hydroxyl group and the amino nitrogen atom to form a tetragonal planar structure;

[0052] After soaking, the titanium rod was rinsed repeatedly with plenty of deionized water five times, then ultrasonically cleaned for five minutes to thoroughly remove physically adsorbed free metal ions. Finally, it was dried with high-purity nitrogen gas to obtain (DOPA)6-PEG5-DBCO / Mn uniformly loaded on the surface. 2+ / Cu 2+ Titanium rods with bimetallic coordination coating;

[0053] (3) Synthesis of (DOPA)6-PEG5-DBCO / Mn 2+ / Cu 2+ / GLP-1RA:

[0054] The preparation method of 10 mmol / L phosphate buffer (pH 7.4) is as follows: Weigh 1.36 g of sodium dihydrogen phosphate (NaH2PO4·2H2O) and 0.71 g of disodium hydrogen phosphate (Na2HPO4), dissolve them in deionized water and bring the volume to 1 L. After stirring thoroughly to dissolve, adjust the pH of the system to 7.4 using 0.1 mol / L sodium hydroxide aqueous solution or hydrochloric acid aqueous solution.

[0055] Weigh 5.0 mg of GLP-1RA peptide and place it in a centrifuge tube. Add 1.0 mL of 10 mmol / L phosphate buffer, which has been pre-adjusted to pH 7.2 with 0.1 mol / L HCl aqueous solution. Gently pipette or vortex at low speed until the peptide is completely dissolved to obtain a GLP-1RA solution with a concentration of 5 mg / mL. Under light-protected conditions in an ice bath, add N-hydroxysuccinimide azidoacetate at a molar ratio of GLP-1RA:N-hydroxysuccinimide azidoacetate of 1:1.6. Stir in the dark for 6 hours and react for 3 hours to specifically acylate the amino group of the unique free lysine side chain at the C-terminus of GLP-1RA, introducing an azide group. Dialyze the reaction solution through a MWCO 3 kDa dialysis bag at 4 °C for 24 hours to remove impurities. Freeze-dry under vacuum at -40 °C to obtain N3-GLP-1RA, which has an azide modification rate of 90% and retains complete receptor binding activity.

[0056] Surface-loaded (DOPA) 6-PEG5-DBCO / Mn 2+ / Cu 2+ Titanium rods with bimetallic coordination coatings were completely immersed in 10 mmol / L phosphate buffer (pH 7.4) containing 5 mg / mL N3-GLP-1RA. A copper-free click reaction was carried out at 25°C in the dark with low-speed shaking at 60 rpm for 12-24 hours, allowing the DBCO groups on the coating surface to cycloaddite with the azide groups of N3-GLP-1RA to form stable 1,2,3-triazole covalent bonds. After the reaction, the titanium rods were removed and rinsed three times sequentially with phosphate buffer and deionized water, followed by ultrasonic cleaning for 3 minutes to remove physically adsorbed unbound peptides. The rods were then dried with high-purity nitrogen to obtain the surface-constructed (DOPA) 6-PEG5-DBCO / Mn. 2+ / Cu 2+ / GLP-1RA is a multifunctional composite-coated titanium-based implant material.

[0057] Comparative Example 1

[0058] Control (pure metallic titanium); the difference from Example 1 is that: in step (1), (DOPA)6-PEG5-DBCO polypeptide is not added, but only titanium rods are used to polish in the order of 400#, 800#, 1600# to 2000# sandpaper. After ultrasonic treatment with anhydrous ethanol and deionized water for 15 min each, it is treated with 5 mol / L sodium hydroxide aqueous solution at 60℃ for 24 h. After taking it out, it is rinsed with deionized water until neutral, and dried with nitrogen gas for later use. The remaining steps are the same as in Example 1.

[0059] Comparative Example 2

[0060] DPD (the surface of titanium metal is modified with (DOPA)6-PEG5-DBCO); the difference from Example 1 is that Mn is not added in step (1). 2+ Cu 2+ The titanium rod surface was modified with (DOPA)6-PEG5-DBCO, and the remaining steps were the same as in Example 1.

[0061] Comparative Example 3

[0062] DPDM (metallic titanium surface using (DOPA) 6-PEG5-DBCO and Mn) 2+ (Ion modification); the difference from Example 1 is that Cu is not added in step (2). 2+ The titanium rod surface was modified with (DOPA)6-PEG5-DBCO and MnCl2 solution, and the remaining steps were the same as in Example 1.

[0063] Comparative Example 4

[0064] DPDC (the surface of titanium metal uses (DOPA) 6-PEG5-DBCO and Cu) 2+ (Ion modification); the difference from Example 1 is that Mn is not added in step (2). 2+ The titanium rod surface was modified with (DOPA)6-PEG5-DBCO and CuCl2 solution, and the remaining steps were the same as in Example 1.

