A ROS-responsive micro-nanostructured titanium implant nanocoating and its preparation method and application

Abstract

The present invention discloses a ROS-responsive micro-nanostructure titanium implant nanocoating and a preparation method and application thereof, and belongs to the field of medical device technology. The ROS-responsive micro-nanostructure titanium implant nanocoating is obtained by self-assembly of a thioether ketone dopamine molecular coating on the surface of a micro-nanostructure implant and a macrophage-targeted functionalized Nb2C MXene nanosheet. The preparation method comprises the following steps: soaking the micro-nanostructure implant in a thioether ketone dopamine solution, adding a macrophage-targeted functionalized Nb2C MXene nanosheet solution under stirring conditions after washing to react, and washing and drying after the reaction to obtain the ROS-responsive micro-nanostructure titanium implant nanocoating. The present invention also discloses the application of the above-mentioned ROS-responsive micro-nanostructure titanium implant nanocoating in the preparation of artificial implants. The present invention solves the problem of periprosthetic bone dissolution faced by orthopedic prosthesis implants, and the preparation method is simple and the materials are easily available.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical devices, and in particular relates to a ROS-responsive micro-nanostructured titanium implant nanocoating and a preparation method and application thereof. Background Art

[0002] Total joint replacement is considered one of the most successful surgeries for treating severe joint diseases. However, for patients after joint replacement, long-term movement and wear of artificial joint prostheses may generate a large number of wear particles, such as titanium and ultra-high molecular weight polyethylene (UHMWPE) particles. At the same time, due to the development of material science, the application of polyethylene liners in knee joint prostheses is currently inevitable in clinical practice. Therefore, UHMWPE particles inevitably gather around the bone-implant interface, seriously affecting the service life of artificial joints, and ultimately leading to aseptic loosening of the prosthesis, requiring revision surgery.

[0003] Due to the oxidative stress response of surrounding cells to the accumulated UHMWPE particles, the level of reactive oxygen species (ROS) in the microenvironment around the prosthesis increases significantly, which not only promotes the formation of osteoclasts, but also hinders the differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts, accelerating the occurrence of periprosthetic bone dissolution. The new two-dimensional nano-Nb2C MXene nanosheets exhibit the ability to spontaneously absorb ROS through their abundant surface oxidation vacancies and reduction properties, which will provide a new strategy for the prevention and treatment of osteolysis.

[0004] Titanium and titanium alloys are usually used as implant materials, but they lack the biological function of promoting bone integration at the prosthesis-bone interface. In the existing technology, physical modification of the surface has gradually become a research hotspot, but the coordinated development of its surface micro-nanostructure morphology and surface modification to achieve titanium implants that inhibit osteolysis and promote bone integration has not been reported.

[0005] Therefore, how to provide a titanium implant that inhibits osteolysis and promotes bone integration is a technical problem that those skilled in the art need to solve urgently. Summary of the invention

[0006] In order to solve the above technical problems, the present invention proposes a ROS-responsive micro-nanostructured titanium implant nanocoating and a preparation method and application thereof.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A ROS-responsive micro-nanostructured titanium implant nanocoating is obtained by self-assembly of a thioetherketone dopamine molecular coating on the surface of the micro-nanostructured implant and macrophage-targeted functionalized Nb2C MXene nanosheets.

[0009] Beneficial effect: The self-assembly process of the nanocoating in the present invention during the preparation process is to use the adhesion effect of the catechol group in the thioether ketone dopamine molecular coating to fix the macrophage-targeted functionalized Nb2CMXene nanosheets on the surface of the micro-nanostructured titanium implant. The obtained ROS-responsive micro-nanostructured titanium implant nanocoating can respond to the high ROS environment derived from periprosthetic bone dissolution, thereby preventing periprosthetic bone dissolution and promoting bone integration at the same time.

[0010] Preferably, the macrophage-targeted functionalized Nb2C MXene nanosheets are obtained by encapsulating Nb2C MXene via electrostatic adsorption of phosphatidylserine and polyethyleneimine.

