Preparation and application of titanium surface / implant self-assembly coating

Abstract

The invention provides preparation and application of a titanium surface / implant self-assembly coating, and relates to the technical field of high polymer materials. The preparation and application of the titanium surface / implant self-assembled coating specifically comprise the following preparation steps of S1, pretreatment and modification of a titanium substrate, S2, preparation of the self-assembled coating, S3, characterization of a test piece and S4, evaluation of cytocompatibility, and the prepared finished product is applied to promotion of early osseointegration of a titanium implant in a complex implantation microenvironment. The success rate and the stability of the implant are improved. CeNPs and EGCG are modified on the surface of the titanium implant through a layer-by-layer self-assembly (LBL) technology, and the CeNPs and EGCG on the surface of the prepared implant can be slowly released along with degradation of a sandwich-shaped coating, so that the antioxidant osteogenic activity is synergistically exerted, the osseointegration demand of an organism in a complex implantation microenvironment is met, and more possibilities are provided for early-stage osseointegration of the implant.

Description

Technical Field

[0001] This invention relates to the field of polymer materials technology, specifically to the preparation and application of a self-assembled coating for titanium surfaces / implants. Background Technology

[0002] Oral implant restorations are affected to varying degrees by the implantation microenvironment in the bone defect area. In the design and preparation of modified implants, in addition to considering their ability to exert osteoinductive activity, it is also important to pay attention to the inhibitory effect of changes in the microenvironment of the bone defect area on peri-implant osseointegration. Therefore, there is an urgent clinical need to find an effective strategy to improve the current situation.

[0003] Titanium implants possess outstanding mechanical properties and good biocompatibility, making them a primary choice for replacing hard tissue and restoring physiological function. However, due to their bioinertness, these implants lack specific bioactive responses after implantation into bone tissue, limiting their ability to regenerate solid tissue in the face of complex in vivo microenvironments. Increasing evidence suggests that multiple biological processes within bone tissue are coordinated by a delicate balance of redox reactions. Oxidative stress plays a crucial role in bone integration, regardless of whether the body is in a physiological or pathological state. Recent studies have shown that mitochondrial damage induced by reactive oxygen species (ROS) is a major factor hindering osteogenic differentiation of mesenchymal stem cells. Therefore, in the design and fabrication of implant modification strategies, in addition to considering their osteoinductive potential, the inhibitory effect of oxidative stress on peri-implant bone integration should also be considered. Advances in implant surface modification technology have enabled the transformation of titanium-based materials into bioactive implants, which not only significantly strengthens the connection between the implant and bone under physiological conditions but also profoundly impacts osteogenic differentiation under ROS-related pathological conditions such as trauma, diabetes, osteoporosis, and aging. Therefore, there is an urgent need to develop titanium implants that can promote early osseointegration in different microenvironments.

[0004] Cerium oxide nanoparticles (CeNPs) have attracted widespread attention in the biomedical field due to their unique biological properties. CeNPs synthesized via bovine serum albumin (BSA) incubation have significant advantages, including a simple synthesis process, excellent environmental friendliness, and high enzyme mimicry activity. Therefore, these CeNPs show great promise for improving the local microenvironment. Epigallocatechin gallate (EGCG) is the most abundant polyphenolic compound in green tea. It provides electron acceptors and donors for hydrogen bonding through its eight phenolic hydroxyl groups, thus exhibiting anti-inflammatory, antioxidant, and antibacterial biological activities. Numerous studies have shown that the regulation of redox balance plays a crucial role in bone biology. Targeting the production of ROS in bone cells holds promise as an important strategy for promoting bone repair.

[0005] Currently, various bioactive components have been modified onto implant surfaces, but no reports have yet described an antioxidant coating synergistically modified with CeNPs and polyphenol EGCG through layer-by-layer self-assembly (LBL). To address the complex implantation microenvironment at bone defect sites and accelerate osseointegration at the implant interface, this study first synthesized CeNPs with both antioxidant and osteogenic properties, and then regulated and optimized the antioxidant assembly units in the implant surface coating. We first treated a large-grit sandblasting and acid etching (SLA) titanium substrate with polyethyleneimine (PEI) to impart a positive charge. Then, we performed LBL modification on the titanium surface using a polyelectrolyte. Hyaluronic acid (HA), carrying a negative charge, carried EGCG, while carboxylated chitosan (CCS), carrying a positive charge, carried CeNPs. CeNPs and EGCG were then self-assembled onto the titanium implant surface via CCS and HA. CeNPs and EGCG are slowly released as the coating degrades, synergistically exerting antioxidant and osteopromoting effects to meet the body's needs for bone integration in a complex microenvironment. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for preparing and applying a titanium surface / implant self-assembly coating, which solves the problem of poor implant-bone integration in complex implantation microenvironments.