[0065] Comparative Example 5

[0066] DPDMC (surface of titanium metal using (DOPA) 6-PEG5-DBCO, Mn) 2+ and Cu 2+ (Ion modification); the difference from Example 1 is that GLP1-RA is not added in step (3), and the surface of the titanium rod is modified only with (DOPA)6-PEG5-DBCO, CuCl2 and MnCl2 solution. The remaining steps are the same as in Example 1.

[0067] Comparative Example 6

[0068] The difference between DPDG (the surface of metallic titanium is modified with (DOPA)6-PEG5-DBCO and GLP-1 click chemical) and Example 1 is that CuCl2 and MnCl2 solutions are not added in step (2), and the surface of the titanium rod is modified only with (DOPA)6-PEG5-DBCO+GLP1-RA. The remaining steps are the same as in Example 1.

[0069] Test Example 1: Characterization

[0070] X-ray photoelectron spectroscopy analysis: The surface chemical composition was analyzed using an X-ray photoelectron spectrometer (ESCALAB 250Xi, Thermo Fisher Scientific, USA). Experimental conditions: Al Kα monochromatic X-ray source (1486.6 eV), power 150 W, vacuum degree <1×10⁻⁶. -9 mbar, analysis region diameter 500 μm. Full spectrum scan: pass energy 200 eV, step size 1 eV, 5 scans. High resolution spectrum: pass energy 20 eV, step size 0.1 eV, scanning C 1s, N 1s, O 1s, Ti 2p and Mg 2p regions. All binding energies were corrected using the C 1s peak (284.8 eV) as an internal standard. Data processing was performed using Avantage software, with Shirley background subtraction and Gaussian-Lorentzian mixture function fitting.

[0071] Figure 1 The X-ray photoelectron spectroscopy (XPS) spectrum of the coating surface is presented in full and high resolution: XPS provides decisive evidence for the successful introduction of each element in each modification step. Full-spectrum scanning confirms the presence of a Ti2p signal from the underlying TiO2 substrate, which gradually diminishes with the coverage of organic and metallic coatings. The appearance and enhancement of C1s, O1s, and N1s signals confirm the deposition of the (DOPA)6-PEG5-DBCO layer and subsequent GLP-1RA coupling. Figure 1 A). Crucially, high-resolution XPS spectroscopy clearly verified the successful loading of metal ions. For the DPDMC and DPDMCG groups, characteristic Cu2p (e.g., Cu2p3 / 2 and Cu2p1 / 2) and Mn2p (e.g., Mn2p1 / 2 and Mn3s) core level spectra were observed, confirming the coexistence of Cu and Mn ions on the surface. Figure 1 B, C).

[0072] AFM (Atomic Force Microscopy) and SEM-EDS (Scanning Electron Microscopy-Energy Dispersive X-ray Spectrometry) characterization: Surface morphology for each group was examined using an atomic force microscope (Dimension Icon, Bruker, Germany) in tapping mode. Experimental conditions: Silicon cantilever probe (TESPA-V2, Bruker), elastic constant 42 N / m, resonant frequency 320 kHz, scanning area 5 × 5 μm, scan rate 0.5 Hz, pixel resolution 512 × 512. At least three different regions of each sample were scanned to ensure representativeness of the results. Data processing used NanoScope Analysis software to calculate surface roughness parameters Ra (arithmetic mean roughness) and Rq (root mean square roughness). Elemental composition of the samples was analyzed using SEM-EDS. Analysis was performed using a Zeiss Merlin Compact field emission scanning electron microscope (Germany) paired with an Oxford Instruments Ultim Extreme silicon drift EDS energy dispersive detector (UK). Test parameters: accelerating voltage 15 kV, working distance 8.5 mm, beam current 0.8 nA; semi-quantitative elemental analysis was performed using the standard-free quantitative method (ZAF correction), and secondary electron morphology images, backscattered electron images, and elemental surface distribution (Mapping) images were acquired simultaneously.

[0073] Figure 2 These are atomic force microscopy (AFM) images and energy-dispersive X-ray spectroscopy (EDS) images of surfaces modified with different coatings: The surface morphology of peptide-metals after different modifications was evaluated by atomic force microscopy (AFM). The results showed that the surface roughness increased significantly after different modifications, especially with the addition of metal ions. Figure 2 A). Energy-dispersive X-ray spectroscopy (EDS) surface distribution maps further demonstrate the uniform distribution of elements on the DPDMCG surface. The merged elemental maps, along with clear signals for Ti, C, O, and N, and the successful co-localization of Cu and Mn, confirm the formation of a uniform and well-integrated multifunctional coating. Figure 2 B, C).