[0011] More preferably, the method for preparing the macrophage-targeted functionalized Nb2C MXene nanosheets comprises the following steps:

[0012] Nb2AlC was etched in a hydrofluoric acid solution for 48 hours, and then peeled and layered in a tetramethylammonium hydroxide solution. After 100W ultrasound, the dispersion was obtained by centrifugation and washing with deionized water. Then, a polyethyleneimine (PEI) solution was added under magnetic stirring to react. After the reaction, the dispersion was centrifuged and the precipitate was dispersed in an ethanol solution. A phosphatidylserine (PS) chloroform solution was added and mixed evenly. Finally, the solvent was evaporated by vacuum drying to obtain a precipitate, which was then washed and dried to obtain the macrophage-targeted functionalized Nb2C MXene nanosheet.

[0013] More preferably, the concentration of the hydrofluoric acid solution is 9 mol / L;

[0014] The ratio of the added amounts of the Nb2C nanosheet dispersion, the polyethyleneimine solution, and the phosphatidylserine chloroform solution is 1:1:10.

[0015] The concentration of the Nb2C nanosheet dispersion is 2 mg / ml;

[0016] The concentration of the polyethyleneimine solution is 5 mg / ml;

[0017] The concentration of the phosphatidylserine chloroform solution is 10 mg / ml.

[0018] More preferably, the stirring time is 15-45 min, preferably 30 min.

[0019] The centrifugal speed is 8000-12000 rpm, and the centrifugal time is 5-10 min.

[0020] The washing is performed by rinsing with anhydrous ethanol, and the number of times of washing is 2-5 times.

[0021] Beneficial effects: The preparation of macrophage-targeted functionalized Nb2C MXene nanosheets in the present invention achieves better macrophage targeting, thereby reducing the side effects of Nb2C MXene nanosheets on other cells in bone tissue and enhancing the ability to inhibit osteoclastogenesis. The electrostatic adsorption self-assembly strategy is adopted, and the ratio of the addition amount of Nb2C nanosheet dispersion, polyethyleneimine solution, and phosphatidylserine chloroform solution is adjusted to 1:1:10 to achieve the optimal yield of macrophage-targeted functionalized Nb2C MXene nanosheets.

[0022] Preferably, the micro-nanostructure implant comprises titanium or titanium alloy and is obtained by processing under 1064nm laser surface direct writing technology (LSDW).

[0023] Beneficial effects: The present invention uses titanium or titanium alloy micro-nano structure implants, making full use of the biological inertness and biocompatibility of titanium or titanium alloy in the body, while having excellent mechanical strength. This implant has performed well in the field of orthopedics and has been widely used in clinical practice. The micro-nano structure produced by 1064nm laser surface direct writing technology not only enhances the surface biocompatibility of the implant, promotes cell adhesion and proliferation, but also improves the bonding force between the implant and the surrounding bone tissue, accelerating the bone healing process.

[0024] A method for preparing a ROS-responsive micro-nanostructured titanium implant nanocoating comprises the following steps:

[0025] The micro-nanostructure implant is immersed in a thioether ketone dopamine solution, washed, and then added with a macrophage-targeted functionalized Nb2C MXene nanosheet solution under stirring to react. After the reaction is completed, it is washed and dried to obtain the ROS-responsive micro-nanostructure titanium implant nanocoating.

[0026] Beneficial effects: The ROS-responsive micro-nanostructured titanium implant nanocoating prepared by the present invention has excellent biocompatibility and bioactivity. Its thioetherketone dopamine molecular coating can respond to changes in ROS and realize the process of intelligent release of Nb2C MXene nanosheets. At the same time, through the introduction of targeted functionalized Nb2C MXene nanosheets, the implant has superior macrophage targeting, which helps to improve tissue compatibility and reduce inflammatory response. This innovative method provides a new way to improve the biocompatibility and therapeutic effect of implants.

[0027] Preferably, the thioether ketone dopamine molecule is obtained by dehydration condensation of the carboxyl group of the ROS-sensitive thioether ketone and the amino group of dopamine having catechol adhesion;

[0028] The molar mass ratio of the ROS-sensitive thioether ketone to dopamine is 1:2;

[0029] Preferably, the concentration of the thioether ketone dopamine solution is 1-5 mg / ml, more preferably 2 mg / ml;

[0030] The concentration of the macrophage-targeted functionalized Nb2C MXene nanosheet solution is 1-3 mg / ml, more preferably 2 mg / ml.