[0007] To achieve the above objectives, the present invention provides the following technical solution: Preparation of a self-assembly coating for a titanium surface / implant, specifically including the following preparation steps:

[0008] S1. Pretreatment and modification of titanium substrate

[0009] S101. Pretreatment of titanium sheets

[0010] Titanium sheets were polished using silicon carbide sandpaper with increasing grit size. The polished titanium surface was then sandblasted and acid-etched to obtain Ti-SLA samples with specific surface morphology.

[0011] S102. Modification of titanium substrate

[0012] The Ti-SLA titanium substrate was immersed in an aqueous solution containing 1.0 mg / mL polyethyleneimine (PEI) for 6 hours, then dried in air for 2 hours, and then thoroughly rinsed with deionized water to obtain the Ti-PEI sample.

[0013] S2. Preparation of self-assembled coatings

[0014] S201. Solution Preparation

[0015] Hyaluronic acid (HA) and carboxylated chitosan (CCS) were dissolved in ultrapure water to form negatively charged and positively charged solutions, respectively. Epigallocatechin gallate (EGCG) of appropriate concentration was mixed with HA to obtain an HA-EGCG composite solution. Cerium oxide nanoparticles (CeNPs) of appropriate concentration were mixed with CCS to obtain a CCS-CeNPs composite solution.

[0016] S202. Coating Assembly

[0017] Ti-PEI titanium substrates were sequentially immersed in HA-EGCG and CCS-CeNPs solutions for 30 minutes each time, and then rinsed twice with ultrapure water. Utilizing the layer-by-layer self-assembly (LBL) principle of polyelectrolytes, HA-EGCG and CCS-CeNPs were alternately deposited on the surface of the titanium substrate to form a sandwich-like coating. The above preparation process was repeated 10 times to obtain the final sandwich-like coating. The final samples were designated as Ti-EGCG and Ti-EGCG@CeNPs, respectively.

[0018] S3. Characterization of the specimen

[0019] S301. Characterization of Chemical Composition

[0020] Fourier transform infrared spectroscopy (FT-IR) was used to characterize carboxylated chitosan (CCS), hyaluronic acid (HA), epigallocatechin gallate (EGCG), and cerium oxide nanoparticles (CeNPs) to determine the chemical structure and composition of these materials.

[0021] S302. Surface morphology observation

[0022] Ti-SLA, Ti-EGCG, and Ti-EGCG@CeNPs specimens were selected, and the surface micromorphology of each group of specimens was observed using scanning electron microscopy (SEM) to verify whether the coating was successfully constructed.

[0023] S303. Hydrophilicity test

[0024] Three samples each of Ti-SLA, Ti-EGCG, and Ti-EGCG@CeNPs were randomly selected, and the hydrophilicity of each experimental group was detected by a water contact angle meter to evaluate the effect of the coating on the hydrophilicity of the titanium surface.

[0025] S4. Cell compatibility assessment

[0026] S401. Cell Culture

[0027] Three samples from different groups were randomly selected, and MC3T3-E1 cells were seeded on the sample surface of a 96-well plate at a density of 5×10³ / well. The cells were cultured in an incubator with 5% CO2 and 37℃ for 1, 3 and 7 days, respectively.

[0028] S402. Cell proliferation detection

[0029] At the corresponding time points, the culture medium was discarded, and 90 μL of α-MEM culture medium and 10 μL of CCK-8 solution were added to each well. The wells were then incubated in the dark for 2 hours. The absorbance value at a wavelength of 450 nm was measured using a microplate reader. The cell proliferation was assessed by the change in absorbance value, and the cell compatibility of the coating was determined.

[0030] Preferably, in step S1, the selected titanium sheets have specifications of Φ5mm×1mm and Φ30mm×1mm.

[0031] Preferably, in S101, the particle sizes of the silicon carbide sandpaper are #600, #800, #1000 and #1200, respectively.

[0032] Preferably, in step S201, the concentrations of both the negatively charged and positively charged solutions are 1.0 mg / mL.

[0033] Preferably, in S202, Ti-EGCG indicates that no CeNPs are incorporated, and Ti-EGCG@CeNPs indicates that CeNPs are present.

[0034] Preferably, in S302, a 1μm scale is used for observation.