[0074] Water contact angle measurement: The static water contact angle was measured using a contact angle meter (DSA100, KRÜSS, Germany). Experimental conditions: ultrapure water droplet volume 2.0±0.1 μL, room temperature (25±1℃), relative humidity 45-55%. A microsyringe was used to gently drop the water droplet onto the sample surface, avoiding impact deformation. Immediately after droplet addition, images were taken, recording the contact angle change every 5 seconds for 2 minutes. Measurements were taken at least 6 different locations for each sample, and the mean and standard deviation were calculated. The Young-Laplace equation was used to fit the droplet profile to accurately calculate the contact angle.

[0075] Figure 3 The image shows the water contact angle measurements of the coated surfaces: these measurements reveal a gradual change in surface hydrophilicity. The water contact angle of the TiO2 surface was 79.7°, which significantly decreased after the modification with the (DOPA)6-PEG5-DBCO layer (DPD: 60.0°). Coordination with metal ions and peptides further enhanced surface wettability, likely due to the hydrated shell of immobilized metal ions, with the bimetallic surface exhibiting the most significant effect. This increased hydrophilicity is beneficial for resisting protein adsorption and cell interactions. Figure 3 ).

[0076] Test Example 2: In vitro cell culture

[0077] Rat bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured from the bone marrow of 20-month-old male SD rats using a method approved by the Ethics Committee of the First Affiliated Hospital of the University of Science and Technology of China. Specifically, rat femurs were isolated under aseptic conditions, and both ends of the femur were gently cut off with ophthalmic scissors. The femoral mesenchymal cavity was repeatedly flushed with PBS using a 5 mL sterile syringe until it turned white. Bone marrow fluid was collected and filtered through a 40 μm cell sieve. The filtered bone marrow fluid was collected and centrifuged at 1500 rpm for 10 minutes. The extracted BMSCs were cultured in α-DMEM medium (Dulbecco modified Eagle medium, Gibico, USA) containing 10% FBS (fetal bovine serum, Gibico, USA), 100 U mL−1 penicillin G, and 0.1 mg mL−1 streptomycin (Gibco, USA) in a cell culture incubator at 37 ℃, 5% CO2, and saturated humidity. When cultured to the third generation, BMSCs cells were identified by flow cytometry using antibodies against CD44, CD73, CD45, CD11b, and CD105 (1:100, Abcam, USA). Results: CD44, CD73, and CD105 were positive (>90%), while CD45 and CD11b were negative (<5%), indicating that the isolated cells were BMSCs cells and suitable for cell experiments.

[0078] RAW 264.7 mouse macrophage cell line was purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in high-glucose DMEM medium supplemented with 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. Cell passage: When the cell confluence reached 80-90%, cells were gently scraped off with a cell scraper, centrifuged at 1000 rpm for 5 minutes, resuspended in fresh medium, and passaged at a ratio of 1:4.

[0079] Test Example 3: Cell Compatibility

[0080] The cell biocompatibility of the biomimetic multifunctional titanium surface modification system was evaluated using the CCK-8 assay (Solarbio, China). Logarithmically growing RAW264.7 cells and BMSCs were prepared into cell suspensions, and cell counts were performed under a microscope at 4 x 10⁻⁶ cells / mL. 3 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured in an incubator. After cell adhesion, extraction medium was added and the plates were cultured for 1, 3, and 7 days, respectively. After the above treatment steps, 100 μL of CCK-8 solution was added to the 96-well plates and incubated at 37°C for 2 hours. The 96-well plates were then placed in a microplate reader (BIO-TEK; USA) to measure the optical density (OD) at a wavelength of 450 nm, and cell viability was calculated using the following formula: Cell viability = (OD scaffold - OD Blank) / (OD control – OD Blank) × 100%. In this formula, OD scaffold is the OD value of the experimental group, OD control is the OD value of the control group, and OD Blank is the OD value of the blank group.

[0081] Figure 4 The CCK-8 biocompatibility assay results show that the coating exhibits excellent biocompatibility, a finding strongly supported by the CCK-8 assay results, which demonstrated time-dependent growth from day 1 to day 5. At different time points, no statistically significant differences were observed between any modified group and the control group for BMSCs and RAW264.7 cells. These results strongly suggest that the introduction of metal ions and GLP-1RA peptide does not impair normal cell growth. Furthermore, the effect of the coating on cytoskeleton structure is a sensitive indicator of cell health and adhesion. Figure 4 A, B).