[0031] Beneficial effects: The preferred thioether ketone dopamine molecules in the present invention exhibit excellent ROS responsiveness and catechol adhesion, and the concentrations of the thioether ketone dopamine solution and the Nb2C MXene solution are selected so that the implant surface is evenly covered and cooperates with each other. Through reasonable ratio and concentration selection, this not only improves the adhesion and stability of the coating, but also optimizes the reaction conditions, ensures the efficiency and repeatability of the preparation process, and lays a solid foundation for the preparation of high-performance ROS-responsive micro-nanostructured titanium implant nanocoatings.

[0032] Preferably, the soaking time is 18-30 hours, preferably 24 hours;

[0033] Preferably, the reaction time is 18-30h, preferably 24h;

[0034] Preferably, the cleaning is performed by washing with anhydrous ethanol;

[0035] The cleaning is performed 2-5 times.

[0036] Beneficial effects: The preferred immersion and reaction time in the present invention ensures that the sulfide ketone dopamine molecules fully react with the Nb2CMXene nanosheets, achieving uniform and stable formation of the coating. By selecting an optimized cleaning agent as anhydrous ethanol and controlling the number of cleanings between 2-5 times, potential residues are effectively removed and the cleanliness of the implant surface is guaranteed. This series of optimization measures not only improves the preparation efficiency, but also ensures the quality and controllability of the resulting ROS-responsive micro-nanostructured titanium implant nanocoating.

[0037] Application of a ROS-responsive micro-nanostructured titanium implant nanocoating in the preparation of artificial implants.

[0038] Preferably, the artificial implant is an artificial implant that inhibits osteolysis and promotes osteointegration.

[0039] Beneficial effects: The ROS-responsive micro-nanostructured titanium implant nanocoating of the present invention has significant advantages in the preparation of artificial implants that inhibit osteolysis and promote bone integration. Through the characteristics of intelligent response to ROS, the coating can achieve precise release of nanosheets in the implant, effectively inhibit the osteolysis process, and improve the stability of the implant to the bone tissue. At the same time, the micro-nanostructure design on the surface of the implant promotes bone cell adhesion and proliferation, which helps to accelerate the bone integration process. This innovative application direction provides new ideas for the design and preparation of artificial implants, and is expected to bring more reliable and lasting therapeutic effects in the field of bone disease treatment and implant repair in clinical practice.

[0040] Compared with the prior art, the present invention has the following advantages and technical effects:

[0041] The present invention guides the functionalized Nb2C nanosheets (PPN) to form a nanocoating (PPN@MNTi) on the surface of a micro-nanostructure implant through the adhesion of catechol groups mediated by ROS-responsive thioether ketone dopamine molecules. The preparation method is simple and easy, and the materials are safe and readily available. The nanocoating provided by the present invention responds to cleavage in a high ROS environment derived from periprosthetic bone dissolution, and locally delivers macrophage-targeted PPN nanosheets to the bone dissolution microenvironment. In addition, the results of osteoclast-related experiments and osteogenic differentiation experiments show that the nanocoating can not only effectively remove reactive oxygen species and inhibit osteoclast formation, but also promote osteogenic differentiation, providing a new method for constructing a prosthesis that has both the function of inhibiting osteolysis and promoting bone integration, solving the problem of periprosthetic bone dissolution currently faced by orthopedic prosthesis implants, and has broad application value in the field of medical biotechnology. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0043] Figure 1 The figure is a schematic diagram of the preparation process of the ROS-responsive micro-nanostructured titanium implant nanocoating of the present invention and the synergistic effect principle of the implant in inhibiting osteolysis and promoting bone integration in an osteolysis mouse model;

[0044] Figure 2 TEM images of Nb2C nanosheets and macrophage-targeted functionalized Nb2C MXene nanosheets (PPN) prepared in Example 1;

[0045] Among them, (a) is Nb2C nanosheet, (b) is macrophage-targeted functionalized Nb2C MXene nanosheet;

[0046] Figure 3The SEM image and main element localization map of the ROS-responsive micro-nanostructured titanium implant nanocoating (PPN@MNTi) obtained in Example 2;

[0047] Figure 4 The Raman and XPS spectra of the ROS-responsive micro-nanostructured titanium implant nanocoating (PPN@MNTi) obtained in Example 2;