[0035] Preferably, the prepared product is used to promote early osseointegration of titanium implants in complex implantation microenvironments, thereby improving the success rate and stability of the implants.

[0036] This invention provides a method for preparing and applying a self-assembly coating for titanium surfaces / implants. It offers the following advantages:

[0037] This invention provides a method for preparing and applying a self-assembled coating for titanium implant surfaces / implants. A sandwich-like coating containing CeNPs and polyphenol EGCG is prepared on the surface of a titanium implant. Polyethylene imine (PEI) provides a positively charged substrate for the coating to adhere to the titanium implant surface. Hyaluronic acid (HA) carries a negative charge and carries EGCG, while carboxylated chitosan (CCS) carries a positive charge and carries CeNPs. CeNPs and EGCG are then modified onto the titanium implant surface using layer-by-layer self-assembly (LBL) technology. The CeNPs and EGCG on the implant surface are slowly released as the sandwich-like coating degrades, thus synergistically exerting antioxidant and osteogenic effects, meeting the body's needs for bone integration in the complex implant microenvironment, and providing more possibilities for early implant osseointegration. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the preparation process of the self-assembled coating on the titanium surface of the present invention;

[0039] Figure 2 This is a schematic diagram of the Fourier transform infrared spectrum of carboxylated chitosan (CCS), hyaluronic acid (HA), epigallocatechin gallate (EGCG), and cerium oxide nanoparticles (CeNPs) of the present invention.

[0040] Figure 3 This is a schematic diagram of SEM detection of titanium surfaces in different experimental groups according to the present invention;

[0041] Figure 4 This is a schematic diagram illustrating the hydrophilicity testing of titanium surfaces in different experimental groups according to the present invention.

[0042] Figure 5 This is a schematic diagram showing the proliferation of MC3T3-E1 cells cultured on titanium surfaces for 1, 3, and 7 days in different experimental groups according to the present invention. Detailed Implementation

[0043] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0044] like Figure 1-5 As shown, this embodiment of the invention provides a method for preparing a self-assembled coating on a titanium surface / implant, specifically including the following preparation steps:

[0045] S1. Pretreatment and modification of titanium substrate

[0046] The selected titanium sheets are Φ5mm×1mm and Φ30mm×1mm in size;

[0047] S101. Pretreatment of titanium sheets

[0048] Titanium sheets were ground using silicon carbide abrasive paper with increasing grit sizes of #600, #800, #1000 and #1200 to achieve a certain roughness on the surface of the titanium sheets. The polished titanium surface was then subjected to sandblasting and acid etching to obtain Ti-SLA samples with specific surface morphology.

[0049] Specifically, through grinding, sandblasting, and acid etching, a micro-nano structure is formed on the surface of the titanium sheet, increasing the surface roughness and helping to improve the adhesion of the coating. The surface morphology after sandblasting and acid etching can promote cell adhesion and proliferation, providing a good foundation for subsequent osseointegration.

[0050] S102. Modification of titanium substrate

[0051] The Ti-SLA titanium substrate was immersed in an aqueous solution containing 1.0 mg / mL polyethyleneimine (PEI) for 6 hours, then dried in air for 2 hours, and then thoroughly rinsed with deionized water to obtain the Ti-PEI sample.

[0052] Specifically, PEI treatment imparts a positive charge to the surface of the titanium substrate, providing conditions for subsequent polyelectrolyte layer-by-layer (LBL) self-assembly, enhancing the stability and uniformity of the coating. At the same time, PEI has good biocompatibility, which can further improve the surface properties of the titanium substrate and provide a favorable environment for cell growth and differentiation.

[0053] S2. Preparation of self-assembled coatings

[0054] S201. Solution Preparation

[0055] Hyaluronic acid (HA) and carboxylated chitosan (CCS) were dissolved in ultrapure water to form negatively charged and positively charged solutions, respectively. Epigallocatechin gallate (EGCG) of appropriate concentration was mixed with HA to obtain an HA-EGCG composite solution. Cerium oxide nanoparticles (CeNPs) of appropriate concentration were mixed with CCS to obtain a CCS-CeNPs composite solution. The concentrations of both the negatively charged and positively charged solutions were 1.0 mg / mL.

[0056] Specifically, HA and CCS, as polyelectrolytes, not only provide charge for self-assembly, but also carry bioactive EGCG and CeNPs, providing the coating with antioxidant and osteogenic functions. The formulation of the solution ensures the uniform dispersion of each component, improving the stability and uniformity of the coating.