[0082] Test Example 4: Immunofluorescence Staining

[0083] BMSCs and RAW264.7 cells cultured on different samples were analyzed by fluorescent staining. In short, cells were treated according to experimental requirements: washed three times with PBS, fixed with 4% paraformaldehyde for 10 min, the paraformaldehyde was discarded, and the cells were washed with PBS. After permeation with 0.1% Triton X-100 (Beyotime, China), the cells were blocked with immunostaining blocking solution (Beyotime; China). Then, the primary antibody of the target molecule was added to the cells and incubated overnight at 4°C. Next, the cells were incubated with the corresponding species-labeled secondary antibody at room temperature in the dark for 1 hour. Cell nuclei were stained with phalloidin (Yeasen, China), and finally mounted with anti-quenching mounting medium. Fluorescently labeled cells were captured using a laser confocal microscope (Zeiss, Germany) in the dark. The fluorescence intensity of the target protein was assessed using Image J (Bethesda, USA).

[0084] Figure 5 Immunofluorescence morphology of the BMSCs cytoskeleton: F-actin and focal adhesion protein were stained with fluorescent dyes on BMSCs. The results showed that all surface-cultured cells exhibited good spreading morphology and a distinct actin cytoskeleton. The DPDMCG group showed the best cytoskeleton spreading, and focal adhesion protein staining was clearly visible on the periphery of the cells, appearing as discrete patches, indicating the formation of mature focal adhesion. Figure 5 A).

[0085] Figure 6 This image shows immunofluorescence staining of macrophages for iNOS and Arg-1: The immunomodulatory capacity of the multifunctional coating was systematically evaluated using macrophage polarization immunofluorescence staining technology. The expression of the M1-related marker iNOS and the M2-related marker Arg-1 in RAW264.7 cells cultured on substrates with different modifications was detected. Fluorescence intensity analysis showed that, compared with the Control group and the DPD group, the expression of iNOS and Arg-1 in macrophages containing Mn... 2+ The bases (DPDM, DPDMC, and DPDMCG) induced a significant increase in iNOS expression, confirming the pro-inflammatory polarization ability of manganese ions. Figure 6 A). Meanwhile, the GLP-1 functionalized groups (DPDG and DPDMGG) showed a significant increase in Arg-1 fluorescence intensity, indicating an anti-inflammatory M2-like phenotypic shift. Figure 6 B). Notably, DPDMCG simultaneously upregulated both M1 and M2 biomarkers, suggesting the existence of a time-regulated immune regulatory profile that is beneficial for sequential sterilization and tissue regeneration phases.

[0086] Figure 7Immunofluorescence images of Arg-1 macrophages compared to the existing anti-inflammatory peptide K23: To further investigate the anti-inflammatory performance of the GLP-1RA peptide and DPDMCG surface-modified system compared to the previously studied and published K23 anti-inflammatory peptide in macrophage metabolic regulation and inflammation control, (DOPA)6-PEG5-DBCO-K23 (DPDK) was synthesized by linking (DOPA)6-PEG5-DBCO to K23 using click chemistry. Immunofluorescence staining was used to evaluate the expression of the M2-related marker Arg-1. The results showed that the fluorescence intensity of Arg-1 in macrophages after DPDK intervention was significantly higher than that in the control group, indicating considerable anti-inflammatory regulatory ability, consistent with published research. Furthermore, the fluorescence intensity of the DPDG and DPDMCG groups was higher than that of the DPDK group, suggesting that the surface-modified system used in this study possesses superior performance. Figure 7 A).

[0087] Test Example 5: Protein Blotting Experiment

[0088] RAW264.7 cells and BMSCs were plated separately and treated differently according to the experimental objectives. The culture plates were then placed on ice and washed three times with pre-chilled PBS. A lysis buffer containing protease inhibitors and RIPA was added to extract total protein from the cells. Protein concentration was determined using a BCA protein assay kit according to the instruction manual. The concentrations of each protein were adjusted with 5× protein loading buffer, incubated in a boiling water bath for 5 minutes, and then stored at -20°C. Proteins were separated by SDS-PAGE gel electrophoresis (FUDE BIOLOGICAL, China). After electrophoresis, the proteins were transferred to a PVDF membrane using a wet transfer method. After transfer, the PVDF membrane was placed in an incubator containing TBST and blocked by incubation with 5% skim milk at room temperature on a shaker for 1 hour. After blocking, the membrane was washed three times with TBST. Subsequently, the primary antibody was diluted according to the instruction manual and added to the incubator, and the membrane was incubated overnight at 4°C on a shaker. After primary antibody incubation, the primary antibody was recovered, and the membrane was washed three times on a shaker for 5 minutes each time with TBST. Secondary antibody was diluted 1:5000 and added to the incubation chamber to completely cover the PVDF membrane. Incubation was carried out at room temperature for 2 hours. After washing the membrane three times with TBST, excess water was blotted with absorbent paper. The membrane was then placed in a light-proof container containing ECL chemiluminescence solution and allowed to react for 2 minutes. Exposure was performed using a chemiluminescent gel imaging system (Proteinsimple, USA), and the grayscale values ​​of the protein bands were analyzed using AlphaEaseFC software.