[0048] Figure 5 The in vitro ROS scavenging performance of the ROS-responsive micro-nanostructured titanium implant nanocoating (PPN@MNTi) obtained in Example 2 was detected by DCFH-DA staining;

[0049] Figure 6 Schematic diagram of the in vitro TRAP staining experiment of the ROS-responsive micro-nanostructured titanium implant nanocoating (PPN@MNTi) inhibiting osteoclastogenesis and the RT-qPCR analysis of BMDMs cell gene expression obtained in Example 2;

[0050] Among them, (a) is a schematic diagram of RT-qPCR analysis of BMDMs cell gene expression, and (b) is an in vitro TRAP staining experiment for inhibiting osteoclastogenesis;

[0051] Figure 7 The in vitro osteogenic differentiation experiment of the ROS-responsive micro-nanostructured titanium implant nanocoating (PPN@MNTi) obtained in Example 2, and the ALP and ARS staining experiment of BMSCs cells in osteogenic differentiation;

[0052] Figure 8 The bone histological analysis of the ROS-responsive micro-nanostructured titanium implant nanocoating (PPN@MNTi) obtained in Example 2 for inhibiting osteolysis in vivo;

[0053] Fig. 9 Micro-CT results of in vivo osteogenesis experiment of the ROS-responsive micro-nanostructured titanium implant nanocoating (PPN@MNTi) obtained in Example 2. DETAILED DESCRIPTION

[0054] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0056] The embodiment of the present invention discloses a ROS-responsive micro-nanostructure titanium implant nanocoating, which is obtained by self-assembly of a thioether ketone dopamine molecular coating on the surface of the micro-nanostructure implant and a macrophage-targeted functionalized Nb2C MXene nanosheet.

[0057] In a preferred embodiment, the macrophage-targeted functionalized Nb2C MXene nanosheets are obtained by encapsulating Nb2C MXene via electrostatic adsorption of phosphatidylserine and polyethyleneimine.

[0058] In a more preferred embodiment, the method for preparing the macrophage-targeted functionalized Nb2C MXene nanosheets comprises the following steps:

[0059] Nb2AlC was etched in a hydrofluoric acid solution for 48 hours, and then peeled and layered in a tetramethylammonium hydroxide solution. After 100W ultrasound, the dispersion was obtained by centrifugation and washing with deionized water. Then, a polyethyleneimine (PEI) solution was added under magnetic stirring to react. After the reaction, the dispersion was centrifuged and the precipitate was dispersed in an ethanol solution. A phosphatidylserine (PS) chloroform solution was added and mixed evenly. Finally, the solvent was evaporated by vacuum drying to obtain a precipitate, which was then washed and dried to obtain the macrophage-targeted functionalized Nb2C MXene nanosheet.

[0060] More preferably, the concentration of the hydrofluoric acid solution is 9 mol / L;

[0061] The ratio of the added amounts of the Nb2C nanosheet dispersion, the polyethyleneimine solution, and the phosphatidylserine chloroform solution is 1:1:10.

[0062] The concentration of the Nb2C nanosheet dispersion is 2 mg / ml;

[0063] The concentration of the polyethyleneimine solution is 5 mg / ml;

[0064] The concentration of the phosphatidylserine chloroform solution is 10 mg / ml. In a more preferred embodiment, the stirring time is 15-45 min, preferably 30 min.

[0065] The centrifugal speed is 8000-12000 rpm, and the centrifugal time is 5-10 min.

[0066] The washing is performed by rinsing with anhydrous ethanol, and the number of times of washing is 2-5 times.

[0067] In a preferred embodiment, the micro-nanostructure implant comprises titanium or titanium alloy and is obtained by processing under 1064nm laser surface direct writing technology.

[0068] The embodiment of the present invention also provides a method for preparing a ROS-responsive micro-nanostructured titanium implant nanocoating, comprising the following steps:

[0069] The micro-nanostructure implant is immersed in a thioether ketone dopamine solution, and then a macrophage-targeted functionalized Nb2C MXene nanosheet solution is added under stirring conditions for reaction. After the reaction is completed, the micro-nanostructure titanium implant nanocoating is obtained by washing and drying.