[0057] S202. Coating Assembly

[0058] Ti-PEI titanium substrates were sequentially immersed in HA-EGCG and CCS-CeNPs solutions for 30 minutes each time, and then rinsed twice with ultrapure water. Utilizing the layer-by-layer self-assembly (LBL) principle of polyelectrolytes, HA-EGCG and CCS-CeNPs were alternately deposited on the surface of the titanium substrate to form a sandwich-like coating. The above preparation process was repeated 10 times to obtain the final sandwich-like coating. The final samples were designated as Ti-EGCG and Ti-EGCG@CeNPs, respectively. Ti-EGCG indicates no CeNPs doping, and Ti-EGCG@CeNPs indicates the presence of CeNPs.

[0059] Specifically, by repeatedly performing the self-assembly process, the thickness and uniformity of the coating are ensured, improving the stability and functionality of the coating. At the same time, the self-assembly technology enables the synergistic release of EGCG and CeNPs, enhancing the coating's antioxidant properties and promoting bone activity, thus meeting the needs of bone integration in complex microenvironments.

[0060] S3. Characterization of the specimen

[0061] S301. Characterization of Chemical Composition

[0062] Fourier transform infrared spectroscopy (FT-IR) was used to characterize carboxylated chitosan (CCS), hyaluronic acid (HA), epigallocatechin gallate (EGCG), and cerium oxide nanoparticles (CeNPs) to determine the chemical structure and composition of these materials.

[0063] Specifically, FT-IR characterization confirmed the chemical structure of each component, ensuring the composition and function of the coating. This provided a scientific basis for subsequent coating performance evaluation and helped in quality control and optimization of the preparation process.

[0064] S302. Surface morphology observation

[0065] Ti-SLA, Ti-EGCG, and Ti-EGCG@CeNPs specimens were selected, and the surface micromorphology of each group of specimens was observed using scanning electron microscopy (SEM) to verify whether the coating was successfully constructed. A scale bar of 1 μm was used during observation.

[0066] Specifically, SEM observation visually verified the successful construction of the coating, ensuring its uniformity and integrity, and providing an important basis for subsequent evaluation of cell compatibility and osseointegration effects.

[0067] S303. Hydrophilicity test

[0068] Three samples each of Ti-SLA, Ti-EGCG, and Ti-EGCG@CeNPs were randomly selected, and the hydrophilicity of each experimental group was detected using a water contact angle meter to evaluate the effect of the coating on the hydrophilicity of the titanium surface.

[0069] Specifically, the improved hydrophilicity helps cell adhesion and proliferation, promotes bone tissue regeneration and repair. The effect of the coating on improving the biocompatibility of titanium surfaces was evaluated by testing the hydrophilicity, providing an important reference for clinical applications.

[0070] S4. Cell compatibility assessment

[0071] S401. Cell Culture

[0072] Three samples from each of the different groups were randomly selected. MC3T3-E1 cells were seeded on the surface of the samples in a 96-well plate at a density of 5×10³ / well. The cells were cultured in an incubator with 5% CO2 and 37℃ for 1, 3 and 7 days, respectively.

[0073] Specifically, cell culture was used to simulate the growth environment of bone tissue in vivo, providing an experimental basis for evaluating the biocompatibility of the coating. By culturing cells at different time points, the effect of the coating on cell activity was evaluated, providing important data for subsequent evaluation of bone integration.

[0074] S402. Cell proliferation detection

[0075] At the corresponding time points, the culture medium was discarded, and 90 μL of α-MEM culture medium and 10 μL of LCK-8 solution were added to each well. The wells were then incubated in the dark for 2 hours. The absorbance value at a wavelength of 450 nm was measured using a microplate reader. The cell proliferation was assessed by the change in absorbance value, and the cell compatibility of the coating was determined.

[0076] Specifically, CCK-8 assays quantified cell proliferation, providing an objective assessment of the coating's cell compatibility. Based on the cell proliferation results, the coating formulation and preparation process can be further optimized to improve the coating's biocompatibility and bone integration.

[0077] The prepared product is used to promote early osseointegration of titanium implants in complex implantation microenvironments, thereby improving the success rate and stability of the implants.

[0078] Specifically, the coating's antioxidant and osteogenic properties significantly improve the bone integration efficiency of titanium implants in complex microenvironments. The coating's bioactive components help form a stable bone-implant interface, enhancing the implant's long-term stability. Furthermore, the coating can play a positive role under various pathological conditions (such as trauma, diabetes, and osteoporosis), improving the implant's adaptability and application range.