[0089] Figure 8Western blot images and quantitative results of macrophage burial-related markers: To verify the regulatory effect of the designed surface modification system on macrophage burial ability, a co-culture system was established for further verification. Macrophages were incubated with apoptotic rat bone marrow mesenchymal stem cells (rBMSCs) irradiated with ultraviolet light at a ratio of 5:1. Western blot images of burial-related proteins (TSP1, MERTK, CD36, and ODC1) showed that, compared with the M1 polarized control group, DPDMCG treatment significantly upregulated all four markers, indicating that phagocytic function was restored through the GLP-1 receptor signaling pathway. Figure 8 A, B).

[0090] Test Example 6: In vitro antibacterial performance evaluation

[0091] Bacterial culture and preparation: *Pseudomonas aeruginosa* (ATCC 27853) and methicillin-resistant Staphylococcus aureus (MRSA, ATCC 43300) were purchased from the China Center for Type Culture Collection. Bacterial activation: The strains were removed from cryovials at -80℃ and inoculated onto Luria-Bertani (LB) agar plates, and incubated at 37℃ for 18-24 hours until single colonies formed. Single colonies were selected and inoculated into LB broth, and cultured at 37℃ with shaking at 180 rpm for 12-16 hours until the logarithmic growth phase (OD600 = 0.6-0.8).

[0092] Bacterial suspension standardization: Collect bacteria cultured to the logarithmic growth phase by centrifugation (3000 rpm, 10 min), wash three times with sterile PBS, and resuspend in PBS. Adjust OD600 to 0.1 using a spectrophotometer, corresponding to a bacterial concentration of approximately 1 × 10⁻⁶. 8 CFU / mL. Further dilute with PBS to a working concentration of 1×10⁻⁶. 6 CFU / mL, prepare fresh before use. Verify actual bacterial concentration using plate count method to ensure experimental accuracy.

[0093] Planktonic bacteria killing experiment: Each group of coated titanium sheets (15 mm in diameter) was placed in a 24-well plate, and 1 mL of bacterial suspension (1×10⁻⁶) was added to each well. 6 (CFU / mL), ensuring the titanium sheet is completely submerged. Six replicates were prepared for each group. The samples were incubated statically at 37°C with 5% CO2 for 24 hours, avoiding shaking to prevent disruption of the bacteria-material interface.

[0094] Bacterial viability assay: After culturing for 24 hours, gently pipette to mix the supernatant, and take 100 μL for serial 10-fold dilution (10... -1 Up to 10 -5100 μL of each dilution was plated onto LB agar plates and incubated at 37°C for 24 hours before colony counting. Bacterial survival rate (%) = (Experimental group CFU / Control group CFU) × 100%. Bactericidal efficiency was expressed as a log reduction value: log 10 (Control group CFU / Experimental group CFU).

[0095] Biofilm formation inhibition assay: The biofilm formation inhibition ability was evaluated using a modified microplate method. Coated titanium sheets were placed in 24-well plates, and 1 mL of fresh LB medium and 100 μL of bacterial suspension (final concentration 1×10⁻⁶) were added. 5 (CFU / mL). Incubate at 37°C for 48 hours, replacing half of the culture medium every 24 hours to maintain nutrient supply and promote the formation of mature biofilm.

[0096] 3D structural analysis of biofilm: The 3D structural information of the biofilm was obtained by Z-axis scanning using a laser confocal microscope. Scanning parameters: Z-axis step size 0.5 μm, scanning range 0-50 μm, pixel resolution 1024×1024. The 3D structure of the biofilm was reconstructed using ImageJ's 3D plugin, and parameters such as biofilm thickness, volume, and coverage were calculated.

[0097] Figure 9 These are bacterial smears and transmission electron microscopy images: the manganese / copper coating effectively inhibits bacterial colonization, a major obstacle in the osseointegration process of infected implants. Bacterial biofilm formation is a major factor leading to refractory orthopedic bacterial infections. Therefore, we evaluated the in vitro antibacterial properties of DPDMCG against two clinically representative bacterial models (Staphylococcus aureus and Pseudomonas aeruginosa). Figure 9 As shown in Figure A, the number of colony-forming units (CFU) in each group was determined using the standard plate count (SPM) method. The results showed that the DPDMC and DPDMCG groups had the lowest bacterial residue levels, indicating potent bactericidal activity. Quantitative analysis results were consistent with these conclusions. Figure 9 B, C). Furthermore, transmission electron microscopy (TEM) was used to further observe changes in the ultrastructure of the bacteria. In the DPDMC and DPDMCG groups, the bacteria exhibited cell membrane rupture and cytoplasmic vacuolation. Figure 9 D, E).