[0070] In a preferred embodiment, the thioetherketone dopamine molecule is obtained by dehydration condensation of the carboxyl group of the ROS-sensitive thioetherketone and the amino group of dopamine having catechol adhesion;

[0071] The molar mass ratio of the thioether ketone to dopamine is 1:2.

[0072] In a preferred embodiment, the concentration of the thioether ketone dopamine solution is 1-5 mg / ml, more preferably 2 mg / ml;

[0073] The concentration of the macrophage-targeted functionalized Nb2C MXene nanosheet solution is 1-3 mg / ml, more preferably 2 mg / ml.

[0074] In a preferred embodiment, the soaking time is 18-30 hours, preferably 24 hours;

[0075] In a preferred embodiment, the reaction time is 18-30h, preferably 24h;

[0076] In a preferred embodiment, the cleaning is performed by washing with anhydrous ethanol;

[0077] The cleaning is performed 2-5 times.

[0078] The embodiment of the present invention also provides an application of a ROS-responsive micro-nanostructured titanium implant nanocoating in the preparation of an artificial implant.

[0079] In a preferred embodiment, the artificial implant is an artificial implant that inhibits osteolysis and promotes osteointegration.

[0080] The raw materials in the examples of the present invention are all purchased from commercial sources.

[0081] Example 1

[0082] The preparation of macrophage-targeted functionalized Nb2C MXene nanosheets includes the following steps:

[0083] (1) Nb2AlC (1 g) and hydrofluoric acid (6 ml) were reacted in a 42°C water bath with magnetic stirring for 48 h, and the resulting product was dispersed in water to obtain a Nb2C nanosheet dispersion with a concentration of 2 mg / ml.

[0084] (2) Add 5 ml of polyethyleneimine (PEI) solution (concentration of 2 mg / ml) to 5 ml of the Nb2C nanosheet dispersion obtained in step (1), stir for 30 min, centrifuge and ultrasonically disperse in an ethanol solution, then add 10 ml of phosphatidylserine (PS) chloroform solution (10 mg / ml) to the above dispersion system. After the organic solvent is evaporated by vacuum drying, it is washed three times with deionized water, and then ultrasonically dispersed in 5 ml of deionized water to form a PPN dispersion with a concentration of 2 mg / ml, thus obtaining macrophage-targeted functionalized Nb2C MXene nanosheets (PS / PEI / Nb2C; PPN), which are stored at 4°C for future use.

[0085] The morphology of Nb2C nanosheets and macrophage-targeted functionalized Nb2C MXene nanosheets was examined by TEM ( Figure 2 ), where (a) is Nb2C nanosheet and (b) is macrophage-targeted functionalized Nb2C MXene nanosheet.

[0086] Example 2

[0087] A preparation method of a ROS-responsive micro-nanostructured titanium implant nanocoating comprises the following steps:

[0088] (1) First, titanium sheets (Ti, length × width × height: 10 × 10 × 2 mm) and titanium rods (Ti, Φ10 × 1 mm) purchased from Jiangsu Byers Company were cleaned in acetone (≥99.5%), ethylene glycol (≥99.5%), and ultrapure water under ultrasonic action for 15 min to obtain clean Ti sheets and clean Ti rods. Then, the clean Ti sheets and clean Ti rods were processed with a line spacing of 20 μm using 1064 nm laser surface direct writing technology to form surface micro-nano structures. After washing with anhydrous ethanol for 3 times, micro-nano structure implants (MNTi) were obtained.

[0089] (2) The above-mentioned micro-nanostructure implant is placed in a solution of thioetherketone dopamine molecules (TK-DA) obtained by dehydration condensation of the carboxyl group of ROS-sensitive thioetherketone and the amino group of dopamine with catechol adhesion, with a concentration of 2 mg / ml. After soaking for 24 hours, the implant is washed and the PPN nanosheet dispersion obtained in Example 1 is added and stirred for 24 hours, wherein the molar mass ratio of thioetherketone to dopamine in the thioetherketone dopamine molecule is 1:2. After the reaction is completed, the solid product is collected, washed with ethanol for 3 times, and then placed in an oven for drying to obtain a ROS-responsive micro-nanostructure titanium implant nanocoating (PPN@MNTi), which is then stored at -20°C in an inert gas atmosphere for later use.