[0079] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a self-assembly coating on a titanium surface / implant, characterized in that, Specifically, the preparation steps include the following: S1. Pretreatment and modification of titanium substrate S101. Pretreatment of titanium sheets The titanium sheet was polished using silicon carbide sandpaper with increasing grit size. Then, the polished titanium surface was sandblasted and acid-etched to obtain a Ti-SLA sample with a specific surface morphology. S102. Modification of titanium substrate The Ti-SLA titanium substrate was immersed in an aqueous solution containing 1.0 mg / mL polyethyleneimine (PEI) for 6 hours, then dried in air for 2 hours, and then thoroughly rinsed with deionized water to obtain the Ti-PEI sample. S2. Preparation of self-assembled coatings S201. Solution Preparation Hyaluronic acid (HA) and carboxylated chitosan (CCS) were dissolved in ultrapure water to form negatively charged and positively charged solutions, respectively. Epigallocatechin gallate (EGCG) of appropriate concentration was mixed with HA to obtain an HA-EGCG composite solution. Cerium oxide nanoparticles (CeNPs) of appropriate concentration were mixed with CCS to obtain a CCS-CeNPs composite solution. S202. Coating Assembly Ti-PEI titanium substrates were sequentially immersed in HA-EGCG and CCS-CeNPs solutions for 30 minutes each time, and then rinsed twice with ultrapure water. Utilizing the layer-by-layer self-assembly (LBL) principle of polyelectrolytes, HA-EGCG and CCS-CeNPs were alternately deposited on the surface of the titanium substrate to form a sandwich-like coating. The above preparation process was repeated 10 times to obtain the final sandwich-like coating. The final samples were designated as Ti-EGCG and Ti-EGCG@CeNPs, respectively. S3. Characterization of the specimen S301. Characterization of Chemical Composition Fourier transform infrared spectroscopy (FT-IR) was used to characterize carboxylated chitosan (CCS), hyaluronic acid (HA), epigallocatechin gallate (EGCG), and cerium oxide nanoparticles (CeNPs) to determine the chemical structure and composition of these materials. S302. Surface morphology observation Ti-SLA, Ti-EGCG, and Ti-EGCG@CeNPs specimens were selected, and the surface micromorphology of each group of specimens was observed using scanning electron microscopy (SEM) to verify whether the coating was successfully constructed. S303. Hydrophilicity test Three samples each of Ti-SLA, Ti-EGCG, and Ti-EGCG@CeNPs were randomly selected, and the hydrophilicity of each experimental group was detected by a water contact angle meter to evaluate the effect of the coating on the hydrophilicity of the titanium surface. S4. Cell compatibility assessment S401. Cell Culture Three samples from different groups were randomly selected, and MC3T3-E1 cells were seeded on the sample surface of a 96-well plate at a density of 5×10³ / well. The cells were cultured in an incubator with 5% CO2 and 37℃ for 1, 3 and 7 days, respectively. S402. Cell proliferation detection At the corresponding time points, the culture medium was discarded, and 90 μL of α-MEM culture medium and 10 μL of CCK-8 solution were added to each well. The wells were then incubated in the dark for 2 hours. The absorbance value at a wavelength of 450 nm was measured using a microplate reader. The cell proliferation was assessed by the change in absorbance value, and the cell compatibility of the coating was determined.

2. The preparation of a titanium surface / implant self-assembly coating according to claim 1, characterized in that: In S1, the selected titanium sheets have specifications of Φ5mm×1mm and Φ30mm×1mm.

3. The preparation of a titanium surface / implant self-assembly coating according to claim 1, characterized in that: In S101, the particle sizes of the silicon carbide sandpaper are #600, #800, #1000 and #1200, respectively.

4. The preparation of a titanium surface / implant self-assembly coating according to claim 1, characterized in that: In S201, the concentrations of both the negatively charged and positively charged solutions are 1.0 mg / mL.

5. The preparation of a titanium surface / implant self-assembly coating according to claim 1, characterized in that: In S202, Ti-EGCG indicates that no CeNPs are incorporated, and Ti-EGCG@CeNPs indicates that CeNPs are present.

6. The preparation of a titanium surface / implant self-assembly coating according to claim 1, characterized in that: In S302, a 1μm scale is used for observation.

7. A self-assembly coating for titanium surfaces / implants, characterized in that: The prepared product is used to promote early osseointegration of titanium implants in complex implantation microenvironments, thereby improving the success rate and stability of the implants.

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