[0098] Figure 10 It is similar to existing antibacterial metal ions Zn 2+ Comparative scanning electron microscope images of bacterial biofilms: for in-depth characterization of the existing antibacterial metal ion Zn. 2+To investigate the damage to bacteria embedded in the biofilm, we conducted supplementary experiments. Scanning electron microscopy (SEM) revealed significant biofilm structural defects in the DPDM and DPDC groups, accompanied by decreased bacterial density, bacterial shrinkage, and widespread bacterial fragmentation. In contrast, the bacteria in the control, DPD, and DPDG groups remained densely distributed, with normal size and smooth surfaces, indicating that the DPD and DPDG groups did not exhibit significant antibacterial activity. The antibacterial effect of the DPDZ group was weaker than that of the DPDM, DPDC, and DPDMCG groups. Figure 10 A, B). This indicates that the designed surface modification system outperforms existing antibacterial metal ion properties.

[0099] Figure 11 It is similar to existing antibacterial metal ions Zn 2+ Comparative scanning electron microscope images of bacterial biofilms: for in-depth characterization of the existing antibacterial metal ion Zn. 2+ To investigate the damage to bacteria embedded in the biofilm, we conducted supplementary experiments. Scanning electron microscopy (SEM) revealed significant biofilm structural defects in the DPDM and DPDC groups, accompanied by decreased bacterial density, bacterial shrinkage, and widespread bacterial fragmentation. In contrast, the bacteria in the control, DPD, and DPDG groups remained densely distributed, with normal size and smooth surfaces, indicating that the DPD and DPDG groups did not exhibit significant antibacterial activity. The antibacterial effect of the DPDZ group was weaker than that of the DPDM, DPDC, and DPDMCG groups. Figure 11 A, B). This indicates that the designed surface modification system outperforms existing antibacterial metal ion properties.

[0100] Test Example 7: In vitro osteogenic differentiation induction

[0101] All grouped materials were sterilized by Co-60 irradiation. Materials were placed in the upper chamber of a Transwell apparatus (Corning, American) and incubated in the lower chamber at 1.0 × 10⁻⁶ cm⁻¹. 4Bone marrow mesenchymal stem cells (BMSCs) were collected. When the cell density reached 60%, the cell culture medium was replaced with osteogenic induction medium every 3 days. After 7 days of culture, the cells were washed 1-2 times with PBS and fixed with 4% paraformaldehyde. The samples were then stained using an alkaline phosphatase (ALP) staining kit (Beyotime, China) according to the manufacturer's instructions. Cell morphology was observed under a light microscope. To quantify ALP activity, after 7 days of culture, the culture medium was discarded, and the cells were co-cultured with 0.1% Triton X-100 for 1 minute. After centrifugation, ALP activity was detected using an AKP / ALP kit (Beyotime, China). After 21 days of culture, BMSCs were fixed with 4% paraformaldehyde and stained with an Alizarin Red staining kit (Cyagen, China) according to the manufacturer's instructions. The cells were then observed under a microscope. To determine the amount of calcium deposits, 1 mL of 10% hexadecylpyridine chloride was added to each well after staining to dissolve the stained calcium nodules. One hour later, the absorbance was measured at 562 nm using an ELISA reader.

[0102] Figure 11 The results of osteogenic differentiation ALP and ARS staining are as follows: First, the bioactivity of the material surface was assessed through direct culture experiments using peptide sheets. Compared with the Control group and the single-modification groups (DPD, DPDM, DPDC), the DPDG group loaded with GLP-1RA and the DPDMCG group loaded with bimetallic ions combined with GLP-1RA showed significantly enhanced alkaline phosphatase (ALP) activity and Alizarin Red (ARS) mineralization nodule formation. In particular, the DPDMCG group showed the highest levels of ALP staining depth and ARS calcium nodule deposition, indicating that Mn 2+ / Cu 2+ Synergistic modification of bimetallic ions and GLP-1RA can significantly promote the direct osteogenic differentiation of BMSCs on the material surface. Figure 11 (A, B). Besides the direct osteogenic effect of functionalized surfaces, we hypothesize that macrophage-mediated remodeling of the local immune microenvironment may provide another pathway for promoting osteogenic formation. Further, by simulating the indirect osteogenic effect mediated by the material under immunomodulatory conditions, BMSCs cultured in the medium treated with the DPDMCG material showed significantly better ALP staining intensity and mineralization capacity than other groups. This indicates that the surface modification system achieves a dual osteogenic enhancement effect through both direct induction and indirect immunomodulation.