[0090] The surface morphology and surface element distribution of PPN@MNTi were obtained by SEM ( Figure 3 ), Raman spectrum shows that TK-DA@MNTi has a -1 There are characteristic peaks at the same time. After depositing Nb2C and PPN on the MNTi substrate, the typical ω4 vibration mode of Nb2C is still retained, which confirms that Nb2C and PPN are successfully grafted on the MNTi surface by TK-DA. XPS analysis of the absorption peaks and element changes of PPN@MNTi and Ti ( Figure 4 ), it can be seen that the PPN@MNTi and Nb2C@MNTi groups show typical Nb 3d peaks compared with the Ti and MNTi groups, which also proves the successful grafting of Nb2C and PPN on the MNTi surface, while the PPN@MNTi group shows the typical P 2d peak of PPN nanosheets compared with the Nb2C@MNTi group, indicating the successful construction of macrophage-targeted functionalized Nb2C MXene nanosheets on MNTi.

[0091] Example 3

[0092] A method for preparing a ROS-responsive micro-nanostructured titanium implant nanocoating is different from Example 2 in that the titanium sheet and titanium rod used in step (1) are replaced with titanium alloy (Jiangsu Baiers Company). The remaining steps and parameters are the same as those in Example 2.

[0093] Comparative Example 1

[0094] A preparation method of Nb2C@MNTi comprises the following steps: The difference from Example 2 is that the PPN nanosheet dispersion used in step (1) is replaced with Nb2C nanosheet dispersion, and the remaining steps and parameters are the same as those in Example 2.

[0095] Technical effect:

[0096] The present invention utilizes bone marrow-derived macrophages (BMDMs) as experimental cells, but is not limited to BMDMs, and various cells of other species are also applicable to the present invention.

[0097] 1. In vitro ROS removal experiment of ROS-responsive micro-nanostructured titanium implant nanocoating (PPN@MNTi)

[0098] Bone marrow-derived macrophages (BMDMs) were induced to differentiate into osteoclasts using α-MEM osteoclast induction medium containing 10% fetal bovine serum, 1% penicillin / streptomycin, osteoclast induction factor M-CSF (30 ng / ml) and RANKL (50 ng / ml) at 37°C and 5% CO2. Subsequently, Ti, MNTi, Nb2C@MNTi obtained in Comparative Example 1, and PPN@MNTi obtained in Example 2 were added to 24-well plates, and 0.5 mL of BMDMs α-MEM osteoclast induction medium cell suspension (2×10 5 cells / ml) for 2 days, RANKL was used to stimulate BMDMs to produce ROS, and then the four samples were stained with the ROS-sensitive probe 2',7'-dichlorofluorescein diacetate (DCFH-DA, 10 μM) for 30 min. The ROS generation of each group was observed by inverted fluorescence microscopy. Figure 5 As shown, the green fluorescence intensity of ROS in the PPN@MNTi group was significantly lower than that in the other groups, indicating that the PPN@MNTi group had better ROS scavenging ability.

[0099] 2. In vitro inhibition of osteoclast differentiation by ROS-responsive micro-nanostructured titanium implant nanocoating (PPN@MNTi)

[0100] The clean Ti sheet (length × width × height: 10 × 10 × 2 mm) obtained in step (1) of Example 2, the Nb2C@MNTi obtained in Comparative Example 1, and the PPN@MNTi obtained in Example 1 were added to the osteoclast induction medium containing 50 ng / ml of RANKL to induce osteoclast differentiation, and were named Ti+RANKL, Nb2C@MNTi+RANKL, and PPN@MNTi+RANKL, respectively. The clean Ti sheet, Ti+RANKL, Nb2C@MNTi+RANKL, and PPN@MNTi+RANKL were added to the 6-well Transwell chamber plate for culturing BMDMs, and 2 mL of BMDMs α-MEM osteoclast induction medium cell suspension (2 × 10 5cells / ml), and the osteoclast induction medium was changed every 2 days for a total of 5 days. Ti is a clean titanium sheet, and each group of samples was treated in the same way for 3 repeated experiments. Subsequently, the tartrate-resistant acid phosphatase (TRAP) staining kit was used for staining. Figure 6 As shown in part a, the TRAP-positive cell area in the PPN@MNTi group was the smallest, indicating that PPN@MNTi had the best effect in inhibiting osteoclastogenesis. Total RNA was extracted using an RNA extraction kit and reverse transcribed to complementary DNA using PrimeScript RT Master Mix. RT-PCR was performed using the Bio-Rad RT-PCR system. The expression levels of DC-STAMP, MMP9, Cathepins K, and TRAP osteoclast-related genes in BMDMs were detected. The primer sequences are shown in Table 1, with GADPH as the internal reference system. The results are shown in Figure 6 As shown in part b, the expression of various osteoclast genes in the PPN@MNTi group was lower than that in other groups, indicating that PPN@MNTi has a good ability to inhibit osteoclast differentiation.

[0101] Table 1

[0102]

[0103]

[0104] 3. In vitro osteogenic differentiation experiment of the ROS-responsive micro-nanostructured titanium implant nanocoating (PPN@MNTi) obtained in Example 2

[0105] The clean Ti sheet (length × width × height: 10 × 10 × 2 mm) obtained in step (1) of Example 2, the clean MNTi obtained in Example 2, the Nb2C@MNTi obtained in Comparative Example 1, and the PPN@MNTi obtained in Example 1 were placed in a 24-well plate and named Ti, MNTi, Nb2C@MNTi, and PPN@MNTi groups, respectively. -8 mol / L) DMEM osteogenic induction medium, and 0.5 mL of BMSCsDMEM osteogenic induction medium cell suspension (5×10 4 cells / ml), and the osteogenic induction medium was changed every 2 days for a total of 7 days. Each group of samples was treated in the same way for 3 repeated experiments. Subsequently, alkaline phosphatase (ALP) and alizarin red solution (ARS) were used as Figure 7 As shown in the figure, the ALP activity and mineralized nodules in the PPN@MNTi group were highly expressed compared with those in other groups, indicating that PPN@MNTi has a good ability to promote osteogenic differentiation.

[0106] 4. In vivo osteolysis inhibition experiment of the ROS-responsive micro-nanostructured titanium implant nanocoating (PPN@MNTi) obtained in Example 2

[0107] The cleaned Ti rod (Φ10×1 mm) obtained in step (1) of Example 2, the Nb2C@MNTi titanium rod obtained in Comparative Example 1, and the PPN@MNTi titanium rod obtained in Example 1 were implanted into the femur of mice, and 10 μl of UHMWPE (1.5×10 7 / μL; Mitsui Petrochemicals) solution was used to induce periprosthetic osteolysis, and they were named Ti+UHMWPE, Nb2C@MNTi+UHMWPE and PPN@MNTi+UHMWPE, respectively. Similarly, the clean Ti rod (Φ10×1mm) obtained in Example 2 was implanted into the femur of mice, and 10μl of normal saline was injected at the same time as the control group. Adult male C57BL / 6J mice aged 6-8 weeks were selected, and the mice were anesthetized with 2% isoflurane during surgery, and the mice were randomly divided into clean Ti rod, Ti+UHMWPE, Nb2C@MNTi+UHMWPE, and PPN@MNTi+UHMWPE groups. Among them, each group of titanium rods was treated in the same way and the experiment was repeated in 3 mice. The intercondylar notch of the distal femur was exposed through medial parapatellar arthrotomy. A hole with a diameter of 1 mm and a depth of 0.5 mm was drilled in the intercondylar notch to enter the medullary cavity. Subsequently, 10 μl of UHMWPE (1.5×10 7 / μL) solution was slowly injected into the medullary cavity to prevent leakage of the suspension. At the same time, Ti, Nb2C@MNTi and PPN@MNTi were implanted in each group respectively. Using the principle of fluid mechanics, the wear particles in the suspension can gradually diffuse to the surface of the implant, and finally the tissue is sutured layer by layer as quickly as possible. Similarly, the difference between the Ti group and the above is that the 10μl UHMWPE used was replaced with 10μl saline, and the other steps and parameters were the same as above.