[0103] Test Example 8: In vivo surface-modified titanium rod implantation experimental modeling

[0104] Male SD rats (2 months old, weighing 200±20g) and male SD rats (20 months old, weighing 450±100g) were purchased from the Animal Experiment Center of the First Affiliated Hospital of the University of Science and Technology of China. All experimental procedures were approved by the Ethics Committee of the First Affiliated Hospital of the University of Science and Technology of China (NO.202602261603000323162). Micro-CT showed that the osteoporosis model of aging was successfully established. Then, titanium rod implantation surgery was performed. The knee joint was fixed with the knee joint flexed at 90°. After layer-by-layer incision of the skin, the knee joint was exposed. A 1.5cm longitudinal incision was made along the medial side of the patella of the knee joint. The medial femoral condyle was bluntly dissected to expose it. A hole was drilled intermittently at low speed (<800rpm) with a 1.8mm drill bit and continuously flushed with physiological saline to prevent heat necrosis. After the hole was enlarged to a diameter of 2.0mm and a depth of 4.0mm, Staphylococcus aureus solution was injected. Then, the titanium rod was vertically implanted to be flush with the joint surface. After layer-by-layer suturing, the area was disinfected with povidone-iodine. Rats were euthanized in batches at 2, 4 and 8 weeks of age, and intact femoral specimens were removed and fixed with 4% paraformaldehyde for further analysis.

[0105] Osteointegration Assessment: The specimen was examined using micro-CT (micro-computed tomography). The micro-CT scanning parameters were set as follows: operating voltage 80 kV, current 385 μA, exposure time 240 ms, and scan thickness 18 μm. After scanning, 3D images were reconstructed using the Feldkamp algorithm with 3D Creator software. After image reconstruction, regions of interest (ROIs) were manually selected layer by layer within the femoral condyle defect to reconstruct 3D images. CTvox was used to analyze bone volume / tissue volume (BV / TV), trabecular width (Th.Th), number of trabeculae (Tb.Th), and bone mineral density (BMD). BV / TV, trabecular width (Th.Th), number of trabeculae (Tb.N), and bone mineral density (BMD) were used to analyze the treatment effect of osteoporotic bone defects. A panoramic image of the entire slice was acquired using a digital slice scanner (KF-PRO-120, KFBIO). Femoral specimens were decalcified with 10% ethylenediaminetetraacetic acid (Sigma Aldrich) for one month after CT scanning, and then paraffin-embedded and sectioned using a Leica SP1600 microtome. All sections were stained with hematoxylin and eosin (HE), Masson's stain, and immunohistochemically. Primary antibodies and corresponding HRP-labeled secondary antibodies were incubated for immunohistochemical (IHC) staining.

[0106] Figure 12The results of three-dimensional reconstruction of bone integration at the bone interface of the rat femoral peptide implant 4 weeks post-surgery are as follows: Micro-CT three-dimensional reconstruction images 4 weeks after implantation show that the unmodified TiO2 group exhibited severe osteolysis and trabecular bone destruction under bacterial infection, with significantly lower bone mass than the uninfected Control group. In contrast, the DPDMCG group demonstrated superior bone repair capacity. Figure 12 A). Quantitative analysis further confirmed this trend. The DPDMCG group showed the highest levels of bone mineral density (BMD), bone volume fraction (BV / TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N) relative to all other groups, significantly higher than the TiO2 group and the single-function modification groups (DPDMC and DPDG). This indicates that the synergistic effect of the bimetallic phenolic network and GLP-1RA effectively reversed infection- and aging-induced bone loss. Figure 12 BE).

[0107] Figure 13 This is an immunohistochemical staining and quantitative analysis of macrophage M1 / M2 phenotypes 5 days post-surgery: To further investigate the polarization state of macrophages in bone tissue at the bone interface caused by the surface-modified implant, dual immunofluorescence staining of CD68+ / CD86+ and CD68+ / CD206+ was performed. Figure 13 A, B). Quantitative analysis of positive area showed that the proportion of CD68+ / CD86+ macrophages was controlled in the DPDMCG group, while the enrichment of CD68+ / CD206+ macrophages was significantly higher than in other infection groups. These observations indicate that the therapeutic effect of DPDMCG is not only related to bacterial control but also to the restoration of the peri-implant immune microenvironment that promotes inflammation resolution and tissue regeneration. Figure 13 C, D).