[0108] Six weeks after surgery, the mice were euthanized, their skin and muscles were stripped, and the femurs containing the implants were removed and fixed with 4% paraformaldehyde for 2 days. The femur specimens were then immersed in a decalcification solution for decalcification for 4 weeks, then paraffin-fixed and sliced. The slices were dehydrated in graded ethanol and TRAP stained using a TRAP staining kit, as shown in Figure 8 As shown in the figure, TRAP staining showed that the osteoclast infiltration in the PPN@MNTi+UHMWPE group was significantly reduced compared with the other groups, indicating that PPN@MNTi has a good function of inhibiting osteoclast formation and significantly inhibits UHMWPE-induced bone dissolution.

[0109] 5. In vivo osteogenesis experiment of ROS-responsive micro-nanostructured titanium implant nanocoating (PPN@MNTi) obtained in Example 2

[0110] The animal surgery process was the same as the in vivo osteolysis inhibition experiment. The mice were euthanized 6 weeks after surgery, the skin and muscles of the mice were peeled off, and the femur containing the implant was removed and fixed with 4% paraformaldehyde for 2 days. Then, the Skyscan 1172 Micro-CT system was used to rotate 0.15° / 180° and scan the bone tissue around the implant at a resolution of 36μm. The Skyscan N Recon software was used for reconstruction, and the CTAN software was used to generate a three-dimensional image. Fig. 9 As shown in the figure, the new bone tissue around the titanium rod in the PPN@MNTi+UHMWPE group was significantly more than that in other groups, indicating that the PPN@MNTi+UHMWPE group has a better ability to promote bone integration in vivo.

[0111] The above are only preferred specific implementations of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A ROS-responsive micro-nanostructured titanium implant nanocoating, characterized in that: It is obtained by self-assembly of thioetherketone dopamine molecule (TK-DA) coating on the surface of micro-nanostructure implant and macrophage-targeted functionalized Nb2C MXene nanosheets; The macrophage-targeted functionalized Nb2C MXene nanosheets are obtained by encapsulating Nb2C MXene with phosphatidylserine and polyethyleneimine through electrostatic adsorption; The thioether ketone dopamine molecule is obtained by dehydration condensation of the carboxyl group of the ROS-sensitive thioether ketone and the amino group of dopamine with catechol adhesion; The molar mass ratio of the ROS-sensitive thioether ketone to dopamine is 1:

2.

2. The ROS-responsive micro-nanostructured titanium implant nanocoating according to claim 1, characterized in that: The micro-nano structure implant comprises titanium or titanium alloy and is obtained by processing under 1064nm laser surface direct writing technology.

3. A method for preparing a ROS-responsive micro-nanostructured titanium implant nanocoating according to any one of claims 1 to 2, characterized in that: The following steps are involved: The micro-nanostructure implant is immersed in a thioether ketone dopamine solution, washed, and then added with a macrophage-targeted functionalized Nb2C MXene nanosheet solution under stirring to react. After the reaction is completed, it is washed and dried to obtain the ROS-responsive micro-nanostructure titanium implant nanocoating.

4. The method for preparing a ROS-responsive micro-nanostructured titanium implant nanocoating according to claim 3, characterized in that: The thioether ketone dopamine molecule is obtained by dehydration condensation of the carboxyl group of the ROS-sensitive thioether ketone and the amino group of dopamine with catechol adhesion; The molar mass ratio of the ROS-sensitive thioether ketone to dopamine is 1:

2.

5. The method for preparing a ROS-responsive micro-nanostructured titanium implant nanocoating according to claim 3, characterized in that: The concentration of the thioether ketone dopamine solution is 1-5 mg / ml; The concentration of the macrophage-targeted functionalized Nb2C MXene nanosheet solution is 1-3 mg / ml.

6. The method for preparing a ROS-responsive micro-nanostructured titanium implant nanocoating according to claim 3, characterized in that: The soaking time is 18-30h; The reaction time is 18-30h.

7. The method for preparing a ROS-responsive micro-nanostructured titanium implant nanocoating according to claim 3, characterized in that: The cleaning is performed by washing with anhydrous ethanol; The cleaning is performed 2-5 times.

8. Use of a ROS-responsive micro-nanostructured titanium implant nanocoating as described in any one of claims 1 to 2 in the preparation of artificial implants.

9. The use according to claim 8, characterized in that: The artificial implant is an artificial implant that inhibits osteolysis and promotes osteointegration.

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