[0108] Figure 14 The results of continuous osteogenic fluorescent staining of bone tissue are as follows: In addition, sequential fluorescent labeling (Calcein / ARS) of hard tissue sections revealed that the DPDMCG group formed a continuous and thick new bone band, with the largest interval between the red and green fluorescent bands, and its new bone area ratio was significantly higher than that of other groups. Figure 14 A, B). These results combined indicate that DPDMCG can effectively reduce the risk of peri-implant infection and significantly improve osseointegration function in osteoporotic bone tissue under infection stimulation.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention; those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention; and all technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a titanium-based prosthetic coating with both antibacterial and cell burial regulation functions, characterized in that the steps include... include: (1) Take (DOPA)6-PEG5-DBCO lyophilized powder and add it to Tris-HCl buffer to prepare a mixture; immerse the titanium rod after the pretreatment step in the mixture and soak it in the dark for 12-24 hours to obtain a titanium rod with (DOPA)6-PEG5-DBCO covalently bonded to its surface. (2) Prepare an aqueous solution of metal ions using manganese and copper salts, immerse the titanium rod with (DOPA)6-PEG5-DBCO covalently bonded to its surface in the solution, and soak it in the dark for 6-24 hours to obtain surface-loaded (DOPA)6-PEG5-DBCO / Mn. 2+ / Cu 2+ Titanium rods with bimetallic coordination coating; (3) The GLP-1RA peptide was modified with an azide group to obtain N3-GLP-1RA, and then an N3-GLP-1RA solution was prepared using phosphate buffer as a solvent; the surface-loaded (DOPA) 6-PEG5-DBCO / Mn 2+ / Cu 2+ The titanium rod with the bimetallic coordination coating is immersed in N3-GLP-1RA solution, and the titanium-based prosthesis coating is obtained after the reaction is carried out in the dark.

2. The method for preparing a titanium-based prosthetic coating with both antibacterial and cell burial regulation functions according to claim 1, characterized in that, The concentration of the Tris-HCl buffer solution is 5-15 mmol / L, and the pH is 8.4-8.

6.

3. The method for preparing a titanium-based prosthetic coating with both antibacterial and cell burial regulation functions according to claim 1, characterized in that, The pretreatment steps include: polishing the titanium rod with sandpaper, ultrasonicating it with deionized water, treating it with sodium hydroxide aqueous solution for 20-28 hours, removing it, cleaning it until neutral, and drying it with nitrogen.

4. The method for preparing a titanium-based prosthetic coating with both antibacterial and cell burial regulation functions according to claim 1, characterized in that, The metal ion aqueous solution is a mixed aqueous solution containing 4-6 mmol / L MnCl2 and 4-6 mmol / L CuCl2.

5. The method for preparing a titanium-based prosthetic coating with both antibacterial and cell burial regulation functions according to claim 1, characterized in that, In step (2), the pH of the aqueous solution of metal ions is pre-adjusted to 7.1-7.3 before immersing the titanium rod on which (DOPA)6-PEG5-DBCO is covalently bonded.

6. The method for preparing a titanium-based prosthetic coating with both antibacterial and cell burial regulation functions according to claim 1, characterized in that, The preparation method of N3-GLP-1RA includes: dissolving GLP-1RA peptide in phosphate buffer to prepare a 2-10 mg / mL solution, adding N-hydroxysuccinimide azide acetate in an ice bath protected from light, stirring and reacting in the dark for 6-8 hours, removing impurities and freeze-drying to obtain the final product.

7. The method for preparing a titanium-based prosthetic coating with both antibacterial and cell burial regulation functions according to claim 6, characterized in that, GLP-1RA: N-hydroxysuccinimide azide acetate molar ratio 1:1.2-1:2.

0.

8. The method for preparing a titanium-based prosthetic coating with both antibacterial and cell burial regulation functions according to claim 1, characterized in that, The N3-GLP-1RA solution is an N3-GLP-1RA solution with a concentration of 4-6 mg / mL, using 8-12 mmol / L phosphate buffer solution at pH 7.3-7.5 as the solvent.

9. The method for preparing a titanium-based prosthetic coating with both antibacterial and cell burial regulation functions according to claim 1, characterized in that, The phosphate buffer solution is prepared by weighing sodium dihydrogen phosphate and disodium hydrogen phosphate, dissolving them in deionized water, and adjusting the pH of the system to 7.3-7.5 using sodium hydroxide aqueous solution or hydrochloric acid aqueous solution.

10. The application of the titanium-based prosthesis coating prepared by any one of the preparation methods of claims 1-9 in the preparation of materials for regulating the metabolism of aging macrophages or for osteointegration.

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